Plant Transcription Factors: Contribution in Development, Metabolism, and Environmental Stress 0323906133, 9780323906135

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Plant Transcription Factors Contribution in Development, Metabolism, and Environmental Stress

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Plant Transcription Factors Contribution in Development, Metabolism, and Environmental Stress Edited by

Vikas Srivastava Department of Botany, School of Life Sciences, Central University of Jammu, Samba, Jammu and Kashmir (UT), India

Sonal Mishra Department of Botany, School of Life Sciences, Central University of Jammu, Samba, Jammu and Kashmir (UT), India

Shakti Mehrotra Department of Biotechnology, Institute of Engineering and Technology, Lucknow, Uttar Pradesh, India

Santosh Kumar Upadhyay Department of Botany, Panjab University, Chandigarh (UT), India

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2023 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-323-90613-5

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Contents List of contributors ............................................................................................................................ xvii About the editors .............................................................................................................................. xxiii Preface ............................................................................................................................................... xxv

Section I Plant transcription factors (TFs) and general aspects CHAPTER 1 Plant transcription factors: an overview of their role in plant life ................................................................................................ 3 1.1 1.2 1.3 1.4 1.5

Aksar Ali Chowdhary, Sonal Mishra, Shakti Mehrotra, Santosh Kumar Upadhyay, Diksha Bagal and Vikas Srivastava Introduction ................................................................................................................ 3 Transcription factors and plant life............................................................................ 4 Transcription factors and stress responses................................................................. 6 Transcription factors and secondary metabolism ...................................................... 9 Conclusion ................................................................................................................ 10 References................................................................................................................. 11

CHAPTER 2 Adaptation of millets to arid land: a special perspective of transcription factors ....................................................................... 21

2.1 2.2 2.3 2.4 2.5 2.6

Alka Bishnoi, Pooja Jangir and Praveen Soni Highlights.................................................................................................................. 21 Abbreviations............................................................................................................ 21 Introduction .............................................................................................................. 22 Distribution of arid land in India and world ........................................................... 23 Millets: climate-smart nutri-cereals ......................................................................... 24 Stress as a limiting factor for crops in the arid zones............................................. 27 Responses of millets to abiotic stresses................................................................... 28 Transcription factors: smart regulators of stress tolerance in millets ..................... 29 2.6.1 WRKY.......................................................................................................... 30 2.6.2 DOF .............................................................................................................. 31 2.6.3 ERF/DREB................................................................................................... 40 2.6.4 NAC ............................................................................................................. 41 2.6.5 bHLH............................................................................................................ 41 2.6.6 ASR .............................................................................................................. 42 2.6.7 bZIP.............................................................................................................. 42 2.6.8 MYB............................................................................................................. 42 2.6.9 SBPs ............................................................................................................. 43

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2.6.10 Other transcription factors ........................................................................... 43 2.7 Harnessing the potential of millet transcription factors .......................................... 43 2.8 Conclusion and future perspectives ......................................................................... 46 Acknowledgments .................................................................................................... 47 Declaration of competing interests .......................................................................... 47 Author contribution .................................................................................................. 47 References................................................................................................................. 47

Section II Plant TFs and development .................................................. 61 CHAPTER 3 Plant transcription factors and root development............................. 63 3.1 3.2 3.3

3.4

Rekha Chouhan, Abhilek Kumar Nautiyal, Nancy Sharma and Sumit G. Gandhi Introduction .............................................................................................................. 63 Plant root architecture and development ................................................................. 64 Transcription factors involved in plant root development ...................................... 64 3.3.1 Root apical meristem ..................................................................................... 64 3.3.2 Lateral roots ................................................................................................... 67 3.3.3 Root hair......................................................................................................... 69 Conclusion ................................................................................................................ 71 Acknowledgments .................................................................................................... 72 References................................................................................................................. 72

CHAPTER 4 The roles of transcription factors in the development of plant meristems .................................................................................. 77 4.1 4.2 4.3 4.4 4.5 4.6

Qingkun Dong and Cui Zhang Introduction .............................................................................................................. 77 Shoot apical meristem.............................................................................................. 77 Axillary meristem..................................................................................................... 79 Flower meristem....................................................................................................... 82 Intercalary meristem................................................................................................. 84 Conclusion and future perspectives ......................................................................... 84 Acknowledgments .................................................................................................... 88 Author contributions................................................................................................. 88 References................................................................................................................. 88

CHAPTER 5 Transcription factors and their role in leaf senescence .................. 93 Jeremy Dkhar and Asosii Paul 5.1 Introduction .............................................................................................................. 93

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5.2 Identification of transcription factor families in senescing leaf transcriptome ............................................................................................................ 94 5.3 Characterization of leaf senescence related TFs families ....................................... 98 5.3.1 No apical meristem (NAM), ATAF1/2, CUP-shaped cotyledon 2 (CUC2) (NAC) TF................................................................... 98 5.3.2 WRKY TF.................................................................................................. 104 5.3.3 APETALA2/Ethylene-responsive element binding protein (AP2/EREBP) superfamily ........................................................................ 107 5.3.4 Basic helix-loop-helix (bHLH) TFs .......................................................... 108 5.3.5 MYB TFs ................................................................................................... 110 5.3.6 Auxin response factor and Auxin/INDOLE-3-acetic acid TFs................. 110 5.3.7 DNA binding-with-one-finger (DOF) proteins ......................................... 112 5.3.8 PSEUDO-response regulators TF .............................................................. 113 5.3.9 VQ TF family............................................................................................. 113 5.3.10 Basic leucine zipper (bZIP) TFs................................................................ 113 5.3.11 Homodomain-leucine zipper (HD-ZIP) TFs ............................................. 114 5.3.12 Plant A/T-rich sequence and zinc-binding protein (PLATZ) TF ............. 115 5.3.13 Growth-regulating factors (GRFS) and GRF-interacting factors (GIFS) ............................................................................................ 115 5.3.14 Teosinte branched 1, Cycloidea, and proliferating cell nuclear antigen binding factor (TCP) TFS............................................................. 116 5.3.15 Homeobox (HB) TFs ................................................................................. 116 5.3.16 C3H (Zn) TFs............................................................................................. 117 5.3.17 GRAS TFs.................................................................................................. 118 5.3.18 CCAAT box-binding TFs .......................................................................... 119 5.3.19 Heat shock factor TFs................................................................................ 119 5.3.20 MADS TFs ................................................................................................. 119 5.3.21 GOLDEN 2, ARR-B, PSR 1 (GARP) family TFs.................................... 120 5.3.22 TRIHELIX TFs .......................................................................................... 120 5.3.23 Arabidopsis response regulator TFs .......................................................... 121 5.3.24 Lateral organ boundaries/asymmetric leaves 2 ......................................... 122 5.3.25 Early flowering 3 (ELF3) TF .................................................................... 122 5.3.26 Ethylene insensitive 3 (EIN3)-like (EIL) TFS .......................................... 122 5.3.27 Brinsensitive 1 (BRI1)-EMS-Suppressor1 (BES1) TF ............................. 123 5.3.28 Calmodulin-binding transcription activator............................................... 123 5.3.29 TIFY TFs.................................................................................................... 124 5.3.30 B-box zinc finger TFs................................................................................ 124 5.4 Conclusion .............................................................................................................. 125 Acknowledgments .................................................................................................. 125 References............................................................................................................... 126

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CHAPTER 6 Plant transcription factors in light-regulated development and UV-B protection.......................................................................... 139 Deeksha Singh, Nevedha Ravindran, Nikhil Job, Puthan Valappil Rahul, Lavanya Bhagavatula and Sourav Datta 6.1 Introduction ............................................................................................................ 139 6.1.1 Transcription factors families involved in light-regulated processes ......... 140 6.1.2 Transcription factors associated with visible light-mediated development in plants .................................................................................. 143 6.1.3 Transcriptional regulation of UV-B signaling in plants ............................. 145 6.1.4 Structural and functional evolution of light-responsive plant transcription factors ..................................................................................... 146 6.1.5 Role of light-regulated transcription factors in other signaling pathways....................................................................................................... 148 6.2 Conclusion .............................................................................................................. 149 References............................................................................................................... 149

CHAPTER 7 Tomato fruit development through the perspective of transcription factors ......................................................................... 159 7.1 7.2 7.3 7.4 7.5 7.6

Vigyasa Singh, Dharitree Phukan and Ujjal Jyoti Phukan Introduction ............................................................................................................ 159 Transcription factors in tomato.............................................................................. 160 MYB transcription factors ..................................................................................... 161 MADS transcription factor..................................................................................... 163 Other transcription factors ..................................................................................... 165 Conclusion and future perspectives ....................................................................... 166 Acknowledgment .................................................................................................... 167 Conflict of interest.................................................................................................. 167 References............................................................................................................... 167

CHAPTER 8 Plant transcription factors and nodule development ...................... 175 Jawahar Singh and Praveen Kumar Verma Introduction ............................................................................................................ 175 CCaMK/CYCLOPS complex................................................................................. 177 AP2-ERF transcription factor (ERN1 and ERN2) ................................................ 179 GRAS transcription factor ..................................................................................... 180 8.4.1 Nodulation signaling pathway 1/2 (NSP1 and NSP2) ................................ 180 8.5 SymSCL1 ............................................................................................................... 181 8.6 NIN and NIN-like proteins .................................................................................... 181 8.7 Structure of NIN and NLPs ................................................................................... 181 8.1 8.2 8.3 8.4

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8.8 Regulation of NIN for rhizobial infection in the epidermis by CYCLOPS ......... 182 8.9 Regulation of NIN by cytokinin-response elements for cell divisions in the pericycle ....................................................................................... 182 8.10 NIN: a master regulator of nodulation .................................................................. 183 8.11 NIN as a negative regulator in systemic control of nodulation ............................ 183 8.12 NIN as a positive regulator in systemic control of nodulation ............................. 184 8.12.1 Lob-domain protein16 ............................................................................... 185 8.12.2 Nodulation pectate lyase 1......................................................................... 185 8.13 Rhizobium-directed polar growth .......................................................................... 186 8.14 Nuclear factor Y..................................................................................................... 186 8.14.1 Short internodes/stylish.............................................................................. 187 8.15 Conclusion and future perspectives ....................................................................... 188 Acknowledgments .................................................................................................. 189 Declaration of competing interest .......................................................................... 189 Contribution ............................................................................................................ 189 References............................................................................................................... 189

Section III Plant TFs and metabolism .................................................. 197 CHAPTER 9 The regulatory aspects of plant transcription factors in alkaloids biosynthesis and pathway modulation............................. 199 9.1 9.2

9.3

9.4

Pravin Prakash, Rituraj Kumar and Vikrant Gupta Abbreviations.......................................................................................................... 199 Introduction ............................................................................................................ 199 Plant transcription factor families involved in alkaloid biosynthesis regulation................................................................................................................ 201 9.2.1 APETALA2/ethylene response factor ......................................................... 201 9.2.2 Basic helix-loop-helix .................................................................................. 205 9.2.3 Basic leucine zipper ..................................................................................... 206 9.2.4 Cys2/His2-type (transcription factor IIIA-type) zinc-finger protein family/Zinc-finger Catharanthus protein (ZCT) family................. 207 9.2.5 Myeloblastosis.............................................................................................. 207 9.2.6 WRKY.......................................................................................................... 208 9.2.7 Other transcription factors ........................................................................... 209 Transcription factor-mediated modulation of alkaloid biosynthesis pathways ........... 209 9.3.1 Overexpression............................................................................................. 209 9.3.2 Downregulation............................................................................................ 210 9.3.3 CRISPR/Cas-mediated genome editing....................................................... 211 Conclusions ............................................................................................................ 212 References............................................................................................................... 212

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CHAPTER 10 Plant transcription factors and flavonoid metabolism .................... 219 Rekha Chouhan, Garima Rai and Sumit G. Gandhi 10.1 Introduction ............................................................................................................ 219 10.2 Plant flavonoids, major subclasses, and biosynthesis ........................................... 220 10.3 Transcription factor families associated with plant flavonoid metabolism .......... 221 10.3.1 Role of basic-helix-loop-helix transcription factors in plant flavonoid metabolism................................................................................. 222 10.3.2 MYB transcription factor family and plant flavonoid metabolism .......... 224 10.3.3 WD40 transcription factors and plant flavonoid metabolism................... 225 10.3.4 Role of basic leucine-zipper transcription factors in plant flavonoid metabolism................................................................................. 226 10.3.5 Role of WRKY transcription factors in plant flavonoid metabolism............ 227 10.4 Conclusions ............................................................................................................ 227 Acknowledgments .................................................................................................. 227 References............................................................................................................... 227

CHAPTER 11 Demystifying the role of transcription factors in plant terpenoid biosynthesis...................................................................... 233 11.1 11.2

11.3

11.4

Ajay Kumar, Parul Sharma, Rakesh Srivastava and Praveen Chandra Verma Introduction ............................................................................................................ 233 Biosynthesis of terpenoids ..................................................................................... 234 11.2.1 Biosynthesis of basic terpenoids precursor (MVA and MEP pathway)........ 235 11.2.2 Biosynthesis of isoprenoid intermediates .................................................. 235 11.2.3 Biosynthesis of terpenes by terpene synthases.......................................... 237 Regulation of terpenoids ........................................................................................ 239 11.3.1 WRKY........................................................................................................ 241 11.3.2 MYB........................................................................................................... 241 11.3.3 bHLH (basic helix
loop
helix) ............................................................... 242 11.3.4 AP2/ERF .................................................................................................... 242 11.3.5 bZIP ............................................................................................................ 243 11.3.6 SPL, YABBY, and other TFs .................................................................... 244 Conclusion .............................................................................................................. 244 Acknowledgment .................................................................................................... 244 References............................................................................................................... 244

CHAPTER 12 The regulatory circuit of iron homeostasis in rice: a tale of transcription factors .......................................................... 251 Pooja Kanwar Shekhawat, Hasthi Ram and Praveen Soni Highlights................................................................................................................ 251

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Abbreviations.......................................................................................................... 251 12.1 Introduction ............................................................................................................ 252 12.2 Iron uptake and transport ....................................................................................... 253 12.3 Major transcription factors involved in iron homeostasis..................................... 255 12.3.1 Regulation of Fe deficiency....................................................................... 255 12.3.4 Regulation of Fe toxicity ........................................................................... 258 12.4 Regulation of the regulators................................................................................... 258 12.4.1 Epigenetic regulation ................................................................................. 259 12.4.2 Regulation at the transcriptional level....................................................... 259 12.4.3 Regulation at the post-transcriptional level............................................... 259 12.4.4 Regulation at the post-translational level .................................................. 259 12.4.5 Regulation by plant hormones................................................................... 260 12.5 Conclusion and future perspectives ....................................................................... 260 Acknowledgments .................................................................................................. 262 Author contribution ................................................................................................ 262 References............................................................................................................... 262

Section IV Plant TFs and Stress........................................................... 269 CHAPTER 13 Impact of transcription factors in plant abiotic stress: a recent advancement for crop improvement.................................. 271 13.1 13.2 13.3 13.4 13.5

13.6

Divya Chauhan, Devendra Singh, Himanshu Pandey, Zeba Khan, Rakesh Srivastava, Vinay Kumar Dhiman and Vivek Kumar Dhiman Introduction ............................................................................................................ 271 Regulatory function of transcription factors in response to abiotic stress............ 271 ABA signaling pathway ......................................................................................... 272 JA signaling pathway ............................................................................................. 274 Transcription factors involved in abiotic stress tolerance..................................... 275 13.5.1 MYB TFs ................................................................................................... 275 13.5.2 NAC TFs .................................................................................................... 275 13.5.3 AP2/ERF TFs ............................................................................................. 276 13.5.4 WRKY TFs ................................................................................................ 277 Conclusion .............................................................................................................. 280 References............................................................................................................... 280

CHAPTER 14 Plant transcription factors and temperature stress ........................ 287 Tingting Zhang and Yang Zhou 14.1 Effect of temperature stress on plant growth ........................................................ 287 14.1.1 Effect of high-temperature stress on plants............................................... 287

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14.1.2 Effect of low-temperature stress to plants................................................. 288 14.2 Transcription factors involved in response to temperature stress......................... 289 14.2.1 HSF transcription factor............................................................................. 289 14.2.2 MYB transcription factor........................................................................... 290 14.2.3 AP2/ERF transcription factors................................................................... 291 14.2.4 WRKY transcription factors ...................................................................... 293 14.3 Conclusions and perspectives ................................................................................ 294 Acknowledgments .................................................................................................. 294 References............................................................................................................... 294

CHAPTER 15 Plant transcription factors and osmotic stress ............................... 301 Tingting Zhang and Yang Zhou 15.1 Effects of osmotic stress on plants and its regulatory mechanism ....................... 301 15.1.1 Stomatal closure......................................................................................... 301 15.1.2 Osmotic regulation mechanism ................................................................. 302 15.1.3 Mechanism of ROS generation and scavenging ....................................... 302 15.1.4 ABA signaling pathway............................................................................. 303 15.2 Transcription factors are involved in regulating osmotic stress ........................... 304 15.2.1 Osmotic stress caused by salt stress .......................................................... 304 15.2.2 Osmotic stress caused by drought stress ................................................... 305 15.2.3 Osmotic stress caused by low temperature ............................................... 305 15.3 Conclusions and perspectives ................................................................................ 306 Acknowledgments .................................................................................................. 307 References............................................................................................................... 307

CHAPTER 16 Transcriptional regulation of drought stress stimulus: challenges and potential for crop improvement ............................. 313 Gyanendra K. Rai, Gayatri Jamwal, Isha Magotra, Garima Rai and R.K. Salgotra 16.1 Introduction ............................................................................................................ 313 16.2 Regulatory role of transcription factors in dry spell tolerance ............................. 314 16.3 Transcription factor and their mechanisms under drought stress ......................... 316 16.3.1 DNA binding with one finger (DOF) ........................................................ 316 16.3.2 WRKY transcription factor........................................................................ 318 16.3.3 Heat shock factor ....................................................................................... 319 16.3.4 Nuclear Factor (NF-Ys) ............................................................................. 320 16.3.5 TCP transcription factor family................................................................. 320 16.3.6 AP2/ERBP.................................................................................................. 322 16.3.7 AREB/ABF family..................................................................................... 323

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16.3.8 NAC transcription factors.......................................................................... 324 16.3.9 MYB/MYC transcription factors ............................................................... 325 16.4 Conclusion and future prospects............................................................................ 327 References............................................................................................................... 327

CHAPTER 17 Plant response to heavy metal stress: an insight into the molecular mechanism of transcriptional regulation ....................... 337 17.1 17.2 17.3 17.4 17.5 17.6 17.7 17.8 17.9 17.10 17.11 17.12 17.13

Mehali Mitra, Puja Agarwal and Sujit Roy Introduction ............................................................................................................ 337 Toxic effects of heavy metals in plants................................................................. 340 Plant signaling in response to heavy metal stress ................................................. 341 MAPK signaling under heavy metal stress ........................................................... 343 Calcium
calmodulin signaling pathway under heavy metal stress ..................... 344 Hormone signaling in response to heavy metal stress .......................................... 345 Reactive oxygen species production and its role in heavy metal stress ............... 346 Role of transcription factors in heavy metal resistance regulation....................... 347 The MYB-family transcription factors under HM stress ...................................... 348 The WRKY-family transcription factors under HM stress ................................... 352 The bZIP-family transcription factors under HM stress ....................................... 353 The AP2/ERF/DREB-family transcription factors under HM stress .................... 354 Conclusion and future perspectives ....................................................................... 355 Acknowledgments .................................................................................................. 355 References............................................................................................................... 355

CHAPTER 18 Plant transcription factors and salt stress ...................................... 369 Tingting Zhang and Yang Zhou 18.1 Effects of salt stress on plants ............................................................................... 369 18.1.1 Osmotic stress ............................................................................................ 369 18.1.2 Ion stress .................................................................................................... 369 18.1.3 Oxidative stress .......................................................................................... 370 18.1.4 Nutritional stress ........................................................................................ 370 18.2 Salt tolerance mechanisms in plants...................................................................... 370 18.2.1 Osmotic regulation mechanism ................................................................. 370 18.2.2 Ion homeostasis mechanism ...................................................................... 371 18.2.3 Reactive oxygen species scavenging mechanism ..................................... 371 18.3 Transcription factors involved in salt stress .......................................................... 372 18.3.1 bHLH transcription factors ........................................................................ 372 18.3.2 bZIP transcription factors .......................................................................... 373 18.3.3 NAC transcription factors.......................................................................... 373

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18.3.4 WRKY transcription factors ...................................................................... 374 18.3.5 MYB transcription factors ......................................................................... 374 18.3.6 Other transcription factors participate in salt stress.................................. 375 18.4 Conclusions and perspectives ................................................................................ 376 Acknowledgments .................................................................................................. 377 References............................................................................................................... 377

CHAPTER 19 Plant transcription factors: important factors controlling oxidative stress in plants .............................................. 383 Shikha Verma, Pankaj Kumar Verma and Debasis Chakrabarty 19.1 Introduction ............................................................................................................ 383 19.2 Oxidative stress and sources .................................................................................. 384 19.2.1 ROS production.......................................................................................... 385 19.3 ROS scavenging ..................................................................................................... 396 19.4 Role of transcription factors in the regulation of stress-responsive genes ........... 397 19.4.1 AP2/ERF family......................................................................................... 398 19.4.2 The bHLH family....................................................................................... 399 19.4.3 MYB family ............................................................................................... 399 19.4.4 The NAC family ........................................................................................ 400 19.4.5 The WRKY family..................................................................................... 400 19.4.6 The bZIP family......................................................................................... 401 19.4.7 The HSF family ......................................................................................... 401 19.5 Conclusion and future prospects............................................................................ 402 Acknowledgments .................................................................................................. 403 References............................................................................................................... 403 Further reading ....................................................................................................... 417

CHAPTER 20 Transcription factors: master regulators of disease resistance in crop plants.................................................................. 419 20.1 20.2 20.3 20.4 20.5 20.6 20.7 20.8

Ravi Ranjan Saxesena, Shreenivas Kumar Singh and Praveen Kumar Verma Introduction ............................................................................................................ 419 Molecular basis of plant
microbe interaction ...................................................... 420 The WRKY family of transcription factors and their functional domain ............ 422 WRKY transcription factors and their role in biotic stress................................... 423 APETELA2/ethylene-responsive factor family of transcription factors............... 427 AP2/ERF family of transcription factors and their role in biotic stress ............... 428 NAC transcription factors and their structural organization ................................. 429 NAC transcription factors and their role in biotic stress ...................................... 430

Contents

20.9 20.10 20.11 20.12 20.13

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bZIP transcription factors....................................................................................... 433 bZIP transcription factors and their role in biotic stress....................................... 433 MYB transcription factor family ........................................................................... 434 MYB transcription factors and their role in biotic stress...................................... 435 Conclusion and future perspectives ....................................................................... 435 Acknowledgments .................................................................................................. 436 Declaration of competing interest .......................................................................... 436 Contribution ............................................................................................................ 436 References............................................................................................................... 436

Index .................................................................................................................................................. 445

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List of contributors Puja Agarwal Department of Botany, Constituent College, Purnea University, Purnia, Bihar, India Diksha Bagal Department of Botany, School of Life Sciences, Central University of Jammu, Samba, Jammu and Kashmir (UT), India Lavanya Bhagavatula Plant Cell and Developmental Biology Laboratory, Department of Biological Sciences, IISER Bhopal, Bhauri, Madhya Pradesh, India Alka Bishnoi Department of Botany, University of Rajasthan, Jaipur, Rajasthan, India Debasis Chakrabarty Molecular Biology and Biotechnology Division, CSIR-National Botanical Research Institute, Lucknow, Uttar Pradesh, India Divya Chauhan Division of Germplasm, ICAR-National Bureau of Plant Genetic Resources, New Delhi, Delhi, India Rekha Chouhan CSIR-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir, India; Guru Nanak Dev University (GNDU), Amritsar, Punjab, India Aksar Ali Chowdhary Department of Botany, School of Life Sciences, Central University of Jammu, Samba, Jammu and Kashmir (UT), India Sourav Datta Plant Cell and Developmental Biology Laboratory, Department of Biological Sciences, IISER Bhopal, Bhauri, Madhya Pradesh, India Vinay Kumar Dhiman Department of Basic Sciences, Dr. YSP UHF Nauni, Solan, Himachal Pradesh, India Vivek Kumar Dhiman Departmnt of Biotechnology, Himachal Pradesh University, Shimla, Himachal Pradesh, India Jeremy Dkhar Plant EvoDevo Laboratory, Agrotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India Qingkun Dong Key Laboratory of Plant Molecular Physiology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Botany, Chinese Academy of Sciences, Beijing, P.R. China

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Sumit G. Gandhi CSIR-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India Vikrant Gupta Plant Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, Uttar Pradesh, India; Academy of Scientific and Innovative Research (AcSIR), CSIR-HRDC Campus, Ghaziabad, Uttar Pradesh, India Gayatri Jamwal School of Biotechnology, S. K. University of Agricultural Sciences & Technology of Jammu, Chatha, Jammu and Kashmir, India Pooja Jangir Department of Botany, University of Rajasthan, Jaipur, Rajasthan, India Nikhil Job Plant Cell and Developmental Biology Laboratory, Department of Biological Sciences, IISER Bhopal, Bhauri, Madhya Pradesh, India Zeba Khan Molecular Biology and Biotechnology Division, CSIR-National Botanical Research Institute, Lucknow, Uttar Pradesh, India Ajay Kumar Molecular Biology and Biotechnology Division, CSIR-National Botanical Research Institute, Lucknow, Uttar Pradesh, India Rituraj Kumar Plant Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, Uttar Pradesh, India; Academy of Scientific and Innovative Research (AcSIR), CSIR-HRDC Campus, Ghaziabad, Uttar Pradesh, India Isha Magotra School of Biotechnology, S. K. University of Agricultural Sciences & Technology of Jammu, Chatha, Jammu and Kashmir, India Shakti Mehrotra Department of Biotechnology, Institute of Engineering and Technology, Lucknow, Uttar Pradesh, India Sonal Mishra Department of Botany, School of Life Sciences, Central University of Jammu, Samba, Jammu and Kashmir (UT), India Mehali Mitra Department of Botany, UGC Centre for Advanced Studies, The University of Burdwan, Golapbag campus, Burdwan, West Bengal, India Abhilek Kumar Nautiyal CSIR-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India

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Himanshu Pandey Department of Biotechnology, Dr. YSP UHF Nauni, Solan, Himachal Pradesh, India Asosii Paul Department of Botany, Nagaland University, Lumami, Nagaland, India Dharitree Phukan ICAR-National Institute for Plant Biotechnology, New Delhi, Delhi, India Ujjal Jyoti Phukan School of Plant Sciences, University of Arizona, Tucson, AZ, United States Pravin Prakash Plant Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, Uttar Pradesh, India Puthan Valappil Rahul Plant Cell and Developmental Biology Laboratory, Department of Biological Sciences, IISER Bhopal, Bhauri, Madhya Pradesh, India Garima Rai CSIR-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir, India Gyanendra K. Rai School of Biotechnology, S. K. University of Agricultural Sciences & Technology of Jammu, Chatha, Jammu and Kashmir, India Hasthi Ram National Institute of Plant Genome Research, New Delhi, Delhi, India Nevedha Ravindran Plant Cell and Developmental Biology Laboratory, Department of Biological Sciences, IISER Bhopal, Bhauri, Madhya Pradesh, India Sujit Roy Department of Botany, UGC Centre for Advanced Studies, The University of Burdwan, Golapbag campus, Burdwan, West Bengal, India R.K. Salgotra School of Biotechnology, S. K. University of Agricultural Sciences & Technology of Jammu, Chatha, Jammu and Kashmir, India Ravi Ranjan Saxesena Plant Immunity Laboratory, National Institute of Plant Genome Research Aruna Asaf Ali Marg, New Delhi, Delhi, India Nancy Sharma CSIR-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir, India Parul Sharma Biological Central Facility, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, Uttar Pradesh, India

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Pooja Kanwar Shekhawat Department of Botany, University of Rajasthan, Jaipur, Rajasthan, India Deeksha Singh Plant Cell and Developmental Biology Laboratory, Department of Biological Sciences, IISER Bhopal, Bhauri, Madhya Pradesh, India Devendra Singh Department of Biotechnology, B.N. College of Engineering and Technology, Lucknow, Uttar Pradesh, India Jawahar Singh Plant Immunity Laboratory, National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, Delhi, India Shreenivas Kumar Singh Plant Immunity Laboratory, National Institute of Plant Genome Research Aruna Asaf Ali Marg, New Delhi, Delhi, India Vigyasa Singh Pharmacology and Toxicology Department, College of Pharmacy, University of Arizona, Tucson, AZ, United States Praveen Soni Department of Botany, University of Rajasthan, Jaipur, Rajasthan, India Rakesh Srivastava Molecular Biology and Biotechnology Division, CSIR-National Botanical Research Institute, Lucknow, Uttar Pradesh, India Vikas Srivastava Department of Botany, School of Life Sciences, Central University of Jammu, Samba, Jammu and Kashmir (UT), India Santosh Kumar Upadhyay Department of Botany, Panjab University, Chandigarh (UT), India Pankaj Kumar Verma Department of Botany, University of Lucknow, Lucknow, Uttar Pradesh, India; French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Israel Praveen Chandra Verma Molecular Biology and Biotechnology Division, CSIR-National Botanical Research Institute, Lucknow, Uttar Pradesh, India Praveen Kumar Verma Plant Immunity Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, Delhi, India

List of contributors

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Shikha Verma Molecular Biology and Biotechnology Division, CSIR-National Botanical Research Institute, Lucknow, Uttar Pradesh, India; French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Israel Cui Zhang Key Laboratory of Plant Molecular Physiology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Botany, Chinese Academy of Sciences, Beijing, P.R. China; College of Life Sciences, University of Chinese Academy of Sciences, Beijing, P.R. China Tingting Zhang School of Horticulture, Hainan University/Key Laboratory for Quality Regulation of Tropical Horticultural Crops of Hainan Province, Haikou, P.R. China Yang Zhou School of Horticulture, Hainan University/Key Laboratory for Quality Regulation of Tropical Horticultural Crops of Hainan Province, Haikou, P.R. China

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About the editors Dr. Vikas Srivastava works as an assistant professor in the Department of Botany, at the Central University of Jammu in India and currently working as ‘Royal Society-Newton International Fellow’ at John Innes Centre, Norwich, United Kingdom. He completed his PhD jointly from the Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP) and Lucknow University, India. Furthermore, he pursued a postdoc at the National Institute of Plant Genome Research (NIPGR) in New Delhi, India. With specializations in various aspects of biotechnology, he has acquired the training and experience to carry out research in diverse fields of plant biology. He has published many articles in journals and books of international repute and worked as a principal investigator for a major project sanctioned by University Grant Commission (UGC) in New Delhi. He is a recipient of various awards and prestigious fellowships. He delivered several invited lectures for institutes of national repute. Dr. Sonal Mishra worked as guest faculty in the Department of Botany at the Central University of Jammu in India. She pursued her PhD jointly from CSIR-CIMAP in Lucknow and Jawaharlal Nehru University in New Delhi. Previously, she completed her postdoc as the Dr. D. S. Kothari-Post Doctoral Fellow (UGC-DSKPDF) and SERB-National Postdoc Fellow (SERB-NPDF) at the School of Biotechnology, University of Jammu in India. She also gained postdoc experience at Jawaharlal Nehru University and NIPGR, in New Delhi, India. With specializations in various aspects of biotechnology, she has acquired the training and experience to carry out research in diverse fields of plant biotechnology and molecular biology. She has published many articles in journals and books of international repute and has received various awards and prestigious fellowships. She has presented her work in several seminars and conferences and received appreciation. Dr. Shakti Mehrotra is a consulting scientist in the Department of Biotechnology at the Institute of Engineering and Technology, Dr. A.P.J. Abdul Kalam Technical University in Lucknow, India. She completed her PhD jointly from CSIR-CIMAP and Lucknow University, India. Furthermore, she pursued her postdoc at the Institute of Engineering and Technology, Lucknow (DBT-PDF) and CSIR-CIMAP (DST Young Scientist). With specializations in various aspects of biotechnology, she has acquired the training and experience to carry out research in the diverse fields of plant biotechnology. At present, she is working in the area of 3D bioprinting and 3D food printing. She has published many articles in journals and books of national and international repute and worked as a principal investigator for major projects sanctioned by the Department of

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About the editors

Science and Technology (DST) and Department of Biotechnology (DBT), Government of India. She is a recipient of various awards and prestigious fellowships. She has delivered several presentations at conferences and workshops and has received appreciation. Dr. Santosh Kumar Upadhyay currently works as an assistant professor in the Department of Botany at Panjab University in Chandigarh, India. Earlier, he was DST-INSPIRE faculty at the National Agri-Food Biotechnology Institute, Department of Biotechnology, Government of India in Mohali, Punjab, India. He completed his doctoral work at the CSIR-National Botanical Research Institute, Lucknow, and was awarded his PhD in biotechnology from UP Technical University, in Lucknow, India. He has been working in plant biotechnology for more than 15 years and currently works in the area of functional genomics. During his doctoral research, he was involved in the characterization of various insect toxic proteins such as lectins, chitin-binding proteins, and others from plant biodiversity. Currently, his research group at PU has characterized numerous important cation transporters, including calcium cation antiporters, monovalent cation transporters, calcium ATPases, and important defense-related protein families such as receptor-like kinases, antioxidant enzymes, of bread wheat. They also characterize long noncoding RNAs related to the abiotic and biotic stress response. He has also demonstrated the method of genome editing in bread wheat using the CRISPR-Cas system for functional genomics studies and has developed a tool Sinder for CRISPR target site prediction. He has authored more than 100 publications, including research papers in leading journals of international repute, national and international patents, book chapters, and books. In recognition of his strong credentials and contributions, he has been awarded the NAAS Young scientist award (2017
18) and NAAS-Associate (2018) from the National Academy of Agricultural Sciences, India, INSA Medal for Young Scientist (2013) from the Indian National Science Academy, India, NASI-Young Scientist Platinum Jubilee Award (2012) from the National Academy of Sciences, India, and Altech Young Scientist Award (2011). He has also been the recipient of the prestigious DST-INSPIRE Faculty Fellowship (2012), SERB-Early Career Research Award (2016) from the Ministry of Science and Technology, Government of India, SBS-MKU Genomics Award (2019) from the Biotech Research Society of India, and several research grants from various funding agencies like SERB, DBT, and CSIR. Dr. Upadhyay also serves as a member of the editorial board and a reviewer of many peer-reviewed international journals.

Preface Transcription factors (TFs) are vital protein molecules involved in biological processes such as growth, development, and the organism’s response to environmental variables. These protein molecules bind to DNA regulatory sequences and regulate the unique expression of each gene in different cell types and during different developmental phases. Analysis and understanding of TF expression and activities can help researchers establish the significance of their roles in various biological processes. Scientific exploration and understanding of TFs and their gene regulation is one of the most dynamic fields of research and technical advancements, giving a wide scope to compile what is known, what is not known, and how the known facts can be harnessed for future research. The principal aim of the book Plant Transcription Factors: Contribution in Development, Metabolism, and Environmental Stress is to provide a resource comprising significant facets of TFs in plant biology. The book intends to provide a methodological reserve highlighting several stirring advancements that may strengthen and expand the reader’s knowledge base of the complexity of transcriptional controls of biological processes. The book comprises four sections and includes chapters that focus on the recent scientific explorations on general aspects of plant TFs and their contribution to plant development, metabolic processes, and stress management. The chapters compile existing key concepts as well as advanced information important for better insight into TF targeting and specificity, the properties of regulatory sequence, and mechanisms of TF action, and present a plant TF information repertoire. The topics selected are diverse from those included in other methodologically oriented books on transcriptional regulation of plant biological processes. Section I of the book includes two chapters (Chapters 1 and 2) that comprise a comprehensive overview of TFs and their role in plant life. Chapter 1 gives general, yet updated information on the types of plant TFs. Chapter 2 discusses the role of plant TFs in the adaption of millet in arid lands. Section II encompasses Chapters 3
8, which focus on transcriptional regulation on plant development in organogenesis (root, meristem, leaves, fruit, and nodule development) and light-regulated developments. This section provides current knowledge on the biological functions performed by various plant TFs and explores the existing molecular data to illustrate how they exert their roles during plant development. Section III consists of four chapters (Chapters 9
12) that comprehensively discuss plant TFs associated with biochemical changes (biosynthesis and regulation of secondary metabolites such as alkaloids, flavonoids, and terpenes) and biochemical developments (such as iron homeostasis). This section sheds light on transcriptional regulation of various biosynthetic pathway genes and signaling cascades to perform highly orchestrated biological functions at the molecular level. Section IV (Chapters 13
20) focuses on plant TFs associated with stress management and mitigation that subsequently leads to stress tolerance and survival of plants. Stress, both biotic and abiotic, causes severe damage to all plants; however, the extent of damage depends upon the growth stage and developmental phase of plants. Chapters in this section cover critical discussions and cross-talks on molecular regulatory strategies of plants, with particular reference to crops to maintain vital physiological activities for their survival under various biotic and abiotic stresses during specific growth stages and developmental phases.

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We express our gratitude to all the authors for their outstanding and cutting-edge contributions to the book. In some instances, this book represents the most current insights, scientific opinions, and perspectives into the transcriptional regulation of various plant processes, underscoring the authors’ generosity in sharing their very recent scientific progress in a bench-side reference format. We would also like to take this opportunity to acknowledge the reviewers of Plant Transcription Factors: Contribution in Development, Metabolism, and Environmental Stress for their generous help and suggestions while reviewing chapter manuscripts. We hope this book is a timely contribution that will serve as an information reserve for the current and next generation of academicians and scientists working to decipher the mysteries of plant biology by studying the intricate nature of transcriptional regulation. Vikas Srivastava Sonal Mishra Shakti Mehrotra Santosh Kumar Upadhyay

SECTION

Plant transcription factors (TFs) and general aspects

I

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CHAPTER

Plant transcription factors: an overview of their role in plant life

1

Aksar Ali Chowdhary1, Sonal Mishra1, Shakti Mehrotra2, Santosh Kumar Upadhyay3, Diksha Bagal1 and Vikas Srivastava1 1

Department of Botany, School of Life Sciences, Central University of Jammu, Samba, Jammu and Kashmir (UT), India 2Department of Biotechnology, Institute of Engineering and Technology, Lucknow, Uttar Pradesh, India 3 Department of Botany, Panjab University, Chandigarh (UT), India

1.1 Introduction Transcription factors (TFs) are the master regulator of gene expression and are mainly associated with plant development, metabolism, and stress management (Yang et al., 2012; Mishra et al., 2013; Srivastava and Verma, 2015; Srivastava et al., 2017; Baillo et al., 2019). TFs perform gene regulatory activities by binding to local or distal cis-elements (commonly known as DNA-binding sites) of genes associated with various biological functions. Once a TF binds on the DNA binding site of a particular DNA sequence, it brings out the activation or repression activity through its activator or repressor domains, respectively. TFs may also have other domains that interact with other proteins, such as other TFs, signaling molecules, etc., which adds to their functional diversity (Phillips and Hoopes, 2008). For instance, the posttranslational modification (phosphorylation) of several TFs has been associated with the MAPK cascade system (Guan et al., 2014). Though TFs are considered the central regulators of the biological functions of plant life, they are also regulated significantly under diverse internal and external stimuli (Srivastava and Verma, 2015; Kumar et al., 2016). Further, many plant TFs are known to regulate multiple functions. For instance, the significance of WRKY and AP2 are reported in stress tolerance and the regulation of plant-specific metabolism (Mishra et al., 2013, 2015; Kumar et al., 2016). A significant portion of the plant genome encodes for TFs, as reported in the case of Arabidopsis, where .5% of the genes in the genome encodes TFs (Arabidopsis Genome Project; Riechmann and Ratcliffe, 2000). Hawkins et al. (2021) generated the genome-scale metabolic pathway databases of 126 algal and plant genomes, including model crops and medicinal plants, considering the significance of TFs. Due to the pivotal role of TFs in the regulation of several molecular events of plant biology, understanding their functional diversity is immensely important and relevant for exploring the molecular biology of plants. Therefore, this book explores plant TFs and their functional potential and significance including updated scientific research on TFs.

Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00003-0 © 2023 Elsevier Inc. All rights reserved.

3

4

Chapter 1 Plant transcription factors: an overview of their role in plant life

1.2 Transcription factors and plant life Several external and internal signals govern plant’s developmental decisions. TFs participate in major regulatory processes that are important for plant’s growth, development, and survival. TFs provide strict regulation at the transcriptional level by modulating the expression of major regulatory pathway genes. Table 1.1 lists some examples of plant TFs involved in regulating different developmental events of plant life. In plant life, seed germination is the initial developmental stage strictly regulated at the molecular level. In addition to dormancy-related factors and plant hormones, the TFs actively regulate seed germination in plants at the molecular level. Some important TFs that control seed germination include bZIP44 (Iglesias-Fern´andez et al., 2013), MADS-box TF AGL21 (Yu et al., 2017), NF-YC TF (Liu et al., 2016), and DOF (DNA-binding with one finger, Ruta et al., 2020). Moreover, a B3-domain TF, FUSCA3 (FUS3), and a GATA-type zinc finger family TF, GATA12, in Arabidopsis thaliana control primary seed dormancy (Chiu et al., 2012; Ravindran et al., 2017). Further, the involvement of two basic helix-loop-helix (bHLH) TFs TCP14 and TCP15 in the regulation of gibberellic acid (GA)-mediated seed germination is also reported (Xu et al., 2020). These TFs interact with EXPANSIN (EXPA) gene promoter and regulate hormone biosynthesis.

Table 1.1 Transcription factors identified for their role in plant development. Plant system

Transcription factors

Function

References

Arabidopsis thaliana

AtWRKY75

Zhang et al. (2018b)

A. thaliana

AtTCP

A. thaliana

AtAP2/ERF2 (DRNL)

A. thaliana

AtGIS

A. thaliana

AtWOX11/12

A. thaliana

AtLBD29

Brassica rapa

BrZFP38

Glycine max L.

GmNAC004

Oryza sativa

OsTCL1

Solanum lycopersicum L.

SlWUS

Overexpression in A. thaliana accelerates flowering Expression in A. thaliana promoted Leaf development Expression in A. thaliana promoted gynoecium development Expression in Nicotiana tabacum regulates the development of glandular trichomes Expression in A. thaliana initiates root primordia/root organogenesis Expression in A. thaliana promoted lateral root emergence Expression in B. rapa promoted flower development Overexpression in A. thaliana promoted lateral root development Expression in A. thaliana induces root hair development Expression in S. lycopersicum assists flower development

Koyama et al. (2017) Dur´an-Medina et al. (2017) Liu et al. (2017)

Hu and Xu (2016)

Porco et al. (2016) Lyu et al. (2020) Quach et al. (2014)

Zheng et al. (2016) Li et al. (2017)

1.2 Transcription factors and plant life

5

Furthermore, the significance of TFs in improving seed germination under stress conditions is also reported. For instance, OsbHLH035 TF is involved in seed germination and seedling recovery after salt stress. The TF is known to regulate both ABA-dependent and ABA-independent activation of OsHKT pathways (OsHKT gene family is associated with salt stress tolerance in rice) (Chen et al., 2018a). Many TFs regulate reproductive development in angiosperms. Flowering is linked with reproduction in angiosperms and regulated by several TFs belonging to different families such as MYB, MADS, WRKY etc. (Kumar et al., 2016; Thomson and Wellmer, 2019; Zhu et al., 2020). In Chrysanthemum morifolium, flowering is regulated by R2R3 MYB and CmMYB2 TFs (Zhu et al., 2020). In A. thaliana, the involvement of R2R3 MYB-like TF MYB30 as a positive regulator of flowering is reported (Liu et al., 2014). Intrinsic crosstalk between MYB30 TF with the FLOWERING LOCUS T (FT) accelerates flowering both in long and short days. Likewise, TaMYB72 identified in wheat was found to be responsible for shortening of flowering time in rice (Zhang et al., 2016a). On the contrary, overexpression of MYB44 and PtrMYB192, other members of the same MYB family, delays flowering in some plants (Jung et al., 2008; Liu et al., 2013). Members of the MYB family also function in association with other TFs; for instance, in Medicago sativa, SPL13 downregulates the expression of MsMYB112, which controls flowering (Gao et al., 2018a). Further, MYB has also been studied for its role in developing flower color. Zhang et al. (2021) have reported PsMYB58 as a positive anthocyanin regulator in tree peony (Paeonia suffruticosa Andr.) flowers. Many investigations have also reported the contribution of MADS TFs in flower development; for example, ZmMADS69 (Liang et al., 2019), LoSVP (Tang et al., 2020), CiMADS43 (Ye et al., 2021), etc. MADS domain TFs (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1)/AGAMOUS-LIKE 20) plays a pivotal role in promoting flowering in Arabidopsis (Borner et al., 2000; Lee et al., 2000). A MADS domain gene (AGL20, AGAMOUS LIKE 20) exhibited activation in shoot apical meristem during its transition to flowering and was regulated by the gibberellin pathway. Further, its constitutive expression in Arabidopsis offers photoperiod-independent flowering (Borner et al., 2000). Similarly, the PeMADS5 reported from bamboo (Phyllostachys edulis) has been found to be responsible for the transition from vegetative to reproductive growth (Zhang et al., 2018a). In addition, APETALA1 (AP1) and LEAFY (LFY) are other significant TFs that promote floral meristem development in Arabidopsis (Mandel et al., 1992; Weigel et al., 1992). Fruit development is a complex process and involves fruit-setting, further cell division and expansion followed by fruit ripening. Fruit ripening is a vital phenomenon addressed mainly for its essentiality in the human diet and for other commercial aspects (Seymour et al., 2013). Fruit ripening involves changes in texture, color, flavor, and fragrance of the fruit. Various TFs regulate these aspects of fruit ripening, and COLORED1, C1, (a Zea mays MYB) was the first TF identified for its role in specifying the purple color of corn kernels (Paz-Ares et al., 1987). In an early study, a strong correlation of MdMYB10 expression and anthocyanin accumulation suggested its role in anthocyanin biosynthesis in apple fruits (Espley et al., 2007). Recently, other MYB family proteins have also been identified for their involvement in ethylene-dependent fruit ripening in tomato (Cao et al., 2020). DNA-binding with one finger or Dof proteins also comprise a well-known TF family associated with fruit ripening (Khaksar et al., 2019). Other vital TFs that regulate fruit ripening and development include FvTCP9; a TEOSINTE BRANCHED 1/CYCLOIDEA/PROLIFERATING4 CELL FACTORS (TCP) family member that governs the fruit ripening process in strawberries (Xie et al., 2020). The significance of NAC1 (SlNAC1) in tomato fruit ripening is also reported by Ma et al. (2014), which inhibited fruit ripening by regulating ethylene synthesis and carotenoid accumulation.

6

Chapter 1 Plant transcription factors: an overview of their role in plant life

In plants, green leaves are the active sites of photosynthesis and thus primarily responsible for plant productivity. The development of leaves depends on plant species, developmental stages, and environmental conditions. However, leaf development is regulated by phytohormones, transcriptional regulators, and tissue mechanical properties (Bar and Ori, 2014). Some of the essential TFs responsible for leaf development are GRF, TCP, and ZIP (Sarvepalli and Nath, 2011; Bou-Torrent et al., 2012; Omidbakhshfard et al., 2015). GRFs, or GROWTH-REGULATING FACTORS, positively affect the lateral organ growth and regulate leaf development (Kim et al., 2003; Omidbakhshfard et al., 2015). Different members of various TF families are involved in leaf development events. For example, the WUSCHEL-RELATED HOMEOBOX (WOX) and YABBY TFs regulate adaxial-abaxial polarity in leaves (Rathour et al., 2020, 2022; Sarojam et al., 2010; Vandenbussche et al., 2009), whereas CINCINNATA-LIKE TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORS (CIN-TCP) family TFs play an essential role in regulating leaf patterning and development (Koyama et al., 2007; Sarvepalli and Nath, 2011; Alvarez et al., 2016). Similarly, the CUP-SHAPED COTYLEDON TFs facilitate organ boundary formation and its organization and play an important role in leaf development (Hasson et al., 2011). Roots facilitate the uptake of water and nutrients from the soil and provide anchorage to the plants in the soil. The TFs that majorly participate in root growth and development belong to the NAC, MYB, bHLH and MADS-box families. NAC1 plays a vital role in regulating lateral root formation in Arabidopsis (Xie et al., 2000). ANR1 and MADS-box TFs regulate the nitrate-induced development of lateral roots (Zhang and Forde, 1998). TFs that participate in root hair formation include bHLH family TFs GLABRA3 (GL3)/ENHANCER OF GLABRA3 (EGL3) (Bernhardt et al., 2003); HB family member GLABRA2 (GL2) (Masucci et al., 1996); MYB family TFs CAPRICE (CPC) (Wada et al., 1997); ENHANCER OF TRY AND CPC (etc1) (Kirik et al., 2004); TRIPTYCHON (TRY) (Schellmann et al., 2002); and WEREWOLF (WER) (Lee and Schiefelbein, 1999).

1.3 Transcription factors and stress responses Many TFs also offer stress tolerance to plants (Table 1.2). They usually bind to the promoters of stress-responsive genes and regulate their expression levels in response to various abiotic and biotic stress conditions in plants. Ng et al. (2018) reported six out of 58 TF families involved in plant response to biotic and abiotic stress. These majorly include AP2/ERF (APETALA2/ethyleneresponsive factor); bHLH (basic helix-loop-helix); MYB (myeloblastosis related); NAC (no apical meristem, NAM; Arabidopsis transcription activation factor, ATAF1/2; cup-shaped cotyledon, CUC2); WRKY; and bZIP (basic leucine zipper). The TF families that majorly control the stress responses in plants include NAC, MYB, WRKY, heat shock factors (HSFs), and APETALA2/ Ethylene responsive element binding factor (AP2/ERF). NAC family members are involved in drought and high salt stress response in rice and Arabidopsis (Shen et al., 2017; Yao et al., 2018). Wang et al. (2018a) have reported that TaNAC30 negatively regulates the wheat resistance to stripe rust (Puccinia striiformis f. sp. tritici (Pst). Its silencing via virus-induced gene silencing inhibited virulent Pst isolates CYR31 colonization and stimulated H2O2 accumulation. In rice, the induction of SNAC3 was observed by drought, heat, salt, and ABA treatment (Fang et al., 2015). Further, its overexpression confers tolerance to heat, drought, and oxidation stress and modulation of ROS

Table 1.2 Exemplification of the contribution of transcription factors in stress challenged plants. Plant system

Transcription factors

Stress

Function

References

Arabidopsis thaliana

AtWRKY30

Heat/ drought

El-Esawi et al. (2019)

A. thaliana

AtWRKY11 and AtWRKY17

ABA/salt/osmotic

A. thaliana

AtJUB1

Drought

Glycine max L.

GmNAC085

Drought

Oryza sativa L.

OsICE1 and OsICE2

Cold

Papaver somniferum

PsAP2

Abiotic/biotic

Saccharum officinarum var. Co740

SoMYB18

Salinity/drought

Solanum lycopersicum L.

SlERF84

Drought/salt

S. lycopersicum L.

SlWRKY3

Salinity

Overexpression in Triticum aestivum L. improved stress tolerance via enhancing antioxidant system, chlorophyll content and expression of stress responsive genes; but lower electrolyte leakage, hydrogen peroxide and malondialdehyde content Expression in A. thaliana improve stress tolerance by enhancing germination percentage and root development Overexpression in Solanum lycopersicum L. improve stress tolerance by maintaining relative water content of leaf, lowering hydrogen peroxide as well as by stimulating expression of SlDREB1, SlDREB2, and SlDELLA Overexpression improve stress tolerance in A. thaliana through reduced transpiration, cell membrane injury and stimulated expression of drought-responsive genes Overexpression in A. thaliana improve stress tolerance by enhancing the expression of coldresponsive genes Overexpression in Nicotiana tabacum enhance tolerance to both abiotic and biotic stress. Expression in tobacco improved stress tolerance by enhancing chlorophyll content, antioxidant activity, and proline content, but reduced accumulation of malondialdehyde Overexpression in A. thaliana enhance stress tolerance through improved ROS-scavenging capability Overexpression in S. lycopersicum L enhance stress tolerance through improved biomass and photosynthesis in transgenic plants, together with increased accumulation of K1 and Ca21but lower Na1 contents in leaves

Ali et al. (2018)

Thirumalaikumar et al. (2018)

Nguyen et al. (2018)

Deng et al. (2017)

Mishra et al. (2015) Shingote et al. (2015)

Li et al. (2018)

Hichri et al. (2017)

(Continued)

Table 1.2 Exemplification of the contribution of transcription factors in stress challenged plants. Continued Plant system

Transcription factors

Stress

Function

References

S. lycopersicum L.

SlNAM1

Chilling

Li et al. (2016)

S. lycopersicum L.

SlWRKY39

Salt/drought/biotic

S. tuberosum L. cv. Spunta

StDREB2

Drought

Sorghum bicolor (Btx623)

SbWRKY30

Drought

Triticum aestivum L.

TaWRKY2

Drought

T. aestivem L.

TaNAC47

Drought/salt/ freezing

T. aestivum L.

TaWRKY93

Salinity/drought/ low temperature

Zea mays (X178)

ZmWRKY106

Drought/heat

Overexpression in tobacco improved stress tolerance by enhancing germination, photosynthesis, and osmolytes contents but minor wilting, reduced ROS in transgenic plants Overexpression in S. lycopersicum L enhances stress tolerance by improving the expression of stress-responsive genes Overexpression in Gossypium barbadense L. enhance stress tolerance through improve biomass, boll number, relative water content, soluble sugars, soluble protein, chlorophyll, proline, gasexchange parameters, and antioxidants enzymes activity, but reduced malondialdehyde, hydrogen peroxide, and superoxide anion Expression in A. thaliana enhance tolerance through improved antioxidant activity, proline content and lower malondialdehyde content Overexpression in transgenic wheat enhance tolerance to stress by improving survival rate, chlorophyll content, soluble sugar and proline content Overexpression in A. thaliana enhances stress tolerance by regulating various stress-responsive genes and changing various physiological indices Overexpression in A. thaliana enhance multiple stress tolerance by maintaining membrane stability osmotic adjustment as well as by regulating transcription of stress-related genes Overexpression in A. thaliana enhances stress tolerance through an improved antioxidant system and regulating stress-related genes via the ABAsignaling pathway

Sun et al. (2015)

El-Esawi and Alayafi (2019)

Yang et al. (2020)

Gao et al. (2018b)

Zhang et al. (2016b) Qin et al. (2015)

Wang et al. (2018b)

1.4 Transcription factors and secondary metabolism

9

homeostasis. Similarly, transgenic Arabidopsis plants overexpressing TaNAC29 also demonstrated enhanced tolerance against salt and drought stress (Huang et al., 2015). MYB family members play important regulatory functions in response to various abiotic and biotic stress conditions. Their regulatory roles in stress responses have been reported in sorghum (Baldoni et al., 2015; Scully et al., 2016); rice (Xiong et al., 2014); maize (Chen et al., 2018b); foxtail millet (Muthamilarasan et al., 2014); and wheat (Shan et al., 2016). TaMYB31 observed the positive regulation of drought resistance in wheat (Zhao et al., 2018). It functions by stimulating genes associated with wax biosynthesis and drought response. Ullah et al. (2020) noticed the induced expression of GhMYB108-like after polyethylene glycol and salt treatments. The significance of WRKY-TFs was observed in response to salt stress (Cai et al., 2017), drought, and heat stress (Wang et al., 2018b; Gao et al., 2018b; El-Esawi et al., 2019). Further, HSFs (Mishra et al., 2019) regulate mainly the temperaturedependent stress conditions in plants such as A. thaliana (Huang et al., 2016) and tomato (Mishra et al., 2002; Giorno et al., 2010). MYB TFs also participate in the regulation of temperature stress response in A. thaliana (Agarwal et al., 2006; Zhao et al., 2017); rice (Su et al., 2010; El-Kereamy et al., 2012); and pear (Li et al., 2012). AP2/ERF family members regulate temperature stress in various plant species (Thomashow, 2010; Akhtar et al., 2012; Lee et al., 2012; Mishra et al., 2015; Yamasaki and Randall, 2016). WRKY TFs regulate temperature stress responses in plants such as wheat (He et al., 2016); Eucalyptus grandis (Fan et al., 2018); Camellia sinensis (Wu et al., 2016); Polygonatum odoratum (Wei et al., 2021); and Medicago sativa (Mao et al., 2020). In a recent study, cold stress regulation and the role of various TFs in plants was reviewed (Mehrotra et al., 2020).

1.4 Transcription factors and secondary metabolism Plant TFs regulate the expression of secondary metabolic pathway genes and thereby affect the biosynthesis of secondary metabolites in plants (Yang et al., 2012; Srivastava et al., 2017; Table 1.3). The TFs participate in regulating the biosynthesis of various important secondary metabolites, including alkaloids, terpenoids, and phenolics. The members of APETALA2/ethylene response factor (AP2/ERF), basic helix-loop-helix (bHLH), basic leucine zipper (bZIP), Cys2/His2-type (transcription factor IIIA-type) zinc finger protein family/Zinc finger Catharanthus protein (ZCT) family, MYB, and WRKY TF families majorly participate in the regulation of several secondary metabolic pathways (Yang et al., 2012; Mishra et al., 2013). MYB TFs regulate biosynthesis of anthocyanins (Paz-Ares et al., 1987; Shin et al., 2016); flavonoids (Saito et al., 2013; Ma and Constabel, 2019); glucosinolates (Celenza et al., 2005); and several other phenylpropanoids and lignins (Tamagnone et al., 1998; Bhatia et al., 2019). AP2/ERF TFs regulate the secondary metabolite biosynthesis in Artemisia annua (Tan et al., 2015), Taxus cuspidata (Dai et al., 2009), Lithospermum erythrorhizon (Zhang et al., 2011), Catharanthus roseus (Singh et al., 2020), tobacco (Sears et al., 2014), Ophiorrhiza pumila (Udomsom et al., 2016), and Solanum lycopersicum (Nakayasu et al., 2018). WRKY TFs majorly regulate the phenylpropanoids, alkaloids, and terpene biosynthetic pathways. The phenolic compound biosynthesis is regulated by WRKY TFs as reported in Medicago truncatula (Naoumkina et al., 2008) and grapevine (Guillaumie et al., 2010). WRKY actively regulate terpenoid indole alkaloid (TIA) biosynthesis in C. roseus (Suttipanta et al., 2011); benzylisoquinoline alkaloid (BIA) biosynthesis in Coptis japonica (Kato et al., 2007)

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Chapter 1 Plant transcription factors: an overview of their role in plant life

Table 1.3 Examples of transcription factors in secondary metabolism. Plant system

Transcription factors

Secondary metabolites

References

Arabidopsis thaliana

AtPAP1

Sitarek et al. (2018)

Artemisia annua

AabHLH112

Catharanthus roseus

CrERF5

C. roseus

CrBIS2

Dendrobium officinale Nicotiana tabacum L. Ophiorrhiza pumila

DobHLH4 NtPMT1

O. pumila

OpWRKY3

Salvia miltiorrhiza

SmbZIP1

S. miltiorrhiza

SmbHLH148

S. miltiorrhiza

SmMYC2

S. tuberosum L.

StWRKY8

Overexpression in Leonurus sibiricus L. increase the production of Chlorogenic acid, neochlorogenic acid, ferulic acid, caffeic acid, and p-coumaric acid Overexpression, A. annua promoted artemisinin production Overexpression enhance anhydrovinblastine, vinblastine, ajmalicine, vindoline and catharanthine content Overexpression in C. roseus enhance the production of vincristine and vinblastine Overexpression in Dendrobium officinale increase linalool production Overexpression in Nicotiana tabacum L. enhance the production of nicotine Overexpression in O. pumila increase camptothecin production Overexpression in O. pumila increase camptothecin production Overexpression in S. miltiorrhiza promoted phenolic acids and tanshinones biosynthesis Overexpression in S. miltiorrhiza promoted caffeic acid, rosmarinic acid, salvianolic acid B, dihydrotanshinone I, cryptotanshinone, and tanshinone I Overexpression in S. miltiorrhiza induce production of salvianolic acid B Expression in S. tuberosum L. increase the production of morphinone, codeine-6glucuronide and morphine-3-glucuronides

OpWRKY2

Xiang et al. (2019) Pan et al. (2019)

Van Moerkercke et al. (2016) Yu et al. (2021) Yang et al. (2016) Hao et al. (2021) Wang et al. (2019) Deng et al. (2020) Xing et al. (2018)

Yang et al. (2017) Yogendra et al. (2017)

and Papaver somniferum (Mishra et al., 2013); taxol biosynthesis in Taxus chinensis (Li et al., 2013); and camptothecin biosynthesis in Ophiorrhiza pumila (Wang et al., 2019). WRKY regulates terpene biosynthesis in A. annua (Han et al., 2014), Panax quinquefolius (Sun et al., 2013), and Hevea brasiliensis (Singh et al., 2017; Wang et al., 2013). Further, LIM has also been reported to regulate the Phenyl-propanoid pathway (Srivastava and Verma, 2017). In addition, other TFs such as basic helix-loop-helix (bHLH), basic leucine zipper (bZIP), Dof, Cys2/His2-type (transcription factor IIIA-type) zinc finger protein family also participate in the regulation of secondary metabolite biosynthesis in plants.

1.5 Conclusion TFs are a vital regulator of many aspects of plant life, and focused research has determined their role in plant development (seed germination to fruit development); stress (abiotic and biotic stress);

References

11

and metabolism (primary and secondary metabolism). MADS, NAC, MYB, WRKY, AP2, and so forth have all been explored and TFs have been proven to play a key role in gene regulation. Considering the high value of TFs for the plant itself and their commercial aspects, the following topics are presented in the upcoming chapters of this book: • • • • • • • • • • • • • • • • • • •

Adaptation of millets to arid land: a special perspective of TFs. Plant transcription factor and root development. The Roles of TFs in the development of plant meristems. Plant transcription factor and leaf senescence. Plant TFs in light-regulated development and UV-B protection. Tomato fruit development through the perspective of TFs. Plant TFs and nodule development. Regulatory aspects of plant TFs in alkaloid biosynthesis and pathway modulation. Plant TFs and flavonoid metabolism. Plant TFs and terpene alkaloids. Regulatory circuit of iron homeostasis in rice: a tale of TFs. Impact of TFs in plant abiotic stress: a recent advancement for crop improvement. Plant TFs and temperature stress. Plant TFs and osmotic stress. Transcriptional regulation of drought stress stimulus: challenges & potential for crop improvement. Plant response to heavy metal stress: an insight into the molecular mechanism of transcriptional regulation. Plant TFs and salt stress. Plant TFs and oxidative stress. TFs: master regulators of disease resistance in crop plants.

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82. Srivastava, V., Verma, P.K., 2015. Genome wide identification of LIM genes in Cicer arietinum and response of Ca-2LIMs in development, hormone and pathogenic stress. PLoS One 10, 0138719. Srivastava, V., Mehrotra, S., Verma, P.K., 2017. Biotechnological interventions for production of therapeutic secondary metabolites using hairy root cultures of medicinal plants. Current Developments in Biotechnology and Bioengineering. Elsevier, pp. 259
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158. Sun, Y., Niu, Y., Xu, J., Li, Y., Luo, H., Zhu, Y., et al., 2013. Discovery of WRKY transcription factors through transcriptome analysis and characterization of a novel methyl jasmonate-inducible PqWRKY1 gene from Panax quinquefolius. Plant Cell, Tissue and Organ Culture 114, 269
277. Sun, X.C., Gao, Y.F., Li, H.R., Yang, S.Z., Liu, Y.S., 2015. Over-expression of SlWRKY39 leads to enhanced resistance to multiple stress factors in tomato. Journal of Plant Biology 58, 52
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Chapter 1 Plant transcription factors: an overview of their role in plant life

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18. Zhang, L., Chen, L., Yu, D., 2018b. Transcription factor WRKY75 interacts with DELLA proteins to affect flowering. Plant Physiology 176, 790
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288. Zhao, Y., Tian, X., Wang, F., Zhang, L., Xin, M., Hu, Z., et al., 2017. Characterization of wheat MYB genes responsive to high temperatures. BMC Plant Biology 17, 208. Zhao, Y., Cheng, X., Liu, X., Wu, H., Bi, H., Xu, H., 2018. The wheat MYB transcription factor TaMYB31 is involved in drought stress responses in Arabidopsis. Frontiers in Plant Science 9, 1426. Zheng, K., Tian, H., Hu, Q., Guo, H., Yang, L., Cai, L., et al., 2016. Ectopic expression of R3 MYB transcription factor gene OsTCL1 in Arabidopsis, but not rice, affects trichome and root hair formation. Scientific Reports 6 (1), 1
12. Zhu, L., Guan, Y., Liu, Y., Zhang, Z., Jaffar, M.A., Song, A., et al., 2020. Regulation of flowering time in chrysanthemum by the R2R3 MYB transcription factor CmMYB2 is associated with changes in gibberellin metabolism. Horticulture Research. 7, 1
10.

CHAPTER

Adaptation of millets to arid land: a special perspective of transcription factors

2

Alka Bishnoi , Pooja Jangir and Praveen Soni Department of Botany, University of Rajasthan, Jaipur, Rajasthan, India

Highlights • • • •

Multistress tolerant millets are the repository of superior alleles of stress-responsive transcriptional regulators. Transcription factors are one of the key players behind the hardiness of millets. Due to the unavailability of annotated genomes and optimized transformation protocols, functional validation of TFs of millets is a challenge for the scientific community. Millets’ transcription factors can prove remarkable in crop improvement programs.

Abbreviations AA ABA ASR AO AP2/ERF ASM bHLH bZIP Ca21 CBF1 Cd CDPKs CRISPR/Cas9 CRT CTD CUC DBD DOF 

Amino acid Abscisic acid Abscisic acid stress ripening Ascorbate oxidase Apetala2/Ethylene response factor Allele-specific marker Basic/helix-loop-helix Basic leucine zipper proteins Calcium C-repeat binding factor1 Cadmium Calcium-dependent protein kinases Clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9 C-repeat C-terminus domain Cup-shaped cotyledon DNA binding domain DNA-binding one zinc finger

Equal contribution.

Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00018-2 © 2023 Elsevier Inc. All rights reserved.

21

22

Chapter 2 Adaptation of millets to arid land

DRE DREB ERF EREBP GRAS HD-Zip HKT1 HSPs MAPKs MNF1 MNB Myb NAC NACRS NAM NF-Y NUE O2 PBF PEG ROS RWC S/T-K SOS RT-qPCR SNP SOS TF(s) SBPs TTF WDS

Dehydration responsive element Dehydration responsive element binding protein Ethylene-response factors Ethylene-responsive element-binding protein Gibberellic acid insensitive, repressor of ga1
3 mutant and scarecrows Homeodomain leucine zipper High-affinity K1 transporter 1 Heat shock proteins Mitogen-activated protein kinases Maize nuclear factor1 MNF binding protein v-myb avian myeloblastosis viral oncogene homolog NAM, ATAF1/2, and CUC1/2 NAC recognized sequence No apical meristem Nuclear factor Y Nutrient use efficiency Opaque2 Prolamin box binding factor Polyethylene glycol Reactive oxygen species Relative water content Serine/threonine kinases Salt overly sensitive pathway Real-time quantitative polymerase chain reaction Single nucleotide polymorphism Salt overly sensitive Transcription factor(s) SQUAMOSA promoter binding proteins Trihelix transcription factors Water deficit stress

2.1 Introduction Abiotic stresses (primarily heat, drought, salinity, cold, and heavy metal stress) are major threats to overall crop health and productivity as they adversely affect the growth and survival of plants. Issues of climate change are causing temperature rise and water scarcity, which is severely affecting agricultural output. Abiotic stresses induce various morphological, physiological, biochemical, and molecular changes in plants. Decreased seed germination, retarded growth, and development, wilting and burning of leaves, ion toxicity, nutrient and water disequilibrium, and reduced photosynthetic efficiency are some of the major impacts on plants (Yadav et al., 2020). Oxidative stress due to formation of ROS (reactive oxygen species) is major secondary stress generated owing to drought, salt, and extreme temperatures. Excessive ROS molecules damage cellular and organelle membranes through lipid peroxidation. Plants make a variety of adjustments at metabolic and physiological levels to cope up with the drastic effects of stresses like an increase in the levels of enzymatic and nonenzymatic antioxidants, accumulation of osmolytes and heat shock proteins (HSPs), ion homeostasis (via SOS/salt overly sensitive pathway), etc. (Madhu, 2021, 2022; Singh, 2019;

2.2 Distribution of arid land in India and world

23

Tyagi, 2017, 2018, 2020, 2021; Yadav et al., 2020; Zhu, 2016). Calcium (Ca21) ions, ROS (at low concentration), and hormones act as important signaling molecules under stress in plants (Baillo et al., 2019; Choudhury et al., 2017; Dixit, 2019; Kaur, 2021; Kaur et al., 2022; Tyagi et al., 2019; Upadhyay, 2021). After perception by sensors downstream signaling components such as CDPKs (calcium-dependent protein kinases), S/T-K (Serine/threonine kinases), and MAPKs (mitogen-activated protein kinases) get activated in an ABA (Abscisic acid) dependent or independent manner (Baillo et al., 2019; Devireddy et al., 2021; Shumayla, 2019, 2022; Taneja and Upadhyay, 2021; Upadhyay, 2022; Zhu, 2016). Stress is managed by the integrated and complex action of two types of genes. The first are those whose products are directly involved in stress mitigation and the second ones are the regulatory genes encoding transcription factors (TFs) or microRNAs, which control the gene expression of the former ones (Lata et al., 2011). In signaling cascades, TFs are the final players activating or suppressing the target genes by binding to their upstream cis-elements (Zhu, 2016; Baillo et al., 2019). The functions of major TF families in the regulation of abiotic stress have been comprehensively discussed in recent reviews (Li et al., 2020; Shahzad et al., 2021). TFs of major cereal crops (rice, wheat) have gained more research focus (Santos et al., 2011; Baillo et al., 2019; Ali et al., 2020). In this chapter, we aim to decipher the role of TFs under various stresses in millets, which are small grain cereals and have been proven to be more stress-resistant (specifically against drought and heat) than other crops (Pathan et al., 2004; Setimela et al., 2007; Lata, 2015; Hadebe et al., 2017; Nagaraju et al., 2020). Sorghum has also been included as it is a hardy crop of arid environment and is among the top five cereal crops of the world. We have highlighted the research gaps and possible future applications of the TFs of millets in crop improvement programs.

2.2 Distribution of arid land in India and world The terms arid lands and drylands are used interchangeably. To understand the agroclimatic signature of these regions of the Earth, UNESCO (1979) proposed a classification on the basis of a ratio of annual precipitation (P) to annual potential evapotranspiration (PET), which is widely accepted as an international standard. According to this measure, there are four categories of the global arid zones. These are hyperarid, arid, semiarid, and semihumid zones. Except for the hyperarid zone, India harbors the rest of all three. UNEP (1992) also categorized dry lands into hyperarid, arid, semiarid, and dry subhumid areas based on aridity ratio (AI, AI 5 P/PET). Over 46% of the total global land area falls under these categories, which is home to about 3 billion people (van der Esch et al., 2017). Another category “very dry area” has been adopted by the FAO with reference to the criteria of the type of soil (FAO, 1995). This approach defines soil according to the presence of soil moisture regimes. Soils of drylands are arid (xerosol) or very arid soil (yermosols). This area in India covers only 4%, which is 145,290 square kilometers of country. Continent-wise distribution shows that a prominent portion of the arid zones is occupied by Africa and Asia continents, which is over 60% of the total global arid zone (Fig. 2.1) (UNEP, 1992). The dryland is a major area for world millet production (B70%). The production of food in drylands is threatened by land degradation, extreme temperatures, rainfall depreciations, groundwater table drop, etc. Fig. 2.2 shows the trend of production, yield, and cultivation area of millets from 1961 to 2019 (FAOSTAT, 2021). The yield of millets has increased over the years despite a decrease in the cultivation area.

24

Chapter 2 Adaptation of millets to arid land

FIGURE 2.1 Continent-wise distribution of total global arid zones. Adapted from UNEP (1992) World Atlas of Desertification. United Nations Environment Programme. Edward Arnold, London.

FIGURE 2.2 Global status of the production, yield, and cultivated area of millets during 1961
2019. Adapted from FAOSTAT (2021) Available at: http://www.fao.org/faostat/en/#data, Food and Agriculture Organization of the United Nations.

2.3 Millets: climate-smart nutri-cereals Minor grain cereals, millets, and sorghum are members of the grass family Poaceae formerly known as Gramineae. This family has been divided into four subfamilies, namely pooideae, bembusoideae, panicoideae, and chloridoideae (Graybosch, 2004). Panicoideae includes sorghum (tribe andropoganeae), pearl millet, and seven other minor millets, namely broomcorn/proso millet, foxtail or Italian millet, fonio

2.3 Millets: climate-smart nutri-cereals

25

millet, little millet, Indian barnyard (sawa) millet, Japanese barnyard millet, and kodo millet (tribe paniceae). Finger millet (tribe chlorideae) and teff (tribe aragrosteae) are members of chloridoideae (Graybosch, 2004). Millets vary in their ploidy level ranging from diploid (such as pearl millet) to hexaploid (fonio, Digitalis iburua) conditions (Tadele, 2016). They are well known for their high nutritional qualities characterized by high levels of micronutrients (high iron, zinc, calcium, and phosphorus), vitamins, antioxidants, polyphenols, bioactive compounds, and free radical scavenging capabilities (Taylor et al., 2014; Kumar et al., 2018) (Fig. 2.3). The nutritional status is regulated by TFs. Expression of genes encoding seed storage proteins, majorly prolamines, are regulated by two crucial TFs, PBF (prolamin box binding factor) and Opaque2 (O2) in cereals (Sood et al., 2016). PBF, a plant-specific Dof (DNA-binding with one finger only), TF shows differential expression in finger millet genotypes having varying grain protein content and color (Gupta et al., 2011). Induced expression of this TF is found in spike tissues. O2 is a bZIP domain containing TF. In finger millet, ECO2 shows differential expression during different seed developmental stages as well as in response to nitrogen content in soil (Gaur et al., 2018). An O2 like TF is also reported in pearl millet, which expresses at early stages of seed development (Marcellino et al., 2004). All of the minor grains are gluten-free and serve as good dietary sources for people suffering from celiac disease. They act as preventing shield against various disorders like anemia, allergies, obesity, eye disorders, aging, diabetes, inflammation, cardiovascular ailments, etc. (Taylor et al., 2014; Kumar et al., 2018). Characterized with comparatively high productivity due to C4 biology, greater water, and nutrient use efficiency, these crops are well adapted to dry hot climates and require minimal agricultural inputs (Goron and Raizada, 2015). With early maturity, these crops are naturally more resistant to various

FIGURE 2.3 Millets are promising cereals to ensure food and health security due to their agronomic and nutritional traits.

26

Chapter 2 Adaptation of millets to arid land

pathogen attacks and abiotic stresses like drought, heat, salt, oxidative, and osmotic stress (Goron and Raizada, 2015; Shivhare and Lata, 2017 Tadele, 2016; Yogeesh et al., 2016; Habiyaremye et al., 2017; Lee et al., 2019; Tari et al., 2013; Mundada et al., 2021) (Table 2.1). The multistress tolerance trait makes them suitable model plants for studying and deciphering the mechanism of stress tolerance. Sequences of the genome of several millets (Zhang et al., 2012; Cannarozzi et al., 2014; Hittalmani et al., 2017; Varshney et al., 2017; Zou et al., 2019; Wang et al., 2021a, b) and sorghum (Paterson et al., 2009) have revealed significant information about the genetic makeup of these hardy crops. This will help the Table 2.1 Description of different millets and sorghum by their taxonomic position, stress resistance, ploidy, and genome size. Stress resistance and agronomic benefits

Botanical name

Common names

Subfamily and tribe

Ploidy level and chromosomes

Pennisetum glaucum

Pearl millet, bulrush millet, bajra

Panicoideae Paniceae

Diploid, 2n 5 2X 5 14

Biotic and abiotic stress specifically terminal drought and heat

1.79 Gb

Graybosch (2004), Tadele (2016), Shivhare and Lata (2017), Varshney et al. (2017)

Setaria italic

Foxtail millet, Italian millet

Panicoideae Paniceae

Diploid, 2n 5 2X 5 18

Drought, salt

490 Mb

Graybosch (2004), Zhang et al. (2012), Goron and Raizada (2015), Tadele (2016)

Panicum miliaceum

Broomcorn millet, proso millet

Panicoideae Paniceae

Tetraploid, 2n 5 4X 5 36

Drought, heat, cold, salt, low inputs, early maturity

923 Mb

Graybosch (2004), Tadele (2016), Habiyaremye et al. (2017), Zou et al. (2019)

Panicum sumatrense

Little millet

Panicoideae Paniceae

Tetraploid, 2n 5 4X 5 36

Abiotic stress

Graybosch (2004), Tadele (2016)

Echinochloa colona

Indian barnyard millet, sawa

Panicoideae Paniceae

Hexaploid, 2n 5 6X 5 36

Early maturity

Graybosch (2004), Tadele (2016)

Echinochloa esculanta

Japanese barnyard millet

Panicoideae Paniceae

Hexaploid, 2n 5 6X 5 36

Early maturity

Graybosch (2004), Tadele (2016)

Paspalum scrobiculatum

Kodo millet

Panicoideae Paniceae

Tetraploid, 2n 5 4X 5 40

Drought

Graybosch (2004), Tadele (2016)

Digitaria exilis

Fonio millet, white fonio, acha

Panicoideae Paniceae

Diploid, 2n 5 2X 5 30;

Heat, drought

Digitalis iburua

Fonio millet, white fonio, acha

Panicoideae Paniceae

Hexaploid, 2n 5 6X 5 54

Heat, drought

Genome sizea

761 Mb

References

Graybosch (2004), Tadele (2016), Wang et al. (2021a, b) Graybosch (2004), Tadele (2016)

2.4 Stress as a limiting factor for crops in the arid zones

27

Table 2.1 Description of different millets and sorghum by their taxonomic position, stress resistance, ploidy, and genome size. Continued Stress resistance and agronomic benefits

Botanical name

Common names

Subfamily and tribe

Ploidy level and chromosomes

Eleusine coracana

Finger millet, ragi, birdsfoot

Chloridoideae Chlorideae

Tetraploid, 2n 5 4X 5 36

Drought, heat, salt, oxidative, osmotic stress

1196 Mb

Graybosch (2004), Goron and Raizada (2015), Tadele (2016), Yogeesh et al. (2016), Hittalmani et al. (2017), Mundada et al. (2021)

Eragrostis tef

Teff, lovegrass

Chloridoideae Aragrosteae

Tetraploid, 2n 5 4X 5 40

Drought, heat, salt, osmotic stress, metal toxicity, waterlogging

672 Mb

Graybosch (2004), Cannarozzi et al. (2014), Tadele (2016), Lee et al. (2019)

Sorghum bicolor

Great millet, jowar, milo

Panicoideae Andropoganeae

Diploid, 2n 5 2X 5 20

Drought, heat, salt

730 Mb

Graybosch (2004), Paterson et al. (2009), Tari et al. (2013)

a

Genome sizea

References

Cells of this column are left blank in the case where the genome is not sequenced yet.

scientific community in mining the potential of these minor grains for further improvement of their agronomic traits.

2.4 Stress as a limiting factor for crops in the arid zones Though millets are stress-tolerant cereals, the environment of arid and semiarid regions is not suitable for their growth and production. Water scarcity and heat are the two dominant abiotic factors that prevail in millet-cultivation areas. Terminal drought significantly reduces their yield (Tadele, 2016). The grain yield of foxtail millet, yellow foxtail (Setaria glauca), proso millet, and little millet decreases by 80%, 70%, 36%, and 20%, respectively (Matsuura et al., 2012). Foxtail millet and yellow foxtail are sensitive to preheading drought while proso millet and little millet are sensitive to both pre and postheading drought. Finger millet is also sensitive to intermittent drought (Maqsood and Ali, 2007). Tef is estimated to suffer a yield loss of 40% (Mengistu and Mekonnen 2011; Wondewosen et al., 2012). In pearl millet, sensitive (H77/833
2) and tolerant genotype (PRLT2/89
33) suffer 60% and 25% yield loss, respectively, due to terminal drought (Aparna et al., 2014). The extent of loss is determined by the degree, duration, and timing of the drought (Bidinger et al., 1987; Mahalakshmi et al., 1987). Drought also reduces the nutritional quality of millet grains. (Saleh et al., 2013; Tadele, 2016).

28

Chapter 2 Adaptation of millets to arid land

FIGURE 2.4 The trend in global temperature change during 1961
2019. Adapted from FAOSTAT (2021) Available at: http://www.fao.org/faostat/en/#data, Food and Agriculture Organization of the United Nations.

High temperatures also adversely affect various physiological and molecular processes, which in turn result in yield loss in millets (Barnab´as et al., 2008; Deryng et al., 2014). As shown in Fig. 2.4, global temperature is continuously rising. It has increased by 1.6 C (FAOSTAT, 2021). Temperature rise is expected to exert a negative effect on millet production (Lipiec et al., 2013). In finger millet, grain yield declines by 75% and 84% under 36/26 C and 38/28 C thermocyclers, respectively, as compared to that at 32/22 C (Opole et al., 2018). In pearl millet, the thermal stress of $ 36/26 C causes floret sterility, which results in 70%
75% reduction in yield (Djanaguiraman et al., 2018). Pistils exhibit more sensitivity to high temperatures compared to pollens. Soil temperature is also a key factor in arid regions, which affects the production of millets. Foxtail millet suffers 60% yield loss at 38 C soil temperature in comparison to 28 C (Aidoo et al., 2016).

2.5 Responses of millets to abiotic stresses Being adapted to arid climate millets withstand a variety of abiotic stresses that affect their overall health and productivity. Millets face significant challenges during seed germination, growth, and development. Abiotic stresses like heat, drought, and salinity significantly interfere with photosynthesis and respiration (Numan et al., 2021). Low water status, altered water use efficiency, and nutrient use efficiency are other significant burdens imposed by stress. Accelerated generation of ROS leads to membrane damage. Altered permeability of membranes leads to ion imbalance and altered osmotic status. Apart from avoidance and escape strategies, millets go through various adjustments at morphological, anatomical, molecular, and biochemical levels for stress tolerance (Numan et al., 2021). Longer and broader roots, and proliferation of lateral roots in millets are crucial for improved water and nutrient use efficiency (NUE) (Nadeem et al., 2020; Numan et al., 2021). The composition of root exudate changes in pearl millet during drought. It contains inhibitors of biological nitrification in soil to improve NUE under drought (Ghatak et al., 2022). Accumulation of osmolytes like proline, glycine betaine, GABA (γ-aminobutyric acid), improved stomatal conductance, and RWC are other major adjustments during drought (Tadele, 2016). Under

2.6 Transcription factors: smart regulators of stress tolerance in millets

29

drought-induced oxidative stress, millets protect themselves by inducing levels of ROS scavenging enzymes like superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase, guaiacol peroxidase, and glutathione reductase (GR) (Bhatt et al., 2011; Lata et al., 2011; Mude et al., 2020; Mundada et al., 2020). A higher APX-to-SOD ratio is associated with drought tolerance in finger millet, which differs with reference to the geographic distribution of genotypes (Bhatt et al., 2011). Drought triggers reduction in chlorophyll pigments in finger millet (Mukami et al., 2019), however, photosynthetic pigments show less degradation in dought tolerant finger millets (Mude et al., 2020). Accumulated proline and soluble sugars help in maintaining water status during drought in finger millet (Mude et al., 2020). Blum and Ebercon (1976) suggested that higher accumulation of free proline amino acid is correlated to recovery rate of plant after drought relief. Proteins involved in cell wall synthesis, signaling, secondary metabolism, and lipid metabolism show increased abundance during drought in pearl millet (Ghatak et al., 2016). Altered expression of genes involved in stress signaling is fundamental for stress tolerance and phytohormone like ABA play an important role in this cascade (Dar et al., 2017; Yang et al., 2019). Sorghum plants show induced expression of some specific genes encoding dehydrin, NADP-malic enzyme, carbonic anhydrase, and plasma membrane intrinsic protein (PIP2
5) to thrive in drought conditions (Fracasso et al., 2016). Drought-tolerant sorghum genotype shows enhanced expression of NADP-malic enzyme, which is involved in stomatal regulation. Photosynthetic machinery especially photosystem II is the most vulnerable to drought (Havaux, 1992). Drought combined with high light causes more photoinhibition and consequently decreases photosynthesis (Masojı´dek et al., 1991). The grain and biological yield are decreased in sorghum due to disturbed photosynthesis when drought is applied after anthesis (Beheshti and Behboodi 2010). High temperature greatly alters relative water content, water potential, and osmotic potential of leaves of sorghum under water limiting conditions (Machado and Paulsen 2001). Sorghum responds to heat stress by accumulating proline and antioxidant enzymes (Gosavi et al., 2014). Heat stress induces differentially accumulation of proteins engaged in metabolism, detoxification, and protein modification functions in sorghum (Ngcala et al., 2020). Pearl millet shows increased net assimilation rate, pronounced uptake of N, P, and K (Ashraf and Hafeez 2004), and cooling effects in the top of plant parts by regulating transpiration to counter heat stress (Shanker et al., 2020). Induction of HSP in sorghum confers thermotolerance (Frova et al., 1991; Gosavi et al., 2014).

2.6 Transcription factors: smart regulators of stress tolerance in millets In field conditions, plants withstand a combination of stresses rather than individual stress and this makes stress response a complex trait. TFs can decrease or enhance the expression of their target genes via specific interaction through their conserved DNA binding domain (DBD) with target sites (i.e., cis-acting elements). TFs are crucial regulators of different processes like metabolic pathways, development, organogenesis, and stress tolerance against salt, drought, heat, heavy metal, extreme temperature, UV light, ozone, oxidative, and osmotic stress (Jeyasri et al., 2021). More than 85 TF families are known but only a limited number of families have been comprehensively studied in relation to stress tolerance (Baillo et al., 2019; Jeyasri et al., 2021). TFs are among the key factors in different millets that control the nature (sensitive vs tolerant) of a genotype of a particular

30

Chapter 2 Adaptation of millets to arid land

species for an abiotic stress (Gelli et al., 2014; Choudhary and Padaria 2015; Tang et al., 2017; Cui et al., 2018; Xu et al., 2019; Abdel-Ghany et al., 2020; Pan et al., 2020). Tolerant genotype owns an effective signal transduction mechanism with a prominent expression of TFs for fine metabolic tuning under harsh conditions as found in foxtail millet (Puranik et al., 2011a). In pearl millet, the TFs of 35 families show differential expression in a terminal drought-tolerant genotype under drought (Shivhare et al., 2020). Similarly, differentially expressed TFs may play a protuberant role in the regulation of tolerance to drought in foxtail millet (Yu et al., 2020). Qin et al. (2020) reported drought-responsive differential expression of 298 and 170 genes in roots and leaves, respectively. In sorghum, 176 TFs belonging to AP2, AUX_ARF, bZIP, MYB, and WRKY TF families exhibit a drought-induced increase in protein level (Sekhwal et al., 2015). In finger millet, TFs show significant upregulation under salt stress in salinity-tolerant genotype (Rahman et al., 2014). In millets and sorghum, research is more focused on WRKY and NAC (NAM, ATAF1/2, and CUC1/2) TFs. Along with other TFs, their role in the adaptation of these small grain cereals is discussed in the upcoming subsections (Fig. 2.5).

2.6.1 WRKY Known to regulate various important biological processes the WRKY proteins constitute a major class of transcription regulators. First reported in Ipomoea batatas (Ishiguro and Nakamura, 1994) the WRKYs were named after the conserved sequence WRKY present at the N-terminus of its DBD (Rushton et al., 1996). This domain is an approximate 56
58 amino acid (AA) long region featured with an N-terminus WRKYGQK sequence and a C-terminus zinc-finger-like motif (Rushton et al., 1995). WRKY TFs have been classified into three classes depending on the number

FIGURE 2.5 A schematic representation of signal transduction mechanism operative under abiotic stress leading to transcription factors mediated stress tolerance.

2.6 Transcription factors: smart regulators of stress tolerance in millets

31

of the hallmark WRKY motif and the differences in the C-terminus domain (CTD). Group I (one WRKY domain) and group II (two WRKY domains) possess similar motif C-X4
5-C-X22
23-HX1-H in the CTD differing from group III (two WRKY domains), which contains C-X7-C-X23-HX1-C motif (Eulgem et al., 2000). Mostly, WRKY proteins exert their regulatory function through the binding on the W box (conserved sequence TTGACT/C) (Eulgem et al., 2000). However, they can also bind on sites other than W box (Sun et al., 2003; Cai et al., 2008). WRKY TFs exert a range of physiological functions in plants along with the response to abiotic stresses as comprehensively discussed by Rushton et al. (2010) and Li et al. (2020). To date, the dynamic role of these proteins has been elucidated through gene expression analysis and functional characterization in many crops and Arabidopsis thaliana, which have been compiled and discussed in detail by Chen et al. (2017). A few recent studies on millets have shown the involvement of WRKY TFs in different abiotic stresses. Xu et al. (2017) reported two genes, SbWRKY1 and SbWRKY2, to be active during drought in sorghum. SbWRKY30 improves drought tolerance of sorghum via regulating SbRD19, a drought-responsive gene, by directly interacting with the W-box of its promoter (Yang et al., 2020). SbWRKY50 acts as a negative regulator of salinity tolerance (Song et al., 2020). It binds to the promoters of two genes related to salt stress management namely, SOS1 and HKT1 (high-affinity K1 transporter 1), and thereby controls ion homeostasis. Expression of SbWRKY71 is induced under drought and salt stress (Qiaoli et al., 2021). 85 SbWRKY genes (Ahmadi et al., 2019) and 94 putative SbWRKY proteins have been identified in Sorghum bicolor through genome-wide analysis (Baillo et al., 2020). Pearl millet genome encodes 97 putative PgWRKY proteins (Chanwala et al., 2020). Their corresponding genes show differential expression patterns under drought and salinity stress. Similarly, 105 SiWRKY and 44 SvWRKY proteins have been identified in Setaria italica and its wild relative Setaria viridis, respectively (Muthamilarasan et al., 2015). Zhang et al. (2017a) reported 103 SiWRKY genes. Yue et al. (2016) reported 32 PmWRKY genes in Panicum miliaceum, out of which 22 genes show abiotic stress responsiveness. In all these studies, the researchers have elucidated the structural, functional, and phylogenetic aspects of the TFs and categorized them into different groups according to the classification recommended by Eulgem et al. (2000). In these reports, the role of different WRKY TFs has been highlighted in abiotic stress responses as evident from the cis-element analysis as well as their induced expression under one or more abiotic stresses (Table 2.2).

2.6.2 DOF The plant-specific DOF (DNA-binding one zinc finger) TF family is another significant family of transcription factors involved in various vital functions. The first identified DOF TFs were MNBla and MNBlb (maize nuclear factor binding proteins) with a shared target site at the promoter of MNF1 (maize nuclear factor1) gene (Yanagisawa and Izui, 1993). The DOF proteins are characterized by a single copy of the highly conserved 52 amino acid long DOF domain present centrally or at the N-terminal end (Yanagisawa, 2004). The CX2CX21CX2C region, which forms one zinc finger, is vital for target sequence recognition. In most cases, the cis-acting element for binding of the DOF proteins is AAAG as well as its complementary counterpart CTTT (Yanagisawa and Schmidt, 1999). However, a variation in the binding site has been reported for a DOF TF (AOBP), which binds at AAGT of the regulatory region of the ascorbate oxidase (AO) gene (Kisu et al., 1998). Depending on the target, they can activate and suppress a particular function. Their structural and

32

Chapter 2 Adaptation of millets to arid land

Table 2.2 Abiotic stress responsiveness of transcription factors of different millets and sorghum as revealed through gene expression studies. TF family

Millet

WRKY

Pennisetum glaucum

Inducing factor

Gene

Response

Reference

Drought

PgWRKY03

Upregulation

Chanwala et al. (2020)

Drought

PgWRKY46 PgWRKY61 PgWRKY96 PgWRKY02 PgWRKY06 PgWRKY52 PgWRKY74 PgWRKY02 PgWRKY18 PgWRKY72 PgWRKY44 PgWRKY59 PgWRKY61 PgWRKY33 PgWRKY62 PgWRKY65 SbWRKY01

Drought

Salt

Drought, Salt

Sorghum bicolor

Setaria italica

Downregulation

Upregulation

Downregulation

Downregulation

Upregulation

Xu et al. (2017)

SbWRKY02 SbWRKY30

Upregulation

Salt

SbWRKY50

Upregulation

Drought, Salt

SbWRKY71

Upregulation

Drought

SbWRKY45

Upregulation

Song et al. (2020) Yang et al. (2020) Qiaoli et al. (2021) Baillo et al. (2020)

Drought, Salt

SbWRKY72 SbWRKY74 SbWRKY75 SbWRKY79 SiWRKY64

Upregulation

Muthamilarasan et al. (2015)

Drought Drought

SiWRKY66 SiWRKY74 SiWRKY82 SiWRKY101 SiWRKY09

Upregulation

Zhang et al. (2017a)

2.6 Transcription factors: smart regulators of stress tolerance in millets

33

Table 2.2 Abiotic stress responsiveness of transcription factors of different millets and sorghum as revealed through gene expression studies. Continued TF family

Millet

Panicum miliaceum

Inducing factor

Salt, Drought, Heat, Cold Salt, Drought, Cold Salt, Cold, Heat Salt, cold

Salt, Heat Drought, cold

Drought, Heat Cold, heat Salt

Drought Cold

Heat

Gene

Response

Reference

SiWRKY19 SiWRKY44 SiWRKY59 SiWRKY85 SiWRKY87 SiWRKY96 SiWRKY102 PmWRKY32

Upregulation

Yue et al. (2016)

PmWRKY29

Upregulation

PmWRKY03

Upregulation

PmWRKY05 PmWRKY10 PmWRKY16 PmWRKY23 PmWRKY27 PmWRKY12 PmWRKY14 PmWRKY18 PmWRKY26 PmWRKY11 PmWRKY10 PmWRKY25 PmWRKY09 PmWRKY28 PmWRKY22 PmWRKY31 PmWRKY07 PmWRKY19 PmWRKY11 PmWRKY22 PmWRKY23 PmWRKY5 PmWRKY7 PmWRKY19

Upregulation Downregulation Upregulation Upregulation

Upregulation Downregulation Upregulation Upregulation Downregulation Upregulation Upregulation Downregulation Upregulation Downregulation

(Continued)

34

Chapter 2 Adaptation of millets to arid land

Table 2.2 Abiotic stress responsiveness of transcription factors of different millets and sorghum as revealed through gene expression studies. Continued TF family

Millet

DREB

Setaria italica cv. Prasad Sorghum bicolor

ERF

Setaria italica

Inducing factor

Response

Reference

Drought, Dehydration, Salt, Cold Salt

SiDREB2L

Upregulation

Pandurangaiah et al. (2016)

SbDREB2A (in root)

Upregulation

Akbudak et al. (2018)

Drought

SbDREB2B (in root) SbDREB2C2 (in root) SbDREB2D (in root) SbDREB2A (in leaf) SbDREB2B (in leaf) SbDREB2C2 (in leaf) SbDREB2D (in leaf) SiAP2/ERF53

Salt

Sorghum bicolor

Gene

Drought, salt Drought

Heat

Combined drought and heat

SiAP2/ERF67 SiAP2/ERF74 SiAP2/ERF92 SiAP2/ERF95 SiAP2/ERF138 SiAP2/ERF166 SiAP2/ERF169 SiAP2/ERF116 SiAP2/ERF58 SiAP2/ERF69 SiAP2/ERF125 SiAP2/ERF92 SiAP2/ERF95 SiAP2/ERF103 SbERF97 SbERF98 SbERF15 SbERF28 SbERF37 SbERF96 SbERF05

SbERF13 SbERF16

Downregulation Upregulation

Lata et al. (2014)

Downregulation Upregulation

Downregulation Upregulation Upregulation

Mathur et al. (2020)

2.6 Transcription factors: smart regulators of stress tolerance in millets

35

Table 2.2 Abiotic stress responsiveness of transcription factors of different millets and sorghum as revealed through gene expression studies. Continued TF family

Millet

Inducing factor

Drought, Heat, and Combined stress

NAC

Pennisetum glaucum

Drought

Salt

Sorghum bicolor

Drought, Salt, Cold, ABA

Gene

Response

SbERF50 SbERF51 SbERF64 SbERF75 SbERF79 SbERF89 SbERF40

Upregulation

SbERF147 SbERF100 SbERF10 SbERF11 PgNAC58 PgNAC64 PgNAC70 PgNAC72 PgNAC29 PgNAC45 PgNAC74 PgNAC94 PgNAC105 PgNAC106 PgNAC142 PgNAC02 PgNAC10 PgNAC36 PgNAC89 PgNAC108 PgNAC113 SbNAC06

Reference

Downregulation Upregulation

Dudhate et al. (2021)

Downregulation

Upregulation

Upregulation

Kadier et al. (2017)

SbNAC17 SbNAC26 SbNAC46 SbNAC56 SbNAC58 SbNAC73 (Continued)

36

Chapter 2 Adaptation of millets to arid land

Table 2.2 Abiotic stress responsiveness of transcription factors of different millets and sorghum as revealed through gene expression studies. Continued TF family

Millet

Inducing factor

Gene

Response

Reference

SbNAC14

Upregulation

Sanjari et al. (2019)

SbNAC35 SbNAC41 SbNAC52 SbNAC73 SbNAC116 SiNAC03

Upregulation

Puranik et al. (2013)

Drought Drought

SiNAC14 SiNAC27 SiNAC28 SiNAC33 SiNAC37 SiNAC52 SiNAC55 SiNAC73 SiNAC76 SiNAC79 SiNAC102 SiNAC105 SiNAC110 SiNAC118 SiNAC120 SiNAC143 SiNAC045 SiNAC29L EcNAC67

Downregulation Upregulation Upregulation

Drought

PmNAC001

Upregulation

Postflowering drought

Setaria italica

Eleusine coracana Panicum miliaceum

Drought, Salt, Cold

PmNAC007 PmNAC023 PmNAC041 PmNAC091 PmNAC093 PmNAC154 PmNAC155 PmNAC172 PmNAC176

Patil (2018) Patil (2018) Shan et al. (2020)

2.6 Transcription factors: smart regulators of stress tolerance in millets

37

Table 2.2 Abiotic stress responsiveness of transcription factors of different millets and sorghum as revealed through gene expression studies. Continued TF family

Millet

bHLH

Sorghum bicolor Pennisetum glaucum

Inducing factor

Gene

Response

Reference

Cold

Sb06g025040

Upregulation

Salt

Downregulation

Chopra et al. (2017) Shinde et al. (2018)

Upregulation

SBP

Pennisetum glaucum

Salt

29 bHLH genes in salinity tolerant line ICMB 01222 26 bHLH genes in salinity susceptible ICMB 081 line 16 bHLH genes in salinity tolerant line ICMB 01222 17 bHLH genes in salinity susceptible ICMB 081 line 2 SBP genes

ASR

Setaria italica

Drought, salt

3 SBP genes SiASR1

Downregulation Upregulation

Drought, salt

SiASR2 SiASR3 SiASR4 SiASR5 SiASR6 SiHDZ4

Upregulation

Chai et al. (2018)

Heat

SiHDZ29 SiHDZ9 SiHDZ14 SiHDZ27 SiHDZ39 SiHDZ45 SiHDZ46 EcbZIP17

Upregulation

Drought

SOBIC.009G244600

Downregulation

Drought

20 MYBs

Upregulation

Chopperla et al. (2017) Abdel-ghany et al. (2020) Xu et al. (2019)

Potassium deficiency

Si008086m.g

Upregulation

Bzip

Setaria italica

ABA

MYB

Eleusine coracana Sorghum bicolor Setaria italica

Upregulation

Shinde et al. (2018) Feng et al. (2016)

Cao et al. (2019) (Continued)

38

Chapter 2 Adaptation of millets to arid land

Table 2.2 Abiotic stress responsiveness of transcription factors of different millets and sorghum as revealed through gene expression studies. Continued TF family

C2H2

Millet

Setaria italica

Inducing factor

Gene

Salt

Si026651m.g Si010608m.g Si012660m.g Si014103m.g Si006790m.g Si026734m.g Si030750m.g Si029872m.g Si036638m.g Si022730m.g Si017704m.g Si017785m.g Si007179m.g Si026803m.g Si022826m.g Si014685m.g Si010342m.g Si013994m.g Si017851m.g SiC2H2_031

Dehydration

Cold

SiC2H2_056 SiC2H2_078 SiC2H2_085 SiC2H2_003 SiC2H2_104 SiC2H2_031 SiC2H2_085 SiC2H2_003 SiC2H2_104 SiC2H2_031 SiC2H2_056 SiC2H2_078 SiC2H2_085 SiC2H2_047 SiC2H2_094 SiC2H2_46

Response

Reference

Downregulation

Upregulation

Downregulation Upregulation Downregulation Upregulation

Muthamilarasan et al. (2014b)

2.6 Transcription factors: smart regulators of stress tolerance in millets

39

Table 2.2 Abiotic stress responsiveness of transcription factors of different millets and sorghum as revealed through gene expression studies. Continued TF family

Millet

NF

Setaria italica

Inducing factor

Gene

Response

Reference

Drought, salt

SiNFYA1 (in leaf)

Upregulation

Feng et al. (2015)

Differential regulation (according to stress type and duration and tissue)

Fan et al. (2021)

Osmotic

SiNFYA2 (in root) SiNFYB7 (in root) SiNFYB8 (in root) SiNFYC1 (in root) SiNFYC3 (in root) SiNFYA3, SiNFYA5 (in stem) SiNFYA9 (in stem)

Salt

SiNFYB2 (in leaf)

Oxidative UV, flood, salt, PEG, heat and cold

SiNFYB8 (in leaf) SbGRAS02, SbGRAS04, SbGRAS11, SbGRAS13, SbGRAS14, SbGRAS27, SbGRAS28, SbGRAS29, SbGRAS31, SbGRAS33, SbGRAS38, SbGRAS79

Drought

Salt, oxidative

SiNFYC12 (in root) SiNFYB8 (in root) SiNFYC12 (in leaf) SiNFYB8 (in leaf and root) GRAS

Sorghum bicolor

functional aspects, interaction with other TF family members, and phylogenetic relationships have been well-reviewed (Yanagisawa, 2002, 2016; Noguero et al., 2013). These proteins are significantly involved in plant growth and developmental processes and stress responses. In millets, DOFrelated studies are limited to bioinformatic analysis. In Setaria italica, the DOF family consists of 35 SiDOF genes (20 intronless; 15 with 1 intron), which express in a tissue-specific manner (Zhang et al., 2017b). Among these, SiDOF7 and SiDOF15 are involved in drought response. Kushwaha et al. (2011) reported the presence of 28 SbDOF genes in the sorghum genome with their diverse functions related to stress, light, hormonal, and developmental responses. The authors revealed that the majority of SbDOF family genes are intronless as found in Arabidopsis and rice. Prediction of the 3D structure of proteins encoded by these genes has enhanced understanding of the function of DOF proteins at the molecular level (Kushwaha et al., 2013; Gupta et al., 2014). Through cis-element analysis, Kushwaha et al. (2011) indicated the involvement of 21 SbDOF genes in abiotic and

40

Chapter 2 Adaptation of millets to arid land

biotic stress responses. Gupta et al. (2014) more specifically suggested the role of different SbDOF genes in heat, drought, salinity, and oxidative stress-related signaling.

2.6.3 ERF/DREB They are mainly involved in cold and drought responses and were first reported in Arabidopsis as CBF1 (C-repeat binding factor1) (Stockinger et al., 1997) and DREB1A (dehydration responsive element binding protein1A), DREB2A (Liu et al., 1998). The plant-specific DREB proteins belong to the ERF (ethyleneresponsive element binding factors) subfamily of AP2/ERF (Apetala2/Ethylene response factor) family. This is also known as the ethylene-responsive element-binding protein (EREBP) family. The DREBs are characterized by a highly conserved AP2/ERF domain (Mizoi et al., 2012). They have been divided into six subgroups (A1 to A6) among which the DREB1 or CBF (A1) and DREB2 (A2) are two major subgroups (Mizoi et al., 2012). DREB1 subgroup proteins are involved in cold stress signaling while DREB2 are involved in heat and drought responses (Mizoi et al., 2012). DREB proteins bind on a 9 bp (TACCGACAT) long C-repeat (CRT) or dehydration responsive element (DRE) of the target gene (Yamaguchi-Shinozaki and Shinozaki 1994). A1 and A2 DREB proteins function independently to ABA. However, their target genes (containing CRT/DRE element in their promoters) are also induced by ABA. It suggests a cross-talk between ABA-dependent and independent pathways (Sarkar et al., 2019). Expansion of the DREB TF family is a result of segmental and tandem duplication in the genome of foxtail millet which harbors 65 DREB genes (Shi et al., 2018). Lata et al. (2011) characterized SiDREB2 and reported its function in salinity and dehydration response in foxtail millet. They also identified an SNP (single nucleotide polymorphism) at the 558th position, which is responsible for differential dehydration tolerance of different foxtail millet accessions. They developed an ASM (allele-specific marker) for SiDREB2, which was later functionally validated for dehydration stress tolerance in terms of relative water content (RWC) (Lata and Prasad, 2014). SiARDP (ABA-responsive DRE-binding protein) plays a key role under drought in foxtail millet (Li et al., 2014). Its expression is in turn regulated by SiAREB (ABA-responsive element-binding protein). SiDREB2L gene responds to drought, salt, and cold stress (Pandurangaiah et al., 2016). Srivasta et al. (2010) revealed overrepresentation of certain motifs related to light response, sugar, and phytohormone signaling (gibberellic acid, ABA, auxin, ethylene, methyl jasmonate) in the promoters of sorghum DREB genes, which suggests a complex signaling network operative under abiotic stress. Yan et al. (2013) reported 52 SbDREB in sorghum. In Sorghum, genes of DREB2 subgroup are induced by cadmium (Cd) and NaCl stress in a tissue-specific manner (Akbudak et al., 2018). Having an affinity with GCC box, the ERF TFs also belong to another major subfamily (ERF) of AP2/ERF family (Mizoi et al., 2012). The role of these transcriptional regulators has been well established in growth and plant development-related processes, hormonal regulation, as well as responses to various stresses. Lata et al. (2014) revealed the chromosomal distribution, structural, phylogenetic, and functional aspects of 171 SiAP2/ERF TFs. They exhibit differential expression patterns under stress indicating their role in drought and salt management. Three genes (SiAP2/ ERF69, SiAP2/ERF103, and SiAP2/ERF120) also show inducibility by ABA. Yan et al. (2013) revealed 105 ERF encoding genes of the AP2/ERF superfamily in sorghum. However, a recent update revealed the presence of 158 SbAP2/ERF genes corresponding to the ERF subfamily (Mathur et al., 2020). They confirmed the role of a few ERF genes in drought, heat, and combined heat and drought through RT-qPCR (real-time quantitative polymerase chain reaction) expression study.

2.6 Transcription factors: smart regulators of stress tolerance in millets

41

2.6.4 NAC Initially, NAC TFs were reported in Petunia hybrida (NAM/no apical meristem) and Arabidopsis thaliana [ATAF1 and 2, CUC1 and 2 (cup-shaped cotyledon) protein] (Souer et al., 1996; Aida et al., 1997). They contain conserved N-terminal DBD (about 150 AA long NAC domain) and diversified CTD. The NAC TFs can induce or suppress the target gene transcription by directly binding on the NACRS (NAC recognized sequence) (Tran et al., 2004; Nuruzzaman et al., 2013). NACs can exert their stress regulatory functions in an ABA-dependent as well as ABAindependent fashion (Puranik et al., 2011b, Dou et al., 2017). As evident from comprehensive reviews (Nuruzzaman et al., 2013; Shao et al., 2015) extensive efforts have been made to investigate structural and functional aspects owing to their potential role in plant abiotic and biotic stress pathways. Arabidopsis has 106 NAC genes (Gong et al., 2004). Dudhate et al. (2021) explored the pearl millet genome for organizational, functional, and phylogenetic features of 151 NAC genes. 36 PgNAC genes show altered expression under drought and salinity stress in a tissue-specific manner. The majority of the genes show enhanced expression in roots, which may be due to higher adversity faced by roots (Dudhate et al., 2021). Total 145 SbNACs (Kadier et al., 2017) and 147 SiNACs (Puranik et al., 2013) have been identified in Sorghum bicolor and Setaria italica, respectively, through bioinformatic study. Seven SbNAC genes (SbNAC06, 17, 26, 46, 56, 58, 73) show activation by salt, cold, drought (PEG/polyethylene glycol), and ABA treatment (Kadier et al., 2017). Fifty SiNAC genes with some genes showing time point and treatment-specific differential expression were found to be involved in drought, salinity, and cold response. Several SiNAC proteins show divergence from the typical general structure of NAC proteins (Puranik et al., 2013). Moreover, SiNAC18 protein regulates stress response via managing oxidative stress in Setaria italica (Dou et al., 2017). Contrary to an earlier report (Kadier et al., 2017), Sanjari et al. (2019) reported 131 putative NAC genes present in the genome of sorghum encode 183 proteins. As studied by the authors, 13 SbNAC proteins belong to the SNAC (stress-responsive NAC) subgroup associated with abiotic stress. Their genes are involved in drought-mediated senescence (SbNAC66, 104), nutrient mobilization (SbNAC34, 37), postflowering drought tolerance (SbNAC14, 35, 41), and negative regulation of drought stress cascade genes of sorghum (SbNAC52, 73, 116). A total of 180 putative pmNAC genes, having tissue-specific expression, have been identified in broomcorn millet (Shan et al., 2020). RNA sequencing data have supported the involvement of 93 out of 180 pmNAC genes in drought signaling in broomcorn millet (Shan et al., 2020). Furthermore, NAC genes show a greater upregulation in tolerant genotypes (Patil, 2018; Sanjari et al., 2019). Structural characterization, transactivation activity, and subcellular localization analysis of some individual millet NAC members have generated a better understanding of their molecular functions related to stress responses (Puranik et al., 2011b; Dou et al., 2017; Patil, 2018; Ardie et al., 2019).

2.6.5 bHLH This family holds the second rank after MYB TFs. bHLH TF family represents those proteins that have a conserved basic helix-loop-helix (bHLH) domain. The HLH region is found at C-terminus whereas the basic part occurs at N-terminus. The basic part is responsible for binding to cis-element. It contains about 15 amino acids and of which six residues are basic in nature. The HLH region is 60 amino acids in length. It is majorly made up of hydrophobic residues and works as a

42

Chapter 2 Adaptation of millets to arid land

dimerization domain (Heim et al., 2003). bHLH protein recognizes hexanucleotide motif familiar as E-box (CANNTG) (Toledo-Ortiz et al., 2003). The first bHLH gene, encoding Lc (leaf color) protein, a regulator of anthocyanin biosynthesis, was identified in maize (Ludwig et al., 1989). In foxtail millet, PPLS1 (purple color of pulvinus and leaf sheath), a bHLH TF, controls the color of pulvinus and leaf sheath (Bai et al., 2020). Wang et al. (2018a, b) identified 149 bHLH genes in foxtail millet and found 8 SibHLH genes exhibit differential responses to drought and ABA.

2.6.6 ASR Abscisic acid stress ripening (ASR) proteins constitute a plant-specific TF family. These proteins contain a characteristic ABA/water-deficit stress (WDS) domain. Interestingly, ASR proteins may also act as molecular chaperones (Konrad and Bar-Zvi 2008). They play a key role in tolerance to abiotic stresses especially drought as well as fruit ripening. ASR binds to C(2
3)(C/G)A sequence (Kalifa et al., 2004), coupling element 1 CACCG, a cis-acting element bound by the ABI4 (ABA insensitive 4) TF (Shkolnik and Bar-Zvi 2008) as well as sugar box (AATAGAAAA) (Jia et al., 2016). The first member of this family was identified in tomato (Iusem et al., 1993). Subsequently, ASR proteins were identified in other plant species (Gonz´alez and Iusem 2014; Virlouvet et al., 2011). However, there is only one report regarding the investigation of this family in millet. Feng et al. (2016) reported six SiARS genes in fox millet and found that SiASR1 enhances drought and oxidative stress by regulating ROS homeostasis.

2.6.7 bZIP Basic leucine zipper (bZIP) proteins constitute one of the largest TF families in plants. These TFs are characterized by a conserved bZIP domain that contains a N-terminus DNA binding region with an affinity for ACGT-containing elements and a leucine zipper region for dimerization at the C-terminus (Fernando, 2020; Wang et al., 2021a, b). In millets, only one bZIP TF has been characterized to date. EcbZIP17 from finger millet shows heat inducibility (Chopperla et al., 2017) and increases heat tolerance in the transgenic tobacco plant (Ramakrishna et al., 2018).

2.6.8 MYB The MYb (v-myb avian myeloblastosis viral oncogene homolog) TF was first discovered in an avian virus (Paz-Ares et al., 1987) while in plants, the first MYB TF ZmMYBC1 was reported in corn (Frampton, 2004). A typical MYB protein contains a DNA binding region, transcriptional activation domain, and the negative regulatory region (Ogata et al., 1996). There are limited studies on MYb TFs in millets. Drought responsive MYB genes are reported in finger millet (Kumari et al., 2017; Jadhav et al., 2018; Bhatt et al., 2021) and sorghum (Zhang et al., 2019). The genome of the foxtail millet contains 209 SiMYB genes (Muthamilarasan et al., 2014a). Eleven SiMYB genes show enhanced transcription during salinity and drought stress. SiMYB3, a low potassium (K1) stressresponsive gene, increases root length and its surface area when it is ectopically expressed in Arabidopsis under K1 deficiency conditions (Cao et al., 2019). One hundred thirty four R2R3 type MYB genes show differential tissue-specific expression in sorghum (Singh et al., 2020). Expression

2.7 Harnessing the potential of millet transcription factors

43

of SbMYB78 shows significant upregulation under combined heat and drought stress in sorghum (Johnson et al., 2014).

2.6.9 SBPs SBPs (SQUAMOSA promoter binding proteins) constitute a plant-specific TF family. They are important for development and abiotic stress responses (Song et al., 2016). Overexpression of SBP contributes to salinity tolerance (Hou et al., 2018). Five unigenes encoding SBPs show differential expression in salinity tolerant genotype of pearl millet (ICMB 01222) (Shinde et al., 2018).

2.6.10 Other transcription factors Apart from the abovementioned TFs, other ones have also been identified in different millets. Foxtail millet genome contains 124, 27, 39, and 47 genes encoding C2H2 type of zinc finger TFs (Muthamilarasan et al., 2014b), trihelix transcription factors (TTF) (Wang et al., 2018a, b), nuclear factor Y (NF-Y) (Feng et al., 2015), and homeodomain leucine zipper (HD-Zip) TFs (Chai et al., 2018), respectively. In HD-Zip TFs, the homeodomain binds to DNA while and a leucine-zipper domain interacts with proteins (Aso et al., 1999; Tron et al., 2001). Expression of SiHDZ29 and SiHDZ45 is induced by salinity and drought as well as ABA (Chai et al., 2018). Nine SiC2H2 genes show differential expression under salinity, dehydration, and cold stress. Expression of SiNFYA1 and SiNF-YB8 are induced by ABA, H2O2, and abiotic stress. These two TFs control droughtresponsive genes and antioxidant enzymes encoding genes and thereby enhance tolerance to abiotic stress. GRAS (Gibberellic acid insensitive, repressor of gal-3 mutant and scarecrow) is a plantspecific TF family. Eighty one SbGRAS genes have been identified in sorghum (Fan et al., 2021). Thirteen SbGRAS members show differential expression in a tissue-dependent manner under abiotic stresses like drought, salinity, cold, heat, flooding, and ultraviolet radiation. SbGRAS03 shows inducibility by all of these stresses in roots. Shinde et al. (2018) identified 935 and 906 TFs encoding unigenes in ICMB 01222 (salinity tolerant) and ICMB 0819 (salinity susceptible) genotypes of pearl millet, respectively. Most of them are the members of the zinc finger (C2H2), AP2-EREBP, MYB, NAC, bHLH, bZIP, WRKY, WD40 (contains conserved domain having tryptophan-aspartate residues), HSF (Heat shock factor), and homeobox-wox families.

2.7 Harnessing the potential of millet transcription factors Improvement of stress tolerance through genetic engineering or genome editing techniques requires prior knowledge of gene function. Overexpression of candidate genes using the transgenic method is a popular reverse genetics approach for their functional validation. Several abiotic stress-responsive WRKY, ERF, bZIP, bHLH, MYB, DREB, and NAC TF genes of major crops like wheat, rice, barley, soybean, and cotton have been functionally validated in transgenics of model and nonmodel crops (Agarwal et al., 2018; Rabara et al., 2014). These transgenic plants have shown increased resistance against drought, salinity, UV light, ozone, chilling, and freezing stress. Similarly, millet genes encoding various TFs have also been explored for their potential in enhancing abiotic stress tolerance (Table 2.3). Various NAC, MYB, bHLH,

44

Chapter 2 Adaptation of millets to arid land

Table 2.3 List of genes from millets and sorghum, functionally validated through transgenesis approach to confer abiotic stresses tolerance.

S. No.

Transcription factor family

Transcription factor

Source millet

1

bHLH

EcbHLH57

2

NAC

EcNAC1

Finger millet Finger millet Finger millet Pearl millet Foxtail millet Foxtail millet Foxtail millet Sorghum Sorghum

EcNAC67 PgNAC21 SiNAC110 SiNAC45 SiNAC18 SbSNAC1 SbSNAC56 3

4

ZIP

MYB

Tobacco

Salinity, drought

Tobacco

Salinity, drought

Rice

Salinity, drought

Arabidopsis

Salinity, ABA

Arabidopsis

Salinity, drought

Babitha et al. (2015a) Ramegowda et al. (2012) Rahman et al. (2016) Shinde et al. (2019) Xie et al. (2017)

Arabidopsis Arabidopsis

Potassium deficiency, ABA Drought

Wang et al. (2015) Dou et al. (2017)

Arabidopsis Arabidopsis

Drought Osmotic stress, ABA Salinity, oxidative stress, dithiothreitol (DDT), tunicamycin Salinity, mannitol, PEG, DTT, heat, drought Drought, ABA

Lu et al. (2013) Kadier et al. (2017) Babitha et al. (2015b)

Finger millet

Tobacco

EcbZIP17

Finger millet

Tobacco

SiMYB56

Foxtail millet Foxtail millet

Rice

SiMYB42 ERF DREB

Increase in stress tolerance

EcbZIP60

SiMYB3

5 6

Ectopic expression in crop/ plant

SbWINL1 PgDREB2A EcDREB2A SbDREB2

Foxtail millet Sorghum Pearl millet Finger millet Sorghum

Arabidopsis and rice Arabidopsis Arabidopsis Arabidopsis Tobacco

References

Ramakrishna et al. (2018) Xu et al. (2020)

Nitrogen deficiency

Ge et al. (2019)

Potassium deficiency Nitrogen deficiency

Cao et al. (2019) Ding et al. (2018) Bao et al. (2017) Agarwal et al. (2010) Singh et al. (2021) Bihani et al. (2011)

Tobacco

Drought Salinity and dehydration Heat

Rice

Drought

2.7 Harnessing the potential of millet transcription factors

45

Table 2.3 List of genes from millets and sorghum, functionally validated through transgenesis approach to confer abiotic stresses tolerance. Continued

S. No.

Transcription factor family

Transcription factor

Source millet

7

ASR

SiASR1

Foxtail millet Foxtail millet Pearl millet

SiASR4 PgASR3

8

NF

SiNF-YA1 and SiNF-YB8 SiNF-YA5

Foxtail millet Foxtail millet

Ectopic expression in crop/ plant Tobacco Arabidopsis Arabidopsis

Tobacco Arabidopsis

Increase in stress tolerance

References

Drought, oxidative stress Salinity, drought

Feng et al. (2016) Li et al. (2017)

Osmotic stress salinity, cold, heat, ABA Salinity, drought, osmotic stress Salinity

Meena et al. (2020) Feng et al. (2015) Huang et al. (2016)

and ZIP genes from different millets improve drought and salinity tolerance as evident by the better performance of their transgenics as compared to wild type. ABA hypersensitive SbNAC56 induces osmotic stress tolerance in Arabidopsis (Kadier et al., 2017). Overexpression of SiMYB3, SiNAC45, and SiMYB42 from foxtail millet results in better performance of the Arabidopsis and rice transgenic plants under nutrient deficiency stress (Cao et al., 2019; Ding et al., 2018; Ge et al., 2019; Wang et al., 2015). Overexpression of SiASR1, SiASR4, and PgASR3 confers increased tolerance to oxidative, and osmotic stress along with drought, heat, cold, and salt stress (Feng et al., 2016; Li et al., 2017; Meena et al., 2020). Similarly, PgDREB2A, EcDREB2A, and SbDREB2 transgenics exhibit enhanced resistance against drought, heat, salinity, and dehydration (Agarwal et al., 2010; Bihani et al., 2011; Singh et al., 2021). Millet TFs modulate transcription of various downstream genes, which are directly or indirectly involved in abiotic stress response. For instance, PgDREB2A induces expression of various downstream genes like dehydrin (NtERD10B), ERF (NtERF5), heat shock factors (NtHSF2), HSPs (Hsp18p), and genes involved in signal transduction and lipid metabolism in transgenic tobacco plants (Agarwal et al., 2010). PgNAC21, a positive regulator of salt tolerance induces expression of ROS scavenger GSTF6 (glutathione s-transferase 6), ABA, cold and, osmotic stress-responsive COR47 (cold-regulated 47), and RD20 (responsive to dehydration 20) genes (Shinde et al., 2019). Heat-inducible EcDREB2A and selected ASRs also induce the expression of ROS scavenger genes (POD, SOD, APX, CAT, and GR) (Feng et al., 2016; Li et al., 2017; Singh et al., 2021). SiNAC18 induces the expression of genes related to ABA (AtRD29A), proline biosynthesis (AtP5CR and AtPRODH), and peroxidase (AtPRX34) (Dou et al., 2017). Water and ion balance are major prerequisites for stress tolerance (Sharma, 2022; Upadhyay, 2022). SiNAC110 manages ion and water homeostasis via upregulation of genes encoding Na1/K1 transporter and aquaporins (Xie et al., 2017). EcbZIP17 and EcbZIP60 mediate stress tolerance by managing ion homeostasis (by downregulation of NCX, which encodes for Na21/Ca21 exchanger) and protein folding capacity. They activate ER stress response genes, namely PDIL (protein disulfide isomerase prevents misfolded

46

Chapter 2 Adaptation of millets to arid land

proteins from aggregation); Bip1 (binding protein 1 causes activation of proteasomal degradation of mis/unfolded proteins); and CRT1 (calreticulin1 is involved in Ca21 homeostasis) (Babitha et al., 2015b; Ramakrishna et al., 2018). SiNF-YA5 enhances the expression of salt-responsive genes NHX1 (Na1/H1 exchanger) and LEA7 (late embryogenesis abundant) (Huang et al., 2016). Transgenic Arabidopsis plants of SiMYB42 show increased expression of nitrate transporter genes (NRT2.1, NRT2.4 and NRT2.5) under nitrogen deficiency (Ding et al., 2018). SiNAC45 enhances the expression of potassium (K) transporter genes, AKT1 and HAK1, under low K conditions (Wang et al., 2015). Apart from other stress-related genes wax, lignin, cutin, and phytohormones are other important contributors to stress tolerance traits in millets. SbWINL1 (sorghum bicolor WIN1-Like 1) induces genes involved in wax and cutin biosynthesis (Bao et al., 2017). Droughtinducible SiMYB56 accelerates lignin and ABA biosynthesis in transgenic rice (Xu et al., 2020). EcbZIP17 transgenics exhibit optimal growth under stress due to enhanced expression of genes related to the brassinosteroid signaling pathway (Ramakrishna et al., 2018). Compared to wild type, the transgenics overexpressing millet TFs are characterized by improved physiological and agronomic traits like higher germination and survival rate, greater root length and number, higher fresh weight, higher biomass, greater number of panicles, high yield, improved photosynthesis and stomatal conductance, high RWC, high proline, increased activity of antioxidant enzymes, less accumulation of ROS, decreased lipid peroxidation, and electrolyte leakage (Agarwal et al., 2010; Bihani et al., 2011; Babitha et al., 2015a; Babitha et al., 2015b; Feng et al., 2016; Huang et al., 2016; Rahman et al., 2016; Kadier et al., 2017; Xie et al., 2017; Cao et al., 2019; Meena et al., 2020; Singh et al., 2021; Wang et al., 2015). In a nutshell, overexpression of these master regulators of millets results in significantly less or negligible impacts of stress on plants than wild-type control and improves their vital functions at the molecular, biochemical, physiological, and phenotypic levels.

2.8 Conclusion and future perspectives Scarcity of water and drastic temperatures changes have posed threat to crop production and yield in arid and semiarid lands. The human race needs more stress-tolerant crops to fulfill the food demand of the overwhelmingly rising population. Adapted to a dry-hot climate and laced with high fibers, nutritional and antioxidant contents, the millets can ensure food security as well as nutritional security for a world fighting hunger, malnutrition, and poverty. We summarized and discussed the role of various TFs in the adaption of millets to arid lands. These smart regulons play vital functions in plant defense against abiotic constraints. Availability of genome sequence and advanced bioinformatic tools have led to the genome-wide identification of millet-specific putative TFs. A database for TFs of foxtail millet (FmTFDb; http://59.163.192.91/FmTFDb/) has been developed. In the future, such TF-specific databases are also expected to be created for other millets. The role of various TFs, namely WRKY, NAC, DOF, DREB, MYB, MYC, bHLH, ASR, and bZIP in millets, is well evident from the published literature. However, these studies are more focused on sorghum and foxtail millet and comparatively less attention has been given to other minor millets. These TFs can induce or suppress a variety of downstream genes in an ABAdependent or independent fashion. The altered expression profile of a TF in response to multiple

References

47

stress treatments suggests its complex role in cross-talk among different stress-associated signaling cascades. Characterization of all the TFs is a tremendous challenge due to their vast diversity. Structural, functional, and phylogenetic analysis through bioinformatic tools have, however, helped immensely in understanding the putative role of the TFs in millets’ stress responses but in planta functional validation of the reported genes with respect to individual and combined abiotic stresses is required to better understand their role at the molecular level. Several TF genes of millets have been functionally validated through heterologous expression in model plants such as Arabidopsis, tobacco, rice, etc. For more reliable insights, the evaluation of the performance of such transgenics in the field conditions is necessary.

Acknowledgments PS would like to acknowledge the Ministry of human resource development, Department of higher education, Government of India for the RUSA 2.0 programme (Thematic Project III), awarded to the Department of Botany, University of Rajasthan. PS would also like to acknowledge the Department of Botany, University of Rajasthan, Jaipur for providing basic infrastructure facilities. AB and PJ acknowledge UGC, India for senior research fellowship and junior research fellowship, respectively.

Declaration of competing interests The authors claim that they have no competing interests and personal relationships that could have influenced the work of this manuscript.

Author contribution PS conceptualized and prepared the framework and proofread the chapter. AB and PJ collected the published literature and wrote the first draft of the chapter. AB and PJ prepared figures and tables. PS also contributed to the writing. All authors approved the chapter.

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SECTION

Plant TFs and development

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CHAPTER

Plant transcription factors and root development

3

Rekha Chouhan1,2, Abhilek Kumar Nautiyal1,3, Nancy Sharma1 and Sumit G. Gandhi1,3 1

CSIR-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir, India 2Guru Nanak Dev University (GNDU), Amritsar, Punjab, India 3Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India

3.1 Introduction The root system is involved in anchoring plants to the soil and serves as a primary source for water and nutrient uptake. Roots are frequently involved in the synthesis and accumulation of many important compounds (Benfey and Scheres, 2000). They contribute significantly to a plant’s ability to adapt to changes in its environment (Jia et al., 2015). The maintenance of a functional root system is thus very essential for plants. Modifications in the number, size, and location of root hairs and radial and distal patterns of root development can drastically affect root functions (Benfey and Scheres, 2000). Numerous factors regulate the development of roots in plants. Different nutrients such as zinc, iron, phosphorus, magnesium, and nitrogen and phytohormones such as abscisic acid (ABA), auxin, and ethylene modulate tissue differentiation as well as growth in roots (Grierson et al., 2014). Arabidopsis thaliana has been widely studied as a model for organ development in plants. A. thaliana root is good for the study of cellular development due to its consistent, simple, and short generation period, abundant germinating seeds, small size, and well-defined pattern of growth and cell division (Dolan et al., 1993; Koizumi and Gallagher, 2013). Studies on A. thaliana have significantly enhanced our knowledge of the molecular aspects of root development and differentiation. Transcription factors (TFs) are important elements that regulate root developmental patterns in response to internal growth factors and external cues. TFs are the proteins that can bind DNA sequences and modulate gene expression. These factors bind to cis-regulatory sequences of target genes and control their transcription. TFs are vital elements of plant signaling pathways that regulate plant responses against biotic and abiotic stimuli besides playing roles in response to internal signals that synchronize developmental processes. The present chapter describes the TF families involved in the development and maintenance of apical root meristem, lateral roots, and root hairs. TFs can act as either positive or negative regulators of root growth and development. The chapter summarizes current knowledge regarding transcriptional regulatory networks associated with the development of plant roots in regards to TFs.

Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00007-8 © 2023 Elsevier Inc. All rights reserved.

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3.2 Plant root architecture and development In most plants, radial organization of roots consists of a set of concentric rings of cells, including outer four layers surrounding the inner vascular tissues. The epidermal and ground tissue layers known as cortex and endodermis border a central stele consisting of vasculature and pericycle. The outer epidermis is made up of two types of cells, one with root hairs and the other without roots hairs. The other layers are composed of only one type of cell. The pericycle consists of cells that can initiate the formation of new lateral roots. The vascular tissues, xylem, and phloem are located radially from the center of the root. The tip of growing roots consists of the root apical merstem. The region consists of four types of stem cells or initials surrounding a quiescent center. The quiescent center consists of slowly dividing cells that maintains the stem cells of the plant’s root tip. An outlying area of living parenchyma cells called the root cap protects the apical meristem as it passes through the soil. The central part of the root cap is known as the columella, which has its own initials (Benfey and Scheres, 2000). According to their cellular activities, the growing root of A. thaliana can be divided into four developmental zones (Dolan et al., 1993; Verbelen et al., 2006). These four zones from root tip to shoot are: the first zone is the apical meristem; the second zone is termed as the transition zone basal meristem where cell division capability still exists but is marked by slow cell growth in terms of both length and width; the third zone is termed as the elongation zone with extensive and rapid cell elongation without growth in width; and the forth zone is termed as the differentiation zone in which cells start to differentiate to their specialized features and cease to expand (Dolan et al., 1993; Verbelen et al., 2006). During embryogenesis, cellular events that lead to the primary root have been well demarcated in reference organism A. thaliana. The root apical meristem differentiates into four primary meristems—procambium, ground meristem, and protoderm, and the root cap. In grasses, the root cap originates from a different meristem known as calyptrogens. Protoderm is the primary meristem that forms the epidermis, ground meristem forms the cortex, and procambium forms the primary xylem and phloem (Baum et al., 2002).

3.3 Transcription factors involved in plant root development During the past few decades, the roles of several TFs in plant developmental pathways have been demonstrated. TFs are involved in regulating many biological processes, including stress response, cellular morphogenesis, signal transduction, etc. (Riechmann et al., 2000). Various TF families are known to control root tissue differentiation and development in response to internal growth regulators and environmental signals. TFs are involved in the development of root apical meristem, lateral roots, and adventitious roots, the components that shape root architecture. GRAS, WOX, bHLH, MYB, KNOX, NAC, MADS-BOX, and AP2 are the major TF families that control different aspects of root growth (Jia et al., 2015(Rathour et al., 2022)).

3.3.1 Root apical meristem Meristems are responsible for plant development and growth (Ioio et al., 2008). The root apical meristem, also known as the root apex, is the smallest region at the root’s tip. All cells in the root

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apical meristem have the ability to divide repeatedly from which primary root tissues are derived (Alejandro et al., 2020). Stem cells and the transit-amplifying cells (TACs) are included in the meristematic region. The primary root of higher plants is formed by a collection of pluripotent, mitotically active stem cells that reside in the root apical meristem (RAM), which serves as the foundation for root growth, development, and regeneration. In A. thaliana, the stem cells of the root meristem self-renew and give rise to daughter cells that segregate in the distal meristem transition zone (Perilli et al., 2012). Some of the major TFs involved in root apical meristem growth are described in subsequent subsections (Table 3.1).

3.3.1.1 WOX TFs and RAM development WUSCHEL-related homeobox (WOX) is a plant-specific TF family involved in plant growth and developmental processes such as embryonic patterning, stem cell maintenance, and organ formation (Vandenbussche et al., 2009; Zhang et al., 2010). It is distinguished by the existence of a conserved 60
66 amino acids long DNA binding homeodomain (HB domain) that attains helix-loop-helixturn-helix secondary structure (Mayer et al., 1998). There are at least 15 WOX genes in A. thaliana that are listed into three clades: ancient clade (WOX10, WOX13, and WOX14); intermediate clade (WOX8, WOX9, WOX11, and WOX12); and modern clade (WUS and WOX1
7) (Fig. 3.1) (Haecker et al., 2004; Vandenbussche et al., 2009; Lin et al., 2013). The functions of WOX in various plants are also preserved and correspond to those of their A. thaliana homologs. The associates of ancient clade govern root development; members of the intermediate clade regulate mainly zygotic as well as early embryo morphogenesis, whereas the member of the modern clade maintains apical stem cells (Schoof et al., 2000; Wu et al., 2005; Sarkar et al., 2007; Breuninger et al., 2008; Etchells et al., 2013; Rathour et al., 2020; Thakku et al., 2018). WOX5 is one of the key regulators maintaining the stem cell niche in plant root tips. WOX5 is expressed in the quiescent center of the root apical meristem, and inhibits the development of columella initials. Furthermore, in association with other TFs including SCARECROW (SCR),

Table 3.1 Transcription factors as a positive regulator of root growth (root apical meristem development). Transcription factors

Gene involved

Mode of action during overexpressed

Mode of action during mutation

WUSCHELrelated homeobox (WOX) TF PLETHORA (PLT) TF

WOX5gene

Dedifferentiation of the columella cells into stem celllike

Terminal differentiation in root distal stem cells

Sarkar et al. (2007), Pi et al. (2015)

PLT gene

Stem cell assembling in the meristem of root, and generation of ectopic roots from the shoot apex Disorganization of the quiescent center (QC), short root phenotype

Reduced size of root meristem; quiescent center marker is also lost

Aida et al. (2004), Galinha et al. (2007) Nakajima et al. (2001)

GRAS TF

SHORT-ROOT (SHR) and SCARECROW (SCR)

Cessation of root growth, loss of stem cell activity and disorganization of the quiescent center

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Chapter 3 Plant transcription factors and root development

FIGURE 3.1 WOX genes classification in Arabidopsis thaliana.

SHORT-ROOT (SHR), PLETHORA1 (PLT1), and PLETHORA2 (PLT2), WOX5 modulates the differentiation of other root meristem initials. WOX5 also binds to the topless (TPL) family of transcriptional corepressors (Oshchepkova et al., 2017). The loss of WOX5 function in the root meristem stem cell niche led to differentiation in the distal and proximal meristems (Sarkar et al., 2007). Ecotopic expression of WOX5 resulted in dedifferentiation of the columella cells into stem cells (Pi et al., 2015).

3.3.1.2 AP2/ERF TFs and RAM development The AP2/ERF (APETALA2/ethylene-responsive element binding factor) TF superfamily contains proteins with one or two AP2 domains. The domain is made up of 60
70 conserved amino acids forming a three-stranded antiparallel β-sheet and an α-helix. The domain is important for binding of these TFs to cis-acting elements for regulation of target genes (Li et al., 2017). During embryonic pattern formation, PLT genes are key effectors for establishing the stem cell niche. PLT1 and PLT2 genes encoding AP2 (APETALA2) class TFs are required for quiescent center specification and stem cell activity. AINTEGUMENTA-LIKE (AIL) family members of AP2/ ERF TFs like PLETHORA1
3 (PLT1
3) and BABYBOOM (BBM/PLT4) have been defined as dominant regulators of root meristem initiation and maintenance (Sablowski and Meyerowitz, 1998). These TFs are abundant in the basal embryo region, followed by the embryonic root primordium, and the stem cell niche of the root meristem. These genes are necessary for the functioning and specification of stem cells in the root meristem. PLT1 and PLT2 double mutants resulted in decrease in meristem size and elimination of quiescent center marker. Overexpression of PLT genes

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led to the proliferation of stem cells in the root meristem. Furthermore, ectopic expression of PLT genes resulted in the development of roots from the shoot apex (Aida et al., 2004; Galinha et al., 2007).

3.3.1.3 GRAS TFs and RAM development The GRAS family of TFs is named after its first three functionally identified genes: Gibberellic acid intensive (GAI), Repressor of GAI (RGA), and SCR (Grimplet et al., 2016). GRAS family proteins have been found in nearly 300 plant species. GRAS proteins are plant-specific TFs that perform fundamental regulatory functions in plant growth and maturation as well as responses to environmental cues. Proteins belonging to this gene family have a highly variable N-terminal region whereas the C-terminal region sequence contains a typical GRAS domain, which is composed of five subdomains, namely LRI, VHIID, LRII, PFYRE, and SAW. Regulatory functions of one-third of the GRAS family members are unknown (Choe et al., 2017). SCR was recognized as the earliest member of the plant-specific GRAS domain family TFs (Di Laurenzio et al., 1996). SHR and SCR are GRAS TFs that regulate Arabidopsis root development and asymmetric cell divisions. In the case of mature roots, SCR expression was confined in the endodermis near or inside the cortex/endodermis boundaries (Wysocka-Diller et al., 2000). SHR protein is generated in stele cells and subsequently travels to the next layer via plasmodesmata, where it is essential for SCR activation and endodermis specification. As a result, SCR prevents SHR from progressing and initiates cell division. According to research findings, A. thaliana stem cell activity is lost due to SHR and SCR mutations, which results in the disfunctionality of the quiescent center, which eventually leads to the short root phenotype due to the shrinkage of proliferating cells in the root meristem (Dhondt et al., 2010; Nakajima et al., 2001). Because of the loss of stem cell activity and the disarray of the quiescent center, SHR and SCR mutations result in the termination of root growth (Dhondt et al., 2010).

3.3.1.4 Negative regulator of root apical meristem UPB1 (UPBEAT1) is a bHLH TF that regulates cell proliferation and differentiation during root growth. The overexpression of UPBEAT1 in A. thaliana lines led to shorter roots and a smaller cortex compared to the wild-type plants. Further, mutations in UPBEAT1 resulted in longer roots and large cortex (Wong, 2010; Manzano et al., 2014; Li et al., 2020).

3.3.2 Lateral roots Lateral roots are the building blocks of the root system that arises from pericycle and exhibit a morphology that is similar to the primary root (Dolan et al., 1993). These roots are important for expanding the contact area of root systems to explore heterogeneous soil environments. Formation of lateral roots involves the developmental process that combines preinitiation, initiation, emergence, and meristem activation of the lateral root primordium (Torres-Martı´nez et al., 2019). Lateral roots originate from pericycle founder cells adjacent to xylem. Auxin-induced signaling leads to irregular division of two neighboring pericycle cells. The small daughter cells further divide and produce a primordium bound by two large cells. The lateral root patterning is closely governed by sequence of divisions that results in a dome-shaped structure to form a new meristem.

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Chapter 3 Plant transcription factors and root development

Table 3.2 Transcription factors as a positive regulator of root growth (lateral root development). Transcription factors NAC (NAM, ATAF1/ ATAF2, and CUC2) TF KNOX (KNOTTED 1like homeobox) TF MADS Box TF

Gene involved

Mode of action during overexpressed

Mode of action during mutation

NAC gene KNOX gene AGL 21 gene

A higher number of lateral roots Increase in lateral roots




Increases length as well as the number of the lateral roots

Shorter and fewer lateral roots




References Hao et al. (2011), Yang et al. (2019) Dean et al. (2004) Lin et al. (2013)

The primordium progressively travels through the outer tissues into the rhizosphere (Swarup et al., 2008). The development of a lateral root defines the “primordium-intrinsic” patterning of de-novo organ tissues and a meristem, along with the interaction between lateral root primordia and superimposing tissues to facilitate movement to cell layers during lateral root emergence. Several TF families are known to regulate the lateral root development in plants (Table 3.2).

3.3.2.1 NAC TFs and lateral root development The NAC TF family is among the most extensively studied plant-specific TF families. The family is named after three identical proteins encoded by NAM, ATAF1/ATAF2, and CUC2 genes (Souer et al., 1996). NAC TFs encompass almost 160 amino acid residues and five N-terminal subdomains, A to E, having similar structure. The subdomains A, C, and D are exceptionally preserved, but the nuclear localization signal involved in the identification process of the NAC TF is restricted to the C and D subdomains only. The NAC protein C-terminus region is enriched with some replicated amino acids such as serine, threonine, and proline and has higher diversification of transcriptional activation region (Aida et al., 1997; Mathew and Agarwal, 2018; Zhang et al., 2018). NAC TFs contribute to plant stress response (high light intensity, osmolarity, and salinity) (Nuruzzaman et al., 2013). They are also involved in various developmental processes, including leaf senescence (Guo and Gan, 2006); flower morphogenesis (Sablowski and Meyerowitz, 1998); hormonal signaling (Ren et al., 2018); stomatal closure (Du et al., 2014); plant height (Kato et al., 2010); fruit ripening (Shan et al., 2012); seed development and germination (Kim et al., 2008; Yang et al., 2019); crop yield and quality (Zhao et al., 2015); formation of the secondary wall (Mitsuda et al., 2005); cell development and metabolism (Kim et al., 2006; Kim et al., 2009); lateral root formation (Zhang et al., 2018) as well as the development of apical shoot (Souer et al., 1996; Aida et al., 1999). The family is found in many plants, including 117 members in A. thaliana, 152 in Glycine max, 151 in Oryza sativa, and 251 in Panicum virgatum (Nuruzzaman et al., 2010; Nuruzzaman et al., 2013; Yan et al., 2017). The overexpression of GmNAC20 in G. max yielded plants with higher tolerance to salinity and freezing stresses. GmNAC20 is also known to accelerate the development of lateral roots in A. thaliana (Hao et al., 2011). Similarly, GmNAC004 has been shown to regulate lateral root length and number under normal conditions and enhance lateral root development under mild water stress in Arabidopsis (Quach et al., 2014).

3.3 Transcription factors involved in plant root development

69

3.3.2.2 KNOX gene family and lateral root development The KNOX (knotted-like) genes belong to a large family of TFs, which consists of a conserved DNA binding domain known as the homeodomain. These genes are present in many organisms, including mammals, insects, and plants, and control pattern formation and growth. A member of the KNOX gene family known as knotted-like 6 Arabidopsis (KNAT6) is expressed at the bottom of the lateral roots. Overexpression of KNAT6 gene has been known to increase the number of lateral roots while decreasing the size of the root. KNAT6 TF is known to inhibit cell division in pericycle cells near growing lateral roots. When exposed to auxin, KNAT6 expression moved to the root tips, which was inhibited by exogenous cytokinin. Due to this movement, KNAT6 embodies an excellent candidate to study the link between its restricted effect during lateral root formation and long-range auxin transport (Dean et al., 2004).

3.3.2.3 MADS Box TFs and lateral root growth The MADS-box TF family plays an essential role in determining plant and animal development (Messenguy and Dubois, 2003). The MADS TF family is named after the discovery of the first four MADS domain proteins: “M” stands for MINICHROMOSOME MAINTENANCE FACTOR 1 from Saccharomyces cerevisiae, “A” for AGAMOUS (AG) from A. thaliana, “D” for DEFICIENS from Antirrhinum majus, and “S” for Serum Response Factor (SRF) from Homo sapiens (Passmore et al., 1988; Sommer et al., 1990; Yanofsky et al., 1990). MADS-box genes contain conserved sequence motifs (Parenicov´a et al., 2003). One hundred and seven MADS-box genes encoding MADS-box protein have been identified in A. thaliana roots, the activities of which are unknown (Parenicov´a et al., 2003). XAL1/AGL12 and XAL2/AGL14 have been reported to play a critical role in the regulation of plant root growth. For lateral root development, ANR1 is the only reported member of this family. It has been observed that the AGL21 MADS-box gene plays a significant role in lateral root development. Overexpression of AGL21 gene increased both the length and number of lateral roots. In contrast, mutant alleles of AGL 21 have shorter and fewer lateral roots (Lin et al., 2013).

3.3.2.4 Negative regulator of lateral root development WRKY75 TF has been reported to negatively regulate the development of lateral roots. The suppression of WRKY75 using RNAi enhanced lateral root length and number in Arabidopsis (Devaiah et al., 2007). MYB (myeloblastosis) TFs also play an essential role in negatively regulating lateral root development in A. thaliana (Ambawat et al., 2013). Mutations in AtMYB93 have been shown to enhance the density and growth of lateral roots (Gibbs and Coates, 2014).

3.3.3 Root hair Root hairs are simple cell extensions formed at the root apical meristem. Root hair makes a direct connection with the soil. It plays a major role in absorbing water and nutrients from the rhizosphere (Dazzo et al., 1984; Mandel and Yanofsky, 1995; Gilroy and Jones, 2000). The modest size of the hairs expedites root penetration at the edges within the soil (Mandel and Yanofsky, 1995). They are involved in the interaction of plants with soil microorganisms, and are directly involved in forming root nodules in legume plants (Dazzo et al., 1984). Root hairs are found only in the zone of

70

Chapter 3 Plant transcription factors and root development

Table 3.3 Transcription factors as a positive regulator of root growth (Root hair development). Transcription factors bHLH (Basic helix loop helix) TF MYB TF

Gene involved RSL1 (ROOT HAIR DEFECTIVE- SIX LIKE-1) RSL2 (ROOT HAIR DEFECTIVE- SIX LIKE-2) CPC gene {controls a negative regulator GL2 (GLABRA 2) function}

Mode of action during overexpressed

Mode of action during mutation

Development of ectopic root hair cells Does not affect the growth of root hairs


Short root hairs Short root hairs


References Yi et al. (2010), Choe et al. (2017) Wada et al. (2002)

maturation, also known as the zone of differentiation, where they grow as outgrowths at the surface of the plant’s roots, which vary between 15
17 mm in diameter and 80
1500 mm in length (Thomas et al., 2016). In A. thaliana, root hairs are approximately 10 μm in diameter and can mature up to 1 mm or more in length. They proliferate at a rate of more than 1 μm per minute, which facilitates studies of cell expansion (Grierson and Schiefelbein, 2002). Root hair is an excellent model system for the study of cell patterning, differentiation, and growth. Understanding the molecular mechanisms is essential for changing root hair morphology in order to generate crops with superior growth traits (Hayat et al., 2010). bHLH and MYB TF families are known to regulate root hair development (Table 3.3).

3.3.3.1 bHLH TFs and root development The basic helix loop helix (bHLH) is the most prominent family of dimerizing transcription factors that govern the expression of the gene through interaction with specific motifs in target genes. RSL (ROOT HAIR DEFECTIVE- SIX LIKE) class II subfamily members encode bHLH (Basic helix loop helix) TFs expressed in root hairs. RSL2 is known to stimulate root hair development. In contrast, a single mutation of RSL2 facilitates shortening of root hairs, and the overexpression of RSL2 does not affect root hair development. RSL4 is also a key regulator of root hair growth. Constitutive expression of RSL4 protein by using CaMV35S promoter showed sufficient root hair growth (Yu et al., 2014). RSL class I positively regulates root hair growth in O. sativa. Loss of RSL class I function resulted in short root hairs compared to wild type O. sativa, whereas the overexpression of RSL class I resulted in ectopic root hair cell formation (Du et al., 2014).

3.3.3.2 MYB TFs and root hair development The MYB (Myeloblastosis) TFs gene family is found in both plants and animals. This family in plants is involved in controlling numerous functions such as plant development, differentiation, metabolism, and biotic and abiotic stress. CPC (CAPRICE) is a R3 type plant MYB TF expressed in atrichoblast root cells (hairless cells) (Mu et al., 2009), and relocated to the nuclei of trichoblasts (root hair cells present on the exterior surface). CPC is a positive regulator of plant root hair growth that stimulates the differentiation of root hair cells by affecting the regulation of GL2 (GLABRA2) (Ioio et al., 2008; Shibata and Sugimoto, 2019).

3.4 Conclusion

71

Table 3.4 Transcription factors as a negative regulator of root growth. Transcription factors

Gene involved

Mode of action during overexpressed

UPB (UPBEAT)

UPB 1

MYB




bHLH

LRL 5(Lotus japonicus Root hair-like) GL2 (GLABRA 2)

Reduction of root hair length


OBF binding protein 4 gene

Reduction of cell number as well as its size

DOF type transcription factor

Shorter roots and cortex compared to wild-type plants Slower development of lateral root

Mode of action during mutation Longer roots compared to control Faster lateral root development
Root hair development


References Wong (2010), Mu et al. (2009) Dolan et al. (1993) Wang et al. (2019)

Pi et al. (2015)

3.3.3.3 Negative regulators of root hair formation The suppression of WRKY75 using RNAi has been shown to increase the length and number of root hairs in Arabidopsis (Devaiah et al., 2007). Overexpression of LRL5 (Lotus japonicus root hair-like) has also been reported to reduce root growth. It functions by suppressing the expression of AtLRL3 (A. thaliana LRL3), which positively regulates root hair development. Overexpression of ZmLRL5, a bHLH TF in Zea mays (maize) roots, has been found to reduce root hair extension (Wang et al., 2019). A homeodomain TF, GL2 (GLABRA 2), is known to inhibit the growth of root hairs. GL2 mutation induces the development of roots hairs altering the differentiation of hairless epidermal cells (Masucci et al., 1996). Another family of plant TFs, DOF (DNA binding with one finger), is a negative regulator of root growth. They contain a conserved region of 50 amino acids with C2C2 type zinc finger-like motif in their DNA binding domain (Table 3.4). DOF type TF, OBF binding protein 4 (OBP4) genes negatively regulate cell proliferation and expansion. Overexpression of OBP4 has been observed to decrease cell number, size, and limit cell growth in A. thaliana (Xu et al., 2016).

3.4 Conclusion Maintenance of a functional root system is essential for plants. This chapter provided useful insights into the TF families associated with plant root growth. Years of research on the A. thaliana model system have brought about comprehensive knowledge on root growth and development. Remarkable improvements have been accomplished in understanding the molecular mechanisms underlying plant root growth. Several TF families have been identified for their contribution in root differentiation and development. GRAS, WOX, AP2, bHLH, MYB, KNOX, NAC, and MADSBOX are the major TF families that control different aspects of root growth and development.

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Acknowledgments SGG thankfully acknowledges support from Council of Scientific and Industrial Research (CSIR, India) funded project MLP110006.

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913.

CHAPTER

The roles of transcription factors in the development of plant meristems

4

Qingkun Dong1 and Cui Zhang1,2 1

Key Laboratory of Plant Molecular Physiology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Botany, Chinese Academy of Sciences, Beijing, P.R. China 2College of Life Sciences, University of Chinese Academy of Sciences, Beijing, P.R. China

4.1 Introduction In higher plants, the shoot architecture is determined by the arrangement and activity of apical, axillary, intercalary, and inflorescence meristems and the subsequent development of stems, leaves, branches, and inflorescences. Meristem is responsible for producing all tissues and organs. Shoot apical meristem (SAM) is a special region where the tip contains a group of stem cells, which will renew themselves through proliferation, and produce new cells for organ differentiation at the same time (Aichinger et al., 2012; Ha et al., 2010; Somssich et al., 2016). During the vegetative stages, SAM give rise to leaf primordia, and develop into inflorescence meristem after the plant receives the flowering signal and thus enters reproductive growth. Inflorescence meristem either directly starts flower meristem, and then further differentiates into flower organs, or forms lateral meristems, such as axillary or intercalary meristem (Hirano et al., 2014; Tanaka et al., 2013). In each meristem, the dynamic balance between stem cell self-renewal and organ/meristem differentiation is very important for the normal development of plants, which makes plants different from animals and humans and can grow for life. The maintenance and differentiation of meristems are regulated by specific protein components such as transcription factors (TFs), peptide signaling and receptors, and plant hormones such as auxin and cytokinin. Transcription factor, also known as trans-acting factors, are DNA-binding proteins that can specifically interact with cis-acting elements in the promoter region of eukaryotic genes. Through the interaction between them and other related proteins, they activate or inhibit the transcription process and are the main regulatory factors of gene expression. TFs play an important role in plant growth and development, fruit ripening, senescence, response, and adaptation to various stresses and defense response (Wang et al., 2016a). According to statistics, about 4% to 7% of genes encode TFs in eukaryotes. In general, the number of TFs is related to the complexity of organisms (Levine and Tjian, 2003).

4.2 Shoot apical meristem All of the postembryonic tissues and organs are derived from meristems in higher plants. Among them, SAM produces all above ground parts by continuously producing new organ primordia. SAM Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00008-X © 2023 Elsevier Inc. All rights reserved.

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is located at the top of the stem, and there is a group of cells in its central area, which are not active in division. These cells are called stem cells (Potten and Loeffler, 1990). Stem cells can divide into two types of cells, one type continue to exist in the original position and maintain cell totipotency and another type leave the central region and move to the peripheral region, thus dividing and differentiating to produce various mature tissues (Snow et al., 1953). SAM is the basis for the growth of stem, axillary buds, and young leaves. During the development of Arabidopsis thaliana, SAM cells proliferate and differentiate, and form leaf primordia around it, and then develop into leaves. When plants change from vegetative growth to reproductive growth, SAM will be transformed into inflorescence meristem and floral meristem. Therefore SAM not only has the main characteristics of promeristem maintaining strong mitotic activity, but also has the characteristics of primary meristem: maintaining cell division ability and differentiating into mature tissue. The maintenance and differentiation of meristem activity depend on various exogenous signals from peripheral cells and the coordinated regulation of internal genes. In Arabidopsis, WUSCHEL-CLAVATA (WUS-CLV) negative feedback loop is the basic mechanism for maintaining homeostasis of stem cell niche in SAM (Aichinger et al., 2012; Brand et al., 2000; Carles and Fletcher, 2003; Ha et al., 2010; Schoof et al., 2000; Somssich et al., 2016). WUS is a TF with homotypic domains, expressed in the organizing center (OC) region of apical meristem and inflorescence meristem, while CLV3 is expressed in the stem cell region of SAM, and its receptor complex is expressed in the central region of tissue and surrounding cells. CLV3 polypeptide migrates from L1/L2 layer to L2/L3 layer to bind its receptor complex composed of CLVATA1 (CLV1), CLVATA2 (CLV2)/CORYNE (CRN) and RECEPTOR PROTEIN KINASE 2 (RPK2), and then inhibits the expression of WUS by inhibiting the expression of protein phosphatases POLTERGEIST (POL) and POLTERGEIST LIKE1 (PLL1) (Clark et al., 1997; Fletcher et al., 1999; Kinoshita et al., 2010; Muller et al., 2008). WUS can also move to L1/L2 layer to directly activate CLV3 expression (Katsir et al., 2011). The genetic mechanism of WUS-CLV is conserved in monocot plants, such as rice and maize (Fornara, 2014; Somssich et al., 2016). In rice, floral organ quantify genes FON1 and FON2 are homologous genes of Arabidopsis CLV1 and CLV3, respectively, and negatively regulate the maintenance of meristem during reproductive growth (Suzaki et al., 2004, 2006). In maize, the orthologous genes of CLV1 and CLV2, THICK TASSEL DWARF1 and FASCIATED EAR2 (FEA2) negatively regulate the maintenance of meristem (Bommert et al., 2005; Somssich et al., 2016; Taguchi-Shiobara et al., 2001). Recently, it has been reported that FON2-LIKE CLE PROTEIN1 (FCP1) in maize also plays a role in the maintenance of meristem (Je et al., 2016, 2018). In brief, the TF WUS and the small polypeptide CLV3 form a signal feedback loop through intercellular transport, which regulates SAM maintenance and differentiation balance by noncellular autonomous behavior (Daum et al., 2014; Fletcher et al., 1999; Mayer et al., 1998; Rodriguez et al., 2016; Yadav et al., 2011). Early genetic experiments have also isolated and identified other genes that regulate the growth and development of SAM, such as TF gene SHOOT MERISTEMLESS (STM), which encodes KNOTTED1-like HOMEOBOX (KNOX) family proteins (Long and Barton, 1998). KNOX is one of the most important regulators in meristem development and function, and can restrict cell differentiation in SAM. Many studies have shown that KNOX plays a role in the formation and maintenance of SAM. Typical representatives are Arabidopsis SAM dysfunction mutant (stm) and maize KNOTTED1 knockout mutant (kn1), both of which lack SAM (Long et al., 1996; Vollbrecht et al., 2000). The embryos of stm mutants develop abnormally, and the mature embryos are deficient in

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SAM. The knockout mutant of STM has no SAM, and leaves only fused cotyledons, which finally produce a few disorganized leaves. Previous studies have shown that STM can maintain cell proliferation by inhibiting the expression of ASYMMETRIC LEAVES 1 (AS1) (Byrne et al., 2002). AS1 protein contains MYB domains and is expressed in cotyledon primordium (Li et al., 2005; Sun et al., 2002). In stm strong mutant, the expression range of AS1 extended to SAM, which indicated that STM inhibits AS1. The AS1-AS2 inhibitor complex in Arabidopsis can induce the cyclization of KNOX promoter and put it in an inhibitory chromatin state, thus inhibiting its expression (Guo et al., 2008). It was found that OsGRF3 and OsGRF10 in rice can bind to the promoter of KNOX family gene OsKN2, thus inhibiting its expression. In Arabidopsis, the upregulated expression of AtGRF4, AtGRF5, and AtGRF6 inhibited the expression of KNOX gene KNAT2, which was accompanied by developmental abnormalities caused by KNOX inhibition (Kuijt et al., 2014). In barley, BGRF1 binds to the intron-specific regulatory element of BKN3 gene and repressed its expression, while OsGRF10 can replace BGRF1 as an inhibitor to repress the expression of BKN3 gene (Osnato et al., 2010). To sum up, the regulation mode of GRF-KNOX in plants is very conserved. It was found that the expression level of CLV3 increased in WUS overexpressed plants, but the expression pattern did not change compared with wild type plants, which indicated that the expression of CLV3 needs other cofactors besides the WUS gene. Further studies have shown that ectopic expression of WUS and STM can produce ectopic meristem in hypocotyl, which indicates that WUS and STM have a synergistic effect in activating CLV3 expression during postembryonic development (Gallois et al., 2002; Lenhard et al., 2002). Recent studies have shown that WUS and STM can bind to each other to form a heterodimer, which binds to the promoter of CLV3. This heterodimer can further enhance the binding strength with CLV3 promoter and activate its expression, thus enhancing the activity of SAM cells (Su et al., 2020).

4.3 Axillary meristem Plant growth has plasticity. In higher plants, the branching plays an important role in producing various plant morphological models. The establishment of plant configuration mainly depends on meristem. During embryogenesis, two groups of multifunctional cells were identified: SAM and RAM (root apical meristem) (Laux and Jurgens, 1997). During the whole postembryonic development, the RAM will form a complete root system, and the aerial structure of the plant is determined by SAM and the axillary meristem (AM) (Muller et al., 2006; Wang and Li, 2008; Ward and Leyser, 2004). The SAM determines the main axis of the plant, while the branching structure of the plant is controlled by the AM (Shimizu-Sato et al., 2009). Taking Arabidopsis as an example, the axillary buds in the vegetative growth period established a bottom-up gradient from the distal end of SAM; on the contrary, during reproductive growth, axillary buds began to appear at the axils of newly formed leaf primordia near SAM, forming a top-down initiation and growth pattern. It was also shown that the formation of axillary buds involves different molecular regulation mechanisms during vegetative and reproductive growth periods. The initiation of AM depends on the stem cell niche in leaf axils, and the establishment of this stem cell niche is related to the boundary area between stem and leaf primordium. Many known genes affecting axillary bud initiation encode

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TFs, which also affect the formation of boundary regions (Wang et al., 2016b). In addition, the initiation of AM is closely related to the polarity of the leaves, because AM only starts to form on the adaxial side of the leaves near SAM. AM can develop into buds, which then remain dormant or outgrow into branches. The quantity and activity of AM indirectly reflect the environmental adaptability of plants and determine the yield of crops. The genes regulating axillary buds were first reported in tomato. Among them, the LATERAL SUPPRESSOR(LS)gene belongs to the GRAS (GAI, RGA, SCR) gene family, which is involved in plant lateral branch formation, gibberellin signal transduction, and many other plant physiological processes. ls mutant had fewer branches, decreased fertility, and abnormal petal development. The LATERAL SUPPRESSOR (LAS) gene in Arabidopsis has high homology with the LS gene (Greb et al., 2003). LAS also encodes a member of the GRAS family TF, which affects the expression of meristem marker gene STM through REVOLUTA (REV) in the vegetative growth stage of Arabidopsis, thus affecting the formation of AM (Greb et al., 2003). In rice and tomato, LAS homologs affect the development of the reproductive growth stage (Li et al., 2003; Schumacher et al., 1999). During the vegetative and reproductive stages, LAS transcripts accumulate specifically in the axils of lateral primordia derived from SAM (Greb et al., 2003). The MONOCULM 1 (MOC1) gene of rice is a homolog of LAS and LS, and the tillers of moc1 decreased significantly. Further experiments proved that MOC1 regulated rice tillering by affecting downstream genes OSH1 and OsTBI (Li et al., 2003). In recent years, it has been reported that the MYB TF family, such as MYB2, MYB37 (RAX1), MYB38 (RAX2), and MYB84 (RAX3) also has an obvious function in plant branching. RAX1 was the first identified AM marker gene (Keller et al., 2006). Compared with the LAS gene, RAX1 was expressed earlier, and the expression pattern of RAX1 was confined to the center of the leaf axils. Genetic results showed that RAXs and LAS/LS are functionally redundant. But molecular evidence showed that the pathway of AM formation is different between them. RAX1 affects the formation of AM by regulating the expression of STM together with CUP-SHAPED COTYLEDON 2 (CUC2). LAS affects the formation of AM by regulating the expression of STM by REV. The latest research results show that LAS and RAX can regulate the formation of AM by jointly regulating ROX. The homologous gene of RAX in tomato is BLIND, which also belongs to the MBY TF family, and the formation of AM is blocked in blind knockout mutant (Jin and Martin, 1999). REVOLUTA (REV) encodes a set of HD-ZIPIII TFs. Different from LAS, REV mutants showed AM deficiency in both vegetative and reproductive growth stages (Otsuga et al., 2001). In addition, REV has a wide expression pattern in many types of tissues of stems and roots. Consistently, the rev mutant not only shows the absence of AM initiation, but also has defects in the development of SAM, leaves, vascular bundles, and roots. The latest research found that AGO10 can promote the development of AM by binding miR165/166, thus promoting the development of AM through the target gene REV. The authors found that the expression of AGO10 gene is precisely regulated by auxin, brassinosteroid, and light signals in a spatiotemporal pattern, thus ensuring that AM will only start in leaf axils of specific stages (Zhang et al., 2020). Auxin responsive factor ARF5 can directly activate the expression of AGO10, while brassinosteroid positive regulator BZR1 and phytochrome interacting factor PIF4 can directly inhibit the expression of AGO10 gene. In young leaf axils, BZR1 and PIF4 inhibited the expression of AGO10 and prevented the initiation of AM. In

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old leaf axils, ARF5 upregulates the expression of AGO10 to promote the initiation of AM (Zhang et al., 2020). The representative genes regulating leaf AM formation in gramineae are LAX PANICLE (LAX) and SMALL PANICLE (SPA). In rice, OsLAX encodes bHLH TF, which is redundant with SPA in tillering regulation (Bennett and Leyser, 2006; Komatsu et al., 2003). In maize, there are genes that play a similar role to OsLAX in regulating the formation and maintenance of AM. The typical one is BA1, which is a homolog to LAX and also encodes a bHLH protein. The development of AM in ba1 mutant was affected during vegetative growth and reproductive growth (Ritter et al., 2002). BIF2 responds to the signal of AM formation in maize, and then directly or indirectly maintains the expression of KN1 in AM, thus playing a positive role in regulating AM formation (Bennett and Leyser, 2006; Kerstetter et al., 1997; McSteen and Hake, 2001). The REGULATOR OF AXILLARY MERISTEM FORMATION (ROX) gene of Arabidopsis is homologous to the LAX1 gene in rice and BA1 gene in maize. The rox mutant has developmental defects on the formation of axillary buds in the vegetative growth stage of Arabidopsis. Mutation of ROX in rax1 and las mutants, two key regulators of AM initiation, can enhance the defect of AM phenotype. In the vegetative growth stage, the expression of ROX depends on RAX1 and LAS, which jointly regulate the formation of AM and are key genes for axillary bud formation (Yang et al., 2012). In addition, NAC domain containing proteins CUC1, CUC2, and CUC3, although functionally redundant in some aspects, perform different functions in boundary formation and AM initiation. CUC genes are expressed specifically at the boundary. During the initiation of AM, CUC1 has no obvious effect, CUC2 has a weak effect, and CUC3 plays a major regulatory role (Hibara et al., 2006). Compared with single mutant, las/cuc3 double mutant showed more obvious phenotype of axillary bud deletion, which indicated that LAS and CUC3 had functional redundancy in axillary bud initiation. Recent studies have found that TF CUC2/3 acts on the initiation of AM, directly binds to the promoter of ubiquitin-dependent peptidase gene DA1, and activates the expression of DA1. Further studies have shown that ubiquitin-specific protease UBP15, the direct substrate of DA1 peptidase, can inhibit the initiation of AM. Genetic analysis showed that CUC2/3, DA1, and UBP15 played a role in the same pathway and regulated the initiation of AM (Li et al., 2020). TB1 is one of the TFs of the TCP family in maize, and the proteins of this family participate in regulating growth and cell division. TB1 gene regulates the inhibition of AM, and then regulates the formation of secondary AM (Doebley et al., 1995; Hubbard et al., 2002). The formation of AM also depends on the expression of STM. Further study showed that STM expression abundance changed from strong to weak and then to strong with the maturation of leaf primordium. The early low abundance of STM expression depends on the low auxin environment in leaf axils. Subsequently, in vivo and in vitro experiments confirmed that REV of HD-ZIPIII TF family can directly bind to STM, upregulate STM expression, and thus promote AM initiation. It was also found that the regulation of STM by REV was tissue-specific, and the histone methylation modification of STM sites in different tissues affected the regulation of STM expression by REV (Shi et al., 2016; Tian et al., 2014). Two functionally redundant AP2 TFs, DORNRO¨ SCHEN (DRN) and DORNRO¨ SCHEN- LIKE (DRNL), control the initiation of AM by regulating STM. The activation of STM expression by DRN/DRNL depends on REV, and vice versa. In addition, another

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REV interacting protein, LITTLE ZIPPER3, is expressed in leaf axils and interferes with DRN/ DRNL-REV interactions, thus controlling STM expression and AM initiation (Zhang et al., 2018). In the Gramineae family, the maize KN1 gene belongs to the class I KNOTTED1-like homeobox (KNOX) gene family, and its coding protein is the first identified protein that has a homeobox domain in plant. The dysfunctional mutant of KN1 gene in maize has similar phenotype to the stm mutant in Arabidopsis, which can only produce cotyledons, but other elements of stems and branches are missing. The KN1 gene is closely associated with meristem and internode growth in gramineae and dicot plants (Kerstetter et al., 1997; Malcomber et al., 2006). At an earlier stage, ATH1 preserve the fate of meristem cells in Arabidopsis leaf axils by maintaining the expression of meristem marker gene STM. In addition, ATH1 protein can interact with STM protein to form a self-activating circuit of the STM gene. Genetic and biochemical data showed that ATH1 can anchor STM to activate the STM gene and other AM regulatory genes (Cao et al., 2020). During reproductive stages, AHL15 acts as an inhibitor of AM maturation. The dysfunctional mutation of AHL15 gene accelerated the maturation of AM. Ectopic expression of AHL15 inhibited the maturation of AM, which indicated that AHL15 and AHL class A genes played a role in the downstream of flowering gene (SOC1, FUL) and upstream of phytohormone gibberellin, which inhibited the maturation of AM and prolonged plant life (Karami et al., 2020).

4.4 Flower meristem Floral organ development refers to the process in which the plant flower primordium develops into mature flower organ, such as calyx, petals, stamens, and carpels. This is a multistage developmental process, which starts from the initiation of flower meristem, then determines and maintains the characteristics of flower meristem, forms flower primordium, recognizes the characteristics of flower organs, terminates the activity of stem cells of flower meristem, and matures flower organs. According to the “ABCDE” model of floral organ development in plants, the characterization of floral organ with four-wheel structure is controlled by five kinds of genes, A, B, C, D, and E (Coen and Meyerowitz, 1991; Theissen et al., 2000), of which most belong to the MADS-box gene family except AP2, which belongs to the class A family (Li et al., 2016). Members of the MADS-box gene family have different regulatory functions (Dreni and Zhang, 2016; Li et al., 2017; Nakatsuka et al., 2016). For example, members of the MIKCc-Type MADS-Box gene family, SUPPRESSOR OF OVERESPRESSION OF CONSTANS1 (SOC1), FLOWERING LOCUS C (FLC1), AGAMOUSLIKE GENE 24 (AGL24), MADS AFFECTING FLOWERING (MAF1/FLM), and SHORT VEGETATIVE PHASE (SVP), play an important role in plant flowering regulation (Balanza et al., 2018; Villarino et al., 2016; Zeng et al., 2018); APETALA1 (AP1), FRUITFUL (FUL), and CAULIFLOWER (CAL) genes function in flower meristem determination (Ferrandiz et al., 2000; Zhang et al., 2019); SEPALLATA 1
3 (SEP1
3), APETALA 3 (AP3), and PISTILLATA (PI) genes play important roles in floral organogenesis (Pelaz et al., 2000).

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SOC1, SVP, AGL24, and SEPALLATA 4 (SEP4) regulate flowering time and directly inhibit TFL1 expression in newborn flower meristem in a redundant way in Arabidopsis. It has been found that SVP, SOC1, AGL24, and SEP4 can interact with AP1 and directly bind to TFL1 gene region to regulate TFL1 expression, among which SVP is the main regulatory factor inhibiting TFL1 expression in inflorescence meristem (Liu et al., 2013). The expression of SVP, SOC1, and AGL24 was downregulated by AP1 to an appropriate level in order to prevent the flower meristem from reversing to inflorescence structure (Liu et al., 2013). SVP, SOC1, and AGL24 keep a moderate expression level at specific developmental stages, and they have dual functions (Yu et al., 2004). In addition, BoFLC2 plays an important role in maintaining vegetative growth of cauliflower, and can also inhibit flowering and inflorescence development (Ma et al., 2017). Recently, it has been found that SOC1 not only affects flowering time, but also promotes lateral branches and inflorescence development in mustard (Tyagi et al., 2019). TERNIMAL FLOWER 1 (TFL1), LEAFY (LFY), and APETALA 1 (AP1) are characteristic genes of flower meristem in Arabidopsis, and the antagonistic interaction among them can regulate the branching mode of inflorescence (Ma et al., 2017). LFY is a “pioneer transcription factor” with the ability to change chromatin state. LFY protein can bind to AP1 DNA sequence located in nucleosome body with strong affinity. Changing the chromatin state of AP1 locus allows AP1 to be transcribed and expressed, and regulates the development of plant flower meristem and flower organ (Jin et al., 2021). TFL1 was specifically expressed in inflorescence meristem and lateral inflorescence meristem, while LFY and AP1 were widely expressed in young flower meristem in Arabidopsis (Winter et al., 2015). Knockout of TFL1 led to early flowering, which induced the transformation of inflorescence meristem and AM into flower meristem, accompanied by ectopic expression of LFY and AP1. AP1 together with its homologs, CAL and FUL, inhibit the expression of TFL1 gene (Parcy et al., 2002), while LFY promote the expression of TFL1 (Serrano-Mislata et al., 2017). In addition, ARGONAUTE1 (AGO1) can inhibit the expression of TFL1 and regulate inflorescence development (Fernandez-Nohales et al., 2014). XAANTAL2 (XAL2/AGL14) can also bind to the regulatory region of TFL1 gene. Overexpression of AGL14 can upregulate the expression of TFL1 and WUS, thus affecting the maintenance and determination of flower meristem and transforming to inflorescence development in Arabidopsis (Perez-Ruiz et al., 2015). The maintenance and termination of floral meristem play a vital role in floral organogenesis and generational alternation. After all the flower organs are produced, the flower meristem will be terminated accurately and programmatically, which is called the termination of flower meristem. This is helpful to ensure the normal reproductive growth and seed development of plants, and can ensure the yield of crops in agricultural application. Many studies have confirmed that AGAMOUS (AG), a floral organ characteristic gene encoding MADS TF, plays an important role in maintaining and terminating the activity of floral meristem cells (Liu et al., 2018; Xu et al., 2017). Through two target genes, CRC and KUN, AG can coregulate and inhibit the expression of WUS, which is the key gene of meristem maintenance, and shut down the cell maintenance program of inflorescence meristem in a specific spatiotemporal manner.

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4.5 Intercalary meristem The intercalary meristem is a type of meristematic tissue located in the base of internodes and leaf blades of monocots. Intercalary meristem supports stem growth independently of the shoot apex in the middle position, thus the name. The growth at this point is referred to as intercalary growth and are common in monocots, especially gramineous plants, such as rice, wheat, and maize. With the activity of the intercalary meristem, plants can achieve jointing and heading, so that the stem can grow taller rapidly. Little is known about the molecular mechanisms that control the growth of intercalary meristem. Ethylene and GA is involved in this regulatory process. A novel GAresponsive TF gene, OsGRF1, was preferentially expressed in rice intercalary meristem. Overexpression of OsGRF1 in Arabidopsis inhibited stem elongation, suggesting that OsGRF1 plays a regulatory role in stem growth (Luo et al., 2005; van der Knaap et al., 2000). In another study, Oscen1 and Oscen2 of the TERMINAL FLOWER1 (TFL1)/CENTRORADIALIS (CEN) gene family showed different expression patterns mainly in secondary meristem. Overexpression of OsCEN1 and OsCEN2 in rice resulted in increased internode number, shorter internode length, altered radial pattern of elongated internode, delayed heading as well as panicle configuration abnormalities. OsCEN1 and OsCEN2 regulate the development of rice plant architecture by stimulating the activity of secondary meristem (Zhang et al., 2005). Because concrete roles of these and many other genes still need to be elucidated, the mechanisms that regulate intercalary meristem activity remain unclear.

4.6 Conclusion and future perspectives SAM stem cells are the basis for the growth and development of aerial parts of plants. Complex gene regulatory networks and signal transduction pathways make stem cells maintain the balance between cell division and differentiation (Fig. 4.1). WUS/CLV3 feedback loop plays an important role in stem cell regulatory network. Meanwhile, it is conserved in plants to some extent; a large number of TFs and chromosome remodeling factors regulate stem cell activity by regulating WUS at the transcription level. Other regulatory cycles coexisting with WUS/CLV3 also function in SAM, and they jointly maintain the stability of stem cell niche through negative feedback. Spatiotemporal and dynamic expression patterns of the factors involved in SAM regulation can explain the proliferation and differentiation behavior of stem cells in detail. At the same time, it can modify the known pathways, while the correlation of different pathways needs more evidence to supplement. In addition, some key TFs regulating meristem have been reported one after another, but most of their upstream regulators and downstream target genes are unknown. Although it is known that the development of meristem is controlled by the interaction of many factors, how external environmental factors such as temperature, nutrients, and light coordinate with endogenous developmental signals such as hormones and TFs to finely regulate meristem development still remain to be further studied. The next work can focus on how transcriptional regulatory networks and epigenetic modifications affect cell fate determination during the establishment, maintenance, and activation of meristem-like cells. Abbreviations of the transcription factors mentioned in this chapter are listed in Table 4.1.

4.6 Conclusion and future perspectives

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FIGURE 4.1 Schematic representations of Shoot Apical Meristem (SAM) and Axillary Meristem (AM) in Arabidopsis, Maize and Rice. The models show multiple transcription factors involved in the regulation of plant meristem development. Arrows and inhibition symbols indicate activation and repression, respectively.

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Table 4.1 Gene symbol and abbreviation. Gene symbol

Common name

AG AGL24 AGO1/10 AHL15 AP1/2/3 ATGRF4/5/6 ATH1 ARF5 AS1/2 BA1 BGRF1 BIF2 BKN3 BL BZR1 CAL CEN CLV1/2/3 CRC CRN CUC1/23 DA1 DRN DRNL FEA2 FLC FON1/2 FUL KN1 KNAT2 KNOX KNU LAS LAX

AGAMOUS AGAMOUS-LIKE GENE 24 ARGONAUTE1/10 AT-HOOK MOTIF NUCLEAR LOCALIZED 15 APETALA1/2/3 GROWTH-REGULATING FACTOR 4/5/6 ARABIDOPSIS THALIANA HOMEOBOX GENE 1 AUXIN RESPONSE FACTOR 5 ASYMMETRIC LEAVES 1/2 BARREN STALK1 BARLEY GROWTH REGULATING FACTOR1 BARREN INFLORESCENCE2 BARLEY KNOX3 BLIND PROTEIN BRASSINAZOLE-RESISTANT 1 CAULIFLOWER CENTRORADIALIS CLVATA1/2/3 CRABS CLAW CORYNE CUP-SHAPED COTYLEDON 1/2/3 DA (LARGE IN CHINESE) 1 DORNRO¨ SCHEN DORNRO¨ SCHEN- LIKE FASCIATED EAR2 FLOWERING LOCUS C FLORAL ORGAN NUMBER1/2 FRUITFULL KNOTTED1 KNOTTED-LIKE FROM ARABIDOPSIS THALIANA 2 KNOTTED1-LIKE HOMEOBOX KNUCKLES LATERAL SUPPRESSOR LAX PANICLE

4.6 Conclusion and future perspectives

87

Table 4.1 Gene symbol and abbreviation. Continued Gene symbol

Common name

LFY LS MAF1/FLM MOC1 MYB2 MYB37 (RAX1) MYB38 (RAX2) MYB84 (RAX3) OSCEN1/2 OSGRF1/3/10 OSH1 OSKN2 OSTBI PI PIF4 PLL1 POL REV ROX RPK2 SEP1
3 SOC1 SPA STM SVP TB1 TD1 TFL1 WUS XAL2 ZPR3

LEAFY THE LATERAL SUPPRESSOR MADS AFFECTING FLOWERING MONOCULM 1 MYB DOMAIN PROTEIN 2 MYB DOMAIN PROTEIN 37 MYB DOMAIN PROTEIN 38 MYB DOMAIN PROTEIN 84 TFL1/CEN-LIKE GENES ORYZA SATIVA GROWTH-REGULATING FACTOR1/3/10 ORYZA SATIVA HOMEOBOX1 ORYZA SATIVA KNOX PROTEIN 2 TEOSINTE BRANCHED1 PISTILLATA PHYTOCHROME INTERACTING FACTOR 4 POLTERGEIST LIKE1 POLTERGEIST REVOLUTA REGULATOR OF AXILLARY MERISTEM FORMATION RECEPTOR PROTEIN KINASE 2 SEPALLATA 1
3 SUPPRESSOR OF OVERESPRESSION OF CONSTANS1 SMALL PANICLE SHOOT MERISTEMLESS SHORT VEGETATIVE PHASE TEOSINTE BRANCHED1 THICK TASSEL DWARF1 TERMINAL FLOWER1 WUSCHEL 1 XAANTAL2 LITTLE ZIPPER3

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Chapter 4 The roles of transcription factors

Acknowledgments We apologize to those authors whose research could not be cited owing to space limits. Research in the Zhang lab is supported by the Strategic Priority Research Program of the Chinese Academy of Sciences, Grant No. XDA26030200 to C.Z.

Author contributions Q.D. and C.Z. wrote the article. All authors read and approved the manuscript.

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CHAPTER

Transcription factors and their role in leaf senescence

5

Jeremy Dkhar1,2 and Asosii Paul3 1

Plant EvoDevo Laboratory, Agrotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India 2Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India 3Department of Botany, Nagaland University, Lumami, Nagaland, India

5.1 Introduction Transcription factors (TFs), as the name suggests, function in the activation or repression of transcription—a process by which the information in a piece of DNA is copied into RNA. They belong to a group of sequence-specific DNA binding proteins that are known to play important roles in the development of a plant and its response to the environment (Riechmann et al., 2000; Zhang, 2003). Transcription factors get turned on or off depending on the tissue type, developmental stage, or the kind of stimuli that acted on the plant (Singh et al., 2019; Zhang, 2003). The first TF was identified in Zea mays (maize) and named COLORED1 (C1) because of its role in specifying the purple color of corn kernels (Paz-Ares et al., 1987). It belonged to the MYB gene family (Paz-Ares et al., 1987). Shortly after the discovery of C1, TFs playing a role in plant growth and development were also identified (Katagiri and Nam-Hai, 1992). For example, the DEFICIENS (DEF A) and AGAMOUS (AG) TFs were found to promote flower development in Antirrhinum majus and Arabidopsis thaliana, respectively (Sommer et al., 1990; Yanofsky et al., 1990), while the KNOTTED-1 (KN1) and the GLABRA 1 (GL1) TFs control leaf and trichome development in maize and A. thaliana, respectively (Vollbrecht et al., 1991; Oppenheimer et al., 1991). Since then, significant efforts have been made to understand how TFs in plants regulate growth and development, including metabolism. Of the 369,434 estimated species of flowering plants (Lughadha et al., 2016), A. thaliana is the most thoroughly studied model organism for plant biology (Koornneef and Meinke, 2010). Twenty years after the publication of its first genome (The Arabidopsis Genome Initiative, 2000), A. thaliana remains the standard reference plant for all biology (Koornneef and Meinke, 2010). The availability of the A. thaliana genome resulted in the identification of the complete set of Arabidopsis TFs. Of the estimated total number of genes (B26,000), the Arabidopsis genome codes for at least 1533 (B5.9%) TFs (Riechmann et al., 2000). With the completion of the A. thaliana genome sequence and the identification of the complete set of Arabidopsis TFs, TFs of other plant species can also be identified and functionally characterized (Zhang, 2003). During growth and development, leaves undergo a series of characteristic processes, which includes initiation at the periphery of the shoot apical meristem, asymmetric outgrowth, expansion, Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00002-9 © 2023 Elsevier Inc. All rights reserved.

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Chapter 5 Transcription factors and their role in leaf senescence

and maturation. In A. thaliana, the underlying factors governing these processes are inherently genetic (Dkhar and Pareek, 2014). Likewise, a highly regulated genetic program controls leaf senescence, the final stage of leaf development (Lim et al., 2007). Leaf senescence is critical to the plants’ fitness as it allows nutrient recycling from the leaves to the reproducing seeds (Lim et al., 2007). This attribute attracted scientists, and research has focused on understanding the molecular mechanism controlling the initiation and progression of leaf senescence. This, in turn, can be used to devise strategies for improving crop plants with higher yield, better quality, or improved horticultural performance (Guo et al., 2021b). These studies have pointed to the crucial role that TFs play in regulating leaf senescence. For example, members of the NAC TF family (i.e., ORE1 and VNI2) were found to promote or delay leaf senescence. NAC and other TFs achieved this through possible interactions with phytohormones and their responses to environmental cues (Luoni et al., 2019). In the present chapter, we present and discuss TFs that are known to play critical roles in the regulation of leaf senescence, a developmental process crucial to the reproductive success and survival of a plant species. As a complement to the text, a list of functionally characterized leaf senescence-related TFs is given in Table 5.1.

5.2 Identification of transcription factor families in senescing leaf transcriptome Transcriptome analysis is a powerful approach used to understand complex biological processes (Paul et al., 2014, 2021). Senescing leaf transcriptomes were reported in several plant species, including Arabidopsis, rice, wheat, maize, cotton, and poplar (Zhang et al., 2014; Lin et al., 2015; Borrill et al., 2019; Breeze et al., 2011; Chai et al., 2019; Li et al., 2019; Lu et al., 2020). A high-resolution time-course microarray analysis identified several TF families to be significantly upregulated during A. thaliana leaf senescence (Breeze et al., 2011). bZIP family members specifically involved in disease resistance, development, abiotic stress response, ABA signaling and phenylpropanoid biosynthesis (Jakoby et al., 2002; Dro¨ge-Laser et al., 2018) were dominantly upregulated in the initial stages of senescence. Another significantly overrepresented upregulated TF family is the large C3H superfamily. Several transcripts of the CCAAT box binding factor family were also upregulated significantly. The CCAAT box binding factors form the heterotrimeric NF-Y binding complex, comprising NF-YA, NF-YB, and NF-YC subunits. NF-Y binding complex were reported to modulate stress responses and flowering time in plants (Mantovani, 1999; Zhao et al., 2017). Interestingly, the NF-YA subunit genes were specifically enriched, with nine of the ten genes in the A. thaliana genome showing upregulation during senescence. The large NAC family exhibited significant early overrepresentation, with 30 members of the family showing altered expression during senescence. NAC TFs have regulatory roles in diverse biological processes, including senescence, defense, and abiotic stress (Puranik et al., 2012; Luoni et al., 2019). TF families showing increased expression in later stages of senescence include the WRKY and AP2EREBP. WRKY TFs are important for senescence (Robatzek and Somssich, 2001; Miao et al., 2004), while most other members are induced during plant-pathogen interaction to regulate salicylic ¨ lker et al., 2007; acid (SA) and jasmonic acid (JA) dependent defense signaling pathways (U Rushton et al., 2010). Members of the AP2-EREBP family are induced by ethylene, JA, SA, wounding, pathogens, and abiotic stresses to modulate stress and disease resistance pathways

5.2 Identification of transcription factor families

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Table 5.1 Functionally characterized leaf senescence-related TFs discussed in the present study. Mode of regulation

Plant species

References

Activator

A. thaliana

Repressor

A. thaliana

AtWRKY6, AtWRKY22, AtWRKY26, AtWRKY30, AtWRKY45, AtWRKY53, AtWRKY75 AtWRKY18, AtWRKY25, AtWRKY54, AtWRKY57, AtWRKY70 DEAR4, AtCRF2

Activator

A. thaliana

Repressor

A. thaliana

Activator

A. thaliana

ANT, CBF2, CBF3, AtCRF6

Repressor

A. thaliana

PIF3, PIF4, PIF5, MYC2, MYC3, MYC4 CIB1 bHLH17, bHLH14, bHLH13 and bHLH03 AtMYBR1; CCA1; LHY

Activator

A. thaliana

Activator Repressor

Glycine max A. thaliana

Guo and Gan (2006), Kim et al. (2009), Balazadeh et al. (2011), Wu et al. (2012), Garapati et al. (2015), Zhu et al. (2015) Yang et al. (2011), Lee at al. (2012b) Robatzek and Somssich (2001), Miao et al. (2004), Zhou et al. (2011), Chen et al. (2017), Guo et al. (2017b), Li et al. (2017b) ¨ lker et al. (2007), Besseau et al. U (2012), Jiang et al. (2014), Potschin et al. (2014) Kwon (2016), Zhang et al. (2020b) Sharabi-Schwager et al. (2010a,b), Zwack et al. (2013), Feng et al. (2016) Song et al. (2014), Yasuhito et al. (2014), Zhu et al. (2015) Meng et al. (2013) Qi et al. (2015)

Repressor

A. thaliana

ARF and Aux/IAA

ARF2, IAA17, IAA29

Activator

A. thaliana

DOF

DOF2.1, CDF4

Activator

A. thaliana

OsDOF24 PRR9 CCA1 ZmVQ52 ABF2, ABF3, ABF4, ABI5, GBF1 MdABI5

Repressor Activator Repressor Activator Activator

Oryza sativa A. thaliana A. thaliana Zea mays A. thaliana

Activator

ABIG1, REV

Activator

Malus domestica A. thaliana

HaHB-4

Repressor

TF family

Gene

NAC

ORE1, NTL4, ATAF1, ANAC016, ANAC019, ANAC029, ANAC055, ANAC059, ANAC072 JUB1, VNI2

WRKY

AP2/ EREBP

bHLH

MYB

PRR VQ bZIP

HD-ZIP

Helianthus annuus

Jaradat et al. (2013), Song et al. (2018) Okushima et al. (2005), Lim et al. (2010), Jiang et al. (2014), Shi et al. (2015) Xu et al. (2020), Zhuo et al. (2020) Shim et al. (2019) Kim et al. (2018a) Song et al. (2018) Yu et al. (2019) Smykowski et al. (2010), Gao et al. (2016), Skubacz et al. (2016) An et al. (2021) Xie et al. (2014b), Liu et al. (2016b) Dezar et al. (2005), Manavella et al. (2006) (Continued)

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Table 5.1 Functionally characterized leaf senescence-related TFs discussed in the present study. Continued TF family

Gene

Mode of regulation

Plant species

References

PLATZ GRF and GIF

ORE15 GRF3, GRF5, GIF1

Repressor Repressor

A. thaliana A. thaliana

TCP

TCP4 BrTCP7

Activator Activator

TCP20 BrTCP2

Repressor Repressor

KN1

Repressor

A. thaliana Brassica rapa var. parachinensis A. thaliana B. rapa var. parachinensis Z. mays

Kim et al. (2018c) Horiguchi et al. (2005), Debernardi et al. (2014), Vercruyssen et al. (2015) Sarvepalli and Nath (2011) Xu et al. (2019)

KNAT1, KNAT2

Repressor

A. thaliana

KHZ1, KHZ2 AtTZF2, AtTZF3 GhTZF1

Activator Repressor Repressor

OsDOS, OsTZF1

Repressor

A. thaliana A. thaliana Gossypium hirsutum O. sativa

GRAS

BrLAS TaSCL14

Repressor Repressor

NF-Y

AtNFYA2, AtNFYA7, AtNFYA10 HaHSFA9 SlFYFL

Repressor

FYF BpMADS4 GLK1, GLK2 TaGT2L1D GhGT31 ARR3, ARR4, ARR5, ARR6, ARR16, ARR2 OsLBD37/ASL39 OsELF3.1 ELF3 EIN3

Repressor Repressor Repressor Activator Activator Activator

H. annuus Solanum lycopersicum A. thaliana Betula pendula A. thaliana T. aestivum G. hirsutum A. thaliana

Repressor Activator Activator Repressor Activator

A. thaliana O. sativa O. sativa A. thaliana A. thaliana

HB

C3H (Zn)

HSF MADS

GARP TRIHELIX ARR

LBD/AS2 ELF3 EIL

Repressor Repressor

B. rapa Triticum aestivum A. thaliana

Danisman et al. (2012) Xiao et al. (2019) Hewelt et al. (2000), Luo et al. (2006) Frugis et al. (2001), Hamant et al. (2002) Yan et al. (2017) Lee et al. (2012b) Zhou et al. (2014) Kong et al. (2006), Jan et al. (2013) Li et al. (2018) Chen et al. (2015) Leyva-Gonz´alez et al. (2012) Almoguera et al. (2015) Xie et al. (2014a) Chen et al. (2011) Hoenicka et al. (2008) Rauf et al. (2013) Zheng et al. (2016) Guo et al. (2017) To et al. (2004), Kudryakova et al. (2008), Ren et al. (2009) Kim et al. (2006) Albinsky et al. (2010) Sakuraba et al. (2016) Sakuraba et al. (2014) Chao et al. (1997), Li et al. (2013)

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97

Table 5.1 Functionally characterized leaf senescence-related TFs discussed in the present study. Continued TF family

Gene

Mode of regulation

Plant species

References

BES1 CAMTA TIFY

BES1 NtER1 SR1 JAZ8, JAZ4, JAZ7

Activator Activator Activator Repressor

A. thaliana N. tabacum A. thaliana A. thaliana

B-BOX (BBX)

GHD2 CmBBX22

Activator Repressor

MdBBX22

Repressor

O. sativa Chrysanthemum morifolium M. domestica

Yin et al. (2002, 2005) Yang and Poovaiah (2000) Nie et al. (2012) Jiang et al. (2014), Yu et al. (2016) Liu et al. (2016a) Liu et al. (2019) An et al. (2021)

(Yamaguchi-Shinozaki and Shinozaki, 1994; Dietz et al., 2010). The data indicate similar transcriptional activity and potential coregulation within diverse TF families during leaf senescence. Using RNA sequencing (RNA-seq) technology, Li et al. (2019) reported 81 differentially expressed TF genes in the flag leaves during the grain-filling stage between wild type and Oryza sativa premature leaf senescence 1 (ospls1) mutant rice. These TFs belonged to 13 gene families, namely HomeoboxZIP, WRKY, AUX-IAA, NAC, HMG, MYB, SPL, PHD-finger, DREB, MADS-box, zinc-finger, HAP3, and PCF. The largest represented family was Homeobox-ZIP (29.2%). Homeobox-ZIP genes such as homeobox-leucine zipper protein HOX1, HOX4, HOX5, HOX7, HOX10, HOX11, HOX15, HOX16, HOX20, HOX22, HOX27, and HOX32 showed high expression levels in the two genotypes. The WRKY TF family accounts for 23.6% of the differentially expressed TFs. WRKY42, WRKY49, and WRKY70 exhibited differential expression in the wild type and mutant ospls1, whereas WRKY24, WRKY39, WRKY44, WRKY53, WRKY67, WRKY70, WRKY71, WRKY72, WRKY74, and WRKY76 showed steady high expression in both genotypes. Approximately 9.7% of differentially expressed TFs belonged to the AUX-IAA family. AUXIN RESPONSE FACTOR 10 (ARF10) was abundantly expressed at the initial grain filling stage in the two genotypes, whereas ARF12 was expressed preferentially in the wild type. The expression of other AUX-IAA members was genotype independent. Around 8.3% of the differentially expressed TFs belonged to the MADS-box family. MADS-box 5 was significantly upregulated in ospls1 flag leaf at 14 days after anthesis. Differential regulation of some members of the DREB, HAP3, HMG, MYB, NAC, PCF, SPL, PHD-finger, and zinc-finger TFs were observed. In wheat, Borrill et al. (2019) found that 2210 TFs were expressed during flag leaf senescence, of which only 341 TFs were expressed differentially. The TF families showed temporal gradient of up- and downregulation throughout the leaf senescence; however, many TF families were up- or downregulated in an initial (3
7 days after anthesis) and later wave (13
19 days after anthesis). CCAAT-HAP2 (NF-YA), pseudo ARR-B, and RWP-RK families were upregulated in the initial wave. The TF families, upregulated in the later wave include CAMTA, GRAS, NAC, and MADS-II. Once upregulated, the TFs continued to be expressed throughout the leaf senescence. Separately, certain TF families were downregulated during the flag leaf senescence. The AS2/LOB, bHLH-TCP, and MADS-I TF families were downregulated in an initial wave occurring at 7 days after anthesis. TF families included the C2C2

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GATA, GARP G2-like, and MADS-II exhibited later wave of downregulation, commencing from 17
19 days after anthesis. Similar to the upregulation of TFs, the downregulation continue throughout the leaf senescence. These data suggested a gradual change in the expression level of specific TFs as the flag leaf senescence progressed. In the natural leaf senescence transcriptome of maize, 233 differentially expressed genes encoding TFs were identified (Zhang et al., 2014). These TFs were distributed into 35 subfamilies, including MYB, bZIP, bHLH, C2H2, AP2/EREBP, and NAC families. Altered expression of 33 MYB and 15 bZIP TF genes suggested the involvement of these two TF families in maize leaf senescence. In yet another transcriptome profiling using the early leaf senescence 5 (ZmELS5) maize mutant having premature leaf senescence (Chai et al., 2019), 170 differentially expressed TFs were identified that belonged to 17 TF families, including bHLH, ERF, MYB, NAC, and WRKY. In particular, genes of the NAC, WRKY, bHLH, MYB, and HD-ZIP TF were significantly induced by premature senescence. RNA-Seq helped identify several TF families that were active at different times during Gossypium hirsutum L. leaf senescence (Lin et al., 2015). Among the 3,624 differentially expressed genes in the senescing leaf transcriptome, 519 transcripts were TFs that belonged to 50 TF families. The WRKY (54 genes), bHLH (44 genes), C3H (43 genes), NAC (42 genes), AP2-EREBP (25 genes), FAR1 (24 genes), DBP (23 genes), SET (22 genes), MYB (21 genes), and HB (17 genes) were the ten largest TF families identified in the transcriptome. Many members of WRKY and C3H families were upregulated early, from around 25 days after the new leaf emergence {leaves started to show yellowing at the tip at around 35 days after leaf emergence (Lin et al., 2015)}. AP2-EREBP, bHLH, DBP, GRAS, MYB, and NAC TFs genes were also differentially regulated during senescence and exhibited upregulation. Transcriptome analysis during seasonal leaf senescence in field-grown Populus trichocarpa identified 881 differentially expressed TF transcripts belonging to 54 TF families (Lu et al., 2020). NAC, WRKY, bZIP, MYB, and C2H2 were the most prominent and active TFs during seasonal leaf senescence. In particular, members of the NAC and WRKY were upregulated in most of the developmental transitions studied. bHLH, ERF, and MYB-related TFs were overrepresented in both up- and downregulated genes (Lu et al., 2020). TFs are key regulatory proteins regulating the precise transcription of genes (Riechmann et al., 2000; Zhang, 2003). Several TFs were reported to play major roles in leaf senescence (Lim et al., 2007; Guo et al., 2021b). The NAC and WRKY subfamilies were found to be the largest TF groups during earlier leaf senescence studies (Guo et al., 2004; Miao et al., 2004), and are therefore better characterized and appear to play central roles in senescence regulation (Zentgraf and Doll, 2019). However, reports of differential regulation of numerous other TF families (Borrill et al., 2019; Breeze et al., 2011; Chai et al., 2019; Li et al., 2019; Lin et al., 2015; Lu et al., 2020; Rathour et al., 2020, 2022; Zhang et al., 2014) during leaf senescence suggested a more complex, intricate, interconnected regulatory network involving several TF families.

5.3 Characterization of leaf senescence related TFs families 5.3.1 No apical meristem (NAM), ATAF1/2, CUP-shaped cotyledon 2 (CUC2) (NAC) TF The NAC (NAM, ATAF and CUC) gene family is the largest group of plant TFs, having more than 100 members in A. thaliana (Puranik et al., 2012). The NAC TF contains a NAC domain at its

5.3 Characterization of leaf senescence related TFs families

99

N-terminal and variable transcription regulatory regions (TRRs) at the C-terminal (Puranik et al., 2012). The NAC domain helps in dimerization and DNA binding, while the TRR region has transcription activator or repressor activity (Puranik et al., 2012). Global transcriptome profiling showed that more than 30 NAC genes have enhanced expressions during natural leaf senescence in A. thaliana. Further, the NAC genes exhibited strong crosstalk with phytohormones and environmental signals, suggesting their importance in regulating the senescence process (Breeze et al., 2011). ORE1, NAC WITH TRANSMEMBRANE MOTIF 1-LIKE 4 (NTL4), A. thaliana ACTIVATING FACTOR 1 (ATAF1), A. thaliana NAC 016 (ANAC016), ANAC019, ANAC029, ANAC055, ANAC059, and ANAC072 were shown to promote leaf senescence, whereas JUNGBRUNNEN 1 (JUB1) and VASCULAR-RELATED NAC-DOMAIN (VND)-INTERACTING 2 (VNI2) delay it (Guo and Gan, 2006; Kim et al., 2009; Balazadeh et al., 2011; Yang et al., 2011; Lee et al., 2012b; Wu et al., 2012; Garapati et al., 2015; Zhu et al., 2015). ORE1 (also called ANAC092 and AtNAC2) is one of the well-studied TFs in leaf senescence. The Arabidopsis oresara1 (ore1, oresara means “long-living” in Korean) mutant was initially identified as a mutant with delayed leaf senescence (Oh et al., 1997). The loss-of-function ore1 mutant display delayed loss of chlorophyll and photochemical efficiency (Fv/Fm) during leaf aging (Kim et al., 2009). The ore1 mutant is the result of a 5-bp deletion in the ORE1 gene (Kim et al., 2009). The ORE1 gene is induced by ETHYLENE INSENSITIVE 2 (EIN2) in an age-dependent manner, but posttranscriptionally and negatively regulated by miRNA164 in young leaf. As the plant age, EIN2 represses miRNA164 expression, resulting in a continuing increase of ORE1 mRNA to promote senescence. The ein2 mutant lacks the agedependent repression of miRNA164. These data supported the importance of ethylene signaling in regulating the age-dependent leaf senescence via the ORE1 TF (Kim et al., 2009). ORE1 can interact with GOLDEN2-LIKE 1 (GLK1) and GLK2 at protein level to disrupt their chloroplast maintenance activity, thereby inducing leaf senescence (Rauf et al., 2013). Furthermore, ORE1 promotes chlorophyll degradation by activating the transcription of chlorophyll catabolic genes namely NON-YELLOW COLORING 1 (NYC1), NON-YELLOWING 1 (NYE1), and PHEOPHORBIDE A OXYGENASE (PAO) (Qiu et al., 2015), as well as directly binding and activating the expression of senescence enhancing genes, namely BIFUNCTIONANUCLEASE 1 (BFN1), SENESCENCE-ASSOCIATED GENE 29/SUGARS WILL EVENTUALLY BE EXPORTER TRANSPORTERS 15 (SAG29/SWEET15) and SEVEN IN ABSENTIA 1 (SINA1), which are involved in nucleic acid degradation, sugar transport, and ubiquitination, respectively (Matallana-Ramirez et al., 2013). Degradation of nucleic acid, proteins, and nitrogen recycling and promotion of sugar transport are distinctive features of leaf senescence to facilitate nutrient remobilization (Balazadeh et al., 2010; Guo et al., 2021b; Matallana-Ramirez et al., 2013; Verma et al., 2011). Moreover, in A. thaliana leaf, ORE1 induces ethylene production by positive feedback through transcriptional activation of a key ethylene biosynthesis gene, 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE 2 (Qiu et al., 2015). ANAC029 [also called NAC-LIKE, ACTIVATED BY AP3/PI (AtNAP)] was positively associated with the senescence process of A. thaliana rosette leaves (Guo and Gan, 2006; Zhang and Gan, 2012). It was shown that an Atnap null mutant had delayed loss of chlorophyll and leaf senescence, whereas inducible AtNAP overexpression in young leaves causes precocious senescence (Guo and Gan, 2006). The expression of the direct target gene, SENESCENCE-ASSOCIATED GENE 113 (SAG113), primarily depends on the binding of AtNAP protein to the 9-bp core sequence (5’-CACGTAAGT-3’) of the SAG113 promoter (Zhang and Gan, 2012). SAG113 encodes a golgi-localized protein phosphatase 2 C. Both AtNAP and SAG113 were induced by ABA and

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leaf senescence. In AtNAP knockout plants, the ABA- and senescence-induced expression of SAG113 was diminished. Further, overexpression of SAG113 in the atnap knockout mutant restored the delayed leaf senescence phenotype to wild type (Zhang and Gan, 2012). As a negative regulator of ABA signal transduction, overexpression of SAG113 was shown to reduce sensitivity of stomatal movement to ABA and specifically suppresses stomata closure in A. thaliana. This resulted in accelerated water loss in senescing leaves, promoting desiccation and precocious leaf senescence (Zhang et al., 2012). Thus ABA, AtNAP and SAG113 together were suggested to form a unique regulatory chain that regulate stomatal movement and water loss during leaf senescence. AtNAP was also reported to directly bind and enhance the transcription of the ABA biosynthetic gene ABSCISIC ALDEHYDE OXIDASE 3, which increased biosynthesis of the senescence-inducing hormone ABA. The increased ABA level induces the expression of chlorophyll catabolic genes, eventually leading to loss of chlorophyll and leaf senescence (Yang et al., 2014). Functional studies demonstrated that the membrane-bound NAC, ANAC016, promotes leaf senescence in A. thaliana (Kim et al., 2013). The anac016 knockout mutant remained green under different senescence-inducing conditions, including natural senescence, darkness, ABA, and methyl jasmonate (MeJA) treatments. Overexpression of ANAC016 in transgenic A. thaliana accelerated leaf yellowing and senescence. Under dark-induced senescence condition, several SAGs, including STAY-GREEN (SGR1), NYE1, NYC1, PHEOPHYTINASE (PPH), WRKY22, JUB1, AtNAP, ORS1, ORE1 and VNI2 were downregulated in anac016 loss-of-function mutant and upregulated in ANAC016 overexpressing plants. Under salt and H2O2 stresses, anac016 mutants exhibited delayed senescence, while ANAC016 overexpressing plants senesced rapidly. ANAC016 was shown to bind both the AtNAP and ORS1 promoters in yeast one-hybrid assays. The observation that the anac016 and atnap loss-of-function mutants have stay-green phenotypes under abiotic stress conditions and dark-induced senescence further supported the placement of AtNAP downstream of ANAC016 (Kim et al., 2013). It was concluded that ANAC016 regulatory mechanism promoting leaf senescence is also required for senescence induced by stress-responsive signaling pathways (Miller et al., 2009; Kim et al., 2013). Garapati et al. (2015) showed that overexpression of the ABA- and H2O2-activated NAC TF, A. thaliana ACTIVATING FACTOR 1 (ATAF1), accelerates both developmental and stress-induced leaf senescence, while the ataf1 knockout mutant has the opposite phenotype. In the ATAF1 overexpressing plants, several senescence marker genes (including ORE1) were highly upregulated while photosynthesis-associated genes (including GLK1) were downregulated. ATAF1 was shown to directly bind to the promoters of the positive senescence regulator ORE1 and the chloroplast maintenance TF GLK1 and regulate their expression. ATAF1 also affects the progression of ABA-induced senescence by exerting a direct positive regulatory effect on the expression of ABA biosynthesis gene, NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3, and transport-related gene, ABC TRANSPORTER G FAMILY MEMBER 40 (Garapati et al., 2015), suggesting that ATAF1 regulates senescence in A. thaliana through gene regulatory network involving target genes that affect photosynthesis, aging-induced programmed cell death, ABA biosynthesis and transport. Recently, ATAF2—known to regulate biotic stress responses—was also shown to promote leaf senescence (Nagahage et al., 2020). ATAF2 overexpression accelerated leaf senescence, whereas the ataf2 mutants exhibited delayed dark-induced leaf senescence. ATAF2 overexpression resulted in the upregulation of several NAC TFs, including ORE1, thereby accelerating leaf senescence (Nagahage et al., 2020).

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101

ORE1 SISTER1 (ORS1)/ANAC059, a member of NAC-d subfamily, also promotes leaf senescence, with overexpression of ORS1 accelerated senescence in transgenic plants, whereas senescence was delayed in the knockout mutants (Balazadeh et al., 2011). Transcriptome analysis of the ORS1 expressing transgenic plants yielded 42 upregulated and 26 downregulated transcripts. Thirty-two (76%) and 24 (57%) of the ORS1 regulated genes were also induced by long-term (4 days) salt stress and 5 hours after H2O2 application, respectively (Balazadeh et al., 2011). ORS1 itself was robustly induced by H2O2. Reactive oxygen species (ROS), particularly H2O2, accumulate during salinity stress (Miller et al., 2009) (Madhu et al., 2022; Tyagi et al., 2019). Kim et al. (2013) reported that the salt- and H2O2-responsive ANAC016 binds the ORS1 promoter. Thus ORS1 and ANAC016 might share a common senescence regulatory network involving salt- and H2O2-dependent signaling pathways. JUNGBRUNNEN1 (JUB1), an H2O2-induced NAC TF, was shown to be a negative regulator of leaf senescence and increased longevity in A. thaliana (Wu et al., 2012). Overexpression of JUB1 delays senescence concomitant with decreasing intracellular H2O2 levels and improving plant’s tolerance to heat and salt stress, whereas jub1
1 knockdown mutant has early senescence and lower tolerance to abiotic stress. JUB1 elevated the expression of several ROS-responsive genes, including GLUTATHIONE S-TRANSFERASE and HEAT SHOCK PROTEIN (HSP), and H2O2 treatment further enhances the genes expression. JUB1 binds directly and activates the AP2 TF DEHYDRATIONRESPONSIVE ELEMENT BINDING PROTEIN2A (DREB2A), which is a master regulator of abiotic stress responses (Liu et al., 1998; Wu et al., 2012). JUB1 recognizes the RRYGCCGT core sequence present in the DREB2A promoter. DREB2A, in turn, functions as a positive regulator of HEAT SHOCK FACTOR A3 (HsfA3) (Schramm et al., 2008) and RESPONSIVE TO DESSICATION 29 A (RD29A) (Liu et al., 1998). HsfA3 TF regulates the expression of HSP genes and is part of a positive feedback loop together with HsfA2 and HsfA1e TFs (Nishizawa-Yokoi et al., 2011). HsfA1e and HsfA2, in turn, also control the transcription of genes encoding HSPs and ROS-scavenging enzymes (Schramm et al., 2006). Based on the finding, JUB1 was suggested to mediate the lowering of the ROS level in plant tissues through the Hsf feedback amplification loop, thereby enhancing the plant’s longevity and abiotic stress tolerance (Wu et al., 2012). In agreement with improved abiotic stress tolerance, metabolite profiling showed increased proline and trehalose levels in JUB1 overexpression plants. Shahnejat-Bushehri et al. (2016) reported that JUB1 could activate the expression of DELLA (GIBBERELLIN INSENSITIVE, GAI and RGA-LIKE 1, RGL1) genes, and the subsequent increment in DELLA protein levels increases stress tolerance by limiting the build-up of stress-induced ROS. The membrane-anchored NAC transcription factor NTL4 mediates drought-induced senescence by promoting ROS production (Lee et al., 2012b). ABA, drought, and heat induce NTL4 expression. In addition to NTL4 gene upregulation, ABA promotes the release of plasma membrane bound NTL4 protein by proteolysis during drought stress. NTL4 directly binds and upregulates the expression of AtrbohA, AtrbohC, and AtrbohE, encoding NADPH oxidases, involved in ROS production to initiate programmed cell death (PCD) during dehydration-induced leaf senescence. Accordingly, transgenic plants overexpressing the active form of NTL4 has accelerated leaf senescence during drought conditions. In contrast, the loss-of-function ntl4 mutant plants have delayed leaf senescence and enhanced tolerance to dehydration. ROS levels were reduced in the ntl4 mutants. These observations indicate that NTL4 fine-tune ROS production during drought to induce leaf senescence in A. thaliana (Lee et al., 2012a). Under heat stress conditions, NTL4 also participate in a positive feedback loop that promoted ROS accumulation to modulate PCD (Lee et al., 2014). The increased

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H2O2 levels during heat stress trigger NTL4 gene expression and NTL4 protein processing. It was suggested that the NTL4-mediated PCD and leaf senescence through ROS might provide an adaptation strategy enabling plants to survive extreme stress conditions (Lee et al., 2012a, 2014). In conclusion, membrane-anchored NTL4 was suggested to be a link connecting abiotic stress responses with senescence. VASCULAR-RELATED NAC-DOMAIN (VND)-INTERACTING2 (VNI2) (also called ANAC083) functions as a transcriptional regulator and negatively regulates leaf senescence through ABA signaling (Yang et al., 2011). The expression of VNI2 was high during senescence and also induced by ABA and salt treatments. However, in the ABA-deficient aba3
1 mutant, the effects of salt on VNI2 expression were reduced significantly, suggesting that VNI2 expression in salt stress was ABA-dependent. Overexpression of VNI2 improves tolerance to salt stress and prolonged leaf longevity, whereas in the VNI2-deficient mutant plants, the salt-induced leaf senescence was significantly accelerated. VNI2 directly binds and upregulates the senescence-induced COLDREGULATED 15 A (COR15A), COR15B, RD29A and RD29B expressions (Yang et al., 2011). Notably, overexpressing RD or COR genes prolonged leaf longevity in transgenic plants (Shi et al., 2017b). The positive regulators of senescence, ORE1 and AtNAP, which are directly activated by EIN3, in turn, was shown to regulate VNI2, which is a negative regulator of leaf senescence (Woo et al., 2019). Thus, both positive and negative regulators might interact to provide a mechanism to fine-tune the process of senescence. The closely related ANAC019, ANAC055, and ANAC072 (RD26) belonged to the same clade of stress-associated NAC proteins and have overlapping expression patterns (Tran et al., 2004; Hickman et al., 2013). They have similar senescence-associated expression patterns during agedependent and salt-induced senescence (Balazadeh et al., 2008; Breeze et al., 2011). Evidence suggests that these three genes are induced under drought, salt and JA treatments (Tran et al., 2004; Zhu et al., 2015), whereas only ANAC072 was induced by cold treatment (Fujita et al., 2004). Moreover, ABA can induce the expression of all three NAC TFs (Tran et al., 2004). ABA signaling regulators AtABF3 and AtABF4 can directly bind to the promoter regions of ANAC019, ANAC055, and ANAC072 and activate their expressions (Hickman et al., 2013). The TF family comprising of C-REPEAT BINDING FACTOR (CBF1), CBF2, CBF3, and CBF4 can bind and activate NAC072 promoter. In contrast, there was no interaction between the ANAC019 and ANAC055 promoters and the CBFs, suggesting that a different control mechanism might be regulating ANAC072 expression (Hickman et al., 2013). AtABF3 and AtABF4 are positive regulators of leaf senescence (Gao et al., 2016), whereas CBF2 and CBF3 delay senescence (Sharabi-Schwager et al., 2010a,b). Zhu et al. (2015) reported that ANAC019/055/072 function downstream of MYC2/3/4 TFs to directly promote the transcription of chlorophyll catabolic genes, namely NYE1, NYE2 and NYC1, during JA-induced chlorophyll degradation. Detached leaves from the anac019 anac055 anac072 loss-offunction triple mutant have a severe stay-green phenotype after MeJA treatment. The JA-induced expression of NYE1, NYE2 and NYC1 was abolished in anac019 anac055 anac072 triple mutant. Importantly, Chromatin Immunoprecipitation (ChIP)-qPCR analyses demonstrated that the ANAC019 protein was associated with NYE1, NYE2, and NYC1 promoter regions that contains the complete DNA-binding sequences for NAC protein. The JA-induced transcription of ANAC019, ANAC055, and ANAC072 was abolished in myc2 myc3 myc4 triple mutant. AtMYC2 was shown to interact directly with ANAC019 at the protein level and synergistically regulate the expression of AtNYE1 (Zhu et al., 2015). In a previous study by Bu et al. (2008), ANAC055 and ANAC019 were

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shown to act downstream of AtMYC2 to regulate JA-signaled defense responses. Yeast one-hybrid and ChIP assays revealed that ANAC019, ANAC055, and ANAC072 were the direct targets of AtMYC2 (Hickman et al., 2013; Zhu et al., 2015). These results suggest that ANAC019, ANAC055, and ANAC072 regulate chlorophyll degradation during JA-induced leaf senescence by acting downstream of MYC2/3/4 TFs. The ANAC019 and ANAC055 regulation of leaf senescence may also involve components of the EIN2-mediated ethylene signaling (Chang et al., 2013; Kim et al., 2014). In a comparative gene expression analysis, ANAC055 and ANAC019 were identified as downstream components of EIN2 (Kim et al., 2014). ANAC055 and ANAC019 transcripts levels were considerably decreased in the mature leaf of ein2 knockout mutant, indicating an EIN2-dependent regulation. Additionally, Chang et al. (2013) showed that EIN3 could directly bind to the promoters of ANAC055, ANAC019, and ORS1 after ethylene treatment in A. thaliana. EIN3 is a downstream component of EIN2 in the ethylene signaling cascade (Guo and Ecker, 2004; Dolgikh et al., 2019). In contrast, ANAC072 expression remains unaltered in ein2 mutants, which suggests that ANAC055/ANAC019 and ANAC072 might trigger senescence through different pathways (Kim et al., 2014). Fujita et al. (2004) showed that ANAC072 is also involved in an ABA-dependent stress-signaling pathway. Transgenic A. thaliana overexpressing ANAC072 were hypersensitive to ABA with rapid loss of chlorophyll, while loss-of-function anac072 mutants were insensitive. In the ANAC072 overexpressed plants, several defense- and senescence-responsive genes, including SAG13, a marker gene of senescence, were significantly upregulated (Fujita et al., 2004). Similarly, Li et al. (2016) reported early leaf-yellowing phenotype in the gain-of-function anac072
2 mutant, whereas the loss-offunction mutant anac072
1 exhibited delayed leaf de-greening under normal or dark-induced conditions. Interestingly, neither ANAC055 nor ANAC019 T-DNA insertion mutants showed any senescence-associated phenotype in normal or dark senescence-inducing conditions (Li et al., 2016). Interestingly, ANAC072 can directly bind and activate the transcriptional expression of chlorophyll catabolic gene NYE1. Thus ANAC072 was suggested to positively regulate leaf senescence via chlorophyll catabolic gene NYE1 (Li et al., 2016). Besides, ANAC072 is implicated as a key regulator of metabolic reprogramming during leaf senescence by controlling the transcription of diverse genes involved in the cellular degradation pathway (Kamranfar et al., 2018). For example, ANAC072 directly binds and upregulates the expression of CHLOROPLAST VESICULATION, encoding a protein crucial for degradation of chloroplastic proteins and genes involved in carbohydrate metabolism and transport such as ALPHA AMYLASE 1, monosaccharide transporter SUGAR-PORTER FAMILY PROTEIN 1, and sucrose efflux transporter SWEET15 (Kamranfar et al., 2018). ANAC072 also activates the transcription of genes involved in phytol degradation, lysine, and γ-aminobutyric acid (GABA) catabolism. Phytol, lysine, and GABA degradations provide needed substrates for cellular respiration during leaf senescence (Kamranfar et al., 2018). Members of the NAC gene family can regulate leaf senescence in a positive or negative manner. Kim et al. (2018b) reported a time-dependent NACs gene regulatory network shift from positive to negative regulation during presenescent stage in A. thaliana. ANAC017, ANAC082, and ANAC090, called the “NAC troika,” are upregulated at the presenescence stage. These NAC genes are negative regulators of senescence, and their transcripts level increases as the leaf approaches senescence. Moreover, knockout mutant plants of the NAC troika showed accelerated senescence. In contrast, overexpression of the NAC troika showed the opposite effects, concomitant with inhibition of senescence-promoting pathways, including SA and ROS stress pathways (Kim et al., 2018b). The NAC troika also downregulates

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the expression of other NAC genes suggesting that these genes may bring about the positive-to-negative regulatory shift during the onset of senescence (Kim et al., 2018b). It was concluded that the regulatory inversion might modulate NAC genes expression to prevent untimely senescence at the presenescent stage.

5.3.2 WRKY TF All WRKY TFs contain the highly conserved WRKYGQK amino acid sequence and the C2H2 or C2HC zinc-finger-like motifs (Rushton et al., 2010; Zhou et al., 2011; Chen et al., 2018). The WRKYGQK sequence and zinc-finger-like motifs are essential for the DNA-binding activity (Rushton et al., 2010; Chen et al., 2018). WRKY TFs usually recognize the W-box binding-motif (TTGACC/T) to regulate target genes expression, although binding to other cis-elements has been suggested (Guo et al., 2017b; Chen et al., 2018; Cui et al., 2020). Seventy-five WRKY genes were reported in A. thaliana (Riechmann et al., 2000). WRKY constitutes the second largest group of TF genes differentially expressed in the senescing leaf transcriptome (Guo et al., 2004; Woo et al., 2019; Lu et al., 2020). Eleven A. thaliana WRKYs, namely AtWRKY6, AtWRKY22, AtWRKY25, AtWRKY26, AtWRKY30, AtWRKY45, AtWRKY53, AtWRKY54, AtWRKY57, AtWRKY70, and AtWRKY75, were reported to function as key regulators of leaf senescence with either positive or negative roles (Chen et al., 2018; Doll et al., 2020). The first WRKY confirmed as a leaf senescence regulator is the A. thaliana WRKY53 (Miao et al., 2004; Zentgraf and Doll, 2019). Overexpression or RNAi knockdown of AtWRKY53 showed senescence-associated phenotypes indicating that AtWRKY53 functions as a positive regulator of senescence (Miao et al., 2004). Genomic pull-down assays identified sixty-three genes as direct targets of AtWRKY53 protein, including eight other members (AtWRKY6, AtWRKY13, AtWRKY15, AtWRKY22, AtWRKY29, AtWRKY18, AtWRKY42, and AtWRKY62) of the WRKY gene family. This indicates that AtWRKY53 regulate senescence-associated genes (SAGs) and acts upstream to other WRKY TFs (Miao et al., 2004). Three CATALASE genes were also identified as the direct targets of AtWRKY53 (Miao et al., 2004). CATALASE are scavenging enzymes that convert two molecules of H2O2 into water and oxygen (Smykowski et al., 2010). On the other hand, AtWRKY53 expression is induced by H2O2 treatment (Miao et al., 2004). Interestingly, AtWRKY18, which was initially reported to be a downstream target gene of AtWRKY53 in the WRKY network (Miao et al., 2004), was reported to regulate the expression of AtWRKY53 (Potschin et al., 2014). AtWRKY18 binds directly to different W-boxes present in the AtWRKY53 promoter to repress its expression. Transgenic A. thaliana plants overexpressing AtWRKY18 exhibited delayed leaf senescence phenotypes, whereas knockout wrky18 mutant plants have accelerated leaf senescence (Potschin et al., 2014). In addition, AtWRKY53 and AtWRKY18 proteins directly interact to form stable AtWRKY18/53 heterodimers, which positively influences AtWRKY53 expression (Potschin et al., 2014). Interestingly, AtWRKY25, a downstream target gene of AtWRKY53, was reported to function as a redox-sensitive upstream regulator of AtWRKY53 expression (Doll et al., 2020). Under nonoxidizing conditions, AtWRKY25 binds to a specific W-box located in the AtWRKY53 promoter and positively regulates AtWRKY53 expression, whereas oxidizing conditions dampened AtWRKY25 action. Overexpression of AtWRKY25 did not promote senescence but increased the longevity of the transgenic A. thaliana plants, whereas the knockout mutant plants have the

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opposite phenotype. This indicates a more complex regulatory function of AtWRKY25 within the WRKY subnetwork of leaf senescence regulation (Doll et al., 2020). The MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE 1 (MEKK1) and singlestranded DNA-binding protein WHIRLY1 were identified to bind the AtWRKY53 promoter and function as an upstream regulator of AtWRKY53 expression during senescence in A. thaliana (Miao et al., 2007, 2013). MEKK1 activates while WHIRLY1 represses AtWRKY53 expression. MEKK1 was further shown to phosphorylate AtWRKY53 in vitro to increase its DNA binding activity (Miao et al., 2007). MEKK1 also enhances the activation potential of AtWRKY25 on the AtWRKY53 promoter (Doll et al., 2020). AtWRKY53 protein level is also tightly controlled by the HECT-domain E3 ubiquitin ligase UPL5 (Miao and Zentgraf, 2010). The UPL5 can polyubiquitinate AtWRKY53 for 26S-proteasomal degradation. Expression of AtWRKY53 and UPL5 was regulated antagonistically during plant development and in response to H2O2 and JA. It was concluded that UPL5-mediated degradation of AtWRKY53 prevented aberrant senescence in the vegetative stage and ensured correct timing of senescence induction when the plant matures (Miao and Zentgraf, 2010). AtWRKY53 was also shown to interact in the nucleus with the JA-inducible protein EPITHIOSPECIFYING SENESCENCE REGULATOR (ESR) to mediate negative crosstalk between senescence and pathogen resistance in relation to SA and JA equilibrium (Miao and Zentgraf, 2007). Expression analyses revealed that AtWRKY53 is positively regulated by SA and repressed by JA. In vitro experiment shows that ESR can inhibit the DNA binding activity of AtWRKY53. It was concluded that interaction with ESR might alter the AtWRKY53 transcriptional activity in the nucleus. Epigenetic programming has also been implicated in the mechanism whereby AtWRKY53 regulates senescence in A. thaliana (Ay et al., 2009). In A. thaliana, the TF AtWRKY75 acts as a positive regulator of senescence by enhancing SA production and repressing ROS removal (Guo et al., 2017b). Knockdown or knockout of AtWRKY75 delayed developmental leaf senescence, while AtWRKY75 overexpression resulted in acceleration of senescence. AtWRKY75 promotes SA production by upregulating the transcription of SA biosynthetic gene SA INDUCTION-DEFICIENT2 (SID2)/Isochorismate Synthase 1 (ICS1) and suppresses H2O2 scavenging by downregulating the expression of CATALASE 2 (CAT2). AtWRKY75 expression was induced by age, H2O2, SA, and multiple plant hormones. However, CAT2 expression in Atwrky75 mutant was further repressed in senescing leaf compared to the young leaf (Guo et al., 2017b), which suggested that in addition to AtWRKY75, other factors are also involved in CAT2 expression and H2O2 production during leaf senescence. Similarly, in Brassica napus a novel WRKY TF gene WSR1 (WRKY regulating SA and ROS 1) was reported to accelerate leaf senescence through increasing SA and ROS production (Cui et al., 2020). AtWRKY45 plays a positive role in age-triggered leaf senescence by influencing the gibberellin (GA) signaling pathway (Chen et al., 2017). AtWRKY45 can interact with the DELLA protein RGL1, a repressor of the GA signaling pathway. Interaction with RGL1 repressed the transcription activation function of AtWRKY45, resulting in attenuation of the downstream target genes expression. In A. thaliana, loss of AtWRKY45 function resulted in increased leaf longevity, whereas overexpression of AtWRKY45 significantly accelerated age-triggered leaf senescence. ChIP assays revealed that AtWRKY45 is bound to the promoters of several SAGs including SENESCENCE 4 (SEN4), SENESCENCE-ASSOCIATED GENE 12 (SAG12), SAG13, and SAG113 to regulate their expressions. Overexpression of RGL1 resulted in significantly increased leaf longevity (Chen et al., 2017). AtWRKY70 encodes a WRKY TF having negative roles during developmental senescence ¨ lker et al., 2007). The Atwrky70 loss-of-function mutants showed early developmental and (U

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dark-induced leaf senescence. Decreased expression of photosynthesis and protein synthesis-related genes and increased expression of SAGs was observed during the leaf senescence. AtWRKY70 also represses the expression of both SA and JA/ethylene-responsive marker genes. Later, Besseau et al. (2012) reported that AtWRKY70 cooperate with AtWRKY54 to function as negative regulators of leaf senescence in A. thaliana. Loss-of-function Atwrky54/Atwrky70 double mutant exhibited a much more precocious senescence phenotype as compared to individual single mutants. AtWRKY54 and AtWRKY70 transcripts showed a slow increase during leaf growth and significant high transient upregulation at the onset of senescence. Moreover, AtWRKY54 and AtWRKY70 were induced neither by H2O2 nor by ozone, unlike AtWRKY30 and AtWRKY53. AtWRKY54 and AtWRKY70 were also found to interact independently with AtWRKY30 at the protein level. AtWRKY30 transcript level was high throughout the senescence process and was therefore suggested to possess possible functions during leaf senescence. However, downregulation of AtWRKY30 in miRNA-WRKY30silenced plants did not show any significant differences in senescence phenotype when compared with the wild type (Besseau et al., 2012). Jiang et al. (2014) reported that auxin and JA antagonistically regulate leaf senescence through AtWRKY57. Exogenous JA can induce senescence, whereas auxin suppresses it. In A. thaliana, loss of AtWRKY57 function accelerated JA-induced leaf senescence, concomitant with upregulation of SAGs in the Atwrky57 mutants. AtWRKY57 binds to the promoters of SEN4 and SAG12 directly and represses their expressions. In the Atwrky57 mutant, exogenous auxin could not stop the JA-induced leaf senescence suggesting that AtWRKY57 is vital for auxin antagonization of JAinduced senescence. At the protein level, AtWRKY57 was induced by auxin but degraded by JA via the 26 S proteasome pathway (Jiang et al., 2014). INDOLE-3-ACETIC ACID INDUCIBLE 29 (IAA29) and JASMONATE ZIM-DOMAIN (JAZ) 4/8, which function as positive and negative regulators, respectively, in JA-induced senescence, interact competitively with the zinc-finger domain of AtWRKY57 (Jiang et al., 2014). Thus AtWRKY57 mediates crosstalk between the JA and auxin signaling pathways during leaf senescence. AtWRKY6 is involved in leaf senescence and plant defense responses (Robatzek and Somssich, 2001). Strong AtWRKY6 expression was observed in the senescent leaf and was also induced by wounding. AtWRKY6 can suppress its own promoter activity and other closely related WRKYs, indicating negative autoregulation (Robatzek and Somssich, 2002). On the contrary, AtWRKY6 positively activate expression of the senescence- and pathogen defense-associated PATHOGENESISRELATED PROTEIN 1 (PR1) and receptor-like protein kinase SENESCENCE-INDUCED RECEPTOR-LIKE SERINE/THREONINE-PROTEIN KINASE (SIRK) promoters. The induction of SIRK is dependent on the AtWRKY6 function. Senescing leaf of Atwrky6 knockout mutants has low SIRK transcripts abundance, while high SIRK expression was observed in the green leaf of AtWRKY6 overexpression lines. Furthermore, AtWRKY6 specifically activated the SIRK promoter by direct interaction at the W-box. AtWRKY6 was suggested to regulate the leaf senescence process via SIRK (Robatzek and Somssich, 2002). AtWRKY22 transcription was induced by darkness and repressed by light, promoting the darkness-induced leaf senescence (Zhou et al., 2011). Dark treated AtWRKY22 overexpression A. thaliana plants showed accelerated senescence, whereas the knockout mutant displayed delayed senescence. AtWRKY22 expression was also strongly induced by H2O2. Li et al. (2017b) found that transgenic plants overexpressing AtWRKY26 exhibit early senescence phenotype, indicating that AtWRKY26 is also a positive regulator of leaf senescence.

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5.3.3 APETALA2/Ethylene-responsive element binding protein (AP2/EREBP) superfamily AP2/EREBP TFs are among the largest and highly conserved gene families and play key roles in plant growth, development, and stress response (Dietz et al., 2010). All AP2/EREBP TFs have a conserved AP2 DNA binding domain of 50 to 70 amino acid residues, consisting of a threestranded antiparallel β-sheet and an α-helix (Allen et al., 1998). This AP2 domain was first reported in the protein sequence of A. thaliana homeotic gene APETALA2 (AP2) (Jofuku et al., 1994; Dietz et al., 2010). Shortly afterwards, ETHYLENE-RESPONSIVE ELEMENT BINDING PROTEINS (EREBPs) were identified from tobacco as DNA-binding proteins essential for the responsiveness of some promoters to ethylene (Ohme-Takagi and Shinshi, 1995; Dietz et al., 2010). The EREBP domain is closely related to the AP2 domain (Dietz et al., 2010). Based on the number of AP2 domains and sequence similarities, the AP2/EREBP genes are grouped into four subfamilies: (1) APETALA2 (AP2), (2) Related to ABI3/VP1 (RAV), (3) Dehydration-Responsive Element Binding protein (DREB), and (4) Ethylene-Responsive Factor (ERF) (Dietz et al., 2010). The AP2 subfamily contains two AP2 domains, while the RAV subfamily consists of an AP2 domain and a B3 DNA binding domain. Both DREB and ERF host a single AP2 domain. DREB genes encode products that recognize the dehydration-responsive element (DRE) with A/GCCGAC core motif sequence (Yamaguchi-Shinozaki and Shinozaki, 1994). The ERF subfamily genes encode TFs that bind the cis-acting element AGCCGCC, known as the GCC box (Hao et al., 1998). AP2 type TF AINTEGUMENTA (ANT), which is a key leaf growth regulator, has been implicated in the regulation of developmental leaf senescence by acting downstream of the AUXIN RESPONSE FACTOR 2 (ARF2) in A. thaliana (Mizukami and Fischer, 2000; Feng et al., 2016). The loss-of-function ant-1 mutant plant displays premature leaf senescence phenotype, whereas in ANT overexpression plant, leaf senescence is delayed. Loss of ANT function reversed the delayed leaf senescence phenotype in the arf2
5 mutant, indicating that ANT acted downstream of ARF2 (Feng et al., 2016). In A. thaliana, ARF2 was shown to positively regulate leaf senescence (Lim et al., 2010). C-REPEAT BINDING FACTORS 1 (CBF1), CBF2, and CBF3 are transcriptional activators belonging to the DREB subfamily. Overexpression of CBF1
3 genes is well-documented for enhancing plant tolerance to cold (Yamaguchi-Shinozaki and Shinozaki, 1994). In addition to conferring cold tolerance, overexpressing CBF2 and CBF3 in A. thaliana significantly delayed the onset of age-dependent and phytohormones-induced leaf senescence (Sharabi-Schwager et al., 2010a,b). Interestingly, overexpression of CBF2 and CBF3 was shown to negatively affect multiple plant hormone signaling pathways in A. thaliana (Li et al., 2017a). Transcript profiling analysis of the CBF2 and CBF3 overexpressing lines revealed altered expression of genes associated with auxin signal transduction and metabolism. In addition, genes involved in the biosynthesis of JA and SA, as well as the signal sensing of brassinolide and SA, were downregulated, while genes associated with gibberellin deactivation were upregulated. Recently, Zhang et al. (2020b) reported the senescence-promoting role of DREB AND EAR MOTIF PROTEIN 4 (DEAR4) from the DREB1/CBF family in A. thaliana. The expression of DEAR4 was induced during leaf senescence and by JA, ABA, darkness, drought, and salt stress. Transgenic plants overexpressing DEAR4 exhibited precocious leaf senescence while the dear4

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knockdown mutant delayed senescence phenotype. DEAR4 overexpressing plant also accumulated significantly more H2O2 compared with wild type. DEAR4 protein acts as a transcriptional repressor and downregulate the transcription of some Cold-Regulated (COR) and Responsive to Dehydration (RD) genes, which were previously reported to promote leaf longevity and abiotic stress tolerance (Shi et al., 2017b). Physical interaction between the drought master regulator DREB2A and the RADICAL-INDUCED CELL DEATH 1 regulates leaf senescence and stress response (Sakuma et al., 2006; Vainonen et al., 2012), suggesting a link between leaf senescence and drought stress. CYTOKININ RESPONSE FACTORS (CRFs) that act downstream of cytokinin perception and signaling pathway have been implicated in the control of leaf senescence in A. thaliana (Raines et al., 2016). The CRFs are closely related AP2/ERF TFs that are transcriptionally induced by cytokinin. Functional studies with loss- and gain-of-function mutants of the numerous CRFs genes revealed that CRFs are positive regulators of leaf senescence. Cytokinin can inhibit and reverse leaf senescence (Gan and Amasino, 1995). Thus the induction of CRFs by cytokinin is in contrast to their role as positive regulators of senescence (Raines et al., 2016). A. thaliana plants overexpressing CRF2 showed accelerated leaf senescence phenotype concomitant with expressions of several pathogenesis-related (PR) genes (Kwon, 2016). Intriguingly, the leaf senescing phenotype of the AtCRF2-transgenic plant was completely suppressed by expressing the bacterial salicylate hydroxylase, NahG gene. Salicylate hydroxylase inhibits SA accumulation. The finding suggested that AtCRF2 might enhance SA biosynthesis, leading to autoimmune responses and senescence (Kwon, 2016). SA has also been reported to negatively regulate the cytokinin signaling pathway to fine-tune the cytokinin responses (Argueso et al., 2012). This finetuning mechanism control by cytokinin and SA equilibrium was suggested to result in the transient expression of AtCRF2 by cytokinin to prevent SA overproduction and autoimmune response which leads to balanced developmental processes and biotic stress responses. Contrary to AtCRF2 role as a positive regulator of leaf senescence and biotic stress response (Kwon, 2016), AtCRF6 negatively regulates developmental senescence and stress responses (Zwack et al., 2013). Transgenic A. thaliana overexpressing AtCRF6 retain more chlorophyll than wild type under dark senescence inducing conditions (Zwack et al., 2013). In contrast, loss-of-function crf6 mutant plant showed accelerated monocarpic senescence. AtCRF6 was highly expressed in the veins of the mature leaf, and the gene expression declined with age. AtCRF6 transcription was also induced by abiotic stress and cytokinin (Zwack et al., 2013). AtCRF6 may therefore be involved in fine-tuning the timing of developmental and stress-induced senescence (Zwack and Rashotte, 2013; Zwack et al., 2013).

5.3.4 Basic helix-loop-helix (bHLH) TFs The basic helix-loop-helix (bHLH) members comprised the second-largest TFs family in eukaryotes after the MYBs (Guo et al., 2021a). The first plant bHLH gene was identified in Z. mays and the genome of the model plant A. thaliana has 162 bHLH genes (Feller et al., 2011; Guo et al., 2021a). bHLH TFs play key regulatory roles in plant growth and development, biosynthetic processes, light response, and stress tolerance (Guo et al., 2021a). Recently, their roles as promoters or repressors of leaf senescence were reported (Lim et al., 2007; Kim et al., 2020; Zhang et al., 2020a).

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Phytochromes regulate light responses in plants by promoting the degradation of PHYTOCHROME INTERACTING FACTORS (PIFs), a family of bHLH TF (Yasuhito et al., 2014). PIF3, PIF4, and PIF5 were shown to participate in the natural- and dark-induced leaf senescence processes (Song et al., 2014; Yasuhito et al., 2014). Loss-of-function mutants of the PIF genes significantly delay age- and dark-induced leaf senescence, whereas transgenic plants overexpressing these genes display early leaf senescence. More studies revealed that PIF4 could bind to the promoter region of the chlorophyll catabolic gene NYE1 and the chloroplast activity maintainer gene GLK2, resulting in their induction and repression, respectively (Song et al., 2014; Shi et al., 2017a). The loss-of-function pif4 mutant also lacks dark-induced ethylene biosynthesis and ethylene-induced leaf senescence (Song et al., 2014). Expression of ethylene biosynthesis genes (ACSs) and ethylene-responsive genes were severely decreased in pif4 mutants suggesting the major role of PIF4 in both ethylene biosynthesis and signaling pathway. More recently, PIF4 was reported to bind to the ORE1 promoter and activate the gene transcription with ABA and ethylene signaling during leaf senescence (Kim et al., 2020). High ambient temperature in the dark was shown to accelerate the inactivation of the light-activated form of phytochrome B (Pfr), which increases PIF4 protein levels (Kim et al., 2020). The increased PIF4 resulted in accelerated dark-induced leaf senescence at high ambient temperature. Similarly, the soybean CRYPTOCHROME-INTERACTING bHLH1 (CIB1) was reported to promote leaf senescence (Meng et al., 2013). CIB1 can activate the transcription of WRKY DNA BINDING PROTEIN53b (WRKY53b) and induce leaf senescence. CIB1 interacts with the E-box (CANNTG) sequences of the WRKY53b promoter; however, the DNA binding activity in the presence of photoexcited CRYPTOCHROME2 (CRY2a) was abolished. Moreover, transgenic soybean overexpressing CRY2a exhibited delayed leaf senescence (Meng et al., 2013). MYC2, MYC3, and MYC4 belonging to bHLH subgroup IIIe can directly promote the expression of chlorophyll catabolic genes PAO, NYC1, and NYE1 or indirectly promote expression via the downstream NAC TFs ANAC072, ANAC 055, and ANAC 019, which can also activate the expression of NYC1, NYE1, and NYE2 in JA-signaling pathways (Zhu et al., 2015). Antagonism interactions between bHLH subgroup IIIe and IIId members are involved in the regulation of jasmonate-induced leaf senescence in A. thaliana (Qi et al., 2015). MYC2, MYC3, and MYC4 of the bHLH subgroup IIIe function redundantly to activate JA-induced leaf senescence. MYC2 can bind and activate the transcription of SAG29. On the other hand, the bHLH subgroup IIId, including bHLH17, bHLH14, bHLH13, and bHLH03, antagonistically binds to the promoter region of SAG29 and repress its transcription to attenuate the MYC2/ MYC3/MYC4 mediated jasmonate-induced senescence. Antagonistic regulation by activators and repressors was suggested to result in fine-tuned control of JA-induced leaf senescence enabling the plants to survive the changing environment (Qi et al., 2015). ROS play an important role in JA-induced leaf senescence (Navabpour et al., 2003; Zhang et al., 2020a). JA induces H2O2 production, and reduced H2O2 content suppresses JA-induced senescence and the transcription of SAGs (Navabpour et al., 2003). Results from a recent study revealed that MYC2, a master regulator in the JA-signaling pathway, was involved in JA-induced H2O2 accumulation by downregulating the transcription of CAT2 through binding to its promoter (Zhang et al., 2020a). Additionally, the loss-of-function myc2 mutant has delayed senescence phenotype with high catalase enzyme activity and reduced H2O2 accumulation compared to the wild type after JA treatment (Zhang et al., 2020a).

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5.3.5 MYB TFs The MYB is the largest TF gene family with around 180 genes in A. thaliana (Riechmann et al., 2000; Feller et al., 2011). All MYB TFs have a conserved 52 amino acids long DNA-binding MYB domain (Feller et al., 2011) and are classified into subfamilies based on the presence of one, two, or three MYB domains (Riechmann et al., 2000; Feller et al., 2011). MYB TFs are involved in secondary metabolism regulation, growth and development, cell fate determination and identity, responses to environmental cues, and phytohormones (Feller et al., 2011). An ABA inducible AtMYBR1/AtMYB44 (R2R3-MYB) was reported to negatively regulate leaf senescence in A. thaliana (Jaradat et al., 2013). Overexpression of the AtMYBR1 resulted in delayed leaf senescence, whereas loss-of-function mybr1 mutant showed enhanced chlorophyll loss and leaf senescence. Many ABA inducible genes were downregulated in the MYBR1 overexpressing plants resulting in increased cell injury upon dehydration. Conversely, mybr1 mutant plants were more tolerant of dehydration with reduced water loss from leaves. MYBR1 can directly interact with PYL8, an ABA receptor suggesting MYBR1 involvement in early ABA signaling (Jaradat et al., 2013). A single-repeat MYB transcription repressor, MYBH, regulated leaf senescence in A. thaliana by controlling auxin homeostasis (Huang et al., 2015). Guo et al. (2017a) suggested that the differential expression of alternatively spliced transcripts of an MYB TF ScMYB2 is an important posttranscriptional regulatory mechanism during ABA- and drought-induced leaf senescence in sugarcane. Yeast one-hybrid assay revealed that AtMYB108 binds to the ANAC003 promoter region, suggesting the involvement of an MYB-NAC regulatory network in dark-stressed induced leaf senescence in A. thaliana (Chou et al., 2018). The R1-MYB transcriptional factor CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1), a core component of the circadian clock controls, was reported to inhibit leaf senescence in A. thaliana (Song et al., 2018; Wang et al., 2018). Mutation of CCA1 causes early leaf senescence phenotype (Song et al., 2018). Intriguingly, CCA1 represses the expression of ORE1, a known positive senescence regulator and upregulate the chloroplast maintenance gene GLK2 by directly binding to the respective gene promoters. At the juvenile stage, the high CCA regulates ORE1 and GLK2 expression to inhibit leaf senescence; but as the plant ages, the declining transcription of CCA1 and GLK2 attenuates the inhibition of leaf senescence and ORE1 accumulation, promoting the initiation of senescence (Song et al., 2018). Interestingly, mutation of LATE ELONGATED HYPOCOTYL (LHY) also accelerated the leaf yellowing phenotype, and the double cca1 lhy mutant further aggravated the early senescence phenotype compared to the single cca1 or lhy mutant (Song et al., 2018). LHY is also a R1-MYB transcription factor and is homologous to CCA1 (Wang et al., 2018). CCA1 also function as a master regulator of ROS homeostasis through transcriptional activation of ROS responsive genes to coordinate time-dependent responses to oxidative stress (Lai et al., 2012). Declining CCA1 during aging might disrupt ROS homeostasis and contribute to senescence initiation.

5.3.6 Auxin response factor and Auxin/INDOLE-3-acetic acid TFs Auxin acts as a suppressor of leaf senescence (Teale et al., 2006). AUXIN RESPONSE FACTOR (ARF) and the Auxin/Indole-3-Acetic Acid (Aux/IAA) TFs are key regulators of auxin-mediated gene expression and were shown to be differentially regulated during leaf senescence in several

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plant species (Teale et al., 2006; Jiang et al., 2014; Li et al., 2019). ARFs bind to auxin-responsive elements found in the auxin-responsive gene’s promoter (Teale et al., 2006; Okushima et al., 2005). Aux/IAA proteins are short-lived nuclear proteins encoded by the auxin early response gene family (Teale et al., 2006; Jiang et al., 2014). Aux/IAA has highly conserved specific domains that interact with ARFs to inhibit the expression of genes activated by ARFs (Teale et al., 2006; Jiang et al., 2014). Studies indicated that ARF2 might be a crucial player in the auxin-mediated regulation of leaf senescence (Ellis et al., 2005; Okushima et al., 2005; Lim et al., 2010). ARF2 was shown to positively regulate leaf senescence in A. thaliana (Okushima et al., 2005; Lim et al., 2010). Loss-offunction arf2 mutant has enhanced leaf growth with delayed leaf senescence (Okushima et al., 2005; Lim et al., 2010). ARF2 transcripts were abundant in senescing leaf, induced by developmental aging or darkness (Ellis et al., 2005). ARF19 and ARF7 transcripts are also abundant in senescing leaf (Lin and Wu, 2004). However, loss-of-function mutations in ARF19 and ARF7 genes do not alter the senescence phenotype (Ellis et al., 2005). Delays in all senescence parameters including membrane ion leakage, photochemical efficiency of photosystem II, chlorophyll content, and expression of SAGs were observed in the arf2 mutant plants (Lim et al., 2010). ARF2 being a repressor of auxin signaling (Okushima et al., 2005; Lim et al., 2010), the lack of its function in the mutant was suggested to lessen the repression of auxin signaling, leading to increased auxin sensitivity and delaying senescence (Lim et al., 2010). Interestingly, ARF2 was recently shown to directly bind to the promoter region of GROWTHREGULATING FACTOR 5 (GRF5) to repress transcription (Beltramino et al., 2021). Furthermore, it was demonstrated that the unrestrained GRF5 expression in the arf2 mutant is responsible for the enhanced growth of arf2 leaves (Okushima et al., 2005; Lim et al., 2010; Beltramino et al., 2021). Previously, GRF5 was reported to modulate cytokinin signaling to stimulate cell division and delay leaf senescence (Vercruyssen et al., 2015) In A. thaliana, the AUX/IAA protein IAA29 was shown to positively regulate JA-induced leaf senescence (Jiang et al., 2014). IAA29 was induced by JA, and its expression was higher in senescent leaves than in normal leaves. IAA29 overexpression lines showed early leaf senescence symptoms compared with wild-type plants. Chlorophyll content reduction and increased cell death were observed in IAA29 overexpression plants. Correspondingly, the transcript levels of SAGs, namely SAG12 and SEN4, were higher in IAA29 overexpression plants than in the wild type. These results suggested that IAA29 positively regulates JA-induced leaf senescence. IAA29 can also interact with AtWRKY57 to antagonize JA-induced leaf senescence (Jiang et al., 2014). AtWRKY57 is a negative regulator in JA-induced leaf senescence (Jiang et al., 2014). Shi et al. (2015) reported an INDOLE-3-ACETIC ACID INDUCIBLE 17 (IAA17) to positively modulate natural leaf senescence through melatonin (N-acetyl-5-methoxytryptamine)-mediated pathway in A. thaliana. Melatonin is a ubiquitous modulator of developmental processes and stress responses in plants (Shi et al., 2015). In A. thaliana natural leaf senescence was delayed by exogenous melatonin treatment. The IAA17 transcripts level was repressed by exogenous melatonin treatment and during developmental aging. Further, overexpression of AtIAA17 resulted in early leaf senescence phenotype with lower chlorophyll content, while the AtIAA17 knockout mutant has the opposite phenotype. Exogenous melatonin can inhibit and markedly delay the senescence phenotype of the AtIAA17 overexpressing plants. AtIAA17 can activate SEN4 and SAG12 transcription, which might have contributed to the process of developmental aging (Shi et al., 2015).

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5.3.7 DNA binding-with-one-finger (DOF) proteins DNA BINDING-WITH-ONE-FINGER (DOF) proteins are TFs unique to plants (Yanagisawa and Sheen, 1998). All DOF proteins have a highly conserved DNA binding domain containing a single C2-C2 zinc finger motif that recognizes the (AT)/AAAG cis-element in the regulatory region of their target genes (Yanagisawa, 1995, 2002). DOF proteins are involved in diverse plant processes, including light, hormone and stress responses, seed development and germination, shoot branching, guard cell development, carbon and nitrogen metabolism (Yanagisawa and Sheen, 1998; Yanagisawa, 2002; Gupta et al., 2015). Studies have reported the divergent involvement of DOF protein family in modulating JAmediated leaf senescence (Shim et al., 2019; Zhuo et al., 2020). In rice, a JA-inducible OsDOF24 delayed leaf senescence (Shim et al., 2019). Gain-of-function mutant osdof24-D, containing an enhancer-trap T-DNA in the OsDOF24 promoter, and transgenic rice plants overexpressing OsDOF24 showed delayed leaf yellowing during natural senescence and dark-induced senescence. Gene expression analysis revealed that senescence-associated, chlorophyll degradation and JA biosynthesis-related genes were downregulated in the osdof24-D mutant in dark treatment. OsDOF24 binds to the promoter region of OsAOS1 (a JA biosynthesis-related gene) in yeast one-hybrid assays. The osdof24-D mutant also exhibited a reduced endogenous JA level. Data demonstrated that OsDOF24 suppresses the initiation of leaf senescence during vegetative growth by disabling JA biosynthetic pathways (Shim et al., 2019). In contrast, overexpression of a JA-inducible DOF2.1 gene in transgenic A. thaliana plant exhibited enhanced dark-induced and age-dependent leaf senescence in addition to the inhibition of root development (Zhuo et al., 2020). Transcriptome and real-time PCR analysis revealed that DOF2.1 globally enhances JA-responsive and leaf senescence-associated genes expression (including MYC2) during JA-induced leaf senescence. DOF2.1 can bind and directly activate the MYC2 promoter in a cotransfection assay with the luciferase gene as a marker. DOF2.1 enhances leaf senescence by promoting MYC2 transcription. In Arabidopsis, MYC2, a bHLH type TF, is a central regulator of JA responses (Dombrecht et al., 2007; Kazan and Manners, 2013). MYC2 binds to the G-box motif in the promoter region of JA-responsive genes to differentially regulate their expression (Dombrecht et al., 2007). Interestingly, MYC2 was also shown to transcriptional activate the JA-inducible expression of DOF2.1 (Zhuo et al., 2020). These results suggested DOF2.1 to act as a promoter of JA-induced leaf senescence through the MYC2-Dof2.1-MYC2 feed-forward transcriptional loop. Recently, Xu et al. (2020) showed that the CYCLING DOF FACTOR 4 (CDF4) TF, belonging to the group II DOF TF family, can positively regulate leaf senescence. CDF4 expression is induced by ABA and ROS, the two well-known leaf senescence inducers. Constitutive and inducible overexpression of CDF4 promotes leaf senescence, while knockdown of CDF4 function delays it. CDF4 increases endogenous ABA accumulation by inducing the expression of the ABA biosynthesis genes 9-CISEPOXYCAROTENOID DIOXYGENASE 2 (NCED2) and NCED3, and suppresses H2O2 scavenging by repressing the expression of the CAT2 gene. Knockout of NCED2 and NCED3 genes and CAT2 overexpression partially rescue premature senescence phenotype caused by CDF4 overexpression. CDF4 was also shown to promote floral organ abscission by upregulating the polygalacturonase PGAZAT gene expression. Based on these data, it was concluded that CDF4, ABA, and ROS levels undergo a gradual increase during the progression of leaf senescence and floral organ abscission, which is driven by their interlinking positive feedback loops (Xu et al., 2020).

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5.3.8 PSEUDO-response regulators TF The Arabidopsis PSEUDO RESPONSE REGULATOR (PRR) gene family is composed of five members, namely PRR3, PRR5, PRR7, PRR9, and TIMING OF CAB EXPRESSION 1 (TOC1), each of which peaks at a specific time of day in a consecutive manner from dawn to dusk (Matsushika et al., 2000; Nakamichi et al., 2010). Recent studies have revealed the shared interaction between the circadian clock and leaf senescence (Kim et al., 2016; Kim et al., 2018a; Song et al., 2018). PRR9, a core circadian component, acts as a key leaf senescence regulator by means of positive regulation of the positive aging regulator gene ORE1 through a feed-forward pathway (Kim et al., 2018a). PRR9, in particular, binds directly to the ORE1 promoter to promote its expression. At the same time, PRR9 also represses miR164 expression. MiR164 is a posttranscriptional repressor of ORE1 (Kim et al., 2009). Consistently, ORE1 overexpression was able to rescue the delayed leaf senescence phenotype of the loss-of-function prr9 mutant. Thus PRR9 regulates senescence by enhancing the ORE1 level via a feed-forward pathway that involves both direct transcriptional regulation and posttranscriptional regulation by miR164. These findings indicate that the circadian clock and leaf senescence processes are closely interconnected (Kim et al., 2018a). Another core circadian component CCA1 can directly bind and suppress ORE1 expression to inhibit leaf senescence in A. thaliana (Song et al., 2018). This implies that ORE1 is an integrator of the circadian clock and the age-dependent senescence. More future studies will help uncover new interaction networks between the leaf senescence systems and the circadian clock.

5.3.9 VQ TF family The VQ TF family contains a unique and conserved VQ (FxxxVQxxTG) motif and play diverse roles in plant defense, growth and development, including leaf senescence (Li et al., 2014; Yu et al., 2019). Yu et al. (2019) reported the overexpression of a Z. mays VQ52 (ZmVQ52) in A. thaliana resulted in accelerated premature leaf senescence. The overexpression line had lower chlorophyll content, higher senescence rate, higher sensitivity to SA and JA, and enhanced tolerance to ABA. Additionally, four Z. mays WRKY TFs, namely ZmWRKY71, ZmWRKY50, ZmWRKY36, and ZmWRKY20 were identified as interacting partners with ZmVQ52 using the maize protoplast expression system. Transcriptome analysis of the ZmVQ52 overexpression plant revealed that ZmVQ52 regulates leaf senescence mostly through modulation of photosynthesis and circadian rhythm pathways.

5.3.10 Basic leucine zipper (bZIP) TFs bZIP proteins are dimeric TFs containing a dimerization domain and a basic motif in the DNA binding domain (Jakoby et al., 2002; Dro¨ge-Laser et al., 2018). In A. thaliana, bZIP proteins were divided into 13 groups (designated A-M) based on sequence similarity of the basic DNA binding domain and the presence of additional conserved motifs (Dro¨ge-Laser et al., 2018). Members of the group A bZIP TFs, namely ABSCISIC ACID-RESPONSIVE ELEMENT BINDING FACTOR 2 (ABF2), ABF3, ABF4, and ABA INSENSITIVE 5 (ABI5), which play core roles in the ABA signaling, have been recently found to directly control genes involved in leaf

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senescence (Gao et al., 2016; Skubacz et al., 2016; Su et al., 2016; Dro¨ge-Laser et al., 2018). Gao et al. (2016) reported that ABF2, ABF3, and ABF4 directly activate the expression of chlorophyll catabolic enzyme genes and SAGs, resulting in the promotion of the ABA-mediated chlorophyll degradation and leaf senescence. Similarly, ABI5 was shown to negatively influence the chlorophyll content, photosynthesis efficiency and enhance leaf senescence via promotion of chlorophyll catabolism genes, STAYGREEN1 (SGR1) and NYC1, and repression of ABA RESPONSIVE PROTEIN (ABR), a gene encoding a LATE EMBRYOGENESIS ABUNDANT protein, which is associated with leaf rescue from the senescence process (Skubacz et al., 2016). Thus ABF2, ABF3, ABF4, and ABI5 were proposed to play positive roles in leaf senescence through its negative impact on the photosynthesis process (Skubacz et al., 2016; Su et al., 2016; Dro¨ge-Laser et al., 2018). Recently, Malus domestica ABI5 (MdABI5) was shown to interact directly with three other transcription factors, namely MdBBX22, MdWRKY40, and MdbZIP44, to regulate the ABAtriggered leaf senescence in apple (An et al., 2021). MdABI5 interaction with the repressor MdBBX22 led to the delay in leaf senescence, while MdWRKY40 and MdbZIP44 accelerated MdABI5 promoted leaf senescence through enhancing the transcriptional activity of MdABI5 on downstream senescence-associated target genes (An et al., 2021). The finding suggested that antagonistic regulation pathways might enable the plants to adjust to external cues efficiently and flexibly. Another bZIP TF, G-BOX BINDING FACTOR 1 (GBF1), belonging to G group was reported to initiate the onset of leaf senescence in A. thaliana through the regulation of CAT2 expression and intracellular H2O2 level (Smykowski et al., 2010). GBF1 binds to the CAT2 promoter region containing the G-box motif (5’-CACGTG-3’) and represses the gene expression. By reducing the H2O2-scavenging activity of CAT2, GBF1 triggers H2O2 accumulation, which functions as a signal to coordinate the senescence program (Smykowski et al., 2010; Dro¨ge-Laser et al., 2018).

5.3.11 Homodomain-leucine zipper (HD-ZIP) TFs Studies have reported the involvement of several HD-ZIP TFs in regulating leaf senescence (Manavella et al., 2006; Xie et al., 2014b; Liu et al., 2016b). Among these TFs, HaHB-4, a class II sunflower HD-ZIP, is involved in leaf senescence and response to abiotic stress (Dezar et al., 2005; Manavella et al., 2006). Ethylene induces HaHB-4 expression during normal leaf senescence. Under drought stress, transgenic A. thaliana plants overexpressing HaHB4 exhibit increased plant survival with inhibition of drought-related senescence (Dezar et al., 2005; Manavella et al., 2006). Transgenic plants expressing HaHB4 exhibit an apparent delay in their entry into developmental senescence at the end of the life cycle under well-watered conditions (Manavella et al., 2006). Microarray and q-PCR data suggested that HaHB4 negatively regulates the expression of genes related to ethylene synthesis and signaling to delay senescence (Manavella et al., 2006). It was concluded that HaHB4 mediates crosstalk between ethylene signaling and water deficit response. The A. thaliana ABA INSENSITIVE GROWTH 1 (ABIG1), belonging to class II HD-ZIP, was reported to relay the ABA-mediated growth inhibition and drought-induced senescence (Liu et al., 2016b). ABIG1 transcript levels were upregulated in drought and ABA treatment. When treated with ABA, loss-of-function abig1 mutant remains greener and produce more leaves, compared to wild-type. During drought stress, abig1 mutants showed fewer senescence leaves. Overexpression

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of ABIG1 mimics ABA application and regulates genes implicated in stress responses, including ABA and ethylene-responsive genes. The class III HD-ZIP protein REVOLUTA (REV), involved in basic pattern formation during leaf development, controls the onset of leaf senescence in A. thaliana (Xie et al., 2014b). REV is a redox-sensitive protein and can positively regulate the expression of AtWRKY53, a key regulator of leaf senescence (Xie et al., 2014b; Zentgraf and Doll, 2019). REV protein is required to activate AtWRKY53 in response to oxidative stress, and the onset of senescence was significantly delayed in REV mutant plants (Xie et al., 2014b). Brandt et al. (2012) reported that REV regulates ABIG1 expression.

5.3.12 Plant A/T-rich sequence and zinc-binding protein (PLATZ) TF Arabidopsis thaliana ORE15 gene encoding a PLATZ TF was shown to enhance leaf growth in young plants, but suppresses leaf senescence in the mature stage (Kim et al., 2018c). ORE15 was preferentially expressed in young leaves and promotes leaf growth by increasing the rate and duration of cell proliferation. It was observed that overexpressing and dominant mutants obtained from activation-tagged lines of A. thaliana had prolonged leaf longevity and enlarged leaf size. Loss-offunction mutant lines exhibited early senescence phenotype than those of the wild type. Microarray, quantitative RT-PCR, chromatin immunoprecipitation, and genetic analysis studies revealed that ORE15 regulates leaf growth and senescence by modulating the GROWTHREGULATING FACTOR (GRF)/GRF-INTERACTING FACTOR regulatory pathway. In the gainof-function oresara15
1D (ore15
1D) mutants, several senescence-associated genes expression were downregulated. In contrast, AtGRF5, AtGIF1/AN3, and cell cycle genes were upregulated, corresponding to the suppression of leaf senescence and enhancement of leaf growth, respectively. The ORE15 TF directly binds to the promoters of AtGRF1 and AtGRF4 (Kim et al., 2018c).

5.3.13 Growth-regulating factors (GRFS) and GRF-interacting factors (GIFS) Studies on growth regulators required for early leaf development point to the potential contribution of leaf growth on senescence (Horiguchi et al., 2005; Debernardi et al., 2014; Vercruyssen et al., 2015). It has been reported that GIFs and GRFs function as transcriptional regulatory complexes and are required for important phases of plant growth and development, including leaf development (Gonzalez and Inze, 2015; Horiguchi et al., 2005 ). Mutations in GRF3 or GRF5 result in leaf size reduction through decreased cell proliferation activity (Debernardi et al., 2014; Vercruyssen et al., 2015). The mutants also display accelerated leaf senescence phenotype (Horiguchi et al., 2005; Debernardi et al., 2014; Vercruyssen et al., 2015). In the overexpressing line, GRF5 was shown to promote chloroplast division, increase chloroplast number per cell, and increase chlorophyll levels. Moreover, GRF5 overexpressing plants also have delayed leaf senescence with increased sensitivity to cytokinins (Vercruyssen et al., 2015). Results suggested that GRF5 and cytokinins synergistically enhance cell division and chlorophyll retention to retard leaf senescence (Vercruyssen et al., 2015). In addition, ANGUSTIFOLIA3 (AN3), a mutant of the GIF1 gene, also showed similar phenotypes as in the grf3 and grf5 mutants with accelerated leaf senescence (Horiguchi et al., 2005; Debernardi et al., 2014).

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5.3.14 Teosinte branched 1, Cycloidea, and proliferating cell nuclear antigen binding factor (TCP) TFS TCP TFs control developmental processes in plants (Schommer et al., 2008; Sarvepalli and Nath, 2011; Danisman et al., 2012). The TCPs are divided into class I and II TCPs, which are proposed to act antagonistically in the control of leaf development via the JA signaling pathway (Danisman et al., 2012). Transgenic A. thaliana plants expressing a hyperactive form of the class II protein TCP4 results in reduced cell proliferation, smaller leaves, and precocious leaf senescence (Sarvepalli and Nath, 2011). The JAGGED AND WAVY (JAW)-D mutant overexpressing miR319a exhibited delayed leaf senescence (Schommer et al., 2008), while overexpression of TCP4 led to premature leaf senescence (Sarvepalli and Nath, 2011). It is known that miR319a regulates leaf senescence through its target genes, the class II TCP transcription factor and partly by altering the JA levels (Schommer et al., 2008). Furthermore, it was shown that TCP4 could influence JA biosynthesis by positively regulating the expression of LIPOXYGENASE 2 (LOX2; Schommer et al., 2008). LOX2 catalyzes the α-linoleic acid conversion to 13(S)- hydroperoxylinolenic acid, a key step of JA biosynthesis (Vick and Zimmerman, 1983). Interestingly, Danisman et al. (2012) reported that the class I TCP20 could also bind to the regulatory sequences of LOX 2 gene promoter and repress expression. In addition, the knockout mutant of TCP20 has increased LOX2 transcripts level, and the earlier leaf senescence phenotype was observed in the loss-of-function tcp9 tcp20 double mutant. Class I TCP9 gene is a target of TCP20 protein and acts downstream of TCP20 (Danisman et al., 2012). Intriguingly, in Chinese flowering cabbages (Brassica rapa var. parachinensis) different class I TCP transcriptional factors, namely BrTCP7 and BrTCP21, were reported to act as positive and negative regulators of leaf senescence, respectively (Xiao et al., 2019; Xu et al., 2019). BrTCP7 is involved in MeJA-promoted leaf senescence by binding to the promoter regions of a JA biosynthetic gene OPDA REDUCTASE 3 and a chlorophyll catabolic gene RED CHLOROPHYLL CATABOLITE REDUCTASE and activating their transcriptions (Xu et al., 2019). Higher transcript levels of BrTCP7 was observed in senescing leaf, and the gene was inducible by MeJA. In contrast, BrTCP21 binds to the promoter of the gibberellic acid (GA) biosynthetic gene BrGA20ox3 and activates its transcription (Xiao et al., 2019). BrTCP21 expression was low in senescing leaf and further decreased after senescence, while GA could keep a higher transcripts level of BrTCP21. GA delays leaf senescence of Chinese flowering cabbage (Xiao et al., 2019). These findings highlight the complexity of TCP transcriptional factors in modulating leaf senescence.

5.3.15 Homeobox (HB) TFs KN1-like protein is a homeobox TF, the overexpression of which increases cytokinin production and leads to leaf senescence inhibition (Vollbrecht et al., 1991; Luo et al., 2006). The maize homeobox gene KNOTTED 1 (KN1) expression has been linked to the accumulation of cytokinin (Hewelt et al., 2000; Sakamoto et al., 2006). Cultured tobacco tissues, overexpressing the maize KN1, were capable of growth in cytokinin free media and have elevated endogenous cytokinin levels (Hewelt et al., 2000). Ori et al. (1999) showed that the ectopic expression of maize kn1 under the control of SAG12 promoter resulted in delayed senescence with increased cytokinin levels in older leaves. Similarly, delayed leaf senescence was observed in tobacco plants overexpressing the maize KN1 under the control of the wound-inducible win3.12 gene promoter (Luo et al., 2006).

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Delayed leaf senescence, the accumulation of specific isopentenyl-type cytokinins, and alteration of leaf morphology and plant architecture were observed when KNAT1 (KNOTTED-like in Arabidopsis), an A. thaliana homolog of maize KN1 was overexpressed in lettuce (Frugis et al., 2001). Transgenic studies by Hamant et al. (2002) found that KNAT2 functions synergistically with cytokinins and antagonistically with ethylene. Increased KNAT2 expression in transgenic plants led to delayed leaf senescence and a higher rate of shoot initiation, two processes induced by hormone cytokinin and inhibited by ethylene. Furthermore, the KNAT2 induced lobbing of leaves was partially reversed by treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid or by the constitutive ethylene response ctr1 mutation. On the contrary, KNAT2 expression suppressed certain phenotypic traits of the ctr1 mutant (Hamant et al., 2002). In addition to increased cytokinin levels, endogenous levels of other hormones such as auxin, ABA, and GAs were altered after overexpression of KNOTTED1 (KN1)-like homeobox (KNOX) genes (Tamaoki et al., 1997; Kusaba et al., 1998; Sakamoto et al., 2006).

5.3.16 C3H (Zn) TFs C3H-type TFs have a zinc-binding domain containing the characteristic C-X6
14-C-X4
5-C-X3-H consensus sequences (Kong et al., 2006; Wang et al., 2008). C3H-ZFPs are widely found in organisms ranging from bacteria to eukaryotes, and they regulate genes at the transcriptional and posttranscriptional levels (Kong et al., 2006; Wang et al., 2008; Jan et al., 2013; Yan et al., 2017). Genome-wide annotation analysis of C3H-ZFPs found 67 and 68 genes in O. sativa and A. thaliana, divided into 8 and 11 subfamilies, respectively (Wang et al., 2008). The C3H-ZFPs exhibit varied expression patterns, suggesting diverse functions, including roles in hormone signaling, stress tolerance, and leaf senescence (Kong et al., 2006; Wang et al., 2008; Zhou et al., 2014; Yan et al., 2017). A nuclear-localized C3H-type ZFP designated Oryza sativa DELAY OF THE ONSET OF SENESCENCE (OsDOS) delayed leaf senescence in O. sativa (Kong et al., 2006). OsDOS expression was downregulated after pollination, panicle development, and during natural leaf senescence. RNAi knockdown of OsDOS resulted in early senescence, but its overexpression led to a marked delay of leaf senescence. A genome-wide expression analysis further revealed that the JA pathway was hyperactive in the OsDOS RNAi transgenic lines but impaired in the OsDOS overexpressing lines. It was proposed that OsDOS acts as a negative regulator of leaf senescence through the suppression of JA biosynthetic genes and JA responses (Kong et al., 2006). OsDOS was also suggested to have posttranscriptional role by interacting with target RNA. Similarly, the Gossypium hirsutum stress-inducible C3H-type ZFP gene GhTZF1 promoted drought tolerance by delaying drought-induced leaf senescence in transgenic A. thaliana (Zhou et al., 2014). Under drought conditions, transgenic A. thaliana plants overexpressing GhTZF1 exhibited higher superoxide dismutase and peroxidase activities, concomitant with lower level of H2O2. Expression analysis revealed that GhTZF1 reduced the expression of oxidative stress-related SAGs under drought stress. The dark- and JA-induced leaf senescence was also attenuated in the overexpressing transgenic plants. GhTZF1 was suggested to delay leaf senescence by inhibiting ROS accumulation (Zhou et al., 2014). A similar response was observed for OsTZF1, a rice C3Htandem ZFP gene with reduced ROS accumulation and attenuation of stress-related genes expression under salt-stress treatment in OsTZF1 overexpressing transgenic plants (Jan et al., 2013).

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OsTZF1 can bind to the U-rich sequences in the untranslated region of mRNAs and therefore was suggested to be associated with RNA metabolism of stress-responsive genes (Jan et al., 2013). Though, the DNA binding activity of GhTZF1 was not reported by Zhou et al. (2014). GhTZF1 is a homolog of A. thaliana AtTZF1 (Pomeranz et al., 2010). AtTZF1 has both plant development and stress response function and acts as a positive and negative regulator of ABA and GA responses, respectively (Pomeranz et al., 2010). As AtTZF1 binds to both DNA and RNA in vitro, it was postulated that it might be involved in transcription in the nucleus and RNA regulation in the cytoplasm (Pomeranz et al., 2010). GhTZF1 might have similar functions. Yan et al. (2017) reported two novel C3H zinc-finger and K-homolog (KH) proteins, KHZ1 and KHZ2, regulating flowering and developmental senescence in A. thaliana. The overexpression of KHZ1 and KHZ2 in transgenic A. thaliana induces early flowering and leaf senescence. In contrast, the loss-of-function khz1 khz2 double mutant has delayed leaf senescence, marked by lateflowering phenotype and increased plant longevity. Further, after treatment with ABA, the detached leaves from double mutant displayed delayed leaf yellowing, while the KHZ1 and KHZ2 overexpressing leaves displayed early senescence. KHZ1 and KHZ2 were shown to be nuclear localized, and possess both RNA-binding abilities and transactivation activities. Lee et al. (2012b) report RNase activity in two A. thaliana C3H ZFPs, namely AtTZF2 and AtTZF3, suggesting that they may be involved in the mRNA turnover process. Subcellular localization assay further demonstrated that AtTZF2 and AtTZF3 were localized in the cytoplasm. Transgenic plants overexpressing AtTZF3 and AtTZF2 showed increased drought tolerance and delayed age-dependent, darkinduced, and JA-induced leaf senescence via altered ABA and JA responses (Lee et al., 2012b).

5.3.17 GRAS TFs GRAS proteins are specific to plants and are named after GIBBERELLIC ACID INSENSITIVE (GAI), REPRESSOR OF GA1
3 mutant (RGA), and SCARECROW (SCR), the first three members identified (Bolle, 2004). GRAS proteins are 400
700 amino acids long and have several conserved motifs in their carboxyl end, including leucine heptad repeat I (LHR I), VHIID, leucine heptad repeat II (LHR II), PFYRE, and the SAW motif (Bolle, 2004; Hirsch and Oldroyd, 2009). The highly variable amino-terminal parts of GRAS proteins are subfamily-specific and determine specificity during molecular recognition events (Bolle, 2004; Hirsch and Oldroyd, 2009). The GRAS protein family is further divided into DELLA, HAM, LISCL, LS, PAT1, SCR, SCL3, and SHR subfamilies. The subfamilies are named either after a common motif or members (Tian et al., 2004). Consistent with their role as transcriptional regulators, most GRAS proteins are nuclear localized; however, some are cytoplasmic proteins (Tian et al., 2004; Hirsch and Oldroyd, 2009). Most GRAS proteins were shown to exert important roles in diverse processes such as hormones signal transduction, phytochrome signaling, shoot meristem maintenance and axillary meristem development, and response to biotic and abiotic stress (Bolle, 2004; Li et al., 2018). Few GRAS TFs modulate leaf senescence (Chen et al., 2015; Li et al., 2018). Overexpression of the BrLAS, a GRAS TF from Brassica rapa resulted in delayed leaf senescence, bolting, flowering time, decreased fertility, and enhanced drought tolerance in transgenic A. thaliana (Li et al., 2018). Transgenic plants also displayed decreased accumulation of ROS, increased antioxidant enzyme activity and increased sensitivity to exogenous ABA. The expression of several stress response genes and SAGs was altered in the transgenic lines. BrLAS is primarily expressed in roots and

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axillary meristems. BrLAS was exclusively localized in the nucleus and expressed primarily in the root and axillary meristem. BrLAS was upregulated by ABA, polyethylene glycol, salt, and H2O2. The virus-induced gene silencing-based silencing of TaSCL14, a member of the LISCL subfamily of GRAS, leads to early leaf senescence, decreased photosynthetic capacity, and reduced resistance to photooxidative stress in Triticum aestivum (Chen et al., 2015).

5.3.18 CCAAT box-binding TFs CCAAT box-binding TFs are conserved among eukaryotes and are named CCAAT-binding factor in mammals, heme activator protein (HAP) in Saccharomyces cerevisiae, and nuclear factor Y (NF-Y) in plants (Mantovani, 1999; Zhao et al., 2017). NF-Y TFs are heterotrimeric complex consisting of NF-YA, NF-YB, and NF-YC subunits (Mantovani, 1999). In A. thaliana, overexpressing AtNFYA2, AtNFYA7, and AtNFYA10 resulted in dwarf late-senescent plants with enhanced tolerance to flooding, nitrogen starvation, freezing, and heat (Leyva-Gonz´alez et al., 2012). Concomitant with delay in dark-induced, nitrogen starvation-induced, and developmental senescence; several SAGs, including POLYUBIQUITIN 10, SALT TOLERANT ZINC FINGER, SENESCENCE 1, and AtWRKY6, were repressed in NF-YA overexpressing plants. Genes encoding for enzymes involved in carbon metabolism and cell wall modification processes were also strongly downregulated, which was in agreement with the dwarf phenotype of the NF-YA transgenic lines (LeyvaGonz´alez et al., 2012). NF-YA was suggested to act as a negative regulator of early stress response genes and control plant growth by modifying carbohydrate metabolism and cell elongation (LeyvaGonz´alez et al., 2012). Previously, transcriptome analysis showed differential regulation of several NF-Y genes during senescence in A. thaliana (Breeze et al., 2011).

5.3.19 Heat shock factor TFs Heat shock factor TFs (HSFs) mainly function as positive regulators for plant tolerance to heat stress (Schramm et al., 2006, 2008). However, reports of their involvement in leaf senescence exist (Almoguera et al., 2015; Fan et al., 2019). Helianthus annuus HSFA9 (HaHSFA9) was shown to enhance tolerance to dark-induced senescence in transgenic tobacco seedlings and delayed the appearance of leaf senescence symptoms during the later stages of vegetative development (Almoguera et al., 2015). HaHSFA9 transgenic tobacco was also found to be tolerant to drastic dehydration and oxidative stress. Interestingly, the combined overexpression of HaHSFA9 and HaHSFA4a improved recovery of the photosynthetic organs of transgenic seedlings after lethal dark treatments; however, the combined overexpression of both HSFs did not lead to the further slowing of dark-induced senescence (Almoguera et al., 2015). Recent transcriptome analysis of dark-induced senescent leaves revealed the upregulation of several HSFs (Fan et al., 2019).

5.3.20 MADS TFs MADS-domain TFs are involved in many plant processes such as meristem specification, flowering transition, flower development, fruit ripening, and winter dormancy (Hoenicka et al., 2008; Xie et al., 2014a; Paul et al., 2014). Overexpressing a tomato MADS-box gene, SlFYFL, was shown to delay leaf and sepal senescence; in addition to slowing fruit ripening, increased storability, and

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longer sepals (Xie et al., 2014a). SlFYFL is an ortholog of A. thaliana FOREVER YOUNG FLOWER (FYF), which has a repressor function controlling floral organ senescence and abscission (Chen et al., 2011). Overexpressing SlFYF delayed leaf senescence in transgenic A. thaliana (Xie et al., 2014a). SlFYFL is expressed in all tissues of tomato with significantly higher expression in mature leaf, fruit of different stages, sepal, and the abscission zone. Ethylene biosynthetic genes such as AMINOCYCLOPROPANE-1-CARBOXYLICACID (ACC) OXIDASE 1, ACC SYNTHASE 1 A, ACC SYNTHASE 2, and ACC SYNTHASE 6 were downregulated significantly in the leaves and sepals of the transgenic plants. SlFYFL was suggested to repress the expression of ethylene biosynthesis genes, and decrease ethylene biosynthesis, thus delaying the senescence of leaf and sepal in the transgenic plant (Xie et al., 2014a). Heterologous overexpression of the birch FRUITFULL-like MADS-box gene BpMADS4 resulted in the prevention of leaf senescence and winter dormancy in Populus tremula (Hoenicka et al., 2008). Transgenic poplar plants maintained under autumn/winter growth conditions showed delayed leaf senescence and dormancy along with maintenance of photosynthetic activity, chlorophyll, and protein contents in leaves.

5.3.21 GOLDEN 2, ARR-B, PSR 1 (GARP) family TFs GOLDEN 2-LIKE (GLK) TFs are members of the GARP family of MYB TFs (Riechmann et al., 2000). GLK TFs, namely GOLDEN 2-LIKE 1 (GLK1) and GLK2, are key regulators of chloroplast development and maintenance in A. thaliana (Waters et al., 2009; Chen et al., 2016). GLKs regulate the expression of photosynthesis-related light-harvesting chlorophyll a/b-binding (LHC) protein genes LHCB2.4, LHCB3, LHCA4, and LHCB4.2 (Waters et al., 2009). Studies have shown that both photosynthesis and chlorophyll content decline in senescent leaves (Vercruyssen et al., 2015; Woo et al., 2019). Overexpressing and knockout mutants revealed GLKs participation in the regulation of leaf senescence (Rauf et al., 2013). Compared with wild type, 35 S:GLK1 and 35 S:GLK2 overexpressing plants showed delayed senescence, while loss-of-function glk1 and glk2 single mutants did not show a significant difference in their senescence behavior (Rauf et al., 2013). It was identified that AtATAF1 regulates leaf senescence by activating the expression of the positive senescence regulator NAC protein ORE1 and by repressing expression of the chloroplast maintenance TF GLK1 (Garapati et al., 2015). Rauf et al. (2013) also showed that ORE1 could interact at the protein level with GLK1 and GLK2 and form GLK1-ORE1 heteromer to repress expression of downstream GLKs target genes (Rauf et al., 2013). The repression of the GLKs transcriptional activity and chloroplast maintenance activity was suggested to initiate the senescence process.

5.3.22 TRIHELIX TFs TRIHELIX TFs have a highly conserved trihelix (helix-loop-helix-loop-helix) domain that binds specifically to GT elements in the promoter regions of target genes (Zhou, 1999; Nagano et al., 2001). TRIHELIX TFs are also termed GT factors. Triticum aestivum GT factor TaGT2L1D was shown to negatively regulate drought tolerance, affect floral organ development and decrease rosette leaf size. Ectopic expression of TaGT2L1D also exhibited slightly early leaf senescence in A. thaliana. Functional analyses demonstrated that TaGT2L1D acts as a transcriptional repressor to suppress the expression of STOMATAL DENSITY

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AND DISTRIBUTION1 (SDD1) in Arabidopsis by binding directly to the GT3 box in its promoter that negatively regulates drought tolerance (Zheng et al., 2016). Guo et al. (2017) identified 39 GT factor genes in Gossypium hirsutum through genome databases search. Eight GhGT genes were found to be senescence-related and were expressed during cotyledon and leaf senescence. Expression analysis also revealed their roles in the responses to diverse stresses and plant hormones. Interestingly, ectopic expression of senescence-related GhGT31 in A. thaliana promotes early flowering. It was concluded that early flowering is always accompanied by premature senescence and that GhGT31 might play an important role in cotton development.

5.3.23 Arabidopsis response regulator TFs ARABIDOPSIS RESPONSE REGULATOR (ARR) TFs function downstream in the cytokinin signaling pathway (Hwang and Sheen, 2001). Based on their structural designs, the ARRs are classified into type A (A-ARR) and type B (B-ARR). The A-ARRs have short C-termini lacking a DNA-binding domain, while the B-ARR are transcription activators with MYB-like domains for DNA binding and a glutamine-rich domain for positive activation of cytokinin-responsive transcription. The type-A ARRs are rapidly induced by cytokinin and function as negative regulators of cytokinin signaling to provide a negative feedback loop that dampens cytokinin response (Hwang and Sheen, 2001). Kim et al. (2006) reported that transgenic plants overexpressing wild type ARR2, a B-type ARR, showed delayed dark-induced and age-dependent leaf senescence. Cytokinin-induced phosphorylation on Asp-80 residue of ARR2 was required for its function. ARABIDOPSIS HISTIDINE KINASE 3, one of the three cytokinin receptors, mediates the cytokinin-induced phosphorylation of ARR2. Intriguingly, the arr2 knockout mutant plant did not show early senescence symptom, despite the arr2 mutants becoming less sensitive to inhibition of hypocotyl growth by cytokinins. Other B-type ARRs were suggested to complement the loss of ARR2 activity in the mutant plants or other leaf longevity pathways independent of ARR2 might exist (Kim et al., 2006). Interestingly, three type B ARRs, namely ARR1, ARR10, and ARR12 proteins were shown to interact directly with CRF6 (Cutcliffe et al., 2011), suggesting that CRFs and type B ARRs probably function together in regulating the transcriptional response to cytokinin during leaf senescence (Zwack and Rashotte, 2013). Although the type B ARR2 promotes leaf longevity by delaying senescence, the type A ARR5 was suggested to facilitate senescence by preventing the cytokinin ability to retard leaf senescence as and when required (Kudryakova et al., 2008; Ren et al., 2009). The type-A ARRs were shown to function as negative regulators of plant response to cytokinin (Hwang and Sheen, 2001). In a study involving transgenic A. thaliana plants transformed with the ARR5-gene promoter-GUS gene construct, the expression of the ARR5-gene promoter (measured in GUS activity) was reduced during natural and dark/detached leaf senescence. However, in the presence of exogenous cytokinin, the GUS activity was maintained during the senescence treatments and even enhanced with the increase in the age of the plants (Kudryakova et al., 2008). Similarly, A. thaliana overexpressing ARR16 displayed an early leaf senescence phenotype with reduced response to cytokinin under dark senescence-induced conditions (Ren et al., 2009). Under dark treatment, ARR16 overexpressing leaf has more rapid loss of chlorophylls compared to wild-type. When treated with cytokinin, ARR16 overexpressing leaf showed significantly reduced response to cytokinin whereas chlorophylls were

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retained in wild-type leaf (Ren et al., 2009). To et al. (2004) reported delayed dark-induced leaf senescence in the loss-of-function arr3, arr4, arr5, and arr6 mutants.

5.3.24 Lateral organ boundaries/asymmetric leaves 2 The LATERAL ORGAN BOUNDARIES (LOB) DOMAIN (LBD)/ASYMMETRIC LEAVES 2 (AS2) gene family members are characterized by their conserved LOB/AS2 domain with a homology region consisting of a leucine-zipper and zinc-finger motif (Katagiri and Nam-Hai, 1992; Albinsky et al., 2010). The LBD37 rice ortholog, OsLBD37/ASL39, is associated with nitrogen remobilization in senescent leaf (Albinsky et al., 2010). Overexpressing OsLBD37/ASL39 in A. thaliana and O. sativa leads to altered nitrogen metabolism, reduced growth, early flowering, and senescence. Metabolite profiling revealed significant accumulation of glutamine, glutamate, asparagine, and GABA in the OsLBD37/ASL39 overexpressing plants. Accumulation of ornithine and arginine, the metabolic products of glutamate, was also observed. Transcripts involved in nitrogen metabolism, namely GLUTAMINE SYNTHETASE, GLUTAMATE DEHYDROGENASE 2, and GLUTAMATE DECARBOXYLASE 1, were strongly upregulated, which was in agreement with the metabolite profiling result. Also, upregulated were genes involved in the phenylpropanoid pathway, lipid metabolism, pathogen responses, and senescence.

5.3.25 Early flowering 3 (ELF3) TF In A. thaliana, EARLY FLOWERING 3 (ELF3) gene plays a central role in controlling the circadian rhythm and photoperiodic flowering (Zagotta et al., 1996; Hicks et al., 2001). The C-terminal region of the 695 amino acid long ELF3 protein has a glutamine/threonine rich sequence containing a nuclear localization signal (Hicks et al., 2001). ELF3 was first identified in A. thaliana as a regulator of floral induction (Zagotta et al., 1996; Hicks et al., 2001). A. thaliana ELF3 also acts as a negative regulator of leaf senescence by repressing the expression of two senescence promoting phytochrome interacting protein genes, namely PIF4 and PIF5 (Sakuraba et al., 2014). The ELF3 overexpressing A. thaliana plants showed delayed senescence, while the loss-of-function elf3 mutants exhibited early senescence phenotype during dark-induced senescence. In contrast, the rice homolog OsELF3.1 positively regulates leaf senescence; loss-of-function oself3.1 mutant display delayed leaf senescence phenotype, whereas OsELF3.1 overexpression resulted in early senescent rice plants (Sakuraba et al., 2016). Several phytohormone-responsive genes (i.e., SAGs, NAC (OsNAP, ONAC106), and WRKY (OsWRKY42)) were differentially expressed in oself3.1 mutant compared to the wild type during leaf senescence. Interestingly, ectopic expression of OsELF3.1 in transgenic A. thaliana resulted in delayed leaf senescence. The results demonstrated that ELF3 and OsELF3.1 had conserved regulatory functions, but the downstream regulatory cascades have opposite effects between Arabidopsis and rice (Sakuraba et al., 2016).

5.3.26 Ethylene insensitive 3 (EIN3)-like (EIL) TFS ETHYLENE INSENSITIVE 3 (EIN3)/EIN3-like (EIL) are a small family of plant-specific TFs that form the core components of ethylene signaling (Dolgikh et al., 2019). There are six EIL genes, namely EIN3 and EIL1
5, in the A. thaliana genome (Chao et al., 1997; Guo and Ecker, 2004).

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EIL proteins have a conservative DNA-binding domain with a unique fold structure at the Nterminal region (Song et al., 2015). EIN3 and EIL act downstream of EIN2 in the ethylene response pathway (Chao et al., 1997; Guo and Ecker, 2004). EIN3 is a positive regulator of leaf senescence (Chao et al., 1997; Li et al., 2013). Knockout ein3 mutant has loss of ethylene-mediated effects, including gene expression, cell growth inhibition the triple response, and accelerated senescence (Chao et al., 1997). EIL1 and EIL2 can rescue the ein3 mutant (Chao et al., 1997). Overexpression of EIN3 or EIL1 results in constitutive ethylene responses (Chao et al., 1997), whereas ein3 eil1 double mutants show complete ethylene insensitivity and are considered the major regulators of ethylene signaling (reviewed in Guo and Ecker, 2004; Dolgikh et al., 2019). In A. thaliana, EIN3/EILs bind to a short DNA sequence in the target gene promoters referred to as EIN3 binding site (Dolgikh et al., 2019). EIN3 downstream direct targets include the positive regulator of leaf senescence such as ORE1 (Li et al., 2013; Rauf et al., 2013) and AtNAP (Guo and Gan, 2006; Kim et al., 2014). Interestingly, EIN3 represses the transcription of three miR164 precursor genes and is also involved in both positive and indirect regulation of the ORE1 gene (Li et al., 2013). ORE1 transcripts are targeted by the microRNA miR164 (Kim et al., 2009).

5.3.27 Brinsensitive 1 (BRI1)-EMS-Suppressor1 (BES1) TF Yin et al. (2002) first reported the gain-of-function mutant bes1 (bri1-EMS-suppressor1), which exhibited constitutive brassinosteroid (BR) response phenotypes that includes constitutive expression of BR-response genes, curly leaves, long and bending petioles and accelerated senescence. BES1 is a positive transcriptional regulator with an atypical bHLH DNA binding motif in the N-terminus and acts downstream of BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRASSINOSTEROIDsINSENSITIVE2 (BIN2) in BR signal transduction pathway (Yin et al., 2002, 2005; Upadhyay and Shumayla, 2022). BES1 interacts with BES1-interacting Myc-like 1, a bHLH protein to synergistically bind to E-box (CANNTG) sequences present in the promoter regions of BR-responsive genes (Yin et al., 2005). Using genome-wide protein-DNA interaction analyses and gene expression profiling, GLK1 and GLK2 were identified as direct target genes of BES1 (Yu et al., 2011). BES1 directly binds and represses the expression of GLK1 and GLK2 and functions to inhibit chloroplast development (Yu et al., 2011). The repression of GLK1 and GLK2 was linked to induction of leaf senescence in A. thaliana (Rauf et al., 2013). Interestingly, ABA and BRs antagonistically regulate senescence via BES1. BES1 was shown to antagonistically modulate ABA signaling (Zhao et al., 2019). The specific interaction of BES1 at the DNA binding bZIP domain of ABI5 drastically suppressed ABI5 binding affinity to the target genes promoter, resulting in reduced transcription of the downstream genes (Zhao et al., 2019). Conversely, the positive leaf senescence regulator ANAC072, belonging to the NAC TF family, negatively regulates BR-induced gene expression by directly binding to BES1 and antagonizing its transcriptional activities (Li et al., 2016; Ye et al., 2017).

5.3.28 Calmodulin-binding transcription activator CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR (CAMTA) binds to gene promoters that contain a CGCG box (Yang and Poovaiah, 2002). CAMTA is reported to be evolutionarily conserved among plants and animals (Finkler et al., 2007; Upadhyay, 2021). The CAMTA family

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was first reported in Nicotiana tabacum (NtER1) during the screening for Calmodulin-binding proteins (Yang and Poovaiah, 2000). NtER1 is an early ethylene upregulated CAMTA gene that was developmentally regulated and acts as a trigger for senescence and death in N. tabacum (Yang and Poovaiah, 2000). NtER1 exhibited significant upregulation in senescing leaves and petals and was also rapidly induced by the senescence promoting hormone, ethylene. Similarly, SIGNAL RESPONSIVE1 (SR1), a CAMTA, was also reported to modulate ethylene-induced senescence by directly regulating EIN3 (Nie et al., 2012). The loss-of-function sr1
1 mutant showed enhanced ethylene-induced senescence, but the gain-of-function sr1
4D mutant was insensitive to ethylene. ChIP assays showed that SR1 directly binds to the EIN3 promoter. Relative transcription of EIN3 in ethylene treated or untreated, sr1
4D, sr1
1, and wild-type plants revealed that EIN3 transcripts were higher in sr1
1 mutant but lower in sr1
4D than in the wild type. Thus SR1 directly binds to the EIN3 promoter and negatively regulates the EIN3 expression (Nie et al., 2012).

5.3.29 TIFY TFs TIFY is a large plant-specific TF family named after the conserved motif (TIF[F/Y]XG) (Vanholme et al., 2007). Depending on the domain composition, TIFY TFs are categorized into four subfamilies, namely TIFY, ZINC-FINGER PROTEIN EXPRESSED IN INFLORESCENCE MERISTEM (ZIM)-like protein (ZML), JASMONATE ZIM domain (JAZ), and PEAPOD (PPD) (Bai et al., 2011; Han and Luthe, 2021). Members of the TIFY, ZML, and JAZ subfamilies are reported in both monocot and dicot plants; however, the PPD subfamily is only present in dicots (Bai et al., 2011). Several members of the TIFY TF family, particularly the JAZs, were implicated in leaf senescence. JAZs are negative regulators of the JA signaling pathway and function via repressing transcriptional activities of downstream TFs (Staswick, 2008). In A. thaliana, JAZ8 and JAZ4 were found to competitively interact with AtWRKY57 for negative regulation of JA-induced leaf senescence (Jiang et al., 2014). AtWRKY57 functions as a node of convergence for JA- and auxinmediated signaling during JA-induced senescence (Jiang et al., 2014). Yu et al. (2016) found that A. thaliana JAZ7 to be involved in inhibiting dark-induced senescence. The knockout mutant of JAZ7 displayed accelerated senescence. The JAZ7 complemented and overexpression lines could rescue the mutant phenotype of dark-induced leaf senescence. Darkness could significantly induce JAZ7 gene expression and protein stability. JAZ7 regulates dark-induced leaf senescence by interacting with CORONATINE INSENSITIVE 1 (COI1; an F-box protein) and MYC2 (Yu et al., 2016). Studies have also demonstrated that COI1 and MYC2 regulate the JA-induced leaf senescence process in A. thaliana (Shan et al., 2010; Zhang et al., 2020a).

5.3.30 B-box zinc finger TFs The B-box (BBX) proteins are a class of zinc finger TFs containing one or more B-box domains; some members also harbor CONSTANS (CO), CO-like, and TOC1 (CCT) domains (Khanna et al., 2009). They act as transcriptional regulators within a number of networks controlling plant growth and developments that include photomorphogenesis of seedlings, flowering regulation by photoperiodic, shade avoidance, and responses to stresses (Gangappa and Botto, 2014). A. thaliana was reported to have 32 BBX genes (Khanna et al., 2009).

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An Oryza sativa CO-like gene, GRAIN NUMBER, PLANT HEIGHT, AND HEADING DATE 2 (GHD2), which positively influences yield, was also shown to regulate leaf senescence and drought resistance (Liu et al., 2016a). Overexpression of Ghd2 resulted in decreased drought tolerance accompanied by accelerated drought-induced leaf senescence in rice. Further, Ghd2 overexpressing rice exhibited earlier developmental and dark-induced leaf senescence, whereas the loss-of-function ghd2 mutant displays the opposite phenotype. The early senescence phenotype and the induction of several SAGs in GHD2 overexpressing transgenic plants suggested that Ghd2 acts as a positive regulator of leaf senescence in O. sativa (Liu et al., 2016a). In contrast, the Chrysanthemum morifolium BBX22 (CmBBX22), an ortholog of AtBBX22, was reported to negatively regulate leaf senescence (Liu et al., 2019). CmBBX22 was induced by ABA and dehydration stress. Ectopic overexpression of CmBBX22 in A. thaliana improves drought tolerance, while the ABA-induced leaf senescence was attenuated. Several SAGs and chlorophyll catabolic genes, namely NYC1, NYE1, NYE2, and SAG29, were repressed in the CmBBX22 overexpressing plants. Based on the findings, CmBBX22 serves as a positive regulator of drought tolerance and negatively modulates leaf senescence (Liu et al., 2019). Recently, a Malus domestica B-box protein MdBBX22 was shown to delay leaf senescence by repressing the transcriptional activity of MdABI5 protein and reducing the transcriptional induction of the MdABI5 gene (An et al., 2021). Direct binding of MdBBX22 with MdABI5 inhibits the transcriptional activation activity of MdABI5 on the chlorophyll catabolic genes MdNYC1 and MdNYE1, resulting in decreased chlorophyll degradation and delayed leaf senescence. MdBBX22 can also interact with MdHY5 to prevent the binding of MdHY5 protein on promoter regions of the MdABI5 gene, which then represses the transcription of MdABI5 (An et al., 2021).

5.4 Conclusion Our understanding of the underlying mechanisms that governs leaf senescence has seen tremendous progress in the last two decades. This has been accomplished through excellent genetic—both forward and reverse—and omics-based technologies studies (Guo et al., 2021b), most of which were covered in this chapter. But this knowledge represents just a fraction of what remains to be discovered about leaf senescence and its regulation. Members of the NAC and WRKY TF families are central to the regulation of leaf senescence. However, the involvement of other TFs is now emerging and the interconnected regulatory network(s) that might exist among them need(s) to be unraveled. These TFs, including NAC and WRKY, appear to act in conjunction with certain plant hormones such as JA, SA, ABA, ethylene, and GA3. The specific roles that these hormones play in regulating the initiation and progression of leaf senescence are now well-established. Still, their joint coordination—if it exists—to influence leaf senescence remains to be elucidated. Further improvement in understanding this complex and biologically significant process can be achieved through the availability of genome sequences of several plant species, especially leafy vegetable crops and perennial trees.

Acknowledgments JD acknowledges Dr. Sanjay Kumar, Director, CSIR-IHBT, Palampur, for providing the necessary facilities needed to prepare this book chapter. JD is grateful to the Council of Scientific & Industrial Research (CSIR), Government of India (MLP 202) and Science and Engineering Research Board (SERB), Government of India

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[(SRG/2020/001139) (GAP 273)] for the financial support. AP acknowledges Department of Botany, Nagaland University, Lumami for support. This manuscript represents CSIR-IHBT communication no. 4964.

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CHAPTER

Plant transcription factors in lightregulated development and UV-B protection

6

Deeksha Singh, Nevedha Ravindran, Nikhil Job, Puthan Valappil Rahul, Lavanya Bhagavatula and Sourav Datta Plant Cell and Developmental Biology Laboratory, Department of Biological Sciences, IISER Bhopal, Bhauri, Madhya Pradesh, India

6.1 Introduction The transcriptional regulation in plants governs several morphological and physiological changes in response to endogenous signals as well as environmental cues. Transcription factors (TFs) bind to cis-elements in the promoter region and regulate the target gene expression, either by acting as an activator or a repressor (Franco-Zorrilla et al., 2014; Badis et al., 2009). Additionally, TFs can bind to other TFs, microproteins, or ncRNA to regulate gene expression during various developmental stages (Song et al., 2021a). These interactions can either enhance their ability to bind to the target promoter leading to transcriptional activation or inhibit the binding onto the promoter, leading to the deactivation of the gene expression (Ye et al., 2004). Many TFs have been identified using methodologies that involve genetic screening and homology-based detection. Based on the protein structure and affinity to bind identical DNA sequences, TFs are categorized into different families, where TFs belonging to the same family are often involved in regulating related phenomena (Jiang et al., 2021). Several studies in past years have helped determining the TFs’ binding sites via several in vitro and in vivo techniques (Harbison et al., 2004; Riechmann, 2000). It was reported that 5% of the Arabidopsis thaliana genome codes for more than 1500 TFs, classified into different families based on structural and domain arrangement. Each TF can bind to several DNA fragments and regulate different processes simultaneously (Chen and Schmid, 2010). Light is a major developmental cue for plants perceived by photoreceptors, including phytochromes, cryptochromes, phototropins, and UVR8 (Cashmore et al., 1999; Rizzini et al., 2011; Briggs and Olney, 2001). These photoreceptors respond to different wavelengths of light and transmit the perceived signals via signaling cascade to TFs, which further regulate the light-mediated responses via modulating the target gene expression. These light-mediated responses include seedling photomorphogenesis, seed germination, shade avoidance, and photoperiod responses. Many light-responsive TFs have been identified based on the gene expression analysis of light-induced plant samples and genetic analyses of mutants that are deficient in their response to light (Jiao et al., 2007).

Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00013-3 © 2023 Elsevier Inc. All rights reserved.

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Some light-responsive TFs respond to only one wavelength of light. In contrast, others react to a spectrum of wavelengths and likewise regulate developmental processes in the respective light conditions (Eckardt, 2007). Plants keep a check on the activity of these TFs by regulating their stability and turnover (Han et al., 2019; Kim et al., 2014; Ling et al., 2017). Plants have employed methods like posttranscriptional modifications, posttranslational modifications, and proteasomal degradation of the TFs, and feedback mechanisms to maintain the homeostasis in the developmental processes (Al-Sady et al., 2006; Xu, 2020; Catal´a et al., 2011).

6.1.1 Transcription factors families involved in light-regulated processes Light-responsive TFs play a vital role in transducing light signals from photoreceptors and modulating downstream gene expression. Fig. 6.1 shows the light and dark-induced TFs that belong to different TF families depending upon their structural variability.

6.1.1.1 Basic helix-loop-helix family The basic helix-loop-helix (bHLH) TFs constitute a large family of TF proteins found in most eukaryotes. They have been shared throughout the kingdoms, including plants, animals, and fungi. The highly conserved bHLH domain contains 60 amino acids (aa) and is present at the N-terminus of the protein. The DNA-binding capacity is attributed to the 15 aa long basic region at the

FIGURE 6.1 Model showing light and dark-dependent transcriptional regulation. Different wavelengths of light activate respective photoreceptors, which in turn induce a set of transcription factors by degrading and destabilizing COP1. In nucleus, these TFs bind to the promoter elements of downstream genes and regulate their transcription. Light-responsive TFs transcribe light-responsive genes that mediate light-mediated development. In dark, COP1 degrades light-responsive TFs. Dark-induced TFs (Zn finger and bHLH) activate dark-induced genes, which triggers dark-induced development. Arrow mark represents localization and different shapes with colors represent TFs playing roles in light signaling. The figure was made using Biorender.

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N-terminus. bHLH TFs can bind specifically to genomic sequences containing G-box, E box, and ACE elements (Chen et al., 2020; Qian et al., 2021). The best-studied and characterized light-regulated bHLH proteinare PIFs (phytochrome interacting factors). PIFs modulate various light-dependent processes like chlorophyll biosynthesis, hypocotyl elongation, temperature and shade responses, and stomatal dynamics. In Arabidopsis, eight PIFs were identified based on a conserved bHLH domain: PIF1, PIF2, PIF3, PIF4, PIF5, PIF6, PIF7, and PIF8 (Job et al., 2018; Pham et al., 2018; Oh et al., 2020). These proteins are lightregulated and directly interact with phytochromes (PHYA, B, C, D, E, in Arabidopsis). PIF1 primarily regulates the PHYB dependent seed germination, whereas PIF3, PIF4, and PIF5 negatively regulate PHYB mediated signaling under red light. Another bHLH protein, HFR1 (long hypocotyl in far-red), is an essential factor in the PHYA signaling under far-red light. HFR1 does not physically interact with PHYA or PHYB, but it makes heterodimer with PIF3 and regulates phytochrome signaling (Zhu et al., 2000). HFR1 is also involved in CRY1 mediated light signaling. Also, MYC2, another bHLH TF regulates interactions between jasmonic acid signaling and phytochrome signaling, and the circadian rhythm. It is involved in shade avoidance response downstream to PHYA (Kazan and Manners, 2013; Robson et al., 2010).

6.1.1.2 Basic leucine zipper family The basic leucine zipper domain-containing (bZIP) TFs are vital for various processes in plants, including light signaling. The basic region in the bZIP domain binds to the DNA and has the nuclear localization signals, whereas the leucine zipper region forms a dimer with other proteins. bZIP TFs specifically recognize G-box, A-box, and C-box cis-acting DNA motifs in the genome. There are about 75 bZIP proteins annotated in Arabidopsis. The genome-wide analysis identified bZIP proteins in rice (89), soybean (160), barely (89), maize (125), tomato (69), and grapevine (55) (Agarwal et al., 2019; Zhang et al., 2018; Li et al., 2015). Functional characterization of bZIP factors unravels their role in various aspects of plant growth and development including, flowering, photomorphogenesis, seed germination, embryogenesis, abiotic and biotic stress responses. Elongated hypocotyl 5 (HY5) and its homolog HYH are the well-studied bZIP TFs in the light signaling pathway (Burko et al., 2020; Gangappa and Botto, 2016). In dark, constitutively photomorphogenic 1 (COP1) act as E3 ubiquitin ligase and ubiquitinates HY5 and HYH for proteasomal degradation (Osterlund et al., 2000a; Oyama et al., 1997). Light positively induces HY5 and HYH, and stabilizes their nuclear localization by cytoplasmic translocation of COP1. HY5 is known to regulate 1/3 of Arabidopsis genes by directly targeting their promoters (Oyama et al., 1997). The protein does not hold the property of transcriptional activation, and thus, with the help of other cofactor proteins such as BBX21 and BBX22, induce the gene expression (Datta et al., 2008).

6.1.1.3 MYB family MYB proteins constitute one of the largest TF families in plants. MYB proteins have been identified in various eukaryotes, including plants and animals (Ambawat et al., 2013). MYB factors related to the c-MYB are regulators of cell cycle progression in multiple kingdoms. The first plant MYB protein COLORED1 (C1) was isolated from maize and is involved in the biosynthesis of anthocyanins. In Arabidopsis, there have been 197 MYB proteins identified so far (Dubos et al., 2010). The highly conserved MYB domain contains up to four imperfect amino acid sequence repeats of about 52 aa long. MYB domain contains a helix-turn-helix structure that binds to the

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major groove of DNA helix. MYB proteins are subdivided into four classes according to the presence of repeats, referred to as 1R-, R2R3-, 3R-, and 4R-MYB. Most MYB proteins regulate secondary metabolite biosynthesis, abiotic stress responses, vegetative state transitions, light responses, and circadian regulation. CCA1 and LHY are well-studied MYB-related proteins that mainly regulate the circadian clock in plants. Another MYB transcription factor, LAF1, is involved in far-red light signaling. LAF1, an R2R3
MYB, acts via PHYA-dependent transcriptional modulation under shade (Ballesteros et al., 2001). This helps explain the vast developmental processes that are being carried out by the MYB family of TFs (Katiyar et al., 2012).

6.1.1.4 Zinc finger proteins The zinc-finger or Zn-ligating TF family is one of the most prominent TF families present in eukaryotes. Zn finger TFs are classified into nine subclasses based on the arrangement of Cys and His residues in their secondary structure, such as Cys2/His2-type (C2H2), C3H, C3HC4, C2HC5, C4HC3, C2HC, C4, C6, and C8 (Miller et al., 1985). Many Zn-ligating TFs have been identified in rice and Arabidopsis. Studies have identified a class of Zn-finger TFs, named as BBX proteins, that are responsible for various light-mediated responses in plants (Gangappa and Botto, 2014). BBX proteins possess a conserved B-box domain at the N-terminal and sometimes a CCT (CONSTANS, CO-like, and TOC1) domain at the C-terminus. The B-box domain comprises 40 aa residues rich in Cys and His (Gangappa and Botto, 2014). There are 32 BBX proteins identified in Arabidopsis classified into five structural groups based on structural domains (Gangappa and Botto, 2014). The B-box domain forms a homo/heterodimer with various factors, including the HY5-PIF transcription module. The first protein identified in the family was BBX1/CO, a master regulator of flowering. BBX proteins play a variety of roles in regulating flowering, seedling development, abiotic stress response, shade avoidance response, etc. Recent evidence suggests that they act as the cofactors to fine-tune the HY5 transcriptional activity on target promoters in photomorphogenesis (Bursch et al., 2020).

6.1.1.5 WRKY The 60 aa long WRKY domain-containing TFs are the most commonly studied TFs in plants (Chen et al., 2019). The WRKYGQK sequence at the N-terminus followed by a novel Zn finger-like motif at the C terminus binds to the W-box or other cis-acting elements. WRKY TFs are well characterized for their role in biotic and abiotic stress responses, trichome development, anthocyanin biosynthesis, and hormone signaling. Recently, WRKY36 was described as a novel UV-B responsive factor that directly interacts with the UVR8 photoreceptor and modulates HY5 transcription under UV-B (Yang et al., 2018).

6.1.1.6 TCP In Arabidopsis, there are 24 TCP (TEOSINTE-LIKE1, CYCLOIDEA, and PROLIFERATING CELL FACTOR) proteins subdivided into class 1 and class 2 based on structural similarities (Li, 2015). The TCP domain constitutes 59 aa long noncanonical bHLH domain that functions as DNA intercalating, nuclear localizing, and protein
protein interaction domain. Initial characterization of higher-order mutants of tcp unravels their roles in leaf development, plant architecture, petal development, axillary growth regulation, and cellular differentiation (Cubas et al., 1999). TCP2 interacts with CRY1 to regulate the expression of HY5 and modulates photomorphogenesis (He et al., 2016).

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Another protein, TCP17, interacts with PIF4 and CRY1 and regulates thermoresponsive hypocotyl growth (Zhou et al., 2019). TCP4 and PIF3 antagonistically regulate the expression of SAUR genes to regulate cotyledon opening in Arabidopsis (Dong et al., 2019).

6.1.2 Transcription factors associated with visible light-mediated development in plants Different photoreceptors in plants perceive the light of varying wavelengths and transmit this signal to various downstream genes, which are a part of transcriptional regulatory networks that regulate plants’ response to different light-dependent cues by their activation or suppression. In Arabidopsis, almost 20% of genes are transcriptionally modulated by light (Ma et al., 2001; Tepperman et al., 2001; Jiao et al., 2005). The light-responsive TFs are identified by their LRE (light-responsive ciselements) binding ability (Jiao et al., 2007). Various TFs are characterized by the phenotypic analysis of their mutants in response to different wavelengths of light. At the same time, many of them are affected by a wide range of wavelengths, specifically, the visible range, which includes red, farred, and blue light (Franco-Zorrilla et al., 2014; Jiao et al., 2007; Jing and Lin, 2020). The microarray analysis using the monochromatic far-red and red light identified that 44% and 25% of the early light-responsive genes are TFs, respectively. Similarly, 64 early responsive TFs are identified in Arabidopsis under blue light (Riechmann et al., 2000; Jiao et al., 2003). Various developmental aspects of plants, starting from seed germination, hypocotyl elongation, floral transition, flowering, etc., are regulated by light. Photomorphogenesis is one of the wellknown processes regulated by light quantity and quality. ELONGATED HYPOCOTYL 5 (HY5), a bZIP transcription factor, is a light-inducible TF factor, which along with its homolog HYH positively regulates light-mediated development (Gangappa and Botto, 2016; Yadav et al., 2020a). PHYTOCHROME INTERACTING FACTORs (PIFs), the bHLH family of TFs, are G-box binding proteins and central positive regulators of skotomorphogenic genes (Chen et al., 2004). Under red light, the Pfr phytochrome is imported to nucleus and interacts with PIF3, which results in the phosphorylation and targeted degradation of PIF3 (Al-Sady et al., 2006; Bauer et al., 2004). PIF3 regulates the expression of other important circadian-related MYB TF genes like CCA1 and LHY (Martı´nez-Garcı´a et al., 2000). Many of the major TFs involved in light signaling need to be regulated to optimize plant development precisely. For example, HY5 is regulated posttranslationally by CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), an E3 ubiquitin ligase present in the cytoplasm. COP1 targets HY5 for degradation in the dark alone or by forming a complex with PHYTOCHROME A SUPPRESSOR 1 (SPA1) (Deng et al., 1992; Osterlund et al., 2000b; Kim et al., 2017; Xu, 2020). Far-red light perceived by PHYA further regulates the R2R3-MYB family of DNA-binding protein family genes like FAR-RED IMPAIRED RESPONSE 1 (FAR1) and FAR-RED ELONGATED HYPOCOTYL 3 (FHY3). These TFs are specific to far-red light and interact with each other (Hudson et al., 1999; Wang and Xing, 2002; Hudson et al., 2003). LONG AFTER FAR-RED LIGHT 1 (LAF1), a R2R3
MYB protein, is also involved in far-red light signal transduction (Ballesteros et al., 2001). LONG HYPOCOTYL IN FAR-RED (HFR1) gene, a bHLH transcription factor, plays a role in PHYA and cryptochrome downstream signaling (Jiao et al., 2007).

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Being light-inducible, different BBX proteins depending upon their structure are involved in several developmental processes. Arabidopsis Group IV BBX proteins (BBX18-BBX25) regulate photomorphogenesis in an HY5-dependent manner. BBX20, 21, 22, 23 act as transcriptional coactivators of HY5, while BBX24, BBX25, BBX28, BBX30, and BBX31 repress the HY5 transcriptional activity (Job et al., 2018; Datta et al., 2008; Yadav et al., 2020b; Bursch et al., 2020; Ravindran et al., 2021; Gangappa et al., 2013a,b; Xu et al., 2016; Zhang et al., 2017; Lin et al., 2018; Heng et al., 2019a). HY5 binds to the promoters of BBX30 and BBX31, and negatively regulates their transcripts levels, and promotes photomorphogenesis (Heng et al., 2019a). BBX32 acts as a negative regulator of photomorphogenesis (Holtan et al., 2011). BBX11 acts as a positive regulator of photomorphogenesis through positive feedback regulation of BBX21 and HY5 (Zhao et al., 2020; Job and Datta, 2021; Song et al., 2021b). All the above mentioned BBX proteins regulate photomorphogenesis in most visible lights, including white, red, far-red, and blue light. At the same time, BBX4 and BBX16 functions downstream of PHYB under only red light conditions to positively regulate photomorphogenesis (Heng et al., 2019b; Datta et al., 2006; Honggui et al., 2013; Zhang et al., 2014). HY5 binds to the promoter of MYB TFs, MYB12, MYB111, MYB75, that in turn interact with HY5. This complex binds to the promoter of early and late anthocyanin biosynthesis genes in a light-dependent manner (Gangappa and Botto, 2016). Myc TFs (bHLH), MYC2, and MYC3 were earlier known to act in the JA-pathway. Recent reports suggest that MYCs regulate the HY5 transcription by binding to its promoter and promote photomorphogenesis (Ortigosa et al., 2020), which is in contradiction to a previous study that reported AtMYC2 acts as a negative regulator of HY5 in a blue light-dependent manner (Yadav et al., 2005; Chakraborty et al., 2019). Nuclear factor Y (NFY) TFs also regulate photomorphogenesis and play a role in hypocotyl elongation. PIF4 is known to interact with NF-YB9 to regulate the expression of IAA19 and thus regulate hypocotyl elongation in the dark. NF-YC subunits like C1, C3, C4, C6, and C9 are also known to positively regulate the photomorphogenesis by interacting with HISTONE DEACETYLASE 15 (HDA15) (Myers et al., 2016; Tang et al., 2017; Zhao et al., 2017). NFYs are known to regulate various light-dependent developmental processes like flowering (Zhao et al., 2017). Group G G-box binding factors (GBF) in Arabidopsis and its homologs in parsley like CPRF1, 3, 4, and 5 are involved in blue light and UV mediated plant development by binding to the promoters of light-responsive genes (Jakoby et al., 2002). COMMON PLANT REGULATORY FACTOR 1 (CPRF1) is light-inducible and positively regulates photomorphogenesis. It is known to repress its transcription by binding to itself. G-BOX BINDING FACTOR 1 (GBF1) until phosphorylated lacks affinity to attach to the G-box. CPRF2 gets phosphorylated under red light, and is further localized to nucleus where it binds to G-box elements to positively regulate photomorphogenesis (Jiao et al., 2007). Several other proteins and TFs are also known to regulate HY5 at its transcriptional and translational levels. WRKY36 binds to the promoter of HY5 and represses its transcription under white light conditions to negatively regulate photomorphogenesis (Yang et al., 2018). CALMODULIN 7 (CAM7) is known to interact with HY5. CAM7, along with HY5, regulates the HY5 expression by binding to the E-box and T/G-box cis-acting elements to promote photomorphogenesis (Xu, 2020; Gangappa and Botto, 2016; Abbas et al., 2014; Kushwaha et al., 2008). The GATA TFs regulate the expression of light-regulated and circadian clock-related genes having GATA motifs in their promoter (Behringer and Schwechheimer, 2015).

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6.1.3 Transcriptional regulation of UV-B signaling in plants Solar radiation consists of 7% of UV radiation. Ozone depletion in recent decades has led to the increased exposure to UV radiation, which is further categorized into UV-A (315
400 nm), UV-B (280
315 nm), UV-C (200
280 nm) (Jenkins, 2009; Kerr and Fioletov, 2008). UV-C is blocked by the atmospheric ozone layer from entering the earth’s surface, while UV-A and UV-B penetrate the ozone and enter the earth’s surface. While low fluence of UV-B is perceived as a developmental signal, high-intensity UV-B is detrimental and causes DNA damage and ROS production (Culligan et al., 2004). UV-B regulates various processes like hypocotyl elongation inhibition, cotyledon opening, flavonoid accumulation, stomatal dynamics, and rosette growth (Yadav et al., 2020a).

6.1.3.1 UV-B signaling and photomorphogenesis UV-B radiation in plants is perceived by UVR8 photoreceptor, which remains as an inactive dimer in the absence of UV-B. On UV-B exposure the inactive dimer is converted to active monomer, which further binds to COP1. This interaction eventually localizes UVR8 into the nucleus, where it regulates the UV-B responsive TFs and their responses. In Arabidopsis, HY5 and HYH are the essential leucine-zipper TFs that act downstream to UVR8. UV-B exposure not only induces the expression of HY5 and HYH but also stabilizes HY5 protein. COP1 degrades HY5 in the dark, but the interaction of UVR8 with COP1 leads to the sequestration of COP1, which promotes the stabilization of HY5 in the nucleus. HY5 binds to the T/G-box cis-acting element of the HY5 promoter and mediates the transcriptional activation of HY5 in response to UV-B (Binkert et al., 2014). Furthermore, HY5, along with UVR8, binds to the UVB target genes and regulates cell elongation and flavonoid production, which governs photomorphogenesis and stress tolerance. For example, HY5 and UVR8 promote the transcription of PFG-MYB, which along with HY5, regulate the expression of CHALCONE SYNTHASE (CHS), a flavonoid biosynthesis gene (Tilbrook et al., 2013). Transcription factors like WRYKY36 and BBX24 negatively regulate the transcriptional activity of HY5 and hence repress its function; contrastingly, BBX31/mip1a positively regulates UV-B signaling and tolerance (Yang et al., 2018; Jiang et al., 2012; Yadav et al., 2019). WRYKY36 binds to the W-box region on the promoter of HY5 and halts its transcription. UV-B activated UVR8 interacts with WRYKY36, and inhibits the binding ability of WRYKY36 onto the promoter of HY5, subsequently leading to HY5 activation (Yang et al., 2018). Many TFs bind with HY5 and regulate its activity positively or negatively in different light conditions. Evidence shows that UV-B radiation upregulates a B-BOX transcription factor STO/BBX24 that binds to both COP1 and HY5. BBX24 negatively regulates the UV-B responsive gene expression by interacting with HY5 and inhibits its binding to its target promoters (Jiang et al., 2012). Yadav et al. (2019) reported that BBX31 in UV-B signaling acts by increasing the accumulation of UV-B protective compounds and flavonoids, and hence positively regulates UV-B signaling and tolerance. Liang et al. (2020) showed that brassinosteroid responsive TFs regulate photomorphogenesis and stress tolerance by interacting with UV-B signaling components, which furthers shows possible crosstalk between the two pathways. BIM1 and BES1 cordially synchronize BR response, hypocotyl elongation, and negatively regulate flavonoid biosynthesis by binding the promoter of PFG-MYBs and inhibiting their expression (Yin et al., 2005). UVR8 interacts with BES1 and inhibits its binding on the promoter

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of plant growth-promoting genes, thereby stopping BR-induced hypocotyl elongation (Liang et al., 2018). Additionally, TF MYB13 regulates auxin response and flavonoid biosynthesis (Qian et al., 2020). The mode of action of MYB13 is UVR8 dependent, unlike that of BES1. UVR8 physically interacts with MYB13 in a UV-B-dependent manner and orchestrates the transcription of a specific set of genes, and positively regulates the UV-B response (Qian et al., 2020).

6.1.3.2 UV stress response in plants Apart from UVR8 mediated UV-B signaling, various other TFs and cell cycle components regulate UV stress tolerance. UV stress distorts the genome integrity by forming pyrimidine dimers and double-strand DNA breaks (Rastogi et al., 2010). To protect genome integrity, plants have SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) TF that activates transcriptional response for cell cycle arrest and programmed cell death (Yoshiyama et al., 2014). Additionally, MAP kinase cascade is induced by UV-B in a UVR8 independent manner, to give tolerance to plants (Ulm et al., 2001; Ulm et al., 2002; Gonz´alez Besteiro and Ulm, 2013). mkp1 mutants are hypersensitive, while mpk3 and mpk6 mutants are tolerant to UV-B stress. MPK1 being a positive regulator of UVB tolerance inactivates MPK3 and MPK6, and hence promotes plant recovery after UV-B stress (Besteiro et al., 2011). The TFs reported in UV-B signaling are also involved in regulating abiotic stress pathways, which suggest the possible crosstalk in the functioning of photoreceptors.

6.1.4 Structural and functional evolution of light-responsive plant transcription factors Being sessile, plants have evolved sophisticated light-regulated transcriptional networks to finetune the developmental changes. Light perceived by the photoreceptors of a developing seedling leads to massive reprogramming in its transcriptome. The light-responsive TFs further act on a range of light-responsive elements (LRE) present in different gene promoters to regulate the expression of target genes positively or negatively (Jiao et al., 2007). During the evolution from aquatic ancestors, land plants have evolved with a wide range of sensory systems that perceive and transduce specific light signals to face the challenges posed by the terrestrial environment (Fern´andez et al., 2016; Li and Mathews, 2016). The aquatic ancestors lived in deep seawater to protect its macromolecules from UV-B radiations (Littler et al., 1985; Henriques et al., 2012; Robson et al., 2019). Researchers have found out that the earliest red algae reside in deep seawater and use phycobilisomes as antenna proteins to harvest light energy. The UV-B photoreceptor, UVR8, might have originated in the chlorophytes, the Viridiplantae groups present in shallow water (Tilbrook et al., 2013; Di et al., 2012). There have been 16 UVR8 orthologs identified in 13 genomes from chlorophytes to angiosperms. All the UVR8 orthologs have W233 and W285 tryptophans and a conserved C27 domain. The earliest diverging chlorophyte Chlamydomonas reinhardtii has the UVR8 ortholog but gymnosperms seem to have lost it (Fern´andez et al., 2016). The identification of orthologs of COP1, SPA, and HY5, the molecular components in the Arabidopsis UVR8 signaling pathway in few chlorophytes, indicates the presence of UVR8 signaling module in the common ancestor of green plants.

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Like Arabidopsis UVR8, chlamydomonas ortholog also monomerizes upon UV-B exposure and interacts with chlamydomonas COP1 (Tilbrook et al., 2016). The expression level of chlamydomonas HY5 is also found to be upregulated upon UV-B radiation (Tilbrook et al., 2016). The land plants absorb red and far-red wavelengths (600
750 nm) of light using photoreceptors’ phytochromes. According to their light stability, the five phytochromes (Phy A, B, C, D and E) of Arabidopsis are classified into two groups (Sharrock and Quail, 1989)
PhyA being lightlabile and others being light-stable. The initial challenge for a land plant in the terrestrial environment during its emergence is the developmental transition from darkness to light (Shi et al., 2016). The signaling pathways of light during dark to light transition in Arabidopsis involve three major light-responsive TFs: HY5/HYH, EIN3 (ETHYLENE INSENSITIVE 3), and PIFs (Shi et al., 2018). A recent transcriptomic analysis of streptophyte algae shows that charophytes have some canonical phytochromes and other regulatory genes that are associated with phytochrome-mediated signaling, further indicating that some phytochrome regulatory modules might have originated before plant terrestrialization (De Vries et al., 2018). There are no homologous phytochrome sequences found in the rhodophyte and chlorophyte genomes (Han et al., 2019). Phylogenetic analyses of phytochrome signaling components FHY1 and PIFs indicate that they have originated in charophytes (Possart et al., 2017; Inoue et al., 2017). Particularly, the presence of SPA, HY5, PIF, and PHY genes simultaneously in the transcriptomes of Cylindrocystis cushleckae and Coleochaete irregularis suggests the charophyte origin of the phytochrome signaling mechanism (Han et al., 2019). The vigorous protrusion of caulonema cells from the protonema networks of Physcomitrella. patens was severely affected in a loss-of-function Δhy5a Δhy5b double mutant both under light and dark conditions. This suggests that the function of HY5-homologs in P. patens is evolutionarily conserved to play the role in protonema-caulonema development (Yamawaki et al., 2011). All the orthologs of FHY1 identified in the 19 genomes, including two orthologs from Chara braunii (belonging to Charophyceae), possess a conserved PhyA binding site at the C terminus. But the positive regulator of FHY1, the FHY3, was inferred to originate in bryophytes. Thus studies suggest that the FHY1-mediated nuclear import of phytochrome might have originated in the charophytes, and the involvement of FHY3 in the feedback loop originated in land plants (Han et al., 2019). The PIF proteins belong to the fifteen-membered subfamily 15 of the Arabidopsis bHLH superfamily. All PIFs in Arabidopsis possess an active phytochrome B binding (APB) site, while PIF1 and PIF3 have an additional active phytochrome A binding (APA) domain (Leivar and Quail, 2011). This codivergence of phytochromes and PIFs might have led to the evolution of shade avoidance response of seed plants under complicated R/FR conditions caused by the taller plant canopy. Among the fifteen members in the PIF family, the remaining non-PIF subfamily members, LONG HYPOCOTYL IN FAR-RED 1 (HFR1) and PHYTOCHROME INTERACTING FACTOR 3-LIKE 1 (PIL1) have photomorphogenesis-related functions, and SPATULA (SPT) functions in regulating seed germination. There are many more members in the family that are yet to be characterized (Leivar and Quail, 2011). EIN3 is a master TF of the ethylene signaling pathway in Arabidopsis that positively regulates ethylene responses. EIN3 is targeted for degradation by EBF that is stabilized by light. A previous study identified the origin of EIN3 in charophytes and demonstrated that the Spirogyra (belonging to Charophyta) EIN3 homolog complements Arabidopsis EIN3 in the ethylene signaling pathway (Van De Poel et al., 2016). None of the EIN3 orthologs were found in chlorophytes or rhodophytes.

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EIN3/EIL1 has a DNA-binding domain (DBD), a V-shaped cleft formed by five α-helices (Chao et al., 1997). This conserved DBD is also found in all EIN3 homologs in other plant species (Kosugi and Ohashi, 2000; Hiraga et al., 2009). The HY5/PIFs/EIN3 TFs are present in Klebsormidium nitens (belonging to Klebsormidiophyceae) and Chara braunii (belonging to Charophyceae) genomes (Han et al., 2019). Since, HY5 was present before charaophytes, all these TFs might have evolved first in charophytes. EBF1 and EBF2 target EIN3 for ubiquitination and all the orthologs of EBF1 and EBF2 coexpress with EIN3 in charophytes (Han et al., 2019). B-BOX proteins are another major group of TFs that are involved in light-dependent development of plants. The 32 membered family in Arabidopsis emerged with the identification of the first B-box protein, CONSTANS (CO). Apart from B-Box domains at the N-terminal, the proteins may possess CCT domain (CO (CONSTANS), COL (CONSTANS-LIKE), and TOC1 (TIME OF CAB1)) at the C terminus. Based on sequence similarity and the domains they contain, B-Box proteins are categorized into five groups. Group I and II have 2 B-Boxes and a CCT domain. Group III has one B-Box and a CCT domain. Group IV and Group V B-Box proteins lack CCT domain and contain 2 and 1 B-Box domains, respectively (Khanna et al., 2009). Furthermore, a phylogenetic study with 214 BBX proteins belonging to 12 plant species from green algae to dicots showed that the B-box consensus sequences of each structural group retained a common and conserved domain topology. The B-Box motifs of 120 bp are thought to be originated from segmental duplication and internal deletion events (Crocco and Botto, 2013). They are found to be important to mediate protein
protein interaction and transcription regulation (Datta et al., 2008; Gangappa et al., 2013b; Datta et al., 2007; Qi et al., 2012). The CCT domain was found to be highly conserved and involved in transcriptional regulation and nuclear protein transport (Crocco and Botto, 2013; Laubinger et al., 2006; Jang et al., 2008; Gendron et al., 2012). In Arabidopsis, 17 of the 32 BBX proteins (BBX1
17) and in rice, 17 of the 30 BBX proteins contain the CCT domain (Griffiths et al., 2003; Huang et al., 2012). The group I BBox proteins, BBX24 and BBX25, possess VP (Valine-Proline) motif that drives the interaction between BBX and COP1, a ubiquitin ligase E3 protein that targets BBX for degradation (Datta et al., 2006; Holm et al., 2001). Even though the amino acid sequence conservation constrained the evolution of BBX proteins in the two B-boxes, it has radiated variation into nuclear localization signal (NLS), VP, and other novel motifs (Crocco and Botto, 2013; Kim et al., 2013). Even though most green algae possess a single B-box motif, the presence of two B-box motifs in the unicellular green alga Chlamydomonas suggests that the duplication of B-box might have taken place much before land colonization of plants, about 450 million years ago, during the Silurian period (Crocco and Botto, 2013). The rapid expansion of the members in the BBX family during evolution, and the high level of conservation across the plant species, suggest that BBX proteins might have played important roles in the adaptation of land plants.

6.1.5 Role of light-regulated transcription factors in other signaling pathways HY5, a positive regulator of light signaling, is also positively regulated by cold temperatures at both transcriptional and posttranslational levels (Catal´a et al., 2011; Toledo-Ortiz et al., 2014). Cold temperature stabilizes HY5 protein by depleting COP1 in the nucleus (Catal´a et al., 2011). HY5 reduces the ROS accumulation to protect photosystems at low temperatures (Catal´a et al., 2011). In plants, both abiotic and biotic stresses trigger apoptosis or cell death. Red light accumulates ROS and promotes cell death during seedling de-etiolation (Gechev et al., 2006; Chen et al., 2013;

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Chai et al., 2015). In a red-light-dependent manner, HY5 promotes the expression of ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1). HY5 also regulates the expression of many WRKY genes that act in defense responses in a light-dependent manner (Lee et al., 2007; Rushton et al., 2010). Apart from stress responses, these TFs also regulate nutrient homeostasis in plants. HY5/HYH positively regulates the expression of two critical genes of nitrogen signaling: NITRATE REDUCTASE 2 (NIA2) encoding for nitrate reductase, which reduces nitrate to nitrite in the cytosol, and NITRITE REDUCTASE 1 (NIR1) encoding for nitrite reductase, which converts nitrate to ammonium (Jonassen et al., 2008; Yanagisawa, 2014; Huang et al., 2015). But HY5 negatively regulates the expression of nitrate uptake genes such as NITRATE TRANSPORTER 1.1 (NRT1.1) and AMMONIUM TRANSPORTER 1;2 (AMT1;2) (Yanagisawa, 2014; Qi et al., 2012). HY5 is involved in maintaining the carbon
nitrogen balance in Arabidopsis plants exposed to fluctuating light conditions. In addition, HY5 regulates sulfate assimilation in a light-dependent manner by inducing the expression of key enzyme ADENOSINE50-PHOSPHOSULFATE REDUCTASE 1 (APR1 and APR2), by directly binding to the promoters (Lee et al., 2011). UV-B spectrum acts as a signal in plants to interpret various environmental cues. In high ambient temperature, UV-B light suppresses the plant responses by UVR8 mediated inhibition of the PIF4 TF (Yin, 2017). Studies have shown that UV-B can mitigate the adverse effects of drought on photosynthetic rate, biomass accumulation, and leaf water content, where the molecular players mediating these responses are not known (Manetas et al., 1997; Poulson et al., 2006).

6.2 Conclusion Light is an integral part of environmental signals perceived by plants to grow, adapt, and survive. Plants being sessile need intricate machinery to sense and respond to various external stimuli; hence they have developed photoreceptors that can sense the light and mediate developmental responses. Downstream to the light-sensing machinery, a well-established network of TFs regulates the physiological responses in plants. Molecular and genomic studies have suggested the possible binding sites and target genes of these TFs. Insights into the structural diversity of TFs lead to their classification based on the presence and absence of domains and their arrangements. The lightresponsive TFs respond to different wavelengths of light by transcriptionally regulating lightresponsive genes. During the evolution from the aquatic plants, terrestrial plants have adapted and developed robust machinery to protect them from harsh conditions by evolving their sensory and signal transduction pathways. Studies are constantly revealing the existing crosstalk of various hormones and light signaling pathways to have a better understanding of mechanisms adapted by plants to develop and survive in their existing environment.

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CHAPTER

Tomato fruit development through the perspective of transcription factors

7

Vigyasa Singh1, Dharitree Phukan2 and Ujjal Jyoti Phukan3 1

Pharmacology and Toxicology Department, College of Pharmacy, University of Arizona, Tucson, AZ, United States 2 ICAR-National Institute for Plant Biotechnology, New Delhi, Delhi, India 3School of Plant Sciences, University of Arizona, Tucson, AZ, United States

7.1 Introduction Fleshy fruit tomato (Solanum lycopersicum) is an essential component of the human diet all around the globe and is cultivated extensively. The species originated in western South America and Central America, then domestication and cultivation occurred in Mexico followed by breeding and hybrid generation in other parts of the world (Bergougnoux, 2014; Nakayama et al., 2021). Botanically tomato is a fruit, a kind of berry since they are formed from ripened flower ovary, has seeds, and helps with the plant’s reproduction process (Liu et al., 2022). However, tomato is also loosely considered a “culinary vegetable” or as a salad because it has a much lower fructose content. Nutritionally it is rich in antioxidant lycopene (Madia et al., 2021; Upadhyay and Singh, 2021). It is also a rich source of vitamin C, potassium, folate, and vitamin K (Salehi et al., 2019). Tomato is widely used as a model plant because its genome is fully sequenced, various mutants are available, and it has a relatively short reproductive cycle (Jaiswal et al., 2020; Ruf and Bock, 2021). Tomato fruit goes through an elaborate synchronized developmental process that controls its ripening process through altering various metabolic pathways as shown in Fig. 7.1, which is of primary importance to farmers, consumers, and researchers. While a lot of information and knowledge is available, its production, yield, and quality have suffered due to various environmental factors. Farmers face huge financial losses due to biotic and abiotic factors (Pico´ et al., 1996; Rosell
o et al., 1996; Jian et al., 2021). It is apparent that understanding the genetic map is key to preventing these losses. Various reports have suggested regulatory mechanisms of pest resistance, pathogen resistance, abiotic stress resistance, plant development, reproductive development, fruit development, and fruit quality improvement in tomato (Thipyapong et al., 1997; Lashbrook et al., 1998; Vleghels et al., 2003; Sun et al., 2004; Alba et al., 2005; Carrari and Fernie, 2006; Park et al., 2007; AbuQamar et al., 2009; Maluf et al., 2010; Orellana et al., 2010; Chevalier et al., 2011; Kohlen et al., 2012; Zhong et al., 2013; Boureau et al., 2016; Gimenez et al., 2016; Bai et al., 2018; Gupta et al., 2020; Shi et al., 2021; Pan et al., 2021; Yang et al., 2021; Shang et al., 2021; Slugina et al., 2021; Upadhyay, 2021). However, it has been suggested that the regulatory mechanism is a huge interconnected network of various factors. Firstly it is regulated because of the genetic makeup and occurrence of characteristic singlenucleotide polymorphisms, then transcriptional and posttranscriptional machinery comes into action Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00011-X © 2023 Elsevier Inc. All rights reserved.

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Unripe Stage I – green

Ripe Stage II – yellow green

Stage III – yellow

Stage IV – orange

Stage V – red

Stage VI – dark red

FIGURE 7.1 Different stages of tomato fruit development and ripening. Tomato fruit development and ripening goes thorough six different stages. From green shoulder phenotype to yellow-orange to ripe red stage it undergoes extensive alterations in genetic behavior to pigment accumulation and metabolite storage.

(Phukan et al., 2016b, Phukan et al., 2017; Liu et al., 2020a,b). This is followed by translational and posttranslational modifications that govern particular function of a gene. Epigenetic modifications and genetic imprinting also regulates various aspects (Cao et al., 2021; Hu et al., 2021). Further study into this is required to understand the huge regulatory crosstalk operating simultaneously or successively in tomato. Therefore in this chapter we have tried to compile the work that has been done to understand the regulatory mechanism of tomato fruit development.

7.2 Transcription factors in tomato We will mainly focus on transcription factors (TFs) because TFs can activate the transcription of various downstream genes through recruiting RNA polymerase. TFs interact with the cis-elements present in the promoter of target genes through their DNA binding domain (DBD) and significantly activate or repress the expression of the target gene (Phukan et al., 2018). This interaction is important because it regulates when the gene is activated, which cells it is expressed and when to shut off. There are various groups of TFs that act in a coordinated manner to direct cell growth, cell division, cell death, cell migration, and cell organization (Babu et al., 2004). According to PlantTFDB and gene annotations from ITAG(v2.3), 1845 TFs (1845 loci) have been identified in S. lycopersicum that are classified into 58 families (http://planttfdb.gao-lab.org, Jin et al., 2017; Tian et al., 2020). Therefore it is evident that there is a huge pool of unexplored TFs that can regulate various factors associated with fruit development and ripening. Regardless of this, a lot of studies has been done in this field, and a comprehensive review of the same will shed some light on the process, and the same can be applied to explore unexplored answers in tomato fruit development and ripening. In this chapter, we will be mainly discussing MYB and MADS-box TFs.

7.3 MYB transcription factors

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7.3 MYB transcription factors The MYB (myeloblastosis) family TFs show large functional diversity and are found in both plants and animals. They have a characteristic MYB DBD consisting of up to four imperfect B52 amino acid sequence repeats (R), each forming three α-helices. They are designated as R1, R2, and R3 and depending on the number of these adjacent repeats MYB TFs are divided into different classes such as 4R-MYB, 3R-MYB, and R2R3-MYB. Most plant MYBs encode R2R3-type MYBs (Dubos et al., 2010). MYB TFs such as S. lycopersicum GOLDEN2-LIKE (SlGLK) from the GARP subfamily play an important role in tomato fruit ripening and development (Powell et al., 2012; Nguyen et al., 2014). It was observed that SlGLK acts in concert with chloroplast development and photosynthesis to regulate this process. Various fruits including tomato initially develop as green photosynthetic tissues (Blanke and Lenz, 1989; Gillaspy et al., 1993; Carrara et al., 2001). SlGLKs are responsible for the normal development of plastids (green coloration) and mutation in it removes the uneven color phenotype and gives uniform ripening (Waters et al., 2008; Powell et al., 2012). In this process, they alter the expression of various fruit photosynthesis and chloroplast development genes. It is common in tomato to show uneven ripening typically in a gradient across the latitudinal (top
bottom) axis of the fruit where SlGLK2 is expressed. The stem end of an unripe tomato remains green or yellowish (causes green shoulder because of higher expression of SlGLK2) while the ripe fruit shows orange, green, or white ripe regions at the top (Nguyen et al., 2014). Overexpression of SlGLK2 resulted in uniformly dark green immature tomato with significantly elevated chlorophyll levels, while cosuppression of SlGLKs mimicked the uniformly ripe mutant as shown in Fig. 7.2 (Powell et al., 2012). It is observed that SlGLK1 and SlGLK2 are functionally similar but expressed differentially leading to tissue-specific leaf phenotypes for SlGLK1 and fruit phenotypes for SlGLK2. SlGLK overexpression results in increased chlorophyll content, chloroplast number, and thylakoid grana stacks in green fruit. Higher sugar/starch content was also observed in the transgenic lines, along with increased content of vitamin C, carotenoids (β-carotene and lutein), and lycopene (Nguyen et al., 2014). It has also been shown that high pigment (hp) mutations has an additive effect on GLK mutation and further improves chlorophyll and carotenoid development in the fruit. Therefore SlGLKs are considered as important regulators that can be used for enhancing nutritional and ripening processes of tomato (Nguyen et al., 2014). During the climacteric fruit ripening process such as in tomato, a sudden burst of respiration and increase in phytohormone level such as ethylene is observed (Seymour et al., 2013). SlMYB70 is a R2R3-type MYB subfamily TF that possesses an EAR repression motif (Cao et al., 2020). It is localized to nucleus and expressed in all the tissues. However, during the ripening process as well as exogenous application of ethylene its accumulation is greatly reduced. SlMYB70 negatively influence fruit ripening as is apparent from the fact that overexpressed and silenced lines show delayed and accelerated ripening, respectively, as shown in Fig. 7.2 (Cao et al., 2020). Also, ethylene production as well as expression of ethylene biosynthetic genes such as SlACS2 (1-aminocyclopropane-1-carboxylic acid synthase 2) and SlACO3 (ACC Oxidase 3) is compromised in the overexpressed lines. SlACS2 and SlACO3 are involved in ethylene mediated fruit ripening in tomato (Hamilton et al., 1990; Oeller et al., 1991; Blume and Grierson, 1997). It has been reported that SlMYB70 specifically interacts with the promoter of SlACS2 and SlACO3 and represses their expression through EAR motif (Cao et al., 2020).

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SlGLK

SlMYB70 Negative regulation SlACS2 Green shoulder

unifrorm green

Ripe

Does not ripe

SlACO3

Green shoulder

Delayed ripening

Early ripening

Ripe

FIGURE 7.2 Ripe phenotype in MYB TF overexpressed and mutant lines. During normal tomato fruit developmental in wild type (WT) SlGLK expression is tightly regulated and leads to a smooth transition of fruit from green shoulder to ripe red phenotype. Overexpression promotes plastid development and prevents the fruit from ripening while mutation in it restores the WT phenotype. Another MYB TF SlMYB70 negatively regulates ethylene biosynthetic genes such as SlACS2 and SlACO3. Therefore mutation in SlMYB70 causes early ripening while overexpression delays the ripening process.

Seeds play a major role in fruit development but seedless fruits are a desirable agronomical trait. Another R2R3-type MYB subfamily TF is SlGAMYBs, which are regulated by GA (AlonsoPeral et al., 2010). TFs associated with hormone responses such as SlGAMYB1/2 are posttranscriptionally modulated by microRNAs such as miRNA159 (Karlova et al., 2013; Liu et al., 2014; da Silva et al., 2017). It has been reported that miR159-targeted SlGAMYB genes are important for ovule development and fruit set. A dynamic regulation is seen in the miR159/GAMYB module during early stages of fruit development and are expressed during flower and ovary development in tomato (da Silva et al., 2017). Downregulation of SlGAMYBs is observed in developing ovaries when SlMIR159 precursor is overexpressed in Micro-Tom tomato cultivar, leading to earlier fruit initiation and parthenocarpy (fruit develops in the absence of fertilization). Ovule development arrest might be because of the altered GA and auxin responsiveness in the ovary. This shows that a coordinated molecular circuit of miR159/GAMYB is necessary for fruit development (da Silva et al., 2017). The cuticle (cutin matrix mainly composed of C16
9/10, 16-dihydroxy fatty acid) provides a waxy barrier that protects the epidermal cells from the harsh environment (Riederer and Muller, 2006; Mintz-Oron et al., 2008). MIXTA-Like TFs expressed mainly in the fruit epidermal tissue are reported to act as positive regulators of conical epidermal cell differentiation in tomato fruit. They regulate various cutin biosynthetic genes and are also involved in deposition and assembly of cuticular lipids (Lashbrooke et al., 2015). They also regulate expression of downstream genes such as Cytochrome P450 SlCYP77A and SlCYP86A to catalyze the formation of major cutin monomers.

7.4 MADS transcription factor

163

Silencing of SlMIXTA-like prevents the fruit from postharvest water loss and fungal infection (Lashbrooke et al., 2015). Therefore MYBs are considered as master regulators of ripening and fruit development in tomato. There are approximately 150 MYB TFs in tomato, so further deep exploration is necessary to understand the complete phenomenon.

7.4 MADS transcription factor MADS-box TFs are an important group of plant TFs that interact with CC[A/T]6GG (CArG-box) cis-element in the promoters of target genes. The name comes from MCM1 (Saccharomyces cerevisiae), AGAMOUS (Arabidopsis thaliana), DEFICIENS (Antirrhinum majus), and SRF (Homo sapiens) (Schwarz-Sommer et al., 1990). They possess a characteristic M (MADS—for DNA binding and dimerization) domain, I (Intervening—for DNA-binding specificity) domain, K (Keratin—for protein
protein interactions) domain, and a C (C terminal—for ternary complex formation and transcriptional activation) domain (Kaufmann et al., 2005). SlRIN (ripening inhibitor) encodes a SEPALATA clade (E-class) MADS-box TF and is an important regulator of tomato ripening that acts along with other MADS-box family proteins such as SlTAGL1 (Tomato Agamous-Like 1) and SlFUL1/2 (Fruitfull 1 and 2). SlRIN specifically targets demethylated sites in the promoters of ripening-related genes such as SlACS4, SlPSY1 (Phytoene synthase 1), SlPG2A (polygalacturonase), SlPL (pectate lyase), and SlCNR (Colorless nonripening) and regulates early ripening processes through ethylene-dependent and -independent pathways (Vrebalov et al., 2002; Itkin et al., 2009; Vrebalov et al., 2009; Bemer et al., 2012; Zhong et al., 2013; Fujisawa et al., 2013; Wang et al., 2014). It is reported that rin mutation is not a null mutation, rather a gain-of-function mutation that does not initiate autocatalytic ethylene burst, fruit color change, ripening, fruit softening, pigment accumulation, and volatile flavor production (Klee and Giovannoni, 2011; Ito et al., 2017; Li et al., 2018). The rin mutant allele encodes a messenger RNA with an in-frame fusion (because of deletion of genomic DNA fragment on chromosome 5) of the adjacent truncated MADS-box TFs, namely SlRIN (having the DBD) and Macrocalyx (SlMC with a repression motif) that translates into RIN
MC chimeric protein or the rin protein (Ito et al., 2008). Ethylene enhances rin transcript accumulation while it does not have much effect on the SlRIN transcript (Li et al., 2018). Chimeric protein rin is a functional TF, and both SlRIN as well as rin localizes to nucleus and interacts with the CArG-boxes present in the promoters of downstream genes. However, SlRIN shows strong activation property while rin shows repressor activity because the transcriptional activation domain in SlRIN is replaced by repressor MC domain in rin. It is shown that inactivation of the rin allele restores the defects in initiation of ripening while overexpression inhibits fruit ripening giving it a yellow colored phenotype (Li et al., 2018). Unlike SlRIN, rin does not form heterodimer with SlFUL1/2, rather forms homodimer and affects transcription of SlACS4, SlPSY1, SlPG2A, SlPL, SlRIN, and SlCNR (Ito et al., 2017). However, in another study it was shown that rin physically interacts with SlFUL1/2, SlMADS1, and SlTAGL1 in the nucleus (Li et al., 2018) (Fig. 7.3). RIN is also reported to interact with SlMBP15, a MADS-box TF FLC/MAF clade member (Yin et al., 2018). It was seen that SlMBP15 expression increases until breaker fruit stage and then declines during fruit ripening. Silencing of SlMBP15 led to delayed fruit ripening, reduced carotenoid as well

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Deleted Genomic DNA RIN

RIN

mRNA

MC In-fusion mRNA

Master regulator complex SlTAGL1

Protein

SlRIN

rin RIN MC SlMBP15

Activator

SlFUL2

SlRIN SlCMB1

Repressor

SlMADS1

Regulates fruit ripening & development Ripe

Promotes ripening

moderate ripening

Inhibits ripening

restore ripening

FIGURE 7.3 Regulation of MADS-box in fruit ripening and development. SlRIN is an important regulator of tomato fruit ripening. Its overexpression promotes ripening while its silencing does not inhibit ripening but causes moderate ripening. It also has an alternate form in which, because of a deletion in the genomic DNA, the resulting protein (rin) is a fusion of RIN and MC (with a repressor domain). It is seen that overexpression of rin, inhibits ripening while silencing of it restores ripening. SlRIN also interacts with various other TFs such as SlTAGL1, SlFUL2, SlMBP15, SlMADS1, and SlCMB1 to coregulate the ripening process. This indicates that the MADS-box TF-mediated ripening process is a complicated network and is tightly regulated to control ripening in tomato.

as ethylene content and affected expression of ripening associated genes (Yin et al., 2018). Alongside SlRIN-interactors, SlFUL1 and SlFUL2 are reported to regulate tomato fruit pigmentation accumulation and ethylene mediated ripening redundantly (Bemer et al., 2012). Similar to SlRIN, SlFUL1 expression is limited in the vegetative tissue while it is significantly increased during fruit ripening process while SlFUL2 is expressed in other tissues as well (Wang et al., 2014). SlFUL2 overexpression inhibited abscission of styles from fruit by regulating ovary development and leading to a prominent pointed tip phenotype at the blossom end of the fruit. SlFUL2 is also reported to induce thinner pericarp and delay postharvest dehydration in tomato. SlFUL1/2 double silenced transgenic plants suppressed tomato fruit ripening and blocked ethylene production through downregulation of ethylene biosynthetic genes such as SlACS2 and SlACO1 (Wang et al., 2014) (Fig. 7.3). Another SlRIN interacting protein MADS-box TF is SlTAGL1, whose mRNA is highly expressed in reproductive structures, developing fruits, and during ripening (Vrebalov et al., 2009). SlTAGL1 silenced lines showed yellow-orange mature fruit, reduced pericarp thickness, and less stylar trichomes. Metabolite contents were also affected with reduced carotenoids, lycopene, and ethylene content while increased Lutein (yellow coloration) content. The silenced lines also affected chloroplast distribution and accumulation of amyloplast-localized starch granule in immature tomato fruit (Vrebalov et al., 2009). “Green Stripe” encodes a methylated isoform of TAGL1 that is involved in chloroplast development and carotenoid accumulation in tomato fruits (Liu et al., 2020a,b). Another MADS-box TF SlMADS1 also interacts with SlRIN and reported to negatively regulate fruit ripening.

7.5 Other transcription factors

165

SlMADS1 transcript showed high expression in flower and fruits. It is observed that the RNAi lines showed enhanced expression of ripening associated genes (such as SlACS2, SlACO3, and SlPSY1), early ripening, and increased carotenoid as well as ethylene content (Dong et al., 2013) (Fig. 7.3). Apart from this various other MADS-box TFs have been reported to play a role in fruit ripening and development such as SlCMB1 (a close relative of SlRIN) that when silenced leads to ripening delay and lower carotenoid as well as ethylene accumulation. Even though transcript expression is low in fruit, it is reported to interact with important fruit development/ripening regulators such as MADS-RIN, TAGL1, MADS1, and SlAP2a (Zhang et al., 2018) (Fig. 7.3). Another MADS-box TF SlFYFL when overexpressed showed delayed fruit ripening (Xie et al., 2014). Therefore MADSbox TFs are regarded as master regulators of fruit development and ripening. However, a lot of unanswered and unexplored questions should be studied to understand the complex regulation of MADS-box TFs.

7.5 Other transcription factors Various other TFs are also reported that play an important role in tomato fruit ripening and development. A NAC TF SlNAC1 expresses highly in early fruit development and during the ripening process. When overexpressed the fruits become soft on maturation with reduced pericarp thickness and develop yellow or orange color. This is because of the reduced expression of various genes involved in ethylene biosynthesis and carotenoid pathway, thereby leading to reduced ethylene and carotenoid, while increased ABA content (Ma et al., 2014). Another NAC TF NOR-like1 is important in tomato fruit ripening because silencing/mutation of NOR-like1 leads to delayed ripening initiation and reduced ethylene, chlorophyll, and lycopene content. It was reported that NOR-like1 specifically interacts with the promoters SlACS2 and SlACS4 (involved in ethylene biosynthesis); SlGgpps2 and SlSGR1 (regulates pigment accumulation); and SlPG2a, SlPL, SlCEL2 as well as SlEXP1 (involved in cell wall metabolism) and activate their expression (Gao et al., 2018). Elongated hypocotyl 5 (SlHY5), a member of bZIP TF family, has been primarily identified as a master regulator of light signaling in Arabidopsis, however, in tomato it regulates carotenoid accumulation giving color and nutritional aspects to the fruit. Loss of function of SlHY5 leads to decreased carotenoid, anthocyanin, and ethylene accumulation as well as altered expression of ripening-related genes. In the mutant translation efficiency of various ripening-related genes such as FUL1, NOR, DML2, and LoxC was decreased, highlighting the fact that SlHY5 is a positive regulator of fruit ripening (Wang et al., 2021). SBP-box TFs also play an important role in tomato fruit development. Mutation in Cnr (colorless nonripening) locus is responsible for naturally occurring epimutant of tomato that harbors the SlSPL-CNR (SQUAMOSA promoter binding protein-like-CNR) SBP-box TF. In the mutant the fruits do not ripen and remain colorless. SlSPL-CNR is a nuclear-localized protein and possess a specific monopartite N-terminal NLS and two zinc-finger motifs (ZFMs) required for Zn binding activity. It was shown that both NLS and ZFM are required to regulate cell death in tomato. SlSPL-CNR physically interacts with SlSnRK1 (SUCROSE-NONFERMENTING1-related kinase 1) that positively regulates ripening because silencing of SlSnRK1 led to reduced expression of ripening-related genes and inhibited ripening (Lai et al., 2020).

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One of the most wanted agronomic traits in tomato is larger fruit size. It is reported that it can be achieved through increasing the final size of floral meristems (FM). A recently identified tomato mutant eno (excessive number of floral organs) displays alterations in FM size that leads to supernumerary organ development in flowers and multilocular fruits (in contrast to bilocular in tomato wild-type species) that are larger and heavier. The mutation was identified in ENO that is an AP2/ ERF TF (subfamily group VIII) and is expressed highly in flower meristematic domes. Some other mutants identified with larger fruits (attributed to more than eight locules) are fasciated (fas) and locule number (lc) (Tanksley, 2004; Barrero et al., 2006). It was observed that eno, lc, and fas Loci exhibit synergistic effects leading to the dramatic increase in FM size, fasciated flowers, and tomato fruit size. lc mutation encodes the SlWUS [CLAVATA (CLV)-WUSCHEL] and it is observed that ENO specifically targets GCC-box present in its promoter that substantially expand SlWUS expression domains and regulation of floral stem-cell homeostasis (Yuste-Lisbona et al., 2020). There are approximately 164 AP2/ERFs TFs in tomato and they are regarded as major regulators of ethylene responses and are involved in a wide array of pathways. This highlights the importance of AP2/ERF TFs in fruit ripening and development in tomato, however, further study is required to understand the entire fruit development phenomenon.

7.6 Conclusion and future perspectives Fruit development and ripening in tomato is a complex and layered regulatory process. TFs such as MADS-box and MYB act as master regulators of this process and can activate various downstream genes associated with carotenoid production, ethylene biosynthesis, delayed ripening, and postharvest quality control. Other TFs and proteins acts in concert with these TFs and assist in the process through forming protein complexes. However, various aspects of this phenomenon are still unanswered and there is an urgent need to address these issues: •

• •

•

What are the upstream components of these fruit regulatory processes? How are these TFs getting phosphorylated and activated? What are the MAPK and upstream kinases that regulate these processes? What are the signaling molecules that trigger these responses? Do environmental factors play any role in this such as abiotic/biotic stress, quality of soil/water, humidity, and rain? How complex is the regulatory network? Do other pathways such as protein homeostasis (autophagy, proteasome machinery), posttranslational modification, and epigenetic regulation play any role in this? How soon TF-mediated fruit improvement can be implemented in actual field trials so that farmers can benefit from this? Will there be an unforeseen side effect associated with this? To answer these questions the following measures could be undertaken:

•

High throughput genome wide studies should be done at a larger scale such as RNA-seq to profile expression of associated genes at every stage of fruit development and different fruit parts should also be taken into consideration. Single cell analysis will further shed some light on the transitioning process. High fidelity ChIP assays such as ChIP-on-chip, ChIP-exo, and RIP-Chip should be performed to identify TF-promoter interactions.

References

• •

•

167

Large-scale coimmunoprecipitation and mass spectroscopy assays should be performed to identify the protein
protein interactors. Importance should be given to identify the whole signaling cascade so that the entire mechanism could be understood. Big consortiums and collaborations and knowledge share will assist in this process. Public databases should be generated with the available information. Proper and extensive field trials should be planned and executed with utmost precautions to avoid any side effects at longer run.

Acknowledgment Vigyasa acknowledges Prof. Hongmin Li and University of Arizona, Tucson for their help and support. Dharitree acknowledges Prof. N. K. Singh and ICAR-NIPB, New Delhi for their help and support. Ujjal acknowledges Dr. Nuria Sanchez Coll and CRAG, Barcelona for their help and support. We also acknowledge the authors and the reports that we might have missed citing inadvertently. We also acknowledge the source of images that we adapted from various platforms.

Conflict of interest The authors declare that there is no conflict of interests.

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CHAPTER

Plant transcription factors and nodule development

8

Jawahar Singh1 and Praveen Kumar Verma2 1

Plant Immunity Laboratory, National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, Delhi, India 2Plant Immunity Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, Delhi, India

8.1 Introduction Nutrient availability in the soil determines plant growth and productivity. Nitrogen (N2) is an essential component of many primary and secondary plant molecules like amino acids, proteins, nucleic acids, chlorophyll, and hormones (Masclaux-Daubresse et al., 2010; Tegeder and MasclauxDaubresse, 2018; Mu et al., 2016). N2 is mainly absorbed as nitrate into the plant root from soil and subsequently reduces to ammonium, the major usable form in plants. Although 78% of the atmosphere is made up of the gaseous N2, it cannot be absorbed directly by plants. Therefore N2 fertilizers, a significant factor for yield gains, proved to be a blessing for human civilization during the “Green Revolution” but damnation later (Schroeder et al., 2013). The atmospheric nitrogen, converted to ammonia by the Haber-Bosch process, contributes to an estimated 2%
4% of energyrelated CO2 emissions (Smith et al., 2020). Besides this, continuous use of N-fertilizer proved to be costly and carries significant environmental risks (Raza et al., 2014). In addition, overuse of synthetic fertilizer leads to nitrogen runoff into groundwater and oceans, causing water pollution by eutrophication (Raza et al., 2014). A more sustainable strategy to reduce our dependency on nitrogen fertilizers is to maneuver a specialized and widely conserved biological process of root endosymbiosis. Plants can associate with various beneficial fungi and bacteria in the soil to facilitate mineral uptake from soil. These symbiotic associations significantly enhance the growth of plants in nutrient-exhausted environments and thus may serve as a natural bio-fertilizer (Fowler et al., 2013; Coskun et al., 2017) (Fig. 8.1). Plants of the Leguminosae family undergo mutualistic interaction with rhizobia to convert the atmospheric nitrogen into ammonia, which plants directly assimilate. In return, rhizobia get carbonrich organic compounds from plants for their survival (Zahran, 1999). This symbiotic relationship does not only enhance the legume’s production but also the subsequent cereal production by improving soil fertility. Besides this, it also significantly reduces the use of chemical fertilizers, reducing global warming and water contamination (Ouma and Asango, 2016). Establishing a successful symbiotic relationship requires an exchange of chemical dialogue between the legume and its partner, rhizobia. Legume’s exudate flavonoid and isoflavonoid into the rhizosphere to induce nod genes in rhizobia (Oldroyd et al., 2011). The expression of rhizobia’s nod genes triggers a set of genes that synthesize the Lipo-Chito-oligosaccharides (LCO) known as Nod factor (NF). Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00020-0 © 2023 Elsevier Inc. All rights reserved.

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FIGURE 8.1 A conceptual model for the transcriptional networks in legumes for nodulation: The transcriptional network in root epidermal cells leads to bacterial infection followed by nodule organogenesis in the root cortex. Nod factor is recognized by the Nod factor receptors and this leads to activation of symbiosis signaling. Outputs from the symbiosis signaling pathway lead to gene induction including the expression of the transcription factors NIN. The NIN gene regulates the expression of NPL1, NFYA1, and LBD16 for rhizobial infection and root cortical cells division for nodule organogenesis. Auxin signaling through STY and UCCA is vital for the initiation of nodule primordia formation for nodulation.

Further, the lysine motif (LysM)-type receptors in coordination with leucine-rich repeats (LRRs) type receptors in plants perceive rhizobial NFs, trigger infection thread (IT) formation, and initiate the nodule organogenesis (Arrighi et al., 2006; Limpens et al., 2003; Madsen et al., 2003; Radutoiu et al., 2003; Smit et al., 2007). Following NF perception by NF receptors, the symbiotic signal is transduced to the common symbiosis signaling pathway (CSSP). Several mutants impaired in the nodulation pathway components were found to be compromised in arbuscular mycorrhiza symbiosis (AMS), which led to identification of a whole set of genes regulating root nodule symbiosis and arbuscular mycorrhiza symbiosis and constituting the so-called CSSP (Gutjahr and Parniske, 2013). CSSP starts with SymRK for Lotus japonicus and DMI2 for Medicago truncatula (Stracke et al., 2002; An´e et al., 2002). Symbiosis receptor kinase (SYMRK) acts upstream of the NF-induced calcium spiking. Calcium spiking activates calcium-calmodulin-dependent protein kinase (CCaMK) inside the nucleus (Madsen et al., 2010; Hayashi et al., 2010; Singh and Parniske, 2012). The CCaMK interacts and phosphorylates CYCLOPS, a coiled-coil protein, which further activates a series of transcription factors (TFs) (Yano et al., 2008).

8.2 CCaMK/CYCLOPS complex

177

Activation of TFs in the nuclei of epidermal and cortical cells leads to the initiation and progression of signal transduction pathways for different developmental stages of nodule organogenesis. Two approaches, namely, the ability of TFs to bind over the promoter of nodulationrelated genes and positional cloning of causative genes for impairment in infection or nodule development, have proven important for TF identification. The first TF described was the NIN (nodule inception) as a major player in the nodulation of L. japonicus (Schauser et al., 1999). Mutation of NIN leads to inhibition of infection and nodule primordia formation. Thus NIN gets induce during the early stages of nodule development and is involved in different stages of nodule formation. Other genes such as CCaMK/CYCLOPS, Ethylene Response Factor Required for Nodulation 1 (ERN1) and ERN2, members of GRAS family Nodulation Signaling Pathway 1 (NSP1) and NSP2, NODULE INCEPTION (NIN), Nuclear Factor-Y (NF-Y), Nodulation Pectate Lyase (NPL), LOB-domain protein gene (LBD16), Rhizobium-Directed Polar Growth (RPG), and SHORT INTERNODES/STYLISH (STY) were later added to the list (Singh et al., 2014; Middleton et al., 2007; Arrighi, 2008; Shrestha et al., 2021; Cerri et al., 2012; Kalo´ et al., 2005; Smit, 2005; Combier et al., 2006; Zanetti et al., 2010; Schiessl et al., 2019; Xie et al., 2012; Liu et al., 2019a) (Table 8.1).

8.2 CCaMK/CYCLOPS complex Nuclear-associated calcium oscillations are a signature of the CSSP. This calcium oscillation is decoded by CCaMK inside the nucleus, which is one of the common components of RN symbiosis and AM symbiosis (Oldroyd et al., 2011; Singh and Parniske, 2012). Binding of Ca21 directly or calmodulin results in CCaMK self-phosphorylation (Miller et al., 2013). Phosphorylated CCaMK then trans-phosphorylates interacting proteins and induces the expression of the downstream genes for symbiosis. Truncation of the autoinhibitory calmodulin and calcium-binding domains of CCaMK, or mutation in the autophosphorylation residue, results in a phospho-mimic version of CCaMK that activates nodule formation even without rhizobia, known as spontaneous nodulation (Hayashi et al., 2010; Gleason et al., 2006). CYCLOPS is one of the major targets that get phosphorylated by CCaMK (Yano et al., 2008). CYCLOPS is a nuclear protein, functions as a transcriptional activator, and is required for both RN symbiosis and AM symbiosis (Singh et al., 2014). The functioning of CYCLOPS as a TF depends on its activation through phosphorylation by CCaMK. CCaMK phosphorylates two serine residues in CYCLOPS, and substituting these two serine to aspartate creates a phospho-mimic mutant that is enough to induce spontaneous nodulation. So, it appears that activation of either CCaMK or CYCLOPS is sufficient for nodulation, and this divulges the essential role of this calcium signaling complex in nodulation. As spontaneous nodulation induced by phospho-mimic mutants of CCaMK and CYCLOPS depends on NIN, that suggests the coordinated action of CCaMK and CYCLOPS and NIN in nodule organogenesis. CYCLOPS binds to the promoter of NIN in a phosphorylationdependent fashion and induces NIN expression (Singh et al., 2014). ERN1, a major regulator of the infection process in L. japonicus and M. truncatula, acts as another direct target of the CCaMK/CYCLOPS complex. ERN1 expression in the epidermis for the infection-associated process is regulated by CYCLOPS, which suggests CYCLOPS and CCaMK act upstream of ERN1.

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Table 8.1 A list of TFs essential for nodule organogenesis, and their interacting proteins. Protein

TF family

CCaMK

Novel

CYCLOPS

Novel

NSP1

GRAS

NSP2

GRAS

SIN1

GRAS

SymSCL1

GRAS

ERN1

AP2/ ERF

ERN2

AP2/ ERF

NIN

RWPRK

NF-YA1

NF-Y

NF-YB1

NF-Y

NF-YC1

NF-Y

STY1/2/3

STY

YUCCA1/ 11 LBD16

YUCCA LBD

Phenotype

References

Autoactive form of CCaMK activates nodule organogenesis even in the absence of rhizobia CYCLOPS mutants shows impaired IT formation Autoactive CYCLOPS induces NIN and trigger nodule formation (nodule-like structures) Mutants does not form infection thread nodule organogenesis upon inoculation with rhizobia Mutants does not form infection thread nodule organogenesis upon inoculation with rhizobia Mutants are defective in infection thread progression and nodule organogenesis RNAi roots characterized with reduction in the size of nodules and decrease in nodule number Mtern1 mutants fail to nodulate. Ljern1 mutants are characterized by balloonshaped root hairs, infection threads do not grow into nodule primordia of cortical cells Mtern2 mutants nodulate but with signs of early senescence

Yano et al. (2008), Singh et al. (2014) Yano et al. (2008), Singh et al. (2014)

Mutants are intracellular infection, nodule organogenesis, and a negative feedback mechanism that controls the number of nodules. Mutants are characterized with defect in nodule meristem Mutants are defective in cell divisions and lateral root formation Mutants impaired in rhizobial infection and nodule formation Mutants are characterized by a drastic reduction in nodule primordial, nodules, and epidermal infection threads. Auxin biosynthesis and cell division for nodule development Mutants are characterized by defect in nodulation

Smit et al. (2005) Kalo´ et al. (2005) Battaglia et al. (2014) Kim and Nam (2013) Middleton et al. (2007), Cerri et al. (2012), Cerri et al. (2016), Yano et al. (2017) Middleton et al. (2007), Cerri et al. (2012), Cerri et al. (2016), Yano et al. (2017) Schauser et al. (1999), Soyano et al. (2014) Kalo´ et al. (2005), Baudin et al. (2015), Laloum et al. (2014) Soyano et al. (2013) Smit (2005) Shrestha et al. (2021)

Shrestha et al. (2021) Soyano et al. (2019)

CYCLOPS can bind directly to a CYC-Response Element (CYC-REERN1) in ERN1 promoter with sequence similarity to the CYC-RENIN, recognized by CYCLOPS in the NIN promoter (Cerri et al., 2017). Hence, CYCLOPS activated by CCaMK activates the transcription of ERN1 and NIN, which consequently activates the expression of genes for the generation and progression of rhizobial ITs (Cerri et al., 2017).

8.3 AP2-ERF transcription factor (ERN1 and ERN2)

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8.3 AP2-ERF transcription factor (ERN1 and ERN2) M. truncatula ERF Required for Nodulation1 (ERN1) encodes an AP2/ERF TF that controls root hair (RH) bacterial infection together with its close homolog ERN2 (Middleton et al., 2007; Cerri et al., 2012; Cerri et al., 2016). The phylogenomics analyses revealed that ERN1 and ERN2 genes were originated as a result of the gene duplication in the common ancestor of Leguminosae plants (Yano et al., 2017; Kawaharada et al., 2017). This duplication event and successive molecular evolution of the ERN1 and ERN2 gene pair is associated with the evolution of RNS in legumes. ERN1 and ERN2 have redundant functions for rhizobial infection. But the entry of ITs into root cortical cells depends only on ERN1 (Cerri et al., 2012). ERN1 and ERN2 have different tissue-specific expressions. ERN2 expresses at initial root infection stages, while ERN1 expresses throughout root nodulation and plays a predominant role during nodulation. ERN1 expression increase within one to three hours of rhizobial inoculation or NF treatment in roots for the early activation of target genes; on the other hand, ERN2 expression constitutively increases approximately six hours of rhizobial inoculation, likely to assure optimal levels at later time points or in coordination with ERN1 (Cerri et al., 2016). The Mtern1 mutants exhibit early symbiotic responses, including rhizobial infection, but failed to nodulate. In contrast, the Mtern2 mutant was found to nodulate but with signs of early senescence (Cerri et al., 2016). Lack of functional ERN1 and ERN2 in Mtern1/ern2 double mutants results in the complete abolition of rhizobial infection and nodule organogenesis (Cerri et al., 2016). Genomic analysis revealed that the ERN2 gene is absent in the genome of L. japonicus (Kawaharada et al., 2017). The molecular function and spatio-temporal expression of the LjERN1 are in harmonization with the ERN2 gene of M. truncatula and other legumes (Kawaharada et al., 2017). The Ljern1 mutants are characterized by balloon-shaped RHs similar to Mtern1/ern2 double mutants (Yano et al., 2017). Double mutants Mtern1/ern2 were incapable of ensnaring rhizobia inside the RHs because of the defective tip curling due to abnormal expansion and loss in its polarity (Yano et al., 2017). Similarly, Ljern1 single mutants are characterized by decreases in RH curling and an increase in balloon-shaped RHs after rhizobial inoculation. This signifies the importance of LjERN1 in RH responses for IT formation (Yano et al., 2017). Therefore the RH phenotype of Ljern1 includes the phenotype of Mtern1/ern2 double mutant, which is in agreement with the lack of ERN2 in L. japonicus. Nevertheless, the phenotype of Ljern1 is weaker in comparison with Mtern1/ern2 (Yano et al., 2017; Kawaharada et al., 2017). ITs do not grow into nodule primordia of cortical cells in Ljern1 mutants. Thus the impairment in IT development is a major Ljern1 phenotype. This phenotype of Ljern1 mutants seems to be an intermediary between Mtern1 single and Mtern1/ern2 double mutants of M. truncatula (Yano et al., 2017). The ITs and nodule primordia do not develop in the double mutant, which insinuates ERN TFs for nodule development. Alternatively, loss of infection events in the root epidermal cells of the double mutant may cause a loss in the stimulation of the meristematic activity of cortical cells (Cerri et al., 2012). ERN1 and ERN2 bind and activate transcription of ENOD11 through a 30-bp regulatory unit (NF-box). Trans-activation assay in Nicotiana benthamiana suggests that ERN1 and ERN2 are sufficient to activate ENOD gene expression. NF-mediated RH response requires both ERN1 and ERN2 in its native condition to assure ENOD11 expression at optimal levels in M. truncatula (Cerri et al., 2012). CYCLOPS regulates the expression of ERN1 for the infection-associated process in the epidermis. Activated CYCLOPS binds directly to a CYC-Response Element (CYC-REERN1) of the

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ERN1 promoter. CCaMK/CYCLOPS-driven expressions NIN and ERN1 were found to be interconnected during the early infection response, RHs curling, and infection chamber formation (Cerri et al., 2017).

8.4 GRAS transcription factor Transcription factors with GRAS-domain belong to the family of plant-specific TFs important for different physiological processes like root development, meristem development and maintenance, and phytohormone signal transduction (Bolle, 2004). GRAS acronym refers to initials of the first three TFs identified in this family: GIBBERELLIC ACID INSENSITIVE: GAI, REPRESSOR OF GAI: RGA, and SCARECROW: SCR (Peng et al., 1997; Silverstone et al., 1998; Di Laurenzio et al., 1996). The size of GRAS proteins ranges from 400 to 770 amino acids. Higher conservation is found in C-terminal regions, containing leucine-rich region I, VHIID, leucine-rich region II, PFYRE and SAW, responsible for interactions with GRAS and other proteins (Bolle, 2004; Richards et al., 2000). A higher level of divergence is found in the N-terminal region of the GRAS proteins compared to the GRAS domain region, and this N-terminal region acts as an activator (Bolle, 2004; Richards et al., 2000). The conserved C-terminal region in the GRAS domain is required for specific association with various signaling molecules acting in different physiological and developmental processes (Bolle, 2004). The LHRs and VHIID domain in the conserved LHRI-VHIID-LHRII region are associated with protein
protein interactions and protein
DNA interactions (Pysh et al., 1999).

8.4.1 Nodulation signaling pathway 1/2 (NSP1 and NSP2) GRAS-type TFs can be categorized into eight categories, which are highly conserved in higher plants (Tian et al., 2004). For example, putative orthologs of NSP1 (class III) and NSP2 (class VII) were found in several higher plant species, including A. thaliana (At3g13840 and At4g08250) and rice (Os03g29480/OsNSP1 and Os03g15680/OsNSP2) (Kalo´ et al., 2005; Smit et al., 2005; Heckmann et al., 2006; Murakami et al., 2007). Furthermore, the trans-complementation of nsp1 and nsp2 knockout mutants of legume with nonlegume NSP1 and NSP2 suggest that NSP1 and NSP2 are functionally conserved across higher plants (Heckmann et al., 2006; Yokota et al., 2010). Several studies have suggested the involvement of GRAS proteins in nodule formation. NSP1 and NSP2, two GRAS proteins, are proven to be specific and indispensable components for RNS in several legume species (Kalo´ et al., 2005; Smit et al., 2005; Heckmann et al., 2006; Murakami et al., 2007). Biochemical and molecular studies suggest that NSP1 interacts with NSP2 to form a heterodimer that binds to a specific cis-regulatory element in the promoter of ENOD11 and ERN1 (Hirsch et al., 2009). NSP1 and NSP2 are crucial for nearly all NF-induced signaling processes, including early nodulin gene expression, nodule organogenesis, and nodule function and positioned downstream of CCaMK (Oldroyd and Long, 2003; Mitra et al., 2004). Although NSP1 is not necessary for mycorrhizal symbiosis, recently, NSP2 was found to facilitate mycorrhizal root colonization (Andre et al., 2011). This indicates that NSP2 functions in rhizobium NF signaling and mycorrhizal root colonization and is justified by the presence of NSP2 orthologs in nonlegume plants (Andre et al., 2011).

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8.5 SymSCL1 MtSymSCL1 is an important GRAS domain protein, highly expressed in the early nodule developments (Kim and Nam, 2013). The spatio-temporal expression suggests that the MtSymSCL1 gene gets transcriptionally activated during rhizobial inoculation and localizes to the nuclei of RHs and epidermis of nodules under its native promoter (Kim and Nam, 2013). These results strongly suggest that MtSymSCL1 has an important function in nodule development. A significant reduction in the nodule number was observed in downregulated MtsymSCL1 RNA interference (RNAi) plants (Kim and Nam, 2013). Moreover, a slight reduction in the size of nodules was observed along with decreased nodule number in MtSymSCL1 RNAi roots. With no impact on the release of rhizobia through ITs and zonation of nodules, MtSymSCL1 contributes to the pattern of nodule occurrence on the roots. Downregulation of MtSymSCL1 leads to deformation in the structure of nodules; for example, MtSymSCL1 RNAi roots contain singlet nodules, whereas nodules in control roots are formed as multiple clusters. However, MtsymSCL1 RNAi neither affects root growth nor RHs curling and growth of ITs. MtSymSCL1 homolog silencing in Arabidopsis by RNAi resulted in inhibition of root growth (Kim and Nam, 2013).

8.6 NIN and NIN-like proteins NIN (NODULE INCEPTION) is a founding member of the NIN-like proteins (NLP) family. The NLP gene family is conserved across the plant species. A. thaliana and rice have nine and six NLPs, respectively. M. truncatula and L. japonicus genome contain one copy of NIN and five copies of NLPs each. NIN is a major TF indispensable for nodulation in legumes (Schauser et al., 1999; Schauser et al., 2005). Root nodule-formation involves two processes: intracellular infection and nodule organogenesis (Marsh et al., 2007). NIN is found to be important for both these processes. The number of nodules in the legume’s root is controlled by a negative feedback mechanism (Soyano et al., 2014; Nishida et al., 2018). Importantly, NIN also plays a vital role in this process (Schauser et al., 1999; Schauser et al., 2005; Marsh et al., 2007; Soyano et al., 2014; Nishida et al., 2018). NIN appears to be one of the major regulators for IT formation as the mutation in the NIN results in excessive RH curling and blockade in IT formation (Schauser et al., 1999). NIN is specifically expressed in the nodule of all studied legume species (Schauser et al., 1999; Marsh et al., 2007; Borisov et al., 2003; Demina et al., 2013; Bu et al., 2020). In contrast, most NLPs express constitutively with preferential expression in certain tissues and developmental stages (Cao et al., 2017; Chardin et al., 2014).

8.7 Structure of NIN and NLPs The NLPs are composed of three functional domains: NRD, RWP-RK, and PB1 (Schauser et al., 2005; Chardin et al., 2014). The N-terminal region of NLPs contains conserved nitrate responsive domain (NRD) to perceive nitrate signals (Schauser et al., 2005). One phosphorylation site in the NRD (S205) has been identified, which undergoes phosphorylation by CPK10, CPK30, and CPK32 (Liu et al., 2017). Nuclear retention of AtNLP7 and its activation for expression of the nitrate-induced gene in response to nitrate depends on the S205 phosphorylation (Liu et al., 2017). The NRD is highly conserved in NLPs

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and partly in the NINs of legumes (Schauser et al., 2005). The NRD of the NIN gene has lost its responsiveness to nitrate, important for N-fixation in legumes (Suzuki et al., 2013). All NLPs contain the highly conserved RWP-RK domain (Chardin et al., 2014). A helix
turn
helix and a basic leucine-zipper with the Arg
Trp
Pro
X
Arg
Lys conserved sequence is the characteristic feature of the RWP-RK domain in all identified NLPs (Schauser et al., 2005). The RWP-RK domain is a prerequisite for binding NLPs to nitrate-responsive cis-elements (NREs) in the promoter of target genes (Konishi and Yanagisawa, 2014). The PB1 domain is required for protein
protein interaction (Korasick et al., 2015; Sumimoto et al., 2007). The homodimerization between two NLPs requires core amino acid residues (i.e., K867, D909, D911, and E913) of the PB1 domains (Konishi and Yanagisawa, 2019). The trans-activation of nitrate-responsive genes does not require NLP homodimerization, but the full activation of nitrate-induced gene expression in nitrate response does require its homodimerization (Konishi and Yanagisawa, 2019). The heterodimerization of AtNLP6/7 with TCP20 in Arabidopsis and MtNIN with MtNLPs in M. truncatula depends on the PB1 domain (Guan et al., 2017; Lin et al., 2018). NLPs are divided into three clades, namely clades 1, 2, and 3. Phylogenetic analysis of NLPs from different legumes and Arabidopsis shows that AtNLP1, AtNLP2, and AtNLP3 are the closest Arabidopsis homologs to NIN. MtNLP1, the closest M. truncatula NLP to NIN, is grouped in a distinct clade with LjNLP1. Most likely, these two subclades result from a duplication event that occurred before the evolution of NFC but after the separation of dicots and monocots (Schauser et al., 2005). AtNLP4 and AtNLP5 with MtNLP1, MtNLP2, and MtNLP3 grouped with AtNLP8 and AtNLP9, whereas MtNLP4 and MtNLP5 grouped with AtNLP6 and AtNLP7.

8.8 Regulation of NIN for rhizobial infection in the epidermis by CYCLOPS Model legumes Medicago and Lotus have been widely used to study the complex expression pattern of NIN in detail. Rhizobial inoculation leads to NIN induction in the epidermis for rhizobial infection demonstrated by in situ hybridizations, promoter: GUS reporter assays, and root hairs (RHs) transcriptome study (Verni´e et al., 2015; Breakspear et al., 2014; Liu et al., 2019b). Following the NF perception, epidermal NIN induction is regulated in a cell-autonomous fashion, activating the CSSP (Singh et al., 2014). NIN promoter contains CYCLOPS-responsive cis-element (CYC-RE) for binding of CYCLOPS. CYCLOPS binding site in the NIN promoter is proximal to the start codon of NIN that primarily regulates rhizobial infection (Singh et al., 2014). In M. truncatula, the CYCLOPS binding site is approximately 3 kb upstream from the NIN start codon (Liu et al., 2019b). Mutation of the CYCLOPS binding site causes a dramatic reduction in the IT formation; however, rhizobial colonies are still formed in tight RH curls (Liu et al., 2019b).

8.9 Regulation of NIN by cytokinin-response elements for cell divisions in the pericycle NIN gets induced in the pericycle for cell division in Medicago (Liu et al., 2019b). Because NFs are immobile molecules and cannot move, a mobile signal gets synthesized after the perception of

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NFs in the epidermis. It gets translocated to the pericycle for activation of NIN for cell division (Liu et al., 2019b; Goedhart et al., 2000). Analysis of two weak nin alleles, daphne in L. japonicus and daphne-like in M. truncatula, leads to identification of remote elements in the NIN promoter for its induction in pericycle (Liu et al., 2019b). In both mutants, NIN expression was induced in the epidermal cells but not in the pericycle, resulting in increased numbers of ITs. As a result, nodule organogenesis is blocked. Both mutants are characterized by an alteration in the NIN promoter primarily by a chromosome translocation (Liu et al., 2019b). A remote cis-regulatory element, called cytokinin-response element (CE), is identified as the region responsible for cytokinin-induced NIN expression in the pericycle and nodule organogenesis. The distance of this CE region from the start codon varies among different legumes (e.g., in Lotus, about 45 kb, and in Medicago, it is 18 kb from the NIN start codon). This CE region consists of six potential binding sites for cytokinin response regulator (RR). Application of exogenous cytokinin failed to induce expression NIN in both daphne and daphnelike mutants, strengthening the inference that this CE region is responsible for cytokinin-induced NIN induction (Liu et al., 2019b). Therefore it appears that at least two main evolutionary changes have occurred to regulate the expression of NIN for nodule development. First, CYCLOPS binding site to regulate the rhizobial infection most likely gained in the ancestor of the NFC (Yano et al., 2008; Singh et al., 2014). Second, it developed cytokinin-regulated NIN expression in the pericycle to initiate nodule organogenesis, exclusive to legume branches (Liu et al., 2019b).

8.10 NIN: a master regulator of nodulation NIN gene is proven to be the master regulator for RNS. NIN competitively inhibits ERN1 in the root epidermis by repressing the expression of early nodulin 11 (ENOD11) and increases the transcript of the cytokinin receptor cytokinin response 1 (CRE1) in the root cortex for the integration of cytokinin signaling and nodule organogenesis (Verni´e et al., 2015). Thus NIN acts as a positive as well as a negative regulator for nodule formation.

8.11 NIN as a negative regulator in systemic control of nodulation Root nodule symbiosis is a costly and complicated process for the plant, so there is a need for a balance between the costs of hosting rhizobia into nodules in the form of nutrients provided to rhizobia for nitrogen benefits. To achieve this balance, an important signaling process takes place in RN symbiosis, called autoregulation of nodulation (AON) (Wang et al., 2018). AON acts systemically via a set of RLKs. External and internal cues have been reported to control the numbers of the nodule. The concentration of nitrate in the soil acts as a major external cue (Nishida and Suzaki, 2018). A regulatory feedback system acts as an internal cue in which earlier formed nodules suppress further nodule formation through two-way communication: root to shoot and shoot to root. This regulatory feedback system is known as AON (Ferguson et al., 2010). Therefore AON represents a stratagem for the legume plants to balance the cost of accommodating rhizobia

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inside nodules and benefits in the form of nitrogen fixed by rhizobia (Wang et al., 2018; Ferguson et al., 2010). Recent reports on AON suggest that NF perception activates the NIN/NIN-like protein (NLP) family, which directly binds to the promoter of nodulation-related CLE peptide-encoding genes and activates their expression to initiate the long-distance (systemic) AON pathway. CLAVATA3/ EMBRYO SURROUNDING REGION (CLE) peptides express in root as ascending signals. CLE peptides move from root to shoot. These mobile CLE peptides activate the LRR-RLK, HAR1 in Lotus, and its homologs SUPER NUMERIC NODULES1/ NODULE AUTOREGULATION RECEPTOR in M. truncatula and soybean (MtSUNN and GmNARK1), respectively in shoots, which further acts as descending signal and move from shoot to root. Activation of this LRR-RLK represses nodule formation in the roots (Krusell et al., 2002; Nishimura et al., 2002; Searle et al., 2003). Besides this, MtSUNN1/LjHAR1/GmNARK1 has been shown to play an important role in maintaining the plant status in the presence of nitrate (Reid et al., 2011; Jeudy et al., 2010; Okamoto and Kawaguchi, 2015). Forward genetics screen for loss of nitrate-suppression of nodulation in L. japonicus resulted in the identification of NITRATE UNRESPONSIVE SYMBIOSIS1 (LjNRSYM1)/NLP4 (Nishida et al., 2018). A reverse genetic screen of NLP mutants in M. truncatula resulted in MtNLP1 identification (Lin et al., 2018). MtNLP1 and LjNRSYM1/NLP4 move to the nucleus in the presence of nitrate to induce their downstream CLE-RS peptides (Nishida et al., 2018; Lin et al., 2018). Legumes use the expression of CLE-RS peptides by NLPs in the root nodule to activate CLAVATA1-like RLKs in the shoot to control the number of nodules in nitrate and rhizobia-dependent fashion (Nishida and Suzaki, 2018). Moreover, Lin et al. (2018) have shown that NLP1 and other NLPs mostly localize in the cytosol and are inactive in response to low nitrate. The NFs induce the expression of NIN for the activation of other nodulation-related gene expression and nodule formation. When nitrate level rises above a certain threshold, NLP1 accumulates in the nucleus, binds with NIN, and reduces the NFsinduced expression of NIN, and inhibits nodule formation (Lin et al., 2018).

8.12 NIN as a positive regulator in systemic control of nodulation Besides negative regulation of nodulation by the AON pathway, C-terminal encoded peptides (CEPs) control rhizobial infections and nodule numbers positively by another independent systemic pathway (Laffont et al., 2020; Mohd-Radzman et al., 2016; Djordjevic et al., 2015). In nitrogendeficient conditions, the expression of CEP peptides increases. Activation of CEP peptide for nodule development is regulated by compact root architecture 2 (CRA2) and LRR-RLK, acting in shoots (Mohd-Radzman et al., 2016). The cra2 mutants are characterized by a reduced number of nodules in legumes (Mohd-Radzman et al., 2016). The exogenous application of synthetic CEP1 peptides or overexpression of CEP1 peptides increases nodule formation in M. truncatula (MohdRadzman et al., 2016; Imin et al., 2013). No obvious change in nodule phenotype due to downregulation of CEP1 and CEP2 genes by RNAi suggests its functional redundancy (Imin et al., 2013). The CEPR1 (CEP receptor 1) in Arabidopsis, the closest homolog of legume CRA2 receptor, regulates the nutrient allocation to roots in systemic signaling pathways by directly binding to CEP peptides (Taleski et al., 2020).

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Laffont et al. (2020) has shown that MtCEP7 has a unique expression pattern during symbiotic association with rhizobia. MtCEP7 is rapidly induced in the root epidermis in response to rhizobia or purified NF or synthetic cytokinin (Laffont et al., 2020). The expression of MtCEP7 depends on the cytokinin receptor MtCRE1 and TF MtNIN. MtNIN is shown to directly bind over the promoter of MtCEP7 and MtCLE13 for their trans-activation. Exogenous application of synthetic MtCEP7 peptides promotes nodule formation systemically through the MtCRA2 receptor (Laffont et al., 2020). Similarly, MtCEP7 downregulation reduces rhizobial infections and nodule formation. Surprisingly, the expression of the MtCEP7 gene under the control of the MtCLE13 promoter mitigates the onset of AON, indicating a fine balance between MtCLE13 and MtCEP7 expression that determines the successful infection in Medicago roots. MtNIN coordinates the expression of CLE and CEP peptides acting in two antagonistic pathways, and their crosstalk is important for tight regulation of nodulation (Laffont et al., 2020). Altogether, these findings suggest that a single regulatory module consisting of cytokinin/MtCRE1/MtNIN regulates two dynamic and antagonistic signaling peptides for fine-tuning the rhizobial infection and nodules number (Laffont et al., 2020).

8.12.1 Lob-domain protein16 Recently, LBD16 (lob-domain protein16) was found as a direct target of NIN (Schiessl et al., 2019). LBD16 is an essential molecular player for lateral root formation that regulates nodule formation by inducing STY and YUCCA genes of the auxin biosynthetic pathway (Schiessl et al., 2019; Soyano et al., 2019). The coexpression of NF-Y subunit genes and LBD16 partially replaces NIN by rescuing nodule organogenesis in the daphne mutant of L. japonicus, a weak nin allele mutant (Soyano et al., 2019). Similarly, LBD16 mutation in the nf-y subunit mutant background enhances defects in the nodulation phenotypes, suggesting LBD16 and NF-Y work together in an additive way to regulate nodulation (Soyano et al., 2019). In Arabidopsis, NLP7 has been shown to regulate LBD16 directly (Alvarez et al., 2020). Therefore the NIN-LBD16 module appears to be evolved before the nodulation and is likely to be adopted from nonlegumes.

8.12.2 Nodulation pectate lyase 1 Cell wall remodeling is important for rhizobial infection (Brewin and Hirsch, 2004). Expression of the cell wall modifying genes is induced during rhizobial infection in a NIN-dependent manner in Medicago and Lotus RHs (Xie et al., 2012; Liu et al., 2019a). Out of all, the cell wall modifying gene NODULATION PECTATE LYASE 1 (NPL1) exclusively expresses during nodulation and is required for ITs formation in Lotus and Medicago (Xie et al., 2012; Liu et al., 2019a). NIN regulates NPL1 expression during the infection (Liu et al., 2019a). NPL1 appears to be evolved due to tandem gene duplication in legumes as its orthologs occur in Glycine max, Lupinus albus, and Arachis ipaensis (Liu et al., 2019a). So, the papilionoid legume subfamily might have conserved the NIN-NPL module for rhizobial infection. Parasponia andersonii (Parasponia) genome contains three putative NPL genes, namely PanNPL8, PanNPL9, and PanNPL10 (van Velzen et al., 2018). However, the most closely related NPL of Parasponia with legume NPL1 is PanNPL8, but it does not express specifically in nodules. The expression of PanNPL9 and PanNPL10 has been only induced fourfold during the initial stages of infection (van Velzen et al., 2018). Relative expression

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Parasponia NPL is lesser at the later stages of symbiosis than legume NPL1 (van Velzen et al., 2018). This indicates that the NIN-NPL module might have been gained within the legume branch.

8.13 Rhizobium-directed polar growth Rhizobium-directed polar growth (RPG) is a nuclear-localized, long coiled-coil protein. NIN regulates the expression of RPG (Liu et al., 2019a). RPG has a nodule-specific expression in L. japonicus, M. truncatula, and Parasponia (Arrighi, 2008; van Velzen et al., 2018; Mun et al., 2016). RPG gene has been characterized for its function in RH curling and IT formation in M. truncatula (Arrighi, 2008). RPG is a direct target of NIN shown by chromatin immunoprecipitation (ChIP) assays with sequencing, ChIP sequencing (ChIP-Seq) in Lotus (Soyano et al., 2014). NLP7 CHIPchip and nlp7 transcriptome suggested that NLP7 does not regulate the expression of RPG homolog (Zhao et al., 2018; Marchive et al., 2013). NIN-dependent and NLP7-independent expression of RPG indicates that an NLP-regulated, RPG expression have evolved before the evolution of nodulation, and the NIN-RPG module evolved early in the NFC. The contribution of NIN for the regulation of RPG expression needs to be studied by using nlp mutants specifically belonging to NIN orthologs clade to confirm its evolution pattern during nodulation further.

8.14 Nuclear factor Y Nuclear factor Y (NF-Y) belongs to the heterotrimeric TF family, made up of three individual proteins, NF-YA, NF-YB, and NF-YC, to regulate the expression of the target gene by binding to the CCAAT element in its promoter (Baudin et al., 2015). Initially, a dimer of NF-YB and NF-YC form in the cytosol, followed by the association of the third subunit, NF-YA, to form a mature, heterotrimeric TF complex. Then, this NF-Y trimeric complex binds to the target gene promoter to regulate their expression either by activation or repression of the target gene (Baudin et al., 2015). The main evolutionary difference between plant and animal NF-Y complexes is the number of genes that encode individual subunits in plants; this is higher in plants than in animals (Petroni et al., 2013; Laporte et al., 2014). In mammals, the NF-Y complex works as key regulators for cell cycle progression to activate genes of the developmental process (Li et al., 2018). Here, we have discussed how the NF-Y complex has evolved through diversification in plants, and how few of them have adapted to regulate plant-specific pathways, like RNS. We further discuss in detail different NF-Y complexes in legumes plant and their regulation. For example, in M. truncatula, MtNF-YA1 and A2, MtNF-YB16, and MtNF-YC1 and YC2 participate in NF-Y complex formation to regulate early symbiotic gene expression for nodule formation (Combier et al., 2006; Baudin et al., 2015; Laloum et al., 2014). In other legumes like L. japonicus and Phaseolus vulgaris, LjNF-YB1 and PvNF-YC1 were characterized as homologs MtNF-YB16 and MtNF-YC2 that play an essential role for RNS (Zanetti et al., 2010; Soyano et al., 2013). Two redundant NF-YA from a legume-specific clad, MtNF-YA1 and MtNF-YA2, regulate NF signaling for rhizobial infection, acting upstream ERN1 and downstream of NIN. Twenty years ago, transcriptome approach was used to identify the first NF-YA1 and its associated miR169 as

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a regulatory module, strongly induced during plant-rhizobia interaction (Combier et al., 2006). Expression of MtNF-YA1 in root epidermis for rhizobial infection and pericycle and cortex for nodule organogenesis was analyzed by transcript profiling through RT-qPCR and promoter: GUS assays. Further, the role of MtNF-YA1 in nodulation was revealed by a reverse genetic approach such as RNAi, overexpression of the miR169a, and a mutant created by the TILLING approach (Combier et al., 2006; Laporte et al., 2014). NF-YA in plants is under tight regulation by alternate splicing similar to animal NF-Ys. The presence of an intron in the 50 leader sequence in almost all NF-YA genes in M. truncatula (except MtNF-YA7) and P. vulgaris suggests that regulation of NFYA by alternate splicing is highly conserved in legumes (Combier et al., 2006; Zanetti et al., 2010). The role of the NF-YA1 gene in nodulation was analyzed by using the nf-ya1
1 mutant of M. truncatula, which is characterized by a defect in nodule meristem (Combier et al., 2006; Laporte et al., 2014). Similarly, downregulation of the MtNF-YA1 ortholog in the L. japonicus results in the absence of a persistent nodule meristem (Soyano et al., 2013). So, it appears that NF-YA regulate organogenesis in determinate as well as indeterminate nodules. Two other subunits of the NF-A complex, NF-YB, and NF-YC and the NF-YA subunit, have been shown to regulate symbiosis between legume and rhizobia (Bu et al., 2020; Rı´podas et al., 2019; Zanetti et al., 2017; Petroni et al., 2013). In P. vulgaris, PvNF-YC1 regulates rhizobial infection and nodule formation through cell cycle genes. In L. japonicus, LjNF-YB1 was identified as a direct target of NIN, and its coexpression leads to an enhanced impact on cell divisions and lateral root formation (Soyano et al., 2013). Interestingly, yeast two-hybrid (Y2H) screening in search of MtNF-YA1 interactors resulted in identification of MtNF-YB and MtNF-YC, which are orthologs to LjNF-YB1 and PvNF-YC1. The occurrence of an orthologs trimer in P. vulgaris and L. japonicus signifies its evolutionarily conserved role in legumes for nodulation (Baudin et al., 2015). In P. vulgaris, Y2H screening results in the identification of PvSIN1 (Scare-crow-like 13 Involved in Nodulation), a GRAS family protein, as an interacting protein of PvNF-YC1. PvSIN1 controls lateral root formation and nodule development (Battaglia et al., 2014). Fonouni-Farde reports that MtDELLA1, 2, and 3 proteins interact and form a complex with MtNF-YA1 and NSP2 to regulate NF signaling and rhizobial infection (Fonouni-Farde et al., 2016). MtNF-YB7 gets upregulated transiently in RHs, 6 hours postrhizobial inoculation (Breakspear et al., 2014). Its expression remains undetectable in nodules compared to other NF-YB subunits such as MtNF-YB16 and MtNF-YB18 (Breakspear et al., 2014). In P. vulgaris, PvNF-YB4, an ortholog of the MtNF-YB7, expresses at the highest level at 24 hours postrhizobial inoculation (Ripodas et al., 2015). These results indicate that members of the NF-YB family function in early rhizobial infection. In nodulation, auxin is indispensable for ITs and nodule organogenesis. Further, NF-YA1 is shown to regulate the expression of auxin biosynthesis genes directly and hence, entry into the cell cycle for rhizobial infection and nodule organogenesis (Shrestha et al., 2021).

8.14.1 Short internodes/stylish The Short internodes/stylish (STY) genes encode zinc-finger TFs that regulate the expression of auxin biosynthesis genes, YUCCA. YUCCA genes encode flavinmonooxygenase-like enzymes. YUCCA4 and YUCCA8 regulate the rate-determining step of tryptophan-dependent auxin biosynthesis (Magnus et al., 2010). NF-YA1 binds to the cis-regulatory element in the promoter region of

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the STY gene to regulate its expression. So, NF-Y regulates auxin biosynthesis genes and stimulates entry into the cell cycle for infection and organogenesis. NF-YA1 and its target STY are essential for nodule emergence. The L. japonicus has nine STY genes, and all are associated with nodule development. The expression of seven STY genes depends on NF-YA1 in L. japonicus. Similarly, seven out of eight STY genes in M. truncatula got induced in nodules, and at least six of them are MtNF-YA1 dependent (Shrestha et al., 2021). Remarkably, all STY genes express initially in dividing cortical cells of young NP and later, at the base and in the vasculature of mature nodules, similar to NF-YA. The triple mutant sty1-2 sty21 sty3-9 are characterized by a drastic reduction in NP, nodules, and eITs; in contrast, microcolonies (MCs) remain unaffected (Shrestha et al., 2021). Out of 21 YUCCA-like genes, only YUCCA1 expresses in uninoculated roots and is significantly induced by rhizobial inoculation in L. japonicus (Shrestha et al., 2021). In contrast, YUCCA11 expresses only in rhizobial inoculated roots. Thus STY regulates YUCCA1 and YUCCA11 expression, and in turn, STYs are regulated by NF-YA1 during rhizobial inoculation. Hence, YUCCA genes, YUCCA1 and YUCCA11, are under NF-YA1-dependent regulation for local auxin biosynthesis and cell division for nodule development (Shrestha et al., 2021).

8.15 Conclusion and future perspectives Legumes-rhizobial symbiosis is a unique model for investigating the evolution of mutualistic plant
microbe interaction. The evolution of the symbiotic relationship between legume and rhizobia depends heavily on NF perception and downstream signaling cascade for rhizobial infection, infection thread formation, and nodule organogenesis. Characterization of TFs and identifying their targets is a key advancement in understanding the evolutionary difference between nitrate signaling in nonlegumes and nodule symbiosis in legumes. Furthermore, it may be used as the blueprint for engineering and improving the nodulation trait in legumes, or even to transfer it to nonlegume crops, which can help the crops to grow without nitrogen fertilizer. The further unearthing of genes involved in the transduction of symbiotic signals downstream of NF perception will help us create more efficient and highly specific symbiotic pairs of crop plants and nitrogen-fixing bacteria. Many regulatory events, including activation of the transcriptional machinery and downstream signaling processes, have been well characterized and explored in model leguminous plants such as L. japonicus and M. truncatula. However, this knowledge is limited in the case of legume crops. Thus it will be interesting to explore evolution in the NF signaling cascade to regulate tight host-symbiont association for N2 fixation. Future studies focusing on conserved and nonconserved mechanisms of NF signaling in various legume-rhizobia interactions, especially crop legumes like chickpea and others, will undoubtedly bring insight into its regulation in nodulation and the evolutionary origin of RNS. Understanding the mechanism of NF signaling from perception through LysM-RLK to downstream signaling cascade and its regulation at transcription and posttranscription level is required from an evolutionary and structural perspective to translate this study from model legumes to crop legumes. The knowledge obtained from these studies and the translation of this knowledge to the crop plants will be breakthrough tools for agriculture in the field.

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Acknowledgments This work is supported by a core grant from the National Institute of Plant Genome Research, New Delhi. JS thanks the Council of Scientific & Industrial Research, Government of India, for the fellowship.

Declaration of competing interest The authors declare that there is no conflict of interest.

Contribution JS and PKV conceptualized this work. JS and PKV wrote the manuscript and read and approved the final version of the manuscript.

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654. Zhao, L., Zhang, W., Yang, Y., Li, Z., Li, N., Qi, S., et al., 2018. The Arabidopsis NLP7 gene regulates nitrate signaling via NRT1.1-dependent pathway in the presence of ammonium. Scientific Reports 8 (1), 1487. Available from: http://www.nature.com/scientificreports.

SECTION

Plant TFs and metabolism

III

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CHAPTER

The regulatory aspects of plant transcription factors in alkaloids biosynthesis and pathway modulation

9

Pravin Prakash1, Rituraj Kumar1,2 and Vikrant Gupta1,2 1

Plant Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, Uttar Pradesh, India 2Academy of Scientific and Innovative Research (AcSIR), CSIR-HRDC Campus, Ghaziabad, Uttar Pradesh, India

Abbreviations AP2/ERF bHLH bZIP ChIP CRISPR/Cas9 EMS IQAs JRE MeJA MEP MIA MYB NIC2 Nrf ORCA RNAi SGA TIA TILLING VIGS ZCT

APETALA2/ethylene response factor basic helix-loop-helix basic leucine zipper chromatin immunoprecipitation clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 ethyl methanesulfonate isoquinoline alkaloids jasmonate-responsive element methyl jasmonate methylerythritol-4-phosphate monoterpenoid indole alkaloid myeloblastosis nicotine2 NF-E2-related factor octadecanoid-derivative responsive Catharanthus AP2-domain RNA-interference steroidal glycoalkaloid terpenoid indole alkaloid targeting-induced local lesions in genomes virus-induced gene silencing zinc finger Catharanthus protein

9.1 Introduction Plants are the prominent source of alkaloids. Plant alkaloids are usually amino acid-derived nitrogen-containing basic organic compounds that are present in about 20% of plant species (Facchini, 2001; Lichman, 2021). Besides plants, the alkaloids are also produced by bacteria Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00019-4 © 2023 Elsevier Inc. All rights reserved.

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(Massingill and Hodgkins, 1967; Singh and Upadhyay, 2021), fungi (Zhang et al., 2012a), and animals (Braekman et al., 1998). In plants, alkaloids are derived from a limited number of amino acid precursors. The amino acids that serve as precursors of alkaloid biosynthesis include lysine (Lys), ornithine (Orn), phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). Ornithine-derived major alkaloids include pyrrolidine and tropane alkaloids (Brauch et al., 2016). Major alkaloids derived from lysine include indolizidine, piperidine, and quinolizidine alkaloids (Bunsupa et al., 2017). Benzylisoquinoline alkaloids are the major alkaloids derived from tyrosine (Lichman, 2021). Terpenoid indole alkaloids (TIAs) are the important alkaloids derived from tryptophan (Ramani et al., 2013). Like other plant secondary metabolites, the alkaloids have significant economic and medicinal value. The plants of Amaryllidaceae, Apocynaceae, Asteraceae, Fabaceae, Papaveraceae, Rubiaceae, Rutaceae, and Solanaceae families serve as major sources of plant alkaloids having significant pharmaceutical values (Dixit et al., 2021a; Krishnan et al., 2021; Upadhyay and Singh, 2021; Yang and Sto¨ckigt, 2010). Plant-based alkaloids that retain significant medicinal values include antineoplastic agents such as camptothecin, Taxol, vincristine, and vinblastine, analgesic compound morphine, antimalarial alkaloid quinine, antihypertensive compounds such as ajmalicine and serpentine, antiarrhythmic alkaloid ajmaline, antimicrobial compound berberine and sanguinarine, and vasodilator agent papaverine. Recently, plant-derived alkaloids have been reported to be effective against coronaviruses as well (Fielding et al., 2020; Majnooni et al., 2021). The biosynthesis of specialized metabolites in plants is a complex process and proceeds through strict regulation at the molecular level. Several genes, regulatory proteins, and small RNAs such as microRNAs (miRNAs) coordinate and control the process of secondary metabolism in plants. Some genes encode for DNA-binding regulatory proteins known as transcription factors (TFs). TFs interact with the cis-elements present on the promoter regions of their target genes and regulate their expression levels. In plants, TFs play a major role in the regulation of several secondary metabolic pathways including the alkaloid biosynthesis pathway. The TFs that participate in alkaloid biosynthesis in plants majorly belong to APETALA2/ethylene response factor, basic helix-loop-helix (bHLH), basic leucine zipper (bZIP), Cys2/His2-type (transcription factor IIIA-type) zinc-finger protein family, MYB, and WRKY families. These TFs are known to regulate the biosyntheis of various economically and medicinally important alkaloids in several plant species such as terpenoid indole alkaloids (TIA) biosyntheis in Catharanthus roseus (van der Fits and Memelink, 2000; Zhou et al., 2010; De Boer et al., 2011; Suttipanta et al., 2011; Li et al., 2013a; Van Moerkercke et al., 2015; Van Moerkercke et al., 2016; Sui et al., 2018; Liu et al., 2019b; Pan et al., 2019; Paul et al., 2020; Singh et al., 2020), nicotine bisynthesis in Nicotiana sp. (Todd et al., 2010; Zhang et al., 2012b; Sears et al., 2014; Yang et al., 2016; Liu et al., 2019a; Sui et al., 2019; Hayashi et al., 2020), camptothecin biosynthesis in Ophiorrhiza pumila (Rohani et al., 2016; Wang et al., 2019), steroidal glycoalkaloids (SGAs) biosynthesis in Solanum lycopersicum (Nakayasu et al., 2018), benzylisoquinoline alkaloids (BIAs) biosynthesis in Coptis japonica (Kato et al., 2007; Yamada et al., 2011), Eschscholzia californica (Yamada et al., 2015), Nelumbo nucifera (Li et al., 2019), and Papaver somniferum (Mishra et al., 2013), Paclitaxel (Taxol) biosynthesis in Taxus cuspidata (Lenka et al., 2015), camptothecin (CPT) biosynthesis in Camptotheca acuminata (Chang et al., 2019), capsaicinoids biosynthesis in Capsicum annuum (Arce-Rodrı´guez and Ochoa-Alejo, 2017; Sun et al., 2019; Zhu et al., 2019), and sesquiterpene pyridine alkaloids biosynthesis in Tripterygium wilfordii Hook.f. (Han et al., 2020).

9.2 Plant transcription factor families involved in alkaloid biosynthesis

201

Several studies have demonstrated the potential of TFs to precisely modulate the plant alkaloid biosynthesis pathways. The TF-mediated modulation allows achieving the enhanced yield of commercially and medicinally important alkaloids either through the generation of stable transgenic plants or hairy root cultures. Genetic manipulation approaches such as TF overexpression, downregulation, and CRISPR/Cas-mediated editing of a candidate TF at the genome level allow the precise and rational modulation of the alkaloid biosynthesis pathway to achieve higher content of these metabolites (Upadhyay, 2021). This chapter highlights information regarding the TFs that regulate alkaloids biosynthesis in plants, and TF-mediated strategies to modulate the biosynthesis of alkaloids of commercial and medicinal importance are discussed.

9.2 Plant transcription factor families involved in alkaloid biosynthesis regulation Transcription factors play an important regulatory role in the biosynthesis of secondary metabolites by controlling the expression of important pathway genes. Table 9.1 provides a list of plant TF families and their members involved in alkaloid biosynthesis.

9.2.1 APETALA2/ethylene response factor The APETALA2/Ethylene-Responsive Factor (AP2/ERF) superfamily TFs harbors about 60 to 70 amino acids (aa) long AP2/ERF domain that interact with the cis-elements present on the promoter region of its target genes (Sakuma et al., 2002; Magnani et al., 2004; Nakano et al., 2006). The AP2 proteins play a major role in plant development and response to various stress conditions (Sakuma et al., 2002; Mizoi et al., 2012). The AP2/ERF families TFs are very well reported to regulate alkaloid biosynthesis-related genes in plants such as C. roseus, Nicotiana tabacum, Solanum tuberosum, and S. lycopersicum. In C. roseus, octadecanoid-derivative responsive Catharanthus AP2-domain (ORCA) family TFs participate in the regulation of terpenoid indole alkaloids biosynthesis. The ORCA TFs predominantly regulate the terpenoid indole alkaloid (TIA) biosynthesis genes in C. roseus. Several members of the ORCA family such as ORCA2 (Menke et al., 1999; Li et al., 2013a), ORCA3 (van der Fits and Memelink, 2000; Zhou et al., 2010), and ORCA6 (Singh et al., 2020) play crucial roles in terpenoid indole alkaloids biosynthesis and regulation in C. roseus. Strictosidine synthase (Str) is a key gene of the TIA pathway. ORCA2 trans-activates the Str gene promoter and regulates jasmonate- and elicitor-responsive expression of Str (Menke et al., 1999). Studies on C. roseus transgenic hairy root lines that overexpress ORCA2 through an ethanolinducible promoter indicate the crucial role of ORCA2 in the downstream segments of the TIA pathway (Li et al., 2013a). ORCA3, a jasmonate-responsive APETALA2 (AP2)-domain harboring TF, was isolated from C. roseus by using the T-DNA activation tagging approach. ORCA3 overexpression upregulated several key genes of the TIA pathway and led to the elevated accumulation of TIAs in C. roseus (van der Fits and Memelink, 2000). In C. roseus hairy root lines, ORCA3 overexpression led to a reduction in catharanthine accumulation, however, MeJA treatment led to a significant increase in catharanthine content (Zhou et al., 2010). TIA pathway gene expression and TIA accumulation was enhanced upon ORCA6 overexpression in C. roseus flower petals (Singh et al., 2020).

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Table 9.1 Transcription factors regulating alkaloid biosynthesis in different plant species. Transcription factor family APETALA2/ Ethylene Response Factor

Transcription factor Octadecanoid-derivative responsive Catharanthus AP2-domain2 (ORCA2) ORCA3 CrORCA3 ORC1 ORCA2 NtERF32 GLYCOALKALOID METABOLISM 9 (GAME9) OpERF2 CR1 (Catharanthus roseus 1) StWRKY8 JRE4

NtERF32, NtERF221/ ORC1 CrERF5 Ethylene Response Factor 91 (ERF91) NtERF189 and NtERF199 NIC2 ERF189 ORCA5 ORCA6 Basic helix-loophelix (bHLH)

NbbHLH1/NbbHLH2 NtMYC2 CjbHLH1 CrMYC2 NtMYC2a and NtMYC2b TcJAMYC1, TcJAMYC2, and TcJAMYC4 bHLH iridoid synthesis 1 (BIS1) EcbHLH1
1 and EcbHLH1
2

Alkaloid biosynthesis pathway

Plant species

References

Terpenoid indole alkaloids

Catharanthus roseus

Menke et al. (1999)

Terpenoid indole alkaloids Catharanthine Nicotine Terpenoid indole alkaloids Nicotine and total alkaloids Steroidal glycoalkaloids

Catharanthus roseus Catharanthus roseus Nicotiana tabacum Catharanthus roseus

van der Fits and Memelink (2000) Zhou et al. (2010) De Boer et al. (2011) Li et al. (2013a)

Nicotiana tabacum

Sears et al. (2014)

Solanum tuberosum and Solanum lycopersicum

C´ardenas et al. (2016)

Camptothecin Terpenoid indole alkaloids Benzylisoquinoline alkaloids Steroidal glycoalkaloids (SGAs) Nicotine

Ophiorrhiza pumila Catharanthus roseus

Udomsom et al. (2016) Liu et al. (2017)

Solanum tuberosum

Yogendra et al. (2017)

Solanum lycopersicum

Nakayasu et al. (2018)

Nicotiana tabacum

Liu et al. (2019a)

Bisindole alkaloids Nicotine

Catharanthus roseus Nicotiana tabacum

Pan et al. (2019) Sui et al. (2019)

Nicotine

Nicotiana sp.

Hayashi et al. (2020)

Terpenoid indole alkaloid Nicotine Terpenoid indole alkaloids Nicotine biosynthesis Nicotine biosynthesis Isoquinoline alkaloids Terpenoid indole alkaloid Nicotine

Catharanthus roseus

Paul et al. (2020)

Nicotiana sp. Catharanthus roseus

Paul et al. (2020) Singh et al. (2020)

Nicotiana sp. Nicotiana sp. Coptis japonica Catharanthus roseus

Todd et al. (2010) Shoji and Hashimoto (2011) Yamada et al. (2011) Zhang et al. (2011)

Nicotiana tabacum

Zhang et al. (2012b)

Paclitaxel (Taxol)

Taxus cuspidata

Lenka et al. (2015)

Monoterpenoid indole alkaloids Isoquinoline alkaloids

Catharanthus roseus

Van Moerkercke et al. (2015) Yamada et al. (2015)

Eschscholzia californica

9.2 Plant transcription factor families involved in alkaloid biosynthesis

203

Table 9.1 Transcription factors regulating alkaloid biosynthesis in different plant species. Continued Transcription factor family

Transcription factor BIS2 NtMYC2a Repressor of MYC2 Targets 1 (RMT1) CrMYC2

Basic leucine zipper (bZIP)

Cys(2)/His(2)-type (transcription factor IIIA-type) zinc finger protein family/Zinc finger Catharanthus protein (ZCT) family Class I TGA

CrGBF1 G-box binding factor 1 (GBF1) CaLMF (Light-Mediated CPT biosynthesis Factor) ZCT1, ZCT2, and ZCT3 ZCT1 and ZCT2

TwTGA1

GATA

CrGATA1

MYB

GmMYBZ2 OpMYB1 CaMYB31

WRKY

CaMYB108 MYB31 CjWRKY1 CrWRKY1 TcWRKY1 PsWRKY CjWRKY1 StWRKY8 NnWRKY40a and NnWRKY40b OpWRKY3

Alkaloid biosynthesis pathway Monoterpenoid indole alkaloid production Nicotine and related pyridine alkaloids Monoterpene indole alkaloids Terpenoid indole alkaloids Terpenoid indole alkaloids Camptothecin

Terpenoid indole alkaloids Monoterpenoid indole alkaloids

Sesquiterpene pyridine alkaloids Terpenoid indole alkaloids Catharanthine Camptothecin Capsaicinoids Capsaicinoids Capsaicinoids Benzylisoquinoline alkaloids Terpenoid indole alkaloids Taxol Benzylisoquinoline alkaloids Benzylisoquinoline alkaloids Benzylisoquinoline alkaloids Benzylisoquinoline alkaloids Camptothecin

Plant species

References

Catharanthus roseus Nicotiana tabacum

Van Moerkercke et al. (2016) Yang et al. (2016)

Catharanthus roseus

Patra et al. (2018)

Catharanthus roseus

Sui et al. (2018)

Catharanthus roseus

Sui et al. (2018)

Camptotheca acuminata

Chang et al. (2019)

Catharanthus roseus

Pauw et al. (2004)

Catharanthus roseus

Chebbi et al. (2014)

Tripterygium wilfordii Hook.f. Catharanthus roseus

Han et al. (2020)

Catharanthus roseus Ophiorrhiza pumila Capsicum annuum Capsicum sp. Capsicum chinense Coptis japonica

Zhou et al. (2011) Rohani et al. (2016) Arce-Rodrı´guez and OchoaAlejo (2017) Sun et al. (2019) Zhu et al. (2019) Kato et al. (2007)

Catharanthus roseus

Suttipanta et al. (2011)

Taxus chinensis Papaver somniferum

Li et al. (2013b) Mishra et al. (2013)

Eschscholzia californica Solanum tuberosum

Yamada et al. (2017)

Nelumbo nucifera

Li et al. (2019)

Ophiorrhiza pumila

Wang et al. (2019)

Liu et al. (2019b)

Yogendra et al. (2017)

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C. roseus ORCA5 overexpression in N. tabacum hairy roots elevates the putrescine Nmethyltransferase (PMT) and quinolinate phosphoribosyltransferase (QPT) gene expression, and N. tabacum ERF189 overexpression in C. roseus hairy roots upregulate STR gene expression levels and positively affects TIA accumulation (Paul et al., 2020). These studies provide evidence regarding the intracluster and mutual regulation of AP2/ERF gene clusters for specialized metabolite biosynthesis in different plant species (Paul et al., 2020). N. tabacum ORC1 is a close homolog of C. roseus ORCA3. ORC1 overexpression positively affects alkaloid biosynthesis in tobacco (De Boer et al., 2011). C. roseus ORCA3 requires only the GCC motif found in the promoters of TIA biosynthesis pathway genes, while N. tabacum ORC1 requires both GCC-motif and G-box elements present in the promoters of nicotine biosynthesis-related genes for its optimum activity (De Boer et al., 2011). Three non-NIC2 locus AP2/ERF genes—NtERF1, NtERF32, and NtERF121—respond to methyl jasmonate (MeJA) treatment and bind to the GCC box-like element. NtERF32 overexpression elevates putrescine N-methyltransferase1a (NtPMT1a) gene expression and upregulates total alkaloids contents in tobacco. RNAi-mediated suppression of NtERF32 downregulates the expression of key genes of the nicotine biosynthesis pathway (i.e., NtPMT1a and NtQPT2), and negatively affected nicotine and total alkaloid content in N. tabacum (Sears et al., 2014). NtERF32, NtERF221/ORC1, and NtMYC2a TFs overexpression significantly increase the nicotine biosynthesis in tobacco. The overexpression of NtERF221/ORC1 under the control of GmUBI3 gene promoter along with MeJA treatment effectively elevated the pyridine alkaloid nicotine biosynthesis ( . nine-fold) in tobacco (Liu et al., 2019a). Genetic manipulation of NtERF189 and NtERF199 alters nicotine biosynthesis in tobacco. Transient overexpression of NtERF189 facilitates the alkaloid production in Nicotiana benthamiana and Nicotiana alata leaf tissues. CRISPR/Cas9-mediated knockout of NtERF189 and NtERF199 drastically reduced the total alkaloid content in tobacco (Hayashi et al., 2020). NtERF91, a non-NIC2 locus MeJA-inducible gene, interacts with NtPMT2 and NtQPT2 gene promoters via GCC-box (cis-element). NtERF91 overexpression elevated the expression levels of nicotine biosynthesis genes and affected the total alkaloids production (Sui et al., 2019). GLYCOALKALOID METABOLISM 9 (GAME9) TF regulates the biosynthesis of steroidal glycoalkaloids (SGAs) in potato and tomato (C´ardenas et al., 2016). Catharanthus roseus 1 (CR1) TF also regulates TIA biosynthesis in C. roseus (Liu et al., 2017). Virus-induced gene silencing (VIGS) of CR1 led to upregulated expression of seven key genes of the TIA pathway. Metabolite analysis indicated the increased vindoline and serpentine content in CR1-silenced lines (Liu et al., 2017). C. roseus AP2/ERF TF CrERF5 overexpression positively affected the TIA biosynthesis pathway genes, and metabolite analysis indicated the enhanced accumulation of secologanin, catharanthine, anhydrovinblastine, and vinblastine contents. Downregulation of CrERF5 through VIGS significantly affected the key genes of the TIA pathway and led to a reduction in secologanin, catharanthine, ajmalicine, anhydrovinblastine, and vinblastine contents in CrERF5silenced plants. It indicates that CrERF5 positively regulates TIA biosynthesis in C. roseus (Pan et al., 2019). OpERF2 is involved in the regulation of camptothecin (CPT) biosynthesis in Ophiorrhiza pumila. RNAi-suppressed O. pumila OpERF2 transgenic hairy root lines indicated reduced expression of genes involved in CPT biosynthesis. The study indicates that OpERF2 positively regulates CPT biosynthesis in O. pumila (Udomsom et al., 2016). Solanum lycopersicum JRE4 that belongs to the JA-responsive ethylene response factor (ERF) transcription factors (JREs) family regulates steroidal glycoalkaloid (SGA) biosynthesis (Nakayasu et al., 2018). Targetinginduced local lesions in genomes (TILLING)-based screening of ethyl methanesulfonate (EMS)-

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mutagenized S. lycopersicum population led to the identification of jre4 mutant. The jre4 mutant lines exhibit reduced expression of SGA biosynthetic genes and lower SGA contents. JRE4 overexpression enhanced the SGA content in tomato. The study suggests the regulatory roles of JRE4 in SGA biosynthesis and defense response in S. lycopersicum (Nakayasu et al., 2018).

9.2.2 Basic helix-loop-helix Basic helix
loop
helix (bHLH) TFs are well kown to regulate various developmental processes in plants. The bHLH family TFs generally undergoes homo- and hetero-dimerization to precisely regulate the transcription of their target genes. MYB proteins are the well-known interacting partners of bHLH TFs to control the expression levels of target genes (Heim et al., 2003; Feller et al., 2011). Several bHLH family TFs regulate secondary metabolite biosyntheis including alkaloid biosynthesis. The bHLH TF family members NbbHLH1/NbbHLH2 (Todd et al., 2010), NtMYC2 (Shoji and Hashimoto, 2011), NtMYC2a and NtMYC2b (Zhang et al., 2012b), and NtMYC2a (Yang et al., 2016) regulate nicotine biosynthesis in tobacco species. N. benthamiana NbbHLH1 and NbbHLH2 interact with G-box cis-elements present in PMT gene promoters. Overexpression of NbbHLH1 and NbbHLH2 elevate nicotine content in N. benthamiana. VIGS- and RNAi-mediated suppression of NbbHLH1 and NbbHLH2 reduce the expression levels of nicotine biosynthesis pathway genes and total nicotine content (Todd et al., 2010). NtMYC2 interact with G-box cis-element present in the promoters of putrescine N-methyltransferase2 (PMT2) and quinolinate phosphoribosyltransferase2 (QPT2) genes, and also upregulate the NIC2-locus ERF genes. Through these two combinatorial ways, NtMYC2 controls jasmonate-inducible nicotine biosynthesis-related genes in tobacco (Shoji and Hashimoto, 2011). N. tabacum NtMYC2a, NtMYC2b, and NtMYC2c encode bHLH TFs and show induced expression upon MeJA treatment. RNAi-mediated suppression of NtMYC2a and NtMYC2b led to a significant reduction in the NtPMT1a and putative nicotine synthase gene NtA662 transcripts. However, the overexpression of NtMYC2a and NtMYC2b did not affect NtPMT1a gene expression. NtMYC2a and MtMYC2b bind to the G-box cis-element present in the NtPMT1a gene promoter. In the absence of JA, NtMYC2a and NtMYC2b form the nuclear complexes with the NtJAZ1 repressors. The study indicated that NtMYC2a and MtMYC2b act as transcriptional activators of nicotine biosynthesis in tobacco (Zhang et al., 2012b). NtMYC2a stimulates the JA biosynthesis-associated genes and elevates NtMYC2a activity upon hightemperature (HT) treatment. NtMYC2a interact with the NtPMT1 gene promoter, and hightemperature promotes NtMYC2a transcription and nicotine biosynthesis (Yang et al., 2016). In Coptis japonica, CjbHLH1 regulates the biosynthesis of isoquinoline alkaloids. Chromatin immunoprecipitation (ChIP) experiment using specific antibodies raised against CjbHLH1 indicated the specific interaction of CjbHLH1 with 30 -hydroxy-N-methylcoclaurine-40 -O-methyltransferase (40 OMT) and canadine synthase (CYP719A1) gene promoters (Yamada et al., 2011). Likewise, EcbHLH1
1 and EcbHLH1
2 regulate isoquinoline alkaloid (IQA) biosynthesis in Eschscholzia californica. RNAi-mediated suppression of EcbHLH1 led to reduced expression of IQA biosynthesis pathway genes, and metabolite analysis indicated a decrease in sanguinarine content (Yamada et al., 2015). TcJAMYC1, TcJAMYC2, and TcJAMYC4 TFs regulate paclitaxel (Taxol) biosynthesis in Taxus cuspidata (Lenka et al., 2015). The bHLH TF family members actively participate in the biosynthesis of terpenoid indole alkaloids (TIAs) in C. roseus. The bHLH TFs that are involved in C. roseus TIA biosynthesis and pathway regulation include CrMYC2 (Zhang et al., 2011),

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bHLH iridoid synthesis 1 (BIS1) (Van Moerkercke et al., 2015), bHLH iridoid synthesis 2 (BIS2) (Van Moerkercke et al., 2016), Repressor of MYC2 Targets 1 (RMT1) (Patra et al., 2018), and CrMYC2 (Sui et al., 2018). C. roseus CrMYC2 interacts with the qualitative sequence present in ORCA3 jasmonate-responsive element (JRE), activates the expression of MeJA-responsive ORCA3, and thus regulates alkaloid biosynthesis (Zhang et al., 2011). bHLH iridoid synthesis 1 (BIS1) overexpression in C. roseus suspension cell cultures led to enhanced biosynthesis of (seco)-iridoid and MIAs. BIS1 trans-activates the promoters of iridoid pathway genes present upstream of loganic acid methyltransferase (LAMT), and does not require JA elicitation or exogenous precursors to boost the MIA biosynthesis (Van Moerkercke et al., 2015). Another bHLH TF, BIS2 from C. roseus, is a jasmonate (JA)-responsive TF. BIS2 overexpression upregulates the expression levels of methylerythritol-4-phosphate (MEP) and iridoid pathway genes. Suppression of BIS2 expression completely terminates the JA-induced upregulation of genes involved in iridoid biosynthesis and MIA accumulation (Van Moerkercke et al., 2016). Repressor of MYC2 target 1 (RMT1), a JAresponsive bHLH TF, is activated by CrMYC2 and BIS1. RMT1 passively represses the CrMYC2 activity. RMT1 together with CrMYC2, BIS1/2 make a bHLH TF network. C. roseus JA signaling component CORONATINE INSENSITIVE 1 (COI1) acts upstream to the bHLH TF network and regulates MIA biosynthesis (Patra et al., 2018). CrMYC2 overexpression induces bZIP factors CrGBFs (CrGBF1 and CrGBF2) and lead to a reduction in alkaloid contents in C. roseus hairy roots. CrGBF1 and CrGBF2 undergo homo- and hetero-dimerization and fine-tune the TIA pathway gene expression (Sui et al., 2018).

9.2.3 Basic leucine zipper Basic leucine zipper domain (bZIP) TFs harbor a 40
80 amino acid long bZIP domain that consists of two motifs: one is the basic region that allows specific interaction of bZIP TFs with its targets and another leucine zipper motif that participate in TF dimerization (Correˆa et al., 2008; Alves et al., 2013). The bZIP TFs regulate the diverse vital and developmental processes in plants. Their implications in plant secondary metabolism, including alkaloid biosynthesis, has also been reported. CaLMF (Light-Mediated CPT biosynthesis Factor) regulates camptothecin biosynthesis in Camptotheca acuminata. Light negatively affects the camptothecin biosynthesis. Under shade conditions, higher expression of CaMLF leads to the downregulated expression of camptothecin biosynthesis genes and negatively affects camptothecin content in leaves (Chang et al., 2019). The silencing of CaMLF completely abolished the expression of camptothecin biosynthesis genes and camptothecin biosynthesis under shade conditions. One study indicated the role of CaLMF in the light-mediated regulation of CPT biosynthesis in C. acuminata (Chang et al., 2019). G-box binding factors (CrGBF1 and CrGBF2) expression is induced by bHLH factor CrMYC2. The homo- and hetero-dimer formation by CrGBF1 and CrGBF2 led to a reduction in the transcriptional activities of key genes of the TIA pathway. CrGBF1 interact with T/G-box cis-element, which is also a preferred binding sequence of CrMYC2 in target gene promoters. CrGBFs acts as a repressor by competing for binding to the same cis-element (T/G-box) and prevent CrMYC2 from binding to its target gene promoters and thus modulating TIA biosynthesis in C. roseus (Sui et al., 2018).

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9.2.4 Cys2/His2-type (transcription factor IIIA-type) zinc-finger protein family/ Zinc-finger Catharanthus protein (ZCT) family In eukaryotes, the C2H2-type zinc-finger proteins usually retain a conserved 25
30 amino acids long sequence: C-X2B4-C-X3-P-X5-L-X2-H-X3-H (Ciftci-Yilmaz and Mittler, 2008; Shimeld, 2008). Mostly C2H2-type zinc-finger proteins in plants harbor a conserved QALGGH sequence within their zinc-finger domains and are known as Q-type zinc-finger proteins. The C2H2-type zinc-finger proteins without any conserved motifs within their zinc-finger domains are known as C-type zincfinger proteins (Agarwal et al., 2007; Liu et al., 2015). C2H2-type zinc-finger family TFs regulate several plant-specific processes including development, biotic stress resistance, and secondary metabolism. A few C2H2-type zinc finger family TFs also participate in the regulation of alkaloid metabolism. C. roseus ZCT1, ZCT2, and ZCT3 belong to Cys2/His2-type (transcription factor IIIAtype) zinc-finger protein family and exhibit induced expression upon treatment with MeJA and yeast extract (Pauw et al., 2004). The ZCT proteins interact with strictosidine synthase (STR) and tryptophan decarboxylase (TDC) gene promoters and repress their activity. ZCT proteins act as repressors of genes regulating alkaloid biosynthesis in C. roseus (Pauw et al., 2004). Zinc-finger Catharanthus protein (ZCT) family TFs, ZCT1 and ZCT2, regulate monoterpenoid indole alkaloid (MIA) biosynthesis in C. roseus. MeJA treatment upregulates the ZCT2 gene expression and ZCT2 bind to the promoter region of hydroxymethylbutenyl 4-diphosphate synthase (HDS) gene (Chebbi et al., 2014). Trans-activation assay indicated that ZCT1 and ZCT2 repress the HDS promoter activity. Studies suggest the role of ZCT1 and ZCT2 TFs in MIA biosynthesis regulation by acting as a repressor of HDS gene activity (Chebbi et al., 2014).

9.2.5 Myeloblastosis MYB (myeloblastosis) TFs harbor a characteristic MYB DNA-binding domain and one to four imperfect repeats of around 52 amino acids (Feller et al., 2011). MYB TFs are involved in the regulation of several plant-specific processes including the regulation of secondary metabolism. The MYB TFs have also been reported to control the alkaloids biosynthesis in plants. Cotransformation of C. roseus hairy roots with a soybean GmMYBZ2 alters the expression of catharanthine biosynthetic pathway genes, which led to a reduction in catharanthine production. GmMYBZ2 upregulates the ZCT1 transcripts and lowers the expression of ASα, STR, and ORCA3 genes, and thus negatively affects catharanthine biosynthesis in C. roseus hairy roots (Zhou et al., 2011). OpMYB1 TF regulates camptothecin biosynthesis in Ophiorrhiza pumila. OpMYB1 overexpression in O. pumila hairy roots resulted in a reduction in the transcripts of the tryptophan decarboxylase (OpTDC) gene, which is involved in the camptothecin biosynthesis pathway. OpMYB1 also suppresses seco-iridoids, monoterpene indole alkaloids, anthraquinone, and chlorogenic acid biosynthesis pathway genes and thus negatively regulates the alkaloids and other secondary metabolites in O. pumila (Rohani et al., 2016). An R2R3-MYB TF, CaMYB31, controls capsaicinoid biosynthesis in Capsicum annuum. CaMYB31 silencing downregulates the expression of genes involved in capsaicinoid biosynthesis and negatively affects capsaicinoid production (ArceRodrı´guez and Ochoa-Alejo, 2017). CaMYB108 TF belongs to the R2R3-MYB family and regulates capsaicinoid biosynthesis in Capsicum sp. Suppression of the transcripts of CaMYB108 through VIGS reduced the expression of capsaicinoid-biosynthetic genes (CBGs) and had an

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adverse impact on capsaicinoid content (Sun et al., 2019). MYB31 regulates capsaicinoid biosynthesis in Capsicum chinense. MYB31 activates the expression of capsaicinoid biosynthesis pathway genes and positively affects capsaicinoid biosynthesis in pepper (Zhu et al., 2019).

9.2.6 WRKY WRKY family of plant TFs harbors one or two conserved DNA-binding WRKY [tryptophan (W)arginine (R)-lysine(K)-tyrosine(Y)] domains. WTKY TFs contain a highly conserved WRKYGQK sequence followed by a CX4
5CX22
23HXH or CX7CX23HXC zinc-finger motif (Rushton et al., 2010). Two WRKY domains are present in group I WRKY TFs, whereas a single WRKY domain lies in group II and III WRKY proteins (Zhang and Wang, 2005; Rushton et al., 2010). The distinct zinc-finger motif (CX7CX23HXC) is a peculiar feature of group III WRKY proteins (Zhang and Wang, 2005; Rushton et al., 2010). The WRKY TFs bind with the W-box (C/TTGACT/C) cis-elements present in the promoter sequences of the target genes and regulate their transcription (Eulgem et al., 2000; Zhang and Wang, 2005). Besides regulating diverse biological processes, the WRKY TFs also regulate plant secondary metabolite biosynthesis. During the past few years, many studies have uncovered the roles of WRKY TFs in alkaloid biosynthesis regulation. Coptis japonica CjWRKY1 regulates benzylisoquinoline alkaloid berberine biosynthesis. RNAi-mediated silencing led to a marked reduction in all the genes involved in berberine biosynthesis, whereas the overexpression of CjWRKY1 in C. japonica protoplasts upregulate the transcripts of all the genes involved in berberine biosynthesis (Kato et al., 2007). The study indicates that CjWRKY1 positively regulates benzylisoquinoline alkaloid “berberine” biosynthesis in C. japonica (Kato et al., 2007). CrWRKY1 regulates TIA biosynthesis in C. roseus. CrWRKY1 overexpression led to upregulation of key genes of TIA pathway like tryptophan decarboxylase (TDC), transcriptional repressors zincfinger C. roseus transcription factors (ZCT1, ZCT2, and ZCT3), and repressed the transcripts of ORCA2, ORCA3, and CrMYC2 (Suttipanta et al., 2011). CrWRKY1 interact with W-box cis-elements present in the TDC gene promoter. C. roseus hairy roots overexpressing CrWRKY1 indicated root-specific enhancement and accumulation of serpentine (Suttipanta et al., 2011). Taxol biosynthesis is regulated by TcWRKY1 in Taxus chinensis. In T. chinensis, 10-deacetylbaccatin III-10 bO-acetyl transferase (DBAT) is a key rate-limiting enzyme involved in taxol biosynthesis. The DBAT gene promoter contains W-box with which the TcWRKY1 interacts (Li et al., 2013b). Overexpression of TcWRKY1 in T. chinensis cell suspension cultures upregulated the expression of DBAT gene transcripts while TcWRKY1-silencing had a negative effect on DBAT expression. The study indicates the role of TcWRKY1 in the regulation of taxol biosynthesis in T. chinensis (Li et al., 2013b). PsWRKY, a wound-induced TF, regulates benzylisoquinoline alkaloid (BIA) biosynthesis in Papaver somniferum. PsWRKY interacts with the W-box cis-element present in the promoters of genes involved in BIA biosynthesis. Yeast one-hybrid assay and protoplast transient analysis indicated the activation of tydc 50 -upstream activating sequence (UAS) by PsWRKY. The study suggests the role of PsWRKY in wound-induced regulation of BIAs biosynthesis in P. somniferum (Mishra et al., 2013). Heterologous expression of Coptis japonica CjWRKY1 in Eschscholzia californica cultured cells upregulated the transcription of BIA pathway genes (Yamada et al., 2017). CjWRKY1 overexpression enhanced the content of several BIAs including 10-hydroxychelerythrine, allocryptopine, chelerythrine, chelirubine, protopine, and sanguinarine (Yamada et al., 2017). Besides C. japonica, CjWRKY1 also regulates BIA biosynthesis in E.

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californica (Yamada et al., 2017). StWRKY8 TF regulates BIA biosynthesis in Solanum tuberosum. StWRKY8 TF interacts with the promoter regions of BIA biosynthetic genes. The higher expression of StWRKY8 upon fungal infection indicates its role in defense responses through an enhanced accumulation of BIAs (Yogendra et al., 2017). NnWRKY40a and NnWRKY40b regulate BIA biosynthesis in Nelumbo nucifera (Li et al., 2019). JA treatment significantly induces the NnWRKY40a and NnWRKY40 expression. NnWRKY40a and NnWRKY40 activate the promoter of key genes of the BIA pathway such as NnTYDC, NnCYP80G, and Nn7OMT to regulate BIA biosynthesis in lotus (Li et al., 2019). Camptothecin biosynthesis is regulated by OpWRKY3 TF in Ophiorrhiza pumila (Wang et al., 2019). OpWRKY3 overexpression led to enhanced camptothecin biosynthesis, while OpWRKY3 silencing led to a reduction in camptothecin biosynthesis in O. pumila hairy roots. The study suggests that OpWRKY3 acts as a potential regulator of camptothecin biosynthesis in O. pumila (Wang et al., 2019).

9.2.7 Other transcription factors A Class I TGA transcription factor, TwTGA1, regulates sesquiterpene pyridine alkaloids biosynthesis in Tripterygium wilfordii Hook.f. TwTGA1 overexpression enhanced the biosynthesis of triptolide and two sesquiterpene pyridine alkaloids (wilforgine and wilforine) (Han et al., 2020). RNAi-mediated suppression of TwTGA1 did not affect the biosynthesis of these metabolites (Han et al., 2020). A GATA family factor, CrGATA1, regulates terpenoid indole alkaloid biosynthesis in C. roseus. C. roseus LLM-domain GATA TF, CrGATA1 exhibits light-inducible expression. CrGATA1 overexpression upregulates the expression of vindoline pathway genes and promotes vindoline biosynthesis. VIGS-assisted suppression of CrGATA1 resulted in reduced expression of vindoline biosynthesis genes and ultimately displayed reduced vindoline content (Liu et al., 2019b). C. roseus phytochrome interacting factor (CrPIF1) acts as a repressor of CrGATA1, and negatively affects the vindoline biosynthesis (Liu et al., 2019b).

9.3 Transcription factor-mediated modulation of alkaloid biosynthesis pathways TFs represent themselves as potential candidates for the successful manipulation of alkaloid biosynthesis pathways in plants. The probable role(s) of a candidate TF in alkaloid biosynthesis could be assessed by using different approaches like its overexpression, downregulation, or editing at the genomic level.

9.3.1 Overexpression The overexpression of candidate TFs may affect the expression of its upstream and downstream pathway genes, and could help to identify the rate-limiting steps of alkaloid biosynthesis pathways. Overexpression of a particular TF could also modulate the metabolite contents of alkaloid biosynthesis pathways. The information gained from such overexpression studies in wild-type and/or mutant backgrounds could be potentially utilized for alkaloid pathway modulations. The TF of

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interest can be fused with a strong constitutive promoter such as CaMV35S to achieve its expression at higher levels in all the plant tissues. However, to decipher the role of a candidate TF in a particular tissue, the use of a tissue-specific promoter is recommended. The use of a suitable binary vector and Agrobacterium strain is also important for successful plant transformation. The overexpression studies of TFs involved in the biosynthesis of various alkaloids such as TIA biosynthesis in C. roseus (van der Fits and Memelink, 2000; Zhou et al., 2010; De Boer et al., 2011; Suttipanta et al., 2011; Li et al., 2013a; Van Moerkercke et al., 2015, 2016; Sui et al., 2018; Liu et al., 2019b; Pan et al., 2019; Paul et al., 2020; Singh et al., 2020), nicotine biosynthesis in tobacco (Todd et al., 2010; Zhang et al., 2012b; Sears et al., 2014; Yang et al., 2016; Liu et al., 2019a; Sui et al., 2019; Hayashi et al., 2020) camptothecin biosynthesis in O. pumila (Rohani et al., 2016; Wang et al., 2019), and C. acuminata (Chang et al., 2019), berberine biosynthesis in C. japonica (Kato et al., 2007), taxol biosyntheis in T. chinensis (Li et al., 2013b), BIA biosynthesis in E. californica (Yamada et al., 2017), and sesquiterpene pyridine alkaloids biosynthesis in T. wilfordii Hook.f. (Han et al., 2020) have provided useful resources for the genetic manipulation of alkaloid biosynthesis pathways. Such resources are extremely useful for carrying out metabolic engineering for generating pharmaceutically valuable plants.

9.3.2 Downregulation 9.3.2.1 RNA-interference The suppression of the transcripts of a candidate TF is again a useful approach to assess its functional roles in planta. RNA-interference (RNAi) is commonly used to downregulate or partially silence the TFs of interest. Several studies demonstrate the potential of the RNAi technique for the modulation of alkaloids biosynthesis including nicotine biosynthesis in tobacco (Todd et al., 2010; Zhang et al., 2012b; Sears et al., 2014), camptothecin biosynthesis in O. pumila (Udomsom et al., 2016; Wang et al., 2019) and C. acuminata (Chang et al., 2019), isoquinoline alkaloids (IQAs) biosynthesis in E. californica (Yamada et al., 2015), MIA biosynthesis in C. roseus (Van Moerkercke et al., 2016), capsaicinoid biosynthesis in pepper (Arce-Rodrı´guez and Ochoa-Alejo, 2017), berberine biosynthesis in C. japonica (Kato et al., 2007), taxol biosynthesis in T. chinensis (Li et al., 2013b), and sesquiterpene pyridine alkaloid biosynthesis in T. wilfordii Hook. f. (Han et al., 2020). Although RNAi is a widely used tool for the effective knockdown of a candidate TF gene, it has some disadvantages like potential off-target effects and the requirement of a time-consuming genetic transformation process.

9.3.2.2 Virus-induced gene silencing Virus-induced gene silencing (VIGS) technique has emerged as an effective approach for functional validation of the gene of interest in plants. VIGS is basically an RNAi-based tool that exerts its effects through the posttranscriptional gene silencing mechanism (Kumagai et al., 1995; Baulcombe, 1999). VIGS utilizes plants’ natural defense mechanism to combat invading viruses (Voinnet, 2001). VIGS has been successfully used to assess the function of several plant genes. It allows for the rapid assessment of gene function as it generates a loss of function phenotypes within a short period (Burch-Smith et al., 2004; Dinesh-Kumar et al., 2003). VIGS has been successfully applied to suppress and assess the function of TFs in the regulation of alkaloid biosynthesis

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including TIA biosynthesis in C. roseus (Liu et al., 2017; Liu et al., 2019b; Pan et al., 2019), nicotine biosynthesis in tobacco (Todd et al., 2010), and capsaicinoid biosynthesis in pepper (Sun et al., 2019). Although VIGS is used to assess the gene function in a rapid time frame and it generally does not transfer the effect to the next generation, few reports are there indicating that it can transfer the silencing effect to the next progeny as well (Senthil-Kumar and Mysore, 2011). Despite being a useful tool, VIGS has some limitations such as compromised efficiency under hightemperature conditions, which usually lasts for few weeks, and thereafter the plants start to recover from the silencing effect; therefore all the molecular and phenotypic analyses are required to be done within that time frame.

9.3.3 CRISPR/Cas-mediated genome editing CRISPR/Cas is a naturally occurring adaptive immune system in microbes that utilizes RNAguided nucleases to counter the invading virus and other genetic elements (Marraffini and Sontheimer, 2008; Horvath and Barrangou, 2010). The type II CRISPR system is one of the commonly used genome editing tools. It consists of a Cas9 endonuclease and a guide RNA (gRNA) encoded by the crRNA array. The gRNA directs the Cas9 endonuclease to a specific genomic locus to introduce double-strand breaks (DSBs). In Streptococcus pyogenes-derived type II CRISPR system, the Cas9 endonuclease requires the presence of the protospacer adjacent motif (PAM) sequence 5’-NGG-30 to specifically recognize and precisely cleave the target DNA (Alok et al., 2018, 2021; Bhalothia et al., 2020; Mojica et al., 2009; Sternberg et al., 2014; Sushmita et al., 2021). CRISPR/Cas-mediated genome editing allows precise and targeted modification in a specific genome (Ran et al., 2013). CRISPR/Cas-mediated genome editing allows the creation of random insertions, deletions, base replacement, knockout, chromosomal rearrangements, and frameshift mutations at a targeted genomic location. To precisely edit a TF of interest, the designing of sgRNAs is crucial. While designing the sgRNAs, it needs to be properly verified that it has no offtargets in the genome. The CRISPR/Cas-mediated genome editing has been successfully applied in plants to modulate the secondary metabolite contents including the biosynthesis of alkaloids (Alagoz et al., 2016; Dixit et al., 2021b; Hayashi et al., 2020). CRISPR/Cas9-mediated knockout of a key gene 40 OMT2 regulating BIA biosynthesis in P. somniferum demonstrated the potential of genome editing in manipulating alkaloid biosynthesis in plants (Alagoz et al., 2016). Metabolite analysis indicated that the accumulation of BIAs including thebaine, codeine, noscapine, papaverine, S-reticuline, and laudanosine were significantly reduced in the CRISPR/Cas9-edited P. somniferum lines (Alagoz et al., 2016). In another study, two TFs, NtERF189 and NtERF199, were effectively knocked out of by using the CRISPR/Cas9 approach. The total alkaloid content was drastically reduced in NtERF189- and NtERF199-edited lines of N. tabaccum (Hayashi et al., 2020). The CRISPR/Cas is a powerful tool to generate stable lines of transgenic plants. This could be useful to generate TF-edited transgenic lines with enhanced alkaloid contents in a wide array of medicinal plants. In addition to the generation of stable transgenic plants through genome editing of a candidate transcription factor, TF-edited hairy root lines is also an effective approach to modulate the bioproduction of plant secondary metabolites through the use of the CRISPR/Cas9 system (Prakash et al., 2021). Although CRISPR/Cas-based genome editing is a useful tool to modulate the alkaloid biosynthesis in plants, there are some limitations of CRISPR/Cas9-based genome editing.

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The off-target effects and the requirement of high-quality reference genome sequence are the major limitations to precisely editing the TFs of interest.

9.4 Conclusions Alkaloids are plant-derived secondary metabolites and have significant pharmaceutical and commercial value. The alkaloids are present in very low amounts in plants. The low content of several important alkaloids in plant species is a major reason for their extremely higher costs. The improvement in the alkaloid content in commercially and medicinally important plants is an active area of research. Genetic tools to modulate the alkaloid yield have been successfully applied in many plant species. TFs play a major role in the regulation of key genes involved in alkaloid biosynthesis in several plant species and represent themselves as potential candidates for the genetic modulation of the alkaloid pathway in plants. Overexpression, silencing, and editing of TFs of interest at the genomic level using the CRISPR/Cas-based tool are promising approaches to rationally modulate the alkaloid biosynthetic pathways and enhance the alkaloid content in plants, which may ultimately lead to higher commercial value and industrial demand. The manuscript communication number alloted by CSIR-CIMAP to this chapter is “CIMAP/ PUB/2021/DEC/104.”

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CHAPTER

Plant transcription factors and flavonoid metabolism

10

Rekha Chouhan1,2, , Garima Rai1, and Sumit G. Gandhi1,3 1

CSIR-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir, India 2Guru Nanak Dev University (GNDU), Amritsar, Punjab, India 3Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India

10.1 Introduction Flavonoids constitute the largest class of plant polyphenolic secondary metabolites, comprising more than 6000 different metabolites (Ferrer et al., 2008; Saito et al., 2013). Flavonoids are extensively distributed among plants and fulfill diverse biological functions. They majorly include plant pigments responsible for characteristic color and aroma in most flowers and fruits. They are also known to control root nodulation, auxin transport, pollen fertility, and protect plants against various biotic (pathogen infections, herbivory) and abiotic stresses (photo-oxidative damage). Flavonoids have also been reported to confer freezing tolerance, heat acclimatization, and drought resistance to plants (Hichri et al., 2011; Panche et al., 2016; Tohge et al., 2017; Dixit et al., 2019; Singh et al., 2019). Furthermore, they are found to serve many benefits to human health. Various pharmacological studies have reported antiinflammatory, antioxidative, anticancerous, hepatoprotective, cardioprotective, neuroprotective, analgesic, antimicrobial, spasmolytic, and free radical scavenging properties of many flavonoids (Hichri et al., 2011; Kumar & Pandey, 2013; Dixit et al., 2021; Upadhyay and Singh, 2021). They have wide range of applications in the nutraceutical, pharmaceutical, and cosmetic industries (Panche et al., 2016). Semisynthetic flavonoids, hydroxyethylrutosides, and inositol-2-phosphatequercetin have been used to treat hypertension and microbleeding (Kumar & Pandey, 2013). For commercial purposes, cost-efficient production of flavonoids is essential. A detailed understanding of the flavonoid biosynthetic pathways and their control would allow the metabolic engineering of plants for the production of metabolites with valuable applications. For this reason, the investigation on transcriptional control of flavonoid production is worthwhile. Transcription factors (TFs) are among the various proteins known to control gene expression by regulating the rate of transcription. TFs ensure that the genes are expressed at the correct time and in the precise amount throughout the life of an organism (Javed et al., 2020). Advancing our knowledge on the transcriptional control of flavonoid synthetic pathways will support the establishment of new biotechnological tools for generating plants with high flavonoid content. 

Equal contribution.

Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00001-7 © 2023 Elsevier Inc. All rights reserved.

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The present chapter discusses plant flavonoids, their biosynthesis, and the major subclasses. It further elucidates the role of various plant TF families in the regulation of flavonoid metabolism.

10.2 Plant flavonoids, major subclasses, and biosynthesis Polyphenolic compounds represent one of the most widely distributed classes of phytochemicals in the plant kingdom. Flavonoids constitute the important polyphenolic plant secondary metabolites. Structurally, flavonoids consist of a 15-C skeleton composed of two benzene rings (A and B) connected through a heterocyclic pyrane ring (C). They have a common diphenylpropane ring structure with two aromatic rings linked through three-carbon chain, displaying a C6
C3
C6 skeleton structure, with the exception of aurones that have C6
C2
C6 structure. Resorcinol or phloroglucinol forms the ring-A synthesized through the acetate pathway. Ring-B is formed from the shikimate pathway. Flavonoids are subdivided into different subgroups depending on the basis of variation of the C ring, including the oxidation level and degree of unsaturation of the central C heterocycle and the occurrence of substitutions (hydroxyl and methyl) on the A and B rings. The six major classes of flavonoids include flavones, flavonols, flavanols, flavanones, anthocyanidins, and isoflavones (Kumar & Pandey, 2013) (Fig. 10.1). The phenylpropanoid pathway is the major pathway for flavonoid biosynthesis, which transforms phenylalanine to 4-coumaroyl-CoA that finally enters the flavonoid biosynthetic pathway

FIGURE 10.1 Major classes of plant flavonoids.

10.3 Transcription factor families associated with plant flavonoid metabolism

221

FIGURE 10.2 A structural outline of the flavonoid biosynthesis process. 4CL, 4-Coumaroyl-coenzyme A ligase; C4H, cinnamate 4-hydroxylase; CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol-4-reductase are enzymes that catalyze key processes; F3H, flavanone 3-hydroxylase; PAL, phenylalanine ammonia-lyase.

(Fig. 10.2). Further, chalcone synthase, the first enzyme of the flavonoid biosynthesis pathway, produces chalcone scaffolds from where all types of flavonoids are produced. A fundamental pathway for flavonoid biosynthesis is conserved in all plants, which depend on the cluster of enzymes like reductases, isomerases, and hydroxylases. These enzymes are responsible for modifying the basic flavonoid skeleton to generate different types of flavonoids (Falcone Ferreyra et al., 2012).

10.3 Transcription factor families associated with plant flavonoid metabolism TFs span all the kingdoms of life. In plants, 58 types of TF families are reported, of which six major TF families are AP2/ERF (APETALA 2/Ethylene Responsive Factor), bHLH (Basic-Helix-Loop Helix),

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Chapter 10 Plant transcription factors and flavonoid metabolism

MYB (Myeloblastosis Related), NAC (No Apical Meristem (NAM), ATAF1/2 (Arabidopsis Transcription Activator Factor), and CUC2 (cup-shaped Cotyledon)) (Ng et al., 2018). Usually, plant TFs contain a DNA-binding region, a nuclear localization signal, an oligomerization site, and a transcription-regulation domain. TFs typically have just one kind of DNA-binding region and oligomerization domain, but they can have many copies. The oligomerization sites are generally next to or partially cover the DNA-binding sites, and their collective tertiary structure affects crucial elements of TF function. Studies have demonstrated that TFs may either positively or negatively regulate the flavonoid biosynthesis. bHLH, MYB, and WD40 are the major plant TF families involved in flavonoid biosynthesis. These families physically interact to form the MBW complex, known to monitor the synthesis and uptake of flavonoids in plants. Furthermore, the AP2/ERF, MADS-box, bZIP, and WRKY TF families have also been linked to flavonoid metabolism in plants (Li et al., 2019a).

10.3.1 Role of basic-helix-loop-helix transcription factors in plant flavonoid metabolism Basic-helix-loop-helix (bHLH) include an important TF family that influence the plant flavonoid metabolism (Table 10.1). Studies have reported the presence of bHLH TFs in Arabidopsis thaliana Table 10.1 bHLH (basic-helix-loop helix) transcription factors that regulate flavonoid biosynthesis in plants. S. no.

bHLH protein

Plant species

Function

References

1

Lc

Zea mays

Anthocyanin synthesis

2

ZmLc

Anthocyanin synthesis

3

ZmC1 (R2R3MYB)
ZmLc(bHLH) AtGL3/ AtbHLH001

Nicotiana tabacum; Petunia hybrida Solanum lycopersicum

Ludwig et al. (1989), Bovy et al. (2002) Lloyd et al. (1992), Bradley et al. (1998)

Production of flavonols and flavanones

Bovy et al. (2002)

Arabidopsis thaliana

Governs the expression of DFR and BAN genes; Synthesis of anthocyanin and procyanidin Flavonoids biosynthesis

Nesi et al. (2000), Baudry et al. (2006)

4

5

AtGL3/ AtbHLH002

Arabidopsis thaliana

6

Arabidopsis thaliana Prunus persica

Anthocyanin synthesis

7

AtMYC3/ AtbHLH005 PpbHLH3

8

AmDEL

Antirrhinum majus

Anthocyanin synthesis

9

SmbHLH13

Solanum melongena

Anthocyanin synthesis

Anthocyanin synthesis

Lea et al. (2007), Maes et al. (2008), Symonds et al. (2011) Niu et al. (2011) Ravaglia et al. (2013) Outchkourov et al. (2014) Babitha et al. (2015)

10.3 Transcription factor families associated with plant flavonoid metabolism

223

Table 10.1 bHLH (basic-helix-loop helix) transcription factors that regulate flavonoid biosynthesis in plants. Continued S. no.

bHLH protein

Plant species

Function

References

10

VvbHLH1

Vitis vinifera

Wang et al. (2016)

11

MtTT8

12 13 14

MdMYC2 MdbHLH33 DhbHLH1

15 16 17

VvbHLH003 VvbHLH1004 SlAN1

Anthocyanin synthesis Flavones synthesis Anthocyanin synthesis

Wang et al. (2018) Wang et al. (2018) Li et al. (2018)

18

SlGL3

Inhibition of anthocyanin accumulation

19

TaMYC1

Medicago truncatula Malus domestica Malus domestica Dendrobium hybrids Vitis vinifera Vitis vinifera Solanum lycopersicum Solanum lycopersicum Triticum aestivum

Regulates the expression of essential enzyme genes (CHS, F3H, DFR, and LDOXCHS,. SCHS,. HCHS,. RCHS,. X) Anthocyanin and procyanidin biosynthesis Anthocyanin synthesis Anthocyanin synthesis Anthocyanin synthesis

Tominaga-Wada et al. (2018) Shoeva (2018)

20

FabHLH25

21

TaPpb1

22

AabHLH1

23 24

MdbHLH74 AcbHLH42

25

PPLS1

Fragaria ananassa Triticum aestivum Anthurium andraeanum Malus domestica Actinidia chinensis Setaria italica

Transcriptional activation of anthocyanin biosynthesis genes Anthocyanin synthesis Regulates anthocyanin synthesis on co-expression with TaPpm1 Regulates anthocyanin synthesis on co-expression with AaMYB3 Inhibition of anthocyanin synthesis Regulates anthocyanin synthesis on co-expression with AcMYB123 Anthocyanin synthesis

Li et al. (2016) An et al. (2016) Xu et al. (2017a) Li et al. (2017)

Zhao et al. (2018) Jiang et al. (2018) Li et al. (2019b) Li et al. (2019c) Wang et al. (2019) Bai et al. (2020)

(164), Brassica napus (440), B. oleracea (268), B. rapa (251), Ginkgo biloba (85), Juglans regia (102), Nicotiana tabacum (190), Oryza sativa (180), and Vitis vinifera L. (191) (Qian et al., 2021). Members of the bHLH superfamily have two highly conserved domains, the N-terminal basic region and the HLH (helix-loop-helix) domain, spanning approximately 60 amino acids. More than half of the bHLH TFs discovered in plants have extremely conserved HER motif (His5-Glu9Arg13), which aids DNA binding in the E box and regulates the transcription of their target genes. The production of dimers necessitates the presence of the HLH region (Jones, 2004). The Lc protein, a bHLH TF originally isolated from maize, has been demonstrated to influence anthocyanin production. Overexpression of the Lc gene in Solanum lycopersicum resulted in a considerable increase in flavonoid accumulation, indicating the importance of bHLH TFs in flavonoid synthesis (Bovy et al., 2002). Plant flavonoid metabolism is mostly regulated by subgroups 1, 2, 5, 13, 14, and 15, among the 32 recognized subgroups of bHLH TFs. AtTT8/AtbHLH042, a bHLH

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Chapter 10 Plant transcription factors and flavonoid metabolism

TF from the IIIf subfamily (corresponding to subfamily 5), can influence the expression of flavonoid biosynthesis genes (DFR and BAN), impacting anthocyanin and procyanidin production. In addition to AtTT8, three additional bHLH proteins, AtGL3, AtEGL3, and AtMYC1, have been linked to flavonoid production. Similarly, MtTT8 is hypothesized to be involved in the regulation of anthocyanin and procyanidin synthesis in Medicago truncatula (Qian et al., 2021).

10.3.2 MYB transcription factor family and plant flavonoid metabolism MYB TFs are among the most diverse families of plant TFs, and consist of an extremely conserved DNA-binding MYB domain. The MYB domain is made up of four substandard amino acid sequence repeats (R), each of which has around 52 amino acids and forms three helices. Each second and third repeat forms a helix
turn
helix shape featuring three evenly spaced hydrophobic residues. The third helix of each repeat inserts in the main groove of the DNA structure (Dubos et al., 2010). Many MYB TFs are known to govern the synthesis and accumulation of plant flavonoids (Table 10.2). PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT1), an A. thaliana R2R3MYB-type TF, is known to activate genes participating in the production of phenolic acids and Table 10.2 MYB transcription factors that regulate flavonoid biosynthesis in plants. S. no.

MYB protein

Plant species

Function

References

1

ZmP1

Zea mays

Flavonoid biosynthesis

2

NtAN2

Anthocyanin biosynthesis

3

MYB11, MYB12, MYB111

Flavone biosynthesis

Li (2014)

4

AtMYB113, AtMYB114, AtMYB175, AtMYB75 VvMYB1a IbMYB1 MdMYB10, MdMYB1, MdMYBA DcMYB6 OjMYB1 FtMYB1, FtMYB2, FtMYB15

Nicotiana tabacum Arabidopsis thaliana Arabidopsis thaliana Vitis vinifera Ipomoea batatas Malus domestica

Chandler et al. (1989), Goff et al. (1990) Pattanaik et al. (2010)

Flavone biosynthesis

Li (2014)

Flavone biosynthesis Flavone biosynthesis Anthocyanin accumulation Anthocyanin biosynthesis Anthocyanin biosynthesis Anthocyanin and procyanidin accumulation Anthocyanin accumulation Anthocyanin synthesis

Li (2014) Li (2014) Xu et al. (2017a)

Catechin biosynthesis Flavonols accumulation

Sheng et al. (2021) Sheng et al. (2021)

5 6 7 8 9 10

Daucus carota Oenathe javanica Fagopyrum tataricum

11

MYB85

Tobacco

12

MYB1

13 14

AtMYB44 AtMYB11, AtMYB22, AtMYB111

Allium cepa, Malus domestica Camellia sinensis Arabidopsis thaliana

Xu et al. (2017b) Feng et al. (2018) Li et al. (2019a)

Bai et al. (2020) Sheng et al. (2021)

10.3 Transcription factor families associated with plant flavonoid metabolism

225

flavonoids (including anthocyanins). Overexpression of Arabidopsis PAP1 gene in transgenic Nicotiana tabacum plants resulted in the formation of anthocyanins in the leaves, flowers, stems, and roots, giving the plants a dark crimson or purple look. Petunia flowers have also been demonstrated to produce more anthocyanins and emit more floral volatiles (benzenoids) due to PAP1 overexpression (Mitsunami et al., 2014). Mainly three types of flavonoids are reported to accumulate in A. thaliana, including flavonols, anthocyanins, and proanthocyanidins (PAs). Three R2R3-MYB proteins, namely, MYB11, MYB12, and MYB111, are known to influence flavonol production in A. thaliana by triggering the early biosynthetic steps (Li, 2014). Furthermore, MYBbHLH-WD40 (MBW) complex participates in the production of anthocyanins and PAs as well as in the activation of the late flavonoid biosynthetic genes (Li, 2014). Overexpression of anthocyanin-associated MYB genes such as AtMYB113, AtMYB114, AtMYB75 (PAP1), AtMYB90 (PAP2) from Arabidopsis, VvMYB1a from Vitis vinifera, IbMYB1 from Ipomoea batatas, and MdMYB10 and MdMYB1/MdM from Malus domestica have been shown to enhance anthocyanin accumulation in different heterologous or homologous plant species (Xu et al., 2017a). The DcMYB6 TF has also been linked to the regulation of anthocyanin production in purple Daucus carota taproots (Xu et al., 2017b). Another MYB TF, OjMYB1, is known to be involved in anthocyanin production in Oenanthe javanica (Feng et al., 2018). In Fagopyrum tataricum, FtMYB1, FtMYB2, FtMYB15, and FtWD40 members of the R2R3-MYB TF family are known to positively control the accumulation of anthocyanin and proanthocyanidins by affecting the flavonoid biosynthesis genes. Further, FtMYB116 is reported to induce the light-dependent synthesis of rutin. In contrast, FtMYB3, FtMYB11, FtMYB13, FtMYB14, FtMYB15, and FtMYB16 TFs negatively regulate flavonoid biosynthesis (Li et al., 2019a). In addition, the upregulation of VviMYBA1, VviMYBA2, VviMYBF1, and VviMYB5a and the downregulation of VviMYBPA1 in Vitis vinifera have been demonstrated to greatly induce the biosynthesis of flavonoids (Chen et al., 2017).

10.3.3 WD40 transcription factors and plant flavonoid metabolism In eukaryotes, WD40 proteins make up a vast gene family. These proteins have a conserved WD40 domain, which is characterized by the presence of many copies (4
16) of WD40 repetitions, each of which contains 44
60 amino acid residues. A GH (Gly-His) dipeptide of 11
24 amino acids is found at the N-terminus of each WD40 repeat, while a WD (Trp-Asp) dipeptide is found at the C-terminus. Each WD40 strand folds into a four-stranded antiparallel sheet (Mishra et al., 2012; Hu et al., 2018). A few WD40 proteins have been identified to participate in the regulation of flavonoid metabolism (Table 10.3). An11 mutant Petunia plants showed significant reduction in anthocyanin content indicating the role of AN11 TF in flavonoid biosynthesis. In Medicago truncatula, MtWD40
1 mutants have shown reduced content of PAs, anthocyanins, flavonols, and benzoic acid. Other WD40 members including Arabidopsis TTG1, Zea mays ZmPAC1, Perilla frutescens PfWD, and Vitis vinifera WDR1 and WDR2 are also related to flavonoid synthesis and accumulation (Hichri et al., 2011). In 2016, Zhu et al. also observed WD40 as the candidate TFs involved in the flavonoid accumulation and transport in Dracaena cambodiana (Zhu et al., 2016). CsWD40 (Camellia sinensis tryptophan-aspartic acid repeat protein) forms a ternary WBM complex with bHLH and MYB TFs, and regulates PA and anthocyanin biosynthesis. The anthocyanins in transgenic petals

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Chapter 10 Plant transcription factors and flavonoid metabolism

Table 10.3 WRKY and WD40 transcription factors that regulate plant flavonoid biosynthesis. S. no.

WRKY/WD40 protein

Plant species

Function

References

1

WDR1, WDR2

Vitis vinifera

2 3

AtTTG1 AN11

Arabidopsis thaliana Petunia hybrida

4

ZmPAC1

Zea mays

de Vetten et al. (1997), Hichri et al. (2011) Walker et al. (1999) Sompornpailin et al. (2002) Carey et al. (2004)

5

TTG1

Arabidopsis thaliana

6

ZmOAC1

Zea mays

7

MtWD40
1

Medicago truncatula

8

CsWD40

9 10 11

MdWRKY11 TaWRKY44 MdWRKY41

Camellia sinensis, Nicotiana tabacum Malus domestica Taraxacum antungense Malus domestica

Flavonoid synthesis and accumulation Flavonoid synthesis Anthocyanin biosynthesis and accumulation Anthocyanin biosynthesis and accumulation Flavonoid synthesis and accumulation Flavonoid synthesis and accumulation Reduction of anthocyanin and flavonols contents Anthocyanin biosynthesis and accumulation Anthocyanin accumulation Luteolin accumulation Inhibition of proanthocyanidin synthesis

Hichri et al. (2011) Hichri et al. (2011) Hichri et al. (2011) Liu et al. (2018) Liu et al. (2019) Li et al. (2021) Mao et al. (2021)

increased significantly when CsWD40 was expressed heterologously in Nicotiana tabacum (Liu et al., 2018).

10.3.4 Role of basic leucine-zipper transcription factors in plant flavonoid metabolism bZIP (basic leucine-zipper) TFs derive their name due to the presence of a highly conserved bZIP domain. The 40
80 amino acid long bZIP domain is made up of two structural regions: a 16-amino-acid basic region and a less conserved leucine-zipper domain. A nuclear localization signal (NLS) precedes an invariant N-x7-R/K-x9 motif for sequence-specific DNA binding in the basic region. The leucine-zipper is made up of hepta repetitions of leucine or other hydrophobic amino acids (e.g., valine, isoleucine, tryptophan, phenylanine, or methionine) that are precisely nine amino acids away from the C-terminus, forming an amphipathic-coiled shape that allows bZIP to dimerize. Through the basic region, dimerized bZIP proteins attach to the main groove of DNA. bZIP TFs attach to DNA sequences with cis-elements like ABRE, G-box (CACGTG), C-box (GACGTC), AACGTT (T box) A-box (TACGTA), and a GCN4 motif, namely TGA(G/C)TCA (Agarwal et al., 2019; Gai et al., 2020). Some members of the bZIP family have been known to play important roles in mediating flavonoid regulation. bZIP TFs are known to facilitate the light-dependent flavonoid biosynthesis. bZIP proteins constitute the light-response unit identified in promoters of various flavonoid synthetic

References

227

genes (Hichri et al., 2011). A UV light-induced VvibZIPC22 TF is known to participate in the regulation of flavonoid biosynthesis in Vitis vinifera. The overexpression of VvibZIPC22 significantly improves the accumulation of flavonols (Malacarne et al., 2016).

10.3.5 Role of WRKY transcription factors in plant flavonoid metabolism The presence of the WRKY domain (WD) distinguishes WRKY TFs from other gene families. The DNA binding activity of WRKY TFs is mediated by WD, which is a 60-amino-acid region. The WD resulting from classical C2H2 zinc fingers exists in mutator transposons by incorporating a WRKY-like motif upstream of the zinc finger. In 2019, Liu et al. observed a significant increase in anthocyanin accumulation in Malus domestica callus with the overexpression of MdWRKY11. MdWRKY11 binds to W-box cis-elements present in the promoters of MdMYB10, MdMYB11, MdUFGT, and MdHY5 (Liu et al., 2019). TaWRKY44 has been shown to enhance the luteolin content in TaWRKY44-transgenic Taraxacum antungense calli in reference to the wild type calli, signifying the role of TaWRKY44 TF in the flavone metabolism (Li et al., 2021). MdWRKY41 has also been shown to inhibit anthocyanin and PA synthesis and accumulation in Malus domestica. The overexpression of MdWRKY41 suppressed the expression of a MYB TF gene (MdMYB12) and certain structural genes (MdLAR, MdUFGT, and MdANR). MdHY5-MdWRKY41-MdMYB regulatory module is known to regulate flavonoid synthesis in Malus domestica fruits (Mao et al., 2021).

10.4 Conclusions MYB, WD40, and bHLH constitute the major TF families known to regulate the synthesis of flavonoids in plants. Flavonoids are widely distributed polyphenolic secondary metabolites that play an essential role in various physiological functions of plants. They impart color to fruits and flowers and determine their nutritional value. In addition, their implications in human health have also been well documented. The insights on transcriptional regulation of plant flavonoids will improve our understanding of molecular mechanisms underlying their biosynthesis and accumulation. It can further assist the production of plant varieties with improved flavonoid content.

Acknowledgments SGG thankfully acknowledges support from Council of Scientific and Industrial Research (CSIR, India) funded project MLP110006.

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2154. Outchkourov, N.S., Carollo, C.A., Gomez-Roldan, V., de Vos, R.C., Bosch, D., Hall, R.D., et al., 2014. Control of anthocyanin and non-flavonoid compounds by anthocyanin-regulating MYB and bHLH transcription factors in Nicotiana benthamiana leaves. Frontiers in Plant Science 5, 519. Panche, A., Diwan, A., Chandra, S., 2016. Flavonoids: an overview. Journal of Nutritional Science 5, e47. Pattanaik, S., Kong, Q., Zaitlin, D., Werkman, J.R., Xie, C.H., Patra, B., et al., 2010. Isolation and functional characterization of a floral tissue-specific R2R3 MYB regulator from tobacco. Planta 231 (5), 1061
1076. Qian, Y., Zhang, T., Yu, Y., Gou, L., Yang, J., Xu, J., et al., 2021. Regulatory mechanisms of bHLH transcription factors in plant adaptive responses to various abiotic stresses. Frontiers in Plant Science 12, 1143. Ravaglia, D., Espley, R.V., Henry-Kirk, R.A., Andreotti, C., Ziosi, V., Hellens, R.P., et al., 2013. Transcriptional regulation of flavonoid biosynthesis in nectarine (Prunus persica) by a set of R2R3 MYB transcription factors. BMC Plant Biology 13 (1), 1
14. Saito, K., Yonekura-Sakakibara, K., Nakabayashi, R., Higashi, Y., Yamazaki, M., Tohge, T., et al., 2013. The flavonoid biosynthetic pathway in Arabidopsis: structural and genetic diversity. Plant Physiology and Biochemistry 72, 21
34. Singh, S.P., Upadhyay, S.K., Pandey, A., Kumar, S., 2019. Molecular Approaches in Plant Biology and Environmental Challenges. Springer. Available from: https://doi.org/10.1007/978-981-15-0690-1_1. Sheng, X., Chen, H., Wang, J., Zheng, Y., Li, Y., Jin, Z., et al., 2021. Joint transcriptomic and metabolic analysis of flavonoids in Cyclocarya paliurus leaves. ACS omega 6 (13), 9028
9038. Shoeva, O.Y., 2018. Complex regulation of the TaMyc1 gene expression in wheat grain synthesizing anthocyanin pigments. Molecular Biology Reports 45 (3), 327
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Sompornpailin, K., Makita, Y., Yamazaki, M., Saito, K., 2002. A WD-repeat-containing putative regulatory protein in anthocyanin biosynthesis in Perilla frutescens. Plant Molecular Biology 50 (3), 485
495. Symonds, V.V., Hatlestad, G., Lloyd, A.M., 2011. Natural allelic variation defines a role for ATMYC1: trichome cell fate determination. PLoS Genetics 7 (6), e1002069. Tohge, T., de Souza, L.P., Fernie, A.R., 2017. Current understanding of the pathways of flavonoid biosynthesis in model and crop plants. Journal of Experimental Botany 68 (15), 4013
4028. Tominaga-Wada, R., Masakane, A., Wada, T., 2018. Effect of phosphate deficiency-induced anthocyanin accumulation on the expression of Solanum lycopersicum GLABRA3 (SlGL3) in tomato. Plant Signaling & Behavior 13 (6), e1477907. Upadhyay, S.K., Singh, S.P., 2021. Bioprospecting of Plant Biodiversity for Industrial Molecules. John Wiley & Sons. Available from: https://doi.org/10.1002/9781119718017. Walker, A.R., Davison, P.A., Bolognesi-Winfield, A.C., James, C.M., Srinivasan, N., Blundell, T.L., et al., 1999. The TRANSPARENT TESTA GLABRA1 locus, which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis, encodes a WD40 repeat protein. The Plant Cell 11 (7), 1337
1349. Wang, F., Zhu, H., Kong, W., Peng, R., Liu, Q., Yao, Q., 2016. The Antirrhinum AmDEL gene enhances flavonoids accumulation and salt and drought tolerance in transgenic Arabidopsis. Planta 244 (1), 59
73. Wang, P., Su, L., Gao, H., Jiang, X., Wu, X., Li, Y., et al., 2018. Genome-wide characterization of bHLH genes in grape and analysis of their potential relevance to abiotic stress tolerance and secondary metabolite biosynthesis. Frontiers in Plant Science 9, 64. Wang, L., Tang, W., Hu, Y., Zhang, Y., Sun, J., Guo, X., et al., 2019. A MYB/bHLH complex regulates tissue-specific anthocyanin biosynthesis in the inner pericarp of red-centered kiwifruit Actinidia chinensis cv. Hongyang. The Plant Journal 99 (2), 359
378. Xu, H., Wang, N., Liu, J., Qu, C., Wang, Y., Jiang, S., et al., 2017a. The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes. Plant Molecular Biology 94 (1
2), 149
165. Xu, Z.S., Feng, K., Que, F., Wang, F., Xiong, A.S., 2017b. A MYB transcription factor, DcMYB6, is involved in regulating anthocyanin biosynthesis in purple carrot taproots. Scientific Reports 7 (1), 1
9. Zhao, Q., Xiang, X., Liu, D., Yang, A., Wang, Y., 2018. Tobacco transcription factor NtbHLH123 confers tolerance to cold stress by regulating the NtCBF pathway and reactive oxygen species homeostasis. Frontiers in Plant Science 9, 381. Zhu, J.H., Cao, T.J., Dai, H.F., Li, H.L., Guo, D., Mei, W.L., et al., 2016. De novo transcriptome characterization of Dracaena cambodiana and analysis of genes involved in flavonoid accumulation during formation of dragon’s blood. Scientific Reports 6 (1), 1
11.

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CHAPTER

11

Demystifying the role of transcription factors in plant terpenoid biosynthesis

Ajay Kumar1, Parul Sharma2, Rakesh Srivastava1 and Praveen Chandra Verma1 1

Molecular Biology and Biotechnology Division, CSIR-National Botanical Research Institute, Lucknow, Uttar Pradesh, India 2Biological Central Facility, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, Uttar Pradesh, India

11.1 Introduction Plants synthesize a huge number of metabolites, grouped as primary metabolites vital for plant survival and secondary metabolites, nonvital for growth and development of the plant. Among these metabolites, terpenoids are the largest class of secondary metabolites ( . 80,000 entities) responsible for the characteristic aroma of plants and also involved in plant defense, environmental interaction, play physiological and ecological roles (Christianson, 2017; Dixit, 2019). Moreover, terpenoids are constituents of some plant hormones like abscisic acid, GAs, cytokinins, and strigolactones, which regulate plant growth and development. Terpenes are hydrocarbon compounds synthesized through combinations of the isoprene units containing five carbons, while the term terpenoids is used when other functional group are added. Terpenoids from plants are categorized as primary terpenoids such as carotenoids, sterols, hormones, and secondary or specialized terpenoids, the most diverse and largest group of plant metabolites, unique to plant species, that ensure inclusive health of the plant through interaction with the environment (Fig. 11.1). They play a crucial role in plant reproduction through the magnetism of pollinators and seed disseminators, plant defense against microbes and herbivores, and in thermo-tolerance (Nagegowda, 2010; Gutensohn et al., 2012). Biosynthesis of specialized terpenoids is species, organ, tissue, and response specific. Terpenes are categorized into different classes based on the number of isoprene units (C5) present in their chemical structure like hemiterpene (C5), mono(C10), sesqui- (C15), di- (C20), sester- (C25), tri- (C30), and tetraterpene (C40) (Dudareva et al., 2006; Chen et al., 2011). A mid-size gene family denoted as terpene synthase is responsible for the biosynthesis of the basic backbone structure of entire terpene metabolites. After various enzymatic modifications such as dehydrogenation, hydroxylation, and glycosylation, terpene structure transforms into terpenoid metabolites (Chen et al., 2011). The spatio-temporal and inducible biosynthesis of specialized terpenoids is the result of tight regulation of consistent biosynthetic genes through transcriptional regulation via transcription factors (TFs) at numerous levels (Srivastava et al., 2014a,b; Srivastava et al., 2018; Pandey et al., 2019). A group of the protein interacts with DNA and binds with the promoter/regulatory regions of the target genes denoted as TFs, and regulates the expression level of the desired gene via Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00016-9 © 2023 Elsevier Inc. All rights reserved.

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Chapter 11 Demystifying the role of transcription factors in plant

Pollinators and seed disseminator

Protection against abiotic Stress

Plant welfare

Defense against pathogens and herbivorous

Plant hormones and pigments

Allopathic Effects

PLANTS TERPENOIDS Perfume industry

Spices

Human welfare Pharmaceutical industry

Food Cosmetics

FIGURE 11.1 Significant roles and applications of plant terpenoids in human and plants welfare.

moderate the activity of RNA polymerase, resulting in altered transcriptional initiation (Vom Endt et al., 2002). TFs can recognize environmental signals (internal and external) and control the expression of corresponding gene expression, thus monitoring the accumulation of terpenoids. Some TFs regulate the expression of the target gene indirectly through the formation of a complex with other cofactors without interacting with DNA. Among the 58 TF families, only a limited number of families that regulate the terpenoid biosynthesis in plants have been identified.

11.2 Biosynthesis of terpenoids Plant terpenoids have extreme diversity in their chemical structures, however, all terpenoids are biosynthesized from the universal five-carbon isoprene unit denoted as IPP and DMAPP (an allylic

11.2 Biosynthesis of terpenoids

235

isomer of IPP). IPP and DMAPP, both precursors, are biosynthesized from two alternate and independent biosynthetic pathways (MVA and MEP), localized in various subcellular organs. The classical mevalonic-acid (MVA) pathway localized in the cytosol produces IPP initiated from acetyl-CoA, and 2-C-methylerythritol 4-phosphate (MEP) pathway produces IPP and DMAPP from pyruvate and glyceraldehyde-3-phosphate in the plastids. These five-carbon isoprene units condense in different manners through the catalytic function of isoprenoids synthase producing isoprenoids intermediate precursor for different downstream enzymes like, terpene synthase. Moreover, Cytochrome P450s also play an essential role in terpenoid skeleton modification and structural diversity.

11.2.1 Biosynthesis of basic terpenoids precursor (MVA and MEP pathway) The MVA pathway produces IPP and DMAPP from acetyl-CoA, and six sequential enzymatic steps are involved during this process; these enzymes are acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-coenzyme A synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase (Liao et al., 2014; Tholl, 2015). Initially, it was reported that the MVA pathway was cytosolic but recent investigations have shown that enzymes from the MVA pathway are also associated with other subcellular compartments such as HMGR associated with ER and MK, PMK, and MVD found in peroxisome and AACT localized in the cytosol, ER, and peroxisomes (Leivar et al., 2005; Reumann et al., 2007; Ahumada et al., 2008; Sapir-Mir et al., 2008; Simkin et al., 2011). IPP is converted into its allylic isomer DMAPP by IPP isomerase enzyme in a vice versa manner, and this enzyme has been shown to have peroxisomal, plastidial, and mitochondrial localization (Wang et al., 2017). The MEP pathway independent from MVA pathway. The MEP pathway biosynthesized IPP and DMAPP, which is initiated from the condensation of pyruvate and glyceraldehyde-3-phosphate and completed in seven sequentially enzymatic steps. MEP pathway includes 1-deoxy-D-xylulose-5phosphate (DXP) synthase (DXS), DXP reductoisomerase (DXR), MCT, CMK, MDS, and HDS (Rohdich et al., 2003). IPP and DMAPP both are produced simultaneously from HMBPP in the last step of the MEP pathway by the action of enzyme HDR. The previous investigation has well proven that enzymes of the MEP pathway are placed on the plastid while genes are located in the nuclear genome (Joyard et al., 2009). A recent study has shown that floral monoterpenes and diterpenes production use enzymes from the MEP pathway while MVA pathway enzymes are utilized in the biosynthesis of sesquiterpenes in plants (Muhlemann et al., 2014) (Fig. 11.2).

11.2.2 Biosynthesis of isoprenoid intermediates Isoprenoids or linear prenyl diphosphate are used as a central precursor of all terpenoid biosynthesis. These are synthesized by fusion of IPP and DMAPP (basic unit of C5: isoprene unit) produced through MVA or MEP pathways, in “head-to-tail” mode by the enzymatic action of prenyltransferases (isoprenoids synthase/prenyl diphosphate synthases) localized in a different compartment. When single molecules of both IPP and DMAPP condense in a head-to-tail manner, by the catalytic activity of plastidial geranyl diphosphate synthase (GPPS) enzyme, GPP (geranyl diphosphate: C10) is produced and used as substrate/precursor for the biosynthesis of monoterpenoids (Nagegowda, 2010). In plants, GPPS is found in two structural forms, (homodimeric and/or heterodimeric).

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FIGURE 11.2 MVA and MEP Biosynthesis pathways for basic terpenoids precursor. Flow charts exposing the biosynthetic pathways of terpenoids in plants catalyzed by different groups of enzymes specifically isoprenoids synthase and terpene synthase. Terpenoid biosynthesis started from two independent pathways, MEP (plastidial) and MAV (cytosolic). MEP pathway starts by condensation of GA-3P and pyruvate and goes through the cascade of a different enzymatic reaction, resulting in the production of IPP and DMAPP, while MVA pathways begin from acetyl-CoA and produce the same products. IPP and DMAPP further condense and produce numerous terpenoids like isoprenoids, monoterpenes, sesquiterpenes, and diterpenes. Abbreviations: AACT, acetoacetyl-CoA thiolase; AcAc-CoA, acetoacetyl-CoA; CAS, cycloartenol synthase; CDP-ME, 4-(cytidine 50 -diphospho)-2-C-methyl- D -erythritol; CDP-ME2P, 4-(cytidine 50 -diphospho)-2-C-methyl-D-erythritol phosphate; CMK, CDP-ME kinase; DMAPP, dimethylallyl diphosphate; dTPS, diterpene synthase; DXP, 1-deoxy-D-xylulose 5-phosphate; DXR, DXP reductoisomerase; DXS, DXP synthase; FPP, farnesyl diphosphate; FPPS, farnesyl diphosphate synthase; GA-3P, glyceraldehyde-3phosphate; GES, geraniol synthase; GPP, geranyl diphosphate; GPPS, geranyl diphosphate synthase GGPP, geranyl geranyl diphosphate; GGPPS, geranyl geranyl diphosphate synthase; HDR, (E)-4-hydroxy-3methylbut-2-enyl diphosphate reductase; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; HMGS, HMG-CoA (Continued)

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237

Homodimeric is composed of two identical peptides/subunits while in the heterodimeric structure one large subunit (LSU) with one small subunit (SSU) is present. The small subunit of heterodimeric GPPS is catalytically inactive while the large subunit works as GGPPS and/or GPPS based on the interaction between LSU and SSU; sometimes it is inactive, while homodimeric GPPS from angiosperms produces GPP as a major product (Schmidt et al., 2010; Rai et al., 2013). Along with GPP, C10 NPP (neryl diphosphate), a cisoid isomer of GPP, is also produced by the same manner of IPP and DMAPP condensation through the catalytic action of a plastidial Z-prenyltransferase, neryl diphosphate synthase (NPPS) (Schilmiller et al., 2009; Akhtar et al., 2013). Both GPP and NPP are utilized in monoterpene formation in plants. Consequently, the C15 farnesyl diphosphate (FPP), produced by coupling of two IPP molecules with one DMAPP molecule in a head-to-tail manner through the catalytic action of FPPS (farnesyl diphosphate synthase), and in the biosynthesis of sesquiterpenoids, triterpenoids, and sterols, this FPP is consumed and used as natural precursor. Normally, FPPSs are localized in cytosol but mitochondrial and peroxisomal localization are also reported in plants (Cunillera et al., 1997). Moreover, when a DMAPP condenses with three molecules of IPP through the catalytic action of geranylgeranyl diphosphate synthase (GGPPS) enzyme geranylgeranyl diphosphate (GGPP) is produced to be served as a precursor of biosynthesis of a diverse class of primary and specialized/secondary terpenoids such as plastid-derived diterpenoids, polyterpenes, chlorophylls, polyprenols, tocopherols, gibberellins, carotenoids, abscisic acid, strigolactones, phytol, phytyl esters, and plastoquinones (Tholl and Lee, 2011; Akhtar et al., 2017). Next, a DMAPP molecule condense with four IPP molecules produces a trans prenyl diphosphate geranylfarnesyl diphosphate (GFPP, C25) that serves as a precursor for sesterterpenoids biosynthesis, by the catalytic action of GFPP synthase (GFPPS) (Tachibana, 1994; Tachibana et al., 2000).

11.2.3 Biosynthesis of terpenes by terpene synthases

L

Terpenes/terpenoids are the most diverse group of secondary metabolites produced by the action of terpene synthase (TPS) enzyme using different prenyl diphosphates as a precursor (Bathe and Tissier, 2019). TPSs are a mid-sized gene family and 30
100 TPSs are found in single plants. These families are original from prenyltransferase/triterpene synthase through sweeping and fusion of protein domain along with gene duplication (Chen et al., 2014; Karunanithi and Zerbe, 2019). The fundamental of enormous structural diversity and promiscuity nature of terpenoids are created predominantly due to the three possible reasons; restructuring of the basic carbon skeletons intermediate ion, utilization of diverse substrates, and multiple product formation by terpene synthases. On the basis of number of carbon (isoprene units) terpenoids are grouped as hemi- (C5), mono(C10), sesqui-(C15), di- (C20), sester- (C25) tri- (C30), and tetra- (C40) terpenes. synthase; HMBPP, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate, HMGR; HDS, (E)-4-hydroxy-3-methylbut2-enyl diphosphate synthase; IPPI, isopentenyl diphosphate isomerase; IPP, isopentenyl diphosphate; MCT, 2-C-methyl- D -erythritol 4-phosphate cytidylyltransferase; MDS, 2-C-methyl-D -erythritol 2,4cyclodiphosphate synthase; MEP, 2-C-methyl-D-erythritol 4-phosphate; MEcPP, 2-C-methyl-D-ery-thritol 2,4cyclodiphosphate; MK, mevalonate kinase; MPDC, mevalonate diphosphate decarboxylase; mTPS, monoterpene synthase; MVAP, mevalonate 5-phosphate; MVAPP, mevalonate 5-diphosphate; PMK, phosphomevalonate kinase; PYS, phytoene synthase; SQS, squalene synthase; sTPS, sesqiterpene synthase.

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11.2.3.1 Hemiterpenes Single isoprene units containing terpenes are the smallest and simplest terpenes denoted as hemiterpenes like isoprene synthesized in oak and conifers (Evidente et al., 2015; Badal and Delgoda, 2017). Isoprene synthase (IspS) enzyme converts DMAPP into isoprene evolved from monoterpene synthases. Isovaleric, tiglic, and angelic acids are the best examples of known hemiterpenoids produced in plants.

11.2.3.2 Monoterpenes Monoterpenes are synthesized from GPP and present in cyclic or acyclic structures containing C10 in molecules. They have a characteristic odor with aromatic properties and are used globally in pharmaceutical, perfume, food, cosmetic, and industries (Booth et al., 2017; Ludwiczuk et al., 2017). Geraniol, menthol, borneol, limonene, linalool, camphor, and eucalyptol are high-value monoterpenoids from plants. Apart from GPP, NPP also consume monoterpenes biosynthesis through the enzymatic reaction of monoterpene synthases (mTPSs).

11.2.3.3 Sesquiterpenes Sesquiterpenes are C15 compounds that exist in cyclic and acyclic structures synthesized from FPP through the enzymatic action of sesquiterpene synthases (sTPSs). They are constituents of floral scents and play a crucial role in plant defense against herbivore and pathogen, and attacks. Sesquiterpenoid lactones are chemically distinct compounds from sesquiterpenoids derived from sesquiterpenes both are used as antimicrobial, anti-inflammatory, and antitumor agents. α-humulene, farnesene, caryophyllene, zingiberene, staleness, and santalols are common sesquiterpenoids, while antimalarial artemisinin and parthenolide belong to the most famous sesquiterpenoid lactones.

11.2.3.4 Diterpenes Diterpenes are C20 hydrocarbons biosynthesized from GGPP originated primarily from the MEP pathway and exist in numerous forms like -linear, bicyclic, tricyclic, tetracyclic, pentacyclic, or macrocyclic. Approximately 13,000 diterpenes have been reported from plants that play a crucial role in both primary (phytohormones) and secondary (phytoalexin) metabolism. The role of diterpenes in human welfare is very well known as the anticancer drugs taxol (paclitaxel) from Taxus brevifolia and steviol (natural sweetener) from Stevia rebaudiana. Cyclization of a polyisoprene diphosphate (C-20: mostly (E,E,E)-GGPP) by the catalytic action of multi-TPSs results in diterpene biosynthesis.

11.2.3.5 Triterpenes Six isoprene units combine and produce triterpene molecules with a C30 carbon skeleton. Triterpenes are mainly derived from FPP originated from MVA pathways, and subsequently two FPP are fused in a “head-to-head” manner and produce squalene used as a precursor of triterpene, where the reaction is catalyzed by squalene synthase (SQS). Next, squalene is converted into 2,3oxidosqualene by enzyme squalene epoxidase (SQE) then undergoes cyclization in triterpenes with different structures by the action of triterpene synthases. Generally, triterpenes are not involved in plant growth and development but play a crucial role in plant defense and enhance the food quality

11.3 Regulation of terpenoids

239

of the crop. The best-known triterpenoids from plants are lupeol, β-amyrin, oleanolic acid, α-amyrin, betulinic acid, and ursolic acid. Withanolides are triterpenoid steroidal lactones derived through sterol pathway that have medicinal and pharmacological properties.

11.3 Regulation of terpenoids Transcription factors play a crucial role in the regulation of genes of expression involved in the secondary metabolite biosynthesis pathway, and recently, most of the research involves revolving around the regulatory mechanism of secondary metabolites (Srivastava et al., 2018; Pandey et al., 2019; Prakash et al., 2021). A number of TFs have been exposed to have a global regulatory function in terpenoid biosyntheses such as myeloblastosis (MYB), WRKY, basic helix
loop
helix (bHLH), APETALA2/ethylene responsive-factor (AP2/ERF), SPL (SQUAMOSA promoter-binding protein-like), basic leucine zipper (bZIP), jasmonate-responsive ERF (JRE), and YABBY (Table 11.1). Table 11.1 Transcription factors in plants terpenoids biosynthesis pathways. TF

Plant

Pathways

Affected gene

References

AabZIP1

Artemisia annua Artemisia annua Artemisia annua Actinidia arguta Artemisia annua Artemisia annua Artemisia annua Aquilaria sinensis Arabidopsis thaliana Salvia sclarea

Artemisinin biosynthesis Artemisinin biosynthesis Artemisinin biosynthesis Monoterpene biosynthesis Artemisinin biosynthesis Artemisinin biosynthesis Artemisinin biosynthesis Sesquiterpene biosynthesis Sesquiterpene biosynthesis MEP-derived terpenoid pathway Triterpenoids biosynthesis Sesquiterpene biosynthesis Monoterpene biosynthesis

ADS, CYP71AV1 MULTIPLE GENES

Zhang et al. (2015); Zhang et al. (2015) Lu et al. (2013)

ADS

Yu et al. (2012)

AaTPS1 ADS

Nieuwenhuizen et al. (2015) Lu et al. (2013)

DBR2

Lv et al. (2019)

ADS

Ma et al. (2009)

ASS1

Xu et al. (2017)

TPS21, TPS11

Hong et al. (2012)

DXS, DXR, GGPPS, CPPS FPPS, SQUALENE SYNTHASE (1)-VALENCENE SYNTHESIS CITTPS16

Alfieri et al. (2018)

AaERF1 AaERF1, AaERF2 AaNAC AaORA1 AaSPL2 AaWRKY1 AsMYC2 AtMYC2 AtWRKY40 BpMYB CitAP2.10 CitERF71

Betula platyphylla Citrus sinensis Citrus aurantium

Yin et al. (2020) Shen et al. (2016) Li et al. (2017) (Continued)

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Chapter 11 Demystifying the role of transcription factors in plant

Table 11.1 Transcription factors in plants terpenoids biosynthesis pathways. Continued TF

Plant

Pathways

Affected gene

References

CrERF5

Catharanthus roseus Catharanthus roseus Catharanthus roseus Crocus sativus

MIA biosynthesis

TDC

Pan et al. (2019)

TIA pathway

ORCA2, ORCA3

Zhang et al. (2011)

TIA pathway

TDC

Suttipanta et al. (2011)

Apocarotenoid biosynthesis Terpene biosynthesis Sesquiterpenoid biosynthesis Sesquiterpene biosynthesis Monoterpene biosynthesis Monoterpene biosynthesis Terpenes biosynthesis

PSY, PDS, BCH, CCD

Ashraf et al. (2015)

TPS10 EAS12

Li et al. (2015a,b) Song et al. (2019)

CADINENE SYNTHASE (CAD1) MTPS (QH6)

Xu et al. (2004)

GPPS, LSU

Reddy et al. (2017)

TPS

Wang et al. (2016)

TIA pathway

STR,SGD

Li et al. (2013a,b)

Diterpenoid biosynthesis Monoterpenes biosynthesis MEP-derived terpenoid pathway Isoprenoid, MVA pathway Monoterpene biosynthesis Terpenes biosynthesis

MOMILACTONE SYNTHASE TPS

Okada et al. (2009)

DXS1, DXR, HDR AACT, HMGS, MVD1

Chenge-Espinosa et al. (2018) Bedon et al. (2010)

MTPS

Spyropoulou et al. (2014)

mTPS

Xu et al. (2018)

Terpene biosynthesis

TPS

Spyropoulou et al. (2014)

Diterpenoid biosynthesis Diterpenoid biosynthesis MEP pathway

C4H1, GGPPS

Deng et al. (2020)

SmCPS1, SmKSL1, SmCYP76AH1 SmDXS, SmDXR

Zhang et al. (2019)

CrMYC2 CrWRKY1 CsULT1 EREB58 ERF2-like GaWRKY1 HY5 MsMYB MsYABBY5 ORCA2 OsTGAP1 PbbHLH4 PIF1/HY5 Sg4C R2R3MYB SlEOT1 SlMYC1 SlMYC1 SmbZIP1 SmERF128 SmWRKY1

Zea mays Nicotiana attenuata Gossypium arboreum Artemisia annua Mentha spicata Mentha spicata Catharanthus roseus Oryza sativa Phalaenopsis bellina Arabidopsis thaliana Spruce spp. Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Salvia miltiorrhiza Salvia miltiorrhiza Salvia miltiorrhiza

Zhou et al. (2015)

Chuang et al. (2018)

Cao et al. (2018)

11.3 Regulation of terpenoids

241

11.3.1 WRKY A WRKY TF is one of the most investigated TF families involved in plant development process and biotic and abiotic stress. The WRKY TF family has a signature domain consisting of 60 highly conserved amino acids denoted as WRKY domain with conserved sequences WRKYGQK. Numerous investigations have shown that WRKY TFs regulate the biosynthesis of plant secondary metabolites in response to jasmonic acid. In contrast, several studies from different plants indicate that WRKY TFs regulate the biosynthesis of terpenes for example monoterpenes in tomatoes; sesquiterpenes, capsidiol in cotton (like gossypol), artimesia (like artemisinin), and nicotiana (like capsidiol); diterpenoids in taxus (such as paclitaxel), salvia (such as tanshinones), and rice (like momilactone A) (Xu et al., 2004; Ishihama et al., 2011; Li et al., 2013a,b; Sun et al., 2013; Akagi et al., 2014; Spyropoulou et al., 2014; Chen et al., 2017; Singh et al., 2017; Cao et al., 2018; Deng et al., 2019). Next, it is well known that WRKY TFs interact with W-box in the promoter’s region of the biosynthetic pathway genes. Examples include: 1. GaWRKY1 from Gossypium arboretum induces the biosynthesis of sesquiterpene phytoalexin (gossypol) through interaction with the promoter region of (1) - δ -cadinene synthase genes (Xu et al., 2004). 2. In A. annua, sesquiterpene synthase gene, ADS, and CYP71AV1 gene from artemisinin biosynthesis pathway are regulated by AaWRKY1 through binding and transactivation of the promoter of these genes promotes the biosynthesis of antimalarial drug artemisinin (Ma et al., 2009). Moreover, AaWRKY1 also regulates HMGR (3-hydroxy 3-methylglutaryl-coa reductase) gene from MVA pathway and DBR2 (artemisinic aldehyde δ11(13) reductase) gene from artemisinin biosynthesis pathway (Ma et al., 2009). 3. WRKY1 from Catharanthus roseus play an important part in regulation of biosynthesis of TIA biosynthesis, but the detailed mechanism of regulation needs more investigation (Suttipanta et al., 2011). 4. Silencing of NaWRKY3 and NaWRKY6 TF in N. attenuata prevented the biosynthesis of cisα-bergamotene sesquiterpene involved in defense against herbivore attack (Skibbe et al., 2008). Apart from the regulation of terpenoid biosynthesis pathways, the WRKY TF family is also involved in the managing of another biosynthesis pathway in plants. 5. TaWRKY1, from Taxus chinensis, manage the expression of 10-deacetylbaccatin III-10 β-oacetyl transferase (DBAT) gene and play a crucial role in the biosynthesis of paclitaxel used as an anticancer drug (Li et al., 2013a,b). 6. Recently, it was investigated that HbEREBP1 and HbWRKY1, from Hevea brasiliensis, play important roles in the regulation of terpenoid compounds (latex production) (Chen et al., 2012; Zhang et al., 2012).

11.3.2 MYB MYB TFs are one of the largest TFs families that have been investigated in plants to reveal their role in plant development, stress response, and regulation of biosynthetic pathways. MYB DNAbinding domain is a signature domain present in different numbers with imperfect repeats of 52 aa in MYB TFs (Feller et al., 2011). This huge MYB family can be grouped into four subfamilies and

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in contrast, different members of the R2R3 family prominently regulate the multiple biosynthetic pathways of plants. Generally, it was believed that MYB TFs regulate phenylpropanoid biosynthesis but some investigations have also shown their involvement in the regulation of terpenoid biosynthetic pathways. C. roseus MYB TF denoted as (CrBPF1) binds to the promoter of STR gene and overexpressing in C. roseus hairy roots alter the expression of numerous genes like ORCA3, CrMYC1, CrMYC2, BIS1, GBF2, and ZCTs but decrease the accumulation of TIAs and serpentine. These results show that CrBPF1 could be controlled by TIA pathway genes through increasing the expression of TIA transcriptional repressors (Li et al., 2015a,b). PtMYB4 and VvMYB5b, two MYB TFs from Pinus taeda and Vitis vinifera, respectively, are managed by the production of phenylpropanoids and terpenoids in plants. SmMYB36 from S. miltiorrhiza inhibits phenolic acid biosynthesis and promotes tanshinone biosynthesis (Ding et al., 2017).

11.3.3 bHLH (basic helix
loop
helix) Another important TF superfamily that also plays a crucial role in the regulation of plant metabolism is basic/helix
loop
helix (bHLH), which consists of highly conserved bHLH signature domain. bHLH and MYB, both TF families, work together through interaction and complex formation, which regulate the expression of downstream pathway genes (Feller et al., 2011). Moreover, various investigations have shown its involvement in the regulation of terpenoid biosynthesis genes and accumulation of terpenoids such as, 1. Two TFs from Medicago truncatula belong to the bHLH class denoted as triterpene saponin biosynthesis activating regulators (MtTSAR1 and MtTSAR2) that regulate the triterpenoid biosynthesis in plants (Mertens et al., 2016). 2. PbbHLH4 of Phalaenopsis bellina enhances the accumulation of monoterpenoids in scentless orchids (Chuang et al., 2018). 3. Another bHLH factor MYC2 manages the accumulation of secondary metabolite directly and/or indirectly. For example, CrMYC2 from Catharanthus roseus controls the expression of ORCA3 gene through binding to cis-elements of the gene promoter, thus regulating the expression of numerous genes of terpenoid indole alkaloid (TIA) biosynthesis and accumulation of TIA (Zhang et al., 2011). Further, in Arabidopsis, AtMYC interacts with DELLA proteins and induces the biosynthesis of sesquiterpene synthase in flowers through incorporating the jasmonate (JA) and gibberellin (GA) hormone signaling pathway (Hong et al., 2012).

11.3.4 AP2/ERF The APETALA2/ethylene response factor (AP2/ERF) family of TFs is the largest TF family in plants. This family comprises 60 conserved amino acid residues with AP2 DNA-binding domain (Mizoi et al., 2012). This family plays a crucial role in the regulation of plant developmental and defense mechanisms by regulating the biosynthesis of terpenoids. Some examples are: 1. ORCA TF belongs to the AP2/ERF TF family and consists of DNA-binding AP2-domain involved in the regulation of secondary metabolism in Madagascar periwinkle (C. roseus). Overexpression of CrORCA3 induces the accumulation of TIAs in suspension cells, and expression analysis has shown that CrORCA3 controls the expression of numerous genes of

11.3 Regulation of terpenoids

2.

3.

4.

5.

243

both the plastidial MEP pathway and TIA pathway (van der Fits and Memelink, 2000). Recently, a number of investigations has been done to prove that AP2/ERF proteins were participating in the regulation of secondary metabolism in different plant species. Two TFs, homologous to Catharanthus ORCA2 and ORCA3 and belonging to the JA-responsive AP2/ ERF family, denoted as AaERF1 and AaERF2, control the expression of two genes amorpha-4,11diene synthase (ADS) and CYP sequiterpene oxidase (CYP71AV1) from artemisinin biosynthesis in A. annua by binding in CRTDREHVCBF2 (CBF2) and RAV1AAT (RAA) motifs in promoters of genes. Further, overexpression of these two TFs enhanced the accumulation of artemisinin and artemisinic acids in the plant (Yu et al., 2012). Subsequently, the AaORA1 TF member of the AP2/ ERF family involved in the defense of A. annua against Botrytis cinerea (necrotrophic plant pathogen) also regulates the biosynthesis of artemisinin (Li et al., 2013a,b). Next, two JA-responsive ERF TFs, AaERF1 and AaERF2, have also been investigated in A. annua, and results indicate involvement of these two TFs in the biosynthesis of sesquiterpene lactone (artemisinin) through interaction with the promoter of amorpha-4,11-diene synthase (ADS) and a cytochrome P450 monooxygenase (CYP71AV1), both crucial genes of artemisinin biosynthesis pathway (Yu et al., 2012). In tomato, GAME9 (glycoalkaloid metabolism 9), a member of the AP2/ERF TF family, and a jasmonate-responsive ERF (JRE) manage steroidal lactone biosynthesis by controlling the expression of the mevalonate pathway and SGA pathway genes including SSR2 (sterol side chain reductase 2) gene from cholesterol biosynthesis (C´ardenas et al., 2016; Thagun et al., 2016). Other AP2/ERFs members from Zea mays (ZmEREB58) induce the expression of TPS10 (sesquiterpene synthase) gene and produce two major products (E)-β-farnesene and (E)-α
bergamotene and S. miltiorrhiza (SmERF128) that regulate the accumulation of diterpenoids tanshinone by altering the expression of SmCPS1 (copalyl diphosphate synthase 1), SmKSL1(kaurene synthase-like 1), and SmCYP76AH1 (cytochrome P450 monooxygenase 76AH1) genes involved in the biosynthesis of tanshinone (Li et al., 2015a,b; Zhang et al., 2019). Moreover, two AP2/ERFs members, CitERF71 and CitERF71 from Citrus sinensis, induce the accumulation of (1)-sesquiterpene valencene and monoterpene E-geraniol, respectively (Shen et al., 2016; Li et al., 2017).

11.3.5 bZIP This TF contains the bZIP domain consisting of two structural features located on a contiguous alphahelix. Plant bZIP proteins specially bind to ACGT core on DNA and specificity is regulated by flanking nucleotides. It has been shown that bZIP TFs also play important roles in terpenoid metabolism; for example, HY5 and AabZIP1 were investigated in A. annua, HY5 incorporate with the promoter of the β-pinene synthase (QH6, monoterpene synthase) producing β-pinene and regulate its rhythmic expression (Zhou et al., 2015), while AabZIP promotes artemisinin biosynthesis through activation of ADS and CYP71AV1 expression regulated by ABA signaling in A. annua (Zhang et al., 2015). Another bZIP TF SmbZIP1 from Salvia miltiorrhiza positively regulates the biosynthesis of phenolic acid by increasing the expression of C4H1 (cinnamate-4-hydroxylase) while negatively regulating the accumulation of tanshinones (diterpenoid) by suppressing the expression of the GGPPS (geranylgeranyl diphosphate synthase) gene (Deng et al., 2020). A bZIP TF, OsTGAP1, regulates the expression of the five genes (OsCPS4, OsKSL4, CYP99A2, CYP99A3, and OsMAS) involved in the biosynthesis of diterpenoid phytoalexin (momilactone) biosynthesis in rice (Okada et al., 2009).

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11.3.6 SPL, YABBY, and other TFs SQUAMOSA promoter-binding proteins like SPL and YABBY TFs were recently investigated with respect to terpenoid metabolism regulation such as AaSPL2, which enhances the biosynthesis of artemisinin by increasing the expression of DBR2 (artemisinic aldehyde 11 reductase) gene through transactivation of the promoter (Lv et al., 2019). Recently, an overexpression and silencing study of Mentha spicata TF MsYABBY5 and ectopic expression in Ocimum basilicum and Nicotiana sylvestris pointed out that MsYABBY5 negatively regulates the biosynthesis of secondary metabolism specifically monoterpenes (Wang et al., 2016). Solanum lycopersicum SlEOT1, belonging to Stylish 1 (AtSTY1) and Short Internode (AtSHI) TFs of Arabidopsis, transactivated the SlTPS5 (linalool synthase) promoter in Nicotiana benthamiana leaves (Spyropoulou et al., 2014). Three AaNAC TFs (AaNAC2, AaNAC3, and AaNAC4) from Actinidia arguta bind to the promoter of terpene synthase1 (AaTPS1), regulate its expression, and responsible for terpinolene production in ripping fruits (Nieuwenhuizen et al., 2015).

11.4 Conclusion Plants respond to environmental cues through diverse receptors and sensors and these signals are further transduced by diverse intermediate signal mediators like Ca12, ROS, etc. to downstream TFs. TFs regulate the gene expression and subsequent biosynthesis of secondary metabolites, specifically terpenoids. Terpenoids may directly inhibit pathogen infection, or participate in ROS scavenging, and terpenoids are also involved in resistance/tolerance against different stresses. TFs can be utilized to enhance terpenoids by optimizing the level of metabolite flux. However, a single TF is often insufficient to regulate the whole biosynthetic pathway of terpenoids, which usually needs regulatory networks consisting of multiple TFs. Recent research improves our understanding of plant terpenoid metabolism and elucidates the regulation of terpenoid production in plants. For example, WRKY TFs regulate the biosynthesis of terpenoids in tomato, cotton, artimesia, nicotiana, taxus, salvia, and rice. MYB, bHLH, AP2/ERF, bZIP, SPL, and YABBY TFs also regulate the accumulation of terpenoids in plants. Recently, databases such as PhytoMetaSyn Medicinal Plant Genome Resource (MPGR) and Medplants Consortium have been developed to accelerate investigations of biosynthesis and regulation of terpenoids

Acknowledgment Authors are also thankful to CSIR, New Delhi for financial support in the form of FBR project MLP0037. Institute’s Manuscript Number is CSIR-NBRI_MS/2022/05/15.

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328, Print ISBN: 978-1-4822-2920-2 eBook ISBN . . .. Srivastava, R., Rai, K.M., Srivastava, R., 2018. Plant biosynthetic engineering through transcription regulation: an insight into molecular mechanisms during environmental stress. Biosynthetic Technology and Environmental Challenges. Springer Nature Singapore Pte Ltd, pp. 51
72, DOI 10.1007/978-981-10-7434-9. Sun, Y., Niu, Y., Xu, J., Li, Y., Luo, H., Zhu, Y., et al., 2013. Discovery of WRKY transcription factors through transcriptome analysis and characterization of a novel methyl jasmonate-inducible PqWRKY1 gene from Panax quinquefolius. Plant Cell, Tissue and Organ Culture (PCTOC) 114, 269
277. Suttipanta, N., Pattanaik, S., Kulshrestha, M., Patra, B., Singh, S.K., Yuan, L., 2011. The transcription factor CrWRKY1 positively regulates the terpenoid indole alkaloid biosynthesis in Catharanthus roseus. Plant Physiology 157, 2081
2093. Tachibana, A., 1994. A novel prenyltransferase, farnesylgeranyl diphosphate synthase, from the haloalkaliphilic archaeon, Natronobacterium pharaonis. FEBS Letters 341, 291
294. Tachibana, A., Yano, Y., Otani, S., Nomura, N., Sako, Y., Taniguchi, M., 2000. Novel prenyltransferase gene encoding farnesylgeranyl diphosphate synthase from a hyperthermophilic archaeon, Aeropyrum pernix: Molecular evolution with alteration in product specificity. European Journal of Biochemistry 267, 321
328. Thagun, C., Imanishi, S., Kudo, T., Nakabayashi, R., Ohyama, K., Mori, T., et al., 2016. Jasmonate-responsive ERF transcription factors regulate steroidal glycoalkaloid biosynthesis in tomato. Plant Cell Physiol 57, 961
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114. Wang, J.Z., Li, B., Xiao, Y., Ni, Y., Ke, H., Yang, P., et al., 2017. Initiation of ER body formation and indole glucosinolate metabolism by the plastidial retrograde signaling metabolite, MEcPP. Molecular Plant 10, 1400
1416. Wang, Q., Reddy, V.A., Panicker, D., Mao, H.-Z., Kumar, N., Rajan, C., et al., 2016. Metabolic engineering of terpene biosynthesis in plants using a trichome-specific transcription factor MsYABBY5 from spearmint (Mentha spicata). Plant Biotechnology Journal 14, 1619
1632. Xu, J., van Herwijnen, Z.O., Dra¨ger, D.B., Sui, C., Haring, M.A., Schuurink, R.C., 2018. SlMYC1 regulates type VI glandular trichome formation and terpene biosynthesis in tomato glandular cells. The Plant Cell 30, 2988
3005. Xu, Y.-H., Liao, Y.-C., Lv, F.-F., Zhang, Z., Sun, P.-W., Gao, Z.-H., et al., 2017. Transcription factor AsMYC2 controls the jasmonate-responsive expression of ASS1 regulating sesquiterpene biosynthesis in Aquilaria sinensis (Lour.) Gilg. Plant and Cell Physiology 58, 1924
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22. Yu, Z.X., Li, J.X., Yang, C.Q., Hu, W.L., Wang, L.J., Chen, X.Y., 2012. The jasmonate-responsive AP2/ERF transcription factors AaERF1 and AaERF2 positively regulate artemisinin biosynthesis in Artemisia annua L. Molecular Plant 5, 353
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CHAPTER

The regulatory circuit of iron homeostasis in rice: a tale of transcription factors

12

Pooja Kanwar Shekhawat1, Hasthi Ram2 and Praveen Soni1 1

Department of Botany, University of Rajasthan, Jaipur, Rajasthan, India 2National Institute of Plant Genome Research, New Delhi, Delhi, India

Highlights 1. Transcription factors belonging to NAC, bHLH, and WRKY families play a crucial role in the regulation of responses to deficiency and toxicity of iron (Fe) in rice. 2. OsIRO2 acts as a prime regulator of Fe deficiency and controls the expression of strategy II-related genes of Fe uptake, transport, and storage. 3. Iron homeostasis-related transcription factors are themselves regulated at epigenetic, transcriptional, post-transcriptional, and post-translational levels involving interconnected loops.

Abbreviations APT ARF bHLH CRE DMAS Fe FD FDH FEP FIT FRO FT IDEF IDS IBP IMA IRO IRT LEA

Adenosine phosphoribosyl transferase Auxin response factor Basic helix-loop-helix protein Cis-regulatory element Deoxymugineic acid synthase Iron Fe deficiency Formate dehydrogenase Fe uptake inducing peptide Fe deficiency-induced transcription factor Ferric reductase oxidase Fe toxicity Fe deficiency-responsive cis-acting element-binding factor Iron deficiency-specific clone Bowman-Birk trypsin inhibitor Iron MAN Iron-related transcription factor Iron-regulated transporter Late embryogenesis abundant

Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00015-7 © 2023 Elsevier Inc. All rights reserved.

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252

MAs NAS NAAT PEZ PRI ROS SAM TFs TOM YS YSL

Chapter 12 The regulatory circuit of iron homeostasis in rice

Mugineic acids Nicotianamine synthase Nicotianamine aminotransferase phenolics efflux zero Positive regulator of iron deficiency response Reactive oxygen species S-adenosyl methionine Transcription factors Transporter of mugineic acid Yellow stripe Yellow stripe-like

12.1 Introduction Iron (Fe) is a key micronutrient necessary for the development of plants. It is essential for respiration, nitrogen assimilation, biosynthesis of chlorophyll and hormone, and defense against pathogens (Hansch and Mendel, 2009). Despite being present in large amounts in soil, it is not readily bioavailable to plants. Plants need 1026
1027 M of Fe concentration in the soil for their optimal growth, whereas bioavailable Fe concentration hardly exceeds 10210 M, especially in calcareous soils (Tsai and Schmidt, 2017). Because of its insoluble form such as ferric hydroxide, Fe is not easily absorbed by plants. Fe deficiency (FD) affects plant growth in terms of reduction in chlorophyll content, photosynthesis rate, root and shoot growth, yield, and nutritional quality (Fig. 12.1). Leaves show interveinal yellowing. An excess amount of Fe is also toxic for plant growth and yield. Leaf bronzing is a characteristic symptom of Fe toxicity. Not only for plants, but Fe is also a

Reduction in yield and nutritional quality

Yield loss

Interveinal yellowing, decrease in photosynthesis efficiency and dry matter accumulation

Leaf bronzing, necrotic lesions, ROS formation, physiological disorder, decline in photosynthesis and decrease shoot growth

Fe deficiency Increase in lateral root density, decrease in primary root elongation and shallower root architecture

Fe toxicity

Fe2+

Fe3+

Reduce root growth, formation of root plaque

Fe3+ Fe2+

Fe2+ Fe3+

Fe2+

Fe2+ Reduction Fe3+

Fe2+

Oxidation

FIGURE 12.1 Rice plants show morphological, physiological, and agronomic defects under Fe deficiency and toxicity conditions.

12.2 Iron uptake and transport

253

vital element for humans. Because of its importance to human health, special biofortification programs of staple crops including rice are underway in many countries for enhancing Fe content in grains. Fe deficiency in humans causes serious issues such as anemia and growth retardation (Sankaran and Weiss, 2015). It is reported that over 2 billion people suffer from anemia, and women and children are more affected by it (Murgia et al., 2012; Briat et al., 2015).

12.2 Iron uptake and transport Plants use two different approaches for Fe acquisition. Nongraminaceous monocots (i.e., except Poaceae) and dicots plants employ a reduction-based mechanism (strategy I), whereas graminaceous plants adopt a chelation-based method (strategy II) (Fig. 12.2) (Marschner et al., 1986; Mori, 1999). Under Strategy I, root cells release protons (via H1-ATPase) in soil, which acidifies the rhizosphere and subsequently causes Fe solubilization (Santi and Schmidt, 2009; Kawakami and Bhullar, 2018). Then, ferric ions (Fe31) are reduced to ferrous ions (Fe21) by the plasma membrane-localized FRO (ferric reductase oxidase) enzyme (Robinson et al., 1999). These ferrous ions are imported through the plasma membrane-localized IRT1 (iron-regulated transporter 1). The components of strategy I are well characterized (Robinson et al., 1999; Henriques et al., 2002; Varotto et al., 2002; Santi and Schmidt, 2009; Vert et al., 2002). Under strategy II, root cells

FIGURE 12.2 Two different Fe uptake strategies exist in plants. Under strategy I, Fe31 present in the soil as ferric hydroxide (insoluble form) gets solubilized through soil acidification by the release of H1 ions. Secreted phenolic compounds induce mobilization of Fe31. Fe31 is reduced to Fe21 by FRO. Fe21 ions are imported via plasma membrane-localized IRT1 transporter. Under strategy II, phytosiderophores (such as mugineic acids), which are natural Fe chelators, are synthesized from SAM precursor. These phytosiderophores are secreted into the soil to bind Fe31 ions. Thereafter, Fe31-phytosiderophore complexes are taken up by YSL or YSL like transporters. Exceptionally, rice plants possess both strategy I and strategy II to uptake Fe21 and Fe31, respectively. HA, H1-ATPase; TOM1, transporter of mugineic acid; SAM, S-Adenosyl methionine; NAAT, Nicotianamine aminotransferase; DMAS, Deoxymugineic acid synthase; FRO, Ferric reductase oxidase; IRT, Iron-regulated transporter; YSL, Yellow stripe like; PEZ, phenolics efflux zero.

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Chapter 12 The regulatory circuit of iron homeostasis in rice

exudate phytosiderophores like mugineic acids (MAs) through TOM1 (transporter of mugineic acid 1) (Nozoye et al., 2011). MAs are natural Fe chelators that bind Fe31 ions with high affinity in the rhizosphere and form Fe31-MAs complexes (Kobayashi et al., 2014; Kobayashi and Nishizawa, 2012b; Shumayla and Upadhyay, 2022). Subsequently, Fe31-MAs complexes are transported inside the root cells via YS (yellow stripe) or YSL (yellow stripe-like) transporters (Curie et al., 2001). Strategy II mainly depends on the biosynthesis and the secretion of phytosiderophores (Fig. 12.2). The precursor of MAs is S-adenosyl methionine (SAM) (Kobayashi and Nishizawa, 2012b; Sauter et al., 2013). Three major enzymes are engaged in the biosynthesis of phytosiderophores namely nicotianamine synthase (NAS), deoxymugineic acid synthase (DMAS), and nicotianamine aminotransferase (NAAT). Rice, a member of the Gramineae (Poaceae) family, is a principal crop and consumed by a large portion of the world’s population. Exceptionally, rice evolved strategy I as well as strategy II for Fe uptake. Rice plants operate strategy I under submerged conditions or acidic soils (Ishimaru et al., 2006). PEZ2 (phenolics efflux zero 2) transporter secretes caffeic and protocatechuic acids and helps in the mobilization of Fe31 ions, which are reduced by FRO enzyme (Gao and Dubos, 2021). OsIRT1 and OsIRT2 are Fe21 transporters in rice. In rice, 2’deoxymugineic acid (DMA) solubilizes and chelates Fe31 ions. OsNAS1, OsNAS2, OsDMAS, OsTOM1, OsYSL15, and OsYSL2 are the components of strategy II in rice (Bashir et al., 2017; Cheng et al., 2007; Gao and Dubos, 2021; Inoue et al., 2003, 2009; Ishimaru et al., 2006; Lee et al., 2009; Sharma et al., 2022; Upadhyay, 2022). As stated earlier, despite the absolute necessity of Fe, if it is present in an excessive amount, it causes cellular damage (Sperotto et al., 2010). It can cause formation of reactive oxygen species (ROS) due to a reaction with oxygen (Fenton reaction) that damages proteins, lipids, and nucleic acids. According to the literature, there are two types of Fe toxicity (FT): true Fe toxicity and indirect Fe toxicity. In the true FT condition, the accumulation of Fe leads to toxic levels in the plant body, while in indirect FT, Fe precipitation occurs on the root apoplast and forms Fe plaque, which leads to multiple nutritional deficiencies (Sahrawat, 2000; Olaleye et al., 2001; Sahrawat, 2005; Stein et al., 2009). Under flooded conditions, rice plants are more prone to FT and the rice yield decreases from 12% to 100% (Sahrawat, 2005). However, various other factors such as soil fertility status, rice genotype (susceptible or tolerant), and intensity of FT stress determine the degree of impact on rice plants (Sahrawat, 2005). To counter FT, various mechanisms operate in rice plants such as the exclusion of Fe from the root, compartmentalization of high Fe concentrations through storage in the inner cavity of ferritin proteins, and activation of antioxidant system to detoxify ROS (Stein et al., 2009; Zhang et al., 2012; Wu et al., 2014, 2017). Plants must be able to sense and respond to Fe stress in the context of Fe deprivation as well as its toxicity conditions. In the last few years, numerous genes entailed in Fe absorption and homeostasis have been characterized in rice along with those encoding TFs which modulate the transcript levels of genes that are Feresponsive (Li et al., 2016). TFs act as molecular switches of target genes to control various biochemical or physiological processes. Along with the DNA binding domain, three other functional domains are found in the structure of a typical TF: an oligomerization domain, a nuclear localization domain, and a domain for transcriptional regulation (Liu et al., 2001). Transcription factors form homo or heterodimers and act as positive or negative regulators. In this chapter, we aim to discuss recent development in rice research regarding the regulatory role of various TFs in Fe homeostasis.

12.3 Major transcription factors involved in iron homeostasis

255

12.3 Major transcription factors involved in iron homeostasis Over the past few years, Fe homeostasis has been the subject of study in rice. For this purpose, numerous reverse and forward genetic studies have been conducted. To date, numerous TFs have been identified and characterized in rice that control the responses under Fe deficiency or toxicity conditions (Table 12.1).

12.3.1 Regulation of Fe deficiency 12.3.1.1 bHLHs bHLH comprises the second largest family (after the MYB) of TFs in plants. bHLH TF contains two domains, a basic region at the N-terminal and a helix-loop-helix (HLH) domain, which is essential for dimer formation (Atchley et al., 1999; Sharker et al., 2020). To bind with DNA, members of the bHLH TF family have conserved motif His5-Glu9-Arg13 (HER) (Atchley and Fitch, 1997; Massari and Murre, 2000; Toledo-Ortiz et al., 2003). These TFs are involved in various responses against stress conditions like drought, cold, osmotic stress, heavy metal stress, salt stress, and iron deficiency (Qian et al., 2021). The first TF of the bHLH family was identified in maize; over time, large numbers of this family have been identified in diverse plants (Xiong et al., 2005; Jaillon et al., 2007; Rushton et al., 2008; Carretero-Paulet et al., 2010; Miao et al., 2020; Zhou et al., 2020; Zhao et al., 2021). Rice contains 180 bHLH TFs (Carretero-Paulet et al., 2010). In rice, bHLH TFs perform a central role in governing uptake, utilization and transport of Fe when it is deficit in soil. These bHLH family members have been further divided into subsets including Ib, IVb, and IVc (Gao et al., 2019; Kobayashi, 2019). bHLH of subset Ib such as OsIRO2 (iron-related transcription factor 2) positively regulates the expression of genes that are associated with Fe uptake, distribution, and storage (Ogo et al., 2007, 2011; Wang et al., 2007, 2013; Yuan et al., 2008; Sivitz et al., 2012). In contrast to the previous subgroup, OsIRO3, a bHLH of the subgroup IVb, suppresses the genes that are related to Fe signaling cascade (Long et al., 2010; Zheng et al., 2010; Tanabe et al., 2019). Members of these two subgroups predominantly express under Fe deficiency (Ogo et al., 2006; Long et al., 2010; Zheng et al., 2010). From recent studies, it is confirmed that OsbHLH060, a bHLH of subset IVc, acts as a transcriptional regulator of subsets Ib and IVb bHLH TFs (Selote et al., 2015; Zhang et al., 2015, 2017; Li et al., 2016; Liang et al., 2017). As mentioned above OsIRO2 is a key Fe deficiency inducible bHLH TF in rice. It increases the transcript levels of genes that are categorized under strategy-II (Ogo et al., 2006, 2007). It binds to core sequence 50 CACGTGG30 present in the promoter of genes such as OsNAS1, OsFDH (formate dehydrogenase), OsNAS3, OsIRT1, OsAPT1 (adenosine phosphoribosyl transferase), and OsIDS3 (iron deficiency-specific clone) (Ogo et al., 2006). OsIRO3 negatively controls Fe deficiency (Wang et al., 2020). It expresses in root, leaves, nodes, and leaf blades during the vegetative phase of rice plants. OsIRO3 binds to the E-box found in the upstream region and represses expression of OsNAS3 in root (Wang et al., 2020). In osiro3 mutant, expression of OsNAS3 increases, which enhances the nicotianamine (NA) level in the root (Wang et al., 2020). The NA further forms DMAs which are important for Fe uptake, transport, and distribution. OsNAS1, OsNAS2, OsTOM1, OsNAAT1, OsIRO2, OsYSL12, and OsYSL15 genes also show significantly higher transcript levels in osiro3 mutant as compared to wild type. OsbHLH156 (also known as OsFIT; Fe

Table 12.1 A list of TFs involved in response to Fe stress in rice. Transcription factor family

Name of transcription factor

Locus ID

NAC family

OsIDEF1

LOC_Os08g01090

OsIDEF2

LOC_Os05g35170

OsWRKY74

WRKY family

bHLH family

ARF family

Expression in particular rice tissue

Type of Fe stress Fe deficiency

Function

Reference Kobayashi et al. (2014) Kobayashi et al. (2021) Dai et al. (2016) Ricachenevsky et al. (2010) Viana et al. (2017) Viana et al. (2017) Viana et al. (2017) Viana et al. (2017) Wang et al. (2020) Kobayashi et al. (2019)

LOC_Os09g16510

Root, leaves, flowers and seeds Root, leaves, flowers and seeds Root and leaves

Fe deficiency

OsWRKY80

LOC_Os03g63810

Shoot and root

Fe toxicity

OsWRKY46

LOC_Os11g02480

Shoot and root

Fe toxicity

Upregulates strategy IIrelated genes Upregulates expression of OsYSL2 Increases accumulation of Fe in root and shoot Regulates Fe toxicity responses Yet to be characterized

OsWRKY113

LOC_Os06g06360

Shoot and root

Fe toxicity

Yet to be characterized

OsWRKY64

LOC_Os12g02450

Shoot and root

Fe toxicity

Yet to be characterized

OsWRKY55-like

LOC_Os03g20550

Shoot and root

Fe toxicity

Yet to be characterized

OsIRO3/ OsbHLH63 OsbHLH059/ OsPRI3

LOC_Os03g26210

Root and shoot

Fe deficiency

LOC_Os02g02480

Root and shoot

Fe deficiency

OsbHLH058/ OsPRI2

LOC_Os05g38140

Root and shoot

Fe deficiency

OsbHLH156/OsFIT

LOC_Os04g31290

Root and shoot

Fe deficiency

OsbHLH56/ OsIRO2 OsbHLH060/ OsPRI1 OsbHLH133

LOC_Os01g72370

Root

Fe deficiency

LOC_Os08g04390

Root and shoot

Fe deficiency

LOC_Os12g32400

Root

Fe deficiency

OsARF16

LOC_Os01g13520

Root and shoot

Fe deficiency

Negatively regulates expression of OsNAS3 gene Increases transcript level of Fe deficiency responsive genes Increases expression of OsYSL2, OsNAS1, and OsTOM1 Mediates nuclear localization of OsIRO2 Increases expression of strategy II-related genes Increases transcript level of OsIRO2 and OsIRO3 Fe distribution between shoot and root Involved in Fe deficiency responses

Fe deficiency

Kobayashi et al. (2019) Wang et al. (2020) Ogo et al. (2006) Zhang et al. (2017) Wang et al. (2013) Shen et al. (2015)

12.3 Major transcription factors involved in iron homeostasis

257

deficiency-induced transcription factor) mainly expresses in root, and its transcript level enhances under iron deficiency (Wang et al., 2019). OsFIT interacts with OsIRO2 and facilitates its nuclear localization (Liang et al., 2020). It is supposed that OsbHLH156 and OsIRO2 may form transcriptional activation complex to increase the transcription of Fe homeostasis-related genes. A loss of function of OsFIT causes a decrease in Fe accumulation, and chlorosis under Fe31 supplementation (Liang et al., 2020). Apart from these TFs, two ubiquitin ligases OsHRZ1 and OsHRZ2 function as negative regulators of Fe deficiency responses in rice as knockdown of these genes leads to increased transcript abundance of Fe deficiency-inducible genes (Kobayashi et al., 2013). Interestingly, OsHRZ1 binds Fe so it is a putative iron sensor. It controls the function of subgroup IVc bHLH TFs OsbHLH058/OsPRI2 and OsbHLH059/OsPRI3, which are positive regulators of Fe deficiency responses. OsHRZ2 may also interact with OsbHLH058 and OsbHLH059 and regulate Fe homeostasis responses (Zhang et al., 2020). OsbHLH058 induces the transcription of numerous Fe responsive genes (e.g., OsYSL2, OsNAS1, and OsTOM1). OsbHLH059 also positively regulates a number of genes that are related to many functions such as internal Fe translocation, Fe21 uptake, Fe31 DMA uptake, and translocation. The OsPRI1 (positive regulator of iron homeostasis) (also designated as OsbHLH60) binds to the promoter of OsIRO2 and OsIRO3 and thereby enhances the expression of genes related to Fe-deficiency response (Zhang et al., 2017). OsHRZ1 regulates the stability of OsPRI1 protein through ubiquitination (Zhang et al., 2017). OsbHLH133 controls the transportation system of Fe from root to young leaves (Wang et al., 2013). OsbHLH133 mutant contains high Fe content in shoot and low Fe content in root.

12.3.1.2 WRKY The presence of the WRKY domain is the characteristic feature of this family (Eulgem et al., 2000). This domain comprises 60 amino acids, with conserved sequence WRKYGQK at N-terminal and zinc-finger motif C-C-H-H/C at C-terminal (Eulgem et al., 2000). In rice genome, 109 WRKY TFs are found (Ross et al., 2007). OsWRKY74 is involved in crosstalk between Fe deficiency and phosphorus starvation (Dai et al., 2016). Root architecture in terms of root number, length, and diameter gets changed in OsWRKY74 overexpressing plants (Dai et al., 2016). OsLTN1 (Leaf tip necrosis 1), a ubiquitin-conjugating enzyme, negatively controls Fe absorption-related genes in rice (Hu et al., 2011, 2015). OsLTN1 is downregulated by OsmiR399 (Hu et al., 2015). OsMiR399 performs a vital function in the regulation of starvation of multiple nutrients including Fe. Interestingly, OsWRKY74 overexpressing transgenic plants show higher expression of OsmiR399, which causes an increase in Fe content (Hu et al., 2015; Dai et al., 2016).

12.3.1.3 NAC The NAC transcription factor family constitutes a large number of plant-specific transcriptional regulators. This family is named after three TFs including NAM (no apical meristem, Petunia), ATAF1
2 (Arabidopsis transcription activation factor), and CUC2 (cup-shaped cotyledon, Arabidopsis). These three TFs contain the same DNA-binding domain (Souer et al., 1996; Aida et al., 1997). NAC proteins contain a highly conserved NAC domain at N-terminal and a highly variable domain at C-terminal (Olsen et al., 2005). This highly variable C-terminal domain is responsible for activation or repression of expression of the target gene (Tran et al., 2004; Hu et al., 2006). OsIDEF1 (iron deficiency-responsive element factor 1), which belongs to the NAC family, recognizes iron-deficiency-responsive element (IDE) 5’CATGC3’ found in the promoters

258

Chapter 12 The regulatory circuit of iron homeostasis in rice

of Fe deficiency-responsive genes (Kobayashi et al., 2009, 2010). At the initial phase of Fe deficiency, OsIDEF1 upregulates genes of strategy I and II related to Fe uptake/utilization like OsIRT1, OsYSL2, OsYSL15, OsNAS1, OsNAS2, OsNAS3, and OsDMAS1. OsIDEF1 also induces the transcription of OsIRO2 and OsIRO3 genes (Kobayashi et al., 2007, 2009, 2014). OsIDEF1 has a proline-rich and histidine
asparagine repeat region (HN region) for binding with divalent metals such as Ni21 (nickel) and Fe21. It may function as a sensor to check cellular Fe status (Kobayashi et al., 2012a). This metal-binding domain is important for the proper functioning of OsIDEF1 as the absence of this domain causes a reduction in rate of germination and seedling growth in rice (Kobayashi et al., 2012a). Under low Fe conditions, OsIMA1 (Iron man) and OsIMA2 play a central role in initiating Fe deficiency responses. These genes are positively upregulated by various TFs such as OsIDEF1, OsbHLH058, OsbHLH059, and by positive feedback (Kobayashi et al., 2021). Another NAC TF OsIDEF2 recognizes CA(A/C)G(T/C)(T/C/A)(T/C/A) IDE2 element as core sequence found in the promoters of Fe deficiency inducible genes (Ogo et al., 2008). The OsYSL2 gene associated with Fe31 nicotianamine transport is upregulated by OsIDEF2. OsIDEF1 and OsIDFE2 constitutively express in root, shoot, and flower in rice plants (Kobayashi et al., 2009).

12.3.1.4 Auxin response factor family Apart from the abovementioned TFs, an ARF (auxin response factor) has been reported to regulate Fe deficiency response. OsARF16 expresses in the shoot and root under Fe starvation conditions (Shen et al., 2015). Mutant plants of this gene show restoration of Fe-deficiency symptoms such as dwarfism, decreased photosynthesis, and reduction in the Fe content. OsARF16 provides a link between the auxin-signaling and the response to Fe deficiency. Fe supply improves root architecture through alteration of auxin distribution (Giehl et al., 2012). However, the exact mechanism by which auxin contributes to Fe deficiency responses in rice is not yet fully explained.

12.3.4 Regulation of Fe toxicity 12.3.4.1 WRKY family Ricachenevsky et al. (2010) identified the first transcription factor OsWRKY80 in rice that shows responsiveness to Fe toxicity conditions. Its transcription is upregulated by Fe toxicity (De Campos Carmona et al., 2021). Viana et al. (2017) found that OsWRKY46, OsWRKY55-like, OsWRKY64, and OsWRKY113 are induced under iron toxicity conditions, especially in iron sensitive genotype. However, these TFs have not been functionally characterized yet. Further studies are required to understand their roles in FT tolerance mechanisms. Recently, novel cis-elements have been identified that are linked to Fe toxicity response in rice (Kakei et al., 2021). In order to avoid Fe toxicity, plants reduce Fe transfer to aerial parts and increase Fe accumulation in the root (Silveira et al., 2007).

12.4 Regulation of the regulators TFs themselves can be regulated at various molecular levels. Phytohormones play a key role in this respect. Here, we describe the regulation of rice TFs engaged in Fe deficiency. In the case of Fe

12.4 Regulation of the regulators

259

toxicity, the mechanism for the regulation of TFs is not known yet and needs to be explored in the future.

12.4.1 Epigenetic regulation The epigenetic mechanisms including alteration of chromatin structure and histone modification are yet to be explored in rice in relation to Fe stress. However, effect of DNA methylation has been reported recently in this regard. DNA methyltransferase is an essential regulator that controls gene expression via methylation of DNA at specific sites (Chen and Zhou, 2013; Galindo-Gonzalez et al., 2018). In a study, it has been found that DNA methylation changes the expression pattern of two important transcriptional regulators OsIRO2 and OsbHLH156 (Sun et al., 2021). Hypermethylation in the promoter region of these genes shows a positive correlation with the transcript level of genes related to Fe transport, binding, and homeostasis. Under Fe scarcity, the application of Aza (5-aza-2-deoxycytidine), a DNA methylation inhibitor, represses the expression of OsIRO2 and OsbHLH156. Moreover, several other genes, OsYSL15 OsHRZ1, OsNAS2, and OsNAAT1, which are upregulated under Fe deficiency are also repressed after treatment with Aza (Sun et al., 2021).

12.4.2 Regulation at the transcriptional level As mentioned earlier OsPRI1, OsPRI2, OsPRI3, and OsIDEF1 positively regulate the transcription of OsIRO2 and OsIRO3 (Zhang et al., 2017).

12.4.3 Regulation at the post-transcriptional level As noted, OsmiR399 is one of the main regulators of deficiency of Fe and other nutrients in rice (Hu et al., 2011, 2015). It targets OsLTN1, which is a negative regulator of Fe absorption-related genes (Hu et al., 2015). Overexpression of OsWRKY74 increases its expression (Dai et al., 2016). However, its role in the regulation of TFs is not known yet.

12.4.4 Regulation at the post-translational level OsIDEF1 expression level positively correlates with Fe-uptake and translocation-related genes such as OsNAS2, OsNAS3, OsYSL2, OsYSL15, OsNAS1, OsIRT1, OsDMAS1, and OsIRO2 under Fe starvation condition (Kobayashi et al., 2009; Zhang et al., 2014). The protein level of OsIDEF1 and OsIDEF2 is controlled by OsIBP1.1 (Bowman-Birk trypsin inhibitor 1.1) and OsIBP1.2 under Fe-sufficient as well as Fe-deficient condition (Zhang et al., 2014). In the case of optimum Fe concentration, OsIDEF1 protein is ubiquitinated and afterward degraded by the 26 S proteasome system. COP9 signalosome subunit CSN6 plays a vital role in this process (Tan et al., 2016). Under Fe-deficiency, degradation of OsIDEF1 protein is hampered by OsIBP1.1 (Zhang et al., 2014). Interestingly, promoters of OsIBP1.1 and OsIBP1.2 genes contain binding sites for OsIDEF1 and OsIDEF2. Under Fe-deficiency conditions, OsIDEF1 induces the expression of OsIBP1.1 gene. OsIBP1.1 protein interacts with OsIDEF1 and prevents its degradation. OsIBP1.1 protein possibly competes at the CSN6 binding site. Under Fesufficient conditions, OsHRZ1 (Hemerythrin motif-containing a really interesting new gene (RING) and zinc-finger protein1) interacts with OsPRI1 transcription factor to degrade it by ubiquitination process

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(Zhang et al., 2014). HRZ proteins contain different Zn-fingers including RING-type, CTCHY-type, rubredoxin-type, and CHY-type. They also contain hemerythrin domains to bind with metals like Fe and Zn (Salahudeen et al., 2009; Hua and Vierstra, 2011). Knockdown lines of OsHRZ1 gene show upregulation of transcription of genes related to Fe uptake and translocation. Although the aforementioned studies have explored the roles of transcription factors and their regulation under Fe stress, it is still necessary to further elucidate their functions and regulatory roles.

12.4.5 Regulation by plant hormones In the plants that use strategy I for Fe uptake, the role of various phytohormones including auxin, cytokinin, ethylene, and brassinosteroids is well-reported in context of Fe homeostasis (Ivanov et al., 2012). However, contrary to this, there are few reports regarding their role in strategy II plants under Fe inadequacy. Rice mutant plants with compromised biosynthesis of brassinosteroids show tolerance to Fe deficiency (Wang et al., 2015). Synthesis of brassinosteroids is inhibited in Fe-deficient conditions, which results in a reduction of endogenous brassinosteroids and facilitates the absorption of Fe (Wang et al., 2015). Under iron-limiting conditions, ethylene enhances the expression of master regulator OsIRO2, which results in increased expression of strategy II components like OsYSL15, OsNAAT, OsTOM1, OsNAS1, and OsNAS2 (Gao et al., 2019). Besides phytosiderophores, the synthesis of ethylene also requires enzymes like ACC oxidases, SAM synthetases, and ACC synthases (Kobayashi and Nishizawa, 2012b; Sauter et al., 2013). A summary of TFmediated regulation of Fe stress in rice is depicted in Fig. 12.3.

12.5 Conclusion and future perspectives Rice and other plants possess key regulators to prevent debilitating conditions such as Fe deficiency and Fe toxicity by tightly controlling Fe uptake, transport, and storage. A major question that is still unanswered is how the plants sense Fe concentration and which proteins are exactly involved in downstream signaling. OsHRZ1 and OsIDEF1 are possible candidates for sensing intracellular Fe, which help in the regulation of Fe homeostasis. Along with these proteins, OsbHLH156, and OsIRO2 are major TFs that positively control Fe deficiency responses. In rice, OsIRO3 is a crucial TF that negatively regulates the Fe uptake, transport, and allocation while its transcript level also gets upregulated under Fe deficiency conditions. OsWRKY55-like, OsWRKY64, OsWRKY113, and OsWRKY46 are probably engaged in the regulation of Fe toxicity. How these TFs function needs to be studied in the future. Other auxiliary proteins involved in the Fe-toxicity signaling cascade are still not identified. In Arabidopsis, NO (nitric oxide), a diffusive molecule has been recognized as a vital regulator for Fe uptake mechanism (Meiser et al., 2011; Yang et al., 2013; Tewari et al., 2021). It induces the expression of TFs such as FIT, AtbHLH38, AtMYB72, and AtbHLH39. In this reference, there is only one recent report in rice that shows that NO treatment alleviates Fe deficiency in rice under alkaline conditions (Li et al., 2021). Its mechanism is yet to be comprehensively investigated in rice. Manipulation of TFs is an alternative method for developing rice that will be more tolerant to iron stress. With the availability of advanced biotechnological techniques, TFs can be targeted to

12.5 Conclusion and future perspectives

261

FIGURE 12.3 Diagrammatic representation of the regulation of Fe stress by various TF. Under Fe deficiency, TFs belonging to ARF, WRKY, NAC, and bHLHs families upregulate the transcript levels of genes related to strategies I and II in rice plants. OsARF16 TF regulates Fe-deficiency responses via auxin signaling and modulates the root architecture system. OsWRKY74 TF increases tolerance against Fe deficiency. It upregulates the expression of OsmiR399 by an unidentified mechanism. OsmiR399 represses OsLTN1, which in turn negatively regulates genes involved in Fe absorption. NAC family TF OsIDEF1 induces the transcription of genes such as OsNAS1, OsNAS2, OsNAS3, OsDMAS, OsYSL15, OsIRT1, OsYSL2, OsIMA1, and OsIMA2 that are engaged in Fe uptake, transport, and storage. The stability of OsIDEF1 protein is controlled by OsIBP1.1 and CSN6. CSN6 promotes OsIDEF1 degradation through 26S proteasome whereas OsIBP1.1 prevents it. Another NAC member, OsIDEF2, upregulates expression of the OsYSL2 gene, which is important for Fe transport to aerial parts. Ubiquitin ligase OsHRZ1 regulates OsbHLH060, OsbHLH058, OsbHLH059, and OsIDEF1 at the posttranslational level. It prevents the expression of these TFs under Fe-sufficient conditions. In rice plants, bHLH family TFs play an important role in maintaining Fe homeostasis. OsbHLH060/OsPRI1, OsbHLH058/OsPRI2, and OsbHLH059/OsPRI3 promote the expression of OsIRO2 and OsIRO3, which are crucial for Fe-deficiency responses. OsIRO2 is the key TF for regulating the transcription of strategy II-related genes. Its nuclear localization is promoted by OsbHLH156 TF. OsIRO3 negatively regulates Fe deficiency responses by hampering the expression of the OsNAS3 gene, which is important for nicotianamine synthesis. OsbHLH133 controls the translocation of Fe from root to shoot. It promotes more Fe accumulation in root as compared to shoot under Fe deficient conditions. OsWRKY64, OsWRKY113, OsWRKY55-like, OsWRKY46, and OsWRKY80 are induced under Fe toxicity condition. In the figure, proteins in colored boxes serve as regulators for TFs. The red lines represent the repression while the black line shows the activation of genes/proteins.

improve crops (Shahzad et al., 2021). A single TF may be involved in the signaling cascade of different pathways. Therefore selection of an appropriate TF is crucial. Using RNAi method, the knockdown of TFs that increase susceptibility towards Fe deficiency or Fe toxicity may lead to the development of Fe stress-resistant rice plants. Another approach of crop improvement is genome editing using tools such as CRISPR/Cas, TALENs, and ZFNs (Alok et al., 2021; Sushmita et al., 2021; Upadhyay, 2021). Genome editing can be used for either gain or loss of function mutation at an appropriate genomic location. Using these tools, the DNA sequence of a

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TF can be edited to explore its role in Fe homeostasis. Before applying the aforementioned tools to achieve Fe-stress tolerance, it is necessary to decipher the signaling cascade operative under Fe stress and the nature of the function (transcription induction or inhibition) a TF performs. Many gaps still exist in the understanding of this signaling cascade in rice that need to be filled in the future.

Acknowledgments Praveen Soni would like to acknowledge the Ministry of human resource development, Department of higher education, Government of India for the RUSA 2.0 programme (Thematic Project III), awarded to the Department of Botany, University of Rajasthan. Praveen Soni also thankfully acknowledges University Grants Commission, India for the Start-up Grant (Grant no. F.30-91/2015 BSR) and Department of Botany, University of Rajasthan, Jaipur for providing basic infrastructure facilities. HR acknowledges DST-INSPIRE Faculty grant from Department of Science and Technology, Government of India, with grant no. DST/INSPIRE/04/2016/001118.

Author contribution Pooja Kanwar Shekhawat wrote the first draft of the chapter. Praveen Soni and Hasthi Ram conceptualized, worked out the outline of the chapter and contributed to editing and proofreading.

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548. Upadhyay, S.K., 2021. Genome Engineering for Crop Improvement. John Wiley & Sons, https://doi.org/ 10.1002/9781119672425. Upadhyay, S.K., 2022. Cation Transporters in Plants. Academic Press. Available from: https://doi.org/10.1016/ C2020-0-02921-X. Varotto, C., Maiwald, D., Pesaresi, P., Jahns, P., Salamini, F., Leister, D., 2002. The metal ion transporter IRT1 is necessary for iron homeostasis and efficient photosynthesis in Arabidopsis thaliana. The Plant Journal 31 (5), 589
599. Vert, G., Grotz, N., D´edald´echamp, F., Gaymard, F., Guerinot, M.L., Briat, J.F., et al., 2002. an Arabidopsis transporter essential for iron uptake from the soil and for plant growth. The Plant Cell 14 (6), 1223
1233. Viana, V.E., Marini, N., Finatto, T., Ezquer Garin, J.I., Busanello, C., Dos Santos, R.S., et al., 2017. Iron excess in rice: from phenotypic changes to functional genomics of WRKY transcription factors. Genetics and Molecular Research 16 (3), gmr16039694. Wang, N., Cui, Y., Liu, Y., Fan, H., Du, J., Huang, Z., et al., 2013. Requirement and functional redundancy of Ib subgroup bHLH proteins for iron deficiency responses and uptake in Arabidopsis thaliana. Molecular Plant 6 (2), 503
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1260. Wang, B., Li, G., Zhang, W.H., 2015. Brassinosteroids are involved in Fe homeostasis in rice (Oryza sativa L.). Journal of Experimental Botany 66 (9), 2749
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236. Wu, L.B., Shhadi, M.Y., Gregorio, G., Matthus, E., Becker, M., Frei, M., 2014. Genetic and physiological analysis of tolerance to acute iron toxicity in rice. Rice 7 (1), 1
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584. Xiong, Y., Liu, T., Tian, C., Sun, S., Li, J., Chen, M., 2005. Transcription factors in rice. A genome-wide comparative analysis between monocots and eudicots. Plant Molecular Biology 59 (1), 191
203. Yang, J.L., Chen, W.W., Chen, L.Q., Qin, C., Jin, C.W., Shi, Y.Z., et al., 2013. The 14-3-3 protein General Regulatory Factor11 (GRF 11) acts downstream of nitric oxide to regulate iron acquisition in Arabidopsis thaliana. New Phytologist 197 (3), 815
824. Yuan, Y., Wu, H., Wang, N., Li, J., Zhao, W., Du, J., et al., 2008. FIT interacts with AtbHLH38 and AtbHLH39 in regulating iron uptake gene expression for iron homeostasis in Arabidopsis. Cell Research 18 (3), 385
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Zhang, J., Liu, B., Li, M., Feng, D., Jin, H., Wang, P., et al., 2015. The bHLH transcription factor bHLH104 interacts with IAA-LEUCINE RESISTANT3 and modulates iron homeostasis in Arabidopsis. The Plant Cell 27 (3), 787
805. Zhang, H., Li, Y., Pu, M., Xu, P., Liang, G., Yu, D., 2020. Oryza sativa positive regulator of iron deficiency response 2 (OsPRI2) and OsPRI3 are involved in the maintenance of Fe homeostasis. Plant, Cell & Environment 43 (1), 261
274. Zhang, H., Li, Y., Yao, X., Liang, G., Yu, D., 2017. Positive regulator of iron homeostasis1, OsPRI1, facilitates iron homeostasis. Plant Physiology 175 (1), 543
554. Zhang, Y., Xu, Y.H., Yi, H.Y., Gong, J.M., 2012. Vacuolar membrane transporters OsVIT1 and OsVIT2 modulate iron translocation between flag leaves and seeds in rice. The Plant Journal 72 (3), 400
410. Zhao, W., Liu, Y., Li, L., Meng, H., Yang, Y., Dong, Z., et al., 2021. Genome-wide identification and characterization of bHLH transcription factors related to anthocyanin biosynthesis in red walnut (Juglans regia L.). Frontiers in Genetics 12, 632509. Zheng, L., Ying, Y., Wang, L., Wang, F., Whelan, J., Shou, H., 2010. Identification of a novel iron regulated basic helix-loop-helix protein involved in Fe homeostasis in Oryza sativa. BMC Plant Biology 10 (1), 1
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15.

SECTION

Plant TFs and Stress

IV

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CHAPTER

13

Impact of transcription factors in plant abiotic stress: a recent advancement for crop improvement

Divya Chauhan1, Devendra Singh2, Himanshu Pandey3, Zeba Khan4, Rakesh Srivastava4, Vinay Kumar Dhiman5 and Vivek Kumar Dhiman6 1

Division of Germplasm, ICAR-National Bureau of Plant Genetic Resources, New Delhi, Delhi, India 2Department of Biotechnology, B.N. College of Engineering and Technology, Lucknow, Uttar Pradesh, India 3Department of Biotechnology, Dr. YSP UHF Nauni, Solan, Himachal Pradesh, India 4Molecular Biology and Biotechnology Division, CSIR-National Botanical Research Institute, Lucknow, Uttar Pradesh, India 5Department of Basic Sciences, Dr. YSP UHF Nauni, Solan, Himachal Pradesh, India 6Departmnt of Biotechnology, Himachal Pradesh University, Shimla, Himachal Pradesh, India

13.1 Introduction Environment fluctuations pose harmful effects on plants at different phonological stages. During signaling events, interactions in the environment are monitored and controlled by regulatory molecules at cellular levels (Srivastava et al., 2014; Takahashi and Shinozaki, 2019). Responses to internal signals between interacting partners are coordinated by transcription factors (TFs) in the developmental processes of plants, which facilitate signaling pathways against biotic as well as abiotic stimuli (Joshi et al., 2016). TFs function with other transcriptional regulators such as chromatin-modifying/remodeling proteins or corepressor/corepressor (Srivastava et al., 2018; Pandey et al., 2019). These TFs regulate the expression of different downstream genes via interacting with the cis-elements present on the promoter region of the genes (Udvardi et al., 2007; Agarwal and Jha, 2010; Srivastava et al., 2018; Agarwal et al., 2020). A study by Riechmann et al. (2000) revealed the expression of 1500 TFs in Arabidopsis thaliana imparting abiotic stress. In plants, TFs account for about 5%
7% of genome coding sequences (Hoang et al., 2017). Expression studies in Arabidopsis suggest different pathways responding differently to environmental stress. Hence, these stress-responsive TFs could act as crucial targets for economical crops to enhance abiotic stress tolerance.

13.2 Regulatory function of transcription factors in response to abiotic stress Environmental stimuli are primarily detected by transcellular membrane sensors that comprise Ca21 binding proteins and sensors, G-protein coupled receptors, and histidine kinases (Kaur et al., 2021a, 2021b, 2022; Taneja and Upadhyay, 2021; Upadhyay, 2021). As a result of such actions, stimulation of Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00005-4 © 2023 Elsevier Inc. All rights reserved.

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secondary messengers (such as kinases) and their partners (e.g., histidine phosphotransferase) occurs via the phosphorylation process and leads the stress signals to the nucleus. In addition, changes in cell shape, turgidity, and solute concentration, along with ROS production, trigger early stress responses in plants. Previous studies have shown the role of several molecules and proteins involved in the stress signal transduction mechanism (Huang et al., 2012; Madhu et al., 2021, 2022; Shumayla et al., 2019; Shumayla and Upadhyay, 2022; Tyagi et al., 2017, 2019, 2021; Upadhyay and Shumayla, 2022) (Fig. 13.1). Further, studies have also shown the involvement of stress hormones such as JA (jasmonic acid) and ABA (abscisic acid) during abiotic stress conditions (Srivastava et al., 2014; Yoon et al., 2020). ABA and JA mediated signaling pathways trigger TFs like bZIP (basic leucine zipper), ABF, whereas JA mediated pathways trigger the bHLH (basic helix-loop-helix) MYC TF (Huang et al., 2012). Here, we highlight abiotic stress-responsive pathways, which are controlled by JA and ABA stress-responsive genes, and their regulation by different TFs.

13.3 ABA signaling pathway The ABA hormone is produced by an enzymatic activity of NCED (9-cis-epoxycartotenoid dioxygenase), cytosolic SDR (short-chain dehydrogenase/reductase), and AAO (abscisic aldehyde oxidase), which helps in the production of xanthoxin, and its conversion to ABA (Singh and Laxmi, 2015). The ABA hormone facilitates plant responses during abiotic stress; for example, stress-induced ABA modulates plant development, leaf senescence, stomatal closure as well as the inhibition of root growth (Singh and Laxmi, 2015). Several studies have suggested that different stress-mediated transcription factors, for instance, bZIP, AP2/ERF, MYC, MYB, and NAC family members, participate in the gene regulation of ABA biosynthesis enzymes, which in turn activate the ABA production during abiotic stresses such as drought and osmosis (Srivastava et al., 2014; Singh and Laxmi, 2015; Fuhrmann-Aoyagi et al., 2021). A study by Takeuchi et al. (2016) reported the role of Arabidopsis CYP707Asis in ABA oxidative catabolism. During ABA biosynthesis, the CYP707As expression is influenced by different abiotic stresses like drought, oxidative stress, and salinity. Hence, loss of CYP707As expression can influence ABA levels, suggesting the importance of CYP707A facilitated ABA degradation to regulate ABA at the cellular level (Zheng et al., 2012; Matilla et al., 2015). Studies on ABA-mediated response have shown ABA biosynthetic gene overexpression such as NCED during stress (Matilla et al., 2015). Other studies have shown stress causes accumulation of ABA in shoots from roots under drought, suggesting that stress signaling induce synthesized of ABA in root is transported from roots to shoots via the xylem sap (Jia and Davies, 2007; Kuromori et al., 2018). The process of ABA signaling involves recognition of ABA by RCAR (Regulatory Component of ABA Receptor) by SNF1-Related Protein Kinases Type 2 s (SnRK2s) and Protein Phosphatase Type 2 (PP2Cs) (Hubbard et al., 2010). A stress-specific transcription response is produced by activation of ABF (ABA-Responsive Element Binding Factor) TF that corresponds to a bZIP TF and therefore regulates the expression profile of ABA-responsive genes (ABRE). Further, SnRK2s trigger phosphorylation, whereas in the absence of ABA, activation of ABFs is blocked by suppression of SnRK2. During abiotic stress, ABA biosynthesis takes place, leading to the formation of the complex (PYR/PYL-PP2C), inhibiting PP2C activity and thus triggering SnRK2 to phosphorylate as well as activate ABFs

13.3 ABA signaling pathway

273

Physical Stress Chemical Stress Salinity Temperature changes Drought Flood Mechanical stress Radiation (UV, Ionisation)

Gaseous Pollutants Aerosols Heavy metals Pesticides Mineral salts

ABIOTIC STRESSES Receptors/Sensors

Signal Transduction

Master Regulators

Signaling Molecules

Ca2+, ROS, cAMP, CDPK, MAPK, PPs, PKs, HsfA1s, etc

Modulation by Transcription Factors bZIP, MYC, bHLH, NAC, AP2/ERF, DRE-B, ERF, CBF1, WRKY, MYB

Re-programming of Stress Responsive Gene Expression Physiological and Morphological Adjustment

Stress Tolerance Plant FIGURE 13.1 A systematic representation of TF mediating abiotic stress-signaling pathways. Different abiotic (physical and chemical) stresses affect plant development and growth. In response to these stresses, plants have established quick feedback to unfavorable circumstances; these feedback processes are regulated by different signal pathways. The different components of stress responses are signal perception (receptors or sensors), signal transduction (such as secondary messenger, kinases, or by other intracellular molecules or ions), modulation of transcriptional regulation, reprogramming of gene expression, and plant adoption. These stepwise cascade mechanisms that occur during abiotic stresses leads to tolerance towards the particular stress conditions.

(Liu et al., 2014). The expressed ABFs interact with ABRE and induce activation TFs such as AP2/ ERFs and NAC under abiotic stress (Nakashima et al., 2014) The genome-wide expression profile is affected along with the defense system of plants to acclimatize and endure the stress, signifying the

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Chapter 13 Impact of transcription factors in plant abiotic

importance of bZIPs and ABFs as important components of plant resistance to stress conditions (Tiwari et al., 2017).

13.4 JA signaling pathway JA metabolism is influenced by environmental stress responses and controls development stages under stress. The JA is a fatty acid-derivate signaling hormone, which is originally isolated from methyl ester called Jasminumgrandiflorum (Ho et al., 2020). The biosynthesis of hormone occurs via the octadecanoid pathway from linolenic acid that comprises various enzymatic components like AOS (allene oxide synthase), AOC (allene oxide cyclase), LOX (lipoxygenase), and OPR (12oxo-PDA reductase) (Creelman and Mullet, 1997). The mechanism takes place by metabolizing the free acid form of JA formed (from the octadecanoid pathway) into methyl jasmonate (MeJA), displaying the activity of jasmonatemethyltransferase (JMT) and jasmonate-amindosynthetase 1 (JAR1) (Mueller et al., 1993). As a result, levels of endogenous JA increase on upregulation of JA biosynthesis genes. For instance, activation of AOC, AOS, and LOX was observed on activation of JA responsive genes during cold stress in rice and Arabidopsis (Ho et al., 2020). Moreover, reduced tolerance to freezing was observed in JA-deficient mutants of LOX2 and AOS, whereas tolerance was enhanced on exogenous JA treatment. Expression studies of transgenic Arabidopsis revealed overexpression of wheat TaAOC1 on improved tolerance to salt stress during JA production (Muchate et al., 2016). These research studies corroborate the view that the metabolism of JA is essential in monitoring tolerance levels in plants under stress conditions. On the other hand, the process for JA transport is not as widely known (Katsir et al., 2008). A study on radioisotopic labeling revealed that transportation of JA happens through both the xylem and phloem (Ruan et al., 2019). In addition, experiments on micrografting showed shoot-to-root flow of JA via the phloem (Hause et al., 2003). Several TFs associated with JA signaling pathway have been recognized and their functions described, including bHLH-type domain type TFs; for example, MYC2, TT8 (Transparent Testa 8), and several others were reported in Guo et al. (2021). The bHLHTF family is widely distributed in eukaryotes including in plants. Arabidopsis constitutes around 162bHLH TFs based on sequence similarity and several bHLH TFs such as AtbHLH122, AtbHLH68, and AtbHLH17 are reportedly involved in stress responses of plants (Weirauch and Hughes, 2011; Guo et al., 2021). Seo et al. (2011a,b) reported on the interaction of rice OsbHLH148 with JAZ proteins, suggesting its role in JA signaling. Further, OsbHLH148 expression was found upregulated on treatment with JA and different abiotic stresses such as cold, salt stress, and drought (Seo et al., 2011a,b) Its overexpression has resulted in enhanced transcript levels of OsDREBs and showed improved tolerance in drought stress conditions. This study supports the finding that bHLH TFs, including MYC2 and OsbHLH, play an important role in plant response and tolerance to drought stress. ABA-mediated plant tolerance was also observed in bHLH TFs that include OsbHLH148 and MYC2 (Seo et al., 2011a,b). It was further seen that OsbHLH148 expression was upregulated by ABA (Seo et al., 2011a,b). Another study on MYC2 mutants and overexpressing plants showed reduced and enhanced sensitivity to ABA, respectively, suggesting MYC2 acts as the positive regulator for ABA signaling (Kazan and Manners, 2013). Further, accumulated evidence suggests that ABA participates in stress regulation with the association of stress-mediation bHLH TFs

13.5 Transcription factors involved in abiotic stress tolerance

275

(Abe et al., 1997; Waseem et al., 2019; Zheng et al., 2019). Moreover, other evidence revealed that JA biosynthesis activation by jasmonic acid methyltransferase stimulates the production of ABA and also reported that ABA receptor PYL6 is associated with MYC2 (Aleman et al., 2016). PYL6 can modify JAZ6 and JAZ8, which have a binding site for MYC2 on their promoter (Aleman et al., 2016). All these findings show the interaction of JA with ABA to regulate stress response and also the involvement of bHLH TF in JA-ABA crosstalk.

13.5 Transcription factors involved in abiotic stress tolerance 13.5.1 MYB TFs The MYB protein family functions as TFs containing a different number of conserved MYB domains that bind to DNA. MYB domain contains nearly 50 amino acids (AA), and MYB TFs are categorized into four subfamilies based on the location and number of MYB repeats like 1R-MYB, R1R2R3MYB, R2R3-MYB, and 4R-MYB. The class of R2R3-MYBs belongs to plant-specific MYB TFs (Smita et al., 2015; Qin et al., 2012). The interaction of MYB domains with other stress-related TFs such as WRKY and MYCs suggests the involvement of MYBs in plant stress response and tolerance to stress. Many studies demonstrate the functional characterization of MYBs during abiotic stress by understanding the system of knock-out and overexpression (e.g., Arabidopsis TF MYB96 is reported for drought tolerance) (Seo et al., 2011a,b). Expression of this TF is regulated by drought and ABA, while overexpression leads to the biosynthesis of cuticular wax, which inhibits loss of waterviathe leaf surface, resulting in increased drought resistance (Seo et al., 2011a,b). Similar results were reported from a study where gene AtMYB96 overexpression showed better drought tolerance via cuticular accumulation in Camelinasativa (Lee et al., 2014). Hence, the abovementioned studies suggest the conserved role of MYB96 in drought tolerance and cuticular wax synthesis. A study on Arabidopsis noted the role of MYB44 in response to drought tolerance. It was also stated that the AtMYB44 expression in guard cells is induced by different stresses like salinity, cold, and dry stress (Jung et al., 2008). All the same, overexpression of AtMYB44 results in high ABA sensitivity and regulates stomatal closure. The AtMYB44 exhibited high sensitivity to drought stress, displaying increased tolerance to drought stress via ABA-dependent stomatal closure (Jung et al., 2008). Moreover, the role of MYBs has also been implicated in salt stress. A study revealed upregulation of Arabidopsis MYB20 in salt stress, whereas AtMYB20 overexpression in transgenic plants induced the plant to salt tolerance (Cui et al., 2013). Although positive regulation of AtMYB20 was noticed, the AtMYB20 suppression caused a hypersensitive response during salt stress. Stress-responsive observations were also identified in different crops including soybean (MYB92, MYB177, and MYB76) and rice (MYB6, MYB4, MYB48
1, and MYB91) (Liao et al., 2008; Yoon et al., 2020). The association between ABA and MYBs is not known, but different research reports show that the expression of several MYBs is influenced by JA and ABA-dependent stress responses (Song et al., 2011; Yoon et al., 2020).

13.5.2 NAC TFs Another important plant TF NAC is an acronym derived from NAM (No Apical Meristem), ATAF1/2 (Arabidopsis Transcription Activator Factor), and Cup-Shaped Cotyledon 2 (CUC2).

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Hence, NACs are among the largest families involved in different biological processes and function as transcriptional regulators in plants. They comprise a conserved NAC domain that falls in the N terminus along with a domain having a regulatory function in the C terminal region. Researchers have reported on the role of NACs in abiotic stress. A study conducted by Jiang and Deyholos (2006) identified 33 NAC genes that showed upregulation in response to salt stress tolerance in Arabidopsis. A total of 40 NACs showed significant expression in response to dry and salinity stress in rice and soybean. These studies indicate the role of NACs in abiotic stress responses (Pinheiro et al., 2009; Rahman et al., 2016). As per the Hong et al. (2016) study, the overexpression of NAC022 in transgenic rice resulted in high sensitivity response. Further, NAC022 was found upregulated during drought and salt stress and displayed salt resistance through an ABA-dependent pathway. Also, the expression of another wheat NAC29 was highly upregulated when wheat plants were exposed to ABA, drought, and salt stress (Xu et al., 2015). Overexpression of this NAC29 in A. thaliana resulted in a hypersensitive phenotype with higher drought and salt tolerance (Huang et al., 2015). Studies conducted on stressresponsive NACs that include ANAC072, ANAC055, and OsNAC9 have depicted their role in abiotic stress (You et al., 2015; Li et al., 2021a). Also, Nakashima et al. (2012) revealed that the role of Early Responsive to Dehydration Stress 1 (ERD1) in abiotic stress coding for a chloroplast ATPdependent protease. This study revealed that the ERD1 expression pattern depends on the CATGTG motif in its promoter. Another study conducted by Tran et al. (2004) describes the interaction of ANAC055, ANAC019, and ANAC072 with the motif that regulates the ERD1 expression. Additionally, characterization of these TF suggested that expression can be affected under different abiotic stress conditions like drought, salinity, and ABA (Li et al., 2012). The results from the study indicated overexpression of these NACs considerably increased drought tolerance. However, mutants lacking seven of twenty-three activities of ANAC072, ANAC019 exhibited reduced tolerance to salinity as compared to control conditions (Bu et al., 2008). Another study on rice OsNAC6 revealed variation in its expression under drought conditions. As a result, overexpression of the OsNAC6 resulted in improved tolerance to dry conditions, while knock-out mutants showed less resistance in control plants. Thus indicating positive regulation of OsNAC6 in a response to abiotic stress (Umezawa et al., 2006). Moreover, genome-wide transcription profiling of this TF resulted in nicotianamine biosynthesis gene higher expression (i.e., NAS2 (Nicotianamine Synthase 2)) under drought conditions and provided high tolerance to stress (Yoon et al., 2020). A NAC TF, TaRNAC1 in wheat, displayed enhanced dehydration tolerance when overexpressed under stress (Chen et al., 2018). The role of the NAC TF and its regulatory interaction with ABA and JA during abiotic stress remains unknown. All these conclusions indicate the role of NACs in JA and ABA-mediated variations in plants under abiotic stress.

13.5.3 AP2/ERF TFs Apetela 2 (AP2) belongs to a class of plant-specific TFs that is comprised of four major families: AP2, Ethylene Responsive Factor, DREB, and related to abscisic acid insensitive 3/RAV (Xie et al., 2019). AP2/ERF domain-containing apetela 2 domain containing nearly 60 amino acids at the N-terminus end with a regulatory sequence in the carboxyl-terminus region that participates in transcriptional regulation (Tiwari et al., 2012). Among these TF families, DREBs have been widely studied in abiotic stress, as they respond positively to cold, heat, salt, and drought tolerance by modifying stress-responsive genes.

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For example, DREB TFs bind to dehydration responsive elements (DRE) present in stress-related genes (Sakuma et al., 2002). The role of the dehydration responsive elements/CBF has been implicated in cold stress conditions (Fernando, 2020). Higher-expression level of this factor increases plant tolerance towards cold stress in Arabidopsis, whereas knock-out mutants show reduced tolerance in freezing stress (Zhao et al., 2016). Expression of osmotic and salt-tolerant genes is induced by dehydration responsive elements 2. Its homolog has been found in soybean dehydration-responsive elements responsible for stress resistance (Chen et al., 2016). A study in mung beans also revealed high expression of DREB2 in dry and saline conditions. The role of homologs of DREB2 is associated with salt stress in many grasses like wheat, maize, and rice where downstream gene products of DREB2A acted as molecular chaperons and played an essential role in detoxification by releasing enzymes of toxins. Further, overexpression of DREB2A and dehydrin was observed in Arabidopsis, where 373 genes showed upregulation (Singh and Laxmi, 2015; Maruyama et al., 2012). Transcriptome studies revealed that the gene regulation of AP2/ERF is affected by different environmental stresses (Desikan et al., 2001). Stimulation of these TFs is influenced by stress or ABA-responsive cis-acting sequences of their promotor positions. A study conducted by Cheng et al. (2012) in Arabidopsis suggets that AtERF53 confers tolerance to drought and heat stress. In addition, AtERF74 showed resistance to dry conditions while AtERF74 knock-out mutant’s harbors decreased tolerance (Yoon et al., 2020). Another study in Arabidopsis showed tolerance to osmotic stress by peanut AP2/ERF AhDREB1 (Zhang et al., 2018). The results from these studies show the role of AP2/ERFs in stress resistance and how their function is conserved in plants. Several researchers have intended to identify and describe stress-related AP2/ERFs in different plants. In rice, stress-related AP2 are OsDREB1, OsERF71, and OsEREBP1 that mediate stress tolerance. A study by Wisniewski et al. (2014) reported that stress-responsive OsDREB71 is involved in lignin biosynthesis. Moreover, lignin biosynthesis is associated with drought stress as lignin is hydrophobic and restricts water-deficit conditions from different plant parts. Indeed, drought-resistant inbred lines showed high levels of lignin content as compared to drought-vulnerable lines in maize (Zheng et al., 2012). These reports indicate that express of OsERF71 is useful against drought stress and also controls lignin production. Different stress AP2/ ERFs have been found in plants like maize DREB2A, tomato SIERF5, and soybean ERF3, based on their function and stress-responsive factor (Yi et al., 2013; Herath, 2018; Mosa et al., 2017). Earlier studies have suggested that stress-responsive DREBs respond to environmental stress in an ABA-independent manner. Nevertheless, evidence of studies shows the response of AP2/ERFs following ABA-dependent mechanisms (Xie et al., 2019; Zhang et al., 2018). Moreover, a study involving the interaction of AP2/ERF TF RAV1 with SNF1-related protein kinase 2 revealed key kinases that regulate ABA sensitivity (Feng et al., 2014).

13.5.4 WRKY TFs WRKY TFs are known to have a conserved motif on their N-terminal region along with a ZNF like motif at their C-terminal (Eulgem et al., 2000). In plants, WRKY TFs are comprised of a large family such that Arabidopsis and soyabean genomes are known to have nearly 74, 103, and 197 WRKYs, respectively (Chen et al., 2016). A study revealed stress-related gene regulation of

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WRKYs and their role in abiotic stresses in plants. Moreover, an expression study using microarray analysis revealed the expression of 18 WRKYs during salinity stress (Jiang and Deyholos, 2006). In addition, functional characterization of WRKYs using knock-out systems was studied during plant stress. It was shown that the expression of AtWRKY25, AtWRKY26, and AtWRKY33 alters in heat stress (Li et al., 2011). However, the survival rates of these mutants were reduced during heat stress. Furthermore, overregulation of WRKY25, WRKY26, and WRKY33 in genetically modified plants resulted in increased tolerance to heat stress, suggesting positive regulation towards heat stress resistance (Li et al., 2011). Previous research reported on the importance of WRKY TFs in controlling development during abiotic stress situations, similar to AP2/ERFs and NACs. For instance, WRKY46 modifies the growth of secondary roots under unfavorable conditions, whereas WRKY22 facilitates dark-regulated senescence (Ding et al., 2015). A study conducted by He et al. (2016) showed overexpression of wheat TaWRKY1 and TaWRKY33 with enhanced drought resistance in Arabidopsis. Many WRKYs have been recognized in other plants and found useful application through characterization. For example, improved tolerance to drought was observed in overexpressed rice OsWRKY11 and OsWRKY45 (Tao et al., 2011). Research performed on Arabidopsis resulted in overexpression of TaWRKY146, which shares homology with TaWRKY46 and exhibited hypersensitive response during drought and salinity stress. Expression studies showed significant upregulation of TaWRKY146 in plant roots and plant leaves under osmotic stress. While in transgenic Arabidopsis, overexpression of TaWRKY146 suggests high contents of proline and soluble sugar and reduced malondialdehyde (MDA) under drought conditions. Overexpression of FaWRKY46 also resulted in enhanced tolerance by scavenging ROS against salinity stress (Lv et al., 2020). Zhang et al. (2016) showed the functional importance of cold stress tolerance during overexpression of WRKY46 in cucumber, which leads to ABA-dependent signaling in plants. Another study on heat stress revealed heat-inducible expression of AtWRKY25 and oxidative stress-related genes having W-box at their promoter position, independent of the SA-mediated biosynthesis (Li et al., 2011). Also, overexpression of WRKY25 elevated the expression of HSP101, HSFA2, HSFB1, and HSFB2A, depicting a key role in the HSFB2 and HSFB1 pathway (Li et al., 2009). A study in Arabidopsis revealed overexpression of GsWRKY20 that resulted in a decreased amount of loss in water from plant tissue and increased resistance towards drought stress, thereby reducing stomatal density. Several researchers have described the functional importance of different WRKY genes in ABA-mediated pathways. For example, the expression of some WRKY genes in rice is induced by OsWRKY11, OsWRKY71, and OsWRKY72. Also, crosstalk between GA and ABA signaling is regulated by OsWRKY51 and OsWRKY71 (Yu et al., 2010). Hence, these investigations suggest that the role of stress-related WRKYs is preserved in different crops and can act as important targets for abiotic stress tolerance in other crops. Further studies have also illustrated the involvement of WRKYs during ABA signaling during abiotic stress, but the mechanism remains unknown. Studies also suggest the expression of WRKYs such as GhWRKY17 and AtWRKY63 influences ABA response suggesting that WRKYs are associated with ABAdependent environmental stress signaling. Table 13.1 lists the TFs that regulate plant tolerance (Table 13.1).

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Table 13.1 TFs that regulate the plant tolerance. TF family

Species

TF member

Role in abiotic stress

References

AP2/ERF

A. thaliana

CBF1, CBF3

Cold and salt stresses

A. hypogaea G. max

DREB1 ERF3

G. max O. sativa O. sativa S. lycopersicum A. thaliana

ERF9 ERF71 ERF3 ERF5 bHLH17

Osmosis tolerance Salt, drought and high temperature stresses Cold and drought stresses Drought stress Drought stress Salt and drought stresses Oxidative and salt stresses

Park et al. (2015), Zhao et al. (2016) Zhang et al. (2018) Zhang et al. (2009)

F. tataricum

bHLH3

A. thaliana F. tataricum

ABF3 bZIP5

O. sativa O. sativa O. sativa

ABF1 bZIP71 bZIP23

A. thaliana A. thaliana C. arietinum C. sinensis G. arboretum G. max

MYB96 MYB20 MYB R2R3-MYB MYB85 MYB76, MYB177 MYB6 MYB48
1 MYB91 ANAC019 ANAC042 NAC06 NAC29 WRKY25 WRKY33 WRKY82

bHLH

bZIP

MYB

NAC

WRKY

O. sativa O. sativa O. sativa A. thaliana A. thaliana G. max T. aestivum A. thaliana A. thaliana H. brasiliensis

Oxidative and drought stresses Drought stress Salinity, drought, and Oxidative stresses Drought stress Salt and drought stresses Salt and drought stresses Cold stress Salt stress Drought stress Drought and cold stresses Drought stress Cold and salt stresses Salt and drought stresses Salt stress Salt stress Drought stress High temperature Salt stress Salt stress High temperature High temperature Drought and salt stresses

Zhai et al. (2017) Lee et al. (2017) Zhang et al. (2013) Pan et al. (2012) Babitha et al. (2013), Jia et al. (2021) Yao et al. (2017) Hwang et al. (2019) Li et al. (2020) Amir Hossain et al. (2010) Liu et al. (2014) Kazan and Manners (2013), Yoon et al. (2020) Lee et al. (2014) Cui et al. (2013) Shui et al. (2021) Liao et al. (2008) Tang et al. (2019) Xiong et al. (2014) Zhu et al. (2015) Li et al. (2012) Shen et al. (2017) Li et al. (2021b) Xu et al. (2015) Li et al. (2011) Li et al. (2011) Kang et al. (2021)

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13.6 Conclusion Heat, cold, salinity, and drought are abiotic stresses responsible for causing yield loss around the world. TFs as target genes could be used to bring a new revolution in the field of biotechnology such that new crops with improved resistance can be generated against biotic/abiotic stresses. Several studies on robust stress (abiotic) tolerance have been developed by enhancing the expression of TFencoding genes in the host plant under monitored growth conditions. However, testing at field trials at different development stages and performance validation of genetically modified crops is still limited under natural conditions, which can limit commercial launch and utilization of resistant varieties. Moreover, data of productivity and phenotypic examination pose a challenge for researchers to study the complex mechanism of pathways involved in determining the behavior of plants under multiple stress situations. Hence, extensive omics data can is required to increase practical knowledge.

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3796. Zhang, B., Su, L., Hu, B., Li, L., 2018. Expression of AhDREB1, an AP2/ERF transcription factor gene from peanut, is affected by histone acetylation and increases abscisic acid sensitivity and tolerance to osmotic stress in Arabidopsis. International Journal of Molecular Sciences 19 (5), 1441. Zhang, Y., Yu, H., Yang, X., Li, Q., Ling, J., Wang, H., et al., 2016. CsWRKY46, a WRKY transcription factor from cucumber, confers cold resistance in transgenic-plant by regulating a set of cold-stress responsive genes in an ABA-dependent manner. Plant Physiology and Biochemistry 108, 478
487. Zhang, H., Zhang, J., Quan, R., Pan, X., Wan, L., Huang, R., 2013. EAR motif mutation of rice OsERF3 alters the regulation of ethylene biosynthesis and drought tolerance. Planta 237, 1443
1451. Zhao, C., Zhang, Z., Xie, S., Si, T., Li, Y., Zhu, J.-K., 2016. Mutational evidence for the critical role of CBF transcription factors in cold acclimation in Arabidopsis. Plant Physiology 171, 2744
2759. Zheng, Y., Huang, Y., Xian, W., Wang, J., Liao, H., 2012. Identification and expression analysis of the Glycine maxCYP707A gene family in response to drought and salt stresses. Annals of Botany 110, 743
756. Zheng, K., Wang, Y., Wang, S., 2019. The non-DNA binding bHLH transcription factor paclobutrazol resistances are involved in the regulation of ABA and salt responses in Arabidopsis. Plant Physiology and Biochemistry: PPB/Societe Francaise de Physiologie Vegetale 139, 239
245. Zhu, N., Cheng, S., Liu, X., Du, H., Dai, M., Zhou, D.X., et al., 2015. The R2R3-type MYB gene OsMYB91 has a function in coordinating plant growth and salt stress tolerance in rice. Plant Science (Shannon, Ireland) 236, 146
156.

CHAPTER

Plant transcription factors and temperature stress

14 Tingting Zhang and Yang Zhou

School of Horticulture, Hainan University/Key Laboratory for Quality Regulation of Tropical Horticultural Crops of Hainan Province, Haikou, P.R. China

14.1 Effect of temperature stress on plant growth Plants often suffer various adverse environmental stresses including heat, cold, salt and drought in their life cycles. Temperature plays a key role in the growth and development of plants. When the environmental temperature is beyond the adaptive range of plants, it will cause stress on plants, affecting photosynthesis, cell membrane fluidity, cell osmotic pressure, protein and nucleic acid structure, ions transportation, receptor signaling and other physiological and metabolic responses (Chinnusamy et al., 2007; Shumayla, 2019; Upadhyay, 2021, 2022; Upadhyay and Shumayla, 2022; Wahid et al., 2007; Zhu et al., 2007).

14.1.1 Effect of high-temperature stress on plants Heat stress is usually divided into two types: one is a long-term temperature slightly higher than the optimal temperature, called “long-term heat stress”; the other is a short-term temperature that is relatively higher than optimal, called “short-term heat stress” (Belehradek, 1957). The damage caused by long-term high-temperature stress to plants is slow, and is manifested as cell water loss and leaf wilting in the early stage, and eventually leads to plant death. However, short-term hightemperature stress causes direct damage to plants and affects cell structure, resulting in a series of physiological obstacles and irreversible damage to plants. When plants are subjected to high-temperature stress, the stomatal movement of plant leaves is directly affected. Under high temperature, the opening angle of leaf stomata increases, which accelerates the water evaporation rate, resulting in a large amount of dehydration in a short period of time. Meanwhile, excessive environmental temperature will increase soil temperature, damage plant roots, and affect absorption of water and nutrients, thus resulting in insufficient water and nutrients in the above-ground parts of plants, yellowing of leaves, wilting of plants, and even death (Huang et al., 2012). Furthermore, high temperature also affects the structure of mesophyll cells and chloroplasts, which can affect photosynthesis in plants (Bayu et al., 2006). Flowering is very sensitive to temperature during plant growth and development and temperature stress can lead to flowering delay or advance. Temperature stress often leads to pollen abortion, pollen and ovary development asynchronously, because pollen is more sensitive to Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00012-1 © 2023 Elsevier Inc. All rights reserved.

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temperature than pistil (Balasubramanian et al., 2006; Sakata et al., 2000). The inflorescences at different developmental stages of poplar were treated at high temperature. It was found that the proportion of macropollens and abortion pollens increased with the extension of the treatment time, and the highest frequency of macropollen appeared in the middle of the first meiotic division (Wang et al., 2017). Cytological observation showed that the chromosome behavior was abnormal during meiosis under high-temperature stress, with parallel, fused, and tripolar spindles. Immunolocalization experiments showed that high-temperature caused irreversible damage to part of the microtubule skeleton during meiosis, which was the cause of the production of macropollen (Wang et al., 2017). The unreduced pollen produced in rose after high-temperature treatment for 48 h was caused by the influence of spindle polarity during the second meiotic division (P´ecrix et al., 2011), and the same phenotype was also found in alfalfa and sweet potato (De Storme and Geelen, 2014). In fact, the temperature tolerance of plants is different, and the sensitivity of pollen of different species to temperature stress is also different. However, in the process of pollen development, the meiosis period, especially the period when the meiosis cytoplasm divides into tetrad, is the most sensitive period to temperature stress, which is related to the conservation of pollen development in different species. High temperature also has an important effect on fruit development. Results have shown that the response of crops to environmental changes was directly reflected in the changes of fruit morphology and yield. When crops are subjected to high-temperature stress, the photosynthetic rate of leaves decreases, which eventually leads to crop yield decrease (Feng et al., 2014; Lin et al., 2016). A previous study showed that the number of panicles per plant, seeds per grain, and dry matter decreased, which led to wheat yield decrease when wheat was subjected to high-temperature stress at flowering and shooting stages (Duffus and Rosie, 1973). Furthermore, researchers have found that the number and diameter of tomato fruits decreased (Din et al., 2015), the leaf area of grapes decreased, and the allocation of dry matter to leaves and stems was inhibited after high-temperature stress (Kadir et al., 2007).

14.1.2 Effect of low-temperature stress to plants According to different temperatures, low-temperature stress can be divided into two types. One is the environmental temperature of 0 C
15 C, called “chilling stress”; second, where the ambient temperature is lower than 0 C, which is called “freezing injury stress” (Shi et al., 2018). Low-temperature stress reduced stomata closure ability and water transport rate of plant leaves, leading to plant wilt (Ball et al., 2004). The chloroplast structure of plants was damaged under low-temperature stress, resulting in the decrease of chlorophyll content and the yellowing of plant leaves (Vranova et al., 2002). Low-temperature stress also reduced the biochemical reaction rate of plants, reduced the ability of plants to absorb water and nutrients, affected the flowering and fruit of plants, and finally showed the decline of fruit yield and quality (Chinnusamy et al., 2007). When plants are subjected to chilling stress, a series of changes will occur in the biofilm system, oxidase system, and osmotic regulatory substances inside the cells to cope with the damage caused by cold or freezing (Wang et al., 2018). Low temperature inhibits the normal metabolism of reactive oxygen species in plant cells, thus causing toxic damage to plants and inhibiting plant growth. Previous studies have found that plants with higher antioxidant capacity are more tolerant to stress (Ball et al., 2004). Under low-temperature stress, the content of reactive oxygen species

14.2 Transcription factors involved in response to temperature stress

289

increases, and plants use a series of antioxidant enzymes such as SOD, CAT, GPX, GR, and APX to remove them, which improved the antioxidant capacity and low-temperature tolerance of plants (Madhu, 2021, 2022; Meng et al., 2017; Tyagi, 2017, 2018, 2019, 2020, 2021). Studies have shown that under low-temperature stress, water loss of cells, and leaf degradation can be alleviated by the continuous accumulation of osmotic regulatory substances such as proline and soluble sugar (An et al., 2012; Wang et al., 2018; Yan, 2019). Meanwhile, exogenous proline can reduce the damage of plants under low-temperature stress (Fu et al., 2019). After the knockout of the proline-rich protein (PRP) synthesis gene OsPRP1, the sensitivity of transgenic rice to low-temperature stress decreased and the survival rate decreased due to the restriction of proline synthesis (Nawaz et al., 2019). Low-temperature stress also affects pollen development. Studies on model plants such as Arabidopsis thaliana and rice have found that the effects of low-temperature stress on pollen development are mainly manifested as decreased fertility, decreased pollen number, abnormal structure, decreased viability, decreased pollen germination rate, decreased seed setting rate, and abnormal degradation of tapetal and callose (Mamun et al., 2006; Oda et al., 2010; Zou et al., 2010). When arabidopsis flower buds were treated at low temperature for 40 h during meiosis, it was found that short-term low-temperature pressure inhibited the formation of microtubules, affected normal cytoplasmic division, and finally induced polyploid production of large numbers of pollen grains (De Storme and Geelen, 2014). The influence of low-temperature stress on pollen development was also reflected in glucose metabolism during pollen development. In the process of glucose metabolism during pollen development, the microspore trophic nuclei begin to slowly synthesize starch after the first mitosis, and the starch content is the highest in the binuclear stage. As pollen matures, starch is gradually converted into soluble sugar (Datta et al., 2002; Pressman et al., 2002).

14.2 Transcription factors involved in response to temperature stress A transcription factor (TF) is a special type of protein, and it is the main regulatory factor in plants under stress environment. Through the interaction of TFs with cis-acting elements in promoters, the transcription process of genes is activated or inhibited, and then the expression of genes is regulated. It is involved in a variety of complex signaling pathways in plants responding to stress (Rabara et al., 2014). In order to survive, plants form a complex and efficient regulatory network to resist and adapt to external stress, in which the regulation of TFs plays a key role. After heat stress occurs, plants can tolerate heat stress to a certain extent by regulating heat shock proteins, abscisic acid (ABA), gibberellic acid (GA), and jasmonic acid (JA). Plant hormones such as indole-3-acetic acid (IAA) are secreted to enhance heat resistance (Ali et al., 2020), and these processes are closely related to heat shock TFs.

14.2.1 HSF transcription factor Plant heat shock TFs are one of the most widely studied TF families in plants. Starting in 1990, Scharf et al. (2012) discovered the TF HSF combined with heat shock response elements through heat shock treatment of tomato cell cultures, and began to carry out HSFs studies in a variety of

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plants. HSF is an important component of plant stress response, which mainly consists of five domains: DNA binding domain (DBD), oligomerization domain (OD), nuclear localization signal (NLS), nuclear export signal (NES), and C-transcriptional activation domain (CTAD). According to the OD types and phylogenetic relationship, HSF in plants can be divided into three subfamilies: group A, B, and C. Compared with group B, there are more abundant amino acid residues in hr-a/B region of groups A and C (Nover et al., 2001; Guo et al., 2015). Many HSF TF family members have been identified in some plants, including 22 in Arabidopsis and 25 in rice (Guo et al., 2008), 56 in wheat (Zhou et al., 2019), and 52 in soybean (Li et al., 2014). Under heat stress, HSF activates and regulates the expression of heat shock proteins (HSP) by identifying heat shock cis-acting elements of HSP genes (Akerfelt et al., 2010; Garbuz, 2017) Transcriptomic analysis has shown that the HSF gene family was upregulated under hightemperature stress in Brassica napus (Zhu et al., 2017). It has been found that tomato overexpressing (OE) SlHsfA1 and plants silencing (CS) SlHsfA1 under normal growth conditions were similar to wild type (WT) tomato in major developmental parameters. However, CS plants died after exposure to an ambient temperature of 45 C for 1 h, while WT and OE plants were not significantly affected during development (Mishra et al., 2002). In situ RNA hybridization, qRT-PCR, Western blot, and other experiments showed that the tomato heat shock factor A2 protein SlHsfA2 and another important member Hsp17-CΠ was regulated during another development and further induced under short-term and long-term heat stress (Giorno et al., 2010). Studies have shown that heterologous expression of tea (Camellia sinensis) heat shock factor A2 gene CsHsfA2 can improve the heat resistance of transgenic yeast (Zhang et al., 2020). AtHSFA6b was found to be involved in plant heat stress resistance as a downstream regulator of ABA-mediated stress response in A. thaliana (Huang et al., 2016). After transferring the peach (Prunus persica) heat shock factor PpHSF5 gene from peach to Arabidopsis, it was found that the heat tolerance of transgenic plant was enhanced, and PpHSF5 may inhibit the growth and development of roots and organic organs (Tan et al., 2021).

14.2.2 MYB transcription factor As one of the largest TF families in plants, MYB TFs exist widely in plants and are involved in plant growth and development regulation and abiotic stress response. The N-terminus of MYB TFs all have conserved DNA binding domains, which are composed of 1
4 incomplete repeats (R), each of which contains about 52 amino acids, forming three α-helixes. The second and the third helical forms a three-dimensional helic-turning-helical (HTH) structure, which combines with the target gene. In each repeat sequence, there are three regularly spaced tryptophan residues, which form hydrophobic clusters in the HTH structure. Hydrophobic clusters may be related to DNA specific recognition (Ogata et al., 1995; Chen et al., 2006). The C-terminal sequence of MYB is not highly conserved and is responsible for the regulation of protein activity (Ramya et al., 2017). The MYB gene family was divided into 1R-MYB/MYB-related, R2R3- MYB, 3R- MYB, and 4R- MYB according to the number of repeated R (Fig. 14.1) (Li et al., 2015). It has been reported in Arabidopsis (Dubos et al., 2010), rice (Katiyar et al., 2012) and other plants. Previous studies have shown that MYB regulates gene expression through competitive binding with cis-acting elements in the promoter region and responds to various stresses (Shangguan et al., 2008; Lai et al., 2014). Studies in rice have shown that overexpression of OsMYB55 enhanced amino acid metabolism

14.2 Transcription factors involved in response to temperature stress

291

FIGURE 14.1 Structure and classification of MYB TFs (Li et al., 2015).

through transcriptional activation and improved rice tolerance to high temperature (El-Kereamy et al., 2012). In wheat, combined with transcriptomic analysis and transgenic technology, it was found that TaMYB80, driven by CaMV35S promoter, enhanced the tolerance of transgenic A. thaliana to high temperature, and this result was related to the increase of abscisic acid (ABA) content (Zhao et al., 2017). AtMYB15 improves freezing resistance by inhibiting the expression of CBF1/ DREB1 (C-repeat/dehydration-responsive element binding TF 1), so it is considered as a negative regulator of freezing resistance (Agarwal et al., 2006). OsMYBS3 is a positive regulator of cold tolerance and can inhibit the DREB1/CBF dependent cold signaling pathway in rice, indicating that different pathways play a gradual role in the adaptation to short/long-term cold stress (Su et al., 2010). It has been shown that low temperature can induce anthocyanin biosynthesis in A. thaliana, which involves regulatory roles of AtMYB3, AtMYB6, and AtMYBL2 (Leyva et al., 1995; Rowan et al., 2009). Anthocyanin accumulation in apples is induced by MYB10 affected by temperature (Lin-Wang et al., 2011). PcMYB10 in pear can positively control the synthesis of anthocyanin induced by low temperature (Li et al., 2012).

14.2.3 AP2/ERF transcription factors The APETALA2/Ethylene responsive element binding factor (AP2/ERF) gene family plays an important role in plant normal growth and development as well as under different stress conditions. AP2/ERF is related to the germination rate of plant seeds, morphological changes of plant organs, ethylene transcription, accumulation of secondary metabolites, response to stress, and signal transduction under stress (Jin et al., 2018; Mizoi et al., 2012; Xu et al., 2011). The AP2/ERF domain is composed of about 60 highly conserved amino acids. According to the number of AP2/ERF-like TF domains and binding sequences, AP2/ERF TFs can be divided into five subfamilies: AP2 (APETALA2), ERF (ethylene-responsive element binding factor), DREB (dehydration responsive element binding), RAV (RELATED TO ABI3/VP1), and Soloist. The AP2 subfamily contains two AP2/ERF domains with high similarity and tandem repetition. The subfamily can be further divided

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into AP2 and ANT (Adenine nucleotide translocator) according to the repeated AP2/ERF domains and the nuclear location sequence. The ERF and DREB subfamilies contain only one AP2/ERF domain, and the difference lies in the different amino acid residues of the conserved sequence. The 14th and 19th positions of the ERF subfamily are alanine and aspartic acid, respectively, while the 14th and 19th positions of the DREB subfamily are valine and glutamic acid, respectively. The ERF and DREB subfamilies can be further divided into six subgroups, B1-B6 and A1-A6, respectively. The RAV subfamily contains one AP2/ERF domain and one B3 domain, with AP2 domain at the C terminal and B3 domain at the N terminal. The Soloist subfamily also contains an AP2/ ERF domain, but its amino acid motif and gene structure are very different from other AP2/ERF subfamilies, and the number of such TFs is small. So far, few studies have been conducted on the TFs of Soloist subfamily (Sakuma et al., 2002; Karanja et al., 2019). DREB is an important TF in the AP2/ERF family that is closely related to abiotic stress. DREB can specifically bind DRE/CRT cis-acting elements to activate the expression of many downstream stress-resistant genes, and is independent of ABA signaling pathway, thus, the resistance of plants to abiotic stress was improved (Rehman and Mahmood, 2015). Studies have shown that overexpression of TaDREB3 can improve the tolerance of transgenic A. thaliana to high temperature (Niu et al., 2020). qRT-PCR in A. thaliana showed that HsfA3 was significantly upregulated in transgenic plants overexpressing DREB2C. Moreover, DREB2C and HsfA3 showed similar transcription patterns in response to HS, and DREB2C specifically activated HsfA3 transcription. Yeast monozygotic experiments also confirmed the interaction between DREB2C and two DREs in the promoter of HsfA3, thus proving that HsfA3 is regulated by DREB2C in the process of participating in response to heat stress (Chen et al., 2010). C-repeat Binding factor (CBF), also known as dehydration responsive element binding factor1 (DREB1), is an important TF involved in abiotic stress response such as low temperature in plants (Lee et al., 2012). When plants are subjected to lowtemperature stress, CBF gene is rapidly induced within 15 minutes (Shi et al., 2018). CBF transcription activators specifically bind to cis-acting elements of COR gene promoter in which the expression of downstream genes is activated and the accumulation of proline and soluble sugar is increased. Thus, plant resistance to abiotic stress such as low temperature can be improved (Thomashow, 2010). There are six CBF/DREB1 genes in A. thaliana, DREB1A/CBF3, DREB1B/ CBF1, DREB1C/CBF2, DREB1D/CBF4, DDF1/DREB1F, and DDF2/DREB1E (Akhtar et al., 2012), in which CBF2/DREB1C negatively regulates the expression of CBF1/DREB1B and CBF3/ DREB1A; these three TFs play a central role in plant response to cold stress (Teige et al., 2004). When soybean CBFs were constitutionally expressed in Arabidopsis, they increased the levels of AtCOR47 and AtRD29a transcripts and increased frost resistance, as measured by reducing leaf frostbite and ion leakage, and GmDREB1A;2 and GmDREB1B;1 constitutive expression in A. thaliana leads to upregulation of downstream genes and stronger freeze resistance (Yamasaki and Randall, 2016). CBF gene expression is regulated by many types of TFs. CBF expression inducer 1 (ICE1) and its homolog ICE2 positively regulate CBF expression and low-temperature tolerance (Ding et al., 2019). MYB15 TF binds to MYB cis-acting elements in CBF/DREB1 promoter and negatively regulates the expression of the latter (Agarwal et al., 2006). The cold-induced C2H2 zinc-finger TF (ZAT12) is also a negative regulator of CBF/DREB1. Studies have shown that the expression of CBF/DREB1 is decreased in ZAT12 overexpressed plants (Vogel et al., 2005) (Table 14.1).

14.2 Transcription factors involved in response to temperature stress

293

Table 14.1 Classification and quantity of AP2/ERF TFs in different species. Species

AP2

ERF

DREB

RAV

Soloist

Total

References

Arabidopsis thaliana Oryza sativa Zea mays Triticum aestivum Hordeum vulgare Populus alba Vitis vinifera Solanum tuberosum

18 26 31 9 8 26 18 49

65 79 84 47 22 91 73 136

57 52 49 57 18 77 36 58

6 7 2 3 4 5 4 2

1 0 1 1 1 1 1 1

147 164 167 117 53 200 132 246

Nakano et al. (2006) Sharoni et al. (2011) Hao et al. (2020) Riaz et al. (2021) Guo et al. (2016) Trupiano et al. (2013) Licausi et al. (2010) Charfeddine et al. (2015)

14.2.4 WRKY transcription factors WRKY is one of the most important and largest TF families in plants (Eulgem et al., 2000), and plays a significant role in many metabolic regulation processes (Rushton et al., 2010). The WRKY proteins contain one or two highly conserved WRKYGQK (Trp-Arg-Lys-Tyr-Gly-Gln-Lys) heptapeptide in the N-terminus, and one or two zinc-finger structure, C2H2 (C-X4
5-C-X22
23-H-XH) or C2HC (C-X7-C-X23-H-X-C) type, located at the C-terminal, which is made up of about 60 amino acids (Eulgem et al., 2000; Rushton et al., 2010). According to the number of conserved WRKY domains and zinc-finger structure, the WRKY gene family could be divided into three main groups: Group I, Group II, and Group III (Eulgem et al., 2000). Group I contains two WRKY conserved domains and a C2H2-type zinc-finger motif. Group III contains one WRKY conserved domain and a C2HC-type zinc-finger motif. Group II aslo contains one WRKY conserved domain, but with a C2H2-type zinc-finger motif. According to the sequence characteristics of DNA binding domains in WRKY proteins, Group II was further divided into five subgrouops, namely IIa, IIb, IIc, IId, and Iie (Eulgem et al., 2000; Rushton et al., 2010). It has been demonstrated that WRKY TFs can specifically recognize and bind to the highly conserved region W-box (C/TTGACT/C) of the target gene promoter in vivo and in vitro experiments (Bakshi and Oelmu¨ller, 2014; Ulker and Somssich, 2004), and further activate or inhibit the expression of downstream target genes at the transcriptional level. Wheat TaWRKY1 and TaWRKY33 were upregulated under heat stress and ABA treatment, and the TaWRKY33 transgenic plants exhibited enhanced tolerance to heat stress (He et al., 2016). qRTPCR showed that FtWRKY7 was significantly upregulated in buckwheat under high-temperature stress (He et al., 2019). Studies have shown that those WRKY genes were not only induced by salt stress, but also low temperature in Eucalyptus grandis (Fan et al., 2018). In Camellia sinensis, CsWRKY can respond to extreme temperature (Wu et al., 2016). It was found that overexpression of Polygonatum odoratum PoWRKY1 not only promoted seed germination and root growth of transgenic plants, but also improved tolerance of transgenic plants to drought and cold stress (Wei et al., 2021). By studying the expression patterns of WRKY genes in alfalfa under different abiotic stress, 27 WRKY genes were found to be involved in abiotic stress responses such as low-temperature and salt stress (Mao et al., 2020). Overexpression of wheat TaWRKY19 gene found that TaWRKY19 could not only activate DREB2A (dehydration-responsive element-binding protein 2A), RD29A

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(desiccation-responsive protein 29A), RD29B (desiccation-responsive protein 29B), and Cor6.6 (cold-regulated protein, a 6.6-kD polypeptide), but also combine with DREB2A and Cor6.6 promotors, which make transgenic plants resistant to freezing stress (Niu et al., 2012). Among 59 VvWRKY genes, 15 WRKY genes were induced by low-temperature stress (Wang et al., 2014).

14.3 Conclusions and perspectives As regulatory proteins, TFs are mainly composed of DNA-binding domains, transcriptional regulatory domains, and oligomeric sites. By identifying and binding cis-acting elements of gene promoters, it is involved in the regulation of plant growth and development, physiological metabolism, and biological and abiotic stress responses. At present, in many abiotic stresses, too high or too low temperature will have a significant impact on plants. Many TFs in plants are involved in various stages of high- and low-temperature stress response, so it is of great significance to study the role of TFs in plants and their specific pathways. Understanding the functions of TFs in high- and lowtemperature stress will help us to further improve the adaptability of plants to temperature stress.

Acknowledgments This study was supported by Hainan Provincial Natural Science Foundation of China (319Ms009, 318QN189), the Education Department of Hainan Province (Hys2020
242, Hnky2021
19), and Startup Funding from Hainan University (KYQD(ZR)1845).

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37645. Akerfelt, M., Morimoto, R.I., Sistonen, L., 2010. Heat shock factors: integrators of cell stress, development and lifespan. Nature Reviews. Molecular Cell Biology 11 (8), 545
555. Akhtar, M., Jaiswal, A., Taj, G., Jaiswal, J.P., Qureshi, M.I., Singh, N.K., 2012. DREB1/CBF transcription factors: their structure, function and role in abiotic stress tolerance in plants. Journal of Genetics 91 (3), 385
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480. An, D., Yang, J., Zhang, P., 2012. Transcriptome profiling of low temperature-treated cassava apical shoots showed dynamic responses of tropical plant to cold stress. BMC Genomics 13, 64. Bakshi, M., Oelmu¨ller, R., 2014. WRKY transcription factors. Plant Signaling & Behavior 9, e27700. Balasubramanian, S., Sureshkumar, S., Lempe, J., Weigel, D., 2006. Potent induction of Arabidopsis thaliana flowering by elevated growth temperature. PLoS Genetics 2 (7), e106. Ball, L., Accotto, G.P., Bechtold, U., Creissen, G., Funck, D., Jimenez, A., et al., 2004. Evidence for a direct link between glutathione biosynthesis and stress defense gene expression in Arabidopsis. The Plant Cell 16 (9), 2448
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CHAPTER

Plant transcription factors and osmotic stress

15 Tingting Zhang and Yang Zhou

School of Horticulture, Hainan University/Key Laboratory for Quality Regulation of Tropical Horticultural Crops of Hainan Province, Haikou, P.R. China

15.1 Effects of osmotic stress on plants and its regulatory mechanism Osmotic stress or water stress can be caused when plants are subjected to drought, high temperature, low temperature, or salt stress (Fig. 15.1), resulting in plant cell dehydration and ion homeostasis disorder (Jakab et al., 2005; Matsui et al., 2008). Thus normal physiological activities of plants are affected, resulting in plants premature senescence or death (Zhu, 2016). Under mild stress, the growth of plants will be significantly inhibited (Schuppler et al., 1998). The total number of plants cells will not change much under water stress, but cells will shrink and their extension will be affected due to cell dehydration, and plants will become dwarfish. It was found that both the isolated cell wall and root cell wall hardened when treated with PEG simulating water stress for 2 minutes (Chazen and Neumann, 1994). In order to adapt to stress, plants generate a series of regulatory mechanisms, such as stomatal closure, pH regulation, osmotic regulation, and reactive oxygen species (ROS) scavenging mechanisms.

15.1.1 Stomatal closure Plant leaves will lose a lot of water, resulting in a decrease in foliar elongation when expose to osmotic stress. According to research findings, the pH of xylem sap of barley increased from 5.9 to

FIGURE 15.1 Cause and regulated mechanism of osmotic stress. Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00014-5 © 2023 Elsevier Inc. All rights reserved.

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6.9 under water stress, and leaf elongation decreased gradually with the increase of pH (Bacon et al., 1998). In order to avoid excessive water loss, plants close their stomata on their leaves to slow transpiration and reduce water loss. Stomatal closure not only effectively reduces water loss, but also reduces the concentration of CO2 required for photosynthesis, thus reducing the photosynthetic rate (Damour et al., 2010). Therefore stomatal closure can also be used as a mechanism to regulate osmotic stress.

15.1.2 Osmotic regulation mechanism Osmotic regulation mechanism is also necessary for plants to cope with stress. Osmotic regulation refers to the accumulation of osmotic substances in cells when plants are subjected to abiotic stress such as drought, high temperature, low temperature, and salt, so as to enhance the water retention ability of plants, stabilize the osmotic pressure balance in plants, and enable plants to tolerate the negative effects of water loss. Osmotic regulation substances are mainly composed of two parts: one kind is polyhydric alcohol and nitrogen-containing compounds (e.g., various special metabolites such as free amino acids, sugars, alcohols, and other organic substances), which play roles in regulating the osmotic potential of the cytoplasm and protecting enzymes, proteins, and biological membrane; the other kind is inorganic ionic substances such as the ion pump on the cell membrane, which can regulate the concentration of inorganic substances inside and outside the cell, change the osmotic potential of the cell, and change the cell morphology and function. Ion pump plays an important role in regulating stomatal circadian clock changes and regulating stomatal switch, and thus affecting metabolic and physiological phenomena such as photosynthesis, transpiration, and water use efficiency (Shen et al., 2018). Betaine, proline, sorbitol, etc., are important osmotic regulators. Exogenous betaine could improve lentil drought tolerance through increasing the content of glutathione (GSH), maintaining the activities of glutathione transferase (GST) and glyoxalase (Gly) and reducing the contents of oxidized glutathione (GSSG) and hydrogen peroxide (H2O2) (Molla et al., 2014). When the exogenous betaine is applied, the accumulation of cold-induced protein WCOR410 in wheat was increased under cold stress, and the expression levels of WCOR410 and WCOR413 were increased accordingly (Allard et al., 1998). Proline is one of the most effective affinity osmotic regulatory substances in plants, with strong water solubility. Increased proline content helps plant cells and tissues retain water and prevent dehydration. Under salt stress and drought stress, soybean transformed with salt-tolerant genes had higher proline content and lower MDA content than wild-type plants (Sun et al., 2021). Sorbitol contains multiple hydroxyl groups and has strong hydrophilic ability, which can effectively maintain cell turgor and effectively resist osmotic dehydration under environmental stress. Ranney et al. (1991) analyzed the content of soluble carbohydrates in cherry leaves under water stress, among which the concentration of sorbitol was the highest. The results showed that the increase in total soluble carbohydrates was mainly caused by the increase in sorbitol.

15.1.3 Mechanism of ROS generation and scavenging When plants experience stress, some changes of membrane lipids in plant cells take place (Pinto et al., 2017), causing a series of changes in plant cells, such as cells losing water, the structures of

15.1 Effects of osmotic stress on plants and its regulatory mechanism

303

proteins and other components being destroyed, relative conductivity increasing, the contents of malondialdehyde, proline, soluble sugar, and soluble protein contents increasing, and ROS accumulating gradually (Duni et al., 2019; Gyur´aszov´a et al., 2020; Verma et al., 2011). Excessive accumulation of ROS will cause serious interference to normal metabolism of cells, including destruction of cell membranes, nucleic acid breakage, protein denaturement, hormone metabolism disorder, etc. (Mittler et al., 2011). ROS is a form in which oxygen is partially reduced or activated. It is a general term for oxygen-containing substances with extremely active properties and strong oxidation capacity, including superoxide anion (O2), hydrogen peroxide (H2O2), hydroxyl radical (  OH), and singlet oxygen (1O2) (Gill and Tuteja, 2010). Antioxidant enzymes including catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione peroxidase (GPX), ascorbate reductase (MDA), glutathione reductase (GR), glutathione S-transferase (GST), and so on, which can remove ROS effectively in plant cells (Fig. 15.1) (Dvoˇra´ k et al., 2021; Madhu et al., 2021, 2022, Tyagi et al., 2017, 2018, 2019, 2020, 2021). In the antioxidant enzyme system throughout the plant cells, SOD constitutes the first line of defense against oxidative stress in the plant, and disproportionates the two superoxide anions to form H2O2 and O2, and H2O2 is then catalyzed by CAT and POD for degradation (Bose et al., 2014). CAT is mainly responsible for scavenging H2O2 in peroxisome, while POD is a key enzyme for scavenging H2O2 in chloroplasts (Willekens et al., 1994, 1997). APX is one of the important antioxidant enzymes in reactive oxygen metabolism of plants, especially the key enzyme in chloroplast scavenging H2O2, and the main enzyme in vitamin C metabolism, belonging to a terminal oxidase, which mainly exists in the chloroplast matrix, thylakoid membrane, microbodies, and cytoplasm (Shafi et al., 2019).

15.1.4 ABA signaling pathway Abscisic acid (ABA), as one of the important hormones in plants, accumulates ABA in response to environmental stress when plants are subjected to abiotic stress. Plants respond to stress mostly through ABA signaling pathway. ABA content is mainly regulated by biosynthesis and catabolic processes, in ABA-dependent or ABA-independent pathways, so that plants can better adapt to different environmental conditions (Negi et al., 2008). In the ABA-dependent pathway, the first step is promoting ABA synthesis, which binds to the PYR/PYL/RCAR complex to produce the PP2Cs complex, which induces the inactivated PP2C/SnRK2s complex to bind to it, and then SnRK2s is released and activated by autophosphorylation. Activated SnRK2s can phosphorylate its downstream TFs, thus regulating the expression of downstream stress-responsive genes (Zhu, 2016). In the ABA-independent pathway, the MAP kinase pathway often plays important roles. The stress signal is transmitted to downstream stress-responsive genes through signal cascade transmission, thus responding to stress (De Zelicourt et al., 2016; Shumayla et al., 2019; Shumayla and Upadhyay, 2022; Upadhyay and Shumayla, 2022). Studies have found that CsTLP8 protein in cucumber negatively regulates osmotic stress through the ABA signaling pathway (Li et al., 2021). Transgenic tobacco seedlings overexpressing wheat TabHLH1 had higher tolerance to osmotic stress compared with wild type. When treated with exogenous ABA and osmotic stress, transgenic plants showed faster stomatal closure, the contents of proline and soluble sugar increased, and the expression levels of ABA receptor genes and the SnRK2 kinase family genes were upregulated, which indicated that TabHLH1 positively

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Chapter 15 Plant transcription factors and osmotic stress

regulated osmotic stress through ABA-dependent pathway (Yang et al., 2016). AtCIPK6 was found to be involved in salt and osmotic stress through an ABA-dependent pathway in Arabidopsis thaliana (Chen et al., 2013). In addition, GhWRKY6-like was found to enhance the tolerance of transgenic Arabidopsis thaliana to salt, drought, and osmotic stress by scavenging ROS and regulating ABA signaling pathways in cotton (Ullah et al., 2018). MMK4 kinase, a member of the MAP kinase family, has been shown to be involved in plant responses to drought and cold stress through an ABA-independent pathway (Jonak et al., 1996).

15.2 Transcription factors are involved in regulating osmotic stress 15.2.1 Osmotic stress caused by salt stress Osmotic stress occurs in the early stage of salt stress and has a direct effect on plants (Fig. 15.1). Under salt stress, a large number of saline ions accumulate in the soil, resulting in lower osmotic pressure in plant cell sap compared with soil solution, which resulted in stomatal closure, inhibition of cell expansion, and slowing growth (Isayenkov and Maathuis, 2019). Under salt stress, the transgenic rice and Arabidopsis thaliana overexpressing the pineapple AcoMYB4 enhanced the sensitivity to osmotic stress, and decreased the expression and activity of several antioxidant enzymes. In addition, overexpressing plants would inhibit ABA biosynthesis by reducing the transcription of ABA synthesis genes and ABA signal transduction factors under osmotic stress. These results suggest that AcoMYB4 negatively regulates osmotic stress by decreasing ABA biosynthesis and signal transduction pathways in cells (Chen et al., 2020). The germination rate of the Arabidopsis det1 mutant seeds was increased under salt and osmotic stress due to the regulation of TFs such as HY5, ABF1, ABF3, and ABF4 (Fernando et al., 2018). Under salt and osmotic stress, the lateral root development was significantly inhibited in atwrky46 mutants, but promoted in AtWRKY46 transgenic plants. Meanwhile, it was found that WRKY46 gene was downregulated under ABA treatment, but upregulated under salt and osmotic stress, suggesting that WRKY46 may participate in salt and osmotic stress through regulating ABA signaling pathway (Ding et al., 2015). Overexpression of MdWRKY30 enhanced the tolerance of transgenic apple callus to salt and osmotic stress through transcriptional regulation of stress-related genes (Dong et al., 2020). Arabidopsis thaliana and tobacco overexpressing pepper CaWRKY27 gene enhanced the sensitivity to salt and osmotic stress. Compared to wild type plants, the root length and whole plant growth of transgenic plants were more obviously inhibited, chlorosis and wilting were more serious, and germination rate of transgenic plants was lower. Pepper mutant silencing the CaWRKY27 gene was found to have increased tolerance to salt and osmotic stress, suggesting that CaWRKY27 negatively regulates salt and osmotic stress (Lin et al., 2019). The pepper CaMADS was induced by low temperature, high temperature, salt, osmotic stress, ABA, SA, MeJA, and CaCl2 treatment. The mads mutants were more damaged than wild type under NaCl and mannitol treatment, and their relative conductivity and MDA contents were increased. The tolerance of overexpressing plants was improved, indicating that MADS TFs play a positive role in pepper response to salt and osmotic stress (Chen et al., 2019). Soybean GmERF75 has also been shown to play an important role in enhancing the osmotic stress tolerance of transgenic Arabidopsis and soybean (Zhao et al., 2019). It was found that the moss ScDREB8 and ScDREB10 were involved in response to osmotic stress,

15.2 Transcription factors are involved in regulating osmotic stress

305

salt stress, cold stress, and high-temperature stress, especially osmotic stress and salt stress (Li et al., 2016). HSF TF is a typical heat shock factor. AtHSFA2 gene was upregulated under salt stress and osmotic stress. Plants overexpressing AtHSFA2 gene have improved tolerance to various stresses (Ogawa et al., 2007).

15.2.2 Osmotic stress caused by drought stress Water stress is directly or indirectly caused by plant water shortage when exposed to drought, which has a serious impact on plant growth and development (Fig. 15.1). Arabidopsis thaliana overexpressing AtMYB109 had more severe damage than wild type plants when treated with PEG simulating the drought stress, while the myb109 mutant had less damage than the wild type, and the overexpressing plants had faster stomatal closure. In addition, it was found that the proline content and expression levels of stress-induced and proline synthesis genes in overexpressing lines were higher than those in WT and myb109 mutant, suggesting that MYB109 is a negative regulator of drought and osmotic stress in Arabidopsis thaliana (So et al., 2020). Studies have found that DREB2A is involved in Arabidopsis thaliana response to drought stress and osmotic stress depending on the ABA pathway (Kim et al., 2011). Overexpression of peanut AP2/ERF family gene AhDREB1 can improve the sensitivity of transgenic plants to ABA and induce the expression of drought-related genes under PEG6000 and ABA treatment, which suggested that AhDREB1 could enhance plant tolerance to drought and osmotic stress in a ABA-dependent signaling pathway (Zhang et al., 2018). Transgenic Arabidopsis thaliana overexpressing of grape VvABF2 was more sensitive to ABA, and showed tolerance to osmotic stress and improved the ability of scavenging ROS under PEG treatment (Liu et al., 2019). The strawberry FvWRKY42 enhanced the salt and drought tolerance of transgenic Arabidopsis thaliana. At the same time, transgenic plants showed increased sensitivity to ABA during seed germination and seedling growth, and stomatal closure increased after ABA and drought treatment. Thus FvWRKY42 is involved in plant responses to salt and drought stress and osmotic stress through ABA pathway (Wei et al., 2018). Compared to the wild type rice, the ric overexpressing JERF3 gene showed higher tolerance to drought and osmotic stress, and the contents of soluble sugar and proline in transgenic rice were significantly higher (Zhang et al., 2010). OsERF101 gene was induced and upregulated under drought and ABA treatment. Overexpressing this gene in plants showed that OsERF101 could regulate drought and osmotic stress in the vegetative and reproductive stages of rice (Jin et al., 2018). The Haloxylon ammodendron HaNAC1 interacting with AtNAC32 could regulate the growth and drought and osmotic stress (Gong et al., 2020).

15.2.3 Osmotic stress caused by low temperature In addition to salt stress and drought stress, osmotic stress is also caused by low temperature (Fig. 15.1). Under low temperature, the water transport rate in plants decreases and stomatal closure slows down, resulting in cell water loss and osmotic stress (Ball et al., 2004). Arabidopsis overexpressing the kiwifruit AchnABF1 gene enhanced cold tolerance. Several key genes related to ABAdependent and ABA-independent pathways in transgenic plants were induced, and the scavenging capacity of ROS was also improved. These results indicated that the TF ABF1 is involved in plant response to low-temperature and osmotic stress (Jin et al., 2021). The Thlaspi caerulescens

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TcWRKY53 gene was found to be induced by low temperature, drought, and salt stress. After transferring the gene into tobacco, it was found that the sorbitol tolerance of transgenic tobacco roots was reduced, indicating that the TF reverse-regulates osmotic stress (Wei et al., 2008). As a typical TF associated with low-temperature stress, CBF can interact with a variety of TFs to participate in plant response to low-temperature stress. It has found that the collaboration between HY5 and MYB15 can precisely regulate the expression of CBFs and further regulate the response of plants to cold stress in tomato (Zhang et al., 2020). Overexpression of the MYC-type bHLH family gene ZjCE2 can improve the tolerance of transgenic plants to cold stress. At the same time, cold stressrelated gene DREB/CBFs was induced to be upregulated under low-temperature treatment, and the scavenging capacity of ROS of transgenic plants was improved. Therefore CE2 can respond to plant low-temperature stress and osmotic stress through DREB/CBF pathway (Zuo et al., 2020). MdNAC029 was found to reduce the cold tolerance of transgenic apple callus. Further studies showed that the TF inhibited the expression of MdCBF1 and MdCBF3 genes by binding to the promoters of the two genes, which showed that MdNAC029 was a negative regulatory factor in apple response to low-temperature stress (An et al., 2018). In addition, studies showed that MdMYB88 and MdMYB124 positively regulated the expression of cold tolerance and cold response genes under low-temperature stress through CBF-dependent and CBF-independent pathways (Xie et al., 2018).

15.3 Conclusions and perspectives During plant growth and development, various abiotic stresses have different effects on plants. Osmotic stress usually appears as a secondary stress. In addition to the osmotic stress caused by salt stress, drought stress, and low-temperature stress alone, osmotic stress is more often caused by the combined effects of various stresses. At present, there are more and more studies on the response of osmotic stress to overexpression of single TF genes by transgenic technology (Table 15.1). Plant TFs often act as positive or negative regulators in response to osmotic stress through ABA-dependent or ABA-independent pathways. A few TFs can interact with other TFs and regulate the expression of stress-related genes and ROS scavenging. These results laid a theoretical foundation for molecular breeding of plant resistance. However, how various abiotic stresses cause osmotic stress remains to be further studied. With the global warming, high-temperature stress has also become an important factor affecting plant growth and development. However, there Table 15.1 Transcription factors involved in osmotic stress. TFs

Type of stress

References

AtHY5, AtABF1, AtABF3, AtABF4, AtWRKY46, MdWRKY30 AtDREB2A, AhDREB1, VvABF2, FvWRKY42 AchnABF1, TcWRKY42, SlHY5, SlMYB15, MdNAC029

Salt for osmotic stress Drought for osmotic stress Temperature for osmotic stress

Fernando et al. (2018), Ding et al. (2015), Dong et al. (2020) Kim et al. (2011), Zhang et al. (2018), Liu et al. (2019), Wei et al. (2018) Jin et al. (2021), Wei et al. (2008), Zhang et al. (2020), An et al. (2018)

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is little research on osmotic stress caused by high-temperature stress, which is also a direction of future research. Although many TFs involved in plant response to osmotic stress have been reported, there are few studies on the complete pathway of plant response to osmotic stress. The solution to these problems is of great significance to the study of plant stress tolerance.

Acknowledgments This study was supported by Hainan Provincial Natural Science Foundation of China (319Ms009, 318QN189), the Education Department of Hainan Province (Hys2020
242, Hnky2021
19), and Startup Funding from Hainan University (KYQD(ZR)1845).

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CHAPTER

Transcriptional regulation of drought stress stimulus: challenges and potential for crop improvement

16

Gyanendra K. Rai1, Gayatri Jamwal1, Isha Magotra1, Garima Rai2 and R.K. Salgotra1 1

School of Biotechnology, S. K. University of Agricultural Sciences & Technology of Jammu, Chatha, Jammu and Kashmir, India 2CSIR-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir, India

16.1 Introduction Plants endure various environmental stresses that can be detrimental for their growth and development. Drought, salt, mechanical wounding, and temperature stresses are key factors that influence plant dispersion in nature, limit plant yield in agriculture, and undermine food security. Therefore plants have evolved various responses to counter these stresses. Plants are vulnerable to both abiotic and biotic stressors throughout their lifespan. Stress mediated through the living organism is termed as biotic stress, which includes fungal, viral, and bacterial infection. Abiotic stress is defined as stress caused by nonliving causes such as drought, salinity, pH, nutrition, and severe temperature (Suzuki et al., 2014). Plants are adversely affected by both types of stress. Abiotic stressors, in particular, affect photosynthesis, growth, carbon partitioning, carbohydrate and lipid metabolism, protein synthesis, gene expression, and osmotic homeostasis, among other cellular activities. Drought stress is one of the key causes of agrarian distress, which not only affects the world economy but also affects the livelihoods of huge populations by affecting crop agronomic growth and production (AghaKouchak et al., 2015). Drought essentially means water shortage caused by singular or cumulative environmental effects like low rainfall, reduced ground water table and increase in temperature leading to water shortage and increase in transpiration rate in plants (Singh and Laxmi, 2015). Drought stress in a crop can be expressed at the field level by reduced precipitation for an extended period that can range from months to years, depending on the crop genotype. Drought stress causes a variety of injuries to plants in terms of physiological, biochemical, and metabolic effects. Once the endurance capacity of a plant decreases, it starts showing visible signs of damage like leaf rolling, wilting, bleaching, and death after a prolonged water deficit (Sahoo et al., 2013). Other physiological changes include reduction in leaf area, leaf abscission, root growth stimulation, alterations in relative water content (RWC), electrolyte leakage (EL), ROS (reactive oxygen species) generation and free radical production (Bartels and Sunkar, 2005). Also, shoot growth is inhibited during drought conditions (Pareek et al., 2010). At the molecular level, when water content Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00017-0 © 2023 Elsevier Inc. All rights reserved.

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of the plant cells decreases, it increases the concentration of solutes. This upsets the chemical equilibrium within the cells and affects the functioning of enzymes eventually leading to decrease in rate of photosynthesis. The water use efficiency (WUE) (measured as the amount of biomass produced per unit of water used by a plant) also decreases (Hatfield and Dold, 2019). Drought also leads to decrease in turgor pressure, change in cell wall, and plasma membrane compositions. Under unfavorable environmental conditions, various stress-response mechanisms have been evolved by plants at different layers, such as cellular signal perception and transduction, inducing expression of specific subsets of defencs genes, and thus activating the overall defense reaction, eventually contributing to phenotype (Fraire-Vel´azquez et al., 2011; Singh et al., 2019). Plants are highly susceptible to osmotic stress during their growth and reproductive stages (especially flowering and seed development) (Samarah et al., 2009a,b,c; Alqudah et al., 2010; Samarah and Alqudah, 2011). In response to any type of stress, a set of stress response genes with the main functions of protecting plants against potential damage get activated. They are translated into proteins that take direct part in either regulating gene expression or participate in signal transduction and metabolic pathways (Agarwal et al., 2006). These proteins can be transcription factors (TFs) or enzymes for osmolyte biosynthesis, water channel proteins and membrane transporters, glutathione S transferase, catalase, superoxide dismutase, APX, fatty acid metabolism enzymes, protease inhibitors, ferritin, lipid transfer proteins, calcium ion binding proteins/transporters, LEA (late embryogenesis abundant) protein, osmotins, heat shock proteins (HSP), and chaperons (Joshi et al., 2016; Wani et al., 2013; Upadhyay, 2021). Identification of drought stress response genes and proteins has been made possible by various transcriptomic, proteomic, and metabolic approaches. The phytohormone abscisic acid is considered a marker for drought stress. Its release leads to closing of leaf stomata and decreased water loss, which further improves the WUE in plants (Yang et al., 2011). However, there are other stress response genes that also get activated even in the absence of abscisic acid (Fig. 16.1) (Aguado et al., 2014). TFs are key regulators of gene activation and repression in all living organisms. They play pivotal roles in plant growth, cell cycling, signaling, and stress response (Gonzalez et al., 2016). TFs are DNA binding proteins that regulate gene expression by interacting with a preinitiation complex of transcription and binding in a sequence-specific manner to the cis-regulatory elements present in the promoter regions of the respective genes (Nuruzzaman et al., 2013; Franco-Zorrilla et al., 2014). In plants, TFs are encoded by approximately 10% of genes at different stages/points to regulate specific signaling-mediated function. In Arabidopsis thaliana, 1500 TFs are reported to be involved in various kinds of stress response (Riechmann et al., 2000). Various TFs databases are available that list major TF families, such as WRKY, MYB, NAC, and AP2/ERF that are crucial regulators of various genes related to different stresses (Table 16.1). Therefore these TFs contribute towards being an ideal choice for genetic engineering in order to enhance resistance of plants against different stress stimuli (Wang et al., 2016). Furthermore, recent efforts to use these TFs in the development of drought-tolerant transgenic plants have been described.

16.2 Regulatory role of transcription factors in dry spell tolerance TFs are regulatory proteins crucial in the conversion of stress-induced signals to cellular responses. A single TF has the ability to control the expression of several genes (regulons). TF constitute a

16.2 Regulatory role of transcription factors in dry spell tolerance

315

FIGURE 16.1 Schematic representation of perception of drought stress signal by TFs and downstream relay of the signals for modulating gene expression for drought stress tolerance in plants.

wide array of proteins (excluding RNA polymerase) that are involved with initiation, regulation, and transcription of genes. TFs can be characterized by the presence of DNA-binding domains that give them the ability to bind to specific sequences of DNA called promoter or enhancer, activation domain, dimerization domain, nuclear export domain, and nuclear localization signal domain. The TFs initiate transcription either by binding to a DNA promoter sequence near the transcription start site or binding to regulatory enhancer sequences present thousands of base pairs upstream or downstream of the gene being transcribed. Various methods are employed to identify and isolate TFs from plants. The whole-genome sequencing projects of recent times have generated a huge number of sequences and various databases specific to TF sequences are maintained. A few such important

316

Chapter 16 Transcriptional regulation of drought stress stimulus

Table 16.1 DNA binding sequences of various TFs. S. No.

TF family

Cis-element sequences

1 2 3 4 5 6 7 8 9 10

bZIP DREB NAC MYC/B TCP WRKY DOF HSF AREB/ABF NF-YA

TACGTA (A-box), GACGTC (C-box), CACGTG (G-box), TGAAAA, GTGAGTCAGT TACCGACAT, AGCCGCC CGT(G/A), CACG CNGTT(A/T) GGNCCCAC, G(T/C)GGNCCC (C/T)TGAC(C/T) T/AAAAG AGAANNTTCT PyACGTGG/TC CCAAT

databases are the PKU Yale collection (Gong et al., 2004) and the TF only library (Mitsuda et al., 2010). These libraries additionally keep TF clones in GATEWAY-compatible cloning vectors to aid researchers in validating and screening the TFs. because of their involvement in plant development or because of their overexpression in response to stress in different plant systems (Wehner et al., 2011). TF genes are also screened using a variety of bioinformatics methods using genome-scale protein or nucleotide sequence libraries. Posttranslational modifications, such as MAPKmediated phosphorylation of bZIP, also play a role in activating TFs for downstream gene expression (Banerjee and Roychoudhury, 2017). When abiotic stressors are no longer present, it is critical for plants to terminate the activity of TFs in order to avoid wasting energy; TFs are then destroyed via the ubiquitin- proteasome system (UPS), the most prevalent method of protein breakdown in all of eukaryotes. The major TFs discovered in plants (Table 16.2) together with their gene regulatory networks that play a role in drought stress tolerance will be described in the following section.

16.3 Transcription factor and their mechanisms under drought stress 16.3.1 DNA binding with one finger (DOF) DOF transcription factor is a plant-specific TF family that regulates vascular tissue formation, abiotic stress, cell cycle, photoperiod regulation, floral organ abscission, redox homeostasis, and secondary metabolite production (Ruta et al., 2020). HSFs have been divided into three types based on the flexible linker length and type of amino acid residues between DBD and OD: HSFA, HSFB, and HSFC. HSFs work by binding to a palindromic binding motif (50-AGAANNTTCT-30) in the heat stress promoter element of eukaryotic HSF-inducible genes, which is located upstream of the TATA box (Scharf et al., 2012). They are engaged in heat stress, as their name implies, but new investigations have revealed their potential relevance in drought stress (Fig. 16.2). Furthermore, in the field, drought stress is frequently coupled with heat stress. As a result, it is feasible to speculate that plants have evolved an intriguing transcriptional control system to mediate drought and thermotolerance at the same time. HSFs can also influence abiotic and phytohormone signaling

16.3 Transcription factor and their mechanisms under drought stress

317

Table 16.2 Gene numbers of water stress TFs in plants (The data was summarized from plant transcription factor database, 2021). Plants

AP2/ERF

NAC

MYB

DOF

HSF

TCP

bZIP

NF-YA

WRKY

Triticum aestivum (Wheat) Oryza sativa subsp japonica (Rice) Oryza sativa subsp indica (Rice) Solanum lycopersicum (Tomato) Glycine max (Soyabean) Saccharum officinarum (Sugarcane) Gossipium hirsutum (Cotton) Hordeum vulgare (Barley) Zea mays (Maize) Arabidopsis thaliana Camelina sativa (Flax seed) Sesamum indicum (Seaseme) Nicotiana tabacum (Tobacco)

43

263

263

52

53

7

186

22

171

22

170

130

37

38

2

140

25

128

27

158

121

28

25

2

94

10

109

27

101

140

33

26

2

70

10

81

99 6

269 44

430 36

97 7

81 13

18 1

352 37

117 4

296 39

59

306

441

118

82

4

224

30

238

34

150

99

40

31

4

156

22

126

54 30 93

189 138 350

203 168 402

52 47 102

49 25 108

6 4 9

216 127 220

36 21 31

161 90 224

43

105

168

38

32

2

127

29

88

93

280

319

83

65

19

210

66

210

FIGURE 16.2 Schematic representation of DOF domains. DOF, DNA binding with one finger.

318

Chapter 16 Transcriptional regulation of drought stress stimulus

pathways at the same time, making them important regulators of stress-responsive genes. Plant HSFs can act as either positive or negative transcriptional regulators for genes that are responsive to drought stress, depending on a variety of variables, such as post-translational modifications, interacting proteins, as well as other factors target which is bound ABA signaling interacts with HSF-mediated gene expression during abiotic stresses. External ABA application, salt, drought, and salt treatment all increased the expression of Arabidopsis thaliana AtHSFA6a. Plant resistance to salt and drought stress was similarly improved by ABA-mediated expression of AtHSF6b. The transgenic lines overexpressing HsfA2 exhibited a greater accumulation of various dehydrationresponsive genes from the raffinose family oligosaccharide (RFO) pathway, such as GALACTINOL SYNTHASE (AtGolS) and RAFFINOSE SYNTHASE (RafS). The overabundance of galactinol and raffinose, which were implicated in the scavenging of hydroxyl radicle produced by oxidative damage, was caused by increased mRNA levels of GolS and RafS (Nishizawa-Yokoi et al., 2008; Panikulangara et al., 2004).

16.3.2 WRKY transcription factor WRKY, as one of the most well-studied plant TFs, regulates a broad range of developmental, physiological, and metabolic processes (Chen et al., 2017). Although the first WRKY TF was discovered in Ipomoea batatas (sweet potato) in the 1990s (Ishiguro and Nakamura, 1994), WRKY genes were once thought to be plant-specific TFs (Baillo et al., 2019). Other eukaryotic creatures, such as fungus, amoebae, and diplomonads, have WRKY proteins in their genetic make-up, according to numerous studies. Numerous WRKY TFs have been experimentally revealed in various plant species, including (Arabidopsis thaliana) (Bao et al., 2018), sugarcane (Saccharum spontaneum) (Li et al., 2020), barley (Hordeum vulgare) (Uluhan et al., 2019), rice (Oryza sativa) (Lilly and Subramanian, 2019), physic nut (Jatropha curcas) (Xiong et al., 2013), maize (Zea mays) (Wang et al., 2018), and so on. The most authenticated and widely recognized classification states that plant-specific WRKY TFs are signified by 60 amino acids in their highly conserved DNA binding region (called the WRKY domain) based on genomic characterization of Arabidopsis (Goyal et al., 2020). Furthermore, WRKY domains comprised of a highly conserved motif WRKYGQK at N terminus that provides a protein-protein interaction interface and a zinc-finger region (either C-X4
5C-X22
23-H-X-H or C-X7-C-X23-H-X-C) at the C-terminus having affinity towards DNA binding (Fig. 16.3) (Eulgem et al., 2000). WRKY proteins, based on WRKY domains present and zincfinger region, have been classified into three major groups (Group I, II, and III) (Brand et al., 2013). However, in S. spontaneum, a new WRKYs Group IV has been hypothesized, as proven by genes with an incomplete domain (just the WRKYGQK motif was found), indicating they may have lost their function as WRKYs (Li et al., 2020). Temperature stress has a major impact on a variety of activities in the plant system, including physiological, enzymatic, metabolic, growth, and developmental processes, resulting in low yield and poor quality produce. Reduced enzymatic activity caused by denatured whole protein structure under severe temperatures causes the entire system to cease, resulting in wilting or senescence of the plant (Zandalinas et al., 2018). The WRKYs group I proteins (such as AtWRKY25, AtWRKY26, and AtWRKY33) contributed to increased significantly thermotolerance by stimulating the heat-induced signal transduction pathway, while double (wrky25/26) and triple (wrky25/26/33) mutants show poor seed germination, poorer membrane integrity, and higher temperature stress

16.3 Transcription factor and their mechanisms under drought stress

319

FIGURE 16.3 Schematic representation of WRKY domains.

susceptibility (Li et al., 2011). TaWRKY1 and TaWRKY33 have constitutive synergistic expression in Arabidopsis (He et al., 2016) and TaWRKY70 has constitutive synergistic expression in T. aestivum (Wang et al., 2017). Furthermore, in T. aestivum, overexpression of TaWRKY008, TaWRKY122, and TaWRKY45 improved tolerance to high-temperature stress (Gupta et al., 2019). Overexpression of VaWRKY12, which was located in the nucleus, reduced cellular damage in Arabidopsis, and grapevine following cold stress (Zhang et al., 2015), whereas overexpression of ZmWRKY106 in Zea mays increased antioxidant activity and improved heat stress tolerance. Drought can directly affect the normal functioning of crop plants by preventing enzymatic activities, the accumulation of soluble matter, the creation of ROS, multiplexes, and the restriction of metabolic pathways, putting agricultural efficiency at risk (Laxa et al., 2019). WRKY TFs have been found and reported to serve a positive effect in regulating defense genes in water-stressed situations. For example, AtWRKY28 in Arabidopsis, positively regulated in response to drought stress (Babitha et al., 2013), whereas WRKY46/54/70 acted as negative regulators under drought stress (Chen et al., 2017).

16.3.3 Heat shock factor Heat shock factors (HSFs) are significant TFs induced by heat stress, as well as other abiotic stimuli such as drought, salt, and cold. HSFs have been found in a wide variety of species, although they are relatively common in plants. The first plant HSF was discovered in the tomato plant, and it was later found in many additional plant species on a genome-wide scale. In Arabidopsis and tomato, for example, 21 and 24 HSF genes have been discovered, respectively (Scharf et al., 2012). The HSF is a modular structure that includes a DNA binding domain (DBD), the most conserved domain with a core helix-turn-helix motif. The oligomerization domain (OD), which connects the DBD via a flexible link, is another key domain (Fig. 16.4). However, the roles of several HSFs under abiotic stressors are still largely unclear (Wu et al., 2018). As a result, additional study on HSFs is needed to improve crop stress tolerance and yield.

320

Chapter 16 Transcriptional regulation of drought stress stimulus

FIGURE 16.4 Schematic representation of HSF domains. HSF, heat shock factor.

16.3.4 Nuclear Factor (NF-Ys) Nuclear factor Y (NF-Y) is present in most eukaryotes and goes by an alternative name CCAAT Binding Factor (CBF)/Heme Activator Protein (HAP). The NF-Y complex consists of the following subunits: (1) NF-YA (CBF-B/HAP2), (2) NF-YB (CBF-A/HAP3), and (3) NF-YC (CBF-C/HAP5). These subunits are important for binding to the CCAAT sequence (Nardini et al., 2013). In Arabidopsis, increase in abscisic acid in response to drought stress induced the expression of AtNF-YA5. However, its mutant form nf-ya5 showed hypersensitivity to drought stress. The overexpression of NF-YA5 enhanced drought tolerance in Arabidopsis (Li et al., 2008). Some other NF-YA factors, for example, AtNF-YA3, AtNF-YA7, and AtNF-YA10 are reported to be positive regulators of drought response (Leyva-Gonzalez et al., 2012). Another factor AtNF-YB2 is also a positive regulator of drought tolerance. It can be induced both in the presence and absence of abscisic acid (Sato et al., 2019). Under drought stress, AtNF-YC3/4/9 is reported to interact with ABF3/ 4 during flowering stage and activate SOC1 expression (Hwang et al., 2019). In the P. trichocarpa genome, 52 NF-Y genes have been identified. They are categorized as NF-YAs (13 in number), NF-YBs (20 in number), and NF-YCs (19 in number). The promoter regions of 11 of PtNF-YA genes out of 13 possess the ABRE box sequence. In plants treated with polyethylene glycol, the expression of PtNF-YA2 and PtNF-YA4 was induced (Liu et al., 2021). Dehydration did not induce the expression of PtNF-YA9 in Arabidopsis thaliana. However, when overexpressed this gene caused closing of stomata in leaves thereby contributing towards higher drought tolerance in ABAdependent manner (Lian et al., 2018). In nutshell, these findings show that drought tolerance through ABA-dependent pathway in plants have NF-Y TFs as important contributors for eliciting downstream effects (Fig. 16.5).

16.3.5 TCP transcription factor family The TCP family is plant-specific and regulates a variety of metabolic activities in plants. The existence of a noncanonical basic bHLH-containing DNA binding domain termed as TCP domain was discovered during phylogenetic study of TCP-TF from various species. This TF family is divided into two classes based on the conserved TCP domain: TCP-P (Class I) and TCP-C (Class II)

16.3 Transcription factor and their mechanisms under drought stress

321

FIGURE 16.5 Schematic representation of NF-Y domains. NF-Y, nuclear factor Y.

FIGURE 16.6 Schematic representation of TCP domains.

(Cubas et al., 1999; Kosugi and Ohashi, 1997). TCP-TF interacts with other proteins, resulting in functional diversity. TCP proteins have been shown to affect phytohormonal regulation, such as the jasmonic acid, auxin, cytokinin, and strigolactone pathways, and to influence cellular processes directly or indirectly (Lucero et al., 2015; Nicolas and Cubas, 2016; Resentini et al., 2015). Several studies have shown that they are involved in leaf shape and pattern modulation (Kieffer et al., 2011), flower and shoot development (Doebley et al., 1995), the circadian cycle (Giraud et al., 2010), and mitochondrial biogenesis (Kieffer et al., 2011). (Welchen and Gonzalez, 2006). GGNCCCAC, G (T/C) GGNCCC are examples of TCP TFs that bind to cis-regulatory elements (Fig. 16.6) (Kosugi and Ohashi, 2002). Apart from the abovementioned roles, a few studies have suggested that certain TCP-TFs play a role in drought stress response (Ding et al., 2019; Lei et al., 2017; Mukhopadhyay and Tyagi, 2015; Zhou et al., 2013). In Arabidopsis, for example, overexpression of OsTCP19 conferred drought stress resistance. During the ABA-induced drought, OsABI4 performed a key role in reaction to stress (Mukhopadhyay and Tyagi, 2015). Higher leaf wax content and enhanced water retention capacity enable Osa-miR319-mediated stress tolerance in transgenic creeping bentgrass (Zhou et al., 2013). Ding et al. (2019) conducted a genome-wide analysis of the TCP-family gene in Zea mays and discovered that ZmTCP42 plays a favorable function in drought stress resistance.

322

Chapter 16 Transcriptional regulation of drought stress stimulus

16.3.6 AP2/ERBP AP2/EREBP stands for the APETALA2/ethylene-responsive element binding protein TFs in plants. They participate in abiotic stress response as well as in phytohormone signaling pathways (ethylene, abscisic acid, cytokinin, and jasmonate) (Ohme-Takagi and Shinshi, 1995; Rashotte and Goertzen., 2010; Shen et al., 2003b; Hu et al., 2013). They possess an AP2/ERF domain composed of 60
70 amino acids (Jofuku et al., 1994; Ohme-Takagi and Shinshi, 1995; Wessler, 2005). It contains two conserved sequence blocks, the YRG element (19
22 amino acids) and the RAYD element (Fig. 16.7). The YRG element has a role to play in providing DNA-binding specificity to the TFs (Okamuro et al., 1997). The AP2/EREBP superfamily is classified into the following four main subfamilies: a) b) c) d)

APETALA2 (AP2), Related to ABI3/VP1 (RAV), Dehydration-Responsive Element Binding protein (DREB) Ethylene-Responsive Factor (ERF)

The ERF and DREB subfamilies have only one AP2 domain in their proteins (Magnani et al., 2004). The AP2/ERF DNA-binding domain of DREB TFs consists of a three-stranded antiparallel β-sheet and a α-helix (Allen et al., 1998). The β-sheet has Arg and Trp residues which help in establishing a contact with DNA (Magnani et al., 2004). The TF N-terminal region is rich in basic amino acids as opposed to the C-terminal region, which is acidic. The N-terminal region functions as a nuclear localization signal (NLS) whereas the C-terminal region might be involved in transactivation activity (Stockinger et al., 1997). The AP2 domain of the DREB subfamily has conserved valine (Val14) and glutamine (Glu19) residues in the DNA binding domain (Sakuma et al., 2002) important for the binding of core sequences of the TF (Liu et al., 1998). In close vicinity to the AP2/ERF DNA binding domain lies a conserved Ser/Thr rich region, which is crucial for phosphorylation (Liu et al., 1998; Agarwal et al., 2006). In Arabidopsis DREBs were further divided groups, A1 A2, A3, A4, A5, and A6 (Sakuma et al., 2002). They act independently of the signaling pathway elicited by abscisic acid. The members of the ERF TFs subfamily, on the other hand, are known to be activated in response to both ABA-dependent and ABA-independent signaling.

FIGURE 16.7 Schematic representation of AP2 domains.

16.3 Transcription factor and their mechanisms under drought stress

323

Table 16.3 Important DREB genes in plants. Gene

Plant

References

AtDREB1A AtDREB1A AtDREB1A AtDREB1A AtDREB1A AtDREB2ACA OsDREB1A OsDREB2B OsDREB1F OsDREB1G ZmDREB2A AhDREB1 CAP2

Arabidopsis Tobacco Wheat Rice Peanut Arabidopsis Arabidopsis Arabidopsis Arabidopsis, Rice Rice Arabidopsis Tobacco Tobacco

Liu et al. (1998) Kasuga et al. (2004) Pellegrineschi et al. (2004) Oh et al. (2005) Bhatnagar-Mathur et al. (2006) Sakuma et al. (2006) Dubouzet et al. (2003) Matsukura et al. (2010) Wang et al. (2008) Chen et al. (2008) Qin et al. (2007) Shen et al. (2003a) Shukla et al. (2006)

The DREB TFs bind to the dehydration-responsive element/C-repeat, A/GCCGAC (DRE/CRT) elements in stress-responsive genes and regulate them in response to stresses induced by extreme temperature variations, water scarcity, and salinity (Mizoi et al., 2012; Yamaguchi-Shinozaki and Shinozaki., 1994; Huang et al., 2012). The DREB2 TF subclass under the DREB subfamily is specifically involved in drought stress response. When there is low availability of water, the concentration of solutes within the cells increases. This is sensed by a histidine kinase receptor present in the plasma membrane. This kinase further activates phospholipase C (PLC). PLC is known to hydrolyze phosphatidylinositol 4, 5-bisphosphat to inositol 1, 4, 5-trisphosphate (InsP3), and diacylglycerol (Mahajan and Tuteja, 2006). These two second messengers amplify the signal many folds downstream. InsP3 causes the release of calcium within the cells thereby increasing the Ca2 1 concentration, which is sensed by the Calcineurin B-like protein (CBL). CBL further activates protein kinases and phosphatases, which in turn activate DREB2 TF. Some of the DREB subfamily genes activated in response to drought are listed in Table 16.3.

16.3.7 AREB/ABF family Abscisic acid response element proteins, as the name suggests, recognize and bind to the PyACGTGG/TC sequence in conserved ABRE (ABA-responsive elements) in the promoter region of stress response genes. They were identified using yeast one hybrid screens (Choi et al., 2000; Uno et al., 2000). The AREB TFs work downstream of the abscisic acid signaling pathway in conjunction with coupling elements (CE) for complete activation of target genes. Some of these genes code for dehydrins (LEA family) that prevent protein aggregation and stabilize membranes. Other TFs of the AREB family include genes coding for ROS detoxification enzymes and regulatory proteins. TFs belonging to this family have a conserved bZIP domain. The N-terminal part of the protein harbors a basic nuclear localization signal (NLS) and a DNA binding domain, whereas the

324

Chapter 16 Transcriptional regulation of drought stress stimulus

FIGURE 16.8 bZIP domain regulation.

C-terminal portion possesses a leucine rich motif important for dimerization (Fig. 16.8) (Wang et al., 2015). These TFs have a role to play in both plant development and abiotic stress management (Llorca et al., 2014). The abscisic acid responsive membrane receptors like PLYs activate type 2 C protein phosphatases, which activate downstream Snf1-related protein kinases 2 (SnRK2s). These kinases phosphorylate ABF TFs then bind to the ABRE cis-elements in the promoter region. Many genes coding for these TFs have been identified that are involved in eliciting drought stress response in plants. Some of these genes are OsZIP16, OsZIP23, OsZIP46, OsZIP71, and OsAREB1 in rice; ABF2, ABF3, and ABF4 in Arabidopsis Thaliana; GmbZIP 44, GmbZIP 62, GmbZIP 78, and GmbZIP 132 in Glycine max; Wlip19 in wheat; and ZmbZIP in Zea mays. Mutations and overexpression studies helped identify these factors in their respective plant species. For example, transgenic plants overexpressing TF genes like OsbZIP16 (Chen et al., 2012a,b), TabZIP60 (Zhang et al., 2015), OsbZIP23 (Todaka et al., 2015), and OsZIP71 (Liu et al., 2014) showed marked improvement in drought tolerance. OsbZIP46CA1, a constitutively expressing mutated version of OsbZIP46, increased drought tolerance in rice plants.

16.3.8 NAC transcription factors NAC stands for a group of three TFs identified in Arabidopsis thaliana. These are NAM (no apical meristem), ATAF1/2, and CUC2 (cup shaped cotyledon) (Shao et al., 2015). Just like bZIP TFs, NAC TFs also have N-terminal and C-terminal domains with distinct functions. The N-terminal has a conserved DNA binding domain (NAC domain) composed of approximately 150 amino acids and the C-terminal has a much less conserved transcriptional regulatory domain (Olsen et al., 2005; Jensen et al., 2010). The NAC domain is divided into five subdomains designated A-E. The subdomain A is required for dimerization (Olsen et al., 2005; Puranik et al., 2012). The N-terminal domain is also important for nuclear localization (Fig. 16.9). The NAC TFs are divided into two classes based on the structure of their N-terminal and C-terminal domains. These are (1) typical NAC TFs and (2) atypical NAC TFs. The typical NAC TFs have a conserved NAC domain and a divergent C-terminal domain (Olsen et al., 2005; Puranik et al., 2012). The atypical NAC TFs have a NAC domain, some conserved motifs in the C-terminal

16.3 Transcription factor and their mechanisms under drought stress

325

FIGURE 16.9 Schematic representation of NAC domains.

region or no presence of the C-terminus domain (Puranik et al., 2012). NTL (NAC with transmembrane motif1-like) is an atypical NAC TF harboring a transcriptional regulatory domain (variable) and a transmembrane region in the C-terminal (Ernst et al., 2004). The transmembrane region is thought to be important for anchoring to plasma membrane or endoplasmic reticulum membrane (Kim et al., 2007a; Liang et al., 2015). Abscisic acid induces zinc-finger homeodomain1 (ZFHD1) TF to which the three NAC proteins bind. Finally the NAC proteins bind to the promoter of early responsive to dehydration stress1 (ERD1) gene, which plays a role in regulating drought response (Fujita et al., 2004; Nakashima et al., 1997; Tran et al., 2004, 2007). Transgenic plants overexpressing NAC genes have shown considerable improvement in drought tolerance. For example, Os01g66120/OsNAC2/6 and Os11g03300/OsNAC10 genes in rice (Nakashima et al., 1997; Jeong et al., 2010) increased their tolerance capacity for drought and high salt conditions. The Os03g60080/SNAC1 gene helped increase grain yield in rice under drought stress (Hu et al., 2013). Similar observations were made for GmNAC085 (Li et al., 2011) and OsOAT (direct target of SNAC2) gene overexpression (You et al., 2013) in plants. Overexpression of NAC encoding genes ANACO19, O551072, TaNAC2, and TaNAC29 in Arabidopsis thaliana also enhanced its drought tolerance capacity. It is to be noted that the transcription of these NAC proteins is not solely induced by water deficit. They also get induced by stimulus from other abiotic stresses like cold and salinity, pathogen attack (Yuan et al., 2019), and biotic stresses. SiNAC, DgNAC1, TaNAC2a, and EcNAC1 are NAC TF genes induced in response to salt and water stress (Puranik et al., 2012; Liu et al., 2011; Ramegowda et al., 2012; Tang et al., 2012).

16.3.9 MYB/MYC transcription factors The myeloblastosis (MYB) family is composed of large number of proteins with diverse functionality in both plants and animals. Most members of this family function as TFs in eukaryotes. They are part of many regulatory networks and play an essential role in generating response to abiotic

326

Chapter 16 Transcriptional regulation of drought stress stimulus

FIGURE 16.10 MYB domains and their function.

and biotic stresses. The first MYB TF (C1) was identified in Zea mays (Paz-Ares et al., 1987). The MYB and MYC TFs are induced in response to ABA-mediated signaling. Structurally, MYB proteins have a conserved DNA binding domain (MYB domain) composed of four repeats (52 amino acids) each forming three α helices. The second and third helix exhibit a helix-turn-helix conformation. This conformation helps the MYB TFs to bind with DNA (via the third helix) by intercalating with its major groove (Fig. 16.10) (Yanhui et al., 2006). Plant MYB proteins are divided into four groups based on the number and position of MYB repeats. These are: (i) (ii) (iii) (iv)

1R-MYB, R2R3-MYB, R1R2R3-MYB, 4R-MYB.

The R2R3-MYB subfamily possesses two adjacent repeats and has the largest number of proteins (Zhang et al., 2012). The 4R-MYB is the smallest class that contains members having four R1/R2-like repeats. MYC2 is another group of TFs that plays a role in abiotic stresses like drought and salinity (Wang et al., 2020; Dombrecht et al., 2007; Verma et al., 2020). These TFs are known to be induced by jasmonic acid signaling. MYC2 TFs are normally suppressed due to their interaction with JAZ proteins. Activation of Jasmonic acid signaling pathway degrades JAZ proteins thereby liberating MYC2 TF, which can then bind to promoter region of JA-responsive genes and induce their expression (Chini et al., 2007; Thines et al., 2007; Yan et al., 2009; Kazan et al., 2013). MYC2 TFs are characterized by the presence of a conserved bHLH domain and therefore belong to a subfamily of bHLH TFs (Yanfang et al., 2018). This bHLH domain is important for the formation of homo- or hetero-dimers with other TFs (Gimenez-Ibanez et al., 2011). The MYC2 TF recognizes and binds to a conserved CACGTG sequence in the G-box of stress-responsive genes (Toledo-Ortiz et al., 2003). The expression of rd22 gene, which is activated in response to drought stress and ABA signaling, is induced by AtMYB2 and AtMYC2 in Arabidopsis (Urao et al., 1993; Abe et al., 2003).

References

327

Another TF AtMYB102 is an important component of pathways functioning in response to drought, salinity, or osmotic stress (Denekamp and Smeekens, 2003). Forty-three out of 156 GmMYB genes identified in glycine max showed a change in expression in response to drought and other abiotic stresses (Liao et al., 2008). A similar observation was made for AtMYB41 gene in Arabidopsis (Lippold et al., 2009). Overexpression studies with MYB15 and MYB96 in Arabidopsis showed that plants exhibited more tolerance towards water and salt stress and reduction in lateral root formation, respectively (Ding et al., 2009; Seo et al., 2009). Similarly in transgenic apple overexpressing Osmyb4 gene there was a marked improvement in drought and cold stress responses (Pasquali et al., 2008).

16.4 Conclusion and future prospects Drought tolerance is a complex trait that involves several genes, a cascade of intricate responses, and crosstalk between genes and signaling molecules. Besides having technological advances, achieving the desired manipulation simultaneously in many genes is difficult. In response to abiotic stress (e.g., dry spell, salinity, heat, cold and mechanical injury) many genes are directed and their gene products function in providing stress tolerance to plants. Understanding the molecular mechanisms of plant reactions to abiotic stresses is vital as it facilitates in exploiting them to improve stress tolerance and productivity. This chapter summarized the role of important plant TFs, namely DOF, WRKY, HSF, NF-Ys, TCP that regulate various stressresponsive gene expression. Using different promoters, these TFs can be genetically modified to produce transgenics that are more resistant to drought, salinity, heat, and cold. However, there is still a plethora of information to be obtained regarding the workings of these TFs. Work is underway to develop transgenic plants expressing these genes that will enhance the level of drought tolerance in the field conditions. Since different TFs can have the same downstream function, this can act as a road block when developing transgenic lines. As a result, functional investigation of these TFs will provide further insight into the complex regulatory networks involved in abiotic stress responses, as well as the crosstalk across distinct signaling pathways during stress adaption.

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161. Shukla, R.K., Raha, S., Tripathi, V., Chattopadhyay, D., 2006. Expression of CAP2, anAPETALA2-family transcription factor from chickpea, enhances growth andtolerance to dehydration and salt stress in transgenic tobacco. Plant Physiology 142, 113
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1391.

CHAPTER

17

Plant response to heavy metal stress: an insight into the molecular mechanism of transcriptional regulation

Mehali Mitra1, Puja Agarwal2 and Sujit Roy1 1

Department of Botany, UGC Centre for Advanced Studies, The University of Burdwan, Golapbag campus, Burdwan, West Bengal, India 2Department of Botany, Constituent College, Purnea University, Purnia, Bihar, India

17.1 Introduction Hypothermia, drought, salinity, and heavy metal stress are the most widespread and negatively affecting stress factors to which agricultural plants are continuously exposed. In the last few years increased anthropogenic activities and rapid urbanization have increased the rate of heavy metal contamination in the environment, which causes toxicity to all living organisms (Kavamura and Esposito, 2010; Miransari, 2011) (Fig. 17.1). Modern agricultural practices like the use of pesticides, fertilizers, and compost wastes have contaminated large areas of land with heavy metals (Yang et al., 2005). Due to their sessile lifestyle plants are continuously exposed to these unfavorable abiotic environmental conditions. These abiotic stressors put constraints on plant growth, which results in the loss of crop productivity (Fig. 17.1). Plants are required to adapt to these stress conditions and respond to the stress factors by activating several regulatory signal transduction pathways, which are very intricate by nature as stressors may affect plants at different and multiple stages of their development (Chinnusamy et al., 2004). All heavy metals are nonbiodegradable matter. Some of them are immobile and thus cannot move from the area in which they have accumulated, while other heavy metals are mobile and can be taken up by plant roots through diffusion, endocytosis, or metal transporters (Alharbi et al., 2018; Ali et al., 2012a, 2015, 2017a, 2018; Ali and Jain, 2004; Ashraf et al., 2010a; Basheer, 2018b; Burakova et al., 2018; Dehghani et al., 2016; Krzesłowska, 2011; Sabir et al., 2015; Upadhyay, 2022). However, some heavy metals like zinc, copper, and nickel act as important micronutrients functioning as cofactors for various enzymes in the different regulatory pathways, whereas heavy nonessential metals like cadmium, lead, arsenic, chromium, and mercury do not play any advantageous role in plant development and even when they accumulate in low concentration they become toxic for plants (Adriano, 1986; Ali et al., 2011, 2012b, 2014, 2016a, 2017b; Ashraf et al., 2010b; Gough et al., 1979; Gu¨cel et al., 2009a; Sharma et al., 2022; Sharma and Ali, 2011). Heavy metals that act as macro- and micronutrients play crucial roles in various physiological and biochemical processes of plants such as DNA synthesis, redox reactions in chloroplast and mitochondria, protein modifications, nitrogen fixation, and sugar metabolism. Reportedly, more than 300 enzymes and Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00004-2 © 2023 Elsevier Inc. All rights reserved.

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FIGURE 17.1 Abiotic and biotic stress factors interrupt normal plant growth and the developmental process by assaulting the plant genome endogenously, which eventually affects plant fertility and crop productivity.

200 transcription factors (TFs) are involved in membrane integrity, reproduction, and Auxin metabolism use Zn as their cofactor (Barker and Pilbeam, 2007; Williams and Pittman, 2010; Ricachenevsky et al., 2013). Plants that face toxicity by nonessential heavy metals show visible effects like chlorosis, stunted growth, and root browning, and nonvisible effects like low biomass accumulation, inhibition of photosynthesis, altered water balance and nutrient assimilation, and senescence, which ultimately leads to plant death (Ozturk et al., 2008, 2015b). Plants encounter the adverse environmental conditions created by heavy metals with various integrated physiological and molecular processes that include complex biochemical and genomic level pathways, depending on the duration and timing of the stress. All the responses of plants are broadly classified as tolerant or avoidance type, which includes alterations in whole plants and at tissuespecific, cellular, physiological, and molecular levels (Krzesłowska 2011). To cope with the adverse environmental condition plants employ specific or combinatorial patterns of intrinsic changes (Farooq et al., 2009). These changes exhibited by the plants include physiological and biochemical adjustments like root growth stimulation, leaf area reduction, leaf wilting and abscission, electrolyte leakage (EL), alterations in relative water content (RWC), production of reactive oxygen species (ROS), and accumulation of free radicals. Due to these modifications in basic cellular processes, cellular homeostasis is disrupted promoting membrane leakage, lipid peroxidation, and inactivation of enzymes that

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ultimately influence cell viability (Bartels and Sunkar, 2005). Molecular responses thus provide stress tolerance to abiotic stresses by perception, signal transduction, gene expression, and metabolic changes (Agarwal et al., 2006; Singh et al., 2019) (Fig. 17.1). At the cellular level, heavy metals impose damage by a variety of mechanisms among which the most common is the production of ROS and induction of oxidative stress. Other mechanisms involve the deactivation of biomolecules by blocking their functional groups (Stohs and Bagchi, 1995). Redox-active metals like Fe and Cu directly generate ROS by redox reactions while metals like Cd, Pb, Ni, Al, Mn, and Zn generate ROS by indirect mechanisms. At a normal physiological level ROS play an important role in cellular functions but increased accumulation deteriorates these functions (Cuypers et al., 2009). The indirect production of ROS includes the activation of ROS-producing enzymes like NADPH oxidases or inhibition of enzyme activity by the loss of cations from their binding sites. Plants primarily activate defense against heavy metal ions by adsorbing them to chelating molecules like phytochelatins or metallothionines or by sequestration of the heavy metal ions in the vacuoles. The defense responses exhibited by plants involve a major contribution of the highly complex signaling network through which the signal perception via upstream receptor and transmission to nucleus occurs involving many defense-related genes at the same time. The major signaling cascades that are induced upon heavy metal stress are calcium signaling, hormone signaling, and MAPK (mitogen-activate protein kinase) signaling pathway among which the most intrinsic and important is the MAPK cascade (Hewage et al., 2020; Yang et al., 2021). This MAPK cascade leads to processes like cell division and cell differentiation, and interacts with hormonal responses giving rise to another set of stress-related genes. The MAPK cascade is connected via phosphorylation and is composed of three-tier phosphorylation module MAPKKKs (Mitogen-Activated Protein Kinase Kinase Kinase), MAPKKs (MitogenActivated Protein Kinase Kinase), and MAPKs (Hamel et al., 2006). MAPK signaling cascade is predominantly conserved within the eukaryotic system (Jonak et al., 2002; Tena et al., 2001). These protein kinases like MAPK, receptor protein kinase, and CDPK are involved in stress signal transduction pathways that are regulated by several regulatory proteins encoded by several genes that in turn alter gene expression to function in stress adaptation (Seki et al., 2003; Shinozaki and YamaguchiShinozaki, 2007). The impact of heavy metals, on the activation of MAPK cascades, ROS-mediated signaling, and nitric oxide (NO) in diverse plant species have been extensively reviewed in previous work (Chmielowska-Bak et al., 2014). It is reported in several studies that the accumulation of stressrelated phytohormones like ABA, ethylene, jasmonic acid, and salicylic acid have increased after exposure to heavy metal stress. In response to heavy metals, the level of phytohormone is altered and cross-talk of phytohormone signaling with other signaling cascade (such as ROS, NO, and MAPK) in plants takes place (Wang et al., 2013a,b). Generally, plant resistance to any of the stress factors is determined by numerous genes that encode the regulatory proteins. The most important of all abiotic stress responses is the transcription factors (TFs) as they are transcriptions of the gene encoding regulatory proteins of the signal transduction pathways (Fig. 17.1). All major families of TFs are more or less involved in stress signaling but some of them contribute to a larger extent. Thus the activation of several TFs upregulates the expression of responsive genes and plants have improved stress tolerance (Fig. 17.1). Heavy metal-mediated toxicity is known to induce an immediate and short-term response, and TFs, belonging to diverse families, play a crucial role in modulating plant response to heavy metalinduced stress through positive and negative regulations of the stress-responsive genes (Roy 2016). Previously, depending on the homology of primary and secondary structures, plant TFs were

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divided into four large groups: (1) group with DNA binding domain known as zinc-finger; (2) group with helix-turn-helix domain; (3) group with domains of main amino acids likely leucine zipper; and (4) groups with β-scaffold domain (Tarchevskii 2001). However, at the present time, plant TFs are classified as families depending upon their variations in the cis-regulatory elements of the promoter region. Thus the TFs that are involved in abiotic stress response are APETALA2/ethylene-responsive factor (AP2/ERF), NAM/ATAF/CUC (NAC), WRKY, myeloblastosis (MYB), and basic leucine zipper (bZIP) families (Huang et al., 2012; Singh et al., 2002). In this chapter, we are mainly focused on the role of TFs of plants during heavy metal contamination along with the function of various signaling pathways, and also discuss the toxicity impaired by heavy metals.

17.2 Toxic effects of heavy metals in plants Accumulation of heavy metals in plants interferes with many morphological, physiological, and biochemical processes that ultimately lead to less crop productivity (Shahid et al., 2015), although the concentration of heavy metals accumulated in soil determines whether the metal will exhibit inhibitory or stimulating effects on plant development (Abolghassem et al., 2018). In general, the heavy metals accumulate in the root cells as they face blockage by Casparian strips or are trapped by the cell wall of root cells. Among heavy metals cadmium, lead, chromium, arsenic, and mercury are the toxic metals causing the greatest issues with public health. In the world of heavy metals, cadmium has received special attention because of its size similarity with calcium. So cadmium interferes with Ca21-mediated processes. Cadmium affects plant cells in various ways, such as depolarization of the plasma membrane of the root epidermis, thus preventing Ca21 influx and retarding root growth (Li et al., 2012b). In Arabidopsis, exposure to an elevated level of Cd inhibits the growth of root hair, by disrupting Ca21 influx and terminal cytosolic Ca21 gradient essential for growth (Fan et al., 2011). Cadmium is mostly found in pesticides that are applied on plants to achieve protection against harmful pathogens in the field. Cadmium retards plant growth due to inhibition of photosynthesis, brown root tips, and chlorotic leaves. Cd affects photosynthesis in more than one way including by decreasing chlorophyll synthesis, inhibiting enzymes that are involved in CO2 fixation, and leading to Fe(II) deficiency by inhibiting Fe(III) reductase. A high level of accumulation of Cd targets nucleolus and leads to chromosomal aberration and fragmentation. Cd affects plant respiratory systems by decomposing the mitochondrial structure and function. Cd inhibits nitrate reductase leading to interference with nitrate absorption and translocation. Cd disrupts the uptake of water along with nutrients like Ca, P, K, and Mg and also promotes water imbalance by disrupting plasma membrane integrity during lipid peroxidation (Nagajyoti et al., 2010). Cd interferes with the redox reactions of the electron transport chain by binding with the sulfhydryl group of structural proteins that leads to conformational changes like misfolding. Lead (Pb) contamination in the soil occurs due to mining, smelting, or natural weather conditions (Ashraf et al., 2015). Reduced root length, reduced growth, and chlorosis are nonspecific Pb-toxicity mediated symptoms. In particular, Pb mediates hormonal changes, reduction in RWC, mineral nutrition, and alterations in membrane permeability and inhibition in enzyme function through binding with a sulfhydryl group. Pb lowers the synthesis of important photosynthetic pigments like chlorophyll, carotenoids, and plastoquinone and also disrupts the ultrastructure of chloroplast leading to blockage in

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photosynthesis. Pb induces a shortage of carbon dioxide by closing stomatal pores along with blocking the ETC and Calvin cycle (Sharma and Dubey 2005). However, the activity of antioxidant enzymes increases with increased lead stress providing effective regulatory pathways to increase tolerance against lead toxicity (Malar et al., 2014). Chromium (Cr) imposes deleterious effects on photosynthesis by lowering the synthesis of essential photosynthetic pigments and anthocyanin, water, and mineral uptake of plants through contaminated groundwater, soil, and sedimentation (Boonyapookana et al., 2002; Shanker et al., 2003). Growth retardation, chlorosis, wilting of the top, and injury of roots are visible effects of Cr toxicity (Ozturk et al., 2015b). Production of negatively affecting metabolites like glutathione and ascorbic acid are increased together with ROS by interfered enzyme activity by chromium (Shanker et al., 2003; Yadav, 2010). However, Cr toxicity increases the production of phytochelatins and histidines that help plants protect themselves from chromium-mediated toxicity (Schmfger 2001). Mercury acts as another toxic element for plants and has no positive physiological effect at all (Hameed et al., 2017). Mercury affects the activity of mitochondria and chloroplast by interrupting the ETC and generate oxidative stress to plant cells by degradation of biomolecules and membrane (Nagajyoti et al., 2010). Mercury visibly affects the area of the plant body where it is absorbed. In general, mercury enters the plant with water and blocks the water flow by binding with the proteins that are present in the water flow channel. Along with the aforesaid heavy metals, arsenic, copper, nickel, and zinc are metabolically and physiologically useful for plants, but when they accumulate in more than required amounts, even they become toxic for plants reducing plant growth and photosynthetic ability. Although Cu plays a major role in carbon assimilation and ATP and photosynthetic pigments synthesis in plants, an overdose of it can cause damage to membranes and macromolecules by creating oxidative stress to plant cells (Yadav, 2010). Cu, Ni, and Zn show some common visible responses like chlorosis, retarded root growth, and ion leakage, necrosis, insufficient nutrient assimilation, and interrupted function of the cell membrane (Bouazizi et al., 2010). Toxic effects of Zn induce Cu and Mn (manganese) accumulation in root and shoot and induce thickened, blunt roots because of restricted cell elongation and division of root cells (Nagajyoti et al., 2010).

17.3 Plant signaling in response to heavy metal stress The evolution of multiple efficient mechanisms that sense, respond, and ultimately help plants to adapt to metal stress occurred as plants, being a sessile organism, are unable to escape from negative environmental factors including metal pollution. Heavy metal stress perception mediates activation of several biochemical, physiological, and molecular modulations of plant cells that include sensing of the stress stimuli at first; transduction and transmission of stress signal into the respective cells; and promoting required and necessary measures to counterbalance the negative effects of stress stimuli (Fig. 17.2). After exposing plants to heavy metal stress it is difficult to measure the changes in signaling pathways at the whole plant level. However, monitoring early responses, such as transcriptomic, oxidative stress, and proteomic changes, or accumulation of metabolites, might be utilized to study sensing and signal transduction changes that take place after plant exposure to

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FIGURE 17.2 Schematic illustrations elucidating heavy metal uptake by plant root cells from the contaminated soil and underground water. Metal stress perception in plant cell occurs by the specific receptor molecules, followed by signal transduction, activation of stress response pathway. This involves mitochondria, chloroplast, and nucleus, that eventually leads to activation of various stress-responsive TF families, leading the regulation and activation of the responsive genes involved in the production of more antioxidants and ROS scavenging. The activation of stress response also leads to metal sequestration in the vacuole or metal exclusion from the cell by metal transporters present in the plant cell. TFs, transcription factors; ROS, reactive oxygen species.

stress (Wang et al., 2020; Pei et al., 2021). The ultimatum of all these stress-responsive activities are the synthesis of metal transporter and metal-binding proteins that help plants to counteract extreme metal stress conditions (Maksymiec, 2007; Peng and Gong, 2014; Singh et al., 2015). Heavy metal stress mediates similar oxidative and dehydration or osmotic stress like other environmental stresses promoting nutrient imbalance, photosynthetic failures, and growth retardation (Chen et al., 2001; Yadav, 2010; Rucinska-Sobkowiak, 2016) and several signal transduction units work to operate different signal transduction pathways depending on the nature of the species and concentration of the stressor. The interplay between these signaling pathways eventually results in the regulation of different families of TFs that activate stress-responsive genes.

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17.4 MAPK signaling under heavy metal stress MAPKs are one of the most important and highly conserved classes of signaling molecules that participate in different stress signaling pathways during the developmental processes of plants (Sinha et al., 2011). MAPK cascade plays a significant role in the induction of the signal transduction pathway used in phytohormone synthesis (Jonak et al., 2002) and redox signaling. MAPKs are mainly involved in the transmission of stress stimuli by regulating several cellular processes (Hamel et al., 2006; Rodriguez et al., 2010). Reportedly, MAPKs are intrinsically involved in metal stress-mediated signaling pathways because they are activated through the perception of specific metal ligands or by the production of ROS by metal ligands (Smeets et al., 2013; Jalmi and Sinha, 2015). The cascade of three-tier components consisting of MAPKKKs, MAPKKs, and MAPKs mediate phosphorylation reaction through the transmission of stress signals from upstream receptors to downstream targets (Hamel et al., 2006) (Fig. 17.2). MAPK signaling cascade is reportedly activated by heavy metals like Cd, Cu, and As whereas not much has been reported yet about the effects of heavy metals like Pb, Zn, and Fe in activating MAPK signaling molecules (Ding et al., 2011; Jonak et al., 2004; Rao et al., 2011; Smeets et al., 2013; Upadhyay and Shumayla, 2022; Yeh et al., 2007). Exposure of Cu and Cd in Medicago sativa (alfalfa) seedlings causes activation of signal transduction cascade in its roots. In this signaling pathway, four MAPKs are activated, including SIMK, MMK2, MMK3, and SAMK (Jonak et al., 2004). When soybean seedlings get exposed to Cd in the early developmental stages, along with MAPK cascade there is induction of ethylene biosynthesis, where expression of genes involved in polyamine metabolism gets upregulated and NO generation also takes place (Chmielowska-Bak et al., 2013). In Brassica juncea, maize-specific involvement of MAP kinase-mediated signaling cascade has been reported after exposure to heavy metal toxicity (Gupta et al., 2009; Wang et al., 2010). The most important MAPKs in Arabidopsis are MPK3 and MPK6, which are activated by the stimuli from CdCl2 and CuSO4 (Asai et al., 2002; Pitzschke et al., 2009; Liu et al., 2010; Sethi et al., 2014). It has been reported that in rice, treatment with Cd and Cu in leaves and roots increases the transcript level of homologs of OsMPK3, OsMPK7, and OsMPK20
4 (Yeh et al., 2007; Rao et al., 2011). When rice root cells are exposed to Cu, accumulation of free cytosolic Ca21, which activates NADPH oxidases and CIPK activity, and eventually causes stimulation of MAP kinase activity, occurs (Yeh et al., 2007). In rice, 50 μM arsenite induces the expression of OsMKK4/OSMPK3 MAPK (Rao et al., 2011). Treatments with CdCl2 and CuCl2 activated four MAPKs, which are SIMK, SAMK MMK2, and MMK3 among which SIMK and SAMK are orthologs of OsMPK6 and OsMPK3, respectively. Copper has shown more rapid MAPK cascade activation by inducing the activities of SIMK, MMK2, and MMK3 than cadmium, which has shown a delayed activation of MAPK (Jonak et al., 2004). Apart from the activation of MAPKs by copper and cadmium, in rice, in response to iron a 42-kDa MAPK activates MBP (myelin basic protein). This iron-triggered MAPK signaling was found to be inactivated when the root cells of rice were pretreated with antioxidant glutathione (GSH) to block ROS-induced MAPK cascade activation and root-cell death, which explained the involvement of ROS in MAPK activation (Tsai and Huang, 2006). Zinc induces oxidative stress, which results in MAPK activation, and lead promotes upregulation of several MAPKs like MAPKKK7, MAPK18, MAPK20, and MAPK6 (Wang et al., 2013a,b). It has been well established that heavy metal stress induces oxidative stress to plants and this oxidative stress induces ROS

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production, which leads to MAPK activation. Several characterized MAPK cascades function downstream to ROS activation and sometimes this signaling cascade shows positive feedback regulation on ROS production (Pitzschke et al., 2009; Jalmi and Sinha, 2015). From the previous studies, it is well established that the MAPK cascade works differently upon different heavy metal perceptions depending on the activation induced by the amount of ROS molecule production (Hasanuzzaman et al., 2020). Together, these observations have indicated a cross-talk between ROS signaling, phytohormones, calcium, and MAP kinase cascade in response to heavy metalmediated toxicity. In response to heavy metal stress complicated and coordinated networks are activated, which includes and integrates various signaling pathways (Fig. 17.2). All these pathways finally lead to the regulation of TFs that in turn activate genes for activation of metal transporters, biosynthesis of chelating compounds, and other protective compounds. All the previous reports have confirmed the intrinsic involvement of MAPK cascade under heavy metal stress, although a detailed study of the signaling pathway is yet to be established.

17.5 Calcium
calmodulin signaling pathway under heavy metal stress Calcium ion (Ca12) acts as a secondary messenger in plant developmental processes or responses to biotic and abiotic stresses (Sanders et al., 2002). The changes in the concentration of the cytosolic Ca12 indicates the perception of heavy metals and activates complex network and signaling pathways (Rudd and Franklin-Tong, 2001) (Fig. 17.2). The chemical stimuli are transferred to the biological response by the increase of free cytosolic calcium ion concentration perceived by calcium-sensing and calcium-binding proteins. There are plenty of calcium-sensing proteins in plant cells like calmodulins (CaMs), calmodulin-like proteins (CMLs), calcineurin B-like proteins (CBLs), and Ca2 1 -dependent protein kinases (CDPKs), which upon sensing the increase in calcium ion concentration triggers different downstream signaling pathway to combat the perceived stress factor (Dodd et al., 2010; Luan et al., 2002; Sanders et al., 2002; Steinhorst and Jo¨rg, 2003; Upadhyay, 2021). Reports have demonstrated that the exogenous application of Ca12 increases the activity of antioxidant enzymes like glutathione reductase, ascorbate peroxidase, and superoxide dismutase, leading to physiological and biochemical changes to combat the adverse effects of heavy metal stress (Ahmad et al., 2015). Cadmium being physiologically and chemically similar to calcium ions binds to Ca12 binding sites in calmodulin, sarcolemma, and troponin C in vitro replacing calcium ion (Choong et al., 2014; Langer and Nudd, 1983; Chao et al., 1984; Ellis et al., 1984). Plants that are exposed to a high level of cadmium stress accumulate an increased level of intracellular Ca12 to activate adaptive mechanisms to decrease the negative effects of high Cd stress (Yang and Poovaiah, 2003). Interestingly, studies on cadmium-exposed Arabidopsis seedlings have explained that intracellular Ca12 mitigates the toxic effects of cadmium by the maintenance of auxin homeostasis indicating cross-talk between the signaling pathways (Zhao et al., 2015). The calcium/calmodulin pathway is also involved in the stress signaling pathway mediated by other heavy metals like lead and nickel (Ahmad et al., 2015). Reportedly, following the cross-talk of the signaling pathways, the Ca2 1 -dependent protein kinases work together with the MAPK pathway to combat the heavy metal toxicity (Takahashi et al., 2011; Wurzinger et al., 2011; Opdenakker et al., 2012). A study suggested the involvement of calcium ions in Pb2 1 -mediated cell death and

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activation of MAPK cascade through the CDPK pathway promoting the function of CDPK-like kinases (Huang and Huang, 2008). Previously an interplay between calmodulin and MAPK cascade has also been reported by explaining the prominent role of Calmodulin in regulating MAPK pathway under heavy metal stress conditions (Tebar et al., 2002). All these previous studies have confirmed the major role of the calcium/calmodulin pathway regarding the maintenance of ion-balance and redox homeostasis in plant cells under heavy metal toxicity, although the detailed study of these signaling networks and cross-talk among them is still not fully understood, unlike in animal systems where these pathways have been elaborately studied under heavy metal stress.

17.6 Hormone signaling in response to heavy metal stress So far several studies have reported the role of phytohormones like auxin, cytokinin, and ethylene in refurbishing the plant root system construction under heavy metal stress. Auxin is one of the most important phytohormones that play important roles in response to abiotic stresses as well as in plant developmental processes. Plant responses to heavy metal stress are directly affected by auxin homeostasis including transport of auxin, maintenance of its stability, and distribution of auxin at the time of heavy metal exposure (Potters et al., 2007) (Fig. 17.2). It has been reported that plants regulate the accumulation of auxin and its location by differential and dynamic regulation of auxinrelated genes in response to metal stress (Wang et al., 2015). Among the heavy metals, copper stress leads to alterations in auxin and cytokinin accumulation in primary and secondary root tips of plants (Lequeux et al., 2010). In Arabidopsis, cadmium toxicity disrupts the stability of auxin homeostasis by changing the expression of several auxin catabolic and biosynthetic genes and by increasing the indole acetic acid oxidase activity (Hu et al., 2013). According to reports it has been established that MAPK signaling is associated with auxin transport and accumulation though there are still uncertainties regarding the regulation of auxin homeostasis by MAPK cascade in response to metal stress (Mockaitis and Howell, 2000). However, studies in rice seedlings have shown interaction of MAPK cascade and auxin signaling under Cd toxicity. It has been explained from the results of previous studies that the expression of the key genes that regulate auxin signaling including YUCCA, ARF, PIN, IAA, and cell cycle regulatory gene are downregulated by the MAPK signaling pathway under Cd stress (Zhao et al., 2014). However, these findings to some extent demonstrate the interaction of both the MAPK and auxin signaling pathway in response to metal stress. The cytokinins that are activated upon the perception of heavy metal stress generally help plants in mitigating the toxic effects of metals like cadmium through reducing the blockage in the synthesis of photosynthetic pigments, thus increasing photosynthetic capacity. Cytokinins also restore the chloroplast membrane and damaged levels of primary metabolites (PiotrowskaNiczyporuk et al., 2012). The alteration in ethylene production and accumulation under metal stress is dependent upon both the type and concentration of the metal (Thao et al., 2015; Keunen et al., 2016). It was experimentally shown that treatment with Mercury (Hg) in rice seedlings upregulated the most important five ethylene synthesis genes—OsACO1, OsACO2, OsACS2, OsACO5, and OsACO6—along with TFs AP2 and ERF1 (Chen et al., 2014; Montero-Palmero et al., 2014). Cd treatment exhibited its role in regulating ethylene synthetic genes ACS2 and ACS6 along with MAPK cascades. MPK3 and MPK6, the two main MAPKs, are reportedly involved in increased

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ethylene synthesis by the phosphorylation of ACS2 and ACS6 (Liu and Zhang, 2012; Li et al., 2012a). The expression of ethylene-responsive TFs ERF1, ERF2, and ERF5 is upregulated upon treatment with Cu, which explains why heavy metals not only regulate ethylene synthesis but are also involved in ethylene signaling (Weber et al., 2006). Also, a link has been established between MAPK-mediated ROS production and ethylene synthesis in Arabidopsis seedlings under Cd stress (Schellingen et al., 2015).

17.7 Reactive oxygen species production and its role in heavy metal stress ROS production and accumulation is one of the negative effects of heavy metal toxicity that depends on the balance between ROS generation and ROS scavenging (Mittler et al., 2004). ROS accumulate in several forms like singlet oxygen (1O2), superoxide anion (O•22), H2O2, and hydroxyl radicals (•OH) by the electron transfer reactions of oxygen when the ETC is active (Sharma and Dietz, 2006). Heavy metals like Cd, Cu, Fe, and Zn can produce ROS directly or indirectly by blocking the antioxidant defense system enzymes. There are several sites for ROS production in plants. For instance, in chloroplast and mitochondria, heavy metal put a limitation on CO2 fixation resulting in over reduction of ETC which leads to the generation of ROS (Mittler et al., 2004; Davidson and Schiestl, 2001; Keunen et al., 2011). ROS are by nature extremely unstable and highly reactive and thus can easily react with essential biomolecules like DNA, proteins, and lipids depending on their chemical characters like reactivity, mobility, redox potential, and half-life that altogether lead to a destructive cellular process, namely “oxidative stress,” in plants (Mittler, 2002; Sharma and Dietz, 2006; Hossain et al., 2012a,b). This oxidative stress causes sometimes reversible or most of the time irreversible damage to the biomolecules. In contrast to this, ROS act as the signaling molecule for many physiological processes of plants that are regulated by antioxidant defense systems, like root hair and cell growth, cell differentiation, and stomatal movement (Foreman et al., 2003; Kwak et al., 2006; Tsukagoshi et al., 2010). Previous reports have shown that the ROS that are produced from NADPH oxidase enzyme upon stress stimuli act as the signaling molecule for activation of defense mechanisms (Davletova et al., 2005; Miller et al., 2008). So from these studies it can be clearly understood that the ROS accumulation, resulting from the combinatorial function of several complex processes like redox reactions, antioxidant systems, and stress signaling pathways, whenever they exceed the scavenging potential of the antioxidants, become damaging molecules generating oxidative stress. The antioxidant defense system of plants works in a specific mechanism to maintain the physiological balance of ROS accumulation blocking them as much as possible to exceed the threshold level of toxicity (Mittler et al., 2004). The antioxidant system of plants is comprised of two parts; one is enzymatic antioxidants containing enzymes like catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), glutathione peroxidase (GPX), glutathione-S-transferase (GST), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR) and the nonenzymatic part containing antioxidants like glutathione, ascorbate, proline, and α-tocopherol (Apel and Hirt, 2004; Hossain et al., 2011, 2012b, 2012a; Madhu et al., 2022; Sharma and Dietz, 2006). These antioxidants play a key role in plant response to biotic and abiotic stress responses by

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maintaining the concentration of ROS at a specific level that can promote plant defense against stress and plant development. The enzymatic components of the antioxidant defense system play a key role in heavy metal toxicity as it has been observed that when ROS formation increases due to heavy metal exposure, there is an increase in the activity of the enzymes CAT, SOD, GR and APX (Bashri and Prasad, 2015; Bharwana et al., 2013; Madhu et al., 2022; Tyagi et al., 2021). Several reports have experimentally demonstrated that heavy metals like Cu, Pb, and As increase the activity of the antioxidant enzymes, and even with the increase in the concentration of the metal the activities of SOD, CAT, GR and APX are influentially increased (Bharwana et al., 2013; Singh et al., 2013; Tyagi et al., 2020; Wang et al., 2004).

17.8 Role of transcription factors in heavy metal resistance regulation Tolerance to heavy metal stress can be achieved at the molecular level by regulating the expression of a gene that is important to plant development. Heavy metal stress induces various genes and proteins to generate the signaling cascade and achieve stress tolerance (Umezawa et al., 2006; Valliyodan and Nguyen, 2006; Manavalan et al., 2009; Tran et al., 2010). These genes are grouped into two categories: the regulatory genes and the functional genes (Tran et al., 2010). The regulatory group of genes encodes various TFs. TFs are key regulators of many developmental processes and defense responses of plants (Yanhui et al., 2006). TFs are those proteins that function together with transcription regulators to employ or block RNA polymerases to target the DNA (Udvardi et al., 2007). The TFs interact with the stress-related genes through the cis-elements of the gene’s promoter region and promote abiotic stress tolerance to plants (Agarwal and Jha, 2010). The regulatory TFs are known to be master regulators and control genes expression and are usually members of multigene families. These TFs can regulate various stress-responsive genes, which can be done cooperatively and/or separately and thus constitute a gene network. However, the genes belonging to the functional category encode metabolic compounds such as alcohols, amines, and sugars, which help in heavy metal stress tolerance. The TFs contain a DNA-binding domain that interacts with cis-regulatory elements present in the proximal-distal region of the promoters of its target genes and the TF interacts with other TFs and regulators via the protein-protein interaction domain (Wray et al., 2003; Shiu et al., 2005). This type of transcriptional regulatory system is referred to as “regulon” (Nakashima et al., 2009). TFs belonging to various families, namely MYB, AP2/EREBP, AREB/ABF, bHLH, bZIP, WRKY, MYC, HSF, DREB1/CBF, NAC, HB, ARID, EMF1, CCAT-HAP2, CCAT-Dr1, CCATHAP5, C3H, C2H2, C2C2-Gata, E2F-DP, ABI3VP1, ARF, AtSR, CPP, E2F-DP, C2C2-Dof, C2C2YABBY, C2C2-CO-like, SBP, MADS, TUB, etc., play important roles in stress response in plants. (Singh et al., 2002; Shiu et al., 2005; Shameer et al., 2009). Transcriptomic analysis of Arabidopsis showed that plant response to environmental stress factors by several independent pathways are made susceptible to stress tolerance at the transcriptional level by a very complicated regulatory network through the 1500 stress-related TFs that are considerably involved in the stress tolerance of Arabidopsis (Riechmann et al., 2000). Plants have evolved with numerous detoxification mechanisms through the activation of several complex signaling pathways in heavy metal stress tolerance. Genome-wide expression analyses have also revealed the modulation of several families of

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TFs upon heavy metal toxicity (Shim et al., 2009; Wang et al., 2010; Smeets et al., 2013). At the initial stage of signal perception, two major signaling cascades, which are MAPK and calciumdependent protein kinase (CDPK) pathways, are activated. Previous studies have demonstrated the role of MAPK signaling cascade in promoting the activation of various downstream TFs during metal toxicity among which MYB, WRKY, bZIP, AP2, ERF, ZAT, and DREB are recognized as potential targets of the MAPK pathway (Roelofs et al., 2008; Li et al., 2016a,b) (Fig. 17.3). Although there is still a lot of complications in the study of TF’s role in protective responses of plants under heavy metal stress which is assumed to be due to the major involvement in the adaptive responses of the aforesaid TF families in other abiotic stresses perceived by plants.

17.9 The MYB-family transcription factors under HM stress The C1 gene from Zea mays was the first identified MYB gene in plants encoding a MYB domain protein that is essential for anthocyanin biosynthesis in maize (Paz-ares et al., 1987). The MYB TFs are characterized by the presence of highly conserved MYB domains that participate in DNA binding. These MYB domains contain repeats designated as R and are comprised of approximately 52 amino acid residues and three alpha-helices among which the second and third form a helixturn-helix structure (Dubos et al., 2010). The MYB family of TFs are one the largest families of TFs and have a diverse function in plants (Dubos et al., 2010). According to the number of adjacent MYB repeats MYB TFs are classified into four groups: 1 R, R2R3, 3 R, and 4 R. The R2R3-MYB domain proteins are specifically found in almost all plants; about 100 R2R3-MYB proteins have so far been reported in monocots and dicots(ref. . . .). The R2R3-MYB TFs play crucial roles in plants in response to numerous environmental stresses. Studies have shown that exposure to Cd upregulates the expression of MYB TFs along with other TFs like WRKY, NAC, DREB, and AP2 at certain time intervals in rice seedlings (Ogawa et al., 2009). The member of the helix-loop-helix TF family, MYB72 and bHLH100 play important role in heavy metal homeostasis, specifically under Cd stress (Sivitz et al., 2012; Palmer et al., 2013; Li et al., 2016b) (Table 17.1). In Arabidopsis, WRKY17, the WRKY-binding proteins, MYB39, MYB45, MYB63, MYB93, and MYB94, play important roles in transcriptional regulation of genes under heavy metal stress. Such response under heavy metal stress is often rapid and transient. In Alpine Penny-cress (Thlaspi caerulescens), MYB28 and WRKY53 showed strong induction after Cd stress (Table 17.1). The TFs belonging to families like WRKY, basic leucine zipper (bZIP), bHLH, MYB, and ethylene-responsive factor (ERF) regulate Cd stress via modulating the expression of specific responsive genes (Wei et al., 2008; Wu et al., 2012). Previous studies demonstrated that in Arabidopsis, MYB TFs like MYB4, MYB28, MYB43, MYB48, MYB72, and MYB124 were expressed at a much higher level upon Cd and Zn exposure (van de Mortel et al., 2008) (Table 17.1). It was reported that Cd inactivates MYB2 by the nitrosylation of the cysteine residues of MYB2 by producing NO (Serpa et al., 2007). Recently it was reported that MYB4 TFs increases Cd tolerance with a developed antioxidant defense system and increased expression of phytochelatin synthase 1 (PCS1) and metallothionein 1 C (MT1C) via binding to their promoter regions (Agarwal et al., 2020). It has also been shown that the transgenic Arabidopsis plants overexpressing MYB4 exhibit enhanced Cd tolerance by improved antioxidant

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FIGURE 17.3 Heavy metal receptors: Heavy metal stress perception via specific receptor molecules, including PDR8, ZIP, heavy metal ATPase, NRAMP, CTR, and CDF, respectively followed by activating of the signaling pathways and induction of oxidative stress response via generation of free radicals, predominantly, the ROS molecules. ROS generation in turn activates several other signaling pathways like MAPK, phytohormone signaling, and calcium-dependent signaling pathways. The associated activation of the TF families like MYB, WRKY, bZIP and AP2/ERF, respectively further modulates the expression of stress responsive genes, leading to stress adaptation through several mophological, physiological, biochemical, cellular and molecular responses in plant cells.

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Table 17.1 Enlistment of transcription factors (TFs) whose overexpression confers heavy metal stress tolerance. Name of TF MYB43, MYB48, MYB124 MYB28, WRKY53 MYB4, MYB28, MYB43, MYB48, MYB72, MYB124

Family of TF MYB MYB & WRKY MYB

Studied plant

Plant response

References

Arabidopsis thaliana Thlaspi caerulescens

Strongly induced on exposure to Cd in the roots Strongly induced on exposure to Cd stress

van de Mortel et al. (2008)

Arabidopsis thaliana & Thlaspi caerulescens

Expressed in much higher level upon Cd and Zn stress

van de Mortel et al. (2008)

Under Cd stress, play role in metal homeostasis

Sivitz et al. (2012), Palmer et al. (2013), Li et al. (2016) Agarwal et al. (2020)

MYB72 & bHLH100

MYB & bHLH

MYB4

MYB

Arabidopsis thaliana

MYB1

MYB

Raphanus sativus

OsMYB45

MYB

Oryza sativa

OsARM1

MYB

Oryza sativa

ERF1 & ERF2

EREBP

BjCdR15

Arabidopsis thaliana & Brassica juncea Arabidopsis thaliana

WRKY6

WRKY

WRKY45

WRKY

Arabidopsis sp.

WRKY22, WRKY25, WRKY29

WRKY

Arabidopsis sp.

Increased Cd tolerance by the improved antioxidant defense and increased expression of PCS1 and MT1C Overexpressing plants were found tolerant to heavy metals by upregulating the expression of stresstolerant and antioxidant genes Hypersensitivity to Cd toxicity due to increased H2O2 content Binds to the promoter region of major As transporters and controls their expression Upregulated by Cd stress and binds to DRE and also several pathogenesisrelated promoters Improves resistance against Cd stress by regulating the expression of various metal transporter genes On exposure to Asv plant exhibits dual WRKY-dependent signaling mechanism that modulates Asv uptake and transposon expression and provides Asv tolerance Expressed in response to Zn and Fe and involved in metal homeostasis Increased expression in roots under Cd and Cu exposure

Hu et al. (2017) Wang et al. (2017)

Singh et al. (2002)

Farinati et al. (2010)

Opdenakker et al. (2012)

17.9 The MYB-family transcription factors under HM stress

351

Table 17.1 Enlistment of transcription factors (TFs) whose overexpression confers heavy metal stress tolerance. Continued Name of TF

Family of TF

WRKY53

WRKY

WRKY13

WRKY

WRKY12

WRKY

WRKY25

WRKY

bZIP62, bZIP1

bZIP

Tamarix hispida

bZIP44, bZIP78

bZIP

Glycine max

OBF5

bZIP

ZIP39

bZIP

Oryza sativa

HsfA4a

HSF

Oryza sativa

ACE1




ERF1, ERF5

ERF

Arabidopsis thaliana Arabidopsis

Involved in transcriptional regulation of glutathione-S-transferase Regulates stress-responsive genes by overexpressing in the endoplasmic reticulum Induces Cd tolerance by upregulating the expression of MT genes Protects the plant against Cu by inducing antioxidant genes Cd stress regulates the expression

DREB1A, DREB1B AtbHLH38, AtbHLH39, AtbHLH100, AtbHLH101 AtbHLH38, AtbHLH39

DREB

Oryza sativa

Cd stress upregulates the expression

bHLH

Arabidopsis

Induced in the roots and leaves in Iron deficient condition

Wang et al. (2007), Yuan et al. (2005), Yuan et al. (2008)

Maintains the Fe ion homeostasis under Cd stress

Wu et al. (2012)

bHLH

Studied plant

Plant response

References

Thlaspi caerulescens Arabidopsis thaliana Arabidopsis thaliana

Increased expression due to Cd toxicity Increases Cd tolerance through binding to PDR8gene Overexpression shows reduced Cd tolerance Upregulated as the main downstream target of MPK4 under Cd stress conditions Upregulated in response to Cd stress

Wei et al. (2008)

Downregulated in response to Cd stress

Sheng et al. (2018) Cao et al. (2019) Smeets et al. (2013) Chmielowska-Bak et al. (2014), Wang et al. (2010) Chmielowska-Bak et al. (2014), Wang et al. (2010) Singh et al. (2002) Takahashi et al. (2011) Shim et al. (2009)

Herbette et al. (2006) Ogawa et al. (2009)

defense system over the wild-type plants whereas the MYB4 knock-out mutant lines show hypersensitivity to Cd toxicity. The study identified two putative MYB4 binding motifs (ACCAACCAA and GGTAGGT) in the promoter region of PCS1 and MT1C, respectively, binding to which MYB4 positively regulates their expression under Cd stress conditions (Agarwal et al., 2020). Along with MYB4, Cd and Zn stress increase the expression of the genes encoding TFs MYB10 and MYB72 (van de Mortel et al., 2008). Interestingly, MYB72 knock-out mutants of Arabidopsis show more

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enhanced metal stress tolerance than the Zn/Cd-hyper accumulator plant Thlaspi caerulescens (van de Mortel et al., 2008). Increased content of MYB72 was observed in the leaves of Thlaspi caerulescens rather than Arabidopsis roots. Also, Cd toxicity increased the expression of the gene encoding MYB28 in Thlaspi caerulescens (van de Mortel et al., 2008). In rice, OsMYB45 mutants show hypersensitivity to Cd toxicity from the wild-type plants due to increased H2O2 content in leaves and reduced CAT activity, which explains the role of OsMYB45 in CD tolerance of rice seedlings (Hu et al., 2017) (Table 17.1). In rice, the As associated transporter genes are regulated by OsARM1 (Arsenite-Responsive MYB1), a rice MYB TF, by binding to the putative MYB domain binding sites in the promoter region of major As transporters like OsLsi1, OsLsi2, and OsLsi6 and controls their expression level (Wang et al., 2017) (Table 17.1). Compromised expression of MYB TFs and reduced activity of MAPK signaling cascade during exposure to Cd toxicity confirms the fact that MYB TFs are downstream targets of MAPk signaling pathway under heavy metal stress. Li et al. (2016a) have shown that MPK4, one of the members of the MAPK cascade, regulates the expression of MYB75, which leads to alteration in the production of photoprotective anthocyanin molecules in plants.

17.10 The WRKY-family transcription factors under HM stress Another large family of TFs in plants are WRKY TFs regulating diverse processes in plants including seed germination, plant growth and development, and plant response to biotic and abiotic stress (Rushton et al., 2010). This WRKY TF family was first discovered from sweet potato (Ipomoea batatas) and extensive study afterward and demonstrated the diverse functions of this TF family. This WRKY TF family generally consists of B60 amino acids and contains a zinc-finger motif at the C-terminal end and the N-terminal end contains the highly conserved WRKYGQK domain, which is responsible for the DNA binding of this TF. Although several binding sites have been identified for WRKY TFs, they invariably bind to the W-box proteins (TTGACT/C) of the promoter region of many genes that encode proteins that participate in plant response to environmental stress. It has been noticed that particularly under Cd and Cu stress in a short period, the expression of genes encoding WRKY TFs increases, such as under short-term Cd and Cu exposure the expression of WRKY22, WRKY25, and WRKY29 increases in roots whereas under 24 h of Cd exposure, the expression of WRKY25 and WRKY29 were reduced (Opdenakker et al., 2012) (Table 17.1). The expression of WRKY53 increases due to Cd treatment in Thlaspi caerulescens (Wei et al., 2008). In Arabidopsis, it has been shown that WRKY13 increases Cd tolerance by transcriptional regulation against Cd accumulation through binding to the PDR8 gene (Sheng et al., 2018). However, WRKY12 overexpressed Arabidopsis shows much-reduced Cd tolerance as WRKY12 binds to the W-box in the cis-element of the promoter region of GSH1 and thus negatively regulates its transcription, reducing Cd tolerance of plants (Cao et al., 2019) (Table 17.1). Previous studies have reported that under heavy metal toxicity, one of the main targets of the MAPK pathway is the WRKY TF family, which exhibits a combinatorial effect. MEKK1-MKK4/MKK5MPK3/MPK6
the flagellin-induced MAPK cascade
activates two close homologs of WRKY22 and WRKY29 (Asai et al., 2002). In Arabidopsis, MAPK pathway members MPK3 and MPK6 promote the biosynthesis of phytoalexin via phosphorylation of WRKY33 (Mao et al., 2011).

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The same trend was observed in tobacco where the MAP kinase SIPK phosphorylates tobacco WRKY1 and activates the defense response system (Menke et al., 2005). MEKK1 interacts with the promoter region of WRKY53 during its transcription and also at the protein level of the TF (Miao et al., 2007). All the WRKY TFs act as the downstream targets of the MAPK cascade under metal toxicity; for example, expression of WRKY25 was upregulated as the main downstream target of MPK4 under Cd exposed conditions (Smeets et al., 2013). Reports have also demonstrated that MAPK cascade member MKS1 induces salicylic acid-dependent activation of TF WRKY25 and WRKY33. This MKS1 directly interacts with MPK4 promoting negative regulation of defense responses of plants against heavy metals (Andreasson et al., 2005). Studies have extensively characterized the function and potential role of the WRKY TF family under heavy metal (HM) stress in model plants but more research is required to put insight into their contribution to crop improvement under such environmental stresses.

17.11 The bZIP-family transcription factors under HM stress Basic leucine zipper (bZIP) constitutes another large family of TFs in plants. A less conserved leucine zipper domain is responsible for the dimerization ability of bZIP TFs, and a basic domain constitutes the highly conserved bZIP dimerization domain. The basic region of the dimerization domain is made up of a nuclear localization signal that is followed by B16 amino acid residues and with an invariant DNA binding motif (Agarwal et al., 2019; Ali et al., 2016). It has been noticed that a different number of bZIP TF exists in different species and overexpression of this family of TFs in transgenic plants has shown increased tolerance to biotic and abiotic stress. There are 78 bZIP members in Arabidopsis (Dro¨ge-Laser et al., 2018), 191 in wheat (Agarwal et al., 2019), and 125 in maize (Wei et al., 2012). According to previous studies the bZIP TFs bind to the cis-element of the promoter region of stress-responsive genes particularly to A-box (TACGTA), C-box (GACGTC), G-box (CACGTG), GLM (GTGAGTCAT), and PB-like (TGAAAA) sequences (Ali et al., 2016). Cadmium toxicity induces the increased activity of bZIP TFs (Ramos et al., 2007). A study has shown that with 6 h exposure to CdCl2, the TFs encoded by the ThbZIP gene were found to get accumulated in the root, stems, and leaves of the transgenic tobacco plants (Wang et al., 2018). The expression bZIP TFs bZIP62 and bZIP1 from and Tamarix hispida are significantly upregulated in response to Cd stress (Chmielowska-Bak et al., 2014; Wang et al., 2010) (Table 17.1). But the expression of bZIP44 and bZIP78 was downregulated in soybean during Cd stress. A bZIP TF BjCdR15 from B. juncea directly regulates cadmium uptake and its transport and finally cadmium accumulation in shoots (Farinati et al., 2010) (Table 17.1). Under Cd stress, a bZIP TF family member OBF5 is involved in the transcriptional regulation of glutathione-S-transferase (Singh et al., 2002). Another novel bZIP gene BnbZIP3 from ramie (Boehmeria nivea) was characterized that when overexpressed improves plant capability to heavy metal stress tolerance by inducing increased root growth (Huang et al., 2016). Although the bZIP TFs are not yet proved to be the direct target of the MAPK pathway under heavy metal toxicity, there is a report that suggests the involvement of MPK3 phosphorylation in localization of VirE1-interacting protein 1 (VIP1) and nuclear trafficking of VirE2/T-DNA complex (Djamei et al., 2007).

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17.12 The AP2/ERF/DREB-family transcription factors under HM stress The APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) was first identified in Arabidopsis though later it was identified in many other species (Jofuku et al., 2007; Sakuma et al., 2002). The AP2/ERF family is responsible for providing resistance to plants under heavy metal toxicity. The ethylene-responsive factor/dehydration-responsive element-binding (ERF/DREB) is a large subfamily belonging to the AP2/ERF family of TFs and this ERF/DREB family is also involved in the response of plants to several biotic and abiotic stresses. Like other families of TFs, Cd stress also modulates the expression of AP2/ERF TFs and DREB TFs get upregulated under heavy metal toxicity. Cadmium stress leads to the regulation of the expression of ERF1 and ERF5 in Arabidopsis and upregulates DREB1A and DREB1B in roots of rice (Herbette et al., 2006; Ogawa et al., 2009) (Table 17.1). Cadmium also induces DREB2A, which specifically binds to the DRE motif of desiccation-responsive Rd29A gene and promotes the expression of Rd29A protein (Suzuki et al., 2001). In the leaves of winter wheat sprout the expression of the gene encoding DREB1 increases due to cadmium exposure (Repkina et al., 2012). This study showed that only 15 minutes of cadmium exposure keeps the expression upregulated for 7 days explaining the role of DREB1 in adaptive responses and protective mechanisms of wheat under Cd stress. Studies have shown that MAPK cascade members, MPK3 and MPK6, regulate the expression of ethylene-insensitive 3 (EIN3), which in turn regulates ERF104, thus having a downstream effect on ethylene signaling (Yoo et al., 2008; Bethke et al., 2009). Other TF family members of basic helix-loop-helix (bHLH) like AtbHLH38, AtbHLH39, AtbHLH100, and AtbHLH101 have shown to be induced in the roots and leaves of Arabidopsis under iron (Fe) deficient condition (Wang et al., 2007; Yuan et al., 2005; Yuan et al., 2008) (Table 17.1). The TFs AtbHLH38 and AtbHLH39 maintain Fe ion homeostasis under Cd stress by promoting the transcription of several iron transporting factors like iron-regulated transporter 2 (IRT2), MTP3, and HMA3 (Wu et al., 2012). Chen et al. (2016) have demonstrated that C2h2-type zinc-finger TF ZAT6 and ZAT12 are direct downstream targets of the MAPK pathway in heavy metal stress. In Arabidopsis, ZAT6 positively regulated Cd tolerance through the glutathionedependent pathway, and ZAT12 expression was affected by short-term exposure though no effect was seen under long treatment of heavy metals. Phytochelatin biosynthesis pathway genes like PCS1, PCS2, GSH1, and GSH2 are positively regulated ZAT6. It has been previously reported that in heavy metal stress MAPK controls the expression of several TF families, which also include the ERF and ZAT families of TFs (e.g., ZAT10 is a direct downstream target of MPK3 and MPK6) (Nguyen et al., 2012). In addition, the TF that participates in plant response to metal stress regulates metallothionein expression by binding the MRE (metal regulatory elements) to the promoter regions of metallothionein encoding genes (Olsson et al., 1995). However, detailed information on plant response to heavy metal stress by activation of TFs is still very limited. And additional research is required to gather more information on transcriptional response in plants under some commonly encountered heavy metals like As, Cd, Pb, and Hg in developing countries. The research data obtained focusing on the transcriptional regulation of plants in response to heavy metal stress will provide insights that might help in enhancing stress tolerance and be employed in breeding and engineering programs that will ultimately help to develop plants with new and desired agronomical traits (Lee et al., 2007; Atkinson and Urwin, 2012).

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17.13 Conclusion and future perspectives Plants are typically the basis of all foodstuff we ingest. Therefore it is important to understand the fundamental mechanisms for the improvement of plant growth and productivity in the context of heavy metal toxicity with future important implications in agriculture, human nutrition, and health. With the growing population, the percentage of contamination in the soil and underground water has increased rapidly. Plants usually collect their required nutrients through uptakes by root and shoot from the soil, which makes it possible to also absorb heavy metals from contaminated soil. In addition, these heavy metals thus localize and accumulate in the stems, leaves, flowers, and seeds, and can be consumed by humans and animals. In plants that produce root vegetables the heavy metals accumulate in them after entering through the root cells. These heavy metals not only affect the plant’s physiology but can also cause massive crop yield reduction and can directly enter into the human and animal food chain. In this chapter, we discussed the role of TF families in plant response to heavy metal toxicity along with the signaling pathways that get activated when plants are affected by heavy metal toxicity. While there are reports in the literature concerning the adverse effects of heavy metal stress in plants, heavy metals affect plants differently in different doses and activate different signaling pathways and TFs, areas of research that are lacking. For instance, the MAPK signaling pathway plays a major role in heavy metal stress by controlling the transcriptional regulation of several downstream TFs. We discussed the function of the MAPK cascade and its interference with different families of TFs, but there are other TF families in plants participating in heavy metal stress response that are well understood. Therefore better understanding of the behavior of signaling networks and TFs specifically under heavy metal toxicity is needed to take full advantage of genetic engineering to improve heavy metal stress tolerance capacity in plants. Genetic engineering of heavy metal-responsive genes (particularly TFs), proteins, and metabolites have shown surprising results, but their full potential remains to be exploited. In the future experiments can be designed that use a multidisciplinary approach with well-integrated “omics,” that is, transcriptomics, metabolomics, proteomics, to ultimately develop significantly improved heavy metal tolerance as well as tolerance to other abiotic stresses in economically important crop plants.

Acknowledgments Authors are thankful for the financial support received from the Council of Scientific and Industrial Research, Govt. of India, (Ref. No. 38(1417)/16/EMR-II, dated:17/05/2016 to SR) and a Start-Up research grant to SR from UGC, Govt. of India (No.F.30
141/2015(BSR) for conducting related research discussed in the chapter. PA is the recipient of senior research fellowship from the above mentioned CSIR, GOI funded project. MM is the recipient of Inspire Fellowship from DST, Govt. of India (DST/INSPIRE Fellowship/2017/IF17001).

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353. Available from: https://doi.org/10.1016/j.jtemb.2005.02.007. Yang, X., Lu, M., Wang, Y., Wang, Y., Liu, Z., Chen, S., 2021. Response mechanism of plants to drought stress. Horticulturae 7, 50. Yanhui, C., Xiaoyuan, Y., Kun, H., Meihua, L., Jigang, L., Zhaofeng, G., et al., 2006. The MYB transcription factor superfamily of Arabidopsis: expression analysis and phylogenetic comparison with the rice MYB family. Plant Molecular Biology 60, 107
124. Yeh, C.M., Chien, P.S., Huang, H.J., 2007. Distinct signaling pathways for induction of MAP kinase activities by cadmium and copper in rice roots. Journal of Experimental Botany 58, 659
671. Available from: https://doi.org/10.1093/jxb/erl240. Yoo, S.D., Cho, Y.H., Tena, G., Xiong, Y., Sheen, J., 2008. Dual control of nuclear EIN3 by bifurcate MAPK cascades in C2H4 signaling. Nature 451, 789
795. Available from: https://doi.org/10.1038/nature06543. Yuan, Y., Wu, H., Wang, N., Li, J., Zhao, W., Du, J., et al., 2008. FIT interacts with AtbHLH38 and AtbHLH39 in regulating iron uptake gene expression for iron homeostasis in Arabidopsis. Cell Research 18, 385
397. Yuan, Y.X., Zhang, J., Wang, D.W., Ling, H.Q., 2005. AtbHLH29 of Arabidopsis thaliana is a functional ortholog of tomato FER involved in controlling iron acquisition in strategy I plants. Cell Research 15, 613
621. Zhao, D., Li, T., Shen, M., Wang, J., Zhao, Z., 2015. Diverse strategies conferring extreme cadmium (Cd) tolerance in the dark septate endophyte (DSE), Exophiala pisciphila: evidence from RNA-seq data. Microbiological Research 170, 27
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1892. Available from: https://doi.org/10.1007/s11738-014-1564-2.

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CHAPTER

Plant transcription factors and salt stress

18 Tingting Zhang and Yang Zhou

School of Horticulture, Hainan University/Key Laboratory for Quality Regulation of Tropical Horticultural Crops of Hainan Province, Haikou, P.R. China

18.1 Effects of salt stress on plants In the natural growth process, plants are often subjected to abiotic stresses such as high salt, heat, cold, and drought from the outside world, among which high salt stress has become one of the most serious environmental stresses affecting plant growth and development (Zhu, 2016). The harm of soil salinity to plants generally refers to the effect of NaCl production (Zhang et al., 2010). The salt in the soil first affects soil compactness and hardness, making it difficult for plants to absorb nutrients and water, resulting in slower plant growth and development, and eventually causing plant death (Zhang et al., 2019). Under high salt environment, plants are subjected to osmotic stress and ion stress and other primary stresses, while salt stress will also bring secondary stresses including oxidative stress and nutritional stress; damage membrane lipid, protein, and nucleic acid, and other cell components; and cause metabolic disorders (Zhu, 2003).

18.1.1 Osmotic stress In the saline environment, the content of salt ions in vitro is high, the osmotic potential of the external environment is lower than that of the cell, and the plants cannot absorb water, resulting in osmotic stress. Osmotic stress mainly occurs in the early stage of plant salt injury. When the salt concentration in the external environment increases to a threshold value, the growth of seedlings decreases significantly. For most plants, the threshold is 40 mM NaCl, and for salt-sensitive plants, such as rice and Arabidopsis, the threshold is even lower, and was mostly caused by the osmotic pressure outside the root (Munns and Tester, 2008; Sharma et al., 2022a).

18.1.2 Ion stress Under normal conditions, the concentration of K1 in plant cells is high, about 100
200 mM, while the concentration of Na1 in cells is low, about 1
10 mM. The K1/Na1 ratio is very important to maintain the metabolism of plant cells and promote plant growth and yield formation. When K1 utilization efficiency of plants is low, moderate Na1 can promote plant growth as a regulatory substance to maintain cell turgor (Rodriguez-Navarro, 2000). Meanwhile, under low potassium Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00010-8 © 2023 Elsevier Inc. All rights reserved.

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conditions, Na1 can also promote the utilization of K1 by the high-affinity K1 absorption system (Spalding et al., 1999). However, when plants are under salt stress, the increase of Na1 in cells will not only affect the ratio of K1/Na1 in cells, but also affect the absorption of K1 in plant cells, resulting in ion toxicity to plants (Blumwald et al., 2000; Upadhyay, 2021, 2022).

18.1.3 Oxidative stress Reactive oxygen species (ROS) is a form of oxygen that is partially reduced or activated. It is a general term for oxygen-containing substances with extremely active properties and strong oxidation capacity. ROS contain superoxide anion (O2), hydrogen peroxide (H2O2), hydroxyl radical (  OH), singlet oxygen (1O2), etc. (Miller et al., 2010). When plants are subjected to salt stress, the excessive accumulation of ROS in cells causes disorder of the antioxidant system in plants and causes oxidative stress damage, such as membrane lipid peroxidation, protein oxidation, enzyme inhibition, and nucleic acid damage (Duni et al., 2019; Gyur´aszov´a et al., 2020).

18.1.4 Nutritional stress In a high-salt environment, the content of Na1 in cells increases and the ratio of Na1/K1 in cells increases rapidly (Banuelos et al., 1998). The Na1 that enters plants will prevent the absorption of K1, Ca21, and other mineral nutrients by plants, resulting in the deficiency of some nutrients and thus interfering with plant metabolism. Then plant growth is inhibited (Maathuis, 2006). On the one hand, excessive Na1 in cells will compete with K1 for K1 selective channel, so that the absorption of nutrient K1 is disturbed. On the other hand, Na1 will adversely affect microorganisms in the soil, inhibit their growth, slow down the growth of roots, and affect the absorption of water and nutrient elements such as P31, Fe31, and Zn21 (Lazof and Bernstein, 1999), thus resulting in nutrient deficiency in plants and producing nutritional stress (Levitt, 1980).

18.2 Salt tolerance mechanisms in plants 18.2.1 Osmotic regulation mechanism In the early stage of salt stress, osmotic stress will cause plant growth and development. When osmotic stress becomes serious, the plant will be in physiological drought or even die. Under osmotic stress, genes involved in the synthesis of osmotic regulatory substances in plants are induced, and small molecular substances such as betaine, sorbitol, and mannitol are produced and accumulated in plants under the action of the synthesis genes of osmotic regulatory substances (Shen et al., 2018). Osmotic regulation substances not only promote plant growth and increase water uptake and water retention, but also play an important role in increasing cell sap concentration and protecting enzyme activity, thus helping plants to better adapt to the environment of low osmotic potential and reduce the threat of salt stress.

18.2 Salt tolerance mechanisms in plants

371

18.2.2 Ion homeostasis mechanism Under salt stress, the selective ability of plants to absorb ions is usually reduced, resulting in the accumulation of toxic sodium and chloride ions in plant cells. In addition, when the salt ions in plant cells exceed the normal content, the excess salt ions will occupy the ion active transport proteins that transport nutrient ions on the cell membrane, so that the plants cannot absorb nutrient ions such as potassium and calcium, resulting in a lack of nutrients and retarding plant growth and development (Zhang and Shi, 2013). Therefore maintaining K1/Na1 homeostasis in cytoplasm is the key to plant response to salt stress. When the plant cannot tolerate the high concentration of salt (mainly NaCl) in the cytoplasm, the excess salt will be transported to vacuoles or isolated in older tissues to protect the plant from salt threat (Zhu, 2003). The sequestration of Na1 into the vacuole is thought to be mediated by the tonoplast Na1/H1 antiporter belonging to the NHX family (Sharma et al., 2022a, 2022b), which is driven by the proton motive force generated by vacuolar H1-ATPase (V-ATPase) and H1-pyrophosphatase (H1-PPase). Under normal conditions, V-ATPase plays an important role in maintaining solute homeostasis, activating secondary transport, and promoting vesicle fusion. The viability of plants under stress depends on the activity of V-ATPase enzyme (Dietz et al., 2001). It has been shown that NHX1 (Na/H exchanger 1) is involved in the formation of Na1 compartments in vacuoles, and that NHX1 and NHX2 mediate K1 entry into vacuoles, which are essential for turgor regulation and stomatal function (Bassil et al., 2011a; Bassil et al., 2011b). The salt overly sensitive (SOS) signaling pathway plays a role in ion homeostasis and salt tolerance. The SOS signaling pathway consists of three major proteins: SOS1, SOS2, and SOS3. The SOS1 protein that plays an important role in regulating Na1 flow at the cellular level functions as a plasma membrane Na1/ H1 antiporter driven by the proton gradient that is generated by the plasma membrane H1-ATPase (Zhou et al., 2015), and it can be considered as a superior salt tolerance determinant (Nawaz et al., 2014). SOS2 gene encode a serine/threonine protein kinase, and are composed of a catalytic domain in the N-terminus and a regulatory domain in the C-terminus. SOS3 is a calcium-binding protein, which can interact with and activate SOS2. The SOS2/SOS3 complex then phosphorylate SOS1 and enhance its activity. Phosphorylated SOS1 transfers Na1 from the cytoplasm to outside, thus reducing sodium toxicity in the cytoplasm (Lin et al., 2009). In addition, the plasma membrane high potassium transporter 1 (HKT1) plays an important role in salt tolerance by regulating the transport of Na1 and K1. Studies have found that HKT1-like transporters protect plants from salt stress by preventing and reducing the accumulation of Na1 and improving plant salt tolerance (Horie et al., 2009; Upadhyay, 2022; Sharma et al., 2022b).

18.2.3 Reactive oxygen species scavenging mechanism Salt stress leads to excessive accumulation of ROS in plants, thus both enzyme system and nonenzyme system are induced in plants to the ROS. The enzyme system includes catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione peroxidase (GPX), glutarine reductase (GR), glutathione S-transferase (GST), etc. (Dvoˇra´ k et al., 2021). Nonenzymatic systems include glutathione (GSH), ascorbic acid (AsA), carotenoids, alkaloids, flavonoids, and α-tocopherol.

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In response to salt stress, plants often use a series of antioxidant enzymes such as CAT, POD, SOD, and APX to remove ROS, thus enhancing salt tolerance. SOD is the first barrier for plants to eliminate ROS (Bose et al., 2014; Madhu et al., 2021, 2022, Tyagi et al., 2017, 2018, 2019, 2020, 2021). Studies show that SOD can catalyze the disproportionation of O2, reducing O2 to H2O2, then CAT and POD can effectively decompose H2O2 into H2O and O2, keeping H2O2 at a low level (Kang et al., 2019). As a major cofactor of various detoxification enzymes, GSH is involved in a variety of physiological processes, such as the regulation of sulfur transport, signal transduction, expression of stress defense genes, etc., and is essential in the antioxidant defense system (Liu et al., 2020). AsA is considered to be the most powerful antioxidant in plant cells. It is synthesized in mitochondria, and can provide electrons in many enzymatic and nonenzymatic reactions, and also can directly clear O2 and OH2 and regenerate and oxidize carotenoids or α-tocopherol, thus playing a protective role in biological membrane (Miyake et al., 1999).

18.3 Transcription factors involved in salt stress 18.3.1 bHLH transcription factors bHLH (basic helix
loop
helix) contains a highly conserved bHLH junction domain composed of about 60 amino acid residues. The N-terminal basic amino acid region is the DNA-binding domain, and the helical-ring-helical region at the C-terminal of the domain is used to form homologous or heterologous dimers (Zhu et al., 2020). According to the protein structure, the bHLH family is generally divided into group A-F. Group A members can bind to the CACCTG or CAGCTG sequences in E-box; Group B members can bind to the CACGTG or CATGTG sequences in E-box or MYC motif; Group C members contain 1
2 PAS domains; Members of Group D have no DNA-binding domain; Group E members can bind to the CACGCG or CACGAG sequences in N-box; Group F only contains COE transcription factors (TFs) (Ma et al., 1994). bHLH TFs regulate the transcription of target genes by binding to G-box/E-box or GCG-box elements in their promoters, and are involved in ABA and mitogen-activated protein kinase (MAPK) signal transduction pathway (Liu et al., 2015). The expression level of Arabidopsis thaliana PRE6 in bHLH family was significantly increased under salt treatment, but decreased under ABA treatment. Gene silencing and overexpression experiments also confirmed that PRE6 was involved in salt response and ABA regulation pathway in A. thaliana (Zheng et al., 2019). The expression levels of the ABA receptor gene NtPYL12 and the sucrose nonfermenting 1-related protein kinase 2 gene in transgenic tobacco overexpressing the wheat TabHLH1 were increased. TabHLH1 promotes stomatal closure by mediating ABA pathway, reduces leaf water loss rate, and improves salt tolerance (Yang et al., 2016). Rice OsbHLH035 alleviates the inhibitory effect of ABA on germination under salt stress by downregulating the ABA synthesis genes OsABA2 and OsAAO3 and upregulating the ABA catabolic gene OsABA8ox1 (Chen et al., 2018). The first Ca21 binding bHLH TF gene was Arabidopsis AtNIG1 gene, which confirmed to be involved in the plant salt stress signaling pathway, and can directly bind Ca21. The physiological indexes including survival rate and dry weight of atnig1
1 mutant plants showed higher salt sensitivity than wild-type A. thaliana under salt stress, indicating that ATNIG1 plays an important positive regulatory role in plant salt stress signaling pathway (Kim and Kim, 2006). Arabidopsis overexpressing the rice

18.3 Transcription factors involved in salt stress

373

OsbHLH2 gene with high homology to Arabidopsis ICE1 can not only enhance salt tolerance but also induce the expression of salt tolerance-related genes such as such as DREB1A/CBF3 (dehydration-responsive element-binding protein 1A/C-repeat/DREB binding factor 3), RD29A (desiccationresponsive protein 29A), COR15A (cold-regulated 15A), and KIN1 (protein kinase) (Zhou et al., 2009).

18.3.2 bZIP transcription factors The basic leucine zipper (bZIP) TF is one of the well-studied TF families in plants. All members of the bZIP TF family contain a highly conserved domain with a length of 60
80 amino acids. It consists of a highly conserved alkaline region that binds DNA and a variable leucine zipper region. The alkaline region is relatively conserved, consisting of about 20 amino acid residues, and contains a fixed nuclear localization structure N-(X)7-R/K that specifically binds to DNA cis-elements. The leucine zipper region is not conserved, consists of one or more repeating regions and contains a large number of hydrophobic residues with oligomeric function. The N-terminal of the zipper region of leucine can be associated with the acid domain to form homologous or heterologous dimer, which then performs transcriptional inhibition and activation functions. The cis-acting elements of bZIP TF are TACGTA (A-box), CACGTC (C-box), CACGTG (G-Box), etc., with a core sequence of ACGT. Salt-responsive bZIP TFs regulate the expression of target genes by binding cis-acting elements such as G-box and ABRE. ABF/AREB belonging to subgroup A in bZIP TFs are ABA-responsive element binding proteins and widely involved in the ABA pathway in response to salt stress. Fagopyrum tataricum FtbZIP83 could interact with FTSNRK2.2/2.3 and enhance the expression of ABA-induced genes AtRD29A, AtRD29B, and AtAIL in transgenic A. thaliana under salt stress (Li et al., 2019). FtSnRK2.6 can also interact with FtbZIP5 to reduce oxidative damage of transgenic A. thaliana under salt stress by regulating ABA signaling pathways (Li et al., 2020). Studies in potato showed that the activity of StbZIP65 promoter could be induced by exogenous methyl jasmonate, and A. thaliana overexpressing this gene showed enhanced salt tolerance (Zhao et al., 2020).

18.3.3 NAC transcription factors NAC protein is a special TF in plants (Ernst et al., 2004). The NAC domain is identified based on a consensus sequence from the NAM (no apical meristem) of Petunia and the ATAF1/2 and CUC2 of A. thaliana, and named from their first letters (Aida et al., 1997). The most important structural feature of NAC is the highly conserved NAC domain composed of about 160 amino acids at the Nterminus. This domain is composed of an α helix, two antiparallel β folds and a helix, and can be divided into five subdomains named from A to E, in which subdomains A, C, and D are more conserved than subdomains B and E (Zhu et al., 2015). The sequence in C-terminal is less conserved and has transcriptional activation function. NAC TFs can form homologous or heterologous dimers through their domains to exert their regulatory functions (Nuruzzaman et al., 2010). NAC TFs are involved in the regulation of ethylene, auxin, and ABA signal transduction pathways by binding to cis-acting elements (CACG) in the NACRS promoter. Apple MdNAC047 induces ethylene accumulation by upregulating the expression of ethylene synthesis genes MdACS1, MdACO1, and TF gene MdERF3, and enhances the tolerance to salt stress by regulating ethylene response (An et al., 2018). GmNAC109 promoted the lateral roots formation of transgenic

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Chapter 18 Plant transcription factors and salt stress

A. thaliana by positively regulating the expression of auxin-responding gene AtAIR3 and negatively regulating TF gene AtARF2, and showed stronger tolerance to high salt stress. The expression of ABA response element binding protein genes AtAREB1 and AtAREB2 and ABA response genes AtABI1 and AtABI5 were significantly upregulated in transgenic A. thaliana overexpressing GmNAC109 under salt stress (Yang et al., 2019). The expression of potato NAC gene StNAC053 was induced by salt stress and ABA, and the salt tolerance of transgenic plants was enhanced. Moreover, it was found that the germination rate of transgenic plants was lower than that of wild type under exogenous ABA treatment. This suggests that NAC TFs may be involved in plant salt tolerance through the ABA pathway (Wang et al., 2021).

18.3.4 WRKY transcription factors WRKY TFs contain one or two WRKY domains consisting of approximately 60 amino acid residues. The N-terminus of the domain is WRKYGQK sequence, which is associated with DNAbinding activity, and the C-terminus is the zinc finger structure of C-X4
5-C-X22
23-H-X1-H or C-X7-C-X23-H-X1-C, which is involved in protein interaction and assisting in DNA binding. According to the characteristics of domain, WRKY are divided into Group I, Group II (IIa, IIb, IIc, IId, and IIe), and Group III (Eulgem et al., 2000; Rushton et al., 2010). WRKY TFs, which bind to the W-box of gene promoters, are involved in abscisic acid (ABA), ethylene, and salt overly sensitive (SOS) signaling pathways and act as intermediate factors in the interaction of different signaling pathways. The members of IIc group play an important role in salt stress response. Overexpression of PeWRKY83 in A. thaliana upregulates the expression of ABA synthesis genes AtAAO3, AtNCED2 and AtNCED3, and plays a positive role in salt tolerance (Wu et al., 2017). Overexpressing the Hevea brasiliensis HbWRKY83 in A. thaliana could enhance the expression level of ethylene signaling pathway TF AtEIN3 and increase the germination rate of A. thaliana under salt stress (Kang et al., 2020). WRKY TFs can negatively or positively regulate salt response in SOS pathway. The sorghum SbWRKY50 can negatively regulate A. thaliana salt response by reducing the expression level of Na1/H1 antitransporter AtSOS1 (Song et al., 2020). FcWRKY40 can directly activate the expression of the serine/threonine protein kinase gene FcSOS2 in the SOS pathway, indirectly regulate the expression of FcSOS1 and FcSOS3 genes, promote Na1 efflux, and positively regulate the response to salt stress. In addition, FcWRKY40 can be induced by ABA, and as a target of the ABA response element binding factor FcABF2. FcWRKY40 may be a key TF in the formation of cross interaction between SOS and ABA pathways (Dai et al., 2018). In peanut, the expression of AhWRKY75, a member of WRKY IIc group, was upregulated under salt stress. Overexpressed AhWRKY75 gene could induce salt tolerance of transgenic peanut by improving the efficiency of ROS scavenging system and photosynthesis of transgenic plants under salt stress (Zhu et al., 2021).

18.3.5 MYB transcription factors The N-terminus of MYB TF contains a highly conserved MYB domain composed of about 52 amino acid residues, which is used to bind DNA, while the C-terminus is a transcriptional regulatory region with diversity, which is used to regulate protein activity (Chen et al., 2006). The MYB gene family was divided into 1R-MYB/MYB-related, R2R3- MYB, 3R- MYB, and 4R- MYB

18.3 Transcription factors involved in salt stress

375

Table 18.1 Numbers of the four MYB subfamilies in different species. MYB protein classes

R2R3MYB

1R-MYB/MYBrelated

3RMYB

4RMYB

Arabidopsis thaliana Oryza sativa

126 109

64 70

5 5

1 1

Populus trichocarpa

192

Not determined

5

0

Vitis vinifera Dendrobium catenatum

108 99

Not determined 32

5 1

1 1

References Dubos et al. (2010) Katiyar et al. (2012) Wilkins et al. (2009) Matus et al. (2008) Zhang et al. (2021)

according to the number of repeated R (Li et al., 2015). The numbers of four subfamilies are different in different species (Table 18.1). A high proportion of MYB TFs in plants respond to salt stress and regulate salt tolerance, and most of them belong to the R2R3-MYB subfamily. Salt-responsive MYB TFs activate or inhibit their temporal and spatial expression by binding cis-acting elements such as MYBCORE in target gene promoters, and participate in ABA and MAPK signal transductive pathways. Some MYB also regulate salt tolerance through ABA-independent pathway. AtMYB73 is a negative regulator of the SOS pathway. The expression levels of Na1/H1 antiporter AtSOS1 and calcineurin binding protein AtSOS3 in atmyb73 mutant were increased under salt stress (Kim et al., 2013). MYB TFs play positive or negative regulatory roles in response to salt stress. The expression of ABA synthesis genes AtNCED3 and AtABA3 was upregulated in Arabidopsis overexpressing the sesame SiMYB75, and the ABA content was also increased (Dossa et al., 2019). The pineapple AcoMYB4 can bind to the promoter of ABA synthesis gene AcoABA1 and AcoABI5, a key factor in the ABA pathway, then negatively regulate salt stress by weakening ABA synthesis and signal transduction pathways (Chen et al., 2020). In poplar, it was found that the expression of R2R3-MYB gene PtrSSR1 could be induced by salt stress, and the salt tolerance of transgenic A. thaliana was improved. Meanwhile, the expression levels of ABA-related genes NCED3 (9-cisepoxycarotenoid dioxygenase3), ABI1 (ABA-insensitive 1), and CBL1 (calcineurin-B like 1) were also upregulated, suggesting that PtrSSR1 could improve the salt tolerance of transgenic A. thaliana by regulating ABA signaling pathway (Fang et al., 2017). Overexpression and gene silencing techniques found that the cotton GhMYB73 could improve the tolerance of transgenic plants under salt stress and ABA treatment through interacting with GhPYL8 and AtPYL8 (Zhao et al., 2019). As a substrate protein of mitogen-activated protein kinase MPK3, AtMYB44 responds to plant salt tolerance by reducing ROS through the MPAK pathway (Persak and Pitzschke, 2014). Therefore, MYB, as the largest TF family in plants, plays an important role in plant response to abiotic stress.

18.3.6 Other transcription factors participate in salt stress In addition, many other TFs also play important roles in the process of plant response to salt stress. Dof (DNA binding with one finger) is a plant-specific TF that contains a zinc finger structure that recognizes the AAAG sequence at the 50 -end in the promoter region of its target gene

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Chapter 18 Plant transcription factors and salt stress

(Yanagisawa, 1995). Dof was first isolated in maize and demonstrated to be involved in C4 plant photosynthesis (Yanagisawa, 2002). The heterologous expression of ThDof1.4 gene from Tamarix hispida can improve the ROS scavenging ability in transgenic tobacco, thereby increasing the tolerance of transgenic tobacco to salt stress (Zang et al., 2019). Overexpression of GhDof1 gene in cotton improved salt and cold tolerance, as well as seed oil content (Su et al., 2017). The tomato SlDof22 increased salt tolerance by binding with the promoter of SlSOS1 gene (Cai et al., 2016). The rice OsDof18 could activate OsRGLP2 gene expression, and then improve transgenic tobacco salt tolerance (Gupta et al., 2016). In addition, OsMADS25 could regulate root length and lateral root density through positively regulating the expression of auxin synthase gene, and adapt to high salt environment (Xu et al., 2018). Both A. thaliana AtHSFA7b and tomato SlDof22 are involved in salt stress response through SOS pathway. AtHSFA7b positively regulates the expression of AtSOS1, AtSOS2, and AtSOS3 genes, and improves plant salt tolerance by maintaining cell ion homeostasis (Zang et al., 2019). Overexpressing OsDREB2A can improve the germination rate and survival rate of rice under salt stress (Mallikarjuna et al., 2011).

18.4 Conclusions and perspectives With the increasing of saline land area, the impact on plant growth and agricultural production of salt stress has increased gradually. TFs play an indispensable role in the salt stress response. Different TF activate or inhibit the expression of stress-related genes by identifying and combined with the cis-elements of target genes (Table 18.2). Plant salt tolerance is regulated by ABA, MAPK, and ethylene synthesis pathway. In addition, genetic engineering can also enhance the adaptability of plants to salt stress by overexpressing these TF genes in plants. Therefore it is of great significance to study the role of plant TFs in response to salt stress, which can not only improve the salt tolerance of plants but also provide a theoretical basis for solving problems in agricultural production.

Table 18.2 Summary of different transcription factors (TFs) involving in salt stress. TFs bHLH

Cis-acting elements

Pathway

NAC WRKY

G-box/E-box/ GCG-box A-box/C-box/Gbox NACRS W-box

MYB

MYBCORE

Ethylene/auxin/ABA Ethylene/ABA/ROS/ SOS ABA/SOS/MAPK

Others

AAAG sequence

Not determined

bZIP

ABA/Ca

21

Example AtPRE6, TabHLH1, OsbHLH035, AtNIG1, OrbHLH2

ABA

FtbZIP83, FtbZIP5, StbZIP65 MdNAC047, CmNAC109, StNAC053 PeWRKY83, HbWRKY83, SbWRKY50, FcWRKY40, AhWRKY75 AtMYB73, SiMYB75, AcoMYB4, PtrSSR1, GhMYB73, AtMYB44 ThDof1.4, GhDof1, SlDof22

References

377

Acknowledgments This study was supported by Hainan Provincial Natural Science Foundation of China (319Ms009, 318QN189), the Education Department of Hainan Province (Hys2020
242, Hnky2021
19), and Startup Funding from Hainan University (KYQD(ZR)1845).

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2758. Bassil, E., Ohto, M.A., Esumi, T., Tajima, H., Zhu, Z., Cagnac, O., et al., 2011a. The Arabidopsis intracellular Na1/H1 antiporters NHX5 and NHX6 are endosome associated and necessary for plant growth and development. Plant Cell 23 (1), 224
239. Bassil, E., Tajima, H., Liang, Y.C., Ohto, M.A., Ushijima, K., Nakano, R., et al., 2011b. The Arabidopsis Na1/H1 antiporters NHX1 and NHX2 control vacuolar pH and K1homeostasis to regulate growth, flower development, and reproduction. Plant Cell 23 (9), 3482
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151. Bose, J., Rodrigo-Moreno, A., Shabala, S., 2014. ROS homeostasis in halophytes in the context of salinity stress tolerance. Journal of Experimental Botany 65 (5), 1241
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741. Chen, H., Lai, L., Li, L., Liu, L., Jakada, B.H., Huang, Y., et al., 2020. AcoMYB4, an Ananas comosus L. MYB transcription factor, functions in osmotic stress through negative regulation of ABA signaling. International Journal of Molecular Sciences 21 (16), 5727. Chen, H.C., Cheng, W.H., Hong, C.Y., Chang, Y.S., Chang, M.C., 2018. The transcription factor OsbHLH035 mediates seed germination and enables seedling recovery from salt stress through ABA-dependent and ABA-independent pathways, respectively. Rice. 11 (1), 50. Chen, Y.H., Yang, X.Y., He, K., Liu, M.H., Li, J., Gao, Z.F., et al., 2006. The MYB transcription factor superfamily of Arabidopsis: expression analysis and phylogenetic comparison with the rice MYB family. Plant Molecular Biology 60, 107
124. Dai, W., Wang, M., Gong, X., Liu, J.H., 2018. The transcription factor FcWRKY40 of Fortunella crassifolia functions positively in salt tolerance through modulation of ion homeostasis and proline biosynthesis by directly regulating SOS2 and P5CS1 homologs. New Phytologist 219 (3), 972
989. Dietz, K.J., Tavakoli, N., Kluge, C., Mimura, T., Sharma, S.S., Harris, G.C., et al., 2001. Significance of the V-type ATPase for the adaptation to stressful growth conditions and its regulation on the molecular and biochemical level. Journal of Experimental Botany 52 (363), 1969
1980. Dossa, K., Mmadi, M.A., Zhou, R., Liu, A., Yang, Y., Diouf, D., et al., 2019. Ectopic expression of the sesame MYB transcription factor SiMYB305 promotes root growth and modulates ABA-mediated tolerance to drought and salt stresses in Arabidopsis. AOB Plants 12 (1), plz081.

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CHAPTER

19

Plant transcription factors: important factors controlling oxidative stress in plants

Shikha Verma1,3, Pankaj Kumar Verma2,3 and Debasis Chakrabarty1 1

Molecular Biology and Biotechnology Division, CSIR-National Botanical Research Institute, Lucknow, Uttar Pradesh, India 2Department of Botany, University of Lucknow, Lucknow, Uttar Pradesh, India 3French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Israel

19.1 Introduction For normal growth and development, plants depend on several factors like light, temperature, water, and nutrients. Any variations in the intensity and quantity of these abiotic factors or adversity of environmental conditions are perceived as abiotic stress, altering normal plant physiology, growth, and productivity. Oxidative stress is one of the most prevalent stress generated by several abiotic and biotic stresses. These stresses may induce the overproduction of reactive oxygen species (ROS) like hydrogen peroxide (H2O2), superoxide (O2•2), and hydroxyl radicals (•OH) and cause oxidative stress (Czarnocka and Karpi´nski, 2018). Specifically, these stresses can impair intracellular oxidation-reduction homeostasis, resulting in the generation of ROS or free radicals and creating oxidative stress. Photosynthesis and aerobic respiration in mitochondria, chloroplasts, and peroxisomes are the primary sites of ROS production (Phua et al., 2021; Suzuki et al., 2012). These ROS have extra electron charge or higher energy due to electron excitation, which causes early aging, reduction in photosynthesis, and increased cell membrane permeability (Bhattacharjee, 2005; Gill and Tuteja, 2010). Thus oxidative stress due to the disastrous effects of ROS is a critical phenomenon in various biological systems. It causes destructive impacts on plants growth and development as it reduces photosynthesis, crop yield, vigor, and germination index of seeds (Foyer and Shigeoka, 2010). Under oxidative stress, plants activate various defense systems to control the accumulation and overproduction of ROS. Although plants beneficially utilize ROS (Inz´e and Van Montagu, 1995), the high production and accumulation of it may trigger a series of action leading to cell death. Thus plants establish the equilibrium between signaling pathways mediated by ROS overproduction and scavenging (Sachdev et al., 2021). Among different plant responses, gene expression regulation is a vital plant mechanism against abiotic stress such as salinity (Ding et al., 2008; Verma et al., 2021), drought (Dash et al., 2014; Ramanjulu and Bartels, 2002; Wang et al., 2011; Tyagi, 2020), heat (Lin et al., 2018; Qian et al., 2019; Tyagi, 2018), cold (Chinnusamy et al., 2007; Wang et al., 2019a,b; Yang et al., 2015), and heavy metals (Gao et al., 2019; Verma et al., 2016a; Verma et al., 2020; Tyagi, 2021). The stress signal transduction pathways play an important role in abiotic stress tolerance by connecting the sensing Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00006-6 © 2023 Elsevier Inc. All rights reserved.

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Chapter 19 Plant transcription factors

mechanism and genetic regulation in plants. Usually, the stress-signaling pathway encompasses signal recognition, signal transduction, and finally, stress response. Recognizing stress signals via plasma membrane receptors of plant cells is the first step in activating a signal cascade for any abiotic stress (Xiong and Zhu, 2001). Different studies have shown that COLD1 (chilling-tolerance divergence 1) (Ma et al., 2015), MSLs (mscs-like proteins) (Haswell, 2007; Kaur, 2020, 2021), OSCA1 (reduced hyperosmolality-induced calcium increase1) (Yuan et al., 2014), GLR (glutamate receptor-like) channels (Grenzi et al., 2021), CNGCs (cyclic nucleotide-gated channels) (Duszyn et al., 2019), GPCRs (G-protein-coupled receptors) (Tuteja and Sopory, 2008), calcium channels (Taneja, 2021; Medvedev, 2005), and histidine kinase (Urao et al., 1999) are the plasma membrane proteins acting as putative stress signal receptors. After recognizing the signal, the sensor transmits the signal downstream through phytohormones and secondary messengers or signaling molecules like ROS and Ca12 (Sm´ekalov´a et al., 2014). These signaling molecules like ROS activate the ROS-modulated protein phosphatases and protein kinase, including calcineurin-B-like proteins (CBLs), CBL-interacting protein kinase (CIPK), mitogen-activated protein kinase (MAPK) cascades, and calcium-dependent protein kinases (CDPKs) (Upadhyay, 2021; Upadhyay and Shumayla, 2022) (Pitzschke and Hirt, 2006). Next, these protein kinases and protein phosphatases trigger phosphorylation/dephosphorylation of transcription factors (TFs) (Chae et al., 2009; Shumayla, 2019). TFs are the genes that directly regulate the expression of functional genes that have a role in cellular defense (Alves et al., 2014; Singh et al., 2002) or indirectly regulate the expression of regulatory genes involving signaling cascades (Fiil et al., 2009; Yang et al., 2013). TFs bind to a specific region in promoters and regulate the expression of targeted genes. Various TFs belonging to different families have been identified in numerous plant species such as NAC, NAM, bHLH, bZIP, MYB, and WRKY (Meraj et al., 2020) (Fig. 19.1). Several studies have shown the importance of TFs in abiotic stress response in salt, heat, cold, and drought stress, including oxidative stress tolerance, as listed in Table 19.1. The present chapter highlights the current understanding of TFs and their potential role in oxidative stress responses and discusses the molecular mechanism and their mode of action in plants.

19.2 Oxidative stress and sources Oxidative stress is associated with a severe and long-term interruption in the balance of redox status or a physiological state where the loss of electrons (oxidation) exceeds the gain of electrons (reduction), leading to oxidative damage and disruptions in cellular signaling (Chaki et al., 2020). Oxidative stress is also designated as a “stress factor” (similar to salinity, drought, and others) that causes damaging of cells and triggers signaling and defense reactions. Oxidative stress is generated by both ROS and the excessive accumulation of free radicals. ROS have partially reduced forms of oxygen and comprise substances containing one or more activated atoms of oxygen that may or may not be radicals (e.g., H2O2 is not a radical) (Apel and Hirt, 2004). On the other hand, free radicals are any chemical species consisting of unpaired electrons that exist independently. In addition, carbon-centerd radicals or transition metals that do not contain oxygen atoms also act as free radicals. The two, ROS and free radicals, oxidize the cell compounds and promote oxidative stress. Depending on the oxygen reduction, three main kinds of ROS are produced. Superoxide (O2•2) is the primary ROS produced when one-electron reduction occurs in molecular oxygen. The formation of this radical had destructive effects on photosynthetic machinery,

19.2 Oxidative stress and sources

385

FIGURE 19.1 The oxidative stress induces ROS generation. ROS acts as a signaling molecule and activates gene transcription in two ways: (A) via TFs that can interact directly with specific DNA domain on promoters of the target gene or (B) via activation of protein kinase cascades, which activates TFs that trigger the transcription of the target gene.

including Photosystem-I (PSI) and Photosystem-II (PSII) (Gill and Tuteja, 2010). Hydrogen peroxide (H2O2) is another ROS produced by superoxide dismutation. Firstly, it forms peroxide and, after neutralization by two protons, forms H2O2 (Habibi, 2014). Then, it communicates signals that lead to biotic and abiotic stress tolerance at low concentration and triggers programmed cell death at higher concentration. Hydroxyl radical (•OH) is the third most prevalent ROS, and is the most reactive and most toxic ROS. The •OH can interact with all forms of biological molecules and cause plant death because no mechanism is found in plants for the elimination of this radical (Garg and Manchanda, 2009).

19.2.1 ROS production ROS are produced in different cell organelles; among these mitochondria, chloroplasts, and peroxisomes are considered the primary ROS production sites. The mitochondrion is the primary producer of ROS under dark conditions, whereas chloroplasts and peroxisomes are the significant producers during day time (Foyer and Noctor, 2003). In chloroplasts, PSI and PSII are the main sites of ROS generation. O2•2 is also produced in PSII by one-electron oxidation of H2O2 at electron donor site

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Chapter 19 Plant transcription factors

Table 19.1 Transcription factors/genes and enzymes involved in oxidative stress resistance TF family

TF/gene

Factors

Function/Mechanism

References

AP1

CHAP1

Oxidative stress

Lev et al. (2005)

bHLH

GSNOR, bHLH100, bHLH39

Iron, nitrosative and oxidative stress

FtbHLH3

Drought and oxidative stress

OsbHLH156

Iron stress

TFEB2, TFE3

Oxidative stress

AtMYC2

Drought stress

AtMYC2

Drought stress

OsbZIP23

Drought and salt stress

OsbZIP62

Drought and oxidative stress

SlbZIP1

Salt and drought stress Salt, drought, heat, and oxidative stress

CHAP1 regulates a set of genes encoding antioxidant proteins Protects root meristem growth and prevents cell death caused by iron toxicity, nitrosative and oxidative toxicity Regulating the expression of critical genes involved in the ABA signaling pathway, proline biosynthesis pathway, and ROS scavenging system Interacts with other TFs that are involved in response to Fe deficiency Promote TFEB/TFE3 activation by stimulating the protein phosphatase PP2A without suppressing mTOR Provides drought resistance by cross-connection between ABA and JA pathways AtMYC2 along with AtMYB2 function as transcriptional activators in the abscisic acid signaling pathway Regulates the expression of various stress-related genes through ABAdependent pathway Involved in ABA signaling pathways and regulates the expression of genes associated with drought stress tolerance Modulates ABA-mediated pathway Induces expression of oxidative stress-related genes like APX1, APX2, APX3, APX4, APX6, CAT1, CAT2, and CAT3 Provides drought tolerance by activation of TaGST1 and expression of other downstream genes Regulates ABA synthesis and promotes root development Provides abiotic stress tolerance by ABA-responsive pathway

Agarwal et al. (2019)

bZIP

TabZIP

TaBZR2

Drought

ZmbZIP4

Abiotic stress

OsABF1

Abiotic stress

Li et al. (2019a)

Yao et al. (2017)

Wang et al. (2020)

Martina and Puertollano (2018) Anderson et al. (2004) Abe et al. (2003)

Xiang et al. (2008)

Yang et al. (2019)

Zhu et al. (2018)

Cui et al. (2019)

Ma et al. (2018) Hossain et al. (2010b)

19.2 Oxidative stress and sources

387

Table 19.1 Transcription factors/genes and enzymes involved in oxidative stress resistance Continued TF family

C2H2

TF/gene

Factors

Function/Mechanism

References

OsABF2

Abiotic stress

Hossain et al. (2010a)

VvABF2

Osmotic stress

ABF2

Abiotic stress

ABF3

Drought stress

ABF4

Drought stress

IbABF4

Drought stress

GmbZIP44, GmbZIP62, GmbZIP78

Cold and salt

GmbZIP132

Salt stress

F39 identical to AtbZIP60

Salt stress

GmFDL19

Salt and drought

SlAREB1

Drought, salt and pathogen defense

ZFP36

Oxidative stress

ZAT6

Heavy metal and oxidative

ZAT7

Salt and temperature

Act as a transcriptional regulator, modulates the expression of abiotic stress-responsive genes through an ABA-dependent pathway Positively regulates enzymatic antioxidant system through ABA-independent pathway ABF2 is an essential component of glucose signaling, interacts with the ABA-responsive elements The critical component in ABA signaling ABF3 and ABF4 together mediate stress-responsive ABA signaling Mediates responses to drought stress via the ABA signaling pathway These TFs function as a negative regulator of ABA signaling and confer salt and freezing tolerance in transgenic Arabidopsis Reduced ABA sensitivity has been found in transgenic Arabidopsis Involved in stress signal transduction and is associated with endoplasmic reticulum stress response Increases activities of antioxidative enzyme and chlorophyll content while reducing malondialdehyde content Regulates abiotic stress and pathogen defense through ABA-dependent pathways Involves in regulating the cross-talk among NADPH oxidase, H2O2, and MAPK in ABA signaling pathway Coordinately activates PC synthesisrelated gene expression and directly targets GSH1 to regulate Cd accumulation and tolerance positively Cross-talk between JA and SA signaling pathways

Liu et al. (2019)

Kim et al. (2004b)

Kim et al. (2004a) Kang et al. (2002) Wang et al. (2019b) Liao et al. (2008b)

Liao et al. (2008a) Fujita et al. (2007)

Li et al. (2017)

Orellana et al. (2010) Zhang et al. (2014)

Chen et al. (2016)

Ciftci-Yilmaz et al. (2007) (Continued)

388

Chapter 19 Plant transcription factors

Table 19.1 Transcription factors/genes and enzymes involved in oxidative stress resistance Continued TF family

DREB

TF/gene

Factors

Function/Mechanism

References

ZAT12,ZAT7

Oxidative stress

Ciftci-Yilmaz et al. (2007), Rizhsky et al. (2004)

ZAT12

Oxidative Stress

ZAT12

Oxidative Stress

ZAT18

Drought stress

GsZFP1

Cold and drought stress

OsZFP36

Oxidative stress

OsZFP213

Salt stress

ZFP179

Salt stress

CBF1/ DREB1

Cold stress

CBF3

Cold stress

CsDREB

Salt and drought stress

APX1, APX2, and FeSOD1 are significantly upregulated in ZAT7 and ZAT12 transgenic Arabidopsis lines Required for cytosolic ascorbate peroxidase-1 expression during oxidative stress in Arabidopsis Interacts with fer-like iron deficiency-induced transcription factor (FIT) linking iron deficiency and oxidative stress responses ZAT18 OE plants suffered less oxidative damage from drought stress Regulates the expression of stressresponse marker genes, including CBF1, CBF2, CBF3, NCED3, COR47, and RD29A in response to cold stress and RAB18, NCED3, P5CS, RD22, and RD29A in response to drought stress A key player involved in abscisic acid-induced antioxidant defense and oxidative stress tolerance in rice Interacts with OsMAPK3 to enhance salt tolerance in rice ZFP179 could increase the cellular ROS-scavenging activity of plant cells to reduce the oxidative stress Cold-induced transcription of CBF/ DREB1s is feedback inhibited by their gene products or by their downstream target genes to provide cold tolerance COR (cold-responsive) genes are activated in response to cold and induces levels of proline, sucrose, glucose, and fructose CsDREB expression is rapidly induced by heat, cold, high salinity, drought, H2O2, and exogenous ABA and enhance tolerance by ABA-dependent and ABAindependent pathways

Rizhsky et al. (2004)

Le et al. (2016)

Yin et al. (2017)

Luo et al. (2012)

Zhang et al. (2014)

Zhang et al. (2018a,b) Sun et al. (2010)

Guo et al. (2002)

Gilmour et al. (2000)

Wang et al. (2017c)

19.2 Oxidative stress and sources

389

Table 19.1 Transcription factors/genes and enzymes involved in oxidative stress resistance Continued TF family

TF/gene

Factors

Function/Mechanism

References

StDREB1

Salt stress

Bouaziz et al. (2015)

StDREB1

Cadmium stress

DREB1A, DREB2A

Cold stress

OsDRAP1

Drought stress

Cold Stress, P. syringae infection

DOF

DEAR1 (DREB and EAR motif protein) CDF3

ERF

SlERF84

Drought and salt stress

APETA A2/ERF

AP2/EREBP

Abiotic stress

ERF

OsBIERF1
4

Abiotic

Increases the expression of the StDHN25 gene by activating the DRE cis-element of its promoter sequence Overexpression of the StDREB genes improves the tolerance of potato plants to Cd by improving plant growth, proline, and antioxidant production leading to low oxidative stress damage Play a pivotal role in dehydration and cold stress-induced gene expression in Arabidopsis Interacting with other stress-related proteins, including OsCBSX3, OsRACK1A, OsRZFP34, OsAPX2, and OsCATC and activate stressresponsive genes which regulate water balance, redox homeostasis, and vascular development directly or indirectly Regulator of freezing stress tolerance pathways, regulates crosstalk between various signaling pathways Regulates a set of genes involved in cellular osmoprotectant and oxidative stress, also induces expression of TFs like CBFs, DREB2A, and ZAT12 It significantly strengthened ROS scavenging capability to decrease ROS accumulation under oxidative stress Members of the AP2/EREBP are implicated in the integration of signals derived from organelles in retrograde feedback loops and stress acclimation OsBIERF1, OsBIERF2, OsBIERF3, and OsBIERF4 act as a positive regulator of abiotic stress tolerance and are involved in various signaling pathways

Abiotic stress

Charfeddine et al. (2017)

Liu et al. (1998)

Huang et al. (2018)

(Tsutsui et al., 2009)

Corrales et al. (2017)

Li et al. (2018b)

Dietz et al. (2010)

Cao et al. (2006)

(Continued)

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Chapter 19 Plant transcription factors

Table 19.1 Transcription factors/genes and enzymes involved in oxidative stress resistance Continued TF family

TF/gene

Factors

Function/Mechanism

References

GmERF3

Drought, salt, Alternaria alternata, Tobacco mosaic virus (TMV) Salt stress and Pseudomonas syringae Environmental stress

GmERF3 as a positive transcription factor might have a role in crosstalk between the biotic and abiotic stress signal pathways in plants

Zhang et al. (2009)

Positive regulator of genes involved in salt tolerance and defense against pathogens A positive regulator of defense genes (ascorbate peroxidase-2 and galactinol synthase 1 and 2) AtHsfA2 modulates expression of stress-responsive genes Regulates the transcription of APX1 and ZAT12

Lee et al. (2004)

CaERFLP1

HSF

AtHSFA2

AtHSFA2

MADS

HSFA4A

Heat and oxidative stress Oxidative stress

CmHSFA4

Salt stress

HSFA1b

Abiotic stress

HsfA4a

Cadmium stress

LlHsfA2b

Heat and oxidative stress

PsHSF1

Oxidative stress

HSFA4A

Oxidative stress

OsMADS25

Salt stress

Plays a role in the cross-talk between ion homeostasis and oxidative stress Controlling different combinations of genes drawn from a core group of developers and stress-associated genes The tolerance was mediated by protection from oxidative damage Activates downstream genes or by dimerization or trimerisation with other HsfA protein harboring activation activity Regulates ROS detoxification by extracellular peroxidases HSFA4A is a substrate of MPK3/ MPK6 signaling, which regulates stress responses in Arabidopsis Essential transcriptional regulator that regulates the root growth and confers salinity tolerance in rice via the ABA-mediated regulatory pathway and ROS scavenging

Nishizawa et al. (2006) Li et al. (2005) Andr´asi et al. (2019), P´erezSalamo´ et al. (2014), Shim et al. (2009) Li et al. (2018a)

Albihlal et al. (2018)

Shim et al. (2009) Xin et al. (2017)

Sheng et al. (2015) P´erez-Salamo´ et al. (2014) Xu et al. (2018)

19.2 Oxidative stress and sources

391

Table 19.1 Transcription factors/genes and enzymes involved in oxidative stress resistance Continued TF family MYB

TF/gene

Factors

Function/Mechanism

References

MdMYB88/ MdMYB124

Cold stress

Xie et al. (2018)

PbrMYB5

Cold stress

SlMYB75

Abiotic stress

TaMyb1D

Drought and oxidative stress

ZmMYB3R

Drought and salt stress

ZmMYB31

Cold and oxidative stress

LeAN2

Cold and oxidative stress

AtMYB15

Salt and drought stress

MYB96

Drought and pathogen

Regulate the expression of CIRCADIAN CLOCK ASSOCIATED 1 (MdCCA1), which modulates the expression of MdCBF genes. MdCBF induce COR genes and promote anthocyanin accumulation, which contributes to H2O2 scavenging Induces antioxidant system (AsA, A.P.X., and proline and enzyme activities of SOD, POD, and CAT) to protect cells from oxidative damage Regulates SlAN2 expression confers enhanced tolerance to abiotic stresses such as cold, high temperature, high light, and oxidative stress Regulates the phenylpropanoid metabolism to enhances drought and oxidative stress tolerance Induces enzyme activities of CAT, POD, and SOD. following stress treatments, which could help to maintain low intracellular ROS levels Positively regulates the expression of CBF genes to enhances chilling and oxidative stress tolerance Promotes accumulation of anthocyanin and enhanced resistance to chilling and oxidative stress Induces the levels of stress-tolerant proteins and genes involved in ABA biosynthesis Encodes enzymes involved in the synthesis of cuticular wax in drought conditions. Plays a vital role in ABA-dependent drought tolerance and enhanced plant’s pathogen resistance by SA biosynthesis and a link interconnecting ABA-SA pathways

Xing et al. (2019)

Jian et al. (2019)

Wei et al. (2017a), Wei et al. (2017b) Wu et al. (2019)

Li et al. (2019b)

Meng et al. (2015)

Ding et al. (2009)

Seo et al. (2011), Seo and Park (2010)

(Continued)

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Chapter 19 Plant transcription factors

Table 19.1 Transcription factors/genes and enzymes involved in oxidative stress resistance Continued TF family

NAC

TF/gene

Factors

Function/Mechanism

References

OsMYB4

Cold, drought stress and viral invasion

Vannini et al. (2007), Vannini et al. (2006), Vannini et al. (2004)

SIAIM1

Abiotic stress

OsNAC6

Cold, drought and pathogen stress

OsNAC6

Drought stress

ONAC066

Drought and oxidative stress

AlNAC4

Oxidative stress

ANAC013

Oxidative

DgNAC1

Salt and drought stress Drought stress

Activates cold-inducible promoters like PAL2, ScD9 S.AD, and COR15a. Increases levels of specific amino acids which play a role in stress response Regulates pathogen resistance and abiotic stress tolerance through regulation of ABA signaling pathway Functions as a transcriptional activator of multiple signaling pathways Upregulates the expression of genes involved in membrane modification, nicotianamine biosynthesis, glutathione relocation, 30 phophoadenosine, 50 phosphosulphate accumulation, and glycosylation, which represent multiple tolerance pathways Increased the contents of proline and soluble sugars, decreased the accumulation of ROS and upregulated the expression of stressrelated genes Regulates the expression of stressresponsive genes including CAT, SOD, LEA5, PLC3, ERD10B, THT1, and TFs like AP2, ZFP during oxidative stress Mediates MRR induced expression of the MDS genes by direct interaction with the MDM cisregulatory element to enhance oxidative stress tolerance Confer salt stress responses by regulating stress-responsive genes Functions as a transcriptional activator and enhance the expression of antioxidant enzyme and drought-responsive genes Reduced the levels of malondialdehyde (MDA) and upregulate the expression of stressresponsive genes

GmNAC085

MusaNAC042

Drought and salt stress

AbuQamar et al. (2009)

Nakashima et al. (2007) Lee et al. (2017)

Yuan et al. (2019)

Khedia et al. (2018)

De Clercq et al. (2013)

Wang et al. (2017a), Zhao et al. (2018) Nguyen et al. (2018)

Tak et al. (2017)

19.2 Oxidative stress and sources

393

Table 19.1 Transcription factors/genes and enzymes involved in oxidative stress resistance Continued TF family

TF/gene

Factors

Function/Mechanism

References

PbeNAC1

Drought and cold stress

Jin et al. (2017)

SNAC1

Oxidative stress

ThNAC13

Salt and osmotic stress

Oxidative stress

WOX

ZmNTL1, ZmNTL2, ZmNTL5 OsWOX13

Drought stress

WRKY

WRKY25

Oxidative stress

WRKY46, WRKY54, WRKY70 OsWRKY45

Drought stress

GmWRKY13, GmWRKY21, GmWRKY54 AtWRKY30

Abiotic stress

TaWRKY2

Drought stress

Interacts with PbeDREB1 and PbeDREB2A to enhance the mRNA levels of stress-associated genes Bind to the promoter of OsSRO1c gene and activate its expression. OsSRO1c has a role in oxidative stress tolerance by promoting stomatal closure and H2O2 accumulation Improves salt and osmotic tolerance by enhancing the ROS-scavenging capability and adjusting osmotic potential Act as positive regulatory components related to H2O2 signaling Enhances tolerance to stress by expression of a gene involved in panicle development showed an enhancement of oxidative stress tolerance in young transgenic panicles Involved in controlling intracellular redox status and intracellular H2O2 level Positively involved in BR-regulated growth and negatively involved in drought responses Regulates stomatal closure and induction of stress-related genes during drought induction. It also regulates ABA sensitivities to the enhanced disease resistance Activate the downstream gene to promotes tolerance against several abiotic stresses Stimulates the antioxidant system to reduce the ROS-induced oxidative damage Induces changes in H2O2 content, soluble sugars, proline content, and chlorophyll content to protect from oxidative and osmotic damages

Drought stress and pathogen entry

Drought stress

You et al. (2013)

Wang et al. (2017b)

Wang et al. (2016)

Minh-Thu et al. (2018)

Doll et al. (2020)

Chen et al. (2017)

Qiu and Yu (2009)

Zhou et al. (2008)

El-Esawi et al. (2019) Gao et al. (2018)

(Continued)

394

Chapter 19 Plant transcription factors

Table 19.1 Transcription factors/genes and enzymes involved in oxidative stress resistance Continued TF family

TF/gene

Factors

Function/Mechanism

References

FcWRKY40

Oxidative stress

Gong et al. (2014)

DgWRKY5

Salt stress

MuWRKY3

Drought stress

PbrWRKY53

Drought stress

PeWRKY83

Salt stress

SlWRKY3

Salt stress

SpWRKY3

Phytophthora infestans

SlDRW1

Oxidative stress, Botrytis cinerea infection Salt stress

Functions in oxidative stress tolerance, possibly by alleviating H2O2 accumulation via regulation of the antioxidant genes Confers greater tolerance to the oxidative stress associated with salt stress Increases antioxidant enzymes activity to reduce oxidative damage Induces the transcript levels of the genes encoding ROS-scavenging enzymes (NtCAT/PbrCAT, NtSOD/ PbrSOD) as well as AsA-GSH cycle genes (DHAR1, MDHAR and APX) involved in AsA synthesis Reduces MDA content than WT plants under salt stress and lead to improved tolerance to oxidative stress caused by salt stress Enhances the expression of genes involved in abiotic stress management, ion and water transport, and cellular detoxification Induces PR gene expression and reduces the ROS accumulation to protect against cell membrane injury leading to enhanced resistance to P. infestans Involves in the JA/ET-dependent signaling pathway leading to defense response against B. cinerea Regulating the expression of genes that are related to ROS scavengings, such as Cu/ZnSOD, CAT1, CAT3, and POD1, or proline biosynthesis and glycol-metabolism, including P5CS1, SS1, SS4, G6DPH, BAM1, and BAM4 Confers pathogen tolerance through the SA pathway, and acts as a positive regulator in plant response to drought and salt stresses through both ABA-dependent and ABAindependent pathways

VvWRKY30

SlWRKY8

Drought, salt, Pseudomonas syringae infection

Liang et al. (2017)

Kiranmai et al. (2018) (Liu et al., 2019)

Wu et al. (2017)

Hichri et al. (2017)

Cui et al. (2018)

Liu et al. (2014)

Zhu et al. (2019)

Gao et al. (2020)

19.2 Oxidative stress and sources

395

Table 19.1 Transcription factors/genes and enzymes involved in oxidative stress resistance Continued TF family

TF/gene

Factors

Function/Mechanism

References

ZmWRKY104

Salt stress

Yan et al. (2021)

BnaWGR1

ROS accumulation and leaf senescence

Positively regulates ZmSOD4 expression to modulate salt-induced O2- accumulation, MDA content, and percent of electrolyte leakage Binds to promoters of RbohD and RbohF and regulates their expression

Yang et al. (2018)

(Edreva, 2005). However, the PSI is considered a significant site for O2•2 generation in the chloroplast (Galvez-Valdivieso and Mullineaux, 2010). On the other hand, peroxisomes are tiny subcellular organelles that produce H2O2 and O2•2 radicals as a part of normal metabolism. The membranes and matrix of peroxisomes are significant sites for O2•2 production (del Río et al., 2006), while beta-oxidation of fatty acids in addition to other metabolic reactions are the causes of H2O2 production (Zhang et al., 2019b). Mitochondria produces a small quantity of H2O2 in normal aerobic respiration, whereas, during abiotic stress conditions, it starts overproducing H2O2 (Gill and Tuteja, 2010). Further, electron leakage in electron transport chains (ETCs) is the primary cause of O2•2 production, which also acts as a significant contributor to oxidative stress in plants (Indo et al., 2007; Zhao et al., 2019). Sites for “electron leakage” are found in mitochondrial complexes I and III as well as in PSI (Blokhina and Fagerstedt, 2010) and PSII in the chloroplast (Edreva, 2005). Nearly 1%
5% of electrons might be leaked out in ETCs, and few electrons may target and activate O2, leading to the formation of O2•2 (Møller, 2001). In non-stressed conditions, O2•2 biosynthesis by plant organelles is a common phenomenon; however, this can increase multifold under stress conditions, generating much higher O2•2 than detoxifying by antioxidants. In addition to electron leakage, several biotic and abiotic stresses also cause ROS and free radical generation. For example, pathogen attack, salinity, heavy metals, herbicides, and xenobiotics induce higher O2•2 production (del Río et al., 2006; Verma et al., 2016b (Tyagi, 2019)). Furthermore, high light intensity and ultraviolet, xenobiotics, and herbicides directly alter the ETCs of chloroplasts, mitochondria, or peroxisomes, leading to enhanced O2•2 generation. Thus electron leakage becomes the prime cause of oxidative stress under these stresses. Hydrogen peroxide (H2O2, HOOH) is the most stable ROS and plays several essential physiological functions in living cells, including signaling (Apel and Hirt, 2004; Veal et al., 2007). H2O2 lacks unpaired electrons (“nonradical”), thus weak acid, and is a comparatively more stable molecule than O2•2, •OH and 1O2. Though, due to the activities of catalases and peroxidases that decompose H2O2, the lifetime of H2O2 in living tissues is very short (,1 s) (Jajic et al., 2015). Furthermore, H2O2 produced in cells may rapidly diffuse outside through aquaporins. The H2O2 produced by the dismutation of O22/ HO2 synthesized by NADPH oxidases and extracellular Class III peroxidases in the apoplast region make other primary sources (Cosio and Dunand, 2009). Apart from these ROS and H2O2, several other nonradical organic peroxides (ROOH, RO2OH, and ROOR) are generated in the cell. These compounds encourage oxidative stress via different ways,

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Chapter 19 Plant transcription factors

like lipid peroxidation chain reactions, and most likely also involve the intra- and extracellular redox signal (Møller et al., 2007). Hydroxyl radical (•OH) is the main cause of oxidative damage to nucleic acids, proteins, and lipid peroxidation during oxidative stress. It is directly involved in oxidative stress signaling and programmed cell death (Demidchik, 2015). For example, •OH radicals are the most prominent inducer of Ca21/K1 channel activation, leading to Ca21 influx and K1 efflux under stress conditions (Demidchik, 2010). It is considered that during stress conditions (particularly under photooxidative stress), overproduction of singlet oxygen takes place. These result in oxidative injuries, programmed cell death, and retrograde signaling (Laloi and Havaux, 2015; Triantaphylide`s and Havaux, 2009).

19.3 ROS scavenging Plants evolved several mechanisms to combat oxidative stress by neutralizing ROS by enzymatic and nonenzymatic factors of the cellular defense system. The expression of gene encoding enzymes, such as glutathione reductase (GR) and ascorbate peroxidase (APX), are found to be higher under water stress recovery, suggesting the protective role of the cellular machinery against stress created by ROS (Pang and Wang, 2010; Madhu, 2021, 2022). On the contrary, plants may trigger the ROS detoxification process under stress conditions. The synthesis of molecular chaperones, proteinases, and ROS scavenging enzymes plays a significant role in stress tolerance and repair of stress-induced damage (Table 19.2). Furthermore, the altered expressions of stressresponsive genes like LEA, dehydrin, GST, GRX, TRX, etc., also play a significant role in combating stress conditions (Kirungu et al., 2020; Verma et al., 2016b; Yang et al., 2014; Zheng et al., 2019). A number of ESTs identified from cDNA libraries of leaf and stem tissues show transcription changes in response to stress. These ESTs encode proteins like peroxidases, thioredoxins, catalases, and oxygen-evolving enhancer proteins. Most of those proteins are upregulated under stress conditions to protect against oxidative stress. In addition to these, an EST with peroxisomal glycolate oxidase (T120) homology was upregulated under abiotic stress (Fernandez et al., 2008), which suggests its involvement in ROS generation under different abiotic stress. On the other hand, genes linked with cellular homeostases like respiration, cellular biogenetic processes and DNA repair, NADH-plastoquinone reductase and catalytic hydrolase are downregulated under abiotic stress may help in combating stress conditions by reallocating energy demand. Furthermore, earlier studies revealed the mechanisms regarding ROS signal transduction pathways, even though the information regarding ROS receptors is still unknown. In order to combat the oxidative stress, activation of ROS scavenging mechanism/antioxidant system and producing essential peptides and proteins are essential. These include ascorbate, tripeptides such as GSH, regulatory proteins that operate in signal transduction pathways, kinases, and TFs. By activating the defense pathway, new transcripts are synthesized within a few hours after the onset of stress, and a steady-state level of stress adaptation is achieved (Sharma et al., 2012). Oxidative stress-responsive expression of TFs may play a crucial role in the sensing and regulation of expression of downstream genes. The expression of different TFs including WRKY, ZAT, RAV, GRAS, and MYB families are found to be enhanced by ROS accumulation (Dharshini et al., 2020). Alternatively, these TFs regulate the expression pattern of the downstream gene by binding with the promoter region of genes. For example, Zat12 induces the

19.4 Role of transcription factors in the regulation of stress-responsive genes

397

Table 19.2 Antioxidant defense mechanism in plants. Antioxidant system APX CAT DHAR MDHAR

GPX GR SOD Ascorbate (AsA) Glutathione (GSH) Tocopherol Proline Carotenoids Flavonoids

Scavenging mechanism

References

AsA is utilized as substrate and electrons are transferred to H2O2, producing water and DHA Scavenges H2O2 by oxidases through beta-oxidation of fatty acids and photorespiration A critical component in the AsA recycling system and regenerates AsA from DHA A FAD enzyme is reduced to MDHA by oxidation of AsA. In mitochondria and peroxisomes, it needs APX to scavenge H2O2 GPX reduces GSH to H2O2. Also, plays an essential role in oxidative signal transduction A key enzyme in the AsA-GSH cycle. Scavenges H2O2 by maintaining GSH/GSSG ratio Dismutation of O2•2 by reducing one O2•2 to H2O2 and another to O2 Scavenge H2O2 in the hydrophilic environment, two molecules of AsA are utilized by APX to reduce H2O2 to H2O It scavenges H2O2, O2•2, and OH- by producing AsA via the AsA-GSH cycle Physical quenching and lipid peroxidation by reducing LOO• (lipid peroxyl radical) Scavenge O2•2 and OH- by NADPH-mediated pentose phosphate pathway activity It play an essential role in quenching O2•2 and protect the photosynthetic system Neutralisation of H2O2, O2•2, and OH-

Asada (1992), Li et al. (2019c), Maruta et al. (2016) Fujiki and Bassik (2021) Rahantaniaina et al. (2017) Garcı´a-Caparro´s et al. (2021)

Ighodaro and Akinloye (2018), Passaia and Margis-Pinheiro (2015) Gill et al. (2013) Bowler et al. (1994), Chaudhry and Sidhu (2021) Hasanuzzaman et al. (2019), Zechmann (2011) Hasanuzzaman et al. (2017; Szalai et al. (2009) Falk and Munn´e-Bosch (2010), Munn´e-Bosch and Alegre (2002) Anwar Hossain et al. (2014), Hayat et al. (2012), Matysik et al. (2002) Cazzonelli (2011), Maoka (2020), Young and Lowe (2018) D’Amelia et al. (2018), Huyut et al. (2017), Oscar and Monica (2018)

expression of cytosolic ascorbate peroxidase1 (Apx1) and plant protection under oxidative conditions (Rizhsky et al., 2004). In addition, a promoter of Apx1 gene and many other genes related to H2O2 signaling accommodate a heat shock TFs (Hsf)-binding motif (Davletova et al., 2005; Miller and Mittler, 2006). For instance, the promoter analyses suggested that AtHsfA1b binds at Hsf-binding site of Apx1 promoter, which regulates the expression of Apx1 gene (Panchuk et al., 2002).

19.4 Role of transcription factors in the regulation of stress-responsive genes Responses to oxidative stress involve a complicated integrated pathway involving gene expression, cofactors, and signaling molecules to coordinate stress responses. The regulation of these components

398

Chapter 19 Plant transcription factors

requires proteins such as TFs that operate in the signal transduction pathways and regulate the expression of downstream genes involved in stress response (Vranov´a et al., 2002). TFs regulate gene expression by binding to specific DNA sequences in the promoter region (i.e., TF-binding sites of respective target genes) (Todeschini et al., 2014). Thus the transcription regulation of genes is directly assisted by a network of TFs and transcription factor-binding sites (TFBS) and plays an essential role in complex signaling networks by regulating many downstream genes to achieve a steadystate for stress adaptation. Moreover, gene induction at the transcriptional level, stress-responsive expression, and temporal and spatial regulation are integral to the plant response to stress adaptation. Oxidative stress impairs the redox equilibrium of the cell, and TFs and stress-responsive downstream genes cross-talk with each other to achieve maximal tolerance against stress. Thus TFs are potent targets for genetic modification in crops for abiotic stress resistance, and several studies have been performed to improve stress tolerance by expressing TFs. In higher plants, six TF families among 58 identified TF families are closely associated with defense signaling and expression of defense-related genes (Ng et al., 2018). These include APETALA2/ethylene-responsive factor (AP2/ERF), basic helix loop helix (bHLH), myeloblastosis related (MYB), basic leucine zipper (bZIP), WRKY, and NAC (a family of three TFs: no apical meristem (NAM), Arabidopsis transcription activation factor (ATAF1/2), and cup-shaped cotyledon (CUC2) (Agarwal et al., 2011; Ambawat et al., 2013; Feng et al., 2020; Heim et al., 2003; Nuruzzaman et al., 2013). These TF families are classified based on a conserved DNA binding domain and their unique and overlapping functions in stress response and plant development.

19.4.1 AP2/ERF family The AP2/ERF TF family is one of the most prominent TFs family involved in stress tolerance in plants. This TF family is characterized by an AP2/ERF DNA binding domain composed of an α-helix motif and three β-sheet strands (for DNA binding). Different subfamilies with distinct DNA binding affinities were categorized based on the presence of conserved motifs outside the AP2 domain (Feng et al., 2020). These subfamilies include AP2, ERF, related to abscisic acid insensitive3/viviparous1 (RAV), and dehydration-responsive element-binding protein (DREB). In addition, these TFs recognize particular cis-elements and possess additional specific conserved motifs involved in protein-protein interaction and protein phosphorylation. The DREB subfamily is the most prominent TF subfamily and has a significant role in cold stress tolerance across the plant kingdom, including non-acclimatized plants like rice and tomatoes (Lata and Prasad, 2011; Moon et al., 2019). The expression of DREB1/CBF has been shown to enhance tolerance in plants against salt and freezing (Kitashiba et al., 2004). On the other hand, DREB2 (DREB2A and DREB2B genes) are induced by salinity and dehydration stress (Nakashima et al., 2000). The expression of ERF6 TF is induced by ROS as well as pathogen and cold stress while suppressed by drought and heat stress. The erf6 knockout mutants study suggested that ERF6 TF regulates the ROS production under both biotic and abiotic stress, affecting the ROS-mediated signaling (Sewelam et al., 2013). A study showed that BpERF13 TF significantly increased cold tolerance and reduced ROS when overexpressed in a woody plant, Betula platyphylla (Lv et al., 2020). The ERF-VII subfamily in Arabidopsis consists of five members and regulates the plant response to hypoxia. In addition to hypoxia, this subfamily also plays a role in other stress like oxidative and osmotic stress. The overexpression of ERF-VII members, RAP2.2, RAP2.3, and RAP2.12 genes, in Arabidopsis results in multiple stress tolerance, including oxidative stress (Papdi et al., 2015).

19.4 Role of transcription factors in the regulation of stress-responsive genes

399

However, the molecular mechanism behind the transcription regulation of AP2/ERF TF under oxidative stress conditions is still needed to explore.

19.4.2 The bHLH family The bHLH TF family represents another large group of TFs. The bHLH TFs are characterized by a basic-helix-loop-helix (bHLH) domain with N-terminus essential DNA-binding domain and C-terminus protein interaction domain. The first plant bHLH TF was identified in Arabidopsis, the calcium-binding NaCl-inducible gene 1 (AtNIG1), involved in salt tolerance (Kim and Kim, 2006). In addition to normal plant growth, development, flower induction, and secondary metabolite biosynthesis, bHLH TFs also have a role in biotic and abiotic stress like salinity, nutrient deprivation, drought, and cold stress. The bHLH-induced drought tolerance in plants is mainly due to abscisic acid (ABA) signaling. For example, overexpression of bHLH122 in Arabidopsis enhances the drought and osmotic stress tolerance by repressing ABA catabolism (Liu et al., 2014a,b). In wheat, TabHLH1 gene is crucial to mediate drought and salt stress by modulating ABA pathway (Yang et al., 2016). In rice, OsbHLH148 improves the drought tolerance by regulating OsJAZ (jasmonate ZIM domain) protein and JA pathway (Seo et al., 2011a,b). The overexpression of finger millet EcbHLH57, homologous to OsbHLH57, increased the salinity, drought, and oxidative stress tolerance in tobacco (Babitha et al., 2015). The Fagopyrum tataricum, FtbHLH3, gene also acts as a positive regulator of drought/oxidative stress tolerance by the ABA-dependent pathway when overexpressed in Arabidopsis (Yao et al., 2017). Moreover, ectopic expression of apple, MdbHLH130, in tobacco showed tolerance to oxidative stress and water deficit by regulating ROS-scavenging and stomatal closures (Zhao et al., 2020). These studies indicated that bHLH genes play significant roles in drought, osmotic, and oxidative stress tolerance and are associated with ABA or JA and ROS scavenging.

19.4.3 MYB family MYB TFs are a universal group, and another large group of TFs found in both plants and animals that contribute to ABA-dependent gene expression. MYB family TFs contain four repeat sequences (R) with three α-helices (H1, H2, and H3). MYB family TFs are classified into four classes: R1, R2R3, R3, and R4, depending on the number of repeats. The R2R3 class is the largest plantspecific group, and functions in primary and secondary metabolisms and biotic and abiotic stress responses (Ambawat et al., 2013). A study suggested that under low-temperature stress, CBFs, a downstream TF of MYB15, can bind to cis-elements present in the promoters region of cold-responsive genes and trigger their expression. Conversely, MYB15 negatively regulates the CBFs expression. As a result, during cold acclimation and freezing tolerance, the expression of CBFs was upregulated in myb15 knockout mutant plants (Agarwal et al., 2006). Another study suggests that ZmMYB31 regulated CBFs and the expression of chilling stress-related genes (AtCBF1, AtCBF2, AtCBF3, and AtGSTU5). In addition, overexpression of ZmMYB31 in Arabidopsis decreased the low-temperature photoinhibition and ROS accumulation to improve low-temperature tolerance (Li et al., 2019a,b,c). Thus ZmMYB31 acts as a positive regulator in chilling-induced oxidative stress by modulating the expression of chilling stress-related genes. In addition, the MYB TF family is involved in

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regulating various responses like plant development, defense, differentiation, metabolism, and biotic and abiotic stress.

19.4.4 The NAC family The NAC family is plant-specific TFs and performs essential regulatory roles in plant developmental and stress response processes. The NAC TF protein contains a 160 amino acid long conserved Nterminal NAC domain and a variable transcription regulatory region pre-existent at C-terminal (Olsen et al., 2005). The DNA binding domain of NAC TF proteins is initially observed in CUC2, NAM, ATAF1, and ATAF2 TFs. Furthermore, the conserved NAC domain contains three highly conserved A, C, and D subdomains and two diverse B and E subdomains. The NAC family TFs play a role in root and shoot apical meristem development (Larsson et al., 2012), hormone signaling (Shahnejat-Bushehri et al., 2016), fruit ripening (Gao et al., 2020a,b; Kou et al., 2021), response to biotic and abiotic stresses (Huang et al., 2017; Wang et al., 2020a,b; Yang et al., 2018a,b; Yang et al., 2019a,b), fiber, and secondary cell wall development (Hussey et al., 2011; Zhang et al., 2018a,b; Zhang et al., 2019a). An earlier study suggested that overexpression of AlNAC4 can improve tolerance under oxidative stress in tobacco (Khedia et al., 2018). Another NAC family, TF ZmNTLs, regulates ROS accumulation and provides stress tolerance under oxidative stress (Wang et al., 2016). Moreover, other NAC family members, SNAC3 and OsNAC2, provide abiotic stress tolerance by regulating ROS accumulation, as OsNAC6 activates the expression of peroxidases involved in tolerance against oxidative stress conditions (Fang et al., 2015; Nakashima et al., 2007; Shen et al., 2017).

19.4.5 The WRKY family The WRKY TF family proteins are characterized by a conserved WRKY DNA binding domain (60 amino acids). The WRKY DNA binding domain contains four β-sheet strands, which form the WRKY motif followed by zinc-finger motif. Three main groups of WRKY TFs were identified, based on the number of WRKY domains and the structure of zinc-finger motif (C2H2 or C2HC) (Rushton et al., 2010). A study showed that a member of the WRKY TF family, WRKY25, is a redox-sensitive TF. WRKY25 overexpression lines showed higher tolerance under H2O2 stress. In addition, this study also found that WRKY25 was also involved in regulating intracellular H2O2 concentration (Doll et al., 2020). Overexpression of another WRKY TF family member, FcWRKY40, in tobacco provides tolerance to oxidative stress. The overexpressing lines with higher expression of CAT and POD accumulated less H2O2 under oxidative stress (Gong et al., 2014). Moreover, the expressions of Arabidopsis AtWRKY6, AtWRKY26, AtWRKY30, AtWRKY40, AtWRKY45, and AtWRKY75 were significantly upregulated under oxidative stress (Cheong et al., 2002). After the mechanical injury, the expression levels of AtWRKY11, AtWRKY15, AtWRKY22, AtWRKY33, AtWRKY40, AtWRKY53, and AtWRKY6 were upregulated (Robatzek and Somssich, 2001). In a similar way, under stress conditions, NaWRKY3 was strongly expressed in tobacco, and knocked out increased sensitivity (Skibbe et al., 2008). In previous studies, UV-B radiation treatment induces OsWRKY89 gene in rice and improved heat tolerance via a thick waxy substance on the leaf surface (Wang et al., 2007). Moreover, overexpression of WRKY25 and WRKY33 in Arabidopsis showed similar phenotypes (i.e., enhanced tolerance of salt stress but more sensitivity to oxidative and ABA treatments) (Jiang and Deyholos,

19.4 Role of transcription factors in the regulation of stress-responsive genes

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2009). This may be due to the transcripts of three Tau class glutathione-S-transferases (GSTs) genes, which were less abundant in wrky33 mutants. Interestingly, Arabidopsis WRKY25 and WRKY33 are responsive to osmotic stress and regulated by oxidative stress. In addition, downstream target genes of WRKY33 include transcripts having a role in ROS detoxification, indicating the involvement of WRKY TFs in both osmotic and oxidative stress response (Jiang and Deyholos, 2009). Furthermore, it was recently found that overexpression of Arabidopsis WRKY30 and WRKY28 increased oxidative stress tolerance, whereas overexpression of WRKY25 attenuated oxidative stress tolerance (Doll et al., 2020). Thus it becomes clear that the plant WRKY family TFs also participate in oxidative stress response, which is probably associated with ROS production during the abiotic stress response. Hence, WRKY TFs emerge as a significant contributor to ROS signaling network.

19.4.6 The bZIP family The bZIP family TFs play an important role in plant metabolism, growth, development, senescence, seed maturation, defense, flower development, and abiotic and biotic stress. The bZIP family proteins have a primary DNA binding region and a leucine zipper region for protein dimerization. The DNA binding domain is linked to nine heptad repeats of leucine, forming a dimer structure. In a recent study, 49 StbZIP genes were identified and analyzed in potato and found that several StbZIP genes were induced by different stress conditions. For example, StbZIP25 gene expression was upregulated under salt stress (Wang et al., 2021). Similarly, genome-wide analysis of bZIP family revealed the involvement of bZIP genes in plant development, fruit ripening, and abiotic stress response in banana (Hu et al., 2016). A recent study identified a TubZIP28 gene that was preferentially expressed in the endosperm of Triticum Urartu; overexpression of TubZIP28 increased the total starch content in common wheat showing the involvement of bZIP in starch storage in grains (Song et al., 2020). A study showed that overexpression of Triticum aestivum, TabZIP in Arabidopsis, improved the tolerance against salinity, drought, heat, and oxidative stress (Agarwal et al., 2019). The overexpression of rice OsbZIP16 in Arabidopsis showed reduced ROS accumulation and conferred drought and oxidative stress tolerance (Pandey et al., 2018). Another study reported that overexpression of OsbZIP62V enhanced drought and oxidative stress tolerance in transgenic rice via regulating ABA signaling (Yang et al., 2019a,b). In pepper, CAbZIP1 gene was upregulated in infected leaves with Xanthomonas campestris pv. vesicatoria. The overexpression of CAbZIP1 in Arabidopsis confers resistance to Pseudomonas syringae pv. tomato DC3000 and also increased tolerance against drought, salt and oxidative stress (Lee et al., 2006). The VqbZIP39 gene isolated from grape (Vitis quinquangularis) enhanced drought, salt and oxidative stress tolerance when overexpressed in Arabidopsis (Tu et al., 2016). The overexpression of sweet potato bZIP TF, IbABF4, in Arabidopsis enhanced ABA sensitivity and also increased drought, salt and oxidative stress tolerance mediating by ABA signaling pathway (Wang et al., 2019a,b). These reports prove that the bZIP TF family plays an important role in oxidative stress tolerance via regulating ABA signaling.

19.4.7 The HSF family Heat stress transcription factors (HSFs) can regulate the expression of genes involved in abiotic stress (Personat et al., 2014). Under abiotic stress, HSFs protect cells from damage via the accumulation of heat shock proteins (Von Koskull-Do¨ring et al., 2007). HSFs contains DNA-binding

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domain (DBD) and oligomerization domain (OD) containing central helix-turn-helix (HTH) motif and heptad repeats of hydrophobic residues (HR-A and HR-B), respectively, in N-terminal, and also contain nuclear localization signal (NLS), nuclear export signal (NES), and activator peptide motif (AHA) in C-terminal. Based on the number of amino acid residues between HR-A and HRB, plant HSFs are classified into three classes: A, B, and C. The Class A and C HSFs have extended HR-A/B regions with insertions of 21 or 7 amino acid residues between HR-A and HR-B region, respectively, whereas Class B HSFs have a compact HR-A/B region. Additionally, class A HSFs contains the AHA activation domains, but class B and class C HSFs are absent. The Class B HSFs has LFGV-tetrapeptide motif at C-terminal and act as a repressor of other TFs (Nover et al., 1996). The HSFS family is further divided into different subclasses, and their detailed function has been reported in several studies. Rice genome sequence showed that rice has 26 OsHSF genes, and expression analysis showed that 22 OsHSF genes were induced by high temperature, 10 OsHSF genes by low temperature, and 14 OsHSF genes by oxidative stress (Mittal et al., 2009). The Arabidopsis AtHSFA1s is a key regulator of heat stress by inducing the expression of several regulators, including other HSFs subclasses and also involved in abiotic stress including oxidative stress (Liu et al., 2011). AtHSFA4A regulates the responses to salt and oxidative stress and acts as a substrate of MPK3/MPK6 signaling pathway (P´erez-Salamo´ et al., 2014). A study showed that cooverexpression of sunflower HaHSFA4a and HaHSFA9 in tobacco enhanced tolerance to severe dehydration and oxidative stress (Personat et al., 2014). These studies suggest that the HSFs family has functional diversity under different abiotic stress conditions including oxidative stress.

19.5 Conclusion and future prospects Various environmental stresses cause oxidative stress and lead to an excess generation of ROS, causing molecular and cellular damage in plants. The scavenging of excessive ROS during oxidative stress requires the action of several enzymes regulated by oxidative signaling. Oxidative stress signaling cascades involve different TFs in regulating the transcription of stress-responsive gene, thus providing oxidative stress tolerance to plants. To date, several TFs have been identified, but plant biology complexity with genome higher ploidy restrict their use in the scientific community. Through the introduction of novel molecular biology tools, evident efforts are made on the involvement of TFs to bestow tolerance under oxidative stress and explain the role of TFs in plant growth and development. This chapter highlighted that many TFs play a significant role in oxidative stress, and their applications are expected to make an immense difference in crop improvement. Though, their interactions and functions are still not sufficient to conquer for the effective implementation of manipulating TFs in the genetic engineering of crops. Additionally, complexion in regulating networks between TFs at different levels may affect the use of TFs in genetic engineering as overexpression or downregulation of certain TFs might influence others signaling cascades. Some TFs have functional redundancy, which makes it difficult for mutation; however, a novel technique, CRISPR-Cas9-mediated genome editing, should be used to knock out the redundant TFs. Genomewide analysis has identified TFs linked with stress tolerance, but a deeper understanding of their functional characterization, crosstalk in signaling pathways, and interactions during oxidative stress is required. The extensive knowledge of TF famililes and their targeted downstream genes can

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provide illumination of the regulatory mechanisms involved in plant oxidative stress tolerance, which can help develop strategies for selecting the ideal candidate TF genes for the generation of oxidative stress tolerance.

Acknowledgments P.K.V. thankfully acknowledges University Grants Commission (UGC), India for DSKPDF (No.F.4
2/2006 (BSR)/BL/17
18/0140), and S.V. thankfully acknowledges CSIR, India for research associateship.

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640. Zhang, Q., Luo, F., Zhong, Y., He, J., Li, L., 2019a. Modulation of NAC transcription factor NST1 activity by XYLEM NAC DOMAIN1 regulates secondary cell wall formation in Arabidopsis. Journal of Experimental Botany 71, 1449
1458. Zhang, Y., Bharathi, S.S., Beck, M.E., Goetzman, E.S., 2019b. The fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase can be a source of mitochondrial hydrogen peroxide. Redox Biology 26, 101253. Zhang, Z., Liu, H., Sun, C., Ma, Q., Bu, H., Chong, K., et al., 2018b. A C2H2 zinc-finger protein OsZFP213 interacts with OsMAPK3 to enhance salt tolerance in rice. Journal of Plant Physiology 229, 100
110. Zhao, Q., Fan, Z., Qiu, L., Che, Q., Wang, T., Li, Y., et al., 2020. MdbHLH130, an Apple bHLH transcription factor, confers water stress resistance by regulating stomatal closure and ROS homeostasis in transgenic tobacco. Frontiers in Plant Science 11, 543696. Zhao, Q., Zhong, M., He, L., Wang, B., Liu, Q.-l, Pan, Y.-z, et al., 2018. Overexpression of a chrysanthemum transcription factor gene DgNAC1 improves drought tolerance in chrysanthemum. Plant Cell, Tissue and Organ Culture (PCTOC) 135, 119
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Zhao, R.Z., Jiang, S., Zhang, L., Yu, Z.B., 2019. Mitochondrial electron transport chain, ROS generation and uncoupling. International Journal of Molecular Medicine 44, 3
15. Zheng, J., Su, H., Lin, R., Zhang, H., Xia, K., Jian, S., et al., 2019. Isolation and characterization of an atypical LEA gene (IpLEA) from Ipomoea pes-caprae conferring salt/drought and oxidative stress tolerance. Scientific Reports 9, 1
21. Zhou, Q.-Y., Tian, A.-G., Zou, H.-F., Xie, Z.-M., Lei, G., Huang, J., et al., 2008. Soybean WRKY-type transcription factor genes, GmWRKY13, GmWRKY21, and GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants. Plant Biotechnology Journal 6, 486
503. Zhu, D., Hou, L., Xiao, P., Guo, Y., Deyholos, M.K., Liu, X., 2019. VvWRKY30, a grape WRKY transcription factor, plays a positive regulatory role under salinity stress. Plant Science 280, 132
142. Zhu, M., Meng, X., Cai, J., Li, G., Dong, T., Li, Z., 2018. Basic leucine zipper transcription factor SlbZIP1 mediates salt and drought stress tolerance in tomato. BMC Plant Biology 18, 1
14.

Further reading Taneja, M, et al., 2021. An introduction to the calcium transport elements in plants. Upadhyay S.K. (Ed.). Calcium transport elements in plants. Academic Press, pp. 1
18. https://doi.org/10.1016/B978-0-12821792-4.00019-9.

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CHAPTER

Transcription factors: master regulators of disease resistance in crop plants

20

Ravi Ranjan Saxesena1, , Shreenivas Kumar Singh1 and Praveen Kumar Verma2 1

Plant Immunity Laboratory, National Institute of Plant Genome Research Aruna Asaf Ali Marg, New Delhi, Delhi, India 2Plant Immunity Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, Delhi, India

20.1 Introduction The global population is expected to reach around 10 billion in 2050. According to the United States Department of Agriculture (USDA), with given population growth, food demand will increase by 70%
100% by the year 2050. To meet, this increasing demand, the agricultural production of developing nations needs to be doubled (Springmann et al., 2018). In the recent past, with the help of technological advancement and the incorporation of novel agricultural practices such as new breeding techniques, mechanized agriculture, and the incorporation of multiple cropping systems, crop yield and productivity have significantly increased (Foley et al., 2011). The USDA-driven research program, “Feed the Future Research Strategy” primarily focuses on the incorporation of innovative research and technologies in the field of agriculture to ensure food security. However, the frequent encounter of crops with biotic and abiotic stresses because of the global warming mediated anomaly in the climate has severely affected their growth and is a major constraint on crop yield. The biotic stresses include infestation of crops with an array of pathogenic microbes like bacteria, viruses, fungi, nematodes, insect pests, and weeds. Collectively, they account for about 20%
30% of the annual loss in the field of agriculture (Oerke, 2006; Oerke and Dehne, 2004). In contrast to animals, plants lack adaptive immunity or the capability to memorize past infections. The continuous pressure of evolution and the threat of pathogenic microbes have helped crops to acquire a plethora of measures such as morphological, physiological, and molecular mechanisms to counterattack microbes. The induction pathogenesis-related (PR) genes are one such mechanism that gets modulated (upregulated or downregulated) upon attack and modulate downstream processes to minimize crop loss. Broadly, plant immunity or defense genes are classified into metabolomics, protein kinases, and transcription factors (TFs) (Cheong et al., 2003; Alves et al., 2015). TFs are regulatory proteins that modulate the expression of downstream genes by facilitating or enhancing the binding efficiency of RNA polymerase on promoter sequences. Generally, these TFs have DNA binding domains (DBD) and activation domains (AD) or repressor domains (RD) in 

Equal contribution.

Plant Transcription Factors. DOI: https://doi.org/10.1016/B978-0-323-90613-5.00009-1 © 2023 Elsevier Inc. All rights reserved.

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their sequences (Liu et al., 1999). TFs show sequence specificity and bind to conserved DNA elements (or motif) present in the promoter sequence of the concerned gene Kummerfeld and Teichmann (2006). On the other hand, the AD or RD of TF mediates positive or negative modulation of genes, and hence the TFs are classified as transactivators or transrepressors. In other words, TFs also act as the on/off switch of gene expression, thereby regulating their function. The transcription and translation processes of genes coding for the TF are spatially separated into the nucleus and cytoplasm, respectively. Post translation, the TFs are translocated back into the nucleus through the membrane-bound nucleoporin complex, where they scan for their targets in the genomic DNA; therefore they are also called diffusible regulatory molecules (Peter and Davidson, 2015). In-plant innate immunity TFs play a key role by regulating the expression of genes involved in pathogen-associated molecular patterns (PAMPs) triggered immunity (PTI), effector-triggered immunity (ETI), phytohormone signaling pathways, and metabolic pathways, which lead to the production of several secondary metabolites such as phenolics and phytoalexins that function as toxins for invading pathogens.

20.2 Molecular basis of plant
microbe interaction As we discuss the applications of TFs in plant immunity, it is important to have a basic understanding of the molecular mechanism of plant
microbe interaction. The interaction between the plant and pathogens starts just after the landing of microbial spores or cells on the host surface. The attacking pathogens get access to the host tissues through natural openings like stomata or through mechanical damage like wounds. Some pathogens directly penetrate the hard-structural coverings such as the cuticle layer and cell wall. Different pathogens use different ways to gain access inside host tissues. The plant pathogenic bacteria enter the host via stomata, hydathodes, or wounds and proliferate in the apoplast. On the other hand, nematodes directly insert their stylate, a tube-like feeding structure into the host cell. Plant cells are penetrated by pathogenic and symbiotic fungal and oomycetes via penetration pegs or haustoria (Jones and Dangl, 2006), whereas host plants use passive defense to counter pathogen attacks. The passive defense includes physical barriers such as cell walls, waxes, and thick cuticles, and chemical compounds that are produced to protect against herbivory and other pathogens. Additionally, plants have an active two-tiered immune system that recognizes invading pathogens and mounts a defense response. The first tier involves recognition of PAMPs through cell membrane attached PAMP/pattern recognizing receptors (PRR) and the immunity generated because of this basal defense is called PTI. The generation of reactive oxygen species (ROS) is the hallmark of PTI, which prevents the further spread of pathogenic microbes to nearby healthy tissues. Next, pathogens subvert the host PTI response by secreting a battery of specialized molecules called effectors into host tissues. Secreted effectors sabotage host immunity by inhibiting the function of key molecules involved in several physiological processes (Tsuda and Somssich, 2015). The compromised host immunity because of the secreted effector is called effector-triggered susceptibility (ETS). In response to ETS, plants employ cytoplasmic nucleotidebinding leucine-rich repeats (NB-LRRs) proteins, which are encoded by the R-gene and which directly or indirectly recognize effector molecules leading to activation of the second-tier immune responses, known as ETI. The ETI triggers several stress responses, such as the hypersensitive

20.2 Molecular basis of plant
microbe interaction

421

response (HR). HR is the localized death of host cells at the site of infection to confine and prevent the spread of the pathogen. It is characterized by local and rapid cell death. It also alters the metabolic reactions in nearby cells and triggers systemic acquired resistance (SAR), which is a nonspecific defense response to protect plants against a variety of pathogens (Fritig et al., 1998). Besides ROS and HR, other immune responses triggered in PTI and ETI include mitogen-activated protein kinase (MAPK) cascades, activation of membrane-localized ion channels, increased cytoplasmic calcium ions (Ca21), phytohormone production, and transcriptional reprogramming of defense genes. The mounting of defense response requires tight regulation and coordination between the two interconnected branches of host immunity (i.e., PTI and ETI) (Imran and Yun, 2020). Perception of pathogens through cell membrane-anchored PRR is translated into a cascade of intracellular signals, which includes molecules or ions such as calcium ions (Ca21), nitric oxide (NO), and ROS (Fig. 20.1). Although ROS are known to damage biomolecules like proteins, DNA, and other macromolecules such as lipids, they are also known to function as signaling molecules and regulate various physiological processes and plant responses to pathogens (Pitzschke et al., 2006; Huang et al., 2019; Torres et al., 2006). Moreover, hormones like abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) constitute an integral part of the intracellular signaling cascade by behaving as powerful second messengers. Plant defense genes alone or in groups play pivotal roles in the complex and tight regulation of plant defense genes so that it is sufficient enough to protect plants from pathogens but also does not cost their general growth and

Stimulus

Signal perception

Signal transduction

Response

FIGURE 20.1 Schematic representation of biotic stress signal transduction pathway in plants. Ca21, Calcium ion; CDPKs, calcium-dependent protein kinases; CIPKs, CBL-interacting protein kinases; ER, endoplasmic reticulum; MAPKs, mitogen-activated protein kinases; TFs, transcription factors.

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development (Tsuda and Somssich, 2015). Thus the regulatory nature of the TFs makes them key targets in developing disease-free plants. In this chapter, we will focus on the regulatory role of major plant TF families associated with crop plant immunity.

20.3 The WRKY family of transcription factors and their functional domain WRKY is one of the major groups of known plant TFs found throughout the green lineage, with 109 identified in rice (Oryza sativa) and approximately 74 WRKY reported in Arabidopsis (Phukan et al., 2016), involved in both biotic and abiotic stress responses. They are distinguished by a 60 amino acid (aa) long WRKY DBD with a four-stranded β-sheet and a zinc-finger motif. The N-terminus of the domain is provided with a signature sequence “WRKYGQK” and the C-terminus incorporates a zinc-finger structure (Rushton et al., 2010). The C-terminal zinc-finger structure is characterized by the conserved sequence Cx4
5Cx22
23HxH or Cx7Cx23HxC (Fig. 20.2). Further, based on the number of WRKY domains present in the sequence, the WRKY TFs are divided into

FIGURE 20.2 Schematic representation of various transcription factors. DBD, DNA binding domain; AP2, apetala 2 domain; NAC domain containing 5 subdomains (A
E), DBS, DNA binding site, TRD, transcriptional repressor domain; BD, binding domain; LZ, leucine zipper domain; R2-R3 MYB, TAD, transcriptional activation domain.

20.4 WRKY transcription factors and their role in biotic stress

423

Group I with two WYKY domains and Group II with one WRKY domain. A third group, which is Group III, is classified on the basis of the presence of a zinc-finger structure in the sequence (Rushton et al., 2010). The Group II WRKY TFs are further categorized as IIa, IIb, IIc, IId, and IIe based on variation in the primary aa sequence. According to the solution structure of the WRKY domain, a protruded N-terminal strand from the protein’s surface facilitates TF binding and contact with the major groove of DNA (Yamasaki et al., 2005). However, the binding studies in Arabidopsis, parsley, and sweet potato show that instead of the N-terminal WRKY domain of the TF, the C-terminal WRKY domain of the TF facilitates site-specific DNA binding (Ishiguro and Nakamura, 1994; Eulgem et al., 1999). Several molecular techniques such as electrophoretic mobility gel shift assay (EMSA) and yeast one-hybrid (Y1H) assay suggest that the W-box, TTGACC/T, is the core region present in the promoter sequence of defense-related genes and is required for the cognate binding of almost all known WRKY TFs (Rushton et al., 1996). However, a few WRKY TFs such as OsWRKY13, HvWRKY46, and NtWRKY12 are shown to bind to promoters with non, W-box sequences as well (Cai et al., 2008; Mangelsen et al., 2008; Sun et al., 2003).

20.4 WRKY transcription factors and their role in biotic stress It is evident from several extensive bioinformatic and functional studies that WRKY TFs play a pivotal role in regulating several plant physiological processes such as biotic stresses. Several members of this multigene family TF are actively involved in transcriptional reprogramming of plant defense genes (Table 20.1). The overexpression knockdown study of WRKY genes suggests that these TFs form an integral component of the plant defense system, including PTI, ETI, and SAR (Eulgem and Somssich, 2007). The nuclear-localized rice TF, OsWRKY71, which gets induced upon treatment of rice seedling with signaling molecules such as SA, methyl jasmonate (MeJA), and upon pathogen infection, suggests its function in rice’s response to the bacterial pathogen Xanthomonas oryzae (Liu et al., 2007). The rice OsWRKY71 overexpression line showed enhanced resistance against the pathogen and constitutive expression of a few marker genes of the defense signaling pathway such as OsNPR1 and OsPR1b, which further supports the role of TF OsWRKY71 in biotic stress. Likewise, functional characterization of nuclear-localized transcriptional activator OsWRKY67 showed that rice plants overexpressing the gene exhibited enhanced resistance against Magnaporthe oryzae and X. oryzae but restricted plant growth. Also, the OsWRKY67 RNA interference (RNAi) line was compromised in immunity against these pathogens (Vo et al., 2018). A similar study on OsWRKY67 by Liu et al. showed that Nipponbare, a susceptible rice variety, overexpressing OsWRKY67 showed enhanced resistance to leaf blast, panicle blast, and bacterial blight by inducing the transcription of certain defense-related genes (Liu et al., 2018). The study also found that TF OsWRKY67 induces the expression of PR1a and PR10 by directly binding to the promoter’s W-box sequence. Thus overall findings suggest that the TF is a promising candidate for developing disease resistance in rice plants. The rice cultivar Mudanjiang 8, which lacks the resistance (R) gene and is highly susceptible to the fungal pathogen Magnaporthe grisea and the bacterial pathogen X. oryzae PXO61, showed significantly enhanced resistance at both the seedling and adult stages to the pathogens upon overexpression of WRKY group II TF, OsWRKY13 (Qiu et al., 2007). An allelic variant of OsWRKY45, the OsWRKY45
1, and OsWRKY45
2, which

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Chapter 20 Transcription factors

Table 20.1 List of WRKY transcription factor gene families in biotic stress tolerance in crop plants. Crop

Pathogen

Disease

Gene

References

Rice

Bacterial

Bacterial blight (Xanthomonas oryzae pv. oryzae)

OsWRKY6, OsWRKY13, OsWRKY30, OsWRKY45, OsWRKY51, OsWRKY67, OsWRKY68, OsWRKY71

Fungal

Sheath blight (Rhizoctonia solani) Rice blast (Magnaporthe oryzae/ Pyricularia oryzae) Brown plant hopper (Nilaparvata lugens) Striped stem borer (Chilo suppressalis) Stripe/Yellow rust (Puccinia striiformis f. sp. tritici) Leaf rust (Puccinia triticina) Russian wheat aphid (Diuraphis noxia) Powdery mildew (Blumeria gramini) Bacterial wilt (Ralstonia solanacearum) Bacterial spot (Xanthomonas axonopodis)

OsWRKY4, OsWRKY80

Liu et al. (2007), Vo et al. (2018), Liu et al. (2018), Qiu et al. (2007), Hu et al. (2016), Seon-Hee et al. (2016), Chen et al. (2018), Hwang et al. (2011), Changhyun et al. (2015), Qiu and Yu (2009), Han et al. (2013), Yang et al. (2016) Peng et al. (2016)

OsWRKY7, OsWRKY22, OsWRKY45, OsWRKY58, OsWRKY62, OsWRKY64, OsWRKY76

Abbruscato et al. (2012), Liu et al. (2016), Masaki et al. (2007), Yokotani et al. (2013), Sureshkumar et al. (2019)

OsWRKY45

Jiayi et al. (2016)

OsWRKY53

Hu et al. (2016)

TaWRKY49, TaWRKY62, TaWRKY70

Wang et al. (2017), Junjuan et al. (2017)

TaWRKY1B

Kumar et al. (2014)

TaWRKY53

Eck et al. (2014)

HvWRKY10, HvWRKY19, HvWRKY28

Meng and Wise (2012)

CaWRKY6, CaWRKY22, CaWRKY27

Dang et al. (2014), Hussain et al. (2018), Hanyang et al. (2015) Wang et al. (2013)

Insects

Wheat

Fungal

Insects

Barley

Fungal

Pepper

Bacterial

CaWRKY58

20.4 WRKY transcription factors and their role in biotic stress

425

Table 20.1 List of WRKY transcription factor gene families in biotic stress tolerance in crop plants. Continued Crop

Pathogen

Disease

Gene

References

Fungal

Phytophthora blight (Phytophthora capsici) Bacterial wilt (Ralstonia solanacearum) Soybean cyst nemadtode (Heterodera glycines) Sheath blight (Rhizoctonia solani) Root knot nematode (Meloidogyne javanica)

CaWRKY08
4, CaWRKY01
10

Cheng et al. (2020)

NtWRKY50

Liu et al. (2017)

GmWRKY53, GmWRKY86, GmWRKY136

Yang et al. (2017)

GhWRKY39
1

Shi et al. (2013)

SlWRKY3, SlWRKY45, SlWRKY70

Chinnapandi et al. (2019), Hagop et al. (2012), Bharathiraja et al. (2017)

Tobacco

Bacterial

Soybean

Nematodes

Mexican Cotton

Fungal

Tomato

Nematodes

encode proteins differing in 10 aa, exhibit contradictory functions in rice response against biotic stresses. Rice plants that overexpressed OsWRKY45
1 were susceptible to the bacterial pathogens X. oryzae pv. oryzae and X. oryzae pv. oryzicola, whereas knock-out plants were resistant. In contrast, the overexpression and knock-out rice line for OsWRKY45
2 was resistant and susceptible, respectively (Tao et al., 2009). The study also showed that rice plants with double overexpression of OsWRKY45
1 and OsWRKY45
2 were resistant to the fungal pathogen M. grisea. The increased resistance of rice plants against bacterial pathogens in OsWRKY45
1 in the knock-out line was because of the upregulation in the expression and accumulation of SA and JA, whereas in the case of the OsWRKY45
2 overexpression line, the resistance was only because of the increased expression of JA (Tao et al., 2009). The positive and negative modulation of the rice WRKY gene in response to biotic stress has been deciphered by loss and gain of function analysis. These coordinated modulations have a threshold amplitude and duration of plant response that is sufficient to protect against invading pathogens while not costing the plants fitness. Among the three most devastating pathogens of rice, namely M. oryzae, X. oryzae, and Rhizoctonia solani, only two WRKY genes, OsWRKY30 and OsWRKY4, are categorized for their defense role against the sheath blight causing pathogen R. solani (Peng et al., 2012, 2016; Wang et al., 2015). Nuclear-localized OsWRKY4 is strongly induced in rice plants infected with the necrotrophic fungal pathogen R. solani. The gene was also shown to be induced upon treatment of rice plants with phytohormones JA and ET. Additionally, experiments suggest that OsWRKY4 contains a transactivation domain and RNAi-mediated suppression of rice OsWRKY4 results in compromised immunity against R. solani. Further, the Y1H assay indicated that OsWRKY4 specifically binds to the W-box of pathogenesisrelated genes PR1b and PR5 and elevates their expression (Wang et al., 2015). The OsWRKY30 is

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another rice WRKY gene that has been characterized for its defense role against R. solani. A transient localization study and a transactivation assay in the yeast system showed that OsWRKY30 is a TF with an intrinsic activation domain. A rapid accumulation of the transcript of OsWRKY30 was observed in rice plants treated with phytohormones, SA, and JA. Also, the overexpression of the gene in rice resulted in enhanced resistance against necrotroph R. solani and the blast fungus M. grisea. Furthermore, characterization of the rice overexpression line revealed that increased plant resistance was caused by induced expression of genes LOX, AOS2, PR3, and PR10, as well as increased accumulation of JA (Peng et al., 2012). Another rice WRKY TF, OsWRKY80, has been characterized recently for its defense role against R. solani. Peng et al. have experimentally shown that the rice OsWRKY80 gene is rapidly induced upon infection with the sheath blight fungus R. solani. The green fluorescent protein (GFP)-tagged TF (OsWRKY80-GFP) localizes in the nucleus of onion epidermal cells and it has a transactivation property as revealed by the GAL4 assay in yeast cells. Further, overexpression of the gene in rice significantly increased resistance to R. solani, and the overexpression line also showed induced expression of OsWRKY4. The suppression of OsWRKY80 through RNAi compromised rice resistance against the pathogen. The induced expression of OsWRKY4 was because of the binding of OsWRKY80 on the W-box in the promoter sequence of OsWRKY4 as revealed by Y1H and transient expression assay in tobacco cells (Peng et al., 2016). Overexpression of OsWRKY53 in rice plants enhanced its defense response against the piercing, sucking herbivore Nilaparvata lugens by activating hydrogen peroxide (H2O2) burst and suppressing ET biosynthesis (Hu et al., 2016). The wheat stripe rust disease, caused by Puccinia striiformis f. sp. tritici (Pst), is a devastating disease of wheat (Triticum aestivum). Two WRKY TFs, TaWRKY49 and TaWRKY62, were characterized for their antagonistic role in high-temperature seedling plant (HTSP) resistance to Pst. Gene silencing studies showed that downregulation of TaWRKY49 enhanced the resistance capability of HTSP, whereas silencing of TaWRKY62 compromised resistance to Pst (Wang et al., 2017). Full-length cDNA of TaWRKY1B identified from wheat cultivar HD2329 showed that in response to a virulent race of leaf rust fungus, TaWRKY1B expression was induced 146-fold in resistance plants and 12-fold in susceptible plants compared to mock treatment, indicating a defense role of WRKY TF in wheat plants in response to biotic stress (Kumar et al., 2014). In the case of barley (Hordeum vulgare), R protein encoded by the mildew resistance locus a (Mla) gene confers resistance against the fungal pathogen Blumeria graminis f. sp. hordei (Bgh) by recognizing and interacting with cognate pathogen-specific effector protein. Among the 60 annotated barley WRKY TFs, 26 HvWRKY were analyzed for their putative roles in Mla-mediated resistance to powdery mildew disease. Further, virus-induced gene silencing (VIGS) of HvWRKY10, HvWRKY19, and HvWRKY28 exhibited compromised immunity of the host against Bgh, indicating a possible role of these TFs in ETI. Although there was no direct interaction between these three TFs and Mla, as revealed by yeast two-hybrid (Y2H) analysis (Meng and Wise, 2012). Several characterized TFs from pepper (Capsicum annuum) that play crucial roles in resistance to biotic stress such as bacterial and fungal pathogens are CaWRKY58, CaWRKY27, CaWRKY6, and CaWRKY22. The nuclear-localized CaWRKY58, which can activate the expression of β-glucuronidase (GUS), shows differential regulation upon pathogen treatment. During the early phase of infection of pepper plants with the bacterial pathogen, Ralstonia solanacearum, CaWRKY58 shows downregulation followed by late upregulation. Additionally, VIGS-mediated targeted gene silencing of CaWRKY58 in pepper plants, exhibited enhanced resistance against

20.5 APETELA2/ethylene-responsive factor family of transcription factors

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R. solanacearum strain FJC100301 with enhanced transcript levels of various defense-related pepper genes (Wang et al., 2013). In a similar study by Dang et al. inoculation of pepper cultivar 76a with R. solanacearum showed induced expression of CaWRKY27, whereas its expression was downregulated in the R. solanacearum treated pepper cultivar, Gui-1
3, which is susceptible to bacterial wilt diseases. The VIGS-mediated silencing of CaWRKY27 resulted in susceptible pepper plants, suggesting the involvement of the gene in host resistance to R. solanacearum pathogen (Dang et al., 2014). Pepper CaWRKY22, which is a member of the subgroup IIe of WRKY TF, is induced upon infection of the pepper plant with R. solanacearum as well as exogenous application of SA and MeJA. Further, pepper plants with targeted nonfunctional CaWRKY22 gene expression, which is done by VIGS, result in enhanced susceptibility of peppers to the pathogen. The chromatin immuno-precipitation (ChIP) analysis revealed that CaWRKY22 directly binds to the promoter of defense-related genes like CaPR1 and CaDEF1 and CaWRKY40. Overall, this finding suggests a direct role of CaWRKY22 in the positive regulation of pepper immunity against the bacterial pathogen R. solanacearum (Hussain et al., 2018). WRKY TFs are also extensively characterized for their active participation in the resistance to plant-parasitic nematodes. The root-knot nematode (RKN) of Meloidogyne species is highly devastating. While deciphering the underlying molecular mechanism of pathogenesis, a tight relationship between WRKY TF and RKN pathogenesis in tomatoes was observed. The promoter-GUS reporter assay revealed induced expression of SlWRKY3 and SlWRKY35 within 5 days of tomato infection by the nematode. Histological analysis of the feeding site revealed higher expression of SlWRKY3 in feeding cells and associated vasculature cells. SlWRKY35 was expressed only in mature feeding cells. Further, the overexpression study suggested that the virulence of the nematode Meloidogyne javanica was compromised in the SlWRKY3 overexpression line, whereas downregulation resulted in the full virulence (Chinnapandi et al., 2019).

20.5 APETELA2/ethylene-responsive factor family of transcription factors The AP2/ERF family is one of the largest groups of plant TFs, and are known to regulate several physiological processes in response to biotic and abiotic stresses. Individual members of this group are characterized by the presence of a conserved 60
70 aa long AP2/ERF domain that facilitates the binding of TFs to the DNA sequence. The AP2/ERF family is further characterized into three subfamilies: ETHYLENE RESPONSIVE FACTORS (ERF), APETALA 2 (AP2), and RELATED TO ABSCISIC ACID INSENSITIVE3/VIVIPAROUS1 (RAV) based on number and presence or absence of specific structural domain. The fourth group is DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEINS (DREBs) (Xie et al., 2019; Licausi et al., 2013; Yamada et al., 2020). Members of the AP2 family contain a tandem repetition of two AP2 domains while proteins belonging to the ERF group have a single AP2 domain (Fig. 20.2). Members of the RAV family possess an ERF domain and a B3 DNA-binding domain. Plant growth and development are regulated by AP2/ERF TFs. These TFs are also known to play a crucial role in response to biotic stress and phytohormone signaling and crosstalk. Generally, AP2/ERF function as transactivators or repressors and regulate transcription of specific target genes by binding in a sequence-specific

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manner to the promoter. The DREBs group of AP2/ERF recognizes core DNA sequence A/GCCGAC in the promoter of stress-related genes and modulates their expression. On the other hand, TFs from the ERF group bind to the AGCCGCC core sequences, which is also known as the GCC-box, to modulate the expression of genes involved in biotic stress (Mizoi et al., 2012).

20.6 AP2/ERF family of transcription factors and their role in biotic stress The AP2/ERF family comprises about 163 members in rice and about 62 in wheat that regulate important functions in response to biotic and abiotic stresses (Zhao et al., 2019). One member of the group, OsEREBP1, acts downstream in a signal transduction pathway that gets activated upon the interaction of rice with the bacterial blight pathogen X. oryzae. The OsEREBP1 overexpression line showed induced expression of lipid metabolism-related genes such as lipase and chloroplastic lipoxygenase. Genes related to jasmonate and ABA biosynthetic pathways were also upregulated. Further, GFP-tagged OsEREBP1 localizes to plastid nucleoids (Jisha et al., 2015). The striped stem borer (SSB), Chilo suppressalis, is an herbivore that feeds on rice, and the role of AP2/ERF in herbivore-induced defense response is largely unknown. A report by Lu et al. suggests that the expression of rice OsERF3 is rapidly upregulated in response to SSB feeding and that OsERF3 mediates resistance in response to C. suppressalis (Lu et al., 2011). The AP2 TF and its role in biotic stress are still unknown. TaAP2
15, a wheat AP2 TF, is found in the nucleus of N. benthamiana and wheat. The barley stripe mosaic virus (BSMV)-mediated VIGS of TaAP2
15 resulted in increased susceptibility of wheat to Pst. Expression analysis of pathogenesis-related genes TaPR1 and TaPR2 showed downregulation, while genes involved in the scavenging of ROS such as TaCAT3 and TaFSOD3D were upregulated. Thus the data altogether confirms the involvement of TaAP2
15 in wheat resistance to Pst (Hawku et al., 2021). A class II ERF TF, SlERF3, was identified in tomatoes and characterized for its role in resistance to R. solanacearum. The overexpression of SlERF3 has a drastic impact on the growth of tomato plants. Therefore for functional characterization of the role of SlERF3 in biotic stress, the ERF-associated amphiphilic repression (EAR) domain of the TF was deleted, and the truncated protein SlERF3ΔRD was expressed by overexpression of the corresponding gene in tomato transgenic plants. Transgenic plants showed induced expression of PR1, PR2, and PR5 genes and plants were resistant to R. solanacearum in comparison to control plants (Pan et al., 2010). A new TF, GmERF3, from the AP2/ERF family was isolated from the legume crop soybean. The identified TF has an AP2/ERF domain of 58 aa and two nuclear localization signal domains in the sequence. The GAL-4 based assay in the yeast system showed that the identified GmERF3 TF is transactivator in nature and localizes to the nucleus of onion epidermal cells. Further, ectopic expression of GmERF3 in tobacco plants triggers the expression of PR genes and also improves plant performance in response to R. solanacearum, Alterneria alternate, and tomato mosaic virus (TMV) (Zhang et al., 2009). Likewise, Dong et al. reported the positive role of soybean GmERF5 in response to root and stem rot diseases caused by Phytophthora sojae. Transgenic soybean plants overexpressing GmERF5 showed improved resistance to the pathogen, and the expression of PR10, PR1
1, and PR10
1 was also induced. The GmERF5 is the first known ERF TF from soybean that contains an EAR motif and plays an

20.7 NAC transcription factors and their structural organization

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important role in pathogen infection response (Dong et al., 2015). An ERF gene, GmERF113, which showed induced expression in response to P. sojae infection, was isolated from the resistant soybean cultivar “Suinong 10” and overexpressed in the susceptible cultivar “Dongnong 50.” The overexpression plants exhibited significant improvement in resistance to the pathogen and also the expression of PR1 and PR10
1 genes were upregulated, suggesting the involvement of GmERF113 in biotic stress (Zhao et al., 2017). Another study carried out by Tian et al. showed that potato StERF3, which contains an EAR domain at its C-terminus, negatively modulates the resistance of potato to Phytophthora infestans. A gene localization study showed that StERF3 localizes to the nucleus. Additionally, the bimolecular fluorescence complementation (BiFC) assay showed that the physical interaction of StERF3 with some cytoplasmic proteins alters its localization from the nucleus to the cytoplasm and nucleus. Silencing of StERF3 in potato has shown improved resistance of potato to P. infestans, whereas the overexpression line was significantly compromised in its resistance property against the pathogen. Potato plants silenced for StERF3 also showed elevated expression of PR1, NPR1, and WRKY1 (Tian et al., 2015). A yeast one-hybrid approach was used to isolate NtERF5, an AP2/ERF family of TF from N. tabacum. In the assay, the TF showed weak binding to the GCC-box present in the promoters of many pathogenesisrelated genes. The expression of NtERF5 is induced in response to infection with Pseudomonas syringae and TMV. Surprisingly, the overexpression of NtERF5 has no effect on plant resistance to P. syringae, but it restricts the spread and size of local hypersensitive-response lesions caused by TMV infection. In the overexpression line, only 10%
30% of the viral mRNA accumulation was observed compared to the wild type. Thus data suggest that NtERF5 regulates the expression of genes involved in plant resistance to TMV infection and viral propagation (Fischer and Dro¨geLaser, 2004). Citrus canker is the devastating disease of sweet orange caused by bacterial pathogens Xanthomonas citri sub spp. citri (Xcc) and leads to heavy loss worldwide. The citrus AP2/ERF family of TF CsAP2
09 localizes to the nucleus and the expression of the corresponding gene is induced in wild-type citrus plants infected with Xcc. The CsAP2
09 overexpression line showed a significant reduction in the diseased lesions and disease index, whereas, in the RNAi line, these parameters were significantly upregulated, suggesting the active role of CsAP2
09 in response to Xcc infection (He et al., 2019). The details of the AP2/ERF family involved in crop plant immunity are given in Table 20.2.

20.7 NAC transcription factors and their structural organization The NAC (NO APICAL MERISTEM, ARABIDOPSIS THALIANA TRANSCRIPTION ACTIVATOR FACTOR 1/2 and CUP SHAPED COTYLEDON 2) family of TFs is among the largest group of plant-specific TFs that have been implicated in diverse processes such as development, abiotic stress, and defense. As revealed by various genome-wide studies the number of identified NAC TFs is significantly large in different plant species. The genome of Arabidopsis thaliana encodes about 106 NAC TFs. Similarly, identified NAC in other plant species includes 149 in rice, 96 in cassava (Manihot esculenta), 167 in banana (Musa acuminata), 101 in soybean (Glycine max), and 74 in grape (Vitis vinifera) (Liu et al., 2018). Structure analysis of a typical NAC TFs indicates the presence of 150 aa long N-terminal NAC domain and a diversified

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Table 20.2 List of AP2/ERF transcription factor gene families in biotic stress tolerance in crop plants. Crop

Pathogen

Disease

Gene

References

Rice

Bacterial

OsEREBP1

Jisha et al. (2015)

Wheat

Insect Fungal

OsERF3 TaAP2
15

Lu et al. (2011) Hawku et al. (2021)

TaPIEP1

Na et al. (2010)

SlERF3, SlERF5 SlERF1

Pan et al. (2010), Li et al. (2011) Pan et al. (2013)

SlERF1

Yang et al. (2021)

GmERF3

Zhang et al. (2009)

Fungal

Bacterial blight (Xanthomonas oryzae) Striped stem borer (Chilo suppressalis) Stripe/Yellow rust (Puccinia striiformis f. sp. tritici) Common root rot (Bipolaris sorokiniana) Bacterial wilt (Ralstonia solanacearum) Rhizopus soft rot (Rhizopus nigricans) Gray leaf spot (Stemphylium lycopersici) Bacterial wilt (Ralstonia solanacearum) Root rot (Phytophthora sojae)

Fungal Viral

Late blight (Phytophthora infestans) Tobacco mosaic virus (TMV)

GmERF5, GmERF113 StERF3 NtERF5

Dong et al. (2015), Zhao et al. (2017) Tian et al. (2015) Fischer and Dro¨ge-Laser (2004)

Tomato

Bacterial Fungal

Soybean

Potato Tobacco

Bacterial

C-terminal transcription regulation domain. Further analysis of the NAC domain showed the presence of five subdomains named A to E (Fig. 20.2). These subdomains have diverse functions ranging from DNA binding to protein-protein interaction and oligomerization. Sequences of C and D subdomains are highly conserved with a net positive charge, and they bind to specific elements present in the DNA sequence. The subdomain A is involved in TF dimerization (Ernst et al., 2004), whereas the C-terminal transcription regulation domain can act as an activator or repressor domain, accordingly modulating the expression of the downstream gene(s).

20.8 NAC transcription factors and their role in biotic stress The function of NAC TFs in plant defense in response to biotic stress has been identified and characterized by gene overexpression or silencing analysis and the generation of knock-out lines in several crop plants (Table 20.3). These TFs function as activators or repressors and modulate processes such as hypersensitive response, stomatal opening, or even target pathogen-secreted effector molecules. The ATAF1 of Arabidopsis, which belongs to the NAC family of TF, imparts penetration resistance against Bgh as the ataf1
1 mutant. Arabidopsis plants showed enhanced penetration by the pathogen. Additionally, Jensen et al. also showed induced expression of barley HvNAC6 upon infection with Bgh. HvNAC6 is highly similar to its rice homolog, OsNAC6, and its

20.8 NAC transcription factors and their role in biotic stress

431

Table 20.3 List of NAC transcription factor gene families in biotic stress tolerance in crop plants. Crop

Pathogen

Disease

Gene

References

Rice

Bacterial

Bacterial Blight (Xanthomonas oryzae) Rice blast (Magnaporthe oryzae/Pyricularia oryzae) Rice dwarf virus (RDV) Yellow rust (Puccinia striiformis f. sp. tritici)

OsNAC58, ONAC66

Liu et al. (2018), Park SRHSKSD-JS-CI-PSHST (2017) Nakashima et al. (2007), Yokotani et al. (2014), Lin et al. (2007), Lijun et al. (2013) Motoyasu et al. (2009) Feng et al. (2014), Wang et al. (2018), Fengtao et al. (2015)

Fungal

Wheat

Viral Fungal

Barley

Fungal

Maize

Fungal

Tomato

Bacterial Viral

Potato

Fungal

Powdery mildew (Blumeria graminis f. sp. tritici) Powdery mildew (Blumeria gramini) Anthracnose leaf blight and stalk rot (Colletotrichum graminicola) Bacterial wilt (Ralstonia solanacearum) Tomato yellow leaf curl virus (TYLCV) Late blight (Phytophthora infestans)

OsNAC6, OsNAC19, ONAC66, OsNAC111 ONAC122, ONAC131 RIM1 TaNAC1, TaNAC4, TaNAC8, TaNAC30, TaNAC21/22 TaNAC6

Zhou et al. (2018)

HvNAC6

Yan-Jun et al. (2013)

ZmNAC41, ZmNAC100

Voitsik et al. (2013)

StNACb4, SlNAC35

Yannan et al. (2020), Wang et al. (2016) Ying et al. (2017)

SlNAC20, SlNAC24, SlNAC47, SlNAC61 StNAC4, StNAC5, StNAC18, StNAC48, StNAC81, StNAC43

Yogendra et al. (2017), Anil Kumar et al. (2013), Lemessa Negasa and Zhengbin, (2020)

silencing in barley results in a compromised ability of the host to resist penetration by the pathogen. The result was further validated through a complementation assay where transient overexpression of the gene restored the penetration resistance capability to Bgh (Jensen et al., 2007). The NAC domain-containing TF ATAF2 from A. thaliana is the host target of the TMV virus, which intends to degrade ATAF2 during infection. Detail studies showed that ATAF2 transcript is induced during infection and the overexpression plants showed significantly lower viral titer accompanied by induced expression of PR1, PR2, and PDF1.2 suggesting the role of ATAF2 in plant immunity (Wang et al., 2009). The overexpression of rice NAC TF OsNAC6, which is a transactivator in nature as revealed in the yeast system, exhibited enhanced resistance to rice blast disease pathogen M. grisea (Nakashima et al., 2007). Rice NAC TFs, OsNAC122 and OsNAC131, showed induced expression of these two genes in rice plants inoculated with M. grisea. A superficial spray of phytohormones SA and MeJA on rice plants also induced the expression of these two genes. The nuclear-localized TFs OsNAC122 and OsNAC131 are transactivators in nature. Next, silencing of OsNAC122 and OsNAC131 through a brome mosaic virus (BMV) based gene silencing approach resulted in compromised immunity of rice against M. grisea infection. Also, the expression of some

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defense-related genes, OsLOX, OsPR1a, and OsWRKY45, was downregulated suggesting the role of OsNAC122 and OsNAC131 TFs in rice plant immunity (Sun et al., 2013). Similarly, the TERN subgroup of NAC TF OsNAC111 localizes to rice cell nuclei and it has a transactivator property as revealed in yeast and rice cells. Further, the transcript of OsNAC111 gets induced upon infection of rice plants with M. oryzae. Also, rice plant overexpressing OsNAC111 showed enhanced resistance in response to the pathogen and the expression of many pathogenesis-related (PR) genes were high compared to control plants (Yokotani et al., 2014). Another NAC TF of rice, ONAC066, is localized to the nucleus and plays an important role in plant immunity. When compared to a control plant, the rice plant infected with blast pathogens showed an induced accumulation of the ONAC066 transcript. And as expected, overexpression of the gene enhanced the resistance of rice against blast and bacterial blight diseases. Also, the metabolomic study in overexpression plants showed a higher accumulation of sugar and amino acids compared to wild-type plants. Data altogether suggests the involvement of ONAC066 in rice immunity against blast and bacterial blight diseases (Liu et al., 2018). NAC TFs that regulate host defense in response to biotic stress are in turn regulated by microRNA (miRNA). These small noncoding miRNAs modulate the expression of target genes by silencing. Through degradome analysis, wheat NAC TF, TaNAC21/22, was identified as the target gene of tae-miR164. TaNAC21/22 is a transactivator and it localizes to the cell nucleus. The gene silencing approach showed that TaNAC21/22 is a negative modulator of wheat immunity to the stripe rust pathogen Pst (Feng et al., 2014). Nuclear-localized TaNAC30 shows transactivation property with the help of its C-terminal domain, and the expression of TaNAC30 increased with the infection of wheat plants with virulent race CYR31 of Pst. Additionally, virus-induced silencing of TaNAC30 enhanced the resistance of wheat against the pathogen. Histological analysis also revealed that silenced wheat plants showed an induced accumulation of H2O2, suggesting that TaNAC30 negatively regulates wheat immunity against Pst (Wang et al., 2018). The NAC TF, TaNAC6, which was identified as a differentially regulated gene in transcriptome analysis in a broad-spectrum wheat resistant line against powdery mildew, localizes to the nucleus and has transactivation property. The stable overexpression line of TaNAC6 showed improved basal resistance against Blumeria graminis f. sp. tritici (Bgt). The downregulation of the gene resulted in compromised immunity of wheat resistant lines, NAU9918 and OEStpk-V, implying a positive role played by TaNAC6 against Bgt (Zhou et al., 2018). Cotton NAC TF GhATAF1 was upregulated upon treatment of the plant with Verticillium dahlia, implying the role of TF in biotic stress. Further molecular characterization studies revealed that GhATAF1 localizes to the nucleus and is a transactivator in nature. In addition, cotton plants overexpressing GhATAF1 were highly susceptible to V. dahlia and B. cinerea with the inhibition of the JA signaling cascade and activation of the SA signaling cascade (He et al., 2016). SlSRN1, a membrane-localized NAC TF from tomato plants, was found to be induced six- to eightfold more in B. cinereal or Pseudomonas syringae pv. tomato (Pst) DC3000 infected plants than in mocktreated plants. The silencing of SlSRN1 resulted in enhanced susceptibility of tomato plants against the two pathogens, suggesting SlSRN1 as a positive regulator of immunity (Liu et al., 2014). There are a few studies that suggest the plant NAC TFs are direct targets of pathogen-secreted effector molecules. A large number of findings indicate that NAC TFs regulate plant immunity by activating the expression of several PR genes. And hence, pathogen-secreted effectors target NAC TF and intervene with their function, thereby rendering plants susceptible. One example is the

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potato NAC TFs, StNTP1 and StNTP2. These two endoplasmic membrane-localized TFs showed induced expression upon treatment with the potato late blight pathogen P. infestans. Silencing of counterparts NTP1 and NTP2 in N. benthamiana severely compromised immunity to P. infestans. Additional molecular characterization showed that treatment of plant with culture filtrate of the pathogen resulted in translocation of StNTP1 and StNTP2 from ER to the nucleus where these proteins were targeted to 26 S proteasome for degradation, thus making the host susceptible (McLellan et al., 2013).

20.9 bZIP transcription factors The basic leucin zipper (bZIP) is among the largest family of plant TFs that control biochemical and physiological processes and play an important role in plant development and its response to abiotic and biotic stresses (Sornaraj et al., 2016). The bZIP domain of the TF, which is 60
80 aa residue long, is composed of two structural features, a highly conserved basic region and a more diversified leucin zipper region (Fig. 20.2). The basic region of the bZIP domain is about 18 aa acid and characterized by an invariant motif of N-x7-R/K-x9, which functions as a nuclear localization signal (NLS) and the region mediates sequence-specific binding of the TF on DNA. The leucin zipper domain, which is less conserved, is composed of heptads of leucin or bulky hydrophobic aa repeated several times and is responsible for bZIP protein homo or heterodimerization (Nijhawan et al., 2007). Each monomer of bZIP TF, which forms α-helices, dimerizes during DNA binding. The N-terminal half of the TF binds to the major groove of the DNA sequence while the Cterminal half interacts with another bZIP monomer to form a classical superimposed coiled-coil structure that appears like a zipper (Nijhawan et al., 2007). In plants, the bZIP TFs are expressed constitutively or in tissue-specific manners to regulate the expression of target genes by functioning as activators or repressors. A significantly large number of bZIP TFs have been identified in different plant species, namely Arabidopsis (Li et al., 2021), potato (Liu et al., 2018), cucumber (Yokotani et al., 2014), tomato (He et al., 2016), and tabacum (Fengtao et al., 2015; Dro¨ge-Laser et al., 2018; Zhao et al., 2020; Baloglu et al., 2014; Li et al., 2015, 2021). To modulate target gene expression, these TFs bind to ACGT core DNA sequences such as TACGTA (A-box), GACGTC (C-box), CACGTG (G-box), TGAAAA (PB-like), and GTGAGTCAT (GLM), which are present in the promoter region (Li et al., 2021; Jakoby et al., 2002).

20.10 bZIP transcription factors and their role in biotic stress Most of the studies on bZIP TFs are largely related to their function in development, seed development, and abiotic stresses in plants. However, their role in plant defense in response to invading phytopathogenic microbes is underexplored. The biotrophic fungus, Phakopsora pachyrhizi, which is the causal agent of Asian Soybean Rust (ARS) is a serious threat to soybean crops. The treatment of soybean plant to phytohormone SA and MeJA and infection to P. pachyrhizi resulted in differential expression of four genes GmbZIPE1, GmbZIPE2, GmbZIP105, and GmbZIP62, which encode the bZIP family of TFs. Further molecular characterization of these TFs showed that GmbZIP62

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TF has strong transactivation activity in the yeast system. Thus detailed molecular work suggests that these bZIP TFs play important roles in plant defense response to P. pachyrhizi (Alves et al., 2015). Pepper Mild Mottle Virus (PMMV) is infectious to capsicum. Differential accumulation of a bZIP TF PPI1 from capsicum was observed in response to infection with PMMV, P. syringae, and X. compestris suggesting a specific role of PPI1 bZIP TF against biotic stress (Lee et al., 2002). Rice plants treated with elicitor chitin showed induced expression of OsTGAP1, which encodes a bZIP TF. Induced expression in response to elicitors suggests that OsTGAP1 is involved in the biosynthesis of diterpenoids as part of plant defense response (Okada et al., 2009). Likewise, tomato bZIP TFs, SlAREB1, overexpression plants showed induced expression of PR genes, protease inhibitors, and catabolic enzymes suggesting the possible role of the TF in tomato response to pathogen invasion (Orellana et al., 2010). The treatment of nonhost resistance pepper plant with soybean pustule pathogen Xanthomonas axonopodis pv. glycines (Xag) followed by cDNA microarray analysis revealed downregulation of two bZIP TFs, suggesting their negative role in plant defense to Xag (Lee et al., 2004). Pepper bZIP TFs, CabZIP1 and CabZIP2, have been shown to play a role in plant response to bacterial pathogens. The overexpression of CabZIP1 in Arabidopsis enhanced its resistance against P. syringae pv. tomato DC3000, whereas, the silencing of CabZIP2 resulted in the compromised immunity of pepper against X. campestris pv. vesicatoria (Lee et al., 2006; Chae Woo et al., 2015). Cassava bacterial blight, which causes leaf wilting, shoot dieback, and necrosis of the stem vascular bundle in tropical crop cassava (Manihot esculenta), is a serious disease caused by Xanthomonas axonopodis pv. manihotis (Xam). The functional role of about 77 genome-wide identified cassava bZIP TF in the host response to biotic stress remains largely unknown. However, a recent gene expression study showed that two cassava bZIP TFs, namely MebZIP3 and MebZIP5, were induced upon treatment of the plant with flg22, Xam, SA, and H2O2. Furthermore, when these nuclear-localized TFs were overexpressed in tobacco, they conferred improved resistance with increased callose deposition. Also, VIGS-mediated silencing of MebZIP3 and MebZIP5 in cassava resulted in reduced resistance of the plant to Xam, the low expression level of PR genes, reduced H2O2 accumulation, and less callose deposition compared to control plants, suggesting a positive effect of MebZIP3 and MebZIP5 in plant defense response (Li et al., 2017).

20.11 MYB transcription factor family The MYB TF family is found in all eukaryotes and is known to regulate a variety of plant processes including biotic and abiotic stress, phytohormone biosynthesis and regulation, development and differentiation, secondary cell wall synthesis, and meristem formation. The first MYB gene to be identified was v-myb from the avian myeloblastosis virus (AMV), which is known for regulating cell apoptosis and differentiation (Klempnauer et al., 1982). The first identified MYB gene from the plant side was C1 (COLORED 1) from maize (Zea mays), which was functionally characterized to be involved in the regulation of anthocyanin biogenesis (Paz-Ares et al., 1987). Since then, a large number of MYB TFs have been identified in different plant species. For example, the A. thaliana genome encodes 198 MYB genes, and the Brassica rapa, Solanum lycopersicum, Brachypodium distachyon, and Oryza sativa genomes contain 256, 127, 122, and 155 MYB genes, respectively (Yanhui et al., 2006; Wang et al., 2015; Li et al., 2016; Chen et al., 2017; Katiyar et al., 2012). These TFs are

20.13 Conclusion and future perspectives

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known to contain about 50
53 aa residues of MYBDBD in their sequence, which forms a helix-turnhelix (HTH). The MYB DBD bind to the major DNA groove in the promoter sequence of the specific gene during the process of transcription (Ogata et al., 1994). Based on MYB repeats, the MYB TFs are classified as 1R-MYB (with one MYB repeat), 2R-MYB (with two MYB repeats), 3R-MYB (with three MYB repeats), and 4R-MYB (with four MYB repeats) (Baldoni et al., 2015). The R2R3 type of MYB domain containing TFs is predominantly found in plants (Fig. 20.2).

20.12 MYB transcription factors and their role in biotic stress In addition to their roles in plant processes such as development, cell wall formation, and meristem formation, several MYB TFs have been implicated for their role in plant response to pathogen attacks. For example, wheat R2R3 MYB TF, TaRIM1, have been functionally characterized for their defense response. The gene silencing and overexpression analysis revealed that TaRIM1 positively regulates wheat defense in response to the necrotrophic fungal pathogen Rhizoctonia cerealis. Additionally, overexpression plants also showed induced accumulation of defense genes such as Defensin, PR10, PR17c, and Chitinase 1 (Shan et al., 2016). An initial ectopic expression of the wheat MYB TF gene TaPIMP1 exhibited enhanced resistance of wheat to Ralstonia solanacearum. Further detailed molecular characterization of the TF through EMSA and Y1H assays showed that TaPIMP1 is a transactivator and it binds to the MYB domain in promoter sequence. The gain and loss of function analysis of TaPIMP1 exhibited that the TF is a positive regulator of wheat resistance to the fungal pathogen B. sorokiniana by modulating the expression of defense-related genes (Zhang et al., 2012). Experimental evidence suggested that the R2R3 MYB subfamily of TF CmMYB19 was isolated from chrysanthemum and experimental evidence suggested its positive role in response to aphid (Macrosiphoniella sanborni) infestation. However, the nuclear-localized CmMYB19 lacks transactivation property in the yeast system and the gene overexpression analysis showed that the aphid’s multiplication was restricted through increased lignin deposition (Wang et al., 2017). Similarly, SlMYB28, a 200 aa long R2R3 MYB identified from tomato, is a negative regulator of tomato resistance to Memisia tabaci, the causal agent of tomato yellow leaf curl virus (TYLCV). The gene silencing study of SlMYB28 showed that the DNA content of the virus was significantly reduced in infected tomato plants (Li et al., 2018). Overexpression analysis revealed that OsMYB21, a candidate gene of QTL qBBR11
4 on chromosome 11, negatively regulates rice resistance to the bacterial blight pathogen Xoo (Chen et al., 2018). The OsMYB21 which is a candidate gene of QTL, qBBR11
4 present on chromosome 11 negatively regulates rice resistance to bacterial blight pathogen Xoo as revealed by overexpression analysis (Yang et al., 2021). Wheat infestation with English grain aphid resulted in induced expression of MYB genes, TaMYB19, TaMYB29, and TaMYB44. VIGS-mediated silencing of these genes resulted in increased aphid infestation and their feeding on phloem. The silencing also resulted in repressed plant phloem-based defense against aphids (Zhai et al., 2017).

20.13 Conclusion and future perspectives The continuous threat of pathogenic microbes to crop plants results in a significant yield loss that needs to be addressed. The cultivars with host resistance are employed, to minimize crop yield

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loss, which is the most effective and economical. However, the conventional breeding approaches to developing cultivars with host resistance have certain limitations due to the genetic complexity of crop plants. Therefore the transgenic approaches through genetic engineering are now gaining more importance to develop stress-tolerant crops. To develop biotic stress-resilient crop plants, the identification of candidate genes and detailed knowledge of the molecular mechanisms of their function and regulation is a crucial step. Since TF is the master regulator of several defense-related genes, they are considered suitable candidates for crop improvement. Several TF families have been identified and characterized for their roles in plants defense. The plant TF families extensively characterized in response to biotic stress are WRKY, AP2/ERF, NAC, bZIP, and MYB. These characterized TFs are incorporated into a wide range of plant species and economically important crops through genetic engineering to cope with several biotic stresses. This chapter summarized the major families of plant TFs and their responses to biotic stresses. This can be accomplished by changing the TF binding site in the promoter sequence through the genome-editing technique CRISPR/Cas9. However, while significant information has been incorporated about the role of TFs in response to biotic stresses, there are still some issues that need to be resolved.

Acknowledgments RRS thankfully acknowledges the SERB-DST, New Delhi, India for providing financial support in the form of an NPDF fellowship.

Declaration of competing interest The authors declare that there is no conflict of interest.

Contribution All authors contributed to the writing of the manuscript, read and approved the final version of it.

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228.

Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A ABA-responsive genes (ABRE), 272
274 ABA signaling pathway, 272
274, 303
304 Abiotic stresses, 22
23, 313, 337
338 Abiotic stress tolerance AP2/ERF TFs, 276
277 MYB TFs, 275 NAC TFs, 275
276 WRKY TFs, 277
279 Abscisic acid stress ripening (ASR) proteins, 42 Active phytochrome A binding (APA) domain, 147 Alkaloid biosynthesis pathways CRISPR/Cas-mediated genome editing, 211
212 overexpression, 209
210 RNA-interference, 210 virus-induced gene silencing, 210
211 Alkaloid biosynthesis regulation, 201
209 APETALA2/ethylene response factor, 201
205 basic helix-loop-helix, 205
206 basic leucine zipper, 206 Cys2/His2-type zinc-finger protein family/Zinc-finger Catharanthus protein (ZCT) family, 207 myeloblastosis, 207
208 WRKY family of, 208
209 Antioxidant enzymes, 303 AP2-ERF transcription factor, 179
180, 291
292, 398
399 APETALA2/ethylene response factor (AP2/ERF), 201
205, 242
243 Apetela 2 (AP2), 276
277 AP2 type TF aintegumenta (ANT), 107 Arabidopsis, 78 A. thaliana, 63, 271 Arid land, 23 abiotic stresses, 22
23 distribution of, 23 millets abiotic stresses, 28
29 climate-smart nutri-cereals, 24
27 stress, limiting factors, 27
28 transcription factors, 29
43 oxidative stress, 22
23 AS1-AS2 inhibitor complex, 78
79 ATAF1/2, 98
104 AtWRKY22 transcription, 106 AtWRKY45, 105 AtWRKY70, 105
106 Autoregulation of nodulation (AON), 183
184 Auxin, 345
346

Auxin response factor family, 258 Axillary Meristem (AM), 79
82, 85f

B Basic-helix-loop-helix (bHLH), 41
42, 108
109, 205
206, 222
224 Basic leucine zipper (bZIP), 42, 113
114, 206, 226
227, 353 BHLH transcription factors, 255
257, 372
373, 399 Biotic stress bZIP transcription factors, 433
434 MYB transcription factor family, 435 NAC transcription factors, 430
433 WRKY transcription factors, 423
427 Biotic stresses, 419 bZIP transcription factors, 243, 373, 401, 433
434

C Calmodulin 7 (CAM7), 144 Catharanthus roseus 1 (CR1) TF, 201
205 CCaMK/CYCLOPS complex, 177
178 ChIP-exo, 166 ChIP-on-chip, 166 Chromatin immunoprecipitation (ChIP) experiment, 205
206 Chromatin Immunoprecipitation (ChIP)-qPCR analyses, 102
103 Chromium (Cr), 341 Chrysanthemum morifolium, 5 Circadian clock-associated 1 (CCA1), 110 CLV3 polypeptide, 78 Common symbiosis signaling pathway (CSSP), 176 Compact root architecture 2 (CRA2), 184 C-repeat binding factor1, 40 CRISPR/Cas-mediated genome editing, 211
212 CsTLP8 protein, 303
304 CUP-shaped cotyledon 2 (CUC2), 98
104, 275
276 Cytokinin response factors (CRFs), 108

D Diterpenes, 238 DNA binding domain (DBD), 29
30, 254, 319, 339
340 DNA binding with one finger (DOF), 31
40, 316
318 DNA synthesis, 337
338 Drought stress, 305 DNA binding with one finger (DOF), 316
318 heat shock factor, 319 MYB/MYC transcription factors, 325
327 NAC transcription factors, 324
325 nuclear factor (NF-Ys), 320
321

445

446

Index

Drought stress (Continued) P2/ERBP, 322
323 P2/ERBPAREB/ABF family, 323
324 physiological changes, 313
314 phytohormone abscisic acid, 314 singular/cumulative environmental effects, 313
314 water use efficiency (WUE), 313
314 WRKY transcription factor, 318
319 Drought-tolerant sorghum genotype, 28
29

E Early Responsive to Dehydration Stress 1 (ERD1), 276 Electron transport chains (ETCs), 385
395 Elongated hypocotyl 5 (SlHY5), 165 Enhanced disease susceptibility 1 (EDS1), 148
149 Epigenetic regulation, 259 ERF/DREB, 40 Ethylene Response Factor Required for Nodulation 1 (ERN1), 176 Ethylene-responsive element-binding protein (EREBP), 40 Exogenous JA, 106 EXPANSIN (EXPA) gene promoter, 4
5

F Flavonoids basic-helix-loop-helix, role of, 222
224 basic leucine-zipper transcription factors, 226
227 biological functions, 219 commercial purposes, 219 MYB transcription factor family, 224
225 pharmacological studies, 219 properties of, 219 subclasses and biosynthesis, 220
221 WD40 transcription factors, 225
226 WRKY transcription factors, 227 Fleshy fruit tomato (Solanum lycopersicum), 159
160 Floral meristems (FM), 166 Flower meristem, 82
83 FON2-like CLE protein1 (FCP1), 78 Foxtail millet, 27 Fruit development, 5

G G-box binding factor 1 (GBF1), 144 Genetic analysis, 81 Genetic and biochemical data, 82 Genomic pull-down assays, 104
105 Gibberellic acid (GA)-mediated seed germination, 4
5 Gramineae, 24
27 GRAS transcription factor, nodulation signaling pathway 1/2, 180 Growth-regulating factors, 6

H Haber-Bosch process, 175 Heat shock factor, 319 Heavy metal stress, 337
338 AP2/ERF/DREB-family transcription factors, 354 bZIP-family transcription factors, 353 calcium
calmodulin signaling pathway, 344
345 hormone signaling, 345
346 MAPK signaling, 343
344 MYB-family transcription factors, 348
352 in plants, 340
341 plant signaling in, 341
342 reactive oxygen species production, 346
347 transcription factors in, 347
348 WRKY-family transcription factors, 352
353 Hemiterpenes, 238 High-temperature seedling plant (HTSP) resistance, 426 High-temperature stress, 287
288 Homeodomain leucine zipper (HD-Zip) TFs, 43, 114
115 HSF transcription factor, 289
290, 401
402 Hydrogen peroxide, 395
396 Hydroxyl radical, 396

I Inflorescence meristem, 77 Intercalary meristem, 84 Ion homeostasis mechanism, 371 Ion stress, 369
370 Iron (Fe) Fe deficiency auxin response factor family, 258 bHLHs, 255
257 NAC transcription factor family, 257
258 WRKY, 257 iron uptake and transport, 253
254 TFs involvement, 255
258 toxicity, regulation of epigenetic regulation, 259 plant hormones, 260 posttranscriptional level, 259 posttranslational level, 259
260 transcriptional level, 259 WRKY family, 258 Isoprenoids, 235
237

J JA signaling pathway, 274
275

K Knotted1-like homeobox (KNOX) family proteins, 78
79

L Lax panicle (LAX), 81

Index

Lead (Pb) contamination, 340
341 Leaf senescence, 93
94, 95t Light-responsive elements (LRE), 146 Lipo-Chito-oligosaccharides (LCO), 175 Lob-domain protein16, 185 Low-temperature stress, 288
289 Lysine motif (LysM)-type receptors, 176

M MADS domain gene, 5 Medplants Consortium, 244 Mercury, 341 Mitogen-activated protein kinase (MAPK), 338
339, 372
373 Mitogen-activated protein kinase kinase kinase 1 (MEKK1), 105 Monoculm 1 (MOC1), 80 Monoterpenes, 238 MYb (v-myb avian myeloblastosis viral oncogene homolog), 42
43, 110, 224
225, 290
291, 325
327, 374
375, 399
400, 434
435 Myeloblastosis, 207
208

N NAC transcription factors, 41, 81, 257
258, 324
325, 373
374, 400 N-fertilizer, 175 NIN and NIN-like proteins A. thaliana and rice, 181 master regulator of nodulation, 183 negative regulator, 183
184 positive regulator, 184
186 regulation by cytokinin-response elements, 182
183 for rhizobial infection, 182 structure of, 181
182 No apical meristem (NAM), 98
104 Nodulation pectate lyase 1, 185
186 Nuclear factor (NF-Ys), 186
188, 320
321 Nutrient use efficiency (NUE), 28
29 Nutritional stress, 370

O Oligomerization domain (OD), 319 Opaque2 (O2), 24
27 ORE1, 99 OsbHLH059, 255
257 OsHKT pathways, 4
5 Osmotic regulation mechanism, 370 Osmotic stress, 369
370 ABA signaling pathway, 303
304 by drought stress, 305 by low temperature, 305
306 osmotic regulation mechanism, 302

447

ROS generation and scavenging, 302
303 by salt stress, 304
305 stomatal closure, 301
302 Overexpression, 209
210 Oxidative stress, 22
23, 338
339, 383
384 ROS generation, 385f ROS scavenging, 396
397 and sources, 384
396 Ozone depletion, 145

P Panicoideae, 24
27 Pathogenesis-related (PR) genes, 108, 419 Pearl millet, 29 P2/ERBP, 322
323 P2/ERBPAREB/ABF family, 323
324 Phenyl-propanoid pathway, 9
10 Phospholipase C (PLC), 323 Photomorphogenesis, 145
146 Photoreceptors, 139 Photosystem-I (PSI), 384
385 Photosystem-II (PSII), 384
385 Phytohormone abscisic acid, 314 PhytoMetaSyn Medicinal Plant Genome Resource (MPGR), 244 Pistils, 28 Plant alkaloids, 199
200 Plant hormones, 260 Plant
microbe interaction, 420
422 Plant resistance, 339
340 Plant root development, 64 lateral roots KNOX gene family, 69 MADS Box TFs and, 69 NAC TFs and, 68 negative regulator of, 69 root apical meristem AP2/ERF TFs and RAM development, 66
67 GRAS TFs and RAM development, 67 negative regulator of, 67 WOX TFs and RAM development, 65
66 root hair bHLH TFs and, 70 MYB TFs, 70 negative regulators of, 71 Posttranscriptional level, 259 Posttranscriptional modifications, 139
140 Posttranslational level, 259
260 Posttranslational modifications, 139
140, 314
316 Programmed cell death (PCD), 101
102 Proteasomal degradation, 139
140 Protein Phosphatase Type 2 (PP2Cs), 272
274 Protoderm, 64 PSEUDO-response regulators TF, 113

448

Index

R

T

Raffinose family oligosaccharide (RFO) pathway, 316
318 Reactive oxygen species (ROS), 22
23, 254, 371
372 Regulatory network, 166 Revoluta (REV), 80
81 Rhizobium-directed polar growth, 186 RIP-Chip, 166 RNA-interference, 210 RNA sequencing data, 41 ROS scavenging, 396
397

Temperature stress, on plants AP2/ERF transcription factors, 291
292 high-temperature stress, 287
288 HSF transcription factor, 289
290 low-temperature stress, 288
289 MYB transcription factor, 290
291 WRKY transcription factors, 293
294 Terpene synthases diterpenes, 238 hemiterpenes, 238 monoterpenes, 238 sesquiterpenes, 238 triterpenes, 238
239 Terpenoids biosynthesis of isoprenoid intermediates, 235
237 MVA and MEP pathway, 235 terpene synthases, 237
239 in human and plants welfare, 234f spatio-temporal and inducible biosynthesis, 233
234 WRKY, 241 TFL1, 83 Tomato culinary vegetable, 159
160 MADS transcription factor, 163
165 MYB transcription factors, 161
163 regulatory mechanisms of, 159
160 ripening process, 159
160 transcriptional and posttranscriptional machinery, 159
160 transcription factors in, 160 Transcriptional level, 259 Transcriptional network, in root epidermal cells, 176f Transcription factor-binding sites (TFBS), 397
398 Transcription factors (TF) ABA signaling pathway, 272
274 abiotic stress, 271
272 in abiotic stress tolerance AP2/ERF TFs, 276
277 MYB TFs, 275 NAC TFs, 275
276 WRKY TFs, 277
279 of alkaloid biosynthesis pathways CRISPR/Cas-mediated genome editing, 211
212 overexpression, 209
210 RNA-interference, 210 virus-induced gene silencing, 210
211 in alkaloid biosynthesis regulation, 201
209 APETALA2/ethylene response factor, 201
205 basic helix-loop-helix, 205
206 basic leucine zipper, 206 Cys2/His2-type zinc-finger protein family/Zinc-finger Catharanthus protein (ZCT) family, 207

S Salinity, 383
384 Salt overly sensitive (SOS) signaling pathway, 371 Salt stress, 304
305 on plants ion stress, 369
370 nutritional stress, 370 osmotic stress, 369
370 oxidative stress, 370 transcription factors, 375
376 Salt tolerance mechanisms bHLH transcription factors, 372
373 bZIP transcription factors, 373 ion homeostasis mechanism, 371 MYB transcription factors, 374
375 NAC transcription factors, 373
374 osmotic regulation mechanism, 370 reactive oxygen species, 371
372 WRKY transcription factors, 374 Senescence-associated gene 113 (SAG113), 99
100 Sesquiterpenes, 238 Setaria italica, 31
40 Shoot apical meristem, 77
79 Shoot apical meristem (SAM), 77, 85f Short internodes/stylish, 187
188 Signaling molecules, 166 SNF1-Related Protein Kinases Type 2 s (SnRK2s), 272
274 Solanum lycopersicum JRE4, 201
205 Sorbitol, 302 Sorghum, 22
23, 28
29 Squalene synthase (SQS), 238
239 SQUAMOSA promoter binding proteins, 43, 244 Steroidal glycoalkaloid (SGA) biosynthesis, 201
205 Stomatal closure, 301
302 Streptococcus pyogenes-derived type II CRISPR system, 211
212 Stress, 22
23 Stress signal transduction pathways, 383
384 Suppressor of gamma response 1 (SOG1) TF, 146 Symbiosis receptor kinase (SYMRK), 176 SymSCL1, 181 Systemic acquired resistance (SAR), 420
422

Index

myeloblastosis, 207
208 WRKY family of, 208
209 AP2/EREBP TFs, 107
108 AP2/ERF family of, 428
429 APETELA2/ethylene-responsive factor family, 427
428 arabidopsis response regulator TFs, 121
122 auxin/indole-3-acetic acid (Aux/IAA) TFs, 110
111 basic helix-loop-helix (bHLH) TFs, 108
109 basic leucine zipper (bZIP) TFs, 113
114 B-box zinc finger TFs, 124
125 brinsensitive 1 (BRI1)-EMS-Suppressor1 (BES1) TF, 123 bZIP transcription factors, 433
434 calmodulin-binding transcription activator, 123
124 CCAAT box-binding TFs, 119 cereal crops, 22
23 chromatin-modifying/remodeling proteins or coactivator/ corepressor, 271 C3H (Zn) TFs, 117
118 DNA-binding proteins, 77 DNA binding sequences of, 316t DNA binding-with-one-finger (DOF) proteins, 112 in dry spell tolerance, 314
316 early flowering 3 (ELF3) TF, 122 ethylene insensitive 3 (EIN3)-like (EIL) TFS, 122
123 genetic screening and homology-based detection, 139 golden 2-likE (GLK) TFs, 120 GRAS TFs, 118
119 growth-regulating factors (GRFS) and GRF-interacting factors (GIFS), 115 heat shock factor TFs, 119 homeobox (HB) TFs, 116
117 homodomain-leucine zipper (HD-ZIP) TFs, 114
115 JA signaling pathway, 274
275 lateral organ boundaries/asymmetric leaves 2, 122 in light-regulated processes basic helix-loop-helix family, 140
141 basic leucine zipper family, 141 MYB family, 141
142 TCP, 142
143 WRKY domain-containing TFs, 142 zinc finger proteins, 142 light-regulated transcription factors, 148
149 MADS TFs, 119
120 MAPK cascade system, 3 MYB TFs, 110 MYB transcription factor family, 434
435 NAC transcription factors, 429
433 nodule organogenesis, 178t in oxidative stress resistance, 386t plant A/T-rich sequence and zinc-binding protein (PLATZ) TF, 115 and plant life, 4
6

449

in plant root development lateral roots, 67
69 root apical meristem, 64
67 root hair, 69
71 PSEUDO-response regulators TF, 113 and secondary metabolism, 9
10 senescing leaf transcriptome characterization of, 98
125 identification of, 94
98 sequence-specific DNA binding proteins, 93 and stress responses, 6
9 stress-responsive genes AP2/ERF family, 398
399 bHLH family, 399 bZIP family, 401 HSF family, 401
402 MYB family, 399
400 NAC family, 400 WRKY family, 400
401 structural and functional evolution of, 146
148 TCP TFs, 116 temperature stress, on plants AP2/ERF transcription factors, 291
292 high-temperature stress, 287
288 HSF transcription factor, 289
290 low-temperature stress, 288
289 MYB transcription factor, 290
291 WRKY transcription factors, 293
294 TIFY TFs, 124 trihelix TFs, 120
121 of UV-B signaling and photomorphogenesis, 145
146 UV stress response in plants, 146 with visible light-mediated development in plants, 143
144 VQ TF family, 113 WRKY family of biotic stress, role in, 423
427 electrophoretic mobility gel shift assay (EMSA), 422
423 yeast one-hybrid (Y1H) assay, 422
423 Zea mays (maize), 93 Trihelix transcription factors (TTF), 43 Triterpenes, 238
239

U United States Department of Agriculture (USDA), 419 UV-B signaling, 145
146

V Vascular-related NAC-domain (VND)-interacting2 (VNI2), 102 Virus-induced gene silencing (VIGS), 201
205, 210
211

450

Index

W Water use efficiency (WUE), 313
314 WD40 transcription factors, 225
226 WRKYGQK amino acid sequence, 104 WRKY proteins, 30
31 WRKY transcription factor, 30
31, 227, 257
258, 293
294, 318
319, 374, 400
401

Wuschel-related homeobox (WOX), 6 WUS/CLV3 feedback loop, 84

Y YABBY TFs, 244 Yellow foxtail, 27