Transgenic Crop Plants: Volume 2: Utilization and Biosafety 3642048110, 9783642048111

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Transgenic Crop Plants

Chittaranjan Kole Charles H. Michler Albert G. Abbott Timothy C. Hall l

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Editors

Transgenic Crop Plants Volume 2: Utilization and Biosafety

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Editors Prof. Chittaranjan Kole Department of Genetics & Biochemistry Clemson University Clemson, SC 29634, USA [email protected]

Prof. Albert G. Abbott Department of Genetics & Biochemistry Clemson University Clemson, SC 29634, USA [email protected]

Prof. Charles H. Michler Director Hardwood Tree Improvement and Regeneration Center at Purdue University NSF I/UCRC Center for Tree Genetics West Lafayette, IN, USA [email protected]

Prof. Timothy C. Hall Institute of Developmental & Molecular Biology Department of Biology Texas A&M University College Station, TX, USA [email protected]

ISBN: 978-3-642-04811-1 e-ISBN: 978-3-642-04812-8 DOI 10.1007/978-3-642-04812-8 Springer Heidelberg Dordrecht London New York Library of Congress Control Number: 2009939124 # Springer-Verlag Berlin Heidelberg 2010 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, 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. Cover design: WMXDesign GmbH, Heidelberg, Germany Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

Transgenic Plants – known also as Biotech Plants, Genetically Engineered Plants, or Genetically Modified Plants – have emerged amazingly fast as a boon for science and society. They have already played and will continue to play a significant role in agriculture, medicine, ecology, and environment. The increasing demands for food, feed, fuel, fiber, furniture, perfumes, minerals, vitamins, antibiotics, narcotics, and many health-related drugs and chemicals necessitate the development and cultivation of transgenic plants with augmented or suppressed trait(s). From a single transgenic plant (Flavr Savr tomato with a longer shelf-life) introduced for commercialization in 1994, we have now 13 transgenic crops covering 800 million ha in 25 countries of six continents. Interestingly, the 13.3 million farmers growing transgenic crops globally include 12.3 million (90%) small and resource poor farmers from 12 developing countries. Increasing popularity of transgenic plants is well evidenced from an annual increase of about 10% measured in hectares but actually of 15% in “trait hectares.” Considering the urgent requirement of transgenic plants and wide acceptance by the farmers, research works of transgenic plants are now being conducted on 57 crops in 63 countries. Transgenic plants have been developed in over 100 plant species and they are going to cover the fields, orchards, plantations, forests, and even the seas in the near future. These plants have been tailored with incorporation of useful alien genes for several desirable traits including many with “stacked traits” and also with silencing of genes controlling some undesirable traits. Development, applications and socio-political implications of transgenic plants are immensely important fields now in education, research, and industries. Plant transgenics has deservedly been included in the course curricula in most, if not all, leading universities and academic institutes all over the world, and therefore reference books on transgenic plants with a class-room approach are essential for teaching, research, and extension. There are some elegant reviews on the transgenic plants or plant groups (including a 10-volume series “Compendium of Transgenic Crop Plants” edited by two of the present team of editors C. Kole and T.C. Hall published by Wiley-Blackwell in 2008) and on many individual tools and

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techniques of genetic transformation in plants. All these reviews could surely serve well the purpose for individual crop plants or particular methodologies. Since transgenic plant development and utilization is studied, taught, and practiced by students, teachers, and scientists of over a dozen disciplines under basic science, agriculture, medicine, and humanities at public and private sectors, introductory reference books with lucid deliberations on the concepts, tools, and strategies to develop and utilize transgenic plants and their global impacts could be highly useful for a broad section of readers. Deployment of transgenic crop plants are discussed, debated, regulated, and sponsored by people of diverse layers of the society, including social activists, policy makers, and staff of regulatory and funding agencies. They also require lucid deliberations on the deployment, regulations, and legal implications of practicing plant transgenics. More importantly, depiction of the positive and realistic picture of the transgenic plants should and could facilitate mitigation of the negative propaganda against transgenic plants and thereby reinforce moral and financial support from all individuals and platforms of the society. Global population is increasing annually by 70 millions and is estimated to grow to eight billion by 2025. This huge populace, particularly its large section from the developing countries, will suffer due to hunger, malnutrition, and chemical pollution unless we produce more and more transgenic plants, particularly with stacked traits. Compulsion to meet the requirements of this growing population on earth and the proven innocuous nature of transgenic plants tested and testified for the last 13 years could substantiate the imperative necessity of embracing transgenics. Traditional and molecular breeding practiced over the last century has provided enormous number of improved varieties in economic crops and trees including wheat and rice varieties that fostered the “green revolution.” However, these crop improvement tools depend solely on the desirable genes available naturally, creatable by mutation in a particular economic species, or their shuffling for desired recombinations. Transgenic breeding has opened a novel avenue to incorporate useful alien genes from not only other cross-incompatible species and genera of the plant kingdom, but also from members of the prokaryotes including bacteria, fungi, and viruses, and even from higher animals including mice and humans. An array of plant genetic engineering achievements starting from the development of insect resistance cotton by transforming the cry genes from the bacteria Bacillus thuringiensis to the present-day molecular pharming that enables the expression of interferon- gene from human in tobacco evidence for this pan-specific gene transfer. Human and animal safety is another general concern related to transgenic food or feed. However, there is no reliable scientific documentation of these health hazards even after 13 years of cultivation of transgenic plants and consumption of about 1 trillion meals containing transgenic ingredients. Utilization of transgenic plants has reduced the pesticide applications by 359,000 tons that would otherwise affect human and animal health besides causing air, water, and soil pollution and also mitigated the chance of consumption of dead microbes and insects along with foods or feeds.

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Gene flow from transgenic crop species to their cross-compatible wild relatives is a genuine concern and therefore required testing of a transgenic crop plant before deployment followed by comprehensive survey of the area for presence of interfertile wild and weedy plants before introduction of a transgenic crop are being seriously conducted. Addition of novel genotypes with transsgenes in the germplasms is increasing the biological diversity rather than depleting it. Using the genetically engineered plants has also eliminated greenhouse gas emission of 10 million metric tons through fuel savings. In fact, 1.8 billion liters of diesel have been saved because of reduced tillage and plowing owing only to herbicide-resistant transgenic crops. Many transgenics are now being used for soil reclamation. Above all, cultivation of transgenic crops has returned $44 billion of net income to the farmers. Perhaps, these are the reasons that 25 Nobel Laureates and 3,000-plus eminent scientists appreciated the merits and safety and also endorsed transgenic crops as a powerful and safe way to improve agriculture and environment besides the safety of genetically modified foods. Many international and national organizations have also endorsed health and environmental safety of transgenic plants; these include Royal Society (UK), National Academy of Sciences (USA), World Health Organization, Food and Agriculture Organization (UN), European Commission, French Academy of Medicine and American medical Association, to name a few. Production, contributions, and socio-political implications of biotech plants are naturally important disciplines now in education, research, and industries and therefore introductory reference books are required for students, scientists, industries, and also for social activists and policy makers. The two book volumes on “Transgenic Crop Plants” will hopefully fill this gap. These two book volumes have several unique features that deserve mention. The outlines of the chapters for these two books are formulated to address the requirements of a broad section of readers. Students and scholars of all levels will obtain a lot of valuable reading material required for their courses and researches. Scientists will get information on concepts, strategies, and clues useful for their researches. Seed companies and industries will get information on potential resources of plant materials and expertise for their own R&D activities. In brief, the contents of this series have been designed to fulfill the demands of students, teachers, scientists, and industry people, for small to large libraries. Students, faculties, or scientists involved in various subjects will be benefited from this series; biotechnology, bioinformatics, molecular biology, molecular genetics, plant breeding, biochemistry, ecology, environmental science, bioengineering, chemical engineering, genetic engineering, biomedical engineering, pharmaceutical science, agronomy, horticulture, forestry, entomology, pathology, nematology, virology, just to name a few. It had been our proud privilege to edit the 23 chapters of these two books those were contributed by 71 scientists from 14 countries and the list of authors include one of the pioneers of plant transgenics, Prof. Timothy C. Hall (one of the editors also); some senior scientists who have themselves edited books on plant transgenics; and many scientists who have written elegant reviews on invitation for quality books and leading journals. We believe these two books will hopefully

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serve the purposes of the broad audience who are studying, teaching, practicing, supporting, funding, and also those who are debating for or against plant transgenics. The first volume dedicated to “Principles and Development” elucidates the basic concepts, tools, strategies, and methodologies of genetic engineering, while the second volume on “Applications and Safety” enumerates the utilization of transgenic crop plants for various purposes of agriculture, industry, ecology, and environment, and also genomics research. This volume also deliberates comprehensively on the legal and regulatory aspects; complies to the major concerns; and finally justifies the compulsion of practicing plant transgenics. Glimpses on the contents of this volume (Volume 2: Transgenic Crop Plants: Applications and Safety) will perhaps substantiate its usefulness. This volume enumerates the application of transgenic technologies in crop plants for particular objectives in the first ten chapters. Biotic stress resistant, specifically insect resistant, transgenics have been developed and commercialized in several crops. An example with Bt-expressing cotton and maize alone, with current market share of about $3.26 billion substantiates their success and popularity (Chap.1). Abiotic stresses, particularly drought, salinity, and temperature extremes, have always been difficult to manipulate. Still success stories are pouring in recently from works mainly in cereals and vegetables (Chap.2). Herbicide-resistant transgenic plants (in cotton and canola) were first deregulated in 1995 and in 2008 more than 80% of the transgenic plants grown globally possess a transgenic trait for herbicide resistance. Chapter 3 details the present and emerging herbicide-resistant transgenic plants. Although the first transgenic trait was developmental, shelf-life in tomato to be precise, transgenics research for these traits are yet to make significant commercial headway but started producing encouraging results (Chaps.4 and 5). Deployment of transgenic plants for biofuel, pharmaceuticals, and other bioproducts has been enunciated in three chapters (Chaps.6, 7, and 9). Transgenic plants have been labeled as a culprit for potential threats to ecology and environment by a few groups of social activists. Chapter 8 addresses these weird concerns with suitable examples of utilization of transgenic plants for phytoremediation, biomonitoring, and the production of bioplastics and biopolymers for amelioration of ecology and environment. Plant genomics has emerged fast within the last three decades and facilitated fine-scale view of the plant genes and genomes. Transgenic plants have provided enormous resources for functional genomics studies and expected to play their roles as more plants systems and genes are targeted (Chap. 10). Scientists practicing transgenics are no less aware of the potential risks of genetic engineering than the few people with antagonistic views. Neither are the regulatory agencies at institutional, state, national, and international level regulatory agencies unaware of the steps to be involved for inspection, monitoring, and approval of transgenic plants for commercial use. Chapter 11 delineates all these aspects with examples from US and other continents and countries. Any original innovation or effort deserves recognition and also an incentive. The scope of patenting and intellectual property rights for materials owned and generated and methodologies implemented have been appreciated and enforced legally. These aspects related to transgenic crop plants have been discussed in Chap.12.

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The concluding chapter (Chap.13) briefs the contributions and concerns with the compliances and compulsion of practicing plant transgenics for science and society. We thank all the 41 scientists from nine countries for their elegant and lucid contributions to this volume and also for their sustained support through revision, updating and fine-tuning their chapters. We also acknowledge for the recent statistics that have been accessed from the web sites of Monsanto Company on “Conversations about Plant Biotechnology” and “International Service for the Acquisition of Agri-Biotech Applications on ISAAA Brief 39-2008: Executive Summary” and used them in this preface and elsewhere in the volume. We enjoyed a lot of our Clemson–Purdue–Texas A&M triangular interaction, constant consultations, and dialogs while editing this book, and also our working with the editorial staff of Springer, particularly Dr. Sabine Schwarz who had been supportive since inception till publication of this book. We will look forward to suggestions from all corners for future improvement of the content and approach of this book volume. Chittaranjan Kole, Clemson, SC Charles H. Michler, West Lafayette, IN Albert G. Abbott, Clemson, SC Timothy C. Hall, College Station, TX

Contents

1

Transgenic Crop Plants for Resistance to Biotic Stress . . . . . . . . . . . . . . . 1 N. Ferry and A.M.R. Gatehouse

2

Transgenic Plants for Abiotic Stress Resistance . . . . . . . . . . . . . . . . . . . . . . 67 Margaret C. Jewell, Bradley C. Campbell, and Ian D. Godwin

3

Transgenic Crops for Herbicide Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Stephen O. Duke and Antonio L. Cerdeira

4

Understanding and Manipulation of the Flowering Network and the Perfection of Seed Quality . . . . . . . . . . . . . . . . . . . . . . . . . 167 Stephen L. Goldman, Sairam Rudrabhatla, Michael G. Muszynski, Paul Scott, Diaa Al-Abed, and Shobha D. Potlakayala

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Biotechnological Interventions to Improve Plant Developmental Traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Avtar K. Handa, Alka Srivastava, Zhiping Deng, Joel Gaffe, Ajay Arora, Martı´n-Ernesto Tiznado-Herna´ndez, Ravinder K. Goyal, Anish Malladi, Pradeep S. Negi, and Autar K. Mattoo

6

Transgenics for Biofuel Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Anjanabha Bhattacharya, Pawan Kumar, and Rippy Singh

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Plant Produced Biopharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 Jared Q. Gerlach, Michelle Kilcoyne, Peter McKeown, Charles Spillane, and Lokesh Joshi

8

Biotech Crops for Ecology and Environment . . . . . . . . . . . . . . . . . . . . . . . . 301 Saikat Kumar Basu, Franc¸ois Eudes, and Igor Kovalchuk

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Contents

9

Algal Biotechnology: An Emerging Resource with Diverse Application and Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Cunningham Stephen and Joshi Lokesh

10

Transgenic Crops and Functional Genomics . . . . . . . . . . . . . . . . . . . . . . . . . 359 Narayana M. Upadhyaya, Andy Pereira, and John M. Watson

11

Deployment: Regulations and Steps for Commercialization . . . . . . . . 391 Kelly D. Chenault Chamberlin

12

Patent and Intellectual Property Rights Issues . . . . . . . . . . . . . . . . . . . . . . . 411 Jim M. Dunwell

13

Transgenic Crop Plants: Contributions, Concerns, and Compulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435 Brian R. Shmaefsky

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479

Contributors

Diaa Al-Abed Edenspace Systems Corporation, Manhattan, KS 66502, USA, [email protected] Ajay Arora Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi 110012, India Saikat Kumar Basu Department of Biological Sciences, University of Lethbridge, 4401 University Drive, Lethbridge, AB, Canada T1K 3M4; Bioproducts and Bioprocesses, Agriculture and Agri-Food Canada, 5403 1st Avenue South, Lethbridge, AB, Canada T1J 4B1 Anjanabha Bhattacharya National Environmental Sound Production Agriculture Laboratory, University of Georgia, Tifton, GA 31794, USA, [email protected] Bradley C. Campbell School of Land, Crop and Food Sciences, The University of Queensland, Brisbane, QLD 4072, Australia Antonio L. Cerdeira Brazilian Department of Agriculture, Agricultural Research Service, EMBRAPA/Environment, C.P. 69, Jaguariuna-SP-13820000, Brazil Kelly D. Chenault Chamberlin USDA-ARS, Wheat, Peanut, and Other Field Crops Unit, 1301 N. Western, Stillwater, OK 74075, USA, kelly.Chamberlint@ars. usda.gov Zhiping Deng Department of Plant Biology, Carnegie Institution of Washington, Stanford, CA 94305, USA Stephen O. Duke Agricultural Research Service, United States Department of Agriculture, P. O. Box 8048, University, MS 38677, USA, [email protected]

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Contributors

Jim M Dunwell University of Reading, Whiteknights, Reading RG6 6AS, UK, [email protected] Franc¸ois Eudes Bioproducts and Bioprocesses, Agriculture and Agri-Food Canada, 5403 1st Avenue South, Lethbridge, AB, Canada T1J 4B1 N. Ferry School of Biology, Institute for Research on Environment and Sustainability, Devonshire Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK, [email protected] Joel Gaffe Laboratoire Adaptation et Pathoge´nie des Microorganismes, LAPM, UMR 5163 CNRS-UJF, Institut Jean Roget BP 170, 38042 Grenoble cedex 9, France A.M.R. Gatehouse School of Biology, Institute for Research on Environment and Sustainability, Devonshire Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK Jared Q. Gerlach Glycoscience and Glycotechnology Group and the Martin Ryan Institute, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland Ian D. Godwin School of Land, Crop and Food Sciences, The University of Queensland, Brisbane, QLD 4072, Australia, [email protected] Stephen L. Goldman Department of Environmental Sciences, The University of Toledo, Toledo, OH 43606, USA, [email protected] Ravinder K. Goyal Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada V8W 3P6 Avtar K. Handa Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907-2010, USA, [email protected] Margaret C. Jewell School of Land, Crop and Food Sciences, The University of Queensland, Brisbane, QLD 4072, Australia Lokesh Joshi Glycoscience and Glycotechnology Group and the Martin Ryan Institute, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland, [email protected] Michelle Kilcoyne Glycoscience and Glycotechnology Group and the Martin Ryan Institute, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland

Contributors

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Igor Kovalchuk Department of Biological Sciences, University of Lethbridge, 4401 University Drive, Lethbridge, AB, Canada T1K 3M4, [email protected] Pawan Kumar National Environmental Sound Production Agriculture Laboratory, University of Georgia, Tifton, GA 31794, USA Joshi Lokesh Glycoscience and Glycotechnology Group and the Martin Ryan Institute, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland, [email protected] Anish Malladi 30602, USA

Department of Horticulture, University of Georgia, Athens, GA

Autar K. Mattoo Sustainable Agricultural Systems Laboratory, The Henry A. Wallace Beltsville Agric Research Center, Beltsville, MD 20705-2350, USA Peter McKeown Genetics and Biotechnology Laboratory, Department of Biochemistry, Biosciences Institute, University College Cork, Cork, Ireland Michael G. Muszynski Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA, [email protected] Pradeep S. Negi Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907-2010, USA Andy Pereira 24061, USA

Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA

Shobha D. Potlakayala Penn State Milton S. Hershey College of Medicine, Hershey, PA 17033, USA, [email protected] Sairam Rudrabhatla Environmental Engineering, College of Science, Engineering and Technology, Penn State University, Middletown, PA 17057, USA, [email protected] Paul Scott Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA, [email protected]; Department of Agronomy, USDA-ARS, Iowa State University, Ames, IA 50011, USA, paul.scott@ ars.usda.gov Brian R. Shmaefsky Lone Star College – Kingwood, HSB 202V, 20,000 Kingwood Drive, Kingwood, TX 77339-3801, USA, Brian.R.Shmaefsky@ lonestar.edu

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Contributors

Rippy Singh National Environmental Sound Production Agriculture Laboratory, University of Georgia, Tifton, GA 31794, USA Charles Spillane Genetics and Biotechnology Laboratory, Department of Biochemistry, Biosciences Institute, University College Cork, Cork, Ireland Alka Srivastava Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907-2010, USA Cunningham Stephen Glycoscience and Glycotechnology Group and the Martin Ryan Institute, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland Martı´n-Ernesto Tiznado-Herna´ndez Fisiologı´a y Biologı´a Molecular de Plantas, Coordinacio´n de Tecnologı´a de Alimentos de Origen Vegetal, Centro de Investigacio´n en Alimentacio´n y Desarrollo, A.C., Hermosillo, Sonora, Me´xico Narayana M. Upadhyaya CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 260, Australia, [email protected] John M. Watson Australia

CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 260,

Abbreviations

1-FFT 1-MCP 1-SST 2,4-D 2D-PAGE 4C3H 4CL 6G-FFT 6-SFT AAFC ABA ABRE Ac ACC AChE ACP ADP ae1 AHK2/3 AL-PCD ALS AMGT AMPA ANVISA AOS AOX ap AP1 AP2

Fructan:fructan 1-fructosyltransferase 1-Methylcyclopropene Sucrose:sucrose 1-fructosyltransferase 2,4-Dichlorophenoxyacetic acid Two-dimensional polyacrylamide gel electrophoresis 4-Coumarate 3-hydroxylase 4-Hydroxycinnamoyl CoA ligase Fructan:fructan 6G-fructosyltransferase Sucrose:fructan 6-fructosyltransferase Agriculture and Agri-Food Canada Abscisic acid ABA responsive element Activator gene 1-Aminocyclopropane-1-carboxylate Acetylcholinesterase Acyl-carrier protein Adenosine di-phosphate amylose extender gene Arabidopsis histidine kinase Apoptotic-like programmed cell death Acetolactate synthase Agrobacterium-mediated gene transfer Aminomethylphosphonic acid National Agency for Health and Surveillance of the Ministry of Health Allene oxide synthase Altenative oxidase apetalla gene APETALA1 gene APETALA2/ Apetela2 gene

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APHIS APX ARF arsC Asc at AtCKX1 Avr AZ BA BADH BAP BAR BBC BC BGI-RIS bla BMR BRS Bt bZIP CAD CAL CAMBIA CaMV CAT CBER CBF cbLCV CBS CCR CDC CDF cDNA CEL CEPA CFIA CFSAN CGH-1 CGIAR cGMP CHO CHO

Abbreviations

Animal and Plant Health Inspection Service Ascorbate peroxidase Auxin response factor Arsenate reductase Ascorbate antherless gene Arabidopsis thaliana cytokinin oxidase gene Avirulence Abscission zone Benzyladenine Betaine aldehyde dehydrogenase 6-Benzylaminopurine Bialaphos resistance gene British Broadcasting Corporation Biotech crop Beijing Genomics Institute- Rice Information System Beta-lactamase Brown midrib Biotechnology Regulatory Service Bacillus thuriengensis Basic leucine zipper Cinnamoyl alcohol dehydrogenase CAULIFLOWER gene Center for the Application of Molecular Biology to International Agriculture Cauliflower mosaic virus Chloramphenicol acetyltransferase/catalase Centre for Biologics Evaluation and Research CRT binding factor Cabbage leaf curl virus Columbia Broadcasting System Cinnamoyl CoA reductase Centers for Disease Control Cycling DOF Factor Complementary-DNA Cellulase Canadian Environmental Protection Act Canadian Food Inspection Agency Centre for Food Safety and Applied Nutrition Cardenolide 16’-O-glucohydrolase Consultative Group on International Agricultural Research Current GMP Chinese hamster ovary Choline dehydrogenase

Abbreviations

chs CIGB CMO CMS CMS CNN CNR CO ConA CONABIA conz1 COR COR CP CpTI CRE CRIIGEN CRT Cry CTNBio CVM CV-N D2GT2A dab DDB DDT DEFRA DET DGDG DHAsc dlf1 DNMA DRE DREB driPTGS Ds DsE DsG DSL dzr1 EA EBV EC EDB

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chalcone synthase gene Cuban Centre for Biotechnology and Genetic Engineering Choline monooxygenase Cytoplasmic male sterility Cellular membrane stability Cable News Network Colorless non-ripening gene Constans gene Concanavalin A National Advisory Commission on Agricultural Biotechnology constans of Zea mays1 gene Cold responsive Cold responsive gene Chloroplast Cowpea trypsin inhibitor Cytokinin response Comite´ de Recherche et d’Information Inde´pendantes sur le Ge´nie C-Repeat Crystal National Technical Commission on Biosafety Centre for Vetinary Medicine Cyanovirin Diacylglycerol acyltransferase 2A delayed abscission gene Damaged DNA binding protein Dichlorodiphenyltrichloroethane Department of Environment, Food and Rural Affairs Detiolated gene Digalactosyldiacylglycerol Dehydroascorbate delayed flowering1 gene Directorate of Agricultural Markets Dehydration responsive element DRE binding protein Direct repeat-induced PTGS Dissociation gene Enhancer trap Ds Gene trap Ds Domestic Substance List delta zein regulator1 gene Environmental assessment Epstein-barr virus European Commission Ethylene dibromide

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EFSA EFSA EIN EIS EMEA EMS En EPA EPA EPSP EPSPS ER ERE ERS EST ETC ETH EU F F1 FAD1 FAD3 FAO FD FDA FFDCA FIFRA fl2 FONSI FPPA Fr FST FT FT-ICR-MS Fuc FucT FUL Fx G3P GA/ GA3 Gal GalNAc GalT GAT

Abbreviations

European Food Safety Authority European Food Standards Agency Ethylene-insensitive gene Environmental Impact Statement European Agency for Evaluation of Medicinal Products Ethylmethane sulfonate Enhancer transposon Eicosapentaenoic Environmental Protection Agency Enolpyruvyl-shikimate-3-phosphate 5-Enolpyruvyl-shikimate-3-phosphate synthase Endoplasmic reticulum Ethylene responsive element Economic Research Service Expressed sequence tag Electron transport chain Ethylene European Union Florigenic signal Fraction 1 anti-phagocytic capsular envelope protein Flavin adenine dinucleotide Omega-3 fatty acid desaturase Food and Agriculture Organization of the United Nations FLOWERING LOCUS D gene Food and Drug Administration Federal Food, Drug and Cosmetic Act Federal Insecticide, Fungicide and Rodenticide Act floury-2 gene Finding of no significant impact Federal Plant Pest Act Fertility restorer gene Flanking sequence tag Flowering Locus T/Flowering Transition gene Fourier-transform ion cyclotron mass spectrometry Fucose Fucosyltransferase FRUITFUL gene Fucoxantine Glycerol-3-phosphate Gibberellic acid Galactose N-Acetylgalactosamine Galactosyltransferase Glyphosate N-acetyltransferase

Abbreviations

GC GCase GCS GDP GE GFLV GFP GI gigz1 GlcNAc GlyBet GM GMHT GMO GMP GMP GMPO GMS GNA GOI GOX GPAT GPX GR GRC GS1 GSH GSSG GST GTN GUS HBcAg HBsAg HBV HCMV hEPO hGM-CSF HIV HMX HR HRC HS HSP HSV

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Glutathione synthetase Glucosyl-N-acylspingosineglycohydrolase g-Glutamylcysteine synthase Gross domestic product Genetic engineering/Genetically engineered Grapevine fanleaf virus Green fluorescent protein Gigantea gene gigantea of Zea mays1 gene N-Acetylglucosamine Glycine betaine Genetically modified Genetically modified herbicide tolerant Genetically modified organism Genetically modified plant Good manufacturing practise Genetically modified plant organism Genic male sterility Galanthus nivalis agglutinin Gene of interest Glyphosate oxidoreductase Glycerol-3-phosphate acyltransferase Glutathione peroxidase Glutathione reductase/ Glyphosate resistant Glyphosate resistant Crop Glutamine synthase gene 1 Glutathione/ Glutamate synthase Glutathione disulfide Glutathione S-transferase Glycerol trinitrate ß-Glucuronidase Hepatitis B core antigen Hepatitis B surface antigen Hepatitis B virus Human cytomegalovirus Human erythropoietin Human granulocyte-macrophage colony-stimulating factor Human immunodeficiency virus Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine Hypersensitive response/ Herbicide resistant Herbicide resistant crop Heat shock Heat shock protein Herpes simplex virus

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HT/Ht I i.p. iAc IBA ICTSD id1 ida IDD IFN IgA IL-12 IMI IPM IPP IPR IPT IR IRGSP ISAAA ISIS ISR JA LB LD LEA Leu LFY LOG LOX1/2/3 lpa1 LPS LRR LTB Lys MAb MAPK MGDG MHBsAg Mi MIP miRNA MPSS mRNA

Abbreviations

Herbicide tolerant/tolerance Inhibitor transposon Intraperitoneal Immobile Ac transposon Indole-3-butyric acid International Center for Trade and Sustainable Development indeterminate1 gene inflorescence deficient in abscission gene ID-domain Interferon Immunoglobulin A Interleukin-12 Imidazolinone Integrated pest management Isopentyl diphosphate Intellectual Property Rights Isopentenyl transferase/ Isopentyl transferase Insect resistant/resistance International Rice Genome Sequencing Project International Service of AgriBiotech Applications Institute for Science and International Security Induced systemic resistance Jasmonic acid Left border of T-DNA Long-day Late embryogenesis abundant Leucine Leafy gene Lonely guy gene Lipoxygenase gene Lysophosphatidic acid receptor Lipopolysaccharide Leucine rich repeats Heat-labile enterotoxin, subunit B Lysine Monoclonal antibody Mitogen-activated protein kinase Monogalactosyldiacylglycerol Middle HBsAg Meloidogyne incognita resistance gene Major intrinsic protein Micro-RNA Massively parallel signature sequencing Messenger-RNA

Abbreviations

MS MS MST MT MTP MTT Mu MuIL-12 MV MVL NAA NAM NAS NAS NBS NCBI NDV NEPA Neu5Ac NIH NIL NMR NO NOI NoV NR NST NUE o2 OECD OFB OMT ORF Ori OSTP PAHs PAL PAMPs PAT PC PCB PCD PCR PCS

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Mass spectrometry Murashige and Skoog (medium) Members of the Landless Rural Workers Movement Metallothionein Metal-tolerance protein Multi-tasking transgenics Mutator transposon Murine IL-12 Methyl viologen Microcystis viridis lectin a-Napthalene acetic acid Napthaleneacetamide National Academy of Sciences Nicotinamine synthase Nucleotide binding site National Center for Biotechnology Information Newcastle disease virus National Environmental Policy Act 5-N-Acetyl-D-neuraminic acid National Institute of Health Near-isogenic line Nuclear magnetic resonance Nitric oxide Notice of Intent Norovirus Nitrate reductase Nac secondary wall thickening promoter factor Nitrogen use efficiency opaque-2 gene Organization for Economic Cooperation and Development Office of Food Biotechnology O-Methyl transferase Open reading frame Origin of replication Office of Science and Technology Policy Polycyclic aromatic hydrocarbon Phenyalanine ammonia lyase Pathogen associated molecular patterns Phosphinothricin-acetyltransferance Phytochelatin Polychlorinated biphenyl Programmed cell death Polymerase chain reaction Phytochlelatin synthase

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PEG PETN PG PG PHB pi PiP PIP PL PLC PLD PLE PME PMP PNT PPO PR Pro PS PSI PSII PTGS Put PVP PVX PyMSP4/5 QPM QTL RAP-DB rasiRNA RB RB rDNA RDX Rf RFLP R-gene rGSII rhCVFVIII rhEPO rhIF RHS RID1 RIL

Abbreviations

Polyethylene glycol Pentaerythritol tetranitrate Polygalacturonase Phosphatidylglycerol Polyhydroxybutyrate pistillata gene Plant Incorporated Protectant Plasma membrane intrinsic protein Pectate lyase Phospholipase C Phospholipase D Phospholipid cleaving enzyme Pectin methylesterase Plant-made pharmaceutical Plant with novel trait Polyphenol oxidase Pathogenesis-related Proline Phytosiderophores Photosystem 1 Photosystem 2 Post-transcriptional gene silencing Putrescine Plant Variety Protection Potato virus X Murine P. yoelii merozoite surface protein 4/5 Quality protein maize Quantitative trait loci Rice Annotation Project-Database Repeat-associated siRNA Non-toxin B-chain from ricin Right border of T-DNA Recombinant-DNA Hexahydro-1,3,5-trinitro-1,3,5 triazine Restorer of fertility gene Restriction fragment length polymorphism Resistance-gene Recombinant Griffonia simplicifolia lectin II Recombinant human clotting factor VIII Recombinant human erythropoietin Recombinant human intrinsic factor Royal Horticultural Society Rice Indeterminate1 gene Recombinant inbred line

Abbreviations

rin Rip RNAi ROIs ROS RT-PCR RWC s.c. SA SA SAGE SAGPyA SAM SAM SAR scFv scN SD SE SENASA Ser SFI1 sh2 siRNA SIV SL SMT soc SOC1 SOD SOliD Spd Spm Spm SQDG ssRNA STP su1 SVN TA TAC TAGI tasiRNA tb1

xxv

ripening inhibitor gene Ribosome inactivating protein RNA-interference Reactive oxygen intermediates Reactive oxygen species Reverse transcriptase-PCR Relative water content Subcutaneously Salicylic acid Splice acceptor Serial analyses of gene expression Secretariat of Agriculture, Livestock, Fisheries and Food S-Adenosylmethionine Shoot apical meristem Systemic acquired resistance Single chain variable fragment Soyacystatin N Short-day Substantial equivalence/ equivalent National Agri-food Health and Quality Service Serine Segestria florentina venom peptide shrunken2 gene Short/Small interfering RNA Simian immunodeficiency virus Selenocysteine lyase Seleno-cysteine methyl transferase suppessor of overexpression of constans gene Suppressor of Overexpression of Constans1 gene Superoxide dismutase Supported Oligo Ligation Detection Spermidine Spermine Suppressor-Mutator transposon Sulfoquinovosyldiacylglycerol Single-stranded RNA Signal transduction pathway sugary1 gene Scytovirin Transcriptional activator Tiller angle control gene The Arabidopsis Genome Initiative Transacting siRNA teosinte branched1 gene

xxvi

TCE TCOH TCP T-DNA TDZ TET TETRYL TF TFL Thr ti TILLING TIP TMV TNT Trp TRV TSCA UAS uf UN UNCTAD US USAID USDA USPTO UV VB Vgt1 Vgt2 VIGS VIP VLP VRO WHO WT WUE XTH Xyl XylT YCF1 YFP ZCN zfl1

Abbreviations

2.4,6-Trichloroethylene Chloral and trichoethanol 2,4,6-Tricholorophenol Transferred-DNA Thidiazuron Transiently expressed transposase N-Methyl-N, 2, 4, 6-tetranitroaniline Transcription factor Terminal Flower gene Threonine Trypsin inhibitor allele Targeting induced local lesions in genomes Tonoplast intrinsic protein Tobacco mosaic virus Trinitrotoluene Tryptophan Tobacco rattle virus Toxic Substances Control Act Upstream activator sequence uniflora gene United Nations United Nations Conference on Trade and Development United States US Agency for International Development United States Department of Agriculture United States Patent and Trademark Office Ultraviolet Vector backbone Vegetative to generative transition1 gene Vegetative to generative transition2 gene Virus-induced gene silencing Vegetative Insecticidal Protein Virus-like particle Variety Registration Office World Health Organisation Wild type Water use efficiency Xyloglucan endotransglucosylase/hydrolase Xylose Xylosyltransferase Yeast vacuolar glutathione Cd transporter Yellow fluorescent protein Zea CENTRORADIALIS gene Zea FLO/LFY1 gene

Abbreviations

zfl2 ZFN ZMM4/5 ZmRap2 g-GCS o3

xxvii

Zea FLO/LFY2 gene Zinc-finger nuclease Zea mays FULL1-like gene Zea mays related to AP2 gene g-Glutamyl cysteine synthetase Omega 3

Chapter 1

Transgenic Crop Plants for Resistance to Biotic Stress N. Ferry and A.M.R. Gatehouse

1.1

Introduction

We couldn’t feed today’s world with yesterday’s agriculture and we won’t be able to feed tomorrow’s world with today’s. – Lord Robert May, President of the Royal Society, March 2002

The human population is everexpanding; conservative estimates predict that the population will reach ten billion by 2050 (United Nations Population Division), and the ability to provide enough food is becoming increasingly difficult (Chrispeels and Sadava 2003). The planet has a finite quantity of land available to agriculture and the need for increasing global food production has led to increasing exploitation of previously uncultivated land for agriculture; as a result wilderness, wetland, forest and other pristine environments have been, and are being, encroached upon (Ferry and Gatehouse 2009). The minimization of losses to biotic stress caused by agricultural pests would go some way to optimizing the yield on land currently under cultivation. For nearly 50 years, mainstream science has told us that this would be impossible without chemical pesticides (Pimental 1997). The global pesticide market is in excess of $30 billion per year (Levine 2007); despite this, approximately 40% of all crops are lost directly to pest damage (Fig. 1.1). These figures are simplified rough estimates; in reality crop losses to biotic stress are extremely difficult to quantify and vary by crop, year, and region.

N. Ferry (*) School of Biology, Institute for Research on Environment and Sustainability, Devonshire Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK e-mail: [email protected]

C. Kole et al. (eds.), Transgenic Crop Plants, DOI 10.1007/978-3-642-04812-8_1, # Springer-Verlag Berlin Heidelberg 2010

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Fig. 1.1 The world agricultural cake

1.2 1.2.1

Biotic Stress due to Insect Pests Crop Losses due to Insect Pests

[A]nimals annually consume an amount of produce that sets calculation at defiance; and, indeed, if an approximation could be made to the quantity thus destroyed, the world would remain skeptical of the result obtained, considering it too marvelous to be received as truth. – John Curtis, 1860

Arthropods are the most widespread and diverse group of animals, with an estimated 4–6 million species worldwide (Novotny et al. 2002). While only a small percentage of arthropods are classified as pests, they cause major devastation of crops, destroying around 14% of the world annual crop production, contributing to 20% of losses of stored grains and causing around US$100 billion of damage each year (Nicholson 2007). Herbivorous insects and mites are a major threat to food production for human consumption. Larval forms of lepidopterans are considered the most destructive insects, with about 40% of all insecticides directed against heliothine species (Brooks and Hines 1999). However, many species within the orders Acrina, Coleoptera, Diptera, Hemiptera, Orthoptera and Thysanoptera are also considered agricultural pests with significant economic impact (Fig. 1.2). Insect pests may cause direct damage by feeding on crop plants in the field or by infesting stored products and so competing with humans for plants as a food resource. Some cause indirect damage, especially the sap-feeding (sucking) insects by transmitting viral diseases or secondary microbial infections of crop plants.

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Fig. 1.2 Phylloxera, a sap-sucking pest of grape, almost devastated the European wine industry in the nineteenth century

1.2.1.1

The Phylloxera Plague

In the late nineteenth century, a phylloxera epidemic destroyed most of the vineyards for wine grapes in Europe, most notably in France. Grape phylloxera (Daktulosphaira vitifoliae, family Phylloxeridae) is a pest of commercial grapevines worldwide, originally native to eastern North America. These minute, pale yellow sap-sucking insects feed on the roots of grapevines. In Vitis vinifera, the resulting deformations and secondary fungal infections can damage roots, gradually cutting off the flow of nutrients and water to the vine. Phylloxera was inadvertently introduced to Europe in the 1860s. The European wine grape V. vinifera was highly susceptible to the pest and the epidemic devastated most of the European winegrowing industry. Some estimates hold that between two-thirds and nine-tenths of all European vineyards were destroyed. Native American grapes Vitis labrusca are naturally Phylloxera-resistant. The grafting of European grape vines onto resistant grape rootstock is the preferred method to cope with the pest problem even today (http://www.calwineries.com). Thus, phylloxera provides a clear example of how a single insect pest can nearly devastate a whole industry. Innumerable examples exist of insect pests that are highly injurious to agricultural production. The most notable for their destructive capacity being the

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Fig. 1.3 Monument to the cotton boll weevil Source: Wikimedia Commons

migratory locust (Locusta migratoria), Colorado potato beetle (Leptinotarsa decemlineata), boll weevil (Anthonomus grandis), Japanese beetle (Popillia japonica), and aphids, which are among the most destructive pests on earth as vectors of plant viruses (many species in ten families of the Aphidoidea), and the western corn rootworm (Diabrotica virgifera virgifera), also called the billion dollar bug because of its economic impact in the US alone. Curiously, one of these pests, the cotton boll weevil, responsible for neardestruction of the cotton industry in North America, is also ultimately responsible for subsequent diversification of agriculture in many regions, thus warranting a monument in the town of Enterprise, Alabama, in profound appreciation of its role in bringing to an end the state’s dependence on a poverty crop (Fig. 1.3). The global challenge facing agriculture is to secure large and high-quality crop yields and to make agricultural production environmentally sustainable. Control of insect pests would go some way towards achieving this goal.

1.2.2

Insecticides

Insecticides have been, and still are, a highly effective method to control pests quickly when they threaten to destroy crops. The chemical nature of the

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insecticides used has evolved over time. In early farming practices, inorganic chemicals were used for insect control; however, with the advances in synthetic organic chemistry that followed the two world wars the synthetic insecticides were born. In the 1940s, the neurotoxic organochlorine, DDT, was the pesticide of choice, but following its indiscriminate use it was reported to bio-accumulate in the food chain where it affected the fertility of higher organisms – such as birds. Rachel Carson first highlighted this in the book Silent Spring published in 1962; while her presumptions have since been proven to be wrong, the book was nevertheless an important signature event in the birth of the environmental movement. This pesticide was subsequently replaced by the comparatively safer organophosphate and carbamate-based pesticides (both acetylcholinesterase inhibitors) and many of these were replaced in turn by the even safer pyrethroid-based pesticides (axonic poisons). Synthetic pyrethroids continue to be used today despite the fact that they are broad-spectrum pesticides. The major limiting factor on the insecticide strategy is the occurrence of resistance in insect populations. In fact, resistance to insecticides has now been reported in more than 500 species (Nicholson 2007). Furthermore, resistance has evolved to every major class of chemical. The underlying causes of insecticide resistance are manyfold. Owing to wide usage and narrow target range, arthropods have been put under a high degree of selection pressure (Feyereisen 1995). Insecticide resistance may be characterized by: (a) Metabolic detoxification (upregulation of esterases, glutathione-S-transferases, and monoxygenases) (b) Decreased target site sensitivity (via mutation of the target receptor) (c) Sequestration or lowered insecticide availability In addition, cross-resistance to different classes of chemicals has occurred because of the fact that many insecticides target a limited number of sites in the insect nervous system (Raymond-Delpech et al. 2005). The five target sites in insects comprise: nicotinic acetylcholine receptors (e.g., imidacloprid), voltagegated sodium channels (e.g., DDT, pyrethroids), g-aminobutyric acid receptors (e.g., fipronil), glutamate receptors (e.g., avermectins), and acetylcholinesterase (AChE) (e.g., organophosphates and carbamates). The world insecticide market is dominated by compounds that inhibit the enzyme AChE. Together, AChE inhibitors and insecticides acting on the voltage-gated sodium channel, in particular the pyrethroids, account for approximately 70% of the world market (Nauen et al. 2001). Unfortunately, as insecticide target sites are conserved between invertebrates and vertebrates, insecticides have undesirable nontarget effects and unacceptable ecological impacts. Insecticides are implicated in the poisoning of nontarget insects, other arthropods, marine life, birds, and humans (Fletcher et al. 2000). The poisoning of nontarget organisms has obvious implications for biodiversity.

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1.2.3

N. Ferry and A.M.R. Gatehouse

Integrated Pest Management and Organic Agriculture

In parallel to the development of modern insecticides, the specific microbial toxins produced by the soil-dwelling bacterium Bacillus thuringiensis (Bt) are increasingly being adopted as biopesticides. In fact, microbial sprays of Bt are used in organic agriculture. This shift is due in part to a demand for increased safety both for humans and for the environment. Organic agriculture is a form of agriculture that relies on crop rotation, green manure, compost, biological pest control, and mechanical cultivation to maintain soil productivity and control pests, excluding or strictly limiting the use of synthetic fertilizers and synthetic pesticides, plant growth regulators, and genetically modified organisms (Directorate General for Agriculture and Rural Development of the European Commission). Integrated pest management (IPM) has also been proposed as a sustainable control system for insects. Several control systems are combined, including the judicious application of chemicals and biopesticides, use of trap crops, biological control, rotation, good husbandry and cultural control to manage all the pests of a particular crop (Gatehouse and Gatehouse 1999). Increasing crop varietal resistance is critical to both IPM and organic agriculture. It is unfortunate that ultimately neither organic agriculture nor IPM will be able to feed the world. In order to feed an increasing world population, more food must be produced in the future and on either the same amount, or less land (Ferry and Gatehouse 2009). Neither of these farming methods will be as productive as will be necessary to meet increased demands (Amman 2009).

1.2.4

Transgenic (Genetically Modified) Crops

Genetically modified (GM) maize and cotton varieties that express insecticidal proteins derived from B. thuringiensis (Bt) have become an important component in agriculture worldwide. At present, 20.3 million hectares of land is planted with insect-protected transgenic Bt cotton and maize (James 2007), with economic benefits from Bt cotton estimated at US$9.6 billion and maize US$3.6 billion (James 2007). Significantly, Phipps and Park (2002) showed that on a global basis GM technology has reduced pesticide use. These authors estimated that pesticide use was reduced by a total of 22.3 million kg of formulated product in 2000 alone.

1.2.4.1

Bacillus thuringiensis Toxins

B. thuringiensis (Bt) is a soil-dwelling bacterium of major agronomic and scientific interest. Whilst the subspecies of this bacterium colonize and kill a large variety of

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Table 1.1 Insecticidal properties of Bt toxins Insect order Cry protein Lepidoptera Cry1A, Cry1B, Cry1C, Cry1E, Cry1F, Cry1I, Cry1J, Cry1K, Cry2A, Cry9A, Cry9I, Cry15A Coleoptera Cry1I, Cry3A, Cry3B, Cry3C, Cry7A, Cry8A, Cry8B, Cry8C, Cry14A, Cry23A Diptera Cry2A, Cry4A, Cry10A, Cry11A, Cry11B, Cry16A, Cry19A, Cry20A, Cry21A Hymenoptera Cry22A Nematodes Cry5A, Cry6A, Cry6B, Cry12A, Cry13A, Cry14A Liver fluke Cry5A

host insects, each strain tends to be highly specific. Toxins for insects in the orders Lepidoptera (butterflies and moths), Diptera (flies and mosquitoes), Coleoptera (beetles and weevils), and Hymenoptera (wasps and bees) (Table 1.1) have been identified (de Maagd et al. 2001), but interestingly none with activity towards Homoptera (sap suckers) have, as yet, been identified, although a few with activity against nematodes have been isolated (Gatehouse et al. 2002). Further, there is little evidence of effective Bt toxins against many of the major storage insect pests. Bt toxins (also referred to as d-endotoxins; Cry proteins) exert their pathological effects by forming lytic pores in the cell membrane of the insect gut. On ingestion, they are solubilized and proteolytically cleaved in the midgut to remove the C-terminal region, thus generating an “activated” 65–70 kDa toxin. The active toxin molecule binds to a specific high-affinity receptor in the insect midgut epithelial cells. Following binding, the pore-forming domain, consisting of a-helices, inserts into the membrane; this results in cell death by colloid osmotic lysis, followed by death of the insect (de Maagd et al. 2001). A number of putative receptors in the insect gut have been identified and include aminopeptidase N proteins (Knight et al. 1994; Sangadala et al. 1994; Gill et al. 1995; Luo et al. 1997), cadherin-like proteins (Vadlamudi et al. 1995; Nagamatsu et al. 1998; Gahan et al. 2001) and glycolipids (Denolf 1996). Transgenic plants expressing Bt toxins were first reported in 1987 (Vaeck et al. 1987) and following this initial study numerous crop species have been transformed with genes encoding a range of different Cry proteins targeted towards different pest species. Since bacterial cry genes (genes encoding Bt toxins) are rich in A/T content compared to plant genes, both the full-length and truncated versions of these cry genes have had to undergo considerable modification of codon usage and removal of polyadenylation sites before successful expression in plants (de Maagd et al. 1999). Crops expressing Bt toxins were first commercialized in the mid-1990s, with the introduction of Bt potato and cotton. Currently, 20.3 million hectares of land is planted with Bt cotton and maize (James 2007). Bt Maize Lepidopteran pests such as European corn borer Ostrinia nubilalis, fall armyworm Spodoptera frugiperda, and corn earworm Helicoverpa zea perennially cause leaf

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and ear damage to corn. The Bt concept was particularly attractive for maize, since it made it possible to combat European corn borer larvae hidden inside the stem of the plant for the first time. Bt maize has now been grown on a large scale for over a decade, particularly in the US. In 2007, insect-resistant Bt maize was grown on 21% of the total maize cultivation area, and Bt maize with a combination of insect and herbicide resistance was grown on a further 28% (James 2007). Various Bt maize varieties are also authorized in the EU. In 2007, there was notable cultivation of Bt maize primarily in Spain, where it was grown on around 75,000 hectares (Ortego et al. 2009). Transgenic corn hybrids expressing the insecticidal protein Cry1Ab from B. thuringiensis (Bt) var. kurstaki were originally developed to control European corn borer, and offer the potential for reducing losses by fall armyworm and corn earworm. Several events of transgenic Bt corn have been developed with different modes of toxin expression (Ostlie et al. 1997). Amongst the most promising events were Bt11 expressing the cry1Ab gene from B. thuringiensis subsp. kurstaki (Novartis Seeds) and MON810 expressing a truncated form of the cry1Ab gene from B. thuringiensis subsp. kurstaki HD-1 (Monsanto Co.). In both events, the endotoxins are expressed in vegetative and reproductive structures throughout the season (Armstrong et al. 1995; Williams and Davis 1997). Crops containing either of these events are collectively referred to as having “YieldGard technology.” Furthermore, a modified cry9C gene from B. thuringiensis subsp. tolworthi strain BTS02618A is expressed in maize (tradename StarLink – marketed by Aventis CropScience). StarLink corn has only been approved in the US for livestock feed use. In recent years, there has been increasing focus on another maize pest, this time a Coleopteran (beetle); the western corn rootworm. Western corn rootworm is one of the most devastating corn rootworm species in North America. Its larvae are root pests and can destroy significant percentages of corn if left untreated. In the US, current estimates show that 30 million acres (120,000 km) of corn (out of 80 million grown) are infested with corn rootworms. The United States Department of Agriculture (USDA) estimates that corn rootworms cause US$1 billion in lost revenue each year. To make matters worse, this pest is extending its geographical range all of the time – including spreading throughout Europe. Bt maize which is resistant to the western corn rootworm has been authorized in the US since 2003 and has been grown on a large scale since. YieldGard Rootworm uses event MON 863 and expresses the Cry3Bb1 protein from B. thuringiensis (subsp. kumamotoensis) in the plant, protecting the plant against root feeding from the western and northern corn rootworm larvae. Products containing both YieldGard Corn Borer (MON810) and YieldGard Rootworm (MON 863) are marketed under the trade name YieldGard Plus (http://www.agbios.com). Corn rootworm-resistant maize is also produced by expression of the cry34Ab1 and cry35Ab1 genes from B. thuringiensis strain PS149B1 (DOW AgroSciences LLC and Pioneer Hi-Bred International, Inc.).

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Bt Cotton Cotton fibers used in textiles around the world come from the seed hairs of Gossypium hirsutum. Cotton develops in closed, green capsules known as bolls that burst open when ripe, revealing the white, fluffy fibers. But cotton is more than just a fiber for textiles. It is also an important source of raw materials used in animal feed and for various processed food ingredients, including cottonseed oil, proteinrich cottonseed meal (mostly used as animal feed) and even leftover fibers can be used as food additives. Lepidopteran, particularly heliothine, pests can have an enormously damaging effect on a cotton crop and controlling these insects in conventional farming involves treatment with a number of insecticide sprays. In 1996, Bollgard1 cotton (Monsanto) was the first Bt cotton to be marketed in the US. Bollgard cotton produces the Cry1Ac toxin from B. thuringiensis (subsp. kurstaki), which has excellent activity on tobacco budworm and pink bollworm. These two insects are extremely important as both are difficult and expensive to control with traditional insecticides and the damage caused by them directly impacts on the harvestable plant organ, the cotton bolls themselves. Bollgard II1 was introduced in 2003, representing the next generation of Bt cottons. Bollgard II contains Cry1Ac plus a second gene from the Bt bacteria which encodes the production of Cry 2Ab (also subsp. kurstaki). WideStrike (a Trademark of DowAgrosciences) was registered for use in 2004, and like Bollgard II, it expresses two Bt toxins but this time Cry1Ac and Cry1F were used in combination. Both Bollgard II and WideStrike have better activity on a wider range of caterpillar pests than the original Bollgard technology. GM Bt cotton has become widespread, covering a total of 15 million hectares in 2007, or 43% of the world’s cotton. Most GM cotton is grown in the US and China, but it can also be found in India, South Africa, Australia, Argentina, Mexico, and Columbia (http://www.agbios.com). Currently, 20% of the cotton grown commercially in China expresses Cry1Ac in combination with a plant protease inhibitor, cowpea trypsin inhibitor (CpTI) (He et al. 2009).

Bt Potato Potato (Solanum tuberosum L.) is a major world food crop. Potato is exceeded only by wheat, rice, and maize in terms of world production for human consumption (Ross 1986). Many commercial potato varieties are highly susceptible to damage by the Colorado potato beetle. In 1999, 93% of the 1.1 million potato acres grown in the US were treated with a total of 2.6 million pounds of insecticide (http://www. usda.gov/). To date, few traditionally bred varieties have been produced with resistance to this major pest. Unfortunately, many of the pesticides currently used are broad-spectrum pesticides, killing not only the target pest but most of its natural enemies as well. The Cry3A d-endotoxin from B. thuringiensis Berliner subsp. tenebrionis is toxic to coleopterans, particularly chrysomelids (Krieg et al. 1983;

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Bauer 1990; MacIntosh et al. 1990). It is insecticidal against the Colorado potato beetle, L. decemlineata (Ferro and Gerlernter 1989). In 1995, Bt.Cry3A (NewLeafTM) potato became the first Bt-crop to be commercialized, although they are currently withdrawn from the US market. 1.2.4.2

Evolution of Resistance in Pest Populations

Perhaps one of the most important issues surrounding the cultivation of Bt crops relates to the evolution of target pest resistance, which could limit the life span of the technology. In the case of Bt toxins, this is a major concern for the organic farming community, since the potential for insect populations to evolve resistance to Bt will not only limit the effectiveness of Bt-expressing crops but also Bt-based biopesticides. Bt resistance in insect pests has been reported to develop within 4–5 generations in the laboratory (Stone et al. 1989). To date, the mechanism of resistance to Cry toxins in insects has been most commonly ascribed to the loss or inactivation of specific toxin-binding sites on midgut cell membranes (Ferre´ and Van Rie 2002). Other resistance mechanisms that have been proposed include a defect in the toxin activation by midgut proteases (Oppert et al. 1994; Sayyed et al. 2001), or an increased repair and/or replacement rate of Cry-damaged midgut cells by stem cells (Forcada et al. 1999). Studies have also revealed evidence for novel resistance mechanisms based on active defensive responses (Rahman et al. 2004; Ma et al. 2005). When one considers the ability of insects to evolve resistance to chemical pesticides (French-Constant 2004), the development of field resistance is inevitable and in fact was recently reported to have already occurred (Tabashnik and Carrie`re 2009). Analysis of monitoring data shows that some field populations of H. zea have evolved resistance to Cry1Ac, the toxin produced by first-generation Bt cotton (Tabashnik et al. 2008). Nonetheless, resistance of H. zea to Cry1Ac has not caused widespread crop failures in the field for several reasons (Tabashnik et al. 2008). First, the documented resistance is spatially limited. Second, from the outset, insecticide sprays have been used to improve the control of H. zea on Bt cotton because Cry1Ac alone is not sufficiently effective to manage this pest. Finally, GM cotton producing two Bt toxins (Cry2Ab and Cry1Ac) was planted on more than one million ha in the US in 2006; control of H. zea by Cry2Ab would limit problems associated with resistance to Cry1Ac (Jackson et al. 2004). Considerable effort has been devoted to delaying the onset of evolution of resistance, e.g., the use of refugia has been required/recommended in most regions growing Bt-crops depending upon the country in question (Tabashnik and Carrie`re 2009). Gene-stacking and integrated pest management should be combined to control this problem. 1.2.4.3

Unexpected Benefits

Interestingly, the expression of Bt has resulted in improved crop quality as a consequence of decreased levels of Fusarium infestation and fumonisin mycotoxin

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production as a direct result of reduced levels of insect pest damage. This benefit is particularly important in food crops such as maize (Glaser and Matten 2003).

1.2.4.4

A Global Technology

Of the global total of 12 million biotech farmers in 2007, over 90% were small and resource-poor farmers from developing countries; the balance of one million were large farmers from both industrialized countries such as Canada and developing countries such as Argentina. Of the 11 million small farmers, most were Bt cotton farmers; 7.1 million in China (Bt cotton), 3.8 million in India (Bt cotton), and the balance of 100,000 in the Philippines (GM maize), South Africa (GM cotton, maize and soybeans often grown by subsistence women farmers) and the other eight developing countries which grew GM crops in 2007. This modest uptake by subsistence farmers contributes towards the Millennium Development Goals of reducing poverty by 50% by 2015 and is a very important development (James 2007).

1.2.5

Other Sources of Insecticidal Molecules

The concept of employing genes encoding Bt toxins to produce insect-resistant transgenic plants arises from the successful use of Bt-based biopesticides. A number of other strategies for protecting crops from insect pests actually exploit endogenous resistance mechanisms (Harborne 1998; Gatehouse 2002a, b). Genes encoding such defensive proteins are obvious candidates for enhancing crop resistance to insect pests.

1.2.5.1

Enzyme Inhibitors

Interfering with digestion, and thus affecting the nutritional status of the insect, is a strategy widely employed by plants for defense, and has been extensively investigated as a means of producing insect-resistant crops (Gatehouse 2002a, b). Insectdigestive proteases tend to fall into four mechanistic classes (serine, cysteine, aspartic or metallo proteases – depending on the enzyme-active site residue). Numerous studies since the 1970s have confirmed the insecticidal properties of a broad range of protease inhibitors from both plant and animal sources (Jouanin et al. 1998; Gatehouse 2002a, b). Proof of concept for exploiting such molecules for crop protection was first demonstrated with expression of a serine protease inhibitor from cowpea (CpTI), which was shown to significantly reduce insect growth and survival (Hilder et al. 1987). These studies were subsequently extended to include a greater range of target pests (Gatehouse et al. 1994; Graham et al. 1995; Xu et al. 1996), and a broader range of inhibitors and plant species, including economically

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important pest species, particularly lepidopterans (Broadway 1997; De Leo et al. 2001). Since many economically important coleopteran pests predominantly utilize cysteine proteases for protein digestion, inhibitors for this class of enzyme (cystatins) have also been investigated as a means for controlling pests from this order. Oryzacystatin, a cysteine protease inhibitor isolated from rice seeds, is effective towards both coleopteran insects and nematodes when expressed in transgenic plants (Leple et al. 1995; Urwin et al. 1995; Pannetier et al. 1997). Similarly, the cysteine/aspartic protease inhibitor equistatin, from sea anemone, is also toxic to several economically important coleopteran pests, including the Colorado potato beetle (Outchkourov et al. 2003). More recent studies have included the stacking of different families of inhibitors to increase the spectrum of activity (Abdeen et al. 2005). A major limitation, however, to this strategy for the control of insect pests arises from the ability of some lepidopteran and coleopteran species to respond and adapt to ingestion of protease inhibitors by either overexpressing native gut proteases, or producing novel proteases that are insensitive to inhibition (Bown et al. 1997; Jongsma and Bolter 1997). Thus, detailed knowledge about the enzyme–inhibitor interactions, both at the molecular and biochemical levels, together with detailed knowledge on the response of insects to exposure to such proteins is essential to effectively exploit this strategy. The concept of inhibiting protein digestion as a means of controlling insect pests has been extended to the inhibition of carbohydrate digestion. For example, inhibitors of a-amylase have been expressed in transgenic plants and shown to confer resistance to bruchid beetles (Shade et al. 1994; Schroeder et al. 1995).

1.2.5.2

Lectins

Lectins, found throughout the plant and animal kingdoms, form a large and diverse group of proteins identified by a common property of specific binding to carbohydrate residues, either as free sugars, or more commonly, as part of oligo- or polysaccharides. Many physiological roles have been attributed to plant lectins, including defense against pests and pathogens (Chrispeels and Raikhel 1991; Peumans and Vandamme 1995). Although some lectins are toxic to mammals, and are thus not suitable candidates for transfer to crops for enhanced levels of protection, this is by no means universal. Many lectins are not toxic to mammals, yet are effective against insects from several different orders (Gatehouse et al. 1995), including homopteran pests such as hoppers and aphids (Powell et al. 1995; Sauvion et al. 1996; Gatehouse et al. 1997). This finding has generated significant interest, not least since no Bts effective against this pest order have been identified to date. One such lectin is the snowdrop lectin (Galanthus nivalis agglutinin; GNA). Both constitutive and phloem-specific (Rss1 promoter) expression of GNA in rice is an effective means of significantly reducing survival of rice brown planthopper (Nilaparvata lugens), and green

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leafhopper (Nephotettix virescens) both serious economic pests of rice (Rao et al. 1998; Foissac et al. 2000; Tinjuangjun et al. 2000). GNA has been expressed in combination with other genes encoding insecticidal proteins, including the cry genes (Maqbool et al. 2001). Although lectins such as GNA, and ConA are not as effective against aphids as they are against hoppers, they nonetheless have significant effects on aphid fecundity when expressed in potato (Down et al. 1996; Gatehouse et al. 1997, 1999) and wheat (Stoger et al. 1999). The precise mode of action of lectins in insects is not fully understood although binding to gut epithelial cells appears to be a prerequisite for toxicity. In the case of rice brown planthopper, GNA not only binds to the luminal surface of the midgut epithelial cells, but also accumulates in the fat bodies, ovarioles and throughout the haemolymph, suggesting that the lectin is able to cross the midgut epithelial barrier and pass into the insect’s circulatory system, resulting in a systemic toxic effect (Powell et al. 1998; Du et al. 2000). As with protease inhibitors, the levels of protection conferred by the expression of lectins in transgenic plants are generally not high enough to be considered commercially viable. However, the absence of genes with proven high insecticidal activity against homopteran pests may well mean that transgenic crops with partial resistance may still find acceptance in agriculture, especially if expressed with other genes that confer partial resistance, or if introduced into partially resistant genetic backgrounds.

1.2.5.3

A Brief Aside; Plant Parasitic Nematodes

Plant parasitic nematodes include several groups causing severe crop losses. The most common genera are: Aphelenchoides (foliar nematodes), Meloidogyne (rootknot nematodes), Heterodera, Globodera (cyst nematodes) such as the potato cyst nematode, Nacobbus, Pratylenchus (lesion nematodes), Ditylenchus, Xiphinema, Longidorus, and Trichodorus. Several phytoparasitic nematode species cause histological damages to roots, including the formation of visible galls (Meloidogyne). Some nematode species transmit plant viruses through their feeding activity on roots. One of them is Xiphinema index, vector of GFLV (grapevine fanleaf virus), an important disease of grapes. Bt toxins, lectins (Burrows et al. 1999) and protease inhibitors have shown some promise for control, particularly the expression of cystatins (Cowgill et al. 2002). For a recent review of the topic, the reader is referred to Fuller et al. (2008).

1.2.5.4

Other Sources of Insecticidal Molecules

Generating insecticidal transgenic crops harboring genes from nonconventional sources is an extremely active area, with amongst others, foreign genes from plants (e.g., enzymes inhibitors and novel lectins) and animal sources including insects

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N. Ferry and A.M.R. Gatehouse

(e.g., biotin-binding proteins, neurohormones, venoms and enzyme inhibitors) being a major focus (Ferry et al. 2006). The development of second-generation transgenic plants with greater durable resistance might result from the expression of multiple insecticidal genes such as the Vip (vegetative insecticidal proteins) produced by B. thuringiensis during its vegetative growth. The benefit of such an approach is a broader insect target range than conventional Bt proteins and the proposed expectation to control current Bt resistant pests due to the low levels of homology between the domains of the two proteins classes (Christou et al. 2006). With Bt toxins as the classical reference, toxins from other insect pathogens provide a potential repository of novel insecticidal compounds. Photorhabdus spp. are bacterial symbionts of entomopathogenic nematodes, which are lethal to a wide range of insects (Chattopadhyay et al. 2004). Photorhabdus toxin expression in Arabidopsis caused significant insect mortality (see for review Ferry et al. 2006). Thus, toxins from other insect pathogens are also opening up new routes to pest control using transgenic-based strategies. Interesting recent developments include the use of novel proteins from insect biological control agents and insect hormones to generate transgenic crops. A teratocyte secretory protein from a hymenopteran endoparasitoid (a parasitic wasp often used in biocontrol programs) has been expressed in transgenic tobacco and shown to increase resistance to lepidopteran pests (Maiti et al. 2003). Similar protection has also been achieved with insect peptide hormones (Tortiglione et al. 2003). Interestingly, they replaced the tomato systemin peptide region of prosystemin (a plant signaling molecule) with the insect peptide and showed that this resulted in the production of biologically active insecticidal peptides. Reliance on the expression of a single gene product for pest control is a relatively short-term strategy that parallels the use of exogenously applied chemical pesticides. Thus, pyramiding (stacking) of genes encoding different Bt toxins has been developed as a method for preventing the onset of evolution of pest resistance, and for conferring greater levels of pest control (Boulter et al. 1990; Maqbool et al. 2001; Zhao et al. 2003). This strategy has now been adopted in commercially available crops (see above). For example, corn lines have recently been developed (Moellenbeck et al. 2001; Ostlie 2001) coexpressing two d-endotoxins from Bt for resistance to corn rootworm. Hybrid proteins have also been developed to enhance and extend the activity of Bt toxins. The use of a single Bt toxin in a crop is limited in that many insects attack a single crop and toxins generally show very high specificity towards a single pest species. Therefore, toxins have been engineered to modify their receptor recognition and pore formation. Each toxin consists of three domains. Domain I is involved in membrane insertion and pore formation. Domains II and III are both involved in receptor recognition and binding. Additionally, a role for domain III in pore function has been found. This approach has proved successful in both enhancing activity (Karlova et al. 2005) and extending host range (Singh et al. 2004). Such hybrid/fusion proteins offer an alternative/ complementary strategy to address potential limitations in conventional transgenic insect pest control.

1 Transgenic Crop Plants for Resistance to Biotic Stress

1.2.5.5

15

Transgenic Plants Expressing Fusion Proteins

The concept of “gene stacking” has recently been extended to the development and use of fusion proteins. Such proteins not only provide a means of increasing durability, but also provide a “vehicle” for more effective targeting of insecticidal molecules, including peptides. It thus offers an alternative/complementary strategy to address potential limitations in conventional transgenic insect pest control. For example, recognition of binding sites in the insect gut is an important factor determining the toxicity of Bt. Enhancing toxin-binding capabilities should thus extend host range and delay resistance in pest populations. Bt is believed to bind primarily to aminopeptidase N or cadherin membrane proteins, while the generation of a fusion protein with the nontoxic B-chain from ricin (RB) was shown to extend the binding of Bt to include specific glycoproteins. Transgenic plants expressing the Bt fused RB demonstrated that the addition of the RBbinding domain provided a wider repertoire of receptor sites within target species and significantly enhanced the levels of toxicity of Bt. For example, survival of the armyworm Spodoptera littoralis, a species of insect not sensitive to Bt, was reduced by ~90% when feeding on transgenic maize expressing the fusion, compared to plants expressing either Bt Cry1Ac alone, or the RB-binding domain. Expression of the fusion protein resulted in the insect becoming susceptible to Bt (Mehlo et al. 2005). This strategy has shown great potential beyond just extending the toxicity of Bt. Zhu-Salzman et al. (2003) have generated fusion proteins with anchor regions to other insecticidal proteins to the insect gut epithelium. Using the legume lectin rGSII, they proposed a system to combat the ability of certain insect species to activate protease inhibitor-insensitive proteolytic enzymes. The soybean cysteine protease inhibitor soyacystatin N (scN) was covalently linked to the GlcNAc-specific legume lectin using a naturally occurring linker region from the potato multicystatin. In this instance, the fusion protein not only has a novel binding ability that is proposed to initiate a concentration effect by localizing the inhibitor at the anterior of the gut, but the fused lectin moiety additionally offers a degree of protection to the insecticidal moiety by blocking the access of scN-insensitive proteases, thereby preventing proteolytic destruction of the cystatin. Not only do fusion proteins have potential for use in transgenic crops, but also to improve the efficacy of biopesticide-based sprays. Neuropeptides potentially offer a high degree of biological activity, and thus provide an attractive alternative pest management strategy. There are major drawbacks to their use, particularly as topical sprays. They are unlikely to be rapidly absorbed through the insect cuticle to their site of action, and are prone to proteolysis and rapid degradation in the environment. Should they survive the application process and are then taken up by the insect, they are then unlikely to survive the conditions of the insect gut or be delivered to the correct targets within the insect. The discovery that snowdrop lectin (GNA) remains stable and active within the insect gut after ingestion, and that it is able to cross the gut epithelium, provided an opportunity for its use as a “carrier molecule” to deliver other peptides to the circulatory system of target insect

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N. Ferry and A.M.R. Gatehouse

species. This strategy effectively delivered the insect neuropeptide hormone, allatostatin, to the haemolymph of the tomato moth Lacanobia oleracea (Fitches et al. 2002). Subsequent expression of the fusion protein in potato further provided proof of concept for the efficacy of fusion proteins as a means of delivery. The results demonstrated significant reduction in mean larval weight when compared to the controls. GNA can be used to deliver insecticidal peptides isolated from the venom of the spider Segestria florentina (SFI1) to the haemolymph of L. oleracea (Fitches et al. 2004). Neither the GNA nor the SFI1 moieties alone were acutely toxic; however, the SFI1/GNA fusion was insecticidal to first stage larvae, causing 100% mortality after 6 days. This spider venom neurotoxin is believed to irreversibly block the presynaptic neuromuscular junctures. Such venom toxins show high degrees of specificity and thus lend themselves to environmentally benign pest management strategies.

1.2.6

Manipulation of Plant Endogenous Defenses

Alternative strategies for protecting crops from insect pests, that are not dependent on the expression of single or stacked genes, seek to exploit the induced endogenous resistance mechanisms exhibited by plants to most insect herbivores. For further information, the reader is referred to Chap. 10 of this volume. Such induced defenses are exemplified by the wounding response, first identified as the local and systemic synthesis of proteinase inhibitors (PIs), which block insect digestion in response to plant damage (Gatehouse 2002a, b). Many transgenic strategies have attempted to exploit the potential overexpression of plant PIs to protect crops from pest damage (Jouanin et al. 1998) but these have relied on the transfer of a single PI gene, and many insects have been able to adapt to this. More recent research has shown that induced defenses also involve the plant’s ability to produce toxic or repellent secondary metabolites as direct defenses, and volatile molecules, which play an important role in indirect defense (Kessler and Baldwin 2002). Insect herbivores activate induced defenses both locally and systemically via signaling pathways involving systemin, jasmonate, oligogalacturonic acid and hydrogen peroxide (Fig. 1.4). Ecologists have long understood that plants exhibit multi-mechanistic resistance towards herbivores, but the molecular mechanisms underpinning these complicated responses have remained elusive (Baldwin et al. 2001). However, recent studies investigating the plant’s herbivore-induced transcriptome, using microarrays and differential display technologies, have provided novel insights into plant–insect interactions. The jasmonic acid cascade plays a central role in transcript accumulation in plants exposed to herbivory (Hermsmeier et al. 2001). A single microarraybased study revealed that the model plant Arabidopsis undergoes changes in levels of over 700 mRNAs during the defense response (Schenk et al. 2000). In contrast, only 100 mRNAs were upregulated by spider mite (Tetranicus urticae) infestation in lima bean (Phaseolus lunatus), although a further 200 mRNAs were upregulated

1

Transgenic Crop Plants for Resistance to Biotic Stress

17

Herbivory physical forces local peptide hormone release eg. prosystemin

insect dervived elicitors

ethylene

systemin plant-plant interactions

pest deterrent/ attraction of natural enemies

receptor binding ABA linolenic acid SA

Voltiles e.g. -green leaf volatiles -C10, C15 terpenoids -indole

octadecanoid pathway methyl jasmonate jasmonic acid auxin, SA (-) ethylene, ABA (+) polygalactouronidase oligalacturonic acid vascular bundle

H202

Insecticidal compounds e.g. Protease Inhbitors

late genes (defence)

Cell wall

NAPDH oxidase

plant-plant interactions (systemic induction of Pls)

early genes signalling

mesophyll cells

Fig. 1.4 The generalized plant-wounding response. Generalized overview of the plant wounding response, and signalling molecules which can modulate it, showing the pathways necessary for both local and systemic induction of insecticidal proteins. (Adapted from: Ferry et al. 2004)

in an indirect response mediated by feeding-induced volatile signal molecules (Arimura et al. 2000). Deciphering of the signals regulating herbivore-responsive gene expression will afford many opportunities to manipulate the response. Signaling molecules such as salicylic acid, jasmonic acid and ethylene do not activate defenses independently by linear cascades, but rather establish complex interactions that determine specific responses. Knowledge of these interactions can be exploited in the rational design of transgenic plants with increased insect resistance (Rojo et al. 2003; De Vos et al. 2005; Giri et al. 2006).

1.2.6.1

A Special Case: Sap-Feeding Insects

While most herbivorous insects cause extensive damage to plant tissues when feeding, many insects of the order Homoptera feed from the contents of vascular tissues by inserting a stylet between the overlying cells, thus limiting cell damage and minimizing induction of a wounding response. In contrast to wounding, plant responses following attack by these insects have been shown to be typical of pathogen attack, with examples of gene-for-gene interactions being known (Walling 2000;

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N. Ferry and A.M.R. Gatehouse

Moran et al. 2002). However, these pathogen-induced pathways can induce expression of many of the genes upregulated by wounding because of pathway cross-talk. Moran and Thompson (2001) demonstrated that phloem feeding by the green peach aphid (Myzus persicae) on Arabidopsis induced expression of genes associated with salicylic acid (SA) responses to pathogens, as well as a gene involved in the jasmonic acid-mediated response pathway. These results suggest stimulation of response pathways involved in both pathogen and herbivore responses. Microarray data has identified genes involved in oxidative stress, calcium-dependent signaling, pathogenesis-related responses, and signaling as key components of the induced response (Moran et al. 2002). It may be that transgenic strategies that activate such signaling cascades could enhance plant resistance to these problematic pests. 1.2.6.2

Indirect Defense (Volatile Production)

The role of plant volatiles in indirect defense has been described as “top-down” defense (Baldwin et al. 2001). Some volatiles appear to be common to many different plant species, including C6 aldehydes, alcohols and esters (green leaf volatiles), C10 and C15 terpenoids, and indole, whereas others are specific to a particular plant species. Many volatiles are preformed and act in herbivore deterrence; furthermore, the wounding response also includes the formation of volatile compounds. Top-down control of herbivore populations is achieved by attracting predators and parasitoids to the feeding herbivore, mediated by these volatile organic compounds (VOCs). For example, genes involved in the biosythesis of the maize VOC bouquet are upregulated by insect feeding (Frey et al. 2000; Shen et al. 2000). In addition, herbivore oviposition has been shown to induce VOC emissions, which attract egg parasitoids (Hilker and Meiners 2002). Herbivoreinduced VOCs can also elicit production of defence-related transcripts in plants near the individual under attack (Arimura et al. 2000; Dicke et al. 2003). Exposure to herbivore-induced volatiles in lima bean results in transcription of genes involved in ethylene biosynthesis (Arimura et al. 2000). Manipulation of volatile biosynthesis can affect insect resistance. Transgenic potatoes in which production of hydroperoxide lyase (the enzyme involved in green leaf volatile biosynthesis) was reduced were found to support improved aphid performance and fecundity, suggesting toxicity of these volatiles to M. persicae (Vancanneyt et al. 2001). In a review of the topic Degenhardt et al. (2003) discuss the potential of modifying terpene emission with the aim of making crops more attractive to herbivore natural enemies. 1.2.6.3

Detoxification and Insect Modulation of the Wounding Response

However, insect pests are able to feed on plants despite their defenses, both constitutive and inducible. Many insects are able to detoxify potentially toxic secondary metabolites, using cytochrome P-450 monoxygenases and glutathioneS-transfereases. These enzymes are induced by exposure to toxic plant secondary

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19

compounds, for example, xanthotoxin (a furanocoumarin) induces P-450 expression in corn earworm (Li et al. 2000). More recently, Li et al. (2002) have shown that corn earworm uses signaling molecules from its plant host, jasmonate and salicylate, to activate four of its cytochrome P450 genes, thus making the induction of detoxifying enzymes rapid and specific. Recent strategies based on RNAi technology have shown that it is possible to overcome these insect responses (discussed later in this chapter).

1.2.7

RNAi

Disrupting gene function by the use of RNAi is a well-established technique in insect genetics based on delivery by injection into insect cells or tissues. The observation that RNAi could also be effective in reducing gene expression, measured by mRNA level, when fed to insects (Turner et al. 2006) has led to two recent articles in which transgenic plants producing double-stranded RNAs (dsRNAs) which are shown to exhibit partial resistance to insect pests. Transgenic maize producing dsRNA directed against V-type ATPase of corn rootworm showed suppression of mRNA in the insect and reduction in feeding damage compared to controls (Baum et al. 2007). Similarly, transgenic tobacco and Arabidopsis expressing dsRNA directed against a detoxification enzyme (Cytochrome P450 gene CYP6AE14) for the breakdown of gossypol (a defensive metabolite) in cotton bollworm caused the insect to become more sensitive to gossypol in the diet (Mao et al. 2007). This approach holds great promise for future development. It is also proving effective for nematode control (Bakhetia et al. 2005). For a recent review on RNAi-mediated crop protection against insect pests the reader is referred to Price and Gatehouse (2008). Advances in our understanding of induced responses in plants and their regulation, has refocused attention on potential exploitation of endogenous resistance mechanisms for crop protection. While plant resistance is an integral component of organic and IPM strategies, it does not afford similar levels of protection as those provided by the use of “direct” protective methods such as the expression of Bt toxins. The goal of the plant breeder, and now the biotechnologist, is to engineer durable multi-mechanistic resistance to insect pests in crops, and increased knowledge of induced defense mechanisms and their molecular control is likely to play an important role in realizing this aim.

1.2.8

Environmental Impact of IR Crops

Almost from the beginning of the production of transgenic crops there have been concerns over their use and introduction into the environment. There is international agreement that GM crops should be evaluated for their safety, including their environmental impact (Dale 2002). During the past 15–20 years, there have been

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extensive research programs of risk assessment, with several areas of major concern identified.

1.2.8.1

Impact on Nontarget Organisms

Assessing the consequences of pest control on nontarget organisms is an important precursor to their becoming adopted in agriculture. The expression of transgenes that confer enhanced levels of resistance to insect pests is of particular significance since it is aimed at manipulating the biology of organisms in a different trophic level to that of the plant. Potential risks to beneficial nontarget arthropods exist. Those groups most at risk include: nontarget Lepidoptera, beneficial insects (pollinators, natural enemies) and soil organisms. Exposure of nontarget Lepidoptera to insecticidal transgene products may occur through both direct consumption of transgenic plant tissues including via consumption of transgenic pollen; many nontarget Lepidoptera are rare butterflies having great conservation value. The case of the Monarch butterfly (Danaus plexippus), a conservation flagship species in the US, highlighted the need for ecological impact research. In a letter to Nature, Losey et al. (1999) claimed that both survival and consumption rates of Monarch larvae fed milkweed leaves (natural host) dusted with Bt pollen were significantly reduced, and that this would have profound implications for the conservation of this species. However, a series of ecologically based studies rigorously evaluated the impact of pollen from such crops on Monarchs and demonstrated that the commercial wide-scale growing of Bt-maize did not pose a significant risk to the Monarch population (Hellmich et al. 2001; Gatehouse et al. 2002). In fact, the initial experiments did not quantify the dose of pollen used, or indeed, if this was a realistic level likely to be encountered in the field; nevertheless, this work highlighted the importance of studying nontarget effects. In a separate field study Wraight et al. (2000) showed that Papilio polyxenes (black swallowtail) larvae were unaffected by pollen from Bt expressing maize event Mon810 at 0.5, 1, 2, 4 and 7 m from the transgenic field edge, highlighting the need for a case-by-case study of organisms considered to be at risk. In addition to the potential direct impacts of Bt toxins on susceptible target insects, as in the case of the Morarch butterfly, some Lepidoptera have been shown to have a reduced sensitivity to the lepidopteran-specific Bt toxins. For example, S. littoralis can survive on maize expressing Cry1Ab (Hilbeck et al. 1998) and thus present a route of exposure to the next trophic level. In the case of Bt Cry3Aa or Cry3Bb expressing potatoes or maize, some Lepidoptera may represent nontarget secondary pests, and whilst not directly affected by the transgene product themselves may again present a route of exposure to the next trophic level, as do other nontarget herbivores. Organisms such as those belonging to the orders Homoptera, Hemiptera, Thysanoptera, and Tetranychidae are not targeted by Bt toxins expressed in transgenic plants; however, they do utilize the Bt crop (Groot and Dicke 2002). The direct effect that this may have on these insects is dependent on the presence of Bt receptors in the first instance, and it is so far unclear whether such receptors are

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present in nontarget organisms (de Maagd et al. 2001). In addition, the fate of the toxin ingested by nontarget herbivores is unclear, since if it retains toxicity then this may have implications at the next trophic level. The impacts of insect-resistant transgenic crops at higher trophic levels have also been considered, where there are concerns over the risks to beneficial arthropod biodiversity (Schuler et al. 1999; Bell et al. 2001); in particular, predators and parasitoids, which play an important role in suppressing insect pest populations both in the field and under specialized cultivation systems (glasshouses). Natural enemies may ingest transgene products via feeding on herbivorous insects that have themselves ingested the toxin from the plant; such tritrophic interactions will be influenced by the susceptibility of the herbivore to the plant protection product. If, as in the case with Bt toxins, the prey item is susceptible to the toxin, then the predator will not come into contact with the toxin as the pest will effectively be controlled, and in target insects the toxin is bound to receptors in the midgut epithelium that are structurally rearranged and may lose their entomotoxicity (de Maagd et al. 2001). In nontarget insects (and resistant insects), the toxins do not bind and may thus retain biological activity. However, the overwhelming weight of evidence from independent laboratory and field studies show that Bt toxins have a limited ability to affect the next trophic level (reviewed in Sanvido et al. 2007; Ferry and Gatehouse 2009; Romeis et al. 2008). Pollinators represent another group of nontarget organisms highlighted as at risk from Bt toxins in GM crops. The current generation of transgenic crops produce Bt toxin in the pollen as well as in the vegetative tissues. Several studies have been conducted to determine toxicity of Bt toxins to pollinators (Vandenberg 1990; Sims 1995, 1997; Arpaia 1997; Malone and Pham-Delegue 2001); generally, they all conclude that neither the adults nor the larvae of bees were affected by Bt toxins. For a comprehensive review of the impact of transgenic crops on pollinators, the reader is referred to two recent reviews (Malone et al. 2008; Malone and Burgess 2009). Finally, nontarget species may come into contact with Bt toxins via the environment. Several studies have shown that Bt toxins released from transgenic plants bind to soil particles (Palm et al. 1996; Crecchio and Stotzky 1998; Saxena et al. 1999). Soil-dwelling and epigeic insects such as Collembola and Carabidae may thus be exposed to the toxins. Several studies (Saxena and Stotzky 2001; Ferry et al. 2007) show no differences in mortality or body mass of bacteria, fungi, protozoa, nematodes and earthworms or carabid beetles exposed to Bt. Exposure to the transgene products, however, does not necessarily imply a negative impact. Most studies to date have demonstrated that crops transformed for enhanced pest resistance have no deleterious effects on beneficial insects (reviewed in Ferry et al. 2003; Romeis et al. 2008).

1.2.9

Conclusions

Ultimately one must consider the impact of transgenic crops and specifically Bt toxins in comparison to other pest control strategies such as conventional crop

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protection using insecticides. While pesticides have no doubt brought vast yield improvements, they have well-documented undesirable nontarget effects (Devine and Furlong 2007). It is worth remembering that whilst potential risks do exist to the environment from the cultivation of GM crops, their potential to decrease reliance on external inputs (less insecticide sprays) and to increase the availability of genetic resources available to breeders is great (Ferry and Gatehouse 2009).

1.3 1.3.1

Biotic Stress due to Weeds Crop Losses due to Weeds

Weeds can compete with productive crops or pasture, or convert productive land into unusable scrub. Weeds are also often poisonous, distasteful, produce burrs, or thorns that interfere with the use and management of desirable plants by contaminating harvests or excluding livestock. Weeds tend to thrive at the expense of the more refined edible or ornamental crops. They provide competition for space, nutrients, water and light, although how seriously they will affect a crop depends on a number of factors. Some crops have greater resistance to competition than others, for example smaller, slower-growing seedlings are more likely to be overwhelmed than those that are larger and more vigorous. Weeds also differ in their competitive abilities, and this can vary according to specific conditions and the time of year. Tall-growing vigorous weeds such as fat hen (Chenopodium album) can have the most pronounced effects on adjacent crops. Chickweed (Stellaria media), a low-growing plant, can happily coexist with a tall crop during the summer, but plants that have overwintered will grow rapidly in early spring and may swamp crops. Simply put, weeds are any plant growing in an area where it is not wanted. Of over 250,000 plant species in the world, only a few hundred are troublesome weeds. Although no single characteristic clearly defines a weed, two attributes are common in the worst weeds: competitiveness and persistence. The world’s worst weeds are shown in Table 1.2 (Chrispeels and Sadava 2003).

1.3.2

Weed Management: Prevention, Control, and Eradication

As weeds are persistent, once they have established in a field they are extremely difficult to eradicate. One of the most successful attempts in the eradication of a weed is provided by witchweed (Striga sp.) in the US. Witchweed is a parasitic plant; its seeds germinate in response to a chemical, strigol, produced by the roots of the host plant. The germinated seedling attaches to the root through special haustoria, as this occurs underground infestation can go unnoticed, consequently serious

1 Transgenic Crop Plants for Resistance to Biotic Stress Table 1.2 World’s worst weeds (from: Chrispeels and Sadava 2003) Plant family Weed examples Grass (Poaceae) Bermuda grass (Cynodon dactylon) Barnyard grass (Echinochloa crus-galli) Johnson grass (Sorghum halepense) Sedge (Cyperaceae) Purple nutsedge (Cyperus rotundus) Yellow nutsedge (Cyperus esculentus) Smallflower umberella sedge (Cyperus difformis) Sunflower (Asteraceae) Canada thistle (Cirsium arvense) Common cocklebur (Xanthium strumarium) Common dandelion (Taraxacum officinale) Buckwheat (Polygonaceae) Wild buckwheat (Polygonum convolvulus) Curly dock (Rumex crispus) Red sorrel (Rumex acetosella) Pigweed (Amaranthaceae) Smooth pigweed (Amaranthus hybridus)

Mustard (Brassicaceae)

Legume (Fabaceae)

Morning glory (Convolvulaceae)

Spurge (Euphorbiaceae)

Goosefoot (Chenopodiaceae)

Mallow (Malvaceae)

Nightshade (Solanaceae)

Spiny amaranth (Amaranthus sinosus) Redroot pigweed (Amaranthus retroflexus) Shepherd’s purse (Capsella bursa-pastoris) Hoary cress (Brassica draba) Wild mustard (Brassica kaber) Black medic (Medicago lupulina) Sensitive plant (Mimosa pudica) Kudzu (Pueraria lobata) Field bindweed (Convolvulvus arvensis) Field dodder (Cuscuta campestris) Swamp morning glory (Ipomoea aquatica) Leafy spurge (Euphorbia esula) Garden spurge (Euphorbia hirta) Spotted spurge (Euphorbia maculata) Common Lamb’s quarters (Chenopodium album) Russian thistle (Salsola iberica) Nettleleaf goosefoot (Chenopodium murale) Venice mallow (Hibiscus trionum) Velvetleaf (Abutilon theophrasti) Arrowleaf sida (Sida rhombifolia) Black nightshade (Solanum nigrum) Jimsonweed (Datura stramonium) Cutleaf groundcherry (Physalis angulata)

23

Crop Wheat

None

Sunflower

Buckwheat

Grain amaranth

Canola

Soybean

Sweet potato

Cassava

Sugarbeet

Cotton

Potato

infestation leading to yield loss occurs before the parasitic plant even appears through the soil. Once the witchweed comes through the soil it becomes photosynthetically active, but still dependent on its host for water. Such parasitic weeds are extremely difficult to control and yield losses of approximately 50% can be expected under drought conditions. In the US, only quarantine of infected areas reduced witchweed infection from 150,000 hectares to a couple of 1,000 hectares, but this quarantine lasted for nearly 50 years (Chrispeels and Sadava 2003)!

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For this reason, preventative weed management is critical. This involves keeping weed seeds and vegetative propagules from getting to the field in the first place and is mainly achieved through seed cleaning and the purchase of certified clean seed. Good management practice and the cleaning of farm equipment moved between different areas are critical in preventative control. Ultimately weed control is achieved by mechanical, biological and predominately chemical means.

1.3.3

Herbicides

The economic importance of weeds is emphasized by the fact that the herbicides comprise as large a share of the world agrochemical market as all other pesticides combined, farmers spend an annual US$10 billion to control weeds in the US alone (Chrispeels and Sadava 2003). Herbicides have evolved over time, and herbicide use has seen a move towards more biodegradable chemicals that only need to be applied at a very low concentration of active ingredient. Thus, the new generation of herbicides are relatively environmentally benign. Many herbicides exploit the differences in plant physiology between the crop species and its weeds (usually the differences between monocots and dicots); they may be systemic or act on contact (Table 1.3). Major crops such as wheat, rice, and maize; and legumes such as soybean, common bean, and peanut come from the same plant families as their weeds, thus creating a control problem. The mode of action of common herbicides is varied (Naylor 2002). For example, inhibition of photosynthesis and light-dependent membrane destruction (acting on photosystems II and I, respectively) are the mode of action of the foliar-acting nonselective herbicides like atrazine, paraquat and diquat. Hormone herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D) induce abnormal plant growth by interring with auxin regulation. The sulfonyl ureas, imidazolines and the environmentally benign, but nonselective glyphosate (acting on 5-enolpyruvylshikimate3-phosphate synthase) inhibit amino acid synthesis. Others include inhibitors of lipid synthesis, inhibition of cell division and pigment synthesis. The advantages of herbicides are clear – they control multiple weed species, control perennial weeds, cause no injury to the crop plant, and can readily be applied to large areas. Table 1.3 Herbicide modes of action Herbicide type Contact herbicides destroy only that plant tissue in contact with the chemical spray. Generally, these are the fastest-acting herbicides. They are ineffective on perennial plants that are able to regrow from roots or tubers Systemic herbicides are foliar-applied and are translocated through the plant and destroy a greater amount of the plant tissue. Modern herbicides such as glyphosate are designed to leave no harmful residue in the soil Soil-borne herbicides are applied to the soil and are taken up by the roots of the target plant Pre-emergent herbicides are applied to the soil and prevent germination or early growth of weed seeds

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Nonchemical weed control includes: cultural weed control practices such as intercropping and rotation; mechanical removal of weeds; and biological control, all of which are labor intensive and unpredictable. Chemical treatment is by far the most effective method, but it is not without problems.

1.3.3.1

Resistance to Herbicides

A common consequence of repeated use of herbicide application is the evolution of resistance in weed populations because of strong selection pressure. The first example of a problematic herbicide resistant weed was reported in the 1970s (Chrispeels and Sadava 2003) when common groundsel resistant to triazine was identified in the US. The mechanism of herbicide resistance is usually a change in the target site with a reduction in herbicide affinity – this may be achieved through a single point mutation and alteration of a single amino acid. By 2001, over 150 weed species had resistance to at least one herbicide. Some biotypes of weeds have resistance to herbicides with different modes of action.

Rigid Ryegrass When a weed evolves resistance to a particular herbicide, the farmer is forced to use an alternative control strategy. This often involves the use of another herbicide with a different mode of action. Subsequent selection pressure may lead to a weed population acquiring multiple herbicide resistance. In nearly all the cases so far, resistance to only one or two chemical families has been reported. Rigid ryegrass is a notable exception. In the most severe cases, biotypes exist that are resistant to nine chemical families with five different modes of action! Resistance is conferred by multiple mechanisms; both altered sites of action and the ability to metabolize particular herbicides, via nonspecific monoxygenases.

1.3.4

Transgenic Crops for Weed Control

The adoption of transgenic herbicide-resistant (HR) crops has made remarkable changes to global agriculture within the last decade. Currently, an estimated 114.3 million hectares of transgenic crops are planted throughout a variety of agroecosystems in 23 developing and industrial countries. Approximately, 90% of the land area with transgenic crops includes a trait for glyphosate resistance (GR) (Owen 2008). While there are a number of herbicide-tolerant genetically modified crops that have been developed for several herbicides with different modes of phytotoxic action, the primary influence in world agriculture are glyphosate-resistant crops

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(GRCs) (Duke 2005; Duke and Powles 2008). More specifically, of the 23 countries that grow plant glyphosate-resistant crops, the US, Canada, Argentina and Brazil are the countries that account for most hectares (Owen 2008). In these countries, the principle GRCs planted include maize (Zea mays), soybean (Glycine max), cotton (G. hirsutum), and canola (Brassica napus). For further information, the reader is referred to Chap. 3 of this volume.

1.3.4.1

Round-up1 Ready Crops

Glyphosate (Round-up1) is a highly effective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, a branch point enzyme in aromatic amino acid biosynthesis. A naturally occurring EPSP synthase gene (cp4) was identified from Agrobacterium sp. strain CP4, whose protein product provided glyphosate tolerance (Fig. 1.5) in plants (Padgette et al. 1995). COO– COO–

SHKP

+

P O

OH

CH2

P O

OH

phosphoenolpyruvate (PEP)

GLYPHOSATE

5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase)

shkG H3PO4

COO–

COO–

H3PO4 CH2

P O

O

OH 5-enolpyruvylshikimate-3-phosphate (EPSP )

CH2

shkH COO–

chorismate synthase

O

COO–

OH chorismate (CHA)

Fig. 1.5 Glyphosate; mode of action. Glyphosate kills plants by interfering with the synthesis of the amino acids phenylalanine, tyrosine and tryptophan. It does this by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which catalyzes the reaction of shikimate-3-phosphate (SHKP) and phosphoenolpyruvate to form 5-enolpyruvyl-shikimate-3-phosphate (EPSP). EPSP is subsequently dephosphorylated to chorismate, an essential precursor in plants for the aromaticamino acids: phenylalanine, tyrosine and tryptophan. These amino acids are used as building blocks in peptides, and to produce secondary metabolites such as folates, ubiquinones and naphthoquinone

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Furthermore, glyphosate detoxification pathways are known in microbes involving glyphosate oxidoreductase (gox) genes (Jacob et al. 1988). These two genes confer glyphosate resistance in selected crops. Crops engineered for resistance to this broad-spectrum herbicide allow control of multiple weeds without injury to the main crop. The economic benefits to farmers of glyphosate-resistant crops are estimated at US$17.5 billion, mainly accrued from reduced sprays and lower inputs of labor. In addition, herbicide-tolerant crops reduce the amounts of active ingredient required for weed control; they also promote the usage of no/low till farming and thus lower fuel consumption with direct benefits to both soil structure and carbon emissions (James 2007).

1.3.4.2

Other Transgenic Herbicide-Tolerant Crops

Traits for resistance to three other classes of herbicides have been developed, but have not reached the same level of popularity as glyphosate resistance. Resistance to oxynil herbicides conferred by the BXN nitrilase from Klebsiella pneumoniae (subsp. ozaenae) (Stalker et al. 1988) was the first trait engineered in cotton (developed by Calgene, Davis, CA [now Monsanto]). Because glyphosate is less expensive and controls more weed species, interest in using the oxynil herbicides has waned and 2004 was the final year of BXN1 cotton sales. BXN canola was commercialized by Rhone-Poulenc Canada (now Bayer Crop Science, Monheim, Germany) and subsequently discontinued. Phosphinothricin acetyltransferase (PAT or BAR) detoxifies phosphinothricin- or bialaphos-based herbicides (glufosinate). The pat gene is native to Streptomyces viridichromogenes and bar is from S. hygroscopicus where they act in both the biosynthesis and detoxification of the tripeptide bialaphos (De Block et al. 1987). Like glyphosate, phosphinothricin herbicides control a broad spectrum of weed species and break down rapidly in the soil so that the problems with residual activity and environmental impact are greatly reduced. Bayer CropScience markets this trait as LibertyLink1 in several species. The pat and bar genes are also popular plant transformation markers in the research community. Finally, BASF (Ludwigshafen, Germany) markets nontransgenic CLEARFIELD1 imidazolinone-resistant canola, wheat, sunflower, corn, lentils, and rice, while DuPont (Wilmington, DE) markets nontransgenic STS1 soybeans with tolerance to sulfonylurea herbicides. A sulfonylurea-tolerant flax variety called CDC Triffid, developed by the University of Saskatchewan (Canada), was grown commercially in Canada in 2000 but is no longer offered. All these crops contain mutagenized versions of the acetohydroxyacid synthase, also called acetolactate synthase (ALS), which are not inhibited by imidazolinone and/or sulfonylurea herbicides (Devine and Preston 2000). Herbicides that inhibit ALS are considered low or very low use-rate herbicides with a good spectrum of weed control and are likely to remain an important part of weed resistance management programs (Castle et al. 2006).

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1.3.5

N. Ferry and A.M.R. Gatehouse

HT Canola

There are two GM traits for herbicide resistance in canola: glyphosate and glufosinate resistance. Resistance to glyphosate herbicides is conferred by the genes 5-enol-pyruvylshikimate-3-phosphate synthase from Agrobacterium sp. CP4 (CP4 epsps) (event GT73, Monsanto) that is targeted to the chloroplast, and a glyphosate oxidoreductasegene from Achromobacter sp. strain LBAA (gox v247) (both Roundup Ready1). Resistance to phosphinothricin herbicides is conferred by phosphinothricin-acetyltransferance (pat gene), and marketed as LibertyLink1 by AgrEvo (now Bayer CropScience). Herbicide-resistant canola (oilseed rape) allows post-emergence application of a single herbicide with a wide spectrum of activity. For effective control, it has to be applied before the weeds reach 10 cm height. This extends the potential time period for spraying (Benbrook 2004). The timing is more flexible and the application of a single herbicide simplifies weed control (Firbank and Forcella 2000). Since many herbicides allow post-emergence applications, HT crops do not generally provide a new option. However, post-emergence spraying with non-GMHT oilseed rape is confined to a short 3–5 week period after crop emergence (Pallutt and Hommel 1998). Thus, GMHT canola offers greater flexibility (Owen 1999). With HT oilseed rape, the intention is to reduce the amount of active ingredient of herbicide used and to rely preferably on one broad-spectrum herbicide only (http://www.canola-councildemo.org). The intent is also to reduce the number of spraying rounds, which helps reduce soil compaction and erosion (Madsen et al. 1999). During the first years of cultivating GMHT oilseed rape, most farmers had reduced active ingredient rates and applications (Champion et al. 2003). GMHT oilseed rape was usually sprayed only once, with an average active ingredient amount that was either lower (Champion et al. 2003) or not significantly different from conventional oilseed rape (Schu¨tte et al. 2004). However, in Canada (where HT canola is widely cultivated), application of herbicide was seen to actually increase (Canola Council of Canada 2001). In fact, after years of continued GMHT oilseed rape cultivation, secondary adverse effects on application rates were reported. This was due to weeds becoming herbicide tolerant on- and offsite (Devos et al. 2004; Hayes et al. 2004). HT oilseed rape volunteers occurred in subsequent rotations (Devos et al. 2004), and multiple tolerances of volunteers to herbicides were recorded because of seed impurities and seed banks after outcrossing events between fields (Hall et al. 2000). Outcrossing to weedy relatives also occurred (Daniels et al. 2005); these weedy relatives, however, have not yet been proved to be fit enough to persist on cultivated fields (Hails and Morley 2005). Nevertheless, improved weed suppression with HT oilseed rape in many agronomic respects has been demonstrated (Westwood 1997; Bohan et al. 2005), and ultimately the technology remains popular with farmers because of its simplicity and reduced costs (Canola Council of Canada 2001; Graef et al. 2007). Genes have always moved between the natural and agroecosystem, and to date – despite the formation of hybrids between HT canola and wild relatives in Canada (Gressel

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2008) – the technology has proven safe and effective (Cerdeira and Duke 2006; Darmency et al. 2007; James 2007; Gressel 2008).

1.3.6

HT Maize

C. album, Amaranthus retroflexus, Abutilon theophrasti and several annual grass species cause economic losses in maize, if left uncontrolled. The option to use glyphosate in these systems is extremely valuable because it is a broad-spectrum herbicide that provides excellent control of a wide range of weed species. Resistance to glyphosate-herbicides in maize is conferred by the epsps gene in event GA21 (the GA21 trait for glyphosate-resistant maize relies on a modified maize epsps gene) (Roundup Ready), developed by DeKalb (now Monsanto) in 1998. This is now being largely replaced by event NK603, Roundup Ready corn 2, with two epsps expression cassettes under the transcriptional regulatory control of the rice (Oryza sativa L.) actin 1 (P-Ract1) and the enhanced cauliflower mosaic virus 35S (P-e35S) promoters to impart fully constitutive expression. Maize has also been developed with resistance to phosphinothricin herbicides via transformation with the pat gene in events T14 and T25, developed by Aventis (now Bayer CropScience), and marketed as LibertyLink. Maize is an open-pollinated, wind-facilitated species and gene flow via pollen is well recognized (Haslberger 2001). Thus, the movement of GM traits is a significant consideration in maize production (Luan et al. 2001; Ma et al. 2004). Generally, the introgression of GR traits in seed maize can be managed successfully (
1% outcross) by establishing isolation distances of 200 m between fields (Ma et al. 2004). However, in typical maize production regions of the US, these isolation distances are not possible and GR trait introgression into non-GR fields is prevalent. The occurrence of the GR transgene in non-GM maize can have significant economic consequences, if the grower of the non-GM maize has a contract to provide a GM-free product. Furthermore, incidences of GM gene introgression in local landraces of maize in Oaxaca, Mexico have been reported (Quist 2001). The implications for transgene occurrence reflect concerns for the maintenance of the genetic resource of the landrace maize. However, the initial report of transgene introgression was followed by a second report that suggested that no transgenes existed in these landraces of maize (Ortiz-Garcia et al. 2005). Regardless, given the adoption of GR maize, the discovery of transgene introgression into landrace maize is likely in the longer term.

1.3.7

HT Cotton

Cotton is a slow-growing plant, and only a limited selection of herbicides can be used for weed control. These two factors sometimes make weed control difficult. Cotton is a semitropical, perennial plant, although it is grown as an annual crop.

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Its growth is especially slow in the northern cotton belt in the US, where it is often planted early into cool soils. Post-emergence broadleaf control is accomplished with herbicides that can injure cotton but that are applied in a directed spray that misses most of the cotton plant; thus, weed control in cotton is particularly difficult. Cotton with resistance to glyphosate herbicides has been developed based on the expression of the CP4 epsps in events MON1445/1698, Monsanto, Roundup Ready1. Resistance to phosphinothricin herbicides is based on the expression of the bar gene in LLCotton25, Bayer CropScience LibertyLink1. Herbicide-tolerant cotton expanded from around 2% of the cotton acreage in 1996 to 26% in 1998, and reached 46% in 2000 (James 2001). In 2008, adoption of GM cotton in the US with either or both herbicide tolerance and Bt reached 86% (James 2007). There are limited reports that cotton demonstrates introgression at low frequencies (Ellstand et al. 1999). Pollen movement in cotton is dependent on insects. Cotton is predominantly self-pollinated and natural outcrossing is typically quite low (Xanthopoulos and Kechagia 2000). Thus, there is a very low probability that the GR transgene would move into non-GR cotton cultivars via pollen flow. While gene flow via GR cotton seed can occur, the consequences of this are not thought to be important.

1.3.8

HT Soybean

Soybean engineered for resistance to glyphosate herbicides has been developed by Monsanto with the CP4 epsps gene, event GTS-40-3-2 and is marketed as Roundup Ready1. Glyphosate-resistant soybeans were one of the earliest transgenic crops brought to market and they have experienced rapid adoption to the point that over 85% of US soybeans and 56% of soybeans globally are now glyphosate-resistant (James 2007). Soybeans are an autogamous (self-fertilizing) species with limited opportunity for pollen-directed gene flow (Palmer et al. 2001). Spontaneous gene flow in cultivated soybeans ranges from 0.02 to 5% depending on distance and is facilitated by thrips (Thrips tabaci) and honeybees (Apis mellifera). While the movement of the GR transgene has been observed in soybeans, there are extremely limited opportunities for this occurrence and pollen-mediated gene flow in GR soybeans is essentially a nonissue (Abud et al. 2004; Owen 2005). However, gene flow by seed is highly probable and represents a significant economic problem (Swoboda 2002; Owen 2005).

1.3.9

Other GMHT Crops

In terms of commercial crops, glyphosate-resistant alfalfa had been developed and was launched in 2006 (James 2007).

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However, of special interest genetically engineered herbicide-resistant crops are being tested as a solution to the parasitic plants Orobanche spp. (broomrapes) and Striga spp. (witchweeds), which parasitize the roots of important crops, heavily reducing yields. Joel et al. (1997) showed that the use of three target-site resistances decimated Orobanche, demonstrating the potential of transgenic herbicide-resistant crops in the control of parasitic weeds. This is potentially of great importance as every year parasitic plant damage to crops accounts for an estimated US$7 billion in yield loss in sub-Saharan Africa, and affects the welfare and livelihood of over 100 million people. For this technology to have a great impact in Africa, crops adapted to African agroecologies need to be available to farmers.

1.3.10

Changes in Agronomic Practice

The adoption of GM Crops, and specifically glyphosate-resistant crops, has resulted in significant changes in agronomic practices. Most obviously, these changes include the increase glyphosate use at the cost of other herbicides and the manner and frequency in which glyphosate is used. In addition, the amount of tillage that is conducted for crop production has significantly changed (Young 2006; Service 2007a, b; Foresman and Glasgow 2008). This reduction in tillage has an important benefit of reducing the use of petroleum-based fuels as well as an implicit gain in time use efficiency by growers. A significant reduction in pesticide use has been attributed to the adoption of herbicide-tolerant (HT) crops (Sankula 2006). Furthermore, the benefits ascribed to herbicide-tolerant crops have dramatically changed the crop cultivars selected by growers and have hastened the development of new transgenic crops for commercial distribution worldwide (Duke 2005; Dill et al. 2008). Despite wide scale commercial plantings of GM soybean, canola, cotton and maize, several herbicide-resistant crops have been developed but have not been commercially introduced. Notably, sugarbeet (Beta vulgaris) cultivars that are resistant to glyphosate were deregulated in 1999, but not commercially offered until 2008 because of concerns about the acceptability of sugar refined from a GM crop (Duke 2005; Gianessi 2005). The development of GM rice (O. sativa) cultivars modified to be resistant to glufosinate herbicide proceeded from 1998 to 2001, but were withdrawn and commercial development terminated because of concerns about market acceptance of the GM rice (Gealy and Dilday 1997; Gealy et al. 2007). Similarly, wheat (Triticum aestivum) glyphosate-resistant cultivars were under development but the program was terminated in 2004 (Dill 2005). While the GR-based wheat production systems demonstrated excellent opportunities for improved weed management, concerns about the acceptance of the flour made from GR wheat cultivars as an export commodity in GM-adverse countries resulted in the decision to halt further development (Stokstad 2004; Howatt et al. 2006). In addition to concerns centered on consumer acceptability, several concerns over potential environmental impact have been raised. These are addressed below.

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1.3.11

N. Ferry and A.M.R. Gatehouse

Weeds, Gene Flow, Invasiveness, and Biodiversity Impacts

There have been significant concerns regarding the potential of GM crops, particularly glyphosate-resistant crops, to become major agricultural problems (by invasion, volunteerism) or to create “superweeds” by cross-pollination (Ellstrand 2003). 1.3.11.1

Gene Flow

While GM crops do have the potential to cross-pollinate other crops and wild relatives, there are four basic elements determining the likelihood and consequences of gene flow: first, the distance of pollen movement from the GM crop; second, the synchrony of flowering between crop and pollen recipient; third, sexual compatibility between crop and recipient; and fourth, ecology of the recipient species (Dale et al. 2002). Research has shown that pollination declines sharply with distance from the pollen source (Lutman 1999) and one could reduce the chances of GM pollen reaching other crops through the use of isolation or buffer zones, although pollen may travel further if plants are insect-pollinated. Ellstrand et al. (1999) reviewed the sexual compatibility of crops with weeds and feral species. For example, oilseed rape (canola), barley, wheat and beans can hybridize with weeds in some countries; however, in the UK, for example, the probability of hybridization with weeds is considered minimal for wheat, low for oilseed rape and barley, and high for sugarbeet. Although sugarbeet can readily hybridize, in the case of herbicide-tolerant varieties of sugarbeet the crop would be harvested before flowering and hence shed no pollen. Indeed, methods have been developed to block expression in the pollen of transgenic plants, including engineering of the chloroplast genome (Heifetz 2000) and transgene mitigation strategies (Gressel 2008). Transgene Mitigation Strategies Transgenic crops may interbreed with nearby weeds, increasing their competitiveness, and may themselves become a “volunteer” weed in the following crop. The desired transgene can be coupled in tandem with genes that would render hybrid offspring or volunteer weeds less able to compete with crops, weeds and wild species. Genes that prevent seed shatter or secondary dormancy, or that dwarf the recipient could all be useful for mitigation and may have value to the crop. Many such genes have been isolated in the past few years (Gressel 1999). Examples include: apomixis (asexual reproduction) as a fail-safe so that the seed is actually of vegetative origin and not from sexual pollination (Zemetra et al. 1998); chromosome-specific cytogenetic fail-safes (the risk of transgenic traits spreading into weeds can be reduced by orders of magnitude by using cytogenetic mapping to locate transgenes and releasing only those transgenic lines in which it is on a genome incompatible with local weeds (Gressel and Rotteveel 2000)); plastome-specific cytogenetic fail-safes (it is possible to introduce some traits into the chloroplast or

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mitochondrial genomes;thus, in species where these genomes are completely maternally inherited, the transgenes cannot move into other species (Daniell et al. 1998)); seed dormancy (the transgenic abolition of secondary dormancy is neutral to the crop but deleterious to weeds (Gressel 1999)); seed ripening and shattering (uniform ripening and anti-shattering genes are harmful to weeds but neutral for some crops (Schaller et al. 1998)), and dwarfing (dwarfing is disadvantageous for weeds, because they can no longer compete with the crop for light (Gressel 1999)). However, these strategies are not as of yet fully developed and in use, with the exception of plastid transformation. 1.3.11.2

Invasiveness

The potential exists for GM crops to become invasive. There has been a great deal of concern that such crops could persist in the wild and disperse from their cultivated habitat. However, recent studies (and experience from the field) have indicated that the ability of GMHT crops to invade and persist was actually no better than that of their conventional counterparts (Crawley et al. 2001). Finally, GM crops persisting in fields after harvest thus becoming a weed in a different crop may be dealt with in two ways; simple treatment with an appropriate herbicide or mitigation technologies that prevent the transgene being carried over to the next generation (Gressel 2008). 1.3.11.3

A Dose of Reality

In order to put these concerns into perspective, one must understand that flow from the agroecosystem to natural ecosystems has always occurred. Gene flow is a continuing process and is the source of biological diversity (Thies and Devare 2007). There has always been gene flow from commercial crops to relatives living in near proximity (Ferry and Gatehouse 2009). In reality, the vast majority of the major cultivated crops have no wild or weedy relatives outside of their centers of origin (Gressel 2008); however, some crops are grown in areas where gene flow may occur and appropriate measures must be taken in these areas.

1.3.12

Coexistence of GM and Non-GM Crops

A pervasive problem that exists with the production of GR crops is their coexistence with non-GM crops (Byrne and Fromherz 2003). The issue of coexistence includes three possibilities: (1) introgression of the trait via pollen (pollen drift), (2) containment of plant products during the production year (grain segregation), and (3) volunteer GRC plants in following years (Owen 2005). While GR crops and non-GM crops can coexist, growers must go to great lengths to accomplish segregation (Anonymous 2007). Grain segregation, while difficult to

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maintain, can be accomplished and will effectively minimize the impact of GR crops on non-GM crops. Given that the mixing of grain is equivalent to loss of intellectual property (IP), this can be costly to growers; thus, appropriate tactics to isolate GR crops from non-GM crops must be in place (Owen 2000). Controlling volunteer GR crops is also relatively easy, depending on the rotational crop, but does require diligence on the part of the grower (Owen 2005). The introgression of the HT trait via pollen movement is another possibility and management of this problem is considerably more difficult, particularly in open-pollinated crops such as maize (Luan et al. 2001; Palmer et al. 2001; Westgate et al. 2003; Abud et al. 2004). A number of factors affect the success of maize pollen movement and subsequent pollination, and generally, the greater the distance between the pollen source and the donor, the less likely is the introgression of the GR trait (Luan et al. 2001; Westgate et al. 2003). However, given the tolerance levels established for some GM traits in non-GM crops, the isolation distances required to mitigate the risks of gene flow are too large to be realistic (Matus-Cadiz et al. 2004). Other open-pollinated crops have also undergone a great deal of scrutiny (Charles 2007; Fisher 2007; Harriman 2007; Weise 2007). It is suggested that the issues of the coexistence of GRCs with non-GM crops will continue to be a concern as long as there are economic differences between the crop cultivar types (Hurburgh 2000; Ginder 2001; Hurburgh 2003). Nevertheless, coexistence guidelines and buffer zone (isolation zone) regulations are in existence (Boller et al. 2004).

1.3.13

Stacked Traits

Stacked products are a very important feature and future trend for GM crops, which meets the multiple needs of farmers and consumers; these are now increasingly deployed by ten countries – US, Canada, the Philippines, Australia, Mexico, South Africa, Honduras, Chile, Colombia, and Argentina (James 2007). In fact, 37% of all GM crops in the US in 2007 were stacked products containing two or three traits that delivered multiple benefits. Currently, there are a number of GM crops that have stacked herbicide resistance and Bt events (Owen 2006). In some events, the pat gene is used as a marker for the Bt – the pat gene also confers resistance for glufosinate herbicide. Interestingly, the Bt trait is combined with a GR hybrid, the resultant GM cultivar is resistant to both glyphosate and glufosinate. Monsanto has announced new transgenic maize cultivars that will combine several Bt events plus two herbicide resistance events. The Bt event functions ecologically differently compared to herbicide-resistant transgenes; the transgene for herbicide resistance can be considered benign to the weed population and has no impact until the herbicide is applied (Owen 2009). In contrast, Bt exerts continuous selection pressure on the target insect whether the insect population is at economic threshold or not. Furthermore, given the potential for the introgression of transgenes into near-relatives, Bt could potentially improve the fitness of compatible weed species (Snow and Pedro 1997). For example, the fitness of canola was increased by the inclusion of Bt (Steward et al. 1997); thus, gene flow

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was kept in check. However, there is no reason to believe that stacked traits will have any greater impact on nontargets than the crops with single traits, and for Bt non-target impacts have been shown to be minimal (Ferry and Gatehouse 2009).

1.3.14

Conclusions

Herbicide-resistant crops, specifically GR crops, have been globally adopted as the basis for the production of maize, soybean, cotton and canola (Dill et al. 2008). Their adoption provides economic benefits to agriculture and has major positive impacts on the environment; specifically, conservation tillage which can reduce soil erosion (Duke and Powles 2008; Shipitalo et al. 2008). However, growers must adopt measures to proactively sustain the technology (Mueller et al. 2005; Johnson and Gibson 2006; Sammons et al. 2007; Christoffoleti et al. 2008) and to steward GR crops to avoid the evolution of GR weeds (Duke and Powles 2008; Owen 2009). While there are tactics that are capable of mitigating some of the other concerns, including the risks of transgene introgression into near-relative plants (Snow and Pedro 1997; Gepts and Papa 2003), an improved process to assess the environmental risk of GR crop technologies and communicating those risks to the lay public should be in place (Owen 2009).

1.4 1.4.1

Biotic Stress due to Plant Pathogens Crop Losses due to Plant Pathogens

Plants are challenged constantly by many different potential pathogens (Table 1.4). There are hundreds of thousands of viral, bacterial, and fungal species in the world and thousands of these are pathogens that infect plants (Chrispeels and Sadava 2003). Any one pathogen can severely depress the yield of a given crop. Pathogens may reduce yield by causing tissue lesions; by reducing leaf, root, or seed growth; or by clogging up vascular tissue and causing wilt. Even in the absence of obvious symptoms, pathogens can still be a major metabolic drain that reduces productivity. They may also cause pre- or postharvest (stored products) damage to the harvested product. Table 1.4 Organisms that cause infectious disease in plants Types of plant pathogens Fungi Ascomycetes e.g., Fusarium, Verticillium Basidiomycetes e.g., Rhizoctonia, Puccinia (rust) Oomycetes e.g., Phytopthora (blight) Bacteria e.g., Xanthomonas, Pseudomonas Phytoplasmas and Spiroplasmas Viruses Viroids and virus-like organisms Nematodes, protozoa, and parasitic plants are also considered plant pathogens

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1.4.1.1

N. Ferry and A.M.R. Gatehouse

Current Disease Control Measures

Disease control in commercial crops is both economically important and environmentally controversial. Disease management includes: quarantine, cultural practices such as modification of the plant environment (e.g., soil management) to reduce pest numbers, host plant resistance, biological control and chemical control. Chemical control can be highly effective. The first fungicide was discovered in the 1880s when Bordeaux mixture (a mixture of copper sulfate and lime) was found to suppress grape downy mildew. Numerous compounds with antifungal or antibacterial activity have since been discovered, most often applied as sprays, dusts or seed coatings. Many older compounds are broad spectrum and toxic with the newer chemistries acting systemically with a narrower target range. Such chemicals are expensive and normally reserved for use on high-value fruit and vegetable crops (Ferry and Gatehouse 2009). They are expensive to manufacture and a great deal of investment must be put into human and ecological safety testing before a product can be released (Chrispeels and Sadava 2003). As with other chemical control strategies, pathogens can evolve resistance to these compounds. Thus, plant breeders have often relied on genetic disease resistance traits to manage pathogens of particular crops. In classical plant breeding, this has relied on crosses between elite crops and wild relatives (that are more genetically diverse) to introduce new disease resistance traits into the crops. Extensive backcrossing of the elite line is then required to eliminate the undesirable traits in the wild relative and thus makes traditional breeding a time-consuming process with a time of about 15 years required before a new resistant variety is available for release to growers. Nevertheless, such traditional plant breeding has had significant successes. For example, the development of F1 hybrid crops (derived from crossing two inbred lines) resulted in vastly improved yields (Chrispeels and Sadava 2003). Despite this enormous success, the F1 hybrids also serve as a warning as to the dangers of largescale monoculture and the need for effective and durable disease resistance. In 1970, the highly successful but genetically uniform F1 maize crops in the US were left devastated by a disease, southern corn leaf blight (Bipolaris maydis Nisik), which caused damage to 710 million bushels of maize (Ullstrup 1972). In extreme cases, crop disease can change political as well as agricultural landscapes!

1.4.1.2

The Irish Potato Famine

The Irish Potato Famine (the Great Hunger) started in 1845, lasted until 1849–1852 and led to the death of approximately one million people through starvation and disease; a further million are thought to have emigrated as a result of the famine. Some estimate that the population of Ireland was reduced by 25% (Kinealy 1995). The cause of the famine was a potato disease commonly known as late blight caused by the oomycete, Phytophthora infestans. Although blight ravaged potato crops throughout Europe, the impact and human cost in Ireland was high as a third of the

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population was entirely dependent on the potato for food; it was also exacerbated by a host of political, social and economic factors. The famine was a watershed in the history of Ireland. Its effects permanently changed the island’s demographic, political and cultural landscape. For both the native Irish and the resulting diaspora, the famine became a rallying point for nationalist movements. The fallout of the famine continued for decades afterwards and Ireland’s population still has not recovered to prefamine levels. 1.4.1.3

The Bengal Famine of 1943

The Bengal famine of 1943 occurred in British administered Bengal. It is estimated that around four million people died from starvation and malnutrition during the period. During the Second World War, the British had just suffered defeat in nearby Burma and British authorities feared a subsequent Japanese invasion of India by way of Bengal, so emergency measures were introduced to stockpile food for British soldiers and prevent access to supplies by the Japanese in case of an invasion. However, in the rice-growing season of 1942 weather conditions were exactly right to encourage an epidemic of the rice disease brown spot following a cyclone and flooding; brown spot in rice is caused by the fungus Helminthosporium oryzae. When food shortages became apparent, the Bengal government reacted to the crisis incompetently refusing to stop the export of food from Bengal to allied soldiers and failing to provide adequate famine relief in Bengal itself. Winston Churchill was Prime Minister at the time and while his involvement in the disaster, and indeed his knowledge of it remains unclear, it has been suggested that in response to an urgent request by the Secretary of State for India to release food stocks for India, Churchill responded with a telegram asking if food was so scarce, “why Gandhi hadn’t died yet” (Mishra 2007). Needless to say, whether this is completely true or is in fact an unfortunate misquote used out of context, the British Administration in India declined in popularity and there is little doubt that this incident fuelled feelings for Indian Independence. Given the vast array of diseases that threaten crops, and the scale of disaster that can ensue, it is a wonder that crops be produced at all?

1.4.2

Plant Defense Against Pathogens

Plants have evolved mechanisms to resist pathogen invasion that consist of different defense layers. Firstly, waxy coatings on epidermal cells provide a physical barrier; secondly, plants contain large amounts of preformed secondary metabolites that have antimicrobial activity (constitutive defenses including glucosides, saponins, alkaloids, antifungal proteins, antifeedants and enzyme inhibitors), these are effective in many cases – but pathogens have evolved enzymes capable of detoxifying these compounds. Often induced defenses are the plants’ last line of defense against pathogens and are sufficient to (partially) ward off invading microorganisms.

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Essentially, plants have two major types of induced disease resistance, basal defense and resistance (R) gene-mediated defense. All plants have basal defense, this is a general immune response to pathogens and other environmental stresses. R-gene-mediated defense is more specific and is only found in certain plant species. R-gene-mediated defense involves recognition of a specific pathogen effector by a plant ligand receptor. These pathogen effectors can suppress plant basal defense, making any plant without the R-gene defense susceptible. The ligand-effector recognition can result in a dramatic immune response such as cell death. In tomato, Solanum lycopersicum, the Mi gene, a member of a large family of R-genes, mediate resistance to potato aphids, whiteflies, and root-knot nematodes (Kaloshian and Walling 2005). Both types of plant defenses (R and basal) involve signaling via three major plant hormones: salicylic acid, jasmonic acid, and ethylene (ETH). In some instances, defense responses are induced distal to the site of infection and this is referred to as systemic acquired resistance (SAR). At least three nonspecific induced defense pathways are described which are triggered by these specific signaling molecules: (a) The salicylic acid (SA)-dependent pathway is induced by necrosis inducing pathogens and triggers systemic acquired resistance (SAR) (b) A second pathway is triggered by nonpathogenic rhizobacteria, it is dependent on jasmonic acid (JA) and ethylene (ETH) and constitutes induced systemic resistance (ISR) (c) JA and ETH regulate a third pathway that is effective against a different set of pathogens and not affected by ISR Most of the inducible defense-related genes are regulated by these signaling pathways (Delaney et al. 1994; Sticher et al. 1997; Van Loon 1997; Reymond and Farmer 1998; Knoester et al. 1998; Ananieva and Ananiev 1999). Defense gene regulation has been extensively studied and severa1 rapid processes characteristic of the hypersensitive response (HR) appear to involve primarily activation of preexisting components rather than changes in gene expression. One of these rapid processes is the striking release of reactive oxygen species.

1.4.2.1

Oxidative Burst

The oxidative burst, a rapid production of reactive oxygen species (ROS), is a welldocumented early plant response to biotic stress (e.g., Apel and Hirt 2004). ROS comprise radicals and other nonradical but reactive species derived from oxygen. Among them, the superoxide anion (O 2 ) and hydrogen peroxide (H2O2) exert various effects on cells, directly or in cooperation with other molecules. In excess, ROS pose a threat to important biomolecules and cell membranes. One of the consequences of ROS activity is oxidative damage of membrane integrity due to lipid peroxidation processes which may result in the generation of highly cytotoxic compounds. Glutathione-S-transferases (GSTs), induced upon pathogen attack, may detoxify lipid peroxides by conjugating them with glutathione (GSH). These

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enzymes can also catalyze the GSH-dependent reduction/inactivation of H2O2, forming glutathione disulfide (GSSG) and increasing GSH synthesis by feedback induction (Marrs 1996). On the other hand, numerous studies indicate an essential role of ROS in plant defense responses to biotic stress. In addition to direct antimicrobial activity and contribution to the strengthening of barriers against pathogens, recent reports point to H2O2 and O 2 as signal transduction agents activating defense pathways and as key mediators in cell death during hypersensitive response (HR) (Grant and Loake 2000). To maintain a balance between negative and beneficial functions of ROS, their levels are strictly controlled by a complex and flexible network of antioxidant systems (Mittler et al. 2004). The major enzymatic ROS-scavenging components of this network are superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX). Superoxide dismutases dismute O 2 to H2O2, an excess of which may be subsequently detoxified by CATs and/or APX. Ascorbate peroxidases, in contrast to CATs localized in peroxisomes, are present in almost all cellular compartments; they exhibit a high affinity to H2O2 and are considered to be responsible for the fine modulation of ROS level (Mittler 2002). Moreover, APX, in cooperation with two main low-molecular antioxidants, ascorbate (Asc) and glutathione (GSH), and enzymes for their regeneration, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase (GR), constitute the ascorbate–glutathione (Asc–GSH) cycle, believed to be the central part of the antioxidant network (Noctor and Foyer 1998). Ascorbate, present at high concentrations in all cellular compartments and capable of direct scavenging of O 2 and hydroxyl radicals, is considered to be the most powerful cell antioxidant (Noctor and Foyer 1998). Ascorbate and glutathione are the major cellular redox buffers, which together with their oxidized forms, dehydroascorbate (DHAsc) and glutathione disulfide (GSSG), enable cells to maintain a redox balance. Changes in the levels or redox state of ascorbate and glutathione pools as well as in H2O2 homeostasis, and thereby in cellular/compartment redox state, are considered to be pivotal signaling events influencing gene expression and modulating the plant defense response (Pastori and Foyer 2002; Foyer and Noctor 2005). Numerous genes are induced during the plant defense response and presumably these function in host plant pathogen defense. Induced defenses include the activation of phytoalexins (including terpenoids, glycosteroids and alkaloids) and PR proteins (including chitinase, b-1,3 glucanase and other antimicrobial proteins) and the production of reactive oxygen species (ROS) leading to a hypersensitive response (HR).

1.4.3

Utilizing Defense Mechanisms

Most conventional breeding strategies have relied on identifying major resistance genes (often in wild or ancestral plants) while fewer have concentrated on breeding for polygenic resistance. New opportunities and strategies to enhance disease

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resistance are afforded by genetic engineering of plants – these may follow the single gene model, or may involve manipulation of defense-activating mechanisms.

1.4.4

Transgenic Disease Resistance

Many transgenic-based approaches exist for conferring enhanced levels of disease resistance, as exemplified by Fig. 1.6, which provides a simplified model. 1.4.4.1

Expression of Single Genes

R-Genes A very effective defense mechanism in plants is gene-for-gene resistance, this induced resistance mechanism is based on the interaction between plant-derived Pathogen

ty

ici en g o th Pa tors c fa

(3) Pathogen mimicry

(1b) Induced defense

(2a) Detection of pathogen

(1a) Constitutive defense (2b) Defense regulation Host cell

Nucleus

Transgenic Strategies. 1a. Constitutive expression of antimicrobial factors, 1b. Pathogen induced expression of one or more genes, 2a. Altering recognition of the pathogen (e.g. R-genes) and 2b. altering downstream regulators, 3. Priming recognition of pathogens (genetic vaccination).

Fig. 1.6 Simplified model of transgenic strategies to enhance plant defense

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resistance (R) gene products and an avirulence (avr)-gene product (elicitor) produced by the pathogen. The interaction is generally very specific and results in the triggering of strong resistance responses including hypersensitive response (HR) and localized cell death at the point of infection. There is no common structure between avirulence gene products, except that most are secreted proteins. Because there would be no evolutionary advantage to a pathogen keeping a protein that only serves to be recognized by the plant, it is believed that the products of avr genes, sometimes known as effector proteins, play an important role in virulence in genetically susceptible hosts. The guard hypothesis model proposes that the R proteins interact, or guard, a protein known as the guardee which is the target of the Avr protein. When it detects interference with the guardee protein, it activates resistance. R-gene specificity (recognizing certain avr gene products) is believed to be conferred by the leucine-rich repeats (LLRs). LRRs are multiple, serial repeats of a motif of approximately 24 amino acids in length, with leucines or other hydrophobic residues at regular intervals. LRRs are involved in protein–protein interactions, and the greatest variation amongst resistance genes occurs in the LRR domain. LRR swapping experiments between resistance genes in flax rust resulted in the specificity of the resistance gene for the avirulence gene changing (Bergelson et al. 2001). Gene-for-gene resistance thus provides obvious targets to engineer disease resistance in transgenic plants. However, pathogens can often breakdown R-gene-mediated resistance if the corresponding avr gene mutates becoming inactivated. Pathogen adaptation is also seen in conventionally bred crops. The use of R-genes is limited by them conferring resistance to only a single pathogen or race of pathogen; however, they can provide effective (even broad spectrum) resistance if transformed by genetic engineering into new species or new genera of plant (Oldroyd and Staskawicz 1998). For example, Rxo1, an R-gene derived from maize, a nonhost of the rice bacterial pathogen Xanthomonas oryzae pv. oryzicola, was successfully transformed into rice and shown to confer resistance against this pathogen (Zhao et al. 2005). Interspecies differences do radically influence R-gene function making it preferable to use R-genes from related species (Ayliffe and Lagudah 2004); however, the transgenic approach circumvents the tedious and time-consuming backcrossing required to introduce a single trait via traditional crop breeding.

Constitutive Expression of Inducible Antimicrobial Factors Several examples of transgenic strategies to enhance plant constitutive defenses are described below. This is an extension of the single gene paradigm that has worked so well for insect-resistant transgenic plants. Expression of Chitinases Chitinases are glycosyl hydrolases catalyzing the degradation of chitin, an insoluble linear b-1,4-linked polymer of N-acetylglucosamine. They are produced by a wide

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range of organisms from microbes to insects and mammals, including plants (Kasprzewska 2003). Plants produce chitinases as defense against chitin-containing fungal pathogens by inhibiting spore germination, germ tube elongation and degrading hyphal tips (Khan et al. 2008). Transgenic plants expressing chitinase ChiC genes have already been produced in several species. In carrot, the tobacco class I ChiC gene has shown resistance to Botrytis cinerea (Punja and Raharjo 1996). Transgenic tobacco transformed with bean class I ChiC exhibited enhanced resistance to Rhizoctonia solani (Broglie et al. 1991) and Alternaria alternata (Lan et al. 2000). The rice chitinase gene (RCC2) exhibited increased resistance to Sphaerotheca humuli in transgenic strawberry (Asao et al. 1997). Peanut (Arachis hypogae) plants transformed with tobacco ChiC gene were resistant to a fungal pathogen (Cercospora arachidicola), the causal organism of leafspot disease (Rohini and Rao 2001). Potato has been transformed with the Streptomyces griseus ChiC gene and resistance to Alternaria solani (causal agent of early blight) demonstrated (Kahn et al. 2008) and cotransfer and expression of ChiC, glucanase, and bialaphos resistance (bar) genes in creeping bent grass conferred resistance to the fungal pathogens Sclerotinia homoeocarpa and R. solani (Wang et al. 2003). Expression of Defensins Defensins are the best characterized cysteine-rich antimicrobial proteins in plants (Broekaert et al. 1995). All known members of this family have four disulphide bridges and are folded in a globular structure that includes three b-strands and an a-helix (Segura et al. 1998). Inhibition of fungal growth by defensins seems to occur by permeabilization of the plasma membrane through binding to a putative receptor (De Samblanx et al. 1997). Genes encoding plant defensins are developmentally regulated, with predominant expression in the outer cell layers, and are induced in response to pathogen infection and stress (Segura et al. 1998). Enhanced tolerance to the fungus Alternaria longipes is observed in transgenic tobacco overexpressing a radish defensin (Rs-AFP2) (Chiang and Hadwinger 1991). In support of this role, Manners et al. (1998) show that the promoter of the plant defensin gene PDF1.2 from Arabidopsis is systemically activated by fungal pathogens and responds to methyl jasmonate, but not to salicylic acid. Expression of Oxalate Oxidase and H2O2 -Generating Enzymes Rapid generation of superoxide and accumulation of H2O2 is a characteristic early feature of the hypersensitive response following perception of pathogen avirulence signals. In germinating barley and wheat seeds as well as in barley leaves challenged with powdery mildew, oxalate oxidase activity has been identified as a generator of H2O2 (Lane et al. 1993; Zhou et al. 1998). Oxalate oxidase utilizes oxalic acid and oxygen as substrates producing H2O2 and CO2. For certain necrotrophic fungi, such as Sclerotinia sclerotiorum, oxalic acid is a major pathogenicity determinant, significantly altering environmental pH (Cessna et al. 2000).

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Pathogen-inducible oxalate oxidase both acts as a generator of H2O2, killing the invading pathogen, and simultaneously detoxifies the acid, which is phytotoxic at high concentrations. Sunflower plants expressing a wheat oxalate oxidase accumulate enhanced levels of salicylic acid and PR1 even in the absence of pathogen infection and display improved tolerance to Sclerotinia infection (Hu et al. 2003). This effect is most likely due to the production of H2O2 from endogenous oxalic acid. Amine oxidases are a class of enzymes mainly found in the plant apoplast that act on a variety of amine substrates, including mono-, di- and polyamines and release the corresponding aldehyde as well as NH3 and H2O2. Amines are present in the plant apoplast and accumulate in response to environmental stress (Bolwell et al. 1999). Thus, in plants several systems are available that can produce H2O2 following pathogen attack. The importance of H2O2 in plant defense has clearly been shown by several groups who have reported increased pathogen resistance in transgenic plants by introducing either H2O2-generating systems or by inhibiting H2O2-degrading systems. The expression of a fungal glucose oxidase resulted in enhanced resistance to P. infestans and Erwinia carotovora in potato (Wu et al. 1995). Similarly, the prevention of H2O2 breakdown resulted in higher levels of H2O2 and increased disease resistance (Chamnongpol et al. 1998). Whether this improved pathogen tolerance is due to the direct antimicrobial effect of H2O2, or due to the fact that the plant defense system is induced by the increased levels of H2O2, was, in either case, not investigated. Inducible Expression of Single Antimicrobial Factors Stilbene Synthase Production of stilbene, a phytoalexin, is a well-characterized defense reaction in grape vine. Stilbene plays an important role in resistance to fungal and bacterial infection in plants. It strongly inhibits the growth of fungi and sprout of spores. Stilbene synthase gene (Vst1) is responsible for the synthesis of resveratrol (trans3,40 5-trihydroxystilbene) when plants are challenged by fungal diseases. Thus, introduction of stilbene synthase genes into transgenic plants can be used to induce synthesis of phytoalexins. Vst1 has been transferred into many plants to enhance fungal resistance including common spring wheat using biolistic transformation, where plantlets with mild resistance to powdery mildew were identified (Leckband and Lo¨rz 1998). Other crops transformed to produce stilbene include: tomato (Thomzik et al. 1997); apple (Szankowski et al. 2003); and Vst1 overexpressed in its native grape (Fan et al. 2008). 1.4.4.2

Activation of Expression of Multiple Genes

Polygenic Resistance There are several types of disease resistance in terms of the effects of genes on the plant and pathogen. However, in terms of the number of genes involved, there are

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two general types of resistance. The first (as described above for R-genes) is called major-gene or single-gene resistance. This type of resistance is well defined and more easily measured; it is sometimes called qualitative resistance because plants are either resistant or susceptible, without intermediate levels. The second type, called polygenic resistance, involves several or many genes. This type of resistance is harder to define; exactly which genes are involved may be unknown. It usually is effective against all races of a pathogen. This type is often called quantitative, because there are intermediate levels ranging from resistant to susceptible. It is also harder to measure than major-gene or single-gene resistance. Often polygenic resistance does not give a plant as high a level of resistance as major gene resistance. Polygenic durable resistance to multiple diseases of a crop would be highly desirable. In nature, most host plant resistance is based on multiple genes and a diverse set of resistant factors. This diverse, polygenic resistance system helps to prevent plant-feeding microorganisms from overcoming the resistance in the host plant. At present, polygenic resistance may be bred into a crop via quantitative trait loci (QTL) analysis, and the use of molecular markers in molecular breeding approaches; promising transgenic strategies (as discussed below) activate polygenic resistance via elicitor-induced resistance and activation of multiple genes.

Detection of Pathogens; Expression of Elicitors and Defense Activators Most plant disease resistance (R) proteins contain a series of leucine-rich repeats (LRRs), a nucleotide binding site (NBS), and a putative amino-terminal signaling domain, termed NBS–LRR proteins. The LRRs of a wide variety of proteins from many organisms serve as protein interaction platforms, and as regulatory modules of protein activation. Genetically, the LRRs of plant R proteins are determinants of response specificity, and their action can lead to plant cell death in the form of the hypersensitive response (HR). It is thought that this halts pathogen growth. In the absence of specific recognition, a basal defense response also occurs, which is apparently driven by pathogen-associated molecular patterns (PAMPS), such as flagellin and lipopolysaccharides (LPS) elicitors of defense responses (Belkhadir et al. 2004). The basal defense response overlaps significantly with R-proteinmediated defense, but is temporally slower and of lower amplitude. However, Takakura et al. 2008 show that expression of a bacterial flagellin gene (N1141) in transgenic rice triggers disease resistance and enhances resistance against blast (Magnaporthe grisea). Furthermore, a number of transgenic plants expressing NBS–LRR proteins under the control of the CaMV 35S promoter have been described (Mindrinos et al. 1994; Ellis et al. 1999; Stokes et al. 2002). For example, Grant et al. (2003) show that an Arabidopsis mutant, adr1 (activated disease resistance), contains both elevated levels of SA and ROIs, accumulates a number of defense-related gene transcripts and exhibits resistance against a number of microbial pathogens. ADR1 encodes a distinct NBS–LRR protein which possesses N-terminal kinase subdomains. Furthermore, transient expression of ADR1 is sufficient to engage defense-related gene expression and establish disease resistance

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in the absence of significant seed yield penalty. ADR1 plants showed striking resistance against both P. parasitica (Noco2) and another biotrophic pathogen, E. cichoracearum (UED1).

Pathogen Phytosensing A related strategy for crop defense involves engineered phytosensors indicating the presence of key plant pathogens to provide an important first line of defense (Mazarei et al. 2008). Plant defense mechanisms are highly regulated on the transcriptional level, and can be induced by chemical elicitors produced by pathogens. These elicitors have been shown to cause changes in gene expression, which initiate a whole plant response from a localized encounter with a pathogenic organism (Metraux et al. 2002). This is controlled by signal transduction pathways, inducible promoters and cis-regulatory elements corresponding to key genes involved in HR, SAR, ISR, and pathogen specific responses, any of which could be useful in building phytosensors. Cis-acting elements are conserved among plant species, which enables them to be used efficiently as synthetic inducible promoters. Employing synthetic promoters with potential inducible elements to engineer plants that can sense the presence of plant pathogens at the molecular level provides novel technologies for monitoring and increasing resistance to diseases (Gurr and Rushton 2005). Identified inducible promoters and cis-acting elements could be utilized in plant sentinels, or “phytosensors,” by fusing these to reporter genes to produce plants with altered phenotypes in response to the presence of pathogens. Mazarei et al. (2008) have employed cis-acting elements from promoter regions of pathogen-inducible genes as well as those responsive to the plant defense signal molecules salicylic acid, jasmonic acid, and ethylene. Synthetic promoters were constructed by combining various regulatory elements supplemented with the enhancer elements from the cauliflower mosaic virus CaMV 35S promoter to increase basal level of GUS expression. Histochemical and fluorometric GUS expression analyses showed that both transgenic Arabidopsis and tobacco plants responded to elicitor and phytohormone treatments with increased GUS expression when compared to untreated plants. Pathogen-inducible phytosensor studies analyzed the sensitivity of the synthetic promoters against virus infection. Transgenic tobacco plants infected with alfalfa mosaic virus showed an increase in GUS expression when compared to mock-inoculated control plants. The end goal of such studies is to engineer transgenic plants for the purpose of early pathogen detection.

1.4.4.3

Regulation of Inducible Defenses

The overexpression of regulatory genes provides another tool to activate plant defenses in response to pathogen attack. For example, transduction of the SA signal requires the function of NPR1 (also known as NIM1), a regulatory protein that was

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identified in Arabidopsis through genetic screens for SAR-compromized mutants (Cao et al. 1994; Delaney et al. 1995; Shah et al. 1997). Mutant npr1 plants accumulate normal levels of SA after pathogen infection but are impaired in their ability to express PR genes and to mount a SAR response. The NPR1 gene encodes a protein with a BTB/BOZ domain and an ankyrin-repeat domain (Cao et al. 1997). Upon induction of SAR, NPR1 is translocated to the nucleus (Kinkema et al. 2000) where it interacts with members of the TGA/OBF subclass of basic domain/leucine zipper (bZIP) transcription factors (Zhang et al. 1999; Despre´s et al. 2000; Zhou et al. 2000; Subramaniam et al. 2001; Fan and Dong 2002) that are involved in the SA-dependent activation of PR genes (Lebel et al. 1998; Niggeweg et al. 2000). Physical interaction between NPR1 and TGA transcription factors has been shown to be required for the binding activity of these factors to promoter elements that play a crucial role in the SA-mediated activation of PR genes (Despre´s et al. 2000; Fan and Dong 2002). In separate studies NPR1 overexpression and enhanced resistance are correlated with either elevated or earlier expression of PR transcripts (Cao et al. 1998). Genes with high sequence similarity to NPR1 are found in Arabidopsis, tobacco, tomato, rice and maize (Campbell et al. 2003). Overexpression of NPR1 in rice has been shown to enhance resistance to bacterial blight (X.o.o) (Chern et al. 2001).

1.4.5

Pathogen Mimicry and Virus Resistance

Numerous reports concern transgenic resistance to plant viruses (Fuchs and Gonsalves 2007) in which RNA-mediated gene silencing is the predominant strategy (viral RNA is degraded and viral DNA is deactivated by methylation). Most of these strategies can be categorized as pathogen mimicry. Transgenes constitutively expressed to provide RNA-mediated virus resistance fall into three main types: 1. Sense or antisense viral sequences 2. Inverted repeats/hairpin RNA of viral sequences, and 3. Sequences of engineered microRNAs targeted against the virus RNA-mediated resistance against viruses was first reported by Lindbo et al. (1993). Since the discovery of what is now generally called RNA silencing in plants, the same specific RNA degradation mechanism has been identified in nearly all eukaryotes (Baulcombe 2004). In plants, genetic and molecular analyses have revealed at least three natural pathways for RNA silencing: cytoplasmic short interfering RNA (siRNA) silencing; endogenous mRNA silencing by microRNA (miRNA); and the silencing associated with DNA methylation and suppression of transcription (Baulcombe 2004). All these pathways involve the cleavage of doublestranded RNA molecules into short 21–26 nuclotide RNAs, known as siRNAs and miRNAs (Baulcombe 2004). To date, it has primarily been the cytoplasmic siRNA silencing pathway that has been exploited by genetic engineering to confer resistance to plant viruses.

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The best documented examples of commercial transgenic resistance involve type (1) resistance, although the mechanism of resistance was not initially known. In the 1990s, the papaya industry in Hawaii suffered a 50% reduction in production because of papaya ringspot virus; the solution came through the expression of viral coat protein in transgenic plants (thought to be immune priming, analogous to vaccination).

1.4.5.1

Papaya Ringspot Virus

Papaya ringspot is a destructive disease (a potyvirus) characterized by yellowing and stunting of the crown of papaya trees, a mottling of the foliage, damage to younger leaves, water-soaked streaking of the petioles (stalks), and small darkened rings on the surface of fruit. Papaya ringspot virus is transmitted from infected papaya trees to healthy trees by the feeding action of various species of aphids, especially the green peach aphid and melon aphid. The virus is transmitted in a nonpersistent manner, meaning that the virus does not multiply within the aphid but is instead carried on its mouth parts and is transmitted from plant to plant while feeding. In 1995, American researchers developed a transgenic papaya resistant to the virus, by expressing a copy of a viral coat protein in the plant (Ling et al. 1991). It was field-tested in Hawaii where it was shown to be effective against the virus. The virus-resistant papaya is now widely used by commercial papaya producers in Hawaii. The presence of small RNA species, presumably siRNA, corresponding to regions of the viral coat protein gene was later shown to be present in transgenic lines resistant to PRSV; thus it is posttranscriptional gene silencing involved in the establishment of resistance (Krubphachaya et al. 2007). Pathogen-derived resistance has also been shown to be effective against maize streak virus (Shepherd et al. 2007), potato leaf roll virus (Vazquez Rovere et al. 2001) and tomato yellow leaf curl (Yang et al. 2004). An important drawback of RNA-based approaches to enhanced resistance is the high level of sequence specificity required for RNA degradation. Viruses containing >10% nucleotide divergence are insensitive to RNA degradation. Another drawback is the size of the transgene, commonly >300 bp, required to trigger efficient RNA silencing. However, Parizotto et al. (2004) has provided experimental evidence using genetically engineered microRNA (miRNA) to show that a 21 nucleotide sequence complementary to GFP mRNA was sufficient to trigger complete GFP silencing in transgenic plants. In the future, similar miRNA-derived strategies could provide resistance to a large number of plant viruses. However, the major technical limitation for technologies based on RNA silencing is that many important plant crop species are difficult or impossible to transform, precluding the constitutive expression of constructs directing production of double-stranded RNA. Moreover, public concerns over the potential ecological impact of virus-resistant transgenic plants have so far significantly limited their use (Fermin et al. 2004). For DNA viruses (particularly, Gemini viruses), transgenic resistance involves both RNA-mediated resistance and mutated viral proteins exerting negative effects on viral replication (Vanderschuren et al. 2007).

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Conclusions

While insect-resistant and herbicide-tolerant transgenic plants are the most widely grown GM crops, very few strategies using genetically engineered plants for disease resistance have had a similar impact (Collinge et al. 2008). Given the effort put into biotechnological strategies for disease resistance over the last two decades, why have so few crops expressing these traits become commercialized crops? The development of molecular markers, which are particularly useful in screening for disease resistance and facilitate conventional breeding, provide a higher commercial incentive for seed companies because of strong public opposition to GM crops. Often, transgenic disease resistant plants have only partial resistance, or resistance to rapid breakdown (single genes), again giving a low economic incentive to developers. At present, clear field results show that transgenic virus resistance is effective; however, there are no signs that commercial bacterial- or fungal-resistant crops will be introduced onto the market at any point soon.

1.5

Transgenic Crops for Resistance to Biotic Stress: Conclusions

Approximately 10.3 million farmers in 22 countries grew transgenic (genetically modified) crops in 2006. Yet this technology remains one of the most controversial agricultural issues of current times. Many consumer and environmental lobby groups believe that GM crops will bring very little benefit to growers and to the general public, and that they will have a deleterious effect on the environment. The human population is currently 6.1 billion and it is predicted to increase to 9–10 billion in the next 50 years (Fig. 1.7). This is at a time when food and fuel are competing for land (Fig. 1.8) and climate change threatens to compromise current resources. Population growth, changing diets, higher transport costs and a drive towards biofuels are forcing food prices up (Fig. 1.9). The UN’s Food and Agriculture Organization (FAO) stated that the food crisis had thrown an additional 75 million people into hunger and poverty in 2007 alone. World Population Growth (billion people) 1950

1975

2000

2025

2050

2.5bn

4.1bn

6.1bn

8.0bn

9.2bn

Fig. 1.7 World population growth

1 Transgenic Crop Plants for Resistance to Biotic Stress Fig. 1.8 Demand for biofuels

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Demand for Biofuels Rise in use of course grains 2005-7 (FAO/OECD).

Total: 80m tonnes

Biofuel use: 47m tonnes

Price Rises in a Single Year (Mar 2007-Mar 2008) Source: Bloomberg, FAO/ Jackson Son & Co 130% 87% 74% 31%

Corn

Rice

Soya

Wheat

Fig. 1.9 Price rises of major food crops (2007–2008)

It is, and will continue to be, a priority for agriculture to produce more crops on less land. The minimization of losses to biotic stress caused by agricultural pests would go some way to optimizing the yield on land currently under cultivation. Traditionally, agricultural production has kept pace, even outstripped human population growth; however, we currently face a set of unique challenges. One of the greatest dangers to agriculture is its vulnerability to global climate change. The expected impacts are for more frequent and severe drought and flooding, and shorter growing seasons. The performance of crops under stress will depend on their inherent genetic capacity and on the whole agroecosystem in which they are

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managed. It is for this reason that any efforts to increase the resilience of agriculture to climate change must involve the adoption of stress-resistant plants as well as more prudent management of crops, animals and the natural resources that sustain their production. Currently, we may be at the limit of the existing genetic resources available in our major crops (Gressel 2008). Thus, new genetic resources must be found and only new technologies will enable this.

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Chapter 2

Transgenic Plants for Abiotic Stress Resistance Margaret C. Jewell, Bradley C. Campbell, and Ian D. Godwin

2.1

Introduction

Modern agricultural crop production relies on the growth of a few of the world’s plant species selected for their superior qualities and suitability as food, animal feed, fiber or industrial end uses. Centuries of selection and, more recently, scientific breeding for adaptation to biotic and abiotic stresses have been necessary to improve yield, yield stability, and product quality in agricultural species. Nevertheless, abiotic stresses remain the greatest constraint to crop production. Worldwide, it has been estimated that approximately 70% of yield reduction is the direct result of abiotic stresses (Acquaah 2007). The ever increasing pressure put on agricultural land by burgeoning human populations has resulted in land degradation, a cultivation shift to more marginal areas and soil types, and heavier requirements for agricultural productivity per unit area. Additionally, climate change has exacerbated the frequency and severity of many abiotic stresses, particularly drought and high temperatures, with significant yield reductions reported in major cereal species such as wheat, maize, and barley (Lobell and Field 2007). In many parts of the world, rainfall has become less predictable, more intense, and, due to increasing temperatures, subject to higher evapotranspiration. For example, in the major grain growing areas of eastern Africa, the predominant rainy season is starting later and ending earlier (Segele and Lamb 2005) with longer dry spells in between (Seleshi and Camberlin 2006). Agricultural practices to improve crop productivity per unit area have, in many cases, accelerated the rate of land degradation, with particularly marked effects in irrigated areas. Irrigation has led to salinity across large tracts of agricultural land,

I. D. Godwin (*) School of Land, Crop and Food Sciences, The University of Queensland, Brisbane, Qld 4072, Australia e-mail: [email protected]

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with cases, such as in India, where it has reportedly led to the loss of seven million hectares from cultivation (Martinez-Beltran and Manzur 2005). In Australia, almost eight million hectares of land are under threat of dryland salinity (Munns et al. 2002). Higher yields are also only sustainable with higher nutrient use, and the heavy demand for fertilizers has caused rising costs for farmers worldwide. The environmental and economic consequences of increased nutrient use have been widely reported. For sustainability of crop production, there is a need to reduce the environmental footprint of food and fiber production, and nutrient use efficient crops are highly sought after. Transgenic approaches are one of the many tools available for modern plant improvement programs. Gene discovery and functional genomics projects have revealed multitudinous mechanisms and gene families, which confer improved productivity and adaptation to abiotic stresses. These gene families can be manipulated into novel combinations, expressed ectopically, or transferred to species in which they do not naturally occur or vary. Hence, the ability to transform the major crop species with genes from any biological source (plant, animal, microbial) is an extremely powerful tool for molecular plant breeding. Transgenic plants can be used as sources of new cultivars (or their germ plasm as new sources of variation in breeding programs) and they are also extremely useful as proof-of-concept tools to dissect and characterize the activity and interplay of gene networks for abiotic stress resistance. In this chapter, we will outline the major yield-limiting abiotic stresses faced by crop plants: drought, salinity, cold, nutrient deficiency, and metal toxicity. Within each section, we will then cite specific examples of transgenic crop approaches to overcome these stresses and also discuss a number of conserved plant stress response mechanisms, which have been demonstrated to confer better adaptation to a number of different abiotic stresses.

2.2

Water Scarcity and Agriculture

Drought is the most significant environmental stress on world agricultural production (Tuberosa and Salvi 2006; Cattivelli et al. 2008) and enormous effort is being made by plant scientists to improve crop yields in the face of decreasing water availability. During the twentieth century, the world’s population tripled from approximately 1.65 to 5.98 billion and population projections of 8.91 and 9.75 billion are expected to occur by 2050 and 2150, respectively. Developing countries in Africa and Asia account for approximately 80% of this growth and, with an estimated 800 million people in these countries already undernourished, the Food and Agriculture Organization (FAO) of the United Nations predicts that a 60% increase in world food production over the next two decades is required in order to sustain these populations. Agriculture accounts for approximately 70% of global water use and irrigation accounts for up to 90% of total water withdrawals in arid nations (World Water

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Council 2008; FAO 2009a). Approximately, 40% of all crops produced in developing countries are grown on irrigated arable land, which accounts for only 20% of the total arable land in these nations (FAO 2009c). The water withdrawal requirement for irrigation is expected to increase by 14% in developing countries by 2030 and strategies to decrease this demand by developing crops that require less irrigation will, therefore, play a vital role in maintaining world food supply. The area of plant drought tolerance research and improvement encompasses an enormous range of environmental, genetic, metabolic, and physiological considerations and an exhaustive discussion of all available avenues for developing droughtresistant crop varieties is beyond the scope of this chapter. Rather, this section attempts to provide an overview of some of the genetic mechanisms that have been manipulated in order to develop transgenic crops with improved drought tolerance and focuses on research that has involved long-term and field-based drought stress treatments performed on agronomic and horticultural crop species at yield determining plant life stages.

2.2.1

Improving Drought Tolerance in Agricultural Crops

All plants require water to complete their life cycle, with most plant cells consisting of at least 70% water on a fresh weight basis. When insufficient water is available, plant water status is disrupted, which causes imbalances in osmotic and ionic homeostasis, loss of cell turgidity, and damage to functional and structural cellular proteins and membranes. Consequently, water-stressed plants wilt, lose photosynthetic capacity, and are unable to sequester assimilates into the appropriate plant organs. Severe drought conditions result in reduced yield and plant death. The overall aim of genetically improving crops for drought resistance is to develop plants able to obtain water and use it to produce sufficient yields for human needs under drought conditions. While advances have been made in developing crops that are genetically improved with traits such as herbicide and pesticide resistance, attempts to improve plant drought resistance have been hindered by the complexity of plant drought resistance mechanisms at the whole plant, cellular, metabolic, and genetic levels. Interactions between these mechanisms and the complex nature of drought itself, adds another layer of intricacy to this problem.

2.2.2

Complexity of Drought and Plant Responses to Drought Stress

Drought (nonavailability of water for crop growth) and water deficit (insufficient plant water status) are variable, complex, and recurring features in most parts of the

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world. Even in areas with very high precipitation, many crops are likely to experience a certain level of water deficit at some stage during the growing cycle. Elucidation of plant drought resistance and response mechanisms has been compounded by the variable levels and forms of drought. Drought can be spatially and temporally variable; terminal, short-term, or sporadic; severe, moderate, or minor; and can occur at rates ranging from very sudden to gradual. In addition, the effects of drought and water deficit on crop productivity vary for different crops, macro- and microenvironments across a single field, plant life stages, and the plant material to be harvested. Furthermore, the effects of drought on crop productivity are often compounded by associated stresses such as heat, salt, and nutrient stress.

2.2.3

Plant Drought Resistance and Response

Plant drought resistance mechanisms can be broadly grouped into avoidance or tolerance mechanisms. Drought avoidance mechanisms are associated with physiological whole-plant mechanisms such as canopy resistance and leaf area reduction (which decrease radiation adsorption and transpiration), stomatal closure and cuticular wax formation (which reduce water loss), and adjustments to sink-source allocations through altering root depth and density, root hair development, and root hydraulic conductance (Beard and Sifers 1997; Rivero et al. 2007). Drought tolerance mechanisms are generally those that occur at the cellular and metabolic level. These mechanisms are primarily involved in turgor maintenance, protoplasmic resistance, and dormancy (Beard and Sifers 1997). Plants respond to water-limiting conditions by altering the expression of a complex array of genes (Fig. 2.1) and, although elucidation of biochemical pathways associated with many of these genes has been the focus of an enormous amount of research over the last two to three decades, the mechanisms by which these genes and their products interact remains relatively poorly understood. Abiotic stress leads to a series of morphological, physiological, biochemical, and molecular changes that adversely affect plant growth and productivity (Wang et al. 2001). Drought, salinity, extreme temperatures, and oxidative stress are often interconnected, and may induce similar cellular damage. For example, drought and/or salinization are manifested primarily as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cell (Serrano et al. 1999; Zhu 2001). Oxidative stress, which frequently accompanies high temperature, salinity, or drought stress, may cause denaturation of functional and structural proteins (Smirnoff 1998). As a consequence, these diverse environmental stresses often activate similar cell signaling pathways (Knight 2000; Shinozaki and Yamaguchi-Shinozaki 2000; Zhu 2001, 2002) and cellular responses, such as the production of stress proteins, upregulation of antioxidants and accumulation of compatible solutes (Vierling and Kimpel 1992; Zhu et al. 1997; Cushman and Bohnert 2000; Wang et al. 2003b).

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Signal sensing, perception, transduction

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Fig. 2.1 Plant responses to abiotic stress

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The Genetic Basis of Drought Tolerance

Expression studies have shown that drought-specific genes can be grouped into three major categories: (1) Genes involved in signal transduction pathways (STPs) and transcriptional control; (2) Genes with membrane and protein protection functions; and (3) Genes assisting with water and ion uptake and transport (Vierling 1991; Ingram and Bartels 1996; Smirnoff 1998; Shinozaki and Yamaguchi-Shinozaki 2000). Plants adapt to drought conditions by tightly regulating specific sets of these genes in response to drought stress signals, which vary depending on factors such as the severity of drought conditions, other environmental factors, and the plant species (Wang et al. 2003b). To date, successes in genetic improvement of drought resistance have involved manipulation of a single or a few genes involved in signaling/regulatory pathways or that encode enzymes involved in these pathways (such as osmolytes/compatible solutes, antioxidants, molecular chaperones/osmoprotectants, and water and ion transporters; Wang et al. 2003b). The disadvantage of this is that there are numerous interacting genes involved, and efforts to improve crop drought tolerance through manipulation of one or a few of them is often associated with other, often undesirable, pleiotropic and phenotypic alterations (Wang et al. 2003b). These complex considerations, when coupled with the complexity of drought and the plant–environment interactions occurring at all levels of plant response to water deficit, illustrate that the task plant researchers are faced with in engineering drought resistant crops is dauntingly multi-faceted and extremely difficult.

2.2.5

Engineering Improved Drought Avoidance in Crops

Most transformation studies to improve plant drought resistance have produced transformants that display a variety of both tolerance and avoidance traits. An exception was demonstrated by Rivero et al. (2007) who manipulated a leaf senescence gene. Leaf senescence is an avoidance strategy and is accelerated in drought-sensitive plants to decrease canopy size. In crop plants, accelerated senescence is often associated with reduced yield and is thought to be the result of an inappropriately activated cell death program. Therefore, suppression of droughtinduced leaf senescence in tobacco plants was investigated as a tool to enhance drought resistance. Transgenic plants were developed by expressing isopentyl transferase (IPT), a key enzyme in the biosynthesis of cytokinin (a leaf senescence inhibitor) under the control of the senescence-associated receptor protein kinase promoter (PSARK). The SARK gene, which is induced during late maturation and drought and decreased during senescence development, encodes a maturation/sensescence-dependent protein kinase. Although transgenic plants wilted under a 15-day glasshouse drought stress treatment, senescence did not occur and a reduced

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photosynthetic capacity was maintained. Following rewatering, transgenic plants recovered full leaf turgor and resumed growth and maximum photosynthetic capacity, while the control plants were unable to recover from the drought stress. Water use efficiency (WUE) of the transgenic plants was also markedly higher than wild-type (WT) plants, and was two to three times higher after rewatering than before the drought treatment. An experiment to assess whether the transgenic plants could produce significant yields under water-limiting conditions determined that biomass and seed yield were not as affected in transgenic plants than in WT controls, although this result was not significant (Rivero et al. 2007). Other drought avoidance/whole-plant traits that have been investigated include stay-green and cuticular biosynthesis. Stay-green is a variable and quantitative trait, which generally refers to delayed senescence. It has not yet been used to successfully produce transgenic plants with increased drought resistance in the field. Cuticular biosynthesis was investigated by transgenic expression of AtMYB41, which encodes an R2R3-MYB transcription factor (TF) in Arabidopsis. AtMYB41 is expressed at high levels in response to drought, abscisic acid (ABA; Sect. 2.2.6.1), and salt treatments, and was demonstrated to have a role in cell expansion and cuticle deposition. The transformation of Arabidopsis with AtMYB41 was associated with undesirable pleiotropic phenotypes including dwarfism, enhanced sensitivity to desiccation, and enhanced permeability of leaf surfaces (Cominelli et al. 2008).

2.2.6

Improving Plant Drought Tolerance

2.2.6.1

Absicisic Acid and Transcriptional Regulation

The plant hormone ABA regulates the plant’s adaptive response to environmental stresses such as drought, salinity, and chilling via diverse physiological and developmental processes. ABA has functional roles ranging from seed maturation processes to lateral root development (McCourt and Creelman 2008; Wasilewska et al. 2008). Under abiotic stress, ABA induces stomatal closure, reduces water loss via transpiration, and induces gene expression (Chandler and Robertson 1994). Gene expression and biochemical studies into ABA synthesis in Arabidopsis and some other model plants have largely elucidated the basic ABA biosynthetic pathway (Schwartz et al. 2003) and many of the key enzymes involved in ABA synthesis have been investigated transgenically in relation to improving plant drought tolerance. For example, transgenic Arabidopsis plants constitutively overexpressing the zeaxanthin epoxidase gene, AtZEP, which encodes an enzyme required for an initial step in ABA synthesis from isopentyl diphosphate (IPP) and b-carotene (Schwartz et al. 2003) showed increased tolerance to drought and salinity stress. The increased drought stress tolerance was attributed to increased leaf and lateral root development, longer primary roots, higher fresh weight, and increased survival compared with control plants following drought treatment.

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Furthermore, compared with WT plants, the rate of water loss was lower, the levels of ABA were higher, the expression of stress responsive genes such as Rd29a was much higher, and stomatal aperture was smaller under salt and/or drought stress. Overall, the increased stress resistance was attributed to increased ABA levels in response to osmotic stress, which resulted in enhanced expression of ABAresponsive stress-related genes (Park et al. 2008). Many of the drought stress response pathways that have been identified to date appear to be under transcriptional regulation and ABA plays a key role in this process (Fig. 2.2). Transcriptional regulation involves interaction between TFs and specific cis-acting elements located within or near the promoter region upstream of expressed genes. Figure 2.2 shows links between responses to low temperature and dehydration stress at the transcriptional level. It can be seen that ABA is involved in both types of abiotic stress. Many transcriptional responses to drought stress have been well characterized and are classified as being ABA-dependent, ABA-independent, or both. ABA is Low Temperature

Dehydration

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CBF1, 2, 3 / DREB1a, b, c DREB2

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DREB2 AREB/ABF

CRT/DRE (Rd29a, Cor15a)

ABRE (Rd29a, Rd29b)

MYCR/MYBR (Rd22, AtADH1)

Fig. 2.2 Plant transcriptional processes induced by dehydration and low temperature stress. Displayed are transcription factors (rounded rectangles) both ABA-dependent (shaded) and ABA-independent (unshaded), posttranscriptional modification such as phosphorylation (elipses), transcription factor binding sites and representative promotors (rectangles), possible regulation points (dotted arrows), and possible cross-talk (bidirectional arrows)

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often used in stress and stress acclimation studies because it is produced in response to stress. ABA induces expression of many genes that are also induced by drought, cold, and salinity when applied exogenously (Shinozaki and Yamaguchi-Shinozaki 1996). There are two types of ABA-dependent transcription. The “direct” pathway involves cis-acting ABA-responsive elements (ABREs), which are directly activated by binding with TFs such as basic-domain leucine zipper (bZIP)-type DNAbinding proteins (Shinozaki and Yamaguchi-Shinozaki 1996; Kobayashi et al. 2008). Alternatively, the “indirect” ABA-dependent transcription pathway involves other cis-acting elements, such as MYC and MYB. These elements are activated through binding with ABA- or drought-inducible TFs, such as basic helix–loop– helix (bHLH)-related protein AtMYC2 and an MYB-related protein, AtMYB2 (Abe et al. 2003). An example of the indirect pathway can be seen in the expression of rd22 from Arabidopsis (Shinozaki and Yamaguchi-Shinozaki 1996). Some genes are induced by drought stress but are not expressed in response to exogenous ABA applications and these genes are the product of ABA-independent STPs. One such gene is rd29a (also known as lti78 and cor78). YamaguchiShinozaki and Shinozaki (Yamaguchi-Shinozaki and Shinozaki 1994) identified a dehydration-responsive element (DRE) in the promoter region of rd29a and the DRE-binding (DREB) protein transcription pathway has since been explored for its important roles in drought, cold, and salinity stress (Shinozaki and YamaguchiShinozaki 1996; Qin et al. 2007). Several C-repeat (CRT) binding factor (CBF)/ DREB proteins have now been identified from the promoter regions of other stressinducible Arabidopsis genes, such as cor15a, kin1, cor6.6 and cor47/rd17, and the CBF/DREB pathway has been shown to be conserved across species (Benedict et al. 2006; Pasquali et al. 2008). CBF/DREB1 and DREB2, belong to the ethyleneresponsive element/apetela 2 (ERE/AP2) TF family; their expression is induced by cold or drought stress and both activate expression of genes possessing a CRT/DRE cis-element (Stockinger et al. 1997; Liu et al. 1998). Likewise, DREB2A positively regulates expression of many abiotic stress-related genes possessing DRE sequences in their 5’-upstream regions. DREB2A overexpression in Arabidopsis confers significant drought tolerance in transgenic plants (Sakuma et al. 2006a, b). DREB genes have been used in transformation of several crops, including wheat and rice, in attempts to increase drought tolerance (Chen et al. 2008; Kobayashi et al. 2008). Although DREs are cis-acting elements that were first thought to activate ABA-independent stress-responsive gene expression, some are also implicated in ABA-dependent expression (Shinozaki and Yamaguchi-Shinozaki 2000). CBF4 is an apparent homolog of the CBF/DREB1 proteins that is thought to be a critical regulator of gene expression in drought stress signal transduction. The action of CBF4 is thought to be through its binding with CRT/DRE elements in promoter regions of drought- and cold-inducible genes (Haake et al. 2002). CBF4 gene expression has been shown to be upregulated in response to drought and ABA; however, constitutive expression of CBF4 was found to result in expression of cold- and drought-induced genes under nonstress conditions and this was associated with retarded growth, shorter petioles, darker green leaves, and delayed time to

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flowering in Arabidopsis seedlings (Haake et al. 2002). Another study showed that CBF4 expression was induced by salt, but not by drought, cold, or ABA (Sakuma et al. 2002). Similar observations, and observations of higher levels of soluble sugars and proline, have been recorded during many CBF overexpression studies, which suggest that the use of constitutively expressed CBF/DREB genes may not be applicable to the development of crops with improved drought tolerance. It is thought that the use of stress-inducible promoters that have low expression levels under non-stress conditions could be used in conjunction with CBF genes to alleviate the retarded growth observed in CBF overexpression studies (Zhang et al. 2004). Many studies have illustrated the potential of manipulating CBF/DREB genes to confer improved drought tolerance. For example, overexpression of CBF1/ DREB1B from Arabidopsis was able to improve tolerance to water-deficit stress in tomato. Furthermore, when driven by three copies of an ABA-responsive complex (ABRC1) from the barley HAV22 gene, transgenic tomato plants expressing CBF1 exhibited enhanced tolerance to chilling, water deficit, and salt stress, and maintained normal growth and yield under normal growing conditions when compared with control plants (Lee et al. 2003a). Other studies have also found that expression of CBF/DREB genes under stress-inducible promoters result in transgenic plants that do not express detectable levels of these genes under non-stress conditions, minimizing growth retardation and other adverse effects (Al-Abed et al. 2007). The CRT/DRE motif also acts as one of the binding sites for the ERF family of TFs (Trujillo et al. 2008). A novel ERF from sugarcane, SodERF3, was found to enhance salt and drought tolerance when overexpressed in tobacco plants. Under drought treatment, transgenic plants were significantly taller than controls and were able to flower under an extended growth period. Furthermore, the absence of observable differences in height, number of leaves, leaf area, leaf weight, and stalk weight between transgenic and control plants illustrates that this gene has potential for engineering drought stress tolerance in plants (Trujillo et al. 2008). Other TFs involved in mediation of ABA-dependent and ABA-independent signal transduction and gene expression include NAC, WRKY, RING finger, and zinc-finger TFs (Seki et al. 2003; Zhang et al. 2004; Chen et al. 2006). Nelson et al. (2007) showed that constitutive expression of a TF from the nuclear factor (NF-Y) family, AtNF-YB1, which belongs to the CCAAT-binding TF family, improved performance of Arabidopsis under drought conditions. Consequently, an orthologous maize TF gene, ZmNF-YB2, was constitutively expressed in maize. Transgenic lines were exposed to both glasshouse-based and field-based drought stress treatments. Transgenic lines exhibited less wilting and faster recovery and re-established growth more rapidly than WT (on average) under glasshouse-based drought treatment. Transgenic lines subjected to field-based drought stress at the late vegetative stage exhibited superior health, higher chlorophyll indices and photosynthetic rates, lower leaf temperatures, higher stomatal conductance, and less yield reduction than WT plants. Furthermore, under favorable conditions, transgenic plants were greener, flowered 1–3 days earlier, and had slightly compressed internodes. Most importantly, the stress adaptation response contributed to a yield advantage in

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transgenic maize grown within drought environments, suggesting that ZmNF-YB2 has a realistic application for use in commercial agriculture under severe waterlimiting conditions (Nelson et al. 2007). Another TF that has been manipulated in order to increase plant drought tolerance is the HARDY (HRD) gene, which has been linked to increased transpiration efficiency related to stomatal adjustment. HRD is an AP2/ERF-like TF isolated from hrd-dominant (hrd-D) Arabidopsis mutants, which displayed vigorous rooting and dark green leaves that were smaller and thicker than WT plants. Karaba et al. (2007) isolated the HRD gene and constitutively expressed it in Arabidopsis under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The thicker leaves, higher root density, and increased root strength were associated with abundant chloroplasts, increased secondary and tertiary root initiation and proliferation, and extra corticle cell layers and more compact, stele bearing vascular tissue, respectively. Furthermore, the mutants survived longer periods of drought stress and could reach full maturity under high levels of salt stress. HRD was also constitutively expressed in rice and transgenic plants displayed no reduction in growth, seed yield, or germination, but had significantly increased leaf canopy with more tillers under normal greenhouse conditions compared with WT controls. Under drought stress, the transgenic plants were of deeper green color (attributable to increased number of bundle sheath cells), displayed distinctive drought tolerance and lower stomatal conductance, had higher net carbon assimilation and photosynthetic rates, and possessed higher root biomass (Karaba et al. 2007). Recently, a novel drought-tolerant gene, HDG11, which encodes a protein from the homeodomain (HD)-START TF family (also known as the Class IV HD-leucine zipper TF family) was identified in Arabidopsis and was found to confer drought resistance via enhanced root growth and decreased stomatal density when constitutively overexpressed in transgenic tobacco (Yu et al. 2008). The constitutive expression of the gene was not associated with retarded growth or any other observable deleterious phenotypic effects and, the gene was also shown to transactivate a number of other genes involved in the drought stress response including ERECTA (Sect. 2.2.6.2.1; Yu et al. 2008). SNAC1 from rice has also been shown to have trans-activation activity. NAC TFs comprise a large gene family with proteins exhibiting a highly conserved N-terminal DNA-binding domain and a diversified C-terminal domain. NAC was derived from the names of the first three described proteins containing the DNA-binding domain, namely, NAM (no apical meristem), ATAF1-2, and CUC2 (cup-shaped cotyledon; Souer et al. 1996; Aida et al. 1997). NAC is a plant-specific TF family with diverse roles in development and stress regulation. When constitutively overexpressed in rice, SNAC1 was found to significantly improve plant resistance to severe drought stress during reproductive and vegetative growth and was not associated with any negative phenotypic effects or yield penalty (Hu et al. 2006). Transgenic plants were more sensitive to ABA and closed more stomata than WT plants but maintained continual photosynthetic activity. There was no difference between root morphologies of transgenic and WT plants indicating that the improved drought resistance was not related to increased root-water uptake.

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The WRKY superfamily of plant TFs has a conserved sequence (WRKYGQK) at their N-terminal ends (Wu et al. 2008b). Transgenic rice seedlings, expressing OsWRKY11 under the control of a rice heat shock protein (HSP) promoter, HSP101, were shown to survive longer and lose less water under a short, severe drought treatment, than WT plants (Wu et al. 2008b). A TFIIIA-type zinc-finger protein gene, ZFP252, was also found to confer improved drought stress resistance in rice. Young transgenic rice plants overexpressing ZFP252 survived longer, displayed less relative electrolyte leakage, and accumulated more compatible osmolytes than WT plants or plants with ZFP252 knocked out during a 14-day period of drought stress (Xu et al. 2008a). A salt- and drought-induced RING-finger protein, SDIR1, was found to confer enhanced drought tolerance to tobacco and rice (Zhang et al. 2008b). Arabidopsis E3 ligase SDIR1 is a positive regulator in ABA signal transduction. Tobacco and rice plants constitutively overexpressing the SDIR1 gene displayed less leaf wilting and rolling, longer survival, and improved recovery under drought conditions than control plants. The mechanism of drought tolerance was thought to be due to decreased stomatal aperture, which increased transpiration efficiency of transgenic plants. Some genes have been shown to suppress expression of drought-response transcription pathways. For example, Jiang et al. (2008) recently characterized SAZ, an Arabidopsis gene from the SUPERMAN (SUP) family of plant-specific zinc-finger genes, which encode proteins containing single C2H2-type zinc-finger motif with a conserved short amino acid sequence and a class II ERF-associated amphiphilic repression (EAR) motif-like TF domain at the carboxy-terminal region. SAZ was found to be rapidly downregulated in response to drought and other abiotic stresses and SAZ gene knockouts resulted in elevated expression of the ABA-responsive genes rd29B and rab18 under stressed and unstressed conditions. This shows that gene knockouts and gene silencing may also be applicable to the development of crops with improved drought resistance.

2.2.6.2

Signal Sensing, Perception, and Transduction

Prior to transcriptional activation of genes, drought stress signals are received and messages conveyed to the appropriate components of the downstream pathway (Xiong and Ishitani 2006). In general, STPs involve perception of stress by specific receptor molecules, which vary in identity, structure, perception, signal relay mechanism, and location within the cell (Xiong and Ishitani 2006). Plant stress STPs often involve secondary messengers, which may modify signals (often via reversible protein phosphorylation) prior to conveying them from receptor molecules to the activators of the appropriate gene expression pathway (Xiong and Ishitani 2006). Other molecules may also be involved in stress STPs and the functions of these include recruitment and assembly of signaling complexes, targeting of signaling molecules, and regulation of signaling molecule lifespan (Xiong and Ishitani 2006).

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The major molecules involved in drought stress signal sensing, perception, and transduction include receptor molecules/osmosensors, phospholipid-cleaving enzymes (PLEs), reactive oxygen species (ROS), mitogen-activated protein kinases (MAPK), and Ca2+ sensors.

Receptor Molecules/Osmosensors Receptor molecules/osmosensors are the initial stress signal perceivers and they convey the signal to the appropriate molecule to initiate STPs. On the basis of analyses of plants and other species, receptor molecules are thought to include receptor-like kinases, two-component receptors, receptor tyrosine kinases, G-protein-coupled receptors, iontropic channel-related receptors, histidine kinases, and nuclear hormone receptors. Receptor molecules that have been identified to date in plants include: ROP10, a small G protein from the ROP family of Rho GTPases, that negatively regulates ABA response in Arabidopsis (Zheng et al. 2002); ATHK1, a putative homolog of the yeast SLN1, which is a functional histidine kinase feeding into the HOG MAPK pathway (Urao et al. 1999; Reiser et al. 2003); NtC7, a receptor-like membrane protein from tobacco (Tamura et al. 2003); and Cre1, a putative cytokinin sensor and histidine kinase from Arabidopsis (Reiser et al. 2003). The ERECTA gene from Arabidopsis is a putative leucine-rich repeat receptorlike kinase (LRR/RLK). It was the first gene to be shown to act on the coordination between transpiration and photosynthesis (Masle et al. 2005). ERECTA was analyzed by its transgenic expression in null-mutants and was shown to have roles in lowering stomatal conductance, controlling leaf photosynthesis and organogenesis, modulation of cell expansion, cell division, cell–cell contact, cell–cell and tissue–tissue signaling, cell proliferation, and inflorescence differentiation. Owing to the range of traits attributed to ERECTA expression, ERECTA is thought to act as a master gene in transpiration regulation (Masle et al. 2005). No known studies have yet involved transgenic expression of ERECTA in economic crops; however, initial studies suggest that this gene may be useful in the design of crops with improved transpiration efficiencies, reduced stomatal limitations, and increased yield potentials.

Phospholipid-Cleaving Enzymes PLEs degrade phospholipid membranes, catalyzing the release of lipid and lipidderived secondary messengers (Chapman 1998; Sang et al. 2001). Phospholipases C (PLC) and D (PLD) are both involved in ABA-mediated signal transduction and drought stress tolerance perception in plants. Phosphatidic acid (PtdOH), a product of the PLC and PLD pathways, is also important in the signaling process (Bartels et al. 2007; Wang et al. 2008a).

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Wang et al. (2008a) successfully produced maize plants constitutively overexpressing ZmPLC1, a phospholipase catalyzing the hydrolysis of 4,5-bisphosphate to form diacylglycerol (DAG) and inositol 1,4,5-trisphosphate, the products of which are the second messengers Ca2+ and PtdOH, respectively. This pathway is important in a wide variety of abiotic stress-responsive processes. Transgenic maize plants carrying the ZmPLC1 gene were shown to have increased photosynthetic activity, reduced anthesis to silking interval (ASI; an indicator of maize yield potential), better recovery, and higher grain yield than WT plants when subjected to 21 days of drought stress at the ten-leaf stage. Because there was no significant difference between stomatal conductances of WT and transgenic plants, the higher photosynthetic rate was attributed to better photochemical activity rather than the improved guard cell signaling. This has also been demonstrated in other studies (Staxen et al. 1999; Hunt et al. 2003; Mills et al. 2004). Sang et al. (2001) showed that overexpression of PLD results in enhanced sensitivity of transgenic tobacco and plays a key role in controlling stomatal movements and plant response to water stress.

Reactive Oxygen Species ROS are generated in plants as photoreaction and cellular oxidation byproducts under normal conditions and can cause cellular damage under water deficit when they accumulate to toxic levels. Some of these species also have important roles in early stress response through activation of cellular defense mechanisms and mitigation of cellular damage. While plant mechanisms must be in place to detoxify high levels of ROS that occur under drought, low levels of these beneficial ROS must also be maintained. Those ROS known to have important signaling roles in plant stress STPs include nitric oxide (NO) and hydrogen peroxide (H2O2).

Mitogen-Activated Protein Kinases MAPKs are enzymes that catalyze reversible phosphorylations, important for relaying signals. They function via cascades, which involve sequential phosphorylation of a kinase by its upstream kinase (Xiong and Ishitani 2006). Recently, the MKK2 pathway was identified in Arabidopsis as having involvement in cold and osmotic stress signal transduction. An example of a MAPK having specific involvement in drought and salt stress is the p44MMK4 kinase from alfalfa (Medicago sativa; Jonak et al. 1996). Phosphatases involved in the sequential phosphorylation of MAPKs and other protein kinases are also important for stress signaling. For example, the ABI1 and AB12 proteins from Arabidopsis have been shown to act in a negative regulatory feedback loop of the ABA signaling pathway (Merlot et al. 2001). Some MAPK and MAPKK proteins have also been shown to activate the Rd29a stress pathway in Arabidopsis (Hua et al. 2006). Other protein kinases involved in stress signaling include calcium-dependent protein kinases (CDPKs),

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kinases from the SNF1 family of protein kinases, and serine-threonine-type protein kinases (Xiong and Ishitani 2006; Bartels et al. 2007). Ca2+ Sensors Ca2+ sensors are important for coupling extracellular signaling to intercellular responses and comprise calmodulin (CaM) and CaM-related proteins (Sneddon and Fromm 1998; Sneddon and Fromm 2001), calcineurin B-like proteins (CBL; also known as SCaBP/SOS3-like calcium-binding proteins; Kudla et al. 1999), and CDPKs (Harmon et al. 2000). Ca2+ sensors that have been attributed with roles in drought tolerance in plants include the CBL1 gene (Kudla et al. 1999) and the AtCAMBP25 protein (Perruc et al. 2004) from Arabidopsis. 2.2.6.3

Stress-Responsive Mechanisms

The outcome of stress signal perception, transduction, and transcriptional up- or downregulation of genes is the production of molecules with various plant protection, repair, and stabilization functions. These molecules can be broadly grouped into five functional groups: (1) detoxification; (2) chaperoning; (3) late embryogenesis abundant (LEA) protein functions; (4) osmoprotection; and (5) water and ion movement. Detoxification To prevent stress injury, cellular ROS need to remain at nontoxic levels under drought stress. Antioxidants involved in plant strategies to degrade ROS include: (1) enzymes such as catalase, superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase; and (2) nonenzymes such as ascorbate, glutathione, carotenoids, and anthocyanins (Wang et al. 2003b). Some proteins, osmolytes, and amphiphilic molecules also have antioxidative functionality (Bowler et al. 1992; Noctor and Foyer 1998). Chaperoning Chaperone functions involve specific stress-associated proteins, which are responsible for protein synthesis, targeting, maturation and degradation, and function in protein and membrane stabilization, and protein renaturation. HSPs, which can be divided into five conserved families, have been shown to have particularly important stress-related chaperone functions in plants (Hendrick and Hartl 1993; Boston et al. 1996; Hartl 1996; Waters et al. 1996; Torok et al. 2001). HSPs, which are induced by heat, have been implicated in plant cell protection mechanisms under drought stress. Protein denaturation occurs under drought stress because decreased

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cellular volume increases the likelihood of degradative molecular interactions (Cho and Hong 2006). HSPs maintain or repair companion protein structure and target incorrectly aggregated and non-native proteins for degradation and removal from cells (Cho and Hong 2006). One such protein, NtHSP70-1, was constitutively overexpressed in tobacco to ascertain its role in plant drought response and tolerance (Cho and Hong 2006). The drought tolerance of transgenic seedlings was increased and their optimum water content was maintained after progressive drought stress (Cho and Hong 2006). Few other studies have involved transforming plants with HSPs; however, HSP24 from Trichederma harzianum was found to confer significantly higher resistance to salt, drought, and heat stress when constitutively expressed in Saccharomyces cerevisiae (Liming et al. 2008).

Late Embryogenesis Abundant Protein Functions LEA proteins are produced in response to dehydration stress and function in water status stabilization, protection of cytosolic structures, ion sequestration, protein renaturation, transport of nuclear targeted proteins, prevention of membrane leakage, and membrane and protein stabilization. LEA and LEA-type genes are found universally in plants. They accumulate in seeds during the late stages of embryogenesis and are associated with the acquisition of desiccation tolerance under drought, heat, cold, salt, and ABA stress (Sivamani et al. 2000; Bartels et al. 2007). They are also present in the biomass tissue of resurrection plants and are upregulated in many desiccation-sensitive plants in response to drought stress (Bartels et al. 2007). LEA proteins are divided into groups based on conserved sequence motifs (Zhang et al. 2000; Wise 2003). Five of these groups have been characterized at the molecular and structural level (Table 2.1); however, recent research indicates that additional groups of LEA and LEA-like proteins are still being identified (Park et al. 2003; Wang et al. 2006; March et al. 2007). Common Table 2.1 The five groups of LEA proteins LEA group Description Group 1 Contain a 20-amino acid motif and are represented by the wheat Em protein, for which gene homologs have been identified in a wide range of plant species Group 2 The most extensively studied group. They contain a lysine-rich 15-amino acid (dehydrins) motif (K-segment; EKKGIMDKIKEKLPG), which is predicted to form an amphipathic a-helix, a tract of contiguous serine residues and a conserved motif containing the consensus sequence DEYGNP in the N-terminal section of the protein Group 3 Contain a characteristic repeat motif of 11 amino acids, which have been predicted to form an amphipathic a-helix with possibilities for intra- and inter-molecular interactions Group 4 Have a conserved N-terminus, which is proposed to form a-helices and a diverse C-terminal region with a random coil structure Group 5 Contain more hydrophobic residues than the other groups, are insoluble after boiling, and are likely to adopt a globular structure Source: Bartels and Salamini (2001), Ramanjulu and Bartels (2002)

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features of LEA proteins generally include hydrophilicity (Garay-Arroyo et al. 2000; Park et al. 2003), heat stability (Close and Gallagher-Ludeman 1989; Ceccardi et al. 1994; Houde et al. 1995; Thomashow 1998, 1999), and transcriptionally regulated and ABA-responsive gene expression (Close and Gallagher-Ludeman 1989). It is generally assumed that they play a role in water-deficit tolerance and the possible functions of LEA proteins include binding and replacement of water (Dure 1993), ion sequestration (Bray 1993), maintenance of protein and membrane structure (Baker et al. 1988), molecular chaperones (Close 1996), membrane stabilization (Koag et al. 2003), and nuclear transport of specific molecules (Goday et al. 1994). One class of LEAs, the dehydrins, which have detergent and chaperone-like properties, stabilize membranes, proteins, and cellular compartments (Close 1996). LEA genes have been manipulated in many plants in order to increase drought resistance. For example, a wheat dehydrin, DHN-5, was ectopically overexpressed in Arabidopsis and transgenic plants displayed superior growth, seed germination rate, water retention, ion accumulation, more negative water potential, and higher proline contents than WT plants under salt and/or drought stress (Brini et al. 2007a). The barley (Hordeum vulgare L.) group 3 LEA gene, HVA1 was constitutively overexpressed in rice plants to increase drought tolerance. Transgenic plants displayed significantly increased tolerance to water deficit and salinity, which was associated with higher growth rates, delayed onset of stress damage symptoms, and improved recovery following stress removal (Xu et al. 1996). A more recent study involving the overexpression of this gene in rice showed that transgenic plants had significantly higher relative water content (RWC), improved turgor, less reduction in shoot and root growth, and improved cell membrane stability under prolonged drought conditions. It was found that HVA1 did not function as an osmolyte and that membrane protection was the mechanism, which inferred drought resistance in rice plants (Chandra Babu et al. 2004). HVA1 was also expressed in Basmati rice under control of either a constitutive rice promoter or a stress-inducible promoter. Transgenic plants exhibited increased stress tolerance in terms of cell integrity and growth, and it was found that inducible expression of HVA1 resulted in transgenic plants that were able to grow normally under nonstress conditions (Rohila et al. 2002). Transgenic wheat plants expressing HVA1 displayed more root fresh and dry weights, and shoot dry weight than WT plants under water-deficit conditions (Sivamani et al. 2000). Similarly, HVA1 overexpressing transgenic mulberry, Morus indica, exhibited improved cellular membrane stability, photosynthetic yield, less photo-oxidative damage, and superior WUE than WT plants under salt and drought stress (Lal et al. 2008). The discussion of expression of HVA1 in mulberry will be continued in Sect. 2.3.5. Other group 3 LEA genes that have been manipulated in order to improve plant drought tolerance include a Brassica napus group 3 LEA gene, which conferred improved salt and drought tolerance when constitutively expressed in Chinese cabbage (Park et al. 2005a), and TaLEA3 from wheat, which increased RWC, leaf water potential, and relative average growth rate of transgenic plants compared to WT plants under drought stress when constitutively overexpressed in the perennial grass Leymus chinensis (Wang et al. 2008b). Two group 4 LEA proteins,

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BhLEA1 and BhLEA2 from the resurrection plant Boea hygrometrica, conferred improved drought tolerance in transgenic tobacco. This was associated with plant cell protection and increased membrane and protein stability during dehydration (Liu et al.). A novel LEA gene from Tamarix androssowii also conferred increased drought tolerance when expressed in transgenic tobacco (Wang et al. 2006).

Osmoprotection Osmoprotection involves the upregulation of compatible solutes (osmolytes) that function primarily to maintain cell turgor, but are also involved in antioxidation and chaperoning through direct stabilization of membranes and/or proteins (Yancey et al. 1982; Bohnert and Jensen 1996; Lee et al. 1997; Hare et al. 1998; McNeil et al. 1999; Diamant et al. 2001). Compatible solutes are low molecular weight, highly soluble compounds that are usually nontoxic at high cellular concentrations. The three major groups of compatible solutes are amino acids (such as proline), quaternary amines (glycine betaine (GlyBet), polyamines, and dimethylsulfonioproprionate), and polyol/sugars (such as mannitol, galactinol, and trehalose; Wang et al. 2003b). Many genes involved in the synthesis of these osmoprotectants have been explored for their potential in engineering plant abiotic stress tolerance (Vinocur and Altman 2005). GlyBet and trehalose act as osmoprotectants by stabilizing quaternary structures of proteins and highly ordered states of membranes. Mannitol serves as a freeradical scavenger. Proline serves as a storage sink for carbon and nitrogen and a free-radical scavenger. It also stabilizes subcellular structures (membranes and proteins), and buffers cellular redox potential under stress. Many crops lack the ability to synthesize the special osmoprotectants that are naturally accumulated by stress tolerant organisms. It is believed that osmoregulation would be the best strategy for abiotic stress tolerance, especially if osmoregulatory genes could be triggered in response to drought, salinity, and high temperature. Therefore, a widely adopted strategy to develop stress-tolerant crops has been to engineer or overexpress certain osmolytes in plants (Bhatnagar-Mathur et al. 2008). GlyBet is a compatible solute that has been extensively studied for its role in drought stress response and increasing the levels of GlyBet in plants via genetic engineering has enhanced the drought tolerance of many model plants (Sakamoto and Alia 1998; Sakamoto and Murata 2000; Mohanty et al. 2002). A two-step enzymatic process accomplishes production of GlyBet in plants. The first step involves conversion of choline to betaine aldehyde by choline monoxygenase (CMO), a stromal enzyme with a Rieske-type (2Fe-2S) center (Brouquisse et al. 1989), and the second step involves betaine aldehyde dehydrogenase (BADH), a nuclear-encoded chloroplast stromal enzyme, which converts betaine aldehyde to GlyBet (Weigel et al. 1986). Quan et al. (2004) reported one of the first attempts to increase the GlyBet expression levels of maize by overexpressing the betA gene, which encodes choline dehydrogenase (CHO), another key enzyme in the choline–betaine aldehyde reaction (Zhang et al. 2008a). The study showed that

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transgenic maize plants were more drought tolerant than WT plants at three different life stages, including the ten-leaf-flowering stage, and also that yields of transgenic plants were less affected by drought stress than WT. Tobacco lacks GlyBet; however, it possess some BADH activity and the transfer of CMO is, therefore, a means of installing the GlyBet pathway in tobacco. Furthermore, because conversion of choline to GlyBet occurs in the chloroplast, it is also possible to use chloroplast genetic engineering to transfer CMO into GlyBet non-accumulators (Zhang et al. 2008a). Zhang et al. (2008a) transformed tobacco with a gene for CMO from beetroot via chloroplast genetic engineering and found that the transgenic plants accumulated GlyBet in leaves, roots, and seeds, and exhibited improved tolerance to toxic choline levels and salt and drought stress. GlyBet accumulation in the chloroplasts may be more effective than in other organelles, such as the nucleus, for abiotic stress protection because of protection and stabilization of chloroplast proteins, membrane, and photosynthesis systems under stress conditions (Zhang et al. 2008a). Lv et al. (2007) found that transgenic cotton plants constitutively overexpressing betA had increased RWCs, increased photosynthesis, better osmotic adjustment, decreased percentage of ion leakage, decreased lipid membrane peroxidation, and increased yield in response to drought stress at the seedling, squaring, and anthesis stages.

Water and Ion Movement Water and ions move through plants via transcellular and intracellular pathways. Aquaporins (major intrinsic proteins; MIPs), which are either tonoplast- (TIP) or plasma membrane- (PIP) localized, facilitate water, glycerol, small molecule, and gas transfer through membranes and, therefore, have a role in water homeostasis (Bartels et al. 2007). Active transport of solutes into the cell and cellular organelles, particularly the vacuole, is another means of cell turgor maintenance as increased solute potential facilitates the passive movement of water into cells and cellular compartments (Li et al. 2008). Successful attempts made in engineering plants expressing genes for enzymes involved in proton pumps that generate energy for tonoplast transport of solutes into vacuoles include the overexpression of the Arabidopsis H+-pyrophosphatase (H+-PPase; AVP1) in Arabidopsis (Gaxiola et al. 2001), upregulation of AVP1 in tomato (Park et al. 2005c), heterologous expression of the Thellungiella halophila vacuolar-H+-PPase (V-H+-PPase; TsVP) in tobacco (Gao et al. 2006), and overexpression of the wheat Na–H+ antiporter, TNHX1, and H+-PPase, TVP1, in Arabidopsis (Brini et al. 2007b; Li et al. 2008). In all the cases, the transgenic plants displayed superior drought and/or salinity resistance compared with WT plants with resistance being attributed to mechanisms such as increased vacuolar H+ to drive secondary uptake of ions into the vacuole and more enhanced development and robustness of root systems (Gaxiola et al. 2001; Li et al. 2005a; Park et al. 2005c; Gao et al. 2006; Brini et al. 2007a, b). Recently, Li et al. (2008) reported that

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heterologous expression of the potassium-dependent TsVP gene from the halophyte T. halophyta in maize under the control of the maize ubiquitin promoter could infer drought tolerance. Under drought stress, transgenic plants had a higher percentage of seed germination, better-developed root systems, more biomass, increased solute accumulation, less cell membrane damage, less growth retardation, shorter ASI, and much higher grain yields than WT plants. Attempts have also been made to improve drought tolerance of plants by altering the expression of aquaporins (Aharon et al. 2003; Porcel et al. 2005; Yu et al. 2005; Peng et al. 2006; Jang et al. 2007; Cui et al. 2008; Miyazawa et al. 2008; Zhang et al. 2008c). Aquaporins facilitate transport of water and other small solutes and ions across membranes via the apoplastic route (Aharon et al. 2003; Cui et al. 2008; Jang et al. 2007; Peng et al. 2006; Zhang et al. 2008c). Research into the role of aquaporins in plant drought tolerance has shown that various aquaporins function differently depending on the severity and type of stress. For example, some aquaporins, such as the Arabidopsis Rd28, and rice RWC3, are upregulated under drought stress and others, such as NtQP1 and AtPIP1, remain unchanged under drought stress (Cui et al. 2008). Additionally, some aquaporins genes, such as AtPIP1b have been shown to diminish the drought tolerance capability of some plants, while others, such as the Vicia faba PIP1, Panax ginseng PgTIP1, Brassica napus BnPIP1, and Brassica juncea BjPIP1, have been shown to improve drought tolerance (Aharon et al. 2003; Yu et al. 2005; Peng et al. 2006; Cui et al. 2008; Zhang et al. 2008c). There is also evidence that overexpression of aquaporins in some plants causes them to respond differently to different stresses. For example, Jang et al. (2007) found that Arabidopsis and tobacco plants overexpressing Arabiopsis PIP’s displayed enhanced water flow and improved germination under cold stress, but exhibited rapid water loss, retarded seedling growth, and inferior germination under drought conditions. It is therefore thought that different aquaporin isoforms are associated with different physiological processes and that plants respond to drought conditions either by increasing aquaporin expression, which facilitates water movement (especially into the tonoplast in order to maintain cell-turgor) or downregulating aquaporin expression to avoid excessive water loss (Aharon et al. 2003; Peng et al. 2006). Overexpression of aquaporins has also been implicated in conferring heavy metal tolerance to transgenic plants by alleviation of metal ion-induced water deficit and oxidative damage caused by metal ions (Zhang et al. 2008c).

2.3 2.3.1

Engineering Salt Tolerance in Plants Impacts of Salinity on Agricultural Production

The damaging effects of salt accumulation in agricultural soils have severely affected agricultural productivity in large swathes of arable land throughout the world. Salt-affected land accounts for more than 6% of the world’s total land area

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(FAO 2009c) and is distributed largely amongst coastal salt marshes or inland desert sands. These have primarily arisen naturally through mineral weathering (which leads to the release of soluble salts such as chlorides of calcium, magnesium and sodium, and, to a lesser extent, sulfates and carbonates) or wind and rain deposition of oceanic water (Szabolcs 1989; Munns and Tester 2008; FAO 2009b). Secondary salinization occurs when irrigation and tree clearing of agricultural land cause water tables to rise and concentrate salts in the root zone (Rengasamy 2006). Approximately 20% of the world’s irrigated land, from which one-third of the world’s food supply is produced, is presently affected by salinity (Ghassemi et al. 1995). With the expected increase in world population, the loss of arable land due to salinity presents a serious challenge to food sustainability and productivity. Removal of salts from the root zone (reclamation) is perhaps the most effective way to ameliorate the detrimental effects of salinity; however, this is a slow and expensive process. The use of plant breeding and genetic engineering technologies to alter the salt tolerance of crops will, therefore, play an important role in maintaining global food production in the future.

2.3.2

Improving Salinity Tolerance of Agricultural Crops

Plants have evolved a complex adaptive capacity to perceive and respond to salt stress. The existence of salt-tolerant flora (halophytes) and differences in salt tolerance between genotypes within the salt-sensitive plant species (glycophytes) give rise to the belief that salt tolerance has a genetic basis (Yamaguchi and Blumwald 2005). As for drought, efforts to improve the salt tolerance of crops have met with limited success because of the physiological and genetic complexity of the trait. Salinity tolerance is a multi-genic trait, with quantitative trait loci (QTL) identified in barley, wheat, soybean, citrus, rice, and tomato (Flowers and Flowers 2005; Jenks et al. 2007). Genetic approaches currently being used to improve salinity tolerance include the exploitation of functional genomics, bioinformatics, and natural genetic variations, either through direct selection in stressful environments or through the mapping of QTLs and subsequent marker-assisted selection (Yamaguchi and Blumwald 2005), or the generation of transgenic plants (Vij and Tyagi 2007).

2.3.3

Physiological Effects of Salinity on Plants and Salinity Tolerance Mechanisms

Salinity imposes a variety of stresses on plant tissues. Two of these are osmotic stress, which results from the relatively high soil solute concentrations, and ion

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cytotoxicity. The decreased rate of leaf growth that occurs after an increase in soil salinity is primarily due to the osmotic effect of the salt around the roots, which inhibits plant water uptake and causes leaf cells to lose water. However, this loss of cell volume and turgor is transient and reductions in cell elongation and also cell division lead to slower leaf appearance and smaller final size over the longer term (Bartels and Sunkar 2005; Munns and Tester 2008). Under prolonged salinity stress, inhibition of lateral shoot development becomes apparent within weeks and, within months, there are effects on reproductive development, such as early flowering and reduced floret number. Concomitantly, older leaves may die while the production of younger leaves continues. The cellular and metabolic processes involved are similar to those occurring in drought-affected plants and are responses to the osmotic effect of salt (Yeo et al. 1991; Munns and Tester 2008). Ion cytotoxicity occurs when salt accumulates to toxic concentrations in fully expanded leaves (which, unlike younger leaves, are unable to dilute high salt concentrations), causing leaf death. Replacement of K+ by Na+ in biochemical reactions leads to conformational changes and loss of protein function, as Na+ and Cl– ions penetrate hydration shells and interfere with noncovalent interactions between amino acids. If the rate of leaf death generated by ion cytotoxicity is greater than the rate at which new leaves are produced, the photosynthetic capacity of the plant will no longer be able to supply the carbohydrate requirement of young leaves, which further reduces their growth rate (Munns and Tester 2008). Halophytes, though taxonomically widespread, are relatively rare amongst the flowering plants and virtually all crop plants are glycophytes (Flowers and Flowers 2005). However, there is considerable variability in the tolerance of glycophytes to salt. Munns and Tester (2008), categorize salinity tolerance under three broad categories: (1) Tolerance to osmotic stress, which immediately reduces cell expansion in root tips and young leaves, and causes stomatal closure; (2) Na+ exclusion from leaf blades, which ensures that Na+ remains at nontoxic concentrations within leaves; and (3) Tissue tolerance to Na+ or Cl–, which requires compartmentalization of Na+ and Cl– at the cellular and intracellular level to avoid accumulation of toxic concentrations within the cytoplasm.

2.3.4

Salt Tolerance Using Transgenic Approaches

2.3.4.1

Osmoprotectants

Osmoprotectants were discussed previously (Sect. 2.2.6.3.4) in relation to their use in developing drought-tolerant crops and the transfer of GlyBet intermediates have improved the drought and salt tolerance of transgenic plants in many cases. Mohanty et al. (2002) demonstrated that Agrobacterium-mediated transformation of an elite indica rice cultivar to increase GlyBet synthesis through the incorporation of the codA gene, which encodes choline oxidase, was an effective way to

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improve salinity tolerance. Challenge studies performed with R1 plants by exposure to salt stress for one week, followed by a recovery period, revealed that in some cases more than 50% of the transgenic plants could survive salt stress and set seed whereas WT plants failed to recover. A more recent example of enhanced GlyBet synthesis experiments involved transformation of maize with the BADH gene, introduced by the pollen-tube pathway (Wu et al. 2008a). Transgenic lines were examined for tolerance to NaCl by induced salt stress and, after 15 days of treatment, most transgenic seedlings survived and grew well, whereas WT seedlings wilted and showed loss of chlorophyll. The amino acid proline is known to occur widely in higher plants and normally accumulates in large quantities in response to environmental stresses (Kavi Kishore et al. 2005; Ashraf and Foolad 2007). In plants, the precursor for proline biosynthesis is l-glutamic acid. Two enzymes, pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR), play major roles in the proline biosynthetic pathway (Delauney and Verma 1993; Ashraf and Foolad 2007). Su and Wu (2004) established that the rate of growth of transgenic rice plants expressing mothbean D1-pyrroline-5-carboxylate synthetase (p5cs) cDNA under either a constitutive or stress-inducible promoter led to the accumulation of p5cs mRNA and proline in third-generation (R2) transgenic rice seedlings. Significantly higher salinity and water-deficit stress tolerance of R2 seedlings were attributed to faster growth of shoots and roots in comparison with non-transformed plants. Stressinducible expression of the p5cs transgene showed significant advantages over constitutive expression in increasing the biomass production of transgenic rice grown in soil under stress conditions. The osmoprotectant role of proline has been verified in other plants such as potato, where salt tolerance, measured by comparing tuber yield of transgenic lines cultivated in a greenhouse and watered with saline water to that of plants watered with normal tap water, had a less significant effect on tuber yield of transgenic plants than WT (Hmida-Sayari et al. 2005). Polyamines, including spermidine (Spd, a triamine), spermine (Spm, a tetramine), and their obligate precursor putrescine (Put, a diamine), are aliphatic amines widely present in living organisms. The polyamine biosynthetic pathway is depicted in Fig. 2.3. Recently, it has been demonstrated that plant polyamines are involved in the acquisition of tolerance to such stresses as high and low temperatures, salinity, hyperosmosis, hypoxia, and atmospheric pollutants (Liu et al. 2007). Furthermore, genetic transformation of several plant species with polyamine biosynthetic genes encoding arginine decarboxylase (ADC), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), or Spd synthase (SPDS) led to improved environmental stress tolerance (Liu et al. 2007). He et al. (2008) tested transgenic apple engineered with (SPDS)-overexpressing transgenic European pear (Pyrus communis L. “Ballad”) for changes in enzymatic and nonenzymatic antioxidant capacity in response to NaCl or mannitol stress. Their research revealed that transgenic plants accumulated more Spd than WT. The transgenic line contained higher antioxidant enzyme activities (less malondialdehyde and H2O2) than the WT, implying that it suffered from less injury and enhanced enzymatic and nonenzymatic antioxidant capacity (He et al. 2008).

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N

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H2N NH2 Putrecine

NH2

NH2 CH3

N

N N

N

+

S N

O

Spermidine synthase

CH3

N

N

N

S O

NH2 H2N

HO

OH

Methylthio-Adenosine

N H Spermidine

HO OH Decarboxylated AdoMet (dcAdoMet)

NH2

Spermine synthase H2N

N H

N H

NH2

Spermine

Fig. 2.3 Polyamine biosynthesis

Mannitol is a primary photosynthetic product that is associated with exceptional salt tolerance. In celery, mannitol metabolism is clearly altered by salt stress, with several lines of evidence indicating a connection between mannitol and salt tolerance, including increases in the capacity for mannitol biosynthesis and accumulation and decreases in catabolism (Williamson et al. 2002). Sickler et al. (2007) showed that Arabidopsis plants transformed with celery’s mannose6-phosphate reductase (M6PR) gene produced mannitol and grew normally in the absence of stress. However, in the presence of salt stress, daily analysis of the increase in growth (fresh and dry weight, leaf number, leaf area per plant, and specific leaf weight) over a 12-day period showed less effect of salt on transformants than WT plants. The daily energy use efficiency for photochemistry by photosystem 2 (PSII) was also measured and demonstrated that, unlike transformed plants, which were not affected, WT plants treated with 100 mM NaCl displayed a reduction in PSII yield after 6 days with a 50% loss in yield after 12 days. Similarly, under atmospheric levels of CO2, growth with 200 mM NaCl caused an increase in sub-stomatal levels of CO2 in WT plants but not in transformants. Trehalose is a disaccharide sugar widely distributed in bacteria, fungi, insects, plants and invertebrate animals. In microbes and yeast, trehalose is produced from glucose by trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP), and functions in sugar storage, metabolic regulation, and protection against abiotic stress (Strom and Kaasen 1993; Wiemken 1990). Trehalose acts as a compatible solute, protecting membranes and proteins and conferring desiccation

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tolerance on cells in the absence of water (Crowe et al. 1984). Ge et al. (2008) illustrated the protective role of trehalose in higher plants. Expression analysis demonstrated that OsTPP1 isolated and cloned from rice, was initiated and transiently upregulated after salt, osmotic, and ABA treatments but slowly upregulated under cold stress. OsTPP1 overexpression in rice enhanced salt and cold stress tolerance. Tolerance of transgenic plants to abiotic stress was examined by observing 2-week-old seedlings exposed to salt. Following one week of exposure, seedlings exhibited salt-induced damage symptoms such as wilted leaves. However, after prolonged salt treatment, transgenic lines were more vigorous and displayed increased leaf greenness and viability over control plants. Generally, two broad themes have emerged from the results of attempts to engineer overexpression of osmoprotectants. The first is that metabolic limitations have been encountered in generating absolute levels of target osmolytes, especially when compared with salt-tolerant halophytes and the second is that the degree to which transformed plants are able to tolerate salinity stress is not necessarily correlative with the levels of osmoprotectants attained.

2.3.4.2

Transporter Genes

Mechanisms that confer salt tolerance vary with the plant species; however, the ability to maintain low cytosolic Na+ is thought to be one of the key determinants of plant salt tolerance (Tester and Davenport 2003). Salt “inclusion” and “exclusion” are recognized as different mechanisms by which higher plants tolerate salinity. The functional removal of Na+ from the cytoplasm of plant cells and the maintenance of low cytosolic Na+ concentrations under salinity conditions (Blumwald et al. 2000) is accomplished by either pumping Na+ out of cells (plasma membrane antiporter) or into vacuoles (vacuolar antiporter) in exchange for H+. Na+/H+ antiporter activity is driven by the electrochemical gradient of protons (H+) generated by the H+ pumps (H+-ATPase) in the plasma membrane or the tonoplast (Chinnusamy and Zhu 2003; Tester and Davenport 2003). In Arabidopsis, active exclusion of Na+ is mediated by the plasma membrane-localized Na+/H+ antiporter, AtSOS1 (Shi et al. 2003). In contrast, the sequestration of excess Na+ into the vacuole is mediated by the vacuolar membrane-localized Na+/H+ antiporter, AtNHX1 (Gaxiola et al. 1999; Shi et al. 2008). In a similar way, overexpression of the S. cerevisiae HAL1 gene (Gaxiola et al. 1992) conferred salt tolerance in yeast by increasing intracellular K+ and decreasing Na+ levels. The successful use of transporter genes has been demonstrated in several plants. He et al. (2005) created transgenic cotton plants expressing AtNHX1 and found that transgenic plants generated more biomass and produced more fibers under salt stress in a greenhouse. It was suggested that the increased fiber yield was due to superior photosynthetic performance and higher nitrogen assimilation rates observed in the transgenic plants compared with WT. Interestingly, the researchers demonstrated that field-grown irrigated AtNHX1-expressing cotton plants produced higher fiber yields (fiber plus seeds) than WT, with an average increase of more

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than 25% per line. Furthermore, the fibers produced by transgenic plants were generally more uniform, stronger, and longer than those of WT. Similarly, Chen et al. (2007) engineered maize plants overexpressing the rice OsNHX1 gene. Transformants accumulated more biomass under greenhouse-based salt stress. Higher Na+ and K+ content was observed in transgenic leaves than in WT when treated with 100–200 mM NaCl, while the osmotic potential and the proline content in transgenic leaves was lower than in WT. Salt stress field trials revealed that the transgenic maize plants produced higher grain yields than WT plants at the vegetative growth stage. Biochemical studies suggest that Na+/H+ exchangers in the plasma membrane of plant cells contribute to cellular sodium homeostasis by transporting Na+ ions out of the cell (Qiu et al. 2002). SOS1 encodes a plasma membrane Na+/H+ exchanger in Arabidopsis (Qiu et al. 2002) and the important role of the plasma membrane Na+/H+ exchangers for plant salt tolerance was supported by the finding that overexpression of SOS1 improved plant salt tolerance (Shi et al. 2003). Zhao et al. (2006) demonstrated that expressing the plasma membrane Na+/H+ antiporter SOD2 from yeast (Schizosaccharomyces pombe) in transgenic rice also increased salt tolerance. Transgenic plants accumulated more K+, Ca2+, and Mg2+ and less Na+ in their shoots compared with non-transformed controls. Moreover, measurements on isolated plasma membrane vesicles derived from the SOD2 transgenic rice plant roots showed that the vesicles had enhanced P-ATPase hydrolytic activity as well as being able to maintain higher levels of photosynthesis and root proton exportation capacity. Martinez-Atienza et al. (2007) identified an AtSOS1 homolog, OsSOS1, in rice, which demonstrated a capacity for Na+/H+ exchange in plasma membrane vesicles of yeast (S. cerevisiae) cells and reduced their net cellular Na+ content. OsSOS1 was also shown to suppress the salt sensitivity of an sos1-1 mutant of Arabidopsis. In relation to the introduction of genes that modulate cation transport systems, many researchers have sought to employ the overexpression of the S. cerevisiae HAL1 gene, which has conferred salt tolerance in yeast by facilitating intracellular K+ accumulation and decreasing intracellular Na+ (Gaxiola et al. 1992; Rios et al. 1997). Rus et al. (2001) established ectopic expression of HAL1 in transgenic tomato plants, and showed that transformants were able to minimize the reduction in fruit production caused by salt stress. Maintenance of fruit production by transgenic plants was correlated with enhanced growth under salt stress of calli derived from the plants. The HAL1 transgene enhanced water and K+ contents in leaf calli and leaves in the presence of salt, which indicates that, similar to the yeast gene, plant HAL1 functions by facilitating K+/Na+ selectivity under salt stress. Ellul et al. (2003), utilizing an optimized Agrobacterium-mediated gene transfer protocol, developed HAL1-expressing watermelon (Citrullus lanatus). Salt tolerance of transgenic plants was confirmed in a semi-hydroponic system on the basis of the higher growth performance of transgenic lines compared to control plants. The halotolerance observed supports the potential usefulness of the HAL1 gene as a molecular tool for genetic engineering salt-stress protection in other crop species.

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93

Detoxifying Genes

The mechanisms of plant detoxification of ROS under drought stress were introduced in Sects. 2.2.6.2 and 2.2.6.2.3. As an antioxidant enzyme, glutathione peroxidase (GPX) reduces hydroperoxides in the presence of glutathione to protect cells from oxidative damage, including lipid peroxidation (Maiorino et al. 1995). Gaber et al. (2006) generated transgenic Arabidopsis plants overexpressing GPX2 genes in cytosol (AcGPX2) and chloroplasts (ApGPX2). The activities toward a-linolenic acid hydroperoxide in ApGPX2- and AcGPX2-expressing plants were 6.5–11.5 and 8.2–16.3 nmol min–1 mg protein–1, respectively, while no activity was detected in the WT plants. Both transgenic lines showed enhanced tolerance to oxidative damage caused by the treatment with H2O2, Fe ions, or methylviologen (MV) and environmental stress conditions, such as chilling with high light intensity, high salinity or drought. The degree of tolerance of the transgenic plants to all types of stress was correlated with the levels of lipid peroxide suppressed by the overexpression of the GPX-2 genes. SOD is the first enzyme in the enzymatic antioxidative pathway and halophytic plants, such as mangroves, reported to have a high level of SOD activity. SOD plays a major role in defending mangrove species against severe abiotic stresses. Prashanth et al. (2008) further characterized the Sod1 cDNA (a cDNA encoding a cytosolic copper/zinc SOD from the mangrove plant Avicennia marina) by transforming it into rice. Transgenic plants were more tolerant to MV-mediated oxidative stress in comparison to WT and withstood salinity stress of 150 mM of NaCl for a period of 8 days while WT plants wilted at the end of the hydroponic stress treatment. Pot-grown transgenic plants tolerated salinity stress better than the WT when irrigated with saline water. In plant cells, APXs are directly involved in catalyzing the reduction of H2O2 to water, which is facilitated by specific electron donation by ascorbic acid. APXs are ubiquitous in plant cells and are localized in chloroplasts (Takahiro et al. 1995), peroxisomes (Shi et al. 2001), and cytosol (Caldwell et al. 1998). Xu et al. (2008b) transformed Arabidopsis plants with a pAPX gene from barley (HvAPX1). The transgenic line was found to be more tolerant to salt stress than the WT. There were no significant differences in Na+, K+, Ca2+, and Mg2+ contents and the ratio of K+ to Na+ between pAPX3 and WT plants, which indicated that salt tolerance in transgenic plants was not due to the maintenance and re-establishment of cellular ion homeostasis. However, the degree of H2O2 and lipid peroxidation (measured as the levels of malondialdehyde) accumulation under salt stress was higher in the WT than in transgenic plants. The mechanism of salt tolerance in transgenic plants was explained by a reduction of oxidative stress injury. Apart from catalase and various peroxidases and peroxiredoxins (Dietz 2003), four enzymes, APX, dehydroascorbate reductase, monodehydroascorbate reductase and glutathione reductase (GR), are involved in the ascorbate-glutathione cycle, a pathway that allows the scavenging of superoxide radicals and H2O2 (Asada 1999). Most of the ascorbate-glutathione cycle enzymes are located in the stroma, cytosol, mitochondria, and peroxisomes (Jimenez et al. 1998). APX and GR, the first and

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last enzymes in this cycle, respectively, are responsible for H2O2 detoxification in green leaves (Foyer et al. 1994). GR has a central role in maintaining the reduced glutathione (GSH) pool during stress (Pastori et al. 2000). Lee and Jo (2004) introduced BcGR1, a Chinese cabbage gene that encodes cytosolic GR into tobacco plants via Agrobacterium-mediated transformation. Homozygous lines containing BcGR1 were generated and tested for their acquisition of increased tolerance to oxidative stress. When ten-day old transgenic tobacco seedlings were treated with 5 to 20 mM MV, they showed significantly increased tolerance compared to WT seedlings. The most drastic difference was observed at a concentration of 10 mM MV. In addition, when leaf discs were subjected to MV, the transgenic plants were less damaged than the WT with regard to their electrical conductivity and chlorophyll content.

2.3.5

Late Embryogenesis Abundant (LEA) Proteins

The LEA proteins were introduced in Sect. 2.2.6.3.3 in relation to their use in improving plant drought tolerance. These proteins have also been used in engineering salt-tolerant crops. Park et al. (2005b) introduced a B. napus LEA protein gene, ME-leaN4 (Wakui and Takahata 2002) into lettuce (Lactuca sativa L.) using Agrobacterium-mediated transformation. Transgenic lettuce demonstrated enhanced growth ability compared with WT plants under salt- and water-deficit stress. After 10-day growth under hydroponic 100 mM NaCl conditions, average plant length and fresh weight of transgenic lettuce were higher than those of WT and the increased tolerance was also reflected by delayed leaf wilting caused by water-deficit stress. Brini et al. (2007a) analyzed the effect of ectopic expression of dehydrin (Dhn-5; Table 2.1) cDNA in Arabidopsis under salt and osmotic stress. When compared to WT plants, the Dhn-5-expressing transgenic plants exhibited stronger growth under high concentrations of NaCl or water deprivation, and showed a faster recovery from mannitol treatment. Leaf area and seed germination rate decreased much more in WT than in transgenic plants subjected to salt stress. Moreover, the water potential was more negative in transgenic than in WT plants and the transgenic lines had higher proline contents and lower water loss rates under water stress. Na+ and K+ also accumulated to a greater extent in the leaves of the transgenic plants. Lal et al. (2008) reported the effects of overexpression of the HVA1 gene in mulberry under a constitutive promoter. HVA1 is a group 3 LEA (Table 2.1) isolated from barley aleurone layers and has been found to be inducible by ABA. Transgenic plants were subjected to simulated salinity and drought stress conditions to study the role of HVA1 in conferring tolerance. Using leaf discs as explants, growth performance under salt-stress and water-deficit conditions were carried out from 8- to 10-month-old transgenic plants. Leaf discs of uniform size were cut and used for simulated salt and water-deficit stress treatments for different durations. After the stress treatments, leaf discs were analyzed for proline content,

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photosynthetic yield, RWC, and cellular membrane stability (CMS). Transgenic plants showed better CMS, photosynthetic yield, less photooxidative damage, and better WUE as compared with the nontransgenic plants under both salinity and drought stress. Under salinity stress, transgenic plants showed a manyfold increase in proline concentration over WT plants and, under water-deficit conditions, proline accumulated only in the nontransgenic plants. Results also indicated that the production of HVA1 proteins enhanced the performance of transgenic mulberry by protecting plasma and chloroplast membrane stability under abiotic stress.

2.3.6

Transcription Factors

The importance of TFs in plant stress response was discussed in Sect. 2.2.5. In addition to binding to cis-acting elements in the promoters of environmental stressresponsive genes, TFs can activate and repress gene expression through interactions with other TFs, thus playing a central role in plant response to environmental stresses (Chen and Zhu 2004). It is generally accepted that activation or ectopic expression of a specific TF can result in expression of many functional genes related to stresses. CBF/DREBs are key regulatory factors that function primarily in freezing tolerance by activating a network of target genes (Fowler and Thomashow 2002; Maruyama et al. 2004). Oh et al. (2007) isolated a barley gene, HvCBF4, whose expression is induced by low temperature stress. Transgenic overexpression of HvCBF4 in rice resulted in an increase in tolerance to drought, high-salinity, and low temperature stresses without stunting growth. Interestingly, under low temperature conditions, the maximum photochemical efficiency of PSII in the dark-adapted state in HvCBF4 plants was higher by 20 and 10% than that in nontransgenic and CBF3/DREB1A-expressing plants, respectively. Using the 60K Rice Whole Genome microarray, 15 rice genes were identified that were activated by HvCBF4. When compared with 12 target rice genes of CBF3/DREB1A, 5 genes were common to both HvCBF4 and CBF3/DREB1A, and 10 and 7 genes were specific to HvCBF4 and CBF3/DREB1A, respectively. Results suggested that CBF/ DREBs of barley acted differently from those of Arabidopsis in transgenic rice. The NAC family of TFs (Sect. 2.2.6.1) has applicability for generating salttolerant crops. Hu et al. (2008) characterized a stress-responsive NAC gene (SNAC2) isolated from upland rice for its role in stress tolerance. Northern blot and SNAC2 promoter activity analyses demonstrated that SNAC2 expression was induced by drought, salinity, cold, wounding and ABA treatment. The SNAC2 gene was overexpressed in japonica rice to test the effect on improving stress tolerance. To test salinity tolerance, germinated positive transgenic and WT seeds were transplanted on Murashige and Skoog (MS) medium containing 150 mM NaCl and the normal MS medium without NaCl as a control. Under saline conditions, transgenic seedlings grew faster and their shoots were significantly longer than WT after 20 days. However, there was no difference in root length or root numbers

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between transgenic and WT seedlings grown under saline conditions and no difference in growth performance was observed between transgenic and WT seedlings in the normal MS medium. Hu et al. (2008) also evaluated germination ability of transgenic lines harboring SNAC2 under salt-stress conditions. After 4 days of germination on the medium containing 150 mM NaCl, only 40% of WT seeds were poorly germinated, whereas more than 70% of transgenic seeds germinated efficiently. In the MS medium, more than 90% of both transgenic and WT seeds germinated well and there was no significant difference in germination rates, suggesting that overexpression of SNAC2 does not affect seed germination under normal conditions. The significantly higher germination rate of transgenic seeds than that of WT under saline conditions further supported the improved salt tolerance of SNAC2-overexpressing plants. DNA chip profiling analysis of the transgenic plants revealed many upregulated genes related to stress response and adaptation such as peroxidase, ornithine aminotransferase, heavy metal-associated protein, sodium/hydrogen exchanger, HSP, GDSL-like lipase, and phenylalanine ammonia lyase. This data suggests that SNAC2 is a novel stress-responsive NAC TF that possesses potential utility in improving stress tolerance of rice. The TFIIIA-type zinc-finger proteins, first discovered in Xenopus, represent an important class of eukaryotic TFs (Miller et al. 1985). More than 40 TFIIIA-type zinc-finger protein genes have been identified from various plants, including petunia, soybean, Arabidopsis, and rice (Kim et al. 2001; Sugano et al. 2003; Mittler et al. 2006; Huang and Zhang 2007) and these genes have been shown to be induced by various abiotic stresses. Xu et al. (2008a) have recently reported the functional analysis of ZFP252 (a salt and drought stress responsive TFIIIA-type zinc-finger protein gene from rice), using gain- and loss-of-function strategies. They discovered that overexpression of ZFP252 in rice increased the amount of free proline and soluble sugars, elevated the expression of stress defense genes, and enhanced rice tolerance to salt and drought stresses compared with ZFP252 antisense and nontransgenic plants. Their findings suggest that ZFP252 plays an important role in rice response to salt and drought stresses and is useful in engineering crop plants with enhanced drought and salt tolerance (Xu et al. 2008a).

2.3.6.1

Signal Transduction Genes

Plant salt-stress-response genes are involved in many plant cellular processes, including physiological metabolism, cell defense, energy production and transportation, ion transfer and balance, and cell growth and division. These genes function through certain coordination mechanisms to maintain the normal growth of plants under salt stress. As discussed previously, components of the STP may also be shared by various stress factors such as drought, salt, and cold (Shinozaki and Yamaguchi-Shinozaki 1999). Signal molecules, H2O2 and NO, are involved in the ABA-induced stomatal closure and gene expression and activities of antioxidant enzymes (Zhang et al. 2006, 2007a). ABA-induced H2O2 production mediates NO generation, which in

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turn activates MAPK and results in upregulation of the expression and activities of antioxidant enzymes (Zhang et al. 2007a). The importance of ABA in plant environmental stress responses was discussed in Sect. 2.2.6.1. The oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycartenoid dioxygenase (NCED) is the key regulatory step of ABA biosynthesis in higher plants. Overexpression of SgNCED1 in transgenic tobacco plants resulted in 51–77% more accumulation of ABA in leaves (Zhang et al. 2008d). Transgenic tobacco plants were shown to have decreased stomatal conductance and transpiration and photosynthetic rates and increased activities of SOD, catalase, and APX activities. H2O2 and NO in leaves were also induced in the transgenic plants. Compared with WT, the transgenic plants displayed improved growth under 0.1 M mannitol-induced drought stress and 0.1 M NaCl induced salinity stress. It was suggested that the ABA-induced H2O2 and NO generation upregulates stomatal closure and antioxidant enzymes and, therefore, increases drought and salinity tolerance in transgenic plants. Salt stress is known to trigger a rapid and transient increase of free calcium concentration in plant cells (Knight 2000; Pauly et al. 2000). As such, Ca2+ signaling processes are one of the earliest events in salt signaling and may play an essential role in the ion homeostasis and salt tolerance in plants ( Zhu 2003; Reddy and Reddy 2004). CBLs represent a unique family of calcium sensors in plants and function as a positive regulator in the salt-stress-response pathway. Extensive studies have progressed toward understanding of Arabidopsis CBLs, yet knowledge of their functions in other plant species is still quite limited. Wang et al. (2007a) have reported the cloning and functional characterization of ZmCBL4, a novel CBL gene from maize. ZmCBL4 encodes a putative homolog of the Arabidopsis CBL4/SOS3 protein. Under normal conditions, ZmCBL4 was shown to be expressed differentially at a low level in various organs of maize plants and its expression was regulated by NaCl, LiCl, ABA and PEG treatments. Expression of 35S::ZmCBL4 not only complemented the salt hypersensitivity in an Arabidopsis sos3 mutant, but also enhanced the salt tolerance in Arabidopsis WT plants at the germination and seedling stages. ZmCBL4-expressing Arabidopsis lines accumulated less Na+ and Li+ as compared with WT plants. Wang et al. (2007a) concluded that the maize CBL gene functions in salt-stress-elicited calcium signaling and thus in maize salinity tolerance.

2.4 2.4.1

Engineering Cold Tolerance in Plants Impacts of Cold Stress on Agricultural Production

Agricultural borders for crop species are defined geographically by occurrences of low temperatures and frost, which cause severe yield losses in marginal areas. Approximately two-thirds of the world’s landmass is annually subjected to temperatures below freezing and half to temperatures below –20 C.

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Most crops of tropical origin, as well as many of subtropical origin, are sensitive to chilling temperatures. Amongst the major world food crops, maize and rice are sensitive to chilling temperatures and yield loss or crop failure of these species can occur at temperatures below 10 C. Many other crops, such as soybean, cotton, tomato, and banana, are injured at temperatures below 10–15 C (Lynch 1990). The temperature below which chill injury can occur varies with species and regions of origin and ranges from 0 to 4 C for temperate fruits, 8 C for subtropical fruits, and about 12 C for tropical fruits (Lyons 1973). Cold acclimation, also known as cold hardening, describes an increase in tolerance over time to cold temperatures and cellular desiccation in response to conditions such as cold temperature, short photoperiods, and mild drought and results from changes in gene expression and physiology (Xin and Browse 2000; Kalberer et al. 2006). Most temperate plants can cold-acclimate and acquire tolerance to extracellular ice formation in their vegetative tissues. Winter-habit plants such as winter wheat, barley, oat, rye, and oilseed rape have a vernalization requirement, which allows them to survive freezing stress as seedlings during winter. However, after vernalization and at the end of the vegetative phase, the cold acclimation ability of winter cereals gradually decreases, making them sensitive to freezing injuries (Fowler et al. 1996; Chinnusamy et al. 2007). Therefore, it is not surprising that the impacts of cold stress on plant life have been comprehensively studied. Many attempts have been made to improve cold resistance of important crop plants; however, progress in achieving frost hardiness of plants either by classical breeding or by gene transfer is difficult because of the fact that cold resistance is not a quality conferred by the product of one gene, but, as for most abiotic stress tolerance mechanisms, is quantitative in nature (Mahajan and Tuteja 2005).

2.4.2

Physiological Effects of Cold Stress on Plants and Cold Tolerance Mechanisms

The symptoms of chilling-induced stress injury in cold-sensitive plants are variable and generally manifest within 48 to 72 h of stress exposure. Observed phenotypic symptoms in response to chilling stress include reduced leaf expansion, wilting, chlorosis, and necrosis (Mahajan and Tuteja 2005). Chilling also severely inhibits plant reproductive development, with species such as rice displaying sterility when exposed to chilling temperatures during anthesis (Jiang et al. 2002). The extent of plant damage caused by exposure to low temperature depends on factors such as the developmental stage, the duration and severity of the frost, the rates of cooling (and rewarming), and whether ice formation takes place intra- or extra-cellularly (Beck et al. 2004). Chilling stress (100 mg/kg for Cd, Cr, Co, Pb; or >1,000 mg/kg for Ni, Cu, Se, As, Al; or 10,000 mg/kg for Zn, Mn in their above-ground dry weight biomass Ability of plants to survive and thrive in heavily contaminated sites contaminated with heavy metals or metalloids

(continued )

Raskin et al. (1994), Simon et al. (1996), Mendez and Maier (2008)

Raskin et al. (1994), Chaney et al. (1997), Salt et al. (1998)

Brooks et al. (1998, 1999), Gratao et al. (2005) Beckett and Davis (1988), Naidu et al. (2003)

Cluis (2004), Suresh and Ravishankar (2004), Pilon-Smits (2005) Bradshaw (1997)

de Crombrugghe (1964), Chaney et al. (1997), Cluis (2004)

Newman et al. (1997), Salt et al. (1998), Trapp and Karlson (2001), Suresh and Ravishankar (2004), PilonSmits (2005), Gifford et al. (2007) Chaney et al. (1997), Salt et al. (1998), Reeves and Baker (2000), Sursala et al. (2002), Gifford et al. (2007)

Salt et al. (1995, 1998), Dietz and Schnoor (2001), Suresh and Ravishankar (2004), Gifford et al. (2007)

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Land farming

Organic pump/Tree pump

Mycofiltration Rhizosphere degradation/ Rhizodegradation

Rhizofiltration/Phytofiltration

Rhizosecretion

Table 8.1 (continued) Terminologies Plant biomonitor/ Phytomonitor Plant-assisted bioremediation

Definitions A plant providing complete quantitative information on environment quality Remediation of soils contaminated with organic pollutants by plant roots in association with rhizosphere-inhabiting microbial communities This is a subset of molecular farming designed to produce and secrete valuable natural products and recombinant proteins from roots Use of live plant roots for the removal of toxic heavy metals and other pollutants from water or any liquid source. Also referred to as phytofiltration Fungal mycelial mats used as biological filters Plant roots and/or root exudates provide a local environment rich in nutrients and enzymes in the rhizosphere that promotes degradation of soil contaminating pollutants by resident microbial communities. Microbial breakdown of organic pollutants in the rhizosphere Trees with dense root systems accumulate greater volume of water, thereby reducing possibilities of surface pollutants to migrate downwards towards the groundwater table and contaminate freshwater resources. Extensively used for regulating run off from agricultural fields and leaching of toxic pollutants from landfill sites Used for land affected by oil pollution. Sludge is ploughed onto topsoil, fertilizers are applied, and grasses mostly rye (Secale cereale) or alfalfa (Medicago sativa) are then sown on it Oil is degraded rapidly in the rooted, aerated and fertilized topsoil zone Trapp and Karlson (2001)

Salt et al. (1998), Trapp and Karlson (2001), Suresh and Ravishankar (2004)

Raskin et al. (1994), Dushekov et al. (1995), Raskin et al. (1997), Salt et al. (1998), Trapp and Karlson (2001), Zayed (2004), Pilon-Smits (2005) Stamets and Sumerlin (undated) Schnoor et al. (1995), Salt et al. (1998), Ramaswami et al. (2003), Suresh and Ravishankar (2004), Pilon-Smits (2005)

Gleba et al. (1999)

Salt et al. (1995)

References Kovalchuk and Kovalchuk (2001, 2003, 2008)

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CUMULATOR HYPER AC

PHYTOVOLATILIZATION PHYTOTRANSFORMATION PHYTODEGRADATION PHYTOCONVERSION

PHYTOREMEDIATION PHYTODECONTAMINATION PHYTODETOXIFICATION PHYTORESTORATION

Less toxic pollutant forms

PHYTOEXTRACTION PHYTOACCUMULATION

Highly toxic pollutant forms PHYTOSTABILIZATION RHIZOFILTRATION PHYTOMINING PHYTOSTIMULATION ORGANIC PUMP

RHIZOSPHERE DEGRADATION

GROUND WATER

Fig. 8.1 Schematic representation of different types of phytoremediation and relationships between them

biological soil treatments, the most common ones are landfarming (see Table 8.1) and ex situ techniques such as biopiles, slurry reactors and composting (Cunningham et al. 1995). Phytoremediation has been considered to be an environmentally compatible, sustainable, easily monitored, efficient and less expensive approach for the removal and detoxification of harmful environmental pollutants, compared to other chemical engineering alternatives (Baker and Brooks 1989; Baker et al. 1994; Nanda Kumar et al. 1995; Raskin et al. 1997; Datta and Sarkar 2004; Gray 2006). Reliable cost estimates associated with phytoremediation have been evaluated earlier by Cunningham et al. (1995) and recently by Pilon-Smits (2005). An important message as indicated by Gratao et al. (2005) is that phytoremediation processes are costeffective and safe alternatives to conventional physical and chemical treatments. Phytoremediation is a process with a lower impact on the surrounding environment and without any disruption of highly fragile and vulnerable ecosystems (Barcelo and Poschenrieder 2003; Zayed 2004). Although a large number of plants (known as hyperaccumulators) are capable of bioaccumulating high concentrations of toxic metals, they generally do not generate sufficient biomass and are not efficient for phytoremediation over a longer period of time. Hence, an alternative solution could be the creation of transgenic plants with greater biomass, faster growth rate, and better phytoremediation characters. Phytoremediation is an advantageous process in the sense that it helps remove toxic components in situ, and there are no direct risks of environmental contamination exposure during handling and transfer of pollutants from the contaminated

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Cost-effective, sustainable, non-intrusive, environment-friendly

Reduction of: Agricultural surface run-offs, loss of top soil, sediment run-offs, soil moisture

Soil stabilization, reclamation, amelioration & revegetation

Restoration of nutritional and biological qualities of contaminated soil

Reduction of soil erosion

Phytoremediation Benefits

Landscaping, increased value of remediated landfills

Wildlife habitat restoration

Immobilization and trapping of pollutants before reaching groundwater

Improvement of the local micro climate

Aesthetic and recreational values

Decontamination of contaminated sites

Biodegradation and detoxification of military munitions compounds Improving qualities of dump sites, landfills, agricultural lands, forests, wetlands, abandoned industrial sites, mining areas, marginal lands

Fig. 8.2 Benefits of phytoremediation

sites. Moreover, the process itself does not generate secondary waste products (Baker and Brooks 1989; Baker et al. 1994; Shann 1995; Chaney et al. 1997; Zayed 2004; Willey 2007). Benefits of phytoremediation have been compared to conventional physical and chemical remediation treatments (Fig. 8.2).

8.2.3

Historical Background

The basic and empirical studies of phytoremediation focused mainly on and around natural plant species capable of detoxifying harmful substances and on their natural properties of hyperaccumulation of toxic metals and environmentally detrimental toxic chemical compounds (Baker and Brooks 1989; Nanda Kumar et al. 1995; Salt et al. 1995; Shann 1995; Chaney et al. 1997; Raskin et al. 1997; Datta and Sarkar 2004). Over the past few decades, extensive investigations have been conducted on different phytoremediation strategies and techniques used by plants (Banuelos et al. 1993; Baker et al. 1994; Salt et al. 1998). The role of a number of phytoremediating species involved in complete and partial degradation of chemical explosives (as reviewed in Hannink et al. 2002), heavy metals and metalloids (Terry et al. 2003), and their biochemical pathways for uptakes has been extensively studied. Basic research associated with phytoremediation was primarily focused on identifying and screening plants capable of

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withstanding heavy metals and radionuclide pollution, such as natural hyperaccumulators. Plants capable of decontamination of polluted sites by rapid biosorption, bioaccumulation, biodegradation, and their conversion (biotransformation) to less toxic, non-bioavailable forms have also been extensively studied and reviewed in primary literature sources (Freeman et al. 2004; Suresh and Ravishankar 2004; Cherian and Oliveira 2005; Pilon-Smits 2005; Denton 2007). A large number of chemicals such as heavy metals, xenobiotics, nitramines and nitraromatics, herbicides, pesticides, fertilizers, complex organic and inorganic salts and compounds, pharmaceutically important compounds, and radioactive chemicals have been reported to be successfully phytoremediated by different plant species and model plants used in the laboratories around the globe (Ellis et al. 2004; Freeman et al. 2004; Suresh and Ravishankar 2004; Cherian and Oliveira 2005; Mezzari et al. 2005; Willey 2007). The promising potential roles played by different metallothionein (MT) and phytochelatin (PC) genes have been reviewed by Eapen and D’Souza (2005). Recently, Hassinen et al. (2007) reported that ~400 plant species have been identified as hyperaccumulators, and the majority of these plants are Brassicaceae (Arabidopsis family) members hyperaccumulating nickel (Ni). A large number of papers are also available on physiology, biochemistry, metabolism, transport mechanisms, sequestration patterns of toxic heavy metals and other pollutant compounds (Baker and Brooks 1989; Baker et al. 1994; Nanda Kumar et al. 1995; Shann 1995; Chaney et al. 1997; Pence et al. 2000; Ellis et al. 2004; Freeman et al. 2004; Denton 2007).

8.2.4

Transgenics Research on Herbs and Shrubs

Compared to natural and conventional phytoremediators, genetically engineered plants or transgenic plants (transgenics) have proved to be a rather handy tool in generating actively phytoremediating plants because of their enhanced ability to effectively reduce, degrade, transform and accumulate toxic pollutants from the environment; their growth rates are rapid, and they have better bioaccumulation characteristics (Cherian and Oliveira 2005; Willey 2007). Conventional plant breeding can only utilize available and rather limited genetic resources within species and genera of plants for phytoremediation. On the contrary, transgenic plants have specific pollutant-detoxifying or binding genes form widely divergent sources and are better equipped to address challenges associated with effective phytoremediation of contaminated sites and water bodies (Chaney et al. 1997). Another important reason for interest in producing transgenics in nondomesticated species is explained by the fact that growth rates of normal phytoremediating plants are slow and are often seasonally variable, and the amount of sequestration and degradation reported for such plants are often low (Terry et al. 2003). Hence, decontamination or detoxification does not always reach the required values accepted and set by regulatory agencies (Suresh and Ravishankar 2004).

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Even worse, at some contaminated sites, the level of pollutants could be at toxic concentration to plants or may be recalcitrant to degradation or bioaccumulation, rendering plant services completely ineffective (Terry et al. 2003). Hence, one of the most direct approaches for making phytoremediation by target plant species more successful is overexpression of genes involved in metabolism, uptake, transport, sequestration, or detoxification of harmful chemical pollutants (Suresh and Ravishankar 2004; Cherian and Oliveira 2005; Willey 2007). Since the genetic diversity among natural phytoremediators is not very high, their ability to clean the environment is comparatively low (Fladung and Ewald 2006). Hence, the idea of transferring genes from bacterial, yeast, and animal members and even from other plants to produce target laboratory plants that are better equipped to be strong phytoremediator has recently gained enormous prominence (Oksman-Caldentey and Barz 2002; Fladung and Ewald 2006; Willey 2007). Terry et al. (2003) have discussed in details the significance of works on overexpression of enzymes catalyzing rate-limiting steps in sulfate assimilation and PC biosysnthetic pathways in transgenic plants exhibiting an increased resistance to selenium (Se) and cadmium (Cd). In addition, conventional contaminated site cleanup technologies have an extensive overhead cost that could only be addressed by promoting transgenic plants as phytoremediators, because they have been recently estimated to be more costeffective than traditional in situ or ex situ processes. They are easier to be used for cleaning up sites located in distant areas, difficult mountainous terrain, or other less inaccessible localities. Moreover, higher adaptability of transgenic phytoremediators to toxic compounds and their better survival rates make them a better choice for cleaning the environment (Suresh and Ravishankar 2004; Cherian and Oliveira 2005; Fladung and Ewald 2006; Willey 2007). Till date, a large number of plants from 45 different plant families have been identified for phytoremediation abilities (Raskin 1996; Salt et al. 1998); the maximum number of phytoremediation species has been reported from the Brassicaceae family (Reeves and Baker 2000; Gratao et al. 2005). The first transgenic plants developed for bioremediation purposes were reported by Misra and Gedamu (1989). These plants expressed the human MT gene to develop tolerance to Cd toxicity. The next major breakthrough in phytoremediation research was reported by Rugh et al. (1996). To increase the tolerance of Arabidopsis thaliana to mercury, they generated transgenic plants overexpressing the mercuric reductase gene. The most important factors for considering plants for phytoremediation as suggested by Newman et al. (1997) and Tong et al. (2004) are: rapid plant growth, large biomass, easy multiple harvesting (3–4 times per year), better than average uptake. Although several plant breeding approaches have been targeted to develop plant varieties demonstrating better phytoremedaiation performance, the success has not been phenomenal. Hence, nowadays the emphasis is being made on genetic engineering and development of transgenic lines for producing better phytoremediating lines in target species (Salt et al. 1998; Clements et al. 2002; Willey 2007). However, it is important to note that transgenes procured from different sources need to be carefully tracked, and their expression needs to be targeted to appropriate

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cell compartments for maximizing the benefits of transgene incorporation (Salt et al. 1998; Clements et al. 2002; Bizily et al. 2003; Pilon et al. 2003). A comprehensive summary of diverse transgenes inserted into different target plant species used in phytoremediation and their corresponding responses after genetic engineering are presented in Table 8.2.

8.2.5

Transgenic Trees in Phytoremediation

Nowadays, trees are extensively used in phytoremediation (Sykes et al. 1999) because of their advantages over other plant species. These advantages are: greater biomass, larger size, strong extensive and proliferating root systems capable of spreading to a considerable depth for efficient phytoremediation, better ability to accumulate pollutants and prevent rapid soil erosion, better ability to withstand low water-stress conditions, and perennial growth habits. Other advantages include easier maintenance, no need for constant monitoring, easy harvesting, effective removal of pollutants from contaminated sites, and better ability to survive in diverse biogeographical and widely fluctuating climatic conditions. Both Clements et al. (2002) and Sykes et al. (1999) suggested introduction of “hyperaccumulation genes” into target tree species for making them more amenable to better phytoremediation in challenging sites and localities, where it was difficult to achieve success using other plant species. Recently, for the first time Doty et al. (2003) reported that the tropical leguminous tree Leucaena leucocephala is an excellent phytoremediating species that can take up and metabolize both toxic organic pollutants 2.4,6-trichloroethylene (TCE) and ethylene dibromide (EDB). The authors reported that this plant’s ability to debrominate makes it an interesting candidate to explore in terms of genetic engineering, if it is not recalcitrant to genetic modifications.

8.3

8.3.1

Phytoremediation of Inorganic Pollutants by Transgenic Plants Mercury

Hg pollution is a serious threat to a wide array of living organisms ranging from humans, animals, plants, and agriculturally important crops to beneficial microbes residing in the soil (Rugh 2001). Hg is commonly released into the environment in its ionic form (Hg+2), and then it is converted into methylmercury mostly by the process of methylation aided by methanogenic anaerobic microbes from aquatic sediment deposits (Meagher 2000). Methylmercury is approximately 200 times more toxic than the ionic form of Hg and is even considered to be more detrimental

A. thaliana A. thaliana A. thaliana A. thaliana A. thaliana (Ac/Ds transposon tagging transformant) A. thaliana A. thaliana (cadl-3 mutant line) Atropa belladonna (hairy root culture) Brassica juncea B. juncea B. juncea B. juncea B. juncea

Increase intake of Fe and better tolerance to Cd Higher Cu accumulation Better tolerance to Cd and accumulation of Cd PCB degradation PCB degradation Detoxification of phenolic compounds like TCP Low CD accumulation; higher transport rate in leaves Rapid TCE metabolism Better Cd accumulation Increase in Zn and Cd uptake Increase in Zn and Cd uptake Increase in Cd tolerance and accumulation Increase in Se tolerance and accumulation

AtNramp3 PsMTA OASTL Lip, MnP Ds transposon + GUS LAC1 TaPCS1 P450 2E1 gshII g-GCS GC gshI SMT

Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae

Brassicaceae Brassicaceae

Solanaceae

Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae

Table 8.2 Summary of transgenes inserted into different target plant species used in phytoremediation and their corresponding engineering Target plant Family Transgene Phytoremediation response(s) reported Arabidopsis halleri Brassicaceae NAS Better Ni hyperaccumulation Arabidopsis thaliana Brassicaceae g-GCS, arsC Increased fresh weight and shoot accumulation of arsenates A. thaliana Brassicaceae merA, merB Better resistance to Hg toxicity A. thaliana Brassicaceae merB Better Hg volatilization A. thaliana Brassicaceae SAT Better tolerance to Ni A. thaliana Brassicaceae AtPCS1 Increase in PC concentration A. thaliana Brassicaceae YCF1 Bigger biomass and higher Cd uptake in leaves A. thaliana Brassicaceae merApe9 Better resistance to Hg contamination A. thaliana Brassicaceae SL Slightly increased Se accumulation and slightly lowered Se incorporation in proteins A. thaliana Brassicaceae SMT Increase in Se tolerance and accumulation A. thaliana Brassicaceae ZAT1 Higher tolerance to Zn

Liang et al. (1999) Bennett et al. (2003) Bennett et al. (2003) Zhu et al. (1999) LeDuc et al. (2004)

Banerjee et al. (2002)

Wang and Chen (2007) Gong et al. (2003)

Sonoki et al. (2007) Sonoki et al. (2007)

Ellis et al. (2004) Van der Zaal et al. (1999) Thomine et al. (2000) Evans et al. (1992)

Bizily et al. (2000) Bizily et al. (2003) Freeman et al. (2004) Lee et al. (2003) Gong et al. (2003) Rugh et al. (1996)

References Becher et al. (2004) Dhanker et al. (2002)

responses after genetic

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Brassicaceae Brassicaceae Brassicaceae

Magnoliaceae merApe9 Magnoliaceae gsh1 Solanaceae MT II

Brassica napa Brassica napus B. napus

Liriodendron tulipifera L. tulipifera Nicotiana benthamiana

AtMHX1 MT1 CUP1 bphC merA, merB

Solanaceae

Solanaceae

Solanaceae Solanaceae Solanaceae Solanaceae

Solanaceae

N. tabacum

N. tabacum

N. tabacum N. tabacum N. tabacum N. tabacum

NtCBP4

CUP1, GUS, HisCUP, HisGUS merA, merB

Solanaceae

N. tabacum

todC1, todC2 TaPCS1 MT merA merA nfs1 onr merA

Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae

N. benthamiana Nicotiana glauca Nicotiana glutinosa Nicotiana tabacum N. tabacum N. tabacum N. tabacum N. tabacum

ACC ACC MT II

GR

Brassicaceae

B. juncea

APS1

Brassicaceae

B. juncea

Better Hg phytovolatilization and phytoaccumulation Better tolerance to Ni and improved hyperaccumulation of Pb Reduce tolerance to Mg and Zn Higher tolerance to Cd Better tolerance to Cu Phytodegradation of PCBs

Increased phytoremediation of toluene Higher Pb and Cd accumulation Increased tolerance to Cd Better resistance to Hg toxicity Better resistance to Hg toxicity Biotransformation of TNTs Better detoxification of nitroglycerin compounds About a fivefold increase in Hg volatilization in roots compared to leaves and shoots Increased accumulation of Cd and Ni

Better phytovolatilization of Hg contaminants Increased accumulation of Cd, Cu, Zn Increased resistance to Cd toxicity

Increased accumulation and tolerance to arsenate Higher heavy metal tolerance (Ni, Zn, Cu, Pb) Increased resistance to Cd toxicity

Increased tolerance to Cd

High accumulation of Se in a plant body

Arazi et al. (1999), Sunkar et al. (2000) Shaul et al. (1999) Pan et al. (1994) Thomas et al. (2003) Chrastilova et al. (2007) Ruiz et al. (2003) (continued )

Bizily et al. (2003)

Pavlikova et al. (2004)

Pilon-Smits et al. (1999) Pilon-Smits et al. (2000) Nie et al. (2002) Stearns et al. (2007) Misra and Gedamu (1989) Rugh et al. (1998) Arisi et al. (2000) Misra and Gedamu (1989) Novakova et al. (2007) Gisbert et al. (2003) Liu et al. (2002) Heaton et al. (1998) Meagher et al. (2000) Hannik et al. (2001) French et al. (1999) He et al. (2001)

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CYP2E1 P450CYP1A1 CYP1A, CYP2B6, CYP2C

Poaceae

Salicaceae

Salicaceae

Solanaceae Solanaceae

Cyperaceae

Brassicaceae Brassicaceae

O. sativa

Populus deltoides

Populus tremula
Populus alba (Hybrid clone) Solanum tuberosum S. tuberosum

Spartina alteriniflora

Thlaspi caerulescens Thlaspi goesingense

ZNT1 TgMTP1

merA, merB

Enhanced ability to withstand Hg toxicity and better phytovolatilization Increase in Zn uptake and tolerance Efficient Ni hyperaccumulation

Better resistance to Hg toxicity and greater biomass generation Increased phytoremediation of different hydrocarbons Herbicide detoxification Herbicide detoxification

Higher Hg phytoremediation in wetland areas Biodegradation of chlorinated aromatic compounds Enhanced resistance to herbicides

merA cbnA

Poaceae Poaceae CYP1A1, CYP2B6, CYP2C9, CYP2C18, CYP2C19 merA9, merA18

Phytoremediation response(s) reported

Transgene

Family

Table 8.2 (continued) Target plant N. tabacum (Chloroplast genome engineering) Oryza sativa O. sativa

Pence et al. (2000) Persans et al. (2001)

Yamada et al. (2002) Ohkawa and Ohkawa (2002) Czako et al. (2006)

Doty et al. (2007)

Ohkawa and Ohkawa (2002) Che et al. (2003)

Heaton et al. (2003) Shimizu et al. (2002)

References

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because of its enormous biomagnification property up in the ecological food chain (Ruiz et al. 2003). Hg, compared to other toxic heavy metals detected in the environment, is extremely detrimental even at considerably low concentrations (Meagher 2000). Methylmercury is exceptionally toxic because of its unique property of hydrophobicity that enables it to migrate inside the cell and then deactivate several key metabolically important enzyme systems (Castoldi et al. 2001; Rugh 2001). A number of bacterial species have been reported that can demethylate methylmercury and transform it back to its less toxic native forms (Rugh et al. 1996; Bizily et al. 2003). Elemental Hg0 is extremely volatile and is readily transformed from the liquid phase to the vapor phase. Bacterial species capable of converting methylmercury ! Hg+2 ! Hg0 are characterized by the presence of a Hg-responsive operon consisting of: (1) Hg-responsive regulatory proteins; (2) transport proteins that bind and carry Hg into the cell; (3) a specific enzyme known as an organomercuric lyase (merB gene) that catalyzes the removal of CH3 group from methylmercury and transforms it to ionic mercury (Hg+2); and (4) mercuric ion reductase (merA gene) transforming ionic mercury to elemental mercury Hg0 (Rugh et al. 1996, 1998). Gene products are expressed only when species is exposed to Hg (Rugh et al. 1996, 1998). In the past, researchers exploited these Hg-responsive operons in bacteria for effective phytoremediation of toxic Hg-contaminated sites (Meagher 2000; Rugh 2001; Suresh and Ravishankar 2004). Earlier attempts to express merA in plant systems were not successful, because the gene was reported to be G + C rich (about 67%), and hence it expressed itself only in bacterial systems (Rugh et al. 1996). In addition, it also had a higher number of CpG motifs (sites for methylation and gene silencing). Rugh et al. (1996) for the first time exploited the merA gene by replacing codons 287–336 and thereby developing a modified gene merApe9 that was efficiently expressed in the A. thailiana system. Transgenic lines with the merApe9 gene produced viable seeds, and the corresponding seedlings survived on agar plates containing 25–100 mM HgCl2 compared to their corresponding non-transformed controls. Hg vapor analysis also confirmed that transgenic lines successfully phytovolatilized ionic mercury to elemental forms with approximately 50 ng Hg0/mg fresh tissue weight. The authors detected that plants expressing merApe9 were also resistant to gold. In an attempt to further improve key technology and extend its application to other plant species in addition to model laboratory plants, Rugh et al. (1998) used bigger biomass generating plants. The researchers further modified the previously developed transgene merApe9 to include an additional 9% of the coding sequence DNA fragment for better codon optimization and effective expression in a particular species of yellow poplar (Liriodendron tulipifera). The gene was delivered into plant embryonic masses using a particle bombardment approach. The authors reported transgenic seedlings growing and surviving on agar plates incorporated with 25 and 50 mM HgCl2 compared to their non-transformed controls and phytovolatilization rates detected in the species were also appreciable. Bizily et al. (1999) reported A. thaliana lines containing the bacterial merB gene and successfully surviving on higher concentrations of HgCl2 and phenyl mercuric

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acetate compared to their controls. The original bacterial merB gene was modified using PCR techniques to contain flanking sites incorporated with consensus plant sequences and restriction sites. Using Western blot analysis, the authors provided substantial evidence that a sufficient amount of the gene product (organomercurial lyase) was synthesized by A. thaliana transgenic lines. Later, Bizily et al. (2000) reported production of A. thaliana lines with enhanced abilities of Hg phytoremediation. In this case, separate transgenic A. thaliana lines carrying merA and merB genes, respectively, were crossed and hybrid F2 plants and screened for expression of both gene products. Plants with double gene expression (merA/merB) survived better in methylmercury-incorporated agar plates containing the concentration of methylmercury higher than 10 mM; compared to plants that expressed either merA or merB separately, which could survive at concentrations up to 5 mM of methylmercury. Western blot analysis confirmed the simultaneous expression of gene products. However, Bizily et al. (2000) suggest that transgenic lines were less hardy compared to control plants in most experimental trials. The authors attributed this to the transgene capable of reducing a wide diversity of ions (particularly merA), many of which being vital for physiology and metabolism of plants. In another study, He et al. (2001) reported detection of an approximately fivefold increase in Hg volatilization in transgenic tobacco (Nicotiana tabacum) roots compared to the above-ground biomass, thus suggesting the organ-specific and species-specific plant response to Hg phytoremediation. An important limiting factor for Hg phytoremediation is the diffusion rate of methylmercury to the cytoplasmically expressed MErB proten (Bizily et al. 2000). It was addressed later by Bizily et al. (2003) in developing a specific merB cassette that targets MerB proteins on the cell wall to avoid the slower diffusion rate of methylmercury in the cytoplasm. The authors showed that transgenic lines having this specific merB construct along with the merA cassette were 10–70 times better than any other competing lines developed. Ruiz et al. (2003) reported for the first time integration of a native operon with merA and merB bacterial genes into the chloroplast genome of tobacco by a single transformation event. The authors detected high levels of tolerance to phenylmercuric acetate (100, 200 and 400 mM) in the stable transgenic lines compared to the controls. These plants were highly resistant to toxic levels of organomercurials and had higher chlorophyll per unit dry leaf weight. The major advantages of this innovative approach have been better transgene expression without the necessity of expensive and laborious codon optimization that is usually necessary for expression in higher plant systems and lower levels of unwanted gene silencing (Ruiz et al. 2003). In addition to extensive work on engineering model plants for Hg phytoremediation, in a fairly recent study, Heaton et al. (2003) reported development of transgenic rice (Oryza sativa) with merA construct delivered through the biolistic gene delivery process. This is the first report on developing a transgenic wetland phytoremediation plant capable of detoxifying toxic mercury form aquatic sediments. Che et al. (2003) reported introduction of the merA gene into another wetland species, eastern yellow poplar (Populus deltoids), and it is good news as they

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confirm that genetic engineering technology is not just restricted to laboratory model plants and actively pursue developing actively remediating plant lines or transgenic plants suitable for specific ecosystems. Future studies need to investigate divergent species representing different ecosystems and habitats, especially those with higher Hg phytoaccumulation and inefficient phytovolatilization of the accumulated Hg for a safer, cleaner and healthier environment. Recently, Czako et al. (2006) reported developing green leaf tissue-specific merA expression cassettes using the wheat rbcS promoter in mature green leaves of A. thaliana and tobacco transgenic lines for enhanced merA gene expression, increased tolerance to Hg toxicity and better phytovolatilization. The authors also reported transgenic lines of wetland species Spartina alteriniflora (salt-water cordgrass/smooth cordgrass) with merA and merB genes under the control of two constitutive promoters 35S and Ubi. These ransgenic plants had higher resistance to both ionic and organic Hg toxicity. Researchers are reported to have been working on a number of other wetland species such as Arundo spp. (giant reed), Phragmites spp. (common reed), Typha spp. (cattail) and Schoenoplectus spp. (common threesquare). If this group is successful in developing a diversity of wetland-specific transgenic plants, it would mean a big step in the decontamination of Hg toxicity in different wetland ecosystems. In a very recent work, Hussein et al. (2007) reported developing transgenic tobacco plants with merA and merB genes engineered through the chloroplast genome. Transgenic lines exhibited a steady growth in the concentration of about 200 mg/g of phenylmercuric acetate or HgCl2 compared to non-transformed lines; and they also showed the ability to phytoaccumulate both organic and inorganic forms of Hg, with better absorption rates of organic Hg over inorganic forms. The authors for the first time reported a 100-fold increase in Hg phytoaccumulation and very rapid rates of phytovolatilization, varying between 2 and 7 days depending upon the chemical form of Hg used under experimental conditions compared to control lines. This is an interesting study that shows high success rate with respect to both Hg phytoaccumulation and phytovolatilization and holds great promise for the development of future transgenics for Hg decontamination.

8.3.2

Cadmium, Lead, Nickel and Zinc

Heavy metals like Cd, Pb, Ni, Fe (iron), zinc (Zn), copper (Cu), and Mg (magnesium) are important sources of environmental pollution because of their toxic effects on human and animal health. They have been under constant investigations by different research groups for their effective phytoremediation (PilonSmits 2005). In the late 1990s, Misra and Gedamu (1989) transferred human metallothionein-II (MT II) into tobacco (N. tabacum) and Brassica napus. Metallothioneins (MTs) represent a broad family of low molecular weight cysteine-rich chelator proteins that bind a number of heavy metals via the thiol group of its cysteine residues and transport them for sequestration into the plant vacuole

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(Pilon-Smits 2005). The transgene expressed the MT protein constitutively, and seeds form self-fertilized transgenic plants were able to survive in agar plates containing up to 100 mM CdCl2. Later overexpressed the metallothionein (MT) gene under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter in tobacco (N. glutinosa) delivered via AMGT. The authors reported that transgenic lines expressing MT grew successfully in the presence of 200 mM CdSO4 compared to non-transgenic lines that failed to withstand concentrations higher than 50 mM CdSO4. PCR analysis of T2 transgenic and non-transgenic seedlings demonstrated a strong correlation between phytotolerance to Cd and the presence of the transgene. This is an interesting strategy of increasing phytoremediation abilities in plants by overexpressing MT genes in the genome of higher plants. Phytochelatins (PCs) represent a big family of heavy metal-inducible peptides that play an important role in intracellular heavy metal detoxification by chelation in different organisms such as microbes, yeasts (Schizosaccharomyces pombe) and higher plants (Arabidopsis); but it has not been reported in animals yet (Ha et al. 1999; Gong et al. 2003). In 2003, Lee et al. reported overexpression of A. thaliana phytochelatin synthase (AtPCS1) back into A. thaliana with an increase (1.3- and 2.1-fold) in PC concentration. However, the authors reported hypersensitivity of transgenic lines to Cd stress measured as related to the proportion of root growth of transgenic plants compared to their wild types. The authors postulated that this might be due to higher concentration of glutathione in transformed plants. On the contrary, Gong et al. (2003) targeted wheat TaPCS1 (involved in PC synthesis) cDNA expression in Arabidopsis under the Arabidopsis alcohol dehydrogenase promoter and also developed special PC-deficient Arabidopsis lines (cadl-3) under the influence of the CaMV 35S promoter. The researchers detected lower Cd accumulation in transgenic plants compared to the original cadl-3 mutant lines and observed a higher rate of Cd transport into leaves. In the same year, Song et al. (2003) reported using a yeast vacuolar glutathione Cd transporter (YCF1) in A. thaliana that resulted in greater biomass and an approximately twofold increase in Pb and Cd tolerance and uptake compared to non-transformed controls. This technology has the potential to be used in developing advanced phytoremediators that can pump heavy metals into safe compartments, requiring only a very low expression of transporters compared to greater quantity of chelating peptides (Tong et al. 2004). Similarly, Bennett et al. (2003) generated transgenic B. juncea lines using microbial g-GCS and glutathione synthetase (GC) (both associated with PC synthesis) separately and reported approximately a 1.5-fold increase in Zn and Cd uptake compared to their corresponding controls. Researchers detected that overexpression of both genes enhanced soil-borne Cd by 25%, and they expected to increase the phytoremediation potential of transgenic lines from 1.5- to 3-folds, compared to their non-transformed control plants. Working on shrub tobacco (N. glauca) in Eastern Spain, Gisbert et al. (2003) genetically modified the species through AMGT using a wheat gene encoding phytochelatin synthase (TaPCS1). The authors reported a significant phytotolerance to Pb and Cd and an increase in root length by approximately 160%. Another significant achievement was reported by Zhu et al. (1999) when they transferred the Escherichia coli gene gshI (involved in PC synthesis) and

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overexpressed it in B. juncea with a fivefold increase in ECS and GC activity and better Cd accumulation (approximately 40–90%). Liang et al. (1999) in their study on Cd phytoremediation overexpressed the gshII gene encoding GC into the cytosol of B. juncea and exhibited higher Cd accumulation compared to wild-type plants. Vacchina et al. (2003) first reported a possible role of nicotinamine (NA) in the process of detoxification of a microelement Ni by Thlaspi caerulescens as a phytoremediator. Another group of researchers (Becher et al. 2004) besides Vacchina et al. (2003) found consistent results by overexpressing the nicotinamine synthase (NAS) gene in Brassicaceae family members such as A. halleri and T. caerulescens. The Cation Diffusion Facilitator (CDF) family homologs such as ZTP1 in T. caerulescens and AhMTP1 in A. halleri have been reported to be constitutively expressed at higher concentrations (Assuncao et al. 2001; Becher et al. 2004). Pence et al. (2000) cloned ZNT1 cDNA (a heavy metal transporter) from T. caerulescens via functional complementation in the yeast strain ZHY3 and found higher and lower affinities for Zn+2 and Cd+2, respectively. The authors also reported that ZNT1 (a heavy metal transporter that forms a complex with heavy metals and carries them inside plant cells for sequestration into the plant vacuole) is heavily expressed in roots and shoots of target plants. A comparative analysis was made between ZNT1 and high-affinity Zn+2 accumulation in roots of T. caerulescens (a hyperaccumulator) and T. arvense (a non-accumulator), and it established an excellent correlation between alterations in ZNT1 expression and speciesspecific Zn accumulation status resulting in overexpression of the target gene in T. caerulescens. This study highlights the pattern of regulation and molecular control mechanisms of heavy metal uptake systems in plants. In another interesting study, Pavlikova et al. (2004) tested four transgenic tobacco lines carrying transgenes: CUP1 (encoding MTs), the GUS reporter gene, HisCUP (CUP combined with a polyhistidine tail) and HisGUS (GUS with a polyhistidine tail) under the constitutive CaMV 35S promoter for Cd, Zn and Ni phytoaccumulation. The GUS line accumulated all the three metals. The HisCUP line showed the best Cd accumulation with the shoot content of Cd enhanced by 90% and the subsequent reduction in root accumulation by 40%. The HisGUS line confirmed the best performance in Ni accumulation, while no significant accumulation was detected for Zn in any of the lines tested. Other lines exhibited higher phytoaccumulation of only one specific metal, as discussed above. Recently, Stearns et al. (2007) reported efficient tolerance to Ni, Zn, Cu, and Pb in canola (B. napus) by transferring to the genome the bacterial 1-aminocyclopropane1-caboxylate (ACC) gene associated with ethylene biosynthesis for inducing better environmental stress response to metal toxicity indirectly.

8.3.3

Arsenic

The first clear evidence of arsenic phytoremediation by transgenic canola (B. napus) was reported by Nie et al. (2002). The researchers expressed the

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Enterobacter cloacae UW4 1-aminocyclopropane-1-caboxylate (ACC) deaminase in canola. The reason for expressing this particular enzyme is that it is associated with lower levels of stress-induced ethylene. Hence, these transgenic lines are expected to tolerate a wide variety of environmental stresses including heavy metal stress and toxicity. Transgenic plants survived quite well while grown in the presence of 2 mM arsenate; they exhibited higher seed germination percentage, enhanced fresh and dry weight of both roots and shoots, and higher concentrations of protein and chlorophyll in plant cells and tissues. The most important improvement reported in transgenic plants compared to their corresponding controls was a fourfold increase in their ability to accumulate arsenate. The authors also explored the role of the plant growth-promoting bacterium E. cloacae CAL2 in both transgenic and control lines with no significant observation with respect to arsenate uptake. They proposed the combined use of transgenic lines along with the growthpromoting bacteria as a better strategy to deal with arsenic contaminated sites. In the same year, Dhanker et al. (2002) were successful in increasing As tolerance in the model plant A. thaliana using two different genes, a bacterial g-glutamyl cysteine synthetase (g-GCS) of the glutathione biosynthetic pathway (involved in PC synthesis) and the arsenate reductase (arsC) from E. coli (for transforming highly toxic arsenates to less toxic arsenites). Constitutive overexpression of the g-GCS gene along with leaf-specific expression of arsC allowed to achieve increased phytoremediation of arsenate by transgenic plants with respect to fresh weight (~fivefold) and shoot accumulation (~threefold), as compared to nontransformed controls. Such advances may play a significant role especially in the parts of world that are adversely affected by severe arsenic pollution like the IndoGangetic plain and the Ganges delta (Ruiz and Romero 2002).

8.3.4

Selenium

One of the most comprehensive and detailed study on Se toxicity in plants was conducted by Banuelos et al. (1997). The authors investigated selenium-induced growth reduction in two different Brassica species (B. juncea and B. carinata). Several other studies have been conducted on Se phytoremediation with particular emphasis on plant physiology and biochemistry, Se toxicity and Se hyperaccumulation (see Suresh and Ravishankar 2004; Cherian and Oliveira 2005). Pilon et al. (2003) studied overexpression of a mouse selenocysteine lyase (SL) that breaks down selenocysteine into elemental selenium and alanine in A. thaliana. The overexpression resulted in a minor increase in the rate of Se accumulation, slightly lowering the amount of Se incorporation in plant proteins. It is very important to note that the researchers reported that chloroplastic SL reduced tolerance to Se, while vacuolar SL enhanced tolerance to Se, indicating the importance of precise localizations of transgenic proteins at the subcellular level (Pilon et al. 2003). Overexpression of metallothionein expressing selenocysteine methyltransferase

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(SMT) from A. bisulcatus to A. thaliana (Ellis et al. 2004) and B. juncea (LeDuc et al. 2004) resulted in an approximately two- to threefold increase in Se tolerance and accumulation in transgenic plants compared to their untransformed controls. This specific enzyme (SMT) is capable of detoxification of selenocysteine to a nonprotein amino acid methylselenocysteine via methylation, thereby reducing chances of toxic incorporation of Se in plant proteins (LeDuc et al. 2004). Recently, Banuelos et al. (2007) reported a twofold increase in Se accumulation and 1.8-fold increase in Se leaf accumulation by transgenic Indian mustard (Brassica juncea (L.) Czern.) overexpressing SL.

8.4

8.4.1

Phytoremediation of Organic Pollutants by Transgenic Plants Organic Hydrocarbons

Organic pollutants are another significant group of environmental toxicants used for rapid phytoremediation (Gratao et al. 2005; Pilon-Smits 2005). In an interesting recent study, Doty et al. (2007) reported the development of a transgenic poplar line from an original base population of the hybrid poplar clone INRA 717-1B4 (P. tremula
P. alba) via overexpression of rabbit CYP2E1 under the control of CaMV 35S. Stable transgenic lines showed excellent phytoremediation of different hydrocarbons (trichloroethylene, vinyl chloride, carbon tetrachloride, benzene and chloroform) from hydroponic solutions, compared to their controls. The plants also exhibited better phytovolatilization for trichloroethylene, chloroform, and benzene. Recently, Novakova et al. (2007) successfully transferred the bacterial todC1 and todC2 genes into N. benthamiana. The todC1 C2 genes were cloned into the plant genome to synthesize ISPTOL (a bacterial component of toluene dioxygenese), causing rapid oxidation of toluene and other organic pollutants. The overall performances of transgenic lines are still in progress to record their phytoremediation ability to degrade toluene and other organic compounds.

8.4.2

Trichloroethylene (TCE)

Doty et al. (2000) for the first time developed a transgenic tobacco line capable of phytoremediating TCE. The authors introduced the mammalian cytochrome P450 E1 gene (CYP2E1) into tobacco leaf disks, and transgenic plants were regenerated. Oxidoreductases of plant and mammalian origins being substantially similar, the mammalian P450 could successfully interact with its tobacco counterpart. Introduction of this specific gene resulted in a significant increase in both TCE and

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ethylene dibromide metabolism. The authors reported a 600-fold increase in TCE metabolism in transgenic lines, compared to their non-transformed controls. This specific gene encodes enzymes that can successfully oxidize a wide diversity of organic pollutants such as TCE, ethylene dibromide (EDB), carbon tetrachloride, chloroform and vinyl chloride, and many others (Aken 2008). Later on, the same group of researchers reported another intriguing finding: they successfully expressed the P450 E1 gene (CYP2E1) in hairy root cultures of the medicinal plant Atropa belladonna (Banerjee et al. 2002).

8.4.3

Polychlorinated Biphenyls (PCB)

PCBs are considered to be a formidable source of environmental pollution, and they are extremely toxic in nature (Sonoki et al. 2007). Chrastilova et al. (2007) transferred the 35S-driven bphC gene derived from the bacterial PCB degradation pathway into tobacco. Significant PCBs degradation was observed in the transgenic lines. Sonoki et al. (2007) also reported PCB degrading transgenic lines of A. thaliana. One of the lines developed contains fungal lignin-degrading enzymes (Lip, MnP), while the other line is an enhancer-trap Ac/Ds transposon tagging transformant of A. thalina containing a nonautonomous mobile Ds transposon linked to the reporter gene GUS. According to the researchers, the minute promoter-driven GUS can only drive gene expression when the Ds transposon GUS is shifted towards the enhancer region of the gene in the Arabidopsis genome. Hence, efficient monitoring of the genes(s) involved in Polyhydroxy butyrate (PHB) degradation is indirectly achieved by monitoring the expression of the reporter gene. Both lines have been found to efficiently degrade PCBs under laboratory conditions.

8.4.4

Phenolic Compounds

Recently, Wang and Chen (2007) reported transferring the laccase enzyme (LAC1) from cotton plants (Gossypium arboreum) into A. thaliana. Transgenic plants were efficient in degrading 2,4,6-tricholorophenol (TCP) by simple oxidation. In another study, Floco and Giulietti (2007) reported developing hairy root cultures of Armoracia lapathifolia using A. rhizogenes for phytoremediation of aromatic compounds like phenol from Argentina. This particular plant species have high concentrations of the enzyme peroxidase (E.C. 1.11.1.7) that is capable of detoxifying phenolic compounds. The authors exposed 30-day-old hairy root cultures to aqueous solutions of phenols of different concentrations (25, 50 and 100 mg/mL). They reported 70% phenol removal in the cultures after 3 h of incubation at all concentrations in the presence of hydrogen peroxide, and approximately 30–55% in the absence of an

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oxidant and co-substrate, suggesting a significant role of a plant peroxidase in phytodetoxification of phenols.

8.4.5

Herbicides, Pesticides and Organic Solvents

Herbicides, pesticides and organic solvents are other significant sources of environmental toxicants that have a detrimental impact on our ecosystems (Gratao et al. 2005). Shimizu et al. (2002) reported transferring the bacterial cbn4 gene from Ralstonia eutropha NH9 into rice (O. sativa) under the constitutive CaMV 35S promoter. Transgenic rice calli were successful in converting 3-chlorocatechol to 2-chloromucote. Such techniques may be suitable for phytoremediation of chlorinated aromatic compounds represented by herbicides, pesticides and several organic solvents. In another related study, Ohkawa and Ohkawa (2002) reported producing transgenic rice and potato (Solanum tuberosum) lines with mammalian cytochrome p450 monooxigenase genes. The researchers introduced five P450 genes in rice, namely, CYP1A1, CYP2B6, CYP2C9, CYP2C18 and CYP2C19. All stable transgenic lines (with the exception of one line) exhibited enhanced tolerance to herbicides metachlor, alochlor and acetochlor (inhibiting protein biosynthesis) and trifluralin (inhibiting cell division). T1 seeds of CYP2C9 showed resistance to such herbicides like chlortoluron, mefenacet, phenylurea herbicide, pyridazinone herbicide etc; while CYP1A1 exhibited resistance to phenylurea herbicide, mefenacet and quizalofopethyl. Rice lines carrying the CYP2C9 gene were resistant to chlorosulfuron and imazosulfuron; however, the line with the CYP2C18 gene did not show any specific resistance to any herbicide. A similar approach was used for generating transgenic lines of potato. Four lines (S1965, S1972, S1974 and T1977), each with three transgenes (CYP1A, CYP2B6 and CYP2C), were selected for testing. The T1977 line showed tolerance to atrazine, chlortoluron, methabenzthiazuron, acetochlor and metolachlor; while the S1972 line had tolerance to chlortoluron and methabenzthiazuron and was susceptible to all these herbicides except acetochlor and metolachlor; the S1974 line was partially tolerant to atrazine and tolerant to acetochlor and metolachlor.

8.4.6

Explosive Chemicals

Bioremediation of explosives is not an innovation; several researchers successfully used different fungal and bacterial members to degrade explosive and ammunition chemicals like TNT (trinitrotoluene), RDX (hexahydro-1,3,5-trinitro-1,3,5 triazine), HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) and TETRYL (N-methyl-N, 2, 4, 6-tetranitroaniline) (Hooker and Skeen 1999; Jhonston 2002).

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In the last few decades, phytoremediation of explosive chemicals has received great attention in a large number of laboratory studies by different research groups (French et al. 1998, 1999). An excellent review by Hannink et al. (2002) covered in details energetic, metabolic, biochemical and transformation mechanisms associated with phytoremediation of explosive chemicals. TNT is one of the most dangerous explosive chemicals that require considerable time for complete biodegradation (Jhonston 2002; Cluis 2004). However, a soil bacteria E. cloacae PB2 has been reported to be able to use TNTs as its primary nitrogen source for growth and metabolism because of the presence of two unique enzymes, pentaerythritol tetranitrate (PETN) reductase and nitroreductase (French et al. 1998). Both these enzymes utilize NADPH as an electron donor source, and thereby can easily reduce TNTs into less toxic compounds. French et al. (1999) introduced the PCR-modified gene encoding PETN reductase (onr) into the tobacco genome with a plant consensus start sequence for better expression in the plant system. The authors reported that seeds from transgenic lines germinated and grew successfully in the presence of 1 mM glycerol trinitrate (GTN) or 0.05 mM TNT, compared to their non-transformed wild types. The resultant transgenic seedlings grown in liquid medium with 1 mM GTN exhibited faster and complete degradation (denitration) of GTN than non-transformed lines. In another related study, expressed the PCR-modified bacterial (E. cloacae NCIMB101011) gene encoding for nitroreductase (nfs1) with a consensus start sequence to facilitate translation in tobacco plants. The nitroreductase enzyme catalyzed the reduction of TNT to hydroxyaminodinitrotoluene, following the subsequent reduction to aminodinitrotoluene derivatives. Transgenic plants expressing nitroreductase exhibited a significant increase in TNT uptake, tolerance, and subsequent detoxification compared to wild-type plants. The ability of plants to metabolize xenobiotic nitrate ester and glycerol trinitrate (nitroglycerin) in sugar beet (Beta vulgaris) cells and cell extracts has been convincingly demonstrated by Goel et al. (1997). Here, it is important to note that the authors suggested that GTNs could not be completely denitrated, they could only be transformed to mono- or dinitrated glycerols. Hence, there are opportunities for future researchers to explore these data and develop new transgenic lines for complete denitration of nitroglycerin compounds. In Fig. 8.3, we have illustrated some of the common pathways of phytoremediation within a plant cell.

8.5

Immunological Approach for Phytoremediation

Immunological approaches for phytoremediation represent entirely new concepts in the field of phytoremediation research. This technology can be called plant immuno-remediation or phytoimmuno-remediation. Drake et al. (2002) for the first time demonstrated the importance of hydroponicaly grown transgenic tobacco plants used for phytoimmuno-remediation. These plants express a murine IgG1

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HMT

PC/MT HM 1

HM 2

C

L

M Tonoplast

C

Cell Wall

M

GC

Cell Membrane

PC/MT HM

ER

HM N Vacuole L

I GSH O GSH

O

Cell Sap

3 3 3 O

Cytoplasm C

C 4

O GSH M

L

M

I GSH

C 4

C

4

I I

Fig. 8.3 Schematic representation of a plant cell and several phytoremediation pathways of target toxic pollutants. C Chloroplast; ER Endoplasmic reticulum; GC Golgi complex; HM Heavy metal; HMT Heavy metal transporters; I Inorganic pollutant; L Lysosome; M Mitochondrion; MT Metallothioneins; N Nucleus and nucleolus; O Organic pollutant; PC Phytochelatins. Pathway 1: PCs and heavy metals form complexes are translocated across the tonoplast and finally sequestered in the vacuole (Gong et al. 2003). Pathway 2: HMTs detoxifies toxic heavy metals by transporting across the vacuole to less toxic forms (Song et al. 2003; Pilon-Smits 2005). Pathway 3: Organic contaminants are phytoremediated by either getting adsorbed on the cell wall during entry or moving into the cytoplasm depending on the nature of pollutants. Within the cell cytoplasm, they are either attacked by series of enzymes and get transformed and degraded or form conjugated complexes with glucose and GSH and get sequestered in the plant cell wall or the vacuole (Pilon-Smits 2005). Pathway 4: Inorganic pollutants may form complexes with nicotinamine and organic acids and get adsorbed on the cell wall; if they can enter the cell cytoplasm, they often form conjugates with PCs and GSH and are finally sequestered in the plant vacuole (PilonSmits 2005)

monoclonal antibody either to neutralize toxic bioactive molecules in the rhizosphere, or to accumulate and concentrate molecules in the above-ground biomass. In their experiment, two different types of transgenic tobacco plants were used. First, a functional antibody was subjected to rhizosecretion and directed to bind with antigen in the surrounding media to generate an immune complex. In the second case, a monoclonal antibody was retained in plant leaves via a

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transmembrane sequence. It is interesting to report that the antigen incorporated in the medium was actively transported to axial plant leaves within a day; there, it underwent sequestration from binding to the antibody on the cellular membrane. The immune complex density remained the same even 3 days following antigen removal form the media. Such innovative technology could empower researchers to develop transgenic species that could phytoremediate any pollutant for which it is possible to develop a monoclonal antibody. It could also contribute to phytodecontamination and phytorestoration of contaminated sites using transgenic plants.

8.6

Limitations of Phytoremediation Research

In spite of its relevance and importance, phytoremediation still has several problems such as reduced growth rate and poor biomass of phytoremediating plants, limited remediation, and high plant mortality rates (Barcelo and Poschenrieder 2003; Cluis 2004; Gray 2006). In addition, there is always a permanent risk of bioaccumulation of toxic elements and compounds by plants and their transmission initially to immediate secondary consumers and subsequently into higher orders of food chains and food webs (Raskin et al. 1994; Barcelo and Poschenrieder 2003; Cluis 2004; Gratao et al. 2005). There are a number of concerns and issues associated with future effectiveness and potential of transgenic phytoremediators from food and feed crops (Dietz and Schnoor 2001; Cluis 2004; Ghosh and Singh 2005; Gratao et al. 2005). Although many plant species have been reported to show uptake, biodegradation and sequestration of several explosive chemicals, such as TNT and RDX residuals, these activities were low. Some chemicals, such as RDX, were only partially degraded or transformed, leaving space for engineered plants to take over in this area in the not-so-distant future (Goel et al. 1997; Dietz and Schnoor 2001; Ghosh and Singh 2005). Among other technical factors associated with phytoremediation, a subtle one is that of a gap between the scientist and the lay person, and misconceptions about benefits of the process and its scientific management (Trapp and Karlson 2001). A list of concerns and challenges haunting the successful development of transgenic lines has been presented in Fig. 8.4. Developing efficient phytoremediators for contaminated sites lab has always been extremely challenging for researchers (Black 1995; Cunningham et al. 1995; Jhonston 2002; McIntyre 2003). Among these challenges are genotype
environment interactions of responding plant species and variability of performance across the years (Cunningham et al. 1995; McIntyre 2003; Zayed 2004; Ghosh and Singh 2005; Willey 2007). Moreover, phytoaccumulation of toxic pollutants is also dependent on root growth of plant species involved. Restricted root growth in natural contaminated sites may or may not allow plants to effectively accumulate toxicants in the above-ground biomass or to immobilize pollutants preventing them from leaching into the groundwater table (Black 1995; Pulford and Watson 2003; Gratao et al. 2005).

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1. Phytotoxic concentrationof pollutants often detrimental to plants 2. Phytoremediation affected by locality, seasonal and climatic variations 3. Phytoremediation success depends on plant species and varieties and specific plant communities 4. Often difficult to establish a successful plant colony for effective and economical remediation 5. Ecological risk associated with introduction of new species 6. Phytoremediation limited by root length and root growth rate in plants

CHALLENGING QUESTIONS CONFRONTING FUTURE AND PROGRESS OF TRANSGENIC LINE DEVELOPMENT FOR PHYTOREMEDIATION

7. Uncertainty regarding production of secondary pollutants and effective disposal of phytoremediating plants 8. Economic and ecological cost of generating transgenic plants

Fig. 8.4 Future of transgenic phytoremediating plants

According to Salt et al. (1998) metal-accumulating plants could be either disposed off or used for metal recovery depending on economics of the process. However, it is important to note that reliable data and information regarding such disposal and success are not easily available (Jhoanston 2002). Although composting and compaction have been suggested by Cunningham et al. (1995) as pretreatment for volume reduction before actual disposal, it is important to make sure that such practices do not promote accidental leaching from compaction (Ghosh and Singh 2005). Incineration of harvested plants from contaminated sites after remediation has been strongly advocated by Ghosh and Singh (2005) to avoid the possibility of secondary waste generation through the application of phytoremediation. However, the impact of such technologies on the environment and ecosystems has not been well documented yet. Lastly, another important question to answer is whether transgenic plants can eventually become a source of secondary pollutants in the process of phytoremediation, and whether their effective disposal becomes another challenge and threat to ecology and environment (Black 1995; McIntyre 2003; Gratao et al. 2005; Willey 2007). It is very important to look for easy recycling of plant biomass to retrieve toxic pollutants and for the opportunity to reuse them after recycling to reduce our ecological footprints on the nature. In addition, it is also very important to look for enhancing the quality of phytovolatilization or phytotransformation

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of toxic pollutants within phytoremediating plants to reduce the possibility of generating secondary pollutants in the long run. The interaction between the plant genome and hyperaccumulation of toxic heavy metals needs to be investigated in further details to understand molecular genetics and physiological aspects of heavy metal uptake in phytoremediating plants (Mejare and Bulow 2001).

8.7

Transgenics in Phytomonitoring and in Biopolymer and Bioplastic Productions

In addition to phytoremediation, transgenics have made a significant impact on some other areas too. Although a detailed discussion would be too voluminous and is beyond the scope of this review, however, we would like to point out some features very briefly in the following paragraphs. One of them deals with the development of transgenic plants for rapid detection of environmental pollution and contamination using efficient molecular screening techniques (plant biomonitoring or phytomonitoring), and the second thriving field deals with the synthesis of bioplastics and biopolymers in transgenic plants.

8.7.1

Transgenic Plants in Biomonitoring

In recent years, substantial progress has been achieved in the realm of transgenic biomonitoring plants (phytomonitors) (Lebel et al. 1993; Kovalchuk et al. 1998, 1999a,b, 2000a,b,c, 2001a,b; Ries et al. 2000; Besplug et al. 2004; Boyko et al. 2006; Li et al. 2006; van der Auwera et al. 2008). One of the most important aspects in the development of transgenic biosensors is the option to customize the assay according to specific biomonitoring requirements (Kovalchuk and Kovalchuk 2008). Two most important assays that are reportedly used in biomonitoring in recent times are the Recombination Reporter Assay and the Point Mutation Reporter Assay (Kovalchuk et al. 2000b, c). The biggest success attributed to the transgenic recombination assay has been its application in detecting radioactive pollution in soil and water (Kovalchuk et al. 2001a, b). Transgenic Arabidopsis and tobacco biomonitoring lines have been reported to be excellent tools for detecting genotoxicity of radioactively contaminated sites (Kovalchuk et al. 1998, 1999a, b). In case of the Point Mutation Reporter Assay, Kovalchuk et al. (2000b, c) has developed a system in which they introduced a stop codon at the 50 -end of the GUS (uidA) gene by means of a single nucleotide substitution that completely inactivated the transgene. Transgenic plants responded to mutagens (either physical or chemical agents) by increasing levels of point mutations. They led to the restoration of the uidA gene activity. Cells where such restorations occurred were visualized as blue sectors on white plants after histochemical staining. Further details of these works are beyond the scope of the current article and are available

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in a series of papers (Hohn et al. 1999; Kovalchuk et al. 1999a,b, 2000a,b,c; Kovalchuk and Kovalchuk 2001, 2003, 2008; Filkowski et al. 2003).

8.7.2

Biopolymer Production in Transgenic Plants

Transgenic plants are catching up rapidly in the areas of biopolymer production. Industrially important biopolymers include proteins, enzymes, recombinant antibodies, vaccines and other biopharmaceutical products (Torres et al. 1999; Doran 2000; Fischer and Emans 2000; Fischer et al. 2000; Giddings et al. 2000; Langridge 2000; Walmsley and Arntzen 2000). Several industrially important proteins have been produced in transgenic plants: human milk proteins in potato (Chong and Langridge 2000; Chong et al. 1997); the oleosin–hirudin fusion protein in canola (Parmenter et al. 1999); phytase in canola (Ponstein et al. 2002); collagens in tobacco (Ruggiero et al. 2000); spider silk protein (spidroins) in tobacco and potato (Gosline et al. 1999); bio-elastic proteins in tobacco and avidin in maize (Giddings et al. 2000); biopolymers in peas and rice and wheat (Frigerio et al. 2000; Perrin et al. 2000; Stoger et al. 2000). Transgenic plants offer the following advantages over microbial and animal expression systems: lower rates of contamination, low production cost, easier and economic protein extraction and purification steps. Lastly, rapid advances in the areas of plant proteomics and protein targeting are also promoting the development of transgenic lines producing different biopolymers (Doran 2000; Giddings et al. 2000; Fischer et al. 2000; Nawrath and Bonetta 2001).

8.7.3

Bioplastics from Transgenic Plants

Transgenic plants producing bioplastics (polyhydroxy butyrate/PHB) are still not a cheaper alternative compared to the microbial PHB production (Scheller and Conrad 2005). According to Scheller and Conrad (2005), PHB production in transgenic plants still needs to reach a comfortable yield target to be economically feasible. Nawrath et al. (1994) first reported PHB production in the model plant A. thaliana. The majority of works on PHB transgenics are restricted to Arabidopsis. However, there are reports of the PHB accumulation in other plants: 5.7% of dry weight has been reported in maize (Zea mays) by Moire et al. (2003); 7.7% in oilseed rape (B. napus) by Houmiel et al. (1999), and 5% in sugar beet (B. vulgaris) by Menzel et al. (2003). Bohmert et al. (2000) reported 4% PHB production in Arabidopsis; however, researchers detected a negative correlation between the PHB accumulation and plant growth. Analysis of T2 plants indicated the loss of PHB synthesis to a biologically significant amount over generations. Among other plants, tobacco plants producing PHB are reported to have stunted growth (Nakashita et al. 2001;

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Lossl et al. 2003). As to fiber-yielding crops, in cotton the amounts of PHB are small – 0.34% fiber weight (John and Keller 1996), in flax – 0.5% fiber weight (Wrobel et al. 2004). Most studies highlighted rapid depletion of other essential plant metabolites because of the increase in PHB production. Recent progress in bioplastic production in transgenic plants is aimed at developing lines with higher PHB production without impacting on growth qualities of targeted plant species (Scheller and Conrad 2005).

8.8

Conclusions and Future Directions of Phytoremediation Research

Overall, it is important to note that in the past few decades, phytoremediation research has moved from its initial emphasis on physiological, biochemical and genetic investigations and screening of phytoremediating species towards advances and applications in plant biotechnology and genetic engineering. The future success of phytoremediation as an emerging agro-industry will depend on how successful we are in acquiring knowledge regarding the plant genome of model plants and applying this knowledge to “real-life situations” and “real-life nondomesticated plant species” to tackle the challenges of complex environmental pollution induced by chemicals. Tree species will certainly have priority over herbs and shrubs because of their better phytoremediation abilities. It has been reported that 64% of contaminated sites contain both organic and inorganic pollutants; hence, it is important to attract our attention towards generating transgenics with multitasking abilities (MTTs). Some authors have been successful in introducing 13 different transgenes in rice using biolistics. Although these transgenes functioned separately, multiple gene insertion with synergistic functions was successful in other plants (Hooker and Skeen 1999). Such approach would be necessary to introduce multiple genes for phytoremediation into a target plant to make it suitable for phytoremediation under diverse conditions and at sites contaminated with mixed toxicants. This approach named as a “molecular tool box” approach by Raskin (1996) could be efficient in dealing with future phytoremediation. Plant omics will help regulate future phytoremediation technologies and strategies to guide us towards a platform where it could be established as a formidable future industry (Fig. 8.5). Phytoremediation research and applications have been previously restricted to North American and European continents only (Raskin 1996; Salt et al. 1998; Pilon-Smits 2005). Now they have been adopted and extensively pursued in emerging giant economies such as India, China, Brazil, Russia, and several smaller EU countries, as well as in Australia and New Zealand (Gratao et al. 2005; Willey 2007). Since the focus on agriculture has shifted back (Raskin 1996), newer initiatives like phytoremediation can open up opportunities as full-scale agroindustries in the long run. The future directions guiding phytoremediation research has been envisioned and highlighted into three separate sections below.

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Fig. 8.5 Phytoremediation and its relationships of with different disciplines

8.8.1

Transgenic Trees: A Better Solution for Phytoremediation

Trees with promising phytoremediation genes from other bacterial, yeast, human and animal sources, and even from other plants could possibly be an essential tool for future phytoremediation of contaminated sites and soils (Barcelo and Poschenrieder 2003; Zayed 2004). It may be a challenging but provocative idea to transfer multiple phytoremediation genes into candidate tree species for an efficient multiphytoremediation approach. This plant would be more desirable than phytoremediating species carrying a single transgene. Such transgenic species can work at two or more different polluted sites contaminated with totally different chemical pollutants, making the process more efficient and cost-effective.

8.8.2

Multidisciplinary Research Approach

Huge progress has been made in the characterization and modification of the chemical nature of soil to facilitate phytoremediation of contaminated sites (Datta and Sarkar 2004) and also in understanding the basic mechanics of pollutant uptake, translocation, detoxification, and storage mechanisms in plants (as reviewed in

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Suresh and Ravishankar 2004; Pilon-Smits 2005). However, there is much still to be investigated to completely identify all the factors that interact in the process of phytoremediation, including soil and site, nature and types of a pollutant affecting contamination, and plants involved in phytoremediation. This realm of research involves a serious multidisciplinary approach, and it needs collaboration among scientists working in different fields (Suresh and Ravishankar 2004; Willey 2007). Although phytoremediating species are slowly turning into crop plants in real life situations, a lab-to-land transition will involve a lot of support research in related disciplines to facilitate and speed up the process. For example, in addition to developing transgenic plant lines, it will also be necessary to engineer plant growthpromoting rhizobacteria and arbuscular mycorrhizal fungi residing in the same contaminated soil to further facilitate the efficiency in the natural cleanup of contaminated sites (Salt et al. 1998). In future, more comprehensive efforts will be necessary to deal with sites contaminated with complex pollutants, pollutants representing different chemical species and products generated by their interactions. A dynamic and integrative approach should be used to address future challenges of phytoremediation.

8.8.3

Applications of Plant Omics for Advancing Phytoremediation Techniques

Research progress in this field with respect to a proteomic and metabolomic approach, and metallomic investigations of metal bindings and metalloproteins are evidently lacking (Azevedo and Azavedo 2006; Garcia et al. 2006). According to Cobbett and Meagher (2002), approximately 80–90% of all phytoremediation genes and gene families have been detected in the genome of A. thaliana. This offers great potential for manipulating and genetic engineering of phytoremediating plant species. It has been estimated that about 5% of the Arabidopsis genome constitute membrane transport proteins involved in the transportation of different ions and metals across the plasma membrane and other organellar plant cell membranes. Several putative metal transporters have not been identified yet, and even a handful of those identified are not fully characterized (Maser et al. 2001; Zayed 2004). According to Heinekamp and Willey (2007), the whole-genome sequence of A. thaliana will give researchers a grand opportunity to identify and analyze all genes associated with phytoremediation. Vacuolar compartmentalization has been suggested to be a second-generation approach to genetic engineering of phytoremediating species (Heinekamp and Willey 2007). According to the authors, targeting the accumulation of toxic metabolites inside plant vacuoles makes it possible to enhance the ability of plants to withstand high toxic concentrations of different target pollutants, and at the same time it enables plants to increase uptake and accumulation within a restricted growth period, thereby maximizing the scale of phytoremediation.

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Simon L, Martin HW, Adriano DC (1996) Chicory (Cichorium intybus L.) and dandelion (Taraxacum officinale Web.) as phytoindicators of cadmium contamination. Water Air Soil Pollut 91(3–4):351–362 Singh H (2006) Mycoremediation: fungal bioremediation. Wiley, NY, USA, pp 1–15 Song WY, Sohn EJ, Martinoia E, Lee YJ, Yang YY, Jasinski M, Forestier C, Hwang I, Lee Y (2003) Engineering tolerance and accumulation of lead and cadmium in transgenic plants. Nat Biotechnol 21:914–919 Sonoki S, Fujihiro S, Hisamatsu S (2007) Genetic engineering of plants for phytoremediation of polychlorinated biphenyls. In: Willey N (ed) Phytoremediation: methods and reviews. Humana Press, New Jersey, USA, pp 3–13 Stabili L, Licciano M, Giangrande A, Fanelli G, Cavallo RA (2006) Sabella spallanzanii filterfeeding on bacterial community: ecological implications and applications. Mar Environ Res 61(1):74–92 Stearns JC, Shah S, Glick BR (2007) Increasing plant tolerance to metals in the environment. In: Willey N (ed) Phytoremediation: methods and reviews. Humana Press, New Jersey, USA, pp 15–26 Stoger E, Vaquero C, Torres E, Sack M, Nicholson L, Drossard J, Williams S, Keen D, Perrin Y, Christou P, Fischer R (2000) Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies. Plant Mol Biol 42:583–590 Sunkar R, Kaplan B, Bouche N, Arazi T, Dolev D, Talke IN, Frans JM, Maathuis FJM, Sanders D, Bouchez D, Fromm H (2000) Expression of truncated tobacco NtCBP4 channel in transgenic plants and disruption of the homologous Arbaidopsis CNGC1 gene confers Pb+2 tolerance. Plant J 24(4):533–542 Suresh B, Ravishankar GA (2004) Phytoremediation- a novel and promising approach for environmental clean up. Crit Rev Biotechnol 24(2–3):97–124 Sursala S, Medina VF, McCutcheon SC (2002) Phytoremediation: an ecological solution to organic chemical contamination. Ecol Eng 18(5):647–658 Sykes M, Yang V, Blankenburg J, AbuBakr S (1999) Biotechnology: working with nature to improve forest resources and products. In: Proc TAPPI Int Pulping Conf, vol 2, April 18–22, 1999, Nashville, TN. TAPPI Press, Atlanta, GA, pp 631–637 Terry N, Sambukumar SV, LeDuc DL (2003) Biotechnological approaches for enhancing phytoremediation of heavy metals and metalloids. Acta Biotechnol 23(2–3):282–288 Thomas JC, Davies EC, Malick FK, Endreszl C, Williams CR, Abbas M, Petrella S, Swisher K, Perron M, Edwards R, Ostenkowski P, Urbanczyk N, Wiesend WN, Murray KS (2003) Yeast metallothionein in transgenic tobacco promotes copper uptake from contaminated soils. Biotechnol Prog 19(2):273–280 Thomine S, Wang R, Ward JM, Crawford NM, Schroeder JI (2000) Cadmium and ion transport by members of a plant metal transporter family in Arabidopsis with homology to Nramp genes. Proc Natl Acad Sci USA 97:4991–4996 Thompson P, Ramer L, Guffey AP, Schnoor JL (1998) Decreased transpiration in poplar trees exposed to 2, 4, 6,-trinitrotoluene. Environ Toxicol Chem 17:902–906 Tong Y-P, Kneer R, Zhu Y-G (2004) Vascular compartmentalization: a second-generation approach to engineering plants for phytoremediation. Trends Plant Sci 9:7–9 Torres E, Vaquero C, Nicholson L, Sack M, Stoger E, Drossard J, Christou P, Fischer R, Perrin Y (1999) Rice cell culture as an alternative production system for functional diagnostic and therapeutic antibodies. Transgenic Res 8:441–449 Trapp S, Karlson U (2001) Aspects of phytoremediation of organic pollutants. J Soils Sediments 1:1–7 Vacchina V, Mari S, Czernic P, Marques L, Pianeli K, Schaumloffe D, Lebru M, Lobinski R (2003) Speciation of nickel in a hyperaccumulating plant by high-performance liquid chromatography-inductively couple plasma mass spectrometry and electrospray MS/MS assisted by cloning using yeast complementation. Anal Chem 75:2740–2745

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Chapter 9

Algal Biotechnology: An Emerging Resource with Diverse Application and Potential Stephen Cunningham and Lokesh Joshi

9.1

Introduction to Algae

Algae include a wide variety of species that range from diatoms, which are microscopic unicellular organisms, to seaweeds extending over 30 m (Fig. 9.1). They constitute a group of approximately 40,000 species, a heterogeneous group that describes a life-form, not a systematic unit; hence, a broad spectrum of phenotypes exists in this grouping. Algae are grouped into six main classes, mainly on the basis of their color (Fogg 1953). Algae are found in fresh or salt water, with a few being terrestrial (e.g., Chrysophyta and Cyanophyta). The eukaryotic algae are placed in the kingdom Protista, classified as euglenoids (phylum Euglenophyta), dinoflagellates (phylum Pyrrophyta) and diatoms (phylum Bacillariophyta). All have chloroplasts and carry out photosynthesis similar to that of plants. Prokaryotic blue-green algae belong to the phlyum Cyanobacteria. Unlike land plants, algae do not have true roots, stems or leaves. Algae of different size and shape not only occupy aquatic ecosystems, but also occur in a number of different habitats, some of which are extreme environments (Hallmann 2007). The environmental conditions have led to the development of adaptive tolerances, such as temperature, salt and pressure selection; such adaptive selection is widely observed in bacteria. Large forms of algae are often referred to as seaweeds or microalgae, which are widely distributed in the ocean, occurring from the tide level to considerable depths, free-floating or anchored, holding an important role in providing marine primary productivity. Eukaryotic green (phylum Chlorophyta), red (phylum Rhodophyta) and brown algae (phylum Phaeophyta) are all grouped as seaweeds. They are often found to produce considerable biomass, evident from natural population

L. Joshi (*) Glycoscience and Glycotechnology Group and the Martin Ryan Institute National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland e-mail: [email protected]

C. Kole et al. (eds.), Transgenic Crop Plants, DOI 10.1007/978-3-642-04812-8_9, # Springer-Verlag Berlin Heidelberg 2010

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344 Fig. 9.1 Demonstration of size diversity among algae species

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Osteococcus tauri Cyanidioschyzon merolae Thalassiosira pseudonana

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Chlamydomanas reinhardtii Phaeodactylum tricornutum

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10 cm Ulva lactuca

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10 m Macrocystis pyrifera 100 m

of brown algae Macrocystis in Northern America and the cultivated populations of artificially cultivated brown algae Laminaria and Undaria in East Asia. Geographically, algae have been used as a food source in the Asian countries, while in Europe algae has been traditionally used for the production of phycocolloids such as agar and carrageenan both from red algae and alginate from brown algae (McHugh 2003; Hallmann 2007). In most cases, algae are used in human and animal foods for their mineral contents or for the functional properties of their polysaccharides. As in the case of terrestrial plants, development of key techniques of genetic manipulation specific for algae was necessary to enable creation of good cultivars and to transform seaweed into multiple, functional marine bioreactors. This has led to the application of algae being maximized in: 1) Eliminating heavy metal pollutants, 2) Reducing factors of eutrophication (e.g., nitrogen and phosphorus),

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3) Improving the feed quality for maricultured animals using transgenic algae delivering/introducing genes encoding immunologically active peptides (molecular pharming), and 4) The production of high-value materials such as oral vaccines and drugs for humans Great strives have been made in the genetic engineering of plants and microorganisms over the last two decades. It is established that an effective transformation model consists of elements permitting an effective transformation methodology, that applicable vectors carry recognizable promoters, and that a screening mechanism to select transformants is in place to isolate the transformants from the endogenous mass. These are sufficient for single-celled organisms; however, multicellular organisms require further factors for successful recombination. This chapter describes the application, development and status of algae as a source of natural and recombinant molecules for industrial and human health applications.

9.2

The Application of Non-Transgenic Algae

9.2.1

Nutritional and Health-Related Properties

Algae have been utilized both historically and currently as food sources for human, animal and mariculture to date. In relation to animal feed, as with human feed, algae have been used to enhance the nutritional content of conventional feed preparations (Spolaore et al. 2006). Recognition as a source of fatty acids such as polyunsaturated fatty acid family (o3) and sterols, has increased their consumption in health-orientated diets (Cardozo et al. 2007). The protein and amino acid nutritional attributes of algae have been reviewed elsewhere by Fleurence (1999) and MacArtain et al. (2007). 9.2.1.1

Polyunsaturated Fatty Acids

Human beings and higher plants lack the required enzymes to synthesize long o3 polyunsaturated fatty acids; therefore, they need to obtain them from external dietary sources. Several o3 polyunsaturated fatty acids including eicosapentaenoic acid (EPA), an important dietary supplement (Yokoyama et al. 2007; Doughman et al. 2007), have been identified in a number of algae species. Although a number of algae species are cultivated as natural sources of these fatty acids, only a few species have demonstrated the potential to be capable of industrial scale production because of low growth rates and low cell number in culture (Wen and Chen 2003). The application of transgenic algae may act as an alternative, like transgenic oilseed crops, providing an alternative sustainable source of these essential oils for human consumption (Abbadi et al. 2001; Doughman et al. 2007).

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Sterols

Sterols are one of the most important chemical constituents of algae and a major nutritional component in the diet of aquacultured organisms. Microalgae are an important component in the diet of many hydrobionts, such as bivalves (Ponomarenko et al. 2004). The ability of bivalves to synthesize or bioconvert sterols de novo varies among different species, but is generally low and sometimes completely absent. This implies that a dietary supply of sterol is necessary for bivalve growth (Soudant et al. 1998). The type of algae used as a food source determines the quality and sterol composition; seasonal shifts also occur. Therefore, this is used as a criterion for species selection for the culture of bivalves (Park et al. 2002). Plant and algae sterols have been shown to reduce cholesterol by blocking absorption, resulting in reduced quantities of cholesterol reaching the liver (Plat and Mensink 2005; Charest et al. 2004). Despite the ability of these sterols to block cholesterol absorption, the human intestine poorly absorbs them (Cater and Grundy 1998).

9.2.1.3

Carotenoids

The natural pigments, carotenoids, are produced in bacteria, algae and plants (Polı´vka and Sundstro¨m 2004). These carotenoids have important biological functional roles including optimal photosynthesis and indeed protection from potential damage arising from UV light exposure. To date, over 600 different carotenoids exercising important biological functions in bacteria, algae, plants and animals have been identified (Polı´vka and Sundstro¨m 2004). Animals lack the ability to synthesize carotenoids endogenously and thus obtain these compounds by nutritional intake. For human nutritional purposes, a number of carotenoids offer provitamin A activity (Mayne 1996). Vitamin A deficiency is a major health risk that has surfaced in the developing countries as the leading cause of preventable blindness in children and also leads to the increased risk of disease and death from severe infections (WHO, Micronutrient deficiencies: http://www.who.int/nutrition/ topics/vad/en/: accessed July 20 2009). They are also biological antioxidants, protecting cells and tissues from free radicals and singlet oxygen. Carotenoids are utilized in pharmaceuticals, dietary supplements, cosmetics, and as food additives. Dunaliella salina and Spirulina maxima have been utilized for cartenoid astaxanthin, a red-orange pigment used widely in the food industry (Meyers and Latscha 1997). The green algae Haematococcus pluvialis has been the focus of biotechnology companies for the commercial development of this carotenoid (Hussein et al. 2006). The potent antioxidant property of astaxanthin has been implicated in its various biological activities demonstrated in both experimental animals and clinical studies, with potential beneficial roles in human health (Hussein et al. 2006). A second group, the phycobiliproteins, consists of proteins with covalently bound phycobilins. Phycobiliproteins, primarily composed of a- and b-polypeptides, are a brilliantly colored group of disc-shaped proteins (Samsonoff and MacColl 2001; Liu et al. 2005). These have been implemented within laboratories as labels for biomolecules (Spolaore et al. 2006).

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9.2.1.4

347

Polysaccharides

Phycocolloids are unique polysaccharides produced by several seaweed species. Carrageenan and agar are sulfated polysaccharides extracted from some Rhodophyceae. The principal feature distinguishing the highly sulfated carrageenans from the less-sulfated agars is the presence of anhydro-d-galactose in the former and l-galactose or anhydro-l-galactose in the latter (Craigie 1990). Agar and carrageenan are extracted from red algae, while alginates are extracted primarily from brown algae. Phycocolloids are used in a wide range of products including foods, pharmaceutics and medical devices (Walker et al. 2005). Carrageenan, extracted from red algae is subdivided into kappa carrageenan, iota carrageenan and lambda carrageenan, each of which has different characteristics. They are used as gelling agents, stabilizers, texturants, thickeners, and viscosifiers for a wide range of food products (Cardozo et al. 2007). Alginates, the salts of alginic acid and their derivatives, are extracted from the cell walls of brown macroalgae. These carboxylated polysaccharides are used for a wide variety of applications within the food industry. Alginates are required for production of dyes. The water absorbing properties of alginates are utilized in slimming aids and in the production of textiles and paper. Calcium alginate has been used in the production of medical products, including burn dressings (Cardozo et al. 2007). Owing to its biocompatibility and simple gelation with divalent cations, it is also used for cell immobilization and encapsulation. Additionally, alginates are widely used in prosthetics and for dental molds. Use within the food sector and as a source of phycocolloids has lead to a need for cultivation for sufficient global demand, with current estimates of global production greater than 6.5 million tons in terms of fresh weight (McHugh 2003). Such large-scale production places this in a similar economic and global importance as land crops (McHugh 2003), with abilities to fix high volumes of carbon dioxide, absorb and consume eutrophied nitrogen and phosphorus transforming them into large amounts of seaweed biomass. In doing so, they serve to protect the marine environment.

9.2.1.5

Lectins

Lectins or agglutinins, defined as proteins that bind to carbohydrates without initiating any further modifications (Weis and Drickamer 1996), have been found widely in all organisms. They are involved in numerous biological processes including host–pathogen interactions, cell–cell communication, cancer, metastasis, differentiation and immune regulation. The presence of lectins in marine algae was first reported by Boyd and colleagues in 1966 (Boyd et al. 1966). Marine algal lectins differ from higher plant lectins (agglutinins) typically in exhibiting (1) lower molecular mass ranging from 4,000 to 25,000 Da, (2) occurrence mainly in monomer form, and (3) independence from divalent cations to maintain their structure (Rogers and Hori 1993). The applications for algal lectins are at an early stage of

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development, though many have emerged as potential therapeutic agents deserving further elucidation. To date, a number of lectins have been discovered in algae, which demonstrate antiviral activity, among them are cyanovirin-N (CV-N), scytovirn (SVN) and Microcystis viridis lectin (MVL) (Zio´łkowska and Wlodawer 2006). Antiviral properties of lectins is due to their interaction with glycans, which in turn disturbs the interactions between proteins of the viral envelope and the cells of the host (Botos and Wlodawer 2005; Balzarini 2006). As a high proportion of current antiviral therapeutics act through inhibition of the viral life cycle, lectins can prevent viral penetration of host cells. Algae lectins with anti-HIV activity have been reviewed previously (Zio´łkowska and Wlodawer 2006). Seaweeds are rarely promoted for the nutritional value of their proteins. Their protein contents differ according to the species and seasonal conditions. To date, little information is available on the nutritional value of algae proteins and on the compounds that affect their digestibility.

9.2.1.6

Antibacterial and Antiviral Properties

A high number of marine algae produce antibiotic and antiviral substances capable of inhibiting bacteria, viruses, and fungi. The antimicrobial activity of aquatic microalgae was first reported for Chorella vulgaris in the 1940’s (Pratt and Fong 1940, 1944). This concept was strengthened with growing literature and reports of the antiviral properties of polysaccharides from marine algae towards mumps virus and influenza B virus in the 1960s. The antibiotic/antiviral properties are dependent on factors including the species of algae, the agent in question, the season, and the growth conditions (Pesando and Caram 1984; Centeno and Ballantine 1999). The antibacterial activity of marine algae has generally been assayed using extracts in various organic solvents (Liao et al. 2003). A number of these chemicals are toxic to microorganisms and therefore may be responsible for the antibiotic activity reported (Ohta 1979). However, this does not reflect the antibacterial activity of marine algae under natural conditions. Earlier investigations have demonstrated the effects of the release of phenolics as antifouling substances, the release of organic substances, which hold both an inhibitory effect on growth of adjacent diatoms or a stimulatory effect depending on source (Liao et al. 2003). Polysaccharide fractions from red algae were found to inhibit a number of viruses including herpes simplex virus (HSV). At that time, these findings did not generate much interest because the antiviral action of the compounds was considered to be largely nonspecific (Witvrouw and De Clercq 1997). Isolation from algae of polysaccharides and sulphated polysaccharides and other compounds with antiviral activity against enveloped viruses increased the interest in algae as a source of antiviral compounds (Schaeffer and Krylov 2000). Enveloped viruses include HIV, HSV type 1 and HSV type 2, influenza A virus, RSV, simian immunodeficiency virus (SIV), pseudorabies virus, bovine herpes virus, and human cytomegalovirus (HCMV) (Schaeffer and Krylov 2000; Zio´łkowska and Wlodawer 2006).

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Anticancer Agents Brown algae grown in the Black Sea, among other global regions such as Japanese coastline, have been demonstrated to be potential sources of antitumor agents (Apryshko et al. 2005). Carotenoid fucoxantine (Fx) extracted from these algae possess antitumor properties, which have been tested using prostate cancer performed in Russia. During these studies, patients typically received dried brown algae Laminaria daily. Dosage of Fx was estimated to be 10–15 mg daily. During treatment, there was gradual improvement of general state and blood indices. Disease course became stabilized and survival rate increased. Similarly, the rate of breast cancer is greatly reduced in populations consuming brown algae (Apryshko et al. 2005). Anti-HIV Activity Most of the research on the anti-HIV activity of marine algae has focused upon red and brown macroalgae (Schaeffer and Krylov 2000). The initial studies using these algae isolated sulfated polysaccharides with antiviral activity and later investigators continued interest in this class of compounds. However, other classes of compounds with anti-HIV activity have been identified including polysaccharides, fucoidan and carrageenans (Schaeffer and Krylov 2000). A number of natural polysulfates isolated from algae and synthetic polysulfates exhibit differential inhibitory activity against different HIV strains, which suggests differences in the target molecules with which these compounds interact (Witvrouw and De Clercq 1997; Table 9.1). They inhibit the cytopathic effect of HIV and also prevent HIV-induced syncytium formation (Zio´łkowska et al. 2006). Antiviral activity increases with increasing molecular weight and degree of sulfation (Witvrouw and De Clercq 1997). Anti-HIV polysaccharides and polyphenols have been isolated from brown algae, Fucus vesiculosus, inhibition of both HIV-induced syncytium and HIV reverse transcriptase (RT) activity at nontoxic levels (Be´ress et al. 1993). The mechanism of this effect remains to be further elucidated. A sulphated polysaccharide isolated Table 9.1 Anti-HIV activity of algal lectins

Lectin Jacalin Myrianthus holstii lectin Urtica diocia agglutinin Concanavalin A N. pseudonarcissus lectin P. tetragonolobus lectin MVL* SVN CV-N GRFT Data from Zio´łkowska et al. (2006) *IC50 rather than EC50 reported

EC50 (nM) >227 150 105 98 96 52 30 (IC50 used) 0.3 0.1 0.04

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from S. horneri demonstrated potent antiviral activity against HSV-I, cytomegalovirus and HIV. Other anti-HIV polysaccharides have been found in Agardhiella tenera, Cochlodinium polykrikoides, and Nothogenia fastigiata. Carrageenans and their cyclized derivatives isolated from G. skottsbergii are potent inhibitors of herpes viruses, and inhibit HIV to a lesser extent. A number of cyanobacteria (blue-green algae) species have been found to produce highly anti-HIV active sulfoglycolipids. In addition to their activity, the sulfoglycolipids constitute part of the chloroplast membrane and are therefore abundant. Their abundance and accessibility should be highlighted for potential future development. Extracts of cultured marine cyanobacteria, Lyngbya lagerheimii and Phormidium tenue, have been screened for anti-HIV compounds leading to the discovery of sulfonic acid-containing glycolipids as a novel class of HIV-1inhibitory compounds (Gustafson et al. 1989). A number of cyanobacteria extracts have been screened for antiviral compounds including Phormidium cebennse, Oscillatoria raciborskii, Scytonema burmanicum, Calothrix elenkinii, and Anabaena variabilis. Compounds in all were found to be anti-HIV and extracts found to contain sulfolipids. Cyanobacteria antiviral polysaccharides have also been demonstrated.

9.3

Application of Transgenic Algae

The ease of growth, biomass content and low cost of production of algae make them immensely attractive for both pharmaceutical and therapeutic compound discovery and for recombinant engineering (Fig. 9.2). In the absence of cell differentiation, algae would provide a much simpler system for genetic manipulations compared with higher plants. Manipulation of algae by metabolic and genetic methods would both permit (1) selection of beneficial pathways redirecting cellular function toward the synthesis of preferred products and (2) introduction of non-algae genes for the generation of algal recombinant protein. The selection of favorable pathways may include increased resistance to environmental or stress changes on the culturing/life cycle of the algae. The potential of this system remains to be optimized as an alternative protein expression system.

9.3.1

Transformation and Engineering

Reports of plant-produced recombinant proteins began to be published in the late 1980s when antibodies were produced in tobacco (Hiatt et al. 1989) and human serum albumin was expressed in tobacco and potato (Sijmons et al. 1990). Genetic transformation of unicellular algae began in the 1970s (Shestakov and Khuyen 1970) with cyanobacteria, followed by in the 1980s with the work in marine algae (Stevens and Parter 1980). Transformation of algae is still an area of research in its

9 Algal Biotechnology: An Emerging Resource with Diverse Application and Potential Potential Resources Produced

Oxygen

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Environmental and Culture Conditions

Recombinant proteins Environmental Conditions

Hydrogen

BioDiesel

Light, Water, CO2

Lipids Photosynthesis

Nutraceuticals

Calvin Cycle Ethanol Nutrition, Carotenoids, Aquaculture

Transgenic Engineering

Carbohydrates

Biomass Nucleus

Gasoline

Organic compounds Nutrients

Fig. 9.2 Downstream potential for algae production, applications and uses

infancy. With continuous expansion of genome information, the application of algal models for the production of proteins will become an attractive alternative to the existing systems of recombinant protein expression. Recombinant engineering technology has evolved globally into an industry sector encompassing food, agriculture, pharmaceutics, biofuel (see sect. 6.1.3), and environmental areas. Recombinant production of proteins now produces a myriad of protein-based industrial and biopharmaceutical products in crop plants, aquatic plants and algae (Giddings et al. 2000). Stability of transformation and efficiency have, to date, been the key determinants upon experimental success. These are relative to the size and complexity of the algae used in each case (Hallmann 2007). Transformation has been focused on transient expression of reported genes, typically antibiotic resistance genes as these confer traits regardless of genotype (Qin et al. 2005; Hallmann 2007).

9.3.1.1

Status of Alga Genome Information

For the development and application of engineered algae for scale-up of endogenous molecules and for their application as a recombinant tool for protein production, highly annotated genome data are required. Complete genome annotation and sequenced expressed sequence tag (EST) mapping is currently available for a number of species. Like other genome projects, data increase almost exponentially. Sequencing and annotation of the 16.5 Mb Cyanidioschyzon merolae genome

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(Matsuzaki et al. 2004), the 34 Mb Thalassiosira pseudonana genome (Armbrust et al. 2004) and the ~120 Mb genome of Chlamydomonas reinhardtii has been completed (Merchant et al. 2003). Genomes of over 50 species are all currently undergoing annotation or nearing sequencing completion, 30 of which are genomes of cyanobacteria (NCBI, Entrez Genome Project: www.ncbi.nlm.nih.gov/sites/ entrez: accessed 20 July 2009). Owing to their small-sized genomes, completed genome information is being produced at a faster rate for blue-green algae.

9.3.1.2

Current Status of Algae Engineering

The genetic engineering and modification of algae is an area, which has received a lot of attention over the last few years (Leo´n-Ban˜ares et al. 2004; Walker et al. 2005). Reports detailing the introduction of DNA into the diatom Phaeodactylum, the green algae Chlamydomonas, and the blue-green algae Synechococcus and Synechocystis have been circulated (Raja et al. 2008). Genetic engineering of the expression of mosquito larvicidal properties in blue-green algae has also been reported (Boussiba et al. 2000). However, at the time of publication there has not been a report on the commercial use of any transgenic algae for the application of a functional transformation system. Literature supports the development of such methodologies demonstrated with the manipulation of diatoms to enhance lipid production (Dunahay 1996), the expression of a functional glucose transporter in the obligate phototrophic Phaeodactylum enabling this diatom to grow on glucose in the dark (Zaslavskaia et al. 2001) and the advancements using genetically modified strains of Chlamydomonas for hydrogen production as an alternative biofuel (Melis et al. 2000). Chlamydomonas is particularly relevant as a model algae system for genetic manipulation and is detailed further below. The potential of algae to be genetically modified, permitting the synthesis of recombinant proteins, opens up alternatives to the current recombinant systems, and presents a simpler model with respect to minimal or removed system contaminants for the use in expression of human antibody and therapeutic proteins.

9.3.1.3

Maintenance in Bioreactors

The ability to culture single-celled plants and aquatic plants in bioreactors offers two advantages over the use of terrestrial plants: (1) the growth conditions can be controlled precisely, insuring optimal growth conditions and batch-to-batch product reproducibility (yield, activity); and (2) growth in bioreactors is contained in-house, thus removing environmental biosafety issues associated with “release” of transgenic terrestrial plants. With respect to their high protein levels and their amino acid composition the red seaweed appear to be an interesting potential source of food proteins. With large scale production in bioreactors possible, this is a developing area of research and industry. Algae may represent functional foods, which remain to be utilized. Use

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of algae, as a food source for mariculture permits the delivery of target genes, influencing both the organism feeding and also downstream consumers. Such an approach to molecular pharming would permit edible therapeutics and health management. The amino acid content is of nutritional value, however, their protein digestibility in vivo remains to be completely elucidated.

9.3.2

Models of Alga Engineering

9.3.2.1

Laminaria japonica

The brown algae, Laminaria japonica, commonly referred to as Kelp is a cultivated seaweed, which has been utilized as both a foodstuff and as a raw material for iodine, mannitol and alginate production (Tseng and Qin 1999). The production of L. japonica is a low-cost, high-yield crop in China. Extensive research has been carried out for the breeding and strain development for the establishment of improved traits. Transformation of L. japonica has been hindered by a lack of knowledge in relation to kelp viruses or symbiotic bacteria, which could be used for direct gene transfer. Therefore, until this point particle bombardment and ultrasound have been the transformation methodologies applied. Research on a virus (ESV-1) infecting Ectocarpus, a filamentous brown algae found as epiphytic to kelp has been performed, including viral genome annotation (Delaroque et al. 2001). This virus infects unicellular spores or gamates, transmitting its genome by mitosis to all cells of the growing host plant (Mu¨ller et al. 1998). The transmission of its genome presents this virus as a potential vector for gene delivery. As no usable promoter from kelp has yet been identified, a number of promoter regions from terrestrial plants, unicellular algae and algal viruses have been introduced without much success. Stable expression has been shown for transforants using two promoters, fcp (diatom fucoxanthin-chorophyll a/c binding protein gene) and the SV40 (simian virus) (Qin et al. 2005). The potential of L. japonica in the production of recombinant protein was further demonstrated by the introduction of the vaccine gene encoding human hepatitis B surface antigen gene (HBsAg) (Qin et al. 2005). Expression levels in transgenic kelp were compared to that of transgenic tobacco. Comparable levels of protein were expressed from both systems (Qin et al. 2005), demonstrating the potential for edible/direct delivery of proteins.

9.3.2.2

Chlamydomonas Reinhardtii

The green algae, Chlamydomonas reinhardtii, has been utilized as a model organism in the study of photosynthesis and light-regulated gene expression. Recently, it has been explored as a potential host for recombinant protein synthesis. Production of several forms of human IgA antibody directed against glycoprotein D and HSV have been reported to date in C. reinhardtii (Mayfield et al. 2003; Franklin and

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Mayfield 2005). The production of these monoclonal antibodies in algae demonstrated that the production is both possible and is comparable to terrestrial plant production. Algae offers several advantages over terrestrial plants including: (1) transgenic forms can be generated quickly (only weeks between generation of initial transformants and their scale-up for production); (2) both the chloroplast and nuclear genome of algae can be genetically transformed by standard methodologies, i.e. microprojectile particle bombardment or electroporation, providing scope for the production of several different proteins simultaneously; and (3) the ability to culture volumes ranging from a few milliliters to 500,000 liters makes such an expression system advantageous at an industrial level (Franklin and Mayfield 2004).

9.4

Summary

The demand for improved systems of production of nutraceuticals and cost-effective protein expression systems (both industrial and pharmaceutical applications) lend themselves to the potential useful capacities of algae. Currently, recombinant expression of proteins is performed in both mammalian and nonmammalian systems routinely. As a system, algae provide a simpler model than plants. Limitations of manufacturing are a bottleneck limited on size, cost, time and levels of purification, especially with reference to mammalian recombinant proteins. The progress being made in relation to sequencing of algal genomes will permit the cloning and manipulation of genes and allow “omics” technologies to be applied. This advancement will aid in the identification of key regulators of metabolism and enable the eventual manipulation of cellular pathways. Such advances in transformation techniques will permit in future more sophisticated forms of recombinant engineering to be performed. Ultimately, algal biotechnology offers the potential to have impact on the advancement of recombinant technologies. This is only the beginning of this field of research and much remains to be achieved to optimize the full potential of algae.

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Lucas S, Medina M, Montsant A, Obornik M, Parker MS, Palenik B, Pazour GJ, Richardson PM, Rynearson TA, Saito MA, Schwartz DC, Thamatrakoln K, Valentin K, Vardi A, Wilkerson FP, Rokhsar DS (2004) The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306:79–86 Balzarini J (2006) Inhibition of HIV entry by carbohydrate-binding proteins. Antiviral Res 71:237–247 Be´ress A, Wassermann O, Tahhan S, Bruhn T, Be´ress L, Kraiselburd EN, Gonzalez LV, de Motta GE, Chavez PI (1993) A new procedure for the isolation of anti-HIV compounds (polysaccharides and polyphenols) from the marine algae Fucus vesiculosus. J Nat Prod 56:478–488 Botos I, Wlodawer A (2005) Proteins that bind high-mannose sugars of the HIV envelope. Progr Biophys Mol Biol 88:233–282 Boyd WC, Almodovar IR, Boyd IG (1966) Agglutinins in marine algae for human erythrocytes. Transfusion 6:82–83 Cardozo KH, Guaratini T, Barros MP, Falca˜o VR, Tonon AP, Lopes NP, Campos S, Torres MA, Souza AO, Colepicolo P, Pinto E (2007) Metabolites from algae with economical impact. Comp Biochem Physiol C Toxicol Pharmacol 146:60–78 Cater NB, Grundy SM (1998) Lowering serum cholesterol with plant sterols and stanols: historical perspectives. J Postgrad Med, 6-14 Centeno POR, Ballantine DL (1999) Effects of culture conditions on production of antibiotically active metabolites by the marine algae Spyridia filamentosa. I. Light. J Appl Phycol 11:217– 224 Charest A, Desroches S, Vanstone CA, Jones PJH, Lamarche B (2004) Unesterified plant sterols and stanols do not affect LDL electrophoretic characteristics in hypercholesterolemic subjects. J Nutr 134:592–595 Craigie JS (1990) Cell Walls. In: Cole KM, Sheath RG (eds) Biology of the Red Algae. Cambridge Univ Press, Cambridge, UK, pp 226–236 Delaroque N, Mu¨ller DG, Bothe G, Pohl T, Knippers R, Boland W (2001) The complete DNA sequence of the Ectocarpus siliculosus Virus EsV-1 genome. Virology 287:112–132 Doughman SD, Krupanidhi S, Sanjeevi CB (2007) Omega-3 fatty acids for nutrition and medicine: considering microalgae oil as a vegetarian source of EPA and DHA. Curr Diabet Rev 3(3):198–203 Dunahay TG (1996) Manipulation of microalgal lipid production using genetic engineering. Appl Biochem Biotechnol 57:223–231 Fleurence J (1999) Seaweed proteins: biochemical, nutritional aspects and potential uses. Trends Food Sci Technol 10:25–28 Fogg GE (1953) The Metabolism of Algae. Methuen, London, UK Franklin SE, Mayfield SP (2004) Prospects for molecular farming in the green algae Chlamydomonas. Curr Opin Plant Biol 7:159–165 Franklin SE, Mayfield SP (2005) Recent developments in the production of human therapeutic proteins in eukaryotic algae. Expert Opin Biol Ther 5:225–235 Giddings G, Allison G, Brooks D, Carter A (2000) Transgenic plants as factories for biopharmaceuticals. Nat Biotechnol 18:1151–1155 Gustafson KR, Cardellina JH, Fuller RW, Weislow OS, Kiser RF, Snader KM, Patterson GM, Boyd MR (1989). AIDS: antiviral sulfolipids from cyanobacteria. J. Natl. Cancer Inst. 81:1254–1258 Hallmann A (2007) Algal transgenics and biotechnology. Transgen Plant J 1:81–98 Hiatt A, Cafferkey R, Bowdish K (1989) Production of antibodies in transgenic plants. Nature 342:76–78 Hussein G, Sankawa U, Goto H, Matsumoto K, Watanabe H (2006) Astaxanthin, a carotenoid with potential in human health and nutrition. J Nat Prod 69:443–449 Leo´n-Ban˜ares R, Gonza´lez-Ballester D, Galva´n A, Ferna´ndez E (2004) Transgenic microalgae as green cell-factories. Trends Biotechnol 22:45–52

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Polı´vka T, Sundstro¨m V (2004) Ultrafast dynamics of carotenoid excited States-from solution to natural and artificial systems. Chem Rev 104:2021–2071 Ponomarenko LP, Stonik IV, Aizdaicher NA, Orlova TY, Popvskaya GI, Pomazkina GV, Stonik VA (2004) Sterols of marine microalgae Pyramimonas cf. cordata (prasinophyta), Atteya ussurensis sp. nov. (Bacollariophyta) and a spring diatom bloom form Lake Baikal. Comp Biochem Physiol (B) 138:65–70 Pratt R, Fong J (1940) Studies on Chorella vulgaris. Am J Bot 27:431–436 Pratt R, Fong J (1944) Chlorellin, an antibacterial substance from Chorella. Science 99:351–352 Qin S, Jiang P, Tseng C (2005) Transforming kelp into a marine bioreactor. Trends Biotechnol 23:264–268 Raja R, Hemaiswarya S, Kumar N Ashok, Sridhar S, Rengasamy R (2008) A Perspective on the Biotechnological Potential of Microalgae. Critical Reviews in Microbiology 34:77–88 Rogers DJ, Hori K (1993) Marine algal lectins: new developments. Hydrobiologia 260:589–593 Samsonoff WA, MacColl R (2001) Biliproteins and phycobilisomes from cyanobacteria and red algae at the extremes of habitat. Arch Microbiol 176:400–405 Schaeffer DJ, Krylov VS (2000) Anti-HIV activity of extracts and compounds from algae and cyanobacteria. Ecotoxicol Environ Saf 45:208–227 Shestakov SV, Khuyen NT (1970) Evidence for genetic transformation in blue-green algae Anacystis nidulans. Mol Gen Genet 107:372–375 Sijmons PC, Dekker BM, Schrammeijer B, Verwoerd TC, van den Elzen PJ, Hoekema A (1990) Production of correctly processed human serum albumin in transgenic plants. Biotechnology 8:217–221 Soudant P, Coz JR, Marty Y, Moal J, Robert R, Samain JF (1998) Incorporation of microalgae sterols by scallop Pecten maximus (L.) larvae. Comp Biochem Physiol (A) 119:451–457 Spolaore P, Joannis-Cassan C, Duran E, Isambert A (2006) Commercial applications of microalgae. J Biosci Bioeng 101:87–96 Stevens SE, Parter RD (1980) Transformation in Agmenellum quadruplicatum. Proc Natl Acad Sci USA 77:6052–6056 Tseng CK, Qin S (1999) Mariculture and genetic transformation of Laminaria japonica in China. In: Xu HS, Colwell RR (eds) Proc Int Symp on Progress and Prospect of Marine Biotechnol. China, Ocean Press, pp 11–16 Walker TL, Purton S, Becker DK, Collet C (2005) Microalgae as bioreactors. Plant Cell Rep 24:629–641 Weis WI, Drickamer K (1996) Structural basis of lectin-carbohydrate recognition. Annu Rev Biochem 65:441–473 Wen ZY, Chen F (2003) Heterotrophic production of eicosapentaenoic acid by microalgae. Biotechnol Adv 21:273–294 Witvrouw M, De Clercq E (1997) Sulfated polysaccharides extracted from sea algae as potential antiviral drugs. Gen Pharmacol 29:497–511 Yokoyama M, Origasa H, Matsuzaki M, Matsuzawa Y, Saito Y, Ishikawa Y, Oikawa S, Sasaki J, Hishida H, Itakura H, Kita T, Kitabatake A, Nakaya N, Sakata T, Shimada K, Shirato K, Japan EPA lipid intervention study (JELIS) Investigators (2007) Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded end point analysis. Lancet 369:1090–1098 Zaslavskaia LA, Lippmeier JC, Shih C, Erhardt D, Grossman AR, Apt K (2001) Trophic conversion of an obligate photoautotrophic organism through metabolic engineering. Science 292:2073–2075 Zio´łkowska NE, Wlodawer A (2006) Structural studies of algal lectins with anti-HIV activity. Acta Biochim Pol 53:617–626 Zio´łkowska NE, O’Keefe BR, Mori T, Zhu C, Giomarelli B, Vojdani F, Palmer KE, McMahon JB, Wlodawer A (2006) Domain-swapped structure of the potent antiviral protein griffiths in and its mode of carbohydrate binding. Structure 7:1127–1135

Chapter 10

Biotech Crops and Functional Genomics Narayana M. Upadhyaya, Andy Pereira, and John M. Watson

10.1

Introduction

The increase in human population, poor performance of crop cultivars under increasingly adverse environmental conditions and a decline in the available land for sustainable crop production are contributing to a shortage of global food supply and increase in its demand. Conventional breeding efforts in crops such as rice over the last three decades have resulted in a doubling of agricultural productivity (Khush 1997). However, for sustained increase in the agricultural productivity, crops which can resist pests, pathogens and tolerate salinity, drought and temperature extremes need to be developed and deployed. The deployment of a handful of gene classes developed as transgenes for insect and/or herbicide resistance in crops such as maize, soybean, canola, cotton, squash, papaya, alfalfa, and sugar beet reduces yield losses and increases agricultural profitability. However, commercial application of transgenes conferring more complex traits such as abiotic stress tolerance, yield, vigor and nutritional quality are yet to be achieved because of the lack of adequate knowledge of the critical genes controlling such traits and the complexity of their behavior under different environmental conditions. Long development times from discovery to commercial release, intellectual property constraints, high development costs, and regulatory constraints and costs are also slowing down the process of commercial deployment (Birch 2000). With continual improvements in transgene delivery, integration and expression modulation, several other transgenes of agronomic importance are being deployed and tested for efficacy in various crop plants. Major crops such as rice, wheat, barley and sugarcane are bound to become biotech crops as more and more novel genes, with high potential to be deployed as transgenes, are discovered, and

N.M. Upadhyaya (*) CSIRO Plant Industry, GPO Box 1600, Canberra ACT 260, Australia e-mail: [email protected]

C. Kole et al. (eds.), Transgenic Crop Plants, DOI 10.1007/978-3-642-04812-8_10, # Springer-Verlag Berlin Heidelberg 2010

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the concerns over the biosafety of their large-scale deployment are addressed scientifically and politically. Mapping and DNA sequencing of plant genomes and analysis of the information content present in genomic sequences commonly termed as “genomics” have the potential to provide valuable insight into genes controlling some of the complex traits mentioned above. Various genome-wide high-throughput “functional genomics” tools and resources are being developed worldwide. The ultimate aim of these approaches is to define the structures of all genes, and the functions of all gene products, as well as all the processes that occur during plant growth and development. Such knowledge could be used effectively, not only in molecular markerassisted breeding, but also in transgenic breeding. Arabidopsis, a model dicot, and rice, a model cereal, have emerged as front-runners with near-complete sequencing of the Arabidopsis ecotype Columbia (TAGI 2000) and two rice genotypes, namely, japonica cv. Nipponbare (IRGSP 2005) and indica cv. 93-11 (Yu et al. 2002, 2005). Genome sequencing efforts are now underway for several other food grain and tuber crops (barley, cassava, maize, mungbean, potato, sorghum and wheat), vegetable crops (tomato, cabbage and field mustard), fruit crops (grape, papaya, orange and apple), oil crops (rapeseed, Indian mustard, black mustard, soybean and castor), forage crops (barrel medic), biofuel crops (jatropha, miscanthus, switchgrass, pine, madhuca, arundo and pongamia) and other commercial crops such as tobacco and cotton (Tables 10.1 and 10.2; http://www.arabidopsis.org/portals/gen Annotation/other_genomes/#sequence). In this chapter, we give a brief overview of transgenic crops, plant genomics and plant functional genomics and then discuss various transgenic strategies being used to establish gene–phenotype relationships and elaborate on how they are facilitating the functional characterization of genes and gene control sequences.

10.2

Transgenic Plants

Transgenic crops have great potential for alleviating some of the production constraints such as insect pests, pathogens, salinity and drought. Since the first demonstration of the introduction and expression of foreign genes in tobacco in 1984, more than 150 plant species in at least 50 plant families have been experimentally transformed and transgenic events reported. Today, 13 transgenic crops are grown commercially in 25 different countries, including 15 developing countries (James 2008). Regulatory approvals for 24 transgenic crops, for the importation for food and feed use and for release into the environment, have been granted in another 30 countries (James 2008). Worldwide acreages of transgenic crops are increasing at the rate of ~12% each year (James 2008). However, the current successes in transgenic crops still have a narrow base with respect to traits (>95% are insect resistance and/or herbicide tolerance traits), crops (>95% are soybean, corn, cotton and canola) and countries (>95% are grown in US, Argentina, Brazil, Canada, India, and China).

Food

Forage legume model

Hordeum vulgare

Medicago truncatula

Barley

Barrel medic

Future

Future

Future

Food

Oil, industrial

Manihot esculenta

Ricinus communis

Cassava

Castor bean

Future

Future

Future

Fruit

Musa acuminata

Banana

Model

Vegetable

Dicot model

Thellungiella halophila

Model

Model

Dicot model

Arabidopsis thaliana

Arabidopsis (thale cress) Arabidopsis relative

Future

Biotech status

Model cereal

Fruit

Malus domestica

Apple

Brachypodium Brachypodium distachyon Broccoli Brassica oleracea

Type

Plant

Common name

na

Yes

na

na

Yes

Yes

Yes

na

Yes

na*

In progress

Sequencing status

62,592

79,444

na

20,449

260,238

501,366

5,524

38,022

18

9

5

8

7

11

7

5

17

Chromosomes (n)

Draft assembly 10

In progress

In progress

In progress

Completed

In progress

In progress

In progress

1,526,124 Completed

256,249

Maps ESTs

16957

13628

12577

18703

9508

9511

15719

18765

9506

NCBI project ID 12882

Biotech Crops and Functional Genomics (continued)

Sequencing group/consortium Haploid genome size (Mb) 750 IASMA, Institoto Agrario S. Michele all’Adige (http://www.ismaa.it/) 120 The Arabidopsis Information resource (http://www.arabidopsis.org/) 260 DOE Joint Genome Institute (http://www.jgi.doe.gov/ sequencing/why/50029.html) 600 The Global Musa Genomics Consortium (http://www.musagenomics.org/) 5,000 International Barley Genome Sequencing Consortium (http://www.public.iastate.edu/ ~imagefpc/IBSC%20Webpage/ IBSC%20Template-home.html) 500 Medicago truncatula Sequencing Resources (http://www. medicago.org/genome/) 300 DOE Joint Genome Institute (http://www.brachypodium.org/) 600 TIGR Brassica oleracea Genome Project (http://www.tigr.org/ tdb/e2k1/bog1/) 760 DOE Joint Genome Institute (http://www.jgi.doe.gov/ sequencing/why/51283.html) 400 J. Craig Venter Institute (http://www.jcvi.org/cms/ research/projects/castorbean-database/overview/)

Table 10.1 Current and future biotech plants for which full-scale genomics studies have been initiated or completed

10 361

Vegetable

Biofuel, grass

Brassica rapa

Arundo donax

Field mustard

Giant cane

Model

Arabidopsis lyrata

Lyreleaf rockress

Dicot model for comparative genomics

Model

Lotus japonicus Model legume

Future

Future

Future

Future

Future

Lotus

Biofuel

Oil, vegetable

Biofuel, tree

Biofuel

Biofuel

Pinus taeda

Jatropha curcas Jatropha tanjorensis Pogamia pinnata Brassica juncea

Future

Future

Future

Future

Biotech status

Loblolly pine

Leaf mustard

Karanj

Jatropha

Jatropha

Model for citrus

Oil, tree

Eucalyptus globulus

Eucalyptus (blue gum)

Japanese bitter Poncirus orange trifoliata

Type

Plant

Common name

Table 10.1 (continued)

na

yes

na

na

na

na

na

na

na

na

na

561

157,951

328,628

193

na

na

1,012

62,344

na

33,398

10,003

Maps ESTs

12

18

11

11

11

9

12

10

11

Chromosomes (n)

In progress

8

Draft assembly 6

In progress

In progress

In progress

In progress

In progress

In progress

In progress

In progress

In progress

Sequencing status

Sequencing group/consortium Haploid genome size (Mb) 600 The International Eucalyptus Genome Network (http://www.fabinet.up.ac.za/ eucagen) 500 The Multinational Brassica Genome Project (MBGP) (http://www.brassica.info/ resource/sequencing.php) 2,744 Nandan Biometrix Ltd (http://www.nandan.biz/) 380 International Citrus Genome Consortium (http://www.citrusgenome. ucr.edu/) 416 Nandan Biometrix Ltd (http://www.nandan.biz/) 416 Nandan Biometrix Ltd (http://www.nandan.biz/) 1,700 Nandan Biometrix Ltd (http://www.nandan.biz/) 1,200 The Brassica Genome Gateway (http://www.brassica.bbsrc. ac.uk/) 30,000 The Pine Genome initiative (http://pinegenomeinitiative. org/) 470 Kazusa DNA Research Institute (http://www.kazusa.or.jp/ lotus/clonelist.html) 230 DOE Joint Genome Institute (http://www.jgi.doe.gov/ sequencing/why/3066.html)

15628

10747

30775

18137

32643

32385

12948

32663

12578

NCBI project ID 12504

362 N.M. Upadhyaya et al.

Potato

Solanum tuberosum

Populus trichocarpa

Food crop

Dicot model, Arabidopsis relative Tree model

Capsella rubella

Pink Shepherd’s purse Poplar

Future

Current

Model

Current

Fruit

Carica papaya

Yes

na

na

na

na

na

Model

Papaya

Moss

Current

Yes

na

Yes

na

na

Model

Future

Current

Future

Future

Oilseed rape

Physcomitrella patens

Moss

Model

Food and fodder

Biofuel

Biofuel tree

Model for comparative genomics Model for Selaginella evolutionary moellen study dorffii Brassica napus Oil

Mimulus guttatus

Madhuca longifolia var. latifolia Miscanthus sinensis Zea mays

Monkey flower

Maize

Maiden grass

Mahwa

In progress

In progress

231,704

89,943

na

77,158

596,249

93,806

20,453

14,587

14

10

19

13

19 (2
)**

8

8

In progress

12

Draft assembly 19

In progress

Draft assembly 9

In progress

Completed

Draft assembly 27

In progress

2,018,337 In progress

na

na

840

480

686

370

1,100

100

510

430

2,400

na

na

The International Populus Genome Consortium (http://www.ornl.gov/ sci/ipgc/) The potato Genome Sequencing Consortium (http://www. potatogenome.net/)

12984

10770

15659

16103

12478

13079

12940

15658

9514

32661

32407

Biotech Crops and Functional Genomics (continued)

Nandan Biometrix Ltd (http://www.nandan.biz/) The Maize Genome Sequencing Project (http://www. maizesequence.org/ overview.html) DOE Joint Genome Institute (http://www.jgi.doe.gov/ sequencing/why/3062.html) DOE Joint Genome Institute (http://www.jgi.doe.gov/ sequencing/why/3141.html) DOE Joint Genome Institute (http://www.jgi.doe.gov/ sequencing/why/3153.html) The Brassica Genome Gateway (http://brassica.bbsrc.ac.uk/) The Hawaii Papaya Genome Project (http://asgpb. mhpcc.hawaii.edu/papaya/) DOE Joint Genome Institute (http:// www.jgi.doe.gov/sequencing/ why/3066.html)

Nandan Biometrix Ltd (http://www.nandan.biz/)

10 363

Current Future

Medicinal crop

Commercial crop

Fruit

Glycine max

Cucurbita pepo Vegetable

Biofuel

Chlorophytum borivilianum Sorghum bicolor

Panicum virgatum

Nicotiana tabacum

Solanum lycopersicum

Safed musli

Soybean

Squash

Switchgrass

Tobacco

Tomato

Oil, vegetable

Food crop

Food

Oryza sativa japonica

Rice

Sorghum

Food

Oriza sativa indica

Rice

Current

Future

Current

Future

Future

Future

Future

Future

Oil, vegetable

Brassica nigra

Rapeseed

Biotech status

Type

Plant

Common name

Table 10.1 (continued)

Yes

Yes

na

na

Yes

Yes

na

Yes

Yes

na

Completed

In progress

Completed

Completed

In progress

Sequencing status

258,830

240,440

436,535

27

In progress

In progress

In progress

In progress

1,386,618 In progress

209,814

na

na

na

1

Maps ESTs

760

Sequencing group/consortium Haploid genome size (Mb) 700 The Brassica Genome Gateway (http://www.brassica. bbsrc.ac.uk/) 375 BGI Rice Information System (http://rice.genomics.org. cn/rice/index2.jsp) 390 International Rice Genome Sequencing Project (http://rgp.dna.affrc.go.jp/IRGSP/) 540 Nandan Biometrix Ltd (http://www. nandan.biz/)

DOE Joint Genome Institute (http://www.phytozome. net/sorghum) 20 1,200 DOE Joint Genome Institute (http://www.phytozome. net/soybean) 20 539 Cucurbit Genomics Database (http://www.icugi.org) 18–36 1,911–2,303 DOE Joint Genome Institute (2–4
) (http://www.jgi.doe.gov/ sequencing/why/50008.html) 24 (2
) 4,500 Tobacco genome Initiative (http://www.tobaccogenome. org/) 12 950 International tomato sequencing Project (http://www.sgn. cornell.edu/about/tomato_ sequencing.pl)

10

14 (2
)

12

12

8

Chromosomes (n)

9509

13234

17453

na

9507

10785

32621

9512

9512

NCBI project ID 18141

364 N.M. Upadhyaya et al.

Fruit and wine

Parasitic weed

Vitis vinifera

Triphysaria versicolor

Wine grape

Yellow owl’s clover

Model

Future

Future

Model

Future

Current

na

Yes

Yes

na

na

Yes

Pending

In progress

In progress

49,006

353,688

21 (3
)

7

9

26 (2
)

Pending

11

Draft assembly 19

1,051,736 Pilot scale

na

207,500

268,779

1,200

500

16,000

350

380

2,100

International cotton genome initiative (http://icgi.tamu.edu/ developing.html) International Citrus Genome Consortium (http://www. citrusgenome.ucr.edu/) DOE Joint Genome Institute (http://www.jgi.doe.gov/ sequencing/why/51280.html) International Wheat Genome Sequencing consortium (http://www.wheatgenome.org/) International Grape Genome Program (http://www. vitaceae.org/index.php/ International_Grape_Genome_ Program) DOE Joint Genome Institute (http://www.jgi.doe.gov/ sequencing/why/3116.html)

*na ¼ not available or not determined or not assigned ** ¼ x denotes ploidy level Data source: NCBI, relevant web pages and Plant DNA C-values database (Bennett and Leitch 2005, http://data.kew.org/cvalues/)

Food crop

Triticum aestivum

Wheat

Flower model

Fruit

Citrus sinensis

Valencia orange

Western Aquilegia columbine formosa

Fibre, oil

Gossypium hirsutum

Upland cotton

15684

12992

9513

18647

9597

12542

10 Biotech Crops and Functional Genomics 365

Biotech status Current Future Future Future Near future Near future Near future Current Current Near future Near future Near future Future Future Current Near future Near future Near future Near future Near future Near future Near future Near future Near future Near future Near future Near future Near future Future Current Near future Near future Near future

Plant

Medicago sativa Prunus dulcis Prunus armeniaca Asparagus officinalis Amomum sp, Elettaria sp Brassica oleracea var capitata Eschscholzia californica Capsicum annuum Dianthus caryophyllus Brassica oleracea var botrytis Cichorium intybus Brassica oleracea var. alboglabra Coffea arabica Phaseolus vulgaris Gossypium arboreum Vigna unguiculata Agrostis stolonifera Cucumis sativus Cuphea Solanum melongena Brassica carinata Linum usitatissimum Lactuca sativa Nuphar advena Vigna radiata Cucumis melo Picea abies Papaver somniferum Prunus persica Petunia axillaris subsp axillaris Ananas comosus Populus tremula Populus tremuloides

Common name

Alfalfa Almond Apricot Asparagus Cardamom Cabbage California poppy Capsicum Carnation Cauliflower Chicory Chinese broccoli Coffee Common bean Cotton Cowpea Creeping bentgrass Cucumber Cuphea hybrid Egg plant Ethiopian mustard Flax Lettuce Lilly Mung bean Musk mellon Norway spruce Opium poppy Peach Petunia Pineapple Poplar Poplar

11,090 3,864 15,105 8,422 na 26,692 9,083 33,311 387 202 53,973 30,759 1,577 83,448 41,768 183,751 9,020 6,662 na 3 2,482 7,929 80,781 20,589 829 5,943 10,217 20,340 79,023 1,696 5,649 37,313 12,813

ESTs Yes na Yes Yes na na na Yes na na na na na na na Yes na na na Yes na na na na na na na na Yes na na na na

Maps Forage Nut Fruit Vegetable Condiment Vegetable Medicinal Vegetable Flower Vegetable Beverage Vegetable Beverage Vegetable Fibre, oil Food Lawn grass Vegetable Ornamental, oil Vegetable Oil Fruit Vegetable Flower Food Fruit Timber tree Medicinal Fruit Flower Fruit Tree Tree

Type

Haploid genome size (Mb) 900 300 300 1,323 na 600 1,103 3,000 613 760 na 760 1,176 630 2,132 588 3,430 370 na 1,100 1,544 686 2,597 2,400 515 931 18,228 3,724 290 1,372 569 na na

Table 10.2 Current and future biotech plants for which full-scale genomics studies are yet to be initiated Chromosome number (n) 8 8 8 10 12 9 6 12 15 9 9 9 11 11 13 11 28 (2
)** 7 8 12 17 15 9 17 11 12 12 11 8 7 25 19 19 13214 12944 20685 16855 na 12577 na 12486 na na na na 10702 12933 12946 31169 na 36671 na 15625 na na 12869 na 17571 na na na 12949 na na 13278 13258

NCBI Project ID

366 N.M. Upadhyaya et al.

Rosa chinensis Rosa hybrid cultivar Rosa luciae Phaseolus coccineus Carthamus tinctorius Beta vulgaris Saccharum officinarum Helianthus annuus Festuca arundinacea Camellia sinensis Nicotiana benthamiana Torenia fournieri Citrullus lanatus Trifolium repens Picea glauca Near future Near future Near future Near future Near future Current Near future Near future Near future Near future Future Near future Near future Near future Near future

1,794 5,563 1,932 391,138 41,011 26,870 246,379 133,682 44,377 6,416 42,658 na 7,891 46 284,329

na na na na na Yes na na na na na na na na na

Flower Flower Flower Flower, food Oil Sugar and herb Food Oil Forage Beverage Narcotics Flower Fruit Forage Wood, ornamental

564 578 na 662 na 760 3,969 3,000 5,629 3,824 3,136 na 424 956 19,796

7 7 7 11 9 9 40 (4
) 17 21 (3
) 15 19 (2
) 17 11 16 (2
) 12

*na ¼ not available or not determined or not assigned ** ¼ x denotes ploidy level Data source: NCBI, relevant web pages and Plant DNA C-values database (Bennett and Leitch 2005, http://data.kew.org/cvalues/)

Rose Rose Rose Runner bean Safflower Sugar beet Sugarcane Sunflower Tall fescue Tea Tobacco relative Torenia Watermellon White clover White spruce

na na na 12925 na 12562 12961 12865 na 31167 12911 na na na 32251

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Long development times from discovery to commercial release, intellectual property and regulatory constraints, and high development and regulatory costs are partly responsible for this (Birch 2000). So far, only transgenic crops and traits with high commercial return potential have been commercialized by large multinational companies in countries which allow the deregulation of genetically modified (GM) crops or have regulatory frameworks in place for commercial releases. In such countries and with such crops, companies, farmers and the consumers (to some extent) have reaped the financial benefits. It is worth noting that 20 countries have banned the importation and/or commercial cultivation of GM crops with another five countries having a moratorium on the same (http://www.centerforfoodsafety. org/geneticall5.cfm). As such, the issue of GM food crops for human consumption is still contentious because of the perceived health and environmental risks associated with the widespread usage of certain transgenes. Commercial deployments of transgenes conferring more complex traits such as abiotic stress tolerance, yield, vigor and nutritional quality are yet to be achieved. This is largely due to the lack of adequate knowledge of the genes controlling such traits and to the complexity of their behavior under different environmental conditions and in different genetic backgrounds. Quite often “green house champion” transgenic lines fail miserably under field conditions. Although very high transformation efficiencies are now achievable in many crop species, they are still highly genotype dependent. This is mainly due to the genotype dependency of tissue culturability and transformability (mainly via Agrobacterium). It is important to note that the proportion of transformation events resulting in an acceptable commercial phenotype is very low. Even with an optimized gene construct (gene promoter-coding region-gene terminator), this frequency could be one in 400 events (Birch 2000). Somaclonal variations introduced during tissue culture, pleiotropic effects of transgene expression, insertional inactivation of endogenous genes, integration position effects and transgene silencing are likely to reduce the frequency of “useful transformation events” (Birch 2000). Recent progress and applications in genomics and functional genomics are not only increasing the number of potential transgenes and gene control sequences but are also improving our understanding of the complexity of gene expression under different environmental conditions and in different genetic backgrounds. Progress with transgenics has been more rapid with rice than with any other cereals due to the very efficient rice tissue culture and transformation systems developed over the past 20 years (Upadhyaya et al. 2000). The use of appropriate embryogenic target tissues, gene delivery methods, gene promoters, selectable marker genes, selection regimes and reporter genes have all contributed to this success. Protoplasts derived from embryogenic callus have been used for gene delivery by either electroporation or polyethylene glycol treatment. Embryo or embryogenic calli have been the target tissues for biolistics and Agrobacteriummediated gene delivery. Although the initial successes were restricted to reporter and selectable marker transgenes, genes of agronomic value such as herbicide, insect, bacterial, fungal and viral resistances have also been introduced into rice and transgenic lines have been studied under field conditions. Transgenic rice is now an

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369

ideal tool for elucidating various aspects of gene expression and regulation, especially of genes from other monocots, which are not as amenable as rice to genetic manipulation. Some of the challenges in producing sustainable transgenic rice currently being addressed include the removal of selectable markers, targeted gene delivery (gene replacement), stability of transgene expression over many generations and the spatial, temporal and developmental control of transgene expression.

10.3

Plant Genomics

As proposed by Hieter and Boguski (1997), genomics can be broadly classified into two disciplines: “structural genomics” and “functional genomics.” Structural genomics corresponds to the initial phase of genome analysis resulting ultimately in the definition of the complete DNA sequence of an organism, while functional genomics makes use of the genome sequence to assess, on a large-scale, the functions of genes as well as their expression and interaction. With the near completion of the sequencing of their genomes (Table 10.3), Arabidopsis and rice are generally accepted as model dicot and monocot species, respectively for genetic and genomic studies. This is because of their small genome sizes (~135 Mb and 430 Mb, respectively), the ease with which they can be grown, transformed and used in genetic experiments, and the similarity of their respective gene orders and gene Table 10.3 Arabidopsis and rice genome sequence assembly and annotation – current status Rice Arabidopsis Rice TIGRb BGIc TAIRa Genotype/ecotype Columbia Japonica indica Cultivar – Nipponbare 93-11 Genome version TAIR8 Release 5 Release 2 Chromosomes 5 12 12 Sequenced genome size (bp) 119,186,497 372,077,801 360,157,649d (374,545,499)e Estimated complete genome size (bp) 134,634,692 388,820,000 NA Unassigned sequences (bp) – – 104,840,190 Predicted genes including transposable 33,282 56,278 (66,710)f 59,660 element (TE) genes and noncoding RNAs (38,963) Predicted non-TE genes 27,235 41,046 (51,286)f 49,088 Mapped Full-length cDNA 13,066 32,775 25,645 ORF cDNA 24,235 – – a The Arabidopsis Information Resource (http://www.arabidopsis.org/) b TIGR = The Institute for Genomic Research. TIGR is now merged with The J. Craig Venter Institute (http://www.jcvi.org/) and TIGR’s Rice Annotation Project is moved to Michigan State University (http://rice.plantbiology.msu.edu/) c BGI (http://rice.genomics.org.cn/index2.jsp) d Genome sizes based on the sum total of assigned contigs e Figures in the parenthesis are the genome sizes as the sum total of genome assigned scaffolds f Figures in the parenthesis are the total genes including splice variants

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sequences with other dicots (e.g., crucifers) or monocot cereals, (e.g., barley, wheat and maize). Thanks to the recent rapid developments in high-throughput nucleic acid sequencing technologies, genome sequencing and/or expressed sequence tag (EST) sequencing efforts are underway for the majority of crop plants (Table 10.1).

10.4

Plant Functional Genomics

Functional genomics is a rapidly expanding field of biological science. Various high-throughput technologies are being employed in the large-scale profiling of genes, mRNAs, proteins and metabolites that participate in various cellular processes during plant growth and development. These include: (1) data mining tools for structural similarities; (2) RNA level expression profiling with ESTs, oligonucleotide or cDNA chips; (3) protein level expression profiling (proteomics); (4) metabolite level expression profiling (metabolomics); (5) gene knock-outs or loss-of-function studies with naturally occurring alleles, induced deletion and insertional mutants; (6) gene expression knock-down (gene silencing) studies; and (7) gain-of-function studies with overexpression or misexpression transgenes.

10.4.1

Gene Predictions Using DNA Sequence Comparison

The most straightforward way of predicting the function of an unknown gene from one organism is by comparison of its DNA sequence with known gene sequences from other organisms, as functionally similar genes normally have sequence similarities at both the DNA and the protein sequence levels. The precision with which sequences can be compared has increased tremendously with recent vast improvements in computing power, gene prediction programs and various other bioinformatics capabilities. Several laboratories have embarked on sequence annotation using this approach (Antonio et al. 2007; Itoh 2007). Computational gene predictions in rice suggest that there could be more than 50,000 rice genes with ~60% having some evidence of expression. For example, according to the International Rice Genome Sequencing Project (IRGSP)’s rice annotation project database (RAP-DB; http://rapdb.dna.affrc.go.jp/), among the 53,461 predicted rice genes 31,439 show evidence of expression and 25,012 are protein-coding loci with fulllength cDNA support. The remainder is based on computer predictions without any evidence of transcriptional activity. The validation of gene functions predicted by sequence comparison needs to be done by other methods to avoid the progressive build up of inaccurate gene function assignments in the genome sequence databases. With the available japonica and indica genome sequences, attempts are being made to unravel allelic variations between these two subspecies using various functional genomics approaches. Transgenic approaches could be used to unravel the function of an unknown gene by overexpression, knock-out or knock-down of that gene as detailed later in this chapter.

10

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10.4.2

371

Gene Expression Studies at the RNA Level

Although there could be more than 50,000 genes in a plant genome, not all of these are transcribed into RNA at any given time, in any given tissue or under any given environmental condition. Even some of the transcribed RNAs are suppressed, broken down or rendered non-translatable. The characterization of all the transcribed genes, referred to as the “transcriptome,” is normally attempted by collecting large numbers of ESTs from diverse cDNA libraries. Currently, there are more than 17 million plant ESTs in the public database (http://www.ncbi.nlm.nih.gov/dbEST/) with ~5 million coming from current transgenic crops and 7 million from future transgenic crops. To date, there are 1,220,876 rice ESTs in the public database. Recent advances in the technology for construction of full-length cDNA libraries have made it possible to produce more than 30,000 rice full-length cDNAs (Kikuchi et al. 2003; Satoh et al. 2007). This has helped in improving the rice genome annotation, gene organization and genome-wide expression profiling. One other significant EST and full-length cDNA collection from indica rice comes from the Beijing Genomics Institute (BGI), which can be viewed through BGI-RIS (http://rice.genomics.org.cn/index2.jsp). Genome-wide expression profiling (including differential expression) of genes in various crop species is being facilitated by high-throughput techniques, such as microarrays, serial analyses of gene expression (SAGE), massively parallel signature sequencing (MPSS) and more recently by ultra-deep sequencing (e.g., 454, Solexa and SOliD technologies). These procedures are typically used to compare two mRNA populations derived from tissues of different developmental stages or those subjected to different environmental stimuli, to yield information on the comparative changes in gene expression in each tissue. The conceptual basis of this method is that genes contributing to the same biological process are likely to exhibit similar expression patterns and thus allow the putative assignment of gene function. With all these new developments in deep sequencing technologies, we are seeing an explosive increase in the RNA expression tag and small RNA datasets from diverse plants under different environmental conditions and experimental treatments. This will help in unraveling the complexities of the transcriptome, including that of non-coding RNAs, in diverse biological systems. Thus, it is now possible to study spatial and temporal RNA expression patterns which could provide insights into their cellular and developmental functions. The regulatory and developmental functions of a transgene could also be studied using these techniques.

10.4.3

Gene Expression Studies at the Protein Level

With recent advances in high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), staining, detection, peptide micro-sequencing and associated computer software, “proteomics” is also emerging as a powerful functional

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genomics tool. Here, instead of looking at gene expression, an assessment is made on the gene product, i.e., the protein. In the 2D-PAGE-based approach, intact proteins are separated by 2D-PAGE and protein abundance is determined by the relative stain intensities of protein spots on the gel. The differential proteome is confirmed by image analysis. The identity of a specific protein is generally determined by mass-spectrometric (MS) analysis of peptides after proteolysis of the protein spot or by protein sequencing after blotting the gel to a membrane. Comparison of the amino acid sequences of fragments of proteins with those predicted from DNA sequences greatly facilitates not only the validation of gene predictions but also provides insight into the cellular and developmental regulation of gene expression. Several public databases of 2D-PAGE-derived plant proteins are already available, such as WORLD-2DPAGE (http://expasy.org/ch2d/2d-index.html), Rice Membrane Protein Library (http://wardlab.cbs.umn.edu/rice/) and the Rice Proteome Database (http://gene64.dna.affrc.go.jp/RPD/), that provide extensive information on the progress of rice proteome research. In addition, progress is being made in detecting post-translational modifications such as glycosylation, lipid attachment, phosphorylation, methylation, disulfide bond formation and proteolytic cleavage. Differential proteome analyzes of a particular transgenic plant and an appropriate non-transgenic plant (i.e., a segregating null and/or wild type plant) could highlight any unintended or flow-through effect of the transgene on the expression of other genes.

10.4.4

Metabolomics

Metabolomics is the comprehensive analysis of low-molecular-weight compounds in biological samples and is emerging as a biochemical phenotyping tool along with transcriptomics and proteomics in functional genomics (Tarpley and Roessner 2007). Technologies used in metabolomics are normally based on the chromatographic separation of complex compound mixtures, using either liquid or gas chromatography and mass-spectrometric detection. Nuclear magnetic resonance (NMR) spectroscopy is also playing a major role in metabolomic approaches. Fourier-transform ion cyclotron mass spectrometry (FT-ICR-MS) can mass-resolve metabolites with a mass accuracy of