GENETIC AND MOLECULAR DISSECTION OF DELLA RELATED GA-SIGNALING PATHWAY IN REGULATING ARABIDOPSIS SEED GERMINATION CAO DONGNI NATIONAL UNIVERSITY OF SINGAPORE 2007 GENETIC AND MOLECULAR DISSECTION OF DELLA RELATED GA-SIGNALING PATHWAY IN REGULATING ARABIDOPSIS SEED GERMINATION CAO DONGNI (B.SC.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSITUTE OF MOLECULAR AND CELL BIOLOGY DEPARTMENT OF BIOLOGICAL SCIENCE NATIONAL UNIVERSITY OF SINGAPORE 2007 Acknowledgements I gratefully acknowledge the Institute of Molecular and Cell Biology (afficiated to Agency of Science, Technology and Research) for their generous financial support I would like to express my deepest gratitude to my supervisor, Associate Professor Peng Jinrong for his invaluable advice, guidance, encouragement and inspiration through out this project I sincerely thank my committee members Associate Professor Yang Xiaohang, Dr He Yuehui, Professor Xie Daoxin and Professor Yang Weicai for their comments and suggestions during my research I would like to extend my special thanks to Professor Nicolas N Harberd for his invaluable suggestions in my research My sincere thanks to all the members of the Functional Genomics Laboratory; Dr Lee Sor Cheng, Ms Cheng Hui, Dr Alamgir Hussain, Dr Cheng Wei, Ms Soo Hui Meng, Dr Chen Jun, Ms Lo Jane, Ms Ruan Hua, Mr Huang Honghui, Dr Guo Lin, Mr Zhang Zhenhai, Mr Wu Wei, Mr Chang Changqing, Dr Yang Shulan, Miss Peiying, Ms Evelyn Ng, Mr Wen Chaoming and Mr Gao Chuan for creating a joyful and conducive working environment and rendering me encouragement, help, discussion and advices I would like to thank my friends who are also doing research in different areas, Shi Yaya, Tang Manli and Ji Liping, for their accompany, encouragement and inspiration during our PhD studies I would like to thank my family members My parents, my sister and brother have given me support and understanding I would like to express my special thanks to my husband Xu Jin, for his love, patience and unconditional support i Table of contents Page Acknowledgements i Table of contents ii Summary x List of abbreviations xiii List of tables xiv List of figures xv List of publications xvi Chapter Introduction 1.1The importance of GA in regulating plant growth and development 1 1.2 DELLA genes encode a group of negative regulators in GA-signaling Pathway 1.3 The important role of RGL2 in regulating seed germination 1.4 Regulation of seed germination by both internal and external regulators 1.5 GA promotion of plant development via targeting the negative regulators DELLAs to degradation via the ubiquitin-proteasome pathway 1.6 Aims, values and scope of this work 10 Chapter Literature review Overview of GA and plant development 12 12 ii 2.1 GA biosynthesis 12 2.1.1 Different forms and metabolism of GA in planta 12 2.1.2 GA biosynthesis in different developmental stages 13 2.1.3 Regulation of GA biosynthesis and catabolism by environmental cues 14 2.1.4 Regulation of GA biosynthesis by other hormone 15 2.1.5 Feedback and feedforward regulation of GA homeostasis 16 2.2 GA signaling components 17 2.2.1 GA-response mutants 17 2.2.2 Positive regulators of GA signaling 17 2.2.2.1 Soluble GA receptor GID1 17 2.2.2.2 F-box proteins essential for GA signaling pathway 18 2.2.2.3 GTP-binding proteins 18 2.2.2.4 PICKLE (PKL) 19 2.2.2.5 PHOTOPERIOD RESPONSIVE (PHOR1) 19 2.2.2.6 MYB transcription factors 20 2.2.3 Negative regulators of GA signaling 21 2.2.3.1 DELLA genes 2.2.3.1.1 Two categories of mutations of GAI gene 21 21 2.2.3.1.2 GAI and RGA together control stem elongation22 2.2.3.1.3 RGL2 is a key factor in GA-regulated seed germination 23 iii 2.2.3.1.4 GA regulates floral development by suppressing the function of RGL1, RGL2 and RGA 23 2.2.3.1.5 “Green Revolution” genes 24 2.2.3.2 SPINDLY (SPY) 24 2.2.3.3 SHORT INTERNODES (SHI) 25 2.2.4 Inhibitor of an inhibitor: GA promote plant development by targeting DELLA to degradation via the ubiquitin-proteasome pathway 2.2.4.1 Structure of DELLA proteins and their degradation 26 26 2.2.4.2 Degradation of DELLA proteins via the ubiquitinproteasome pathway 28 2.2.4.3 Posttranslational modification and degradation 28 2.3 GA signaling and seed germination 2.3.1 Seed germination and dormancy 31 31 2.3.1.1 Dormancy 31 2.3.1.2 Seed germination 31 2.3.1.3 Factors affect seed germination and dormancy 32 2.3.2 GA signaling pathway plays a key role in regulating seed germination in Arabidopsis Chapter General materials and methods 32 37 3.1 Plant materials and growth conditions 37 3.2 Genotyping of mutant lines 37 3.3 Germination assays 38 3.4 Scanning electron microscope (SEM) 38 iv 3.5 Chemical solutions and growth media 41 3.6 General methods for gene cloning 41 3.6.1 Polymerase chain reaction (PCR) 41 3.6.2 Purification of DNA from agarose gel 42 3.6.3 Isolation of plasmid DNA from E.coli 42 3.6.4 Ligation of DNA fragments into plasmid vectors 42 3.6.5 Transformation of bacteria with plasmids 43 3.6.5.1 Preparation of E.coli competent cells for heat-shock transformation 43 3.6.5.2 Transformation of E.Coli cells using heat-shock method 43 3.7 DNA sequencing 44 3.8 Plant genomic DNA extraction 44 3.9 Plant total RNA extraction 44 3.9.1 RNA extraction from seeds 44 3.9.2 RNA extraction from other tissue types 45 3.9.3 RNA extraction from BY2 cells 45 3.8.4 Removal of genomic DNA 45 3.10 Isolation of mRNA from total RNA 46 3.11 Microarray analysis 46 3.11.1 First-cycle, first-strand cDNA synthesis 46 3.11.2 First-cycle, second-strand cDNA synthesis 47 3.11.3 First-cycle, IVT amplification of cRNA 47 v 3.11.4 First-cycle, cleanup of cRNA 47 3.11.5 Second-cycle, first-strand cDNA synthesis 48 3.11.6 Second-cycle, second-strand cDNA synthesis and purification 48 3.11.7 Synthesis, purification and quantification of biotin-labeled cRNA for two-cycle target labeling assays 49 3.11.8 Fragmenting the cRNA for target preparation 49 3.11.9 Target hybridization, washing, staining, and scanning 49 3.12 Ontology analysis and cross-comparing DELLA-dependent transcriptomes 50 3.13 Probe labeling 51 3.13.1 DNA probes 51 3.13.1.1 PCR amplification 51 3.13.1.2 Probe purification 52 3.13.1.3 Concentration estimation 52 3.13.2 RNA probes 53 3.12.2.1 Template preparation 53 3.13.2.2 In vitro transcription 53 3.14 RT-PCR 54 3.14.1 First strand cDNA synthesis 54 3.14.2 RT-PCR analysis of DELLA transcripts in imbibed seeds 55 3.14.3 RT-PCR confirmation of candidate genes identified from microarray analysis 55 vi 3.14.4 RT-PCR to assay tissue specific expression of candidate DELLAregulated genes 56 3.14.5 RT-PCR to assay expression of GA 20-oxidase in Arabidopsis leaves 56 3.14.6 RT-PCR to assay expression of GA 20-oxidase in BY2 cells 57 3.15 Virtual northern 57 3.15.1 DNA gel electrophoresis 58 3.15.2 Transfer of DNA from gel to membrane 58 3.15.3 Hybridization 58 3.15.4 Antibody hybridization and detection 58 3.16 Northern blot hybridization 59 3.16.1 Preparation of formaldehyde-denatured RNA gel 59 3.16.2 Sample preparation and electrophoresis 59 3.16.3 RNA transfer from gel to nylon membrane 59 3.16.4 Hybridization 59 Chapter Genetic study of the roles of four DELLA proteins in light- and GAregulated seed germination 79 4.1 Introduction 79 4.2 Results 80 4.2.1 RGL2 is the predominant repressor of seed germination in the light 80 4.2.2 GAI, RGA and RGL1 enhance the function of RGL2 to repress seed germination 81 vii 4.2.3 RGL2 functions with RGA and GAI to repress seed germination in the dark 84 4.2.4 Combination of loss-of-function of GAI, RGA, RGL1 and RGL2 leads to both light- and GA-independent seed germination 90 4.2.5 Combination of loss-of-function of GAI, RGA, RGL1 and RGL2 results in embryos with elongated epidermal cells 95 4.3 Discussion 98 Chapter Identification of DELLA-dependent transcriptomes involved in seed 103 germination 5.1 Introduction 103 5.2 Results 105 5.2.1 Identification of DELLA-dependent transcriptomes for seed germination 105 5.2.2 Ontology analysis of DELLA-dependent transcriptomes for seed germination 109 5.2.3 DELLAs regulate distinct transcriptomes to control seed germination and floral development 111 5.2.4 Novel GAMYB genes and other transcription factors 115 5.2.5 DELLAs maintain the low metabolic activity in ga1-3 mutant 117 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genes encode a group of negative regulators in GA- signaling Pathway 1.3 The important role of RGL2 in regulating seed germination 1.4 Regulation of seed germination by both internal and. .. RGA and GAI to repress seed germination in the dark 84 4.2.4 Combination of loss -of- function of GAI, RGA, RGL1 and RGL2 leads to both light- and GA- independent seed germination 90 4.2.5 Combination... To investigate whether DELLA genes are involved in the flowing of the light signal to GA and to seed germination and to evaluate the roles of each DELLA protein in regulation of seed germination,