ROLE OF BRASSINOSTEROID CATABOLISM IN

ARABIDOPSIS DEVELOPMENT

By

KULBIR SINGH

A dissertation submitted in partial fulfillment of

the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY

Department of Crop and Soil Sciences

MAY 2013

ii

To the Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of KULBIR

SINGH find it satisfactory and recommend that it be accepted.

Michael Neff, Ph.D., Chair

Scot Hulbert, Ph.D.

Camille Steber, Ph.D.

iii

ACKNOWLEDGEMENTS

I am grateful to my major advisor Dr. Michael Neff for giving me the opportunity to work in his

lab, for his continuous support, patience, and motivation. I believe that his scientific training and

guidance will always help in my future scientific career as well in other areas of life. I am also

grateful to my graduate committee members, Dr. Camille Steber, Dr. Scot Hulbert and Dr.

Andris Kleinhofs for their valuable suggestions, critical analysis of my research and their

academic support.

I would also like this opportunity to thank all members of the Neff lab for their valuable

suggestions, encouragement and affectionate attitude. My special thanks to Pushpa Sharma for

all her help and contributions during the course of my research work. Special thanks to Dan

Dreesmann and the staff of Plant growth facilities for managing and taking care of the facilities

which helped me to complete my research work in time. My thanks also go to the faculty, office

staff and students of the Department of Crop and Soil Sciences for their friendly attitude and

assistance.

My respectful thanks are due to my parents, my brother and sister-in-law, and my nephew

Keerat who always inspired me to continue my research. The moral support, encouragement and

sacrifice of my loving wife Aman remained my source of inspiration during my PhD. Last but

not the least; I would like to earnestly thank all my friends in Pullman for being an incessant

source of love, support and comfort during my stay in Pullman.

iv

ROLE OF BRASSINOSTEROID CATABOLISM IN

ARABIDOPSIS DEVELOPMENT

Abstract

by KULBIR SINGH, Ph.D.

Washington State University

May 2013

Chair: Michael Neff

BAS1 (phyB-4 ACTIVATION TAGGED SUPPRESSOR 1) and SOB7 (SUPPRESSOR OF

phyB-4 7) are brassinosteroid-catabolizing P450s in Arabidopsis thaliana that

synergistically/redundantly modulate photomorphogenic traits such as flowering time. This study

investigates the role of BAS1 and SOB7 in photomorphogenesis by studying null-mutant genetic

interactions with the photoreceptors phyA, phyB and cry1 with regard to seed-germination and

flowering time. The removal of BAS1 and/or SOB7 rescued the low germination rate of the phyA-

211 phyB-9 double-null mutant. With regard to floral induction, bas1-2 and sob7-1 showed a

complex set of genetic interactions with photoreceptor-null mutants. Histochemical analysis of

transgenic plants harboring BAS1:BAS1-GUS and SOB7:SOB7-GUS translational fusions

revealed overlapping and distinct expression patterns. BAS1’s expression in the shoot apex

increased during the phase transition from short-to-long-day growth conditions and requires

phyB in red light.

Application of this kind of genetic analysis approach to study other BR catabolic genes

required availability of the loss-of-function mutants. Our analysis shows that the ben1-1 (bri1

enhanced 1-1) mutant (T-DNA intronic insertion mutant of BEN1) was not suitable for multiple

mutant analyses which involve combining two or more T-DNA insertion mutations. We

v

generated a genetic triple-mutant from a cross between the bas1-2 sob7-1 double-null (T-DNA

exonic insertion mutants) and ben1-1. The single ben1-1 line behaved as a transcript null.

However, in the triple-mutant background ben1-1 was reverted to a partial loss-of-function

allele. The enhanced expression of BEN1 remained stable when the ben1-1 single-mutant was re-

isolated from a cross with the wild-type. In addition, the two genetically identical pre-triple and

post-triple ben1-1 mutants also differed phenotypically. Restriction endonuclease analysis with

methylation sensitive enzymes demonstrated that the recovered ben1-1 mutant was

epigenetically different from the original ben1-1 allele.

In vitro biochemical analyses demonstrate that ATST4a has catalytic activity with BRs.

Taking a genetic approach in this study, we show that atst4a1-1 (a T-DNA insertion null mutant

of ATST4a) did not display reduced seedling hypocotyl growth inhibition in white light.

Additionally, overexpression of the ATST4a gene in transgenic lines did not result in any

characteristic BR-deficient phenotypes. These observations suggest that ATST4a is an atypical

BR catabolic gene.

vi

TABLE OF CONTENTS

ACKNOWLEDGEMENTS………………………………………………………….................iii

ABSTRACT…………………………………………………………………………………...…iv

LIST OF TABLES……………………………………………………………………………....ix

LIST OF FIGURES…………………………………………………………………………..…ix

DEDICATION…………………………………………………………………………………..xi

CHAPTER ONE

INTRODUCTION……………………………………………………………………….1

LITERACTURE CITED………………………………………………………………17

TABLES AND FIGURES………………..…………………………………………….38

CHAPTER TWO

Genetic interactions between brassinosteroid-inactivating P450s and photomorphogenic

photoreceptors in Arabidopsis thaliana

ABSTRACT……………………………………………………………………………..43

INTRODUCTION……………………………………………………………………...44

MATERIALS AND METHODS………………………………………………………48

RESULTS……………………………………………………………………………….51

vii

DISCUSSION…………………………………………………………………………...56

ACKNOWLEDGEMENTS……………………………………………………………59

LITERATURE CITED………………………………………………………………...59

TABLES AND FIGURES……………………………………………………………...67

CHAPTER THREE

The ben1-1 brassinosteroid-catabolism mutation is unstable due to epigenetic modifications

of the intronic T-DNA insertion

ABSTRACT……………………………………………………………………………..78

INTRODUCTION……………………………………………………………………...79

MATERIALS AND METHODS………………………………………………………81

RESULTS……………………………………………………………………………….83

DISCUSSION…………………………………………………………………………...88

ACKNOWLEDGEMENTS……………………………………………………………92

LITERATURE CITED………………………………………………………………..92

TABLES AND FIGURES……………………………………………………………..96

CHAPTER FOUR

The Arabidopsis gene ATST4a in not a typical brassinosteroid catabolic gene

ABSTRACT……………………………………………………………………………107

INTRODUCTION…………………………………………………………………….108

viii

MATERIALS AND METHODS……………………………………………………..110

RESULTS……………………………………………………………………………...112

DISCUSSION………………………………………………………………………….113

ACKNOWLEDGEMENTS …………………………………………………………..115

LITERATURE CITED……………………………………………………………….116

FIGURES………………………………………………………………………………120

CHAPTERS FIVE

SUMMARY AND FUTURE DIRECTIONS………………………………………...126

LITERATURE CITED……………………………………………………………….130

ix

LIST OF TABLES

1. Biochemical analysis of ATST4a BR catalytic activity……………………………….38

2. Percentage of seed germination in white light or darkness.………………………….67

3. Table of PCR primer sequences used in the study…………………………………..105

LIST OF FIGURES

CHAPTER ONE

1. BR biosynthesis pathway.................................................................................................39

2. Fluence rate response shows genetic redundancy between BAS1 and SOB7 for

hypocotyl growth repression in white light (A) and flowering time (B).....................40

3. Reactions catalyzed by the known BR catabolic enzymes………………………...…41

CHAPTER TWO

1. Adult phenotype of the genotypes used in this study…………………………………68

2. Genetic interactions between BAS1, SOB7, PHYA and PHYB to control flowering

time…………………………………………………………………………………...….69

3. Expression patterns of BAS1 and SOB7………………………………………………71

4. Histochemical GUS analysis of BAS1 expression in the shoot apex during the

transition to flowering…….…………………………………………………………....72

5. BAS1 expression in the shoot apex in red-light is dependent on the presence of

functional phyB…………………………………………………………………………73

6. Model based on the interpretation of the genetic interactions between

photomorphogenic photoreceptors and BAS1 and SOB7……………………………74

x

7. Genetic and molecular analysis of BAS1:BAS1-GUS and SOB7:SOB7-GUS

lines………………………………………………………………………………………75

CHAPTER THREE

1. ben1-1 mutation is unstable in the bas1-2 sob7-1 background………………………96

2. The BEN1 transcript levels stay steady in the re-isolated ben1-1 single null

lines………………………………………………………………………………………97

3. Re-isolated ben1-1 lines are phenotypically different from the original ben1-1

lines………………………………………………………………………………………98

4. The ben1-1 is a leaky mutant based on RT-PCR analysis and the enhanced T-DNA-

containing-intron splicing in recovered ben1-1 is not induced by BR feedback

regulation. ……………………………………………………………………………..100

5. The kanamycin resistance trait, conferred by the T-DNA resistance marker NPTII,

is absent in the bas1-2 sob7-1 ben1-1 and the recovered ben1-1 line……………….101

6. Genomic DNA in the NOS Promoter region of the T-DNA insertion in the recovered

ben1-1 shows methylation…………………………………………………………….102

7. The size and sequence of the T-DNA insertion remains unchanged in the pre-triple

and post-triple ben1-1 lines…………………………………………………………...103

8. Hypothetical model for RNAi and chromatin structure dependent intron

splicing............................................................................................................................104

CHAPTER FOUR

1. RT-PCR analysis of the atst4a1-1 T-DNA insertion line……………………………120

2. atst4a1-1 mutant do not show a hypocotyl-elongation phenotype in both dark and

continuous white light conditions…………………………………………………….121

xi

3. Overexpression of ATST4a do not lead to BR-deficient phenotypes in

Arabidopsis……………………………………………………………………………..122

4. Hypocotyl-elongation response of atst4a1-1 and ATST4a-OX T3 lines to dark and

continuous white light conditions…………………………………………………….123

5. Hypocotyl-elongation response of atst4a1-1 and ATST4a-OX T3 lines to BL

treatment……………………………………………………………………………….124

6. ATST4a-OX T3 lines did not display any feedback regulation of BR biosynthesis and

catabolic genes DWF4 and BAS1……………………………………………………..125

xii

Dedicated to my Parents…

1

CHAPTER ONE

INTRODUCTION

Brassinosteroids (BRs): BRs are growth-promoting hormones essential for normal development

of plants (Sasse, 2003; Kutschera and Wang, 2012). Genetic screens for Arabidopsis seedlings

with de-etiolated morphology when grown in the total darkness (short, thick hypocotyl,

expanded cotyledons, young primary leaves and high levels of anthocyanins) have led to the

identification of many BR biosynthetic genes. DE-ETIOLATED2 (DET2) (Chory et al. 1991) and

CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM (CPD) (Szekeres et al. 1996)

were among the first genes isolated in the above-mentioned screens. Other genes such as

DWARF 1 (DWF1) (Feldmann et al. 1989;Takahashi et al. 1995; Klahre et al. 1998; Choe et al.

1999a), DWF4 (Choe et al. 1998), DWF7/STE1 (Choe et al. 1999b) and HYPERSENSITIVE TO

ABSISIC ACID AND AUXIN (SAX1) (Ephritikhine et al. 1999a; Ephritikhine et al. 1999b) were

isolated in similar screens. Isolation of these genes in screens suggests that BRs are involved in

photomorphogenesis. Figure 1 shows important enzymes and the reactions they catalyze in the

BR biosynthesis pathway. For review on BR biosynthesis pathways and enzymes see: Fujioka

and Yokota, 2003; Zhao and Li, 2012.

Initially, the BR biosynthetic pathway was elucidated by analysis of endogenous BRs and

the conversion products of potential intermediates in Catharanthus roseus cell cultures. After

the identification of BR biosynthetic mutants in Arabidopsis, pea and tomato, these alleles

became the main tool for understanding BR biosynthetic pathways (Fujioka and Yokota, 2003).

Genetic and molecular studies of BR-insensitive mutants and their modifiers led to the

identification of several key components of the BR signal-transduction pathway, including the

2

receptor. For review on recent advances in BR signaling see: Ye et al. 2012; Kim and Wang

2010; Li 2010; Yang et al. 2011.

Various studies demonstrate that BR biosynthetic and BR perception genes are ubiquitously

expressed with increased transcript accumulation in actively growing tissues (Shimada et al.

2003; Bancos et al. 2002; Friedrichsen et al. 2000). The expression of DWF4 and CPD has been

demonstrated to play an important role in controlling tissue-specific BR levels (Kim et al. 2006;

Mathur et al. 1998). These genes are also important targets of feedback regulation (Tanaka et al.

2005; Guo et al. 2010). The receptor of brassinosteroids, BR-INSENSITIVE-1 (BRI-1) is also

ubiquitously expressed, with increased transcript accumulation in actively growing tissues

(Friedrichsen et al. 2000).

Hormone levels in a given tissue can be affected by the relative rates of de novo synthesis,

destruction, inactivation, and transportation within the plant (Bandurski et al. 1995). The balance

of these processes determines the endogenous level of any hormone in plant tissues. Recent

evidence suggests that unlike other plant hormones, BRs do not undergo long distance transport

within plants. For example, grafting experiments in pea and tomato demonstrate that BRs are not

transported long distances within plants (Reid and Ross, 1989; Symons and Reid, 2004; Montoya

et al. 2005). Experiments by Salvadi-Goldstein et al. (2007) involving tissue-layer-specific

expression of BR biosynthetic and perception genes in Arabidopsis demonstrate that BRs are not

efficiently transported even short distances. These studies suggest that every tissue maintains its

levels of BRs by autonomously regulating biosynthesis and catabolism.

Role of BRs in growth and development in plants: BRs are known to play diverse roles in

plant growth and development. Growth promotion is the most characteristic function of BRs in

3

plants. The most classical demonstration of this property is the second-internode growth

response induced in Phaseolus vulgari by application of exogenous BRs (Grove et al. 1979).

These growth responses are partially accomplished by the regulation of the downstream genes

involved in the various aspects of cell expansion (Goda et al. 2002; Shigeta et al. 2011). For

example early targets of BR responses include TCH4 which encodes xyloglucan

endotransglycosylase (XET) and is implicated in cell wall loosening during cell expansion (Xu et

al. 1995). Other mechanisms, such as reorientation of cortical microtubules in Arabidopsis

hypocotyl cells (Wang et al. 2012) and regulation of plasma membrane ion channels (Zhang et

al. 2005) are also part of BR-induced cell expansion. BRs are also known to stimulate cell

division. BR treatment of cell cultures of Chlorella vulgaris accelerates cell division and causes

an increase in the levels of nucleic acids and proteins (Bajguz 2000).

Apart from a general role in cell division and expansion, recent studies show that BRs are

also involved in various developmental aspects of plants including; determination of lateral

organ boundaries (Gendron et al. 2012; Bell et al. 2012), stomata development (Kim et al. 2012;

Gudesblat et al. 2012; Kong et al. 2012), shoot gravitropism (Gupta et al. 2012; Vandenbussche

et al. 2013), fruit development (Symons et al. 2006), vascular differentiation (Yamamoto et al.

1997), cell wall homeostasis (Wolf et al. 2012) and biosynthesis (Xie et al. 2011), leaf

development (Nakaya et al. 2002; Zhiponova et al. 2013), seed germination (Steber and

McCourt, 2001; Xi et al. 2010; Sandhu et al. 2012), sex determination (Hartwig et al. 2011),

fertility (Ye et al. 2010; Xi and Yu 2010), determination of the root meristem size (Gonzalez-

Garcia et al. 2011; Hacham et al. 2011), plant circadian clock (Hanano et al. 2006) and

chromatin modification (Shigeta et al. 2011; Sui et al. 2012). BRs also play a role in plant

responses to environmental stresses through their involvement in immunity (For review see:

4

Wang, 2012) and stress tolerance aspects (Kagale et al. 2007; for review see, Divi and Krishna,

2009; Divi et al. 2010). The involvement of BRs in diverse aspects of plant development has

encouraged research into the use of BRs as a potential biotechnology tool to improve crop

productivity (Wu et al. 2008; Gudesblat and Russinova, 2011; Oh et al. 2011; Vriet et al. 2012;

Kutschera and Wang, 2012).

Plants with altered BR levels or signaling, also display photomorphogenic phenotypes such

as altered seed germination, light-mediated hypocotyl growth and flowering (Turk et al. 2005;

Sandhu et al. 2012). A brief introduction to the photomorphogenesis as relevant to this study is

given in the next section.

Photomorphogenesis: Because of the sessile nature of plants, adaptation to changing

environmental conditions is critical. One of the most important abiotic factors affecting plant

growth is light. Light regulates multiple aspects of plant growth such as germination, plant

architecture and transition to flowering. Not only quantity and quality, but also direction of light

is very important for plants. Plants perceive light quality and quantity via a suite of signal-

transducing photoreceptors to facilitate adaption to their ambient environment. In Arabidopsis

there are five red/far-red-absorbing phytochromes (phyA through phyE), two blue-light-

absorbing cryptochromes, (cry1 and cry2) and two blue/UV-light-absorbing phototropins (phot1

and phot2) (For review see: Chory 2010).

Koornneef (1980) pioneered research in light-signaling by employing large scale mutation

screens in Arabidopsis using hypocotyl growth assays to detect mutants compromised for light-

perception (Koornneef et al. 1980). In these studies, mutants were placed into five unique

complementation groups (hy1, hy2, hy3, hy4 and hy5). These mutants display elongated

5

hypocotyls by virtue of their reduced sensitivity to white light when compared to the wild type.

Detailed analysis of these mutants by various laboratories associated these mutations with

chromophore biosynthesis (hy1 and hy2) (Parks and Quail, 1991), PHYB (hy3) (Somers et al.

1991; Reed et al. 1993; Reed et al. 1994) and CRY1 (hy4) (Ahmad and Cashmore, 1993) and a

downstream transcription factor (hy5) (Oyama et al. 1997; Ang et al. 1998; Chattopadhyay et al.

1998).

Based on the observation that hy3 displays normal hypocotyl inhibition in far-red light and

that hy1 and hy2, which lack activity of all phytochromes, were insensitive to both white and far-

red light, it was suggested that there was at least one yet unidentified phytochrome dedicated to

the perception of far-red light. Based on this knowledge, PHYA mutants were identified by virtue

of their elongated hypocotyls in continuous far-red light (Nagatani et al. 1993; Parks and Quail,

1993; Whitelam et al. 1993). Subsequently, PHYC, PHYD and PHYE were also identified based

on their homology to the already identified phytochromes (Sharrock and Quail, 1989; Clack et

al. 1994). Later CRY2 was also cloned on the basis of its homology to CRY1 (Lin et al. 1998).

CRY2 loss-of-function mutants confer wild-type hypocotyl phenotypes under high intensity blue

light but exhibit elongated hypocotyls in dim blue light when compared to the wild-type (Lin et

al. 1998). These Arabidopsis photoreceptors have overlapping and distinct functions, which help

to fine-tune plant responses to dynamic light conditions (Neff and Chory, 1998; for review see,

Franklin and Quail, 2010).

phyA is the most important far-red-light sensor in Arabidopsis. A role of phyA as the major

far-red sensor is supported by the observation that far-red-light grown PHYA loss-of-function

mutants phenocopy dark-grown wild-type plants (Nagatani et al. 1993; Parks and Quail, 1993;

Whitelam et al. 1993; Neff and Chory, 1998). phyB is a major regulator of seedling deetiolation

6

in response to both white and red light, with null alleles conferring elongated hypocotyls in both

conditions (Somers et al. 1991; Reed et al. 1993; Reed et al. 1994). cry1 and cry2 regulate

deetiolation in response to medium and low intensity blue-light, respectively (Ahmad and

Cashmore, 1993; Lin et al. 1998). In addition to seedling deetiolation, some of these

photoreceptors also mediate floral induction in responses to changing light conditions.

Arabidopsis is a facultative long-day-plant and employs photoperiodic flowering pathways to

accelerate flowering under long-day conditions. phyA plays a vital role in the photoperiodic

flowering pathway by perceiving changes in day length. PHYA-null mutant plants are insensitive

to floral induction by day-length extensions or night-break light treatments for short-day-grown

plants, both of which mimic long-day growth conditions (Johnson et al. 1994; Reed et al. 1994).

In addition, under long-day growth conditions, PHYA-null mutant plants display a late-flowering

phenotype when compared to the wild type (Johnson et al. 1994; Neff and Chory, 1998). phyB,

on the other hand, inhibits flowering in Arabidopsis. Loss of phyB accelerates flowering under

both long- and short-day conditions (Goto et al. 1991; Halliday et al. 1994; Whitelam and Smith,

1991).

BR- and photomorphogenesis-mediated development: As mentioned before, BRs have been

implicated in the modulation of photomorphogenic traits. For example, a study examining

genome wide targets of PIL5 (PHYTOCHROME INTERACTING FACTOR-3 Like 5), a major

negative regulator of phytochrome regulated seed germination, have identified genes involved in

BR signaling, indicating a connection between BR signaling and phytochromes during seed

germination (Oh et al. 2004; Oh et al. 2009). PIFs (PHYTOCHROME INTERACTING

FACTORS) are nuclear transcription factors which are known to interact with phytochromes

(Monte et al. 2007; Bae and Choi, 2008). Recent evidence also suggests that brassinosteroids

7

play a role in flowering, a developmental process predominately controlled by light. For

example, BRs affect flowering by repressing the expression of FLOWERING LOCUS C (FLC), a

negative regulator of the autonomous flowering pathway (Domagalska et al. 2007). In addition,

bri1-EMS SUPPRESSOR 1 (BES1), a transcription factor required for BR-dependent gene

expression, interacts in vivo with EARLY FLOWERING 6 (ELF6) and RELATIVE OF EARLY

FLOWERING 6 (REF6) (Yu et al. 2008). ELF6 and REF6 are transcription factors and were

initially isolated in a genetic screen for mutants with altered flowering phenotypes (Noh et al.

2004).

The link between BRs and light-mediated morphogenesis has also been suggested based on

the deetiolated morphology of dark-grown BR biosynthetic mutants, suggesting that BR

signaling is essential for normal growth in the dark (skotomorphogenesis) (Li et al. 1996;

Szekeres et al. 1996; Nagata et al. 2000; Asami et al. 2004). It has also been suggested that light

inhibits skotomorphogenesis and promotes photomorphogenesis in part by inhibiting BR levels

or signaling (Li et al. 1996; Chory and Li, 1997; Kang et al. 2001). However, studies of BR

measurements during deetiolation do not indicate any changes in BR levels during this response

(Symons et al. 2008). Light-induced decrease in BR sensitivity has also been suggested as a

complementary mechanism for light-regulation of BR signaling (Turk et al. 2003). In fact, light-

dependent reduction in BR sensitivity has been reported in the case of Arabidopsis and rice

(Bancos et al. 2006; Jeong et al. 2007).

Microarray-based studies that targeted the effects of BRs on transcript profiles in Arabidopsis

suggest that BRs may be acting upstream in addition to acting downstream of light-signaling

pathways (Goda et al. 2002; Song et al. 2009; Sun et al. 2010). In addition, BRs and

photomorphogenesis may interact by converging on common target genes and pathways (Lee et

8

al. 2007; Hu et al. 2009; Luo et al. 2010; Sun et al. 2010; Yu et al. 2011). One such recent study

has shown that the BR-activated transcription factor BZR1 (BRASSINAZOLE-RESISTANT 1)

and PIF-4 (PHYTOCHROME-INTERACTING FACTOR 4) interact in vivo to regulate

hundreds of common target genes and are genetically interdependent in regulating cell expansion

in the Arabidopsis hypocotyl in response to BRs, darkness and heat (Oh et al. 2012). For review

on BR signaling and its role in photomorphogenesis see: Wang et al. 2012.

One of the goals in the Neff lab is to study the role of phytohormones in photomorphogenic

development of Arabidopsis. For this purpose, we focus our studies on the phyB-mediated

development. In addition to the early-flowering phenotype, phyB mutants are severely

pleiotropic, demonstrating widespread importance of phyB in Arabidopsis development (for

review see: Franklin and Quail, 2010). To identify downstream components of phyB signaling,

various loss-of-function genetic approaches have been employed, including the identification of

mutants that either mimic or suppress phyB-null phenotypes (Reed et al. 1998; Reed et al. 2000).

To complement these loss-of-function approaches, we utilized an activation-tagging screen to

identify gain-of-function downstream components that may act in a redundant manner (Weigel et

al. 2000). PHYB-4 ACTIVATION-TAGGED SUPPRESSOR #1-DOMINANT (bas1-D) and

SUPPRESSOR OF PHYB-4 #7-DOMINANT (sob7-D) were both identified in a gain-of-function

activation-tagging screen for suppressors of the photomorphogenic phenotypes conferred by a

weak mutation of phyB, phyB-4 (Neff et al. 1999; Turk et al. 2005).

BAS1 and SOB7: BAS1 and SOB7 are members of the cytochrome P450 monooxygenase

superfamily (P450s). Members of the P450 superfamily catalyze oxidation of a diverse array of

plant metabolites. Reactions catalyzed by P450s are highly substrate specific, to an extent that

9

even close P450 family members may have widely diverse biochemistries as well as substrate

requirements (for review see: Schuler et al. 2006).

Over-expression of either BAS1/CYP734A1 or SOB7/CYP72C1 suppresses the long-

hypocotyl phenotype of phyB-4 and also confers a BR-deficient phenotype typified by de-

etiolated 2-1 (det2-1), a BR biosynthetic mutant. Molecular, biochemical and genetic analyses

have shown that in spite of the sequence similarity at both DNA and protein levels, BAS1 and

SOB7 each catabolize their own specific substrates with unique biochemistries. BAS1

hydroxylates brassinolide (BL), the most active BR in Arabidopsis, and its immediate precursor

castasterone (CS) to their respective inactive C-26 hydroxy products (Turk et al. 2005). SOB7,

on the other hand, is not a C-26 hydroxylase and seems to act on precursors of BR biosynthesis

(Turk et al. 2003; Turk et al. 2005; Thornton et al. 2010). BR levels are elevated in the bas1-2

sob7-1 double-null mutant when compared to the wild type or either single null allele (Turk et al.

2005). BAS1 and SOB7 also affect developmental processes, such as hypocotyl growth in

response to light and flowering (Figure 2), in a synergistic/redundant fashion. In a manner

quantitatively similar to phyB-null mutants, the bas1-2 sob7-1 double null flowers earlier than

the wild type in both long- and short-day growth conditions, demonstrating a role for BR

inactivation in floral induction (Turk et al. 2005). It is possible that the effect of BRs on

flowering is independent of light. However, another possibility is that, the light is regulating

flowering by modulating BR levels directly through BR-inactivation.

Other BR inactivating pathways:

BR Glucosylation: UGT73C5 was the first of two steroid glucosyltransferases to be identified

in Arabidopsis (Popppenberger et al. 2005; Husar et al. 2011). UGT73C5 belongs to a subset

10

of the UDP-glucosyltransferases family. Members of this family are known to be involved in

glucosylation of plant hormones such as auxins, cytokinins and abcisic acid (for review see:

Bowles et al. 2006). In most cases the conjugation of hormones with sugars makes them

inactive. Steroid glucosylating UGTs are also found in mammals and insects. UGT73C5

(At2g36800) was identified by gene over-expression conferring a characteristic BR-deficient

morphology. Plants overexpressing UGT73C5 accumulate 23-O glucosides of BRs to a much

higher levels than control plants. No 23-O-glucosides of BRs were detectable in antisense

RNAi lines of UGT73C5 demonstrating that UGT73C5 is required to convert BRs to

glucosides. Antisense RNAi line seedlings are less sensitive to hypocotyl growth inhibition by

red and blue light but not to white light, suggesting a role for UGT73C5 in

photomorphogenesis. UGT73C5 transcript accumulation is up regulated in the presence of

DON (a fungal toxin). In fact, UGT73C5 was initially identified in a yeast cDNA expression

screen for identifying genes in Arabidopsis that are capable of metabolizing DON

(Poppenberger et al. 2003). The Arabidopsis over expression lines of UGT73C5 also display

BR deficient phenotype as adults in addition to expressing tolerance to DON. This suggests a

broader catalytic activity of UGT73C5 with a function in detoxification.

A second glucosyltransferase, UGT73C6 (At2g36790) was recently identified in

Arabidopsis as having a BR catabolic activity (Husar et al. 2011). UGT73C6 is the closest

homologue of UGT73C5 (Li et al. 2001). UGT73C6 was previously characterized as a

flavonol-3-O-glycoside-7-O-glucosyltransferase (Jones et al. 2003). Over-expression of

UGT73C6 also results in dwarf plants typical of a BR deficiency. Similar to the case of

UGT73C5, CS and BL fed plants overexpressing UGT73C6 accumulate 23-O-glucosides of

these BRs to a much higher levels than control plants. Further analysis also detected

11

modification of BL-23-O-glucosides to BL-malonylglucosides. Interestingly, malonylation is

catalyzed by acyltransferases of the BAHD family and is considered to protect glucosides from

degradation.

Transcript accumulation of UGT73C5 and UGT73C6 is not induced in response to

externally applied BL in plants (Husar et al. 2011). However, both these genes show

transcriptional response to toxins and various xenobiotics (Poppenberger et al. 2003). The

physiological relevance of UGT73C6 in BR metabolism still remains to be fully determined

(Husar et al. 2011).

BR Acylation: Recently, two members of the putative BAHD (for benzylalcohol O-

acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-

hydroxycinnamoyl/benzoyltransferase, and deacetylvindoline 4-O-acetyltransferase)

acyltransferase family showing BR catalytic activity were also identified among the gain-of-

function mutants generated by activation-tagging (Ahn et al. 2007; Yu et al. 2008; Roh et al.

2012; Wang et al. 2012) and full-length cDNA-overexpressor screens (FOX) (Ichikawa et al.

2006; Schneider et al. 2012; Choi et al. 2012) in Arabidopsis. Members of BAHD family are

involved in acylation modification of a diverse array of secondary metabolites in plants

(D’Auria, 2006). The modification may alter one or more of the substrate properties such as

volatility, solubility and activity. The Arabidopsis genome is known to contain 64 BAHD family

members, which can be classified into five major clades (D’Auria, 2006; Yu et al. 2009). As

with the P450s, BAHD family members are highly diverse in their substrate preferences and

sequence similarity provides poor predictability of the substrates.

BIA1 (BRASSINOSTEROID INACTIVATOR1; Roh et al. 2012), also known as ABS1

12

(ABNORMAL SHOOT1; Wang et al. 2012) is encoded by the At4g15400 gene in Arabidopsis.

Two activation-tagging alleles of BIA1, bia1-1D and bia1-2D exhibit typical BR deficiency

phenotypes. BR measurement in bia1-1D plants shows reduced endogenous levels of various

BRs including CS. The expressions of the known BR-feedback regulated genes are also altered

in bia1-D plants in accordance with reduced BR levels. The near RNA-null line of At4g15400,

bia1-3 confers increased hypocotyl length in the dark when compared to the wild type (Col-0).

Quantitative transcript accumulation analysis of BR-related genes in dark grown seedlings of

bia1-3 show altered expression of BR-related genes when compared to the wild type, suggesting

higher BR levels in dark grown bia1-3 seedlings. BIA1 transcript accumlation in seedlings is also

higher in the dark when compared to continuous white light. The BIA1 protein is expressed

mainly in the root elongation zone with increased expression in darkness compared to growth in

light. The sub-cellular localization of BIA1 protein is cytosolic. In addition, the BIA1 gene

expression is feed-back regulated in response to exogenously applied BL as well as in mutants

with altered endogenous BR levels. Genetic analysis of the overexpression as well as knockdown

of BIA1 in Arabidopsis strongly suggests its involvement in BR homeostasis.

PIZ (PIZZA; Schneider et al. 2012), also known as BAT1 (BR-RELATED

ACYLTRANSFERASE1; Choi et al. 2012) is encoded by At4g31910. Overexpression of PIZ

causes characteristic BR-deficient phenotypes. The dwarf phenotype in PIZ overexpressors can

be partially rescued with the application of exogenous BL indicating that the 35S:PIZ

phenotypes are caused by BR deficiency. Alteration in the expression of many BR-feedback

regulated genes in 35S:PIZ plants confirms lower BR levels. Interestingly, 35S:PIZ plants show

more reduction in the concentration of earlier BR precursor than the more active downstream

BRs, CS and BL. Feeding experiments with heterologously expressed HIS-tagged PIZ shows

13

that PIZ can use BL, CS and TY (Typhasterol) as substrates for acylation. PIZ is mainly

expressed in roots as detected by the QRT-PCR. However, PIZ1 T-DNA insertion knockout lines

piz1 and piz2 do not display any defects in development of roots or other organs. Additionally,

PIZ expression is not feed-back regulated by alteration of endogenous BR concentration or

signaling.

At4g31910/PIZ was also characterized as BAT1 by Choe et al. 2012. Similar to 35S:PIZ, the

endogenous BR levels of CS precursors are also reduced in BAT1 overexpression line. BR levels

in null allele bat1-1 are not significantly different than the wild type. The bat1-1 allele confers a

larger inflorescence stem as well as increased number of vascular bundles when compared to the

wild type. BAT1 is localized in the endoplasmic reticulum. GUS transcriptional fusions with the

BAT1 promoter show activity in the root tip, root maturation zone, lateral root primordial,

cotyledon and leaf vein vasculature, and phloem of the inflorescence stem. BAT1 transcript

accumulation is significantly induced by exogenous auxin treatment and not BL treatment in

phytohormone response assays. Chromatin immuno-precipitation shows that the BAT1 promoter

is bound by ARF19 (Auxin Response Factor 19). The overall genetic and biochemical analysis

suggests that BAT1 is involved in BR homeostasis and may also have a role in auxin signaling.

BR Sulfonation: A soluble sulfotransferase ATST4a (At2g14920) capable of catalyzing O-

sulfonation of BRs has also been identified in Arabidopsis (Table 1; Marsolais et al. 2007).

Soluble sulfotransferases form a superfamily of enzymes whose members are found in plants,

mammals, and bacteria. Mammalian steroid sulfotransferases are known to be involved in

hormone transport and inactivation (Tong et al. 2005). The Brassica napus soluble

sulfotransferases BNST3 and BNST4 have broad catalytic activity toward plant steroids

(Rouleau et al. 1999; Marsolais et al. 2004). Sulfotransferase genes from Brassica napus are

14

induced by salicylic acid, ethanol and other xenobiotics, which suggest a role in detoxification.

ATST4a was cloned on the basis of homology to ATST1. ATST1 is an ortholog of BNST

proteins and displays similar catalytic activities and substrate specificity.

Recent evidence shows that ATST1 is also involved in the sulfonation of salicylic acid in

response to pathogen stress (Baek et al. 2010). In vitro protein expression and sulfotransferase

assay studies demonstrate that ATST4a has catalytic activity with BRs. Catalytic activity of

ATST4a is specific for biologically active end products of the BR biosynthetic pathway,

including CS, BL, related 24-epimers and the naturally occurring (22R, 23R)-28-homoBRs. The

expression of ATST4a is largely root specific and is down regulated by the cytokinin hormone

trans-zeatin. However, no genetic study involving loss of function or over expression was done

to show that BR inactivation is the in vivo function of ATST4a. Biochemical data clearly

demonstrates the role of ATST4a in BR inactivation. But genetic studies coupled with in vivo

biochemical data are essential to test the hypothesis that BR inactivation is the endogenous

function of ATST4a.

BR Reduction: Another putative BR deactivating gene, BEN1 (At2g45400), was identified in an

activation-tagging screen for extragenic modifiers of bri1-5, a weak mutant allele of BR receptor

encoding gene BRI1. BEN1 belongs to a small family of proteins in Arabidopsis that also

includes the dihydroflavonol reductase, DFR, and the anthocyanidin reductase, BAN, both

involved in the flavonoid biosynthesis pathway. The over-expressor mutant BRI1-5

ENHANCED1-1DOMINANT (ben1-1D) has a characteristic BR deficient phenotype (Yuan et al.

2007). Molecular, genetic and physiological characterization of BEN1 demonstrates a role in BR

metabolism. BR levels are slightly elevated in the ben1-1 null allele when compared to the wild

type. Adult ben1-1 plants have organ-elongation phenotypes when compared to the wild type.

15

BEN1 is mainly expressed in the root elongation zone in a pattern similar to SOB7. BEN1

expression is up-regulated 4-5 times in white light when compared to growth in the dark.

Seedlings lacking BEN1 are also less responsive to light inhibition of hypocotyl growth

suggesting a role in seedling photomorphogenesis and BR inactivation. However, feeding

experiments failed to detect any metabolic product of BEN1 activity (Yuan et al. 2007). Genetic

data strongly favors the hypothesis that BEN1 is involved in BR inactivation though the

substrates and products of BEN1 activity are not known. Figure 3 shows the reactions catalyzed

by various BR catabolic genes discussed above.

This work: Independent evolution of multiple BR inactivating pathways indicates the

importance of this process in plant growth and development. Therefore, identifying the

contributions of enzymes and pathways related to the inactivation of these hormones is important

for understanding BR-mediated development. The research presented in this dissertation is

concerned with the genetic analysis of the role of four BR catabolic genes in growth and

development of Arabidopsis.

Chapter Two, which has been published in the journal “G3: Genes, Genomes, Genetics”,

investigates the role of BAS1 and SOB7 in photomorphogenesis (Sandhu et al. 2012). Genetic

interactions for seed germination and flowering time were studied between BAS1, SOB7 and the

photoreceptors PHYA, PHYB and CRY1 using null-mutant combinations. Our results indicate

that both BAS1 and SOB7 contribute to the rate of seed germination in a manner that is

genetically independent and/or downstream of PHYA and PHYB. BAS1 and SOB7 also show

complex genetic interactions with PHYA and PHYB for flowering time. For example, bas1-2 and

sob7-1 single-nulls have a mutually distinct pattern of genetic interactions with a phyA null. In

contrast, the early-flowering phenotype of the bas1-2 sob7-1 double null requires functional

16

phyB. Furthermore, we show that BAS1 and SOB7 have both unique and overlapping expression

patterns in Arabidopsis, and that BAS1 expression in the shoot apex in red light is dependent on

the presence of functional phyB. Recent scientific evidence has shed some light on the molecular

and genetic basis of the association between BRs and photomorphogenesis. Research conducted

in Chapter Two is also a step in this direction. Research in this area will also lead to the better

understanding of photomorphogenesis itself.

Chapter Three reports the genetic analysis of the T-DNA insertion mutant ben1-1. Our study,

which has been submitted to the journal “G3: Genes, Genomes, Genetics”, shows that the ben1-1

mutation is unstable due to epigenetic modifications of the intronic T-DNA insertion. As a part

of our study, we generated a genetic triple-mutant from a cross between the bas1-2 sob7-1

double-null (T-DNA exonic insertion mutants of BAS1 and SOB7) and ben1-1. Our results show

that, the complete loss-of-function ben1-1 mutation was transformed to a partial loss-of-function

mutation in the bas1-2 sob7-1 ben1-1 (triple-mutant) background showing enhanced levels of the

wild-type-spliced transcript. Interestingly, the enhanced expression of BEN1 remained stable

when the ben1-1 single-mutant was re-isolated from a cross with the wild-type. In addition, the

two genetically identical pre-triple and post-triple ben1-1 mutants also differed phenotypically in

terms of seedling-development characteristics. The size of the T-DNA insertion and the NPTII

gene sequence did not change in the pre-triple and post-triple ben1-1 mutants, indicating that it

did not play a role in the transformation. However, a previously functional NPTII T-DNA marker

gene (which encodes kanamycin resistance) was no longer functional in the triple mutant nor in

the recovered ben1-1 allele. Restriction endonuclease analysis with methylation sensitive

enzymes followed by genomic PCR showed that the methylation status of the T-DNA is different

between the original and the recovered ben1-1.

17

Chapter four reports the results of genetic manipulation of ATST4a in Arabidopsis. In this

study, we have used both loss-of-function and over-expression genetic approaches to further

explore the role of ATST4a in Arabidopsis. Our results show that atst4a1-1, a T-DNA insertion

null mutant of ATST4a does not display reduced seedling hypocotyl growth-inhibition in white

light. Additionally, overexpression of ATST4a gene in transgenic lines does not result in

characteristic BR-deficient phenotypes despite the lack of feed-back regulation of DWF4 and

BAS1 expressions. These observations suggest that ATST4a is an atypical BR catabolic gene.

Chapter Two shows that molecular genetic approach can help in understanding complex

processes such as flowering as well as interactions between biological pathways. Chapter Three

shows the need to continue the effort of generating additional genetic resources for basic studies.

Chapter Four suggests the need for further study to fully understand the role of ATST4a in

Arabidopsis development. Delineating the overall role of BRs and its catabolism in plant

physiology and development is likely to include a similar molecular genetic approach as

described in this study.

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Table 1. Biochemical analysis of ATST4a BR catalytic activity. Marsolais et al. 2007.

Substratea

K m (μM) V

max (pkatal mg

−1

) V max

K m

−1

(pkatal mg−1

μM−1

)

Brassinolide 16 5 0.3

Castasterone 14 3 0.2

24-Epibrassinolide 43 10 0.2

(22R, 23R)-28-Homobrassinolide

19 34 1.8

(22R, 23R)-28-Homocastasterone

7 19 2.7

39

Figure 1: BR biosynthesis pathway. Copied from Bishop and Koncz, 2002.

40

Figure 2. Fluence rate response shows genetic redundancy between BAS1 and SOB7 for

hypocotyl growth repression in white light (A) and flowering time (B). Copied from Turk et

al. 2005.

41

Figure 3: Reactions catalyzed by the known BR catabolic enzymes.

CHAPTER TWO

Genetic interactions between brassinosteroid-inactivating P450s and photomorphogenic

photoreceptors in Arabidopsis thaliana

Publication citation: Sandhu, K. S., Hagely, K., and Neff, M.M., (2012) Genetic interactions

between brassinosteroid-inactivating P450s and photomorphogenic photoreceptors in

Arabidopsis thaliana. G3. 2(12):1585-93

Other people who contributed to this work: Genetic mutant combinations generated by Dr. Ed

Turk, flowering analysis done by Katherine Hagely (co-author), and GUS constructs generated

by Dr. Leeann Thornton.

43

ABSTRACT

Plants use light as a source of information via a suite of photomorphogenic photoreceptors to

optimize growth in response to their light environment. Growth-promoting hormones such as

brassinosteroids can also modulate many of these responses. BAS1 and SOB7 are

brassinosteroid-catabolizing P450s in Arabidopsis thaliana that synergistically/redundantly

modulate photomorphogenic traits such as flowering time. The role of BAS1 and SOB7 in

photomorphogenesis has been investigated by studying null-mutant genetic interactions with the

photoreceptors phyA, phyB and cry1 with regard to seed-germination and flowering time. The

removal of BAS1 and/or SOB7 rescued the low germination rate of the phyA-211 phyB-9 double-

null mutant. With regard to floral induction, bas1-2 and sob7-1 showed a complex set of genetic

interactions with photoreceptor-null mutants. Histochemical analysis of transgenic plants

harboring BAS1:BAS1-GUS and SOB7:SOB7-GUS translational fusions under the control of

their endogenous promoters revealed overlapping and distinct expression patterns. BAS1’s

expression in the shoot apex increases during the phase transition from short-to-long-day growth

conditions and requires phyB in red light. In summary, BAS1 and SOB7 displayed both simple

and complex genetic interactions with the phytochromes in a plant-stage specific manner.

44

INTRODUCTION

In angiosperm plant species, the timing of flowering plays an important role in the success of

sexual reproduction. Precision in the timing of reproduction requires a flowering mechanism that

is both flexible and robust. Flexibility ensures that the timing of flowering leads to good seed set

and survival under a variety of circumstances. Robustness ensures that the mechanism is strong

enough to trigger flowering in the majority of members in a population. Cues that regulate floral

induction include both environmental (external) and developmental (internal) factors. Being

sessile and photosynthetic, light is a major environmental factor for plants. Light-mediated

development, or photomorphogenesis, plays an important role in the optimization of flowering

time (Mockler et al. 2003). Internal factors that affect flowering time include hormones such as

brassinosteroids (BRs), as well as developmental factors, which include age. In plants, these

external and internal cues are perceived by various reproductive pathways, which ultimately

converge to ensure a proper flowering response (Srikanth and Schmid, 2011). One of the

fundamental questions in plant biology relates to how plants integrate light and hormone signals

to optimize growth and development in constantly changing environmental conditions.

Plants perceive light quality and quantity via a suite of signal-transducing photoreceptors to

facilitate adaption to their ambient environment. In Arabidopsis there are five red/far-red-

absorbing phytochromes (phyA through phyE), two blue-light-absorbing cryptochromes, (cry1

and cry2) and two blue/UV-light-absorbing phototropins (phot1 and phot2) (Chory 2010). phyA

is the most important far-red-light sensor in Arabidopsis. A role of phyA as the major far-red

sensor is supported by the observation that far-red-light grown PHYA loss-of-function mutants

phenocopy dark-grown wild-type plants (Nagatani, Reed and Chory, 1993; Parks and Quail,

1993; Whitelam et al. 1993; Neff and Chory, 1998). phyB is a major regulator of seedling

45

deetiolation in response to both white and red light, with null alleles conferring elongated

hypocotyls in both conditions (Somers et al. 1991; Reed et al. 1993; Reed et al. 1994). cry1 and

cry2 regulate deetiolation in response to medium and low intensity blue-light, respectively

(Ahmad and Cashmore, 1993; Lin et al. 1998). In addition to seedling deetiolation, some of these

photoreceptors also mediate floral induction in responses to changing light conditions.

Arabidopsis is a facultative long-day-plant and employs photoperiodic flowering pathways to

accelerate flowering under long-day conditions. phyA plays a vital role in the photoperiodic

flowering pathway by perceiving changes in day length. PHYA-null mutant plants are insensitive

to floral induction by day-length extensions or night-break light treatments for short-day-grown

plants, both of which mimic long-day growth conditions (Johnson et al. 1994; Reed et al. 1994).

In addition, under long-day growth conditions, PHYA-null mutant plants display a late-flowering

phenotype when compared to the wild type (Johnson et al. 1994; Neff and Chory 1998). phyB,

on the other hand, inhibits flowering in Arabidopsis. Loss of phyB accelerates flowering under

both long- and short-day conditions (Goto et al. 1991; Halliday et al. 1994; Whitelam and Smith

1991).

In addition to the flowering phenotype, phyB mutants are severely pleiotropic, demonstrating

its widespread importance in Arabidopsis development (for review see Franklin and Quail,

2010). To identify downstream components of phyB signaling, various loss-of-function genetic

approaches have been employed, including the identification of mutants that either mimic or

suppress phyB-null phenotypes (Reed et al. 1998; Reed et al. 2000). To complement these loss-

of-function approaches, we utilized an activation-tagging screen to identify gain-of-function

downstream components that may act in a redundant manner (Weigel et al. 2000). PHYB-4

ACTIVATION-TAGGED SUPPRESSOR #1-DOMINANT (bas1-D) and SUPPRESSOR OF

46

PHYB-4 #7-DOMINANT (sob7-D) were both identified in a gain-of-function activation-tagging

screen for suppressors of the photomorphogenic phenotypes conferred by a weak mutation of

PHYB (Neff et al. 1999; Turk et al. 2005). BAS1 and SOB7 are members of the cytochrome

P450 monooxygenase superfamily (P450s). Members of the P450 superfamily catalyze oxidation

of a diverse array of plant metabolites. Reactions catalyzed by P450s are highly substrate

specific, to an extent that even close P450 family members may have widely diverse

biochemistries as well as substrate requirements (For review see, Schuler et al. 2006).

Over-expression of either BAS1/CYP734A1 or SOB7/CYP72C1 suppresses the long-

hypocotyl phenotype of phyB-4 and also confers a BR-deficient phenotype typified by de-

etiolated 2-1 (det2-1), a BR biosynthetic mutant. Molecular, biochemical and genetic analyses

have shown that in spite of the high sequence similarity at both DNA and protein levels, BAS1

and SOB7 each catabolize their own specific substrates with unique biochemistries. BAS1

hydroxylates brassinolide, the most active BR in Arabidopsis and its immediate precursor

castasterone to their respective inactive C-26 hydroxy products (Turk et al. 2005). SOB7, on the

other hand, is not a C-26 hydroxylase and seems to act on precursors of BR biosynthesis (Turk et

al. 2003; Turk et al. 2005; Thornton et al. 2010). BR levels are elevated in the bas1-2 sob7-1

double-null mutant when compared to the wild type or either single null allele (Turk et al.,

2005). BAS1 and SOB7 also affect developmental processes, such as flowering, in a

synergistic/redundant fashion. In a manner quantitatively similar to phyB-null mutants, the bas1-

2 sob7-1 double null flowers earlier than the wild type in both long- and short-day growth

conditions, demonstrating a role for BR inactivation in floral induction (Turk et al. 2005).

BRs are growth-promoting hormones essential for normal development of plants (Sasse

2003). BRs affect plant growth and development by altering the expression of hundreds of BR

47

responsive genes (Goda et al. 2002). In addition to their general role in cell division and

expansion (Bajguz 2000), BRs are also involved in tissue-specific development (Symons et al.

2006; Yamamoto et al. 1997). Unlike most plant hormones, however, BRs are not transported

within or between plant tissues, implying that levels of BRs are regulated locally through both

biosynthesis and catabolism (Reid and Ross, 1989; Symons and Reid, 2004; Montoya et al.

2005; Salvadi-Goldstein et al. 2007). BR catabolism, therefore, can play a significant role as a

regulatory point for BR-mediated development. In fact, apart from BAS1 and SOB7, there are at

least five more enzymes with unique biochemistries leading to BR inactivation in Arabidopsis

(Husar et al. 2011; Masrsolais et al. 2007; Poppenberger et al. 2005; Yuan et al. 2007).

Independent evolution of multiple BR inactivating pathways further indicates the importance of

this process in plant growth and development. Therefore, identifying the contributions of

enzymes and pathways related to the inactivation of these hormones is important for

understanding BR-mediated development.

The observation that photomorphogenic photoreceptors, along with and BAS1 and SOB7,

affect common developmental processes suggests that in at least some cases these pathways act

in an interdependent manner. In the present work, we describe the contribution of BAS1 and

SOB7 in the modulation of seed germination and flowering time in Arabidopsis. Genetic

interactions for seed germination and flowering time were studied between BAS1, SOB7 and the

photoreceptors PHYA, PHYB and CRY1 using null-mutant combinations. Our results indicate

that both BAS1 and SOB7 contribute to the rate of seed germination in a manner that is

genetically independent and/or downstream of PHYA and PHYB. BAS1 and SOB7 also show

complex genetic interactions with PHYA and PHYB for flowering time. For example, bas1-2 and

sob7-1 single-nulls have a distinct pattern of genetic interactions with a phyA null. In contrast,

48

the early-flowering phenotype of the bas1-2 sob7-1 double null requires functional phyB.

Furthermore, we show that BAS1 and SOB7 have both unique and overlapping expression

patterns in Arabidopsis, and that BAS1 expression in the shoot apex in red light is dependent on

the presence of functional phyB.

MATERIALS AND METHODS

Plant material: All mutants used in this study, phyA-211 (Reed et al. 1994), phyB-9 (Reed et al.

1993), cry1-103 (Liscum and Hangarter 1991), bas1-2 and sob7-1 (Turk et al. 2005), were in the

Columbia (Col-0) background. The phyA-211 mutation was identified by amplifying genomic

DNA with primers 5'-GTC ACA AGA TCT GAT CAT GGC-3', 5'-AAC AAC CGA AGG GCT

GAA TC-3', 5'-TTA TCC ACA GGG TTA CAG GG-3', and 5'-GCA TTC TCC TTG CAT CAT

CC-3', followed by resolution of 1243- and 1136-bp fragments for the wild type and a 1243-bp

fragment for the phyA-211 mutant. The PCR-based markers used to identify the phyB-9 and

cry1-103 mutants are described by Ward et al. (2005). Identification of bas1-2 and sob7-1 is

described in Turk et al. (2005). Photoreceptor mutants were crossed with bas1-2 sob7-1, and

multiple mutant combinations were isolated in F2 populations.

Due to the use of a large number of higher order null-mutant combinations in this study, it

was not appropriate to use a sibling wild-type Col-0 derived from any one cross as a control.

Therefore, a common Col-0 strain was used as a wild-type control in all the experiments for

uniformity. To address variation due to environmental growth conditions all genotypes were

grown at the same time under the same growth chamber conditions, and seeds harvested for

phenotypic analysis.

49

For generating BAS1:GUS translational fusion lines, the BamHI/NcoI fragment from pED10

(described in Turk et al. 2003) was cloned into the BamH1/Nco1 site of pCAMBIA1305.1 vector

(CAMBIA, CANBERRA, AUSTRALIA). This construct was transformed into bas1-2 sob7-1

double-null plants. Multiple transgenic lines segregating at a 3:1 ratio (hygromycin

resistant/sensitive ratio) in the T2 generation were identified as single insertion lines. The entire

SOB7 gene from ~ 2.1-Kb upstream of the start codon to the last base before the stop codon was

cloned in frame with uidA gene in pCAMBIA1305.1 vector. This construct was also transformed

into bas1-2 sob7-1 double-null plants. The segregation ratio was studied to identify multiple

single locus T-DNA insertion lines.

Seed sterilization, plating, and growth conditions: Seeds were surface sterilized by 15 minute

agitation in 70% (v/v) ethanol with 0.05 % (v/v) TritonX-100 followed by 5 minutes of agitation

in 95% (v/v) ethanol with 0.05 % (v/v) TritonX-100 before being air-dried on 90-mm filter paper

in a sterile petri dish. Sterilized seeds were plated on growth media plates containing 1% (w/v)

phytagel (Sigma Life Sciences, St. Louis, MO) with 1.5% (w/v) sucrose and 1/2X Linsmaier and

Skoog basal media (Phytotechnology Laboratories®; Shawnee Mission, KS). Plates were

incubated in darkness at 4°C for four days. Germination was induced with a red-light treatment

at a fluence rate 60-70 µmol m-2

sec-1

for 16 h at 25°C before being transferred to the appropriate

light or dark conditions for a total of five days at the same temperature. White light was provided

by 8 fluorescent tubes (F17T8 17WT; GE Fairfield, CT) and two incandescent tubes (T10 FR25

130V; Satco, Brentwood, NY) in a temperature-controlled growth chamber (Model E-30B;

Percival Scientific, Perry, IA).

Flowering-time analysis: Since transplanting seedlings to soil can cause stresses that alter

flowering time, all seeds were directly sown in pots containing a pre-watered soil mix (Sunshine

50

Mix4 (Aggregate) LA4, Green Island Distributers Inc.; Riverhead, N.Y). These pots were then

incubated in darkness for four days at 4ºC to induce near-uniform germination. Pots were then

transferred to growth chambers with white light (200 µmol m-2

sec-1

) set at 21ºC and 60-70 %

humidity. After a week of growth, seedlings were thinned to one per pot by clipping using small

scissors, since removal of whole seedlings causes root damage to neighboring seedlings, which

in turn can also lead to altered flowering time. We have found that this approach gives the most

uniform and repeatable flowering time results for each genotype. Flowering time for both long-

day and short-day grown plants was calculated by the number of days until the floral stem was

0.5 cm above the basal rosette. In long-day grown plants, flowering time was also calculated by

the total number of primary rosette and cauline leaves present at bolting. This latter approach

was not usable with short-day grown plants due to senescence of older leaves during the

prolonged growth period for some genotypes.

Statistical analysis: All statistical results were obtained from at least three independent

experiments. Each independent experiment showed the same statistical trend. Results are

presented as mean values for the combined data. Error bars represent the standard error of the

mean (SE). A student’s unpaired two-tailed t-test was used to calculate p-values that allowed

identification of statistically significant differences between two genotypes in a given

experiment.

Histochemical GUS analysis: Plant material was incubated overnight in a GUS staining

solution containing 100 mM sodium phosphate, pH 7.0, 10 mM EDTA, 0.5 % v/v Triton X-100,

0.5 mM potassium ferri- and ferrocyanide and 1 mM X-GlucA (Research Product International

Corp.; Mount Prospect, IL). After staining, plant material was treated with 70% ethanol for 1h,

followed by 100% ethanol overnight, to remove chlorophyll before photographing. For

51

histological GUS analysis of BAS1 expression in the shoot apex, shoot apices of transgenic

plants were dissected with twin blades and GUS stained as described above. For embedding,

tissues were rinsed in 0.1M phosphate buffer (pH 7.0) and embedded in Tissue-Tek® OCT

compound for sectioning. Embedded tissues were stored at -20ºC until sectioned. Longitudinal

sections were cut using a Leica Cryocut 1800 (Leica Reichert-Jung 1800 Cryostat; Holly, MI)

and image analyzed by Olympus BH-2 Light microscope at the Washington State University

Francschi Microscopy and Imaging Center.

Transcript analysis: Total RNA was isolated, using the RNeasy Plant Kit (Qiagen, Valencia,

CA), from four-day-old seedlings grown in continuous white light (45 µmol m-2

sec-1

). On-

column DNase digestion was performed using the RNase-Free DNase Set (Qiagen, Valencia,

CA), to eliminate genomic DNA contamination. Total cDNA was synthesized using

SuperScriptIII First-Strand Synthesis System (Invitrogen, Carlsbad, CA). BAS1 transcript was

amplified using primers 5'-GCT TAA AAC GTT GAG TAT GAT C-3' and 5'- TCC TCA TGA

TTG GTC AAT CTC -3'. SOB7 transcript was amplified using primers 5'- CCT GAA AGT CGT

AAC AAT GAT C -3' and 5'- GTT TTC GGA TGA TCA AAT GAG C -3'. ACTIN2 was used as

an internal control in RT-PCR. ACTIN2 transcript was amplified using primers 5'-GGT CGT

ACA ACC GGT ATT GTG CTG G-3' and 5'-CTG TGA ACG ATT CCT GGA CCT GCC-3'.

The linear range of amplification for each gene transcript was determined by comparing samples

obtained using different numbers of cycles. Lack of genomic and foreign DNA contamination

was ascertained by using all RNA samples and water as a template in a PCR reaction.

RESULTS

52

Removal of BAS1 and SOB7 rescues the low germination rate of a phyA phyB double-null

mutant: Flowering-time analysis required seeds to be directly sown on the soil. Since the

number of days to flowering were calculated based on the day of planting, it was important to

know if the genotypes in this study conferred any germination phenotypes. For this purpose, a

seed germination study was conducted.

In both light and dark conditions the bas1-2 and sob7-1 single and bas1-2 sob7-1 double null

did not show a significant difference in germination rates when compared to the wild type Col-0

(in white light, p = 0.18, 0.64 and 0.27 respectively; in dark, p = 0.45, 0.33 and 0.29

respectively). The phyA-211 phyB-9 double mutant conferred a lower germination percentage

rate, both in darkness and after a prolonged white light treatment (Table 1). In contrast, the phyA-

211 phyB-9 bas1-2 and phyA-211 phyB-9 sob7-1 triple-null mutants displayed significantly

higher germination rates than phyA-211 phyB-9 in both white light (p = 0.0025 and 0.0018

respectively) and dark treatments (p = 0.0001 and 0.0002, respectively). The phyA-211 phyB-9

bas1-2 sob7-1 quadruple-null displayed the highest germination rate, which was similar to the

wild type in both dark and light treatments (p = 0.79 and 0.52 respectively). These results

suggest that BAS1 and SOB7 are acting downstream and/or in parallel with phyA and phyB for

modulating seed germination.

BAS1 and SOB7 show genetic interactions with photoreceptors for flowering time: To

further examine genetic interactions between the BR-inactivating enzymes and

photomorphogenic photoreceptors, we measured flowering time in multiple null mutant

combinations of bas1-2 and sob7-1 with phyA-211, phyB-9, cry1-103, and phyA-211 phyB-9

grown in both long- (16 h light: 8 h darkness) and short-day (8 h light: 16 h darkness) conditions.

53

Adult plant phenotypes of the various genotypes grown for three weeks under long-day

conditions are shown in Figure 1.

Flowering time analysis demonstrated complex genetic interactions between these various

genes for determination of flowering time. These genetic interactions were maintained in both

long- and short-day growth conditions (Figure 2). The genetic state of CRY1 did not have any

impact on floral induction or the early-flowering phenotype conferred by the loss of BAS1 and

SOB7. In contrast, the bas1-2 mutation suppressed the phyA-211 late-flowering phenotype in

both long and short days. However, the sob7-1 mutation had the opposite effect on flowering

time in combination with phyA-211. In addition, phyB-9 bas1-2 sob7-1 triple-null and phyA-211

phyB-9 bas1-2 sob7-1 quadruple-null plants did not flower earlier than the phyB-9 single-null

and phyA-211 phyB-9 double-null controls respectively, suggesting that the bas1-2 sob7-1 early-

flowering phenotype requires functional phyB.

BAS1 and SOB7 have distinct and overlapping expression patterns: To gain further insight

into the function of BAS1 and SOB7, we generated translational GUS fusion lines of BAS1 and

SOB7 under the control of their native promoters. These BAS1:BAS1-GUS and SOB7:SOB7-

GUS transgenes were expressed in the bas1-2 sob7-1 double-null mutant background. Single

locus insertion lines were isolated and characterized via molecular and genetic analyses (Figure

S1). Transformation into the bas1-2 sob7-1 null background provided transgene stability by

reducing the chance of co-suppression from the endogenous transcript. In addition, it also

provided an opportunity for functional analysis of the transgenic lines by complementation

analysis of the double-null hypocotyl elongation phenotype.

54

The hypocotyl elongation phenotype of bas1-2 sob7-1 was rescued by BAS1:BAS1-GUS

translational fusions in accordance with the expression level from the transgene (Figure S1A, B).

However, all SOB7:SOB7-GUS lines except one have a similar hypocotyl-elongation phenotype

as the double mutant regardless of the level of gene expression (Figure S1C, D). This

observation can be explained on the basis that BAS1 and SOB7 are not completely redundant for

hypocotyl growth (Turk et al. 2005). Since the pSOB7:SOB7-GUS was transformed into bas1-2

sob7-1 double-null background, the pSOB7:SOB7-GUS transgenic lines shown in Figure S1C

are still lacking BAS1 activity. Therefore, the restoration of SOB7 to its wild-type-expression

level by the transgenic construct does not rescue the bas1-2 sob7-1 hypocotyl phenotype

completely. Only when the expression from the transgenic pSOB7:SOB7-GUS construct is

significantly higher than the wild type level, as is the case in line #3.3, does it significantly

shorten the hypocotyl of the bas1-2 sob7-1 double-null line.

Histochemical GUS analysis was performed on a set of representative lines. In white-light-

grown seedlings, BAS1 expression was observed in the shoot apex and root tip (Figure 3). In

contrast, SOB7 expression was seen only in the root elongation zone (Figure 3). In juvenile and

adult plants, BAS1 expression was present in the shoot apex before flowering, and in the flowers

and developing embryos after flowering (Figure 3). SOB7 expression was present in the

transition zone between the root and the shoot, as well as in developing anthers, the vasculature

of rosette leaves and hydathodes of cauline leaves (Figure 3). Overall, BAS1 and SOB7 had

some overlapping but mostly distinct expression patterns.

The early-flowering phenotype of bas1-2 sob7-1 and expression of BAS1-GUS in the shoot

apex suggests that BAS1 is involved in regulating BR levels in the shoot apical meristem, which

may in turn affect the vegetative to floral phase transition. To test this hypothesis transgenic

55

plants carrying the pBAS1:BAS1-GUS construct were grown in short-days for four weeks. After

four weeks, half of the plants were shifted to long days while the remaining half stayed in the

short days. After two additional days, tissue from plants that stayed in the short days for the

entire time and plants that were shifted to the long days were collected together. As a result, at

the time of collection, all plant tissues were the same age. These harvested tissues were

immediately used for histological analysis. Histochemical GUS analysis demonstrated a change

in the expression pattern of BAS1 during this phase transition from short-day to long-day growth

conditions (Figure 4). In short-day-grown plants (Figure 4A), expression was only visible at the

base of the shoot apex, whereas after floral induction via transfer to long-day growth conditions

BAS1-GUS expression was present throughout the shoot apex (Figure 4B). This observation,

that the change in BAS1 expression pattern is correlated with floral induction suggests a role for

BAS1 in flowering.

BAS1 expression in the shoot apex in red light is dependent on the presence of functional

phyB: Based on the observation that BAS1-GUS expression is present in the shoot apex and that

the early-flowering phenotype of bas1-2 sob7-1 is dependent on the presence of functional phyB,

we hypothesized that phyB signaling is regulating BAS1 expression in the shoot apex which in

turn affects flowering time. To test this hypothesis multiple pBAS1:BAS1-GUS transgenic lines

in the bas1-2 sob7-1 background were crossed with the phyB-9 bas1-2 sob7-1 triple null to

isolate BAS1-GUS translational fusions in both the wild type PHYB and phyB-9 mutant genetic

backgrounds. BAS1-GUS fusions in the bas1-2 sob7-1 and phyB-9 bas1-2 sob7-1 background

were grown in continuous red-light for five days. BAS1-GUS expression was examined in the

shoot apex by histochemical GUS analysis in the genotypes. Using this approach, it was

observed that the BAS1-GUS expression in the shoot apex was much more prominent in the wild

56

type PHYB background than the phyB-9 background (Figure 5). This observation suggests that

BAS1 expression at the shoot apex is modulated by the presence of functional phyB. It also

suggests a possible cause for the dependence of the early-flowering phenotype of bas1-2 sob7-1

on functional phyB.

DISCUSSION

As previously reported, the phyA-211 phyB-9 double mutant displayed a lower germination rate

than the wild type (Poppe and Schafer 1997), which was rescued by the removal of BAS1 and

SOB7 (Table 1). This implies that both are acting either downstream of or in parallel with PHYA

and PHYB to modulate seed germination presumably by changing levels of active BRs. In

comparison to germinating seeds, flowering-time phenotypes in adult plants cannot be

interpreted solely based on the changing of overall BR levels.

Flowering-time analysis of mutants blocked in BR biosynthesis (e.g. det2, dwf4 and cpd) or

BR perception (bri1) suggests a positive role for BRs in floral induction (Azpiroz et al. 1998;

Chory et al. 1991; Domagalska et al. 2007; Li and Chory 1997). However, transgenic

Arabidopsis plants having constitutive overexpression of DWF4, with higher levels of active

BRs and increased organ size, do not display early flowering, suggesting that the interplay

between BRs and flowering is not simply a matter of altering whole-plant hormone levels (Choe

et al. 2001). The bas1-2 sob7-1 double mutant contains higher BR levels and flowers earlier than

the wild type, demonstrating a role for these BR-inactivating enzymes in floral induction (Turk

et al. 2005).

57

Our results show that the bas1-2 mutation suppresses the late-flowering phenotype of phyA-

211, whereas the sob7-1 mutation does not (Figure 2), suggesting that these two BR-inactivating

genes can have distinct roles in plant development. Unlike BAS1, SOB7 is not expressed in the

apical meristem in seedlings as well as adult plants (Figure 3). Since the transition to flowering

occurs at the shoot apical meristem, these differences in expression can, in part, explain the

distinct genetic interactions that bas1-2 and sob7-1 have with phyA-211.

In addition, we also observed that in short-day grown-plants that have not been induced to

flower, BAS1 expression is confined mainly to the basal region of shoot apex (Figure 4A). In

contrast, after floral induction, BAS1 expression is seen throughout the shoot apex (Figure 4B).

The expression of BAS1 at the base of the shoot apex may be involved in excluding BRs from

the shoot apical meristem to prevent an early transition to flowering. Interestingly, a GA

catabolic enzyme encoding gene, OsGA2ox1, implicated in vegetative to floral phase transition

in rice, also shows a similar expression pattern (Sakamoto et al. 2001).

The bas1-2 sob7-1 double mutant also displayed a genetic interaction with phyB-9 with

regard to flowering time (Figures 2A, B). The observation, that both the phyB-9 single-null and

phyB-9 bas1-2 sob7-1 triple-null flower at the same time, suggest that the early-flowering

phenotype of bas1-2 sob7-1 requires functional phyB. Loss of BAS1 expression in the phyB-9

background in red-light (Figures 5B, D) also suggests a possible molecular basis for the genetic

interaction of BAS1 and SOB7 with PHYB in regard to floral induction. Another possible

explanation is that BAS1 expression in the shoot apex is dependent on certain morphological and

physiological attributes that are lacking in the phyB-9 mutant background. In this case, the

requirement of functional phyB would be indirect rather than direct. Examples of two such

morphological attributes that are altered in phyB-9 seedlings grown in red light are smaller

58

cotyledons and petioles when compared to the wild type (Neff and Chory, 1998; Neff and Van

Volkenburgh, 1994), a phenotype that is obvious in the Figure 5. In contrast to PHYA and PHYB,

the genetic state of CRY1 had no impact on the early-flowering phenotype conferred by the bas1-

2 sob7-1 double null. These results demonstrate that higher-order null-mutant analysis can be

used to differentiate the roles of different photoreceptors in BAS1- and SOB7-mediated

development.

To fully understand the role of BAS1 in flowering, it will be necessary to study the changes

in its expression pattern in relation to cellular or tissue-specific BR levels in the shoot apex,

during floral induction, in both the wild type and phyB-9 mutant backgrounds. However, we

currently lack the technology to accurately measure BR levels in small tissues and organs such as

the shoot apical meristem. Such an advancement, which could include a DR5-like reporter

system for BR levels (Ulmasov et al. 1997), would further help in understanding the overall role

of BR catabolism in plant growth and development.

A simplified model describing the suggested roles of BAS1 and SOB7 in seed germination

and floral induction is shown in Figures 6A and 6B respectively. BRs are known to have a

positive effect on seed germination (Steber et al. 2001). Therefore, germination may include an

increase in BR levels via the regulation of BR catabolism. phyA and phyB also affect

germination (Poppe and Schafer 1997). These two processes are either acting independently,

interdependently or both. As shown in the Figure 6A, BAS1 and SOB7 may act downstream of

and/or in parallel to phyA and phyB to promote germination.

In the shoot apex, the transition to flowering includes changes in BAS1’s expression pattern

(Figure 4), suggesting that there might be a cause and/or effect relationship between the two

59

events. phyB plays an inhibitory role in flowering (Goto et al. 1991; Halliday et al. 1994;

Whitelam and Smith 1991). phyB is required for the early flowering phenotype conferred by the

bas1-2 sob7-1 double mutant (Figure 2) and alters the expression pattern of BAS1 (Figure 5).

Therefore, it is possible that phyB modulates BAS1 expression to inhibit phase transition in the

shoot apex. Figure 6B depicts a possible mechanism for the inhibition of flowering at the shoot

apex with regard to genetic interactions between phyB and BAS1. In addition to BAS1 and

SOB7, at least five more genes (BEN1, UGT73C5, UGT73C6, ATST4a and BIA1) have been

suggested to play a role in BR catabolism (Husar et al. 2011; Masrsolais et al. 2007;

Poppenberger et al. 2005; Yuan et al. 2007). Delineating the overall role of BRs and BR

catabolism in plant physiology and development is likely to include a similar molecular genetic

approach as described in this study.

ACKNOWLEDGMENTS

The authors would like to thank Drs. Edward Turk and Leeann Thornton for initiating the crosses

used to identify the large number of null mutant combinations used in this study. We also thank

members of the Neff lab for the critical review of this manuscript. This research was supported

by the National Science Foundation 0758411 (M.M.N.). We are also grateful for support from

the Department of Energy DE-PS02-09ER09-02 (M.M.N.).

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TABLES, FIGURES AND LEGENDS

Table 1: Percentage of seed germination in white light or darkness. Imbibed seeds were

incubated at 4ºC in the dark for four days before being treated with white light for six days (three

replicates) or with white light for one day followed by five days in darkness (six replicates) at

25ºC. Values in brackets represent the se of the mean.

Wild type bas1-2 sob7-1 bas1-2 sob7-1

Shifted to dark 95.6 (1.1) 97.2 (1.8) 92.8 (2.5) 97.2 (1.0)

White light 97.7 (1.1) 91.1 (4.0) 96.7 (1.9) 96.7 (3.3)

phyA phyA bas1-2 phyA sob7-1 phyA bas1-2

sob7-1

Shifted to dark 86.7 (5.7) 94.9 (1.9) 95.5 (1.9) 96.7 (1.2)

White light 95.5 (2.2) 94.4 (4.0) 94.4 (4.0) 100 (0)

phyB phyB bas1-2 phyB sob7-1 phyB bas1-2

sob7-1

Shifted to dark 95.5 (3.3) 94.4 (1.8) 93.3 (2.6) 95.5 (2.2)

White light 95.5 (1.1) 91.1 (2.9) 96.7 (1.9) 90.0 (6.9)

phyA phyB phyA phyB

bas1-2

phyA phyB

sob7-1

phyA phyB

bas1-2 sob7-1

Shifted to dark 20.4 (5.8) 83.9 (3.7) 87.2 (4.7) 95.0 (1.7)

White light 35.6 (6.7) 85.6 (2.9) 87.8 (2.2) 98.9 (1.1)

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Figure 1: Adult phenotype of the genotypes used in this study. Wild-type and single-, double-

and multiple-mutant genotypes were grown in long-day conditions for three weeks before being

photographed. Scale bar= 1cm.

bas1 Sob7 bas1 sob7

phyA phyA bas1-

phyA sob7

phyA bas sob7

phyB phyB bas1

phyB sob7 phyB bas1 sob7

cry1

phyA phyB

cry1 bas1 cry1 sob7 cry1 bas1 sob7

phyA phyB bas1

phyA phyB sob7

phyA phyB bas1 sob7

Col-0

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Figure 2: Genetic interactions between BAS1, SOB7, PHYA and PHYB to control flowering

time. BAS1 but not SOB7 mutant suppresses the late-flowering phenotype of PHYA mutants in

both long day (A and B) and short days (C). phyB-9 bas1-2 sob7-1 triple- and phyA-211 phyB-9

bas1-2 sob7-1 quadruple-null did not flower earlier than the phyB-9 single- and phyA-211 phyB-

9 double null respectively suggesting that the bas1-2 sob7-1 early-flowering phenotype requires

functional phyB (A and B). Three replications with ten plants per replication were used for

flowering analysis. Error bars represent SE.

70

71

Figure 3: Expression patterns of BAS1 and SOB7.

GUS staining patterns of the seedlings or tissues from BAS1:BAS1-GUS and SOB7:SOB7-GUS

transgenic plants at various developmental stages. Scale bar = 1.0 mm.

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Figure 4: Histochemical GUS analysis of BAS1 expression in shoot apex during transition

to flowering. Longitudinal section through the shoot apex of four-week-old transgenic plant

expressing BAS1: BAS1-GUS grown in short day conditions (A). Longitudinal section through

the shoot apex of four-week-old transgenic plants expressing BAS1: BAS1-GUS after shifting to

long day conditions for two days (B). Scale bar = 0.1 mm.

(A) BAS1-GUS 30 SD

(B) BAS1-GUS 28 SD+2 LD

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Figure 5: BAS1 expression in the shoot apex in red-light is dependent on the presence of

functional phyB. Transgenic seedlings carrying identical transgenic event of pBAS1: BAS1-

GUS construct in the bas1-2 sob7-1 background (A and C) and phyB-9 bas1-2 sob7-1 triple-null

background (B and D). Seedlings were grown in 45 µmol m-2

sec-1

of red light for five days

before histochemical GUS analysis. Scale bar = 1 mm.

(A) BAS1-GUS (bas1-2 sob7-1) (B) BAS1-GUS (phyB-9 bas1-2 sob7-1)

(C) BAS1-GUS (bas1-2 sob7-1) (D) BAS1-GUS (phyB-9 bas1-2 sob7-1)

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Figure 6: Model based on the interpretation of the genetic interactions between

photomorphogenic photoreceptors and BAS1 and SOB7. (A) In the seed, BAS1 and SOB7 act

downstream and/or in parallel of phyA and phyB to promote germination. (B) In the shoot-apex,

phyB modulate BAS1 expression to inhibit phase transition. The dashed arrows indicate the

suggested interactions based on this study.

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Figure S1: Genetic and molecular analysis of BAS1:BAS1-GUS and SOB7:SOB7-GUS

lines. Genetic and molecular analysis of transgenic plants expressing BAS1:BAS1-GUS (A and

B) and SOB7:SOB7-GUS (C and D) in bas1-2 sob7-1 background. Seedlings were grown in 45

µmol m-2

sec-1

of white light for four days before being digitized and measured. For transcript

analysis total RNA was isolated from four-day-old seedlings grown in similar conditions as used

for hypocotyl growth analysis.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Col-0 bas1-2sob7-1

15.3 41.4 72.11 107.6Hyp

oco

tyl le

ng

th (

mm

)

(A) pBAS1:Bas1-GUS lines

76

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Col-0 bas1-2sob7-1

3.3 11.5 45.2 58.8Hyp

oco

tyl le

ng

th (

mm

) (C) pSOB7:SOB7-GUS lines

CHAPTER THREE

The ben1-1 brassinosteroid-catabolism mutation is unstable due to epigenetic modifications

of the intronic T-DNA insertion

(Sandhu, K.S., Sharma, P., Neff, M.M., The ben1-1 brassinosteroid-catabolism mutation is

unstable due to epigenetic modifications of the intronic T-DNA insertion (Submitted))

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ABSTRACT

Loss-of-function genetic analysis plays a pivotal role in elucidating individual gene function

as well as interactions among gene networks. The ease of gene tagging and cloning provided by

T-DNA insertion mutants have led to their heavy use by the Arabidopsis research community.

However certain aspects of T-DNA alleles require caution, as highlighted in this study of an

intronic insertion mutant (ben1-1) in the BEN1 (BRI1-5 ENHANCED 1) gene. As a part of our

analysis of brassinosteroid catabolic enzymes, we generated a genetic triple-mutant from a cross

between the bas1-2 sob7-1 double-null (T-DNA exonic insertion mutants of phyB-4

ACTIVATION TAGGED SUPPRESSOR 1 and SUPPRESSOR OF phyB-4 7) and ben1-1. As

previously described, the single ben1-1 line behaves as a transcript null. However, in the triple-

mutant background ben1-1 was reverted to a partial loss-of-function allele showing enhanced

levels of the wild-type-spliced transcript. Interestingly, the enhanced expression of BEN1

remained stable when the ben1-1 single-mutant was re-isolated from a cross with the wild-type.

In addition, the two genetically identical pre-triple and post-triple ben1-1 mutants also differed

phenotypically. The previously functional NPTII T-DNA marker gene (which encodes

kanamycin resistance) was no longer functional in the recovered ben1-1 allele, though the length

of the T-DNA insertion and the NPTII gene sequence did not change in the pre-triple and post-

triple ben1-1 mutants. Restriction endonuclease analysis with methylation sensitive enzymes

followed by genomic PCR showed that the methylation status of the T-DNA is different between

the original and the recovered ben1-1. These observations demonstrate that the recovered ben1-1

mutant is epigenetically different from the original ben1-1 allele.

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INTRODUCTION

Loss-of-function mutants play an essential role in genetic analysis. Mutation-tagging

approaches, such as mutagenesis by T-DNA insertion, takes advantage of Agrobacterium-

mediated plant transformation where a modified Ti plasmid is integrated into the plant genome

(Krysan et al. 1999). In plant species that are amenable to transformation by Agrobacterium,

community projects have been undertaken to generate large collections of genomic T-DNA

insertion libraries (Sessions et al. 2002; Alonso et al. 2003; An et al. 2005; Thole et al. 2011).

The ease of gene tagging and cloning provided by T-DNA insertion mutants have led to their

heavy use in Arabidopsis research. However certain aspects of T-DNA-insertion-generated

alleles require caution, as highlighted in this study of an intronic insertion mutant (ben1-1) in the

BEN1 (BRI1-5 ENHANCED 1) gene.

BEN1 (At2g45400) was identified in an activation-tagging screen for extragenic modifiers of

the semi-dwarf phenotype of bri1-5, a weak mutant allele BRI1 (BRASSINOSTEROID

INSESITIVE-1), which encodes the brassinosteroid (BR) receptor. Though the mechanism of

BEN1 activity is not known, genetic data supports the hypothesis that BEN1 is involved in BR

inactivation. The BEN1 over-expressor mutant BRI1-5 ENHANCED1-1DOMINANT (ben1-1D)

has a characteristic BR-deficient phenotype (Yuan et al. 2007). A transcript-null mutant ben1-1

with a T-DNA insertion in the 2nd

intron of BEN1 has elevated BR levels when compared to the

wild type. BEN1 expression is up-regulated in seedlings grown in white light when compared to

those grown in the dark. ben1-1 seedlings are also less responsive to light-mediated inhibition of

hypocotyl growth, suggesting a role in seedling photomorphogenesis (Yuan et al. 2007).

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BR inactivation also involves members of the cytochrome P450 gene family, BAS1 (phyB-4

ACTIVATION TAGGED SUPPRESSOR 1) and SOB7 (SUPPRESSOR OF phyB-4 7) (Neff et al.

1999; Turk et al. 2003; Turk et al. 2005). Over-expression of either BAS1/CYP72B1/CYP734A1

or SOB7/CYP72C1 suppresses the long-hypocotyl phenotype of phyB-4 and also confers a BR-

deficient phenotype (Neff et al. 1999; Turk et al. 2003; Turk et al. 2005). BAS1 hydroxylates

brassinolide, the most active BR in Arabidopsis, and its immediate precursor castasterone to their

respective inactive C-26 hydroxy products (Turk et al. 2005). BAS1 transcript accumulation is

strongly feed-back regulated in a positive manner by BR levels (Tanaka et al. 2005). SOB7, on

the other hand, is not a C-26 hydroxylase and seems to act on precursors of BR biosynthesis

(Turk et al. 2005; Thornton et al. 2010). BR levels are elevated in the bas1-2 sob7-1 double-null

mutant (first-exon T-DNA insertion alleles) when compared to the wild type or either single null

allele. BAS1 and SOB7 also affect developmental processes, such as hypocotyl elongation and

flowering, in a synergistic/redundant fashion (Turk et al. 2005). Independent evolution of

multiple BR-inactivating pathways indicates the importance of this process in plant growth and

development. Therefore, identifying the contributions of enzymes and pathways related to the

inactivation of these hormones is important for understanding BR-mediated development

(Sandhu et al. 2012). Genetic interactions among the mutants of BAS1, SOB7 and BEN1 would

indicate their specific roles in Arabidopsis development.

As a part of our analysis of BR catabolic enzymes, we generated a genetic triple-mutant from

a cross between the bas1-2 sob7-1 double-null and ben1-1. Our results show that the full loss-of-

function ben1-1 mutation was transformed to a partial loss-of-function mutation in the bas1-2

sob7-1 ben1-1 (triple-mutant) background showing enhanced levels of the wild-type-spliced

transcript. Interestingly, the enhanced expression of BEN1 remained stable when the ben1-1

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single-mutant was re-isolated from a cross with the wild-type. In addition, the ben1-1 single-

mutant isolated back from the triple mutant was phenotypically different from the original ben1-

1 allele in terms of seedling-development. The size of T-DNA insertion and the NPTII gene

sequence did not change in the pre-triple and post-triple ben1-1 mutants. However, the

previously functional NPTII T-DNA marker gene (which encodes kanamycin resistance) was no

longer functional in the recovered ben1-1 allele. Restriction endonuclease analysis with

methylation sensitive enzymes followed by genomic PCR showed that the methylation status of

the T-DNA is different between the original and the recovered ben1-1. Our study shows that the

ben1-1 BR-catabolism mutation is unstable due to epigenetic modifications of the intronic T-

DNA insertion.

MATERIALS AND METHODS

Plant material: All mutants used in this study, ben1-1 (Yuan et al. 2007), bas1-2 and sob7-1

(Turk et al. 2005), were in the Columbia (Col-0) background. ben1-1 was crossed with bas1-2

sob7-1, and multiple mutant combinations were isolated from populations of F2 individuals

derived from self-pollinated F1 plants. The ben1-1 allele was characterized by amplifying

genomic DNA with gene specific PCR primers GSP1 and GSP2, and T-DNA specific PCR

primers LBb1.3, and PRT2 (Table S1). Molecular-genetic analysis of the bas1-2 and sob7-1

alleles is described in Turk et al. (2005).

ben1-1 was isolated from the triple-mutant bas1-2 sob7-1 ben1-1 background by crossing

with the wild-type (Col-0) and allowing the F1 population to self-pollinate. The subsequent F2

82

population was screened with PCR markers specific for the bas1-2, sob7-1 and ben1-1 T-DNA

insertions. An individual F2 plant homozygous for the wild-type SOB7 allele and heterozygous

for bas1-2 and ben1-1 T-DNA insertions was thus identified. The F3 progeny of this individual

was further screened to identify and recover multiple single ben1-1 F3 lines (#11.4 and #11.5) as

well as a bas1-2 sob7-1 ben1-1 triple-mutant line. The homozygous F4 seeds derived from the

identified individual F3 lines were used for quantitative RT-PCR and hypocotyl growth analysis.

Exogenous hormone treatment: The stock solution of brassinolide (BL) was dissolved in 95 %

ethanol (v/v). BL treatment was performed by adding the BL stock solution to the seedling

growth media to a final concentration of 100 nM. An equal amount of 95 % ethanol were added

to the negative-hormone control media.

Hypocotyl measurement: Procedures for seed sterilization, plating, growth conditions and

hypocotyl measurement were as described in Turk et al. (2003).

Transcript analysis: Total RNA was isolated, using the RNeasy Plant Kit (Qiagen, Valencia,

CA), from five-day-old seedlings grown in continuous white light (25 µmol m-2

sec-1

). On-

column DNase digestion was performed using the RNase-Free DNase Set (Qiagen, Valencia,

CA), to eliminate genomic DNA contamination. Total cDNA was synthesized using

SuperScriptIII First-Strand Synthesis System (Invitrogen, Carlsbad, CA). The complete BEN1

transcript was amplified using primers PRT1 and PRT2. ACTIN2 was used as an internal control

in RT-PCR (Table S1). The linear range of amplification for each gene transcript was determined

by comparing samples obtained using different numbers of cycles. Lack of genomic and foreign

DNA contamination was ascertained by using all RNA samples and water as a template in a PCR

reaction.

83

Real-time quantitative RT-PCR analysis: For real-time quantitative RT-PCR analysis, Applied

Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems; Foster City, CA) was used.

BEN1 transcript was amplified using primers PQ1 and PQ2. BAS1 transcript was amplified using

primers PQ3 and PQ4 (Table S1). The internal control ubiquitin gene (At5g15400) was amplified

using PCR Primers UBQ1 and UBQ2 (Table S1). PCR thermocycling program profile used was

as following: initial denaturation at 95◦C for 20 s, followed by 40 cycles of 95

◦C for 3 s, 60

◦C for

30 s. Melt curve profile used melt curve analysis was as following: 95◦C for 15 s, 60

◦C for 1 min,

95◦C for 30 s and 60

◦C for 15 s.

Amplification of T-DNA insertion: The T-DNA insertion fragment was amplified using

PrimeSTAR® GXL polymerase (Clonetech, Mountain view, CA). PCR thermocycling program

profile used was as following: 35 cycles of 98◦C for 10 s, 64

◦C for 15 s, and 68

◦C for 10 min.

RESULTS

The ben1-1 T-DNA insertion mutation is unstable in the bas1-2 sob7-1 background: A bas1-

2 sob7-1 ben1-1 triple mutant line was isolated from a cross between a bas1-2 sob7-1 double-

and a ben1-1 single-mutant (Figure 1A, B and C). Transcript levels of all three genes were

examined to confirm that the triple mutant was a transcript-accumulation-null at all three loci.

Surprisingly, quantitative RT-PCR analysis showed that the triple mutant had significantly

enhanced levels of BEN1 transcript when compared to the single ben1-1 parental line, which

showed very low levels of BEN1 transcript accumulation (Figure 1D). Sequencing confirmed

that the BEN1 cDNA from both the wild type and the triple mutant was identical, indicating that

84

the T-DNA-containing 2nd

intron in the ben1-1 allele in the triple mutant had spliced in a wild-

type manner. These observations show that the ben1-1 null mutation is unstable in the bas1-2

sob7-1 background.

The BEN1 transcript levels stay steady in the re-isolated ben1-1 single mutant lines: The

bas1-2 sob7-1 ben1-1 triple mutant was back crossed to the wild type and in the segregating F3

progeny single ben1-1 mutant lines (# 11.4 and # 11.5) were re-isolated (Figure 2A).

Interestingly, BEN1 transcript levels in the two re-isolated ben1-1 lines were still enhanced

relative to the original ben1-1 line and similar to the bas1-2 sob7-1 ben1-1 triple mutant (Figure

2B). The unstable nature of the ben1-1 mutation in the bas1-2 sob7-1 ben1-1 background, and

the transmission of this phenotype in the re-isolated ben1-1 single-mutant lines, suggests that the

ben1-1 locus was modified either genetically or epigenetically in a heritable manner during the

creation of the triple mutant.

Re-isolated ben1-1 single-null lines are phenotypically different from the original ben1-1

lines: To test the hypothesis that the re-isolated ben1-1 mutant lines are also phenotypically

different from the original ben1-1 allele, fluence-rate response analysis of the hypocotyl-

elongation response was studied in these lines (Figure 3A). The two re-isolated ben1-1 lines

showed attenuated hypocotyl-elongation phenotypes when compared to the two original ben1-1

lines (Figure 3A). At all three fluence rates of white light, the re-isolated lines displayed

significantly shorter hypocotyls than the original ben1-1 lines (P< 0.05). There were no

significant differences in hypocotyl-length between the original and the re-isolated ben1-1 lines

in the dark. Hypocotyl-elongation responses to exogenous BL treatments were also examined in

these lines (Figure 3B and C). In white-light, two original ben1-1 lines showed reduced

hypocotyl-elongation when compared to the wild type, whereas the behavior of the two re-

85

isolated ben1-1 lines were similar to the wild type (Figure 3B). The two original and re-isolated

ben1-1 lines did not respond differently to BL treatment in darkness (Figure 3C).

BEN1 transcript accumulation is not feed-back regulated by BRs: It is possible that the

original ben1-1 was a leaky mutation to start with and that the elevated BR levels in the bas1-2

sob7-1 genetic background further increased BEN1 transcript accumulation which amplified the

leakiness of ben1-1. To test this hypothesis, first, a modified RT-PCR approach was used to

detect the rare wild-type-spliced BEN1 transcripts in the original ben1-1 line. As was previously

published, after 35 PCR cycles no product was detected in the ben1-1 line (Yuan et al. 2007;

Figure 4A). However after 50 PCR cycles, a BEN1 amplification product could be detected,

showing the presence of low level wild-type-spliced BEN1 transcript in the original ben1-1 line

(Figure 4A).

To further test the hypothesis that the BR feed-back regulation in the bas1-2 sob7-1

background was causing the instability of the ben1-1 mutation, BEN1 transcript levels were

examined in the wild-type and the bas1-2 sob7-1 double-null line using quantitative RT-PCR

(Figure 4B). BEN1 wild-type-spliced transcript levels were also examined in response to

exogenous BL treatment in the wild type and the original ben1-1 line (Figure 4D). Results

showed that when compared to the wild type, BEN1 transcript levels were not significantly

enhanced in the bas1-2 sob7-1 background (Figure 4B). Moreover, unlike BAS1 (Figure 4C)

exogenous BL treatment did not result in any increase in BEN1 transcript accumulation levels in

the wild type (Figure 4D). In addition, exogenous BL treatment also did not result in an

increased accumulation of wild-type spliced BEN1 transcript in the original ben1-1 line. These

observations clearly show that BR-mediated feed-back regulation in the bas1-2 sob7-1

background is not responsible for the ben1-1 mutation instability in the triple mutant.

86

T-DNA trans-interactions likely cause ben1-1 instability: Given that BEN1 transcript

accumulation is not feed-back regulated by changing levels of BRs, it is possible that T-DNA

trans-interactions between T-DNA insertions located in the bas1-2, sob7-1 and ben1-1 alleles

can also lead to ben1-1 mutation instability in the triple-mutant. Epigenetic silencing of the T-

DNA-located resistance markers often indicates the presence of T-DNA trans-interactions

(Daxinger et al. 2008).

To test this hypothesis, NPTII resistance marker gene function in the ben1-1 T-DNA was

examined by planting seeds of the wild type, original ben1-1, triple-mutant and the re-isolated

ben1-1 (# 11.4) lines on kanamycin containing plant growth media. Seedlings of all the lines

except the original ben1-1 line showed complete kanamycin sensitivity (Figure 5A). This

observation suggests the presence of T-DNA trans-interactions resulted in silencing of the

resistance marker in the triple- and the re-isolated ben1-1 single-mutant lines. Another possible

explanation for these observations is that the NPTII locus had been mutated during the crossing

and recombination process. To test this second possibility, the NPTII gene was amplified and

sequenced from both the original and the re-isolated ben1-1 lines (Figure 5B and C). The NPTII

gene sequence and primary structure was found to be unaltered in the original and the re-isolated

ben1-1 lines. These observations confirm that the loss of the kanamycin resistance marker in the

triple-mutant and the re-isolated ben1-1 lines is the effect of the presence of T-DNA trans-

interactions in the triple-mutant.

The ben1-1 T-DNA is differentially methylated in the original and the re-isolated ben1-1

lines: Silencing of the NPTII resistance marker gene in the triple-mutant and re-isolated ben1-1

lines suggest that this genomic region has been differentially modified by methylation in the

triple-mutant and the re-isolated ben1-1 T-DNAs. To test this hypothesis, genomic DNA from

87

the wild type, triple-, original and re-isolated ben1-1 single-mutant lines was digested with CpG

methylation sensitive and insensitive restriction enzymes (REs). The restriction-site-specific

sequences for the CpG methylation sensitive enzymes SacII and SmaI are present in the NPTII

promoter and in proximity to the left border of the ben1-1 inserted T-DNA respectively (Figure

6A). The restriction-site-specific sequence for the CpG methylation insensitive enzyme EcoRI is

located adjacent to the SmaI RE site (Figure 6A). Following digestion with REs, genomic DNA

was used as a PCR template in which primers flanking the corresponding RE sites were used to

prime the amplification (Figure 6A). Results of the PCR amplification showed that the triple

mutant and re-isolated ben1-1 T-DNA were more resistant to RE digestion by both the CpG

methylation sensitive REs when compared to the original ben1-1 T-DNA (Figure 6B), whereas

there were no differences among these genotypes with regard to EcoRI digestion (6B). These

observations demonstrate that the ben1-1 T-DNA in the triple- and the re-isolated ben1-1 single-

mutant is differentially methylated in both the promoter region of NPTII gene as well as in the

adjacent regions.

T-DNA size and structure remains unchanged in the original and the re-isolated ben1-1

lines: In another scenario, a shortening of T-DNA insertion due to unequal recombination during

the creation of the triple-mutant could also result in enhanced BEN1 transcript levels when

compared to the original ben1-1 line. To test this hypothesis, the inserted T-DNA was amplified

from the original and the re-isolated ben1-1 lines using primers anchored in the 2nd

and 3rd exons

of the BEN1 gene (Figure 7A). The PCR results show that, using wild-type DNA as a PCR

template gave a band of the size expected in the case of no T-DNA insertion (Figure 7B). On the

other hand, both the original and re-isolated ben1-1 lines showed a large band of the same size,

indicating the presence of approximately two T-DNA insertions in each case (Figure 7B). To

88

further test the possibility of any re-arrangement of the T-DNA in original versus the re-isolated

ben1-1, the large T-DNA containing amplification product of the extended PCR (Figure 7B) was

purified and cut using two different restriction enzymes. Identical restriction pattern of the two

amplification products showed the absence of any T-DNA re-arrangement between the original

and the re-isolated T-DNA insertions (Figure 7C). These experiments suggest that the T-DNA

size and structure have not changed between the original and the re-isolated ben1-1 lines.

DISCUSSION

As a part of our analysis of BR catabolic enzymes, we generated a genetic triple mutant from

a cross between the bas1-2 sob7-1 double-null and ben1-1. Surprisingly, the originally stable

ben1-1 mutation was converted to a partial loss-of-function mutation in the triple mutant

background showing increased levels of the wild-type-spliced transcript (Figure 1D). To study

the genetics of ben1-1 instability, we re-isolated the ben1-1 mutation by back crossing the triple

mutant with the wild type (Figure 2). Molecular analysis of re-isolated ben1-1 lines showed that

the unstable state of the ben1-1 mutation in the triple mutant is maintained in an otherwise wild-

type background. The original and the re-isolated ben1-1 single lines also differed

phenotypically (Figure 3). The original ben1-1 single-mutant line displays an aberrant hypocotyl-

elongation phenotype in white light (Yuan et al. 2007). When compared to the original ben1-1

allele, the re-isolated ben1-1 lines displayed significantly different hypocotyl-elongation

phenotypes in white-light fluence-rate response experiments as well as in a BL dose-response

treatment (Figure 3A, B and C). These genetic and molecular observations suggest that the

89

changes in the re-isolated ben1-1 are of an epigenetic nature. However, the exact mechanism

leading to the enhanced T-DNA-containing intron-splicing is unknown.

In the case of intronic T-DNA insertion mutations, the presence of wild-type-spliced

transcript is not uncommon (Wang 2008). Additionally, conditional mutant instability has been

observed in an intronic T-DNA insertion mutation in the OPR3 (OPDA REDUCTASE 3) gene

(Chehab et al. 2011). opr3 is an intronic T-DNA insertion mutant of OPR3 which does not

produce any detectable jasmonic acids (JAs) and acts as a complete loss-of-function allele under

normal conditions. However, the opr3 allele becomes unstable when mutant plants are infected

with a fungus (Chehab et al. 2011). This suggests that environment may play a role in the

phenomenon of T-DNA insertion mutant instability.

To test the hypothesis that the epigenetic transformation of ben1-1 in the triple mutant could

be due to elevated BR content in the bas1-2 sob7-1 genetic background, we studied BEN1

expression in the bas1-2 sob7-1 double-null. BEN1 expression was not significantly affected in

the bas1-2 sob7-1 background (Figure 4B), suggesting that BEN1 is not strongly feed-back

regulated by changes in BR levels. This suggests that elevated BR levels in bas1-2 sob7-1 (Turk

et al. 2005) did not result in ben1-1 instability in the triple-mutant background (Figure 4B).

Another possible explanation is that BEN1 is not expressed in the same tissues where both BAS1

and SOB7 are expressed. bas1-2 and sob7-1 single-null mutants do not have altered BR levels.

As BAS1 and SOB7 expression patterns do not overlap completely (Sandhu et al. 2012), it is

possible that all tissues in the bas1-2 sob7-1 double-null do not have altered BR levels. Analysis

of the publically available microarray data suggests that BEN1 transcript accumulation is not

significantly affected by BL treatment (Winter et al. 2007). To complement the microarray data

analysis, we also studied BEN1 expression in the wild type and the original ben1-1 single-mutant

90

in response to exogenous BL (Figure 4D). BAS1 expression was also studied in the same

experiment to compare BEN1 gene expression response to a known BR feedback regulated gene.

The results of gene expression analysis also showed that BEN1 expression is not affected by BL

treatment and hence it is highly unlikely that BR feed-back regulation is responsible for the

ben1-1 instability (Figure 4C and D).

Since the mutagenic T-DNA is the same in bas1-2, sob7-1 and ben1-1 (Alonso et al. 2003), a

possible cause of ben1-1 instability could be the effect of T-DNA trans-interactions. As

documented previously, T-DNA trans-interactions can result in the loss of antibiotic resistance

(Daxinger et al. 2008). In fact, the original ben1-1 mutant seedlings showed kanamycin

resistance, whereas the re-isolated lines as well as the triple were completely sensitive to

kanamycin (Figure 5A). Interestingly, the loss of kanamycin resistance also showed an

epigenetic pattern of inheritance. Similar loss of kanamycin resistance is observed in the case of

yuc1-1 (yucca1-1) and ag-TD (agamous-TDNA insertion mutant) interactions (Gao and Zhao,

2012). Silencing of homologous genes is known to be an effect of T-DNA trans-interactions

(Daxinger et al. 2008). Homology induced silencing is also associated with a phenomenon

known as RNA-directed DNA methylation in the region of RNA-DNA homology (Wasseneger

2000). In fact, the promoter region of the re-isolated ben1-1 line T-DNA showed cytosine

methylation, which is not the case with the original ben1-1 T-DNA (Figure 6). The T-DNA

methylation was also observed in the region adjacent to the NPTII locus (Figure 6). The spread

of DNA methylation to adjacent parts of the RNA-DNA homology region has previously been

documented (Wassenger 2000). Another possibility is that apart from the NPTII gene, the mRNA

transcribed from other parts of the T-DNA is also present in the ben1-1 mutant.

91

T-DNA trans-interactions have also been associated with instability of an intronic T-DNA

insertion mutant (Gao and Zhang, 2012). However, currently there is no evidence which supports

that the mutant instability is caused directly by T-DNA trans-interactions. Plausibly, one of the

factors involved in mutant instability is the efficient splicing of the T-DNA containing intron.

The splicing efficiency could be affected by a change in the size of T-DNA insertion due to

events such as unequal recombination. However, the size of T-DNA insertion and structure is

unchanged in the pre- and post-triple ben1-1 mutant lines (Figure 7). The most apparent

difference in the original and the re-isolated ben1-1 lines is the presence of methylation in the re-

isolated ben1-1 T-DNA region (Figure 6). RNA-induced DNA methylation has been associated

with chromatin modification (Aufsatz et al. 2002a; Aufsatz et al. 2002b). Chromatin

modifications in turn have been linked to the regulation of pre-mRNA splicing by many studies

(for review see: Hnilicova and Stanek, 2011). For example, alternative splicing in the CD44 gene

required proper recruitment of argonaute RNAi proteins to the CD44 transcribed regions

(Ameyar-Zazoua et al. 2012). Therefore, it is possible that the methylation of the T-DNA region

is altering the splicing efficiency of the T-DNA containing intron (Figure 8).

However, more studies are required to test the association between T-DNA trans-

interactions, T-DNA methylation and the T-DNA containing intron splicing. To further analyze

the role of T-DNA trans-interactions, ben1-1 mutation stability can be studied in the genetic

background of non-BR related transgenics (harboring T-DNAs expressing kanamycin

resistance). Further studies can also analyze the nature of chromatin modifications in the original

vs the recovered ben1-1 T-DNA. Primarily, this study indicates the need for more caution while

using T-DNA insertional mutants. Future directions for this study will include isolation of

92

additional mutations in the BEN1 locus. An EMS mutagenesis approach (Street et al. 2008) using

the activation-tagged mutant ben1-D will be used to isolate true null-alleles of BEN1.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Jia Li for providing ben1-1 seeds on our request. We also

thank William R. Buckley and other Neff lab members for their critical review of this

manuscript. This research was supported by the National Science Foundation 0758411

(M.M.N.). We are also grateful for support from the Department of Energy DE-PS02-09ER09-02

(M.M.N.).

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proteins SOB3/AHL29 and ESC/AHL27 are negative modulators of hypocotyl growth in

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homeostasis in Arabidopsis is ensured by feedback expressions of multiple genes involved in its

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Arabidopsis thaliana. Plant J. 51: 220-233.

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TABLES, FIGURES AND LEGENDS

Figure 1: ben1-1 mutation is unstable in the bas1-2 sob7-1 background. (A) Graphical

depiction of BEN1 gene structure and location of T-DNA insertion in the ben1-1 allele. (B)

Depiction of the crossing scheme used to isolate bas1-2 sob7-1 ben1-1 triple-null. (C) Example

of the genetic analysis of ben1-1 allele. (D) Quantitative RT-PCR analysis of BEN1 expression

in the wild type, ben1-1 and triple null. Error bars indicate the standard error of the mean (SE).

97

Figure 2: The BEN1 transcript levels stay steady in the re-isolated ben1-1 single-null lines.

(A) Depiction of the crossing scheme used to re-isolate ben1-1. (B) Quantitative RT-PCR

analysis of the BEN1 transcript in the five-day-old seedlings of Col-0, original ben1-1, bas1-2

sob7-1 ben1-1, F3# 11.4 (re-isolated line) and F3# 11.5 (re-isolated line) shows that the

transcript accumulation levels in the re-isolated lines (F3# 11.4 and F3# 11.5) remain stable at

the same level as that of triple null line. Error bars indicate the standard error of the mean (SE).

98

Figure 3: Re-isolated ben1-1 single-null lines are phenotypically different from the original

ben1-1 lines. (A) Fluence-rate analysis of the Col-0, original ben1-1 line #1 and #2, and re-

isolated ben1-1 lines at three different white-light fluence rates. The hypocotyl length of the re-

isolated ben1-1 is not significantly different from original ben1-1 (p> 0.5) in the dark. At all

three white-light fluence rates, the hypocotyl length of the re-isolated ben1-1 is significantly

different from original ben1-1 lines (p<0.05 in all cases). (B) Hypocotyl-elongation response of

the re-isolated and original ben1-1 lines to the exogenous BL treatment (100 nm BL) in white-

light. In white light (24 µmol m-2

sec-1

) the hypocotyl-elongation response of the re-isolated

ben1-1 is similar to the wild-type. The hypocotyl elongation is suppressed by BL treatment in the

original ben1-1 lines when compared to the wild type. (C) In the dark the hypocotyl-elongation

response of the re-isolated ben1-1 is not significantly different from original ben1-1 (p> 0.5). To

calculate the percentage of BL-negative-control hypocotyl length (as in B and C), each seedling

value in BL treatment experiment was normalized to the average of the same genotype in BL

negative control experiment. The resulting group of values was used to calculate standard error

of the mean (SE).

99

100

Figure 4: The ben1-1 is a leaky mutant based on RT-PCR analysis and the enhanced T-

DNA-containing-intron splicing in recovered ben1-1 is not induced by BR feedback

regulation. (A) Primer pair, PRT1 and PRT2 was used to amplify full-length coding region of

BEN1 transcript. Amplification of ACTIN2 transcript was used as a cDNA loading control (Lane

1 and 2). At 35 cycles no product is detected in the ben1-1 lane (Lane 5) as compared to the Col-

0 lane (Lane 4). After 50 cycles BEN1 transcript is detectable in the ben1-1 lane (Lane 8). Primer

sequence information is given in Table S1. (B) BEN1 expression is not affected in the bas1-2

sob7-1 double-null background relative to the wild type background. (C) Exogenous BL

treatment induces BAS1 expression in Col-0 and ben1-1 background. (D) Exogenous BL

treatment did not induce BEN1 expression in Col-0 and ben1-1 background. Error bars indicate

the standard error of the mean (SE).

101

Figure 5: The kanamycin resistance trait, conferred by the T-DNA resistance marker

NPTII, is absent in the bas1-2 sob7-1 ben1-1 and the recovered ben1-1 line. (A) Seedlings

were grown on the plant media containing kanamycin for seven days before photographing. (B)

The graphic depiction of the location of the primers used to amplify NPTII from the T-DNA. (C)

The gel image shows that genomic PCR amplifies bands of equal size from original ben1-1 and

the recovered ben1-1 lines.

102

Figure 6: Genomic DNA in the NOS Promoter region of the T-DNA insertion in the

recovered ben1-1 shows methylation. (A) Graphic depiction of restriction enzyme sites and the

primer pair locations in the T-DNA used for the methylation study. (B) Genomic PCR

amplification from the undigested (-) and the restriction digested (+) DNA of the Col-0, original

ben1-1, recovered ben1-1 (11.4), and the bas1-2 sob7-1 ben1-1 (triple). Primer pairs MT1 and

MT2 were used to test methylation at the SacII site. Primer pairs MT3 and MT4 were used to test

methylation at the SmaI site. Primer pairs MT3 and MT4 were also used to amplify genomic

DNA digested with non-methylation sensitive EcoRI. PCR amplification, by using SacII site and

SmaI/EcoRI flanking PCR primers (Table S1), is seen only from the digested genomic DNA of

the recovered ben1-1.

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Figure 7: The size and sequence of the T-DNA insertion remains unchanged in the pre-

triple and post-triple ben1-1 lines. (A) Graphic depiction of the location of the 2nd

intron

flanking primer pair used for amplification in the ben1-1 allele. (B) The genomic PCR using the

2nd

intron flanking primers amplify equal size band from the original ben1-1 and the recovered

ben1-1 line. (C) The original and the recovered ben1-1 alleles show identical restriction digestion

pattern. The PCR amplification product from gel image (B) was gel purified and restriction

digested with HindIII and KpnI.

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Figure 8: Hypothetical model for RNAi and chromatin structure dependent intron splicing.

Argonaute protein is guided by the associated siRNA and recruited by chromatin component

proteins associated with chromatin marks (H3K9me3). This complex slows down the movement

of RNA polymerase II and possibly affects the splicing decisions by the spliceosome. Figure

adapted from Ameyar-Zozoua et al. 2012.

105

Supplemental Information:

Table S1. Table of PCR primer sequences used in the study.

Primer Name Primer Sequences

GSP1 5'-TTTATATCTTCGTCGTTGCCT-3'

GSP2 5'-ACCTTTTGCAACTGGCTTTT-3'

LBb1.3 5'-ATTTTGCCGATTTCGGAAC-3'

PRT1 5'-ATGGTGAGAGAAGAACAAGA-3'

PRT2 5'-TTAAAGAAATCCCCTTGCTTG-3'

PQ1 5'-ATCTCTCGCCATGCTCTTTG-3'

PQ2 5'-CTTCATCTCCACCGACGAAC-3'

UBQ1 5'-GAAATGCATGGAGACGGATT-3'

UBQ2 5'-TTGGTCTCTGCTCCCACTCT-3'

ACTIN2 RT-F 5'-GGTCGTACAACCGGTATTGTGCTGG-3'

ACTIN2 RT-R 5'-CTGTGAACGATTCCTGGACCTGCC-3'

PQ3 5'-TCTAAACAAGGAATTCGAGGTC-3'

PQ4 5'-AACCAGAAATGTAGCACCGT-3'

NPT-Fv/MT1 5'-ACAAGCCGTTTTACGTTTGG-3'

NPT-Rv 5'-TCATTTCGAACCCCAGAGTC-3'

MT2 5'-ATACTTTCTCGGCAGGAGCA-3'

MT3 5'-TTCGCAAGACCCTTCCTCTA-3'

MT4 5'-GTTTTCCCAGTCACGACGTT-3'

CHAPTER FOUR

The Arabidopsis gene ATST4a in not a typical brassinosteroids catabolic gene.

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ABSTRACT

Brassinosteroids (BRs) homeostasis is maintained in part by this hormone’s catabolism.

Presence of multiple brassinosteroids (BRs) catabolic pathways in Arabidopsis suggests the

importance of this process in plant growth and development. In vitro biochemical analyses

demonstrate that ATST4a has catalytic activity with BRs. However, unlike other BR catabolic

pathways the genetic evidence to suggest such a function for the ATST4a pathway in planta is

scant. In this study, we have used both loss-of-function and over-expression genetic approaches

to further explore the role of ATST4a in Arabidopsis. This study shows that atst4a1-1, a T-DNA

insertion null mutant of ATST4a did not display reduced seedling hypocotyl growth inhibition in

white light. Additionally, overexpression of ATST4a gene in transgenic lines did not result in any

characteristic BR-deficient phenotypes. These observations suggest that ATST4a gene encodes

an atypical BR catabolic enzyme.

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INTRODUCTION

Catabolism is an important aspect of brassinosteroid (BR) homeostasis. Discovery of several

independent BR catabolic pathways and their enzymes in Arabidopsis also suggest the

importance of this process in plant growth and development (Neff et al. 1999; Turk et al. 2003;

Turk et al. 2005; Poppenberger et al. 2005; Yuan et al. 2007; Husar et al. 2011; Roh et al. 2012;

Schneider et al. 2012). These BR catabolic pathways were mainly discovered and latter

characterized using genetic manipulation of the genes involved. In some cases, in vivo and in

vitro biochemical assays of the enzymes encoded by these genes were also used to confirm their

role in BR catabolism. Genetic analyses of knockout or knockdown mutants of most of these BR

catabolic genes show a reduction in hypocotyl-elongation inhibition in white light in Arabidopsis

seedlings (Neff et al. 1999; Turk et al. 2005; Popppenberger et al. 2005; Yuan et al. 2007; Roh

et al. 2012). The knockout or knockdown approach may in some cases fail to provide any

evidence of involvement in BR catabolism due to genetic redundancy or yet other unknown

reasons (Husar et al. 2011). However, in all cases, in planta overexpression of BR catabolic

genes leads to characteristic BR-deficient phenotypes at both seedling and adult stages of

Arabidopsis (Neff et al. 1999; Turk et al. 2003; Turk et al. 2005; Takahashi et al. 2005;

Nakamura et al. 2005; Poppenberger et al. 2005; Yuan et al. 2007; Husar et al. 2011; Roh et al.

2012; Schneider et al. 2012). These BR-deficient phenotypes typified by a BR biosynthetic

mutant de-etiolated 2-1 (det2-1), include small organ size, small round and dark green leaves,

and delayed flowering (Chory et al. 1991; Azpiroz et al. 1998; Domagalska et al. 2007; Li and

Chory 1997).

Members of the sulfotransferase protein superfamily are known to perform various metabolic

functions in plants (Klein and Papenbrock, 2004; Kopriva et al. 2012) and some family members

109

are also known to be metabolically active in hormonal pathways (Gidda et al. 2003; Baek et al.

2010). ATST4a (AT2G14920) encodes a sulfotransferase member in Arabidopsis (Klein and

Papenbrock, 2004). ATST4a was cloned on the basis of homology to ATST1 (Marsolais et al.

2007). ATST1 is an ortholog of BNST (steroid sulfotransferase from Brassica napus) proteins

and displays similar catalytic activities and substrate specificity. Recent evidence shows that

ATST1 is also involved in the sulfonation of salicylic acid in response to pathogen stress (Baek

et al. 2010). In vitro protein expression and sulfotransferase assay studies demonstrate that

ATST4a has catalytic activity with BRs (Marsolais et al. 2007). Catalytic activity of ATST4a is

specific for biologically active end products of the BR biosynthetic pathway, including

castasterone, brassinolide, related 24-epimers and the naturally occurring (22R, 23R)-28-

homobrassinosteroids. The addition of a sulfate group to the steroid molecules may cause a

change in their activity and therefore play a role in steroid homeostasis (Strott et al. 2002). These

observations suggest that ATST4a is involved in BR homeostasis via catabolism in Arabidopsis.

Although biochemical analysis can provide important clues, use of genetic approaches can

potentially further our understanding of endogenous gene functions. In this study, we have used

both loss-of-function and over-expression genetic approaches to further explore the role of

ATST4a in Arabidopsis. This study shows that atst4a1-1, a T-DNA insertion null mutant of

ATST4a did not display reduced seedling hypocotyl growth inhibition in white light.

Additionally, overexpression of the ATST4a gene in transgenic lines did not result in any

characteristic BR-deficient phenotypes, and did not cause a predicted feed-back-regulation

change in the transcript accumulation of DWF4 and BAS1 (Tanaka et al. 2005). These

observations suggest that ATST4a gene encodes an atypical BR catabolic enzyme.

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MATERIAL AND METHODS

Plant Material: All plant material used in this study was in Col-0 background. For loss-of-

function analysis of ATST4a, a T-DNA insertion line GK-177E08 with an insertion in the

ATST4a ORF was obtained from GABI-Kat (Kleinboelting et al. 2012). The T-DNA line was

backcrossed twice to the Col-0 to clean the background of any unwanted mutations. The T-DNA

insertion was followed through the crosses using the following set of primers: gene specific

primers- 5'-ATG GAT GAA AAA GAT AGA CC-3' and 5'-TTA GAA TTT CAA ACC GGA

ACC-3' and T-DNA specific primer 5'-ATA TTG ACC ATC ATA CTC ATT G-3'. For over-

expression analysis, ATST4a coding sequence including start and stop codon were cloned in

pENTR/D/TOPO vector (Invitrogen, Carlsbad, CA). The cloned DNA was sequenced to make

sure that there were no mutations. The coding sequence containing fragment was further

subcloned into a binary vector pEARLYGATE100 by gateway®

cloning using LR clonase

(Invitrogen). The binary vector was transformed into atst4a1-1 to increase the frequency of

transgenic over expressers by avoiding RNAi interference from the endogenous transcript. Single

locus insertion lines based on a 3:1 ratio of resistant to susceptible were selected for further

analysis. For generating ATST4a:GUS translational fusion lines, the 3.0 Kb genomic fragment

containing the ATSt4a promoter and gene was cloned in frame with uidA gene in the

pCAMBIA1305.1 vector (CAMBIA, CANBERRA, AUSTRALIA). This construct was

transformed into st4a1-1 plants. Multiple transgenic lines segregating at a 3:1 ratio (hygromycin

resistant/sensitive ratio) in the T2 generation were identified as single insertion lines.

Transcript analysis: Total RNA was isolated, using the RNeasy Plant Kit (Qiagen, Valencia,

CA) from five-day-old seedlings grown in continuous white light (25 µmol m-2

sec-1

). On-

111

column DNase digestion was performed using the RNase-Free DNase Set (Qiagen, Valencia,

CA), to eliminate genomic DNA contamination. Total cDNA was synthesized using

SuperScriptIII First-Strand Synthesis System (Invitrogen, Carlsbad, CA). ATST4a transcript was

amplified using primers 5'-ATG GAT GAA AAA GAT AGA CC-3' and 5'-TTA GAA TTT

CAA ACC GGA ACC-3'. ACTIN2 was used as an internal control in RT-PCR. ACTIN2

transcript was amplified using primers 5'-GGT CGT ACA ACC GGT ATT GTG CTG G-3' and

5'-CTG TGA ACG ATT CCT GGA CCT GCC-3'. The linear range of amplification for each

gene transcript was determined by comparing samples obtained using different numbers of

cycles. Lack of genomic and foreign DNA contamination was determined by using all RNA

samples and water as a template in a PCR reaction.

Real-time quantitative analysis of transcript levels: Applied Biosystems 7500 Fast Real-Time

PCR System (Applied Biosystems; Foster City, CA) was used for real-time quantitative RT-PCR

analysis. ATST4a transcript was amplified using primers 5'-TCA CTC GAG CGG AGG ATT

AC-3' and 5'-GAG TAC GAG GCT CCG CTT T-3'. Internal control ubiquitin gene

(AT5G15400) was amplified using PCR Primers 5'-GAA ATG CAT GGA GAC GGA TT-3' and

5'-TTG GTC TCT GCT CCC ACT CT-3'. PCR thermocycling program profile used was as

following: initial denaturation at 95◦C for 20 s, followed by 40 cycles of 95

◦C for 3 s, 60

◦C for 30

s. Melt curve profile, which was used for melt curve analysis was as following: 95◦C for 15 s,

60◦C for 1 min, 95

◦C for 30 s and 60

◦C for 15 s.

Exogenous hormone treatment: The stock solution of brassinolide (BL) was dissolved in 95 %

ethanol (v/v). BL treatment was given by adding BL stock solution to the seedling growth media

to a final concentration of 100 nM.

112

Hypocotyl measurement: Procedures for seed sterilization, plating, growth conditions and

hypocotyl measurement were done as described in Turk et al. (2003).

RESULTS

The T-DNA insertional mutant of ATST4a displayed a wild-type response to light-induced

inhibition of hypocotyl growth: A T-DNA insertion line (designated atst4a1-1) with an

insertion in ATST4a gene was obtained from GABI-Kat (Kleinboelting et al. 2012). Genetic

analysis by gene- and T-DNA-specific primers indicated that the T-DNA is inserted in the

coding sequence of the ATST4a gene (Figure 1A and B). Histochemical analysis of the ATST4a-

GUS translational fusion lines indicated that ATST4a is expressed in both white-light and dark

grown seedlings (Figure 2A). This observation suggested that ATST4a may play a role in

hypocotyl growth. To test this hypothesis, hypocotyl growth was studied in the wild type and the

atst4a1-1 lines. Hypocotyl growth in dark and two white-light fluence rates showed no

significant differences between wild type and atst4a1-1 lines (Figure 2B). This suggests that

ATST4a does not play a role in light-induced hypocotyl inhibition.

Arabidopsis plants overexpressing ATST4a cDNA display wild-type phenotypes at both

seedling and adult stages: To test the hypothesis that the constitutive overexpression of ATST4a

will lead to BR-deficient phenotypes; multiple transgenic lines overexpressing ATST4a cDNA

were generated. These ATST4a-overexpression transgenic lines were characterized by

quantitative RT-PCR analysis. Three independent transgenic lines displaying high levels of

ATST4a expression (Figure 3A) were selected for further phenotypic analysis. Adult ATST4a-Ox

113

transgenic lines did not display any BR-deficit phenotypes (Figure 3B). Hypocotyl-growth was

also studied in the homozygous T3 ATST4a-Ox, wild-type and the atst4a1-1 seedlings in both

dark and white-light (Figure 4A and B). The hypocotyl length in the atst4a1-1 line was not

significantly different than the wild type (p>0.5). The three overexpression lines displayed

significantly shorter hypocotyl lengths than the wild type (p<0.01). The hypocotyl-growth

response of wild type, ATST4a-OX and atst4a1-1 seedlings was not altered by BL treatment in

the dark (Figure 5A). In white light, the hypocotyl length in the atst4a1-1 line was not

significantly different than the wild type (p>0.5) (Figure 5B). The T3-52 and T3-64

overexpression lines displayed significantly shorter hypocotyl lengths than the wild type

(p<0.01) (Figure 5B.)

Expression of the known BR-feedback regulated genes is not affected in ATST4a loss-of-

function and ATST4a-OX lines: Overexpression of BR catabolic genes often causes alterations

in the transcript levels of BR-feedback regulated genes. In these conditions, the transcript

accumulation of BR biosynthesis genes, such as DWF4, is enhanced whereas that of catabolic

genes, such as BAS1, is reduced. This response is often vectorially opposite in the loss-of-

function lines. To test this hypothesis, gene expression of BR-feedback regulated genes, BAS1

and DWF4 was studied in wild type, atst4a1-1 and T3 ATST4a-Ox seedlings. Quantitative RT-

PCR analysis showed that transcript accumulation of both BAS1 and DWF4 was not significantly

different in these lines (p>0.05 for all pair-wise comparisons)(Figure 6A and 6B).

DISCUSSION

114

Biochemical in vitro analysis of ATST4a suggests its role in BR catabolism (Marsolais et al.

2007). However, not enough genetic data is available to support this role of ATST4a’s in BR

catabolism in Arabidopsis. This study uses both loss-of-function and gain-of-function genetic

approaches to further analyze the role of ATST4a in Arabidopsis. Mutants of BR catabolic genes

in most cases are compromised in light-mediated hypocotyl growth inhibition (Neff et al. 1999;

Turk et al. 2005; Poppenberger et al. 2005; Yuan et al. 2007; Roh et al. 2012). However, despite

the observation that ATST4a is expressed in the seedling hypocotyl (Figure 2A), atst4a1-1 did

not display any defect in light-mediated hypocotyl inhibition (Figure 2B). This is not surprising

since mutants of some known BR catabolic genes such as UGT73C6 and PIZ1 also do not

display hypocotyl phenotypes (Husar et al. 2011; Schneider et al. 2012).

More surprisingly, the overexpression of ATST4a cDNA in Arabidopsis did not result in BR-

deficient phenotypes at adult plant stages (Figure 3A and B). Moreover, seedlings of the

transgenic lines overexpressing ATST4a cDNA did not show any dramatic increase in the light-

induced inhibition of hypocotyls (Figure 4). This observation is significant since all the known

BR catabolic genes characterized so far result in dramatic BR-deficient phenotypes both at

seedling and at adult stage when overexpressed in Arabidopsis (Husar et al. 2011; Poppenberger

et al. 2005; Roh et al. 2012; Turk et al. 2003; Turk et al. 2005; Yuan et al. 2007; Schneider et al.

2012). This suggests that ATST4a overexpression did not reduce BR content in the transgenic

lines. In addition, quantitative transcript analysis of the feed-back regulated genes DWF4 and

BAS1 also suggests that BR content is not reduced in the ATST4a-OX lines when compared to

wild type (Figure 6). Overall, this study shows that despite its in vitro preference for BR

substrates, ATST4a does not behave like a typical BR catabolic gene in planta.

115

In vitro biochemical analysis can give important clues to the function of an enzyme.

However enzyme activity and function at a tissue or cellular level may be dependent on certain

in vivo conditions and therefore difficult to assess. For example, the unique metabolic

environment within a cell or a tissue may provide crucial cofactors or in another case the

substrate preference may be different in an in vivo vs. the in vitro environment. Sulfotransferases

can display preference for a diverse range of substrates under in vitro conditions; however, this

may not fully reflect their endogenous functions (Kopriva et al. 2012). For example, biochemical

analysis of BNST3 suggests that it is involved in catabolizing 24-epibrassinosteroids; however,

over-expression of BNST3 do not lead to BR-deficient phenotypes in Arabidopsis, suggesting

that BR catabolism is not its endogenous function (Marsolais et al. 2004). Another explanation

for lack of BR-deficient phenotypes in overexpression lines is that of post-translational

regulation of ATST4a. It is also possible that the overexpressed ATST4a protein is not properly

localized in the plant cells. Given these observations, more studies are required to assign

biological function to ATST4a.The Brassica napus steroid sulfotransferases are inducible by

ethanol, xenobiotics and low oxygen stress and may be involved in plant stress response

(Marsolais et al. 2004). Therefore, it is possible that ATST4a also has a similar role in

Arabidopsis. This can be studied by observing growth of wild type, ATST4a loss-of-function and

overexpression lines in response to various stresses. Another direction would be to identify the

transcriptional regulators of ATST4a by using its promoter in a yeast-one-hybrid screen. They

may help identify the biological pathways in which ATST4a might be involved.

ACKNOWLEDGMENTS

116

We thank William R. Buckley and other Neff lab members for their critical review of this

manuscript. This research was supported by the National Science Foundation 0758411

(M.M.N.). We are also grateful for support from the Department of Energy DE-PS02-09ER09-02

(M.M.N.).

LITERATURE CITED

Azpiroz, R., Y. Wu, J. C. LoCasio and K. A. Feldmann, 1998 An Arabidopsis brassinosteroid

dependent mutant is blocked in cell elongation. Plant Cell 10: 219-230.

Baek, D., P. Pathange, J. S. Ching, J. Jiang, L. Gao et al., 2010 A stress-inducible

sulphotransferase sulphonates salicylic acid and confers pathogen resistance in Arabidopsis.

Plant Cell Environ. 33: 1383-1392.

Chory, J., P. Nagpal and C. A. Peto, 1991 Phenotypic and genetic-analysis of Det2, a new mutant

that affects light-regulated seedling development in Arabidopsis. Plant Cell 3: 445-459.

Domagalska, M. A., F. M. Schomburg, R. M. Amasino, R. D. Vierstra, F. Nagy et al., 2007

Attenuation of brassinosteroid signaling enhances FLC expression and delays flowering.

Development 134: 2841-2850.

Gidda, S. K., O. Miersch, A. Levitin, J. Schmidt, C. Wasternack et al., 2003 Biochemical and

molecular characterization of a hydroxyjasmonate sulfotransferase from Arabidopsis thaliana. J.

Biol. Chem. 278: 17895-17900.

Husar, S., F. Berthiller, S. Fujioka, W. Rozhon, M. Khan et al., Overexpression of the UGT73C6

alters brassinosteroid glucoside formation in Arabidopsis thaliana. BMC Plant Biol. 11: 51.

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Klein, M., and J. Papenbrock, 2004 The multi-protein family of Arabidopsis sulphotransferases

and their relatives in other plant species. J. Exp. Bot. 55: 1809-1820.

Kleinboelting, N., G. Huep, A. Kloetgen, P. Viehoever and B. Weisshaar, GABI-Kat

SimpleSearch: new features of the Arabidopsis thaliana T-DNA mutant database. Nucleic Acids

Res. 40: D1211-1215.

Kopriva, S., S. G. Mugford, P. Baraniecka, B. R. Lee, C. A. Mathewman et al., Control of sulfur

partitioning between primary and secondary metabolism in Arabidopsis. Front. Plant Sci. 3: 163.

Li, J. M., and J. Chory, 1997 A putative leucine-rich repeat receptor kinase involved in

brassinosteroid signal transduction. Cell 90: 929-938.

Marsolais, F., C. H. Sebastia, A. Rousseau and L. Varin, 2004 Molecular and biochemical

characterization of BNST4, an ethanol-inducible steroid sulfotransferase from Brassica napus,

and regulation of BNST genes by chemical stress and during development. Plant Science 166:

1359-1370.

Marsolais, F., J. Boyd, Y. Paredes, A. M. Schinas, M. Garcia et al., 2007 Molecular and

biochemical characterization of two brassinosteroid sulfotransferases from Arabidopsis, AtST4a

(At2g14920) and AtST1 (At2g03760). Planta 225: 1233-1244.

Nakamura, M., T. Satoh, S. Tanaka, N. Mochizuki, T. Yokota et al., 2005 Activation of the

cytochrome P450 gene, CYP72C1, reduces the levels of active brassinosteroids in vivo. J. Exp.

Bot. 56: 833-840.

118

Neff, M. M., S. M. Nguyen, E. J. Malancharuvil, S. Fujioka, T. Noguchi et al., 1999 BAS1: A

gene regulating brassinosteroid levels and light responsiveness in Arabidopsis. Proc. Natl. Acad.

Sci. USA 96: 15316-15323.

Schneider, K., C. Breuer, A. Kawamura, Y. Jikumaru, A. Hanada et al., 2012 Arabidopsis

PIZZA Has the Capacity to Acylate Brassinosteroids. PLoS One 7.

Poppenberger, B., S. Fujioka, K. Soeno, G. L. George, F. E. Vaistij et al., 2005 The UGT73C5

of Arabidopsis thaliana glucosylates brassinosteroids. Proc. Natl. Acad. Sci. USA 102: 15253-

15258.

Poppenberger, B., W. Rozhon, M. Khan, S. Husar, G. Adam et al., CESTA, a positive regulator

of brassinosteroid biosynthesis. EMBO J. 30: 1149-1161.

Roh, H., C. W. Jeong, S. Fujioka, Y. K. Kim, S. Lee et al., Genetic evidence for the reduction of

brassinosteroid levels by a BAHD acyltransferase-like protein in Arabidopsis. Plant Physiol.

159: 696-709.

Strott, C. A., 2002 Sulfonation and molecular action. Endocr. Rev. 23: 703-732.

Takahashi, N., M. Nakazawa, K. Shibata, T. Yokota, A. Ishikawa et al., 2005 shk1-D, a dwarf

Arabidopsis mutant caused by activation of the CYP72C1 gene, has altered brassinosteroid

levels. Plant J. 42: 13-22.

Turk, E. M., S. Fujioka, H. Seto, Y. Shimada, S. Takatsuto et al., 2003 CYP72B1 inactivates

brassinosteroid hormones: an intersection between photomorphogenesis and plant steroid signal

transduction. Plant Physiol. 133: 1643-1653.

119

Turk, E. M., S. Fujioka, H. Seto, Y. Shimada, S. Takatsuto et al., 2005 BAS1 and SOB7 act

redundantly to modulate Arabidopsis photomorphogenesis via unique brassinosteroid

inactivation mechanisms. Plant J. 42: 23-34.

Yuan, T., S. Fujioka, S. Takatsuto, S. Matsumoto, X. Gou et al., 2007 BEN1, a gene encoding a

dihydroflavonol 4-reductase (DFR)-like protein, regulates the levels of brassinosteroids in

Arabidopsis thaliana. Plant J. 51: 220-233.

120

FIGURES AND LEGENDS

Figure 1: Genetic analysis of the atst4a1-1 T-DNA insertion line. (A). Graphical

representation of the location of T-DNA insertion in the ATST4a ORF. Arrows show the location

of the gene and T-DNA specific primers used for genetic screening. (B). A gel image showing

results of genomic PCR amplification using ATST4a gene specific and T-DNA specific primers.

121

Figure 2: The atst4a1-1 mutant does not show a hypocotyl-elongation phenotype in both

dark and continuous white light conditions. (A) ATST4a is expressed in the hypocotyls of the

white-light and dark grown seedlings as shown by the histochemical analysis of the pATST4a:

ATST4a-GUS expressing lines. Scale bar= 2mm. (B) Fluence rate analysis of hypocotyl growth

of atst4a1-1 in dark and at two different fluence rates, WL-3.5(white light=3.5 µmol m-2

sec-1

)

and WL-10 (white light=10 µmol m-2

sec-1

) did not show any altered hypocotyl-elongation

response when compared to Col-0. Three replications of five-day-old seedlings were used to

measure hypocotyl growth. Error bars indicate standard error (SE).

122

Figure 3: Overexpression of ATST4a does not lead to BR-deficient phenotypes in

Arabidopsis. (A) The ATST4a over-expression lines show ~1000 fold levels of ATSt4a transcript

as compared to the Col-0. Error bars indicate SE. (B) Adult phenotypes of the three-week-old

Arabidopsis plants overexpressing ATST4a cDNA.

123

Figure 4: Hypocotyl-elongation response of atst4a1-1 and ATST4a-OX T3 lines to dark and

continuous white light conditions. Fluence rate analysis of hypocotyl growth of ATST4a-OX T3

lines in dark (A) and in continuous white light (10 µmol m-2

sec-1

) (B) did not show dramatic

differences in hypocotyl-elongation response when compared to Col-0. Three replications of

five-day-old seedlings were used to measure hypocotyl growth. Error bars indicate SE.

124

Figure 5: Hypocotyl-elongation response of atst4a1-1 and ATST4a-OX T3 lines to BL

treatment. Seedlings were grown in continuous light for five days on media containing BL at

100 nM concentration in Dark (A) and white light (25 µmol m-2

sec-1

). Hypocotyl-elongation

response of atst4a1-1 and ATST4a-OX T3 lines do not show dramatic differences when

compared to Col-0 in both dark and light conditions. Three replications of five-day-old seedlings

were used to measure hypocotyl growth. Error bars indicate SE.

125

Figure 6: ATST4a-OX T3 lines did not display feedback regulation of BR biosynthesis and

catabolic genes DWF4 and BAS1. Quantitative RT-PCR analysis of the DWF4 (A) and BAS1

(B) transcript in Col-0, atst4a1-1, and three single locus insertion ATST4a-OX T3 lines show that

ATST4a over-expression lines did not show significantly altered levels of DWF4 and BAS1

transcript as compared to the Col-0. Error bars indicate SE.

126

CHAPTER FIVE

SUMMARY AND FUTURE DIRECTIONS

BR inactivation is an emerging field in plant biology. Biochemically, there are more possible

ways to modify or inactivate a BR molecule than the ones discovered to date (Bajguz 2007).

Therefore, it is likely that we will continue to discover novel mechanisms for BR inactivation in

the future. In addition, not much is known about the regulation of BR inactivation and how

plants use BR inactivation in their development and adaptation. The goal of my doctoral research

was to study the unique and overlapping roles in Arabidopsis development played by some of the

known BR catabolic genes.

To achieve this goal, Chapter Two explored the role of BAS1 and SOB7 in

photomorphogenic development of Arabidopsis. This study uses a molecular genetic approach to

delineate the role of BRs and BR catabolism in plant development. In summary, BAS1 and SOB7

displayed both simple and complex genetic interactions with the phytochromes in a plant-stage

specific manner (Sandhu et al. 2012). The removal of BAS1 and/or SOB7 rescued the low

germination rate of the phyA-211 phyB-9 double-null mutant. With regard to floral induction,

bas1-2 and sob7-1 showed a complex set of genetic interactions with photoreceptor-null mutants.

Histochemical analysis of transgenic plants harboring BAS1:BAS1-GUS and SOB7:SOB7-GUS

translational fusions under the control of their endogenous promoters revealed overlapping and

distinct expression patterns. BAS1’s expression in the shoot apex increases during the phase

transition from short-to-long-day growth conditions and requires phyB in red light.

This study is important for the following reasons. Firstly, this is the first kind of study which

specifically examines the role of BR catabolism in Arabidopsis developmental. It has been

127

suggested that light promotes photomorphogenesis in part by inhibiting BR levels or signaling

(Li et al. 1996; Chory and Li, 1997; Kang et al. 2001). However, this hypothesis lacks support

by direct evidence. A major contribution of this study is that it shows the regulation of a BR

catabolic gene (BAS1) by a photomorphogenic pathway (phyB) in the shoot apex. A key future

experiment will be to study whether this is a direct regulation of BAS1 transcript accumulation or

if the change in BAS1 expression is caused by cell-specific changes in BR content. Such studies

will require technical improvements in the ability to measure BRs in small tissues and organs.

Another contribution of this study lies in the evolution of redundant biological pathways.

Previous analysis from the Neff lab has shown that BAS1 and SOB7 affect developmental

processes, such as flowering, in a synergistic/redundant fashion (Turk et al. 2005). However, the

existence of multiple BR catabolic pathways suggests that it is unlikely that these pathways are

completely redundant. Research in Chapter Two shows divergence of BAS1 and SOB7, in terms

of both gene expression patterns and their interactions with other developmental pathways. For

example, phyA bas1-2 flowers early, whereas phyA sob7-1 flowers later than the single phyA.

The study therefore provides the critical evidence for the notion that the multiple BR catabolic

pathways have functional diversity in Arabidopsis.

The application of the genetic analysis approach used in Chapter Two requires availability of

loss-of-function mutants of the BR catabolic genes. Chapter Three focuses on the

characterization of the T-DNA insertion mutant ben1-1. In summary, our analysis shows that the

ben1-1 mutant is not suitable for multiple mutant analyses which involve combining two or more

T-DNA insertion mutations.

128

As a part of our analysis of brassinosteroid catabolic enzymes, we generated a genetic triple-

mutant from a cross between the bas1-2 sob7-1 double-null (T-DNA exonic insertion mutants of

BAS1 and SOB7) and ben1-1. The single ben1-1 mutant behaves as a transcript null for BEN1.

However, in the triple-mutant background ben1-1 was reverted to a partial loss-of-function allele

showing enhanced levels of the wild-type-spliced transcript. Interestingly, the enhanced

expression of BEN1 remained stable when the ben1-1 single-mutant was re-isolated from a cross

with the wild-type. In addition, the two genetically identical pre-triple and post-triple ben1-1

mutants also differed phenotypically. The previously functional NPTII T-DNA marker gene

(which encodes kanamycin resistance) was no longer functional in the recovered ben1-1 allele

though the length of the T-DNA insertion and the NPTII gene sequence did not change in the

pre-triple and post-triple ben1-1 mutants. Restriction endonuclease analysis with methylation

sensitive enzymes followed by genomic PCR showed that the methylation status of the T-DNA

is different between the original and the recovered ben1-1. These observations demonstrate that

the recovered ben1-1 mutant is epigenetically different from the original ben1-1 allele.

Future work in this direction will involve isolation and characterization of at least one novel

loss-of-function mutant of BEN1. EMS mutagenesis of the activation tagging line ben1-D can be

used for generating novel mutations in the BEN1 locus. The ben1-D line displays a semi-dwarf

seedling phenotype. The M2 progeny of the EMS mutagenized ben1-D line can be screened for

suppressors of the semi-dwarf phenotype. The identified suppressors will be test crossed with the

WS-2 line to identify the intragenic suppressors. Progeny of a cross between an intra-genic

suppressors and the wild-type parent will display a wild-type phenotype. On the other hand, the

progeny from a cross between an extra-genic suppressors and the wild-type parent will display a

semi-dwarf seedling phenotype. The future goal of the research described in Chapter Three is to

129

isolate and characterize a true loss-of-function mutation in the BEN1 gene, which can be used for

creation of multiple mutants lacking two or more BR catabolic genes. The isolation of true loss-

of-function mutant of BEN1 will also aid in examining its full role in other biological pathways.

Another important future direction will be to isolate transcription factors involved in the

regulation of BR catabolic genes. Using a yeast-one-hybrid approach Dr. Hao Peng has isolated a

transcription factor ATAF2, which binds to promoters of both BAS1 and SOB7. Molecular and

genetic analysis shows that ATAF2 is involved in the regulation of BAS1 and SOB7 expression

in Arabidopsis. Similar approaches can be used to isolate and characterize transcription factors,

which regulate expression of other BR catabolic genes. BEN1 will be a high priority for yeast-

one-hybrid analysis. The ben1-1 single-mutant has elevated BR levels, which indicates its

importance in maintaining BR homeostasis in Arabidopsis (Yuan et al. 2007). BAS1 gene

expression is up-regulated by BL application (Bancos et al. 2002). However, BAS1 expression is

not up-regulated in the ben1-1 background, which has elevated BR levels (Chapter Three, Figure

4C). This suggests that these two genes have distinct spatio-temporal expression patterns.

Therefore, these two genes are likely to be part of different biological pathways.

This study also contributes to the further characterization of the ATST4a. In vitro biochemical

analyses demonstrate that ATST4a has catalytic activity with BRs (Masolais et al. 2007).

However, unlike other BR catabolic pathways, the genetic evidence to suggest such a function

for ATST4a pathway in planta is scant. In this study, we have used both loss-of-function and

over-expression genetic approaches to further explore the role of ATST4a in Arabidopsis. This

study shows that atst4a1-1, a T-DNA insertion null mutant of ATST4a did not display reduced

seedling hypocotyl growth inhibition in white light. Additionally, overexpression of the ATST4a

gene in transgenic lines did not result in any characteristic BR-deficient phenotypes.

130

Overexpression of ATST4a did not cause a predicted feed-back-regulation change in the

transcript accumulation of DWF4 and BAS1. These observations suggest that ATST4a is an

atypical BR catabolic gene. Given these observations, more studies are required to assign as in

planta biological function to ATST4a. The detailed analysis of atst4a1-1 mutant under various

growth conditions might give a hint to its functions. Previous reports on steroid sulfotransferase

suggest their role in environmental stress responses (Marsolais et al. 2004; Marsolais et al.

2007).

Understanding BR inactivation will impact many fields of basic sciences including hormone

biology, role of redundant biological pathways, and evolution of biological pathways. Detailed

understanding of BR inactivation and its role in plant growth and development will also enable

crop scientists to use this growth-promoting hormone as a plant biotechnology tool.

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Molecular and biochemical characterization of two brassinosteroid sulfotransferases from

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Turk, E. M., S. Fujioka, H. Seto, Y. Shimada, S. Takatsuto, S. Yoshida, H. C. Wang, Q. I.

Torres, J. M. Ward, G. Murthy, J. Y. Zhang, J. C. Walker and M. M. Neff, 2005 BAS1 and SOB7

act redundantly to modulate Arabidopsis photomorphogenesis via unique brassinosteroid

inactivation mechanisms. Plant J. 42: 23-34.

Yuan, T., S. Fujioka, S. Takatsuto, S. Matsumoto, X. Gou, K. He, S. D. Russell, and J. Li, 2007

BEN1, a gene encoding a dihydroflavonol 4-reductase (DFR)-like protein, regulates the levels of

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