A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

ANALYSIS OF RGA-LIKE (RGL2), A GIBBERELLIC ACID RESPONSE REGULATOR, IN THE FLOWER DEVELOPMENT OF ARABIDOPSIS THALIANA TAN EE LING (B.Sc. HONS, NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS This stint in research has truly been an eye-opener. It has exposed me to various cultures through the relationships I have developed with people from different countries. It has also taught me patience and perseverance in honing my research skills, as well as developing thinking skills in a more systematic manner. Firstly, I would like to give my sincere and grateful thanks to my main supervisor, Associate Professor Prakash Kumar, for his guidance, advice and supervision throughout my years of research, ever since I started out research as a wee honors student in his Plant Morphogenesis lab. I also greatly appreciate the ideas and advice of my co-supervisor, Assistant Professor Yu Hao, who was always ready to listen and help in troubleshooting. I also thank him for imparting valuable techniques and skills relevant to my research. I am thankful to my thesis committee advisors, Associate Professors Sanjay Swarup and Loh Chiang Shiong, for their invaluable advice, encouragement and ideas. I would like to acknowledge the National University of Singapore for providing the research scholarship that made this research stint possible. I wish to acknowledge Associate Professor Peng Jinrong from the Institute of Molecular and Cell Biology for providing the starting materials for this research work. I would also like to thank A/P Peng and Professor Bertrand Seraphin for their kind permission in allowing me to reproduce some images from their earlier papers. I am deeply grateful for the technical help from the staff at the Electron Microscopy Unit. I thank Mdm Loy Gek Luan for her kindness and technical assistance in the SEM work. I also sincerely appreciate the patience and help from Mr Loh Mun Seng for teaching me how to perform and improve on sectioning ii techniques. I am also truly grateful to Mr Chong Ping Lee who never hesitated to help me and promptly responded to my email queries whenever I encountered technical problems with plant growth chambers. I would also like to give my thanks to Mrs Ang-Lim Swee Eng for helping me source temporary space to grow my plants and help in getting photography equipment. I thank Mr Yan Tie and Ms Kho Say Tin for assistance in photo imaging and mass spectrophotometry, respectively. I give my deepest appreciation to my lab mates who inculcated a warm lab environment that made carrying out research work in a lab, interesting, fun and stimulating. Firstly, I wish to say a big thank you to my lab colleagues, Lianlian and Jaclyn, who provided a fun and estrogen-loaded lab environment. I thank the both of them for constantly supplying and ensuring there was food present for me, which was crucial to satisfying my hunger pangs during lab work. I give my sincere thanks to Yifeng who helped me troubleshoot experiments and shared his techniques in research work. I thank Mandar for his useful discussions on protein experiments. I like to say a big thanks to Edwin for cerebral discussions during tea breaks. I am also deeply appreciative to Yuhfen, Serena, Carol, Weekee, Dileep, Liu Chang, Lailai, Wangyu and Sulee who have helped me in one task or another. I am indebted to my husband, Chico, for his wonderful improvised ideas and unwavering support in whatever I do. I am tremendously thankful for his help in some technical aspects of my work, and for being my constant pillar of strength. Lastly, I am truly grateful and immensely appreciative to my family for their constant support, love, encouragement (with a bit of nagging thrown in) and understanding in whatever I do, that made all things possible. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS . i TABLE OF CONTENTS . iv SUMMARY vii LIST OF TABLES . ix LIST OF FIGURES .x LIST OF ABBREVIATIONS xiii CHAPTER .1 GENERAL INTRODUCTION .1 CHAPTER .5 LITERATURE REVIEW .5 2.1 Introduction 2.2 Regulation of gibberellins in plants .6 2.2.1 The gibberellin biosynthesis pathway 2.2.2 The gibberellin signaling pathway and its regulatory proteins 10 2.3 A soluble receptor for gibberellin is finally characterized .14 2.4 The GRAS superfamily of putative transcription factors and DELLA proteins 15 2.5 RGL genes and their implications in plant development .16 2.6 Characterization of RGL genes 17 2.7 RGL2 and seed germination .20 2.8 DELLA proteins are important integrators of multiple phytohormone signaling pathways 22 2.9 The ubiquitin 26S proteasome pathway is involved in the degradation of DELLA proteins 24 2.10 The DELLA proteins ‘relief of restraint’ model 26 2.11 The postulated model on GA signaling pathway and potential interactions among the DELLA proteins 28 2.12 Various strategies to isolate potential downstream genes and interacting coeffectors of RGL2 31 2.12.1 The Glucocorticoid Receptor (GR) domain tag – an inducible gene expression system 31 2.12.2 The Tandem Affinity Purification tag (TAP™ tag) system for isolation of interacting factors 34 2.12.3 A rapid one-step protein purification system in plants using a StrepII tag .37 CHAPTER .40 ANALYSIS OF RGL2 EXPRESSION DURING FLORAL TRANSITION AND DIFFERENT STAGES OF FLOWER DEVELOPMENT IN ARABIDOPSIS .40 3.1 Introduction 40 3.2 Materials and methods .40 3.2.1 Plant materials and growth conditions .40 3.2.2 GUS histological analysis 41 iv 3.2.2.1 Tissue fixation and staining………………………………….41 3.2.2.2 Dehydration………………………………………………….41 3.2.2.3 Clearing………………………………………………………42 3.2.2.4 Infiltration……………………………………………………42 3.2.2.5 Embedding and sectioning………………………………… .42 3.2.2.6 Deparaffination and rehydration…………………………… 43 3.2.3 In situ hybridization .43 3.2.3.1 Probe preparation…………………………………………….43 3.2.3.2 Carbonate hydrolysis…………………………………………44 3.2.3.3 Fixation………………………………………………………45 3.2.3.4 Dehydration………………………………………………….45 3.2.3.5 In situ section pre-treatment…………………………………46 3.2.3.6 In situ hybridization………………………………………….46 3.2.3.7 Post hybridization washing and detection………………… .47 3.3 Results 48 3.3.1 GUS histological analysis of rgl2-5 floral tissues .48 3.3.2 In situ hybridization analyses of wild-type Arabidopsis floral tissues 48 3.4 Discussion 52 CHAPTER .57 CONSTITUTIVE AND CONDITIONAL OVEREXPRESSION OF RGL2 IN ARABIDOPSIS .57 4.1 Introduction 57 4.2 Materials and methods .57 4.2.1 Plant growth and conditions .57 4.2.2 Bacterial strains and plasmids 57 4.2.3 Plant genomic DNA extraction 58 4.2.4 Isolation of full-length RGL2 cDNA by PCR .61 4.2.5 Cloning of the full-length RGL2 cDNA .63 4.2.6 Preparation of E. coli competent cells for heat-shock transformation.64 4.2.7 Transformation of E. coli competent cells .66 4.2.8 Screening of clones containing the desired fragments .66 4.2.9 DNA sequencing and analysis .68 4.2.10 Manipulation of the modified pGreen vectors .69 4.2.11 Cloning full-length RGL2 gene into modified pGreen binary vector 70 4.2.12 Cloning full-length RGL2 gene into modified pGreen-GR binary vector 72 4.2.13 Preparation of Agrobacterium tumefaciens competent cells for transformation 72 4.2.14 Electroporation of Agrobacterium tumefaciens .74 4.2.15 Agrobacterium-mediated transformation of Arabidopsis by floral dip75 4.2.16 Quick DNA preparation using Shorty buffer .76 4.2.17 Genetic analysis and characterization of the transformants .77 4.2.18 Extraction of total RNA from Arabidopsis 77 4.2.19 First-strand cDNA synthesis for Reverse-Transcription Polymerase Chain Reaction (RT-PCR) .79 4.2.20 Expression analysis of transgenic overexpression lines 79 4.2.21 Scanning electron microscopy .80 4.2.22 Steroid hormone treatment of transgenic Arabidopsis plants 80 4.3 Results 81 v 4.3.1 4.3.2 4.3.3 4.3.4 Cloning full-length RGL2 into modified pGreen vectors 81 Genomic DNA screening of transgenic Arabidopsis plants 86 Phenotypic analyses of transgenic Arabidopsis plants 90 Analysis of floral tissues of transgenic lines using Scanning Electron Microscopy 93 4.3.5 Conditional analysis of steroid hormone treated transgenic plants .96 4.4 Discussion 98 CHAPTER .102 INVESTIGATION OF POTENTIAL RELATIONSHIPS AMONG RGL2, FLORAL ORGAN IDENTITY GENES AND DELLA GENE FAMILY MEMBERS .102 5.1 Introduction 102 5.2 Materials and methods .104 5.2.1 Plant materials and growth conditions .104 5.2.2 Agrobacterium-mediated plant transformation 104 5.2.3 Characterization of transgenic plants .104 5.2.4 Steroid hormone treatment of transgenic mutant plants 104 5.2.5 Expression analyses of different floral organ identity genes in the rgl2 mutant background .105 5.2.6 Cloning full-length RGL2 into pGreen-TAP binary vector .106 5.2.7 Cloning full-length RGL2 into pGreen-GR-TAP binary vector……106 5.2.8 Cloning full-length RGL2 into pGreen-StrepII binary vector 108 5.2.9 Characterization of transgenic plants carrying the fusion constructs 110 5.2.10 Extraction of proteins from transgenic lines 112 5.2.10.1 Purification via StrepII tag………………………………….112 5.2.10.2 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS- PAGE)……………………………………………….114 5.2.10.3 Silver staining of polyacrylamide gels…………………… .114 5.2.10.4 Western blot analysis……………………………………….117 5.3 Results 118 5.3.1 Characterization of ga1-3 rga rgl2 RGL2::GR transgenic plants 118 5.3.2 Effect of RGL2 on the floral organ identity genes analyzed using the steroid inducible system .126 5.3.3 Characterization of transgenic plants and effectiveness of various protein tagging approaches – TAP™ tag .130 5.3.4 Characterization of transgenic plants and effectiveness of another protein tagging approach – StrepII tag .137 5.4 Discussion 146 GENERAL CONCLUSIONS AND FUTURE PERSPECTIVES 156 REFERENCES 158 APPENDICES . I vi SUMMARY In this study, the role of a Gibberellic Acid (GA) regulator named RGA-Like (RGL2) in Arabidopsis thaliana flower development was analyzed using molecular genetics approaches. The GA signaling components and their interactions with one another, as well as crosstalk with other phytohormone signaling components, have only been discovered recently. The functions of a small group of transcriptional regulators found in GA signaling of Arabidopsis, called the DELLA proteins, named for their distinctive five amino acids domain, have been recently analyzed. The first two DELLA proteins, RGA (Repressor of GA1) and GAI (Gibberellic Acid Insensitive), were characterized as negative regulators of the GA signaling pathway. The remaining three DELLA proteins called RGL proteins were later found to have effects on seed germination (RGL1 and RGL2) with the exception of RGL3, which has not been characterized yet. So far, the GA signaling pathway involving these proteins and their interacting factors are not clear. This study focused on the potential role of RGL2 in floral development, as the combination of RGA and RGL2 has shown effects on floral development (Cheng et al., 2004; Yu et al., 2004). Using GUS staining and in situ hybridization techniques, the spatial expression of RGL2 in floral development was examined. It was found that RGL2 was expressed in floral primordia from stage to stage of floral development. Furthermore, RGL2 was also expressed in the placenta of the carpel, at the base of the stigma, and in the style, filaments and petals. A prior study had shown that RGL2 was a negative regulator of seed germination in loss-of-function experiments. By generating overexpression transgenic lines and transgenic lines possessing a fusion protein with a steroid hormone-inducible gene expression system, the function of RGL2 in flower development was further examined. Although overexpression of RGL2 in wild-type vii Arabidopsis did not produce substantial aberrations or major phenotypic changes in the floral tissues, its overexpression in ga1-3 rga rgl2 caused obvious floral defects, indicating that RGL2 is a negative regulator of flower development. Last but not least, the final objective was to identify potential interacting co-effectors of RGL2, as it was classified as a putative transcriptional regulator as other DELLA proteins. Using a controlled gene inducible expression system, the translocation of RGL2 into the nucleus led to potential downregulation of AP2, a Class A floral organ identity gene. Other floral organ identity genes were not substantially affected. Various protein tagging strategies were also employed to isolate possible co-effectors of RGL2, which could have modified RGL2 activity, and thus affected RGL2 regulation of target genes. After trial and error attempts on experimenting with the various tags, we found that the TAP™ tagging strategy was not feasible with RGL2, as it hindered its biological function. Instead, the StrepII tag was successfully utilized. Amino acid sequencing of proteins pulled-down with the tagged RGL2 revealed that a potential kinase or serine/threonine protein kinase could be an interacting factor with RGL2. This is a potential co-effector of RGL2 as RGL2 has a GA-response specific domain (DELLA domain), which has a poly serine/threonine string of residues in the Nterminal region. This region could be targeted by the putative kinase for phosphorylation. viii LIST OF TABLES Table 4.1. List of primers used in experiments mentioned in Chapters and 62 Table 4.2. Culture media, antibiotics and chemicals used for growing bacteria. 65 Table 5.1. Components used in SDS-PAGE .115 Table 5.2. Reagents and chemicals used for SDS-PAGE 116 Table 5.3. Preparation of SDS gel – loading buffer .116 Table 5.4. Reagents and chemicals used in Western blotting 119 Table 5.5. Changes in gene expression level of floral organ identity after mock and dexamethasone treatment on floral tissues of ga1-3 rga rgl2 RGL2::GR. 129 ix LIST OF FIGURES Figure 2.1. Model showing the homeostatic regulation of GA biosynthesis .8 Figure 2.2. Comparison in phenotype between an Arabidopsis wild-type plant (left panel, red arrow) and a ga1-3 GA biosynthesis mutant (right panel). Both plants were photographed weeks after germination. (Scale bars = cm) Figure 2.3. Amino acid sequence alignment of the predicted DELLA proteins .18 Figure 2.4. The DELLA subfamily of the GRAS regulatory proteins .19 Figure 2.5. The ‘relief of restraint’ model. ………………………………………… 27 Figure 2.6. Postulated model of GA signaling in Arabidopsis. .30 Figure 2.7. The mechanism of the steroid inducible activation system .33 Figure 2.8. Overview of the TAP™ tag purification strategy. 35 Figure 2.9. An overview of the StrepII tag affinity purification strategy. ………… 39 Figure 3.1. GUS histological analysis of rgl2-5 showing staining in connective tissue and stamens 49 Figure 3.2. GUS histological analysis of rgl2-5 showing stainings in petals and stigma .50 Figure 3.3. GUS histological analysis of rgl2-5 showing staining in the carpel. 51 Figure 3.4. In situ hybridization of wild-type Arabidopsis inflorescence apex .53 Figure 3.5. In situ hybridization of floral meristems from wild-type Arabidopsis 54 Figure 3.6. In situ hybridization on the cross sections of the wild-type floral buds (ecotype Landsberg erecta). .55 Figure 4.1. The binary vector pGreen-HY102 35S::GR::TAP. .59 Figure 4.2. The binary vector pGreen-HY103 35S::TAP 60 x * **** * ** ***** *** * ** ** ** ***** ** ** ** gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| AGAAGCTTGAACAACTTGAGATGGTCTTGTCTAATGAT---GATGTTGGT AGAAGCTTGAACAGCTTGAAATGGTCTTGTCCAATGAT---ATTGCCTCT ACAAGCTTGAACAGTTAGAGATGGTTCT------TGGT---GATGGAATC AGAAACTCGAGCAGCTTGAAGTTATGATGTCTAATGTTCAAGAAGACGAT TGAAACTCGAACAATTAGAGACGATGATGAGTAATGTTCAAGAAGATGGT ** ** ** ** * ** * * ** * * 364 397 327 380 446 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| TCTA---CTGTCTTAAACGACTCTGTTCATTATAACCCATCTGATCTCTC TCTAGTAATGCCTTCAATGACACCGTTCATTACAATCCTTCTGATCTCTC TCGA---ATCTTTCTGATGAAACTGTTCATTACAATCCTTCTGATCTCTC CTTTCTCAACTCGCTACTGAGACTGTTCACTATAATCCGGCGGAGCTTTA TTATCTCATCTCGCGACGGATACTGTTCATTATAATCCGTCGGAGCTTTA ** * ***** ** ** ** * ** ** * 411 447 374 430 496 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| TAACTGGGTCGAGAGCATGCTTTCTGAGCTGAACAACCCGGCTTCTTCGG CGGTTGGGCTCAGAGCATGCTCTCGGATCTTAATTAC------TACCCGG TGGTTGGGTCGAAAGCATGCTCTCGGATCTTGACCCG-------ACCCGG CACGTGGCTTGATTCTATGCTCACCGACCTTAATCCTCCGTCG------TTCTTGGCTTGATAATATGCTCTCTGAGCTTAATCCTCCTCCTCTTCCGG *** * ***** * ** ** * 461 491 417 473 546 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| ---ATCTTGACA----CGACCCGAAGTTGTGTGGATAG--------------ATCTTGACC----CGAACCGGATTTGCG---------------------AT-TCAAGA----AAAGCCTGAC----------------------------TCTAACG-------------------------------------CGAGTTCTAACGGTTTAGATCCGGTTCTTCCTTCGCCGGAGATTTGTGGT * 492 515 435 480 596 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| --------ATCCGAATACGATCTCAGAGCAATTCCGGGTCTTTCTGCGTT --------ATCTGAGACCAATCACAGA-CGACGATGAGTGTTGCAGT-----------TCAGAGTACGATCTTAGAGCTATTCCTGGCTCTGCAGTGTA ----------CCGAGTACGATCTTAAAGCTATTCCCGGTGACGCGATTCT TTTCCGGCTTCGGATTATGACCTTAAAGTCATTCCCGGAAACGCGATTTA * ** * * * * * * * 534 553 476 520 646 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| TC--CAAAGGAAGAGGAAGTCTTTGACGAGGAAG----CTAGCAGCA---------AGCAATAGTAA---------------------CAGCAACA--TC--CACGTGACGAGCACGTGACTCG-------------TCGGAGCA--CAATCAGTTCGCTATCGATTCGGCTTCTTCGTC---TAACCAAGGCG-GC TCAGTTTCCGGCGATTGATTCTTCGTCTTCGTCGAATAATCAGAACAAGC * * 575 572 508 566 696 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| -----------------------------------------------A-G -----------------------------------------------A-G -----------------------------------------------A-G GGAGGAGAT--------------------------ACGTATACT---ACA GTTTGAAATCATGCTCGAGTCCTGATTCTATGGTTACATCGACTTCGACG * 577 574 510 587 746 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| AGGATCCG----ACTCGGATCGT------------GGTGCGAATCGTCGG AGGATTCG----ACTCGGTCCTT------------GGTGTGACTCAGTGA AGGACGAG----AATTGAATC--------------GGAGTTATCC----AACAAGC-----GGTTGAAATGCTCAAACGGCGTCGTGGA-AACCACTAC GGTACGCAGATTGGTGGAGTCATAGGAACGACGGTGACGACAACCACCAC * * * * * * * 611 608 537 631 796 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| ---A--------------CGAGTCAACTCGGTCCGTGGTGCTCGTTGACT CCAG--------------CGAGTCAACTCGTTCCGTGGTGCTTATCGA-----------------------TCTACGCGCTCTGTGGTGGTTTTGGATT AGCGACGGC---------TGAGTCAACTCGGCATGTTGTCCTGGTTGACT GACAACGACGGCGGCGGGTGAGTCAACTCGTTCTGTTATCCTGGTTGACT ** ** ** ** * * * ** 644 642 565 672 846 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| CTCAGGAGACCGGAGTTAGACTTGTCCACGCACTAGTGGCGTGCGCTGAG ----GGAGACAGGAGTTAGACTCGTTCAGGCGCTAGTGGCCTGCGCCGAG CTCAAGAAACTGGAGTGCGTTTAGTCCACGCGCTATTAGCTTGTGCTGAA CGCAGGAGAACGGTGTGCGTCTCGTTCACGCGCTTTTGGCTTGCGCTGAA CGCAAGAGAACGGTGTTCGTTTAGTCCACGCGCTTATGGCTTGTGCAGAA ** * ** ** * * ** ** ** ** * ** ** ** ** 694 688 615 722 896 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| GCGATTCACCAGGAGAATCTCAACTTAGCTGACGCGCTGGTGAAACGCGT GCGGTTCAGCTGGAGAATCTGAGCCTCGCGGATGCTCTCGTCAAGCGCGT GCTGTTCAACAGAACAATTTGAAGTTAGCCGACGCGCTCGTGAAGCACGT GCTGTTCAGAAAGAGAATCTGACTGTAGCGGAAGCTCTGGTGAAGCAAAT GCAATCCAGCAGAACAATTTGACTCTAGCGGAAGCTCTTGTGAAGCAAAT ** * ** * *** * * * ** ** ** ** ** ** * * 744 738 665 772 946 gi|30678851|ref|NM_111216.2| GGGAACACTCGCGGGTTCTCAAGCTGGAGCTATGGGAAAAGTCGCTACGT 794 II gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| GGGATTACTCGCGGCTTCTCAAGCCGGAGCCATGGGGAAAGTCGCTACCT GGGGTTACTCGCGTCCTCTCAAGCTGGTGCTATGAGGAAAGTCGCGACTT CGGATTCTTAGCCGTTTCTCAAATCGGAGCGATGAGAAAAGTCGCTACTT CGGATGCTTAGCTGTGTCTCAAGCCGGAGCTATGAGAAAAGTGGCTACTT ** * ** ****** ** ** *** * ***** ** ** * 788 715 822 996 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| ATTTTGCTCAAGCCTTGGCTCGTCGTATTTACCGTGATTACACGGCGGAG ACTTCGCCGAAGCCCTAGCTCGTCGAATTTACCGGATTCATCCTTCCGCACTTCGCTGAAGGGCTTGCGAGAAGGATTTACCGTATTTACCCTCGAGAACTTCGCCGAAGCTCTCGCGCGGCGGATTTACCGTCTCTCTCCGTCGCAACTTCGCCGAAGCTTTAGCGCGGCGGATCTACCGTCTCTCTCCGCCGCA* ** ** *** * ** * * ** ***** * 844 837 764 871 1045 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| ACGGACGTTTGCGCGGCGGTGAACCCATCTTTCGAAGAGGTTTTGGAGAT -----------CGCCGCCATTGATCCTTCCTTCGAAGAGATTCTTCAGAT -----------CGATGTCGCGTTGTCTTCGTTTTCGGACACTCTTCAGAT -----------GAGTCCAATCGACCACTCTCTCTCCGATACTCTTCAGAT -----------GAATCAGATCGATCATTGTCTCTCCGATACTCTTCAGAT * * ** * * **** 894 876 803 910 1084 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| GCACTTTTACGAGTCTTGCCCTTACCTGAAGTTCGCTCATTTCACGGCGA GAACTTCTACGACTCGTGTCCCTACCTGAAATTCGCTCATTTCACGGCCA TCATTTCTATGAGTCTTGTCCGTATCTCAAGTTTGCGCATTTTACGGCGA GCACTTCTACGAGACTTGTCCTTATCTCAAGTTCGCTCACTTCACGGCGA GCACTTTTACGAGACTTGTCCTTATCTTAAATTCGCTCACTTCACGGCGA * ** ** ** * ** ** ** ** ** ** ** ** ** ***** * 944 926 853 960 1134 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| ACCAAGCGATTCTAGAAGCTGTTACGACGGCGCGTAGAGTTCACGTCATT ATCAGGCGATTCTAGAAGCTGTTACGACGTCGCGTGTCGTACACGTAATC ATCAAGCGATACTTGAGGTTTTTGCTACGGCGGAGAAGGTTCATGTTATT ATCAAGCGATTCTCGAAGCTTTTCAAGGGAAGAAAAGAGTTCATGTCATT ACCAAGCGATTCTCGAAGCTTTTGAAGGTAAGAAGAGAGTACACGTCATT * ** ***** ** ** * * ** * ** ** ** ** 994 976 903 1010 1184 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| GATTTAGGGCTTAATCAAGGGATGCAATGGCCTGCTTTAATGCAAGCTTT GATCTAGGGCTTAATCAAGGTATGCAATGGCCGGCGTTAATGCAAGCCTT GATTTAGGACTTAACCATGGTTTACAATGGCCGGCTTTGATTCAAGCTCT GATTTCTCTATGAGTCAAGGTCTTCAATGGCCGGCGCTTATGCAGGCTCT GATTTCTCGATGAACCAAGGTCTTCAATGGCCTGCACTTATGCAAGCTCT *** * * * ** ** * ******** ** * ** ** ** * 1044 1026 953 1060 1234 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| AGCTCTCCGACCCGGTGGACCTCCGTCGTTTCGTCTCACCGGAATCGGAC AGCTCTCCGACCCGGTGGTCCACCGTCGTTTCGTCTCACCGGCGTTGGGA TGCTTTACGTCCTAATGGTCCACCGGATTTTCGGTTAACCGGGATCGGTT TGCGCTTCGACCTGGTGGTCCTCCTGTTTTCCGGTTAACCGGAATTGGTC TGCGCTTCGAGAAGGAGGTCCTCCAACTTTCCGGTTAACCGGAATTGGTC ** * ** ** ** ** ** ** * ***** * ** 1094 1076 1003 1110 1284 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| CACCGCAGACGGAGAATTCAGATTCGCTTCAACAGTTAGGTTGGAAATTA ATCCGTCGA------ATCGAGAAGGGATTCAAGAGTTAGGTTGGAAGCTA ATTCGTTAA-------CCGATA-----TTCAAGAAGTTGGTTGGAAACTT CACCGGCACCGGATAATTTCGATTATCTTCATGAAGTTGGGTGTAAGCTG CACCGGCGCCGGATAATTCTGATCATCTTCATGAAGTTGGTTGTAAATTA ** * **** * * ** ** ** * 1144 1120 1041 1160 1334 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| GCTCAATTCGCTCAGAACATGGGCGTTGAATTCGAATTCAAAGGCTTAGC GCTCAGCTGGCTCAAGCCATCGGCGTCGAATTCAAATTCAATGGTCTAAC GGTCAGCTTGCGAGTACTATTGGTGTCAATTTCGAATTCAAGAGCATTGC GCTCATTTAGCTGAGGCGATTCACGTTGAGTTTGAGTACAGAGGATTTGT GCTCAGCTTGCGGAGGCGATTCACGTAGAATTCGAATACCGTGGATTCGT * *** * ** ** ** * ** * * * * * 1194 1170 1091 1210 1384 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| CGCTGAGAGTTTATCGGATCTTGAACCCGAAATGTTCGAAACCCGACC-GACGGAGAGGTTATCCGATTTAGAACCGGATATGTTCGAGACCCGAAC-TTTAAACAATTTGTCTGATCTTAAACCGGAAATGCTAGACATTAGACC-GGCTAACACTTTAGCTGATCTTGATGCTTCGATGCTTGAGCTTAGACCAA TGCTAACAGCTTAGCCGATCTCGATGCTTCGATGCTTGAGCTTAGACCGA * * ** * *** * * * *** * ** ** * 1242 1218 1139 1260 1434 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| -CGAATCTGAAACCTTAGTGGTTAATTCGGTATTTGAGCTCCACCGGTTA -CGAATCGGAGACTCTAGTGGTTAATTCGGTTTTCGAGCTTCACCCGGTT -CGGTTTAGAATCAGTGGCGGTTAACTCGGTCTTCGAGCTTCATCGCCTC GTGAGATTGAATCTGTTGCGGTTAACTCTGTTTTCGAGCTTCACAAGCTC GCGATACGGAAGCTGTTGCGGTGAACTCTGTTTTTGAGCTACATAAGCTC * ** * * * *** ** ** ** ** ***** ** * 1291 1267 1188 1310 1484 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| TTAGCCCGATCCGGTTCAATCGAAAAGCTTCTCAATACGGTTAAAGCTAT TTATCCCAACCCGGTTCGATCGAAAAGCTGTTAGCGACGGTTAAGGCGGT TTAGCTCATCCCGGTTCCATCGATAAGTTTTTATCGACAATCAAATCAAT TTGGGACGACCTGGTGCGATCGATAAGGTTCTTGGTGTGGTGAATCAGAT 1341 1317 1238 1360 III gi|30677937|ref|NM_126218.2| TTAGGTCGTCCCGGTGGGATAGAGAAAGTTCTCGGCGTTGTGAAACAGAT 1534 ** * * *** ** ** ** * * * ** * gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| TAAACCGAGTATCGTAACGGTGGTTGAGCAAGAAGCGAACCACAACGGAA TAAACCGGGTCTCGTAACAGTGGTGGAACAAGAAGCGAACCATAACGGTG CCGACCGGATATAATGACTGTGGTCGAGCAAGAAGCAAACCATAACGGTA TAAACCGGAGATTTTCACTGTGGTTGAGCAGGAATCGAACCATAATAGTC TAAACCGGTGATTTTCACGGTGGTTGAGCAAGAATCGAACCATAACGGAC **** * * ** ***** ** ** *** * ***** ** * 1391 1367 1288 1410 1584 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| TCGTCTTCCTCGATAGGTTCAACGAAGCGCTTCATTACTACTCGAGCTTG ACGTTTTCTTAGACCGGTTTAACGAAGCGCTTCACTATTACTCGAGCTTG CCGTATTTCTCGATCGGTTCACGGAATCGCTACATTACTATTCGAGCTTA CGATTTTCTTAGATCGGTTTACTGAGTCGTTGCATTATTACTCGACGTTG CGGTTTTCTTAGACCGGTTTACTGAATCGTTACATTATTATTCGACTCTG * ** * ** **** * ** ** * ** ** ** **** * 1441 1417 1338 1460 1634 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| TTTGACTCGCTCGAAGACAGTTATAGTTTACCGAGTCAAGACCGAGTTAT TTCGACTCGCTCGAAGATGGTGTTGTGATACCGAGTCAAGACCGAGTCAT TTCGACTCGCTCGAGGGC---------CCGCCAAGCCAAGACCGAGTGAT TTTGACTCGTTGGAAGGTG------TACCGAGTGGTCAAGACAAGGTCAT TTTGATTCGTTGGAAGGAG------TTCCGAATAGTCAAGACAAAGTCAT ** ** *** * ** * * ****** ** ** 1491 1467 1379 1504 1678 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| GTCAGAAGTGTACTTAGGGAGACAGATACTCAACGTTGTTGCGGCGGAAG GTCGGAGGTTTACTTAGGGAGACAGATATTGAACTTGGTGGCGACGGAAG GTCGGAGTTATTCCTAGGACGGCAGATACTAAACCTTGTGGCATGCGAAG GTCGGAGGTTTACTTGGGTAAACAGATCTGCAACGTTGTGGCTTGTGATG GTCTGAAGTTTACTTAGGGAAACAGATTTGTAATCTGGTGGCTTGTGAAG *** ** * * * * ** ***** ** * ** ** ** * 1541 1517 1429 1554 1728 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| GGTCCGATCGGGTCGAGCGGCACGAGACGGCTGCACAGTGGAGGATTCGG GAAGCGATAGGATCGAGCGACACGAGACGCTGGCTCAGTGGCGAAAACGT GAGAAGACCGGGTAGAGAGGCATGAGACTTTAAATCAGTGGAGAAACCGG GACCTGACCGAGTTGAGCGTCATGAAACGTTGAGTCAGTGGAGGAACCGG GTCCTGACAGAGTCGAGAGACACGAAACGTTGAGTCAATGGGGAAACCGG * ** * * *** * ** ** ** ** *** * * ** 1591 1567 1479 1604 1778 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| ATGAAATCCGCTGGGTTTGACCCGATTCATCTCGGATCTAGCGCGTTTAA ATGGGATCCGCCGGGTTTGACCCGGTTAACCTCGGATCAGACGCGTTTAA TTCGGTTTAGGAGGATTTAAACCGGTTAGTATCGGTTCGAACGCGTATAA TTCGGGTCTGCTGGGTTTGCGGCTGCACATATTGGTTCGAATGCGTTTAA TTTGGTTCGTCCGGTTTAGCGCCGGCACATCTTGGGTCTAACGCGTTTAA * * ** ** * * ** ** **** *** 1641 1617 1529 1654 1828 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| ACAAGCGAGTATGCTTTTATCGCTTTACGCTACCGGAGATGGATACAGAG GCAAGCGAGTTTGCTATTGGCGTTATCTGGCGGTGGAGATGGATACAGAG GCAAGCAAGCATGTTGTTGGCACTTTATGCCGGGGCTGATGGGTATAATG GCAAGCGAGTATGCTTTTGGCTCTGTTCAACGGCGGTGAGGGTTATCGGG GCAAGCGAGTATGCTTTTGTCTGTGTTTAATAGTGGCCAAGGTTATCGTG ***** ** ** * ** * * * * * ** ** * 1691 1667 1579 1704 1878 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| TTGAAGAAAATGACGGATGTTTAATGATAGGGTGGCAGACGCGACCACTC TGGAGGAGAACGACGGAAGCCTAATGCTTGCGTGGCAAACGAAACCTCTA TGGAAGAGAATGAAGGTTGTTTGTTGCTTGGATGGCAAACGCGACCGCTT TGGAGGAGAGTGACGGCTGTCTCATGTTGGGTTGGCACACACGACCGCTC TGGAGGAGAGTAATGGATGTTTGATGTTGGGTTGGCACACTCGTCCACTC * ** ** * * ** * * ** * * ***** ** ** ** 1741 1717 1629 1754 1928 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| ATCACAACCTCGGCGTGGAAACTCGC-----------CTGAGTCGCGGCG ATCGCTGCATCGGCGTGGAAACTAGC---------GGCGGAGTTGCGGCG ATTGCAACATCTGCGTGGCGAATCAA---------TCGTGTGGAATAAAA ATAGCCACCTCGGCTTGGAAACTCTCCACCAATTAGATGGTGGCTCAATG ATTACCACCTCCGCTTGGAAACTCTC---------GACGGCGGCGTACTG ** * * ** ** *** * * * * 1780 1758 1670 1804 1969 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| G--TAGAGA-TGACTCGCCTGAAACCGGGAAAAAC-AATAAATGTTTTAA G--TAGATA-CG--TCGTCATAAAGAGGAGAAGAA-AA-----------A A--TAAATAATGGGAAAAGTGAAAATGTGCTATAT-ACTTTATTGCATTG AATTGATCTGTTGAACCGGTTATGATGATAGATTT---CCGACCGAAGCC AGTTTGACT-CGAAGCATACGACGGTGGTGGAGTCGAGTCGAGTGAATTT * * * * 1826 1791 1717 1851 2018 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| AAAATTAGGAAAAGAGACCGTAACTTTAGTTATGTTTTTACTTTTTAACC AGACTTAGCGAACGTGACCTTATGTTT--TTATTTTACTTTAACTTACCC CTGATAAAGAAAAAAAGTCCCACGTTTTCCAAATTTTATGAATTCTAAAT AAACTAAATCCTACTGTTTTTCCCTTT--GTCACTTGTTAAGATCTTATC GAGATTGAGATCAGTGGACCGGTGAT---GACATATGTTCGGACCAAGAC * * * * 1876 1839 1767 1899 2065 gi|30678851|ref|NM_111216.2| CGAAGTTTTTGTGTG---TTTAACCTTTTTGCCT-AAATGTTTACAACTT 1922 IV gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| CAGTAGTTTCGTTT----TGTGACAATTTCGCCCGAAATATTCCGTGCCT TT-TGTTCACTTGT----CACGAGATTTTGACCTCGCATAAATAGACTAT TTTCATTATATTAGGTAATTGAAAAATTTTAATCTCGCTTTGGAGAGTTT CTAAACCGAACTG---AATCGAACCGTTTTGCCTTTTGTTTATTTTATTT * * *** * * 1885 1812 1949 2112 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| TAT-CTTTTTGGACCTTGTGCGTATCTTTG----AGAGTT---AAGAGAA TATACTTTTGGGACCCAGTTGGTTCGTTGGTCGTGGAGAT---TCGAGAA TAC---GTCAGGGTCAGGCCAATGAAATGATTTTTTATC----------TTTTTT-TTTGCATGT--GACATTGGAGGGTAAATTG----GATAGGCAG ATTTTCGTTCACTTGTTTAAAATTCTTATATATATCGTTTTGGTAGGTCA * * 1964 1932 1848 1992 2162 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| CGAGTAAAAAATCT---TGTATCG-TAGATCGAGCTAAGTAGTTTTCAAT CGAGGAACATGTGTGTATGTAACAACAGCACGAGC-AAGT-GTTTTCA-T -------------------------------------------------AAATAGAAGTATGTGTTACCAAGT---ATGTGCAATTGGTTGAAATAA-TTTTTAATTTATGCCTTTTTGGGATCAATTTTTAATAGGCTGAGTTTG-- 2010 1979 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| AAATGGAAGGATAACGATTCTGTATGTTTTTTACTTGATCCAATATATAT AGTTTGAATAAATATGAAAGAA-ATGACGTTTATTTT-------------------------------------------------------------AATCATCTTGAGTGTCACCATCTATAAAATTCA--TTGTAATGACTAATG TATTTATTAATAAATTATC-TTTATGAATTTTAAACTAAAACTATGTTTT 2060 2015 gi|30678851|ref|NM_111216.2| gi|30686387|ref|NM_121755.2| gi|30697338|ref|NM_105306.2| gi|30684069|ref|NM_101361.2| gi|30677937|ref|NM_126218.2| GAATTTATTT---------------------------------- 2070 --------------------------------------------------------------------------------------AGCCTGATTAAACTGTCTCTTATGATAATGTGCTGATTCTCATG 2129 AATCTCATTTAAAAAAAAATTAATATCAAGTTTTATTAATCTC- 2302 2037 2210 2085 2259 V RGL2 Genbank Accession No. NM_111216 (At3g03450) ATGAAGAGAGGATACGGAGAAACATGGGATCCGCCACCAAAACCACTACCAGCTTCTCGT 60 RGL2 FWD FL TCCGGAGAAGGTCCTTCAATGGCGGATAAGAAGAAGGCTGATGATGACAACAACAACAGC 120 AACATGGATGATGAGCTTCTTGCTGTTCTTGGCTACAAGGTTCGATCTTCTGAGATGGCT 180 RGL2RVS-PCR GAAGTAGCACAGAAGCTTGAACAACTTGAGATGGTCTTGTCTAATGATGATGTTGGTTCT 240 ACTGTCTTAAACGACTCTGTTCATTATAACCCATCTGATCTCTCTAACTGGGTCGAGAGC 300 ATGCTTTCTGAGCTGAACAACCCGGCTTCTTCGGATCTTGACACGACCCGAAGTTGTGTG 360 GATAGATCCGAATACGATCTCAGAGCAATTCCGGGTCTTTCTGCGTTTCCAAAGGAAGAG 420 RGL2F-640 GAAGTCTTTGACGAGGAAGCTAGCAGCAAGAGGATCCGACTCGGATCGTGGTGCGAATCG 480 TCGGACGAGTCAACTCGGTCCGTGGTGCTCGTTGACTCTCAGGAGACCGGAGTTAGACTT 540 GTCCACGCACTAGTGGCGTGCGCTGAGGCGATTCACCAGGAGAATCTCAACTTAGCTGAC 600 GCGCTGGTGAAACGCGTGGGAACACTCGCGGGTTCTCAAGCTGGAGCTATGGGAAAAGTC 660 GCTACGTATTTTGCTCAAGCCTTGGCTCGTCGTATTTACCGTGATTACACGGCGGAGACG 720 GACGTTTGCGCGGCGGTGAACCCATCTTTCGAAGAGGTTTTGGAGATGCACTTTTACGAG 780 TCTTGCCCTTACCTGAAGTTCGCTCATTTCACGGCGAACCAAGCGATTCTAGAAGCTGTT 840 RGL2F-1055 ACGACGGCGCGTAGAGTTCACGTCATTGATTTAGGGCTTAATCAAGGGATGCAATGGCCT 900 GCTTTAATGCAAGCTTTAGCTCTCCGACCCGGTGGACCTCCGTCGTTTCGTCTCACCGGA 960 ATCGGACCACCGCAGACGGAGAATTCAGATTCGCTTCAACAGTTAGGTTGGAAATTAGCT 1020 CAATTCGCTCAGAACATGGGCGTTGAATTCGAATTCAAAGGCTTAGCCGCTGAGAGTTTA 1080 TCGGATCTTGAACCCGAAATGTTCGAAACCCGACCCGAATCTGAAACCTTAGTGGTTAAT 1140 TCGGTATTTGAGCTCCACCGGTTATTAGCCCGATCCGGTTCAATCGAAAAGCTTCTCAAT 1200 ACGGTTAAAGCTATTAAACCGAGTATCGTAACGGTGGTTGAGCAAGAAGCGAACCACAAC 1260 RGL2F-1470 GGAATCGTCTTCCTCGATAGGTTCAACGAAGCGCTTCATTACTACTCGAGCTTGTTTGAC 1320 VI TCGCTCGAAGACAGTTATAGTTTACCGAGTCAAGACCGAGTTATGTCAGAAGTGTACTTA 1380 GGGAGACAGATACTCAACGTTGTTGCGGCGGAAGGGTCCGATCGGGTCGAGCGGCACGAG 1440 ACGGCTGCACAGTGGAGGATTCGGATGAAATCCGCTGGGTTTGACCCGATTCATCTCGGA 1500 TCTAGCGCGTTTAAACAAGCGAGTATGCTTTTATCGCTTTACGCTACCGGAGATGGATAC 1560 AGAGTTGAAGAAAATGACGGATGTTTAATGATAGGGTGGCAGACGCGACCACTCATCACA 1620 ACCTCGGCGTGGAAACTCGCCTGA 1644 RGL2 RVS FL * ¬ Primers RGL2 FWD FL and RGL2 RVS FL amplify the whole coding region of RGL2. Refer to Table 4.1 for the different enzyme cut sites integrated for various cloning experiments. ¬ Primers sequences highlighted in yellow with black arrows are primers used for sequencing of RGL2 gene in the various constructs. ¬ The primer RGL2RVS-PCR was also used together with 35S PRO primer to check for successful ligation of RGL2 into the various constructs. ¬ Sequences in bold are the primers RGL2 FWD RT-128 AND RGL2 RVS RT527 which are used for the RT-PCR experiments. The product length is approximately 400 bp. VII RGL2 – 1645 bp full length cDNA RGL2 FWD FL RGL2F-640 RGL2-1055 RGL2-1470 This schematic scale diagram illustrates the use of the internal primers (forward direction) to sequence and verify the RGL2 PCR fragment. Arrows represent the approximate length of the sequence read. The internal reverse primers used (as highlighted in the sequence above) are not represented here. VIII IX X XI XII LOCUS NM_126182 2151 bp mRNA linear PLN 04-NOV-2005 DEFINITION Arabidopsis thaliana ATP binding / kinase/ protein serine/threonine kinase AT2G01210 mRNA, complete cds. ACCESSION NM_126182 REGION: 111 2261 VERSION NM_126182.2 GI:42568852 KEYWORDS . SOURCE Arabidopsis thaliana (thale cress) ORGANISM Arabidopsis thaliana Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core eudicotyledons; rosids; eurosids II; Brassicales; Brassicaceae; Arabidopsis. COMMENT PROVISIONAL REFSEQ: This record has not yet been subject to final NCBI review. This record has been curated by TAIR. This record is derived from an annotated genomic sequence (NC_003071). The reference sequence was derived from AT2G01210.1. On Feb 17, 2004 this sequence version replaced gi:18379175. FEATURES Location/Qualifiers source 2151 /organism="Arabidopsis thaliana" /mol_type="mRNA" /db_xref="taxon:3702" /chromosome="2" /ecotype="Columbia" gene 2151 /locus_tag="AT2G01210" /note="synonym: F10A8.9, F10A8_9" /db_xref="GeneID:814649" CDS 2151 /locus_tag="AT2G01210" /go_component="endomembrane system" /go_function="ATP binding; kinase activity; protein serine/threonine kinase activity" /go_process="protein amino acid phosphorylation; transmembrane receptor protein tyrosine kinase signaling pathway" /note="leucine-rich repeat transmembrane protein kinase, putative" /codon_start=1 /product="ATP binding / kinase/ protein serine/threonine kinase" /protein_id="NP_178230.1" /db_xref="GI:15226197" /db_xref="GeneID:814649" /translation="MLASLIIFVALLCNVTVISGLNDEGFALLTFKQSVHDDPTGSLN NWNSSDENACSWNGVTCKELRVVSLSIPRKNLYGSLPSSLGFLSSLRHLNLRSNRFYG SLPIQLFHLQGLQSLVLYGNSFDGSLSEEIGKLKLLQTLDLSQNLFNGSLPLSILQCN RLKTLDVSRNNLSGPLPDGFGSAFVSLEKLDLAFNQFNGSIPSDIGNLSNLQGTADFS HNHFTGSIPPALGDLPEKVYIDLTFNNLSGPIPQTGALMNRGPTAFIGNTGLCGPPLK DLCQGYQLGLNASYPFIPSNNPPEDSDSTNSETKQKSSGLSKSAVIAIVLCDVFGICL VGLLFTYCYSKFCACNRENQFGVEKESKKRASECLCFRKDESETPSENVEHCDIVPLD AQVAFNLEELLKASAFVLGKSGIGIVYKVVLENGLTLAVRRLGEGGSQRFKEFQTEVE AIGKLKHPNIASLRAYYWSVDEKLLIYDYVSNGNLATALHGKPGMMTIAPLTWSERLR IMKGIATGLVYLHEFSPKKYVHGDLKPSNILIGQDMEPKISDFGLARLANIAGGSSPT IQSNRIIQTDQQPQERQQHHHKSVSSEFTAHSSSGSYYQAPETLKMVKPSQKWDVYSY GIILLELIAGRSPAVEVGTSEMDLVRWVQVCIEEKKPLCDVLDPCLAPEAETEDEIVA VLKIAISCVNSSPEKRPTMRHVSDTLDRLPVAGD" ORIGIN atgttggcct cgctgatcat cttcgttgct cttctctgca atgtgactgt cataagtggc 61 ttaaacgacg aaggctttgc gctcttaacg tttaaacaaa gcgttcacga tgatcctaca 121 ggttctctta acaattggaa ctcatcagac gagaacgcct gttcgtggaa cggtgtaact 181 tgcaaagaac tcagggtggt gtctctaagc attccaagga agaatcttta cggttctctc 241 ccttcgtctc tagggtttct ctccagtctc cgtcacttga atctccggag caataggttt 301 tatgggtcgt tgcctattca gcttttccat cttcagggac tccaaagtct agtcctttac 361 ggcaattcct ttgatgggtc attgtcggaa gagatcggta agctgaagct tcttcagacc 421 ttagatttgt cacagaatct cttcaacggg tcgttaccac tttcgattct gcaatgcaac 481 agacttaaaa cactcgatgt cagcagaaac aacttgtctg gtcctttgcc tgatgggttt XIII 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 ggctcagcgt atccccagcg cacttcaccg ctcactttca ccaacagcgt ggatatcaac gattctgata gtcattgcca tactgctact tccaagaaaa gaaaacgtgg gagctgctaa gtggttctag agattcaagg gctagtcttc tccaacggta ttgacatggt catgagttta ggacaggaca ggaggatctt gagaggcaac ggatcctact gtttactctt gtggggacat ccattgtgtg gttgctgttc atgaggcacg tcgtctctct acattggaaa gctcaatccc ataatctctc ttatcggcaa ttggcctcaa gtactaactc ttgttctctg caaagttctg gggcttctga agcattgcga aggcgtcagc agaacgggct agtttcagac gagcttatta acctcgcaac ctgaacggtt gtcccaagaa tggaacctaa ctccgaccat aacaccatca atcaagcacc atgggatcat cggaaatgga atgttcttga ttaagattgc tttctgatac ggagaaactc cctctccaac acctgccctt cggtcccatt cactggtcta tgcctcttac tgaaaccaaa cgacgtgttt cgcttgtaac gtgtctgtgt cattgttccc tttcgttctg cacactggcc agaagttgaa ttggtctgtc cgccctccac gaggataatg atacgtccat gatctcagat acagtctaac caagagcgta tgagactctc tttactggaa tctagtgcgg tccttgtttg aatcagttgc gctcgacaga gacctcgcct cttcaaggaa ggagatcttc cctcaaaccg tgcgggcctc ccttttatcc cagaaatcaa ggtatctgcc cgcgagaacc ttccgtaaag ctcgacgctc gggaagagcg gtacggagat gccataggga gatgaaaagc ggaaagccgg aaagggattg ggagatctca tttggactag agaatcatcc tcttcggagt aagatggtaa ttgatagcgg tgggtacagg gctcctgagg gtgaatagca ttacccgtgg tcaaccaatt ctgctgattt ctgaaaaggt gtgctcttat cattgaagga caagcaacaa gtggtttgag ttgtgggttt aattcggagt acgaatccga aggttgcgtt gaatcggaat tgggtgaagg aactaaaaca ttctcatcta ggatgatgac ctacagggct agccaagcaa cacggttagc aaacggatca tcactgctca aaccttcaca gtaggtctcc tatgcattga cagagacgga gtccagagaa ccggcgattg caacggctcc ctctcataat ttacattgat gaacagagga cctttgccaa cccacctgag caaaagcgcc gcttttcact cgagaaagaa aacgccatct taacctggag tgtctacaaa agggtctcag tcctaacatt cgactatgtt catcgctccg tgtttatctg cattctcatt taacatcgct acagccgcaa ctcttcttct gaagtgggat cgcagtggag ggagaagaaa agacgagatt aaggcctacc a XIV LOCUS NM_102342 2109 bp mRNA linear PLN 04-NOV-2005 DEFINITION Arabidopsis thaliana ATP binding / kinase/ protein serine/threonine kinase AT1G25320 mRNA, complete cds. ACCESSION NM_102342 REGION: 132 2240 VERSION NM_102342.1 GI:18395640 KEYWORDS . SOURCE Arabidopsis thaliana (thale cress) ORGANISM Arabidopsis thaliana Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core eudicotyledons; rosids; eurosids II; Brassicales; Brassicaceae; Arabidopsis. COMMENT PROVISIONAL REFSEQ: This record has not yet been subject to final NCBI review. This record has been curated by TAIR. This record is derived from an annotated genomic sequence (NC_003070). The reference sequence was derived from AT1G25320.1. FEATURES Location/Qualifiers source 2109 /organism="Arabidopsis thaliana" /mol_type="mRNA" /db_xref="taxon:3702" /chromosome="1" /ecotype="Columbia" gene 2109 /locus_tag="AT1G25320" /note="synonym: F4F7.29, F4F7_29" /db_xref="GeneID:839116" CDS 2109 /locus_tag="AT1G25320" /go_component="endomembrane system" /go_function="ATP binding; kinase activity; protein serine/threonine kinase activity" /go_process="protein amino acid phosphorylation; transmembrane receptor protein tyrosine kinase signaling pathway" /note="leucine-rich repeat transmembrane protein kinase, putative, similar to putative receptor-like protein kinase GI:4262228 from (Arabidopsis thaliana)" /codon_start=1 /product="ATP binding / kinase/ protein serine/threonine kinase" /protein_id="NP_564228.1" /db_xref="GI:18395641" /db_xref="GeneID:839116" /translation="MSVGSLLFMFLLIWNFNGELNALNDEGFALLTLKQSISKDPDGS LSNWNSENQNPCSWNGVTCDDNKVVVSLSIPKKKLLGYLPSSLGLLSNLRHLNLRSNE LSGNLPVELFKAQGLQSLVLYGNFLSGSIPNEIGDLKFLQILDLSRNSLNGSIPESVL KCNRLRSFDLSQNNLTGSVPSGFGQSLASLQKLDLSSNNLIGLVPDDLGNLTRLQGTL DLSHNSFSGSIPASLGNLPEKVYVNLAYNNLSGPIPQTGALVNRGPTAFLGNPRLCGP PLKDPCLPDTDSSSTSHPFVPDNNEQGGGGSKKGEGLSKTAIVAIVVCDFIGICIVGF LFSCCYLKICARRNSVDEEGYVLEKEGKEKKGSFCFRRDGSESPSSENLEPQQDLVLL DKHIALDLDELLKASAFVLGKGGNGIVYKVVLEDGLTVAVRRLGEGGSQRCKEFQTEV EAIGKLRHPNIVSLKAYYWSVEEKLLIYDYIPNGSLTNALHGNPGMVSFKPLSWGVRL KIMRGISRGLVYLHEFSPKKYVHGSLKLSNILLGQDMEPHISDFGLMHLSSIAGTLES TTVDRPSNKTASSIGSSANLSSFYLAPEATKATVKPSQKWDVYSFGVILLEMITGRLP IVFVGKSEMEIVKWIQMCIDEKKEMSDILDPYLVPNDTEIEEEVIAVLKIAMACVSTS PEKRPPMKHIADALTQICLQ" ORIGIN atgtcggtgg gatctctttt attcatgttt cttcttatct ggaacttcaa tggcgaattg 61 aatgctctaa acgacgaagg gtttgctctt ttaactctga agcagtctat ttcaaaagat 121 ccagatggtt ctttaagcaa ttggaactca gagaatcaaa acccttgttc ttggaatgga 181 gtcacttgtg atgataacaa ggttgttgtc tctcttagca tcccaaagaa gaaactttta 241 ggttatcttc cttcttctct tggtttactc tctaatctcc gtcatttgaa tttgagaagc 301 aatgagctta gtgggaactt acctgttgag ctttttaagg ctcaaggtct tcaaagtttg 361 gttctttatg ggaatttctt gtctgggtcc attccaaatg agattggtga cttaaagttt 421 ctgcaaattt tggatctttc tcgtaattct cttaatgggt ctattccaga atcggttttg XV 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 aagtgtaata agtggatttg attggtcttg tctcataatt tatgtcaatc aacagaggac ccttgtttgc gagcaaggtg attgtggttt ttgaagattt gggaaagaaa gaaaatcttg gatgagctgc aaagttgtcc caaagatgca atcgttagtc attcctaatg cctctgtctt cttcacgagt ttgggacagg gctggaacat ggatcatctg aagccatcac ggaagattac atgtgtatcg gacacagaga actagccccg cttcagtga ggcttaggag gtcagtcttt ttcctgatga cgtttagtgg tagcttacaa caactgcatt cagatacgga gaggagggtc gtgatttcat gtgcacgtcg agaaaggttc agccacaaca ttaaggcttc tcgaagatgg aggagtttca ttaaagctta gaagccttac ggggagttcg ttagtcctaa atatggagcc tggaatcaac caaacttgag agaaatggga cgattgtttt atgagaagaa ttgaagaaga agaaacgtcc ctttgatttg ggcttctttg tttggggaat ctcgattcca caacttgagt cttggggaat tagttcttca aaagaaagga tggaatctgc taatagtgtg cttctgtttc agatcttgtt agctttcgtt tctaacggtc gacagaagtt ttattggtca caatgcactc gttaaagata aaagtatgtt tcatatctcg tacagttgac ctcattttat tgtatactca cgtgggtaaa agagatgtca ggtcatcgcc accgatgaag agtcagaata cagaagcttg ttgactagat gctagtttgg ggaccaatcc ccaagactct acttctcacc gaaggtttaa attgtgggat gatgaggaag agaagagatg ctgttggata cttggaaaag gctgttagga gaagcaattg gttgaagaga cacggaaatc atgaggggaa catggatctc gatttcggac cgaccatcca ctggctcctg ttcggagtga tcggaaatgg gacattttgg gtgttgaaaa catatcgctg atctcactgg atctttcttc tgcaaggaac ggaatttgcc cacaaaccgg gtggccctcc cttttgtacc gtaaaactgc ttctcttctc gctatgtgtt gatcagagtc aacatattgc gcgggaacgg gactgggaga ggaagctaag aacttctcat ctggaatggt tctcaagagg tgaaactcag ttatgcacct acaagaccgc aagccactaa tcttgctaga agatagtcaa atccttattt ttgcaatggc atgctttgac ttctgttcct taacaatctt tcttgatttg tgagaaagtt tgctttggtg attgaaggat ggataacaac tattgttgca ttgttgttac ggagaaagaa cccttcttcg tttggattta gattgtgtat aggaggttcc gcatccgaat ctacgactac gtcattcaag gctggtgtat caatatactg ctctagcatt ctcatcgatt agcaacagtg gatgataacg gtggatacaa ggtgcctaat ttgtgttagt ccaaatttgc XVI CLUSTAL W (1.83) multiple sequence alignment of the putative kinases. gi|15226197|ref|NP_178230.1| gi|18395641|ref|NP_564228.1| -MLASLIIFVALLCNVT-VISGLNDEGFALLTFKQSVHDDPTGSLNNWNS 48 MSVGSLLFMFLLIWNFNGELNALNDEGFALLTLKQSISKDPDGSLSNWNS 50 :.**:::. *: * : **********:***: .** ***.**** gi|15226197|ref|NP_178230.1| gi|18395641|ref|NP_564228.1| SDENACSWNGVTCKELR-VVSLSIPRKNLYGSLPSSLGFLSSLRHLNLRS 97 ENQNPCSWNGVTCDDNKVVVSLSIPKKKLLGYLPSSLGLLSNLRHLNLRS 100 .::*.********.: : *******:*:* * ******:**.******** gi|15226197|ref|NP_178230.1| gi|18395641|ref|NP_564228.1| NRFYGSLPIQLFHLQGLQSLVLYGNSFDGSLSEEIGKLKLLQTLDLSQNL 147 NELSGNLPVELFKAQGLQSLVLYGNFLSGSIPNEIGDLKFLQILDLSRNS 150 *.: *.**::**: *********** :.**:.:***.**:** ****:* gi|15226197|ref|NP_178230.1| gi|18395641|ref|NP_564228.1| FNGSLPLSILQCNRLKTLDVSRNNLSGPLPDGFGSAFVSLEKLDLAFNQF 197 LNGSIPESVLKCNRLRSFDLSQNNLTGSVPSGFGQSLASLQKLDLSSNNL 200 :***:* *:*:****:::*:*:***:*.:*.***.::.**:****: *:: gi|15226197|ref|NP_178230.1| gi|18395641|ref|NP_564228.1| NGSIPSDIGNLSNLQGTADFSHNHFTGSIPPALGDLPEKVYIDLTFNNLS 247 IGLVPDDLGNLTRLQGTLDLSHNSFSGSIPASLGNLPEKVYVNLAYNNLS 250 * :*.*:***:.**** *:*** *:****.:**:******::*::**** gi|15226197|ref|NP_178230.1| gi|18395641|ref|NP_564228.1| GPIPQTGALMNRGPTAFIGNTGLCGPPLKDLCQGYQLGLNASYPFIPSNN 297 GPIPQTGALVNRGPTAFLGNPRLCGPPLKDPCLPDTDSSSTSHPFVPDNN 300 *********:*******:**. ******** * . .:*:**:*.** gi|15226197|ref|NP_178230.1| gi|18395641|ref|NP_564228.1| PPEDSDSTNSETKQKSSGLSKSAVIAIVLCDVFGICLVGLLFTYCYSKFC 347 EQGGGGSK------KGEGLSKTAIVAIVVCDFIGICIVGFLFSCCYLKIC 344 .*. * ****:*::***:**.:***:**:**: ** *:* gi|15226197|ref|NP_178230.1| gi|18395641|ref|NP_564228.1| ACNR---ENQFGVEKESKKRASECLCFRKDESETPS-ENVE-HCDIVPLD 392 ARRNSVDEEGYVLEKEGKEKK-GSFCFRRDGSESPSSENLEPQQDLVLLD 393 * *: : :***.*:: .:***:* **:** **:* : *:* ** gi|15226197|ref|NP_178230.1| gi|18395641|ref|NP_564228.1| AQVAFNLEELLKASAFVLGKSGIGIVYKVVLENGLTLAVRRLGEGGSQRF 442 KHIALDLDELLKASAFVLGKGGNGIVYKVVLEDGLTVAVRRLGEGGSQRC 443 ::*::*:************.* *********:***:************ gi|15226197|ref|NP_178230.1| gi|18395641|ref|NP_564228.1| KEFQTEVEAIGKLKHPNIASLRAYYWSVDEKLLIYDYVSNGNLATALHGK 492 KEFQTEVEAIGKLRHPNIVSLKAYYWSVEEKLLIYDYIPNGSLTNALHGN 493 *************:****.**:******:********:.**.*:.****: gi|15226197|ref|NP_178230.1| gi|18395641|ref|NP_564228.1| PGMMTIAPLTWSERLRIMKGIATGLVYLHEFSPKKYVHGDLKPSNILIGQ 542 PGMVSFKPLSWGVRLKIMRGISRGLVYLHEFSPKKYVHGSLKLSNILLGQ 543 ***::: **:*. **:**:**: ****************.** ****:** gi|15226197|ref|NP_178230.1| gi|18395641|ref|NP_564228.1| DMEPKISDFGLARLANIAGGSSPTIQSNRIIQTDQQPQERQQHHHKSVSS 592 DMEPHISDFGLMHLSSIAG----TLES----TTVDRPSNK-------TAS 578 ****:****** :*:.*** *::* * ::*.:: .:* gi|15226197|ref|NP_178230.1| gi|18395641|ref|NP_564228.1| EFTAHSSSGSYYQAPETLK-MVKPSQKWDVYSYGIILLELIAGRSPAVEV 641 SIGSSANLSSFYLAPEATKATVKPSQKWDVYSFGVILLEMITGRLPIVFV 628 .: : :. .*:* ***: * ***********:*:****:*:** * * * gi|15226197|ref|NP_178230.1| gi|18395641|ref|NP_564228.1| GTSEMDLVRWVQVCIEEKKPLCDVLDPCLAP-EAETEDEIVAVLKIAISC 690 GKSEMEIVKWIQMCIDEKKEMSDILDPYLVPNDTEIEEEVIAVLKIAMAC 678 *.***::*:*:*:**:*** :.*:*** *.* ::* *:*::******::* gi|15226197|ref|NP_178230.1| gi|18395641|ref|NP_564228.1| VNSSPEKRPTMRHVSDTLDRLPVAGD 716 VSTSPEKRPPMKHIADALTQICLQ-- 702 *.:******.*:*::*:* :: : XVII [...]... small subfamily of GRAS regulatory proteins called the DELLA subfamily, which includes RGA and GAI that have a unique conserved DELLA domain at the Nterminus (D – aspartic acid; E – glutamic acid; L – leucine; A – alanine) (Richards et al., 2001) It was found that a 17 amino acid deletion in this domain causes a GAinsensitive dwarf phenotype This was important for the inactivation of RGA and GAI by the. .. application of GA 7 GA12/53 GA9/20 GA4/1 (bioactive) GA51/29 GA34/8 GA receptor GA 20-oxidase and GA 2-oxidase GA 3 hydroxylase GA response Figure 2.1 Model showing the homeostatic regulation of GA biosynthesis Feedback inhibition is shown by the T-bar GA 20-oxidase and GA 3 -hydroxylase transcripts are negatively regulated by GA activity The arrows indicate feedforward upregulation Adapted from Yamaguchi... is a dwarf mutant, which was impaired in a regulator that has orthologs in wheat and maize (Peng and Harberd, 1993) The last mutant that will be often mentioned is rga (repressor of ga1-3) The corresponding gene has been characterized as a transcriptional regulator that represses GA signaling (Silverstone et al., 1997b) The latter two GA signaling mutants are members of the GRAS (GAI, RGA and SCARECROW)... confirmed that GID1 plays a major role as a soluble GA receptor which mediates GA signaling in rice 2.4 The GRAS superfamily of putative transcription factors and DELLA proteins As mentioned briefly earlier, RGA and GAI belong to a huge class of transcriptional regulatory proteins named GRAS family The founding members of GRAS are GAI, RGA and SCARECROW (Pysh et al., 1999) There are at least 38 GRAS family... signaling response mutants are the rga and gai mutants RGA was first identified because of the ability of recessive rga alleles to partially suppress the phenotype of ga1-3 (Silverstone et al., 1997b; Silverstone et al., 1998) Cloning and characterization of RGA reveal that it is one of the first known transcriptional regulators that could suppress GA signaling in Arabidopsis The gai mutant in Arabidopsis... N-terminal regions containing the DELLA and VHYNP amino acid domains, which are however, absent in the other GRAS family members The two domains are critical for GA signaling, based on research data from the gai mutant and other rga/gai orthologs in wheat and maize (Olszewski et al., 2002) The 5 DELLA genes also possess high sequence similarity in the C-terminal regions in their gene structure Thus, it was... gibberellin signaling pathway and its regulatory proteins The well-established studies of mutants’ defective biosynthesis of GAs and the utilization of biochemical techniques have revealed the developmental role of GAs On the other hand, the studies on GA signaling effects are more recent The perception and transduction of active GA signals to different locations in plants explain how the synthesized GAs, coupled... enzymes can catalyze the same reaction for different, but structurally similar substrates E.g., GA 3 -hydroxylase is critical in catalyzing GA9 to GA4, and GA20 to GA1, both end products that are bioactive GAs (Figure 2.1) As differences in the level of GAs can affect and alter plant development, plants have evolved a mechanism to control the levels of GAs being synthesized It seems that GA levels are controlled... by a negative feedback mechanism that affects the amount of transcripts coding for the GA biosynthesis genes (Harberd et al., 1998) Silverstone et al (199 7a) has elucidated the role of GA1 in GA developmental regulation GA1 codes for the enzyme that is involved in the first committed step in GA biosynthesis Arabidopsis GA1 mRNA and protein levels in plant tissues were extremely low as revealed from the. .. domain This domain consists of aspartic acid (D), glutamic acid (E), leucine (L), leucine (L) and alanine (A) - (DELLA) (Richards et al., 2001) Previous studies have shown that this domain is required for the inactivation of RGA and GAI by the GA signal, because a 17 amino acid deletion in this domain in either protein causes a GA-insensitive dwarf phenotype It has been hypothesized that these mutant