FUNCTIONAL CHARACTERIZATION OF GIANT KILLER IN FLOWER DEVELOPMENT AND MERISTEM REGULATION IN ARABIDOPSIS THALIANA NG KIAN HONG (M. Sc., NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisor Dr Toshiro Ito for giving me the opportunity to work in his laboratory, and also his unflagging support during the course of my study. I am also grateful to all the members of Dr Toshiro Ito laboratory, past and present, for providing me a kind and helpful working environment. I wish to thank Dr Suresh Jesuthasan for giving me a chance to work as a rotation student in his laboratory, and thanks to Feng Bo for mentoring me through my rotation project. I am also grateful to Dr Tadashi Sakata for his help in my experiments. I would like to thank my thesis advisory committee members Profs. Frederic Berger, Naweed Naqvi and Sanjay Swarup for their invaluable comments and suggestions. Special thanks to Siou Ting, Serena, Phing Chian, Ting Gang, Huay Mei, Tsui Han, Siang Yee, Wan Zhong and Zhou Jie for their help and support. I am also grateful to Temasek Life Sciences Laboratory and the Singapore Millennium Foundation for financial support. Thanks to Wan Yi, Phing Chian, Norman and Eng Seng for critical reading of my thesis. Last but not least, I wish to thank my parents and sister for their unwavering support and understanding all these years. ii TABLE OF CONTENTS Acknowledgements…………… .………………………………………………………ii Table of contents…………… .……………………………………………… .iii Summary………………………………………………………………… .ix List of Tables and Figures……………… .…………………………………… .xi List of Abbreviations…………………………….……………………………………xiii CHAPTER 1: Introduction…………………………………………………… .1 1.1 General Introduction…………………………………………………… ………… .1 1.2 Regulation of shoot apical meristem, inflorescence meristem and floral meristem in Arabidopsis thaliana …… ………………………………….4 1.3 Regulation of flower development in Arabidopsis thaliana………………………….9 1.4 Patterning and differentiation of lateral organs in Arabidopsis thaliana 13 1.4.1 The patterning of lateral organs…………….……………………………13 1.4.2 Regulation of abaxial-adaxial polarity………… .………………………14 1.4.3 Regulation of apical-basal polarity…………………………………… 18 1.4.4 Downstream target genes of AGAMOUS……………………………….19 1.5 Functions of AT-hook DNA binding proteins during development…………… .…20 1.6 Objective of the study……………………………………………………………….23 1.7 Significance of this study………………………………………………………….23 CHAPTER 2: Materials and Methods………………………………………… … 25 2.1 Materials……………………………………………………………………………25 iii 2.1.1 Plant materials………………………………………………… ……… 25 2.1.2 Bacterial strains………………………………………………………….25 2.2 Agrobacterium-mediated plant transformation………………………………………26 2.2.1 Preparation of Agrobacterium tumefaciens competent cells for electroporation-mediated gene transfer…………………………….…….26 2.2.2 Agrobacterium transformation…………………………… .……………26 2.2.3 Plant transformation…………………………………………… .………27 2.3 Dexamethasone treatment……………………………………………………………27 2.4 Plant observation and photography………………………………………………… 28 2.5 Scanning electron microscopy……………………………………………………….28 2.6 Generation of RNAi silencing lines………………………………………………….28 2.7 Extraction of plant genomic DNA…………………………………………… .……29 2.8 Expression analysis………………………………………………………………… 30 2.8.1 RNA isolation………………………………………… .……………….30 2.8.2 Reverse transcription…………………………………………………….31 2.8.3 Real-time PCR……………………………………………………… ….31 2.9 RNA in situ hybridization………………………………………………………….32 2.9.1 In vitro transcription……………………………………………………32 2.9.2 Fixation of floral tissues………………………………………………….33 2.9.3 Dehydration and wax embedding 33 2.9.4 Tissue sectioning……………………………………………………… 34 2.9.5 Pre-hybridization……………………………………………………… 35 2.9.6 Hybridization…………………………………………………………….35 iv 2.9.7 Post-hybridization 36 2.10 ETT promoter analysis…………………………………………… ………………37 2.10.1 Constructs………………………………………………….…………….37 2.10.2 Transgenic plants and DEX treatments…………………………………38 2.10.3 GUS staining…………………………………………………………… 38 2.11 Antigen purification and generation of polyclonal antibodies……… ……………39 2.12 Chromatin immunoprecipitation……………………………………………… ….39 2.13 Western blotting……………………………………………………………… .….42 2.14 South-western blotting (in vitro MAR binding assay)…………………………… 43 2.15 Isolation of nuclear matrix…………………………………………………………44 2.16 Immunofluorescence staining and confocal microscopy………………………… 45 CHAPTER 3: Characterization of GIANT KILLER as a multifunctional modulator in reproductive patterning and differentiation 46 3.1 Introduction………………………………………………………………………….46 3.2 Results……………………………………………………………………… .…… 47 3.2.1 Nucleotide and protein sequences of GIANT KILLER……………….…47 3.2.2 AGAMOUS directly regulates the expression of GIK in developing flowers………………………… .……………………………………….50 3.2.3 Expression of GIK in inflorescence meristem and developing flowers….53 3.2.4 GIK protein expression in Arabidopsis tissues and its subcellular localization……………………………………………………………….56 3.2.5 Overexpression of GIK leads to reproductive defects …………… .… .59 v 3.2.6 Loss of function of GIK causes partial disruption of reproductive ……… development .…………………………………………… 62 3.2.7 GIK directly regulates ETT expression in flowers…………… ………65 3.2.8 GIK is a bona fide matrix protein and binds to ETT putative MARs in vitro and in vivo………………… ……………………………………69 3.2.9 MAR is essential for GIK-mediated ETT downregulation…………… 74 3.2.10 GIK-mediated ETT repression is associated with dynamic change of dimethylated histone H3 at lysine 9…………………………… …….…77 3.2.11 Overexpression of GIK enhances weak mutant phenotype of ETT… ….80 3.2.12 GIK regulates a set of carpel regulators in flowers……………….… .…82 3.2.13 GIK binds to MAR regions of CRC, JAG and KNU promoters…………87 3.3 Discussion…………………………………………………………… .……………90 3.3.1 The balancing act of GIK on patterning and differentiation………… …90 3.3.2 Regulation of ETT by GIK……………………………………………….92 3.3.3 Evolutionary convergence on MAR-binding proteins with AT-hook motif……………………………………………………….… 94 CHAPTER 4: Characterization of GIANT KILLER as a dynamic regulator in meristem control and maintenance……………………………………………………97 4.1 Introduction…………………………………………….……………………………97 4.2 Results……………………………………………………………….………………98 4.2.1 Overexpression of GIK leads to floral and inflorescence meristems defects………… .………………………… ……………………… 98 vi 4.2.2 Loss of function of GIK leads to partial disturbance of meristem regulation…………… .…………………………………….………….100 4.2.3 Ectopic expression of GIK decreases WUS signals in meristems and induces ectopic formation of stem cell organizing centers……………102 4.2.4 Ectopic GIK activity downregulates WUS expression in inflorescences……………………………………………….………… 105 4.2.5 GIK binds directly to WUS promoter …………………………………108 4.2.6 GIK-mediated negative modulation of WUS is associated with change of dimethylated H3K9………………………….………… …………110 4.3 Discussion……………… ………………………………………………………112 CHAPTER 5: Characterization of GIANT KILLER2 in flower and meristems development……………………………………………………………………………116 5.1 Introduction…………………………………………………………………………116 5.2 Results………………………………………………………………………………116 5.2.1 Protein sequence alignment for GIK1 and GIK2.………………………116 5.2.2 Expression of GIK2 in inflorescence meristem and developing flowers …………………………………………………… ………… 119 5.2.3 Silencing of GIK2 causes reproductive and meristem defects similar to loss of function of GIK…………… .……………………….…………121 5.3 Discussion………………………………………………………………………… 123 CHAPTER 6: Concluding remarks……………………….…………………………124 vii References…………………………………………… .………………………………126 Appendix…………………………………………… .………………………… 152 viii SUMMARY The Arabidopsis homeotic gene AGAMOUS (AG) encodes a MADS domain transcription factor, and specifies reproductive organ identity during flower development. Microarray studies have highlighted that more than 1,000 genes are expressed downstream of AG. Nevertheless, very few of these potential target genes have been functionally characterized. My thesis work was initiated based on a bioinformatics screen looking for direct binding targets of AG. In the first part of my study, I demonstrate that GIANT KILLER (GIK) is a direct target of AG in the regulation of reproductive organs patterning and differentiation in Arabidopsis thaliana through binding assay and expression analysis. The GIK protein contains an AThook DNA binding motif that is widely found in chromosomal proteins and that binds to nuclear matrix attachment regions of DNA. Overexpression and loss of function of GIK cause wide-ranging defects in patterning and differentiation of reproductive organs. I show that GIK directly regulates the expression of several key transcriptional regulators, including ETTIN/AUXIN RESPONSE FACTOR 3, which pattern the gynoecium by binding to the matrix attachment regions of target promoters. In addition, I provide evidence that overexpression of GIK is closely associated with a dynamic change of a repressive histone mark in the ETT promoter. The results from the first part of my study suggest that GIK acts as a molecular node downstream of the homeotic protein AG, regulating patterning and differentiation of reproductive organs through modulation of multiple genes expression. ix The second part of my thesis work revolves around the role of GIK in the regulation of inflorescence and floral meristems development. I show that ectopic GIK activity is responsible for a breakdown of meristem homeostasis. I further demonstrate that GIK directly modulates the expression of WUSCHEL, an important gene involved in stem cells maintenance. The last part of my work center on the pilot study of a close member of GIK, the GIK2. I show that GIK2 has a very similar expression pattern to GIK in inflorescence meristems and reproductive organs. I further show that GIK2 may share a redundant function with GIK in the regulation of flower development and meristem regulation. In conclusion, my studies have suggested that GIK acts as a multifunctional chromatin organizer that could modulate and refine the expression of a large number of genes. x LIST OF TABLES AND FIGURES Table 1: List of the genes tested in the time-course analysis upon GIK induction……………………………………………………………84 Figure 1: Inflorescence meristem and floral meristem in Arabidopsis thaliana…….8 Figure 2: The ABCE model of flower development……………………………….12 Figure 3: The axes of polarity in lateral organs and the gynoecium .17 Figure 4: Nucleotide and protein sequences of GIK…………… .……………… 48 Figure 5: AG positively regulates GIK expression through direct binding to its genomic CArG sequence………….…………………… ………………51 Figure 6: RNA in situ hybridization of GIK in inflorescence meristem and developing flowers……………………………………………………….54 Figure 7: GIK protein expression and subcellular localization………………….…57 Figure 8: Overexpression of GIK disrupts normal reproductive development…….60 Figure 9: Loss of function of GIK leads to partial disruption of reproductive development…………………………………………………………… 63 Figure 10: GIK negatively regulates ETT expression in flowers……………………67 Figure 11: GIK is a nuclear matrix protein that binds to putative matrix attachment regions of ETT genomic DNA in vitro and in vivo…………….……… 71 Figure 12: MAR is essential for GIK-mediated ETT downregulation………………75 Figure 13: GIK-mediated ETT repression is associated with dynamic change of dimethylated histone H3 at lysine 9……………………………….…… 78 Figure 14: Overexpression of GIK enhances heterozygous and homozygous xi ett-3 mutants……………………………………………………… .……81 Figure 15: GIK regulates multiple carpel regulators……………………….……… 85 Figure 16: GIK binds to the MAR regions of CRC, JAG and KNU promoters in vivo…………………………………………………… .…………….88 Figure 17: Overexpression of GIK disrupts the integrity of floral and inflorescence meristems development…………………………………………… .… 99 Figure 18: Loss of function of GIK leads to partial disturbance of floral meristem regulation………………………………… …………………………101 Figure 19: Ectopic expression of GIK decreases WUS activity in meristems and induces ectopic stem cell organizing centers… .…………………… 103 Figure 20: Ectopic GIK activity downregulates WUS……………………………106 Figure 21: GIK binds directly to WUS promoter in vivo…………… .…………109 Figure 22: GIK-mediated WUS repression is associated with change of dimethylated H3K9………………………………………… …………111 Figure 23: Protein sequence alignment for GIK and GIK2…………………… ….118 Figure 24: GIK2 shows similar expression pattern to GIK in inflorescence meristem and developing flowers………………… …….…………….120 Figure 25: Silencing of GIK2 causes partial disruption of reproductive development and meristem regulation…………………………….……122 xii LIST OF ABBREVIATIONS AG AGAMOUS AGF1 AT-HOOK PROTEIN OF GA FEEDBACK REGULATION1 AGL AGAMOUS-LIKE AHL22 AT-HOOK MOTIF NUCLEAR LOCALIZED PROTEIN22 ANT AINTEGUMENTA AP1 APETALA1 AP2 APETALA2 AP3 APETALA3 ARF AUXIN RESPONSE FACTOR AS1 ASYMMETRIC LEAVES1 AS2 ASYMMETRIC LEAVES2 BP BREVIPEDICELLUS BSA bovine serum albumin CAL CAULIFLOWER CLV CLAVATA ChIP chromatin immunoprecipitation CRC CRABS CLAW CTAB mixed alkyltrimethyl-ammonium bromide CUC CUP-SHAPED COTYLEDONS CYC cycloheximide DAD1 DEFECTIVE IN ANTHER DEHISCENCE1 xiii DEPC diethyl pyrocarbonate DEX dexamethasone DIG digoxigenin DMSO dimethyl sulphoxide DTT dithiothereitol EDTA ethylenediamine tetraacetic acid ESC ESCAROLA ETT ETTIN FM floral meristem FIL FILAMENTOUS FLOWER FT FLOWERING LOCUS T GIK GIANT KILLER GIK2 GIANT KILLER2 GR glucocorticoid receptor GUS beta-glucuronidase INO INNER NO OUTER JAG JAGGED KAN KANADI KNOX KNOTTED1-LIKE HOMEOBOX KNU KNUCKLES LFY LEAFY LUG LEUNIG MAR matrix attachment region xiv MU MU-LIKE TRANSPOSASE NaOAc sodium acetate NUB NUBBIN OD optical density PBS phosphate buffered saline PCR polymerase chain reaction PFK PHOSPHOFRUCTOSE KINASE PHB PHABULOSA PHV PHAVOLUTA PI PISTILLATA PIN PINFORMED PMSF phenylmethylsulphonylfluoride REV REVOLUTA RNAi RNA interference RAM root apical meristem SAM shoot apical meristem SDS sodium dodecyl sulphate SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis SEM scanning electron microscopy SEP SEPALATA SPL SPOROCYTELESS SSC saline sodium citrate SHP SHATTERPROOF xv SPT SPATULA STM SHOOT MERISTEMLESS STY STYLISH SOB3 SUPPRESSOR OF PHYB-4#3 TE Tris-EDTA TEMED N,N,N’N’-tetramethylenediamine TUB TUBULIN WUS WUSCHEL YAB YABBY YUC YUCCA X-GLUC 5-bromo-4-chloro-3-indolyl-beta-glucuronic acid, cyclohexylammonium salt xvi [...]...LIST OF TABLES AND FIGURES Table 1: List of the genes tested in the time-course analysis upon GIK induction……………………………………………………………84 Figure 1: Inflorescence meristem and floral meristem in Arabidopsis thaliana ….8 Figure 2: The ABCE model of flower development …………………………… .12 Figure 3: The axes of polarity in lateral organs and the gynoecium .17 Figure 4: Nucleotide and protein sequences of GIK……………... promoter in vivo…………… ……… 10 9 Figure 22: GIK-mediated WUS repression is associated with change of dimethylated H3K9………………………………………… ……… 11 1 Figure 23: Protein sequence alignment for GIK and GIK2…………………… … .11 8 Figure 24: GIK2 shows similar expression pattern to GIK in inflorescence meristem and developing flowers………………… …….…………… .12 0 Figure 25: Silencing of GIK2 causes partial disruption of reproductive development. .. inflorescence meristems development ………………………………………… … 99 Figure 18 : Loss of function of GIK leads to partial disturbance of floral meristem regulation ……………………………… ……………………… 10 1 Figure 19 : Ectopic expression of GIK decreases WUS activity in meristems and induces ectopic stem cell organizing centers… …………………… 10 3 Figure 20: Ectopic GIK activity downregulates WUS………………………… 10 6 Figure 21: GIK binds directly to... reproductive development and meristem regulation ………………………….… 12 2 xii LIST OF ABBREVIATIONS AG AGAMOUS AGF1 AT-HOOK PROTEIN OF GA FEEDBACK REGULATION1 AGL AGAMOUS-LIKE AHL22 AT-HOOK MOTIF NUCLEAR LOCALIZED PROTEIN22 ANT AINTEGUMENTA AP1 APETALA1 AP2 APETALA2 AP3 APETALA3 ARF AUXIN RESPONSE FACTOR AS1 ASYMMETRIC LEAVES1 AS2 ASYMMETRIC LEAVES2 BP BREVIPEDICELLUS BSA bovine serum albumin CAL CAULIFLOWER CLV CLAVATA... lysine 9……………………………….…… 78 Figure 14 : Overexpression of GIK enhances heterozygous and homozygous xi ett-3 mutants……………………………………………………… …… 81 Figure 15 : GIK regulates multiple carpel regulators……………………….……… 85 Figure 16 : GIK binds to the MAR regions of CRC, JAG and KNU promoters in vivo…………………………………………………… …………….88 Figure 17 : Overexpression of GIK disrupts the integrity of floral and inflorescence meristems... direct binding to its genomic CArG sequence………….…………………… ……………… 51 Figure 6: RNA in situ hybridization of GIK in inflorescence meristem and developing flowers……………………………………………………….54 Figure 7: GIK protein expression and subcellular localization………………….…57 Figure 8: Overexpression of GIK disrupts normal reproductive development ….60 Figure 9: Loss of function of GIK leads to partial disruption of reproductive... reproductive development ………………………………………………………… 63 Figure 10 : GIK negatively regulates ETT expression in flowers……………………67 Figure 11 : GIK is a nuclear matrix protein that binds to putative matrix attachment regions of ETT genomic DNA in vitro and in vivo…………….……… 71 Figure 12 : MAR is essential for GIK-mediated ETT downregulation………………75 Figure 13 : GIK-mediated ETT repression is associated with dynamic change of. .. FLOWERING LOCUS T GIK GIANT KILLER GIK2 GIANT KILLER2 GR glucocorticoid receptor GUS beta-glucuronidase INO INNER NO OUTER JAG JAGGED KAN KANADI KNOX KNOTTED1-LIKE HOMEOBOX KNU KNUCKLES LFY LEAFY LUG LEUNIG MAR matrix attachment region xiv MU MU-LIKE TRANSPOSASE NaOAc sodium acetate NUB NUBBIN OD optical density PBS phosphate buffered saline PCR polymerase chain reaction PFK PHOSPHOFRUCTOSE KINASE PHB PHABULOSA... ChIP chromatin immunoprecipitation CRC CRABS CLAW CTAB mixed alkyltrimethyl-ammonium bromide CUC CUP-SHAPED COTYLEDONS CYC cycloheximide DAD1 DEFECTIVE IN ANTHER DEHISCENCE1 xiii DEPC diethyl pyrocarbonate DEX dexamethasone DIG digoxigenin DMSO dimethyl sulphoxide DTT dithiothereitol EDTA ethylenediamine tetraacetic acid ESC ESCAROLA ETT ETTIN FM floral meristem FIL FILAMENTOUS FLOWER FT FLOWERING LOCUS... PHAVOLUTA PI PISTILLATA PIN PINFORMED PMSF phenylmethylsulphonylfluoride REV REVOLUTA RNAi RNA interference RAM root apical meristem SAM shoot apical meristem SDS sodium dodecyl sulphate SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis SEM scanning electron microscopy SEP SEPALATA SPL SPOROCYTELESS SSC saline sodium citrate SHP SHATTERPROOF xv SPT SPATULA STM SHOOT MERISTEMLESS STY STYLISH . ………… 1 1. 2 Regulation of shoot apical meristem, inflorescence meristem and floral meristem in Arabidopsis thaliana …… ………………………………….4 1. 3 Regulation of flower development in Arabidopsis thaliana ……………………….9. Results…………………………………………………………………………… 11 6 5.2 .1 Protein sequence alignment for GIK1 and GIK2.…………………… 11 6 5.2.2 Expression of GIK2 in inflorescence meristem and developing flowers …………………………………………………… ………… 11 9 5.2.3 Silencing. …………………………………………………… 11 2 CHAPTER 5: Characterization of GIANT KILLER2 in flower and meristems development ……………………………………………………………………… 11 6 5 .1 Introduction……………………………………………………………………… 11 6 5.2 Results…………………………………………………………………………… 11 6