ABSTRACT Title of Document: MOLECULAR MECHANISMS OF PLANT RESPONSES TO COLD, HEAT AND SALT STRESSES IN ARABIDOPSIS Qingmei Guan, Doctor of Philosophy, 2013 Directed By: Dr. Jianhua Zhu, Assistant Professor, Department of Plant Science and Landscape Architecture Abiotic stresses, such as temperature extremes and salinity adversely affect plant productivity and distribution worldwide. Resistant or susceptible to stresses is a complex trait because more than one stress may occur simultaneously, for example, salinity is accompanied with ion toxicity and water deficit. To survive in a fixed environment, plants have to adjust their metabolisms and developmental programs to adapt to the stress or acclimate to the transitory stress. The responses of plants to different abiotic stresses are extremely complex, involving stress perception, signaling transduction, and response induction. We took a forward genetic analysis approach and identified three novel proteins in the reference plant Arabidopsis thaliana, Regulator of CBF Gene Expression 1 (RCF1), Regulator of CBF Gene Expression 3 (RCF3), and Short Root in Salt Medium 3 (RSA3), which are critical for plant tolerance to cold, heat and salinity, respectively. RCF1 is a cold-inducible DEAD box RNA helicase protein which is localized in the nucleus. RCF1 is a positive regulator for chilling and freezing tolerance. RCF1 functions to maintain proper splicing of pre-mRNAs because many cold-responsive genes are mis-spliced in rcf1-1 mutant plants under cold stress. RCF3 encodes a KH-domain containing putative RNA-binding protein. RCF3 is a negative regulator of most heat stress transcription factors (HSFs). Consistent with the overall increased accumulation of heat-responsive genes, the rcf3 mutants are heat-tolerant. RSA3, a xylogluscan galactosyltransferase, is essential for salt stress tolerance. rsa3-1 mutant plants are hypersensitive to NaCl and LiCl but not to CsCl or to general osmotic stress. RSA3 controls expression of many genes including genes encoding proteins for reactive oxygen species (ROS) detoxification under salt stress. RSA3 functions to maintaining the proper organization of actin microfilaments in order to minimize damage caused by excessive ROS. miRNAs play important regulatory roles in plants by targeting messenger RNAs (mRNAs) for cleavage or translational repression. We determined role of the heat-inducible miR398 in plant heat stress tolerance. Our results suggest that plants use a previously unrecognized strategy to achieve thermotolerance, especially for the protection of reproductive tissues. This strategy involves the down-regulation of two copper/zinc superoxide dismutase (CSDs) and their copper chaperone CCS through the heat-inducible miR398. MOLECULAR MECHANISMS OF PLANT RESPONSES TO COLD, HEAT AND SALT STRESSES IN ARABIDOPSIS By Qingmei Guan Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2013 Advisory Committee: Professor. Caren Chang Associate Prof. Gary D. Coleman Associate Prof. June M. Kwak, Dean’s Rep. Associate Prof. Shunyuan Xiao Assistant Prof. Jianhua Zhu, Chair © Copyright by Qingmei Guan 2013 Preface This dissertation is composed of an overview, four chapters, a conclusion and 19 appendices. Each chapter is structured in manuscript format with an abstract, introduction, results, discussion, and materials and methods. As such, descriptions of some methods are repeated. Ideas and facts presented in introduction sections may be expressed in a similar manner for chapter 2 and chapter 3. Supplemental materials are presented in the corresponding appendices and indicated with syntax for each appendix (i.e. supplemental materials in chapter 1 are designated as Appendix A). A comprehensive bibliography is located at the end of document. ii Dedication This work was dedicated to my parents because of all the great things they have been doing for me; this work was dedicated to my closest friend and husband, Xinke Xue, for his entire understanding, tolerance and support; this work was dedicated to my lovely little girl, Jiner, who is the most important person for me in the world. iii Acknowledgements I would like to thank my supervisor Dr. Jianhua Zhu, for his great and continued support. Without him, this work would never have been done. His thoughtful advice, encouragement, and knowledgeable ideas guided me to be a plant scientist whom I am today. I would also appreciate for his help in improving my writing skills. I would like to thank my committee members for their great suggestions and encouragement. They always have good ideas and they are a great source for critical thinking. I am very grateful to Chinese Scholarship Council (CSC) which partially provided my stipend. Without this scholarship, I would not have such a chance to study at UMD. I want to express my thanks to the following co-authors, Jianmin Wu, Xiaoyan Lu, Changlong Wen, Renyi Liu, Chenglin Chai, Haitao Zeng, Changhua Jiang, Yanyan Zhang, Wenbo Li and Zhenyu Wang for their contribution to this work. I also want to thank my lab mates who helped me: Lixin Li, Ozlem Cekic, Xiule Yue, Xiaohui Hu, Rongrong Wang, and Zhengpei Yao. I would like to express my special thanks to Dr. Gary Coleman, Dr. Heven Sze, Dr. Anne Simon and Dr. Priscila Chaverri and their lab members as we used their lab equipment. iv Table of Contents Preface ........................................................................................................................... ii Dedication .................................................................................................................... iii Acknowledgements ...................................................................................................... iv Table of Contents .......................................................................................................... v List of Figures ............................................................................................................ viii List of Abbreviations.................................................................................................... x Overview ....................................................................................................................... 1 Chapter 1: A DEAD Box RNA Helicase Is Critical for Pre-mRNA Splicing, Cold- Responsive Gene Regulation, and Cold Tolerance in Arabidopsis .............................. 4 Abstract ..................................................................................................................... 4 Introduction ............................................................................................................... 5 Results ....................................................................................................................... 8 Isolation of the rcf1-1 Mutant…………………………………………………... 8 RCF1 Is Required for Plant Tolerance to Chilling and Freezing Stresses............ 9 RCF1 Controls Gene Expression under Cold Stress…………............................11 RCF1 Encodes a Cold-Inducible DEAD Box RNA Helicase.............................16 RCF1 Is Not Involved in mRNA Export.............................................................19 RCF1 Is Required for Proper Splicing of Pre-mRNAs for Cold-Responsive Genes Including Positive and Negative Regulators of CBFs and for Cold Tolerance..............................................................................................................22 Overexpression of RCF1 in Arabidopsis Increases Tolerance to Chilling and Freezing Stresses..................................................................................................28 Discussion.…...........................................................................................................31 Methods................................................................................................................... 39 Plant Materials and Growth Conditions…...........................................................39 Chilling- and Freezing-Tolerance Assays............................................................40 Genetic Mapping and Complementation ............................................................ 41 RCF1 Subcellular Localization and Overexpression of RCF1, PRR5, AtSK12, CIR1, and SPFH..................................................................................................42 Microarray Analysis, Tilling Array Analysis, and Real-Time RT-PCR Analysis .............................................................................................................................43 Northern Hybridization Analysis........................................................................45 ATPase Activity Assay.......................................................................................46 Poly(A) RNA in situ Hybridization Assay..........................................................47 Chapter 2: A KH Domain-Containing Putative RNA-Binding Protein Is Critical for Heat Stress-Responsive Gene Regulation and Thermotolerance in Arabidopsis ....... 49 Abstract ................................................................................................................... 49 Introduction ............................................................................................................. 50 Results ..................................................................................................................... 52 Identification of the rcf3-1 Mutant.......................................................................52 RCF3 Encodes a KH Domain-Containing Putative RNA-Binding Protein.........52 v The rcf3 Mutations Do not Alter Expression of Endogenous CBF2 and CBF3, and the rcf3-1 Mutant Plants Are Tolerant to HS................................................55 The rcf3-1 Mutation Affects Expression of HSFs under HS...............................56 The rcf3-1 Mutation Affects Expression of DREB2s under HS..........................56 RCF3 Negatively Regulates Expression of HSPs under HS...............................58 Effect of rcf3-1 Mutation on Expression of CBK3, CaM3, and HSBP................58 The rcf3-2 Mutation Affects Expression of HS-Responsive Genes and RCF3 Restores Effects of rcf3-1 Mutation on Expression of HSFA2 and HSP17.6 ..........................................................................................................................60 Overexpression of RCF3 Leads to Down-Regulation of HS-Responsive Gene ...........................................................................................................................60 Discussion ............................................................................................................. ..60 Methods................................................................................................................. ..66 Plant Materials and Growth Conditions...............................................................66 Thermotolerance Assays......................................................................................67 Genetic Mapping and Complementation ........................................................... .67 Subcellular Localization of RCF3.......................................................................68 Real-Time RT-PCR Analysis..............................................................................68 Chapter 3: Heat Stress Induction of MiR398 Triggers a Regulatory Loop That Is Critical for Thermotolerance in Arabidopsis...............................................................71 Abstract ................................................................................................................... 71 Introduction ............................................................................................................. 72 Results ..................................................................................................................... 75 miR398 Is Induced by Heat Sress........................................................................75 Loss-of-Function Mutants csd1, csd2, and ccs Show Enhanced Heat-Responsive Gene Expression and Are more Heat-Tolerant...................................................80 ROS Accumulation in CSD1, CSD2, and CCS Transgenic Plants and Loss-of- Function csd1, csd2, and ccs Mutant Plants.... ...................................................85 HSFs Are Responsive to Oxidative Stress...........................................................88 HSFA1b and HSFA7b Bind Directly to the Promoter Regions of miR398b.......89 Discussion ............................................................................................................... 89 Methods.................................................................................................................. .96 Plant Materials and Growth Conditions .............................................................. 96 Generation of miR398b:GUS and miR398c:GUS constructs...............................97 Generation of CSD1:CSD1, CSD1:mCSD1, CSD2:CSD2, CSD2:mCSD2, CCS :CCS, and CCS:mCCS Constructs.......................................................................97 Real-Time RT-PCR Analysis..............................................................................98 Determination of Reactive Oxygen Species (ROS) Levels.................................99 Chromatin Immunoprecipitation (ChIP) Assays.................................................99 Small RNA Northern Hybridization Analysis .................................................. 100 Chapter 4: A Bi-Functional Xyloglucan Galactosyltransferase Is an Indispensable Salt Stress Tolerance Determinant in Arabidopsis............................................................102 Abstract ................................................................................................................. 102 Introduction ........................................................................................................... 103 Results ................................................................................................................... 106 Isolation of the rsa3-1 Mutant...........................................................................106 vi Accumulation of ROS in rsa3-1........................................................................108 Molecular Cloning of RSA3...............................................................................108 RSA3 Controls Gene Expression under Salt Stress............................................112 Actin Microfilaments Organization Is Disrupted in rsa3-1 under Salt Stress...114 Discussion ............................................................................................................. 116 Methods................................................................................................................. 119 Plant Material and Growth Conditions..............................................................119 Quantification of ROS.......................................................................................121 Positional Cloning of RSA3 and Gene Complementation of rsa3-1.. ...............121 RSA3::GUS Construct and GUS Assay..................................... .......................122 Microarray Analysis and Real-Time RT-PCR Analysis....................................122 Visualization of Actin Microfilaments..............................................................124 Chapter 5: Conclusions and Perspectives ................................................................. 126 Appendices ................................................................................................................ 129 Bibliography ............................................................................................................. 162 vii