Genetic and molecular dissection of DELLA related GA signaling pathway in regulating arabidopsis seed germination

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 5.2.6 Distinct approaches are utilized to control cell growth and cell wall modification during seed germination and floral development 118 5.2.7 DELLAs act as convergence point for phytohormone signaling 118 5.2.8 DELLA-independent or -partially-dependent GA-regulated genes123 viii Lang, GA (1996) Plant Dormancy (Oxford, UK: CAB International) Lange T (1998) Molecular biology of gibberellin synthesis Planta 204: 409–419 Lee SC, Cheng H, King KE, Wang W, He Y, Hussain A, Lo J, Harberd NP, Peng JR (2002) Gibberellin regulates Arabidopsis seed germination via RGL2, a GAI/RGA-like gene whose expression is up-regulated following imbibition Genes Dev 16: 646-658 LeClere S, Tellez R, Rampey RA, Matsuda SP, Bartel B (2002) Characterization of a family of IAA-amino acid conjugate hydrolases from Arabidopsis J Biol Chem 277: 20446–20452 Liscum E, Reed JW (2002) Genetics of Aux/IAA and ARF action in plant growth and development Plant Mol Biol 49: 387–400 Lee MM, Schiefelbein J (1999) WEREWOLF, a MYB-related protein in Arabidopsis, is a position-dependent regulator of epidermal cell patterning Cell 24: 473–483 Lee MM, Schiefelbein J (2001) Developmentally distinct MYB genes encode functionally equivalent proteins in Arabidopsis Development 128: 1539–1546 Lorenzo O, Chico JM, Sanchez-Serrano JJ, and Solano R (2004) JASMONATEINSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated defense responses in Arabidopsis Plant Cell 16: 1938– 1950 176 Luschnig C, Gaxiola RA, Grisafi P, Fink GR (1998) EIR1, a root-specific protein involved in auxin transport, is required for gravitropism in Arabidopsis thaliana Genes Dev 12: 2175–2187 Martin DN, Proebsting WM, Parks TD, Dougherty WG, Lange T, Lewis MJ, Gaskin P, and Hedden P (1996) Feed-back regulation of gibberellin biosynthesis and gene expression in Pisum sativum L Planta 200: 159–166 Martin DN, Proebsting WM, Hedden P (1997) Mendel's dwarfing gene: cDNAs from the Le alleles and the function of the expressed proteins Proc Natl Acad Sci USA 94: 8907-8911 McCarty DR (1995) Genetic control and integration of maturation and germination pathways in seed development Annu Rev Plant Physiol Plant Mol Biol 46: 71-93 McGinnis KM, Thomas SG, Soule JD, Strader LC, Zale JM, Sun TP, and Steber CM (2003) The Arabidopsis SLEEPY1 gene encodes a putative F-box subunit of an SCF E3 ubiquitin ligase Plant Cell 15:1120–1130 McGrath KC, Dombrecht B, Manners JM, Schenk PM, Edgar CI, Maclean DJ, Scheible WR, Udvardi MK, Kazan K (2005) Repressor- and activator-type ethylene response factors functioning in jasmonate signaling and disease resistance identified via a genome- 177 wide screen of Arabidopsis transcription factor gene expression Plant Physiol 139: 949– 959 Millar A, Gubler F (2005) The Arabidopsis GAMYB-like genes, MYB33 and MYB65, are microRNA-regulated genes that redundantly facilitate anther development Plant Cell 17: 705–721 Mitsunaga S, Tashiro T, Yamaguchi J (1994) Identification and characterization of gibberellin-insensitive mutants selected from among dwarf mutants of rice Theor Appl Genet 87:705–12 Monte E, Amador V, Russo E, Martı´ nez-Garcı´ a J, Prat S (2003) PHOR1: a U-box GA signaling component with a role in proteasome degradation? J Plant Growth Regul 22: 152–162 Nakajima M, Nakajima M, Shimada A, Takashi Y, Kim YC, Park SH, Ueguchi-Tanaka M, Suzuki H, Katoh E, Iuchi S, Kobayashi M, Maeda T, Matsuoka M, Yamaguchi I (2006) Identification and characterization of Arabidopsis gibberellin receptors Plant J 46: 880–889 Nambara E, Akazawa T, McCourt P (1991) Effects of the Gibberellin Biosynthetic Inhibitor Uniconazol on Mutants of Arabidopsis Plant Physiol 97:736-738 178 Neuteboom LW, Ng JM, KuyperM, Clijdesdale OR, Hooykaas PJ, van der Zaal BJ (1999) Isolation and characterization of cDNA clones corresponding with mRNAs that accumulate during auxin-induced lateral root formation Plant Mol Biol 39: 273–287 Ogas J, Cheng JC, Sung ZR, and Somerville C (1997) Cellular differentiation regulated by gibberellin in the Arabidopsis thaliana pickle mutant Science 277: 91–94 Ogas J, Kaufmann S, Henderson J, Somerville C (1999) PICKLE is a CHD3 chromatinremodeling factor that regulates the transition from embryonic to vegetative development in Arabidopsis Proc Natl Acad Sci USA 96: 13839–13844 Ogawa M, Kusano T, Katsumi M, Sano H (2000) Rice gibberellin-insensitive gene homolog, OsGAI, encodes a nuclearlocalized protein capable of gene activation at transcriptional level Gene 245: 21–29 Ogawa M, Hanada A, Yamauchi Y, Kuwahara A, Kamiya Y, Yamaguchi S (2003) Gibberellin biosynthesis and response during Arabidopsis seed germination Plant Cell 15: 1591–1604 Oh E, Kim J, Park E, Kim JI, Kang C, Choi G (2004) PIL5, a phytochrome-interacting basic helix-loop-helix protein, is a key negative regulator of seed germination in Arabidopsis thaliana Plant Cell 16:3045–3058 179 Oh E, Yamaguchi S, Hu J, Yusuke J, Jung B, Paik I, Lee HS, Sun TP, Kamiya Y, Choi G (2007) PIL5, a phytochrome-interacting bHLH protein, regulates gibberellin responsiveness by binding directly to the GAI and RGA promoters in Arabidopsis seeds Plant Cell 19(4):1192-208 Ohta M, Matsui K, Hiratsu K, Shinshi H, Ohme-Takagi M (2001) Repression domains of class II ERF transcriptional repressors share an essential motif for active repression Plant Cell 13: 1959–1968 Olszewski N, Sun TP, Gubler F (2002) Gibberellin signaling: Biosynthesis, catabolism, and response pathways Plant Cell 14: S61–S80 Parinov S, Sevugan M, Ye D, Yang WC, Kumaran M, Sundaresan V (1999) Analysis of flanking sequences from dissociation insertion lines: A database for reverse genetics in Arabidopsis Plant Cell 11: 2263–2270 Peng JR, Carol P, Richards DE, King KE, Cowling RJ, Murphy GP, Harberd NP (1997) The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses Genes Dev 11: 3194-3205 Peng JR, Richards DE, Hartley NM, Murphy GP, Devos KM, Flintham JE, Beales J, Fish LJ, Worland AJ, Pelica F et al (1999a) “Green Revolution” genes encode mutant gibberellin response modulators Nature 400: 256-261 180 Peng J, Richards DE, Moritz T, Cano-Delgado A, Harberd NP (1999b) Extragenic suppressors of the Arabidopsis gai mutation alter the dose-response relationship of diverse gibberellin responses Plant Physiol 119: 1199–1207 Peng JR, Harberd NP (2002) The role of GA-mediated signalling in the control of seed germination Curr Opin Plant Biol 5: 376–381 Penfield S, Josse EM, Kannangara R, Gilday AD, Halliday KJ, Graham IA (2005) Cold and light control seed germination through the bHLH transcription factor SPATULA Curr Biol 15: 1998–2006 Perazza D, Vachon G, Herzog M (1998) Gibberellins promote trichome formation by upregulating GLABROUS1 in Arabidopsis Plant Physiol 117: 375–383 Petit JM, Briat JF, Lobreaux S (2001) Structure and differential expression of the four members of the Arabidopsis thaliana ferritin gene family Biochem J 359: 575–582 Phillips AL, Ward DA, Uknes S, Appleford NEJ, Lange T, Huttly AK, Gaskin P, Graebe JE, Hedden P (1995) Isolation and expression of three gibberellin-20-oxidase cDNA clones from Arabidopsis Plant Physiol.108: 1049-1057 181 Pysh LD, Wysocka-Diller JW, Camilleri C, Bouchez D, Benfey PN (1999) The GRAS family in Arabidopsis: sequence characterization and basic expression analysis of the SCARECROW-LIKE genes Plant J 18: 111-119 Raynal M, Guilleminot J, Gueguen C, Cooke R, Delseny M, Gruber V (1999) Structure, organization and expression of two closely related novel Lea (late-embryogenesisabundant) genes in Arabidopsis thaliana Plant Mol Biol 40: 153–165 Richards DE, Peng JR, Harberd NP (2000) Plant GRAS and metazoan STATs: one family? Bioassays 22: 573-577 Richards DE, King KE, Ait-ali T, Harberd NP (2001) How gibberellin regulates plant growth and development: a molecular genetic analysis of gibberellin signaling Ann Rev Plant Physiol Pl Mol Biol 52: 67-88 Riechmann JL, Heard J, Martin G, Reuber L, Jiang C, Keddie J, Adam L, Pineda O, Ratcliffe OJ, Samaha RR, et al (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes Science 290: 2105–2110 Rock CD, Zeevaart JA (1991) The aba mutant of Arabidopsis thaliana is impaired in epoxy-carotenoid biosynthesis Proc Natl Acad Sci USA 88:7496–7499 182 Roos MD, Hanover JA (2000) Structure of O-linked GlcNAc transferase: Mediator of glycan-dependent signaling Biochem Biophys Res Commun 271: 275–280 Ross JJ, Mackenzie-Hose AK, Davies PJ, Lester DR, Twitchin B, Reid JB (1999) Further evidence for feedback regulation of gibberellin biosynthesis in pea Physiol Plant 105: 532-538 Ross JJ, O'Neill DP, Smith JJ, Kerckhoffs LHJ, Elliott RC (2000) Evidence that auxin promotes gibberellin A1 biosynthesis in pea Plant J 21: 547–552 Sasaki A, Itoh H, Gomi K, Ueguchi-Tanaka M, Ishiyama K, Kobayashi M, Jeong DH, An G, Kitano H, Ashikari M, and Matsuoka M (2003) Accumulation of phosphorylated repressor for gibberellin signaling in an F-box mutant Science 299: 1896–1898 Schiefelbein J (2003) Cell-fate specification in the epidermis: a common patterning mechanism in the root and shoot Curr Opin Plant Biol 6:74-78 Shi L, Olszewski NE (1998) Gibberellin and abscisic acid regulate GAST1 expression at the level of transcription Plant Mol Biol 38: 1053–1060 183 Shinomura T, Nagatani A, Chory J, Furuya M (1994)The Induction of Seed Germination in Arabidopsis thaliana Is Regulated Principally by Phytochrome B and Secondarily by Phytochrome A Plant Physiol 104: 363-371 Shinomura T, Nagatani A, Hanzawa H, Kubota M, Watanabe M, Furuya M (1996) Action spectra for phytochrome A- and B-specific photoinduction of seed germination in Arabidopsis thaliana Proc Natl Acad Sci USA 93:8129–8133 Silverstone AL, Chang CW, Krol E, Sun TP (1997) Developmental regulation of the gibberellin biosynthetic gene GA1 in Arabidopsis thaliana Plant J 12: 9–19 Silverstone AL, Ciampaglio CN, Sun TP (1998) The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway Plant Cell 10: 155-169 Silverstone AL, Jung HS, Dill A, Kawaide H, Kamiya Y, and Sun TP (2001) Repressing a repressor: Gibberellin-induced rapid reduction of the RGA protein in Arabidopsis Plant Cell 13: 1555–1565 Staswick PE, Serban B, Rowe M, Tiryaki I, Maldonado MT, Maldonado MC, Suza W (2005) Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid Plant Cell 17: 616–627 184 Steber CM, Cooney SE, McCourt P (1998) Isolation of the GA-response mutant sly1 as a suppressor of ABI1-1 in Arabidopsis thaliana Genetics 149:509-521 Stracke R, Werber M, Weisshaar B (2001) The R2R3-MYB gene family in Arabidopsis thaliana Curr Opin Plant Biol 4: 447-456 Sun TP, Kamiya Y (1994) The Arabidopsis GAl locus encodes the cyclase ent-kaurene synthetase A of gibberellin biosynthesis Plant Cell 6:1509-1518 Sun TP and Gubler F (2004) Molecular mechanism of gibberellin signaling in plants Annu Rev Plant Biol 55: 197–223 Sundaresan V, Springer P, Volpe T, Haward S, Jones JDG, Dean C, Ma H, Martienssen R (1995) Patterns of gene action in plant development revealed by enhancer trap and gene trap transposable elements Genes Dev 9: 1797-1810 Swain SM, Tseng TS, Olszewski NE (2001) Altered expression of SPINDLY affects gibberellin response and plant development Plant Physiol 126: 1174–1185 Talon M, Koornneef M, Zeevaart JAD (1990) Endogenous gibberellins in Arabidopsis thaliana and possible steps blocked in the biosynthetic pathways of the semidwarf ga4 and ga5 mutants Proc Natl Acad Sci USA 87: 7983-7987 185 Telfer A, Bollman KM, Poethig RS (1997) Phase change and the regulation of trichome distribution in Arabidopsis thaliana Development 124: 645–654 Thomas SG, Phillips AL, Hedden P (1999) Molecular cloning and functional expression of gibberellin 2-oxidases, multifunctional enzymes involved in gibberellin deactivation Proc Natl Acad Sci USA 96: 4698–4703 Thomas SG and Sun TP (2004) Update of gibberellin signaling: a tale of the tall and the short Plant Physiol 135: 668–676 Thornton TM, Swain SM, Olszewski NE (1999) Gibberellin signal transduction presents the SPY who O-GlcNAc’d me Trends Plant Sci 4: 424–428 Tian C, Wan P, Sun S, Li J, Chen M (2004) Genome-wide analysis of the GRAS gene family in rice and Arabidopsis Plant Mol Biol 54: 519–532 Tseng TS, Swain SM, Olszewski NE (2001) Ectopic expression of the tetratricopeptide repeat domain of SPINDLY causes defects in gibberellin response Plant Physiol 126: 1250–1258 Tyler LS, Thomas SG, Hu JH, Dill A, Alonso JM, Ecker JR, Sun T-P (2004) DELLA proteins and gibberellin-regulated seed germination and floral development in Arabidopsis Plant Physiol 135: 1008-1019 186 Ueguchi-Tanaka M, Fujisawa Y, Kobayashi M, Ashikari M, Iwasaki Y, Kitano H, Matsuoka M (2000) Rice dwarf mutant d1, which is defective in the α-subunit of the heterotrimeric G protein, affects gibberellin signal transduction Proc Natl Acad Sci USA 97: 11638–11643 Ueguchi-Tanaka M, Ashikari M, Nakajima M, Itoh H, Katoh E, Kobayashi M, Chow TY, Hsing YI, Kitano H, Yamaguchi I, Matsuoka M (2005) GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin Nature 437: 693–698 Ullah H, Chen JG, Young JC, Im KH, Sussman MR, Jones AM (2001) Modulation of cell proliferation by heterotrimeric G protein in Arabidopsis Science 292: 2066–2069 Uno Y, Furihata T, Abe H, Yoshida R, Shinozaki K, Yamaguchi-Shinozaki K (2000) Arabidopsis basic leucine zipper transcription factors involved in an abscisic aciddependent signal transduction pathway under drought and high-salinity conditions Proc Natl Acad Sci USA 97: 11632-11637 van Huizen R, Ozga JA, Reinecke DM (1997) Seed and hormonal regulation of gibberellin 20-oxidase expression in pea pericarp Plant Physiol.115:123–128 Vierstra RD (2003) The ubiquitin/26S proteasome pathway, the complex last chapter in the life of many plant proteins Trends Plant Sci 8:135–42 187 Wang JW, Wang LJ, Mao YB, Cai WJ, Xue HW, Chen XY (2005) Control of root cap formation by microRNA-targeted auxin response factors in Arabidopsis Plant Cell 17: 2204–2216 Wang XQ, Ullah H, Jones AM, Assmann SM (2001) G protein regulation of ion channels and abscisic acid signaling in Arabidopsis guard cells Science 292: 2070–2072 Willige BC, Ghosh S, Nill C, Zourelidou M, Dohmann EM, Maier A, Schwechheimer C (2007) The DELLA Domain of GA INSENSITIVE Mediates the Interaction with the GA INSENSITIVE DWARF1A Gibberellin Receptor of Arabidopsis Plant Cell 19:1209-20 Wen CK, Chang C (2002) Arabidopsis RGL1 Encodes a Negative Regulator of Gibberellin Responses Plant Cell 14: 87-100 Willert K, Nusse R (1998) Beta-catenin: A key mediator of Wnt signaling Curr Opin Genet Dev 8: 95–102 Wilson RN, Heckman JW, Sommerville CR 1992 Gibberellin is required for flowering in Arabidopsis thaliana under short days Plant Physiol 100: 403–8 Wilson RN, Somerville CR (1995) Phenotypic suppression of the gibberellin-insensitive mutant (gai) of Arabidopsis Plant Physiol 108: 495–502 188 Xu YL, Li L, Wu K, Peeters AJM, Gage DA, Zeevaart JAD (1995) The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression Proc Natl Acad Sci USA 92: 6640-6644 Xu YL, Li L, Gage DA, Zeevaart JAD (1999) Feedback regulation of GA5 expression and metabolic engineering of gibberellin levels in Arabidopsis Plant Cell 11: 927–935 Yadav V, Mallappa C, Gangappa SN, Bhatia S, Chattopadhyay S (2005) A basic helixloop-helix transcription factor in Arabidopsis, MYC2, acts as a repressor of blue lightmediated photomorphogenic growth.Plant Cell 17: 1953-66 Yamaguchi S, Smith MW, Brown RGS, Kamiya Y, Sun TP (1998) Phytochrome regulation and differential expression of gibberellin 3β-hydroxylase genes in germinating Arabidopsis seeds Plant Cell 10: 2115–2126 Yamaguchi S, Kamiya Y, Sun T (2001) Distinct cell-specific expression patterns of early and late gibberellin biosynthetic genes during Arabidopsis seed germination Plant J 28: 443-453 Yamauchi Y, Ogawa M, Kuwahara A, Hanada A, Kamiya Y, and Yamaguchi S (2004) Activation of gibberellin biosynthesis and response pathways by low temperature during imbibition of Arabidopsis thaliana seeds Plant Cell 16: 367–378 189 Yu H, Ito T, Zhao YX, Peng JR, Kumar P, Meyerowitz EM (2004) Floral homeotic genes are targets of gibberellin signaling in flower development Proc Natl Acad Sci USA 101: 7827-7832 Yuan M, Shaw PJ, Warn RM, Lloyd CW (1994) Dynamic reorientation of cortical microtubules, from transverse to longitudinal, in living plant cells Proc Natl Acad Sci USA 91: 6050–6053 190 ... 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. .. 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,