Functional characterization of giant killer in flower development and meristem regulation in arabidopsis thaliana 1

FUNCTIONAL CHARACTERIZATION OF GIANT KILLER IN FLOWER DEVELOPMENT AND MERISTEM REGULATION IN ARABIDOPSIS THALIANA NG KIAN HONG M.. In the first part of my study, I demonstrate that GI

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

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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

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

v

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

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

vii

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

References……… ………126 Appendix……… ……… 152

ix

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

AT-hook 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

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

xi

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

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

xiii

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

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

xv

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

SPT SPATULA

STM SHOOT MERISTEMLESS

STY STYLISH

SOB3 SUPPRESSOR OF PHYB-4#3

TE Tris-EDTA

TEMED N,N,N’N’-tetramethylenediamine

TUB TUBULIN 2

WUS WUSCHEL

YAB YABBY

YUC YUCCA

X-GLUC 5-bromo-4-chloro-3-indolyl-beta-glucuronic acid, cyclohexylammonium

salt