Plant Photomorphogenesis: Methods and Protocols (Methods in Molecular Biology, 2297) 1071613693, 9781071613696

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Methods in Molecular Biology 2297

Ruohe Yin Ling Li Kaijing Zuo Editors

Plant Photomorphogenesis Methods and Protocols

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-bystep fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

Plant Photomorphogenesis Methods and Protocols

Edited by

Ruohe Yin School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China

Ling Li Key Laboratory of Urban Agriculture (South) Ministry of Agriculture, Plant Biotechnology Research Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China

Kaijing Zuo School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China

Editors Ruohe Yin School of Agriculture and Biology Shanghai Jiao Tong University Shanghai, China

Kaijing Zuo School of Agriculture and Biology Shanghai Jiao Tong University Shanghai, China

Ling Li Key Laboratory of Urban Agriculture (South) Ministry of Agriculture, Plant Biotechnology Research Center, School of Agriculture and Biology Shanghai Jiao Tong University Shanghai, China

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-1369-6 ISBN 978-1-0716-1370-2 (eBook) https://doi.org/10.1007/978-1-0716-1370-2 © Springer Science+Business Media, LLC, part of Springer Nature 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface This book aims at providing readers with protocols for research in plant photomorphogenesis. The chapters in this book include a broad range of topics, including assays for shade avoidance responses, assays for light-dependent protein–protein interactions, photobody detection with immunofluorescence and the super-resolution imaging method, photoreceptor containing protein complex isolation from plants, detection of homodimer and monomer of photoreceptor UVR8 with immunoblotting analysis, assays for seedling greening, procedures for studying skotomorphogenesis, phenotypic study of photomorphogenesis at seedling stage, expression of cryptochrome in insect cells, and more. Each chapter includes a list of the necessary materials and reagents, detailed reproducible experimental procedures, and notes on avoiding known pitfalls. This book, Plant Photomorphogenesis: Methods and Protocols, serves as a guideline to researchers or students who are new to the field, and a stepping-stone for experienced researchers to further their skills in this fast-developing field. Shanghai, China

Ruohe Yin Ling Li Kaijing Zuo

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Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1 Setting Up an Arabidopsis LED Culture Module that Simulates Plant Neighbor Proximity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Carlos D. Crocco 2 Photobody Detection Using Immunofluorescence and Super-Resolution Imaging in Arabidopsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Giorgio Perrella, Anna Zioutopoulou, Andrew Hamilton, and Eirini Kaiserli 3 Analysis of Shade-Induced Hypocotyl Elongation in Arabidopsis . . . . . . . . . . . . . 21 ˜o Yetkin C ¸ aka Ince and Vinicius Costa Galva 4 Isolation of UVR8 Protein Complexes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Yan Liu and Xi Huang 5 Phenotypic Study of Photomorphogenesis in Arabidopsis Seedlings . . . . . . . . . . . 41 Chuanwei Yang, Famin Xie, and Lin Li 6 Experimental Procedures for Studying Skotomorphogenesis in Arabidopsis thaliana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Huanhuan Jin, Hong Li, and Ziqiang Zhu 7 Global Identification for Targets of Circadian Transcription Factors in Arabidopsis and Rice Using Chromatin Immunoprecipitation Followed by Sequencing (ChIP-seq) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Shuxuan Xu, Jing Huang, Jian Jin, and Wei Huang 8 Co-immunoprecipitation Assays to Detect In Vivo Association of Phytochromes with Their Interacting Partners . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Pengyu Song, Shaoman Zhang, and Jigang Li 9 Detection of UVR8 Homodimers and Monomers by Immunoblotting Analysis in Tomato . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Guoqian Yang, Xiaorui Liu, and Li Lin 10 Characterization of Seedling Greening Process in Plant Photomorphogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Wanqing Wang, Yuhong Li, and Rongcheng Lin 11 Protoplast System for Studying Blue-Light-Dependent Formation of Cryptochrome Photobody. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Xiangguang Lyu, Hongyu Li, and Bin Liu 12 Uncover the Nuclear Proteomic Landscape with Enriched Nuclei Followed by Label-Free Quantitative Mass Spectrometry . . . . . . . . . . . . . . . . . . . . 115 Yan Wang, Zhuang Lu, and Lei Wang

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Strategies to Study Dark Growth Deficient or Slower Mutants in Chlamydomonas reinhardtii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Huanling Yang, Fei Han, Yue Wang, Wenqiang Yang, and Wenfeng Tu Co-immunoprecipitation Assay for Blue Light-Dependent Protein Interactions in Plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jingyi Zhang and Shengbo He Detecting Blue Light-Dependent Protein–Protein Interactions by LexA-Based Yeast Two-Hybrid Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xiaolong Hao and Ling Li Express Arabidopsis Cryptochrome in Sf9 Insect Cells Using the Baculovirus Expression System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xu Li, Yawen Liu, and Hongtao Liu Semi-In-Vivo Pull-Down Assay for Blue Light-Dependent Protein Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xu Li, Yawen Liu, and Hongtao Liu Tobacco System for Studying Protein Colocalization and Interactions . . . . . . . . . Jingyi Zhang and Shengbo He

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors CARLOS D. CROCCO • IFEVA, Facultad de Agronomı´a, Universidad de Buenos Aires y Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas, Buenos Aires, Argentina VINICIUS COSTA GALVA˜O • Center for Integrative Genomics, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland ANDREW HAMILTON • School of Medicine, Dentistry and Nursing, College of Medical, Veterinary and Life Sciences, Bower Building, University of Glasgow, Glasgow, Scotland, UK FEI HAN • Key Laboratory of Photobiology, Institute of Botany (CAS), Beijing, China; University of Chinese Academy of Sciences, Beijing, China XIAOLONG HAO • Laboratory of Medicinal Plant Biotechnology, College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou, China SHENGBO HE • Department of Cell and Developmental Biology, John Innes Centre, Norwich, UK JING HUANG • State Key Laboratory for Conservation and Utilization of Subtropical grobioresources, College of Life Science and Technology, Guangxi University, Nanning, Guangxi, China WEI HUANG • State Key Laboratory for Conservation and Utilization of Subtropical grobioresources, College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, China XI HUANG • State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, China YETKIN C ¸ AKA INCE • Center for Integrative Genomics, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland HUANHUAN JIN • College of Life Sciences, Nanjing Normal University, Nanjing, China JIAN JIN • State Key Laboratory for Conservation and Utilization of Subtropical gro-bioresources, College of Life Science and Technology, Guangxi University, Nanning, Guangxi, China EIRINI KAISERLI • Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, Bower Building, University of Glasgow, Glasgow, Scotland, UK HONG LI • College of Life Sciences, Nanjing Normal University, Nanjing, China HONGYU LI • Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China JIGANG LI • State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China LIN LI • State Key Laboratory of Genetic Engineering and Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, China LING LI • Key Laboratory of Urban Agriculture (South) Ministry of Agriculture, Plant Biotechnology Research Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China XU LI • National Key Laboratory of Plant Molecular Genetics (NKLPMG), CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences, Shanghai, P. R. China

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YUHONG LI • Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China; University of Chinese Academy of Sciences, Beijing, China LI LIN • Key Laboratory of Urban Agriculture Ministry of Agriculture, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China RONGCHENG LIN • Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China; University of Chinese Academy of Sciences, Beijing, China BIN LIU • Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, P. R. China HONGTAO LIU • National Key Laboratory of Plant Molecular Genetics (NKLPMG), CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences, Shanghai, P. R. China XIAORUI LIU • Key Laboratory of Urban Agriculture Ministry of Agriculture, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China YAN LIU • State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, China YAWEN LIU • National Key Laboratory of Plant Molecular Genetics (NKLPMG), CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences, Shanghai, P. R. China ZHUANG LU • Key Laboratory of Plant Molecular Physiology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Botany, Chinese Academy of Sciences, Beijing, People’s Republic of China XIANGGUANG LYU • Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China GIORGIO PERRELLA • Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, Bower Building, University of Glasgow, Glasgow, Scotland, UK; ENEA Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Trisaia Research Center, Rotondella, Italy PENGYU SONG • State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China WENFENG TU • Key Laboratory of Photobiology, Institute of Botany (CAS), Beijing, China; University of Chinese Academy of Sciences, Beijing, China LEI WANG • Key Laboratory of Plant Molecular Physiology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Botany, Chinese Academy of Sciences, Beijing, People’s Republic of China WANQING WANG • Biochemical Engineering College, Beijing Union University, Beijing, China YAN WANG • Key Laboratory of Plant Molecular Physiology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Botany, Chinese Academy of Sciences, Beijing, People’s Republic of China YUE WANG • Key Laboratory of Photobiology, Institute of Botany (CAS), Beijing, China; University of Chinese Academy of Sciences, Beijing, China FAMIN XIE • State Key Laboratory of Genetic Engineering and Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, China SHUXUAN XU • State Key Laboratory for Conservation and Utilization of Subtropical grobioresources, College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, China CHUANWEI YANG • State Key Laboratory of Genetic Engineering and Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, China

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GUOQIAN YANG • Key Laboratory of Urban Agriculture Ministry of Agriculture, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China HUANLING YANG • Key Laboratory of Photobiology, Institute of Botany (CAS), Beijing, China; University of Chinese Academy of Sciences, Beijing, China WENQIANG YANG • Key Laboratory of Photobiology, Institute of Botany (CAS), Beijing, China; University of Chinese Academy of Sciences, Beijing, China; The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, China JINGYI ZHANG • Department of Cell and Developmental Biology, John Innes Centre, Norwich, UK SHAOMAN ZHANG • State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China ZIQIANG ZHU • College of Life Sciences, Nanjing Normal University, Nanjing, China ANNA ZIOUTOPOULOU • Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, Bower Building, University of Glasgow, Glasgow, Scotland, UK

Chapter 1 Setting Up an Arabidopsis LED Culture Module that Simulates Plant Neighbor Proximity Carlos D. Crocco Abstract Competition for light between neighboring plants has important consequences for plant fitness and crop productivity. Studies on the molecular mechanisms of plant responses to neighbor proximity have been largely based on the model species Arabidopsis thaliana grown under controlled light environments. These controlled conditions commonly use fluorescent tubes for the main light source for photosynthesis and filtered light form incandescent bulbs to adjust the ratio of red (R) to far-red (FR) radiation. However, both of these types of bulbs are being discontinued and replaced by more efficient sources based on light emitting diodes (LEDs). For that reason, there is a need to evaluate alternative light sources, which can phenocopy the physiological and molecular results obtained with traditional lighting systems. Here we evaluate a custom-made LED culture module that can be used to effectively evaluate shade-avoidance responses, yielding results that, in Arabidopsis, are comparable to those obtained using traditional lighting systems. Key words Phytochrome, Shade avoidance, Far-red, LED, Light competition

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Introduction FR radiation (730 nm) reflected by green tissues is a key signal that plants use to detect the proximity of future competitors. This signal is perceived by phytochromes (phy), a family of reversible photoreceptors [1]. phys alternate between two photo-convertible isomers, a biologically inactive R-absorbing form (Pr), and a biologically active FR-absorbing form (Pfr). A low R:FR ratio reduces the proportion of Pfr in plant tissues, and consequently triggers a specific signal transduction pathway leading to the upregulation of a suite of transcription factors, including PIL1, ATHB2, HFR1, PAR1, and PAR2, and genes encoding auxin-responsive proteins, like IAA19 and IAA29 [2–4]. The activation of these transcriptional cascades promotes a set of physiological responses known as the shade-avoidance syndrome (SAS), which involves adaptive changes in plant morphology that improve plant competitive ability in dense stands [1]. The SAS is characterized by increased stem and

Ruohe Yin et al. (eds.), Plant Photomorphogenesis: Methods and Protocols, Methods in Molecular Biology, vol. 2297, https://doi.org/10.1007/978-1-0716-1370-2_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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petiole elongation, leaf epinasty, reduction of branching, and early flowering [5, 6]. Under laboratory conditions, a classic way to mimic the effect of neighbor proximity is based on adding to the main white light (WL) source used to provide light for photosynthesis, FR radiation in order to decrease the R:FR ratio. These light treatments are often obtained by using fluorescent tubes to provide WL, and incandescent bulbs coupled with filters to add FR radiation [2, 3]. These systems will present some problems in the near future, given that the incandescent bulbs are no longer manufactured and the fluorescent tubes are being rapidly replaced by commercial WL LEDs. After testing a set of widely available white-light emitting LEDs, in combination with commercial FR LEDs, we set up a LED culture module to evaluate Arabidopsis responses to neighbor proximity, which yields morphological and transcriptomic SAS outputs that are comparable to the ones obtained with traditional light sources.

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Materials A LED culture module to simulate the proximity of neighboring plants was assembled with seven “warm white” LED tubes (Philips 18 W 840-T8 1200mm) hanged above the plants and one GreenPower FR LED unit (Philips—GP LED production FR 120) positioned on one of the sides (Fig. 1a). Currently, there is a wide variety of commercial white LEDs segmented in cold, neutral, or warm white emission spectra but not all of them are suitable to grown plants and perform experiments related with photomorphogenic processes (see Note 1). Warm white LEDs provide the plants with a balanced light spectrum and Arabidopsis plants develop normally under these light sources throughout all of their phenological stages. To mimic the effect of neighboring plants, we placed the plants inside the LED culture module at 10, 30, or 50 cm from the FR LED (Fig. 1a; see Note 2). These positions simulate the presence of neighbors at three different degrees of proximity, where the R:FR ratio is modified for each position without seriously affecting the total amount of light for photosynthesis received by the plants (100 μmol m 2 s 1, between 400 and 700 nm). Spectral quality for each light source was measured using an Ocean Optics USB4000 micro-spectroradiometer (Fig. 1b). PAR and R:FR ratio were measured (Fig. 1c) using a SpectroSense2 attached with a SKR-1850SS2 light sensor (Skye Instruments).

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Fig. 1 LED culture module. (a) Diagram of the LED culture module composed by a lateral GreenPower FR LED bar and seven white warm LED tube from the top. Plants were placed at three positions (10, 30, and 50 cm) from the FR LED to perform different R:FR ratios degrees. (b) Spectral photon fluencies of white warm LEDs (black line) and FR LEDs (red line). (c) Table with the R:FR ratios obtained for each treatment placing the sensor laterally toward the FR LED source

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Methods We grew A. thaliana (Col-0) plants for 3 weeks under warm white LEDs (100 μmol m 2 s 1) in short-day conditions (8 h light–16 h dark) at 22  C. Then, A. thaliana plants were placed inside the LED culture module at 10, 30, or 50 cm from the FR source. Equivalent modules in which the FR source was inactivated were used as controls (control treatment). After 6 days, we measured morphological parameters such as rosette diameter, petiole length, and petiole angle (Fig. 2). The plants placed at 10, 30, or 50 cm showed a promotion of rosette diameter growth (Fig. 2a) and petiole length (Fig. 2b) compared to control plants (R:FR ratio > 1), which correlate well with the progressive reduction of the R:FR ratio (0.55, 0.35 and 0.15) under the LED culture module. Additionally, the FR treatments caused robust leaf epinastic responses compared to the control treatment (Fig. 2c). These data indicate that the LED culture module could mimic different degrees of neighbor proximity promoting rosette growth, petiole

Carlos D. Crocco

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Fig. 2 Phenotypes obtained in the LED culture module. Three-week Arabidopsis plants grown under white LEDs were placed for 6 days inside de the LED culture module at 10, 30, or 50 cm from the FR LED bar (0.15, 0.35, and 0.55 R:FR ratio, respectively), or they were kept in white LED (R:FR > 1; control treatment). Graphics represent a negative correlation between the promotion of (a) rosette diameter, (b) petiole length, (c) leaf epinasty (petiole angle) and the decrease of R:FR ratio obtained in the LED culture module (0.15, 0.35, and 0.55) or control treatment (R:FR > 1). Bars indicate mean  SD (n  7). Asterisk indicate differences from control treatment (R:FR > 1) by Student’s t test (***P  0.005)

elongation, and petiole angle in a R:FR quantitative manner (see Note 3). Upon exposure of plants to low R:FR conditions, a complex signaling network is activated, which involves the upregulation of a set of transcription factors such as ATHB2, PIL1, HFR1, and PAR2 [4]. We quantified the transcript levels of these genes in 3-week A. thaliana plants exposed for 5 h to the various FR (0.55, 0.35, and 0.15 R:FR ratio) and control treatments (R:FR ratio > 1) in the LED culture module. FR supplementation in the LED culture module induced the transcription of these low R:FR-marker genes (Fig. 3a). Additionally, genes encoding two auxin-responsive proteins (IAA19 and IAA29) are upregulated by supplemental FR radiation, which is consistent with previous reports [3, 4] (Fig. 3b). These data indicate that SAS signaling could be modulated by positioning the plants at different distances from the FR source. Here we constructed and evaluated a LED culture module that simulates the proximity of neighboring plants generating a reduction of the R:FR ratio without affecting the total amount of PAR. The results obtained with this LED system are comparable in terms of morphological and transcriptional responses with those achieved with previous light arrangements that simulated competition with neighboring plants (Crocco et al., 2010–2014).

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Fig. 3 Quantitative RT-PCR analysis. (a) Relative expression levels of PIL1, HFR1, ATHB2, and PAR2 in 3-week Arabidopsis plants (col) in response to 5 h inside the LED culture module at the different positions from FR source (0.15, 0.35, and 0.55 R > FR ratio) or kept under control white LED treatment (WL—R:FR > 1). (b) Relative transcripts levels of IAA19 and IAA29 under the same treatments described above. Values are means  SD of three biological replicates (each composed by three-member pools) normalized to UBC and IPP2A transcript levels. Asterisk indicate differences from control treatment (WL) by Student’s t test (*P  0.05, **P  0.01 and ***P  0.005)

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Notes 1. Arabidopsis grown under warm white light LED express a phenotype consistent with these one obtained under fluorescent white light tubes. Cold or neutral white light LEDs induce a strong inhibition on rosette growth promotion and petiole elongation when Arabidopsis plants are supplemented with lateral FR LED. We strongly recommend not using cold or neutral light LED to evaluate shade-avoidance responses. 2. Lateral position of the FR LED source is decisive to evaluate the SAS phenotypes gating by a decrease of the R:FR ratio. In many laboratories, the FR light sources are implemented from the top together with the white light tubes. Although both positions of the FR light source reduce the R:FR ratio, plants supplemented with FR from the top could experiment high irradiance responses triggered by prolonged exposure to FR. Assembling a lights system where the FR light is supplemented on one side will be key to design properly the experimental conditions to evaluate vegetation proximity responses in shade-avoiding plants. 3. Three-week-old Arabidopsis grown under short days are able to express the classic SAS phenotypes (i.e., petiole elongation

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and rosette growth promotion) after 2 days under the LED culture module, but those phenotypes are maximized when the plants grow between 5 and 7 days under these FR enrichment treatments.

Acknowledgments I thank Carlos Ballare´ for their critical reading of the manuscript and many helpful suggestions, and Ana Medina for help with the experiments. This work is supported by the Agencia Nacional de Promocio´n Cientı´fica y Te´cnica, Universidad de Buenos Aires, and CONICET (Argentina). References 1. Ballare´ CL, Pierik R (2017) The shadeavoidance syndrome: multiple signals and ecological consequences. Plant Cell Environ 40:2530–2543 2. Crocco CD, Holm M, Yanovsky MJ, Botto JF (2010) AtBBX21 and COP1 genetically interact in the regulation of shade avoidance. Plant J 64:551–562 3. Crocco CD, Locascio A, Escudero CM, Alabadı´ D, Bla´zquez MA, Botto JF (2015) The transcriptional regulator BBX24 impairs DELLA activity to promote shade avoidance in Arabidopsis thaliana. Nat Commun 6:6202 4. Sessa G, Carabelli M, Sassi M, Ciolfi A, Possenti M, Mittempergher F, Becker J,

Morelli G, Ruberti I (2005) A dynamic balance between gene activation and repression regulates the shade avoidance response in Arabidopsis. Genes Dev 19:2811–2815 5. Smith H, Whitelam GC (1997) The shade avoidance syndrome: multiple responses mediated by multiple phytochromes. Plant Cell Environ 20:840–844 6. Ballare´ CL, Sa´nchez RA, Scopel AL, Casal JJ, Ghersa CM (1987) Early detection of neighbour plants by phytochrome perception of spectral changes in reflected sunlight. Plant Cell Environ 10:551–557

Chapter 2 Photobody Detection Using Immunofluorescence and Super-Resolution Imaging in Arabidopsis Giorgio Perrella, Anna Zioutopoulou, Andrew Hamilton, and Eirini Kaiserli Abstract Light triggers changes in plant nuclear architecture to control differentiation, adaptation, and growth. A series of genetic, molecular, and imaging approaches have revealed that the nucleus forms a hub for photoinduced protein interactions and gene regulatory events. However, the mechanism and function of lightinduced nuclear compartmentalization is still unclear. This chapter provides detailed experimental protocols for examining the morphology and potential functional significance of light signaling components that localize in light-induced subnuclear domains, also known as photobodies. We describe how immunolabeling of endogenous proteins and fluorescent in situ hybridization (FISH) could be combined with confocal imaging of fluorescently tagged proteins to assess co-localization in Arabidopsis nuclei. Furthermore, we employ a super-resolution imaging approach to study the morphology of photobodies at unprecedented detail. Key words Photobodies, Light signaling, Arabidopsis, Nuclear localization, Immunofluorescence, Super-resolution imaging

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Introduction The nucleus forms a key cellular compartment where environmental and endogenous signals integrate to regulate plant architecture. Light, in particular, is known to trigger the nuclear import and clustering of many photoreceptors and core signaling components in subnuclear compartments named photobodies [1, 2]. One of the best characterized photoreceptor families that form photobodies are the red/far-red light absorbing phytochromes. The kinetics, wavelength-specificity, and reversibility of photobody formation are well characterized thanks to elegant fluorescence imaging, genetics, and modeling approaches [3–8]. Arabidopsis nuclear photobodies have been hypothesized to act as sites of protein regulation (stability, post-translational modifications, desensitization) and gene expression; however, there is very little information on the precise molecular function, composition, and

Ruohe Yin et al. (eds.), Plant Photomorphogenesis: Methods and Protocols, Methods in Molecular Biology, vol. 2297, https://doi.org/10.1007/978-1-0716-1370-2_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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Fig. 1 Overview of the workflow for the preparation of Arabidopsis thaliana leaf nuclei followed by immunolabeling (left) or Fluorescent In Situ Hybridization (FISH) (right). See main text for details. O/N: overnight

three-dimensional morphology of these structures [9–11]. Studies on protein clustering in nuclear photobodies have been majorly based on monitoring the co-localization or in vivo interactions of fluorescently tagged proteins of interest by confocal microscopy. Here, we describe a combinatorial approach to investigate the function of photobodies in planta by employing immunolabeling, FISH, and fluorescent-tagged confocal imaging in Arabidopsis. We have optimized a workflow (Fig. 1) combining

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Fig. 2 Examples of photobody co-localization using a combination of immunofluorescence and conventional confocal imaging of FP-tagged proteins in Arabidopsis. (A) Confocal images of Arabidopsis nuclei from a transgenic line expressing TZP-GFP; (B) Arabidopsis nuclei from a transgenic line expressing SCL33-GFP was used as a negative control of a nuclear protein that does not form photobodies; (C, D, E) immuno-detection of phytochrome B using Alexa Fluor 568 anti-mouse; (C) immuno-detection of phyB in a phyAphyB double mutant, respectively, used as negative controls assessing the specificity of the antibodies. (F, G) Overlay of green and red signals; (H, I, J) DAPI counterstaining of Arabidopsis nuclei. Arrows show co-localization areas. Imaging was performed on isolated nuclei obtained from fixed Arabidopsis leaf tissue of 12-day old plants exposed to white light. Scale bars: 10 μm

immunofluorescence (IF) (Subheading 3.2) and FISH (Subheading 3.3) protocols on isolated nuclei (Subheading 3.1) from wildtype and transgenic lines expressing GFP-tagged light signaling components. We use as a case study a light signaling component that is known to co-purify with multi-protein complexes [12, 13] and form photobodies [8], Tandem Zinc-finger Plus3 (TZP), to assess if it co-localizes with endogenously expressed photoreceptors (Fig. 2) as well as core chromatin components (Fig. 3), transcriptional machinery (Fig. 3) or nucleic acids (Fig. 4). Based on the experimental approach and data obtained from this study, we can conclude that we can indeed successfully apply a combination of the three aforementioned imaging techniques (IF, FISH, FP-imaging)

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Fig. 3 Examples of immunofluorescence studies to assess the co-localization of chromatin and transcriptional marks with nuclear photobodies in Arabidopsis nuclei. (A to C) Confocal images of TZP-GFP nuclear bodies in Arabidopsis (green channel); (D) immuno-detection of the phosphorylated CTD of Pol II using Alexa Fluor 568 anti-mouse; (E, F) immuno-detection of histone modifications H3K9K14Ac and H3K9me2 using Alexa Fluor 568 anti-rabbit; (G, H, I) overlay of green and red channels; (J, K, L) DAPI counterstaining of Arabidopsis nuclei. Arrows show co-localization areas. Imaging was performed on isolated nuclei obtained from fixed leaf tissue from 12-day old Arabidopsis plants exposed to white light. Scale bars: 10 μm

to further dissect the in vivo function of nuclear photobodies in Arabidopsis. In the case of TZP, we further confirm its absolute co-localization with endogenous phyB nuclear bodies (Fig. 2a, d, f). Partial co-localization was also observed between TZP-GFP photobodies and the phosphorylated form of RNA Pol II (Fig. 3a, d, g, j), which is a mark of active transcription and with poly(A) + RNA (Fig. 4b, c, d, f). Very little or no co-localization was observed between TZP-GFP photobodies and histone marks of H3K9K14 acetylation (Fig. 3b, e, h, k) or H3K9 dimethylation (Fig. 3c, f, i, l). To extend our understanding of the morphology and structure of nuclear photobodies, we describe an optimized protocol used to perform super-resolution imaging on intact plant leaf tissue (Subheading 3.4). Once more, we used transgenic Arabidopsis lines

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Fig. 4 RNA FISH on isolated nuclei from WT and TZP-GFP plants. (A, C) Detection of poly(A) + RNA Cy3 labeled oligo (dT)70 in nuclei from WT and transgenic lines expressing TZP-GFP, respectively; (B, C, D) confocal image of TZP nuclear bodies and overlay with the signal from the poly(A) + RNA Cy3 probe; (E, F) DAPI counterstaining of WT and TZP-GFP Arabidopsis nuclei. Arrows show co-localization areas. Imaging was performed on isolated nuclei obtained from fixed leaf tissue from 12-day old plants exposed to white light. Scale bars: 10 μm

expressing TZP-GFP and showed that TZP photobodies form donut-shaped ring structures (Fig. 5b) that resemble mammalian promyelocytic leukemia (PML) bodies [14]. We believe that the protocols described in this chapter will be widely applicable to study the morphology, co-localization, and possible subnuclear function of photobodies, but also other nuclear body-forming proteins in plants.

2 2.1

Materials Equipment

2.1.1 Equipment for Sample Preparation

1. Eppendorf tubes (1.5 mL). 2. Falcon tubes (15 mL and 50 mL). 3. Refrigerated microcentrifuge. 4. Dark incubation chamber for microscope slides: plastic box wrapped in black electrical tape with wet paper towel being humidified with no excess of liquid. Plastic pipettes are placed in the box to support the microscope slides. 5. Regular microscope glass slides or frosted microscope slides. 6. Glass coverslips 22
50 mm. 7. Razor blades.

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Fig. 5 Comparison of TZP-GFP photobody morphology obtained by confocal microscopy (A) or 3D-SIM using an OMX microscope (B). Imaging was performed on tissue obtained from fixed leaves from 12-day old plants grown in white light. Scale bars: 1 μm

8. Plastic Petri dishes and multi-well serological plastic plates. 9. Filters (40 and 100 μm). 10. Filter tips. 2.1.2 Equipment for Imaging

1. Confocal laser scanning microscope (CLSM) was performed using a Leica SP8 inverted microscope with an Apochromat 63
oil immersion objective lens and tunable excitation from 470–670 nm. 2. Three-dimensional structured illumination (3D-SIM) was performed on an OMX Blaze microscope using a 488 nm solidstate laser. Images were acquired using an Apochromat 100
oil immersion objective lens and back-illuminated 512
512 electron microscopy charge-coupled device cameras. Image acquisition and analysis was performed as previously described [15, 16].

2.2

Plant Material

Arabidopsis lines expressing an FP-tagged protein of interest and/or an antibody for detecting native proteins are required for co-localization using immunofluorescence or FISH. The following

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Arabidopsis thaliana Columbia (Col-0) lines were used in this study: wild-type (WT), 35SproTZP-GFP [8], 35SproSCL33-GFP, and phyAphyB (phyA-211 phyB-9). The Gateway technology (Life Technologies) was used to generate translational fusions of homozygous transgenic Arabidopsis lines expressing 35SproSCL33-GFP (primers used for amplifying SCL33 from cDNA: SCL33_for 50 A TGAGGGGAAGGAGCTACA 30 and SCL33_rev 50 CTGGCTT GGTGAACGG 30 ) using the binary vectors previously described [17]. Tissue from twelve-old seedlings was used for this study (see Note 1). 2.3 Reagents and Solutions for Nuclei Isolation

Nuclear Extraction Buffer (NEB) [18]: 10 mM Tris–HCl pH 9.5; 10 mM KCL; 0.5 M Sucrose; 4 mM Spermidine; 10 mM Spermine; 0.1% 2-mercaptoethanol (see Note 2). Fixation solution: 8% formaldehyde is added fresh to the NEB buffer after mincing the tissue.

2.4 Reagents and Solutions for Immunostaining and RNA FISH

10
KPBS: 1.28 M NaCl; 20 mM KCl; 80 mM Na2HP04; 20 mM KH2P04, pH 7.2. Blocking solution: 1% BSA Fraction V in 1
KPBS 0.1% Triton X-100. Nuclease-free water. 20
SSC (Saline Sodium Citrate: 3 M NaCl, 300 mM Na3C6H5O7, pH 7.2) in sterile water. Stock Solutions: 100% deionized formamide, 20
SSC, 100% dextran sulfate, nuclease-free dH20. Hybridization mixture: 50% deionized formamide, 2
SSC, 10% dextran sulfate, adjusted final volume with nuclease-free dH20. A blocking reagent (e.g., 20 μg/mL E. coli tRNA or 20 μg/mL salmon sperm DNA) should be used.

2.5 Reagents and Solutions for DNA Staining

DAPI (40 -6-Diamidino-2-phenylindole) (Molecular Probes) (Stock solution: 5 mg/mL in KPBS). Prolong Gold Antifade.

3

Methods The workflow for the isolation of nuclei required for immunofluorescence and FISH is described in Fig. 1. Super-resolution imaging of fluorescently tagged proteins can be performed on fixed but intact sections of leaf tissue.

3.1 Isolation of Nuclei from Arabidopsis Leaf Tissue

Nuclei preparation was performed based on protocols developed by Dr. Olga Pontes [19] with specific modifications. 1. Arabidopsis seeds were sterilized in 50% sodium hypochlorite solution for 3 min and washed 4–5 times with sterile water. Seeds were then sown on ½ Murashige and Skoog and 0.8%

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agar plates and stratified in the dark for 3 days at 4  C. Seedlings were grown for 12 days at 22  C in long day conditions, (18 h light/6 h dark, white light at 80 μmol m2 s1) and plant material was collected at ZT 0.5. (see Note 1). 2. Tissue homogenization can be performed by finely chopping 1 gr of seedlings using a razorblade on a Petri dish kept on ice. Once the tissue is thinly cut, add 1–1.5 mL of NEB buffer (see Note 2). After adding the buffer, keep chopping the leaves until you obtain a fine suspension to release the nuclei in solutions. 3. To fix the tissue add an equal volume of freshly prepared 8% formaldehyde to the homogenized leaves, transfer to a 15 mL Falcon tube, and keep the solution on ice for 20 min. Mix every 5 min. 4. Apply the 100 μM filter to a 15 mL Falcon tube and pass the homogenate through the filter. Resuspend the solution while filtering. Repeat the same procedure with the 40 μM filter. 5. Centrifuge the flow-through for 3 min at 2500
g at 4  C. 6. Remove the supernatant and resuspend the pellet in 1 mL of NEB. Centrifuge for 3 min at 2500
g at 4  C. 7. Repeat this step 3–4 times until pellet appears to be of a creamy color. 8. Finally resuspend the pellet in 40 μL of NEB. The nuclei can be stored in solution at 4  C for 1 month (see Notes 3 and 4). 3.2 Immunolabeling of Isolated Nuclei 3.2.1 Antibodies

The following antibodies were used in this protocol (see Figs. 2 and 3): Primary antibodies: l

Anti-RNA polymerase II CTD YSPTSPS (phospho-Ser 5) mouse monoclonal antibody.

l

Anti-H3K9K14Ac rabbit polyclonal antibody.

l

Anti-H3K9me2 rabbit polyclonal antibody.

l

Anti-phyB mBA1 mouse monoclonal antibody (a kind gift from the Nagatani lab [20]). Secondary antibodies:

3.2.2 Antibody Incubation

l

Alexa Fluor 568 goat anti-mouse IgG.

l

Alexa Fluor 568 goat anti-rabbit IgG.

– Spread 5–10 μL of the nuclei preparation on a frosted microscope slide and allow air-dry in a flow hood for 5–10 min. – Wash the slides three times with 1
KPBS 0.1% Triton (see Note 5).

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– Add 200 μL of blocking solution to the slides and cover with a 22
50 mm coverslip. Alternatively, parafilm can be used to spread the solution on the samples. – Incubate the samples in the humid chamber for 30 min at 37  C (see Note 7). – Wash the Blocking solution 3
5 min with 1
KPBS 0.1% Triton. – Add 100 μL of the desired primary antibody at the recommended concentration in blocking solution, cover with a 22
50 mm coverslip, and incubate overnight at 4  C (see Note 6). 3.2.3 Detection and Staining

– Remove the primary antibody solution. – Wash slides 3
5 min with KPBS solution (see Note 5). – Add blocking solution and incubate at 37  C for 30 min. – Remove blocking solution and add 100 μL of the appropriate Alexa 568 secondary antibody (1:250 in KPBS solution) and incubate in the dark humid chamber for 2 h at 37  C (see Note 7). – Wash 3
10 min with KPBS. – Mount the slides with DAPI (300 nM final concentration) and incubate for 5 min in darkness at room temperature. – Wash 3
30 s with KPBS. – Add 5–10 μL of Prolong Gold Antifade and cover with a 22
50 mm coverslip. Directly proceed to imaging or store the slides at 4  C for a maximum duration of 2 days (see Notes 7– 9). – Imaging was performed using an inverted confocal Leica SP8 microscope with an Apochromat 63
oil immersion objective lens and tunable excitation from 470–670 nm. Images were acquired with a single section and a line average of 16. For GFP detection an excitation line of 488 nm was used and GFP emission was collected between 499 and 539 nm. Immunostaining with Alexa 568 was detected by excitation at 552 nm emission between 583 and 694 nm (see Figs. 2 and 3).

3.3 RNA Fluorescence In Situ Hybridization (FISH)

This specific protocol is used to monitor endogenous poly (A) + RNA as well as retaining the GFP signal of the transgenic protein of interest. Pepsin or proteinase K digestion is deliberately omitted to retain the fluorescence signal of the genetically encoded GFP (or any FP) and does not compromise the RNA FISH.

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3.3.1 In Situ Hybridization

– Spread 5–10 μL of the nuclei preparation on a frosted microscope slide and let air-dry in a flow hood for few minutes. – Wash the slides three times with 2
SSC in nuclease-free water. – Add 50 μL of the hybridization mixture (see Subheading 2.4) to each slide containing 0.1 μM of a poly(A) + RNA 5’ Cy3-labeled oligo (dT)70 probe (synthesized and purified by Sigma [21]) (see Fig. 4 and Note 10). – Cover with a 22
50 mm coverslip. – Hybridize at 37  C for 4 h up to O/N in a dark humid chamber (see Note 4). – Wash the slides 3 times
5 min with 200 μL of 2
SSC at 37  C in the dark (see Note 4). – Mount the slides with DAPI (300 nM final concentration) and incubate for 5 min in darkness at room temperature. – Wash 3
30 sec with 2
SSC. – Add 5–10 μL of Prolong Gold Antifade and cover with a 22
50 mm coverslip. – Store the slides at 4  C in the darkness or proceed directly to imaging (see Notes 7–9).

3.3.2 Detection and Imaging

3.4 Super-Resolution Imaging of Nuclear Photobodies in Arabidopsis

Imaging was performed using an inverted confocal Leica SP8 microscope with an Apochromat 63
oil immersion objective lens and tunable excitation from 470–670 nm. For GFP detection an excitation line of 488 nm was used and GFP emission was collected between 499 and 539 nm. The signal emitted by the Cy3-labeled poly(A) + RNA probe was examined with the use of a laser excitation of 552 nm and emission was collected between 549 and 593 nm (see Fig. 4). 1. Carefully detach individual leaves from 12-day-old Arabidopsis plants expressing an FP-tagged protein of interest grown under constant white light (120 μmol m2 s1). 2. Incubate the leaf tissue in a solution containing 8% formaldehyde for 20 min using 15 mL ice-cold Falcon tubes (Leaf tissue fixation). 3. Wash the fixed leaf tissue with ddH20 water. 4. Keep the leaf tissue hydrated in serological plastic plates at room temperature. 5. Use a razor blade to slice a small (3 mm
3 mm) and flat section of a leaf and mount it in the middle of a regular frosted slide and cover with a 22
50 mm coverslip (see Note 11). 6. 3D-SIM was performed by an OMX Blaze microscope using a 488 nm solid-state laser. Images were acquired using an

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Apochromat 100
oil immersion objective lens and backilluminated 512
512 electron microscopy charge-coupled device cameras. Image acquisition and analysis was performed as previously described [15, 16]. The super-resolution image shown in Fig. 5B was analyzed and exported from Fiji.

4

Notes 1. It is important to use young and healthy seedlings. We use 12-day-old seedlings, but alternatively even younger plants can be used. 2. Fixative and buffers have to be prepared fresh. NEB can be prepared in advance but without Spermidine, Spermine, and 2-mercaptoethanol that will be added on the day of the experiment. 3. If using immunostaining or FISH on transgenic lines expressing an FP-tagged protein, check the integrity of the FP signal of the protein of your interest and the quality of the nuclei before continuing with each procedure. Spread 5 μL of the nuclei preparation on a frosted microscope slide and let air-dry in a flow hood for few minutes. Wash the nuclei with 1
KPBS and mount with DAPI. 4. When using FISH protocols, keep slides always in the dark, even at high temperatures. Avoid direct contact with light. 5. All the washes are performed at room temperature unless stated otherwise. We apply the buffer washes directly on the slide. We do not use Coplin jars to minimize loss of nuclei. 6. For the primary antibodies used in this protocol we recommend the following dilutions in 1
KPBS solution: Anti-RNA polymerase II CTD YSPTSPS

1:250

Anti-H3K9K14Ac

1:500

Anti-H3K4me3

1:500

Anti-H3K9me2

1:500

Anti-phyB

1:250

Mutants of the respective protein should be included as a negative control assessing the specificity of the antibodies. Alternatively, non-inducive photobody conditions (e.g. darkness) or lack of secondary antibody could be used. 7. Always keep the slides in a humid chamber to preserve the quality of the signal.

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8. For detection by confocal microscopy we recommend using 0.13–0.16 mm in thickness 22
50 mm glass coverslips. 9. For optimal results image acquisition needs to be performed as soon as the samples are labeled (Day 2). Alternatively, the slides can be kept in the dark overnight at 4  C. 10. For poly(A) + RNA FISH use nuclease-free water and filter tips. 11. For optimal 3D-SIM images the sample needs to be in the middle of the slide and really close to the cover slip. The FP-tagged protein of interest would have to be expressed well. Agarose mounting could be used to further flatten the leaf tissue. Application of Prolong Gold Antifade was not required for maintaining FP fluorescence.

Acknowledgments Many thanks to Dr. Markus Posch (University of Dundee) for his help in performing super-resolution imaging on plant tissue. We are grateful to Prof. Akira Nagatani for providing us with an anti-phyB antibody. The authors would also like to thank the COSTINDEPTH action for organizing and funding G.P. to participate at the “3D FISH and Image Analysis” training workshop at Clermont-Ferrand, France. E.K. is grateful to the BBSRC for the New Investigator Grant Award BB/M023079/1 and the John Grieve Bequest for supporting her Lectureship. A.Z. is funded by a PhD studentship from the College of Medical, Veterinary and Life Sciences at the University of Glasgow. References 1. Van Buskirk EK, Decker PV, Chen M (2012) Photobodies in light signaling. Plant Physiol 158(1):52–60. https://doi.org/10.1104/pp. 111.186411 2. Kaiserli E, Perrella G, Davidson ML (2018) Light and temperature shape nuclear architecture and gene expression. Curr Opin Plant Biol 45(Pt A):103–111. https://doi.org/10. 1016/j.pbi.2018.05.018 3. Rausenberger J, Hussong A, Kircher S, Kirchenbauer D, Timmer J, Nagy F, Schafer E, Fleck C (2010) An integrative model for phytochrome B mediated photomorphogenesis: from protein dynamics to physiology. PLoS One 5(5):e10721. https:// doi.org/10.1371/journal.pone.0010721 4. Kircher S, Gil P, Kozma-Bognar L, Fejes E, Speth V, Husselstein-Muller T, Bauer D, Adam E, Schafer E, Nagy F (2002)

Nucleocytoplasmic partitioning of the plant photoreceptors phytochrome A, B, C, D, and E is regulated differentially by light and exhibits a diurnal rhythm. Plant Cell 14 (7):1541–1555 5. Kircher S, Kozma-Bognar L, Kim L, Adam E, Harter K, Schafer E, Nagy F (1999) Light quality-dependent nuclear import of the plant photoreceptors phytochrome A and B. Plant Cell 11(8):1445–1456 6. Yu X, Sayegh R, Maymon M, Warpeha K, Klejnot J, Yang H, Huang J, Lee J, Kaufman L, Lin C (2009) Formation of nuclear bodies of Arabidopsis CRY2 in response to blue light is associated with its blue light-dependent degradation. Plant Cell 21(1):118–130. https://doi.org/10.1105/ tpc.108.061663

Antibacterial Evaluation of Medicinal Plants 7. Chen M, Galvao RM, Li M, Burger B, Bugea J, Bolado J, Chory J (2010) Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes. Cell 141 (7):1230–1240. https://doi.org/10.1016/j. cell.2010.05.007 8. Kaiserli E, Paldi K, O’Donnell L, Batalov O, Pedmale UV, Nusinow DA, Kay SA, Chory J (2015) Integration of light and photoperiodic signaling in transcriptional nuclear foci. Dev Cell 35(3):311–321. https://doi.org/10. 1016/j.devcel.2015.10.008 9. Perrella G, Kaiserli E (2016) Light behind the curtain: photoregulation of nuclear architecture and chromatin dynamics in plants. New Phytol 212:908. https://doi.org/10.1111/ nph.14269 10. Feng CM, Qiu Y, Van Buskirk EK, Yang EJ, Chen M (2014) Light-regulated gene repositioning in Arabidopsis. Nat Commun 5:3027. https://doi.org/10.1038/ncomms4027 11. Legris M, Klose C, Burgie ES, Rojas CC, Neme M, Hiltbrunner A, Wigge PA, Schafer E, Vierstra RD, Casal JJ (2016) Phytochrome B integrates light and temperature signals in Arabidopsis. Science 354 (6314):897–900. https://doi.org/10.1126/ science.aaf5656 12. Huang H, Yoo CY, Bindbeutel R, Goldsworthy J, Tielking A, Alvarez S, Naldrett MJ, Evans BS, Chen M, Nusinow DA (2016) PCH1 integrates circadian and light-signaling pathways to control photoperiod-responsive growth in Arabidopsis. elife 5:e13292. https://doi.org/10.7554/eLife.13292 13. Huang H, Alvarez S, Bindbeutel R, Shen Z, Naldrett MJ, Evans BS, Briggs SP, Hicks LM, Kay SA, Nusinow DA (2016) Identification of evening complex associated proteins in Arabidopsis by affinity purification and mass spectrometry. Mol Cell Proteomics 15 (1):201–217. https://doi.org/10.1074/mcp. M115.054064

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14. Hattersley N, Shen L, Jaffray EG, Hay RT (2011) The SUMO protease SENP6 is a direct regulator of PML nuclear bodies. Mol Biol Cell 22(1):78–90. https://doi.org/10.1091/mbc. E10-06-0504 15. Posch M, Khoudoli GA, Swift S, King EM, Deluca JG, Swedlow JR (2010) Sds22 regulates aurora B activity and microtubulekinetochore interactions at mitosis. J Cell Biol 191(1):61–74. https://doi.org/10.1083/jcb. 200912046 16. Hands KJ, Cuchet-Lourenco D, Everett RD, Hay RT (2014) PML isoforms in response to arsenic: high-resolution analysis of PML body structure and degradation. J Cell Sci 127 (Pt 2):365–375. https://doi.org/10.1242/ jcs.132290 17. Karimi M, Inze D, Depicker A (2002) GATEWAY vectors for agrobacterium-mediated plant transformation. Trends Plant Sci 7 (5):193–195 18. Pontes O, Li CF, Costa Nunes P, Haag J, Ream T, Vitins A, Jacobsen SE, Pikaard CS (2006) The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center. Cell 126(1):79–92. https://doi.org/10.1016/j.cell.2006.05.031 19. Li CF, Pontes O, El-Shami M, Henderson IR, Bernatavichute YV, Chan SW, Lagrange T, Pikaard CS, Jacobsen SE (2006) An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana. Cell 126(1):93–106. https://doi.org/10.1016/j.cell.2006.05.032 20. Oka Y, Matsushita T, Mochizuki N, Suzuki T, Tokutomi S, Nagatani A (2004) Functional analysis of a 450-amino acid N-terminal fragment of phytochrome B in Arabidopsis. Plant Cell 16(8):2104–2116. https://doi.org/10. 1105/tpc.104.022350 21. Huang S, Deerinck TJ, Ellisman MH, Spector DL (1994) In vivo analysis of the stability and transport of nuclear poly(A)+ RNA. J Cell Biol 126(4):877–899

Chapter 3 Analysis of Shade-Induced Hypocotyl Elongation in Arabidopsis Yetkin C¸aka Ince and Vinicius Costa Galva˜o Abstract The presence of neighbor or overtopping plants is perceived by changes in light quality, which lead to several growth and developmental changes known as shade avoidance syndrome (SAS). Among them, the analysis of hypocotyl elongation is an important SAS physiological output that has been successfully used to investigate photoreceptors and downstream signaling components. Here we describe the experimental setup and growth conditions used to investigate photoreceptors and their signaling mechanisms through the analysis of hypocotyl elongation in laboratory, using simulated low R/FR ratio, low blue light, and true/deep shade conditions. Key words Hypocotyl, Low R/FR ratio, Low blue, Shade avoidance, True shade

1

Introduction Growing in shaded environments, such as under dense canopy or in communities surrounded by many competitors, constitutes a major threat for plant growth and development. The presence of neighboring plants or overtopping canopy elicits multiple morphological and developmental responses collectively known as the shade avoidance syndrome (SAS) [1–3]. SAS responses include hypocotyl, stem, and petiole elongation [4–7], hyponastic leaf movement [8, 9], enhanced phototropic response [10], and premature transition to flowering [11]. Such responses are triggered by changes in the light environment via the activity of photoreceptors through two different mechanisms. The first is a consequence of the reduction in red/ far-red ratio (low R/FR ratio) perceived predominantly via the phytochrome B (phyB) photoreceptor because of reflected FR light from neighboring plants (known as neighbor detection). In addition to neighbor detection, plants growing under dense vegetation access very limited light, which is strongly depleted in R and blue (B) wavelengths absorbed for photosynthesis at the canopy,

Ruohe Yin et al. (eds.), Plant Photomorphogenesis: Methods and Protocols, Methods in Molecular Biology, vol. 2297, https://doi.org/10.1007/978-1-0716-1370-2_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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and enriched in transmitted FR light. This condition is known as true shade or deep shade and leads to the combined effect of low R/FR ratio perceived by phytochromes and low B perceived by cryptochromes (cry) [12–15]. Experiments to investigate the role of plant photoreceptors and signaling components have been successfully performed in laboratory conditions using simulated shade in plant growth incubators [16]. Although these experiments by no means substitute the experiments in natural conditions, they have been particularly useful to uncover the molecular mechanisms underlying specific variables, such as light and temperature. In particular, the analysis of hypocotyl elongation of the model plant Arabidopsis thaliana is a very convenient physiological output to dissect photoreceptors function and signaling pathways in simulated shade. Such experiments are easily performed using relatively simple and inexpensive lab resources and require relatively restricted space. Nevertheless, despite its simplicity, hypocotyl elongation is a highly sensitive trait affected by many external factors. As such, experiments should be carefully planned and controlled in order to prevent variability between samples either within or between experiments. Here we describe a protocol routinely used for the analysis of hypocotyl elongation in the plant model Arabidopsis using simulated shade environment in plant growth incubators. More specifically, we describe the experimental setup to investigate the relative contribution of phytochromes (low R/FR ratio), cryptochromes (low B), and the co-action of both photoreceptors in true shade (low R/FR ratio and low B). Alternatively, this protocol can be easily adapted for hormones and/or chemicals treatments, which are important factors mediating SAS downstream of photoreceptors [1, 5, 10, 13, 14, 17].

2

Materials 1. 1.5 mL tubes. 2. 1000 μL tips and pipette. 3. 100% ethanol and 70% ethanol (v/v) supplemented with 0.01% Triton-X100. 4. Rotating wheel. 5. Sterile hood. 6. Sterile square petri dish (100  100  20 mm). 7. Sieve for seed size selection (250, 280 and 300 μm). 8. Micropore tape. 9. Autoclave. 10. Scissors and forceps.

Hypocotyl Elongation in Shade

23

11. Solid 1/2 strength Murashige and Skoog (MS/2) plant growth medium: 2.2 g MS salt mixture, 0.5 g MES, adjust pH to 5.7 with NaOH and complete to 1000 mL with deionized water. Add 1.6% (w/v) plant agar for vertical plates and 0.8% for horizontal plates (see Note 1). Autoclave 121  C at 1.5 atm for 20 min and store at room temperature (RT) or at 4  C (see Note 2). 12. Microwave. 13. Square plate racks for experiments at vertical position (custommade, Fig. 1). 14. Nylon net filters 160 μm, 9  9 cm (see Note 3). 15. CLF Climatics plant growth incubators equipped with fluorescent light bulbs and FR light-emitting diodes (LED) light source (see Note 4).

Fig. 1 Spectra of white light (high R/FR ratio), neighbor detection (low R/FR ratio), low B, and true shade (low R/FR ratio and low B) light conditions. Measurements were performed with OceanOptics USB2000+ spectrometer in CLF incubators equipped with fluorescent light bulbs and FR LED light sources. Blue light depletion in low B and true shade were achieved using two layers of yellow polyester LEE filter

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Yetkin C¸aka Ince and Vinicius Costa Galva˜o

16. Infrared light source (see Note 5). 17. Yellow polyester filter (LEE Filters, 010 Medium Yellow, 002–199). 18. OceanOptics USB2000+ spectrometer. 19. iButton® Hygrochron Temperature/Humidity Logger. 20. IR or regular digital camera (see Note 5). 21. Computer software for image analysis.

3

Methods In this section we describe a convenient method for the analysis of hypocotyl elongation in Arabidopsis in three different shade light environments using plates at the vertical position (see Note 6 for comments on horizontal plates). We recommend avoiding conditions variation between experiments in order to minimize eventual phenotypic variation.

3.1 MS/2 Plates Preparation

1. Prepare MS/2 plates immediately before sowing to prevent medium dehydration. Either starting from freshly autoclaved or melting stored MS/2 medium (1.6% agar) in microwave (see Note 1), cool it down to 50–60  C using a water bath. 2. Pour 50 mL MS/2 medium into sterile square petri dish and remove bubbles using a pipette in order to ensure a homogeneous medium surface. Allow it to solidify for 15–20 min under the sterile hood and cover it immediately after solidification to prevent dehydration. 3. For experiments using plates at the vertical position, place one autoclaved nylon mesh (9  9 cm) on the solidified medium using sterile forceps and ensure that no air bubbles are present between nylon mesh and the MS/2 medium (see Note 6). Remove bubbles using a sterile flat-tip forceps.

3.2 Seeds Sterilization and Plating

1. After harvesting, sieve seeds and sort them out by size (usually 250, 280, and 300 μm). Discard the seeds that are not within this size range and plant debris before proceeding to sterilization (see Note 7). 2. For seed surface sterilization, add 750 μL 70% ethanol (v/v) supplemented with 0.01% Triton-X100 in 1.5 mL tubes and incubate for 20 min in rotating wheel at RT. 3. Spin down the seeds and remove 70% ethanol (v/v) with 0.01% Triton-X100. Add 750 μL 100% ethanol and incubate using a rotating wheel for 10 min at RT. 4. Remove the 100% ethanol and keep the seeds in sterile hood until they are completely dry.

Hypocotyl Elongation in Shade

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Fig. 2 Overview of the experimental setup used for the analysis of hypocotyl elongation. After stratification of seeds for 3 day at 4  C in complete darkness, plates are transferred to WL (high R/FR ratio) for de-etiolation for 4 days. Finally, plates are either transferred to different light conditions (low R/FR ratio, low B, or true shade) or kept in WL (high R/FR ratio) as control for 3 days

5. Add 750 μL sterile H2O and let seeds to swell for 25–30 min at RT before sowing. 6. Take 300 μL seeds using 1000 μL pipette and sow ~50 seeds per biological replicate per condition (WL control and light treatment) by touching with the tip the nylon net filter. Leave at least 1 cm horizontally and 1 cm vertically between seeds (Figs. 1c and 2a) (see Note 8). 7. Remove excess seeds using the pipette tip or sterile toothpick. 8. Seal the plates with micropore tape and wrap them with several layers of aluminum foil (or store in complete darkness) for stratification at 4  C for 3 days. 3.3 Growth Incubator Preparation and Seedlings De-etiolation

3.3.1 Germination and De-etiolation

After seeds stratification, the hypocotyl elongation experiment is performed in 2 steps: the first consists of seed germination and seedling de-etiolation in normal light (WL, high R/FR ratio) for 4 days. After de-etiolation the light treatment is performed using one of the following conditions in addition to the WL control for 3 additional days (Figs. 3 and 4): (a) neighbor detection (low R/FR ratio), (b) low B, and (c) true shade (low R/FR ratio and low B) (Table 1). 1. Set up the growth incubator photoperiod to long-days (16 h light, 8 h dark) and white light intensity of 130 μmol m 2 s 1 and R/FR ratio of 1.1–1.2 (high R/FR) using the spectroradiometer (Fig. 3). Set constant day and night temperature to 21  C. 2. (Optional) Because of the high sensitivity of hypocotyl elongation to temperature we recommend checking the temperature before starting the experiment, as well as monitoring it throughout the experiment using data loggers (e.g., iButton®

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Fig. 3 Custom-made square plate rack used for experiments performed at the vertical position. The dimensions of wooden stand (black) are 14 cm  58 cm. On top of the stand 7 custom-made plastic plate holders are placed with a distance of 6 cm

Hygrochron Temperature/Humidity Logger) with sampling rate of at least one measurement every hour. 3. Transfer the plates directly from stratification at 4  C to the plant growth incubator at ZT 0–2 and place them vertically on plates rack. Germinate and de-etiolate seedlings for 4 days in WL (high R/FR ratio) (Fig. 4). 4. (Optional) After 4 days de-etiolation, take pictures of the plates (at ZT 0–2) before starting the light treatment (Fig. 4) (see Note 5). 5. (Optional) For experiments using hormones and/or chemical treatments. After taking pictures, gently transfer meshes and seedlings using two sterile forceps to freshly prepared MS/2 plates containing the appropriate filtered hormone and/or

Hypocotyl Elongation in Shade

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Fig. 4 Representative pictures of seedlings at the end of light treatment (7 days) showing the position of seedlings on the nylon mesh (a) and the distance designated as hypocotyl length for measurements (two dots and dashed arrow (b)

Table 1 Light intensities (μE m 2 s 1) of photosynthetically active radiation (PAR, between 400–700 nm), blue (400–500 nm), Red (640–700 nm), far-red (700–760 nm), and R/FR ratio of the light treatment using OceanOptics USB2000+ spectrometer in CLF incubators equipped with fluorescent light bulbs and FR LED light sources. Blue light depletion in low B and true shade were achieved using two layers of yellow polyester LEE filter Condition

PAR

Blue

Red

Far-red

R/FR ratio

White light

96.12

21.10

5.28

4.95

1.08

Low R/FR

92.91

20.03

5.01

63.58

0.08

Low B

52.83

2.26

3.08

4.13

0.75

Low B + low R/FR

54.35

2.34

4.39

39.79

0.11

chemical under sterile hood. Be careful to not dislodge the seedlings on the mesh during transfer (see Note 9). 6. Return plates to the plant growth incubators for light treatment. 3.4

Shade Treatment

All light treatments described below should be started at ZT 0–2 in order to allow sufficient hypocotyl growth for 3 additional days. In addition to the specific shade treatment (neighbor detection, low B, or true shade) we use the same number of seedlings for the WL control (Fig. 4). Before returning plates to the incubators, start

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light treatment as described below and confirm it using a spectroradiometer. 3.4.1 Neighbor Detection (Low R/FR Ratio)

The neighbor detection experiments (low R/FR ratio) are performed using the same WL intensity (130 μmol m 2 s 1) used for de-etiolation. Light treatment is performed using continuous supplemental FR in order to reach R/FR ratio of approximately 0.1 (Fig. 3; see Note 10).

3.4.2 Low Blue (Low B)

The low B experiments are performed using yellow polyester filter for depleting the blue light wavelengths (Fig. 3). Maintain the same WL intensity used for de-etiolation (130 μmol m 2 s 1) and cover fluorescent light bulbs with 2 layers of yellow polyester LEE filters. Ensure that the filter is not dislodged during the experiment.

3.4.3 True Shade (Low B and Low R/FR Ratio)

True shade experiments are performed using a combination of supplemental FR light and depletion of blue light wavelengths with yellow polyester filter. To this end, cover the fluorescent light bulbs with two layers of yellow filter and switch on the continuous FR light in order to reach 0.15–0.20 R/FR ratio.

3.5 Hypocotyl Measurements

1. After 3 days of light treatment, take pictures of plates in control WL and shade treatment (Fig. 2a). 2. Annotate 7-day-old seedlings that are well positioned for the measurements. 3. Measure seedlings hypocotyl length (4- and 7-day-old) using image analysis software as the length between shoot apex and beginning of the root (see Note 11) (Fig. 2b). 4. The hypocotyl length can be represented either as final length in after light treatment (final length in day 7) or the elongation in response to light treatment (the difference between elongation in day 4 and 7). For hypocotyl elongation, calculate the elongation of individual seedlings and the average of each genotype/condition (see Note 12). 5. Different methods can be used for the statistical analysis. For single genotype control vs. treatment experiments, the statistical Student’s t test is widely used. Nevertheless, for analyses of several genotypes and conditions, we recommend ANOVA together with Tukey’s HSD test.

4

Notes 1. Hypocotyl measurements are performed using plates either at vertical or horizontal positions. We prefer using plates at vertical position because it offers several advantages: (a) it does not

Hypocotyl Elongation in Shade

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require seedling manipulation for pictures, (b) it is possible to take pictures of the same seedlings at different time points, and (c) it requires less space. It is important to note, however, that the plates for experiments at vertical position are prepared with MS/2 medium with 1.6% plant agar and 0.8% for horizontal plates. 2. Solid MS/2 medium is stored at RT or at 4  C for several weeks after autoclaving. Melt the medium using a microwave and pour the plates prior to sowing. Cool down the medium to 50–60  C in water bath before adding hormones and/or chemicals. 3. Nylon meshes are very convenient for hypocotyl elongation experiments using plates at the vertical position as it prevents hypocotyl bending due to contact with the MS/2 medium surface. Moreover, meshes allow the fast transfer to fresh plates containing MS/2 medium supplemented with hormones and/or chemicals. It is possible to reuse meshes for several experiments after washing them several times with deionized water and autoclaving (121  C at 1.5 atm for 20 min). Nevertheless, we strongly recommend using a mesh for a specific chemical treatment to avoid eventual carry over from previous experiments. 4. There are different FR light-emitting diodes (LED) sources commercially available. For our experiments we use Gro LEDs peaking at 748 nm. 5. Infrared light source is required for taking pictures using IR camera. Pictures taken using this system allow a good contrast between hypocotyls and the medium, which makes easier to use automated software specifically written for hypocotyl measurements. However, this is not a strict requirement for imaging; regular cameras are also useful for measurements using software such as ImageJ. Otherwise, it is possible to customize regular cameras using infrared light source. 6. It is possible to perform hypocotyl elongation experiments using plates at the horizontal position. Such experiments are performed using MS/2 medium with 0.8% plant agar without nylon net filters and pictures taken (or plates scanned) after transferring and stretching seedlings onto agar 0.8%. 7. Avoid using old seeds and/or coming from different sources. Preferably, use freshly propagated seeds of plants growing at the same time and at the same condition. After harvesting, sieve seeds for size sorting and removing plant debris reduce the phenotypic variability arising from seeds that have not been properly developed and it facilitates sowing by pipetting.

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8. It is also convenient to sow seeds using a toothpick. After seeds sterilization, use the tip of an autoclaved wooden toothpick soaked into sterile H2O to sow. 9. Filter sterilized (Minisart® High Flow Syringe Filter 16,532-K, 0.22 μm Polyethersulfone) hormones and/or chemicals can be added to the MS/2 media after cooling down. Hormones and chemicals stock solutions should be prepared and stored according to manufacturer instructions. 10. We recommend checking the light intensities using a spectroradiometer before every experiment. 11. Measurement of hypocotyl length only on seventh day of growth may be enough to compare genotypes of which growth are the same for the first 4 days. 12. We use a customized MATLAB script developed in the Fankhauser lab to measure hypocotyl lengths. However, there are several software available to measure hypocotyl lengths including ImageJ and HYPOTrace [18].

Acknowledgments We are very grateful to Prof. Christian Fankhauser for the time VCG and YI spent preparing the manuscript and using lab equipment for figures illustration. We thank Adriana Beatriz Arongaus, Alessandra Boccaccini, Anne-Sophie Fiorucci, Ana Lopez Vazquez and Martina Legris for comments on the manuscript. We thank Oliver Michaud for assistance plotting light spectra in MatLab. VCG was supported by EMBO long-term fellowship (ALTF 293-2013). References 1. de Wit M, Galvao VC, Fankhauser C (2016) Light-mediated hormonal regulation of plant growth and development. Annu Rev Plant Biol 67:513–537 2. Casal JJ (2013) Photoreceptor signaling networks in plant responses to shade. Annu Rev Plant Biol 64:403–427 3. Fiorucci AS, Fankhauser C (2017) Plant strategies for enhancing access to sunlight. Curr Biol 27(17):R931–R940 4. Lorrain S, Allen T, Duek PD, Whitelam GC, Fankhauser C (2008) Phytochrome-mediated inhibition of shade avoidance involves degradation of growth-promoting bHLH transcription factors. Plant J 53(2):312–323 5. Li L, Ljung K, Breton G, Schmitz RJ, PrunedaPaz J, Cowing-Zitron C, Cole BJ, Ivans LJ,

Pedmale UV, Jung HS, Ecker JR, Kay SA, Chory J (2012) Linking photoreceptor excitation to changes in plant architecture. Genes Dev 26(8):785–790 6. Kozuka T, Kobayashi J, Horiguchi G, Demura T, Sakakibara H, Tsukaya H, Nagatani A (2010) Involvement of auxin and brassinosteroid in the regulation of petiole elongation under the shade. Plant Physiol 153 (4):1608–1618 7. de Wit M, Ljung K, Fankhauser C (2015) Contrasting growth responses in lamina and petiole during neighbor detection depend on differential auxin responsiveness rather than different auxin levels. New Phytol 208 (1):198–209

Hypocotyl Elongation in Shade 8. Michaud O, Fiorucci AS, Xenarios I, Fankhauser C (2017) Local auxin production underlies a spatially restricted neighbor-detection response in Arabidopsis. Proc Natl Acad Sci U S A 114(28):7444–7449 9. Pantazopoulou CK, Bongers FJ, Kupers JJ, Reinen E, Das D, Evers JB, Anten NPR, Pierik R (2017) Neighbor detection at the leaf tip adaptively regulates upward leaf movement through spatial auxin dynamics. Proc Natl Acad Sci U S A 114(28):7450–7455 10. Goyal A, Karayekov E, Galvao VC, Ren H, Casal JJ, Fankhauser C (2016) Shade promotes phototropism through phytochrome B-controlled auxin production. Curr Biol 26 (24):3280–3287 11. Halliday KJ, Koornneef M, Whitelam GC (1994) Phytochrome B and at least one other phytochrome mediate the accelerated flowering response of Arabidopsis thaliana L to low red/far-red ratio. Plant Physiol 104 (4):1311–1315 12. de Wit M, Keuskamp DH, Bongers FJ, Hornitschek P, Gommers CMM, Reinen E, Martinez-Ceron C, Fankhauser C, Pierik R (2016) Integration of phytochrome and cryptochrome signals determines plant growth during competition for light. Curr Biol 26 (24):3320–3326 13. Pedmale UV, Huang SC, Zander M, Cole BJ, Hetzel J, Ljung K, Reis PAB, Sridevi P, Nito K,

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Nery JR, Ecker JR, Chory J (2016) Cryptochromes interact directly with PIFs to control plant growth in limiting blue light. Cell 164 (1–2):233–245 14. Keuskamp DH, Sasidharan R, Vos I, Peeters AJ, Voesenek LA, Pierik R (2011) Blue-lightmediated shade avoidance requires combined auxin and brassinosteroid action in Arabidopsis seedlings. Plant J 67(2):208–217 15. Keller MM, Jaillais Y, Pedmale UV, Moreno JE, Chory J, Ballare CL (2011) Cryptochrome 1 and phytochrome B control shade-avoidance responses in Arabidopsis via partially independent hormonal cascades. Plant J 67 (2):195–207 16. Fankhauser C, Casal JJ (2004) Phenotypic characterization of a photomorphogenic mutant. Plant J 39(5):747–760 17. Tao Y, Ferrer JL, Ljung K, Pojer F, Hong F, Long JA, Li L, Moreno JE, Bowman ME, Ivans LJ, Cheng Y, Lim J, Zhao Y, Ballare CL, Sandberg G, Noel JP, Chory J (2008) Rapid synthesis of auxin via a new tryptophandependent pathway is required for shade avoidance in plants. Cell 133(1):164–176 18. Wang LY, Uilecan IV, Assadi AH, Kozmik CA, Spalding EP (2009) HYPOTrace: image analysis software for measuring hypocotyl growth and shape demonstrated on Arabidopsis seedlings undergoing photomorphogenesis. Plant Physiol 149(4):1632–1637

Chapter 4 Isolation of UVR8 Protein Complexes Yan Liu and Xi Huang Abstract The fundamental mechanism of light regulated plant development involves photoreceptors and their interacting proteins which act as light signaling intermediate factors. In Arabidopsis thaliana, UV RESIS TANCE LOCUS 8 (UVR8) is responsible for the perception and the initiation of UV-B light signal. To data, only a few proteins have been revealed as the components of UVR8 protein complexes, limiting our understanding of the molecular mechanisms by which UV-B light input is interpreted to orchestrate numerous physiological outputs in plants. Therefore, it is necessary to isolate and identify the components of UVR8 protein complexes at a global level, in order to uncover novel UV-B light signaling factors and pathways. In this chapter, we provide a protocol for the isolation of UVR8 protein complexes. Basically, co-immunoprecipitation (co-IP) assay is employed to enrich UVR8 and its associating proteins in vivo. This method can be used coupling with specific treatments and is compatible with successive biochemical analysis. Key words UV-B, UVR8, Protein complex, Light signaling, Co-immunoprecipitation

1

Introduction Sessile plants utilize light not only as the energy source for photosynthesis but also as an informational cue that regulates a variety of developmental programs via light signal transduction. Light signals are perceived by multiple photoreceptors that monitor the quality, quantity, duration, and direction of the ambient light [1]. As an essential developmental process at the seedling stage, photomorphogenesis is physiologically characterized by the development of short hypocotyls, open and expanded cotyledons, and green chloroplasts [2]. Although UV-B light (280–315 nm) makes up less than 0.5% of the solar energy that reaches the earth’s surface, appropriate UV-B irradiation exerts beneficial effects on plants via promoting various biological processes including photomorphogenesis and stress acclimation [3, 4]. Initially identified in Arabidopsis thaliana, UV RESISTANCE LOCUS 8 (UVR8) is a long-sought UV-B light receptor and is conserved among plant species [5]. Upon irradiation with photomorphogenic UV-B, dimeric UVR8 perceives

Ruohe Yin et al. (eds.), Plant Photomorphogenesis: Methods and Protocols, Methods in Molecular Biology, vol. 2297, https://doi.org/10.1007/978-1-0716-1370-2_4, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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UV-B light via its own tryptophan-based chromophore, and monomerizes by disrupting the critical intermolecular bonds shaped by its own arginine residues [6, 7]. The monomeric UVR8 interacts directly with CONSTITUTIVELY P HOTOMORPHOGENIC 1 (COP1) to initiate UV-B signaling [3]. COP1 functions in the form of COP1– SUPPRESSOR OF PHYA (SPA) complexes. They serve as E3 ubiquitin ligases or substrate receptors of CULLIN4–DAMAGED DNA BINDING PROTEIN 1 (CUL4–DDB1)-based E3 ubiquitin ligases to target photomorphogenesis promoting transcription factors including E LONGATED HYPOCOTYL 5 (HY5) for degradation, in order to repress light signaling [8, 9]. In this manner, COP1 is a central repressor of traditional photomorphogenesis triggered by far-red and visible light. Intriguingly, upon UV-B irradiation, COP1 directly interacts with monomerized UVR8 to promote the nuclear accumulation of UVR8 [3, 5, 10, 11]. Their affinity determines the efficiency of photomorphogenic UV-B signaling output [12]. Meanwhile, the association of UVR8-COP1 complex leads to the dissociation of COP1-SPA complex from CUL4-DDB1. This reorganization of protein complexes enables a functional switch of COP1 from repressing to promoting photomorphogenesis [13]. Hence, the assembly of UV-B induced UVR8 protein complexes is the root of UVR8 mediated UV-B light signal transduction. REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2, two UV-B inducible and UVR8 interacting proteins, repress UV-B induced photomorphogenesis via negative feedback regulation [14]. Under UV-B light, they disrupt the UVR8-COP1 interaction [15, 16], mediate HY5 degradation in the form of CUL4-DDB1-RUP1/RUP2 E3 ligase, and are targeted by COP1 for degradation [17]. When the UV-B light is removed, RUP1 and RUP2 facilitate UVR8 redimerization and inactivation [18]. In addition to the initiation and termination of UV-B light signaling, UVR8 has been recently found to be directly involved in gene expression regulation via interacting with multiple transcription factors. Based on yeast two-hybrid screen, three transcription factors, WRKY DNA-BINDING PROTEIN 36 (WRKY36), BRI-EMS-SUPPRESSOR 1 (BES1), and BES1-INTERACTING MYC-LIKE 1 (BIM1), have been characterized as UVR8 interacting proteins. Unlike its UV-B specific interaction with COP1, UVR8 physically interacts with WRKY36 in the nucleus in a UV-B independent manner. Nuclear localized UVR8 affects the DNA binding activity of WRKY36. UVR8 represses the binding of WRKY36 to the HY5 promoter to modulate HY5 transcription and hypocotyl elongation, establishing UVR8-WRKY36-HY5 as a novel UV-B signaling pathway [19]. UVR8 can also physically interact with BIM1 and the functional dephosphorylated BES1.

Isolation of UVR8 Protein Complexes

35

UV-B light induces the accumulation of the UVR8-BES1 complex in the nucleus where UVR8 represses the DNA binding activity of BES1 and BIM1 to inhibit hypocotyl elongation and plant growth. Therefore, the UVR8-BES1 complex functions as a signaling module for the crosstalk of light and BR pathways in plant growth and development [20]. In the above studies, a variety of approaches have been developed to detect UV-B dependent or independent interactions of UVR8 with other proteins in vitro and in vivo, including yeast two-hybrid (Y2H) assay, luciferase complementation imaging (LCI) assay, bimolecular fluorescence complementation (BiFC) assay, in vitro pull-down assay, and co-immunoprecipitation (co-IP) assay. Particularly, co-IP assay is an effective strategy to analyze the formation of protein complexes in vivo [21, 22]. It can be carried out coupled with specific light conditions or chemical treatments, and can be followed by SDS-PAGE, immunoblotting, and mass spectrometry. We describe here a protocol for the isolation of UVR8 protein complexes formed with or without photomorphogenic UV-B irradiation. In this specific case, YFP-UVR8 protein extracted from the reported proUVR8-YFP-UVR8/uvr8–6 transgenic seedlings [13] is used as a bait to co-immunoprecipitate its associating proteins (Fig. 1). FLAG-UVR8 constitutively overexpressing UVR8 is an alternative suitable material to co-immunoprecipitate UVR8 protein complexes [13]. Representative results can also be referred to our previous studies [12, 13].

Fig. 1 Detection of UVR8–COP1 complex by co-IP assay. In vivo co-IP assays using 4-day-old seedlings grown under –UV-B and + UV-B by anti-GFP antibodies. Immunoblot analysis was performed by anti-GFP and antiCOP1 antibodies. Anti-RPN6 was used as a un-immunoprecipitated and loading control

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2 2.1

Yan Liu and Xi Huang

Materials Seed Sterilization

2.2 Protein Extraction

15% Sodium hypochlorite solution. 1. 1 M Tris–HCl, pH 7.5. 2. 5 M NaCl. 3. 500 mM EDTA. 4. 100 mM Na3VO4. 5. 1 M β-glycerophosphate. 6. 1 M NaF. 7. 20% Tween20. 8. 100 mM PMSF. 9. Lysis buffer: 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 2 mM Na3VO4, 25 mM β-glycerophosphate, 10 mM NaF, 0.05–0.1% Tween 20. Store at 4  C. Add 1 protease inhibitor and 1 mM PMSF before use (see Note 1).

2.3 Beads Washing and Elution

1. 1  TBS. 2. Acid-eluting solution: Dissolve 0.75 g glycine (FW: 75.07) and 2 mL of 5 M NaCl in about 90 mL of Milli-Q water, and adjust pH to 2.5 with about 330 μL of HCl to get a 100 mL of working solution. 3. Neutralization buffer: Dissolve 24.22 g Tris in about 90 mL of Milli-Q water, and adjust pH to 9–9.5 with approximately 1.8 mL of HCl, and then fulfill with Milli-Q water to 100 mL. 4. 5SDS protein loading buffer: Add 5 mL of 0.5 M Tris–HCl, pH 6.8, 2.5 mL of glycerol, 1 g SDS, 0.5 mL of β-mercaptoethanol, 1 mL of 1% Bromo Phenol Blue, and 0.771 g DTT, and fulfill with Milli-Q water to 10 mL.

3

Methods

3.1 Preparation of Plant Materials

1. Prepare 50 μL of mature and dry Arabidopsis seeds (producing approximately 400–600 mg of 4-day-old seedlings) for each line grown under each light condition (see Note 2). Therefore, we prepare 100 μL of proUVR8-YFP-UVR8/uvr8–6 seeds as experimental groups (50 μL for –UV-B and another 50 μL for +UV-B). The equal amount Col seeds are prepared as control groups. Surface-sterilize seeds with 1 mL of 15% sodium hypochlorite solution for no more than 12 min (see Note 2) and wash with sterile water for 6 times.

Isolation of UVR8 Protein Complexes

37

2. Sow the surface-sterilized seeds on solid Murashige and Skoog medium supplemented with 1% sucrose for biochemical assays and cold-treated at 4  C for 4 days. 3. For photomorphogenic UV-B treatment, seedlings were grown under continuous white light (3 μmol·m 2·s 1, measured by HR-350 Light Meter, Hipoint) supplemented with Philips TL20W/01RS narrowband UV-B tubes (1.5 μmol·m 2·s 1, measured by UV-297 UV-B Light Meter, HANDY) under a 350-nm cutoff (half-maximal transmission at 350 nm) filter ZUL0350 (–UV-B; Asahi spectra) or a 300-nm cutoff (half-maximal transmission at 300 nm) filter ZUL0300 (+UV-B; Asahi spectra). 4. Col and proUVR8-YFP-UVR8/uvr8–6 seedlings are grown under –UV-B and + UV-B for 4 days. Flash freeze plant tissues in liquid nitrogen, and immediately store them at 80  C, if you stop the following steps (see Note 3). 3.2 Extraction of Total Proteins

1. For each co-IP sample, grind 200–300 mg of the frozen seedlings (producing approximately 1 mg of total proteins) using mortar and pestle (see Note 4) prechilled with liquid nitrogen to fine powder, and homogenize the powder in 500 μL of ice-cold lysis buffer. 2. Incubate the lysis buffer/plant extract mixture on ice for 5–10 min (see Note 5). 3. After incubation, spin the lysis buffer/plant extracts at 13,500  g for 15 min at 4  C to spin down the cell-debris pellet, and keep the supernatant. Repeat once. 4. Determine the protein concentration by Bradford 1  Dye Reagent (Bio-Rad) (see Note 6). 5. Aliquot 50 μL of total proteins of each sample as the input fraction and check the expression of proteins of interest by immunoblot analysis.

3.3 Prewashing of Beads

1. Wash 20 μL of Dynabeads Protein G (see Note 7) for each co-IP sample twice with 1 mL of 1  TBS. 2. Wash beads with 1 mL of acid-eluting solution, and repeat once. 3. Wash beads twice with 1 mL of 1  TBS until the pH recovers to 7–7.5 (see Note 8).

3.4 Coimmunoprecipitation

1. Add 2–4 μL anti-GFP antibodies (Life Technologies) (see Note 7) to 500–1000 μg total soluble protein solution and incubate the tubes on a rotator at a slow speed for 2 h at 4  C under the same condition (–UV-B or + UV-B) (see Note 9) as where the seedlings were grown.

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2. Add 20 μL of Dynabeads Protein G (Life Technologies) to each sample and continue to incubate on a rotator at a slow speed for 2 h (see Note 10) at 4  C under the same condition (–UV-B or + UV-B) as where the seedlings were grown. 3. Pellet the Dynabeads Protein G conjugates at 4  C. Aliquot 50 μL of the supernatant as the flow-through fraction to determine the immunoprecipitation efficiency (see Note 11). 3.5 Washing and Eluting Protein from Beads

1. Wash the beads bound with UVR8 protein complexes with 1 mL of ice-cold lysis buffer (see Note 12) containing 150 mM NaCl (see Note 13), and repeat twice. 2. For acid elution, add 500 μL of acid-eluting solution and incubate for 5 min (see Note 14). Spin down the beads and collect the supernatant. Repeat once. 3. Add 30–40 μL of neutralization buffer to recover pH to 7–7.5. 4. Add 20 μL of StrataClean Resin and incubate for 5 min. 5. Spin down the resin, and resuspend in 2  SDS protein loading buffer, boil for 10 min (see Note 15), and analyze by immunoblot analysis.

4

Notes 1. The phosphatase inhibitors (Na3VO4, β-glycerophosphate and NaF), MgCl2, and proteasome inhibitors (MG132, etc.) are optional. DTT or other reducing reagents, which may affect IgG binding efficiency, is not recommended. Buffer ingredients can be optimized for each specific case. 2. Do not sterilize seeds in 15% sodium hypochlorite solution for longer than 12 min as this will damage seed coat and reduce germination ratio. Do not place more than 100 μL of dry seeds in a 1.5 mL microcentrifuge tube. Due to seed imbibition, the volume of seeds with water will get close to 1.5 mL during sterilization, making it difficult to sterilize and wash sufficiently. Prepare several 1.5 mL microcentrifuge tubes if more seeds are needed. 3. Fresh or frozen tissues can be used for the experiment. Tissues can be stored at 80  C for several months. 4. Complete disruption of plant cell walls and plasma membranes is required for total protein extraction. For plant tissue lysis, grinding with liquid nitrogen is a well-accepted method. It is important to prechill the mortar and pestle with liquid nitrogen to avoid thawing the plant materials. The grinding step should be carried out as soon as possible.

Isolation of UVR8 Protein Complexes

39

5. Make sure that grinded tissue powder thaws thoroughly in ice-cold lysis buffer before the cell debris is removed. 6. Prepare BSA samples (e.g., 1 μg, 2 μg, and 3 μg of BSA) to generate a standard curve for protein concentration determination. 7. The amount of antibodies and beads should be optimized according to their binding efficiency and the expression level of targeted proteins. 8. Check with pH paper if you work with this for the first time. 9. During co-immunoprecipitation, the –UV-B sample should be prevented from exposure to UV-B light. 10. To avoid unspecific binding and high background in subsequent immunoblot analysis, total incubation time more than 4 h is not recommended. In addition, purified antibody, clear and soluble protein extracts without cell debris, and prewashed beads will help reduce unspecific binding. 11. If the protein of interest is easily detected in the flow-through fraction, indicating low immunoprecipitation efficiency, you may adjust the amount of antibody and beads. 12. Gently invert the tubes for a couple of times before centrifugation. 13. You may optimize the washing step by increasing the concentration of NaCl (e.g., 200–500 mM NaCl). It can also help determine the complex association is tight or not. 14. Besides acid elution, alternative eluting methods can be considered in specific study cases, such as competitive elution using a peptide, and denatured elution by SDS loading buffer before boiling. 15. Though usually used for enzyme clean up, StrataClean Resin is used here to enrich the eluted proteins. References 1. Galvao VC, Fankhauser C (2015) Sensing the light environment in plants: photoreceptors and early signaling steps. Curr Opin Neurobiol 34:46–53 2. Kami C, Lorrain S, Hornitschek P, Fankhauser C (2010) Light-regulated plant growth and development. Curr Top Dev Biol 91:29–66 3. Favorry JJ, Stec A, Gruber H et al (2009) Interaction of COP1 and UVR8 regulates UV-B-induced photomorphogenesis and stress acclimation in Arabidopsis. EMBO J 28:591–601 4. Kliebenstein DJ, Lim JE, Landry LG, Last RL (2002) Arabidopsis UVR8 regulates

ultraviolet-B signal transduction and tolerance and contains sequence similarity to human regulator of chromatin condensation 1. Plant Physiol 130:234–243 5. Rizzini L, Favory JJ, Cloix C et al (2011) Perception of UV-B by Arabidopsis UVR8 photoreceptor. Science 332:103–106 6. Christie JM, Arvai AS, Baxter KJ et al (2012) Plant UVR8 photoreceptor senses UV-B by tryptophan-mediated disruption of crossdimer salt bridges. Science 335:1492–1496 7. Wu D, Hu Q, Yan Z et al (2012) Structural basis of ultraviolet-B perception by UVR8. Nature 484:214–219

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8. Osterlund MT, Hardtke CS, Wei N, Deng XW (2000) Targeted destabilization of HY5 during light-regulated development of Arabidopsis. Nature 405:462–466 9. Chen H, Huang X, Gusmaroli G et al (2010) Arabidopsis CULLIN4-damaged DNA binding protein 1 interacts with CONSTITUTIVELY PHOTOMORPHOGENIC 1-SUPPERESSOR 0F PHYA complexes to regulate photomorphogenesis and flowering time. Plant Cell 22:108–123 10. Yin R, Skvortsova MY, Loubery S (2016) COP1 is required for UV-B-induced nuclear accumulation of the UVR8 photoreceptor. Proc Natl Acad Sci U S A 113:E4415–E4422 11. Qian C, Mao W, Liu Y et al (2016) Dual-source nuclear monomers of UV-B light receptor direct photomorphogenesis in Arabidopsis. Mol Plant 9:1671–1674 12. Huang X, Yang P, Ouyang X et al (2014) Photoactivated UVR8-COP1 module determines photomorphogenic UV-B signaling output in Arabidopsis. PLoS Genet 10:e1004218 13. Huang X, Ouyang X, Yang P et al (2013) Conversion from CUL4-based COP1 SPA E3 apparatus to UVR8-COP1-SPA complexes underlies a distinct biochemical function of COP1 under UV-B. Proc Natl Acad Sci U S A 110:16669–16674 14. Gruber H, Heijde M, Heller W et al (2010) Negative feedback regulation of UV-Binduced photomorphogenesis and stress acclimation in Arabidopsis. Proc Natl Acad Sci U S A 107:20132–20137

15. Ouyang X, Huang X, Jin X et al (2014) Coordinated photomorphogenic UV-B signaling network captured by mathematical modeling. Proc Natl Acad Sci U S A 111:11539–11544 16. Yin R, Arongaus AB, Binkert M, Ulm R (2015) Two distinct domains of the UVR8 photoreceptor interact with COP1 to initiate UV-B signaling in Arabidopsis. Plant Cell 27:202–213 17. Ren H, Han J, Yang P et al (2019) Two E3 ligases antagonistically regulate the UV-B response in Arabidopsis. Proc Natl Acad Sci USA doi 116:4722. https://doi.org/10. 1073/pnas.1816268116 18. Heide M, Ulm R (2013) Reversion of the Arabidopsis UV-B photoreceptor UVR8 to the homodimeric ground state. Proc Natl Acad Sci U S A 110:1113–1118 19. Yang Y, Liang T, Zhang L et al (2018) UVR8 interacts with WRKY36 to regulate HY5 transcription and hypocotyl elongation in Arabidopsis. Nat Plants 4:98–107 20. Liang T, Mei S, Shi C et al (2018) UVR8 interacts with BES1 and BIM1 to regulate transcription and photomorphogenesis in Arabidopsis. Dev Cell 44:513–523 21. Dwane S, Kiely PA (2011) Tools used to study how protein complexes are assembled in signaling cascades. Bioeng Bugs 2:247–259 22. Markham K, Bai Y, Schmitt-Ulms G (2007) Co-immunoprecipitations revisited: an update on experimental concepts and their implementation for sensitive interactome investigations of endogenous proteins. Anal Bioanal Chem 389:461–473

Chapter 5 Phenotypic Study of Photomorphogenesis in Arabidopsis Seedlings Chuanwei Yang, Famin Xie, and Lin Li Abstract Light is one of the most important environmental factors, serving as the energy source of photosynthesis and a cue for plant developmental programs, called photomorphogenesis. Here, we provide a standardized operation to measure physiological parameters of photomorphogenesis, including in hypocotyl length, cotyledon size, and anthocyanin content. Key words Photomorphogenesis, Hypocotyl lengths, Cotyledon sizes, Anthocyanin contents

1

Introduction Light is crucial for plants. Besides providing the source of energy for photosynthesis, light also acts as informational signal to trigger photomorphogenic development processes, including seed germination, seedling de-etiolation, leaf expansion, stem elongation, shade avoidance response, circadian rhythms, flowering time, and so on [1–3]. The light spectrum ranging from UV-B to far-red are perceived by an array of photoreceptors (UVR8, cryptochromes, and phytochromes) and interpreted by a series of signaling intermediate factors for final changes in plant architecture [4, 5]. The remarkable phenotypic changes of young seedling from skotomorphogenesis (etiolation) to photomorphogenesis (de-etiolation) are inhibiting hypocotyl elongation, promoting growth of the cotyledons, opening of the apical hook, and accumulating anthocyanin [6]. Depending on the biology scientific problems to be solved, various light intensities or different light qualities may need to be set up. In general, monochromatic light (blue/red/far-red) can induce the de-etiolation process. With the increase of light intensity, the effect of photomorphogenesis is more obvious, such as shorter hypocotyl and more anthocyanin contents [6].

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To ensure reproducibility and comparability of the obtained data, it is crucial to set up stable light sources and do the standardized operation. Usually, a chamber with different individually programmable light-emitting diode (LED) levels could provide diverse monochromatic color (blue, red, far-red) with the different light intensity. The photoreceptor mutants exhibit a lightinsensitive phenotype, such as phyA mutants, phyB mutants, and cry mutants are blind to far-red, red, and blue light, respectively [1]. Therefore, when measuring the phenotype in different light conditions, the photoreceptor mutants are usually used as a control. Here we illustrate how to set up light parameters and the methods to measure physiological parameters of seedlings including in hypocotyl length, cotyledon size, and anthocyanin content.

2 2.1

Materials Plant Culture

1. Wide type or mutants of Arabidopsis seeds. 2. Ethanol: 100% and 75%. 3. Murashige and Skoog (MS). 4. Agar. 5. Filter papers. 6. Tricolor LED chamber. 7. Illuminometer.

2.2 Measurement Physiological Parameters

1. Scanner. 2. ImageJ software. 3. Tweezers. 4. HCl, methanol, and chloroform. 5. Spectrophotometric measurements.

3 3.1

Methods Growth Condition

Growth of Arabidopsis in experimental settings such as examination on shoot phenotypes of young seedlings is usually conducted on solid media. If a few seed lines need to be sterilized at a time, 75% ethanol sterilization is a practical method. Larger numbers of lines can be sterilized easily using chlorine gas or liquid bleach with less manipulation. This section described here is to use 75% ethanol sterilization and the medium (MS agar media) in plate for sterile growth conditions. Adaptation to other sterile formats is straightforward, and most experimental additives (as hygromycin) can be easily incorporated in the preparation (see Note 1)

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1. Wide type or mutants of Arabidopsis seeds were ethanolsterilized. Mix with 75% alcohol about 15 min and transfer seeds to clean filter papers with 100% alcohol. After sterilization, spread the dry seeds directly on 1/2 MS medium ( pH ¼ 5.7) plate with 0.8–1% agar and without sucrose (see Note 2). 2. Put the plates in dark for 4 days at 4  C to vernalize seeds (see Note 3). 3. A poststratification treatment consisting of a 1.5 h white light or red light was provided before incubation at 22  C in dark or monochromatic light conditions for 4 days (see Note 4). 4. When you detect the phenotypes of Arabidopsis under shade conditions, these plates usually should be placed under white light for 3 or more days until seedlings are fully de-etiolated, and then transferred them to shade light for another 5 days [7, 8]. 3.2 Set up the Monochromatic Light

Please refer to Fig. 1 for the selection of light intensity under different monochromatic light conditions. White or shade conditions can be simulated by different composition of three monochromatic lights. This system used to investigate shade avoidance response is to reduce the ratio of R/FR while keeping PAR constant by adding far-red light (Fig. 2) [9] (see Note 5).

3.3 Measurement Hypocotyl Length of Seedlings

At present, there is an advanced imaging system (Dynaplant; http:// www.yphbio.com/DynaPlant.asp) that can monitor the changes in hypocotyls in real time [9, 10]. Here we introduce a simpler and more universal measurement method. 1. Transfer seedlings to new agar plates gently in horizontal direction by tweezers. At least 20 cotyledons were measured in each set of experiments to analyze the hypocotyl length (see Note 6). 2. Scan the plates with seedlings on a flatbed scanner, and save the scanned image as *.tiff format. 3. Download the ImageJ software and install it properly (https:// imagej.nih.gov/ij/). Open the image file by the ImageJ software. 4. To obtain the physical length (mm) instead of the pixels value, please set the scale according to the pixel value to a known distance. Choose the “straight line” to make a line that corresponds to a known distance. Then select “Analyze—set scale” command, enter the number into “Distance in Pixels” and “Known Distance,” fill the unit in “Unit of Length,” and then choose the “Global” and click “ok.”

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Fig. 1 Phenotypes of seedlings grown in different monochromatic light. (a) Seedlings were grown under continuous red light conditions for 4 days. (b) Seedlings were grown under continuous far-red light conditions for 4 days. (c) Seedlings were grown under continuous blue light conditions for 4 days

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Fig. 2 Light spectral composition of white light and shade treatments. The continuous LED white light condition is: Red  25 μmol/m2/s; Blue  27 μmol/m2/s; and Far-red  5 μmol/m2/s. The simulated shade is: Red  25 μmol/m2/s; Blue  27 μmol/m2/s; and Far-red  60 μmol/m2/s. The ratio of R to FR is approximately 0.4

5. Choose the “Segmented Lines” button and make a line from the root/shoot junction to the branch point of petiole. And then click the “Analyze—Measure.” 6. Repeat this procedure with all seedlings in the image file. Choose “File” -"Save as” in the results window and generate an appropriate file name and save in Excel format. 7. For statistical analysis generate mean values and standard deviations from all length data and perform a Student’s t-test to evaluate if the data is significant. Data are evaluated as significantly different when the P-values are below 0.05. 3.4 Measurement Cotyledon Size of Seedlings

1. Cut off the cotyledons and position them to new agar plates gently in horizontal direction by tweezers. At least 20 cotyledons were measured in each set of experiments to analyze the cotyledon area graphically. 2. Scan the plates with seedlings on a flatbed scanner (HP 8270), and save the scanned image as *.tiff format. 3. Open the image file using the ImageJ software. 4. To obtain the physical area (mm2) instead of the pixels value, please set the scale according to the pixel value to a known distance. Choose the “straight line” to make a line that corresponds to a known distance. Then select “Analyze—set scale” command, enter the number into “Distance in Pixels” and “Known Distance,” fill the unit in “Unit of Length,” and then choose the “Global” and click “ok.” 5. Choose the “free hand selection” button and encircle the irregular cotyledons. Click on “Analyze—set measurements” and check “Area,” “Min & Max Gray value,” “Integrated density,” and “Mean Gray value” and click “ok.” Then go back to “analyze” and click “measure.”

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6. Repeat this procedure with all cotyledons in the image file. Choose “File” -"Save as” in the results window and generate an appropriate file name and save in Excel format. 7. For statistical analysis generate mean values and standard deviations from all areas data and perform a Student’s t-test to evaluate if the data is significant. Data are evaluated as significantly different when the P-values are below 0.05. 3.5 Measurement Anthocyanin Content of Seedlings

4

For the anthocyanin determinations, about 50 mg seedlings grown in different light conditions were collected, frozen in liquid nitrogen, and ground to a fine powder. Total plant pigments were extracted overnight in 0.6 mL of 1% (vol/vol) HCl in methanol. After addition of 0.2 mL of H2O, chlorophyll was extracted with an equal volume of chloroform. The quantity of anthocyanins was determined by spectrophotometric measurements of the aqueous phase (A530  A657) and normalized to the total fresh weight of tissue used in each sample.

Notes 1. Physiological experiments were repeated three times, and only a representative one is shown. 2. Before starting, make a careful plan on how many plants will be needed for doing a reliable statistical analysis of the experiments and then prepare the appropriate amounts of seeds. In this section, the medium usually does not contain sucrose, because plants grow more vigorously and quickly on media containing 1–2% of sucrose and sucrose as an energy can affect the photomorphogenesis [11, 12]. 3. Its purpose is to increase the content of the hormone gibberellin in the seeds and ensure consistent germination. 4. This section describes standard dark operations. If you need to use real dark conditions, please refer to: a poststratification treatment consisting of a 5-min R pulse (Rp) followed by 3 h dark incubation and a terminal 5-min far-red (FR) pulse (Rp + 3 h D+ FRp) was provided before incubation at 22  C in dark or monochromatic light for 4 days [13]. 5. The advantage of this approach is that plants can be exposed to low R/FR without altering other features, which is called neighbor detection. 6. The hypocotyl is very fragile and easy to break; you can clamp the cotyledon slightly to avoid damaging the hypocotyl of seedlings.

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References 1. Lymperopoulos P, Msanne J, Rabara R (2018) Phytochrome and Phytohormones: working in tandem for plant growth and development. Front Plant Sci 9:1037 2. Yang C, Li L (2017) Hormonal regulation in shade avoidance. Front Plant Sci 8:1527 3. Liang T, Yang Y, Liu H (2019) Signal transduction mediated by the plant UV-B photoreceptor UVR8. New Phytol 221:1247–1252 4. Paik I, Huq E (2019) Plant photoreceptors: multi-functional sensory proteins and their signaling networks. Semin Cell Dev Biol:30574–30578 5. Franklin KA, Whitelam GC (2005) Phytochromes and shade-avoidance responses in plants. Ann Bot 96:169–175 6. McNellis TW, Deng XW (1995) Light control of seedling morphogenetic pattern. Plant Cell 7:1749–1761 7. Huang X, Zhang Q, Jiang Y, Yang C, Wang Q, Li L (2018) Shade-induced nuclear localization of PIF7 is regulated by phosphorylation and 14-3-3 proteins in Arabidopsis. elife 7 8. Yang C, Xie F, Jiang Y, Li Z, Huang X, Li L (2018) Phytochrome a negatively regulates the shade avoidance response by increasing auxin/ indole acidic acid protein stability. Dev Cell 44 (1):29–41

9. Jiang Y, Yang C, Huang S, Xie F, Xu Y, Liu C, Li L (2019) The ELF3-PIF7 interaction mediates the circadian gating of the shade response in Arabidopsis. iScience 22:288–298 10. Zhang X, Ji Y, Xue C, Ma H, Xi Y, Huang P, Wang H, An F, Li B, Wang Y, Guo H (2018) Integrated regulation of apical hook development by transcriptional coupling of EIN3/ EIL1 and PIFs in Arabidopsis. Plant Cell 30 (9):1971–1988 11. Saijo Y, Sullivan JA, Wang H, Yang J, Shen Y, Rubio V, Ma L, Hoecker U, Deng XW (2003) The COP1-SPA1 interaction defines a critical step in phytochrome A-mediated regulation of HY5 activity. Genes Dev 17(21):2642–2647 12. Qiu Y, Li M, Kim R, Moore C, Chen M (2019) Daytime temperature is sensed by phytochrome B in Arabidopsis through a transcriptional activator HEMERA. Nat Commun 10:140 13. Leivar P, Monte E, Oka Y, Liu T, Carle C, Castillon A, Huq E, Quail PH (2008) Multiple phytochrome-interacting bHLH transcription factors repress premature seedling photomorphogenesis in darkness. Curr Biol 18 (23):1815–1823

Chapter 6 Experimental Procedures for Studying Skotomorphogenesis in Arabidopsis thaliana Huanhuan Jin, Hong Li, and Ziqiang Zhu Abstract Seedlings grown in darkness exhibit distinct morphologies comparing with light-grown seedlings. Elongated hypocotyls, closed yellow cotyledons, and the formation of apical hooks are typical characteristics for etiolated seedlings, which are collectively named skotomorphogenesis. Various plant hormones and environmental factors are essential for maintaining skotomorphogenesis. Due to the diverse morphological outcomes in etiolated seedlings grown under different treatments, studies on skotomorphogenesis are of particular importance to reveal the molecular mechanisms underlying plant response to environmental cues. Here, we detailed experimental procedures to facilitate researchers who are investigating etiolation growthrelated studies. Key words Skotomorphogenesis, Arabidopsis thaliana, Ethylene, Jasmonate

1

Introduction Most terrestrial plants start their life cycle in subterranean darkness, where they have to develop long hypocotyls and closed cotyledons to secure their meristems undamaged during soil penetration. These morphological phenotypes for seedlings grown in darkness are also named etiolation growth or skotomorphogenesis [1, 2]. Interestingly, etiolation growth is extremely plastic and sensitive to exogenous or endogenous signal cues. For example, light or jasmonate suppresses skotomorphogenesis by inhibiting hypocotyl elongation and promoting cotyledon opening [2– 5]. Blocking gibberellin biosynthesis or signaling also represses skotomorphogenesis [6], suggesting that gibberellins are positive regulators for etiolation growth. Gaseous plant hormone ethylene strongly inhibits hypocotyl and root elongation and promotes the exaggerated apical hook formation (Fig. 1) [7–9]. Therefore, etiolation growth is an elegant tool for studying the crosstalk between plant hormones and environmental factors [10]. However, etiolation growth usually emerges rapidly (less than 5 days in Arabidopsis

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Fig. 1 Phenotypes of seedlings grown under different conditions. Six-day-old etiolated seedlings grown with or without 50 μM JA or 10 μM PAC treatment. Seedlings grown on 10 μM ACC are 4 days old. For light treatment, 6-day-old seedlings grown under continuous light are shown

thaliana). Non-experts might find unequal germination or phenotypic variations when they are going to observe the phenotypes. Conclusions could not be made from these frustrated results. Another technical problem in skotomorphogenesis research is the difficulty to extract total RNA or protein samples from minimal quantities of materials. As we know, water contents in etiolated seedlings are relatively high, which makes it not easy to obtain enough RNA or protein samples from few tissues. In this chapter, we will use A. thaliana as materials to introduce our protocols on phenotype observation, RNA isolation, protein extraction, and hormone cross-talk studies.

2

Materials The A. thaliana seeds, including the wild-type control (Col-0), and mutants (hls1–1, ein3 eil1, EIN3-FLAG/ein3 eil1) were described before [10, 11]. All solutions are prepared with ultrapure water (Milli-Q Reference, attaining a sensitivity of 18 MΩ.cm at room temperature).

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2.1 Solutions for Phenotypic Analysis

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1. Bleach solution: Prepare 10% NaClO and 0.1% Triton X-100 in a 100 mL container. Keep this solution out of direct light irradiation. 2. 1-Aminocyciopropane-1-carboxylic Acid (ACC) stock solution: Dissolve 0.0101 g ACC in 10 mL of water to prepare 10 mM stock solution. Sterilize the solution by passing through the 0.22 μm filter, then aliquot it in 1 mL each and store in 20  C. 3. Methyl jasmonate (MeJA) solution: Dissolve 23 μL of MeJA in 1 mL of absolute ethanol to prepare 100 mM stock solution (see Note 1). 4. Paclobutrazol (PAC) stock solution: Dissolve 0.029 g PAC in 10 mL of water to prepare 10 mM stock solution. Store in 20  C. 5. Murashige and Skoog medium: Add about 800 mL of water to a 1 L beaker. Weigh 4.4 g of Murashige and Skoog Basal Medium and 10 g of sucrose and then transfer them to the beaker. Mix and make up to 1 L with water. Adjust pH with KOH to 5.8 and then add 8 g of Agar. Autoclave it at 121  C for 20 min (see Note 2). 6. After the temperature of MS medium decreases to about 60  C, add ACC stock solution, MeJA stock solution, or PAC stock solution to prepare MS medium with 50 μM MeJA, 10 μM ACC, or 1 μM PAC, respectively. Mix thoroughly and place the medium carefully into round-shaped petri dishes. For 9 cm diameter petri dishes, 20 mL of medium is poured into each petri dish.

2.2 Materials for RNA Isolation

1. TRIzol Reagent is purchased from Thermo Fisher. 2. Ethanol: 70% ethanol prepared in RNase-free water. 3. 1.5 mL RNase-free microcentrifuge tubes. 4. 0.2 mL RNase-free PCR tubes. 5. 0.1–10 μL, 1–200 μL, and 100–1000 μL RNase-free pipette tips.

2.3 Reagents for Reverse Transcriptional PCR

1. RQ1 RNase-free DNase kit (#M6101, Promega), contains RQ1 RNase-free DNase, stop solution, and RQ1 DNase10  reaction buffer. 2. Reverse transcriptional PCR kit (#M170B, Promega), contains M-MLV 5  reaction buffer, recombinant RNasin ribonuclease inhibitor, M-MLV RT, and nuclease-free water. 3. Oligo (dT)18 primer: Dissolve two OD260 of Oligo (dT)18 primer in 132 μL of nuclease-free water to prepare 0.5 μg/μL stock solution.

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2.4 Solutions for qRT-PCR

1. AceQ qPCR SYBR Green Master Mix (#Q141, Vazyme).

2.5 Materials for Protein Extraction

1. Ethylenediamine tetraacetic acid (EDTA) (1 M, pH 8.0): Add about 40 mL of water to 100 mL glass beaker. Weigh 29.2 g EDTA and transfer it to the beaker. Mix and make up to 100 mL with water. Adjust pH with NaOH to 8.0.

2. Primers are designed on the website Primer 3 (http://wwwgenome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi).

2. Tris–HCl (1.2 M, pH 6.8): Add about 50 mL of water to 100 mL glass beaker. Weigh 14.5 g Trizma base and transfer it to the beaker. Mix and adjust pH with HCl. Make up to 100 mL with water. 3. Lysis buffer: Add about 50 mL of water to 100 mL glass beaker. Add 10 mL of EDTA (1 M, pH 8.0), 10 mL of Tris– HCl (1.2 M, pH 6.8), 10 mL of β-mercaptoethanol, and 5 mL of glycerol in the beaker. Weigh 4 g of sodium dodecyl sulfate (SDS) (see Note 3) and 5 mg of bromophenol blue. Mix and make up to 100 mL with water. Store at 4  C. 2.6

Instruments

1. Plant growth chamber. 2. PCR machine. 3. Real-Time Fluorescent Quantitative PCR machine. 4. Spectrophotometer. 5. Centrifuge. 6. Gel imaging system. 7. Dissecting microscope. 8. Electrophoresis apparatus.

3

Method

3.1 Phenotypic Analysis

1. The A. thaliana seeds are surface-sterilized with bleach solution for 5 min. 2. Wash seeds with sterile water for five times. 3. Sow sterilized seeds on Murashige and Skoog (MS) medium with or without MeJA, ACC, or PAC with sterilized pipette tips (see Note 4 and Fig. 2). 4. Keep plates at 4  C in darkness for 3 days. 5. Exposure plates with continuous white light (80–90 μmol 2 s 1) for 3 h to synchronize seed germination. 6. Some plates are wrapped in aluminum foil to keep them in darkness; others are irradiated with continuous white light. Plates are kept in a growth chamber (80–90 μmol 2 s 1) at 22  C for indicated days.

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Fig. 2 Place A. thaliana seeds on the indicated medium. (a) Add equal volume of seeds and water in a 1.5 mL microcentrifuge tube, mix, and draw up the mixture with pipette. (b) The pipette tip containing seeds and water. (c) Sow seeds on MS medium with the pipette

Fig. 3 Illustration of etiolated seedlings. Four-day-old etiolated seedlings (Col-0) are grown with or without 10 μM ACC. Hypocotyl and root are marked. The bending angle of the apical hook is calculated as 180 + α or 180 -α for seedlings grown on MS medium with or without ACC, respectively

7. Etiolated seedlings are imaged under a dissecting microscope (Fig. 1). 8. Measure hypocotyl lengths (see Note 5 and Fig. 3), apical hook angles (Fig. 3) [12], and root lengths (see Note 6 and Fig. 3) with the ImageJ software (http://rsbweb.nih.gov/ij/). 3.2

RNA Isolation

1. Prepare etiolated seedlings as described above in Subheadings 3.1, step 1–5. Seedlings are kept in darkness and cultivated at 22  C for 4 days. In order to study the genes with high expression levels in hypocotyls and apical hooks, we intend to harvest materials only from aerial tissues. We quickly freeze etiolated seedlings with liquid nitrogen (see Note 7), and then scrape tissues with precooled spoon to collect these tissues in aluminum foil (Fig. 4) and freeze them immediately in liquid nitrogen. However, for collecting tissues with roots, it is recommended to grow seedlings vertically, which will save time for harvesting seedlings. 2. Grind the etiolated seedlings thoroughly with precooled mortars and pestles.

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Fig. 4 Illustration on how to collect aerial tissues. (a) Items used for tissues collection (disposable paper cup, spoon, aluminum foil and medium plate). (b) Use one disposable paper cup to directly pour liquid nitrogen onto the medium surface. (c) Seedlings are immediately frozen. (d) Scrape tissues with spoon. (e) Collect tissues in aluminum foil. (f) Wrapped aluminum foil are stored in liquid nitrogen for downstream manipulation

3. Transfer the powder into an RNase-free tube and immediately add 1 mL of TRIzol reagent to the same tube (see Note 8). 4. Vortex the tube thoroughly to mix the powder with TRIzol reagent and then keep it under room temperature for 15 min for lysing nucleoprotein complex. 5. Centrifuge at 12,000  g for 10 min at 4  C. 6. Transfer the supernatant to a new RNase-free tube. Add in 200 μL of chloroform. 7. Thoroughly mix the tube and keep it under room temperature for 5 min to remove protein. 8. Centrifuge at 12,000  g for 10 min at 4  C. 9. Transfer 400 μL of supernatant to a new RNase-free tube. Add in 500 μL of isopropanol to precipitate RNA. 10. Gently mix the tube up-and-down and keep it under room temperature for 10 min. 11. Centrifuge at 12,000  g for 10 min at 4  C. Discard the supernatant carefully (see Note 9). 12. Add 1 mL of cold ethanol (70%) to wash the precipitate. 13. Centrifuge at 12,000  g for 10 min at 4  C. Discard the supernatant and dry the precipitate. 14. Add 30 μL of RNase-free water to dissolve RNA.

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Fig. 5 Agarose gel electrophoresis analysis for RNA extracted from etiolation seedlings. Four-day-old etiolated seedlings of Col-0, hls1–1, and ein3 eil1 are grown with or without 10 μM ACC. After extraction, RNA is detected by agarose gel electrophoresis. The positions of 28S and 18S RNA are marked

15. Measure the RNA quantity through spectrophotometric analysis. Determine the RNA quality by agarose gel electrophoresis (Fig. 5). Store the RNA samples in 80  C if not used immediately. 3.3 Removal of DNA from RNA Samples Prior to Reverse Transcriptional PCR

1. Add the following components to a sterile RNase-free 0.2 mL PCR tube: 2 μg of RNA, 1 μL of RQ1 RNase-free DNase 10  reaction buffer, 2 μL of RQ1 RNase-free DNase (see Note 10). Add nuclease-free water to reach the final volume 10 μL. 2. Incubate at 37  C for 30 min (see Note 11). 3. Add 1 μL of RQ1 DNase stop solution to stop the reaction. 4. Incubate at 65  C for 10 min to inactivate DNase. 5. 10 μL of the treated RNA is ready for downstream RT-PCR.

3.4 Reverse Transcription

1. Set up the first step of reverse transcription reaction as follows: 10 μL of DNase-treated RNA, 1 μL of Oligo (dT)18 primer, and 4 μL of nuclease-free water (see Note 12). 2. Incubate at 70  C for 15 min. 3. Cool the tube immediately on ice to prevent reforming secondary structure, and then briefly spin down the tube to collect the solution. 4. Add the following components to the product of the first step: 5 μL of M-MLV 5 reaction buffer, 1.25 μL of dNTPs (10 mM), 0.65 μL of recombinant RNasin Ribonuclease Inhibitors, 1 μL of M-MLV RT, and 2.1 μL of nuclease-free water to the final volume of 25 μL.

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Table 1 Two-step PCR amplification procedure Procedure Pre-denaturation Circular reaction Dissolve curve

Cycle number 1 40 1

Temperature 

95 C 95  C 60  C 95  C 60  C 95  C

Time 5 min 10 s 30 s 15 s 60 s 15 s

5. Mix gently and spin briefly to collect the solution at the bottom of the tube. 6. Incubate at 42  C for 60 min, and then 70  C for 15 min. 7. Cool the tube immediately on ice. 3.5 qRT-PCR Analysis

1. Set up the reaction as follows: 10 μL of AceQ qPCR SYBR Green Master Mix, 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 2 μL of cDNA (see Note 13), and 7.2 μL of sterile water. 2. Choose ACTIN2 as a reference gene to normalize expression levels. 3. Set up the two-step PCR amplification procedure at LightCycler 96 as indicated in Table 1. 4. Analyze raw data and draw column chart comparing the expression of target genes in different samples in SigmaPlot 10.0.

3.6 Protein Extraction

Ethylene stabilizes the transcription factor ETHYLENE INSENSI TIVE 3 (EIN3) protein levels for eliciting various ethylene responses [13]. Here, we detail the protein extraction procedures with the ethylene-triggered EIN3 protein accumulation as an example. 1. Prepare A. thaliana seedlings (Col-0 and EIN3-FLAG/ein3 eil1) as described above in Subheading 3.1, steps 1–5. 2. After irradiating with continuous white light for 3 h, these plates are kept in darkness and cultivated in a growth chamber at 22  C for 4 days. Etiolated Col-0 seedlings are also collected as negative control. 3. Prepare treatment (100 μM ACC). Add 15 mL of autoclaved MS liquid solution and 150 μL of ACC (10 mM) in a sterilized centrifuge tube (50 mL) to prepare 100 μM treatment solution. Mix thoroughly and add 4 mL of the solution into each well in one 6-well plate.

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Fig. 6 Seedling treatment. (a) Items used for seedlings treatment. (b) Gently pull the etiolated seedling out of medium. (c) The etiolated seedlings pulled out of medium. (d) Incubate seedlings in treatment solution

4. After harvesting, immediately incubate etiolated seedlings into treatment (see Note 14 and Fig. 6). About one hundred of etiolated seedlings incubated in one treatment represent as one sample. 5. Wrap these plates in aluminum foil to keep them in darkness. Incubate in a growth chamber at 22  C for different time points. 6. Seedlings are rapidly collected from the well and put on a paper towel to absorb water, then transferred into a 1.5 mL microcentrifuge tube. Tubes are immediately frozen in liquid nitrogen (see Note 15). 7. Add 120 μL of lysis buffer into the tube (see Note 16). 8. Boil seedlings in the lysis solution at 100  C for 10 min. Mix once per 3 min [14]. 9. After sample temperature decreases to room temperature, then centrifuge it at (13,000  g) for 5 min. Supernatants are transferred to a new tube (see Note 17). 10. Perform the following SDS-PAGE gel electrophoresis, membrane transferring, and immuno-blot in a standard way (Fig. 7).

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Fig. 7 Immunoblot examples. Four-day-old etiolated EIN3-FLAG/ein3 eil1 seedlings are treated with 100 μM ACC for the indicated time points. The level of EIN3 is detected with anti-FLAG antibody (#F1804, SigmaAldrich). Actin is also detected to ensure equal loading

Fig. 8 Apical hook phenotypes. The apical hook phenotypes of etiolated seedlings grown on MS, 10 μM ACC, 50 μM JA, or 10 μM ACC plus 50 μM JA for 4 days 3.7 Hormone Interaction Analysis

Previous research has shown that phytohormone ethylene and jasmonate act antagonistically in the regulation of apical hook development [15]. Ethylene positively regulates apical hook formation, while jasmonate antagonizes ethylene’s effect [7, 15]. Following protocol is depicted to study the crosstalk between ethylene and jasmonate in the regulation of apical hook development. Similar protocols could be applied for investigating other hormones’ co-actions in etiolated seedlings. 1. Prepare MS medium with or without 10 μM ACC or 50 μM MeJA or 10 μM ACC plus 50 μM MeJA. 2. Surface-sterilized seeds are placed on indicated medium with sterile pipette tips described in Subheadings 3.1, steps 1–5. 3. Keep plates in darkness at 4  C for 3 days. 4. Image seedlings and measure hook angles as described above in Subheadings 3.1, steps 7–8 (Fig. 8).

4

Notes 1. MeJA solution should be freshly prepared before using. 2. The solution volume is less than 2/3 volume of a conical flask. 3. For personal safety, wear a mask when weighing sodium dodecyl sulfate.

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4. Add distilled water into 1.5 mL microcentrifuge tube. The volume is equal to seeds. Mix and draw up the mixture with pipette. Remove and hold the pipette tip by fingers. To ensure seeds spreading evenly, gently touch the pipette tip to medium and sow seeds one by one. 5. More than 30 seedlings are recorded in each experiment. The hypocotyl lengths are measured from the joint point of two cotyledons to the point of hypocotyl-root junction. 6. The root lengths are measured from the point of hypocotylroot junction to the root tip. 7. During collecting seedlings, liquid nitrogen is always kept to maintain seedlings in a frozen environment. 8. When using TRIzol reagent, caution should be taken. TRIzol can cause serious chemical bures and permanent scarring. Proper protective items should be used. 9. Be careful to avoid precipitate loss after RNA precipitation. 10. Use 1 unit of RQ1 RNase-free DNase per microgram of RNA. For RNA less than 1 μg, use 1 unit of RQ1 RNase-free DNase per reaction. 11. Carry out this step on PCR machine. 12. Do not alter the ratio of primer to RNA. 13. Reverse transcriptional product could be diluted in 10 times. 14. To ensure the seedlings are not injured, gently clamp the junction of hypocotyl and root, and then pull the whole etiolated seedling out of medium. To avoid the interference of light, the whole operation is carried out in darkroom under green light. Make sure these seedlings are immersed in the solution. 15. Carry out this step in darkroom with green light. 16. The lysis buffer should completely submerge the plant tissues. 17. The solution can be directly loaded into gel after centrifuging.

Acknowledgments We thank the Fok Ying Tong Education Foundation (161023), the Natural Science Foundation of Jiangsu Higher Education Institutions (18KJB180010), and the Priority Academic Program Development of Jiangsu Higher Education Institutions for their financial supports.

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References 1. Sinclair SA, Larue C, Bonk L, Khan A, CastilloMichel H, Stein RJ, Grolimund D, Begerow D, Neumann U, Haydon MJ, Kr€amer U (2017) Etiolated seedling development requires repression of photomorphogenesis by a small cell-wall-derived dark signal. Curr Biol 27:3403–3418 2. Gommers CMM, Monte E (2018) Seedling establishment: a dimmer switch-regulated process between dark and light signaling. Plant Physiol 176:1061–1074 3. Liscum E, Hangarter RP (1993) Lightstimulated apical hook opening in wild-type Arabidopsis thaliana seedlings. Plant Physiol 101:567–572 4. Ma L, Gao Y, Qu L, Chen Z, Li J, Zhao H, Deng XW (2002) Genomic evidence for COP1 as a repressor of light-regulated gene expression and development in Arabidopsis. Plant Cell 14:2383–2398 5. Turner JG, Ellis C, Devoto A (2002) The jasmonate signal pathway. Plant Cell 6(Suppl): S153–S164 6. Alabadı´ D, Gil J, Bla´zquez MA, Garcı´a-Martı´nez JL (2004) Gibberellins repress photomorphogenesis in darkness. Plant Physiol 134:1050–1057 7. Vriezen WH, Achard P, Harberd NP, Van Der Straeten D (2004) Ethylene-mediated enhancement of apical hook formation in etiolated Arabidopsis thaliana seedlings is gibberellin dependent. Plant J 37:505–516 8. De Grauwe L, Vandenbussche F, Tietz O, Palme K, Van Der Straeten D (2005) Auxin, ethylene and brassinosteroids: tripartite control of growth in the Arabidopsis hypocotyl. Plant Cell Physiol 46:827–836

9. Zhong S, Shi H, Xue C, Wei N, Guo H, Deng XW (2014) Ethylene-orchestrated circuitry coordinates a seedling’s response to soil cover and etiolated growth. Proc Natl Acad Sci U S A 111:3913–3920 10. Lehman A, Black R, Ecker JR (1996) HOOKLESS1, an ethylene response gene, is required for differential cell elongation in the Arabidopsis hypocotyl. Cell 85:183–194 11. Alonso JM, Stepanova AN, Solano R, Wisman E, Ferrari S, Ausubel FM, Ecker JR (2003) Five components of the ethylene response pathway identified in a screen for weak ethylene-insensitive mutants in Arabidopsis. Proc Natl Acad Sci U S A 100:2992–2997 12. Vandenbussche F, Petra´sek J, Za´dnı´kova´ P, Hoyerova´ K, Pesek B, Raz V, Swarup R, Bennett M, Zazimalova´ E, Benkova´ E, Van Der Straeten D (2010) The auxin influx carriers AUX1 and LAX3 are involved in auxinethylene interactions during apical hook development in Arabidopsis thaliana seedlings. Development 137:597–606 13. Chao Q, Rothenberg M, Sokano R, Roman G, Terzaghi W, Ecker JR (1997) Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENEINSENSITIVE3 and related proteins. Cell 89:1133–1144 14. Tsugama D, Liu S, Takano T (2011) A rapid chemical method for lysing Arabidopsis cells for protein analysis. Plant Methods 7:22–29 15. Zhang X, Zhu ZQ, An FY, Hao DD, Li PP, Song JH, Yi CQ, Guo HW (2014) Jasmonateactivated MYC2 represses ETHYLENE INSENSITIVE3 activity to antagonize ethylene-promoted apical hook formation in Arabidopsis. Plant Cell 26:1105–1117

Chapter 7 Global Identification for Targets of Circadian Transcription Factors in Arabidopsis and Rice Using Chromatin Immunoprecipitation Followed by Sequencing (ChIP-seq) Shuxuan Xu, Jing Huang, Jian Jin, and Wei Huang Abstract The circadian clock is a self-sustaining 24 h timekeeper which enables plants to anticipate periodic environmental changes and optimize the biological activities for most beneficial time during day/night cycles. As in many organisms, the sustained circadian rhythmicity in plant relies on network of transcriptional/translational feedback loops (TTFLs) of transcription factors at the core of the oscillator. Over the past years, ChIP-seq has become an indispensable method to uncover the clock network through identifications of circadian transcription factors binding sites on a genome-wide scale. Here, we show how to use ChIP-seq to analyze the occupancy of circadian transcription factor in Arabidopsis. In addition, we briefly describe some modifications of protocol applied to rice (Oryza sativa). Key words Circadian clock, Chromatin immunoprecipitation followed by sequencing (ChIP-seq), ChIP-qPCR, Arabidopsis thaliana, Rice

1

Introduction Circadian clocks have been found in almost all living organisms on earth. The 24 h endogenous rhythms driven by circadian clock improve plant performance by allowing plants to predict and prepare for the coming environmental changes and by optimally scheduling biological processes at most appropriate time during the diurnal cycle [1, 2]. In Arabidopsis, the circadian clock mechanisms involve multiple interlocked feedback loops in which TIMING OF CAB EXPRESSION 1 (TOC1) and CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) act as master transcription factors to modulate a large number of core clock components and key players in output processes [3–6]. Over past 20 years, a number of new circadian genes and interlocking feedback loops were characterized and integrated into clock model through classic genetic approach. These important circadian components function at the core of Arabidopsis circadian

Ruohe Yin et al. (eds.), Plant Photomorphogenesis: Methods and Protocols, Methods in Molecular Biology, vol. 2297, https://doi.org/10.1007/978-1-0716-1370-2_7, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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clock including LATE ELONGATED HYPOCOTYL (LHY), LUX ARRHYTHMO (LUX), EARLY FLOWERING 3 (ELF3), ELF4, PSEUDO-RESPONSE REGULATORS (PRRs), REVEILLES(RVEs), ZEITLUPE (ZTL), GIGANTEA (GI), and so forth, most of which are transcription factors [2]. Recently, contributions from the new advance and applications of omics approaches such as ChIP-seq method have dramatically enriched our understanding of the mechanisms of clock gene networks in Arabidopsis. For instance, the genomic wide binding profiles of TOC1 [4], CCA1 [6, 7], PRR5 [8], PRR7 [9], and PRR9 [10] had already been identified by ChIP-seq. However, much of the existing knowledge about plant circadian clock was gained from Arabidopsis, and the architecture of crop circadian clock still remains poorly understood. In addition, recent works suggested that circadian clock transcription factor always control important agricultural relevant traits in crops [11–14]. Therefore, understanding the genome-binding profiles of crop circadian TFs may potentially benefit the genetic manipulation of master clock regulators in crops improvement. ChIP-seq is a vital tool to study global mapping of protein-DNA interactions, dynamic of chromatin structure/histone modifications, DNA methylation and cooperative or antagonistic interplay between different cofactors in gene regulation, all of which are critical for fully deciphering circadian TF regulatory network [15]. In general, the ChIP-seq procedure includes several steps: (a) DNA-protein cross-linking, (b) shearing DNA into short fragments, (c) immunoprecipitation with antibodies, and (d) sequencing (Fig. 1). Here, we provide a ChIP-seq protocol to identify the targets of transcription factors in Arabidopsis and rice. The original protocol has been successfully applied to identify the global binding sites of the central circadian gene TOC1 in Arabidopsis [4]. This modified version is suitable for both rice and Arabidopsis. Following this protocol, we have successfully identified the binding sites of AP1 in Arabidopsis and OsTOC1 in rice (Unpublished data).

2

Materials

2.1 Plant Tissue Fixation

1. 0.2 M Sodium phosphate buffer pH 7: 0.2 M disodium hydrogen phosphate solution mix with 0.2 M sodium dihydrogen phosphate solution to pH 7. 2. MC buffer: 0.1 M sucrose, 10 mM sodium phosphate, pH 7, 50 mM NaCl. 3. Fixation Buffer: MC buffer add 1% formaldehyde and 0.05% Triton X-100. 4. Glycine buffer: 2.5 M glycine.

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1. M1 buffer: 0.1 M NaCl, 10 mM sodium phosphate, pH 7, 1 M 2-methyl 2,4-pentanediol, add 10 mM β-mercaptoethanol, 1 mM PMSF, and Protease Inhibitor cocktail before use. 2. M2 buffer: 0.1 M NaCl, 10 mM sodium phosphate, pH 7, 1 M 2-methyl 2,4-pentanediol, 10 mM MgCl2, 0.5% Triton X-100, add 10 mM β-mercaptoethanol, 1 mM PMSF, and Protease Inhibitor cocktail before use.

Fig. 1 Flowchart of the ChIP-seq. The figures (a) and (b) show procedures of ChIP-seq

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Fig. 1 (continued)

3. M3 buffer: 0.1 M NaCl, 10 mM sodium phosphate, pH 7, add 10 mM β-mercaptoethanol, 1 mM PMSF, and Protease Inhibitor cocktail before use. 4. Nuclei lysis buffer: 10 mM EDTA, 50 mM Tris–HCl, pH 8.0, 1% SDS. add 1 mM PMSF and Protease Inhibitor cocktail before use. 5. Complete Protease Inhibitor cocktail. 2.3 Immunoprecipitation

1. ChIP dilution buffer: 16.7 mM Tris–HCl, pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% SDS, add 1 mM PMSF and Protease Inhibitor cocktail before use. 2. IP buffer: 50 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 10 μM ZnSO4, 1% Triton X-100, and 0.05% SDS. 3. Protein A agarose beads. 4. GFP antibody from Thermo Fisher Scientific.

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1. Low Salt Wash Buffer: 150 mM NaCl: 1.5 mL, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.0, 0.1% SDS, add 1 mM PMSF and Protease Inhibitor cocktail before use. 2. High Salt Wash Buffer: 500 mM NaCl, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.0, 0.1% SDS, add 1 mM PMSF and Protease Inhibitor cocktail before use. 3. LiCl Wash Buffer: 0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris–HCl, pH 8.0, add 1 mM PMSF and Protease Inhibitor cocktail before use. 4. TE buffer: 10 mM Tris–HCl, pH 8.0, 1 mM EDTA, add 1 mM PMSF and Protease Inhibitor cocktail before use. 5. Reverse buffer: 10 mM Tris–HCl, pH 8.0, 10 mM EDTA, 300 mM NaCl, 0.5% SDS. 6. Elution buffer: 0.1 M NaHCO3, 1% SDS. 7. RNAse (DNAse free). 8. Proteinase K. 9. QIAquick PCR Purification Kit.

2.5 ChIP-seq Library Construction

1. ChIP-seq Sample Prep Master Mix. 2. NEBNext Multiplex Oligos for Illumina. 3. AMPure XP beads. 4. QIAquick PCR Purification Kit. 5. MinElute PCR Purification Kit.

3

Methods

3.1 Experimental Design

The antibody quality is critical for the ChIP-seq experiment. Therefore, we suggest using monoclonal antibody with high specificity in recognizing the TF in plants. Using antibodies against the epitope tags fused to the TF is another option to perform the experiment. In this case, the function of the fusion protein must be verified before experiments. We strongly recommend using transgenic plants expressing the TF genomic fragment fused to the epitope tags such as GFP in the TF knockout mutant background. If the knockout mutant can be rescued, the transgenic plants would be the ideal materials for the experiment. These transgenic plants have several advantages for ChIP-seq. 1. In TF knockout mutant, the exogenous tagged TF protein will not be completed by the endogenous TF. 2. The subcellular localization and the diurnal fluorescent fluctuations of the GFP-tagged TF could be easily traced by a confocal

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microscopy, providing a confirmation whether the tagged protein can behave normally as the native protein in wild type. 3. Unlike the coding sequence fused with tags, the genome fragment fused with tags is still subjected to the complex expression regulation by many factors, such as long noncoding RNAs (lncRNAs) which could be the natural antisense transcripts (NATs) to clock genes, or alternative splicing, which has been defined as a fundamental regulatory mechanism of circadian clock. Transgenic plants expressing the functional TF genomic fragment can maximally mimic the native protein, making it an ideal material for the experiment. 4. Tissue harvested at the lowest TF expression time is an ideal negative control for ChIP-seq and ChIP-qPCR. 3.2 Fixation of Plant Material

1. Collect 1–2 g 10-day-old young Arabidopsis seedlings (1–2 g 3-week-old rice leaves) into a new 50 mL tube with 25 mL precooling fixation buffer on ice (see Notes 1 and 2). 2. Vacuum infiltrate (800 mbar) on ice, release vacuum after 10 min, mix tissue, and re-apply vacuum. Repeat for 1 time (20 min in total) (for rice leaves vacuum 40 min in total). 3. Stop the cross-link reaction by addition 1.25 mL 2.5 M glycine buffer and vacuum for additional 10 min. 4. Wash the seedlings thrice with MC buffer and dry it thoroughly with filter paper. Then frozen it in liquid nitrogen. 5. Store the tissue in

3.3 Chromatin Isolation

80  C freezer.

1. Be sure to prepare M1, M2, M3, and lysis buffer in advance before this step and before use keep these buffers cooled on ice; simultaneously all protease inhibitors should be added instantly (see Note 3). 2. Grind the frozen tissue in mortar by adding liquid nitrogen to a fine powder. Transfer the samples in 25 mL cold M1 buffer in 50 mL tube on ice (15–30 min) (see Note 4). 3. The slurry was filtered slowly through two layers of Miracloth into a new 50 mL tube and the solution was centrifuged for 15 min at 2900  g at 4  C. 4. Remove supernatant gently and wash the pellet three times with 5 mL of M2 buffer by resuspending and centrifuging for 10 min at 2900  g at 4  C. 5. Remove supernatant gently and rinse the pellet once with 5 mL of M3 buffer by resuspending and centrifuging for 10 min at 2900  g at 4  C. 6. Remove supernatant gently and the nuclear pellet was resuspended in 200 μL lysis buffer, transfer to a new 2 mL tube, and incubate for 10 min on ice (see Note 5).

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7. Add 800 μL dilution buffer and split the sample carefully in Diagenode tubes (do not transfer air bubble in tubes). 8. Precooling Bioruptor (Diagenode) in advance. Sonicate chromatin with 5 cycles by 30 s on and 30 s off to shear chromatin DNA into fragments smear around 300–500 bp in size (see Note 6). 9. Centrifuge the chromatin for 10 min at 13,000  g speed at 4  C and transfer the supernatant to a new 1.5 mL tube. 10. Take 20–50 μL sonicated chromatin for checking sonication efficiency and another supernatant stored at 80  C. 3.4 Check Sonication Efficiency

1. Add 150 μL reverse buffer in 50 μL sonicated chromatin from above and incubate at 65  C at least 6 h. 2. Add 1.5 μL RNase and incubate for 30 min at 37  C. 3. Add 1.5 μL proteinase K and incubate for 2 h at 55  C. 4. Add 200 μL Phenol:chloroform:isoamyl alcohol (25:24:1), centrifuge for 10 min at 13,000  g, and transfer the supernatant to a new 1.5 mL tube. 5. Optional step: Add 200 μL chloroform, centrifuge for 10 min at 13,000  g, and transfer the supernatant to a new 1.5 mL tube. 6. Add 3 μL glycogen, 20 μL of 3 M NaAc pH 5.4 and 500 μL 100% EtOH. Mix it and incubate at 80  C for 1 h. 7. Centrifuge at 13,000  g for 20 min at 4  C and remove supernatant carefully. 8. Add 1 mL cold 70% EtOH and centrifuge at 13,000  g for 5 min. 9. Remove supernatant gently and dry the pellet. 10. Add 20 μL ddH2O and resuspend pellet. 11. Check the sonicated chromatin DNA fragments smear around 200–500 bp in size on a 1.5% agarose gel and choose the best sonication conditions.

3.5 Washing and Blocking the Beads, Pre-clearing the Sonicated Chromatin

1. Subpackage 200 μL slurry of protein A agarose beads in new tube and wash the protein A agarose beads five times with 1 mL IP buffer, centrifuge at 2000  g for 1 min at 4  C, and remove supernatant (see Note 7). 2. Add final 0.5 mg/mL lipid-free BSA concentration in IP buffer. Incubate at 4  C for 20 min, centrifuge at 2000  g for 1 min at 4  C, and remove supernatant. 3. Add 3  volume of IP buffer in the beads to make a 25% slurry and remove 40 μL 25% slurry in two tubes for immunoprecipitation.

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4. Dilute sonicated chromatin to 1.9 mL with IP buffer from Subheading 3.3, step 9. Centrifuge at 13,000  g for 10 min at 4  C and transfer supernatant to a new tube. 5. Add 100 μL beads (25% slurry; equivalent to 25 μL settled resin) from Subheading 3.5, step 3, to the sonicated chromatin solution, incubate at 4  C for 90 min on rotating wheel, centrifuge at 13,000  g for 1 min at 4  C, and transfer supernatant to a new tube. 6. Take 40 μL of sonicated chromatin from above in a new tube and keep as INPUT. 3.6 Immunoprecipitation

1. Transfer 900 μL pre-cleared chromatin from Subheading 3.5, step 5, in two new tubes. 2. Add 5 μL anti-GFP in one tube and another is using as negative control without antibody. Incubate both for 1 h at 4  C on rotating wheel. 3. Add in two tubes 40 μL beads (25% slurry; equivalent to 10 μL settled resin) from Subheading 3.5, step 3, and incubate for 6 h at 4  C on a rotating wheel.

3.7 Reverse Cross-Linking

1. Centrifuge the solution at 2000  g for 5 min at 4  C and remove supernatant, wash the beads with the following washing buffer: Low Salt Wash Buffer, High Salt Wash Buffer, LiCl Wash Buffer, and twice TE buffer by shaking on a rotator at 4  C for 5 min and remove supernatant gently. 2. Add 300 μL fresh Elution buffer in two samples to release the protein-DNA complexes and shaking at 1400 rpm at 65  C for 1 h. 3. Centrifuge the slurry at 14,000  g for 5 min and transfer supernatant to new tubes. 4. Add TE buffer in input DNA from Subheading 3.5, step 6, to the same volume. 5. Add 12 μL 5 M NaCl in 300 μL samples form Subheading 3.7, steps 3 and 4, incubate at 65  C at least 6 h. 6. Add 6 μL 0.5 M EDTA, 12 μL 1 M Tris–HCl (pH 6.5), 1.5 μL RNase and incubate for 30 min at 37  C. 7. Add 1.5 μL proteinase K and incubate for 1 h at 55  C. 8. Purify DNA using QIAquick PCR Purification Kit or the same as steps of check sonication efficiency to purify DNA. 9. Quantify the DNA using Qubit Fluorometer. 10. Validate IP efficiency by q-PCR (Fig. 2).

3.8 ChIP-seq Library Construction

1. End Repair: Combine the reagent according to the protocol of ChIP-seq Sample Prep Master Mix (NEB) (Table 1).

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Fig. 2 TOC1 accumulation at CCA1 promoter. ChIP-qPCR assays of TOC1 in WT (black) and proTOC1::TOC1-YFP (TMG) (white). PP2A is reference gene and represented as means  SEM Table 1 Reaction mixture for ending repair In PCR tube combine ChIP DNA

1–40 μL

NEBNext end repair reaction buffer

5 μL

NEBNext end repair enzyme mix

1 μL

H2O

To 50 μL

2. Incubate for 30 min at 20  C in thermal cycler. 3. Purify DNA use QIAquick PCR Purification Kit, elute in 48 μL of EB (44 μL needed for next step). 4. A-Tailing: Combine the reagent according to the protocol of ChIP-seq Sample Prep Master Mix (NEB) (Table 2). 5. Incubate for 30 min at 37  C in thermal cycler. 6. Purify DNA use MinElute PCR Purification Kit, elute in 22 μL of EB (19 μL needed for next step). 7. Adaptor Ligation: Combine the reagent according to the protocol of ChIP-seq Sample Prep Master Mix (NEB) (see Note 8 and Table 3). 8. Incubate for 15 min at 20  C in thermal cycler.

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Table 2 A-Tailing reaction mixture In PCR tube combine End-repaired DNA

44 μL

NEBNext dA-tailing reaction buffer

5 μL

Klenow fragment

1 μL

Table 3 Adaptor ligation reaction mixture In PCR tube combine dA-tailed DNA

19 μL

Quick ligation reaction buffer

6 μL

1.5 μM adaptor

1 μL

Quick T4 DNA ligase

4 μL

9. Add 1.8 μL USER enzyme to the ligation mixture and incubate for 15 min at 37  C in thermal cycler. 10. Purifying: according to the protocol of AMPure XP beads (see Note 9). 11. Add 54 μL AMPure XP beads in samples and mix it by pipetting. Incubate for 5 min at room temperature. 12. Place on magnetic stand and wait 5 min. Remove and discard supernatant. 13. Add 200 μL 80% EtOH in beads, incubate on magnetic stand for 30 s, remove and discard supernatant. Repeat once time. 14. Air dry on magnetic stand for about 10 min until EtOH has evaporated. 15. Add 102 μL H2O and mix it by pipetting. Incubate at room temperature for 2 min. 16. Place on magnetic stand and wait 3 min. Remove and save 100 μL supernatant to a new tube. 17. Selection: according to the protocol of AMPure XP beads (see Note 9). 18. Add 80 μL (0.8) AMPure XP beads to the sample and mix it by pipetting. Incubate for 5 min at room temperature. 19. Place on magnetic stand and wait 5 min. Transfer supernatant to a new tube and discard beads.

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Table 4 PCR reaction mixture In PCR tube combine Adaptor-ligated DNA

23 μL

Universal PCR primer (25 μM stock)

1 μL

Index primer (25 μM stock)

1 μL

2 PCR master mix

25 μL

20. Add 20 μL (0.2) AMPure XP beads to the sample and mix it by pipetting. Incubate for 5 min at room temperature. 21. Place on magnetic stand and wait 5 min. Remove and discard supernatant. 22. Add 200 μL 80% EtOH in beads, incubate on magnetic stand for 30 s, remove and discard supernatant. Repeat once time. 23. Air dry on magnetic stand for about 10 min until EtOH has evaporated. 24. Add 25 μL EB and mix it by pipetting. Incubate at room temperature for 2 min. 25. Place on magnetic stand and wait for 3 min. Remove and save 23 μL supernatant to a new tube. 26. PCR Enrichment: Combine the reagent according to the protocol of NEBNext Multiplex Oligos for Illumina (Table 4). 27. PCR cycling conditions: denaturation at 98  C for 30 s, followed by 18 cycles of denaturation at 98  C for 10 s, annealing at 65  C for 30 s, and elongation at 72  C for 30 s and then for 5 min at 72  C, hold in 4  C. 28. Last Selection: according to the protocol of AMPure XP beads. 29. Add 60 μL (1.2) AMPure XP beads to the PCR reaction solution and mix it by pipetting. Incubate for 5 min at room temperature. 30. Place on magnetic stand and wait 5 min. Remove and discard supernatant. 31. Add 200 μL 80% EtOH in beads, incubate on magnetic stand for 30 s, remove and discard supernatant. Repeat once time. 32. Air dry on magnetic stand for about 10 min until EtOH has evaporated. 33. Add 22 μL EB and mix it by pipetting. Incubate at room temperature for 2 min. 34. Place on magnetic stand and wait 3 min. Remove and save 20 μL supernatant to a new tube. 35. Quantify the DNA using Qubit Fluorometer.

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3.9 ChIP-seq Data Analysis

4

Massive ChIP-DNA data could be obtained from ChIP-seq, Clean reads from IP and input/ control (without antibody) libraries were mapped to a reference genome by Bowtie2, which is an ultrafast and memory efficient tool for aligning sequencing reads to long reference sequence [16, 17]. Model-based analysis of ChIP-seq (MACS) was used for the peak-calling, which is available online (http://liulab.dfci.harvard.edu/MACS/) [18]. Mot Motif searching is performed by MEME-ChIP, which is also an online tool (http://meme-suite.org/tools/meme-chip) [19, 20]. The peak could be selected as associated with gene, if the peak located within the region from the upstream 3000 bp of TSS to the downstream 1000 bp of TES.

Notes 1. The expression levels of circadian TFs peak at different times of the day. The TFs may recognize DNA motifs in a tissue-specific manner by interacting with tissue-specific cofactors. Thus, it is important to choose the sampling time and tissue. 2. Ambient light conditions may have the global influence on the binding of TF to the chromatins. If the samples should be harvested in darkness, using a lamp or a light equipped with a green filter would be a choice. For Arabidopsis, 1 g of 10-day-old young seedlings is necessary. For rice, 1–2 g of 3-week-old leaves is necessary. The samples should be washed with distilled water, and cut into small pieces. 3. The buffer should be kept at 4  C. If necessary, protease inhibitor should be added into the buffer just before use. 4. Keep the powder frozen before transferring it to the M1 buffer. 5. Low-adhesion tubes are recommended. 6. Depending on the material, the time for fixation and sonication could be variable; e.g., the fixation and sonication time should be longer for the rice leave samples than the Arabidopsis seedling samples. 7. Be careful when transferring supernatant. Any agitation can make the beads sticking to the pipette tip. 8. Different adaptors should be used for multiple libraries construction when these libraries are used for sequencing at the same time. 9. Be careful to avoid touching the magnetic beads with the pipette tip when transferring the supernatant.

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Acknowledgments We would like to thank Dr. Liangfa Ge, Dr. Zhicheng Dong, Dr. Liang Chen and Dr. Shenxiu Du for critical reading of the manuscript and valuable suggestions. This work was supported by 1000-Talents Plan for young researchers of China (to W.H.), Guangdong Province Universities and Colleges Pearl River Scholar Funded (to W.H.), the Research Team Project of the Natural Science Foundation of Guangdong Province (2016A030312009) and the NSFC-Guangdong Joint Fund(U1701232). References 1. Greenham K, McClung CR (2015) Integrating circadian dynamics with physiological processes in plants. Nat Rev Genet 16(10):598–610. https://doi.org/10.1038/nrg3976 2. Sanchez SE, Kay SA (2016) The plant circadian clock: from a simple timekeeper to a complex developmental manager. Cold Spring Harb Perspect Biol 8(12). https://doi.org/10. 1101/cshperspect.a027748 3. Pruneda-Paz JL, Breton G, Para A, Kay SA (2009) A functional genomics approach reveals CHE as a component of the Arabidopsis circadian clock. Science 323(5920):1481–1485. https://doi.org/10.1126/science.1167206 4. Huang W, Perez-Garcia P, Pokhilko A, Millar AJ, Antoshechkin I, Riechmann JL, Mas P (2012) Mapping the core of the Arabidopsis circadian clock defines the network structure of the oscillator. Science 336(6077):75–79. https://doi.org/10.1126/science.1219075 5. Gendron JM, Pruneda-Paz JL, Doherty CJ, Gross AM, Kang SE, Kay SA (2012) Arabidopsis circadian clock protein, TOC1, is a DNA-binding transcription factor. P Natl Acad Sci USA 109(8):3167–3172. https:// doi.org/10.1073/pnas.1200355109 6. Nagel DH, Doherty CJ, Pruneda-Paz JL, Schmitz RJ, Ecker JR, Kay SA (2015) Genome-wide identification of CCA1 targets uncovers an expanded clock network in Arabidopsis. P Natl Acad Sci USA 112(34): E4802–E4810. https://doi.org/10.1073/ pnas.1513609112 7. Kamioka M, Takao S, Suzuki T, Taki K, Higashiyama T, Kinoshita T, Nakamichi N (2016) Direct repression of evening genes by CIRCADIAN CLOCK-ASSOCIATED1 in the Arabidopsis circadian clock. Plant Cell 28 (3):696–711. https://doi.org/10.1105/tpc. 15.00737 8. Nakamichi N, Kiba T, Kamioka M, Suzuki T, Yamashino T, Higashiyama T, Sakakibara H,

Mizuno T (2012) Transcriptional repressor PRR5 directly regulates clock-output pathways. P Natl Acad Sci USA 109 (42):17123–17128. https://doi.org/10. 1073/pnas.1205156109 9. Liu T, Carlsson J, Takeuchi T, Newton L, Farre EM (2013) Direct regulation of abiotic responses by the Arabidopsis circadian clock component PRR7. Plant J 76(1):101–114. https://doi.org/10.1111/tpj.12276 10. Liu TL, Newton L, Liu MJ, Shiu SH, Farre EM (2016) A G-box-like motif is necessary for transcriptional regulation by circadian pseudoresponse regulators in Arabidopsis (vol 170, pg 528, 2016). Plant Physiol 170(2):1168–1168. https://doi.org/10.1104/pp.16.000890 11. Turner A, Beales J, Faure S, Dunford RP, Laurie DA (2005) The pseudo-response regulator Ppd-H1 provides adaptation to photoperiod in barley. Science 310(5750):1031–1034. https://doi.org/10.1126/science.1117619 12. Koo BH, Yoo SC, Park JW, Kwon CT, Lee BD, An G, Zhang ZY, Li JJ, Li ZC, Paek NC (2013) Natural variation in OsPRR37 regulates heading date and contributes to Rice cultivation at a wide range of latitudes. Mol Plant 6(6):1877–1888. https://doi.org/10.1093/ mp/sst088 13. Bendix C, Marshall CM, Harmon FG (2015) Circadian clock genes universally control key agricultural traits. Mol Plant 8(8):1135–1152. https://doi.org/10.1016/j.molp.2015.03. 003 14. Muller NA, Wijnen CL, Srinivasan A, Ryngajllo M, Ofner I, Lin T, Ranjan A, West D, Maloof JN, Sinha NR, Huang SW, Zamir D, Jimenez-Gomez JM (2016) Domestication selected for deceleration of the circadian clock in cultivated tomato. Nat Genet 48 (1):89. https://doi.org/10.1038/ng.3447 15. Solomon MJ, Larsen PL, Varshavsky A (1988) Mapping protein-DNA interactions in vivo

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with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene. Cell 53 (6):937–947 16. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10(3):R25. https:// doi.org/10.1186/gb-2009-10-3-r25 17. Langmead B, Salzberg SL (2012) Fast gappedread alignment with bowtie 2. Nat Methods 9 (4):357–359. https://doi.org/10.1038/ nmeth.1923

18. Feng J, Liu T, Qin B, Zhang Y, Liu XS (2012) Identifying ChIP-seq enrichment using MACS. Nat Protoc 7(9):1728–1740. https:// doi.org/10.1038/nprot.2012.101 19. Machanick P, Bailey TL (2011) MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics 27(12):1696–1697. https://doi.org/ 10.1093/bioinformatics/btr189 20. Ma WX, Noble WS, Bailey TL (2014) Motifbased analysis of large nucleotide data sets using MEME-ChIP. Nat Protoc 9 (6):1428–1450. https://doi.org/10.1038/ nprot.2014.083

Chapter 8 Co-immunoprecipitation Assays to Detect In Vivo Association of Phytochromes with Their Interacting Partners Pengyu Song, Shaoman Zhang, and Jigang Li Abstract The red (R)/far-red (FR) light absorbing phytochromes are one of the major photoreceptor classes in plants. Phytochromes exist in two distinct but interconvertible forms: the R light-absorbing Pr form and the FR light-absorbing Pfr form. Upon photoactivation by light, phytochromes physically interact with their partners to transduce the light signal. Co-immunoprecipitation (Co-IP) is one of the most efficient techniques to study these protein-protein interactions in vivo. However, the co-IP procedure for phytochromes needs to be modified to allow their formation of Pr or Pfr form. Here, we describe a detailed co-IP procedure to examine which form of phytochrome (Pr or Pfr) is preferentially associated with their interacting partners in vivo. Key words Protein-protein interaction, Co-immunoprecipitation, Phytochrome, Pr and Pfr forms, Light signal transduction

1

Introduction Co-immunoprecipitation (Co-IP) is one of the most crucial and popular techniques to study protein-protein interactions in vivo. Co-IP was first developed from the immunoprecipitation technique which utilizes the antigen-antibody interaction to precipitate or isolate the specific proteins from a cell lysate or mixture, while an interacting protein or protein complex precipitated with the target protein is captured by co-IP [1–3]. The antibody used in the co-IP assay is always the most important factor that influences the final result, although an antibody against the protein of interest is usually difficult to generate. To get over this obstacle, transgenic plants expressing tagged proteins (short peptide fused to either C- or N- terminal of the specific protein) could be used in co-IP assays and the antibodies against these tags are commercially available. Examples of tags used frequently in plant research include green fluorescent protein (GFP),

Ruohe Yin et al. (eds.), Plant Photomorphogenesis: Methods and Protocols, Methods in Molecular Biology, vol. 2297, https://doi.org/10.1007/978-1-0716-1370-2_8, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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FLAG, Myc, HA, etc. However, it should be noted that tag itself may interfere with the structure of the target protein, which may affect the interactions detected by co-IP assays. Co-IP can be simply divided into four steps: (1) preparation of cell extracts; (2) coupling of antigen to antibody-beads; (3) purification of the precipitated proteins or protein complexes; and (4) analysis of the precipitated proteins or protein complexes. The extraction condition needs to be mild enough to ensure the protein complexes as intact as possible; therefore lysis buffer should contain nonionic detergents like NP-40 or Triton X-100. In a classical procedure, the antibody is first added to the total lysate to recognize the target protein, then agarose beads conjugated with protein A/G which specifically bind IgG are employed to precipitate the protein complexes [4, 5]; however, the usage of commercially available agarose gel coupled with antibodies may reduce the loss of proteins of interest during the washing steps and improve the efficiency of co-IP procedure. Plants have evolved several different classes of photoreceptors to monitor light quality and quantity. Among the best characterized photoreceptors, phytochromes (phys), mainly absorbing red (R)/far-red (FR) light while partially absorbing blue (B)/ultraviolet-A (UV-A) wavelengths, play fundamental roles in perceiving the light environment and guiding plant development, and avoiding adversity like canopy shade. There are five phytochrome photoreceptors in Arabidopsis thaliana, designated phytochrome A (phyA) to phyE. According to their stability, phytochromes can be categorized into two groups: light-labile type I (phyA) and light-stable type II (phyB to phyE) [6]. Phytochromes exist in two distinct but interconvertible forms, i.e., the R light-absorbing Pr form and the FR light-absorbing Pfr form, and the Pfr form is generally considered to be the biologically active form. Phytochromes are synthesized in their inactive Pr form in the cytosol in darkness; upon light irradiation, they convert into the Pfr form and translocate into the nucleus, where they trigger a signaling cascade that alters the expression of many target genes which ultimately leads to the modulation of various physiological and developmental responses [6–8]. phyB can enter the nucleus by itself under R light irradiation, whereas phyA nuclear accumulation requires two plant-specific proteins, FAR-RED ELONGATED HYPOCOTYL1 (FHY1) and FHY1-LIKE (FHL) [9, 10]. A group of basic helix-loop-helix (bHLH) transcription factors, named PHYTOCHROME -I NTERACTING FACTORS (PIFs), accumulate in dark-grown seedlings and repress photomorphogenesis; upon light exposure, they are rapidly phosphorylated and degraded, and this process is regulated by phytochromes and requires their direct interactions [6–8]. PIF3 was shown to be preferentially associated with the Pfr form of phytochromes in yeast cells and in vivo [11, 12] (Fig. 1); however, FHY1 and FHL

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A R phyA Pr

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B phyA Pr

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Fig. 1 Schematic illustration of co-immunoprecipitation of PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) and TANDEM ZINC-FINGER/PLUS3 (TZP) with phytochrome A (phyA). (a) R light irradiation converts the Pr form of phyA to the Pfr form, which interacts preferentially with Myc-tagged PIF3 in vivo. (b) Flag-tagged TZP interacts with both the Pr and Pfr forms of phyA with similar affinity in vivo

were shown to be preferentially associated with the Pr form of phyA in vivo [12], although they can only interact with the Pfr form of phyA in yeast cells [9]. In addition, a recent study showed that TANDEM ZINC-FINGER/PLUS3 (TZP), a new positive regulator of phyA signaling, interacts with both Pr and Pfr forms of phyA at similar strength in vivo [13] (Fig. 1).

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Here, we describe a co-IP procedure designed to examine which form of phytochrome (Pr or Pfr) is preferentially associated with their interacting partners in vivo. The conclusion from this assay would contribute to a better understanding of their interaction and shed more light on the molecular mechanisms of phytochrome signaling.

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Materials

Stock Solution

Prepare all the solutions using ultrapure water (obtained by purifying deionized water and to attain a sensitivity of 18 MΩ-cm at 25  C) (see Note 1). 1. 1 M Tris–HCl, pH 7.5. Dissolve 12.114 g Tris base in water to a volume of 90 mL. Adjust pH to 7.5 with HCl. Add water up to 100 mL. Autoclave at 121  C for 15 min. Store at room temperature (RT). 2. 1 M Tris–HCl, pH 6.8. Dissolve 12.114 g Tris base in water to a volume of 90 mL. Adjust pH to 6.8 with HCl. Add water up to 100 mL. Autoclave at 121  C for 15 min. Store at RT. 3. 5 M NaCl. Dissolve 29.22 g NaCl with water to final volume of 100 mL. Autoclave at 121  C for 15 min. Store at RT. 4. 0.5 M EDTA, pH 8.0. Add 14.612 g EDTA in water to a volume of 900 mL. Add solid state NaCl and agitate well to adjust pH to 8.0. Make it up to 100 mL with water (see Note 2). Autoclave at 121  C for 15 min. Store at RT. 5. 1 M MgCl2. Dissolve 20.33 g MgCl2·6H2O with water to final volume of 100 mL. Autoclave at 121  C for 15 min. Store at RT. 6. 0.1 M PMSF (100). Dissolve 0.1742 g phenylmethanesulfonyl fluoride (PMSF) with isopropanol to a final volume of 10 mL (see Note 3). Store at 20  C. 7. 20 mM MG132 (500). Dissolve 5 mg MG132 with 525.65 μL DMSO. Store at 20  C. 8. 2 SDS loading buffer. Add 12.5 mL of 1 M Tris–HCl pH 6.8, 4 g sodium dodecyl sulfate (SDS), 20 mL glycerol, 20 mg Bromophenol blue (BPB), 3.1 g DTT in water and make it up to a final volume of 100 mL (see Note 4).

2.2

Pre-lysis Buffer

Prepare 50 mL water and add these stock solutions as indicated order: 5 mL of 1 M Tris–HCl pH 7.5, 3 mL of 5 M NaCl, 1 mL of 1 M MgCl2, 200 μL of 0.5 M EDTA pH 8.0, 100 μL NP-40. Make it up to 100 mL with water. Store at 4  C.

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Methods The procedure below is for small-scale experiments (can be scaled up as needed) and should be performed on ice or 4  C and in a dark room under dim green light unless otherwise mentioned (see Note 5).

3.1 Protein Extraction

1. Collect 0.5–1.0 g plant cell materials and grind them in liquid nitrogen with pestle. 2. Transfer the fine powder into a pre-cooled 2 mL tube and add 1 mL lysis buffer (add 10 μL of 100 cocktail protease inhibitor, 2 μL of 500 MG132, 10 μL of 100 PMSF for every mL of pre-lysis buffer in advance). Gently agitate the extract on a rotator at 4  C for 10 min (see Note 3). 3. Centrifuge at 13,000  g, 4  C for 15 min. Transfer the supernatant to a new microcentrifuge tube. 4. Repeat step 3 once and then go to step 5. 5. Place the tube horizontally on ice and put them in a R or FR light chamber (as each experiment needed) for 5 min of monochromatic light exposure. Gently rotate the tube several times in the middle of the light treatment (see Note 6). 6. Take out 100 μL of each sample to serve as the total control. Add 100 μL of 2 SDS loading buffer and mix well. Denature at 100  C for 15 min and put on ice for 5 min. Store at 20  C for later use (see Note 7).

3.2 Immunoprecipitation

1. Prepare protein A beads or antibody covalently coupled beads with pellet around 5% (v/v) of the extract (see Note 8). Wash the beads with 1 mL lysis buffer for 3 times. Each time pellet the beads by centrifuging at 4  C, 3000  g for 30 s and remove the supernatant (see Note 9). 2. Add appropriate amount of antibodies (e.g., 1:200 of total volume of the extract solution). Incubate on a spinning wheel at 4  C for 2 h. (Alternatively, for antibody covalently coupled beads, directly add the beads to the solution, incubate on the spinning wheel at 4  C for 2 h and skip step 3.) (see Note 10). 3. Add the protein A beads to the mixture and incubate on the spinning wheel at 4  C for 2 h (see Note 9).

3.3 Purification of Immune Complexes

1. Centrifuge the mixture at 4  C, 3000  g for 30 s. Remove the supernatant and add 1 mL lysis buffer to resuspend the pellet beads gently. Incubate on the spinning wheel at 4  C for 10 min. 2. Repeat step 1 for another three times. 3. Centrifuge the mixture at 4  C, 3000  g for 1 min. Remove the supernatant, and add appropriate amount of 2 SDS

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loading buffer (e.g., 200%–300% of the beads pellet volume) and mix well. 4. Denature at 100  C for 15 min. Put on ice for 5 min. 5. Centrifuge at 13,000  g for 1 min (see Note 7). 3.4 Immunoblot Analysis

Analyze 15 μL aliquots of both total control (see Subheading 3.1, step 6) and the immunoprecipitated samples (see Subheading 3.3, step 5) by standard SDS-PAGE and immunoblot assay. The in vivo associations of TZP-phyA and PIF3-phyA by co-IP assays are shown in Fig. 2 and 3, respectively.

Fig. 2 The co-IP assay data showing that TANDEM ZINC-FINGER/PLUS3 (TZP) associates with both Pr and Pfr forms of phytochrome A (phyA) in vivo with similar affinity. The total proteins extracted from 4-day-old seedlings grown under continuous FR light were treated with either 5 min of R light only (R) or 5 min of R light followed by 5 min of FR light (R + FR). TZPp:TZP-3Flag transgenic lines [13] and Anti-Flag M2 Affinity Gel (Sigma-Aldrich) were used in the assay. Anti-phyA [13] and anti-Flag (Sigma-Aldrich) antibodies were used to detect phyA and TZP-3Flag, respectively. Anti-RPN6 was used as a negative control

Fig. 3 The co-IP assay data showing that PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) preferentially associates with the Pfr form of phytochrome A (phyA) in vivo. The total proteins extracted from 4-day-old etiolated seedlings were treated with either 5 min of R light only (R) or 5 min of R light followed by 5 min of FR light (R + FR). 35S::PIF3-Myc transgenic lines [14] and anti-Myc beads (Sigma-Aldrich) were used in the assay. Anti-phyA [13] and anti-Myc (SigmaAldrich) antibodies were used to detect phyA and PIF3-Myc, respectively. AntiRPN6 was used as a negative control

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Notes 1. Using heatable glass bottle to prepare stock solution is helpful as mild heating (e.g., 40–60  C) can accelerate the dissolving rate. However, adjusting pH should be operated after the solution is cooled to RT. Pre-added water in the bottle has to be limited in case the final volume exceeds the demanded volume after adding all the reagents. 2. The EDTA is hard to dissolve completely unless the pH is higher. Adding solid NaOH can make the dissolving more easily, but NaOH solution is not preferred in this case. 3. Put on a mask to avoid the toxic effect while preparing or using PMSF solution. Because the half-life period of PMSF in water is short, adding PMSF solution to the lysis buffer right before the assay is performed. 4. When preparing 2 SDS loading buffer, SDS needs to completely dissolve prior to BPB is added because the blue pigment of BPB brought to the solution makes it difficult to observe whether SDS is dissolved. 5. The dark room, where all the steps are performed, should be utilized strictly to avoid any light exposure other than the artificial dim green light. 6. The ice could be held in a shallow container (e.g., a culture dish) to allow an appropriate distance of the sample from the light source. 7. Low temperature may cause SDS partially precipitate out of the solution. Thaw the denatured protein solution and redissolve SDS thoroughly at RT prior to subsequent immunoblot analysis. 8. The beads ought to be mixed well before distributing aliquots. 9. If the abundance of the protein of interest is too low to perform an effective co-IP, scale up the experiment extent and divide the protein extracts into several microcentrifuge tubes. Keep the volume of every aliquot of beads below 50 μL in each tube for better efficiency. 10. Antibody or antibody-beads incubation can be performed overnight at 4  C for better interaction between antibody and antigen; however, prolonged incubation may increase the risk of protein degradation.

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Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (31970262 and 31770321) and the Recruitment Program of Global Youth Experts of China to J.L. References 1. Phizicky EM, Fields S (1995) Protein-protein interactions: methods for detection and analysis. Microbiol Rev 59:94–123 2. Golemis E (2002) Protein-protein interactions: a molecular cloning manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p 682 3. Lee C, Rosato E (eds) (2007) Circadian rhythms: methods and protocols. Humana Press, Totowa, NJ 4. Kessler SW (1975) Rapid isolation of antigens from cells with a staphylococcal protein A-antibody absorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol 115:1617–1624 5. Akerstrom B, Brodin T, Reis K, Bjorck L (1985) Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies. J Immunol 135:2589–2592 6. Li J, Li G, Wang H, Deng XW (2011) Phytochrome signaling mechanisms. Arabidopsis Book 9:e0148 7. Jiao Y, Lau OS, Deng XW (2007) Lightregulated transcriptional networks in higher plants. Nat Rev Genet 8:217–230 8. Franklin KA, Quail PH (2010) Phytochrome functions in Arabidopsis development. J Exp Bot 61:11–24

9. Hiltbrunner A, Viczia´n A, Bury E, Tscheuschler A, Kircher S, To´th R et al (2005) Nuclear accumulation of the phytochrome a photoreceptor requires FHY1. Curr Biol 15:2125–2130 10. Hiltbrunner A, Tscheuschler A, Viczia´n A, Kunkel T, Kircher S, Sch€afer E (2006) FHY1 and FHL act together to mediate nuclear accumulation of the phytochrome a photoreceptor. Plant Cell Physiol 47:1023–1034 11. Shimizu-Sato S, Huq E, Tepperman JM, Quail PH (2002) A light-switchable gene promoter system. Nat Biotechnol 20:1041–1044 12. Shen Y, Zhou Z, Feng S, Li J, Tan-Wilson A, Qu L-J et al (2009) Phytochrome a mediates rapid red light-induced phosphorylation of Arabidopsis FAR-RED ELONGATED HYPOCOTYL1 in a low fluence response. Plant Cell 21:494–506 13. Zhang S, Li C, Zhou Y, Wang X, Li H, Feng Z et al (2018) TANDEM ZINC-FINGER/ PLUS3 is a key component of phytochrome a signaling. Plant Cell 30:835–852 14. Park E, Kim J, Lee Y, Shin J, Oh E, Chung W-I et al (2004) Degradation of phytochrome interacting factor 3 in phytochrome-mediated light signaling. Plant Cell Physiol 45:968–975

Chapter 9 Detection of UVR8 Homodimers and Monomers by Immunoblotting Analysis in Tomato Guoqian Yang, Xiaorui Liu, and Li Lin Abstract The UV RESISTANCE LOCUS 8 (UVR8) is a photoreceptor mediating photomorphogenic responses to UV-B. UVR8 exists as homodimer in plants and UV-B induces dissociation of dimeric UVR8 into monomers to initiate responses. The monomer/dimer status of UVR8 is reversible and a dynamic photoequilibrium is established in plants according to the ambient light conditions. Here we describe a method to detect UVR8 homodimer and monomer by immunoblotting method from tomato (Solanum lycopersicum) plants. The feature of this method is that protein samples are not boiled prior to loading on an SDS-PAGE gel, which allows the detection of UVR8 homodimer and monomers simultaneously with a single antibody. Key words UVR8, Homodimer, Monomer, Western blotting, Tomato

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Introduction Ultraviolet-B (UV-B, 280–315 nm) of the solar spectrum is an important environmental signal that can regulate plant growth and development [1, 2]. Plant photoreceptors including phytochromes, cryptochromes, and phototropins have been well studied; however, the mechanism for UV-B receptor UV RESISTANCE LOCUS8 (UVR8) has only been recently revealed. UVR8 was originally characterized in the model plant Arabidopsis thaliana [3]. Interestingly, intrinsic tryptophan residues of UVR8 act as chromophores for UV-B perception [3]. Arabidopsis UVR8 contains 440 amino acid residues [4]. UVR8 is composed of sevenbladed β-propellers, a C27 domain, and N- and C-terminal ends [5–7]. UVR8 is highly conservative in plants and mediates a variety of responses to UV-B. The angiosperm, bryophyte, stone pine, and green algae plants UVR8 share a high amino acid sequence similarity, especially the location and number of key amino acids, including salt bridge network tryptophan and arginine, suggesting an important function of UVR8 during evolution [3, 8].

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In the absence of UV-B, UVR8 exists as a dimer in plants, and the conformation of UVR8 is regulated by UV-B [3, 6, 9]. Photoreception induces dissociation of dimeric UVR8 into monomers to initiate responses, and the monomer/dimer status of UVR8 is dynamic and reversible. In fact, UVR8 establishes a dimer/monomer photo-equilibrium in plants according to light conditions in both controlled laboratory conditions and natural field conditions [10]. Heilmann et al. further found that the UVR8 dimer can be depolymerized into monomers at a very rapid rate under the induction of UV-B, indicating that UVR8 can directly sense UV-B and undergo a conformational change [11]. The UVR8 monomers interact with key signaling protein E3 ubiquitin ligase CONSTITU TIVELY PHOTOMORPHOGENIC 1 (COP1) and activate the expression of UV-B response genes (including HY5) in the nucleus [4, 9, 12–14]. To control the signaling response, the negative regulators RUP1 and RUP2 interact directly with UVR8 and facilitate the reversion of UVR8 monomers to homodimers [15] (Fig. 1). The UVR8 conformational change is critical for adjusting UVR8 activity in plants, as it can quickly adjust the balance between active form (monomer) and inactive form (homodimer) according to UV-B levels. To monitor the relative amounts of UVR8 dimer and monomer following UV-B treatment of intact Arabidopsis seedlings, a western blotting assay was developed by Rizzini et al. [3]. Rizzini

UV-B Dimer

Monomer

Nucleus

Fig. 1 The UVR8 photocycle in plants. The UVR8 homodimer is monomerized instantly in response to UV-B irradiation, with UV-B absorption proceeding via a tryptophan-based chromophore. The UVR8 seven-bladed beta-propeller domain interacts directly with COP1 WD40 domain to initiate UV-B signaling. The activated UVR8-COP1 signaling pathway induces expression of RUP1 and RUP2, which regenerates the UVR8 homodimer and inactivates the signaling pathway, forming a negative feedback loop

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et al. studied the properties of UVR8 protein under non-heatdenatured conditions and found that UV-B irradiation of the gel was necessary for the antibody to immunize UVR8 dimer [3]. It is shown that under non-heat-denatured conditions in vitro, the conformation of UVR8 dimer is activated by UV-B in the gel and changes, so that it releases the antigenic site inside the protein and is recognized by the antibody [3]. The special feature of this method is that protein samples are not boiled prior to loading on an SDS-PAGE gel. Intermolecular interactions between monomers to maintain the dimer are sufficiently strong to resist denaturation by SDS under these conditions [16]. Hence, the UVR8 dimer and monomer are clearly resolved on the gels and are visualized by incubating an immunoblot of the gel with an anti-UVR8 antibody [16]. As a model species in the Solanaceae, the tomato has been extensively used in genetic analyses and fleshy fruit development [17–19]. Although immunoblot analysis of UVR8 was extensively studied in Arabidopsis, little is known about the dimer/monomer status of tomato UVR8 (SlUVR8) gene. Here we described the methodology of western blotting applied in SlUVR8 research, focusing specifically on sample preparation and sample treatment. The experimental procedure was successful and produced similar results as those in Arabidopsis [15]. As shown in Fig. 4, essentially all the UVR8 protein is in the dimer status before UV-B exposure, and it is then rapidly converted to the monomer state in response to UV-B treatment (0.5 h). A decrease in the amount of monomer and a concomitant increase in the dimer are seen within 30 min subsequently after plant seedlings are transferred to white light. Virtually all the UVR8 protein is reverted to the dimer state after 1 h in white light. The total amount of UVR8 (protein gel blots created in parallel using heat-denatured aliquots of the same protein samples) does not appear to change significantly over the time course, at least up to 6 h following the end of illumination, demonstrating that UVR8 levels remain stable during the recovery phase.

2

Materials

2.1 Technical Equipment

Light growth chamber, grinder, power supply equipment, vertical electrophoresis apparatus, electrical transfer device, PVDF membrane, Whatman 3 mm filter paper, and blot image acquisition system. Other tools: UV-B lamps (broadband), tweezers, sponge pads, scissors, gloves, small plastic or glass containers, and shallow plates.

2.2 Buffers and Solutions

1. Acrylamide mix 30%: Acrylamide 29 g, N,N0 -methylene-bisacrylamide 1 g, add water to 100 mL. Stored in a brown bottle

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at 4  C in the dark. The pH should not exceed 7.0 and the solution needs to be prepared every few months. Discard it once precipitate is present. 2. SDS solution 10% (w/v): 0.1 g SDS, add 1 mL deionized water and stored at room temperature. 3. Separation gel buffer: 1.5 mmol/L Tris–HCl (pH 8.8): 18.15 g Tris and 48 mL 1 mol/L HCl are mixed and diluted with deionized water to a final volume of 100 mL. After filtration, stored at 4  C. 4. Stacking gel buffer: 0.5 mmol/L Tris–HCl (pH 6.8): 6.05 g Tris is dissolved in 40 mL of water, adjusted to pH 6.8 with about 48 mL of 1 mol/L HCl, and diluted with water to a final volume of 100 mL. After filtration, stored at 4  C. 5. TEMED stock solution: stored at 4  C. 6. Ammonium persulfate solution 10% (w/v), prepare it before use. 7. SDS-PAGE loading buffer: 0.5 mol/L Tris buffer (pH 6.8) 8 mL, glycerin 6.4 mL, 10% SDS 12.8 mL, 2-mercaptoethanol 3.2 mL, 0.05% bromophenol blue 1.6 mL, water 32 mL. Mix the protein sample in a ratio of 1:1 or 1:2. 8. Tris-glycine electrophoresis buffer (10  running buffer): 30.2 g Tris, 144 g glycine, 10 g SDS, dissolved in distilled water to 1000 mL to obtain 0.25 mol/L Tris-1.92 mol/L glycine electrode buffer. Dilute to 1  before use. 9. Protein extraction buffer: 50 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 0.10% Tween 20, Protease inhibitors (Roche tablet, 1 tablet for 10 mL buffer). 10. Transfer buffer: Prepare 1 L of transfer buffer, weigh 2.9 g of glycine, 5.8 g of Tris base, 0.37 g of SDS, and add 200 mL of methanol, add water to a total of 1 L. 11. TBS: 20 mmol/L Tris–HCl (pH 7.5), 500 mmol/L NaCl. Weigh 24.23 g Trizma HCl, 80.06 g NaCl, mix in 800 mL deionized water. Adjust pH to 7.6 with pure HCl. Top up to 1 L. TBST: For 1 L: 100 mL of TBS 10  + 900 mL deionized water +1 mL Tween20. 12. Blocking solution: 10% nonfat milk in TBST. 13. Diluent solution: 5% nonfat milk in TBST. 14. Ponceau S staining buffer: Ponceau S 2 g, trichloroacetic acid 30 g, sulfosalicylic acid 30 g, add sterilized water to 100 mL. Dilute 10-folds into 1  Ponceau S working solution. 15. 100 mmol/L Tris–HCl (pH 9.5). 16. 100 mmol/L NaCl. 17. 50 mmol/L Tris–HCl (pH 7.5).

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2.3 Plant Materials and Gene Identifier

The tomato (cultivar “Ailsa Craig,” accession LA2838A, Tomato Genetic Resource Center, TGRC, University of California, USA) seeds were used in all experiments and stored in a 4  C refrigerator. The Sol Genomics Network (SGN) locus identifier for UVR8 is Solyc05g018620.

2.4

Primary antibody: Tom-Dimer-α-UVR8 (Santa Cruz, 1:5000 dilution before use). Secondary antibody: HRP-Goat Anti-Rabbit (1:10,000 dilution before use).

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Antigens

Methods To avoid contamination, wear latex gloves during all the steps of the protocol.

3.1 Sample Preparation

The tomato (AC) seeds were firstly sterilized in absolute ethanol for 20 s, rinsed 3 times with sterile water, placed in 1/2 Murashige and Skoog (MS) medium, and vernalized in darkness at 4  C for 48 hrs. Then the seeds are cultured in a 26  C light growth chamber (in a weak white light field) for 5–7 days and subjected to UV-B treatment (broadband UV-B with maximal emission at 311 nm, Philips, USA) (Fig. 2).

Fig. 2 Sample preparation for the tomato. The seeds are sterilized and cultured in a 26  C growth chamber (in the weak white light field) for 5 days to reach a height of 2–3 cM (a, b) before subjecting to a broadband UV-B treatment (c)

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Time course analysis of UVR8 monomerization in plants were then conducted after exposure to UV-B for the indicated times. First, the seedlings were exposed to broadband UV-B for 30 min and then transferred to white light (3.6 μmol m2 s1) to recover for 0.5 h, 1 h, 2 h, and 6 h, respectively. Control seedlings were cultured on 1/2 MS medium without being irradiated. All operations are at room temperature unless otherwise stated. Prepare the protein samples as follows: 1. Collect tomato seedlings with a 1.5 mL tube, grinding into powder, and homogenize samples under 20  C (see Note 1). 2. Add 1 mL protein extraction buffer to each tube. 3. Mix buffer with the fine plant powder only for 1 min; leave on ice till the other samples are ready. 4. Centrifuge at 4  C for 10 min with 14,000  g; take the clear supernatant, if necessary centrifuge once more with only 5 min. Now the clear extract can be measured with the Bradford method for protein concentration. To ensure loading volume is consistent across the samples in a gel, all samples were normalized to the same concentration. 5. For UVR8 homodimer detection: Take one small aliquot (30–40 μg) and directly add protein loading buffer to the aliquot and keep it on ice till loading. 6. For UVR8 monomer detection: Mix the protein sample with 5  loading buffer, then denatured at 95 for 7 min. Then place it on ice before loading it to the gel. 3.2 Gel Electrophoresis

1. Pouring SDS-Polyacrylamide Gels. Clean and assemble glass plates to the gel holder according to the manufacturer’s instructions (see Notes 2 and 3). Prepare 5 mL 8–10% separating gel, and rapidly inject it into the gap between the glass plates from the upper left corner. Leave sufficient space for the stacking gel and overlay the acrylamide solution with 500 μL absolute ethanol. Place the gel in a vertical position at room temperature. After the separating gel is polymerized in ~20 min, prepare the stacking gel and inject the stacking gel solution to the gap between the glass plate after carefully removing the absolute ethanol. Insert the comb immediately to the stacking gel without introducing air bubbles. 2. Running the Gel. Remove the prepared gel carefully from the gel holder, place it in an electrophoresis apparatus, and pour the 1  SDS running buffer into the electrophoresis tank, and slowly pull out the comb (see Note 4). Load 20 μL sample of the processed protein to each well with protein standards

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Broadband UV-B Lamp 20-50 cM Transfer buffer Gel

Container with ice

Fig. 3 Schematic diagram for the treatment of protein gels with UV-B. The gel immersed with running buffer is put on a container filled with ice. Then the gel is irradiated for 15 min under broadband UV-B before the proteins were electrophoretically transferred onto a PVDF membrane. The distance from gel to UV-B lamp is about 20-50 cM

(5 μL/well) (see Notes 5 and 6). Run the gel at 70 V until the sample has entered the separating gel and then continue at 100 V till the bromophenol blue reached the bottom of the separation gel (after about 1.5 h). 3.3 UV-B Treatment of Protein Gels

3.4

Protein Transfer

After gel electrophoresis, open the gel plates with a spatula and transfer the protein gel to a plastic container with a thin layer of SDS-PAGE running buffer to avoid drying of the gel (see Note 7). Then irradiate the gel for 15 min under the broadband UV-B before the proteins were electrophoretically transferred onto a PVDF membrane according to standard procedures using a Mini Trans Blot Cell (Bio-Rad Laboratories, 7.5  10 cm blotting area) (Fig. 3). 1. Cut a piece of PVDF membrane (see Note 8) according to the size of the gel, and the size of the membrane should be slightly smaller than that of the gel. Immerse the membrane in methanol for 30 s and then rinse twice with distilled water before transferring it to the transfer buffer. 2. Then sandwich the irradiated gel and membrane between sponges and double filter papers. Open the transfer cassette in a shallow tray, place a sponge pad soaked in the transfer buffer on the black sieve plate of the transfer cassette, then place the 3 mm filter paper soaked with the transfer buffer above the sponge pad; carefully place the gel on the 3 mm filter paper; place the PVDF membrane on top of the gel while taking care to remove the air bubbles; place a 3 mm filter paper that has also been soaked with the transfer buffer solution above the membrane; place another soaked sponge pad and clamp the

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transfer cassette tightly (sponge/paper/gel/membrane/ paper/sponge) (see Note 9). 3. Put the transfer cassette into the transfer tank in the correct direction. The black sieve plate of the transfer cassette face to the black end of the transfer tank, while the white sieve plate of the transfer cassette face to the white end of the transfer tank. The blot should be on the cathode and the gel on the anode. Then fill up the tank with transfer buffer and an ice pack, incubating on a magnetic stirrer (see Note 10). Turn on the power and maintain a constant voltage of 105v for 1 h at 4  C. After electrotransfer and disassembling the apparatus, the dot membrane is then processed immediately for immunoblotting. 3.5 Blocking the Membrane

Place the dot membrane (see Note 11) in a small rectangular plastic box, rinse twice with 1  TBST, then add 15 mL of fresh 10% skim milk, and shake on a rotary shaker for 1 h at room temperature (see Note 12).

3.6 Antibody Incubation

1. Dilute the primary antibody to 1/5000 with 5% skim milk (see Note 13); transfer the membrane from the blocking solution to the antibody dilution and incubate it overnight at 4  C on a shaker. 2. Wash the dot membrane 5 times with TBST on a shaker at room temperature for 5 min each time to remove residual primary antibody. 3. Prepare the secondary antibody dilution of 1/10,000 with 5% skim milk and incubate with the membrane for 1 h at room temperature (see Note 14). 4. Wash the dot membrane 4 times with TBST on a shaker at room temperature for 5 min each time (see Note 15). Then go forward with ECL detection.

3.7 Imaging and Data Analysis

1. We utilize the ECL Plus Western detection kit (Enhanced Chemiluminescet detection) to detect signals (see Note 16). Transfer 350 μL of ECL solution A and solution B from the refrigerator to two 1.5 mL tubes and place them in the dark. After the two solutions are returned to room temperature, mix them thoroughly in a ratio of 1:1. Place the dot membrane in the ECL mixture for 5 min. Finally, spread the dot membrane on the heat seal film and transfer it to a CCD camera-based imager. Capture the chemiluminescent signals according to the manufacturer’s recommendation (see Note 17). 2. Following signal capturing, immerse the dot membrane in the Ponceau S staining solution (see Note 18) and shake on a shaker for 5–10 min; then rinse the blotting membrane with distilled water for 3–5 times until protein bands are clearly visible. The experiment results are shown in Fig. 4.

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Recovery -UVB +UVB 0.5h 70 kDa -

1h

2h

6h D

50 kDa M Non-heat-denatured samples 50 kDa SIUVR8 Heat-denatured samples

Fig. 4 Time course analysis of UVR8 monomerization/dimerization in tomato seedlings by immunoblotting. Plants were exposed to broadband UV-B for 30 min and then transferred to white light for the indicated times before extracts were made. Extract samples were prepared for electrophoresis without boiling prior to SDS-PAGE and immunoblotting (a). The gel blot of heat-denatured proteins showed equal UVR8 protein amounts (b). The UVR8 dimer and monomer are indicated

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Notes 1. Keep samples at low temperature during the grinding process by freezing the tube with liquid nitrogen and precool the grinder in the 80  C refrigerator. 2. The experiment is performed using equipment from BioRad; however, suitable equipment is also available from other manufacturers. 3. Both glass plates must be tightly settled on the holder. If not tightened, the buffer will leak slowly, thus causing the “smiling gel.” Once the buffer is found to be leaking, add excess buffers between the two glass plates every 10–15 min. 4. There is usually some acrylamide debris left in the wells after removing the combs. So aspirating the wells with 1 SDS running buffer is necessary after assembling the glasses into the running tanks. 5. Pay attention to the size of comb (5.08 mm for 10 wells and 3.35 mm for 15 wells) and do not overload wells to their maximum capacity as an overflow of the sample to the adjacent well would affect the outcome of the experiment. 6. We find that the bands of the TureColor pre-stained protein are clearly separated and easy for observation as the marker contains 8 purified pre-stained proteins from 15 kDa to 130 kDa (15, 20, 25, 35, 50, 70, 100, 130 kDa) with the orange 70 kDa band corresponding to the UVR8 dimer. 7. Transferring the protein gel to a thin layer of SDS-PAGE running buffer to avoid drying of the gel is very important

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during UV-B treatment of protein gels, as the UVR8 dimer fails to form monomers in the gas phase in the absence of UV-B and under UV-B illumination (see ref. [20]). 8. We choose PVDF membrane for transferring proteins but not nitrocellulose because PVDF membrane has a high binding capacity for proteins. 9. The Whatman 3 mm filter paper could be reused for several times before it is broken. Do not reuse it once a hole appears in the middle. 10. It is better to refrigerate the buffer to 4  C in advance. Though the transfer buffer may be reused two to several times if stored at 4  C, it is better to use fresh buffers to avoid poor results. 11. We usually cut away the upper right corner of the PVDF membrane to mark the orientation. 12. It is better to prepare the blocking milk solutions fresh. We used 10% non-fat milk as the blocking solutions are cheaper and could produce good results for our antibodies. 35% BSA (fraction V) dissolved in TBST could also be used as the blocking solutions. Blocking for longer than 1.5 h at RT is not recommended as it may result in over-blocking which can increase the background. 13. The primary antibody dilution is of 1:5000; too high concentration may result in the high background while too low will results in faint bands. The primary antibody solution could be reused for 3–5 times. 14. The secondary antibody dilution ratio may range from 1:20,000 to 1:10,000, and avoid reusing secondary antibodies. 15. To increase the signal/noise ratio, the washing times can be increased to six times without decreasing the overall signal. 16. ECL plus is a luminol-based chemiluminescence kit that is catalyzed by horseradish peroxidase (HRP) and emits intense fluorescence with a sensitivity of 1–10 pg. 17. Expose the membrane with a short exposure time in the beginning, then followed by longer exposures if necessary. We usually expose the membrane for from 1 s to 5 min. 18. The Ponceau S staining solution can be reused many times to stain the blots until it no longer gives proper staining results. Another method of preparing Ponceau S staining solution: 0.2% (w/v) Ponceau, 3% (v/v) acetic acid or 2% (w/v) Ponceau S, 30% trichloroacetic acid, 30% 5-sulfosalicylic acid.

Acknowledgments This work was supported by the National Natural Science Foundation of China (31870261), a startup fund for Youngman Research at SJTU (18X100040018), the Shanghai Pujiang Program

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(18PJ1404400), the Natural Science Foundation of Shanghai (18ZR1419600), and the Agri-X Interdisciplinary Fund of Shanghai Jiao Tong University (Agri-X2017006). References 1. Jenkins GI (2009) Signal transduction in responses to UV-B radiation. Annu Rev Plant Biol 60:407–431 2. Tilbrook K, Arongaus AB, Binkert M, Heijde M, Yin R, Ulm R (2013) The UVR8 UV-B photoreceptor: perception, signaling and response. The Arabidopsis Book/American Society of Plant Biologists 11 3. Rizzini L, Favory J-J, Cloix C, Faggionato D, O’Hara A, Kaiserli E, Baumeister R, Sch€afer E, Nagy F, Jenkins GI (2011) Perception of UV-B by the Arabidopsis UVR8 protein. Science 332 (6025):103–106 4. Brown BA, Cloix C, Jiang GH, Kaiserli E, Herzyk P, Kliebenstein DJ, Jenkins GI (2005) A UV-B-specific signaling component orchestrates plant UV protection. Proc Natl Acad Sci U S A 102(50):18225–18230 5. Christie JM, Arvai AS, Baxter KJ, Heilmann M, Pratt AJ, O’Hara A, Kelly SM, Hothorn M, Smith BO, Hitomi K (2012) Plant UVR8 photoreceptor senses UV-B by tryptophanmediated disruption of cross-dimer salt bridges. Science 335(6075):1492–1496 6. Wu D, Hu Q, Yan Z, Chen W, Yan C, Huang X, Zhang J, Yang P, Deng H, Wang J (2012) Structural basis of ultraviolet-B perception by UVR8. Nature 484(7393):214 7. Jenkins GI (2014) The UV-B photoreceptor UVR8: from structure to physiology. Plant Cell 26(1):21–37 8. Ferna´ndez MB, Tossi V, Lamattina L, Cassia R (2016) A comprehensive phylogeny reveals functional conservation of the UV-B photoreceptor UVR8 from green algae to higher plants. Front Plant Sci 7:1698 9. Favory JJ, Stec A, Gruber H, Rizzini L, Oravecz A, Funk M, Albert A, Cloix C, Jenkins GI, Oakeley EJ (2009) Interaction of COP1 and UVR8 regulates UV-B-induced photomorphogenesis and stress acclimation in Arabidopsis. EMBO J 28(5):591–601 10. Findlay KM, Jenkins GI (2016) Regulation of UVR8 photoreceptor dimer/monomer photoequilibrium in Arabidopsis plants grown under photoperiodic conditions. Plant Cell Environ 39(8):1706–1714

11. Heilmann M, Jenkins GI (2013) Rapid reversion from monomer to dimer regenerates the ultraviolet-B photoreceptor UV RESISTANCE LOCUS8 in intact Arabidopsis plants. Plant Physiol 161(1):547–555 12. Yin R, Ulm R (2017) How plants cope with UV-B: from perception to response. Curr Opin Plant Biol 37:42–48 13. Yin R, Skvortsova MY, Loube´ry S, Ulm R (2016) COP1 is required for UV-B–induced nuclear accumulation of the UVR8 photoreceptor. Proc Natl Acad Sci U S A 113(30): E4415–E4422 14. Yin R, Arongaus AB, Binkert M, Ulm R (2015) Two distinct domains of the UVR8 photoreceptor interact with COP1 to initiate UV-B signaling in Arabidopsis. Plant Cell 27 (1):202–213 15. Heijde M, Ulm R (2013) Reversion of the Arabidopsis UV-B photoreceptor UVR8 to the homodimeric ground state. Proc Natl Acad Sci U S A 110(3):1113–1118 16. Heilmann M, Velanis CN, Cloix C, Smith BO, Christie JM, Jenkins GI (2016) Dimer/monomer status and in vivo function of salt-bridge mutants of the plant UV-B photoreceptor UVR 8. Plant J 88(1):71–81 17. Lippman Z, Tanksley SD (2001) Dissecting the genetic pathway to extreme fruit size in tomato using a cross between the small-fruited wild species Lycopersicon pimpinellifolium and L. esculentum var. Giant Heirloom Genetics 158(1):413–422 18. Seymour GB, Chapman NH, Chew BL, Rose JK (2013) Regulation of ripening and opportunities for control in tomato and other fruits. Plant Biotechnol J 11(3):269–278 19. Consortium TG (2012) The tomato genome sequence provides insights into fleshy fruit evolution. Nature 485(7400):635 20. Camacho IS, Theisen A, Johannissen LO, Dı´az-Ramos LA, Christie JM, Jenkins GI, Bellina B, Barran P, Jones AR (2019) Native mass spectrometry reveals the conformational diversity of the UVR8 photoreceptor. Proc Natl Acad Sci U S A 116(4):1116–1125

Chapter 10 Characterization of Seedling Greening Process in Plant Photomorphogenesis Wanqing Wang, Yuhong Li, and Rongcheng Lin Abstract Seedling deetiolation is a hallmark of the photomorphogenic response, and successful conversion of protochlorophyllide (Pchlide) into chlorophyllide during initial light exposure is critical for plant survival and growth. Here we describe the seedling deetiolation process of two typical mutants pif3 and flu by analysis of the cotyledons greening, Pchlide content, and reactive oxygen species (ROS) production and summarize a set of general methods for the research of seedling greening. Key words Seedling deetiolation, Pchlide, ROS, Cell death

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Introduction Seedling greening, which allows seedling to become photosynthetically competent and autotrophic, is the most remarkable change of plant during the switch from skotomorphogenesis to photomorphogenesis. In preparation for this switch, dark-grown seedlings accumulate the chlorophyll precursor protochlorophyllide (Pchlide) and the size of the Pchlide pool must be stoichiometrically linked to the amount of NADPH:protochlorophyllide oxidoreductase (POR) enzymes. Once the seedling is exposed to light, the dark-accumulated Pchlide is rapidly reduced by the light-dependent POR enzyme to synthesize chlorophyllide [1, 2]. However, overaccumulation of free Pchlide results in cotyledon photobleaching or even cell death, so appropriate accumulation and quick reduction of Pchlide are crucial for seedling survival. For the successful seeding greening, plants have evolved efficient mechanisms to carefully regulate Pchlide content in the dark. This protocol describes the step-by-step methods of characterization the seedling greening process, including calculating seedling greening rate, analyzing Pchlide levels, determining cell death, and visualizing reactive oxygen species (ROS). This protocol has

Ruohe Yin et al. (eds.), Plant Photomorphogenesis: Methods and Protocols, Methods in Molecular Biology, vol. 2297, https://doi.org/10.1007/978-1-0716-1370-2_10, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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been used successfully in our previous studies and readers may look details of the published data and images [3–5].

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Materials

2.1 Plant Materials and Growth Conditions

The wild-type, mutant, or transgenic Arabidopsis thaliana seeds were surface sterilized with 1.0% sodium hypochlorite (see Note 1). Seeds were sown onto Murashige and Skoog (MS) medium (see Note 2) and were incubated at 4
C in darkness for 3 d (see Note 3). Germination was induced under white light (100–120 μmol m2 s1) for 6 h at 22
C (see Note 4), then plates were wrapped in several layers of aluminum foil and kept in darkness at 22
C for 5 d (see Note 5).

2.2 Reagents and Equipments

Prepare all solutions using ultrapure water and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise).

2.2.1 Common Solutions

50 mM Tris-acetate buffer pH 5.0. Add about 10 mL water to a 100 mL glass beaker. Weigh 0.61 g Tris and transfer to the beaker. Add water to a volume of 90 mL. Mix and adjust pH with acetate (see Note 6). Make up to 100 mL with water. 10 mM potassium phosphate, pH 7.8. Weigh 17.41 g K2HPO4 and transfer to a 100 mL glass beaker containing about 40 mL of water. Add water to a volume of 100 mL and mix to prepare 1 M K2HPO4 stock solution. In the same way, dissolve 13.6 g KH2PO4 in 100 mL of water to prepare 1 M KH2PO4. Extract 9 mL of 1 M K2HPO4 and 0.9 mL of 1 M KH2PO4 to 90 mL of water and mix (see Note 7). 50 mM Tris–HCl, pH 7.5. Weigh 0.61 g Tris and dissolve in 90 mL of water. Adjust pH with HCl. Make up to100 mL with water. 10 mM Tris–HCl, pH 7.2. Dissolve 0.12 g Tris in 100 mL of water and adjust pH with HCl.

2.2.2 Seed Sterilization Solution and MS Medium

1. Seed Sterilization Solution: 1.0% NaClO, 0.02% Triton X-100. Add about 40 mL water to a 100 mL graduated cylinder. Pour 10 mL 8% NaClO and 80 μL 20% Triton X-100 to the cylinder. Make up to 80 mL with water. Store at 4
C in a bottle wrapped with aluminum foil (see Note 8). 2. MS Medium: 1 MS, 1% sucrose, 0.8% agar, pH 5.7 ~ 5.8. Weigh 4.43 g MS and 10 g sucrose and transfer to a 1 L glass beaker containing about 900 mL of water. Add 0.5 g MES and adjust pH with KOH. Make up to 1 L with water. Finally, add 0.8% (w/v) agar and autoclave in an appropriately sized conical flask.

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1. Extraction Solution: 80% acetone (see Note 9). Store in a bottle wrapped with aluminum foil at 4
C. 2. Equipment: a fluorescence spectrophotometer F-7000.

2.2.4 Cell Death Detection Trypan Blue Staining

1. Staining Solution: 0.25 mg/mL trypan blue, 25% lactic acid, 23% water-saturated phenol, 25% glycerol. Dissolve 0.1 g trypan blue in 100 mL of ddH2O. Extract 10 mL trypan blue to a 50 mL glass bottle. Add 9.3 mL phenol, 10 mL lactic acid, and 10 mL glycerol and mix (see Note 10). 2. Destaining Solution: 2.5 g/mL chloral hydrate. Dissolve 25 g chloral hydrate in 10 mL of ddH2O (see Note 11).

Electrolyte Leakage Measurement 2.2.5 Reactive Oxygen Species Measurement Diaminobenzidine (DAB) Staining Nitroblue Tetrazolium (NBT) Staining

Equipment: a conductivity meter.

1. Staining Solution: 0.1 mg/mL DAB, 50 mM Tris-acetate buffer pH 5.0 (see Note 12). 2. Destaining Solution: ethanol/glacial acetic acid/glycerol (3:1:1). Add 30 mL ethanol, 10 mL glacial acetic acid and 10 mL glycerol to a 50 mL tube and mix. 1. Staining Solution: 1 mg/mL NBT, 0.1 mg/mL NaN3, 10 mM potassium phosphate, pH 7.8 (see Note 13). 2. Destaining Solution: the same as DAB destaining solution.

Singlet Oxygen Fluorescence

1. Staining Solution: 10 μM Singlet Oxygen Sensor Green (SOSG, Invitrogen), 50 mM Tris–HCl, pH 7.5. Prepare 5mM SOSG stock solution in 1 mL of methanol. Extract 4 μL SOSG stock solution to 1 mL of 50 mM Tris–HCl and store the remaining dye at 30
C (see Note 14). 2. Equipment: a confocal microscope.

H2DCFDA Fluorescent Dye

1. Staining Solution: 10 μM 20 ,70 -dichlorodihydrofluorescein diacetate (H2DCFDA), 100 mM Tris–HCl, pH 7.2. Dissolve 0.48 g H2DCFDA in 1 mL of dimethyl sulfoxide (DMSO). Extract 1 μL H2DCFDA to 10 mL of 10 mM Tris–HCl and mix. Store the remaining dye at 30
C (see Note 15). 2. Destaining Solution: 0.6% Triton X-100, 10 mM Tris–HCl pH 7.2. 3. Equipment: DMI4500 fluorescence microscope equipped with a charge-coupled device camera.

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Methods Carry out all procedures at room temperature unless otherwise specified.

3.1 Greening Rate Measurement

1. Seedlings (about 50 to 80 seedlings of each genotype) were grown in darkness for different days and exposed to continuous white light (100–120 μmol m2 s1) for 2 days (see Note 16). 2. Count the number of dark-green cotyledons and calculate the percentage of green cotyledons (greening rate) (see Note 17).

3.2 Pchlide Content Analysis

1. 50 seedlings are grown in darkness for 5 days, or then illuminated for 5 to 10 min (see Note 18). 2. Harvest seedlings to a 1.5 mL conical centrifuge tube and resuspend samples in 500 μL of ice-cold 80% acetone under green light (see Note 19). 3. Wrap in several layers of aluminum foil and keep at 4
C overnight (see Note 20). 4. Centrifuge at 5000  g for 5 min (see Note 21). 5. Transfer the supernatant to a cuvette, measure excitation at 440 nm, and scan from 600 to 700 nm with a bandwidth of 1 nm using a fluorescence spectrophotometer under green light (see Note 22).

3.3 Cell Death Detection

1. Expose 5-d-old etiolated seedlings to continuous light (100–120 μmol m2 s1) for 2 days.

3.3.1 Trypan Blue Staining

2. Submerge more than 10 seedlings in 1 mL of trypan blue staining solution (prewarmed to 65
C) (see Note 23). 3. Vacuum infiltrate for 5 min in a desiccator, interval 5–10 min and then reinfiltrate (repeat 2-3) (see Note 24). 4. Boil for 2–4 min, alternatively microwave until boiling for 10 s (see Note 25). 5. Replace staining solution with a chloral hydrate solution for destaining (see Note 26). 6. Slowly shake for 4–8 h and multiple exchanges of destaining solution (see Note 27). 7. Equilibrate samples in 70% glycerol for several hours. 8. Mount samples on slides and photograph on a dissecting microscope (see Note 28).

3.3.2 Electrolyte Leakage Measurement

1. Transfer 5-d-old dark-grown seedlings (about 200 seedlings of each genotype) to white light (100–120 μmol m2 s1) for 12 h (see Note 29). 2. Submerge the seedlings in 5 mL of distilled water in a glass tube (see Note 30).

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3. Measure the conductivity of the solution every 4 h with a conductivity meter, setting gear:1999 μs (see Note 31). 4. After 24 h, boil the samples for 25 min. 5. Cool down to room temperature and measure the maximum electrolyte content. 6. Determine the electrolyte leakage rate by calculating the percentage of the transient conductivity to the maximum content. 3.4 ROS Histochemical Staining

1. Expose 5-d-old etiolated seedlings to continuous light (100–120 μmol m2 s1) for 2 days.

3.4.1 DAB Staining

2. Submerge more than 10 seedlings in 1 mL DAB staining solution (see Note 32). 3. Vacuum infiltrate for 5 min, interval 5–10 min and then reinfiltrate (repeat 2-3). 4. Incubate in the dark for 24 h. 5. Replace staining solution with destaining solution and boil for 10 min (see Note 33). 6. Store in 60% glycerol. 7. Mount samples on slides and photograph with a dissecting microscope.

3.4.2 NBT Staining

1. Expose 5-d-old etiolated seedlings to continuous light (100–120 μmol m2 s1) for 2 days (see Note 34). 2. Submerge more than 10 seedlings in 1 mL NBT staining solution. 3. Vacuum infiltrate for 5 min, interval 5–10 min and then reinfiltrate (repeat 2-3). 4. Stain for 30 min under bench light. 5. Replace staining solution with destaining solution and boil for 10 min. 6. Store in 60% glycerol. 7. Mount samples on slides and photograph with a dissecting microscope.

3.4.3 Singlet Oxygen Fluorescence Determination

1. Transfer 5-d-old dark-grown seedlings (more than 10 seedlings of each genotype) to a 1.5 mL centrifuge tube in the dark. 2. Immerse samples in 300 μL SOSG reagent. 3. Incubate in the dark for 2 h. 4. Transfer samples to light for 3 h or different periods. 5. Mount samples on slides and photograph through a fluorescence microscope with a GFPA (535 nm) interference filter in the objective, UV light excitation, excitation 480 nm, emission 532 nm (see Note 35).

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3.4.4 ROS Fluorescence Determination

1. Expose 5-d-old etiolated seedlings to continuous light (100–120 μmol m2 s1) for 2 days. 2. Submerge more than 10 seedlings in 1 mL H2DCFDA staining solution. 3. Incubate in the dark for 10 min (see Note 36). 4. Wash with destaining solution more than 1 h to remove excess H2DCFDA (see Note 37). 5. Mount samples on slides and observe fluorescence on a fluorescence microscope with a chloroplast fluorescence interference filter (660 nm) in the objective, excitation 500 nm, emission 535 nm.

4

Notes 1. For the complete surface sterilization, seeds were soaked in 1% NaClO disinfection solution and slowly shaken for 5 min, then washed with distilled water for 3 times. 2. Make sure the quantity and quality of seeds are consistent. To minimize errors, seeds of each genotype had better be harvested at the same time. Wear gloves when sowing seeds. 3. Wrap the plates with aluminum foil and keep at 4
C at least 3 days and no more than 5 days to ensure complete vernalization of seeds. 4. Light-induce time is at least 3 h and no more than 12 h. We find that 6 to 9 h is an appropriate irradiation time. 5. Before packaged, note the direction of the plates and do not reverse. Then, wrap the plates with three layers of aluminum foil to avoid light leak. 6. Concentrated acetate (17 N) can be used at first to narrow the gap from the starting pH to the required pH. From then on it would be better to use a series of acetate (e.g., 6 and 1 N) with lower ionic strengths to avoid a sudden drop in pH below the required pH. 7. Use 1 M stock solution to prepare potassium phosphate buffer and store the mother liquor at 4
C. 8. Seed disinfectant solution can be stored at 4
C for 1 month. Sodium hypochlorite is easy to see the light decomposition. In our laboratory we make the seed disinfectant solution fresh about every month. 9. Acetone is a volatile and irritating liquid. Vapor is irritating to eyes and mucous membranes and acts as an anesthetic in very high concentrations. Wear a mask and gloves when adding acetone. Tighten the cap after use.

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10. Trypan blue can be prepared in high concentration. We prepare 1 mg/mL stock solution and make the lactic acid-phenoltrypan blue solution fresh every few weeks. Wear gloves for all handlings (see ref. 6). 11. The chloral hydrate can be dissolved best at 60–65
C. It is a nearly saturated solution, cooling or water loss will result in crystallization. Wear a mask and gloves when preparing chloral hydrate. 12. The DAB staining solution can be prepared in large batches, frozen in aliquots, and used indefinitely. Remove the required amount, bring to room temperature, and use for dyeing. 13. Prepare the NBT staining solution by reference to the DAB. Sodium azide can be prepared to high concentrated stock solution and store at 30
C. Wear gloves when weighing sodium azide. 14. SOSG can be prepared in high concentration. We prepare 5 mM stock solution in methanol (see ref. 7), wrap with aluminum foil and frozen at 30
C. 15. H2DCFDA can be prepared in high concentration. We prepare 100 mM stock solution, wrap with aluminum foil and frozen at 30
C (see ref. 8). 16. The percentage of green cotyledons (greening rate) can decrease with darkness prolonging and light intensity enhancing. According to the greening characterization of each seedling, choose appropriate treatment condition. For the research of pif3 and flu, we grow seedlings in darkness for 5 d, and then induce deetiolation process under white fluorescent light (100–120 μmol m2 s1). 17. Greening rate was calculated by counting the percentage of dark-green cotyledons. White, yellowish, or light-green cotyledons were classified as the impaired greening and cannot be counted. 18. Ensure the germination rate of each genotype; we cannot accurately calculate the number of seedlings in darkness. 19. Prepare the extraction solution in advance, accurately sub-pack in aliquots and place in a covered carton. Rapidly transfer seedlings to extraction solution and avoid sticking the MS medium. Immerse seedlings in extraction solution, ensure complete extraction, and then rapidly put back in the carton. All handlings must be in complete darkness under a dim-green safe light. The illuminated samples need firstly be frozen in liquid N2, then transfer to darkness and add acetone. 20. In the process of wrapping, gently operate to avoid samples sticking on the wall of tube.

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21. Before centrifugation, vortex mixes the samples for 10 s under green light. 22. Transfer supernatant as more as possible. According to its volume, choose suitable cuvette. Keep measurement as fast as possible to avoid the luminous damage from fluorescence spectrophotometer. 23. Gently put the seedlings into each tube and keep the seedling intact to get the best quality photos. Keep staining volume as small as possible. 24. Swirl desiccator to remove air bubbles from cotyledons. Alternatively, you can pull vacuum using a lyophilizer. 25. Place tubes into boiling water bath so well bottoms just touch the water. Dyeing time cannot be too long and keep within 4 min. Avoid cover opening during boiling. 26. Avoid touching the samples when replacing the destaining solution. Add chloral hydrate to just cover the seedlings. Wear a mask to avoid breathing vapors. 27. Seedlings can be stored in chloral hydrate for days to weeks. IF storing, make sure that the tube is tightly sealed. If water evaporates from chloral hydrate, there will be crystals forming in and on the tissue. 28. Gently operate to avoid seedlings damage and intertwining. The dead cells are painted blue. The numbers of cell death increase with darkness prolonging and light intensity enhancing. 29. Carefully transfer seedlings to distilled water, avoid samples damage to increasing conductivity. 30. Make sure the samples completely immerse in the distilled water. 31. Before every measurement, gently mix the distill water. Do not touch the wall of tube when measuring. Wash the probe after each measurement. Other measurements were done with a conductivity cell. 32. DAB is used for the detection of hydrogen peroxide (H2O2) accumulation in cotyledons. When H2O2 is present, it forms brown precipitate in the tissue. The production of ROS can increase with darkness prolonging and light intensity enhancing. 33. Open the pipe cover and release the air in time to avoid destaining buffer spilling during boiling. 34. NBT is used for the detection of superoxide (O2 –) accumulation in cotyledons. In the presence of O2 –, it is reduced to blue precipitate in the tissue. l

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35. SOSG is a detection reagent that is highly selective for singlet oxygen. This indicator initially exhibits weak blue fluorescence, but in the presence of singlet oxygen, it emits a green fluorescence (excitation/emission maxima ~480/532 nm) (see ref. 7). Fluorescence intensity was determined by Image J software, and the background was subtracted. 36. The H2DCFDA fluorescent probe is commonly employed and may react with several ROS, including hydrogen peroxide, hydroxyl radicals, and peroxynitrite. Upon oxidation by ROS, the nonfluorescent H2DCFDA is converted to the highly fluorescent 20 , 70 -dichlorofluorescein. Dyeing time cannot be too long. It is best to keep within 10 min lest stomatal guard cells are excessively tinted (see ref. 8). 37. Wrap samples with aluminum foil in the process of washing. Gently shake to keep seedlings intact. Wash more than 1 h to ensure the excess H2DCFDA is removed.

Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (31325002,31870213) to R.L. References 1. Reinbothe S, Reinbothe C, Lebedev N, Apel K (1996) PORA and PORB, two light-dependent protochlorophyllide-reducing enzymes of angiosperm chlorophyll biosynthesis. Plant Cell 8:763–769 2. Mochizuki N, Tanaka R, Grimm B, Masuda T, Moulin M, Smith AG, Tanaka A, Terry MJ (2010) The cell biology of tetrapyrroles: a life and death struggle. Trends Plant Sci 15:488–498 3. Chen D, Xu G, Tang W, Jing Y, Ji Q, Fei Z, Lin R (2013) Antagonistic bHLH/bZIP transcription factors form transcriptional modules that integrate light and reactive oxygen species signaling in Arabidopsis. Plant Cell 25:1657–1673 4. Tang W, Wang W, Chen D, Ji Q, Jing Y, Wang H, Lin R (2012) Transposase-derived proteins FHY3/FAR1 interact with PHYTOCHROME-INTERACTING FACTOR 1 to regulate chlorophyll biosynthesis by modulating HEMB1 during deetiolation in Arabidopsis. Plant Cell 24:1984–2000

5. Xu G, Guo H, Zhang D, Chen D, Jiang Z, Lin R (2015) REVEILLE1 promotes NADPH:Protochlorophyllide oxidoreductase a expression and seedling greening in Arabidopsis. Photosynth Res 126:331–340 6. Keogh RC, Deberall BJ, McLeod S (1980) Comparison of histological and physiological responses to Pbakospora pacbyrbiziin resistant and susceptible soybean. Trans Br Mycol Soc 74:329–333 7. Flors C, Fryer MJ, Waring J, Reeder B, Bechtold U, Mullineaux PM, Nonell S, Wilson MT, Baker NR (2006) Imaging the production of singlet oxygen in vivo using a new fluorescent sensor, Singlet Oxygen Sensor Green. J Exp Bot 57:1725–1734 8. Joo JH, Wang SY, Chen JG, Jones AM, Fedoroff NV (2005) Different signaling and cell death roles of heterotrimeric G protein a and b subunits in the Arabidopsis oxidative stress response to ozone. Plant Cell 17:957–970

Chapter 11 Protoplast System for Studying Blue-Light-Dependent Formation of Cryptochrome Photobody Xiangguang Lyu, Hongyu Li, and Bin Liu Abstract Cryptochromes (CRYs) belong to an ancient and conserved class of blue-light receptor regulating circadian clock and development in animals and plants. Arabidopsis CRY2 form physiologically active homodimers in response to blue light treatment and further oligomerize into photobodies, which are expected to be the foci harboring protein interaction, phosphorylation, and ubiquitination. Here we describe two efficient methods developed to test the formation of blue-light-dependent photobodies of CRY-GFP fusing proteins using the mesophyll protoplasts of Arabidopsis or soybean, respectively. Key words Mesophyll protoplast, Cryptochrome, Photobody, Transient expression, Arabidopsis, Soybean

1

Introduction Blue light receptor cryptochromes (CRYs) were firstly discovered in Arabidopsis and then found in other plants, animals, and microbes [1–3]. They are structurally homologue to photolyase but have no photolyase activity [4]. Arabidopsis genome encodes two CRYs, CRY1 and CRY2, which regulate photomorphogenesis and photoperiodic flowering time, respectively, or in a partially redundant manner [5–8]. CRYs mediate blue light signals by homodimerizing and then interacting with protein partners including COP1 (constitutive photomorphogenic 1), SPA (suppressor of PhyA-105), and CIBs (cryptochrome-interacting basic helix-loop-helixes) to manipulate expressions of blue light-responsive genes [9– 12]. Photobody is defined as the nuclear body/domain consisting of photoexcited photoreceptor in plants [13]. Light irradiation triggers rapid formation of photobody whose number and size are strictly regulated with the dosage of received photons [14]. Multiple plant photoreceptors including Phytochromes (PhyA to E), CRYs, and possibly UVR8 (UV resistance locus 8) can form photobodies [15–19]. Interestingly, Soybean GmCRY1s were

Ruohe Yin et al. (eds.), Plant Photomorphogenesis: Methods and Protocols, Methods in Molecular Biology, vol. 2297, https://doi.org/10.1007/978-1-0716-1370-2_11, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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Fig. 1 Analyzing the formation of GmCRY2a photobodies in Arabidopsis mesophyll protoplasts. The 35S:: GmCRY2-YFP construct was transiently expressed in Arabidopsis mesophyll protoplasts. The transformed protoplasts were kept in dark or exposed to blue light (30 μmol m
2 s
1) for 5 min and then observed using the confocal laser scanning microscope. Scale bar ¼ 10 μm

recently found to accumulate into photobody-like structures in cytoplasm concomitantly with the formation of photobodies in the nucleus in response to blue light treatment [20]. Taken that the functional and regulating mechanisms of photobodies are still largely unclear, it is highly demanded to establish a quick, efficient, and standard protocol to study the process of photobody formation at a cellular level. Here we described two transient expression methods to study the formation of CRY-photobodies in response to blue light treatment using the mesophyll protoplasts prepared with leaves of Arabidopsis and soybean, respectively (Fig. 1 and Fig. 2). A good performance can achieve 90% transfection efficiency using protoplasts of Arabidopsis and 80% using that of soybean [20]. Two methods are majorly different in the solutions and selection of leave tissues. Researchers are suggested to start with our recommended procedures and adjust parameters and steps according to specific experimental requirements and plant species.

2 2.1

Materials Plant Cultivation

Cultivate Arabidopsis thaliana L. accession Columbia (Col-0) and soybean (Glycine max L. Merr.) cultivar Williams 82 in growth cabinets under 8-h-light (120–180 μmol m
2 s
1)/16-h-dark conditions at 25  C/22  C (light/dark), respectively (see Note 1).

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Fig. 2 Analyzing the formation of GmCRY2a photobodies in soybean mesophyll protoplasts. The 35S:: GmCRY2-YFP construct was transiently expressed in soybean mesophyll protoplasts. The transformed protoplasts were kept in dark or exposed to blue light (30 μmol m
2 s
1) for 5 min and then observed using the confocal laser scanning microscope. Scale bar ¼ 5 μm 2.2 Solutions for Isolation of Arabidopsis Mesophyll Protoplasts

1. 0.2 M KCl: Weigh 1.49 g KCl, make up to 100 mL with water, and sterilize using a 0.45-μm filter.

2.2.1 Stock Solution

3. 1 M CaCl2: Weigh 14.70 g CaCl2-2H2O, make up to 100 mL with water, and sterilize using a 0.45-μm filter.

2. 2 M NaCl: Weigh 11.69 g NaCl, make up to 100 mL with water, and sterilize using a 0.45-μm filter.

4. 0.2 M MES-KOH (pH 5.7): Weigh 4.27 g MES and transfer to the cylinder. Add water to a volume of 80 mL and stir until absolute dissolution. Adjust pH to 5.7 with 1 M KOH, make up to 100 mL with water, and sterilize using a 0.45-μm filter. 5. Digestion solutions: 20 mM MES-KOH (pH 5.7) containing 1.5% (W/V) Cellulase “Onozuka” R-10 (Yakult, catalog number: L0012), 0.4% Macerozyme R-10 (Yakult, catalog number: L0021), 0.4 M mannitol, 20 mM KCl, 10 mM CaCl2, and 0.1% BSA. Mix 1 mL 0.2 M MES-KOH (pH 5.7), 0.15 g Cellulase “Onozuka” R-10, 0.04 g Macerozyme R-10, 0.728 g D-Mannitol, and 1 mL 0.2 M KCl. Warm the solution at 55  C for about 10 min and cool it to room temperature (see Note 2). Add 0.1 mL 1 M CaCl2 and 0.1 g BSA and make up to 10 mL with water. Filter the final enzyme solution through a 0.45-μm filter (see Note 3). 6. W5 solutions: 2 mM MES-KOH (pH 5.7), 154 mM NaCl, 125 mM CaCl2, 5 mM KCl. Mix 3.85 mL 2 M NaCl, 1.25 mL 0.2 M KCl, 0.5 mL 0.2 M MES-KOH (pH 5.7), and 6.25 mL 1 M CaCl2-2H2O. Make up to 50 mL with sterile water.

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2.3 Solution for Isolation of Soybean Mesophyll Protoplasts

1. 0.2 M KH2PO4: Weigh 2.72 g KH2PO4, make up to 100 mL with water, and sterilize using a 0.45-μm filter. 2. 1 M KNO3: Weigh 10.11 g KNO3, make up to 100 mL with water, and sterilize using a 0.45-μm filter. 3. 1 M MgSO4: Weigh 24.65 g MgSO4-7H2O, make up to 100 mL with water, and sterilize using a 0.45-μm filter. 4. 0.1 M KI: Weigh 1.66 g KI, make up to 100 mL with water, and sterilize using a 0.45-μm filter. 5. 0.1 M CuSO4: Weigh 1.60 g CuSO4, make up to 100 mL with water, and sterilize using a 0.45-μm filter. 6. 1 M KOH: Weigh 5.61 g KOH, make up to 100 mL with water. 7. 0.15 M MgCl2: Weigh 1.52 g MgCl2-6H2O, make up to 50 mL with water, and sterilize using a 0.45-μm filter. 8. CPW buffer (pH 5.7): 0.2 mM KH2PO4, 1 mM KNO3, 10 mM CaCl2, 1 mM MgSO4, 1 μM KI, and 0.1 μM CuSO4. Mix 1 mL 0.2 M KH2PO4, 1 mL 1 M KNO3, 10 mL 1 M CaCl2, 1 mL 1 M MgSO4, 10 μL 0.1 M KI, and 1 μL 0.1 M CuSO4. Add water to a volume of 900 mL. Mix well and adjust to pH 5.7 with 1 M KOH. Make up to 1 L with sterile water. Store at 4  C. 9. CPW9M solution: 9% (W/V) mannitol in CPW buffer. Weight 4.5 g mannitol and transfer to 30 mL CPW solution. Mix well and make up to 50 mL with CPW buffer. 10. CPWE solution: 1.0% (W/V) Celluase “Onozuka” RS (Yakult, catalog number: L0011) and 0.2% (W/V) Pectolase Y-23 (Yakult, catalog number: L0042) in CPW9M solution. Weight 0.1 g RS and 0.02 g Y-23, transfer to 5 mL CPW9M solution, and mix well. Warm the solution at 55  C for about 10 min and cool it to room temperature. Make up to 10 mL with CPW9M buffer and sterilize using a 0.45-μm filter. 11. MMG solution: 4 mM MES-KOH solution (pH 5.7), 0.4 M mannitol, 15 mM MgCl2. Weight 0.728 g D-Mannitol and dissolve in 5 mL water. Add 1 mL 0.15 M MgCl2 and 0.2 mL MES-KOH (pH 5.7). Make up to 10 mL with water.

2.4 Transient Transfection

Transfection solution: 0.1 M CaCl2, 0.2 M mannitol, and 40% (w/v) PEG 4000. Weigh 4 g PEG4000, 0.364 g D-Mannitol, dissolve in sterile water. Add 1 mL 1 M CaCl2. Make up to 10 mL with sterile water (see Note 4).

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Methods

3.1 Isolation and Transfection of Arabidopsis Mesophyll Protoplast

1. Collect 0.2 g healthy and well-expanded leaves from 3- to 4-week-old plants. Usually, true leaves from five to seven are suitable for protoplast experiments (see Note 5). 2. Cut leaves into approximately 0.5 mm strips with propulsive force using sharp razor blade without tissue damage at cutting site (see Note 6). 3. Transfer leaf strips into 10 mL digestion solutions immediately and submerge completely. 4. Digest leaf strips in the enzyme solution with shaking  50 g at room temperature in dark condition for about 3–4 h until most of protoplasts are released from leaf strips (see Note 7). Check the separated protoplasts under the microscope (see Note 8). 5. Separate the enzyme solution from undigested material by filtration through a stainless-steel mesh sieve (200 wires per inch) and collect the filtered solution into a new 50 mL roundbottomed tube on ice. 6. Centrifuge at 100  g for 2 min at 4  C to collect the protoplasts. Remove supernatant as much as possible (see Note 9). 7. Resuspend the protoplasts gently with the same volume of the precooling W5 solution. 8. Centrifuge at 100  g for 2 min at 4  C to collect the protoplasts. Discard the supernatant. 9. Resuspend the protoplasts with the same volume of the precooling W5 solution again. Keep the protoplasts on ice for 30 min. 10. Centrifuge at 100  g for 2 min to collect the protoplasts. Discard the supernatant. 11. Resuspend the protoplasts in 3 mL MMG buffer. 12. Centrifuge at 100  g for 2 min to collect the protoplasts. Discard the supernatant. 13. Resuspend the protoplasts at 2  105/mL in MMG buffer. 14. Add 10 μL plasmid DNA (10–20 μg, less than 10Kb in size) to 2 mL sterile tube (see Note 10). 15. Add 100 μL protoplasts and mix well with softly tapping. 16. Add 110 μL transfection solution, mix the components completely by gently pipetting up and down. 17. Keep the transfection mixture under the dark condition at room temperature, and incubate approximately 15 min.

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18. Add 440 μL W5 solution into transfection mixture, then mix the suspension carefully by inverting the tube to terminate the reaction. 19. Centrifuge at 100  g for 2 min, then remove the supernatant. 20. Add 500 μL W5 solution and resuspend the protoplasts. Centrifuge at 100  g for 2 min and remove the supernatant. Repeat this step three times. 21. Resuspend the transfected protoplasts carefully with 1 mL W5 solution and transfer into the tissue culture plate prior to incubation in dark condition at 24  C. 3.2 Isolation and Transfection of Soybean Mesophyll Protoplast

1. Collect 0.2 g fully expanded simple leaves of 10-day-old plants or trifoliate leaves of 17-day-old plants grown under short day conditions (see Note 11). 2. Divide the leaves into two parts along veins with sharp razor blade, and then cut leaves into approximately 0.5 mm strips and avoid tissue damage at cutting site (see Note 12). 3. Transfer the leaves strips quickly into a conical flask containing 10 mL CPWE solution. 4. Incubate at room temperature with shaking at 50 rpm/min on a gyratory shaker for about 5 h in dark. 5. Check for the number of intact and independent protoplasts under the microscope (see Note 13). 6. Filter the digestion solution through three layers of medical gauze into a 50 mL round-bottomed tube. 7. Centrifuge at 100  g for 5 min at 4  C to pellet the protoplasts. Remove as much supernatant as possible. 8. Resuspend the pelleted protoplasts in 1 mL precooling CPW9M solution by gently pipetting up and down. Dilute the protoplasts with an additional 9 mL precooling CPW9M solution (see Note 14). 9. Centrifuge at 100  g for 3 min at 4  C and discard the supernatant. Repeat resuspending and centrifugation three times. 10. Resuspend the pelleted protoplasts in precooling CPW9M solution and keep on ice for 30 min. 11. Centrifuge at 100  g for 3 min and remove the supernatant. Resuspend with 3 mL MMG solution. 12. Centrifuge at 100  g for 3 min, and remove the supernatant. Resuspend protoplasts at a concentration about 105–106/mL in MMG solution. 13. Add 10 μL plasmid DNA (10–20 μg, less than 10Kb in size) to 2 mL sterile tube.

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14. Add 100 μL protoplast solution and mix well. 15. Add 110 μL PEG solution to the mixture. Mix by inverting the tube gently. 16. Incubate the mixture in dark at 25  C for 15 min. 17. Dilute the transfection mixture with 500 μL CPW9M solution, and mix the suspension gently by inverting the tube to terminate the reaction. 18. Centrifuge at 100  g for 2 min and remove the supernatant carefully. 19. Resuspended the protoplasts with 1 mL CPW9M solution. 20. Centrifuge at 100  g for 2 min and remove the supernatant carefully. Repeat resuspending and centrifugation three times. 21. Resuspend the transfected protoplasts in 1 mL CPW9M solution and transfer into the tissue culture plate prior to incubate in dark condition at 25  C. 3.3 Detecting the Formation of Cryptochrome Photobodies

1. Keep the protoplasts transfected with the 35S::GmCRY2-YFP construct in dark condition overnight (see Note 15). 2. Expose the protoplast to blue light at a fluence rate of 30 μmole m
2 s
1 for 5 min. 3. Observe the protoplasts using a confocal laser scanning microscope at 488 nm wavelength. 4. Process the image using the ZEN 2009 Light Edition software (Figs. 1 and 2).

4

Notes 1. Healthy and vigorous leaf is the prerequisite to produce highquality mesophyll protoplasts. Therefore, plants need to be cultivated carefully away from abiotic and biotic stresses including drought, flooding, extreme temperature and light, mechanical damage, diseases, and pests. 2. Moderate high temperature of 55  C can help to dissolve the enzymes and inactivate the trace amount of Dnase and protease. Gently vortexing the solution 2–3 times can also enhance enzyme solubility. 3. The enzyme solution should be freshly prepared to ensure a high enzymatic activity. 4. The freshly prepared transfection solution is required for a high transfection efficiency. However, to ensure complete dissolution of PEG, the transfection solution should be prepared at least 1 h before transfection.

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5. The fresh and just fully expanded leaf is the best tissue to produce high-quality mesophyll protoplasts. 6. Generally, 10 leaves digested in 10 mL digestion solutions were able to produce enough protoplasts for the transformations of ten independent constructs. 7. The protoplast isolation efficiency is markedly different between different ecotypes and genotypes. The incubation time can be optimized according to different experiments. 8. The round shape indicates that the protoplast is intact and in good quality. The average diameter of Arabidopsis mesophyll protoplast is approximately 30–50 μm. 9. The protoplasts are fragile and easy to be physically broken. We usually cut off the sharp end of pipet tip and resuspend the pellets by gently pipetting up and down. 10. A good quality of plasmid DNA is essential for a successful protoplast transfection. The trace amount of residual ethanol or salt in the plasmid solution can cause damage of the protoplasts and reduce transfection efficiency. Commercial Mini Plasmid Kit is recommended for the extraction of high-quality plasmid DNA. 11. The selection of the just fully expanded leaves at the proper developmental stage is critical for high transfection efficiency. We have experienced low transfection efficiency using the protoplasts made of younger or older leaves. Generally, simple leaf of 10-day-old plants and trifoliate leaf of 17-day-old plants are suit for protoplast isolation. 12. The yield is up to approximately 1  107 protoplasts per gram fresh weight. Typically, one simple leaf or 2/3 trifoliate leaf digested in CPWE solution is enough for the transformations of ten independent constructs. 13. The average diameter of soybean mesophyll protoplast is approximately 15–20 μm, which is about half that of Arabidopsis. 14. The CPW9M solution was first reported for isolating Petunia leaf protoplasts in 1973 [21]. We found that CPWM solution is also compatible well with the preparation of soybean mesophyll protoplasts. The W5 solution is suitable for Arabidopsis but not for soybean, probably because soybean mesophyll protoplasts suspended in W5 solution tended to aggregate. 15. Generally, cryptochrome fused at the N-terminal of fluorescent protein (GFP or YFP) is easy to form photobody or photobody-like structure than that fused at the C-terminal of fluorescent protein [17, 22].

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References 1. Ahmad M, Cashmore AR (1993) HY4 gene of A. thaliana encodes a protein with characteristics of a blue-light photoreceptor. Nature 366 (6451):162–166 2. Griffin EA, Staknis D, Weitz CJ (1999) Lightindependent role of CRY1 and CRY2 in the mammalian circadian clock. Science 286 (5440):768–771 3. Xiangguang L et al (2020) GmCRY1s modulate gibberellin metabolism to regulate soybean shade avoidance in response to reduced blue light. Mol Plant. https://doi.org/10.1016/ j.molp.2020.11.016 4. Brautigam CA et al (2004) Structure of the photolyase-like domain of cryptochrome 1 from Arabidopsis thaliana. Proc Natl Acad Sci U S A 101(33):12142–12147 5. Exner V et al (2010) A gain-of-function mutation of Arabidopsis cryptochrome1 promotes flowering. Plant Physiol 154(4):1633–1645 6. Ono D, Honma S, Honma K (2013) Postnatal constant light compensates Cryptochrome1 and 2 double deficiency for disruption of circadian behavioral rhythms in mice under constant dark. PLoS One 8(11):e80615 7. Liu B et al (2010) Searching for a photocycle of the cryptochrome photoreceptors. Curr Opin Plant Biol 13(5):578–586 8. Wang Q et al (2016) Photoactivation and inactivation of Arabidopsis cryptochrome 2. Science 354(6310):343–347 9. Wang H et al (2001) Direct interaction of Arabidopsis cryptochromes with COP1 in light control development. Science 294 (5540):154–158 10. Liu HT et al (2008) Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis. Science 322 (5907):1535–1539 11. Liu B et al (2011) Arabidopsis cryptochrome 1 interacts with SPA1 to suppress COP1 activity in response to blue light. Genes Dev 25 (10):1029–1034

12. Lian HL et al (2011) Blue-light-dependent interaction of cryptochrome 1 with SPA1 defines a dynamic signaling mechanism. Genes Dev 25(10):1023–1028 13. Van Buskirk EK, Decker PV, Chen M (2011) Photobodies in light signaling. Plant Physiol 158(1):52–60 14. Chen M, Chory J (2011) Phytochrome signaling mechanisms and the control of plant development. Trends Cell Biol 21(11):664–671 15. Van Buskirk EK, Decker PV, Chen M (2012) Photobodies in light signaling. Plant Physiol 158(1):52–60 16. Van Buskirk EK et al (2014) Photobody localization of Phytochrome B is tightly correlated with prolonged and light-dependent inhibition of hypocotyl elongation in the dark. Plant Physiol 165(2):595–607 17. Zuo ZC et al (2012) A study of the blue-lightdependent phosphorylation, degradation, and Photobody formation of Arabidopsis CRY2. Mol Plant 5(3):200–207 18. Kircher S et al (1999) Light quality-dependent nuclear import of the plant photoreceptors phytochrome A and B. Plant Cell 11 (8):1445–1456 19. Favory JJ et al (2009) Interaction of COP1 and UVR8 regulates UV-B-induced photomorphogenesis and stress acclimation in Arabidopsis. EMBO J 28(5):591–601 20. Xiong L et al (2019) A transient expression system in soybean mesophyll protoplasts reveals the formation of cytoplasmic GmCRY1 photobody-like structures. Sci China Life Sci 62(8):1070–1077 21. Frearson EM, Power JB, Cocking EC (1973) The isolation, culture and regeneration of Petunia leaf protoplasts. Dev Biol 33 (1):130–137 22. Yu X et al (2007) Arabidopsis cryptochrome 2 completes its posttranslational life cycle in the nucleus. Plant Cell 19(10):3146–3156

Chapter 12 Uncover the Nuclear Proteomic Landscape with Enriched Nuclei Followed by Label-Free Quantitative Mass Spectrometry Yan Wang, Zhuang Lu, and Lei Wang Abstract Treated with light pulse or under certain diurnal conditions, photoreceptors can translocate into nucleus followed by conformation change. Many critical components of light signaling pathways also majorly function in nucleus. Hence, it is beneficial to establish a combined method to uncover and compare the nuclear proteomic landscape among the mutants of light signaling components. Here we describe an optimized method to isolate nucleus with seedlings growing under light/dark cycles for further characterizing the nuclear proteome with label-free quantitation by liquid chromatography mass spectrometry (LC–MS). Key words Nuclear protein isolation, Mass spectrometry, Quantitative proteomics, SWATH

1

Introduction Nascent phytochromes are biosynthesized in cytosol as inactive Pr form, and transferred into biological active Pfr form upon light treatment, then translocate into nucleus to change the expression of downstream target genes [1–4]. Phytochrome A (phyA) could be directly recruited to specific promoters through its nuclear interacting partners [5]. Very recently, blue light receptor Cryptochrome 2 was also shown to directly bind DNA and active gene expression in the presence of blue light via cooperating with CIB1 (cryptochrome-interacting basic-helix-loop-helix 1) [6]. Moreover, ultraviolet-B (UV-B) light receptor UVR8 (UV RESISTANCE LOCUS 8) can trigger regulatory changes in gene expression by affecting PIF4 stability or interact with WRKY36 to transduce UV-B light signaling [7–9]. Altogether, numerous nuclear events are essential for transducing environmental dynamic light information, such as proteolysis to affect protein stability and protein-

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protein interaction to form the complex which is required for DNA binding capability. On the other hand, diurnal rhythm of light/dark cycles is also critical and sufficient for entraining endogenous circadian clock. A few core circadian components, including Evening Complex formed by ELF4-ELF3-LUX, TIC (TIME FOR COFFEE), ZTL (ZEITLUPE), and LWD1/2 (LIGHT REGULATED WD1/2), play dual roles in both mediating light input to core oscillator and acting within the oscillator itself [10, 11]. Furthermore, protein stability of many core circadian components can be modulated by light signaling [12]. By contrast, nucleocytoplasmic partitioning of phytochromes displayed a robust diurnal rhythmic pattern. For instance, the amount of intranuclear Phytochrome B (phyB) speckles significantly increased about 10 min before light-on signal in the light/dark cycles growing plants [1]. Hence, uncovering quantitative nuclear proteomic landscape is essential for elucidating the molecular mechanisms of plant adaption to dynamic light signals. Here, we describe a feasible protocol for isolating nucleus with seedlings grown under light/dark cycles, and also introduce how to characterize the nuclear proteome with a label-free quantitation analysis.

2

Materials Prepare all solutions with ultrapure water. Prepare and store all reagents at 4
C. All operations must be carried out at 4
C (unless indicated otherwise). Two-week-old Arabidopsis seedlings were harvested at ZT0 (Zeitgeber time, ZT0 represents the light on) and ZT12, and quickly frozen in liquid nitrogen. Store the material in the 70
C freezer immediately or start the nucleic isolation as below. At least 10 mL powder should be used after grinding in liquid nitrogen for each sample. Grind the sample using mortar and pestle in liquid nitrogen. Store the samples in a 50 mL tube and keep the tubes on dry ice until use.

2.1 The Plant Nuclei Isolation/Extraction Kit

The reagents in the kit as follows: Nuclei Isolation Buffer 4  (NIB), Sucrose 2.3 M, TRITON X-100 10% solution, and Nuclei PURE Storage Buffer (see Note 1).

2.2

100 mM Tris–HCl pH 7.5, 1 mM EDTA (Ethylene Diamine Tetraacetic acid), 10% Glycerol, 75 mM NaCl, 0.05% SDS (Sodium dodecyl sulfate), 0.1% Triton X-100, 1 mM PMSF, supplemented with protease inhibitor Aprotinin, Pepstatin A, Leupeptin, Antipain, Chymostatin, Proteosome inhibitor mixture, NaF (sodium fluoride), and Na3VO4 (sodium orthovanadate), as listed previously [13–15].

Extraction Buffer

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2.3 Preparation of SDS-PAGE Gel

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1. Prepare a 12% acrylamide gel (1 mm thick), make the separating gel as follows: Separating gel (5 mL): 2 mL 30% Acrylamide/Bis-acrylamide (37.5:1), 1.72 mL ddH2O, 1.25 mL 4  separating buffer, 25 μL 10% APS (Ammonium persulfate), 5 μL TEMED. 2. Add ethyl-alcohol to cover the separating gel, to avoid bubble, waiting about 30 min then discard it. 3. Stacking gel (5 mL): 0.66 mL 30% Acrylamide/Bis-acrylamide (37.5:1), 3.04 mL ddH2O, 1.25 mL 4  stacking buffer, 16 μL 10% APS, 16 μL TEMED. 4. 6  SDS protein loading buffer (45 mL): 1.75 mL ddH2O, 6.25 mL 1 M Tris–HCl (pH 6.8), 25 mL Glycerol, 20% SDS, 2 mL 0.5% bromophenol blue. To each 900 μL add 100 μL β-mercaptoethanol to make work solution freshly.

2.4

Equipment

TripleTOF 5600+ Mass Spectrometer (Sciex), Eksigent NanoLC Ultra nanoflow high-performance liquid chromatography (HPLC) system, refrigerated high-speed centrifuge, centrifuge, freeze dryers, and test tubes (50 mL, 2 mL, 1.5 mL).

2.5

Reagents

Dithiothreitol (DTT), Miracloth, LC–MS grade Acetonitrile, LC– MS grade water, LC–MS grade formic acid, and Bradford Quant Kit. Mobile phase A: 0.1% FA (formic acid). Add 1 μL FA to 1 L water, mix, and store at 4
C. Mobile phase B: 100% ACN (Acetonitrile), 0.1% FA. Add 1 μL FA to a 1 L ACN, mix, and store at 4
C.

2.6

Software

Analyst version1.7, ProteinPilot version 4.5, PeakView version2.2, MS/MSALL with SWATH™ Acquisition MicroApp version2.0, MarkerView version1.2.1, excel template of Variable Window Calculator Version 0.2112513.

3

Methods For a brief flowchart of enriching nuclei followed by SWATH labelfree quantitation analysis, see Fig. 1.

3.1

Cell Lysis

Carry out reagents on the ice (except stated specifically), prepare appropriate dry ice and precooling the centrifuge to 4
C. 1. Prepare 10 mL of liquid nitrogen ground tissue powders of Arabidopsis wild type and the corresponding mutant into 50 mL test tubes, respectively, and keep them on the dry ice. 2. Add 25 mL lysis buffer of 1  Nuclei Isolation Buffer1 (NIB1) (1 mL NIB1: 250 μL of 4  NIB, 1 μL of 1 M DTT, 10 μL of

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Fig. 1 A brief flowchart of enriching nuclei followed by SWATH label-free quantitation analysis

100 mM PMSF, 2 μL of 1 M NaF, 2 μL of 1 M Na3VO4, dd H2O complement, mix well), and homogenized completely (see Note 2). 3. Prepare double layers of Miracloth to 50 mL tube, and the homogenate was filtered through it. The flow-through was spun at 1260 g for 5 min at 4
C (see Note 3). 4. Remove the Miracloth from the tube, centrifuge again for another 5 min, 1260  g at 4
C (see Note 4), and then discard the supernatant (see Note 5). 5. Add 10 mL cytosolic protein extraction buffer NIBA (1  NIB1 add 30 μL/mL 10% Triton X-100, and all the protease and proteosome inhibitors), mix it vigorously by pipetting (see Note 6), and centrifuge with 2880  g for 10 min at 4
C. 6. Transfer 40 μL supernatant to new tubes with prepared 20 μL of 6  SDS protein loading buffer as the cytoplasmic fraction, and then discard the supernatant and resuspend the pellets completely in 5 mL NIBA, and keep on ice for 5 min. 7. Centrifuge at 2880  g for 10 min at 4
C to harvest nuclei. 8. Decant the supernatant gently. Washing pellets with 1 NIBA containing 0.3% Triton X-100 briefly until no green color on the pellets (see Note 7). 9. Centrifuge at 2880  g for 10 min at 4
C. 10. Repeat 8 and 9 until no any green color on the pellet (optional). 11. Decant the supernatant gently and resuspend the pellets completely in 2.4 mL NIBA. Take several microliters of the cells in buffer and check them under the microscope to check the intact of nuclei as shown in Fig. 2 (see Note 8). 3.2 Semi-Pure Preparation of Nuclei

1. Prepare 1.5 M sucrose and mix well before used as a cushion (see Note 9). 2. Prepare three 2 mL tubes with 1 mL 1.5 M sucrose as cushion, and apply 800 μL resuspended pellets in NIBA buffer (Subheading 3.1, step 11) on top of each tube. Centrifuge at 13,000  g for 10 min at 4
C (see Note 10).

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Fig. 2 Quality check of purified nuclei with fluorescence microscope. Imaging of the isolated nuclei (left panel). DAPI staining and merged photos were used to show the specific localization of nuclei (middle panel and right panel, respectively). Bars ¼ 30 μm

3. Aspirate the upper green phase and the sucrose cushion layer by using pipette tips to avoid disturbing the pellets of nuclei. 4. Wash the pellets twice, resuspend with 1 mL of NIBA, centrifuge at 12,000  g for 5 min at 4
C, and pool the pellets from the three tubes (see Note 11). 3.3 Nuclear Protein Extraction

1. Prepare a working nuclear protein extraction buffer as follows: 100 mM Tris–HCl pH 7.5, 1 mM EDTA, 10% Glycerol, 75 mM NaCl, 0.05% SDS, 0.1% Triton X-100, 1 mM PMSF, supplemented with protease inhibitor Aprotinin, Pepstatin A, Leupeptin, Antipain, Chymostatin, Proteosome inhibitor mixture, NaF, and Na3VO4, all the protease inhibitors need to be doubled (see Note 12). 2. The final nuclear pellets (Subheading 3.2, step 4) were resuspended in 300 μL nuclear protein extraction buffer, and then were sonicated with amplitude 30 sec/30 sec for 8 min using high mode on ice water with bioruptor. 3. After sonication, centrifuge at 12,000  g for 10 min at 4
C. Transfer supernatant to new 1.5 mL tubes and centrifuge for another 10 min, collect all the supernatant. 4. As quality controls for the fractionation, transfer 10 μL of nuclear extracts and mix with 5 μL 6  SDS protein loading buffer as nuclear protein. 5. Detect the cytosolic and nuclear protein by western blot. The tubulin and histone H3 were taken as the cytoplasmic and nuclear marker, respectively. Use a 12% SDS-PAGE gel for separation (Fig. 3). 6. Detect the protein concentration of nuclear extracts using the Bradford 2-D Quant Kit, and make the standard curve with

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Fig. 3 Quality check of nucleus extracts with immunoblot for the following label-free quantitation by LC–MS. Histone H3 was used as a nuclear protein marker, whereas tubulin was taken as a cytosolic marker. The protein extracts were loaded onto 12% denaturalized gel for separation

BSA. Take 20 μL nuclear extracts into one action of the kit, and calculate the samples’ total nuclear protein content of wild-type and mutant samples, respectively (see Note 13). The final protein amount should be over 40 μg for the followed SWATH assay. 3.4 Reduce the Nuclear Proteins and Block Cysteine

1. Take 20 μg of nuclear extracts, add reducing reagent (TECP) to make the end concentration to 0.5 mM, gently vortex to mix, then spin. Incubate the samples at 60
C for 1 h. 2. For each sample, add IAM (MMTS, cysteine-blocking reagent), to the end concentration of 25 mM, briefly vortex to mix, then spin, and incubate the samples at room temperature for 10 min. 3. Add LC–MS grade water to each tube to the final volume no more than 400 μL, briefly vortex and spin, transfer the sample to a 10 kD ultrafilter tube. Centrifuge at 12,000  g for 10 min at 4
C. 4. Repeat step 3 for 4 times. Finally remain about 30 μL buffer in each 10 kD ultrafilter tube.

3.5 Digest the Proteins with Trypsin

1. Transfer the filter to a new collect tube, add 10 μL 1 M TEAB to the center of each tube, and then add 2 μg of the Trypsin enzyme (LC–MS grade). Vortex to mix, then spin. Incubate at 37
C overnight (about 12 h to 16 h). 2. Centrifuge at 12,000  g for 30 min at 4
C. Add 100 μL LC– MS grade water for twice. Spin to collect the digested samples to the bottom of the tube. The final total volume should be about 300 μL. 3. Lyophilize samples at 40
C and centrifuge at 2000  g with freeze dryers.

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3.6 Label-Free Protein Quantitation Analysis Using SWATH Method 3.6.1 Spectral Library Building

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Samples were separated to 2 equal parts and individually used for spectral library building and quantitation analysis.

1. Peptide sample preparation: lyophilized peptides were dissolved in 30 μL mobile phase A, vortex for 30 sec. Centrifuge the sample at 18000  g for 30 min at 4
C. 10 μL supernatant was extracted for following LC–MS analysis. 2. LC–MS: sample was desalted online on a 100 μm  20 mm, 5 μm trap column and eluted on an analytical 100 μm  150 mm, 1.9 μm column. Both trap column and analytical column were filled with Reprosil-pure 120 C18-AQ phase. Peptides were separated by a gradient formed by mobile phase A and B, from 5 to 30% of mobile phase B in 75 min at a flow rate of 300 nL/ min. Eluent from the column was sprayed using the NanoSpray Source into mass spectrometer. Data-dependent acquisition was performed for protein identification where the MS was acquired from 350–1500 m/z (250 msec accumulation time). MS/MS was acquired from 100–1500 m/z (50 msec) on top 40 precursors passing the selection criteria. 3. Database searching: All the biological repetition raw LC–MS data were combined searching in the ProteinPilot software using the Paragon database search algorithm and the integrated false discovery rate (FDR) analysis function. The software used only unique peptide sequences as evidence for protein identification. The data were searched against uniprot Arabidopsis thaliana database (ver.20161107). The samples were described using the following parameters in the Paragon method: Sample Type—identification; Cys Alkylation— MMTS; Digestion—Trypsin; Special Factors—no selection; Species—None. ID Focus—Biological Modifications; Search Effort—Thorough; Detected Protein Threshold—0.05 (10.0%). For FDR determination, data were searched against concatenated databases by in silico on-the-fly reversal for decoy sequences automatically by the software. Only proteins at 1% global FDR were used for further analysis. 4. Spectral library building: The database searching result file (. group) was imported into MS/MSALL with SWATH™ Acquisition MicroApp in PeakView and create a spectral library.

3.6.2 Quantitative Analysis Using SWATH Method

1. Variable window optimization: One raw LC–MS data of any biological repetition transfer to peaklist format using PeakView software and was imported to excel template of Variable window calculator to optimize the precursor ion window in dataindependent acquisition mode.

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2. LC–MS: Each sample was performed on the same LC condition showed in spectral library building session. SWATH acquisition on MS was performed for quantitative analysis, Q1 was stepped from 400–1250 m/z (60 variable precursor ion window steps) and high-sensitivity MS/MS was acquired from 100–1500 m/z (100 msec accumulation time). 3. Data processing: All data were processed using the MS/MSALL with SWATH™ Acquisition MicroApp 2.0 in PeakView using parameters as follows: Number of peptides per protein-6; Number of transitions per peptides-6; Peptide confidence threshold % (0–99)-95; False discovery rate threshold % (0–100)-1.0; Exclude modified peptides and exclude shared peptides were checked; XIC extraction window (min)-10; XIC width (Da)-0.05. Quantitative data of the proteins and peptides were exported to MarkerView software for statistical analysis using t-test.

4

Notes 1. The CelLytic PN kit should be stored at 2–8
C, and only can be diluted to working concentration right before each experiment. 2. Keep NIB1 on the ice always. DTT should be added freshly before every extraction. To homogenize the tissue powder, use 1 mL pipette tip to agitate the samples and briefly pipette. Do not vortex it. 3. Make the Miracloth fixed to the tube when centrifugation. To do that, we usually make extra size of Miracloth to cover the edge of the tube, and tighten the tube cap. 4. Remove the Miracloth, and it should be dry but not immersed into solution. It is important to centrifuge again to achieve the maximum of nuclei amount. It is alternative to skip to take out the Miracloth, instead of centrifuging at 1260  g at 4
C for 10 min. 5. Remove the supernatant gently and quickly. Do not touch the pellet with pipette tip. And the pellets should be in green color. In this step, the pellet majorly is comprised of the intact plant cells. 6. Carefully and briefly to resuspend, do not vortex in case of damaging nuclei. The pipette tips should be cut off about 0.5 cm to avoid damage the nucleus. At this step, adding all protein inhibitors in 1 NIB buffer is highly recommended. 7. The pellet color should be white and kind of gray, but no any green color. Otherwise, need to repeat steps 8 and 9.

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8. Each type of plant requires a different percentage of Triton X-100 for proper cell lysis. In Arabidopsis, 0.3% as the final concentration of Triton X-100 is suggested. 9. Use the 2.3 M sucrose freshly dilute to 1.5 M with 1 NIB buffer. 10. The ratio of cushion to sample is about 4 to 3. 11. At this stage the nuclei pellets can be stored in 100 μL of Nuclei PURE Storage Buffer and stored at 70
C for up to 3 months. 12. All the protease inhibitor should be added freshly before every extraction. Keep all the operations on the ice. 13. Make the standard curve at first, and choose a proper concentration to measure the sample.

Acknowledgments This work is supported by fundings from National Natural Science Foundation of China (No. 31570292) and from Chinese Academy of Sciences to L.W. (QYZDB-SSW-SMC011 and XDB27030206). References 1. Kircher S, Gil P, Kozma-Bognar L, Fejes E, Speth V, Husselstein-Muller T, Bauer D, Adam E, Schafer E, Nagy F (2002) Nucleocytoplasmic partitioning of the plant photoreceptors phytochrome A, B, C, D, and E is regulated differentially by light and exhibits a diurnal rhythm. Plant Cell 14:1541–1555 2. Wang H, Deng XW (2004) Phytochrome signaling mechanism. The Arabidopsis Book 3: e0074.1 3. Qiu Y, Pasoreck EK, Reddy AK, Nagatani A, Ma W, Chory J, Chen M (2017) Mechanism of early light signaling by the carboxy-terminal output module of Arabidopsis phytochrome B. Nat Commun 8:1905 4. Zhang S, Li C, Zhou Y, Wang X, Li H, Feng Z, Chen H, Qin G, Jin D, Terzaghi W, Gu H, Qu LJ, Kang D, Deng XW, Li J (2018) TANDEM ZINC-FINGER/PLUS3 is a key component of Phytochrome a signaling. Plant Cell 30:835–852 5. Chen F, Li B, Li G, Charron JB, Dai M, Shi X, Deng XW (2014) Arabidopsis phytochrome a directly targets numerous promoters for individualized modulation of genes in a wide range of pathways. Plant Cell 26:1949–1966 6. Yang L, Mo W, Yu X, Yao N, Zhou Z, Fan X, Zhang L, Piao M, Li S, Yang D, Lin C, Zuo Z

(2018a) Reconstituting Arabidopsis CRY2 signaling pathway in mammalian cells reveals regulation of transcription by direct binding of CRY2 to DNA. Cell Rep 24(585–593):e584 7. Christie JM, Arvai AS, Baxter KJ, Heilmann M, Pratt AJ, O’Hara A, Kelly SM, Hothorn M, Smith BO, Hitomi K, Jenkins GI, Getzoff ED (2012) Plant UVR8 photoreceptor senses UV-B by tryptophan-mediated disruption of cross-dimer salt bridges. Science 335:1492–1496 8. Hayes S, Sharma A, Fraser DP, Trevisan M, Cragg-Barber CK, Tavridou E, Fankhauser C, Jenkins GI, Franklin KA (2017) UV-B perceived by the UVR8 photoreceptor inhibits plant Thermomorphogenesis. Curr Biol 27:120–127 9. Yang Y, Liang T, Zhang L, Shao K, Gu X, Shang R, Shi N, Li X, Zhang P, Liu H (2018b) UVR8 interacts with WRKY36 to regulate HY5 transcription and hypocotyl elongation in Arabidopsis. Nature Plants 4:98–107 10. Ding Z, Millar AJ, Davis AM, Davis SJ (2007) TIME FOR COFFEE encodes a nuclear regulator in the Arabidopsis thaliana circadian clock. Plant Cell 19:1522–1536 11. Wang Y, Wu JF, Nakamichi N, Sakakibara H, Nam HG, Wu SH (2011) LIGHT-

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REGULATED WD1 and PSEUDORESPONSE REGULATOR9 form a positive feedback regulatory loop in the Arabidopsis circadian clock. Plant Cell 23:486–498 12. Choudhary MK, Nomura Y, Wang L, Nakagami H, Somers DE (2015) Quantitative circadian Phosphoproteomic analysis of Arabidopsis reveals extensive clock control of key components in physiological, metabolic, and signaling pathways. Mol Cell Proteomics 14:2243–2260 13. Fujiwara S, Wang L, Han L, Suh SS, Salome PA, McClung CR, Somers DE (2008) Post-

translational regulation of the Arabidopsis circadian clock through selective proteolysis and phosphorylation of pseudo-response regulator proteins. J Biol Chem 283:23073–23083 14. Wang Y, He YQ, Su C, Zentella R, Sun TP, Wang L (2020) Nuclear localized O-fucosyltransferase SPY facilitates PRR5 proteolysis to fine-tune the pace of Arabidopsis circadian clock. Mol Plant 13(3):446–458 15. Wang L, Fujiwara S, Somers DE (2010) PRR5 regulates phosphorylation, nuclear import and subnuclear localization of TOC1 in the Arabidopsis circadian clock. EMBO J 29:1903–1915

Chapter 13 Strategies to Study Dark Growth Deficient or Slower Mutants in Chlamydomonas reinhardtii Huanling Yang, Fei Han, Yue Wang, Wenqiang Yang, and Wenfeng Tu Abstract Photosynthesis is the most important chemical reaction on the earth, and about 60% of the CO2 is fixed by algae through photosynthesis. Photosynthetic organisms including algae experience half of the entire life in the dark due to diel cycles, and dark metabolism is critical and necessary for photosynthetic organisms to restart photosynthesis when receiving light again. Briefly, dark metabolism provides necessary materials and energy for restoring photosynthesis, reoxidizes NADH to form NAD+, rationally stores photosynthates, and maintains correct redox balance. Chlamydomonas reinhardtii grows under both autotrophic and heterotrophic conditions, making it an ideal organism to study photosynthesis, dark metabolism, and light dark transitions as well. In addition, it provides a good model to identify key molecular components and elucidate the molecular regulatory mechanisms of heterotrophic, which provides new clues to understand how photosynthetic organisms restart photosynthesis from the dark. Chlamydomonas mutants with dark growth deficiency or slower growth phenotypes are likely caused by the inefficient uptake and transport of acetate, the damaged proteins of mitochondrial electron transport chain, the malfunctioned mitochondrion, the redox state alteration in the dark or failed communication between mitochondrion and other organelles, the imbalanced redox or the disrupted distribution of the photosynthetic products. Here we summarize the methods and strategies for analyzing these mutants in Chlamydomonas reinhardtii. Key words Chlamydomonas Mitochondria

1

reinhardtii,

Dark

metabolism,

Photosynthesis,

Respiration,

Introduction The day-night cycle makes photosynthetic microorganisms spend half of their time in darkness and generate energy for survival exclusively through dark metabolism [1]. Under dark conditions, algae assimilate extracellular organic substrates (acetate, glucose, glycerol, etc.) for ATP to grow heterotrophically, reasonably utilize and distribute photosynthetic products [2]. By elucidating the metabolic pathways associated with dark and their association with light-dominant metabolisms in the diurnal cycles, we can fully understand the net carbon cycle and the overall energy budget of photosynthetic organisms. Compared to photosynthesis, it is

Ruohe Yin et al. (eds.), Plant Photomorphogenesis: Methods and Protocols, Methods in Molecular Biology, vol. 2297, https://doi.org/10.1007/978-1-0716-1370-2_13, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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difficult to study dark metabolisms and even more difficult to study the effects of dark metabolisms to photosynthesis during the darklight transition. Chlamydomonas reinhardtii is a unicellular, soildwelling (and aquatic) green alga that grows photoautotrophically, mixotrophically and heterotrophically, and has unique metabolic characteristics and significant metabolic flexibility for balancing redox equivalents and generating ATP when it experiences different conditions. Chlamydomonas uses CO2 for photoautotrophic growth, acetate for heterotrophic growth, and the two carbon sources for mixotrophic growth, which is applied to study the defective mutants under different conditions. With the availability of the sequenced nuclear, chloroplast, and mitochondrial genomes [3, 4] and the application of various advanced biological techniques [5–8], Chlamydomonas has become a powerful single-cell eukaryotic model organism for various studies, such as the photosynthesis regulation [9, 10], nutrients deprivation responses [11, 12], fermentation and biohydrogen production [13–15], circadian clock [16, 17], chloroplast transformation [18, 19], chloroplast biogenesis [20–22], CO2-concentrating mechanisms (CCM) [23–25], light signal transduction [26–29], symbiosis with bacteria [30, 31], flagellar assembly and transportation [32–35], lipid metabolisms [36–39], and pigment metabolisms [40], and some pioneered studies, e.g., the vitamin-C-derived DNA modification recently found in Chlamydomonas, which regulates the acclimation to high light conditions through LHCSR3 [41, 42]. In particular, it can assimilate acetate to grow under dark conditions, while maintaining a functional thylakoid membrane system which is capable of restoring a complete photosynthetic capability when shifted to the light [2], as a result, Chlamydomonas has become an ideal model for dissecting the mechanism of dark metabolism. It has been discussed previously that darkness can function as a signal in plants [43], and skotomorphogenesis has been studied extensively [44]. In addition, respiration deficient mutants of Chlamydomonas have been well summarized [45]. It was found recently that genes have different expression patterns over the course of the day according to their biological functions, and fermentation, rather than respiration, was the preferred pathway for pyruvate metabolism at night by analyzing the diurnal transcriptome, combined with the pigment measurements, selected metabolites, and physiological parameters [46]. Mitochondrion is an indispensable organelle evolved from ancestral proteobacteria after symbiosis. It is the main site of aerobic respiration and plays critical roles in producing energy. The mitochondrial electron transport chain (mETC) eventually produces ATP through a series of redox processes [47]. Many Chlamydomonas mutants defective in dark heterotrophic growth are resulted from the disruption of the genes encoding mitochondrial functional proteins. Most of Chlamydomonas mutants lose the

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capacity to grow or grow slowly in the dark because of the failure to uptake or assimilate acetate as a carbon source for heterotrophic growth [48], the dysfunctional mitochondria [49], or the inefficient communication between mitochondria and other organelles such as chloroplast [26, 50]. A number of Chlamydomonas “dark death” mutants that are defective in dark growth due to the malfunctioned mitochondria have been identified, including that missing or defective in specific components associated with mETC complex I-IV, or that affecting the correct assembly of these complexes. For example, dark growth deficiency or retard phenotypes in the dum (dark uniparental minus) series mutants, with disruptions in the complex I, III, IV in the respiratory chain [51–57], are resulted from the decreased rate of acetate assimilation accompanied by a decreased rate of respiration and ATP synthesis. In Chlamydomonas, the mitochondrial proteome contains about 350 proteins [58], while the mitochondrial genome encodes only 12 proteins and 7 out of them functions in mETC [59, 60]. Therefore, most of the proteins involved in mitochondrial function (including respiration) are encoded by nuclear genome and translocated to mitochondria through the mitochondrial protein transport complexes consisting of Transport Inner Membrane (TIM) and the Transport Outer Membrane (TOM) [61]. However, the functions of many mitochondria localized proteins have not been dissected. The most critical process of acetate assimilation is the glyoxylate cycle, which is very important for the occurrence of gluconeogenesis [62]. Chlamydomonas does not appear to have an obvious glyoxysome, but it does appear to contain components for glyoxylate cycle [63, 64]. In the most basic scheme of the glyoxylate cycle, the isocitrate lyase (ICL) converts isocitrate to succinate, which enters the mitochondria to participate in the tricarboxylic acid (TCA) cycle [65–67]. Succinate is then converted to malate in mitochondria, and this malate is exported to the cytosol, then converted to oxaloacetate by cytosolic malate dehydrogenase (MDH). Oxaloacetate is catalyzed to phosphoenolpyruvate (PEP) by PEP carboxykinase, and PEP is eventually assimilated in the synthesis of soluble carbohydrates via glycogenesis [63, 68]. The TCA cycle consists of very important metabolic pathways in mitochondria, which is the final hub of the three nutrients (sugars, lipids, and amino acids), and the source of NADH and FADH2 for mitochondrial respiration [69]. Mitochondria use reducing power generated by glycolysis, pyruvate dehydrogenase complex (PDC), and TCA cycle to build electrochemical transmembrane gradients to drive ATP synthesis, which provides energy to the cellular life activities [70]. Mitochondrial respiration is usually inhibited to some extent in the dark and has some consequences. On the one hand, the TCA cycle and glycolysis will be restricted due to the increase of NADH levels in mitochondria and cytoplasm

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resulted from the hindrances of electron transfer to oxygen. On the other hand, ATP consumption in dark metabolism may promote glycolysis of starch/sucrose [2, 71]. Researches on the glyoxylate cycle, TCA cycle, and mETC have become non-negligible directions in the study of Chlamydomonas dark mutants. Studies have shown that mutations in the genes involved in mitochondrial metabolic pathways lead to dark growth defective in Chlamydomonas. For example, icl1 fails to grow in the dark and grows slower under mixotrophic conditions, which is caused by the inefficient utilization of acetate and decreased respiration rate. In addition, reduced β-oxidative activity and decreased levels of gluconeogenesis and glyoxylate cycle enzymes were also found in the dark grown icl1 [48]. Mitochondrion plays a vital role in providing energy for a variety of cellular activities and performs a wide range of functions in concert with other organelles. Studies have shown that the mitochondrial respiratory system of plants shows flexible adaptability to the changes of light environments. To date, multiple interactions and communications between chloroplasts and mitochondria have been reported [72–75] (Fig. 1), including: (1) redox-shuttle machineries to transport reducing equivalents [76–78]; (2) C/N exchange [79, 80]; (3) photorespiration [81]; (4) energy (ATP)

Cross Talk Light

Respiration

Photosynthesis

Chloroplast

ATP Sucrose State transition Signal molecule

Mitochondrion

C/N assimilation Photorespiratory metabolism CO2-concentration mechanism Reducing equivalents equilibrium

Fig. 1 Interactions between chloroplasts and mitochondria in Chlamydomonas reinhardtii

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balance [82]; (5) sucrose [65, 73]; (6) mitochondrial respiratory chains regulating photosynthesis genes [83]; (7) algal unique CCM affected by mitochondria [84]; (8) state transition [50, 85]; and (9) alternative oxidase (AOX) to dump extra electrons. Studies have shown that malfunctioned mitochondria can be partially compensated by regulating the photosynthetic activity through state transition. Briefly, in mitochondrial mutants lacking respiratory complexes and ATP leads to state 2 [86]. Therefore, mutants with respiratory defects or dark metabolism disruption can be screened by measuring the maximum photochemical efficiency of photosystem II. The chloroplast localized FDX5 regulates dark metabolism through regulating lipid metabolism, the structure of the thylakoid membrane, the respiration in mitochondria, and possibly the redox state of the cells [86]. It was also found that the soluble guanylate cyclase CYG12 is related to the NO signaling, and its mutant stain cyg12 has a phenotype that does not grow in the dark [87]. More and more evidences have proved that dark metabolism is very important. Based on the introduction mentioned above, initial phenotype screen is a key step in the study of Chlamydomonas dark mutants. Here, we summarize the methods to study respiration deficient or impaired and growth deficient or retarded mutants in the dark, which is likely to identify the key molecular components of heterotrophic growth, elucidate the molecular regulation mechanisms in the dark, and understand the relationship between photosynthesis and dark metabolism. Dark growth deficiency or slower growth is likely caused by many defectives, such as biochemical, biophysical, physiological, or metabolic disorders (Fig. 2). First, Chlamydomonas wild-type and the mutant cells are cultivated in the dark heterotrophic condition with light autotrophic or mixotrophic conditions as control to confirm the defective or slower growth in the dark. A series of phenotype analyses are conducted, including cell size, cell density, growth curve, cell dry weight, chlorophyll content, chlorophyll a/b ratio, and so on. Then oxygen consumption/uptake is measured to check whether the mitochondrial respiration is disrupted. The phenotypes are likely caused by the failed uptake or utilization of acetate as the carbon source, the destruction of the mitochandrial complexes, the uncoupling of the mETC, the inactivation of the enzymes in TCA cycle, as well as the inefficient accommodation of acetyl-CoA and other necessary metabolites from glyoxylate cycle and TCA cycle. In addition, it may be caused by the discordant communication in terms of metabolites such as reducing equivalents and energy between mitochondria and other organelles (in particular, the chloroplast). The general research routes on the dark mutants are shown in details (Fig. 2).

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The strains do not grow or grow slowly in the dark

Phenotype analysis

Morphologic analysis

Acetate uptake/assimilation

Localization analysis

Mechanism

Chloroplast

Biophysical analysis

Respiratory rate

Peroxisome

Biochemical analysis

Metabolism

Mitochondrion Cell size

Acetate uptake rate

O2 uptake rate

G3P

ATP Spectrum

Cell density Growth curve Acetate concentration

O2 evolution rate

NAD(P)H

Lipids

mETC subunits

Starch

Cell dry weight Redox potential Chlorophyll content Acetate assimilation rate Chlorophyll a/b ratio

Effects of inhibitors on respiratory rate

Redox-shuttling

Chlorophyll fluorescence

Amino acids Organic acids

Fig. 2 Strategies to study dark growth deficient or slower growth mutants in Chlamydomonas reinhardtii

2 2.1

Materials Glassware

Beaker (1000 mL, 2000 mL), measuring cylinder (50 mL, 100 mL and 1000 mL), sterile Erlenmeyer flask (50 mL, 100 mL and 1000 mL), glass rod, and cuvette.

2.2 Compostable Materials

Sterile petri dish, aluminum foil, parafilm, filter paper, pipette and pipette tips, rubber band, hemocytometer, Eppendorf tube, centrifuge tubes, toothpick, syringe, lab spoon, forceps, scissors, and weighing paper.

2.3

Apparatus

Clark-type oxygen electrode Chlorolab 2, Dual-PAM-100, JTS-10 spectrophotometer, Hitachi F-7500 fluorescence spectrophotometer, Shimadzu GC-2014 gas chromatograph, Waters e2695 high-performance liquid chromatography (HPLC), three-electrode system, UV-visible spectrophotometer, illumination growth chamber, pH meter, magnetic stir place, oven, microwave oven, gel system, electronic balance and balance, centrifuge, hood, high temperature steam sterilizer, vortex, electron microscopy, electrophoresis system, PCR machine, shaker, ultra-low temperature freezer, and glove box.

2.4 Chemicals and Reagents

The amino acids and organic acids standards used for gas chromatography-mass spectrometer (GC-MS) and HPLC experiments are chromatographic grade.

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Sodium hyposulfite, sodium bicarbonate, agar, Coomassie brilliant blue, iodine and potassium iodine solution, methanol, 80% aqueous acetone, glacial acetic acid, chemicals and reagents used to prepare TAP medium (refers to Subheading 3.1).

3

Methods In this section, we describe the main strategies for screening the dark growth deficient or slower growth mutants in Chlamydomonas reinhardtii. This part is just a general introduction. It is not necessary to follow the steps completely; some adjustments and adaptation according to the actual phenotype of the mutants and some experimental results are also welcomed.

3.1 Tris-AcetatePhosphate (TAP) Medium Preparation [88]

1 M Tris-base:Dissolve 121.14 g Tris-base with 1000 mL distilled water. 1000 mL

Concentration

Beijerinck’s Solution (100
) (see Note 1): NH4Cl

40 g

750 mM

CaCl2 · 2H2O

5g

34 mM

MgSO4 · 7H2O

10 g

64 mM

Phosphate Solution (1000
) (see Note 2): K2HPO4

108 g

620 mM

KH2PO4

56 g

411 mM

EDTA-Na2

50.0 g

134 mM

H3BO3 (boric acid)

11.14 g

180 mM

ZnSO4 · 7H2O

22.0 g

77 mM

MnCl2 · 4H2O

5.1 g

26 mM

FeSO4 · 7H2O

5.0 g

18 mM

COCl2 · 6H2O

1.6 g

6.7 mM

CuSO4 · 5H2O

1.6 g

6.4 mM

(NH4)6Mo7O24 · 4H2O

1.1 g

0.89 mM

Trace Elements Solution

The chemicals are added one at a time (except EDTA-Na2) to 550 mL milli-Q H2O in a 2000 mL beaker and the mixture is heated up to approximately 70  C. In a second beaker, EDTANa2 is added to 250 mL milli-Q H2O and heated until dissolved (see Note 3). Then the EDTA-Na2 solution is mixed with salt solution (not vice versa) and the combined solution is heated to a

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boil. Let the combined solution maintain temperature at 70–75  C to adjust pH to 6.5–6.8 with 20% KOH (see Note 4). The combined solution is diluted with milli-Q H2O to 1000 mL. The bottle with combined solution is covered by a cotton plug (not parafilm!) for two weeks until the color changes from green to purple (see Note 5). The trace elements is filtered and stored in refrigerator at 4  C (see Note 6). Per 1000 mL TAP medium: 20 mL 1 M Tris-base 10 mL 100
Beijerinck’s Solution 1 mL 1000
Phosphate Solution 1 mL Trace Elements Solution 1 mL Acetic Acid The stocking solutions are added seriatim and autoclaved (121  C at 103 kPa for 20 min). Per 1000 mL TP medium: 20 mL 1 M Tris-base 10 mL 100
Beijerinck’s Solution 1 mL 1000
Phosphate Solution 1 mL Trace Elements Solution. The pH is adjusted to 7.2–7.4 with 1.5 mL concentrated HCl and with acetic acid excluded. For TAP/TP solid medium: 12 g agar is added to 1000 mL medium before autoclave. 3.2 Growth Conditions

Chlamydomonas strains are grown at 25  C in TAP medium under continuous growth light (50 μE m2 sec1) [88]. In some circumstances, the cells are synchronized by day and night cycles (12 h light/12 h dark or 16 h light/8 h dark). Briefly, the cells are streaked on TAP agar plates for 5–6 days for single colony growing, and then the picked single colonies are inoculated into the TAP liquid culture. The Chlamydomonas cells are refreshed twice to stationary phase, then reinoculated into new TAP liquid medium until the cells are cultivated to early-exponential phase for further experiments (see Note 7).

3.3 Phenotypic Analyses of the Mutants on TAP Agar Plates

The Chlamydomonas parental and mutant strains in robust status are recommended. Briefly, the initial cell concentration for solid TAP inoculation is 1
106 cells/mL, and 10 μL of the dilutions are dropped on agar plates with replications (see Note 8) and grown in the dark for 7–10 days. The cells are then checked whether turn into green or not to indicate the growth. The Chlamydomonas cells are cultivated under dark heterotrophic condition and with mixotrophic condition as control (Fig. 3). In addition, inoculation can be performed with an initial chlorophyll concentration of 0.5 μg/ mL. Chlorophyll determination was performed as described by Porra et al. (1989) (see Note 9) [89].

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Light

At mid-exponential phase Dark

Fig. 3 Experimental flows to show the cells inoculation and cultivation in the light and dark in Chlamydomonas reinhardtii 3.4 Phenotype Analyses of the Mutants in TAP Liquid Media

Phenotypic analyses of the mutants can also be accomplished in TAP liquid media, which is indicated by growth curves including cell numbers or chlorophyll amounts. The growth curves are conducted for about 7–10 days. Briefly, cells with a concentration of 1
105 cells/ml are inoculated in a sterile conical flask containing 50–100 ml (as needed) liquid TAP medium, and aluminum foil is used to avoid light, with the mixotrophic culture under standard light conditions as control (see Note 10). Phenotypic analyses are performed for 7–10 days, and cell number and chlorophyll content are counted and measured (Fig. 3). Cell densities are monitored by a cell counter or hemocytometer (see Note 11). Other physiological properties such as cell size, cell dry weight, and chlorophyll a/b ratio, and biochemical analyses such as lipid contents can be detected as needed. The biomass analyses were determined as described before [90]. For the mutants losing the growth capacity in the dark, all of the rest experiments are performed using the samples transited into the dark from the light.

3.5 Measurement of Oxygen Consumption and Evolution Rate Under Dark Heterotrophic Conditions

After the Chlamydomonas cells are cultivated to the mid-exponential phase with normal growth light, the cells are split into two for growth in the dark and light, respectively. The rate of oxygen uptake and consumption are determined using a Clark-type oxygen electrode Chlorolab2 at 1 h, 6 h, 12 h, 24 h, and 48 h, respectively (see Note 12). The time points of the measurements can be adjusted as needed. Sometimes, Membrane Inlet Mass Spectrometry (MIMS) is applied to check the rate of oxygen consumption and evolution [91, 92]. Furthermore, the effect of Potassium cyanide (KCN) and Salicylhydroxamic acid (SHAM) on total respiratory O2 consumption rates can also be tested by a Clark-type oxygen electrode. Chlamydomonas protein localization can be predicted online by PredAlgo [93]. The mitochondria localized proteins responsible

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3.6 Analysis of Mitochondrial Protein Localization Under Dark Heterotrophic Conditions

for dark heterotrophic growth, which are likely involved in mETC, are subunits or assembly factors for the complex assembly or regulators for the correct functionality maintenance. Mitochondrial complex can be extracted for analysis by Blue native (BN)-PAGE with consideration of the contamination by chloroplast proteins during the extraction, and the extraction procedure can be followed as described before (see Note 13) [94]. Proteomic analyses combined fractionation is conducted to confirm the localization as well if necessary [95].

3.7 Quantification and Qualification of Metabolites Under Dark Heterotrophic Conditions

Many mutants deficient in dark heterotrophic growth are caused by the failure of acetate uptake and assimilation. Acetate is analyzed by HPLC [96] or quantified by an acetate determination kit [87], and acetate uptake rate is monitored by checking radioactivity. In addition, other metabolites affecting the dark heterotrophic growth, including starch, fatty acids, organic acids, amino acids, and so on, should be considered. Starch is purified with Percoll as described before [97] or measured by the kit from Sigma-Aldrich (see Note 14). The neutral lipids are determined by Nile Red fluorescence or transmission electronic microscopy (TEM) [98]. Amino acids and organic acids are determined by HPLC (see Note 15).

3.8 Measurements of the Chlorophyll Fluorescence Parameters Under Dark Heterotrophic Conditions

Chlorophyll fluorescence is used for screening mutants with mitochondrial respiratory defects, which may affect the reorganization of the photosynthetic activity and more particularly through state transition (state 2). Mutants impaired in respiration or altered in dark metabolism can be screened by measuring the maximum photosynthetic efficiency (FV/FM) through chlorophyll fluorescence analyses [99].

3.9 Determination of Middle Point Redox Potential Under Dark Heterotrophic Conditions

Mitochondria are the main source of energy for ATP and NAD (P)H. It is necessary to detect the levels of ATP and NAD(P)H which reflect the accumulation of reducing power and energy in cells. In some cases, the damaged mitochondrial phosphorylation in the mutants may change the level of cellular ATP concentration. The ATP level can be determined as described previously [100], and cellular ATP concentration can be determined with the Enliten luciferase/luciferin kit. Determination of internal NADH oxidation and external oxidation of NAD(P)H is carried out as described before [101]. The NAD/NADH and NADP/NADPH assays are mostly based on monitoring the changes in NADH or NADPH absorption at 340 nm, while the traditional methods suffer low sensitivity and high interference. Abcam’s Colorimetric NADPH Assay Kit may provide a convenient method for detecting NADPH. Some of the mitochondrial redox proteins are involved in electron transfer, and the middle point redox potentials can be measured using cyclic voltammetry. Cyclic voltammetry is conducted using a conventional three-electrode system connected to the BAS 00-watt potentiostat (see Note 16) [102, 103].

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Notes 1. Three compounds are dissolved seriatim and the stocking solution is stored in refrigerator at 4  C after autoclave. 2. When TAP medium is prepared, it appears milky white, which is normal. The medium turns to be clear after acetic acid is added. 3. EDTA-Na2 solution is added to salt solution (not the other way) and the combined solution is boiled, then cooled down before pH adjustment. 4. It is very critical to adjust pH of the combined solution at 70–75  C. 5. The combined solution is stirred occasionally to help it to be oxidized. 6. Full oxidation of trace elements solution is recommended; otherwise the precipitation affects the real concentrations of elements in the solution. 7. Chlamydomonas cells are flexible and easily affected by the physiological state and environmental cues. It is recommended to refresh Chlamydomonas cells twice to stationary phase, then the cells are reinoculated and grown until middle log phase for further phenotypic and other analyses. 8. In order to present a more pronounced phenotype in the dark, 10 μL of cells diluted with three serial 1:10 ratio titration are spotted on plates with technical replicates. More than three biological replicates are recommended. 9. Porra and colleagues described ways to extract chlorophyll and equations for the determination of chlorophylls. For Chlamydomonas cells, two solvents, methanol and acetone, are usually used. No matter which extraction method is used, light should be avoided during the operation to prevent chlorophyll degradation, which may cause measurement errors. 10. Because Chlamydomonas cells are prone to hypoxia, four strategies are applied to keep the culture in a complete dark and aerobic state: (1) the volume of the culture is less than 1/10 of the growing container; (2) the growing container is covered with aluminum foil loosely to increase aeration; (3) the growing container is wrapped tightly with aluminum foil to avoid light; (4) the culture is stirred thoroughly and vigorously or bubbled with air to avoid hypoxia. 11. When counting cells, 10 μL of 0.25% (w/v) iodine in ethanol per 1 mL is used to prevent the cells from moving. 12. The electrode must be wiped and cleaned with specialized polish before being installed. Sharp metal objects should be avoided to protect the electrodes; otherwise the acquired

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signals would fluctuate and drift, and in serious cases, irreparable damage will be caused. After calibration, the reaction chamber is washed with distilled water for 5–6 times with special care to avoid damage. The oxygen electrode is very sensitive to temperature and the whole measurements should be conducted with stable temperature. When adding samples, avoid introducing bubbles, as bubbles will cause signal instability. 13. The isolated Chlamydomonas chloroplast is easily contaminated by mitochondria and vice versa. 14. Iodine and potassium iodide is applied, and the observed purple blue color under the microscope is used to indicate the amount of the accumulated starch. 15. The thorough collection of samples is particularly important for the detection of the metabolites. The supernatant and precipitates are for external and internal metabolites, respectively, and should be separated thoroughly to avoid mutual contamination. Frequent freezing and thawing of samples should be avoided. 16. All experiments are carried out in a glove box without oxygen.

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spectroscopy. Biochim Biophys Acta 975:384–394 90. Vigeolas H, Duby F, Kaymak E et al (2012) Isolation and partial characterization of mutants with elevated lipid content in Chlorella sorokiniana and Scenedesmus obliquus. J Biotechnol 162(1):3–12 91. Chaux F, Burlacot A, Mekhalfi M et al (2017) Flavodiiron proteins promote fast and transient O2 photoreduction in Chlamydomonas. Plant Physiol 174(3):1825–1836 92. Radmer RJ, Kok B (1976) Photoreduction of O2 primes and replaces CO2 assimilation. Plant Physiol 58(3):336–340 93. Tardif M, Atteia A, Specht M et al (2012) PredAlgo: a new subcellular localization prediction tool dedicated to green algae. Mol Biol Evol 29(12):3625–3639 94. Van Lis R, Atteia A, Mendoza-Herna´ndez G et al (2003) Identification of novel mitochondrial protein components of Chlamydomonas reinhardtii. A proteomic approach. Plant Physiol 132(1):318–330 95. Terashima M, Specht M, Naumann B et al (2010) Characterizing the anaerobic response of Chlamydomonas reinhardtii by quantitative proteomics. Mol Cell Proteomics 9 (7):1514–1532 96. Masset J, Hiligsmann S, Hamilton C et al (2010) Effect of pH on glucose and starch fermentation in batch and sequenced batch mode by new isolated strain of hydrogen producers of Clostridium butyricum. Int J Hydrog Energy 35:3371–3378

97. Delrue B, Fontaine T, Routier F et al (1992) Waxy Chlamydomonas reinhardtii: monocellular algal mutants defective in amylose biosynthesis and granule-bound starch synthase activity accumulate a structurally modified amylopectin. J Bacteriol 174(11):3612–3620 98. Doebbe A, Keck M, Russa ML et al (2010) The interplay of proton, electron, and metabolite supply for photosynthetic H2 production in Chlamydomonas reinhardtii. J Biol Chem 285(39):30247–30260 99. Massoz S, Larosa V, Horrion B et al (2015) Isolation of Chlamydomonas reinhardtii mutants with altered mitochondrial respiration by chlorophyll fluorescence measurement. J Biotechnol 215:27–34 100. Gans P, Rebeille F (1990) Control in the dark of the plastoquinone redox state by mitochondrial activity in Chlamydomonas reinhardtii. Biochim Biophys Acta 1015:150–155 101. Svensson ÅS, Rasmusson AG (2001) Lightdependent gene expression for proteins in the respiratory chain of potato leaves. Plant J 28 (1):73–82 102. Terauchi AM, Lu SF, Zaffagnini M et al (2009) Pattern of expression and substrate specificity of chloroplast ferredoxins from Chlamydomonas reinhardtii. J Biol Chem 284(38):25867–25878 103. Avila L, Wirtz M, Bunce RA et al (1999) An electrochemical study of the factors responsible for modulating the reduction potential of putidaredoxin. J Biol Inorg Chem 4 (5):664–674

Chapter 14 Co-immunoprecipitation Assay for Blue Light-Dependent Protein Interactions in Plants Jingyi Zhang and Shengbo He Abstract Co-immunoprecipitation (CoIP) assay has been used as a powerful and routine technique to detect in vivo protein-protein interactions. Not only can it probe stable interactions, but also it is applicable for semiquantitative and inducible protein associations. Here we describe the protocol for detecting blue lightdependent protein interactions, particularly for blue light receptor cryptochrome-mediated complex formation. In addition, we present some notes which may be helpful for common Co-IP study as well. Key words CoIP, CRY, Arabidopsis, Inducible interactions

1

Introduction Co-immunoprecipitation (CoIP) assay has been extensively used to study in vivo protein-protein interactions [1, 2]. It essentially examines if proteins are in the same complex in vivo rather than if they directly interact with each other. Therefore, CoIP assay usually comes with in vitro studies such as pull-down and yeast 2-hybrid that probe physical interactions [3, 4]. The principle of CoIP assay is like fishing where a bait is used to capture a prey. An antibody specific to the bait protein can be covalently conjugated to beads or indirectly bind to the beads through a bridge protein which is usually Protein A or G. Beads coated with Protein A or G in a covalent manner are commercially available and widely used for CoIP or antibody purification. Protein A and G recognize and bind antibodies with high affinity and different specificity to distinct isotypes. Therefore, isotypes of the antibody being used must be taken into consideration. The beads with antibody on specifically recruit the bait protein and the unspecific binding will be washed away using appropriate buffer, then the prey proteins associated with the bait protein are eluted and probed with antibodies specific to prey proteins through Western blot.

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Protein-protein interactions are mostly dynamic in vivo, and in some cases are inducible or conditional. For example, phytohormones induce the interactions of their receptors with downstream partners, which is highly dependent on the concentrations of phytohormones and critical for plant growth and development [5– 10]. Here, we use blue light receptor as an example to demonstrate how to investigate blue light-dependent interactions in vivo through CoIP assay. Plants perceive light signals using photoreceptors. Specific photoreceptors have been identified to sense distinct wavelengths of lights, with phytochrome, cryptochrome/phototropin, and UVR8 sensing red/far-red light, blue light/UV-A, and UV-B, respectively [11–15]. In Arabidopsis, cryptochromes including CRY1 and CRY2 are excited by blue light and transduce blue light signal by interacting with downstream partners. The consequence of the blue light-induced physical contacts of CRY with its partners is modulation of enzymatic activity or stability of the interactors, thus reprogramming gene expression profile and adjusting plant growth and development to surrounding environment [16–18]. Therefore, as the trigger of these profound changes, the dynamic interactions of CRY with its partners are worth studying through CoIP assay.

2

Materials

2.1 Put out Seeds on Plates

1. Transgenic line: 35S-Myc-SPA1 [16, 19].

2.2 Coimmunoprecipitation

1. Lysis buffer: 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.2% Triton-X-100. Before use, add Pefabloc (final concentration 1 mM), and complete protease inhibitor cocktail.

2. Seeds-sterilizing solution: Prepare immediately before use. Dilute 10% Bleach to 2% with water.

2. Bradford assay kit. 3. Antibodies: anti-Myc and anti-CRY1 [16, 20]. 4. Protein A agarose beads (see Note 1). 5. Heating block: thermomixer (0  C–99  C).

3

Methods

3.1 Put out Seeds on Plates

1. Put 35S-Myc-SPA1 seeds into 50 mL Falcon tube. Usually 50 μL seeds are used for one sample. 2. Add 30–40 mL 2% Bleach.

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3. Use a pipette tip to dip a droplet of Tween-20 into the Falcon tube containing seeds and Bleach. 4. Vortex to disperse seeds in the solution. 5. Shake the Falcon tube on a shaker for 15 min. 6. Wash out the Bleach solution and Tween-20 with sterile water in a flow hood. Usually wash 5 or 6 times with 30–40 mL water for each time. 7. Pour away water and leave some behind after the final wash. 8. Resuspend seeds with residual water and pipette seeds to plates with media. 9. Use appropriate volume of water to spread out seeds with pipetting. 10. Remove water by pipetting. 11. Seal the plates. 3.2 Grow and Collect Seedlings

1. Stratify seeds on plates in 4  C for 3 days. 2. Transfer the plates with seeds to white light for about 12 h. 3. Wrap the plates with 3 layers of foil to avoid light (see Note 2). 4. Allow seedlings to grow for 7 days before exposing the plates to different light conditions (see Note 3). 5. Collect seedlings in liquid nitrogen with tweezers (see Note 4).

3.3 Coimmunoprecipitation

1. Grind seedlings in liquid nitrogen to fine powder with mortar and pestle. During grinding, do not let samples warm up and be hydrated in case of protein degradation by proteases in the absence of protease inhibitors. 2. Collect powder in an appropriate container (see Note 5). Less than 1 mL is ideal as immunoprecipitation is usually done in 1.5 mL Eppendorf tubes which can reduce loss of beads and are easy to handle. 3. Add equal or double volume of lysis buffer to the powder (see Note 6). It is critical to add lysis buffer as quickly as possible to avoid hydration and protein degradation. 4. Vortex to homogenize samples. It is important to resuspend sample powder thoroughly in case of any clots being formed. 5. Get rid of debris by centrifugation at 12,000 rpm for 15 min at 4  C. 6. Transfer the supernatant to a fresh tube. 7. Quantify the protein concentration by Bradford assay. A kit for Bradford assay can be used. BSA is used for generating the standard curve. Usually 1 μL of sample is added to the reaction and lysis buffer is used as the blank.

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8. Transfer equal amount of total proteins to fresh tubes and add lysis buffer to equal volume among samples. 9. Add anti-CRY1 antibody to the samples and incubate at 4  C with gentle rotation for 1 h. This is to allow antibody (here is anti-CRY1 antibody) to recognize and bind the protein of interest (here is CRY1). There is no need to illuminate the sample with blue light as it seems that photoexcited CRY1 interacting with its partners is fairly stable and that the complex containing CRY1 will not come apart in several hours. 10. Pre-wash Protein A agarose beads with 1 mL of lysis buffer for 3 times (see Note 7). 11. Add the samples with the antibody to the beads and incubate at 4  C with gentle rotation for another hour. 12. Wash the beads with 1 mL of lysis buffer for 3 times (see Note 8). Make all the beads completely resuspended. 13. Add SDS loading buffer to the beads and boil the beads at 100 ˚C for 5 min (see Note 9). 14. Use the eluates for SDS-PAGE and Western blot with antiCRY1 and anti-Myc antibodies, respectively.

4

Notes 1. There are mainly two types of beads suitable for CoIP. Agarose and magnetic beads are commercially available. Generally, magnetic beads will give lower background binding than agarose beads. Either Protein A or Protein G can be covalently conjugated to beads. Protein A and G have different binding affinities for antibodies of distinct isotypes. Some frequently used tag beads such as Myc, flag, and HA beads are also commercially available. 2. This is for the later treatments with different light conditions. It is important not to stack plates as this may lead to different temperatures on each plate, especially when plates are placed on a shelf with airflow underneath and lights are on above the plates. 3. This can be done in different spectra of lights or different light intensities. 4. This should be done in dim green safe light. Samples collected could be weighed before being put into liquid nitrogen. 5. Normally less than 1 mL of sample powder should be enough for a CoIP experiment. The sample powder could be collected into a 2-mL Eppendorf tube.

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6. Depending on the volume of the samples and the expression level of the protein of interest, 1–2 mL of lysis buffer is usually used for each sample. 7. 20 uL of beads are usually used for each sample. Beads can be washed in a batch and aliquoted to individual tubes after wash. 8. This step can vary with different proteins under study and has to be tested experimentally. The volume of the buffer used, salt concentration, detergent concentration, and times for washing can be adjusted. 9. There are a number of ways for eluting proteins from beads. The SDS loading buffer plus boiling can elute most of the proteins if not all including both specific and unspecific proteins. Acid elution with glycine buffer elutes proteins involving protein-protein interactions, which is quite efficient although not as SDS loading buffer plus boiling, while improving the specificity of elution [16, 19]. The most specific method is antigen competition for elution [18]. For example, flag peptide can be used to competitively elute flag-tagged proteins from the flag beads. This method can vastly improve the specificity while avoiding elution of the heavy and light chains of antibodies from the beads.

Acknowledgments We thank Dr. Danmeng Zhu and Dr. Yuqiu Wang from Peking University, China for valuable advising on CoIP assay. References 1. Identification of associated proteins by coimmunoprecipitation (2005) Nat Methods 2 (6):475–476 2. Lin JS, Lai EM (2017) Protein-protein interactions: co-immunoprecipitation. Methods Mol Biol 1615:211–219. https://doi.org/10. 1007/978-1-4939-7033-9_17 3. Paiano A, Margiotta A, De Luca M, Bucci C (2019) Yeast two-hybrid assay to identify interacting proteins. Curr Protoc Protein Sci 95(1): e70. https://doi.org/10.1002/cpps.70 4. Izumi KM (2001) The yeast two-hybrid assay to identify interacting proteins. Methods Mol Biol 174:249–258. https://doi.org/10.1385/ 1-59259-227-9:249 5. McSteen P, Zhao Y (2008) Plant hormones and signaling: common themes and new developments. Dev Cell 14(4):467–473. https:// doi.org/10.1016/j.devcel.2008.03.013

6. Weijers D, Wagner D (2016) Transcriptional responses to the auxin hormone. Annu Rev Plant Biol 67:539–574. https://doi.org/10. 1146/annurev-arplant-043015-112122 7. Wang ZY, Bai MY, Oh E, Zhu JY (2012) Brassinosteroid signaling network and regulation of Photomorphogenesis. Annu Rev Genet 46 (46):701–724. https://doi.org/10.1146/ annurev-genet-102209-163450 8. Hwang I, Sheen J, Muller B (2012) Cytokinin signaling networks. Annu Rev Plant Biol 63 (63):353–380. https://doi.org/10.1146/ annurev-arplant-042811-105503 9. Lumba S, Cutler S, McCourt P (2010) Plant nuclear hormone receptors: a role for small molecules in protein-protein interactions. Annu Rev Cell Dev Biol 26(26):445–469. https://doi.org/10.1146/annurev-cellbio100109-103956

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10. Waters MT, Gutjahr C, Bennett T, Nelson DC (2017) Strigolactone signaling and evolution. Annu Rev Plant Biol 68(68):291–322. https:// doi.org/10.1146/annurev-arplant-042916040925 11. Kong SG, Okajima K (2016) Diverse photoreceptors and light responses in plants. J Plant Res 129(2):111–114. https://doi.org/10. 1007/s10265-016-0792-5 12. Briggs WR, Christie JM (2002) Phototropins 1 and 2: versatile plant blue-light receptors. Trends in Plant Science 7(5):204–210. Pii S1360–1385(02)02245–8. https://doi.org/ 10.1016/S1360-1385(02)02245-8 13. Cashmore AR, Jarillo JA, Wu YJ, Liu DM (1999) Cryptochromes: Blue light receptors for plants and animals. Science 284 (5415):760–765. https://doi.org/10. 1126/science.284.5415.760 14. Quail PH (2002) Phytochrome photosensory signalling networks. Nat Rev Mol Cell Biol 3 (2):85–93. https://doi.org/10.1038/nrm728 15. Rizzini L, Favory JJ, Cloix C, Faggionato D, O’Hara A, Kaiserli E, Baumeister R, Schafer E, Nagy F, Jenkins GI, Ulm R (2011) Perception of UV-B by the Arabidopsis UVR8 protein. Science 332(6025):103–106. https://doi. org/10.1126/science.1200660 16. Lian HL, He SB, Zhang YC, Zhu DM, Zhang JY, Jia KP, Sun SX, Li L, Yang HQ (2011) Blue-light-dependent interaction of cryptochrome 1 with SPA1 defines a dynamic

signaling mechanism. Genes Dev 25 (10):1023–1028. https://doi.org/10.1101/ gad.2025111 17. Liu B, Zuo ZC, Liu HT, Liu XM, Lin CT (2011) Arabidopsis cryptochrome 1 interacts with SPA1 to suppress COP1 activity in response to blue light. Genes Dev 25 (10):1029–1034. https://doi.org/10.1101/ gad.2025011 18. Xu F, He SB, Zhang JY, Mao ZL, Wang WX, Li T, Hua J, Dui SS, Xu PB, Li L, Lian HL, Yang HQ (2018) Photoactivated CRY1 and phyB interact directly with AUX/IAA proteins to inhibit auxin signaling in Arabidopsis. Mol Plant 11(4):523–541. https://doi.org/10. 1016/j.molp.2017.12.003 19. Zhu DM, Maier A, Lee JH, Laubinger S, Saijo Y, Wang H, Qu LJ, Hoecker U, Deng XW (2008) Biochemical characterization of Arabidopsis complexes containing CONSTITUTIVELY PHOTOMORPHOGENIC1 and SUPPRESSOR OF PHYA proteins in light control of plant development. Plant Cell 20(9):2307–2323. https://doi.org/10.1105/ tpc.107.056580 20. Sang Y, Li QH, Rubio V, Zhang YC, Mao J, Deng XW, Yang HQ (2005) N-terminal domain-mediated homodimerization is required for photoreceptor activity of Arabidopsis CRYPTOCHROME 1. Plant Cell 17 (5):1569–1584. https://doi.org/10.1105/ tpc.104.029645

Chapter 15 Detecting Blue Light-Dependent Protein–Protein Interactions by LexA-Based Yeast Two-Hybrid Assay Xiaolong Hao and Ling Li Abstract The LexA-based yeast two-hybrid system is one of the most powerful techniques used to detect blue lightdependent protein–protein interactions. In Arabidopsis, many protein–protein interactions in blue light signaling pathway were identified using this system. Here we present an easy and efficient method of the LexA-based yeast two-hybrid assay for testing protein–protein interactions in a blue light-dependent manner. Key words LexA, Yeast two-hybrid assay, Blue light-dependent, Protein–protein interaction, Blue light signaling pathway

1

Introduction The Yeast Two-Hybrid (Y2H) system developed by Stanley Fields and Ok-kyu Song has become to be a powerful technique which is used widely for detecting protein-protein interactions [1–4]. It is a powerful tool to rapidly identify novel proteins interactions with a specific protein of interest, so the Y2H is always the first option when we need to verify protein-protein interactions in blue light signaling pathway [5–8]. In Arabidopsis, some protein-protein interactions which are dependent on blue light were confirmed by using the LexA-based yeast two-hybrid system, for example, the interaction between the CRY1 (Arabidopsis blue-light receptor cryptochrome 1) and the COP1 (constitutive photomorphogenic 1 protein), the CRY1 and the COP1-interacting protein SPA1 (S UPPRESSOR OF PHYTOCHROME A) [9–11]. Thus, we focus on introducing the LexA-based assay for detecting the blue lightdependent protein-protein interactions in this protocol. The principle of yeast two-hybrid assay is based on the fact that eukaryotic transcriptional regulator contains a DNA-binding domain (DNA-BD) that binds to a specific promoter sequence and an activation domain (AD) that directs the RNA polymerase

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II complex to transcribe the gene adjacent of the DNA-binding site [1, 12–14]. In the LexA-based yeast two-hybrid system, the DNA-BD is provided by the entire prokaryotic LexA protein which normally binds to LexA operators and the AD is an 88-residue acidic peptide that activates transcription in yeast [15– 17]. When bait and prey fusion proteins interact, the DNA-BD and AD are brought into proximity to activate transcription of two different and independent reporter genes (LEU2 and LacZ) under the control of multiple LexA operators (The LEU2 and LacZ reporter genes are preceded by six copies and eight copies of the LexA operator, respectively) [4, 18–20]. The integrated LEU2 nutritional reporter gene allows the Leu-auxotrophic host cell EGY48 to grow on SD medium lacking Leu when transformed with plasmids encoding interacting hybrid proteins. When LacZ reporter gene within p8op-lacZ plasmid is activated, the cells produce β-galactosidase whose activity can be monitored using the colony-lift filter β-galactosidase assay [21, 22]. The promoters differ in the sequences flanking the LexA operators and this sequence difference helps to minimize false positives. Furthermore, the LexA DNA BD does not contain a nuclear localization signal that also reduces the potential false positive interactions. We here summarize the protocol for identification of the interactions with interest protein using the LexA-based yeast two-hybrid assay. Before yeast transformation, we need to construct bait and prey vectors and prepare the EGY48 yeast competent cells. Then we co-transform bait, prey, and reporter plasmids into the competent cells using polyethylene glycol (PEG)/LiAc-based method. After incubation at 30  C until colonies appear, we test positive colonies using the leucine auxotrophy and colony-lift filter β-galactosidase assay under blue light conditions.

2

Materials

2.1 Plasmids and Yeast Strain

1. Plasmids: Three plasmids (bait, prey, and reporter vectors) need to be prepared in the LexA-based yeast two-hybrid system. For bait and prey constructs, candidate genes were PCR amplified and cloned into pLexA (Original name pEG202) and pB42AD (Original name pJG4–5), respectively [16]. All bait constructs and prey constructs were confirmed by the DNA sequencing. p8op-lacZ (Original name pSH18–34) is used as the reporter vector which was co-transformed into the EGY48 yeast strain together with the bait and prey constructs (see Note 1) [17]. 2. Yeast strain: The Saccharomyces cerevisiae strain varies depending on the different laboratories and yeast two-hybrid systems. In this protocol, EGY48 yeast strain is recommended to use (see Note 2).

LexA-Based Yeast Two-Hybrid Assay

2.2

Stock Solution

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1. 10  TE stock solution: 0.1 M Tris–HCl, 10 mM EDTA, adjust to pH 7.5, autoclave for 15 min and store at room temperature (RT). 2. 10  LiAc stock solution (1 M): Dissolve 10.2 g lithium acetate in 100 mL of deionized water, adjust to pH 7.5 with dilute acetic acid, autoclave for 15 min, and store at RT. 3. 50% (w/v) PEG3350 stock solution: Dissolve 50 g PEG3350 in about 30 mL of deionized water, stir until it dissolves and make up the volume to 100 mL, autoclave for 15 min, and store at RT (see Note 3). 4. 40% (w/v) Galactose stock solution: Dissolve 40 g galactose in 100 mL of deionized water, autoclave for 15 min (see Note 4). 5. 40% (w/v) Raffinose stock solution: Dissolve 40 g Raffinose in 100 mL of deionized water, autoclave for 15 min (see Note 4). 6. 10  BU salts stock solution: 70 g/L Na2HPO4l7H2O, 30 g/ L NaH2PO4, adjust to pH 7.0, autoclave for 15 min, and store at RT (see Note 5). 7. X-Gal stock solution: Dissolve 20 mg 5-Bromo-4-chloro-3indolyl β-D-galactopyranoside in 1 mL N, N-dimethylformamide (DMF), store in the dark at 20  C.

2.3

Media

1. YPD medium: 10 g/L Yeast extract, 20 g/L peptone, 20 g/L glucose, adjust to pH 6.5, autoclave for 15 min. Add 20 g/L Agar for plates only. 2. SD/-His/ Trp/-Ura plate medium: 6.8 g/L Yeast nitrogen base without amino acids, 0.70 g/L amino acids with -His/ Trp/-Ura Dropout Supplements, 20 g/L glucose, 20 g/L agar, adjust to pH 5.8 and autoclave for 15 min, pour plates, and store at RT. 3. SD/Gal/Raf/-His/ Trp/-Ura/+BU liquid medium: 6.8 g/ L Yeast nitrogen base without amino acids, 0.70 g/L amino acids with -His/ Trp/-Ura Dropout Supplements, adjust to pH 5.8 and autoclave for 15 min. After autoclaving, cool to ~55  C, then add: 50 mL/L 40% galactose stock, 25 mL/L 40% raffinose stock, 100 mL/L of 10  BU salts stock, store at RT (see Note 6). 4. SD/Gal/Raf/-His/ Trp/-Ura/ Leu plate medium: 6.8 g/ L Yeast nitrogen base without amino acids, 0.60 g/L amino acids with -His/ Trp/-Ura/ Leu Dropout Supplements, 20 g/L agar, adjust to pH 5.8 and autoclave for 15 min. After autoclaving, cool to ~55  C, then add: 50 mL/L 40% galactose stock, 25 mL/L 40% raffinose stock, pour plates, and store at RT. 5. SD/Gal/Raf/-His/ Trp/-Ura/+X-Gal/+BU salts plate medium: 6.8 g/L Yeast nitrogen base without amino acids,

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0.70 g/L amino acids with -His/ Trp/-Ura Dropout Supplements, 20 g/L agar, adjust to pH 5.8 and autoclave for 15 min. After autoclaving, cool to ~55  C, then add: 50 mL/L 40% galactose stock, 25 mL/L 40% raffinose stock, 4 mL/L of 20 mg/mL X-Gal, 100 mL/L of 10  BU salts stock, pour plates, and store in dark at 4  C (see Note 7). 2.4 Buffers and Reagents

1. 1  TE/LiAc solution (10 mL): 1 mL 10  TE stock, 1 mL 10  LiAc stock, 8 mL deionized water. Prepare fresh just prior to use. 2. PEG/LiAc solution (10 mL): 8 mL 50% PEG3350 stock, 1 mL 10  TE stock, 1 mL 10  LiAc stock. Prepare fresh just prior to use. 3. Carrier DNA (10 mg/mL): Single-stranded deoxyribonucleic acid from salmon testes. Boil the solution for 10 min and then cool rapidly in an ice for bath at least 5 min prior to use (see Note 8). 4. Dimethyl sulfoxide (DMSO) (see Note 9).

2.5

Equipment

1. Incubator: For all of yeast two-hybrid assays, yeast cells were grown under darkness condition or blue light condition of 35 μmol·m 2·s 1 at 30  C (see Note 10). 2. Shaking incubator: Incubate the yeast in shaking incubator at 30  C, 200 rpm. 3. Spectrophotometer: Measure the OD of yeast culture at 600 nm.

3

Methods

3.1 Preparation of Competent Yeast Cells

1. Streak one YPD plate medium with EGY48 yeast cells from a frozen yeast stock. Incubate the plate upside down at 30  C until colonies appear (~3 days) (see Note 11). 2. Inoculate a single colony of the EGY48 yeast strain (diameter 2–3 mm) with a sterile inoculation loop from a fresh YPD plate (2 mm in diameter on medium without X-Gal. 15. On plates containing X-Gal, you can distinguish variations in blue color intensity of the positive colonies by 3 days after

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plating; the color variations may or may not reflect the strength of the protein-protein interaction. By 5 days, all positive colonies will appear to have the same color intensity. Beyond 5 days, there is an increased risk of false positive results due to the sensitivity of the LacZ reporter system. Colonies grown on X-Gal containing medium will be somewhat smaller than those grown without X-Gal. References 1. Fields S, Song O (1989) A novel genetic system to detect protein–protein interactions. Nature 340:245 2. Mendelsohn AR, Brent R (1994) Biotechnology applications of interaction traps/twohybrid systems. Curr Opin Biotechnol 5:482–486 3. Fields S (2009) Interactive learning: lessons from two hybrids over two decades. Proteomics 9:5209–5213 4. Xing S, Wallmeroth N, Berendzen KW, Grefen C (2016) Techniques for the analysis of protein-protein interactions in vivo. Plant Physiol 171:727–758 5. McNellis TW, Torii KU, Deng X (1996) Expression of an N-terminal fragment of COPI confers a dominant-negative effect on light-regulated seedling development in Arabidopsis. Plant Cell 8:1491–1503 6. Ahmad M, Jarillo JA, Smirnova O, Cashmore AR (1998) The CRY1 blue light photoreceptor of Arabidopsis interacts with phytochrome a in vitro. Mol Cell 1:939–948 7. Liu H, Yu X, Li K, Klejnot J, Yang H, Lisiero D et al (2008) Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis. Science 322:1535–1539 8. Zuo Z, Liu H, Liu B, Liu X, Lin C (2011) Blue light-dependent interaction of CRY2 with SPA1 regulates COP1 activity and floral initiation in Arabidopsis. Curr Biol 21:841–847 9. Yang H, Tang R, Cashmore AR (2001) The signaling mechanism of Arabidopsis CRY1 involves direct interaction with COP1. Plant Cell 13:2573–2587 10. Lian H, He S, Zhang Y, Zhu D, Zhang J, Jia K et al (2011) Blue-light-dependent interaction of cryptochrome 1 with SPA1 defines a dynamic signaling mechanism. Genes Dev 25:1023–1028

11. Liu B, Zuo Z, Liu H, Liu X, Lin C (2011) Arabidopsis cryptochrome 1 interacts with SPA1 to suppress COP1 activity in response to blue light. Genes Dev 25:1029–1034 12. Fritz CC, Green MR (1992) Fishing for partners. Curr Biol 2:403–405 13. Fields S, Sternglanz R (1994) The two-hybrid system: an assay for protein-protein interactions. Trends Genet 10:286–292 14. Luban J, Goff SP (1995) The yeast two-hybrid system for studying protein-protein interactions. Curr Opin Biotechnol 6:59–64 15. Ebina Y, Takahara Y, Kishi F, Nakazawa A, Brent R (1983) LexA protein is a repressor of the colicin E1 gene. J Biol Chem 258:13258–13261 16. Golemis EA, Brent R (1992) Fused protein domains inhibit DNA binding by LexA. Mol Cell Biol 12:3006–3014 17. Brent R, Finley RL Jr (1997) Understanding gene and allele function with two-hybrid methods. Annu Rev Genet 31:663–704 18. Ma J, Ptashne M (1987) A new class of yeast transcriptional activators. Cell 51:113–119 19. James P, Halladay J, Craig EA (1996) Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144:1425–1436 20. James P (2001) Yeast two-hybrid vectors and strains. Methods Mol Biol 177:41–84 21. Gyuris J, Golemis E, Chertkov H, Brent R (1993) Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75:791–803 22. Estojak J, Brent R, Golemis EA (1995) Correlation of two-hybrid affinity data with in vitro measurements. Mol Cell Biol 15:5820–5829

Chapter 16 Express Arabidopsis Cryptochrome in Sf9 Insect Cells Using the Baculovirus Expression System Xu Li, Yawen Liu, and Hongtao Liu Abstract The Bac-to-Bac® Baculovirus Expression System provides a rapid and efficient method to generate recombinant cryptochrome (CRY) proteins with chromophore flavin (FAD), which showed blue light response in vitro. Key words Arabidopsis, Cryptochrome, FAD, Blue light response, Baculovirus expression system, Sf9 insect cell

1

Introduction Cryptochromes are photolyase-like blue light receptors first discovered in Arabidopsis thaliana but later found in other plants, microbes, and animals [1–4]. Plant Cryptochromes mediate bluelight inhibition of hypocotyl elongation, photoperiodic promotion of floral initiation, and blue light regulation of the circadian clock, tropism growth, shade avoidance, etc. [1, 5–7]. The PHR domains of Arabidopsis CRY1 and CRY2 bind noncovalently to chromophore FAD which is important for CRY’s function [8–11]. However, E. coli expressed CRYs lack FAD. The His-tagged CRY1 and CRY2 proteins were expressed well in Sf9 cells with FAD by using the Bac-to-Bac Baculovirus expression system (Invitrogen) [12– 16], which provide a more detailed study of this photoreceptor, such as protein-protein interaction in vitro or the protein structure analysis.

2

Materials 1. Bluo-gal solutions are made by dissolving the dry powder in dimethyl sulfoxide (DMSO) to make 20 mg/mL stock

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2.1 Prepare Recombinant Bacmid and Baculovirus

solution. Weigh 0.2 g Bluo-galto 9 mL water, make up to 10 mL water. 2. A 200 mg/mL stock of IPTG is made by mixing 2 g of IPTG with 8 mL of water until dissolved. 3. pFastBac™ Vectors (pFastBacHTA was used in this protocol). 4. 15–20 μL MAX Efficiency® DH10Bac™ Competent E. coli for each recombinant Bacmid construction. 5. S.O.C. Medium and LB agar plate with 100 μg/mL Bluogal,40 μg/m LIPTG, and antibiotics (50 μg/mL kanamycin, 7 μg/mL gentamicin, and 10 μg/mL tetracycline). Made fresh. 6. Sf-900 II SFM with penicillin and streptomycin (50 units/mL penicillin, 50 μg/mL streptomycin). 7. 1 M Tris–HCl, pH 8.0 or 7.5. Add about 50 mL water to a glass beaker. Weigh 12.1 g Tris–HCl and transfer to the beaker. Add water to a volume of 90 mL. Mix and adjust pH with HCl. Make up to 100 mL with water. Store at 4
C. 8. 5 M NaCl, Weigh 292.2 g NaCl to 900 mL water. Mix well and make up to 1 L with water. Store at 4
C. 9. 0.5 M EDTA stock, Weigh 37.7 g [ (NaOOCH2)2. NCH2CH2.N. (CH2COOH)2.2H2O, EDTA-2Na] and transfer to a beaker, add water to a volume of 150 mL water, mix, and adjust pH to 8.0 with NaOH, make up to 200 mL with water. Store at 4
C. 10. 1 M DTT stock. Weigh 3.09 g DTT to a 50 mL beaker, add 20 mL 0.01 M NaOAc (pH 5.2), sterilize by 0.22 μm filter. Leave one aliquot at 4
C for current use, store the remaining aliquots at 20
C. 11. Solution I (15 mM Tris–HCl pH 8.0, 10 mM EDTA, 100 μg/ mL RNase A). 12. Solution II (0.2 N NaOH, 1% SDS). 13. 3 M Potassium acetate, pH 5.5. Weight 29.4 g potassium acetate to 90 mL water, adjust pH to 5.5 with acetic acid, make up to 100 mL with water. Sterilize and store at room temperature.

2.2

Purify Proteins

1. Buffer: 50 mM Tris–HCl (pH 7.5), 0.5 M NaCl, 0.5% TX-100, 20 mM βMEP, 1 mM PMSF. 2. Wash buffer: 50 mM Tris–HCl (pH 7.5), 0.5 M NaCl, 10% glycerol, 20 mM βMEP, 20 mM imidazole. 3. Elution buffer: 50 mM Tris–HCl (pH 7.5), 0.5 M NaCl, 10% glycerol, 20 mM βMEP, 500 mM imidazole, 100 mM EDTA.

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4. Desalting buffer: 50 mM Tris–HCl (pH 7.5), 0.1 M NaCl, 10% glycerol, 20mM βMEP. 5. Storage buffer: 50 mM Tris–HCl pH 7.5, 0.1 M NaCl, 50% glycerol, 5mM DTT.

3

Method

3.1 Generating the Recombinant Bacmid

1. Fuse the coding sequence of CRY1 or CRY2 to the C terminus of the His tag, at the EcoRI and XhoI restriction sites of the vector pFastBacHTA donor plasmid. 2. Transform the pFastBacHTA-CRY1/2 construct (app.1 ng) into DH10Bac competent cells by heat shock the mixture in 42
C water bath for 45 s. After incubate the mixture with S.O.C. medium at 37
C for 4 h, spread the diluted mixture to the plates (containing Bluo-gal, IPTG and antibiotics, containing kanamycin, gentamicin, tetracycline), and incubate for at least 24 h at 37
C. 3. Pick 10 white candidates and streak to fresh plates to verify the phenotype. Select a single colony confirmed (by PCR) to set up a liquid culture for isolation of recombinant bacmid DNA. 4. Transfer 1.5 mL of culture to a 1.5-mL micro centrifuge tube and centrifuge at 10,000  g for 5 min. Remove the supernatant and resuspend (by gently vortexing or Pipetting up and down, if necessary) each pellet in 0.3 mL of Solution I. Add 0.3 mL of Solution II and gently mix. Incubate at room temperature for 5 min. The appearance of the suspension should change from very turbid to almost translucent. 5. Slowly add 0.3 mL of 3 M potassium acetate (pH 5.5), mixing gently during addition. A thick white precipitate of protein and E. coli genomic DNA will form. Place the sample on ice for 5–10 min. 6. Gently transfer the supernatant to the tube containing isopropanol. Mix by gently inverting tube a few times and place on ice for 5 to 10 min. Centrifuge the sample for 15 min at 15,000  g at room temperature. 7. Remove the supernatant and add 0.5 mL of 70% ethanol to each tube. Invert the tube several times to wash the pellet. Centrifuge for 5 min at 15,000  g at room temperature. (Optional: repeat step 7). 8. Air-dry the pellet at room temperature and dissolve the DNA in 50 μL TE (see Note 1).

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3.2 Producing the Recombinant Baculovirus

1. Seed 9  105 cells per 35-mm well (of a 6-well plate) in a 2 mL of Sf-900 II SFM containing penicillin/streptomycin at 0.5 final concentration. Allow cells to attach at 27
C for at least 1 h. 2. For each transfection sample, prepare complexes as follows: Solution A: For each transfection, dilute ~l μL of mini-prep bacmid DNA into 100 μL Sf-900 II SFM without antibiotics. Solution B: For each transfection, dilute~8 μL CellFectin Reagent into 100 μL Sf-900 II SFM without antibiotics. Combine solution A and B (total volume ~210 μL). Mix gently and incubate for 25–45 min at room temperature. 3. Add ~210 μL DNA-lipid mixture dropwise onto the cells from step 1. Incubate cells at 27
C for 5 h. 4. Remove the transfection mixture and add 2 mL of Sf-900 II SFM medium containing antibiotics. Incubate cells in a 27
C incubator for 72 h+. Visually inspect the cells daily for signs of infection (granular appearance, cell detachment); once the cells appear infected, harvest the virus (see Note 2). 5. Then harvest P1 virus by transferring the supernatant (2 mL) to a sterile tube. Clarify by centrifugation for 5 min at 500  g and transfer the virus containing to a fresh tube (store at 4
C, protected from light). Storage of an aliquot of the viral stock at 70
C is also recommended. 6. From the initial transfection, viral titers of 2  107 to 4  107 pfu/mL can be expected. Use 0.5 mL viral stock to infect a 50-mL culture at 2  106 cells/mL (MOI of 0.1) to amplify virus. Harvest virus at 48–72 h post-infection. Usually, P3 is highest usable passage.

Innoculum required ðmLÞ :

3.3 Expressing and Purifying the Recombinant Protein

desired MOI ðpfu=mLÞ  ðtotal number of cellsÞ Titer of viral innoculum ðpfu=mLÞ

1. Use P3 virus to infect 300 mL culture at 2  106 cells/mL. Collect cells at 48–72 h post-infection by centrifugation for 10 min at 500  g (see Note 3). 2. Lysis cell pellet in equal volume of cell pellet (for example add 3 mL lysis buffer in 3 mL cell pellet). 3. Incubate on ice for about 30 min, then sonicate for 5  5 s at 30% amplitude until the solution became watery. 4. The lysate was passed twice through an 18-gauge needle and was spun at 16, 000 g for 1 h at 4
C. 5. The supernatant was filtered through a 0.22-μm filter unit attached to a syringe (see Note 4).

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Fig. 1 CRY1 and CRY2 expressed and purified from insect cell. (a) SDS-PAGE gel with Coomassie stain showing purified CRY1, CRY2 proteins. Lane 1, 2, 3 indicate sf9 cells infected by virus carrying CRY1, infected by virus carrying CRY2, uninfected, respectively. Lane 4 and 5 indicate purified CRY1 and CRY2. Lane 7 and 8 indicate concentrated CRY1 and CRY2. (b) The absorption of FAD was recorded. Purified CRY1 and CRY2 bind stoichiometric FAD

6. Mix s/n with Ni beads which has been equilibrated with lysis buffer at cold room for 2 h. 7. Wash the resin 5 times with wash buffer and spin at 2000 g for 3 min to remove wash buffer. 8. Pack resin into small column and elute with elute buffer 1 mL each time at 10–15 min interval. 9. Collect all samples and go to desalting column to remove imidazole and EDTA. Dialysis against storage buffer. 10. The purified CRY1/2 protein can be used for photospectrometry analysis or pull-down assay, etc. (Fig. 1a, b).

4

Notes 1. Avoid repeated freeze-thaw; the recombinant bacmid can be stored at 4
C in TE buffer for up to 2 weeks. 2. Use insect cell only from a 3- to 4-day-old suspension culture in mid-log phase with viability of >95% for transfection. Do not add antibiotics during transfection.

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3. To obtain the highest expression of the protein, you may optimize transfection efficiency by varying virus concentrations, and cell density. 4. The CRY proteins are easy to aggregate; filter the supernatant through a 0.22-μm filter before adding to Ni beads; avoid exposure to blue light during purification (purify in red light condition).

Acknowledgments This work was supported by the National Natural Science Foundation of China (31730009, 31721001). References 1. Ahmad M, Cashmore AR (1993) HY4 gene of A. thaliana encodes a protein with characteristics of a blue-light photoreceptor. Nature 366:162–166 2. Lin C, Ahmad M, Chan J et al (1995) CRY2, a second member of the arabidopsis cryptochrome gene family. Plant Physiol 110:1047 3. Cashmore AR (2003) Cryptochromes: enabling plants and animals to determine circadian time. Cell 114:537–543 4. Partch CL, Sancar A (2005) Cryptochromes and circadian photoreception in animals. Methods Enzymol 393:726–745 5. Guo H, Yang H, Mockler TC et al (1998) Regulation of flowering time by Arabidopsis photoreceptors. Science 279 (5355):1360–1363 6. Somers DE, Devlin PF, Kay SA (1998) Phytochromes and cryptochromes in the entrainment of the Arabidopsis circadian clock. Science 282:1488–1490 7. Tsuchida-Mayama T, Sakai T, Hanada A et al (2010) Role of the phytochrome and cryptochrome signaling pathways in hypocotyl phototropism. Plant J 62(4):653–662 8. Lin C, Robertson DE, Ahmad M et al (1995) Association of flavin adenine dinucleotide with the Arabidopsis blue light receptor CRY1. Science 269:968–970 9. Malhotra K, Kim ST, Batschauer A et al (1995) Putative blue-light photoreceptors from Arabidopsis thaliana and Sinapis alba with a high degree of sequence homology to DNA photolyase contain the two photolyase cofactors but

lack DNA repair activity. Biochemistry 34:6892–6899 10. Banerjee R, Schleicher E, Meier S et al (2007) The signaling state of Arabidopsis cryptochrome 2 contains flavin semiquinone. J Biol Chem 282:14916–14922 11. Bouly JP, Schleicher E, Dionisio-Sese M et al (2007) Cryptochrome blue light photoreceptors are activated through interconversion of flavin redox states. J Biol Chem 282:9383–9391 12. Bac-to-Bac® Baculovirus Expression System (2010) An efficient site-specific transposition system to generate baculovirus for high-level expression of recombinant proteins. Version F 13. Liu H, Yu X, Li K et al (2008) Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis. Science 322(5907):1535–1539 14. Li X, Wang QYXH et al (2011) Arabidopsis cryptochrome 2 (CRY2) functions by the photoactivation mechanism distinct from the tryptophan (trp) triad dependent photoreduction. Proc Natl Acad Sci U S A 108 (51):20844–20849 15. Gao J, Wang X, Zhang M et al (2015) Trp triad-dependent rapid photoreduction is not required for the function of Arabidopsis CRY1. Proc Natl Acad Sci U S A 112:9135–9140 16. Liu YW, Li X, Ma D et al (2018) CIB1 and CO interact to mediate CRY2 regulation of flowering. EMBO Reports. https://doi.org/10. 15252/embr.201845762

Chapter 17 Semi-In-Vivo Pull-Down Assay for Blue Light-Dependent Protein Interactions Xu Li, Yawen Liu, and Hongtao Liu Abstract Cryptochromes are photolyase-like blue-light receptors found in all major evolutionary lineages (Ahmad and Cashmore, Nature 366:162–166, 1993; Lin, Plant Physiol 110:1047, 1996; Cashmore, Cell 114:537–543, 2003; Partch and Sancar, Methods Enzymol 393:726–745, 2005). Arabidopsis cryptochrome 1 (CRY1) and cryptochrome 2 (CRY2) mediate primarily blue-light inhibition of hypocotyl elongation and photoperiodic control of floral initiation (Ahmad and Cashmore, Nature 366:162–166, 1993; Somers et al., Science, 282:1488–1490, 1998; Guo et al., Science 279 (5355):1360–1363, 1998; Yu et al., Arabidopsis Book 8:e0135, 2010). It has been proposed that phototransduction of cryptochromes involves the blue-light-dependent protein interactions, such as AtCRY2-CIB1 (CRYPTOCHROME-IN TERACTING BASIC-HELIX-LOOP-HELIX 1), AtCRY1-PIF4 (PHYTOCHROME INTERACTING FACTOR 4) modules, sequentially mediate gene expression and plant growth (Liu et al., Science 322 (5907):1535–1539, 2008; Ma et al., Proc Natl Acad Sci U S A 113 (1):224–229, 2016; Wang et al., Science 354:343–347, 2016). Cryptochromes also showed blue light response in vitro when expressed in Sf9 insect cells using the baculovirus expression system, thus the wavelength-specific CRY2CIB1 interaction can also be observed in Semi-in-vivo pull-down assay (Li et al., Proc Natl Acad Sci U S A 108 (51):20844–20849, 2011; Liu et al., EMBO Reports, 2018). Here, we describe the detailed process of blue light-dependent CRY2-CIB1 interaction in Semi-in-vivo conditions. Key words Blue light dependent, Protein interaction, Photoreceptor, Cryptochrome, CIB1, Semi-invivo

1

Introduction Light inducible protein–protein interactions are powerful tools to manipulate biological processes. For example, phytochromes and PIFs have been used as the dimerizer pair to make the red light controlled system [1]. CRY2 undergoes blue light-specific interaction with the bHLH protein CIB1.A blue light triggered protein translocation system and a DNA recombination system in living cells were made based on it [2, 3].As a blue light receptor, AtCry2 showed blue light activity in vitro when expressed in Sf9 insect cells

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[4, 5]. The conditions from this optimization step can rapidly be applied to blue light-dependent protein interactions in vitro.

2 2.1

Materials Prepare Proteins

1. The semi-in-vivo CRY2–CIB1 interaction was carried out in lysates of insect cells expressing CRY2 and Escherichia coli cells expressing CIB1. 2. Protein A/G agarose (Abmart). 3. Sonication (model VC505; Sonics & Materials, Inc.). 4. BCA Protein Assay system (Pierce). 5. Anti-CRY2 (or anti-CIB1)polyclonal rabbit antibody was prepared against the C-terminus of CRY2 (CIB1)protein expressed in E. coli. 6. 1 M Tris–HCl, pH 7.8.Add about 50 mL water to a glass beaker. Weigh 12.1 g Tris–HCl and transfer to the beaker. Add water to a volume of 90 mL. Mix and adjust pH to 7.8 with HCl. Make up to 100 mL with water. Store at 4  C. 7. 5 M NaCl, Weigh 292.2 g NaCl to 900 mL water. Mix well and make up to 1 L with water. Store at 4  C. 8. 200 mM PMSF (phenylmethanesulfonyl fluoride) stock. Weight 0.174 g PMSF to 5 mL absolute ethyl alcohol. Store at 20  C. 9. 0.84 M IPTG stock. Weight 200 mg IPTG to 1 mL water. 10. XB buffer [50 mM Tris (pH 7.8), 500 mM NaCl, 0.5% Triton X-100, 1 mM PMSF, 4.7 mM β-ME]. 11. WB buffer [50 mM Tris (pH 7.8), 500 mM NaCl, 0.1% Triton X-100, 1 mM PMSF]. 12. SDS loading buffer[25.2 g Glycerol, 0.02 g Bromophenol Blue, 4 g SDS, 20 mL 1 M Tris–HCl (pH 6.8), 3.1 g DTT, to 50 mL ddH2O, store at 20  C]. 13. Light condition, blue or red LED (20 μmol m (20 μmol m 2 s 1).

2.2 Make SDS-PAGE Gel

2

s 1), red light

1. Solution 1:100 mL (Acrylamide 29.2 g, Bisacrylamide 0.8 g, Add ddH2O to 100 mL. Use filter paper to filter. Store at 4  C). 2. Solution 2:200 mL (1.5 M Tris pH 8.8 (measure Tris 36.3 g) Add ddH2O to 200 mL. Autoclave). 3. Solution 3:100 mL (0.5 M Tris pH 6.8 (measure Tris 6 g) Add ddH2O to 100 mL. Autoclave).

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4. Solution 4:10%SDS (measure SDS 10 g, Add ddH2O to 100 mL. Autoclave). 5. AP: Ammonium persulfate, measure 0.1 g to 1 mL ddH2O. 6. For gel making:

2.3

Western Blot

10% Separation Gel

5 mL

Solution 1

1.667 mL

Solution 2

1.25 mL

Solution 4

0.05 mL

ddH2O

2.017 mL

10% AP

50 μL

TEMED

5 μL

4% Stacking Gel

2 mL

Solution 1

0.266 mL

Solution 3

0.5 mL

Solution 4

0.02 mL

ddH2O

1.22 mL

10% AP

15 μL

TEMED

1.5 μL

1. SDS-PAGE gel running buffer: Weight 3 g Tris, 14.4 g Glycine, 1 g SDS, add ddH2O to 1000 mL. 2. Transfer buffer: Weigh 3 g Tris, 14.4 g Glycine, add ddH2O to 1000 mL. 3. PBST: Weigh 8 g NaCl, 0.2 g KH2PO4, 0.2 g KCl, 2.14 g Na2HPO4·7H2O to 1000 mL water, add 1 mL Tween20. 4. Bio Rad Electrophoresis Chambers and Wet/Tank Blotting Systems.

3

Method

3.1 Generating Recombinant Proteins

1. Fuse the coding sequence of CRY2 to the C terminus of the His tag, at the EcoRI and XhoI restriction sites of the vector pFastBacHTA. Transform the recombinant plasmid to DH10Bac competent cells to get recombinant bacmid DNA. 2. Transfect Sf9 cells with the recombinant bacmid DNA at 27  C incubator. Harvest and amplify the recombinant baculovirus in

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darkness. Collect the infected cells for the following assay (see Note 1). 3. Fuse the coding sequence of CIB1 to the C-terminus of His tag in the vector pCold-TF (Takara) at the EcoRI and SalI restriction sites. Express the fusion proteins in E. coli (strain BL21) with 0.5 mM IPTG. 3.2 Semi-In-Vivo Pull-Down Assay

1. Prepare anti-CRY2/IgG-coupled protein A/G agarose resin before each pull-down experiment by incubating 5 μL antiCRY2 antiserum with 30 μL protein A/G agarose (Abmart) in 120 μL XB buffer [50 mM Tris (pH 7.8), 500 mM NaCl, 0.5% Triton X-100, 1 mM PMSF, 4.7 mM β-ME] at 4  C for 2 h. 2. Cells expressing CRY2 and CIB1 were lysed by sonication (model VC505; Sonics & Materials, Inc.) for 5  5 s at 30% amplitude until the solution became watery (see Note 1). 3. Pass the lysate twice through an 18-gauge needle and spun at 15,000  g for 1 h at 4  C. 4. Filter the supernatant through a 0.22-μm filter unit attached to a syringe (see Note 2). 5. Measure the protein concentration of the lysate using the BCA Protein Assay system. Dilute the lysate to 1 μg protein/μL in XB buffer. 6. Mix one-milliliter lysates of cells expressing CRY2 and CIB1 with 20 μL protein A/G agarose to preclear the cell lysate (see Note 3). 7. After a brief spin, mix the supernatant with a 125-μL suspension of the anti-CRY2/IgG-coupled protein A/G agarose prepared earlier the same day and rotate gently for the indicated time period in darkness, in blue light (20 μmol· m 2· s 1), or in red light (20 μmol· m 2· s 1) at 4  C. 8. After incubation, collect the agarose beads by spinning at 1000 rpm for 3 min, wash with 1 mL ice-cold WB buffer, and wash again three times with 600 μL WB buffer. 9. Suspend the agarose beads in 30 μL (2) SDS/PAGE loading buffer and boil for 5 min. Then 10 μL of the mixture was analyzed by SDS/PAGE and immunoblot, along with 0.2% of the input.

3.3 SDS/PAGE and Immunoblot

1. For SDS-PAGE: 90v for stacking gel, 150v for separating gel. 2. Transfer to nylon membrane through Bio Rad Wet/Tank Blotting Systems (see Note 4). 3. 5% Milk/PBST block the nylon membrane for 1–2 h. 4. Wash with PBST 1–2 times, 10 s.

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Fig. 1 The in vitro pull-down assay showing blue light-dependent formation of the CRY2-CIB1 complex

5. The anti-CIB1polyclonal rabbit antibody was prepared against the CIB1 protein expressed in E. coli. The first antibody is diluted in PBST or 1% milk/PBST and incubate for 1–2 h. 6. Wash with PBST for 10 min, 3 times or 6 min, 3 times, then incubate in second antibody for 1 h. Wash with PBST for 10 min, 3 times or 6 min, 3 times. Then develop film. Figure 1 shows the formation of CRY2-CIB1 complex depends on blue light, consistent with the in vivo response, this is the first time a blue light-specific protein–protein interaction is observed in vitro for a plant cryptochrome.

4

Notes 1. Avoid exposure of the insect cell with CRY to blue light before doing the protein–protein interaction. Harvest and amplify the infected cells in darkness. Collect and lysis the infected cells in red light, to keep CRY in inactive form. 2. Filter the supernatant through a 0.22-μm filter before to use is very important. This step can preclean the aggregated CRY protein. 3. Preclear the lysates of cells expressing CRY2 and CIB1 with 20 μL protein A/G agarose to avoid nonspecific binding of CRY2 and CIB1. 4. Put the nylon membrane and gel in transfer buffer for 5 min, then make the sandwich for transfer. From button to up: black color stands for cathode, then put filter paper (if it is thick one, use one layer; if it is thin one, use two layers), then your gel, then your nylon membrane, then filter paper, then red color which stands for anode. All progress should be done in the transfer buffer, and need to remove bubbles. 90v for 50 min, or 8v for overnight.

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Acknowledgments This work was supported by the National Natural Science Foundation of China (31730009, 31721001). References 1. Shimizu-Sato S, Huq E, Tepperman J et al (2002) Light-switchable gene promoter system. Nat Biotechnol 20:1041–1044 2. Kennedy M, Huges R, Peteya L et al (2010) Rapid blue-light-mediated induction of protein interactions in living cell. Nat Methods 6: 973–975 3. Liu H, Gomez G, Lin S et al (2012) Optogenetic control of transcription in zebrafish. PLoS One 7(11):e50738 4. Li X, Wang Q, Yu XH et al (2011) Arabidopsis cryptochrome 2 (CRY2) functions by the photoactivation mechanism distinct from the tryptophan (trp) triaddependent photoreduction. Proc Natl Acad Sci U S A 108 (51):20844–20849 5. Liu YW, Li X, Ma D et al (2018) CIB1 and CO interact to mediate CRY2 regulation of flowering. EMBO Rep 19. https://doi.org/10. 15252/embr.201845762 6. Ahmad M, Cashmore AR (1993) HY4 gene of A. thaliana encodes a protein with characteristics of a blue-light photoreceptor. Nature 366:162–166 7. Lin C, Ahmad M, Chan J et al (1996) CRY2, a second member of the arabidopsis cryptochrome gene family. Plant Physiol 110:1047 8. Cashmore AR (2003) Cryptochromes: enabling plants and animals to determine circadian time. Cell 114:537–543

9. Partch CL, Sancar A (2005) Cryptochromes and circadian photoreception in animals. Methods Enzymol 393:726–745 10. Somers DE, Devlin PF, Kay SA (1998) Phytochromes and cryptochromes in the entrainment of the Arabidopsis circadian clock. Science 282:1488–1490 11. Guo H, Yang H, Mockler TC et al (1998) Regulation of flowering time by Arabidopsis photoreceptors. Science 279 (5355):1360–1363 12. Yu X, Liu H, Klejnot J et al (2010) The cryptochrome blue light receptors. Arabidopsis Book 8:e0135. https://doi.org/10.1199/tab. 0135 13. Liu H, Yu X, Li K et al (2008) Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis. Science 322(5907):1535–1539 14. Ma D, Li X, Guo Y et al (2016) Cryptochrome1 interacts with PIF4 to regulate high temperature-mediated hypocotyl elongation in response to blue light. Proc Natl Acad Sci U S A 113(1):224–229 15. Wang Q, Zuo Z, Wang X et al (2016) Photoactivation and inactivation of Arabidopsis cryptochrome 2. Science 354:343–347

Chapter 18 Tobacco System for Studying Protein Colocalization and Interactions Jingyi Zhang and Shengbo He Abstract Transient protein expression in a heterologous system has been very useful in many research fields. As a plant expression system, tobacco has some unique advantages including big leaves, simple infiltration and transformation, high activity in expressing transgenes, and easy sampling for microscopy. Because of these advantages, tobacco system has been extensively used for many purposes, such as large-scale expression and purification of proteins of interest, protein colocalization, protein degradation, protein-protein interaction assays including co-immunoprecipitation (CoIP), fluorescence resonance energy transfer (FRET), and bimolecular fluorescence complementation (BiFC), transcription regulation, plant-pathogen interactions, and functional verification of small RNAs. A large number of publications have used this system and generated critical results to support their conclusions. The results obtained from tobacco system are highly reproducible and mostly consistent with those generated from traditional techniques, indicating its reliability. Here we describe a protocol for studying protein-protein interactions in tobacco system, which could be applied to multiple experimental purposes as the procedure of tobacco leaf infiltration is basically shared among them. Key words Nicotiana benthamiana, Infiltration, Agrobacteria, Protein colocalization, Protein interaction

1

Introduction It is desirable for scientists to quickly test their hypothesis before throwing much effort to it. However, traditional genetic manipulation is time-consuming, which is even true for the extensively studied model plant Arabidopsis thaliana [1], let alone relatively less studied plant species, for which agrobacterium-mediated stable transformation typically takes at least 3 months not including callus induction from cocultivation to transplanting transformants [2– 6]. In addition, stable transformation sometimes gives weak expression of transgenes especially for those genes whose overexpression would be lethal or harmful for the plants [7]. Such weak expression of transgenes makes some experiments such as co-immunoprecipitation (CoIP) difficult to handle. Moreover, it is not very

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straightforward to create stable transgenic plants expressing multiple transgenes. To do this, people often have to transform plants sequentially with different constructs, which is even more timeconsuming and laborious [8]. Alternatively, people could create special vectors which are compatible with multiple expression cassettes [9]. However, such vectors tend to be very big and are not easy to handle for cloning. Transient expression system can overcome these issues and allows researchers to timely and reliably get a sense of whether their ideas are likely to be true. As the most frequently used system, cells with unlimited proliferative ability have been extensively utilized and to some extent are indispensable for animal studies. For plant studies, tobacco transient expression system has many favorable features. Typically, 2–3 days post-infiltration will give strong expression of the proteins under study and almost all types of proteins can be expressed in tobacco leaves including transcription factors, photoreceptors, hormone receptors, and pathogen effectors [7, 10–14]. More strikingly, up to 6 different proteins or even more can be expressed simultaneously in the same leaf cells [7, 15], allowing this system to be more versatile. The results obtained from tobacco system are reproducible and reliable for most cases if not all as long as bona fide negative and positive controls are nicely designed. Simple procedure is desirable including easy-doing laboratory routines such as agrobacteria transformation and culture, OD adjustment, and chemical induction of transformation-favoring genes. Tobacco plants are as easily grown as Arabidopsis and can grow in the same conditions as Arabidopsis. Not much space is required to grow tobacco plants as only plants at vegetative phase will be used for infiltration. Tobacco leaves have high ability to express exogenous genes carried by plasmids. Tobacco system has been documented to mimic in vivo biological processes and is therefore reliable [15]. As transgenes tend to be silenced by plant defense system, p19 has been introduced to compromise plant defense system and vastly elevate protein expression level [15].

2

Materials

2.1 Agrobacteria Transformation

1. Constructs [11]: 35S-ARF6-CFP, 35S-CRY1-YFP, 35S-CFP, 35S-GUS-NLS-YFP, 35S-cLUC-flag, 35S-ARF6-flag, 35S-Myc-CRY1, pSOUP-p19. 2. Agrobacteria strain GV3101 (see Note 1). 3. Liquid nitrogen. 4. LB medium: dissolve 10 g Peptone, 5 g Yeast Extract, and 5 g Sodium Chloride in 1 L of water and autoclave.

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5. Antibiotics: dissolve Rifampicin in DMSO and make the stock as 50 mg/mL (1000
); dissolve Gentamycin, Spectinomycin, and Kanamycin in water and make the stocks as 50 mg/mL (1000
). 6. Spectrophotometer. 2.2

Infiltration

1. Acetosyringone: dissolve Acetosyringone in DMSO and prepare stock solution as 100 mM. 2. MES-KOH: dissolve MES in water and prepare stock solution as 0.5 M. Use KOH to adjust pH to 5.6. 3. 1 mL syringe (see Note 2).

2.3 Confocal Microscopy and CoIP

1. Confocal microscope. 2. Lysis buffer: 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.2% Triton-X-100. Before use, add Pefabloc (final concentration 1 mM), and complete protease inhibitor cocktail. 3. Elution buffer: lysis buffer with 0.5 μg/μL 3
flag peptide. 4. Antibodies: anti-Myc and anti-flag. 5. Anti-flag magnetic beads for immunoprecipitation. 6. Heating block: thermomixer (0  C–99  C).

3

Methods

3.1 Transform Agrobacteria and Make Liquid Culture (See Note 3)

1. Add plasmids to the competent cells (GV3101), mix well by tapping the tube on the side, and incubate on ice for 30 min. 2. Freeze the cells in liquid nitrogen for 1 min and transfer to 37 ˚ C water bath or heating block for 3 min. 3. Remove the tubes from 37 ˚C and leave on ice. In a flow hood, add 800 μL LB medium to the cells and incubate at 28 ˚C for 3 h. 4. Spin cells down at 12,000 rpm for 30 s. Pour away supernatant and leave about 100 μL behind. Resuspend cells by pipetting and spread out the cells on plates containing Rifampicin, Gentamycin, and the resistance from the plasmids. Put the plates in an incubator at 28 ˚C for 2–3 days. 5. Pick single colonies in LB medium to make liquid culture and shake at 220 rpm and 28 ˚C overnight.

3.2

Adjust OD600

1. Take 600 μL liquid culture to a tube and add equal volume of 40% glycerol to make a glycerol stock for future use. Store the glycerol stock at 70 ˚C.

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2. Pellet the rest of liquid culture by centrifugation at 4,000 rpm (3,200
g) for 10 min at room temperature. 3. Discard the supernatant and resuspend the pellet with 5 mL of Murashige and Skoog (MS) medium (see Note 4). 4. Transfer an aliquot of the suspension to a fresh tube and add an appropriate volume of MS medium to adjust Agrobacteria harboring individual constructs to OD600 ¼ 0.6 (see Note 5). 5. Add acetosyringone (AS) and MES (pH 5.6) to a final concentration of 200 μM and 10 mM, respectively. 6. Incubate at room temperature for 3 h. 3.3

Infiltration

1. Mix Agrobacteria harboring different constructs as needed in a ratio (see Note 6). For ARF6-CRY1 colocalization, mix 35S-ARF6-CFP, 35S-CRY1-YFP, and pSOUP-p19 in a ratio of 1:1:1. As negative controls, mixtures of 35S-CFP/35SCRY1-YFP/pSOUP-p19 and 35S-ARF6-CFP/35S-GUSNLS-YFP/pSOUP-p19 were made in the same ratio, respectively. For ARF6-CRY1 CoIP, mix 35S-ARF6-flag, 35S-MycCRY1, and pSOUP-p19 in a ratio of 1:1:1. As the negative control, mixture of 35S-cLUC-flag/35S-Myc-CRY1/ pSOUP-p19 was made in the same ratio. 2. Select healthy tobacco leaves for infiltration (see Note 7). 3. Draw regions for infiltration with a marker pen and label the regions. Multiple regions could be drawn for different constructs on the same leaf. Avoid big veins. 4. Infiltrate the abaxial side of tobacco leaves in the pre-drawn regions with 1 mL syringe (see Note 8). Make sure change gloves between constructs in case of any cross-contamination. 5. Leave the infiltrated tobacco plants in shade overnight and put back to normal growth conditions next day. 6. After 2–3 days, leaves or individual regions can be harvested for downstream experiments (see Note 9).

3.4 Confocal Microscopy for Protein Colocalization

1. Put the infiltrated leaf samples on slides, with abaxial side up, and drip some water to the samples and put the coverslips on (see Note 10). 2. Put the slides under a confocal microscope and examine the fluorescence protein-tagged proteins of interest under appropriate channels (see Note 11).

3.5

CoIP

1. Grind leaf samples in liquid nitrogen to fine powder with mortar and pestle. During grinding, do not let samples warm up and be hydrated in case of protein degradation by proteases in the absence of protease inhibitors.

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2. Collect powder in an appropriate container. Depending on the expression abundance of the proteins of interest in tobacco leaves, sample amount for CoIP varies. Typically, 0.1–1 mL of powder is collected in 1.5 mL Eppendorf tubes and can be stored at 70 ˚C for future use. 3. Add equal or double volume of lysis buffer to the powder (see Note 12). It is critical to add lysis buffer as quickly as possible to avoid hydration and protein degradation. 4. Vortex to homogenize samples. It is important to resuspend sample powder thoroughly in case of any clots being formed. 5. Get rid of debris by centrifugation at 12,000 rpm (11,000
g) for 15 min at 4  C. 6. Transfer the supernatant to a fresh tube. 7. Pre-wash anti-flag magnetic beads with 1 mL of lysis buffer for 3 times. Use 20 μL beads for each sample (see Note 13). 8. Add the samples to the beads and incubate at 4  C with gentle rotation for 30 min–1 h. 9. Wash the beads with 1 mL of lysis buffer for 3 times (see Note 14). Make all the beads completely resuspended. 10. Add 50 μL of elution buffer and incubate in a thermomixer at 1,200 rpm, 4  C for 10 min (see Note 15). 11. Use the eluates for SDS-PAGE and Western blot with anti-flag and anti-Myc antibodies, respectively.

4

Notes 1. Other strain (e.g., EHA105) is suitable for this experiment as well. 2. Although syringes of bigger sizes may be also used, we found 1 mL syringes are very handy and can ease the infiltration. 3. Glycerol stock can also be used but it will be better to recover glycerol stock on fresh medium with antibiotics. 4. 10 mM MgCl2 can be used as well. 5. After overnight culture, the OD600 can reach up to 1.5. Depending on how much you need, you may need to make a larger liquid culture by using the first liquid culture. 6. This can vary a lot with different experimental purposes. The protein abundance is positively correlated with how much agrobacteria is infiltrated. The protein stability should be also considered. For short-lived proteins, their percentages in the mixture can be increased to get more proteins to be expressed. Proteosome inhibitor MG132 can be injected into leaves 12 h before collecting samples as well if the proteins expressed are

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degraded by 26S Proteosome. P19 is always included to suppress plants’ silencing mechanism. At least 6 different cultures can be mixed, and the proteins are expressed to a level detectable by Western blot. 7. Four–six-week-old plants are suitable for infiltration. Different leaves growing in the same plant often express quite distinct levels of proteins, with some relatively old leaves producing much less proteins. Always select young leaves if they are not too small or veiny to be easily infiltrated. 8. This needs some experience and practice. The more you do it, the better and quicker you will be. Because leaves are prone to damage, you may often make holes on leaves and leek the culture without infiltrating leaves. Try to infiltrate a big area on leaves in one go, which will reduce the damage. 9. Usually most proteins will be highly expressed 2–3 days after infiltration, although the expression peaks for some proteins may be earlier or later. If this really matters for your experiments, you have to determine the best time windows for your protein expression experimentally. 10. There is no need to peel the epidermal layer for imaging. Tobacco leaves are very nice to look at under microscope. 11. GFP/RFP and CFP/YFP are frequently used pairs for colocalization study with no much spectral overlap between them. Nevertheless, attention should be always paid to spectral overlap between fluorescence proteins in case of any misleading results. 12. Depending on the volume of the samples and the expression level of the protein of interest, 1–2 mL of lysis buffer is usually used for each sample. Small tags such as flag, Myc, and HA are often used for transient protein expression in tobacco leaves. Commercially available magnetic beads for these tags are highly recommended as the corresponding antibodies are generally of high specificity and affinity compared to homemade antibodies and magnetic beads give much lower nonspecific binding than agarose beads. 13. 20 μL of beads are usually used for each sample. Beads can be washed in a batch and aliquoted to individual tubes after wash. 14. This step can vary with different proteins under study and has to be tested experimentally. The volume of the buffer used, salt concentration, detergent concentration, and times for washing can be adjusted. 15. There are a number of ways for eluting proteins from beads. The SDS loading buffer plus boiling can elute most of the proteins if not all including both specific and unspecific proteins. Acid elution with glycine buffer elutes proteins involving

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protein-protein interactions, which is quite efficient although not as SDS loading buffer plus boiling, while improving the specificity of elution [16, 17]. The most specific method is antigen competition for elution [7]. For example, flag peptide can be used to competitively elute flag-tagged proteins from the flag beads. This method can vastly improve the specificity while avoiding elution of the heavy and light chains of antibodies from the beads. References 1. Clough SJ, Bent AF (1998) Floral dip: a simplified method for agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6):735–743 2. Ishida Y, Hiei Y, Komari T (2007) Agrobacterium-mediated transformation of maize. Nat Protoc 2(7):1614–1621. https:// doi.org/10.1038/nprot.2007.241 3. Ishida Y, Hiei Y, Komari T (2013) High efficiency wheat transformation mediated by agrobacterium tumefaciens. In Vitro Cell Dev-An 49:S24–S25 4. Hayta S, Smedley MA, Demir SU, Blundell R, Hinchliffe A, Atkinson N, Harwood WA (2019) An efficient and reproducible Agrobacterium-mediated transformation method for hexaploid wheat (Triticum aestivum L.). Plant Methods 15(1). https://doi. org/10.1186/s13007-019-0503-z 5. Hiei Y, Komari T (2008) Agrobacteriummediated transformation of rice using immature embryos or calli induced from mature seed. Nat Protoc 3(5):824–834. https://doi. org/10.1038/nprot.2008.46 6. Sahoo KK, Tripathi AK, Pareek A, Sopory SK, Singla-Pareek SL (2011) An improved protocol for efficient transformation and regeneration of diverse indica rice cultivars. Plant Methods 7. https://doi.org/10.1186/17464811-7-49 7. Scarpeci TE, Frea VS, Zanor MI, Valle EM (2017) Overexpression of AtERF019 delays plant growth and senescence, and improves drought tolerance in Arabidopsis. J Exp Bot 68(3):673–685. https://doi.org/10.1093/ jxb/erw429 8. Oh E, Zhu JY, Bai MY, Arenhart RA, Sun Y, Wang ZY (2014) Cell elongation is regulated through a central circuit of interacting transcription factors in the Arabidopsis hypocotyl. Elife 3. https://doi.org/10.7554/eLife. 03031 9. Castel B, Tomlinson L, Locci F, Yang Y, Jones JDG (2019) Optimization of T-DNA

architecture for Cas9-mediated mutagenesis in Arabidopsis. PLoS One 14(1). https://doi. org/10.1371/journal.pone.0204778 10. Xu F, He SB, Zhang JY, Mao ZL, Wang WX, Li T, Hua J, Dui SS, Xu PB, Li L, Lian HL, Yang HQ (2018) Photoactivated CRY1 and phyB interact directly with AUX/IAA proteins to inhibit auxin signaling in Arabidopsis. Mol Plant 11(4):523–541. https://doi.org/10. 1016/j.molp.2017.12.003 11. Mao ZL, He SB, Xu F, Wei XX, Jiang L, Liu Y, Wang WX, Li T, Xu PB, Du SS, Li L, Lian HL, Guo TT, Yang HQ (2019) Photoexcited CRY1 and phyB interact directly with ARF6 and ARF8 to regulate their DNA-binding activity and auxin-induced hypocotyl elongation in Arabidopsis. New Phytol 225:848. https:// doi.org/10.1111/nph.16194 12. Huh SU, Cevik V, Ding PT, Duxbury Z, Ma Y, Tomlinson L, Sarris PF, Jones JDG (2017) Protein-protein interactions in the RPS4/ RRS1 immune receptor complex. PLoS Pathog 13(5). https://doi.org/10.1371/journal. ppat.1006376 13. Aung K, Xin X, Mecey C, He SY (2017) Subcellular localization of Pseudomonas syringae pv. tomato effector proteins in plants methods. Mol Biol 1531:141–153. https://doi.org/10. 1007/978-1-4939-6649-3_12 14. Zhang JY, He SB, Li L, Yang HQ (2014) Auxin inhibits stomatal development through MONOPTEROS repression of a mobile peptide gene STOMAGEN in mesophyll. P Natl Acad Sci USA 111(29):E3015–E3023. https://doi.org/10.1073/pnas.1400542111 15. Liu LJ, Zhang YY, Tang SY, Zhao QZ, Zhang ZH, Zhang HW, Dong L, Guo HS, Xie Q (2010) An efficient system to detect protein ubiquitination by agroinfiltration in Nicotiana benthamiana. Plant J 61(5):893–903. https:// doi.org/10.1111/j.1365-313X.2009. 04109.x 16. Zhu DM, Maier A, Lee JH, Laubinger S, Saijo Y, Wang H, Qu LJ, Hoecker U, Deng

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17. Lian HL, He SB, Zhang YC, Zhu DM, Zhang JY, Jia KP, Sun SX, Li L, Yang HQ (2011) Blue-light-dependent interaction of cryptochrome 1 with SPA1 defines a dynamic signaling mechanism. Genes Dev 25 (10):1023–1028. https://doi.org/10.1101/ gad.2025111

INDEX A

H

Agrobacteria ......................................................... 168–171 Anthocyanin contents ...............................................41, 42 Antibodies ........................................................... 9, 12, 14, 15, 17, 18, 36, 37, 39, 58, 62, 64, 65, 68, 69, 75, 76, 79–81, 84, 86, 90, 92, 141, 142, 144, 145, 162, 165, 169, 171–173 Arabidopsis thaliana ............................................... 3, 4, 8, 13, 22, 33, 49–59, 76, 83, 96, 106, 121, 155, 167 Auxotrophy.................................................. 148, 151, 153

Hypocotyl lengths.............................................27, 28, 30, 42, 43, 53, 59 Hypocotyls...................................................21–30, 33–35, 41, 43, 46, 49, 53, 59, 62, 155

B

Jasmonate ........................................................... 49, 51, 57

Baculovirus expression system............................. 155–160 Blue light ...........................................................24, 27, 28, 42, 44, 105–107, 111, 115, 142, 144, 147, 148, 150, 151, 153, 155, 160, 161, 164, 165

L

C Cell death ............................................................... 95, 102 ChIP-qPCR ...............................................................66, 69 Chlamydomonas reinhardtii................................. 125–136 Chromatin immunoprecipitation followed by sequencing (ChIP-seq) ............................61–73 Circadian clock ..................................................61, 62, 66, 116, 126, 155 Co-immunoprecipitation (CoIP)35, 37–39, 75–81, 142, 143, 167 Co-immunoprecipitation (CoIP) assay........................ 141 Colony-lift filter β-galactosidase assay ....... 148, 151, 153 Cotyledon sizes ............................................................... 42 Cryptochromes..................................................22, 41, 83, 105–112, 115, 142, 147, 155–160, 165

D Dark metabolism .................................125, 126, 128, 134

E Ethylene..................................................... 49, 56, 57, 116

F Far red light................................................. 7, 43, 44, 142

I Immunofluorescence .................................................. 7–18

J

LexA-based Yeast Two-Hybrid (Y2H) system ............. 35, 147–154 Light competition .....................................................4, 145 Light emitting diodes (LEDs)................................2–5, 29 Light signaling ................................................................ 34 Low blue.......................................................................... 28 Low R/FR ratio ....................................21, 22, 24, 25, 28

M Mass spectrometry ................................ 35, 115–123, 133 Mesophyll protoplasts .......................................... 106–112 Mitochondria.............................. 127–129, 133, 134, 136

N Nuclear localization ...................................................... 148

P Pchlide ................................................................ 95, 97, 98 Photobodies ...............................................7–18, 105–113 Photomorphogenesis ........................................33, 34, 41, 46, 76, 95–103, 105 Photoreceptors ..................................................1, 7, 9, 21, 22, 33, 41, 42, 76, 83, 105, 142, 155, 168 Photosynthesis..................................................... 2, 21, 33, 41, 125, 126, 128 Phytochromes (phy) .........................................1, 7, 9, 21, 22, 41, 75–83, 105, 115, 116, 142, 147, 161

Ruohe Yin et al. (eds.), Plant Photomorphogenesis: Methods and Protocols, Methods in Molecular Biology, vol. 2297, https://doi.org/10.1007/978-1-0716-1370-2, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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176 Index

AND

PROTOCOLS

Polyethylene glycol (PEG)/LiAc-based method............................................................... 148 Protein colocalization .......................................... 167–173 Protein-protein interaction................................. 115, 154, 155, 165

Skotomorphogenesis......................................... 41, 49–59, 95, 126 Soybean................................................................ 105–108, 110–112 Super-resolution imaging ........................................... 7–18

Q

T

Quantitative proteomics ...................................... 115–123

Tomato ......................................................................83–92 Transient expression............................................. 106, 168 True shade .......................................................... 22, 24–28

R Reactive oxygen species (ROS) .............................. 95, 97, 99–100, 102, 103 Respiration....................................................126–129, 134

S Seedling de-etiolation ...............................................25, 41 Semi-in-vivo pull down assay .............................. 161–165 Sf9 insect cell ........................................................ 155–160 Shade avoidance .........................................................1, 21, 41, 43 Shade-avoidance responses (SAR).................................... 5 Shade‐avoidance syndrome (SAS) ...............................1, 2, 4, 5, 21, 22

U Ultraviolet-B (UV-B)........................................ 33–37, 39, 41, 83–89, 91, 92, 115, 142 UV resistance locus 8 (UVR8)................................33–39, 41, 83–92, 105, 115, 142 UVR8 homodimer ....................................................85, 88 UVR8 monomer ............................................................. 88

Y Yeast ......................................................................... 34, 35, 76, 77, 141, 147–154, 168