A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

xiii CHAPTER 1 ...1 GENERAL INTRODUCTION...1 CHAPTER 2 ...5 LITERATURE REVIEW ...5 2.1 Introduction...5 2.2 Regulation of gibberellins in plants ...6 2.2.1 The gibberellin biosynthes

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

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

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 1

GENERAL INTRODUCTION 1

CHAPTER 2 5

LITERATURE REVIEW 5

2.1 Introduction 5

2.2 Regulation of gibberellins in plants 6

2.2.1 The gibberellin biosynthesis pathway 6

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 3 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

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 4 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

4.3.1 Cloning full-length RGL2 into modified pGreen vectors 81

4.3.2 Genomic DNA screening of transgenic Arabidopsis plants 86

4.3.3 Phenotypic analyses of transgenic Arabidopsis plants 90

4.3.4 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 5 102

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

SUMMARY

In this study, the role of a Gibberellic Acid (GA) regulator named RGA-Like

2 (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 1 to stage 7 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

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 N-terminal region This region could be targeted by the putative kinase for phosphorylation

LIST OF TABLES

Table 4.1 List of primers used in experiments mentioned in Chapters 4 and 5 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

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 3 weeks after germination (Scale bars = 1 cm) 9 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

Figure 4.3 The completed construct pGreen-HY103 (modified) 35S::RGL2 .71

Figure 4.4 The completed construct pGreen-HY102 (modified) 35S::RGL2::GR .73

Figure 4.5 Expected band sizes of RGL2 and GR in PCR amplification 82

Figure 4.6 RGL2 was cloned into pGEM®-T Easy vector as an intermediate step .84

Figure 4.7 Excision of TAP from the pGreen GR-TAP vector backbone .85

Figure 4.8 PCR amplification results of RGL2 clones 87

Figure 4.9 PCR screening of overexpression transgenic lines 35S::RGL2 .89

Figure 4.10 PCR screening of 35S::RGL2::GR overexpression lines 91

Figure 4.11 Phenotypes of 35S::RGL2 overexpression lines .92

Figure 4.12 Phenotypes of 35S::RGL2::GR transgenic lines .94

Figure 4.13 SEM images of sepal cells from transgenic overexpression lines 95

Figure 4.14 RGL2 expression in constitutive and conditional overexpression lines .97 Figure 5.1 The completed construct pGreen-HY103 35S::RGL2::TAP 107

Figure 5.2 The completed construct pGreen-HY102 35S::RGL2::GR::TAP 109

Figure 5.3 The completed construct pGreen-LC101 35S::RGL2::StrepII .111

Figure 5.4 The triple mutant ga1-3 rga rgl2 120

Figure 5.5 Analyses of RGL2 expression in ga1-3 rga rgl2 transgenic plants 122

Figure 5.6 First optimization of application of dexamethasone .123

Figure 5.7 Independent ga1-3 rga rgl2 transgenic line showing strong complementation of RGL2::GR 124

Figure 5.8 Overall view of complementation analyses 125

Figure 5.9 Time course expression of floral organ identity genes after induction of RGL2 expression .128

Figure 5.10 PCR screening of RGL2::GR::TAP transgenic lines .131

Figure 5.11.Floral tissues of independent transgenic lines of ga1-3 rga rgl2

RGL2::GR::TAP .133

Figure 5.12 Phenotypes of ga1-3 rga rgl2 RGL2::GR::TAP transgenic lines treated with dexamethasone 135

Figure 5.13 PCR screening of transgenic lines expressing RGL2::TAP .136

Figure 5.14 Phenotypes of overexpression of RGL2::TAP in different backgrounds. .138

Figure 5.15 Verification of 35S::RGL2::StrepII construct and protein function 140

Figure 5.16 Silver-stained acrylamide gel of protein samples after purification 143

Figure 5.17 Screenshots of MS sequencing results .145

LIST OF ABBREVIATIONS

Chemicals and reagents

AEBSF 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride

BCIP 5-bromo-4-chloro-3-indolyl phosphate, toluidine salt

DEPC diethylpyrocarbonate

dNTP deoxyribonucleic acid triphosphate

EDTA ethylenediaminetetraacetic acid

EGTA ethylene glycol-bis[β-aminoethyl ether]-N, N, N’N’-tetraacetic acid

IPTG isopropyl-1-thio-β-D-galactopyranoside

NBT 4-nitro blue tetrazolium chloride

PBS phosphate buffered saline

SDS sodium dodecyl sulfate

SSC standard saline citrate

Units and measurements

g gram(s) or gravitational force, according to the intended meaning

h hour

CaMV cauliflower mosaic virus

cDNA complementary deoxyribonucleic acid

DNA deoxyribonucleic acid

mRNA messenger ribonucleic acid

RNA ribonucleic acid

oligo-dT oligodeoxythymidylic acid

PCR polymerase chain reaction

T-DNA transfer of DNA

CHAPTER 1

GENERAL INTRODUCTION

Growth and development of plants are subjected to many intrinsic and extrinsic influences Plant hormones, in particular, are crucial and vital to the survival

of plants These hormones are involved in many stages throughout the life cycle of

plants Gibberellic Acids (GAs), or gibberellins, are one such important class of plant

hormones They are involved in many aspects of plant development, including seed germination, hypocotyl elongation, floral induction, leaf expansion, apical dominance, floral development, fruit maturation and internode elongation

Like all plant hormones, the influence of gibberellins on growth regulation is controlled in two ways The first one is by controlling the quantity of available GA by modifying the biosynthesis and metabolism The second pathway of regulation is by altering the perception and signal transduction of active GA Early research on mutants defective in GA biosynthesis revealed the developmental role of gibberellins

An important GA-deficient biosynthesis mutant used for this project is the ga1-3

mutant (Koornneef and van der Veen, 1980) This mutant has a deletion of a gene

GA1 that encodes ent-CDP synthase, which is a crucial enzyme that is involved in GA

biosynthesis The mutant phenotype of ga1-3 is severe Compared to the wild-type

Arabidopsis, ga1-3 shows dwarfed and bushier statures (because of the reduced apical

dominance), darker green leaves, delayed flowering in short days, and retarded growth of floral organs, especially the male organ, stamens (Koornneef and van der Veen, 1980)

Investigation of GA signal transduction mutants has revealed three important

players in GA signaling The first, spy (spindly), is a recessive

GA-constitutive-response slender mutant The corresponding gene is a negative regulator of stem elongation and mutations in this gene results in early flowering and stem elongation

(Jacobsen and Olszewski, 1993) The next important GA signaling mutant, gai (gibberellic acid insensitive), 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) family of regulatory proteins These proteins are unique only to plants and are involved in many aspects of plant development Currently, there are 38

known Arabidopsis GRAS family members and all contain a conserved central region

and a C-terminal region in their protein sequences The diversity lies in the acidic terminal region Within this large family of transcriptional regulators, there lies a small subfamily of GRAS regulatory proteins called the DELLA subfamily, which includes RGA and GAI that have a unique conserved DELLA domain at the N-terminus (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 GA-insensitive dwarf phenotype This was important for the inactivation of RGA and GAI

N-by the GA signal Functional orthologs of RGA and GAI were also isolated from

economically important food crops Thus the DELLA genes were deemed important

in agriculture, especially during the era of the ‘Green Revolution’ (Peng et al., 1999b)

In a previous study, loss of function mutants, rga and gai, could not,

individually or in combination, rescue the germination and floral development defects

of ga1-3 in the absence of exogenous GA (Dill and Sun, 2001) It was hypothesized

that additional genes or signaling components were necessary in modulating these two aspects of plant development in GA signaling Previous studies have also revealed

that apart from RGA and GAI, the other DELLA members were RGA-Like 1 (RGL1),

RGA-Like 2 (RGL2) and RGA-Like 3 (RGL3) These 5 DELLA gene family members

possess two highly conserved 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 postulated by Lee et al (2002) that the RGL genes, which shared high homology with RGA and GAI, could be implicated in seed germination and flower development The specific role of RGL2 in flower development had not been

fully understood and may have functional redundancy, due to the high homology

shared among these RGL genes

The following progress revealed that the DELLA proteins are important in flower development and seed germination Recently, it was found that in the GA signaling pathway, DELLA proteins regulated floral homeotic genes in flower development (Yu et al., 2004) Synergistic effects of DELLA proteins in flower

development and seed germination in Arabidopsis were also observed (Tyler et al., 2004) Loss of function of RGA, GAI, RGL2 and RGL1 DELLA genes led to light- and gibberellin-independent seed germination in Arabidopsis (Cao et al., 2005) Most

importantly, a GA receptor was recently isolated from rice A GA-insensitive dwarf

mutant of rice named gid1, was identified due to loss of function of a soluble receptor

mediating GA signaling (Ueguchi-Tanaka et al., 2005) With these findings in consideration, the following objectives were proposed

The main research objectives in this project were:

1 to analyze RGL2 expression during floral transition and different stages of flower development in Arabidopsis

2 to study the constitutive and conditional overexpression of RGL2 in

Arabidopsis

3 to investigate potential relationships among RGL2, floral organ identity genes

and DELLA gene family members

This project focussed on studying the effect of RGL2 on floral development using

molecular genetics approaches This would shed light on how RGL2 may interact with other DELLA genes to affect the floral organ identity genes, and provide insights

into the understanding of GA signaling pathway in Arabidopsis floral development

CHAPTER 2

LITERATURE REVIEW

2.1 Introduction

All botany students would have learned the importance of the different classes

of plant hormones affecting various aspects of plant development Gibberellic acids/gibberellins (GAs) are an important group of plant hormones It was discovered

as an active compound first isolated from an ascomycete Fusarium species at the sexual stage, called Gibberella fujikuroi This was discovered in Japan in the 1930s

where rice farmers noticed that a particular fungal species caused a serious disease in rice plants They described the plants afflicted with the disease as bakanae-byo, or

“foolish seedling disease” The symptoms of these infected plants included excessive growth in height of the rice seedlings, and a decline in seed production in mature plants The active compound, gibberellin, was subsequently discovered and named after the disease-causing fungus (Eckardt, 2002)

This new class of essential endogenous plant hormones was instrumental in regulating various aspects of plant development These include the promotion of seed germination, leaf expansion, cell elongation, hypocotyl and stem elongation, apical dominance, regulation of flowering time, fruit maturation and floral development (Dill and Sun, 2001; Sun 2000; Richards et al., 2001; Harberd et al., 1998)

Gibberellins are a group of tetracyclic diterpenoid carboxylic acids Only a few are known to have biological functions even though there are over a hundred identified gibberellins (Yamaguchi and Kamiya, 2000) Gibberellins are regulated by the biosynthesis and metabolism pathways, as well as by the perception and

transduction of active GA signals These pathways interact and interlink to ensure that GAs are regulated at the proper endogenous quantities in plants

2.2 Regulation of gibberellins in plants

2.2.1 The gibberellin biosynthesis pathway

The biosynthesis of gibberellins is regulated by both endogenous and environmental signals The biosynthesis pathway is controlled by three classes of enzymes namely, terpene cyclases, Cyt P450 monooxygenases, and 2-oxoglutarate-dependent dioxygenases (Hedden and Kamiya, 1997) These enzymes are involved in the creation of bioactive GAs in the following process Diterpenes and carotenoids have a common precursor that is geranylgeranyl diphosphate (GGDP) This compound is converted to a tetracyclic hydrocarbon, ent-kaurene, by two terpene cyclases, copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) This process is carried out in plastids In the next stage, ent-kaurene is oxidized by Cyt P450 monooxygenases to form GA12-aldehyde, which happens in the endoplasmic reticulum In the last stage, GA12-aldehyde is converted to bioactive GAs by 2-oxoglutarate dependent dioxygenases, that are possibly cytosolic enzymes, including

GA 7-oxidase, GA 20-oxidase, GA 3β-hydroxylase, and GA 2-oxidase The genes that encode for these GA biosynthesis enzymes were isolated from pumpkin where it was found that the endosperm from immature pumpkin seeds contained a high concentration of GA biosynthetic enzymes (Hedden and Kamiya, 1997) Subsequently, the genes involved in GA biosynthesis were also isolated from

Arabidopsis using molecular genetics approach The GA1, GA3 and GA4 genes were

found to encode for CPS, ent-kaurene oxidase and GA 3β-hydroxylase, respectively, which are all critical enzymes in the generation of bioactive GAs (Helliwell et al., 1998) Some of these enzymes can catalyze the same reaction for different, but structurally similar substrates E.g., GA 3β-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 (1997a) 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 results showing cellular localization of GA1 by GA1 promoter-GUS reporter gene expression and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) The promoter activity was limited to specific cell types, namely, shoot apices, root tips, anthers and immature seeds This organ/tissue-specific

expression of GA1 correlated to the mutant phenotype of ga1 The ga1-3 is a GA deficient mutant where the function of GA1 is lost due to a large deletion leading to dramatic developmental abnormalities The severe phenotype of ga1-3 include dwarfism, darker green leaves and bushier stature than wild-type Arabidopsis

(reduced apical dominance), ungerminated seeds, delayed flowering in short days, and abnormal flowers with retarded growth of floral organs (Figure 2.2) These phenotypes can be reverted back to wild-type characteristics upon exogenous application of GA

Figure 2.1 Model showing the homeostatic regulation of GA biosynthesis

Feedback inhibition is shown by the T-bar GA 20-oxidase and GA 3β-hydroxylase transcripts are negatively regulated by GA activity The arrows indicate feedforward upregulation Adapted from Yamaguchi and Kamiya (2000)

GA12/53

GA9/20

GA4/1 (bioactive)

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 3 weeks after germination (Scale bars = 1 cm)

Other ga1 mutant alleles possess less dramatic phenotypic aberrations compared to ga1-3 For instance, ga1-6 mutation results also in a dwarf phenotype

albeit less severe, and can germinate and set seed in the absence of exogenous GA

The ga1-6 mutation is caused by an amino acid substitution in ent-CDP synthase and thus reduced enzyme activity Compared to the ga1-3 mutant, the ga1-6 mutant contains higher levels of endogenous GA In this study, ga1-3 is the only GA

biosynthesis mutant used for genetic crossing and other major investigations

2.2.2 The 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 with the GA signaling pathway, control GA-response gene expression to affect plant development The first model used to study the GA response genes was the cereal aleurone (Lovegrove et al., 2000) The expression of α-amylase gene, the most abundant hydrolase found in the aleurone layer, was directly induced by GA, which was used as an indicator for measuring the

GA response in this tissue GA-response mutants are classified into two groups The first phenotypic category comprises the elongated slender mutants that have longer petioles, paler green leaves, taller stems and lower fertility as compared to wild-type plants This kind of phenotype is similar to wild-type plants being excessively treated with a high dose of GA The second category is the GA unresponsive dwarf mutants that look similar to the GA biosynthesis mutants, except that the defects are not

reverted back to wild-type plants when exogenous GA is applied They are semi- or extreme dwarfs that also possess impaired seed germination, compact dark green leaves, delayed flowering, and aberrant flower development (Sun, 2000) The following section will discuss briefly some of the key components in the GA signaling pathway, which comprise several recently isolated positive and negative

regulators of GA signaling Some crucial positive regulators include DWARF1 (D1) (Mitsunaga et al., 1994) and GA-Insensitive Dwarf 2 (GID2) (Sasaki et al., 2003) from rice and SLEEPY1 (SLY1) (Steber et al., 1998) from Arabidopsis Their

corresponding mutants possess a semidwarf characteristic, and their phenotypes

cannot be rescued by exogenous GA application The study on D1 revealed that it

codes for a putative α-subunit of the heterotrimeric G proteins in rice A null mutation

of the gene encoding the α-subunit of the G protein in Arabidopsis (GPA1) displayed

a 100 –fold less responsiveness to GA (Ullah et al., 2002) This case, which

investigated the role of heterotrimeric G proteins in Arabidopsis seed germination

regulation, as well as heterotrimeric G proteins in GA signaling in other plant species (Iwasaki et al., 2003; Fujisawa et al., 2001), shows the importance of these proteins in regulating different aspects of plant development

GID2 and SLY1 code for homologous F-box proteins The mutant sly1, is a

GA-insensitive dwarf, and the role of the wild-type allele in Arabidopsis is a positive

regulator of GA signaling These F-box proteins are subunits of the SCF complex (Skp1, Cullin, F-box components), which is a class of the ubiquitin E3 ligases This complex also contains a RING-domain protein, the Rbox (McGinnis et al., 2003) The C-terminal end of F-box proteins is important, as it is the crucial protein-protein interacting region responsible for recruiting target proteins to the SCF E3 complex for ubiquitination and ultimate degradation by the 26S proteasome Such SCF-mediated

proteolysis is important in regulating plant developmental processes, which include floral development, light-receptor signaling, circadian rhythm, senescence, and hormone signaling (Sun and Gubler, 2004) It seems that GID2 and SLY1 may modulate GA signaling responses by controlling the stability of negative regulators such as SLENDER RICE1 (SLR1) and RGA, respectively

PICKLE (PKL) and Photoperiod-Responsive 1 (PHOR1) are two other putative positive regulators of GA signaling that are involved in other aspects of plant

development PKL could encode a putative chromatin-remodeling factor (Ogas et al.,

1997) The phenotype of its loss-of-function mutant is similar to that of other response mutants However, the mutant possesses a unique characteristic compared with other GA-biosynthesis or GA-response mutants in that the primary root meristem

GA-possessed features of embryonic tissue It seems that PKL could possibly mediate

GA-induced root differentiation during germination by repressing the expression of embryonic identity genes (Ogas et al., 1997) PHOR1 plays a role in leaf elongation under short day conditions in potato (Amador et al., 2001) Knockout analysis of the gene resulted in a semidwarf phenotype, as well as a reduced GA response in elongating internodes, while overexpressing this gene resulted in a phenotype similar

to GA constitutively expressed elongated mutants PHOR1 possesses seven armadillo repeats, which are also present in armadillo and β-catenins, proteins involved in Wnt

signaling in Drosophila and vertebrates While PHOR1 has been shown to activate

transcription of GA-induced genes, it also possesses a U box domain that is highly conserved among proteins involved in the ubiquitin proteasome pathway, indicating its role in proteasome degradation (Monte et al., 2003)

In addition, there are several important negative regulators of GA signaling

The first example is a recessive GA-response slender mutant named spindly (spy),

which resembles the wild-type plant treated with exogenous GA (Jacobsen and Olszewski, 1993; Jacobsen et al., 1998) This characteristic suggests a defect in the

negative regulation of GA signaling The spy mutant could germinate even in the

presence of paclobutrazol, which is a triazole derivative that inhibits GA biosynthesis

at the kaurene oxidase reaction Furthermore, additional alleles of this mutant were

found to be suppressors of ga1-3 and gai (Jacobsen and Olszewski, 1993) SPY

encodes a protein with a high similarity to O-linked GlcNAc (N-acetyl-glucosamine) transferases (OGTs), which are a class of enzymes that are known to regulate protein activity via glycosylation of serine or threonine residues The protein, SPY, is a negative regulator of stem elongation (Jacobsen and Olszewski, 1993) It has been

speculated that SPY plays a pivotal role in influencing GA signaling via O-linked

GlcNAc modification of the DELLA proteins

The next two examples of GA 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 (Peng et al., 1997), together with Reduced height 1-3 (Rht1-3) mutant in wheat, and D8 and D9 in maize, are semi-dominant GA response mutants

These mutants accumulate high levels of bioactive GAs in the plants However, because these mutants were gain-of-function mutants, their roles were unclear until the loss-of-function (null) alleles were isolated These mutants could partially restore

the phenotype of ga1-3 to the wild-type phenotype, and are able to survive on media

containing paclobutrazol Sequence analysis of RGA and GAI reveal that they are 82% identical at the amino acid level and possess features typical of transcriptional

regulators These features include nuclear localization signals, homopolymeric serine and threonine sequences, leucine heptad repeats and an SH2 (Src Homology 2 phosphotyrosine domain)-like domain (Lee et al., 2002) Another possible negative

regulator of GA signaling is SHORT INTERNODES (SHI) in Arabidopsis Its

loss-of-function mutant showed a GA-insensitive dwarf phenotype The predicted protein

is a RING-finger protein with a zinc finger motif, which suggests a possible role in ubiquitin-targeted proteolysis and transcriptional regulation (Thomas and Sun, 2004)

2.3 Identification and characterization of a soluble gibberellin receptor

When the gibberellin signaling pathway was initially characterized in the late nineties, the various signaling components were gradually elucidated However, a membrane bound or soluble GA receptor was elusive for quite a long time The mechanism of GA perception was not clearly understood, until the GA receptor was

finally discovered recently by Ueguchi-Tanaka et al (2005) Gibberellin Insensitive

Dwarf1 (GID1) was a GA-insensitive dwarf mutant of rice and its corresponding gene

encoded for a soluble GA receptor, which was an unknown protein that has some sequence similarity to hormone-sensitive lipases A fusion protein of GFP::GID1 was observed to be localized in the nucleus To further confirm the function of GID1, a glutathione S-transferase (GST) fused to GID1 was analyzed and it showed high affinity for biologically active GA, while a mutated fusion protein of GST-GID1 had

no GA-binding affinity at all Yeast two-hybrid interactions revealed that GID1 was bound to the RGA ortholog in rice, SLR1, and that this interaction depended on the presence of GA The overexpression of GID1 in transgenic plants also revealed a phenotype observed in a plant with GA-hypersensitive responses This study thus

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 members in the Arabidopsis genome All of them possess a highly

conserved central domain with the amino acids: valine, histidine, isoleucine, isoleucine and aspartic acid (VHIID), and a C-terminal region comprising of: arginine, valine, glutamic acid and arginine (RVER) The divergence lies in the N-terminal region for these proteins RGA and GAI contain a conserved sequence near their N-termini, named the DELLA 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 proteins turn into constitutive repressors of GA signaling (Dill et al., 2001)

Functional orthologs of RGA and GAI have been isolated from other economically important plant species They include Rht from wheat, D8 from maize,

SLENDER1 from barley (SLN1) and SLENDER RICE1 (SLR1) (Gubler et al., 2002;

Ikeda et al., 2001; Peng et al., 1999b) All these proteins were characterized to be

repressors of GA signaling as well, indicating that the RGA/GAI function is conserved among monocotyledons and dicotyledons These DELLA proteins are considered to be important in agriculture as their applicability includes the high yielding wheat varieties used in the ‘Green Revolution’ These varieties of wheat were successful because they were resistant and hardy compared to other varieties

This was especially clear in the dwarf mutant carrying the rht-1 allele that possessed

reduced height qualities (Peng et al., 1999b)

A study by Dill and Sun (2001) generated homozygous rga/gai double mutant lines in the wild-type and ga1-3 backgrounds This study revealed that rga and gai

null alleles had synergistic effects on a number of GA mediated processes

Loss-of-function also affected feedback regulation of GA biosynthesis gene GA4 This study

highlighted that GA signaling can control GA biosynthesis through a feedback mechanism and that quantitative differences in GA signaling can determine degree of derepression of the pathway in different cells Thus, proper balance must be struck between bioactive GA signal and activity of GA signaling, which is important for proper plant development

2.5 RGL genes and their implications in plant development

An important aspect mentioned earlier revealed that neither rga-24 nor gai-t6,

individually or in combination, could rescue the germination and flower development

defects in the ga1-3 background This indicated that there were other factors having

an influence on these two aspects of development In the DELLA subfamily of the GRAS regulatory proteins, there are three other members named RGA-Like 1 (RGL1), RGL2 and RGL3 in addition to RGA and GAI These five members possess two highly conserved N-terminal regions (regions I and II) containing the DELLA

domain in their open reading frames (Figure 2.3) Their C-terminal domains also share high homology The sequences of RGL1, RGL2 and RGL3 are closely related

to those of RGA and GAI

Sequence similarity between RGL1 and RGL2, RGL1 and RGL3, RGL2 and

RGL3 is 59%, 58% and 68%, respectively The three RGL genes show 58%, 55% and

54% sequence similarity to GAI, respectively, and 55%, 57% and 53% sequence similarity to RGA, respectively (Sun and Gubler, 2004)

The three RGL proteins show such high amino acid sequence conservation, especially in regions I and II, implying that these three members may also be GA response regulators (Figure 2.4) Some recent studies on isolating knockout mutants

of RGL genes using reverse genetics have explored the roles of the RGL genes in

GA-regulated germination and floral development (Lee et al., 2002; Wen and Chang,

2002; Cheng et al., 2004) The functions of the RGL genes have been gradually

revealed in multiple mutant backgrounds because of their potential functional

redundancy In particular, the mutant phenotypes are more visible in the ga1-3 background because the RGL genes, like RGA and GAI, are more active in the GA

deficient conditions

2.6 Characterization of RGL genes

RGL1 has been found to be a negative regulator of gibberellin responses according to Wen and Chang (2002) Gain-of-function and loss-of-function

phenotypes of rgl1 mutants were isolated Their phenotypes were similar to gai and

rga with some important distinctions Gain-of-function, co-suppression and loss-of-

function lines gave rise to different phenotypic changes The overexpression of RGL1

Figure 2.3 Amino acid sequence alignment of the predicted DELLA proteins

Shown here is the alignment of RGA, GAI, RGL1, 2 and 3 of Arabidopsis (Lee et al.,

2002) The GA response regions are in regions I and II, as predicted by Peng et al

(1999b) (Reproduced with permission from Peng J)

Figure 2.4 The DELLA subfamily of the GRAS regulatory proteins

This structure shows the N-terminal DELLA domain from which the name of this subfamily of proteins is derived This region contains two highly conserved motifs called the DELLA and VHYNP (grey areas) A polymeric serine/threonine (poly S/T) region also exists in the DELLA domain The C-terminal or GRAS domain, containing the Leucine heptad repeats (LHR1 and LHR2), the nuclear localization signal (NLS) and the SH2-like domain, are conserved among GRAS family members

Adapted from Sun and Gubler (2004)

with a 17 amino acid deletion containing the DELLA sequence, resulted in flowers that were severely underdeveloped and male sterile (Wen and Chang, 2002) A co-suppression line that reverted to wild-type phenotype retained the transgene, and under GA-deficiency, exhibited resistance to paclobutrazol effects of inhibition of seed germination, stem elongation, delayed flowering and defective flower

development Using in situ hybridization studies, RGL1 expression was detected in

developing ovules and anthers throughout microspore development However, this

aspect was not thoroughly investigated RGL3 showed a similar expression pattern to

RGL1, but its detailed function is thus far unknown On the other hand, the remaining RGL gene, RGL2, has been reported, and its role in seed germination has been

clarified

2.7 RGL2 and seed germination

A study by Lee et al (2002) revealed that RGL2 was a GA signaling

component acting as a negative regulator of seed germination The RGL2 gene

expression was studied following the onset of imbibition in both the wild type and

ga1-3 mutant seeds The transcript level of RGL2 decreased rapidly in wild-type

germinating seeds once the radicle protrusion took place However, in imbibed,

non-germinating ga1-3 mutant seeds, the transcript levels remained high It was suggested that RGL2 prevents germination after imbibition and GA promotes germination through downregulation of RGL2 It was also proposed that GA regulated stem elongation via RGA and GAI, while seed germination was controlled via RGL2

Although these three regulators show high similarity in their amino acid sequences, this study showed that they function in different GA-mediated signaling pathways

However, phenotypes of the double mutant rgl2-1 ga1-3 and paclobutrazol resistance

analysis on seed germination showed that RGL2, but not RGL1, was a negative

regulator of seed germination This was contradictory to a previous study on RGL1

(Wen and Chang, 2002)

Lee et al (2002) also revealed that RGL2 had no or little effect on stem

elongation or leaf expansion using paclobutrazol resistance studies The mutant allele

rgl2-1 also did not suppress the dwarfism phenotype of ga1-3 Using RNA gel blot

analysis, RGL2 transcripts were detected in inflorescences, with high levels in young flower buds and significant levels in siliques In situ GUS staining patterns of rgl2-5

heterozygotes were also studied from the young seedlings to 40-day-old plants GUS staining was observed in almost all the floral organs with particularly strong staining

in stamen filaments, the top of the style, and sepals GUS staining was also detected at the base of young developing seeds in siliques It was proposed that RGL2 may act as

a negative regulator that represses the expression of genes encoding hydrolyzing enzymes, and such repression can be released in the presence of GA (Lee et al., 2002) Alternatively, RGL2 may act as a negative regulator that prevents cell expansion by other means

Since there were discrepancies in the studies reported in separate papers by Wen and Chang (2002) versus Lee et al (2002), the exact functional mechanism of

RGL2 is still not clear In the former study, no RGL2 expression was detected in inflorescences On the contrary, the latter study found high levels of RGL2 expression

in young inflorescences In a more recent study by Tyler et al (2004), it was found

that RGA and GAI were expressed ubiquitously in all tissues, but transcript levels of

RGL genes were elevated in germinating seeds, flowers and siliques RGL2 was

verified as the major repressor of seed germination, which overruled the findings by

Wen and Chang (2002) Yeast two hybrid interaction studies also revealed that RGL2 was quickly degraded by GA treatment in imbibed seeds, which was mediated by a F-box protein, SLY1 With these findings in mind, the aim of our project was to

specifically analyze the RGL2 expression during floral transition and different stages

of flower development from floral meristem development to silique formation The

RGL2 gene expression pattern in the backgrounds of different floral organ mutants

would also be explored Constitutive and conditional overexpression of RGL2 in

Arabidopsis would also be examined These studies would help to further elucidate

the role of RGL2 in the GA signaling pathway in Arabidopsis flower development

2.8 DELLA proteins are important integrators of multiple phytohormone

signaling pathways

In plant development, interactions or cross-talks among plant hormones are crucial to the balance and regulation of these hormone signals to ensure that plants develop and grow in the right way These intricate relationships can be illustrated by the DELLA proteins, which are of no exception to receive cross-talk signals from other plant growth hormones There are two recent case studies showing that the degradation of DELLA proteins is not just regulated by the GA signal but are also affected by two other plant hormones, auxin and ethylene In the first study by Fu and Harberd (2003), auxin that exuded from the shoot apical meristem, influenced root growth by regulating the cellular responses to GA GA is able to control the growth of roots by antagonizing the effects of the DELLA proteins RGA and GAI The experimental procedures of surgical ablation, as well as genetic disruption to decrease the flow of auxin from the shoot apical meristem, which in turn affected the polarity

of auxin transport, showed that the responsiveness of GA-deficient roots to exogenous

GA was drastically reduced This was a result of increased DELLA protein stability,

as they became more resistant to GA-induced destabilization once auxin transport was disrupted Root growth that lacked GAI and RGA was less constrained by inhibitors

of the polar auxin transport system than root growth containing GAI and RGA This interesting ‘relief of restraint’ effect indicates that the disruption of auxin flow and transport results in a delay in GA-induced disappearance of GAI and RGA in the nucleus, and an increased restraint on growth of roots

In a separate study, the effect of ethylene on plant development via the DELLA proteins was explored (Achard et al., 2003; Vriezen et al., 2004) The findings from this study revealed that ethylene had inhibitory effects on root growth, which was dependent on the DELLA proteins They found that roots lacking the two DELLA proteins, GAI and RGA, were resistant to the growth-inhibiting effects of ethylene Furthermore, ethylene application on transgenic GFP::RGA plants delayed GA-induced disappearance of the fusion protein On the other hand, plants lacking the ethylene signaling kinase, CTR1, caused a constitutive delay in the GA-induced disappearance of GFP::RGA fusion protein A third finding revealed that a prominent characteristic of ethylene response from etiolated seedlings, the apical hook, was much dependent on GA signaling as well as the release of DELLA protein-mediated growth restriction Thus, the findings from the above studies showed that both auxin and ethylene responses are also attributed to DELLA proteins While auxin promoted GA-dependent DELLA protein degradation, ethylene inhibited DELLA protein degradation This is a sure sign that these proteins are important in receiving and integrating signals from other classes of phytohormones as well for their critical roles

in plant development

2.9 The ubiquitin 26S proteasome pathway is involved in the degradation of

DELLA proteins

In recent years, the ubiquitin proteasome pathway has been found to be an important part of cellular regulation in eukaryotes The regulation of protein degradation at the precise moment is important for plant development, such as embryogenesis, hormone signaling and senescence (Moon et al., 2004) In brief, the ubiquitin pathway works in the following manner The molecule, ubiquitin, is attached to lysine residues in the substrate protein, thereby targeting it for degradation

in the 26S proteasome pathway Ubiquitin is conjugated to a substrate with the help of three enzymes, ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3) The 26S proteasome is a multisubunit complex made up of a 20S core protease flanked on each end by a 19S regulatory component Once the protein is degraded in the proteasome into short peptides, the amino acids are recycled (Yang et al., 2004) The E3 ubiquitin ligases consist of a large family of highly diversified proteins, which include the RING domain proteins One of the small classes of proteins that have the RING domain in multisubunits is the SCF complex The SCF class of E3 ligases has been thoroughly studied in plants The SCF

is named after three of its four subunits, namely, SKP1 (ASK in plants for

Arabidopsis SKP1), CDC53 (also known as Cullin), and the F-box protein The last

subunit is called the RING finger protein RBX1 (for Ring-Box 1) The SCF complex

is known to target transcription factors involved in development and signal transduction (Itoh et al., 2003) The F-box proteins, whose conserved motifs bind to

ASK/SKP, are the largest superfamily of proteins in the Arabidopsis genome Their

N-terminus consists of the F-box motif, and the remaining part is heavily involved in protein-protein interactions needed for substrate binding It has been suggested that the ubiquitin proteasome pathway destabilizes DELLA proteins via polyubiquitination by an SCF E3 ubiquitin ligase and ultimate destruction in the proteasome A number of recent studies have delved into this aspect

The cloning of GID2 and SLEEPY1 (SLY1) from rice and Arabidopsis,

respectively, unveiled the F-box components from a SCF complex (Gomi et al., 2004;

Fu et al., 2004) that interacted specifically with DELLA proteins (Dill et al., 2004; Gomi et al., 2004; Fu et al., 2004) The GID2 and SLY1 proteins contained a conserved F-box interaction domain that could associate with the SKP1 component of

the SCF complex Loss of function of GID2 or SLY1 resulted in the accumulation of rice/Arabidopsis DELLA proteins SLR, GAI or RGA, respectively (McGinnis et al.,

2003; Sasaki et al., 2003; Dill et al., 2004) Further analysis into the intricate interactions among these proteins also showed that a lack of GAI/RGA or SLR1 suppressed the dwarfing phenotype resulted from the lack of SLY1 or GID2 Observations from these studies suggested that GID2 and SLY1 were F-box components of a SCF complex targeting specifically DELLA proteins for proteasome destruction (Alvey and Harberd, 2005)

Protein modification plays a part in the interaction of the SCF complex with the target proteins Normally, one of the most common processes is phosphorylation

of the recognition domain of the targeted protein (Patton et al., 1998; Jackson and Eldridge 2002) There is solid evidence that phosphorylation of the DELLA proteins enhanced the interaction with SCFSLY1 or SCFGID2 (Fu et al., 2004; Gomi and Matsuoka, 2003; Gomi et al., 2004) Furthermore, the rice DELLA protein SLR1 was phosphorylated when GA was present (Sasaki et al., 2003) Another recent study