ANALYSIS OF RGA-LIKE 2 (RGL2), A GIBBERELLIC
ACID RESPONSE REGULATOR, IN THE FLOWER
DEVELOPMENT OF ARABIDOPSIS
THALIANA TAN EE LING
(B.Sc. HONS, NUS)
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
iii
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

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
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
vi
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

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

vii
SUMMARY
In this study, the role of a G
ibberellic 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 (R

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.
ix

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



xi
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
GA gibberellic 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
TBA tert-butanol
TBS Tris buffered saline
TE Tris-EDTA
TEMED N, N, N’N’ - tetramethylethylenediamine
Tris Tris(hydromethyl)-aminomethane
X-GAL 5-bromo-4-chloro-3-indolyl-β-galactopyranoside
xiv
Units and measurements
bp base pairs
ºC degrees Celsius
g gram(s) or gravitational force, according to the intended meaning
h hour
kb kilo base pair
kDa kilo Dalton
kV kilovolt
l liter
M mole per liter
mg milligram

RNA ribonucleic acid
oligo-dT oligodeoxythymidylic acid
PCR polymerase chain reaction
RT reverse-transcription
T-DNA transfer of DNA
1
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

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
3
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-


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
6
transduction of active GA signals. These pathways interact and interlink to ensure that
GAs are regulated at the proper endogenous quantities in plants.


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 GA
9

to GA
4
, and GA
20
to GA
1
, 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 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).

GA
12/53

GA
9/20

GA
4/1

(bioactive)
GA receptor
GA response
GA 20-oxidase
and
GA 3β-
hydroxylase
GA
51/29

GA 2-oxidase
GA

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