Financial support from Fundação para a Ciência e Thesis.pdf · Firstly, in a case-study approach, the functional relevance of an alternative splicing event in an E3 ubiquitin ligase

Financial support from Fundação para a Ciência e

a Tecnologia, Ministério da Educação e Ciência,

Portugal, through grant SFRH/BD/28519/2006

awarded to Sofia Domingues de Carvalho

Work performed at the

Laboratory of Plant Molecular Biology

Instituto Gulbenkian de Ciência

Oeiras, Portugal

Supervisor

Paula Duque, PhD

“é preciso dizer rosa em vez de dizer ideia

é preciso dizer azul em vez de dizer pantera

é preciso dizer febre em vez de dizer inocência

é preciso dizer o mundo em vez de dizer um homem”

Mário Cesariny

vii

ACKNOWLEDGEMENTS

Firstly, I wish to thank Paula Duque, my supervisor, who

guided me into the beauty of splicing research and its underneath,

still unveiled, world. This journey comes to an end after innumerous

long and pleasant discussions, hard but insightful defeats, and

accomplished successes that we achieved together. I thank her

also for precious advice and suggestions during the writing of this

thesis.

I would like to thank Miguel Godinho-Ferreira and Jocelyne

Demengeot, members of my thesis advisory committee, for

guidance during this PhD, the tough scientific discussions and their

kind support.

I thank Mathias Zeidler and Jutta Rösler, our collaborators

in Germany, for receiving me in the lab to make use of the light

experiments set-up. The very short period I spent in Giessen

turned out to be an extraordinary time, during which we shared

exciting scientific discussions.

The Plant Molecular Biology laboratory had several

members during the time of my PhD. I thank Raquel Carvalho and

Estelle Remy for the great discussions, support and all the fun

shared at work. To the past members, I especially thank Inês

Barbosa, Rita Saraiva and Teresa Maia, whom I had the chance to

work with very closely. Finally, to Vera Nunes, our plant technician,

for her endless care of our Arabidopsis plants and plant growth

facilities.

I wish to thank our lab neighbors, for sharing of knowledge,

experience and lab material. In particular, to the plant groups at our

Institute: Plant Stress Signaling, Plant Genomics, and Cell

viii

Biophysics and Development, but also to our bay neighbors, the

Evolution Genomics group.

This work was financially supported by a grant from

Fundação para a Ciência e a Tecnologia (SFRH/BD/28519/2006)

and by the Instituto Gulbenkian de Ciência, a wonderful place

providing excellent conditions to do science. I had the chance to

meet several people who undoubtedly left a mark during the period

of my PhD. Special thanks to Thiago Carvalho, for the great

discussions and for reminding me that doing science always gets

much easier when we keep smiling.

I would like to thank my former supervisor before I arrived at

the Instituto Gulbenkian de Ciência, Filip Rolland, who opened my

heart to plant research. Thank you also to two former professors

during my undergraduate studies, Isabel Sá-Correia and Marília

Mateus, who helped me in initiating this PhD.

I wish to thank Clube Futebol Benfica, and the Fofó players

for sharing the best stress reliever therapy: soccer playing.

I thank Jorge and Linda for their endless care and support

in every decision I have made.

For guiding me at every moment, thanks to Cata and Guida.

Para a avó Dora e a avó Marta, por me mostrarem que

todas as preocupações são relativas e que o passar do tempo

ajuda sempre a encontrar soluções.

For their love and precious close presence, I thank

Francisco. Ana. Daniela. Susana. Sandra. Ricardo. Catarina.

ix

RESUMO

O splicing do ARN é um passo essencial da expressão

génica em eucariotas durante o qual os intrões são removidos com

precisão do mARN precursor (pré-mARN) e os exões ligados entre

si, originando uma molécula madura de mARN. A existência de

vários exões por gene possibilita que a maquinaria de splicing

processe o mesmo pré-mARN de modos diferentes e remova

seletivamente sequências intrónicas distintas. Torna-se assim

possível que se gerem vários transcritos, e possivelmente mais do

que uma proteína, a partir de um único gene. Estas vias de splicing

alternativo têm-se revelado um mecanismo-chave na geração de

diversidade proteómica e complexidade funcional. A prevalência

do splicing alternativo em muitos genomas, incluindo os de plantas

superiores, sugere que este fenómeno desempenha um papel

importante em processos biológicos.

De modo a adaptarem-se às alterações do meio ambiente,

as plantas, enquanto organismos sésseis, desenvolveram

elevados níveis de plasticidade de desenvolvimento e tolerância ao

stress. Estes são, em última análise, regulados a nível do genoma

e é provável que a excecional versatilidade associada à regulação

génica pelo splicing alternativo desempenhe um papel

proeminente na resposta das plantas ao meio ambiente. No

entanto, a relevância biológica deste mecanismo de regulação

pós-transcricional em plantas encontra-se ainda pouco estudada.

Esta tese teve como objectivo aprofundar a compreensão

do papel biológico do splicing alternativo no crescimento e

desenvolvimento das plantas, utilizando Arabidopsis thaliana como

organismo modelo. Começou-se por investigar a relevância

x

funcional de um caso de splicing alternativo num gene que codifica

uma E3 ligase de ubiquitina envolvida na degradação proteica pelo

proteassoma 26S. Numa segunda abordagem, descobriu-se a

função in vivo e os alvos endógenos de um fator de splicing

específico de plantas envolvido na regulação do splicing

alternativo.

O gene XBAT35 de Arabidopsis, que codifica uma E3

ligase do tipo RING envolvida em degradação proteica mediada

pela molécula de ubiquitina, sofre splicing alternativo e gera dois

transcritos ubíqua e constitutivamente expressos. As duas

variantes de splicing resultam de um caso de omissão de um exão,

o que leva à exclusão de um sinal de localização nuclear (SLN) e

determina localizações intracelulares diferentes para as duas

isoformas. De facto, ensaios de expressão transiente em células

de cebola e em protoplastos isolados de Arabidopsis revelaram

que a isoforma contendo o SLN é translocada para o núcleo, onde

acumula em speckles, enquanto a isoforma sem o SLN permanece

no citoplasma. Comprovou-se através de ensaios de ubiquitinação

in vitro que ambas as proteínas são E3 ligases funcionais. No

sentido de se encontrar alvos de ubiquitinação das isoformas

XBAT35, efetuou-se um ensaio de dois híbridos em levedura.

Utilizando como presa uma biblioteca de cADN obtida a partir de

plântulas de Arabidopsis e como isco quatro fragmentos distintos

de XBAT35, tendo-se isolado cinco candidatos a substratos. Por

último, para investigar as funções biológicas desta E3 ligase,

geraram-se dois alelos mutantes homozigóticos e linhas

transgénicas de ARNi, com redução significativa do nível de

expressão do gene XBAT35. A caraterização fenotípica destes

alelos revelou que a E3 ligase XBAT35 desempenha um papel no

controlo do desenvolvimento do gancho apical mediado pelo

xi

etileno. É de realçar que ensaios de complementação indicaram

que ambas as variantes de splicing de XBAT35 têm atividade na

regulação da curvatura do gancho apical mas, aparentemente,

com uma contribuição diferencial para esta resposta. Em paralelo,

iniciou-se também o isolamento de um mutante com perda de

função de um homólogo de XBAT35, o XBAT34, de forma a

investigar a possibilidade de existência de redundância funcional

entre os dois genes.

As proteínas SR são fatores de splicing essenciais que

desempenham papéis fundamentais na modulação do splicing

alternativo, ao ligarem-se a sequências específicas do pré-mARN

e influenciarem a seleção dos sítios de splicing de forma

dependente da sua concentração. A família de genes SR em

Arabidopsis contém 18 membros, dez dos quais não têm ortólogos

em animais, o que indica que poderão ter adquirido funções

específicas associadas ao reino das plantas. Este estudo relata o

isolamento e a caracterização do primeiro mutante com perda de

funcionalidade de uma proteína SR em plantas. A disrupção do

gene SCL30a, específico de plantas, afeta os últimos estádios do

desenvolvimento da semente, o que resulta na produção de

sementes mais pequenas e de dormência mais acentuada. A

proteína SCL30a regula também as respostas à luz durante o

desenvolvimento precoce da planta, uma vez que os hipocótilos do

mutante se apresentam mais pequenos do que os de plantas

selvagens na presença de luz na zona do vermelho, do vermelho-

extremo e do azul. Apesar de a germinação de sementes em

condições ótimas não se mostrar afetada, as sementes mutantes

são hipersensíveis ao ácido abscísico (ABA) e ao stress salino e

osmótico durante a germinação. Esta hipersensibilidade é revertida

xii

na presença de um inibidor da biossíntese do ABA e análises

epistáticas revelaram de forma inequívoca que a SCL30a atua na

via do ABA. No entanto, não parece que a perda ou o aumento de

função da SCL30a alterem os níveis de ABA em sementes.

Notavelmente, plantas transgénicas sobreexprimindo SCL30a

produzem sementes maiores e mais tolerantes ao stress salino e

osmótico durante a germinação. Assim, a proteína SR SCL30a de

Arabidopsis constitui um novo regulador da via de sinalização do

ABA que modula características específicas das sementes,

incluindo o tamanho, a dormência e os níveis de tolerância à

salinidade elevada e à seca durante a germinação. Por último,

para identificar alvos endógenos desta proteína de ligação ao

ARN, efetuou-se uma análise global por deep sequencing ao

transcritoma de sementes selvagens, mutantes em SCL30a e com

sobreexpressão de SCL30a na presença de stress salino.

Em resumo, o trabalho apresentado nesta tese mostra que

o splicing alternativo determina a localização intracelular de duas

isoformas funcionais do gene XBAT35, que codifica uma nova E3

ligase de ubiquitina em Arabidopsis. No futuro, a validação dos

possíveis substratos da XBAT35 através de ensaios de co-

imunoprecipitação e de ubiquitinação in vitro deverá permitir

discernir os mecanismos moleculares subjacentes aos papéis

fisiológicos desta enzima. Esta tese representa também uma

importante contribuição para a elucidação da relevância biológica

do splicing alternativo na resposta das plantas ao stress, ao atribuir

à proteína SR SCL30a de Arabidopsis um papel na tolerância à

salinidade elevada e à seca. Análises de genética reversa e

ensaios de imunoprecipitação de ARN poderão vir ainda a

demonstrar que os transcritos identificados por deep sequencing

são alvos funcionais diretos da SCL30a, revelando assim os

xiii

mecanismos moleculares que governam o modo de ação desta

nova proteína de ligação ao ARN.

ABSTRACT

RNA splicing is an essential step in eukaryotic gene

expression during which introns are precisely removed from the

precursor-mRNA (pre-mRNA) and exons joined together to form

the mature mRNA molecule. The presence of numerous exons per

gene enables the splicing machinery to process the same pre-

mRNA differently by selectively removing different intronic

sequences, thus generating multiple transcripts, and eventually

more than one protein, from a single gene. Such alternative

splicing pathways have emerged as a key mechanism for

generating proteome diversity and functional complexity. The

prevalence of alternative splicing in many genomes, including

those of higher plants, suggests that this mechanism plays crucial

roles in biological processes.

To adapt to an environment in constant change, plants, as

sessile organisms, have evolved high degrees of both

developmental plasticity and stress tolerance, which are ultimately

regulated at the genome level. The exceptional versatility

associated with gene regulation by alternative splicing is likely to

play a prominent role in plant responses to environmental cues, but

the biological significance of this posttranscriptional regulatory

mechanism in plants remains poorly understood.

This thesis aimed at gaining insight into the biological roles

of alternative splicing in plant growth and development, using the

xiv

model organism Arabidopsis thaliana. Firstly, in a case-study

approach, the functional relevance of an alternative splicing event

in an E3 ubiquitin ligase gene involved in protein degradation by

the 26S proteasome was investigated. As a second approach, we

uncovered the in vivo function and endogenous targets of a plant-

specific splicing factor implicated in the regulation of alternative

splicing.

The Arabidopsis XBAT35 gene, which encodes a RING E3

ligase involved in ubiquitin-mediated protein degradation,

undergoes alternative splicing generating two constitutively and

ubiquitously expressed transcripts. The two splice variants arise

from an exon-skipping event, which excludes a nuclear localization

signal (NLS) and determines dual targeting of the encoded splice

isoforms. Indeed, transient expression assays in onion cells and

Arabidopsis protoplasts indicate that the NLS-containing isoform is

targeted to the nucleus, where it accumulates in speckles, while the

isoform lacking the NLS localizes in the cytoplasm. Importantly,

both isoforms are functional E3 ligases, as determined by in vitro

ubiquitination assays. A yeast two-hybrid system was employed in

the search for ubiquitination targets of the XBAT35 isoforms. Using

as prey a cDNA library from Arabidopsis seedlings and four

different XBAT35 fragments as baits, five candidate substrates

were identified. Finally, to investigate the biological functions of this

E3 ligase, we generated two homozygous insertion alleles as well

as transgenic RNAi lines exhibiting significantly reduced expression

of the XBAT35 gene. Phenotypical characterization of these loss-

of-function lines revealed a role for the XBAT35 E3 ligase in

ethylene-mediated control of apical hook development.

Interestingly, complementation experiments indicated that both

XBAT35 splice forms are active in regulating apical hook curvature

xv

but appear to contribute differentially to this response. In addition,

the isolation of a loss-of-function mutant for the close XBAT35

homolog, XBAT34, was initiated to assess functional redundancy

between these two genes.

SR proteins are essential splicing factors that play major

roles in the modulation of alternative splicing by binding specific

sequences in the pre-mRNA and influencing splice site selection in

a concentration-dependent manner. The Arabidopsis SR protein

gene family contains 18 members, ten of which share no orthologs

in metazoans, suggesting that they have evolved functions specific

to the plant kingdom. The present study reports the isolation and

characterization of the first loss-of-function mutant for a plant SR

protein. Disruption of the plant-specific SCL30a SR gene affects

the later stages of seed development, resulting in the production of

smaller seeds displaying enhanced dormancy. Moreover, SCL30a

regulates light responses during early seedling development, as

the mutant displays shorter hypocotyls under red, far-red and blue

light when compared to wild-type plants. Although seed

germination under optimal conditions is not affected, mutant seeds

are hypersensitive to abscisic acid (ABA), as well as to salt and

osmotic stress during germination. The mutant’s germination salt

and osmotic stress oversensitivity is rescued by an inhibitor of ABA

biosynthesis, and epistatic analysis unequivocally places SCL30a

in the ABA pathway. However, loss or gain of SCL30a function do

not appear to alter ABA levels in seeds. Remarkably, ectopic

expression of SCL30a in transgenic plants leads to the production

of larger seeds with enhanced salt and osmotic stress tolerance

during germination. Thus, the plant-specific SCL30a SR protein

defines a novel negative regulator of the ABA signaling pathway

xvi

that modulates specific seed traits including size, dormancy and

germination tolerance to high salinity and drought. Finally,

endogenous targets of this RNA-binding protein were identified by

means of global deep-sequencing transcriptome analysis of wild-

type, SCL30a-mutant and SCL30a-overexpressing seeds under

salt stress.

In conclusion, the work presented here reveals a role for

alternative splicing in the differential subcellular localization of two

functional isoforms encoded by XBAT35, a novel Arabidopsis E3

ubiquitin ligase gene. Future validation of the identified XBAT35

substrate candidates by co-immunoprecipitation and in vitro

ubiquitination assays should provide invaluable clues on the

molecular mechanisms underlying the physiological roles of the

XBAT35 enzyme. Furthermore, this thesis represents a major

contribution towards the elucidation of the biological significance of

alternative splicing in plant stress responses, by ascribing a role in

ABA-dependent salt and drought tolerance to the Arabidopsis

SCL30a SR protein. Reverse genetics and RNA-

immunoprecipitation analyses hold much promise for the validation

of the transcripts pinpointed by deep-sequencing as functional,

direct SCL30a targets, thus uncovering the molecular mechanisms

underlying the mode of action of this novel plant-specific RNA-

binding protein.

xvii

LIST OF ABBREVIATIONS

aa amino acid

ABA abscisic acid

ACC 1-aminocyclopropane-1-carboxylate

ANK ankyrin

bp base pair

CDS coding sequence

Da Dalton

DNA deoxyribonucleic acid

DNG did not germinate

ESE exonic splicing enhancer

ESR exonic splicing regulator

ESS exonic splicing silencer

GUS β-glucuronidase

hnRNP heterogeneous ribonucleoprotein

IAA indole-3-acetic acid

ISE intronic splicing enhancer

ISR intronic splicing regulator

ISS intronic splicing silencer

KO knockout

LB left border

mRNA messenger RNA

nt nucleotide

PTB polypyrimidine tract-binding

Pre-mRNA precursor mRNA

PTC premature stop codon

MAPK mitogen-activated protein kinase

NMD nonsense-mediated decay

xviii

NLS nuclear localization signal

OX overexpression

RH relative humidity

RING really interesting new gene

RNA ribonucleic acid

RNAi RNA-interference

RNP ribonucleoprotein

RRM RNA-recognition motif

RS arginine/serine

SCL SC35-like

snRNP small nuclear ribonucleoprotein

SR protein serine/arginine-rich protein

TAIR The Arabidopsis Information Resource

UB ubiquitin

UTR untranslated region

WT wild type

YFP yellow fluorescent protein

xix

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ....................................................................... vii

RESUMO ............................................................................................. ix

ABSTRACT ........................................................................................ xiii

LIST OF ABBREVIATIONS................................................................... xvii

TABLE OF CONTENTS ........................................................................ xix

CHAPTER 1: GENERAL INTRODUCTION ..................................................1 1.1. Pre-mRNA Splicing in Plants .......................................................4

1.1.1. Structure of exons and introns ...........................................4 1.1.2. The splicing reaction ..........................................................7 1.1.3. Alternative splicing ...........................................................13 1.1.4. The SR protein family ......................................................21

1.1.4.1. The SCL30a SR protein ...........................................29 1.2. Protein Ubiquitination in Plants ..................................................31

1.2.1. E3 ligase classification and activity ..................................35 1.2.2. The XBAT family of RING E3 ligases ...............................38 1.2.3. Alternative splicing of E3 ligases......................................42

1.3. Thesis Outline............................................................................44 1.4. References ................................................................................46

xx

CHAPTER 2: XBAT35, A NOVEL ARABIDOPSIS RING E3 LIGASE

EXHIBITING DUAL TARGETING OF ITS SPLICE ISOFORMS, NEGATIVELY

REGULATES ETHYLENE-MEDIATED APICAL HOOK CURVATURE ...............63 2.1. Abstract .....................................................................................65 2.2. Introduction................................................................................66 2.3. Results.......................................................................................69

2.3.1. Alternative splicing of the XBAT35 pre-mRNA excludes

nuclear localization signal ..........................................................69 2.3.2. The XBAT35 gene is ubiquitously expressed in

Arabidopsis................................................................................70 2.3.3. Exon skipping determines the subcellular localization of

two XBAT35 isoforms ................................................................73 2.3.4. Both the nuclear and cytoplasmic XBAT35 isoforms are

active E3 ubiquitin ligases..........................................................74 2.3.5. Loss of XBAT35 function causes hypersensitivity to

ethylene-mediated control of apical hook curvature ...................75 2.3.6. Both XBAT35 isoforms function in ethylene control of

apical hook curvature.................................................................83 2.3.7. Four photosystem proteins and an unknown protein are

putative XBAT35 interacting partners ........................................85 2.3.8. The XBAT35 and XBAT34 duplicated genes display

overlapping expression patterns ...............................................87 2.4. Discussion .................................................................................90 2.5. Materials and Methods...............................................................95

2.5.1. Plant materials and growth conditions..............................95 2.5.2. Generation of XBAT35-RNAi lines and complementation

of the xbat35-1 mutant...............................................................96 2.5.3. Phenotypical analyses .....................................................97 2.5.4. RNA extraction and RT-PCR analysis..............................98 2.5.5. Subcellular localization of XBAT35-YFP fusion proteins 100

xxi

2.5.6. Expression of recombinant XBAT35.1 and XBAT35.2

proteins and in vitro ubiquitination assays................................101 2.5.7. Yeast manipulations and two-hybrid assay ....................101

2.6. References ..............................................................................105

CHAPTER 3: THE ARABIDOPSIS SCL30a SR PROTEIN CONFERS ABA-

DEPENDENT SALT AND OSMOTIC STRESS TOLERANCE DURING SEED

GERMINATION...................................................................................111 3.1. Abstract ...................................................................................113 3.2. Introduction..............................................................................114 3.3. Results.....................................................................................118

3.3.1. The SCL30a gene generates three splice variants and is

markedly induced during seed germination..............................118 3.3.2. The scl30a-1 mutant displays seed-specific phenotypes

and hypersensitivity to ABA, salt and osmotic stress during

germination..............................................................................122 3.3.3. SCL30a is a novel component of the ABA pathway .......128 3.3.4. SCL30a-overexpressing plants produce larger seeds

exhibiting abiotic stress tolerance during germination ..............133 3.3.5. SCL30a regulates ABA signaling under salt stress ........135 3.3.6. SCL30a affects the expression and splicing pattern of

several Arabidopsis genes.......................................................136 3.3.7. The SCL33 and SCL30a duplicated gene pair displays

similar expression patterns in Arabidopsis ...............................151 3.4. Discussion ...............................................................................154 3.5. Materials and Methods.............................................................165

3.5.1. Plant materials and growth conditions............................165 3.5.2. Generation of transgenic plants .....................................166 3.5.3. Gene expression analyses.............................................168

xxii

3.5.4. Phenotypical analyses ...................................................169 3.5.5. Determination of ABA content ........................................172 3.5.6. Deep-sequencing...........................................................172

3.5.6.1. Mapping reads to the Arabidopsis genome.............173 3.5.6.2. Transcript prediction and expression level

estimation............................................................................173 3.5.6.3. Comparison with TAIR annotation and testing for

differential expression .........................................................174

3.5.6.4. Splice junction analysis ..........................................175 3.5.6.5. Transcript expression ratio analysis........................175

3.6. References ..............................................................................176

CHAPTER 4: CONCLUDING REMARKS AND FUTURE

PERSPECTIVES.................................................................................185

4.1. Introduction..............................................................................187

4.2. Alternative Splicing of the XBAT35 RING E3 Ligase ................188

4.3. Biological Roles of the Plant-Specific SCL30a SR Protein .......193

4.4. Conclusions .............................................................................198

4.5. References ..............................................................................199

APPENDIX I ......................................................................................203

CHAPTER 1 GENERAL INTRODUCTION

Chapter 1 – General Introduction

3

Complex molecular signaling pathways control overall plant

physiology and morphology and guide organismal growth throughout

a life cycle. These pathways are precisely regulated, ensuring correct

development, and at the same time relatively flexible, allowing for

unique adaptive features of these sessile organisms, which confer the

developmental plasticity and stress tolerance required to

appropriately respond to environmental cues.

It is of fundamental interest to investigate the mechanisms

that control plant growth. Besides the general biological interest, plant

manipulation and the generation of transgenic plants more tolerant to

certain adverse conditions can help feed the world population, whose

resources are becoming limited due to its increase in number and to

climate changes that affect crop yield.

Different plant species have been used as models for genetic

studies to dissect plant developmental signaling pathways, but the

mustard Arabidopsis thaliana has in recent years established itself as

the most powerful system. It is small, easy to manipulate and

transform by agroinfilitration, it has a short life cycle, it self-fertilizes or

can be cross-pollinated, and its seed yield is high. In addition, a large

number of mutant lines are publicly available, and the Arabidopsis

genome sequence has been available to the research community for

over ten years (The Arabidopsis Genome Initiative, 2000).

The signaling pathways that define plant development are

ultimately determined at the genome level. During gene expression,

from the starting DNA molecule to the final product as an expressed

protein, different layers of regulation exist. The combination of

regulatory mechanisms at the transcriptional and posttranscriptional

levels, but also at that of epigenetics or mRNA transport and

translation, allows for perfect fine-tuning of protein activity. Using the

model organism Arabidopsis thaliana, the work presented in this


4

thesis focused on the biological relevance of a specific

posttranscriptional regulatory mechanism, alternative pre-mRNA

splicing.

1.1. Pre-mRNA Splicing in Plants

During DNA transcription, the precursor-mRNA (pre-mRNA) is

formed. It is constituted by alternate sequences called exons and

introns. As non-coding sequences, introns are removed from the pre-

mRNA and exons, as coding sequences, are joined together in order

to form the mature mRNA, which will be later translated into protein.

The process of intron removal and exon ligation is called pre-mRNA

splicing. It is a tightly regulated process that involves the activity of

hundreds of proteins, ensuring that the correct sequences are spliced

out from the pre-mRNA.

1.1.1. Structure of exons and introns

Splicing is conserved among eukaryotes and the basic

splicing mechanism seems to be similar in plants and metazoans.

However, in many aspects the splicing machinery diverges among

these organisms, likely due to specific features of their intronic

sequences.

Introns are found in all eukaryotes but the number and size of

exons and introns, as well as how they are recognized by the splicing

machinery, varies between organisms. Over 80% of the genome of

higher plants contains introns, and some genes can even contain

more than 40 introns (reviewed in Lorkovic et al., 2000). Plant introns


5

are usually shorter than those found in vertebrates, with their size

ranging from 60 to 10,000 kb, although around two thirds are shorter

than 150 bp (reviewed in Lorkovic et al., 2000).

Exon/intron boundaries share consensus sequences, known

as canonical splice sites, which are common to plants and

vertebrates: the 5’ splice site is AG/GTAAGT and the 3’ splice site

TGCAG/G, where the dash bars represent exon/intron junctions

(reviewed in Brown and Simpson, 1998 and Lorkovic et al., 2000).

The underlined base pairs (GT-AG) are the most conserved. In

animals, two other intronic consensus sequences, that can be

considered as part of the 3’ splice site, exist: the branch point, located

20 to 40 bp upstream of the 3’ junction, and a 10 to 15 bp-long

polypyrimidine-rich sequence, the polypyrimidine tract, downstream of

the branch point (reviewed in Lorkovic et al., 2000 and Hertel, 2008).

Plant introns do not contain a polypyrimidine tract but instead a

uridine-rich region. They also include a branch point, with a loose

consensus sequence and an adenosine nucleotide conserved among

plants, vertebrates and yeast. Despite the conserved sequences

described, plant introns are often incorrectly processed in mammalian

nuclear extracts (van Santen and Spritz, 1987; Reddy, 2001) and

animal introns are not recognized in plant nuclei (Barta et al., 1986;

van Santen and Spritz, 1987; Wiebauer et al., 1988), indicating that

plant introns possess particular cis-acting sequences that are

differently recognized by the splicing machinery.

Another class of introns, the AT-AC or U12-introns, has been

identified. It occurs at a very low frequency (0.1% of all introns) and

contains non-canonical splice sites (AT-TC) as well as distinct

conserved branch point sequences (Patel and Steitz, 2003; Lorkovic

et al., 2005). U12 introns also possess more UA-rich sequences and


6

their structure is conserved among vertebrates and plants but is

absent from yeast (reviewed in Lorkovic et al., 2000).

Splice site recognition depends on the nucleotide length of

exonic and intronic sequences. Two mechanisms have been

proposed for exon and intron selection by the splicing machinery: the

exon definition and the intron definition models (reviewed in Lorkovic

et al., 2000; Ram and Ast, 2007; and Hertel, 2008). According to the

exon definition model, splice sites flanking the exon are selected and

splicing factors interact along the exon. In the intron definition model,

splice sites at the 5’ and 3’ ends of introns are bound and splicing

factors interact across the intron. In mammals, the much shorter

exonic sequences when compared to introns probably evolved the

spliceosome machinery to recognize short exonic sequences rather

than intronic ones. Larger exons are usually skipped during splicing

but are retained when the flanking introns are small, while shorter

introns (up to 250 bp) promote the retention of exons with weak splice

sites. As their introns are shorter, it is likely that in plants the intron

definition model is preferred by the splicing machinery. Similarly,

yeast possesses very short introns and likely prefers the intron

definition model (reviewed in Ram and Ast, 2007). Thus, intron size

provides important information on the tendency of the splicing

machinery to include a particular exon. Furthermore, the upstream

intron is usually more influent than the downstream one in the

selection of the flanked exon, possibly due to the coupling of pre-

mRNA splicing with DNA transcription (reviewed in Hertel, 2008).

Plant introns are 15% richer in U nucleotides than exons,

whereas the latter are 15% richer in CG. The UA- or U-rich

sequences found in plant introns also appear at higher frequency

than in introns from vertebrates or yeast, which may explain some of

the specific properties of the splicing reaction in plants (reviewed in


7

Brown and Simpson, 1998; Simpson and Filipowicz, 1996; and

Lorkovic et al., 2000). These U-rich sequences are important for

correct splice site selection and intron processing and, unlike the

polypirimidine tract, which is located downstream of the branch point

in animals, they are distributed all along introns (Simpson et al.,

2004).

Despite the clear definition of exon/intron junctions and the

branch point, these splice sites sequences are short and degenerate

and do not allow for precise fine-tuning of the splicing reaction

(reviewed in House and Lynch, 2008 and Shepard and Hertel, 2010).

Indeed, exons and introns contain additional cis-acting splicing

enhancers and silencers, which serve as binding sites for essential

splicing factors that promote accurate splicing events. While exons

contain exonic splicing regulators (ESRs), intronic splicing regulators

(ISRs) are found in introns. These sequence elements constitute

binding sites for different families of splicing factors, which also

interact with the splicing machinery and modulate its activity, thus

helping define the splice sites.

1.1.2. The splicing reaction

The removal of an intron from the pre-mRNA occurs by a two

step transesterification reaction (Fig. 1.1). In the first reaction, the

phosphosdiester bond at the 5’ splice site is cleaved via a

nucleophilic attack by the 2-hydroxyl group of the adenosine

nucleotide at the branch point. The 5’ splice site is fragmented and

the intron lariat formed through a phosphodiester bond between the

intron’s 5’ end and the branch point adenosine. The second step

results in cleavage of the 3’ splice site by a nucleophilic attack to its


8

phosphate group of the 3-hydroxyl group of the 5’ exon fragment. The

two exons are then joined together and the released intron lariat is

degraded for nucleotide recycling (reviewed in Brown and Simpson,

1998; and Fedor, 2008).

Figure 1.1. The process of intron removal. (A) Schematic representation of 5’ and 3’ exons (black and gray boxes, respectively) flanking an intron, with indication of the 5’ and 3’ splice sites and the conserved adenosine residue at the branch point. (B) The two-step reaction leading to intron removal and joining of exons. In the first step, the transesterification reaction occurs at the 5’ splice site and the intron lariat is formed, whereas in the second step the 3’ splice site is attacked, exons are ligated and the intron lariat released. Adapted from Simpson and Filipowicz (1996) and Lorkovic et al. (2000)

The process of intron removal during pre-mRNA splicing is

achieved by a large protein complex known as the spliceosome.

Splicing accuracy depends on the capacity of the spliceosome

machinery to efficiently assemble and recognize correct splice sites

on the pre-mRNA (reviewed in Simpson and Filipowicz, 1996; and


9

Brown and Simpson, 1998). Furthermore, the pre-mRNA also

undergoes conformation rearrangements essential for correct splice

site selection (reviewed in Hertel, 2008).

The spliceosome is composed by more than 300 proteins

(reviewed in Jurica and Moore, 2003). Its core components are five

U-type (uridine-rich) small nuclear ribonucleoproteins (snRNPs) – U1,

U2, U4, U5 and U6 – that are highly conserved from yeast to

mammals and plants (reviewed in Brown and Simpson, 1998; and

Lorkovic et al., 2000). The spliceosome complex is responsible for

intron excision and exon joining, but a recent report has also

described a role for the U1 snRNP in transcript stabilization

independently of the splicing reaction (Kaida et al., 2010).

Additional non-U snRNP proteins can be found in the

spliceosome, such as hnRNPs (heterogeneous RNPs), SR

(serine/arginine-rich) proteins, and DEAD- or DEAH-box containing

proteins (reviewed in Brown and Simpson, 1998). The DEAD- or

DEAH-box containing proteins are RNA-dependent ATPases or ATP-

dependent RNA helicases that mediate RNA conformational changes

during spliceosome assembly and disassembly. hnRNPs and SR

proteins bind the RNA and contain additional specific domains – rich

in either glycine residues or serine/arginine repeats, respectively –

which are involved in protein-protein interactions.

Splicing starts with a first approach to the pre-mRNA by the

U1 and U2 snRNPs and other splicing regulators that initiate the

recognition of exons and introns. The U1 snRNP binds first to the 5’

splice site. In mammals, the U2AF65 subunit of the U2AF (U2 auxiliary

factor) splicing factor binds the polypirimidine tract while another U2

auxiliary factor (SF1/BBP) binds the branch point (reviewed in Brown

and Simpson, 1998; Hertel, 2008; and House and Lynch, 2008). The

resulting RNP complex is called the E complex and triggers initiation


10

of the splicing reaction (Fig. 1.2). Simultaneously, U2AF35 binds near

the 3’ splice site and helps promote binding of U2AF65 to the

polypirimidine tract, by directly interacting with the second U2AF

subunit, in a process that also involves the activity of SR proteins.

Plants possess both subunits of U2AF but given their lack of

polypirimidine tracts, binding of U2AF to the introns seems to rely on

a conserved recognition of the 3’ splice site and stabilization of the

large subunit at the branch point (reviewed in Brown and Simpson,

1998). The splicing reaction continues with the recruitment of the U2

snRNP to the branch point and the conversion of the E complex into

the prespliceosomal A complex. The remaining three U snRNPs (U4,

U5 and U6) assemble and join the A complex, resulting in formation

of the B complex. The latter then undergoes conformational

rearrangements to form complex C. Splicing is achieved at this

moment and after lariat release and exon junction the spliceosome

disassembles, with the mRNA being exported to the cytosol for

translation into protein. Also at this stage, a nuclear surveillance

mechanism detects aberrant transcripts, targeting them to

degradation by nonsense-mediated decay (NMD).

Splicing of U12-dependent introns is achieved by the minor

U12-type spliceosome (Patel and Steitz, 2003; Lorkovic et al., 2005).

Similarly to the main U2-type spliceosome, it assembles with five

snRNP subunits, but only U5 is common between the two

spliceosomes. U12-type assembly resembles that described for the

U2-type spliceosome although U11 and U12, the analogues of U1

and U2, respectively, can also form dimers and bind the branch point.

The U12-type spliceosome functions in a similar way in plants and

vertebrates, suggesting that U12-dependent minor introns and their

splicing machinery existed in eukaryotes before plants and

metazoans diverged (Patel and Steitz, 2003; Lorkovic et al., 2005).


11

Figure 1.2. Spliceosome activity in plants. Schematic representation of U2-type spliceosome assembly, according to the preferred model in plants, the intron definition model. U snRNPs assemble sequentially along the pre-mRNA to form complexes E, A, B and C, after which the exons are ligated, the intron lariat is released and the spliceosome disassembles. Adapted from Lorkovic et al. (2000), Jurica and Moore (2003), and House and Lynch (2008)

The non-snRNP factors acting in splicing are generally

conserved among metazoans but appear in higher number in plants

(reviewed in Lorkovic et al., 2000). They participate in constitutive

splicing by recruiting the spliceosome machinery to correct splice

sites and by bridging interactions between spliceosome components,

helping in spliceosome assembly (Wu and Maniatis, 1993). They also

interact with the cap-binding complex at the transcript’s 5’ end and

with the polyadenylation machinery at the 3’ end in order to promote


12

splicing at the first and last exons. Moreover, they can bind ESRs

and/or ISRs in the pre-mRNA.

ESRs and ISRs can be either enhancers (ESEs and ISEs) or

silencers (ESSs and ISSs). The hnRNP splicing factors usually bind

silencers – when binding ESSs they inhibit spliceosome assembly by

multimerization along the exon or by blocking snRNP recruitment to

nearby splice sites (reviewed in Hertel, 2008). The polypyrimidine

tract-binding (PTB) protein family, a class of hnRNPs, binds ISS

motifs and represses inclusion of the adjacent exon in the splicing

product. It is likely that binding to this region also affects the activity of

U2AF65 at the same site. Arabidopsis possesses PTB-related proteins

whose activity during splicing has been shown (Stauffer et al., 2010).

SR proteins usually bind ESEs or ISEs and aid in spliceosome

assembly by recruiting the U1 snRNP to the 5’ splice site and U2AF

to the 3’ splice site, eventually bridging an interaction between the

components located at both splice sites (reviewed in Hertel, 2008 and

Reddy and Ali, 2011). They also facilitate the incorporation of the

U4/U6.U5 tri-snRNP in the spliceosome and promote U2 and U6

interactions (Roscigno and Garcia-Blanco, 2005).

Binding of splicesomal components to the pre-mRNA and to

other splicing factors is based on weak interactions, allowing for a

flexible modulation of the proteins involved. This idea is consistent

with the fact that several splicing factors influence splice site

recognition in a concentration-dependent manner (Lopato et al.,

2002; Solis and Patton, 2010). Furthermore, cis-acting ESRs and

ISRs can be closely located near the same exon/intron boundaries,

suggesting that the splicing product is a result of a large interplay

between trans-acting enhancers and repressors (reviewed in Hertel,

2008).


13

Spliceosome assembly occurs simultaneously with

transcription and the growing pre-mRNA is immediately bound to the

splicing machinery (Prasanth et al., 2003; Das et al., 2006).

Furthermore, some proteins acting during transcription also interact

and/or co-localize with spliceosome-related factors, indicating a close

connection between these two steps. For instance, in humans,

localization of Cdc2-related kinase with RS domain, CrkRS, overlaps

with that of RNA polymerase II and with splicing compartments (Ko et

al., 2001). In Arabidopsis, the cyclin-dependent kinase 2 (CDKC2)

phosphorylates RNA polymerase II and co-localizes with the SR34

SR protein and with cyclophilin CypRS64, whose activity has also

been linked to the splicing machinery (Lorkovic et al., 2004b; Kitsios

et al., 2008). Also, Cyp59 is a mutidomain cyclophilin that interacts

with the SCL33 SR protein and with the C-terminus of RNA

polymerase II (Gullerova et al., 2006). Besides its functional coupling

with transcription, the splicing process has also been linked to

chromatin remodeling and histone modifications (Zhang et al., 2011),

transcriptional elongation (Das et al., 2006), and mRNA stabilization

(Kaida et al., 2010), transport (Huang and Steitz, 2001; Delestienne

et al., 2010) or translation (Sanford et al., 2004), suggesting the

existence of common factors regulating different steps of gene

expression.

1.1.3. Alternative splicing

The spliceosome can process differently the same pre-mRNA

by selecting distinct exonic sequences to be joined together, in a

process known as alternative splicing. This mechanism allows a

single gene to encode multiple transcripts and, if these are indeed


14

targeted for translation, also several protein isoforms, upscaling the

genome capacity. There are five common types of alternative splicing

– exon skipping, alternative 5’ or 3’ splice site choice, mutual

exclusion of exons and intron retention (Fig. 1.3). Intron retention is

one of the most ancestral events and the most abundant (41%) in

plants (Ner-Gaon et al., 2004; Barbazuk et al., 2008).

Figure 1.3. Common types of alternative splicing events. Schematic representation of exons (boxes) and introns (horizontal lines) within a pre-mRNA structure, and representation of the selected splice sites in the most common types of alternative splicing events (left). Indication of the resulting mRNA structures (right). Adapted from Barbazuk et al. (2008)


15

The protein isoforms resulting from alternative splicing can

have different specificities, such as binding properties, stability or

intracellular localization. Moreover, it is also common to observe the

inclusion of premature stop codons (PTCs) in alternative splice forms,

which may trigger mRNA degradation by NMD. It was recently

suggested that 13% of the Arabidopsis intron-containing genes are

targeted for NMD (Kalyna et al., 2012). This number has been

estimated to range from 20 to 30% in humans (Lewis et al., 2003). It

was also recently reported that Arabidopsis splice variants resulting

from intron retention are not NMD sensitive, in contrast with

transcripts with alternative 5’- and 3’-splice sites at UTRs, which

appear to be more prone to NMD (Kalyna et al., 2012).

The hundreds of proteins active during splicing can act

synergistically or compete for a particular pre-mRNA binding site or

for an interaction with another specific protein. Different combinations

of all the components involved account for the occurrence of

regulated alternative splicing. For instance, in zebrafish depletion of

the U1C subunit of the U1 snRNP results in aberrant alternative

splicing profiles, but it can be relatively compensated by an increase

in the amount of some SR proteins (Rosel et al., 2011). Several

splicing regulators display different tissue-, developmental- and

stress-specific expression patterns, which can provide a means of

regulating the splicing pattern of a particular gene target and

concomitantly the activity of its encoded protein(s) (Stauffer et al.,

2010).

Alternative splicing has been widely described in multicellular

organisms (reviewed in Ram and Ast, 2007). In the human genome,

95% of the genes are now predicted to undergo alternative splicing,

indicating that this posttranscriptional mechanism provides a powerful

means of generating functional complexity and proteome diversity


16

(Pan et al., 2008; Wang et al., 2008). In plants, this number was

recently estimated to be 42% (Filichkin et al., 2010). Differences in

the estimates between organisms can be explained by the fewer

expressed sequence data available in plants, as suggested by the

fact that the estimated fraction of alternatively-spliced Arabidopsis

genes was initially estimated to be only 5% (Brett et al., 2002) but

tends to increase with every report released (Iida et al., 2004; Ner-

Gaon et al., 2004; Haas et al., 2005; Nagasaki et al., 2005; Wang and

Brendel, 2006; Filichkin et al., 2010). In addition, since alternative

splicing in plants is known to be regulated by developmental and

stress cues (Tanabe et al., 2006; Palusa et al., 2007), splice variants

that are only expressed during a specific developmental stage or in

response to a particular external signal are likely to be absent from a

sample used in a genome-wide approach. However, it is also

plausible that fewer genes undergo alternative splicing in plants due

to differences at the level of splicing machinery activity or of exonic

and intronic structures. In the context of this hypothesis, it is

interesting to note that, until recently, alternative splicing was thought

to be absent from yeast. However, in Saccharomyces cerevisiae the

ubiquitin-like molecule Hub1, which helps in the assembly of the tri-

snRNP complex prior to formation of complex B, has been found to

affect splice site selection and therefore alternative splicing (Mishra et

al., 2011). This indicates that novel splicing factors can emerge in the

future and supports the notion that the poor knowledge on plant

alternative splicing is probably just a consequence of fewer studies in

these organisms.

In humans, defects in splicing result in several diseases, such

as cancer (Karni et al., 2007) or Alzheimer’s (Twine et al., 2011). In

fact, the database of human disease alleles (www.hgmd.org)

indicates that 16% of the point mutations listed are within splice sites


17

(reviewed in Hertel, 2008). Experimental evidence for the

physiological importance of alternative splicing in plants has begun to

emerge in the past decade, paralleled by a considerable growth in the

number of reports of individual genes undergoing alternative splicing.

In 2003, Kazan reviewed the biologically relevant examples described

at the time and predicted the potential of upcoming data. In this

review, the need to address four key issues in future alternative

splicing research were underscored: to identify alternative splicing

events using large-scale approaches, to determine developmental-

and stress-specific conditions that exert an effect on splicing profiles,

to understand the mechanisms of splice site selection, and to

functionally characterize alternative splice forms. While the first two

topics have since then been widely addressed, knowledge on the last

two remains scarce. In the absence of an in vitro plant splicing assay,

which has still not been established despite the efforts of several

research groups, current research should probably focus on the

functional characterization of individual splice forms. Indeed, several

recent reports have addressed this question in Arabidopsis. The

SR45 gene, which encodes an SR-related splicing factor, undergoes

alternative splicing and its disruption results in shorter roots, delayed

growth and flowering, altered leaf shape and abnormal flowers (Ali et

al., 2007), as well as increased sensitivity to abscisic acid (ABA) and

sugars (Carvalho et al., 2010). Interestingly, while one SR45 splice

form is able to complement the narrow petal phenotype but not the

defective root growth, the second isoform rescues root growth but not

the floral defects, clearly ascribing distinct roles to the two SR45

splice forms during plant development (Zhang and Mount, 2009).

Nevertheless, both isoforms appear to be active in sugar responses

during early seedling establishment (Carvalho et al., 2010). The

phytochrome interacting factor 6 (PIF6) gene generates two


18

alternatively-spliced transcripts (Penfield et al., 2010). The full-length

splice variant encodes a transcription factor harboring also a

phytochrome-interacting domain, whereas the second transcript is

shorter and does not include the DNA-binding domain. Interestingly,

transgenic plants overexpressing each of the alternative transcripts

revealed that the shortest splice variant accounts for a role of PIF6 in

the control of seed dormancy in Arabidopsis, whereas both isoforms

are active in light regulated-hypocotyl growth, especially under red

light (Penfield et al., 2010). In another recent study, the identification

of a novel splicing factor, suppressor of abi3-5 (SUA), allowed the

annotation of a developmentally-regulated alternative splicing event in

the ABA signaling ABI3 gene, a transcription factor that positively

regulates seed dormancy and represses germination (Sugliani et al.,

2010). The ABI3 splicing event generates two transcripts, one

encoding the full-length protein and the shortest encoding only two of

the four functional domains. Importantly, the latter isoform

accumulates at the end of seed maturation and counteracts the effect

of the full-length protein, resulting in the initiation of seed germination

(Sugliani et al., 2010). In yet another example, alternative splicing of

the Arabidopsis inderminate domain 14 (IDD14) gene also results in

two splice forms. While the full-length transcript encodes the IDD14α

transcription factor associated with the promotion of starch

accumulation, the shortest, IDD14β, lacks the DNA-binding domain

and interacts with IDD14α, inhibiting its activity (Seo et al., 2011). The

expression of this shortest splice variant is induced by cold stress and

allows for controlled starch accumulation under these conditions (Seo

et al., 2011).

In addition to developmental processes, alternative splicing in

plants has often been linked to the response to abiotic stress. Given a

large interest within the research community in understanding plant


19

responses to stress cues, this issue has also been recently

addressed. Two major lines of evidence support a role for plant

alternative splicing under stress conditions (reviewed in Duque et al.,

2011). Arabidopsis genes related to stress responses are more prone

to undergo alternative splicing (Zhou et al., 2003, Wang and Brendel,

2006) and stress conditions exert a huge impact on the splicing

profile of alternatively-spliced plant genes (Iida et al., 2004, Filichkin

et al., 2010). An additional interesting observation relating splicing

with plant stress responses is the fact that several genes with roles in

splicing display stress-responsive splicing patterns, with the SR

protein gene family being a good example (Palusa et al., 2007;

reviewed in Reddy and Ali, 2011). Moreover, the activity of splicing

regulators has been linked to stress responses. For instance, a

mutation in the U6 snRNP-specific Sm-like protein LSM4 results in

hypersensitivity to salt stress during germination in Arabidopsis

(Zhang et al., 2011), and correct splicing of the nad2 subunit 2 of

mitochondrial complex I, promoted by the pentatricopeptide ABO5, is

needed for proper ABA signal transduction (Liu et al., 2010).

Apart from Arabidopsis, the biological significance of

alternative splicing has been examined in several other plant species.

In rice, the dehydration-responsive element binding protein 2,

DREB2B, gene undergoes alternative splicing and generates two

splice variants (Matsukura et al., 2010). The shortest and non-

functional isoform is more abundant under control conditions but less

under stress, whereas the active form, which acts as a transcription

factor, is dramatically more expressed under drought and heat-shock

stress conditions. Importantly, overexpression of this isoform in

transgenic rice plants results in enhanced target gene expression and

stress tolerance (Matsukura et al., 2010). This mechanism allows the

maintenance of a basal DREB2B expression level, thus facilitating


20

rapid responses to unfavorable external conditions (Matsukura et al.,

2010). Other reports still fail to provide extensive insight into the

functional relevance of alternative splice forms. For instance, different

splicing profiles have been identified in members of the rice sulphate

transporter family under sulphate starvation, suggesting the

involvement of alternative splicing in the control of sulphur status,

which is essential for protein and lipid structures as well as for many

cofactors and coenzymes during plant development and stress

responses (Kumar et al., 2011). Also in rice, the OsBWML1 MAPK

kinase gene generates three splice variants that display differential

relative expression levels in various tissues and upon stress

induction, with the resulting three isoforms being differently localized

in the cell (Koo et al., 2007). In maize, ZmrbohB, encoding a nicotine

adenine dinucleotide phosphate oxidase with a role in the generation

of reactive oxygen species, undergoes tissue-, developmental- and

stress-regulated alternative splicing (Lin et al., 2009). Moreover, cold

acclimation in cotton involves alternative splicing of phospholipase

Dα, resulting in altered levels of the intermediate signaling molecule

phosphatidic acid, whose activity is associated with the stability of

bilayer membranes (Kargiotidou et al., 2010). In another example, the

sunflower sf21C, a member of a small plant gene family related to the

human N-myc downstream-regulated gene family whose activity has

been linked to cell growth and differentiation as well as hormonal and

stress responses, undergoes alternative splicing in an organ-specific

manner, but the functional significance of this occurrence is unknown

(Lazarescu et al., 2010). Interestingly, the latter study identified novel

splice sites distinct from those recognized by the U2 or U12-type

spliceosomes. Finally, Citrus clementina possesses an acidic

chitinase that displays two isoforms upon pathogen attack or

treatment with jasmonic acid (Del Carratore et al., 2011). The longer


21

splice variant arises from an intron retention event that introduces a

PTC and it was suggested that the resulting shorter isoform facilitates

rapid defense responses (Del Carratore et al., 2011).

Together, the examples described above clearly demonstrate

the potential that alternative splicing may have on controlling many

biological processes in several plant species, underscoring the

importance of further research exploring this hypothesis.

1.1.4. The SR protein family

SR proteins are highly conserved splicing factors present in

both metazoans and plants. They display a consensus structure

consisting of one or two N-terminal RNA-recognition motifs (RRMs)

and a C-terminal RS domain rich in arginine and serine residues. The

RRM binds the pre-mRNA at ESRs or ISRs and therefore determines

pre-mRNA recognition specificity. The RS domain is responsible for

the interaction with other proteins during spliceosome recruitment to

splice sites and also helps in pre-spliceosome assembly by binding

the 5’ splice site and the branch point (reviewed in Ram and Ast,

2007; Barta et al., 2010; and Duque, 2011). As ESRs are found in

both constitutively and alternatively-spliced exons, SR proteins are

thought to play roles during both constitutive and alternative splicing

(Golovkin and Reddy, 1999; Lorkovic et al., 2004b; Tanabe et al.,

2006; reviewed in Hertel, 2008; Solis and Patton, 2010).

Apart from splicing-related functions, SR proteins may fulfill

other roles in different biological processes such as transcription (Das

et al., 2006; Kitsios et al., 2008), mRNA export (Huang and Steitz,

2001; Pendle et al., 2005; Rausin et al., 2010), transcript stability and

quality control (Zhang and Krainer, 2004) or translation (Sanford et


22

al., 2004; Delestienne et al., 2010). Interestingly, recent data also

suggest a link between alternative splicing and DNA methylation,

which is likely to involve the activity of SR proteins (Li et al., 2008;

reviewed in Reddy and Ali, 2011). In support of this notion, a report

on the analysis of the Human Epigenome Project data indicates that

DNA methylation appears to be increased in sequences containing

multiple ESEs (Anastasiadou et al., 2011).

Knowledge on the mode of action of SR proteins stems mainly

from studies in animal cells but it has been considered to be similar to

that operating in plants, since several plant SR proteins have been

identified as U1-70K interactors (Golovkin and Reddy, 1999; Lorkovic

et al., 2004b; Tanabe et al., 2006). Although, the establishment of an

efficient in vitro splicing reaction for plants has hitherto failed, a few

plant SR proteins have efficiently complemented HeLa S100

mammalian splicing-deficient cell extracts (Lopato et al., 1996; Lopato

et al., 1999a; Ali et al., 2007; Barta et al., 2008).

Arabidopsis possesses 18 SR protein members (Barta et al.,

2010), rice 22 (Reddy and Ali, 2011), soybean 25 (Reddy and Ali,

2011), and Brachypodium 17 (International Brachypodium Initiative,

2010), whereas only 12 SR proteins are found in humans (Manley

and Krainer, 2010) and 7 in C. elegans (Longman et al., 2000). These

differences in SR protein number between plants and metazoans may

reflect divergences at the level of splice site recognition and/or

splicing regulation. On the other hand, no SR proteins exist in S.

cerevisiae and only two are found in S. pombe, indicating that SR

proteins have emerged with multicellularity (Barbosa-Morais et al,

2006; reviewed in Ram and Ast, 2007). The lack of SR splicing

regulators in yeast is consistent with the very short introns observed

in these organisms, likely as a means of optimizing splicing regulation


23

and attenuating erroneous splice site choices (reviewed in Ram and

Ast, 2007).

Plant SR proteins can be divided into subfamilies, according

to their structural domain organization (Barta et al., 2010). In

Arabidopsis, six subfamilies (SR, SC, RSZ, SCL, RS2Z and RS) can

be identified, three of which (SCL, RS2Z and RS) are specific to

plants and the other three true orthologs of human SR proteins (Fig.

1.4). Within each subfamily, the size and number of exons and introns

of the protein-encoding genes are also conserved (Kalyna and Barta,

2004). In addition, some members have arised from gene duplication

events, raising the possibility of functional redundancy (Kalyna and

Barta, 2004). However, several duplicated gene pairs show different

tissue and/or developmental-specific expression patterns, suggesting

specific roles for each pair member (Palusa et al., 2007).

Reversible phosphorylation by several kinases and

phosphatases at the RS domain is a fundamental mechanism in the

regulation of SR protein activity, as it determines interactions with

other RS-domain-containing, spliceosome-related components

(Lorkovic et al., 2004b; Tripathi et al., 2010; reviewed in Stamm,

2008). The splicing reaction is therefore affected by the

phosphorylation status of SR proteins, but this posttranslational

mechanism can also influence the cellular mobility of SR proteins,

resulting in altered effects on mRNA stability, export or translation

(Tillemans et al., 2005; Sanford et al., 2005; Ali and Reddy, 2006; de

la Fuente van Bentem et al., 2006; Rausin et al., 2010). Information

on the plant kinases and phosphatases acting on SR proteins is

scarce, but future work on their characterization may provide valuable

clues in the dissection of the biological processes in which SR

proteins are involved (reviewed in Kersten et al., 2006). In

Arabidopsis, the Clk/Sty-type kinase AFC2 was found to


24

phosphorylate SR33, SR45, RSZ21 and RSZ22 (Golovkin and

Reddy, 1999), while in tobacco the LAMMER kinase PK12

phosphorylates several SR proteins (Savaldi-Goldstein et al., 2000).

Also, a microarray-based proteomic approach in Arabidopsis has

identified several SR proteins as possible targets for mitogen-

activated protein kinases, MAPKs (Feilner et al., 2005).

Figure 1.4. The Arabidopsis SR protein family. Schematic representation of the structure of the 18 Arabidopsis SR proteins and their classification into six subfamilies. Same letters indicate duplicated gene pairs, and the nomenclature of the mammalian orthologs is shown. RRM: RNA-recognition motif. ψRRM: RRM with an additional SWQDLKD motif. RS: arginine/serine-rich. SR: serine/arginine dipeptides rich. SP: serine/proline-rich. The SCL subfamily contains an additional N-terminal domain rich in arginine, proline, serine, glycine and tyrosine residues. ZnK: zinc-knuckle. Adapted from Kalyna and Barta (2004), Barta et al. (2010), and Reddy and Ali (2011)


25

Phosphorylation is a fundamental mechanism to regulate

overall SR protein family activity. However, other specific protein

domains contained within each subfamily can account for additional

regulatory mechanisms. For instance, the SC35-like subfamily

contains residues rich in arginines, prolines, serines, glycines and

tyrosines in its N-terminus. Methylation at arginine residues by protein

arginine methyltransferases (PRMTs) is a regulatory mechanism that

has been described for RNA-binding proteins, including SR proteins,

which may affect their recruitment to splice sites, activity and/or

mobility (reviewed in Yu, 2011). In fact, two human SR proteins,

SFRS9/SRp30c and SF2/ASF, have been shown to be methylated as

a means of controlling their role in mRNA stabilization and export to

the cytosol (Bressan et al., 2009; Sinha et al., 2010). In yeast, Hmt1

methylates the U1 Snp1 specific protein and the levels of Snp1

methylation affect its association with the SR-like protein Np13 and

splicing efficiency (Chen et al., 2010). Finally, in Arabidopsis

PRMT5/SKB1 methylates the U6 snRNP-specific Sm-like protein

LSM4, and mutations in PRMT5/SKB1 result in severe defects in

splicing (Zhang et al., 2011).

Many studies on plant SR proteins have addressed the

interaction with other proteins as well as their intracellular localization

and dynamics. The U1 snRNP 70K protein interacts with the

Arabidopsis SR45, SR34, RSZ21, RSZ22 and SCL33, suggesting an

important role for SR proteins in 5’ splice site recognition (Golovkin

and Reddy, 1999; Lorkovic et al., 2004b; Tanabe et al., 2006). On the

other hand, RS2Z33 interacts with SR34, RSZ21, RSZ22, members

of the SCL subfamily and SC35 (Lopato et al., 2002). Futhermore,

cell imaging revealed plant SR proteins to be generally localized in

the nucleus either in speckles, also known as splicing factor


26

compartments, or in the nucleoplasm, a feature slightly divergent from

their animal counterparts (Lorkovic at al., 2004a; Tillemans et al.,

2005; Tanabe et al., 2006). Speckles are clusters of interchromatin

used as storage, assembly and modification sites for SR proteins

when transcription rates are low. SR proteins diffuse to the

nucleoplasm upon transcription, in order to associate with the pre-

mRNA and participate in its splicing, and shuttle back to the nuclear

speckles when transcription slows down (Huang and Steitz, 2001;

Fang et al., 2004; Tillemans et al., 2006). Some observations suggest

particular shapes for nuclear speckles depending on the cell type or

the SR protein analyzed (described for RSp31, SR34, RSZ22 and

RS2Z33), but no explanation for this has been proposed (Tillemans et

al., 2005; Lorkovic et al., 2008). Shuttling between cellular

compartments is also dependent on the phosphorylation status of SR

proteins (Rausin et al., 2010; Sanford et al., 2005; reviewed in

Stamm, 2008). Indeed, the mobility of these RNA-binding factors is

tightly related to transcription and phosphorylation (Ali and Reddy,

2006; Kitsios et al., 2008; reviewed in Reddy and Ali, 2011). In

support of their role in mRNA transport and stability during translation,

several animal SR proteins have been observed in the cytoplasm

(Caceres et al., 1998; Delestienne et al., 2010). The Arabidopsis

RSZ22 is so far the sole such case described in plants (Rausin et al.,

2010).

Cell imaging has also provided data showing co-localization

between plant SR proteins (Lorkovic et al., 2008; reviewed in Reddy

and Ali, 2011). Interestingly, it is more common to observe co-

localization between SR members of the same subfamily. SR proteins

also share intracellular localization and/or interact with other splicing

factors or other proteins, such as transcription-related factors

(Bourquin et al., 1997; Ko et al., 2001; Kitsios et al., 2008). In human


27

cells, the PKA C subunit, involved in the transcriptional activation of

cAMP-responsive elements, co-localizes with SC35, phosphorylates

SR proteins and is involved in splicing regulation (Kvissel et al.,

2007). In Arabidopsis, SR34 colocalizes with TGH, a putative RNA-

processing protein, whose loss of function results in several

developmental defects (Calderon-Villalobos et al., 2005).

SR proteins not only play central roles in alternative splicing,

but SR genes also often generate multiple splice variants themselves

(Tanabe et al., 2006; reviewed in Reddy and Ali, 2011). In fact, from

the 18 Arabidopsis proteins, only two (RSZp22a and SCL28) seem to

encode a single transcript (reviewed in Duque, 2011). In this gene

family, the most common alternative splicing event appears to be

intron retention, whereas exon skipping is the less frequent. It is usual

to observe the inclusion of PTCs in several of the alternatively-spliced

forms of SR proteins, which are presumably but not necessarily

targeted to degradation by NMD (Pendle et al., 2005; Lareau et al.,

2007; Palusa and Reddy, 2010). For instance, studies using a UPF3

mutant background, which is impaired in NMD decay, revealed that

not all of the PTC-containing SR splice variants increased their

steady state levels, suggesting that not all of them are degraded by

this quality control mechanism (Palusa and Reddy, 2010).

Importantly, the splicing pattern of SR protein genes is tissue- and

developmental-specific (Tanabe et al., 2006; Palusa et al., 2007).

Furthermore, SR proteins can regulate splicing of several genes

including of their own pre-mRNA, a feature that has also been

described for other splicing factors (Stauffer et al., 2010). In

Arabidopsis, RS2Z33 plays a role in the regulation of its own splicing

pattern as well as of SR30 and SR34 (Kalyna et al., 2003), while

SR30 also influences its own splicing and that of other SR proteins

(Lopato et al., 1999b). Interplay between these different levels of SR


28

protein gene expression regulation, including the inclusion of PTCs in

alternative splice forms to control SR protein activity, may contribute

to their ability to select for correct splice sites of a given set of target

genes.

The bias of plant alternative splicing to stress-related genes

and the existence of a higher number of SR proteins in plants suggest

that SR proteins are important mediators of stress responses in

plants. In agreement, the splicing pattern of different SR proteins is

not only developmentally-regulated but also responsive to external

stress stimuli (Tanabe et al., 2006; Palusa et al., 2007). Moreover, SR

protein phosphorylation can also be regulated by external cues,

pointing to a mechanism of relaying stress signals to these splicing

factors. For instance, the MAPK kinases MPK3 and MPK6, which

phosphorylate SR proteins, are important mediators in stress

signaling (Feilner et al., 2005), and the LAMMER kinase PK12 is

involved in ethylene signaling (Savaldi-Goldstein et al., 2000).

Furthermore, SR protein dynamics and re-localization have also been

shown to be affected by external stress signals (Delestienne et al.,

2010; Rausin et al., 2010; reviewed in Lorkovic and Barta, 2004).

Despite the described work on SR proteins, few functional

studies exist in plants and analyses of loss-of-function mutants for

plant SR proteins are yet to be reported. This largely contrasts with

human studies, where SR proteins have been identified as

fundamental elements in the control of correct gene expression. For

instance, the human papillomavirus E2 transcription factor alters the

expression of SF2/ASF, SRp20 and SC35 in infected cells

(McFarlane and Graham, 2010) and gene expression of SF2/ASF is

upregulated in different types of cancer, resulting in splicing

deregulation – SF2/ASF is considered an oncoprotein with potential

to be targeted in cancer therapy (Karni et al., 2007). In Arabidopsis,


29

overexpression of SR30 and RS2Z33 cause changes in alternative

splicing of other SR protein genes and general morphological and

developmental abnormalities (Lopato et al., 1999b; Kalyna et al.,

2003). In addition, heterologous expression of portions containing the

RS domain of two Arabidopsis SR-like proteins (RCY1 and SRL1)

has been found to confer salt stress tolerance in yeast (Forment et

al., 2002). Disruption of the SR-related SR45 gene results in aberrant

growth (Ali et al., 2007) and altered responses to sugars and ABA

(Carvalho et al., 2010).

1.1.4.1. The SCL30a SR protein

The Arabidopsis SCL30a belongs to the plant-specific SC35-

like (SCL) subfamily of SR proteins (Barta et al., 2010) (Fig. 1.5).

Together with SCL33 it arose from a genomic interchromosomal

duplication (Kalyna and Barta, 2004) (see Figure 1.4). The SCL30a

sequence harbors an RRM similar to the one found in the Arabidopsis

and the mammalian SC35 factors. However, its N-terminal end

contains an extension rich in arginine, proline, serine, glycine and

tyrosine residues, which places it, together with the other SCL

members, in a plant-specific subfamily (Barta et al., 2010; Manley and

Krainer, 2010). Importantly, the mammalian SC35 (recently renamed

SRSF2; Manley and Krainer, 2010) has been shown to regulate

alternative splicing in vivo (Merdzhanova et al., 2008; Shi et al.,

2008).

Very few reports on the characterization of SCL30a or other

members of its subfamily are available in the literature. Members of

the SCL subfamily were found to co-localize in tobacco protoplasts

(Lorkovic et al., 2008). In particular, SCL30a is found in nuclear


30

speckles and has been reported to interact with itself and many other

SR proteins, such as SCL28, SCL30, SC35, RSZ21, SR30, SR34

and RS2Z33 (Lopato et al., 2002; Lorkovic et al., 2008). Intriguingly,

SCL30a does not appear to interact with the fourth member of the

SCL subfamily, SCL33, which is its duplicated gene pair and interacts

with SCL28 and SCL30. The SCL30a SR protein also co-localizes

partially with RS2Z33 and SC35 (Lorkovic et al., 2008).

Figure 1.5. The SCL30a SR protein. Schematic representation of the annotated genomic structure of the SCL30a gene (boxes represent exons and lines introns, with the coding sequence shown in black) and the corresponding encoded protein. RRM: RNA-recognition motif. SR: serine/arginine dipeptides rich. Adapted from www.arabidopsis.org and Barta et al. (2010)

The SCL30a gene has been reported to be highly expressed

in different tissues during Arabidopsis development, such as in roots,

the stem, leaves, and inflorescences, and to a lesser extent in pollen

(Palusa et al., 2007). Despite the annotation of a single splice variant

(www.arabidopsis.org, see Figure 1.5), SCL30a has been reported to

undergo alternative splicing (Palusa et al., 2007) and its splicing

pattern appears to be altered by heat but not by cold, glucose, or

auxin (Palusa et al., 2007), nor by ABA or salt (Palusa et al., 2007;

Zhang et al., 2011).


31

1.2. Protein Ubiquitination in Plants

Ubiquitin is a highly conserved small protein present in all

eukaryotes (Hershko and Ciechanover, 1986). The covalent

attachment of ubiquitin to other molecules is known as protein

ubiqutination. It allows specific proteins to be targeted for degradation

and constitutes the major proteolytic system in plants (reviewed in

Smalle and Vierstra, 2004). Protein degradation is essential for

removing abnormal and short-lived regulatory proteins and for amino

acid recycling for further protein synthesis, but it also allows the

regulation of protein activity in response to stimuli that direct growth

and development and/or responses to stress.

The covalent attachment of polymers of the 76-amino acid

ubiquitin protein to a specific substrate is performed by three groups

of enzymes (E1, E2, and E3) through a conjugation cascade

(reviewed in Hershko and Ciechanover, 1998). The ubiquitin molecule

has the same amino acid sequence in all higher plants, differing in

only two residues in yeast and in three residues in animals, and is

ubiquitously expressed as its name indicates (reviewed in Smalle and

Vierstra, 2004). Target substrates can be localized in several cellular

compartments, such as the cytoplasm or the nucleus, they can be

membrane-localized or even found in the endoplasmic reticulum

(reviewed in Smalle and Vierstra, 2004 and Haglund and Dikic, 2005).

Substrate ubiquitination can be regulated by different signals, via the

stimulation of cell surface receptors by external ligands or by protein

phosphorylation (reviewed in Hicke and Dunn, 2003 and Haglund and

Dikic, 2005).

The ubiquitination pathway starts with the ATP-dependent

activation of an ubiquitin molecule by an E1 ubiquitin-activating

enzyme, resulting in direct binding of a glycine residue at the ubiquitin


32

C-terminus to a cysteine residue in E1 via a thioesther linkage – see

Figure 1.6 (reviewed in Hershko and Ciechanover, 1998 and Smalle

and Vierstra, 2004). The activated ubiquitin molecule is then

transferred to an E2 ubiquitin-conjugating enzyme by a

transesterification reaction. Lastly, the ubiquitin-E2 intermediate

delivers the ubiquitin molecule to the target substrate using the

activity of an E3 ubiquitin-protein ligase. Here, the glycine residue at

the C-terminal end of ubiquitin is attached to a lysine residue in the

target protein.

Figure 1.6. The ubiquitination/26S proteasome pathway. Schematic representation of the attachment of a ubiquitin monomer (UB) to a target substrate, through a conjugation cascade performed by the E1, E2 and E3 enzymes. The cycle repeats itself when polyubiquitination is needed. If the chain gets attached at lys-48, the target substrate will be recognized by the 26S proteasome and subsequently degraded. Adapted from Pickart (2001) and Vierstra (2003)

The ubiquitination cycle can be achieved only once, resulting

in a monoubiquitinated substrate, which will be targeted for


33

destruction in the lysosome/vacuole or serve as a signaling

intermediate in the control of other processes, such as DNA repair

and methylation, histone modifications or transcription (Kraft et al.,

2008; reviewed in Hicke and Dunn, 2003 and Haglund and Dikic,

2005). However, it has been more common to observe several

repetitions of the ubiquitination cycle, during which a lysine residue in

the last ubiquitin molecule attached is used as acceptor. After at least

four repeats of the cascade, a polyubiquitin chain will be attached to

the substrate (Thrower et al., 2000). Seven lysine residues – lys-6,

lys-11, lys-27, lys-29, lys-33, lys-48 and lys-63 – can be used as

acceptors but the last two have been the most described (reviewed in

Haglund and Dikic, 2005). If the chain is bound at the ubiquitin

molecule’s lysine residue 63, activation of downstream events related

to protein kinase activation, stress responses or DNA damage repair

will occur. In contrast, attachment of the polyubiquitin chain at lysyl-48

results in recognition of the target protein by the 26S proteasome,

which will degrade the substrate in an ATP-dependent manner,

releasing in the end the ubiquitin monomers (reviewed in Hershko

and Ciechanover, 1998). Attachment at lys-48 is the best

characterized polyubiquitin mechanism. It is likely that only a subset

of the polyubiquitin chain is recognized by the downstream

components, which may explain how the number of ubiquitin

monomers in the chain and the lysine residues used for ubiquitin

binding affect recognition by the corresponding regulated pathways

(Thrower et al., 2000).

More than 5% of the Arabidopsis proteome is involved in the

ubiquitin/26S proteosome pathway, highlighting its importance in the

maintenance of protein homeostasis (reviewed in Smalle and

Vierstra, 2004). Moreover, this number is more than twice that of

occurring in yeast and humans, suggesting that the sessile growth


34

habit of plants, and their need to rapidly adapt growth and

development to changing external conditions, resulted in an

ubiquitination pathway with a larger spectrum of action. In support of

this notion, disruption of ubiquitination pathway-related components

results in dramatic effects on plant growth and development under

stress conditions (reviewed in Moon et al., 2004).

Arabidopsis expresses two E1 isoforms, 37 E2 enzymes, and

has approximately 1,300 genes encoding putative E3 ligases (Hatfield

et al., 1997; Kraft et al., 2005; reviewed in Mazzucotelli et al., 2006).

The E1 enzymes are ubiquitously expressed in Arabidopsis and their

high catalytic efficiency may explain the sufficiency of such a low

number of copies in ensuring the activation of the downstream

ubiquitination cycle (Pickart, 2001). It is possible that E1 activity is

regulated by posttranslational modifications, such as phosphorylation,

but also by a different intracellular localization of both enzymes

(Hatfield et al., 1997). Indeed, it has been suggested that one E1 is

nuclear and the second cytoplasmic (Hatfield et al., 1997). E2

enzymes, classified into 12 subfamilies, are also broadly expressed in

Arabidopsis tissues (Kraft et al., 2005). Obviously, a single E2 can

interact with different E3 ligases, possibly by flexible modulation of

the three-dimensional structure of E2s and the small sets of surface

residues that specify protein interaction with E3 enzymes (reviewed in

Pickart, 2001). E3 ligases confer specificity to the ubiquitination

pathway by recognizing the substrates that will be ubiquitinated,

which explains the existence of such a high number of E3 when

compared to E1 and E2 enzymes. In plants, E3 ligases have been

implicated in photomorphogenesis, organ morphogenesis, flowering,

senescence, hormone signaling, cold sensing, self incompatibility,

wax biosynthesis and removal of misfolded polypeptides, as well as

responses to biotic and abiotic stresses (reviewed in Moon et al.,


35

2004, Smalle and Vierstra, 2004, and Dreher and Callis, 2007).

However, substrates for most of the E3 ligases remain to be

identified. Future research in this field may uncover additional

biological processes involving the activity of E3 ligases and the

ubiquitination pathway.

1.2.1. E3 ligase classification and activity

In Arabidopsis, analysis of the structure of E3 ligase-encoding

genes has allowed clustering into nine subfamilies (reviewed in

Mazzucotelli et al., 2006). The homology to E6-AP C-terminus

(HECT), plant U-box (PUB), CULLIN (CUL), Arabidopsis Skp1-related

(ASK), bric-a-brac, tramtrack and broad complex (BTB), CULLIN4-

damaged DNA-binding protein (CUL4-DDB) and anaphase promoting

complex (APC) are the less represented subfamilies, ranging from

five (CUL4-DDB) to 81 (BTB) gene members. The other two

subfamilies – really interesting new gene (RING) and cyclin F proteins

(F-box) – possess around 500 and 700 members, respectively. The

U-box members are related to those harboring RING domains and it

is acceptable to consider a single subfamily (RING/U-box) comprising

both motifs (Aravind and Koonin, 2000).

E3 ligases can act as monomers or as multisubunit complexes

that include members from the nine referred subfamilies (reviewed in

Smalle and Vierstra, 2004 and Mazzucotelli et al., 2006). One of the

following two motifs is always found in E3 ligases, irrespective of their

monomeric or protein complex structure: a HECT or a RING/U-box

motif. E3 ligases may contain binding sites for both the substrate and

the E2, highlighting their ability to act as single E3 units in the

ubiquitination cascade. The HECT or RING/U-box motif present in a


36

particular E3 ligase generally defines its mode of action, since RING

E3s generally function as adaptors between the E2 and the target

protein, whereas HECTs form a covalent bond with the ubiquitin

molecule before transferring it to the substrate. In addition, RING/U-

box E3s, in contrast to the HECTs, can form multisubunit complexes

with other ligases in order to form an active E3 ligase. These

multidomain complexes can be either CULLIN- or RING-based,

depending on the motif identifiable in the catalytic module, the domain

responsible for E2 binding. The catalytic module of E3 ligases binds

the second domain found in these complexes – the adaptor module,

responsible for substrate recognition. A single catalytic module can

bind different adaptors, increasing the specificity of the E3 ligase

complexes. Furthermore, a single E3 ligase can target different

substrates, depending for instance on target protein availability. For

example, the Arabidopsis AIP2 E3 ligase targets the germination

regulator transcription factor ABI3 for degradation, but both genes

display low overlap of tissue- and developmental-specific expression

patterns (Zhang et al., 2005). Whereas AIP2 is mostly expressed in

vegetative and reproductive tissues, ABI3 is preferentially seed-

specific, suggesting that AIP2 may have other non seed-related

protein targets (Zhang et al., 2005). On the other hand, ubiquitination

of a single substrate may depend on the activity of multiple E3s.

Degradation of the kip-related protein 1 (KRP1), involved in the

control of cell cycle progression, is dependent on both SCFSKP2b and

the RING RKP E3 ligases (Ren et al., 2008).

The large representation of RING motif-containing proteins in

the Arabidopsis group of E3 ligases and their capacity to act as

monomers or in multidomain complexes, but also the fact that they

are expressed in various Arabidopsis tissues, suggests major roles

for RING ligases in the ubiquitination system and concomitantly in


37

plant physiology (reviewed in Mazzucotelli et al., 2006). The RING

finger motif was first identified in humans (Freemont et al., 1991). It

was named based on the fact that it contains an exquisite structure

with four pairs of zinc ligands (small repeats of cysteine and histidine

residues) that bind coordinately two zinc ions in a cross-brace

structure. Furthermore, in contrast with the known DNA-binding zinc

finger domains, RING domains are protein-protein interaction

modules (Lorick et al., 1999). In Arabidopsis, analysis of RING E3

ligase genes and their encoded proteins revealed the presence of

modified RING-finger domains (11% of the total number of RING

domains), which differ from the canonical RING-finger in the number

of zinc ligands and/or the distance between the corresponding

cysteine and histidine residues (Stone et al., 2005). RING E3 ligases

can be classified into eight subfamilies, according to the RING-

domain type: RING-H2, -HCa, -HCb, -C2, -v, -D, -S/T and –G. The

RING-D domain structure is specific to Arabidopsis, but the remainder

can be found in other organisms. Protein members from all

Arabidopsis RING-type subgroups except RING-S/T have been

shown to exhibit in vitro polyubiquitination activity (Stone et al., 2005).

Furthermore, some RING-motif E3 ligases also display

autoubiquitination capacity, which may constitute a regulatory

mechanism to control their activity (reviewed in Pickart, 2001).

Analysis of other domains in their protein structures allowed the

classification of RING ligases into 30 subfamilies, eight of which are

plant-specific. The two largest classes possess either no additional

domain or a transmembrane domain. In addition, ubiquitin and nucleic

acid-binding or protein-protein interaction domains are identifiable. As

the RING domain is responsible for binding an E2 enzyme, the

protein-protein interaction domains are most likely responsible for


38

binding the target substrate to be ubiquitinated (Stone et al., 2005;

reviewed in Smalle and Vierstra, 2004).

The best characterized E3 ligase in Arabidopsis is the RING-

motif-containing COP1. COP1 is a negative regulator of

photomorphogenesis whose activity, based on a microarray analysis,

seems to affect direct or indirectly more than 20% of the genome,

probably through its activity on the controlled degradation of several

transcription factors, thus influencing their target gene expression (Ma

et al., 2002; reviewed in Moon et al., 2004). cop1 mutant seedlings

exhibit short hypocotyls and photosynthetic activity when developing

in the dark, both characteristics of light-grown seedlings (Deng et al.,

1991). Under dark conditions, COP1 is found in the nucleus, where it

targets for degradation activators of photomorphogenesis, such as

the transcription factor long hypocotyl 5, HY5, and the photoreceptor

A, phyA (Osterlund et al., 2000; Seo et al., 2004). In the light COP1

shuttles back to the cytoplasm, and the nuclear inducers of light

responses get stabilized. It should be noted that COP1 has also been

widely studied in humans, where it degrades the tumor suppressor

p53 protein (Dornan et al., 2004).

1.2.2. The XBAT family of RING E3 ligases

Members of the Arabidopsis XBAT family of RING E3 ligases

are orthologous to the rice XB3 protein, which was isolated in a yeast

two-hybrid assay through its interaction with the innate immunity-

associated XA21 protein (Nodzon et al., 2004). The XBAT family

comprises five members, XBAT31-35, which can be grouped into

three classes with levels of similarity ranging from 23% to 73%,

Figure 1.7A (Nodzon et al., 2004).


39

XBAT E3 ligases contain a RING-HCa domain at the C

terminus preceded by an additional recognizable domain at the N-

terminus with ankyrin repeats – see Figure 1.7B (Stone et al., 2005).

BLAST searches have revealed the existence of this protein structure

in humans, mouse, C. elegans, Drosophila and Xenopus (Stone et

al., 2005). Further analyses revealed similar proteins in several plant

species, including Artemisia desertorum, Lilium longiflorum, Medicago

truncatula, Oryza sativa, Populus trichocarpa, Solanum tuberosum,

and Vitis vinifera (Prasad and Stone, 2010). The RING-HC type is the

second most represented within Arabidopsis RING ligases and also

includes COP1. Whereas the RING domain is responsible for E2

binding, the protein-protein interaction ankyrin repeats motif appears

to be involved in substrate recognition (Bork, 1993; Lorick et al.,

1999). According to Stone and coworkers (2005), the family of RING-

HCa domain E3 ligases containing ankyrin repeats comprises seven

members – five of which, as mentioned above, compose the XBAT

family. The other two members, At3g28880 and At5g13530 (keep on

going, KEG), share the XBAT structure but whereas the KEG gene

contains an additional serine/threonine kinase domain at the N-

terminus (Stone et al., 2005), At3g28880 encodes a protein with a low

level of similarity (less than 20%) to the XBAT members

(www.arabidopsis.org).

In vitro ubiquitination capacity has been shown for XBAT32,

XBAT33, XBAT35 and KEG (Nodzon et al., 2004; Stone et al., 2005;

Stone et al., 2006). Negative results were reported for XBAT31 and

XBAT34, although this could reflect the use of inappropriate

ubiquitination components for these particular ligases in the in vitro

assays (Stone et al., 2005).

To date, biological functions have only been uncovered for


40

Figure 1.7. The XBAT family of RING E3 ligases. (A) Phylogenetic tree of XBAT family members, according to their predicted amino acid sequences. (B) Schematic representation of the conserved structure of the XBAT family of RING E3 ligases. ANK: ankyrin repeats (the number of repeats varies between XBAT members). RING: zinc-finger domain. Adapted from Nodzon et al. (2004) and Stone et al. (2005)

KEG and one XBAT member – XBAT32. Disruption of KEG impairs

early seedling growth, as well as further development and entry into

the mature flowering stage (Stone et al., 2006). Mutant KEG

seedlings are also more sensitive to sugars, ethylene and ABA.

Responses to ABA may rely on the stabilization of ABI5, a central

transcription factor acting in signal transduction of this hormone

(Stone et al., 2006). On the other hand, XBAT32 positively regulates

lateral root development by interfering with the levels of the ethylene

phytohormone (Nodzon et al., 2004; Prasad et al., 2010). Importantly,

XBAT32 was found to ubiquitinate in vitro not only itself but also two

enzymes involved in ethylene biosynthesis – aminocyclopropane-1-

carboxylic acid synthase 4 (ACS4) and 7 (ACS7) – and its loss of

function results in higher levels of ethylene in roots and concomitantly

root growth arrest (Nodzon et al., 2004; Prasad et al., 2010).

Consistently, disruption of XBAT32 also results in shorter hypocotyls

of dark-gown seedlings relative to the wild type (Prasad et al., 2010).

Both auxin and ABA are able to partially rescue the defect in lateral

root emergence in the XBAT32 mutant, suggesting that XBAT


41

proteins can act in different hormone signaling pathways, thus

controlling multiple physiological processes. In agreement with this

notion, XBAT32 also appears to be involved in ethylene-mediated

responses to salt stress (Prasad and Stone, 2010).

The involvement of the rice XB3 in innate immunity and of

XBAT32 in ethylene and auxin signaling, two hormones not only

implicated in plant growth but also in disease resistance, raises the

hypothesis of conserved functionalities between XBAT members and

their orthologous proteins (Nodzon et al., 2004). XBAT32 expression

was also found outside the root system, such as in leaves, stems and

anthers, and disruption of its activity results in delayed growth of

aerial organs, suggesting a role for XBAT32 in other processes during

plant development (Nodzon et al., 2004). Interestingly, the Lily

ankyrin repeat-containing protein (LIANK), homologous to the XBAT

family, possesses in vitro E3 ligase activity and is required for pollen

germination and tube growth (Huang et al., 2006). Huang and

coworkers (2006) refer to the XBAT32 expression in anthers, an

observation that was not explored by the group reporting it (Nodzon

et al., 2004), underscoring the importance of considering conserved

functionalities. Surprisingly, LIANK is associated with membrane-

enclosed organelles but possesses no amino acid sequence pointing

to its localization in transmembrane regions. It was proposed that the

ankyrin repeat domain interacts with proteins specific to these

organelles, thus targeting the protein to the observed intracellular

localization (Huang et al., 2006).

In Arabidopsis, testing of overlapping activities between XBAT

members has been initiated for XBAT32, XBAT34 and XBAT35 but

has only been addressed through analyses of single mutant lines

(Prasad et al., 2010). Individual disruption of XBAT34 or XBAT35

does not result in aberrant lateral roots nor in altered ABA responses,


42

in contrast to what is observed for XBAT32 (Prasad et al., 2010).

XBAT34 and XBAT35 are the closest members in the XBAT family

(68% identity, 73% similarity) and homologous proteins exist in Oryza

sativa, Populus trichocarpa and Vitis vinifera, but none has been

characterized (Nodzon et al., 2004; Prasad and Stone, 2010).

Interestingly, XBAT34 and XBAT35 may have arisen from a genome

duplication event (Nodzon et al., 2004; Stone et al., 2005), which

appears to be common within the RING family, and although

functional redundancy can be expected, the possibility of unique roles

for duplicated genes should not be excluded.

1.2.3. Alternative splicing of E3 ligases

While 5% of the Arabidopsis proteome is predicted to play a

role in the ubiquitination/26S proteasome system, more than 10%

appears to be regulated by this mechanism, which accounts for

around 2600 substrates (reviewed in Smalle and Vierstra, 2004). The

smaller number of E3 ligases compared to that of target substrates is

consistent with the capacity of one E3 ligase to recognize multiple

substrates. Several studies have shown that the functions of E3

ligases, and hence the recognition of their substrates, can be tightly

regulated by specific intracellular compartmentalization, degradation,

oligomerization and posttranslational modifications (reviewed in

Haglund and Dikic, 2005). A mechanism that may control the

described mechanisms and exert a strong regulatory effect on the

activity of E3 ligases is alternative splicing (reviewed in Mazzucotelli

et al., 2006). This phenomenon may allow increasing the range of E3

ligase action towards their substrates. However, very few reports

exist on the biological relevance of alternative splicing of E3 ligase


43

genes, which has been barely addressed in Arabidopsis.

Interestingly, from the XBAT family, both XBAT31 and XBAT35 genes

undergo alternative splicing (www.arabidopsis.org), but its functional

significance has not been investigated.

The human RBCK1 interacts with protein kinase C and

possesses E3 ligase activity, but its activity is inhibited when

interacting with RBCK2, its alternative splice variant that lacks the

RING domain (Tatematsu et al., 2008). In Drosophila, the E3 ligase

D-Cbl gene, which plays a role in eye development, also generates

two splice variants by alternative splicing (Wang et al., 2010). Despite

the fact that the encoded isoforms possess similar structures, the

longer isoform, D-CblL, is involved in restricting Epidermal Growth

Factor Receptor (EGFR) signaling, whereas D-CblS is associated

with Notch signaling restriction (Wang et al., 2010). More recently,

alternative splicing of the rat gene encoding the RING-CH 10 E3

ligase was shown to generate two isoforms, both of which are

expressed in elongating and elongated spermatids, but while the

longer protein is associated with microtubules, the shortest is

cytoplasmic (Iyengar et al., 2011). Moreover, the microtubule-

associated protein is a functional E3 ligase, whereas the second

isoform acts most likely as an adaptor or scaffold protein, as it lacks

the RING-finger domain (Iyengar et al., 2011).

In plants only two cases of alternatively-spliced genes

encoding E3 ligases have been reported. Retention of the 3’ intron in

one of the splice forms of ARI15, a member of the ARIADNE RING

subfamily, may account for altered mRNA stability, localization and/or

translational efficiency (Mladek et al., 2003). Importantly, COP1 has

also been described to undergo alternative splicing, generating an

alternative splice form, COP1b, with a deletion in the WD-40 domain,

when compared to the full-length protein (Zhou et al., 1998).


44

Interestingly, the COP1b isoform has a dominant-negative effect on

the protein activity of the full-length COP1, thus interfering with the

suppression of photomorphogenesis in the dark (Zhou et al., 1998).

1.3. Thesis Outline

The work presented in this thesis aimed at exploring the

biological relevance of alternative splicing in higher plants. To this

end, a two-fold approach was undertaken, using the model organism

Arabidopsis thaliana. Firstly, the physiological significance of an exon

skipping event, the least common type of alternative splicing in

Arabidopsis, in an E3-ligase-encoding gene was investigated.

Secondly, we have functionally characterized an RNA-binding protein

belonging to the SR protein family, which plays key roles in

alternative splicing, and identified some of its endogenous targets.

In addition to this introductory Chapter 1, this thesis comprises

three more chapters:

Chapter 2. XBAT35, a novel Arabidopsis RING E3 ligase

exhibiting dual targeting of its splice isoforms, is involved in

ethylene-mediated regulation of apical hook curvature

XBAT35 encodes an E3 ligase that undergoes alternative

splicing, generating two splice variants. The encoded proteins both

contain the functional RING-finger and ankyrin repeat domains, but

the shortest isoform lacks a nuclear localization signal as a result of

an exon-skipping event. The work described in this Chapter

demonstrates control of XBAT35 protein localization by alternative

splicing. Furthermore, loss-of-function approaches revealed a role for


45

this E3 ligase in ethylene control of apical hook formation in etiolated

seedlings. Both XBAT35 isoforms are functional in this response, but

appear to differentially contribute to apical hook exaggeration in the

dark. Finally, using a yeast two-hybrid assay, we isolated five putative

XBAT35 ubiquitination substrates.

Chapter 3. The Arabidopsis SCL30a SR protein confers ABA-

dependent salt and osmotic stress tolerance during seed

germination

This Chapter reports the first functional characterization of a

loss-of-function mutant for a plant SR protein. Loss- and gain-of-

function approaches, as well as epistatic analyses, uncovered a role

for the Arabidopsis SCL30a as a negative regulator of the ABA

signaling pathway under salt and osmotic stress during seed

germination. Furthermore, SCL30a was found to regulate seed size

and dormancy, as well as light responses during early seedling

development. Importantly, endogenous transcripts targeted by the

SCL30a SR protein were identified by means of deep-sequencing-

based global transcriptome analysis of wild-type, SCL30a-mutant and

SCL30a-overexpressing seeds germinated under salt stress.

Chapter 4. Conclusions and future perspectives

This last Chapter discusses the major findings of this doctoral

thesis in light of the biological importance of alternative splicing in

higher plants, suggesting perspectives for future research.


46

Most of Chapter 2 is the reproduction of the following publication:

Carvalho, S.D., Saraiva, R., Maia, T.M., Abreu, I.A., and Duque, P.

(2012). XBAT35, a novel Arabidopsis RING E3 ligase exhibiting dual

targeting of its splice forms, is involved in ethylene-mediated

regulation of apical hook curvature. Mol Plant (DOI:

10.1093/mp/sss048).

A substantial part of Chapter 3 constitutes a manuscript currently

under preparation.

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CHAPTER 2 XBAT35, A NOVEL ARABIDOPSIS RING E3 LIGASE

EXHIBITING DUAL TARGETING OF ITS SPLICE ISOFORMS, IS

INVOLVED IN ETHYLENE-MEDIATED REGULATION OF

APICAL HOOK CURVATURE

Most of this Chapter is the reproduction of the following publication:

Carvalho, S.D., Saraiva, R., Maia, T.M., Abreu, I.A., and Duque P.

(2012). XBAT35, a novel Arabidopsis RING E3 ligase exhibiting dual

targeting of its splice isoforms, is involved in ethylene-mediated

regulation of apical hook curvature. Mol Plant, DOI:

10.1093/mp/sss048.

Sofia D. Carvalho was the main responsible for the experimental work

and data analysis presented in this Chapter.

Acknowledgements to Vera Nunes for technical assistance in the

plant growth facilities, to Pedro Lima for cellular imaging support, and

to Nam-Hai Chua and Simon Møller for providing the pBA vector and

the Arabidopsis cDNA library in yeast, respectively.

Chapter 2 – XBAT35 regulates apical hook curvature

65

2.1. Abstract

The Arabidopsis XBAT35 is one of five structurally-related

ankyrin repeat-containing RING E3 ligases involved in ubiquitin-

mediated protein degradation, which plays key roles in a wide range

of cellular processes. Here we show that the XBAT35 gene

undergoes alternative splicing, generating two transcripts that are

constitutively expressed in all plant tissues. The two splice variants

derive from an exon skipping event that excludes an in-frame

segment from the XBAT35 precursor mRNA, giving rise to two

protein isoforms that differ solely in the presence of a nuclear

localization signal (NLS). Transient expression assays indicate that

the isoform lacking the NLS localizes in the cytoplasm of plant cells,

whereas the other is targeted to the nucleus, accumulating in nuclear

speckles. Both isoforms are functional E3 ligases, as assessed by in

vitro ubiquitination assays. Two insertion mutant alleles and RNA-

interference (RNAi) silencing lines for XBAT35 display no evident

phenotypes under normal growth conditions, but exhibit

hypersensitivity to the ethylene precursor 1-aminocyclopropane-1-

carboxylate (ACC) during apical hook exaggeration in the dark, which

is rescued by an inhibitor of ethylene perception. Independent

expression of each XBAT35 splice variant in the mutant background

indicates that the two isoforms may differentially contribute to apical

hook formation but are both functional in this ethylene-mediated

response. Thus, XBAT35 defines a novel player in ethylene signaling

involved in negatively regulating apical hook curvature, with

alternative splicing controlling dual targeting of this E3 ubiquitin

ligase to the nuclear and cytoplasmic compartments.


66

2.2. Introduction

Alternative splicing, which by selectively joining different splice

sites generates multiple transcripts from the same precursor mRNA

(pre-mRNA), provides a key posttranscriptional regulatory mechanism

for expanding proteomic diversity and regulating gene expression in

higher eukaryotes. In the past decade, alternative splicing has

emerged as a major feature of several genomes and been assigned

crucial biological relevance, such as in human disease (Ward and

Cooper, 2010). Based on next generation sequencing analyses, at

least 42% of the Arabidopsis intron-containing genes have been

recently estimated to undergo alternative splicing (Filichkin et al.,

2010), which is likely to participate in important plant functions,

including the response to environmental stress (Ali and Reddy, 2008;

Duque, 2011). This versatile gene regulation mechanism can

modulate transcript abundance by imposing changes in the 5’ or 3’

untranslated regions, thus affecting mRNA stability. In addition, by

introducing premature stop codons, alternative splicing can be

coupled to nonsense-mediated mRNA decay (NMD) to downregulate

expression of specific transcripts. However, and unlike transcriptional

control, alternative splicing can also change the structure of

transcripts and thereby influence almost all functional aspects of their

encoded proteins, such as binding properties, stability, enzymatic

activity, posttranslational modifications or subcellular localization.

The ubiquitin/26S proteasome pathway constitutes a tightly

regulated and highly specific system for protein degradation that

ensures the proteomic plasticity essential for a wide range of cellular

processes. In this pathway, polymers of the 76-amino acid, 8-kDa

ubiquitin protein are covalently attached, through a conjugation


67

cascade performed by three groups of enzymes, to a specific

substrate, targeting it for proteolysis by the 26S proteasome.

Posttranslational modification by ubiquitin has also been implicated in

several non-proteolytic processes, such as protein activation,

trafficking and sorting, which utilize either monoubiquination or

different ubiquitin-ubiquitin linkages within the polyubiquitin chain

(Mukhopadhyay and Riezman, 2007). The multistep ubiquitination

reaction is initiated with the ATP-dependent binding of ubiquitin to the

E1 activation enzyme. The activated ubiquitin is then transferred to an

E2 conjugating enzyme, which binds an E3 ligase that facilitates

transfer of ubiquitin to the substrate. This E3 enzyme is responsible

for recruiting the target protein for ubiquitination and hence confers

specificity to the pathway. In fact, the Arabidopsis genome encodes

only two E1 isoforms, around 40 E2 enzymes, and over 1,300

putative E3 ligases (Smalle and Vierstra, 2004). The existence of

such a large number of E3 ligases suggests high specificity of their

target recognition, underscoring their importance for downstream

signaling pathways.

In Arabidopsis, the largest class of E3 ligases is characterized

by the presence of a Really Interesting New Gene (RING) domain, in

which eight conserved Cys and His residues coordinate two zinc ions

in a cross-brace structure (Barlow et al., 1994). The RING domain is

a particular type of zinc finger that is usually involved in protein-

protein interactions instead of DNA binding and constitutes the E2-

binding domain essential for catalyzing E3 ligase activity of RING-

containing proteins (Stone et al., 2005). It can be found in complex E3

ligases, in which binding of the substrate occurs on a distinct protein,

or in monomeric E3 ligases, which include both the substrate

recognition and the RING E2-binding domains in a single protein.


68

A subgroup of Arabidopsis monomeric RING E3 ligases

contains two to nine N-terminal ankyrin (ANK) repeats (Stone et al.,

2006), which are among the most common protein-protein interaction

motifs (Mosavi et al., 2004) and may function as the substrate-binding

domain of these E3 ligases (Stone et al., 2005). Five of the

Arabidopsis RING-ANK proteins have been named XBAT as they are

structurally related to the rice (Oryza sativa) XB3 (Nodzon et al.,

2004), an ubiquitin ligase required for disease resistance mediated by

the receptor-like kinase XA21 (Wang et al., 2006). To date, only one

of the XBAT proteins has been assigned a biological role. XBAT32

(for XB3 ortholog 2 in Arabidopsis) positively regulates lateral root

development (Nodzon et al., 2004) via downregulation of ethylene

biosynthesis (Prasad et al., 2010), and may also be implicated in

ethylene-mediated responses to abiotic stresses, such as high salinity

(Prasad and Stone, 2010).

In the present study, we characterized the Arabidopsis

XBAT35 gene and found that exon skipping during processing of its

pre-mRNA generates two transcripts that are ubiquitously expressed

in Arabidopsis and encode proteins with in vitro E3 ubiquitin ligase

activity. Using fluorescent fusion proteins in transient expression

assays, we show that alternative splicing controls the subcellular

localization of the two isoforms, with one being targeted to the

cytoplasm and the other to the nucleus of plant cells. Finally, loss of

XBAT35 function results in ethylene hypersensitivity during apical

hook exaggeration in etiolated seedlings, indicating that this RING E3

ligase plays a role in negatively regulating ethylene-mediated control

of apical hook curvature in Arabidopsis.


69

2.3. Results

2.3.1. Alternative splicing of the XBAT35 pre-mRNA excludes

nuclear localization signal

Consistent with the current genome annotation, we detected

two RT-PCR products using primers specific for the Arabidopsis

thaliana XBAT35 gene (At3g23280). Sequencing of the amplified

bands showed that they correspond to two alternative XBAT35

transcripts generated by an exon skipping event, reportedly the least

common type of alternative splicing in Arabidopsis (Wang and

Brendel, 2006). As depicted in Figure 2.1, the XBAT35 gene contains

ten exons and nine introns. Constitutive splicing of the corresponding

pre-mRNA results in the longer splice variant (XBAT35.1), which is

predicted to encode a protein isoform of 462 amino acids with three

recognizable domains — an N-terminal ankyrin repeat region

harboring two ankyrin motifs, a putative bipartite nuclear localization

signal (NLS) roughly in the middle of the protein, and a zinc-finger

RING domain in the C-terminal end (Fig. 2.1). Hence, XBAT35 is a

putative E3 ubiquitin ligase, in which it is anticipated that the RING

domain functions in binding the E2 conjugating enzyme, while the

ankyrin repeats interact with the proteolytic substrate(s).

Importantly, the lower XBAT35-specific band corresponding to

the shorter splice variant (XBAT35.2) encodes a protein of 438 amino

acids and derives from skipping of the 72-bp exon 8, whose

corresponding 24 amino acids include the entire NLS sequence. As

the alternatively-spliced fragment is in frame, the XBAT35.2 splice

variant encodes a protein isoform that is structurally identical to

XBAT35.1, except that it lacks the bipartite nuclear targeting

sequence (Fig. 2.1).


70

Figure 2.1. Alternative splicing of the XBAT35 gene. Schematic representation of the genomic structure of the XBAT35 gene (boxes indicate exons with UTRs in grey and lines between boxes represent introns), the coding sequence of the two splice variants, XBAT35.1 and XBAT35.2, generated by alternative splicing (the skipped exon is shown in black), and the two corresponding predicted protein isoforms (ANK, ankyrin repeat; NLS, nuclear localization signal; RING, Really Interesting New Gene domain). The inset shows RT-PCR amplification of the two XBAT35 transcripts using primers F1 and R1, whose location is indicated by the arrows in the genomic scheme.

2.3.2. The XBAT35 gene is ubiquitously expressed in

Arabidopsis

To initiate the characterization of the A. thaliana XBAT35

gene, RT-PCR analyses were used to investigate its expression in

different tissues, in response to several external stimuli, and during

two photoperiod regimes. As shown in Figure 2.2, the two XBAT35-

specific bands were amplified in all tissues analyzed. In general, the

longer transcript was detected at slightly higher levels than the


71

shorter mRNA, with no striking changes in the ratio between the

expression levels of the two transcripts being observed.

Figure 2.2. Expression patterns of the XBAT35 gene. (A) RT-PCR analysis of XBAT35 transcript levels in whole seedlings and different tissues from wild-type (Col-0) plants. (B) XBAT35 expression levels in rosette leaves of 5-week old plants grown under long day (LD) or short day (SD) photoperiod regimes, represented by the diagrams on the right (white and black boxes indicate light and dark cycles, respectively, while dashed lines indicate the time points at which the plant material was harvested). (C) Expression of XBAT35 in 2-week old seedlings exposed to cold (4ºC, 36 h) or heat (37ºC, 3 h) stress, NaCl (250 mM, 6 h), glucose (6%, 9 h), ABA (3 µM, 48 h), IAA (10 µM, 6 h), ACC (10 µM, 6 h) or AgNO3 (100 µM, 6 h). For each condition, treatment effectiveness was confirmed using the indicated positive control gene. The cyclophilin (ROC1) and the actin2 (ACT2) genes are shown as loading controls. Results are representative of at least three independent experiments.


72

The XBAT35 transcripts were detected at considerable levels

in both whole seedlings and various organs of Arabidopsis (ecotype

Col-0) plants, with expression being highest in young (one-week old)

seedlings, flowers and senescent leaves (Fig. 2.2A). Furthermore, we

assessed steady state levels of the XBAT35 transcripts at different

time points during two distinct 24-h light/dark cycles. Both under long

(16-h photoperiod) and short (8-h photoperiod) day conditions, gene

expression was found to be lower at the end of the dark phase,

peaking 4-6 h after the onset of the light period (Fig. 2.2B). These

results indicate ubiquitous expression of the Arabidopsis XBAT35

RING gene.

As E3 ubiquitin ligases are known to play important roles in

plant developmental plasticity and stress response (Stone and Callis,

2007), we also examined the effect of several external signals, such

as different hormones or stress cues, on the expression of XBAT35

(Fig. 2.2C). The gene was clearly regulated by temperature stress,

with its expression being induced by cold and repressed by heat.

Moreover, high salinity stress imposed by 250 mM NaCl also

downregulated the levels of the XBAT35 transcripts, while high

glucose concentrations markedly induced their expression. Finally,

the exogenous application of the phytohormones abscisic acid (ABA)

and indole-3-acetic acid (IAA) had no significant effect on the levels of

XBAT35 mRNAs, which were also unaltered upon treatment with

either the ethylene precursor 1-aminocyclopropane-1-carboxylate

(ACC) or AgNO3, an inhibitor of ethylene signaling (Fig. 2.2C). Thus,

expression of the XBAT35 gene appears to be modulated by glucose

as well as by temperature and salt stress.


73

2.3.3. Exon skipping determines the subcellular localization of

two XBAT35 isoforms

Owing to the presence of an NLS in the predicted full-length

XBAT35 protein, the expectation was that the XBAT35.1 isoform

would be targeted to the nucleus. On the other hand, exclusion of the

NLS sequence due to an exon skipping event suggested distinct

compartmentalization of XBAT35.2. To examine subcellular

localization of the two XBAT35 splice forms, constructs in which the

coding sequence of each of the isoforms was individually placed

under the control of the cauliflower mosaic virus (CaMV) 35S

promoter and upstream of the yellow fluorescent protein (YFP)

sequence were first transiently transformed into a heterologous

system (Fig. 2.3A). Unlike the control YFP protein, which was

distributed throughout the cytoplasm and the nucleus of onion

epidermal cells, the XBAT35.1-YFP fusion protein was found

exclusively in nuclei, apparently accumulating in nuclear speckles. In

striking contrast, the XBAT35.2-YFP fusion lacking the NLS was

observed in the cytoplasmic compartment of onion cells. These

observations were then confirmed upon transient transfection of the

XBAT35-YFP constructs into isolated Arabidopsis protoplasts (Fig.

2.3B), where nuclear staining clearly demonstrated not only

localization of the XBAT35.1 fusion protein in the nucleus, but also

exclusion of the cytoplasmic XBAT35.2-YFP from this compartment.

These results indicate that alternative splicing of the XBAT35

pre-mRNA determines distinct subcellular localization of the two

isoforms, with XBAT35.1 being targeted to the nucleus and XBAT35.2

localizing in the cytoplasm of plant cells.


74

Figure 2.3. Subcellular localization of the two XBAT35 splice isoforms. Representative images of onion cells (A) or isolated Arabidopsis protoplasts (B) transiently transformed with YFP fusions of XBAT35.1 and XBAT35.2 under the control of the 35S promoter. YFP alone was used as a control (A and B) and DAPI as a nuclear marker (B). Scale bars = 50 µm.

2.3.4. Both the nuclear and cytoplasmic XBAT35 isoforms are

active E3 ubiquitin ligases

Proteins containing RING zinc-finger domains are known to

typically function as E3 ligases (Lorick et al., 1999), and XBAT32 has

been identified as an Arabidopsis E3 ligase (Nodzon et al., 2004;


75

Prasad et al., 2010). Moreover, a functional analysis of the

Arabidopsis RING-type E3 ligase family where 64 recombinant

proteins were tested previously reported that the At3g23280 gene

encodes a protein possessing in vitro E2-dependent ubiquitination

activity (Stone et al., 2005), but it is unclear which of the two XBAT35

isoforms was analyzed. In order to determine whether the XBAT35

splice forms function as E3 ligases, we examined their capacity to

direct ubiquitination in vitro. To this end, the two isoforms were

independently expressed as His-tagged versions in Escherichia coli

and the recombinant proteins affinity purified from the soluble fraction.

Figure 2.4 shows that, in the presence of rabbit E1 and the human

E2 UbcH5b, both the longer (XBAT35.1) and the shorter (XBAT35.2)

splice versions were able to execute ubiquitination, as evidenced by

the appearance of high molecular mass proteins visualized by

western blot analysis using anti-His antibody. As negative controls,

omission of ATP, E1 or E2 from the reaction resulted in inability of

either isoform to catalyze polyubiquitination (Fig. 2.4). Hence, both

the nuclear and the cytoplasmic XBAT35 splice forms are active E3s

capable of in vitro E1- and E2-dependent protein ubiquitination.

2.3.5. Loss of XBAT35 function causes hypersensitivity to

ethylene-mediated control of apical hook curvature

We next used a reverse genetics approach to gain insight into

the in vivo function of the XBAT35 gene and generated mutant and

transgenic plants showing reduced XBAT35 expression. After

screening various T-DNA insertion collections for XBAT35 mutants,

we identified two lines, SALK_104813 (named xbat35-1) and

FLAG_202C07 (named xbat35-2), carrying the insertions in exon 10,


76

Figure 2.4. In vitro E3 ubiquitin ligase activity of the two XBAT35 isoforms. His-tagged recombinant versions of XBAT35.1 and XBAT35.2 were incubated with His-tagged ubiquitin in the presence or absence of rabbit E1, human E2 UbcH5b or ATP for 2h at 30ºC. Samples were resolved by 10% SDS-PAGE and XBAT35 proteins detected by western-blot analysis using anti-His antibody. The blot on the left shows each isoform run alone. The molecular mass (kDa) of protein markers is shown on the left of the blots.

509 bp upstream of the stop codon, and in the 5’ untranslated region

(UTR), 215 bp upstream of the start codon, respectively (Fig. 2.5A).

PCR-based genotyping using T-DNA- and gene-specific primers (Fig.

2.5B) followed by sequencing of the obtained products allowed

verification of the insertion sites and the isolation of lines homozygous

for the T-DNA insertion. RT-PCR analysis of XBAT35 transcript levels

in the xbat35-1 homozygous line using gene-specific primers flanking

the insertion detected no bands in the mutant, while both XBAT35

splice variants (or only XBAT35.1, when using a primer annealing in

exon 8) were amplified in the corresponding Col-0 wild type (Fig.

2.5C). However, gene expression analysis using primers upstream of


77

the T-DNA insertion revealed the presence of both alternative

transcripts in xbat35-1 albeit at lower levels than the wild type (Fig.

2.5C), indicating that this mutant generates truncated versions of both

splice variants. In contrast, no expression of the XBAT35 gene was

detected in the FLAG line generated in the Wassilewskija (Ws) background (Fig. 2.5C), suggesting that xbat35-2, harboring the T-

DNA insertion in the 5’ UTR, is a true null mutant. In addition, we

generated transgenic RNA-interference (RNAi) lines to silence

XBAT35 expression. The RNAi construct was generated using a 522-

bp fragment encompassing exons 2 to 7 of the XBAT35 coding region

and expressed in the xbat35-1 mutant background under the control

of the strong constitutive CaMV 35S promoter. Ten independent

transgenic plants were obtained, two of which (lines 3 and 7)

exhibited complete depletion of the XBAT35 transcripts (Fig. 2.5D).

The insertion mutant alleles and RNAi silencing lines were

then submitted to detailed phenotypical analysis. Under greenhouse

conditions, no phenotypes were observed for any of the XBAT35

loss-of-function lines generated. Furthermore, despite significant

regulation of XBAT35 transcript levels in response to exogenous

glucose and NaCl as well as heat and cold stresses (see Fig. 2.1),

germination and early seedling development of the loss-of-function

lines were unaffected in the presence of the sugar, and no evident

phenotype was observed for the xbat35-1 mutant under temperature

or high salinity stress (data not shown).

XBAT32, the only of the five XBAT genes with known in vivo

function, has been shown to regulate lateral root formation (Nodzon

et al., 2004) by affecting the ethylene pathway (Prasad et al., 2010).

In the latter report, Stone and co-workers analyzed the same SALK


78

Figure 2.5. Isolation of XBAT35 loss-of-function lines. (A) Schematic representation of the XBAT35 gene showing the site of insertion and orientation of the T-DNA in xbat35-1 and xbat35-2 insertion lines (boxes indicate exons with UTRs in grey and the alternatively-spliced exon in black, lines between boxes represent introns, and arrows indicate the location of XBAT35- and T-DNA-specific primers used in genotyping and mRNA analysis). (B) PCR-based genotyping of homozygous xbat35-1 and xbat35-2 lines. (C) RT-PCR analysis of XBAT35 transcript levels in xbat35-1 and xbat35-2 seedlings. The ubiquitin10 (UBQ10) and cyclophilin (ROC1) genes were used as loading controls. (D) RT-PCR analysis of XBAT35 transcript levels in two independent XBAT35 RNAi silencing lines and the xbat35-1 insertion mutant. Cyclophilin (ROC1) is shown as a loading control.

insertion line isolated in the present study and found no changes in

lateral root production. In agreement, our analysis of the xbat35-1

mutant revealed no lateral root phenotype or defects in primary root

elongation. We therefore decided to investigate a possible role for the

XBAT35 E3 ligase in ethylene signaling by focusing on the two other

components of the ethylene-mediated triple response observed in

dark-grown Arabidopsis seedlings — shortening of the hypocotyl and

exaggeration of the apical hook (Fig. 2.6).


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Figure 2.6. Ethylene phenotype of XBAT35 loss-of-function lines. (A) Hypocotyl length of Col-0, xbat35-1, two independent XBAT35-RNAi lines, Ws and xbat35-2 seedlings grown 3 days in the dark under control conditions (white bars) or in the presence of 10 µM ACC (gray bars) or 25 µM ACC (black bars) supplemented or not with 100 µM AgNO3 (means ± SE, n = 40-60). (B) Apical hook curvature of Col-0, xbat35-1, two independent XBAT35-RNAi lines, Ws and xbat35-2 seedlings grown 3 days in the dark under control conditions (white bars) or in the presence of 10 µM ACC (gray bars) or 10 µM ACC supplemented with 100 µM AgNO3 (black bars). The ctr1-1, ein3-1 and hls1-1 hook mutants were included as controls. The inset illustrates the determination of the hook angle (means ± SE, n = 40-60). Asterisks indicate significantly different values (* P < 0.05; ** P < 0.01; *** P < 0.001) from the corresponding wild type according to Student’s t test.

When etiolated seedlings were grown in control media,

hypocotyl length appeared unaffected in XBAT35 loss-of-function

lines except for the xbat35-2 knockout, which displayed slightly longer


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hypocotyls than wild-type Ws seedlings (Fig. 2.6A). However, no

differences in hypocotyl length were observed between any of the

genotypes in the presence of 10 or 25 µM of ACC. Hence, the

ethylene precursor repressed hypocotyl elongation similarly in all

lines except for xbat35-2, which showed a slightly higher ACC-

mediated inhibition than the corresponding wild type, suggesting

subtle ethylene hypersensitivity of this mutant allele during hypocotyl

elongation. Blocking ethylene perception by supplementing the media

with the ethylene receptor antagonist silver nitrate (AgNO3) rescued

hypocotyl growth inhibition in all mutant and silencing lines (Fig.

2.6A), confirming that the observed effect on hypocotyl elongation is

ethylene-specific. Also under these conditions, the xbat35-2 mutant

displayed significantly longer hypocotyls than the Ws wild type,

indicating that this phenotype is independent of the ethylene

phytohormone. This particular defect could be ecotype-related, but

most likely results from another mutation in the xbat35-2 allele that is

unrelated to the insertion in the XBAT35 gene.

We next analyzed ethylene-controlled apical hook curvature of

dark-grown seedlings. In the absence of the ethylene precursor ACC,

no significant differences in hook curvature were observed for any of

the mutant or silencing lines, except for a slight reduction observed in

xbat35-2 (Fig. 2.6B). By contrast, in the presence of 10 µM ACC,

xbat35-1, xbat35-2 and the two RNAi lines all showed significantly

enhanced curvature of the apical hook in comparison with the

corresponding wild types (Fig. 2.6B). Addition of AgNO3 suppressed

bending of the apical region of the hypocotyls in all genotypes —

despite the observation of residual degrees of hook curvature,

probably as a result of incomplete blocking of ethylene signaling —

with no differences being observed between genotypes under these

conditions. We also included in this assay three known hook mutants


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derived from the Col-0 ecotype. As expected, the constitutive

ethylene response mutant, ctr1, displayed an exaggerated hook in the

absence of ACC, which was unaffected in the other two conditions

tested, while the hookless mutant hls1-1 never showed an apical

hook and the ethylene-insensitive ein3-1 displayed substantially

reduced hook curvature in both the absence and presence of the

ethylene precursor (Fig. 2.6B). Taken together, these results show

that loss of XBAT35 function results in ethylene hypersensitivity

during apical hook formation in etiolated seedlings, and thus that the

XBAT35 ubiquitin ligase is involved in negatively regulating ethylene-

mediated control of apical hook exaggeration in Arabidopsis.

In addition to ethylene, both auxin and brassinosteroids have

been implicated in the regulation of apical hook development

(Schwark and Schierle, 1992; De Grauwe et al., 2005;

Vandenbussche et al., 2010), Gibberellins (GAs) are also required for

apical hook formation, as a block in GA synthesis or signaling leads

to a hookless phenotype (Achard et al., 2003; Alabadi et al., 2004;

Vriezen et al., 2004). Moreover, jasmonates have been reported to

suppress the ethylene-induced apical hook (Ellis and Turner, 2001).

Therefore, we also assessed the effects of IAA, the predominant

naturally occurring auxin, the brassinosteroid epibrassinolide (EBR),

methyl jasmonate (MeJA) and the GA biosynthesis inhibitor

paclobutrazol (PAC) on apical hook curvature of the XBAT35 loss-of-

function alleles. However, apart from one of the RNAi lines, which

showed considerable hook tightening under EBR, no significant

differences were observed among the six genotypes (Fig. 2.7),

suggesting that the XBAT35 protein does not target signaling of these

other phytohormones during early development of dark-grown

seedlings.


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Figure 2.7. Effect of auxin, brassinosteroids, jasmonate and paclobutrazol on apical hook curvature of XBAT35 loss-of-function lines. Apical hook curvature of Col-0, xbat35-1, two independent XBAT35-RNAi lines, Ws and xbat35-2 seedlings grown 3 days in the dark under control conditions (white bars) or in the presence of 1 µM IAA, 100 nM EBR, 20 µM MeJA or 0.01 µM PAC (light gray to black bars, respectively). The inset illustrates the determination of the hook angle (means ± SE, n = 40-60). Asterisks indicate significantly different values (* P < 0.05; *** P < 0.001) from the corresponding wild type under each condition according to Student’s t test. DNG, did not germinate.

We also sought to determine whether XBAT35 gene

expression would be induced in mutants displaying impaired apical

hook tightening in response to ethylene, but RT-PCR analysis of etr1-

1, hls1-1 and ein3-1 seedlings revealed no appreciable changes in

the expression of XBAT35 (Fig. 2.8).

Figure 2.8. XBAT35 expression levels in ethylene-related mutants. RT-PCR analysis of XBAT35 transcript levels in Col-0, xbat35-1 and the ethylene-signaling mutants etr1-1, hls1-1 and ein3-1. The ubiquitin10 (UBQ10) gene was used as a loading control.


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Consistent with the XBAT35 expression pattern observed in

the ethylene-related mutants, publicly available microarray data

(www.genevestigator.com) show no significant misregulation of

XBAT35 expression in the etr1-1 nor in the ein2-1 ethylene-

insensitive mutants.

2.3.6. Both XBAT35 isoforms function in ethylene control of

apical hook curvature

To investigate the biological significance of the nuclear and

cytoplasmic XBAT35 isoforms and gain insight into the physiological

relevance of alternative splicing of this RING E3 ligase gene, we next

expressed each of the splice variants in the XBAT35 mutant

background. To this end, the coding sequences of both XBAT35.1

and XBAT35.2 were independently cloned under the control of the

35S promoter and infiltrated into xbat35-1 plants. Six independent

transgenic lines expressing either XBAT35.1 or XBAT35.2 were

isolated and characterized with regard to ethylene control of apical

hook curvature in etiolated seedlings. The results obtained with three

representative lines expressing the transgenes at different levels (Fig.

2.9A) are presented in Figure 2.9.

As shown in Figures 2.9B and 2.9C, both XBAT35.1 and

XBAT35.2 complementation lines exhibited complete restoration of

wild type apical hook curvature in the presence of 10 µM ACC.

Interestingly, despite overall similar expression levels in the two

genotypes, only one of the XBAT35.1 transgenic lines showed the

opposite phenotype of XBAT35 loss of function lines, i.e., reduced

hook tightening upon ACC treatment when compared with the wild

type, while all XBAT35.2 transformants exhibited this overexpression


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Figure 2.9. Complementation of the xbat35-1 ethylene-mediated apical hook curvature phenotype. (A) RT-PCR analysis of XBAT35 expression in light-grown seedlings of the wild-type (Col-0), mutant (xbat35-1) and six independent complementation lines expressing either the XBAT35.1 (cXBAT35.1a-c) or XBAT35.2 (cXBAT35.2a-c) splice variants. Ubiquitin10 (UBQ10) is shown as a loading control. (B) Apical hook curvature of Col-0, xbat35-1 and the six complementation lines grown 3 days in the dark under control conditions (white bars) or in the presence of 10 µM (black bars) ACC (means ± SE, n = 40-60). Asterisks indicate significantly different values (* P < 0.05; ** P < 0.01; *** P < 0.001) from the wild type according to Student’s t test. (C) Representative images of Col-0, xbat35-1, cXBAT35.1a and cXBAT35.2a seedlings grown in the dark under control conditions or in the presence of 10 µM ACC. Scale bar = 1 mm.


85

phenotype (Figs. 2.9B and 2.9C). Taken together, these results

confirm that disruption of the XBAT35 gene is responsible for the

identified xbat35 apical hook mutant phenotype and demonstrate that

both splice isoforms are biologically active in this specific ethylene-

mediated response. Furthermore, XBAT35.2 was clearly more

effective in conferring an overexpression phenotype, suggesting a

differential contribution of the two isoforms to XBAT35 RING ligase

control of apical hook exaggeration.

2.3.7. Four photosystem proteins and an unknown protein are

putative XBAT35 interacting partners

In order to identify putative XBAT35 ubiquitination targets, we

performed a yeast two-hybrid assay based on the GAL4 system. Four

different XBAT35 constructs (Fig. 2.10A), encoding either the full-

length isoforms or N-terminal and C-terminal fragments of the protein,

were cloned into pGBKT7, the vector carrying the GAL4-binding

domain, and transformed into the Y187 yeast strain. Absence of

reporter gene autoactivation by the bait constructs was confirmed by

co-transformation with the pGADT7-T control construct (Fig. 2.10B).

A cDNA library from Arabidopsis seedlings was cloned into the

pGADT7 vector harboring the GAL4 activation domain and

transformed into Saccharomyces cerevisiae AH109 strain. Yeast

mating was performed using the cDNA library and the XBAT35.1

construct-containing strains. Plasmid DNA was extracted from the

isolated colonies (around 100), different clones were selected based

on band size differences and retransformed for further protein

interaction testing.


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Figure 2.10. XBAT35 constructs used in the yeast two-hybrid assay. (A) Schematic representation of the four XBAT35 fragments used as bait proteins: the full-length of each of the two XBAT35 isoforms and two truncated versions of XBAT35.1, excluding either the RING domain or the ankyrin repeats and the nuclear localization signal. (B) Analysis of the effective stringency of the selective media on inhibiting yeast growth in the absence of positive protein interactions. All constructs listed on the left (pGBKT7 vector) were tested against the pGADT7-T vector. According to the Matchmaker system, 53 and Lam were used as positive and negative controls, respectively, as the first interacts with the T fragment whereas the second does not. OD; optical density (600 nm).

After four assays of protein interaction analyses with

XBAT35.1 and two cycles with the three remaining XBAT35

constructs, ten clones were selected based on their capacity to allow

yeast growth on the selective media in all the assays performed.


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Plasmid DNA sequencing of these colonies allowed the identification

of five putative targets, listed in Table 2.1. These five clones positively

interacted with the four XBAT35 fragments. From the list depicted in

Table 2.1, four proteins are active during photosynthesis, whereas the

fifth has no protein function annotated yet, although according to the

TAIR website (www.arabidopsis.org) it is predicted to play roles in the

response to karrikin, a signaling molecule present in burned plant

tissues that triggers seed germination in many angiosperms.

Table 2.1. Putative XBAT35 targets isolated from the yeast two-hybrid assay.

Locus ID Gene Description

At1g15820 LHCB6 Light harvesting complex of photosystem II

At3g54890 LHCA1 Light harvesting complex of photosystem I

At5g64040 PSAN Photosystem I subunit located in the thylakoid lumen

AtCg00340 PSAB D1 subunit of photosystem I and II reaction centers

At1g25275 Unknown protein, component of the endomembrane system, involved in responses to karrikin

2.3.8. The XBAT35 and XBAT34 duplicated genes display

overlapping expression patterns

XBAT34 (At4g14365) is another member of the XBAT family

of E3 ligases arising from a genome duplication event together with

XBAT35 (Nodzon et al., 2004; Stone et al., 2005). This raised the

question of possible functional redundancy between the two XBAT

members. We therefore assessed the tissue- and developmental-

specific pattern of XBAT34 expression in wild-type Col-0 plants (Fig.

2.11A).


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Compared to XBAT35 (see Figure 2.2), the expression of

XBAT34 is restricted to fewer tissues (leaves, flowers and roots),

albeit at constant levels, except in cauline leaves where higher

transcript levels were observed, and in roots where the expression

level was weak. XBAT34 gene expression was undetectable in young

seedlings and, later during development, in siliques. The overlap of

XBAT34 and XBAT35 expression in some of the Arabidopsis tissues

analyzed may explain the absence of broader phenotypes in the

XBAT35 loss-of-function lines described above (see Figures 2.5 and

2.6). Hence, we attempted to generate XBAT34 mutant lines for

subsequent generation of a XBAT34/XBAT35 double mutant.

Screening of different T-DNA insertion collections led to the

identification of three insertion lines in XBAT34: SALK_056294

(named xbat34-1), GABI_Kat 708C04 (named xbat34-2) and

FLAG_357A03 (named xbat34-3) – Fig. 2.11B. PCR-based

genotyping allowed the isolation of a homozygous line for xbat34-1

(Fig. 2.11C), but comparison of XBAT34 expression levels by RT-

PCR in the wild type and the insertion line revealed that we were in

the presence of an overexpression line (Fig. 2.11D). This probably

occurred as a result of the insertion of the T-DNA segment in the

XBAT34 gene 5’ UTR region, 450bp upstream of the start codon (Fig.

2.11B). In xbat34-2, the T-DNA insertion site is predicted to be

located at the end of exon 10, 30 bp upstream of the stop codon, but

we were unable to isolate a homozygous line possibly due

phenomenon of a T-DNA insertion in tandem. Also for xbat34-3 we

failed to isolate a homozygous insertion line. Although the insertion

site is predicted to be in exon 8, 640 bp upstream of the stop codon,

no T-DNA fragment was found at this position nor in any other gene

location.


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Figure 2.11. Expression pattern of the XBAT34 gene and isolation of a homozygous insertion line. (A) RT-PCR analysis of XBAT34 transcript levels in whole seedlings and different tissues from Col-0 plants. (B) Schematic representation of the XBAT34 gene showing the predicted insertion sites and orientation of the T-DNA in xbat34-1, xbat34-2 and xbat34-3 insertion lines (boxes indicate exons with UTRs in grey, lines between boxes represent introns, and arrows indicate the location of XBAT34- and T-DNA-specific primers used in genotyping and mRNA analysis). (C) PCR-based genotyping of a homozygous xbat34-1 line. (D) RT-PCR analysis of XBAT34 transcript levels in xbat34-1 seedlings and in two independent XBAT35 RNAi silencing lines. Ubiquitin10 (UBQ10) and cyclophilin (ROC1) were used as loading controls.

Finally, we asked whether the lines generated by RNAi in

order to repress XBAT35 expression (see Figure 2.5) would also

affect expression of the XBAT34 gene. Although we observed

downregulation of XBAT34 expression levels in these lines (Fig.

2.11C), we considered it insufficient to justify extensive phenotypical

characterization. As we were unable to isolate any bona fide loss-of-


90

function mutant for XBAT34, it was not possible to generate a double

mutant for the two XBAT genes and consequently assess their

functional redundancy.

2.4. Discussion

XBAT35 belongs to a subgroup of five structurally-related

Arabidopsis RING ankyrin repeat-containing E3 ligases that, with the

exception of XBAT32 (Nodzon et al., 2004; Prasad et al., 2010;

Prasad and Stone, 2010), has remained uncharacterized. Here we

show that the XBAT35 gene negatively regulates specific ethylene-

mediated responses and that alternative splicing of its pre-mRNA

generates two transcripts, both of which appear to be constitutively

and ubiquitously expressed in Arabidopsis and to produce functional

proteins with different subcellular localization.

In agreement with in silico expression analyses from the

Genevestigator and BAR databases, and unlike XBAT32, which

displays a tissue-specific expression pattern (Nodzon et al., 2004),

our results indicate that XBAT35 is expressed in all Arabidopsis

vegetative tissues as well as during development. Expression levels

appear to be higher at early developmental stages, as well as in

flowers and old leaves, two tissues in which the phytohormone

ethylene is well known to stimulate senescence. Furthermore,

XBAT35 is expressed in plants grown both under long- and short-day

photoperiod, with transcript levels showing a peak around 5 h upon

the onset of the light cycle. Interestingly, a global transcriptome

analysis listed XBAT35 as a circadian regulated gene, finding clock-

regulated genes to be overrepresented among hormone signaling

components (Covington et al., 2008) amidst mounting evidence for


91

crosstalk between circadian clock and classical hormone pathways,

including ethylene signaling, in the regulation of plant physiology and

growth (Robertson et al., 2009). However, transcription of the

XBAT35 gene is unaffected by the ethylene precursor ACC and two

other major phytohormones, ABA and IAA. By contrast, XBAT35

transcript levels appear to be modulated by temperature and high

salinity stress, as well as by high levels of exogenously applied

glucose. Despite the gene’s ubiquitous expression in Arabidopsis,

XBAT35 loss-of-function lines do not display developmental or

morphological defects when grown under standard growth conditions

nor altered response to glucose, high salt or temperature stress

during early seedling development. The absence of evident

phenotypes under these conditions may be a consequence of the

overlapping activity of other redundant XBAT proteins in various plant

tissues. In particular, XBAT34 shows the strongest homology to

XBAT35, with 68% identity and 73% similarity (Nodzon et al., 2004),

and a similar expression pattern in most of the Arabidopsis tissues.

Only during the earlier stages of development and later in

senescence do the genes show a different pattern of expression. The

generation of double mutant lines in the two genes was hindered by

the inability to isolate a loss-of-function mutant for XBAT34, thus

preventing conclusive assessment of functional redundancy between

these two genes.

Nevertheless, insertion mutants and RNAi silencing lines

displaying either high reduction or complete depletion of XBAT35

transcript levels exhibit a very specific phenotype — hypersensitivity

to ethylene control of apical hook curvature. When dicotyledonous

seeds germinate under darkness, a hook structure forms at the top of

the hypocotyl, protecting the apical meristem from injury during

seedling emergence from the soil (Darwin and Darwin, 1881).


92

Exaggeration of apical hook curvature in etiolated seedlings results

from differential cell division and elongation (Raz and Koornneef,

2001) and is part of the classical ethylene triple response, along with

inhibition of hypocotyl and root elongation (Kieber et al., 1993).

Arabidopsis mutants impaired in ethylene signaling, such as etr1,

ein2, or ein3, do not show enhanced hook curvature upon ethylene

treatment (Roman et al., 1995), whereas the ethylene overproducing

mutant, eto1, and the constitutive ethylene response mutant, ctr1,

develop an exaggerated hook in the absence of ethylene (Guzman

and Ecker, 1990; Kieber et al., 1993). Strikingly, all four XBAT35 loss-

of-function lines analyzed in this study display exaggerated tightening

of the apical hook in response to the ethylene precursor ACC. Since

no ethylene-related phenotypes were observed in the absence of

ACC, it is unlikely that, as its homolog XBAT32, XBAT35 is involved

in the inhibition of ethylene biosynthesis. Nevertheless, the

exaggerated hook phenotype is dependent on active ethylene

signaling, as blocking ethylene perception with AgNO3 prevents hook

formation in all XBAT35 mutant and silencing lines. Apical hook

development is known to involve close crosstalk between auxins and

ethylene, which influence hook formation and exaggeration,

respectively (Vandenbussche et al., 2010). Brassinosteroids (De

Grauwe et al., 2005), gibberellins (Vriezen et al., 2004) and

jasmonate (Ellis and Turner, 2001) have also been implicated in

apical hook development. However, mutations in XBAT35 did not

alter the response to any of these plant hormones, suggesting that

XBAT35’s control of hook curvature is ethylene-specific. Moreover,

similarly to previously described hookless ethylene mutant

phenotypes uncoupling apical hook from root and hypocotyl

responses (Guzman and Ecker, 1990), the two latter triple response

components are unaffected by loss of XBAT35 function indicating that


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this ubiquitin ligase is specifically implicated in ethylene-mediated

differential growth responses in etiolated seedlings. Taken together,

our results provide evidence that the XBAT35 RING E3 ligase is a

negative regulator of ethylene-mediated control of apical hook

exaggeration, whose mode of action presumably involves the

ubiquitin-dependent degradation of positive ethylene signaling

components.

Importantly, the present study has revealed that the two

XBAT35 transcripts encode isoforms that are deployed to different

subcellular compartments. Owing to skipping of the 72-bp exon 8 in

the XBAT35 pre-mRNA, an NLS is excluded from the shorter splice

form. Accordingly, transient expression of YFP fusion proteins in a

heterologous system and Arabidopsis protoplasts indicates that the

longer isoform, XBAT35.1, is targeted to the nucleus, while the

shorter, XBAT35.2, is localized in the cytoplasm. Furthermore,

XBAT35.1-YFP appears to accumulate in the nucleoplasm in nuclear

speckles. The exact nature of these bodies remains unclear, but it is

interesting to note that both another extensively studied RING E3

ligase, COP1 (Stacey and von Arnim, 1999), and splicing factors such

as arginine/serine-rich (SR) proteins (Lorkovic et al., 2008), which are

established key players in the regulation of alternative splicing, have

been found to localize to nuclear speckles in Arabidopsis.

In plants, a few examples of alternative splicing resulting in

altered compartmentalization of isoforms have been previously

described. In pumpkin cells, alternative 5’ splice site selection

controls the inclusion of a peroxisome targeting signal in the

hydroxypyruvate reductase (HPR) protein resulting in two isoforms,

one localizing in leaf peroxisomes and the other in the cytosol (Mano

et al., 1999). Exon inclusion introduces two putative transmembrane

domains in the tomato protein phosphatase 5 (PP5), targeting the


94

longer isoform to the endoplasmic reticulum and the shorter to the

nucleus and cytoplasm (de la Fuente van Bentem et al., 2003).

Moreover, during expression of the OsBWMK1 gene, a member of

the rice mitogen-activated protein kinase (MAPK) family, alternative

transcription start sites combined with a splicing event in the first

intron generate two predominantly cytoplasmic isoforms and a third

localized in the nucleus (Koo et al., 2007). Finally, the Arabidopsis

gene encoding protein isoaspartyl methyltransferase 2 (PIMT2) has

been shown to undergo a complex transcriptional and splicing

regime, generating at least eight transcripts, which produce

catalytically competent protein isoforms that are deployed to various

subcellular compartments (Dinkins et al., 2008).

The ubiquitous and constitutive co-expression of the two

XBAT35 splice variants in Arabidopsis, together with the fact that they

are both translated upon transient expression in plant cells,

suggested that the two transcripts produce functional proteins in vivo.

Furthermore, recombinant His-tagged versions of both the nuclear

and the cytoplasmic XBAT35 proteins display E3 ubiquitin ligase

activity, as assessed by in vitro ubiquitination assays. Although

different subcellular compartments typically fulfill distinct biochemical

roles, many enzyme activities are required in more than one

compartment (Small et al., 1998), and ubiquitin-mediated proteolysis

in plants has been well established to occur in the cytoplasm and the

nucleus, with all but two (Lin et al., 2008; Gu and Innes, 2011) of the

RING E3 ligases reported to date being shown to be either cytosolic

or nuclear proteins. The high structural similarity between XBAT35.1

and XBAT35.2, which differ solely in 24 amino acids that include the

entire NLS, indicates that the two E3 ligase isoforms may be

performing similar functions in the nucleus and the cytoplasm.

Indeed, ectopic expression of either isoform in the XBAT35 mutant


95

background rescues the hook exaggeration phenotype, clearly

showing that both proteins are functional in ACC-dependent apical

hook formation. Furthermore, overexpression of each isoform

appears to have different phenotypical impact in transgenic plants,

suggesting a differential contribution of the nuclear and cytoplasmic

XBAT35 splice forms to ethylene signaling. This could stem from

differences in the efficiency of ubiquitin-dependent protein

degradation or the targeting of distinct sets of proteins in both

compartments. A first step towards the identification of XBAT35

targets was performed based on the yeast two-hybrid system.

However, the putative proteins isolated may arise from unspecific

interactions and conclusions on the degradation substrates of the

XBAT35 E3 ligase will require further biochemical tests, such as in

vitro ubiquitination and co-immunoprecipitations assays.

2.5. Materials and Methods

2.5.1. Plant materials and growth conditions

Seeds from wild type Arabidopsis thaliana (L.) Heyhn,

ecotypes Columbia-0 (Col-0) or Wassilewskija (Ws), or from the ctr1-

1 (Kieber et al., 1993), etr1-1 (Bleecker et al., 1988), hls1-1 (Guzman

and Ecker, 1990) and ein3-1 (Roman et al., 1995) mutant lines were

surface-sterilized for 10 min in 50% (v/v) bleach and 0.07% (v/v)

Triton X-100 under continuous shaking and then washed 3 times in

sterile water. Following stratification for 3 days in the dark at 4ºC (to

break dormancy), the seeds were sown in petri dishes containing 1X

Murashige and Skoog (MS) salts (Duchefa Biochemie), 2.5 mM MES

(pH 5.7), 0.5 mM myo-inositol and 0.8% (w/v) agar, and placed in a


96

growth chamber under 16-h (long-day) or 8-h (short-day) photoperiod

(90 µmol.m-2.s-1 white light) at 22ºC (light period)/18ºC (dark period)

and 60% RH. After 3 weeks, plants were transferred to soil in

individual pots.

Seeds containing T-DNA insertions in the XBAT35

(At3g23280) gene, Salk_004132 (Col-0 ecotype) and FLAG_004132

(Ws ecotype), or in XBAT34 (At4g14365), Salk_056294, GABI_Kat

708C04 (Col-0 ecotype) and FLAG_357A03, were obtained from the

Nottingham Arabidopsis Stock Centre (NASC), the Genomanalyse im

Biologischen System Pflanze (GABI) or the Institut National de la

Recherche Agronomique (INRA) and grown as described above. The

exact location of the insertions was verified by PCR using XBAT35-

and XBAT34- specific primers and primers annealing at the left

border of the T-DNA (see Table 2.2), which also allowed genotyping

to identify homozygous lines for the insertions.

2.5.2. Generation of XBAT35-RNAi lines and complementation of

the xbat35-1 mutant

To silence expression of the XBAT35 gene by RNAi, first-

strand XBAT35 cDNA was obtained from total RNA extracted from 2-

week old wild-type Col-0 seedlings using M-MLV Reverse

Transcriptase (Promega) and a poly-T primer. A 522-bp fragment of

the coding region of the XBAT35 cDNA was amplified by PCR using

primers 5’-TGTGTCTCGAGGTATGAACTCCGAGCTATTTGAT-3’

and 5’-TAGGAGAATTCAAGCTTTTGTGGAGTTATCAACGATCATC-

3’ — which added an XhoI and both an EcoRI and a HindIII restriction

sites, respectively — and subcloned into the pSK-int vector in the

sense and antisense orientations separated by the actin11 intron 3


97

spacer region. This segment was then transferred into the binary

pBA002 vector under the control of the 35S promoter before being

introduced into Agrobacterium tumefaciens strain EHA105 for floral

dip transformation (Clough and Bent, 1998) of homozygous xbat35-1

plants. Ten T1 transformants were selected on BASTA-containing

medium, grown to maturity, and selfed. Gene expression was

assessed at the T2 generation, and phenotypic analyses were carried

out in T3 plants.

For genetic complementation of the xbat35-1 mutant, the

coding sequences of XBAT35.1 and XBAT35.2 were amplified by

PCR using primers 5’-

GGCGCGCCGTCTTCTTTCTGATCTGTGAGC-3’ and 5’-

TTCGATTAATTAATCAGACACGGTACAGCTTAATAAC-3’ — which

introduced an AscI and a PacI restriction site, respectively — and

independently cloned into pBA002 under the control of the 35S

promoter. Each of the constructs was then introduced into xbat35-1

plants and transformants selected as described above. Phenotypical

complementation was assessed in the six lines obtained for each

transformation.

2.5.3. Phenotypical analyses

Plants of different genotypes were sown and grown

simultaneously under identical conditions. Seeds from fully matured

siliques of dehydrated plants of the same age were collected and

stored in the dark at room temperature. All assays were performed

using seeds from comparable lots stored for 1-24 months.

For assessment of ethylene phenotypes, seeds were surface-

sterilized and stratified as described above, sown in petri dishes


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containing MS medium (1X MS salts, 2.5 mM MES pH 5.7, 0.5 mM

myo-inositol and 0.8% agar) supplemented or not with the appropriate

concentrations of ACC (A3903; Sigma-Aldrich) or AgNO3, and placed

in a growth chamber (16-h photoperiod; 22ºC/18ºC) for 6 h before

transfer to complete darkness at room temperature for 3 days.

Hypocotyl length and apical hook angle of etiolated seedlings were

then scored using the ImageJ software (http://rsbweb.nih.gov/ij). The

degree of hook curvature was measured as 180º minus the angle

between the apical bended part of the hypocotyl and its basal axis, or

180º plus this angle when the apical part crosses the basal axis (see

inset in Fig. 2.6B). In each condition, 40-60 seedlings of each

genotype were analyzed. All assays were repeated at least three

times with similar results. The same procedure was followed for hook

curvature assessment in the presence of IAA (I2886; Sigma-Aldrich),

EBR (E1641; Sigma-Aldrich), MeJA (392707; Sigma-Aldrich) or PAC

(P0922; Duchefa).

2.5.4. RNA extraction and RT-PCR analysis

The expression patterns of the XBAT35 and XBAT34 genes

were determined in the Col-0 ecotype using whole seedlings and

different tissues of adult plants. Rosette leaves of 5-week old plants

grown under different photoperiod regimes were used to assess

XBAT35 expression under these conditions. For XBAT35 expression

analysis under the presence of exogenous compounds, 2-week old

Col-0 seedlings grown in control MS medium were transferred for

different time periods to plates supplemented with 6% (w/v) glucose

(9 h), 3 µM ABA (mixed isomers, A1049; Sigma-Aldrich) (48 h) or 250


99

mM NaCl (6 h), or placed for 6 h under continuous shaking in MS

liquid medium containing 10 µM ACC (A3903; Sigma-Aldrich), 10 µM

IAA (I2886; Sigma-Aldrich) or 100 µM AgNO3 before harvesting of the

plant material. For temperature stress, 2-week old seedlings were

placed at 4ºC for 36 h or 37ºC for 3 h. Finally, for analysis of XBAT35

transcript levels in xbat35 or ethylene-signaling mutants and

transgenic RNAi or complementation lines, or of XBAT34 expression

in the xbat34-1 mutant and transgenic RNAi lines, 10-day old

seedlings grown in MS medium were used.

Total RNA was extracted from all plant tissues with Tri

Reagent (T924; Sigma-Aldrich) according to the manufacturer's

protocol. All RNA samples were digested with DNAse I (Promega)

and phenol-chloroform purified before reverse transcription with M-

MLV Reverse Transcriptase (Promega) and a poly-T primer. XBAT35,

XBAT34, the marker genes RD29A, HSP18, RBCS, IAA5, ERS2 and

TAT3, as well as the housekeeping ROC1, ACT2 and UBQ10 genes,

were amplified by PCR using Paq5000 DNA polymerase (Stratagene)

with the gene-specific primers shown in Table 2.2. The RT-PCR

analyses shown in Figures 2.2, 2.6 and 2.9 were performed with

XBAT35 primers F1 and R1. The RT-PCR shown in Figure 2.11A was

carried out with XBAT34 primers F2 and R. Preliminary PCRs were

carried out with different number of cycles to determine the linear

range of amplification. Based on these analyses, 20-30 cycles were

chosen for cDNA detection, depending on the gene (see Table 2.2).

Amplified DNA fragments were separated and visualized in 1.5%

agarose gels.


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2.5.5. Subcellular localization of XBAT35-YFP fusion proteins

The coding sequences of XBAT35.1 and XBAT35.2 were

amplified by PCR using primers 5’-

GGCGCGCCGTCTTCTTTCTGATCTGTGAGC-3’ and 5’-

CCTTAATTAAGACACGGTACAGCTTAATAACC-3’ — which

introduced an AscI restriction site and a PacI site while deleting the

stop codon, respectively — and independently introduced upstream

of the YFP sequence into pBA-YFP, a pBA002 derivative equipped

with the YFP coding sequence via the MluI and SpeI sites.

The constructs harboring the coding sequences of the two

XBAT35 isoforms were then independently used for direct

bombardment of onion cells with 1.6-mm diameter gold particles

coated with the constructs using a helium-driven biolistic particle

delivery system (PDS-1000/He, BioRad, Hercules, CA, USA). After

incubation at room temperature in darkness for 16-20 h, YFP

fluorescence was visualized in onion tissues using a Zeiss

AxioImager microscope (Carl Zeiss AG, Germany).

Arabidopsis protoplasts were isolated as described by Yoo et

al. (2007), independently transformed with the two XBAT35-YFP

constructs by the polyethylene glycol method (Abel and Theologis,

1994), and kept overnight at room temperature. Imaging was then

performed in an Olympus IX71 inverted microscope, using a 40x, 1.3

numerical aperture oil immersion objective. DAPI and YFP were

excited by a xenon arc lamp (Applied Precision) with 360/40 and

500/20 band pass filters. Emission was acquired with 457/50 and

535/30 band pass filters for DAPI and YFP respectively, using a 16-

bit cooled EMCCD Photometrics Cascade 1K camera.


101

2.5.6. Expression of recombinant XBAT35.1 and XBAT35.2

proteins and in vitro ubiquitination assays

The XBAT35.1 and XBAT35.2 coding sequences were

amplified with primers 5’-

GAGTAACATATGATGGGACAACAGCAATCAAAAGG-3’ and 5’-

TAATCCTCGAGTCAGACACGGTACAGCTTAATAAC-3’ and cloned

into the NdeI and XhoI sites of pET28a (Novagen) to produce in-

frame N-terminal fusions with the His tag. The two recombinant

protein isoforms were then expressed in Escherichia coli strain

BL21(DE3) and purified using a single affinity purification step in a

HisTrap HP column (GE). After purification, the protein buffer was

exchanged for 20 mM phosphate buffer (pH 7.5), 200 mM NaCl, 5%

(v/v) glycerol, 1 mM DTT and 1 mM EDTA.

For the ubiquitination assay, each reaction (15-µL final

volume) contained 5 µg of His-tagged recombinant ubiquitin (U5507,

Sigma-Aldrich), 0.05 µg rabbit E1 (E-302, BostonBiochem), 0.11 µg

E2 UbcH5b (E2-622, BostonBiochem), 300 ng purified His-XBAT35.1

or His-XBAT35.2, 2 mM ATP, 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2,

50 mM KCl and 1 mM DTT. After incubation at 30ºC for 2 h, the

reaction was stopped by adding 5x SDS-PAGE loading buffer and 10

µL were analyzed by electrophoresis on 10% SDS-PAGE gels.

Ubiquitinated proteins were detected by western blotting using anti-

His antibody.

2.5.7. Yeast manipulations and two-hybrid assay

RNA was extracted from wild-type Col-0 7-day old seedlings

and first strand cDNA synthesized. Following the MATCHMAKER


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GAL4 Two Hybrid System protocol (CLONTECH Laboratories), a

cDNA library was cloned into the pGADT7 vector and the XBAT35

constructs into pGBKT7. Primers 5’-

AAGGAGGATCCATGGGACAACAGCAATCAAAAG-3’ and 5’-

TTGGTGGATCCGACACGGTACAGCTTAATAACC-3’ (BamHI

restriction sites) were used to amplify the full-length XBAT35.1 and

XBAT35.2. Similarly, primers 5’-

AAGGAGGATCCATGGGACAACAGCAATCAAAAG-3’ and 5’-

TTGGTGGATCCCGAAGGTGCGAATTTAAGACG-3’ (BamHI

restriction sites) were used to amplify the N-terminal XBAT35

fragment and primers 5’-

AAGGAGGATCCACTGAAGGTGACAGCCAACAG-3’ and

TTGGTGGATCCGACACGGTACAGCTTAATAACC-3’ (BamHI

restriction sites) for the C-terminal fragment.

Saccharomyces cerevisiae strains (AH109 and Y187) were

grown at 30ºC in YPD rich medium – 1% (w/v) yeast extract (Merck),

2% (w/v) bacto-peptone, 2% (w/v) D-glucose, 1.5% (w/v)

bacteriological agar, pH adjusted to 6.3 with 4 M NaOH. When in

minimal medium, yeast strains were grown in synthetic defined (SD)

medium containing 6.7 g/L yeast nitrogen base, and the amino acid

supplements 0.5 g/L arginine, 0.5 g/L methionine, 0.75 g/L tyrosine,

0.75 g/L isoleucine, 0.75 g/L lysine, 1.25 g/L phenylalanine, 2.5 g/L

glutamic acid, 3.75 g/L valine, 2.5 g/L aspartic acid, 2 mg/L threonine,

4 mg/L serine, 25 mg/L uracil, and 25 mg/L adenine (ADE), 1 mg/L

histidine (HIS), 1 mg/L leucine (LEU), and 0.5 mg/L tryptophan (TRP),

5 g/L ammonium sulphate, 2% (w/v) D-glucose, 1.5% (w/v)

bacteriological agar, pH adjusted to 6.3 with 4 M NaOH. 3-

aminotriazole was added to the media.

Yeast transformation was performed according to the lithium

acetate/ssDNA/PEG-based protocol (Gietz et al., 1995). Overnight 3


103

mL YPD grown cultures were spun down, washed once with 0.1 M

lithium acetate, resuspended in the same solution and incubated at

room temperature for 10 min. Transformation mix was prepared with

50 µL of the cell suspension, 300 µL PLI (8 vol. PEG 3350, 1 vol. 1 M

lithium acetate, 1 vol. water), 5 µL ssDNA (Invitrogen), previously

boiled for 5 min and chilled on ice, and 1 µg of plasmid DNA, and

incubated at 42ºC for 25 min. Cells were plated on the appropriate

selective media and grown for 3-4 days at 30ºC.

Yeast mating was performed by growing 10 mL of the bait

(Y187) strain in SD-TRP medium until an OD600 > 0.8. A frozen 1-mL

aliquot of the library AH109 strain (5 x 107 cells/mL) was thawed at

room temperature. YPD medium (9 mL) was inoculated with 1 mL of

the bait overnight culture, 100 µL of the library cell suspension and 50

µL/mL kanamycin, and incubated at 30°C with gentle shaking (50

rpm) for 22 h. The presence of diploids was confirmed under standard

light microscopy. A volume of 300 µL of the mating mixture was

plated on SC-TRP-LEU-HIS (around 30 plates per mating) and

incubated at 30°C for 5 days. Colonies were then transferred into new

SC-TRP-LEU-HIS-ADE and grown for 3 days.

For plasmid DNA extraction from yeast, 3-mL cultures were

grown overnight in selective media, spun down, resuspended in 100

µL STET buffer, containing 8% (w/v) sucrose, 50 mM EDTA pH 8.5,

50 mM TRIS-HCl pH 8, and 5% (v/v) Triton X-100, mixed with 0.3 g

glass beads (450 µm size, Sigma) and vortexed for 10 min. STET

buffer (100 µL) was added to the mix solution, which was then

incubated at 95°C for 3 min, and briefly chilled on ice. After spin

down, 100 µL of the supernatant were transferred to 60 µL of 7.5 M

ammonium acetate and incubated at -80°C for 1 h. The solution was

spun down and 120 µL of the supernatant were added to 240 µL of


104

ice-cold ethanol, incubated at -20°C for 30 min, spun down, and the

pellet washed with 70% (v/v) ethanol and resuspended in 25 µL

water. 10 µL were used for bacteria transformation and 5 µL for

agarose gel visualization.

Table 2.2. Sequences of the primers used for PCR and RT-PCR analyses.

Name Gene ID Primer name

Primer sequence # cycles

F1 5’-CGATACTTAGTGAT GATCG-3’ 27 or 35*

R1 5’-CCAAAGGCACTTG CTTTTCC-3’ 27 or 35*

F1a 5’-CAGGATGAAGAAA CGAAGGG-3’ 27

F2 5’-GGTTAATGAATGGT TTATAATATCC-3’ 35*

R2 5’-CATAACCCACGAG CAAAAGC-3’ 35*

F3 5’-ATGGGACAACAGC AATCAAAAG-3’ 27

XBAT35 At3g23280

R3 5’-CGAAGGTGCGAAT TTAAGACG-3’ 27

F1 5’-ATGGGGCAACAAC AATCACAG-3’ 30 or 35*

R 5’-CAAGAATGGTGATA CCTTTGTGG-3’ 30 or 35* XBAT34 At4g14365

F2 5’-ATGGGGCAACAAC AATCACAG-3’ 30

rd29aF 5’-GGAAGAGTCGGCT GTTTCAG-3’ RD29A At5g52310

rd29aR 5’-GTGCTCTGTTTTGG CTCCTC-3’

25

hspF 5’-TCCAAGCATTTTTG GAGGAC-3’ HSP18 At5g59720

hspR 5’-GAACCACAACCGT AAGCACA-3’

25

rbcsF 5’-GGCTAAGGAAGTT GACTACC-3’ RBCS At5g38410

rbcsR 5’-ACTTCCTTCAACAC TTGAGC-3’

20


105

iaaF 5’-TCTTGGACTCGAAA TCACCG-3’ IAA5 At1g15580

iaaR 5’-AACATCTCCAGCAA GCATCC-3’

30

ersF 5’-AGCAGACTGAGAC GAGTTTGC-3’ ERS2 At1g04310

ersR 5’-TCAATGGAAGCAC ACACACC-3’

30

tatF 5’-TGCTCGTGCTGTC TATAGCG-3’ TAT3 At2g24850

tatR 5’-AGGATCGTTCATG GCGATCC-3’

30

rocF 5’-GTCTGATAGAGATC TCACGT-3’ 25

ROC1 At4g38740

rocR 5’-AATCGGCAACAAC AACAGGC-3’ 25

ubqF 5’-GATCTTTGCCGGA AAACAATTGG-3’ 23

UBQ10 At4g05320

ubqR 5’-TAGAAAGAAAGAG ATAACAGG-3’ 23

actF 5’-TTTGCAGGAGATG ATGCTCCC-3’ 25

ACT2 At3g18780

actR 5’-GTCTTTGAGGTTTC CATCTCC-3’ 25

LBf 5’-CTACAAATTGCCTT TTCTTATCGAC-3’ 35*

T-DNA segment

LBc1 5’-CAAACAGGATTTT CGCCTGCTGGGG-3’ 35*

* number of cycles used for PCR-based genotyping of the xbat35-1, xbat35-2 and xbat34-1 insertion lines.

2.6. References

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CHAPTER 3 THE ARABIDOPSIS SCL30a SR PROTEIN CONFERS ABA-

DEPENDENT SALT AND OSMOTIC STRESS TOLERANCE

DURING SEED GERMINATION

A substantial part of this Chapter constitutes a manuscript currently

under preparation.

Sofia D. Carvalho was the main responsible for the experimental work

and data analysis presented in this Chapter.

Ji He and Nick Krom were responsible for the deep-sequencing raw

data analysis and first input on the reads obtained.

Acknowledgements to Vera Nunes for technical assistance in the

plant growth facilities, to Jutta Rösler for support during the

phenotypical analyses under selective light conditions, to Nam-Hai

Chua for providing the pBA vector, and to Jörg Becker for guidance

during the deep-sequencing data analysis.

Chapter 3 – SCL30a is involved in salt and osmotic stress tolerance

113

3.1. Abstract

Members of the highly conserved SR (serine/arginine-rich)

protein gene family are RNA-binding proteins that play key roles in

the regulation of pre-mRNA splicing and other aspects of RNA

metabolism. Here, we report the functional characterization of the

Arabidopsis SCL30a SR protein and show that it is involved in

abscisic acid (ABA)-dependent regulation of important seed traits as

well as germination under abiotic stress conditions. Analysis of the

SCL30a expression pattern using RT-PCR and reporter gene

experiments revealed that this SR gene is ubiquitously expressed in

vegetative tissues and markedly induced during the initial stages of

seed germination. Furthermore, in addition to the full-length

transcript, two splice variants containing premature stop codons are

produced during early developmental stages. A loss-of-function

mutant for SCL30a, scl30a-1, produces smaller seeds showing

enhanced dormancy that, despite germinating normally under optimal

conditions, are hypersensitive to the exogenous application of the

stress hormone ABA and high NaCl or mannitol concentrations. This

oversensitivity to salt and osmotic stress during seed germination is

rescued by an inhibitor of ABA biosynthesis, with epistatic analyses

confirming that the observed seed and germination phenotypes are

dependent on a functional ABA pathway. Importantly, transgenic

plants overexpressing SCL30a produce larger seeds exhibiting

enhanced tolerance to salt and drought stress. Measurement of

endogenous ABA levels in wild-type, scl30a-1 and SCL30a-

overexpressing seeds indicate that SCL30a does not affect

biosynthesis of the phytohormone neither under control or high

salinity conditions. Finally, we identified endogenous target


114

transcripts of SCL30a via global deep-sequencing transcriptome

analysis. Thus, the SCL30a SR protein defines a novel negative

regulator of the ABA signaling pathway controlling seed size and

dormancy as well as germination under salt and osmotic stress.

3.2. Introduction

Alternative splicing precisely selects different splices sites on

a single precursor mRNA (pre-mRNA), allowing the removal of

different non-coding intronic sequences and the retention of distinct

coding exonic sequences in the resulting mature transcripts. This

posttranscriptional regulation mechanism allows the generation of

multiple transcripts from a same gene and eventually different protein

isoforms, providing an important means of gene expression regulation

and generation of proteomic diversity. Ongoing work on the

annotation of the Arabidopsis genome estimates that at least 42% of

the genes undergo alternative splicing (Filichkin et al., 2010), with

different approaches, mostly at the genome-wide level, allowing

detection of altered alternative splicing profiles in various plant

tissues, at different developmental stages, and upon exposure to

specific stress conditions (Zhou et al., 2003; Iida et al., 2004; Filichkin

et al., 2010). This suggests that alternative splicing controls many

physiological and morphological aspects during plant development.

However, in contrast to humans, where alternative splicing has been

widely studied and is currently known to play a role in many diseases

(reviewed in Kalsotra and Cooper, 2011), the biological relevance of

alternative splicing in plants has been poorly addressed, with very few

functional reports available.


115

The pre-mRNA is spliced by the spliceosome, a dynamic and

highly regulated protein complex composed of five central small

nuclear ribonucleoproteins (snRNPs) and numerous non-snRNP

proteins. Serine/arginine-rich (SR) proteins are essential non-snRNP

spliceosomal factors conserved in higher eukaryotes that bind the

pre-mRNA and help in spliceosome assembly (Wu and Maniatis,

1993; Lorkovic et al., 2004; Shen and Green, 2004) by recruiting core

components of the splicing machinery to nearby splice sites (Wu and

Maniatis, 1993), thus influencing splice site selection in a

concentration-dependent manner (Graveley, 2000). The activity of SR

proteins in pre-mRNA splicing has been largely addressed in

mammals but not in plants, mostly due to the inability so far to

establish an in vitro plant splicing assay. Interestingly, genes

encoding SR proteins commonly undergo alternative splicing

themselves, generating multiple splice variants with premature stop

codons that are targeted for degradation by nonsense-mediated

decay (NMD) or encode truncated protein isoforms (Lareau et al.,

2007; Palusa and Reddy, 2010). Moreover, several plant SR proteins

have been found to regulate their own splicing and/or that of other

splicing factors (Lopato et al., 1999; Isshiki et al., 2006) and to

interact and/or colocalize with other SR members (Lopato et al.,

2002; Lorkovic et al., 2008). Besides their indisputable role during

splicing, SR proteins are now known to interfere with other aspects of

gene expression, such as DNA organization, methylation and histone

modifications (Zhang et al., 2011), transcription (Gullerova et al,

2006; Lin et al., 2008), mRNA export (Huang and Steitz, 2001),

stability and quality control (Zhang and Krainer, 2004) or translation

(Sanford et al., 2004), underscoring their more general role in

ensuring correct gene expression.


116

SR proteins typically possess two recognizable functional

domains: one (or two) RNA-Recognition Motifs (RRM) at the N-

terminus and a C-terminal arginine/serine-rich (RS) domain. While the

RRM binds the pre-mRNA, the RS domain is responsible for protein-

protein interactions with other components of the spliceosome (Wu

and Maniatis, 1993). Reversible phosphorylation at the RS domain is

the best described mechanism involved in the control of SR protein

localization, mobility and activity (Graveley, 2000; de la Fuente van

Bentem et al., 2006; Tillemans et al., 2006), but other regulatory

mechanisms, such as methylation at arginine residues, have been

recently reported in spliceosomal-related proteins, including human

SR proteins (Zhang et al., 2011; reviewed in Yu, 2011). The

conserved structure of SR proteins allows the identification of 18

members in Arabidopsis (Barta et al., 2010) and 22 in rice (Reddy

and Ali, 2011), while only 12 SR proteins are present in humans

(Manley and Krainer, 2010). The striking difference between these

numbers may account for divergences in splice site recognition and

intron excision in plants and metazoans. Moreover, in Arabidopsis

more than half of the SR proteins are specific to plants, showing no

orthologs in mammals (Barta et al., 2010).

SR proteins are thought to be active in ensuring correct plant

growth and development and stress responses, fundamentally due to

the identification of cell-, tissue- and stress-specific expression

patterns (Fang et al., 2004; Palusa et al., 2007; Duque, 2011; Reddy

and Ali, 2011), but few functional studies providing evidence for this

assumption are available. Reports in the literature addressing the in

vivo role of individual SR proteins are limited to overexpression

studies. Transgenic plants overexpressing SR30 and RS2Z33

affected the splicing profiles of other SR genes and caused altered


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morphologies and growth defects (Lopato et al., 1999; Kalyna et al.,

2003), but the response to stress was not investigated. Nevertheless,

two Arabidopsis SR-like proteins, RCY1 and SRL1, were found to

confer salt tolerance upon heterologous expression in yeast or

expression of portions of the full-length proteins in stably transformed

Arabidopsis plants (Forment et al., 2002). More recently, the

Arabidopsis SR-related splicing factor SR45, which had been

previously involved in plant growth and development (Ali et al., 2007),

was shown to be a negative regulator of ABA and sugar signaling

(Carvalho et al., 2010).

SCL30a belongs to the plant-specific SCL (SC35-like)

subfamily of Arabidopsis SR proteins. Using single-cell transient

expression assays, SCL30a has been shown to localize to the

nucleus (Lorkovic et al., 2008) and to interact with the RS2Z33 SR

protein (Lopato et al., 2002). Moreover, SCL30a expression has been

found in different Arabidopsis tissues (Palusa et al., 2007), generating

multiple splice variants (Palusa et al., 2007; Zhang et al., 2011).

The ABA phytohormone controls different aspects of plant

development and abiotic stress responses. In seeds, ABA regulates

embryo and seed development, dormancy establishment and the

transition to germination (reviewed in Finkelstein et al., 2008;

Holdsworth et al., 2008). Upon exposure to drought and high salinity

stress conditions, ABA is involved in blocking seed germination and,

later during development, in controlling stomatal aperture, thus

preventing excessive water loss from leaves (reviewed in Cutler et al.,

2010 and Hubbard et al., 2010).

Based on a reverse genetics approach, this study provides

evidence that the SCL30a SR protein is a novel negative regulator of

the ABA pathway, relying on this hormone to regulate seed-specific


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traits, such as size and dormancy, as well as germination under high

salt and osmotic conditions. Moreover, we found that this splicing

factor controls light responses during early seedling development.

Finally, by means of a transcriptome-wide approach based on an

RNA deep-sequencing analysis, we show that the SCL30a RNA-

binding protein controls the expression and splicing of several genes.

3.3. Results

3.3.1. The SCL30a gene generates three splice variants and is

markedly induced during seed germination

As a first approach to the characterization of the Arabidopsis

SCL30a gene (At3g13570), we investigated its developmental and

tissue-specific expression pattern by RT-PCR. In vegetative tissues,

SCL30a was expressed both in young seedlings and at later

developmental stages, with its mRNA being detected in different

aerial tissues, such as leaves, the stem, flowers and siliques, but also

in roots (Fig. 3.1A). When embryonic tissues were analyzed,

although SCL30a transcript levels were undetectable in dry seeds,

gene expression was clearly observed after 3 d of seed imbibition and

increased sharply during the first hours of seed germination upon

transfer to 22ºC in light (Fig. 3.1A).

To obtain a more detailed profile of SCL30a expression, we

generated transgenic plants stably expressing the β-glucuronidase

(GUS) reporter gene under the control of the SCL30a endogenous

promoter. To this end, a DNA fragment corresponding to the 2206 bp


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Figure 3.1. SCL30a expression pattern and promoter activity in Arabidopsis. (A) RT-PCR analysis of SCL30a transcript levels in embryonic and vegetative tissues of wild-type (Col-0) plants. The cyclophilin gene (ROC1) is shown as a loading control. (B) to (N) Histochemical observation of GUS activity in transgenic Col-0 plants carrying the SCL30a promoter-GUS fusion construct. ProSCL30a:GUS expression in two-week old seedlings (B), the primary root tip (C), a lateral root primordium (D), mature and immature flowers (E), developing embryos (F-K), the embryo (L) and testa (M) from imbibed mature seeds, and seeds germinated for 1-2 d (N). Scale bars = 100 µM.

immediately upstream of the SCL30a start codon was fused to the

GUS sequence and transformed into wild-type plants. Consistent with

the established RT-PCR expression pattern, the SCL30a promoter

was active throughout plant development (Figs. 3.1B-1N). GUS

staining was mainly observed in vascular tissues and actively dividing

cells, such as in the shoot meristem and young leaves (Fig. 3.1B),

the primary root tip (Fig. 3.1C) and lateral root primordia (Fig. 3.1D).


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At the reproductive phase, SCL30a appears to be particularly

expressed in the pistil tip, the vasculature tissue of sepals, the

stamen filaments and in pollen grains (Fig. 3.1E) of developing

flowers. In embryonic tissues, the SCL30a promoter was found to be

active from the early – globular and heart (Figs. 3.1F-1H) – to the

later – torpedo and mature embryo (Figs. 3.1I-1K) – stages of seed

development. Finally, in mature imbibed seeds GUS staining was

detected in the whole embryo (Fig. 3.1L) as well as strongly in the

seed coat (Fig. 3.1M), but appeared restricted to the radicle tip during

germination (Fig. 3.1N).

An interesting feature of both animal and plant pre-mRNAs

encoding SR proteins and other splicing components is that they

appear to be particularly prone to alternative splicing themselves. In

fact, this RNA processing mechanism has been shown to dramatically

increase the complexity of the Arabidopsis SR protein gene family

transcriptome (Ali et al., 2007; Palusa and Reddy, 2010), prompting

us to examine the splicing pattern of the SCL30a gene. Although only

one transcript has been annotated (www.arabidopsis.org), Reddy and

coworkers have reported the amplification of five SCL30a-specific

bands by RT-PCR (Ali et al., 2007; Palusa and Reddy, 2010). With

the same primer pair, we consistently amplified five bands of different

relative intensities using cDNA from either imbibed/germinating seeds

or young seedlings as a template. However, cloning and sequencing

of these PCR products showed that only three corresponded to

alternative SCL30a transcripts, with the two faintest bands being

unspecific.

Comparison of the three SCL30a-specific sequences with that

of the genomic fragment revealed that, as predicted in the current

genome annotation, the gene contains six exons. The shortest and by


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Figure 3.2. Alternative splicing of the SCL30a gene and isolation of the scl30a-1 loss-of-function mutant. (A) Schematic representation of the SCL30a gene showing the site of insertion and orientation of the T-DNA in the scl30a-1 mutant (boxes indicate exons with UTRs in grey, lines between boxes represent introns, and arrows indicate the location of SCL30a- and T-DNA-specific primers), and structure of the three identified splice variants as well as the corresponding predicted protein isoforms (RRM, RNA-recognition motif; RS, arginine/serine-rich domain). (B) PCR-based genotyping of an scl30a-1 homozygous line. The location of the F3, R2 and LBc primers is shown in (A). (C) RT-PCR analysis of SCL30a transcript levels in wild-type (Col-0) and mutant (scl30a-1) 5-d old seedlings using primers flanking the T-DNA, and up- or downstream of the insertion site. The location of the F1, F4, R1 and R3 primers is shown in (A). The ubiquitin 10 (UBQ10) gene was used as a loading control.

far most expressed transcript (Figs. 3.1A and 3.2C), SCL30a.1,

arises from constitutive splicing of the pre-mRNA and is predicted to

encode the full-length protein, which displays a typical SR protein

structure organization with one N-terminal RNA recognition motif

(RRM) followed by an RS domain (Fig. 3.2A). A second splice

variant, SCL30a.2, includes a short cryptic exon between exons 3

and 4, while the longest transcript, SCL30a.3, derives from retention


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of intron 3. Both the SCL30a.2 and SCL30a.3 mRNAs harbor a

premature termination codon (PTC) in the cryptic exon or third intron,

respectively, and therefore encode putative severely truncated

proteins containing as recognizable domains only a portion of the

RRM (Fig. 3.2A).

Taken together, the above results indicate that the SCL30a

gene displays ubiquitous expression in vegetative tissues, is highly

induced during seed imbibition/germination, and produces three

alternative transcripts during early seedling development. While the

most expressed SCL30a mRNA encodes the full-length protein, the

other two transcripts are putative NMD targets or may alternatively be

translated into nonfunctional proteins or truncated proteins with

altered functions.

3.3.2. The scl30a-1 mutant displays seed-specific phenotypes

and hypersensitivity to ABA, salt and osmotic stress during

germination

To initiate the functional characterization of SCL30a, we

obtained a T-DNA mutant line, SALK_041849, carrying the insertion

in the gene’s second exon, 510 bp downstream of the start codon

(Fig. 3.2A). PCR-based genotyping with primers specific for SCL30a

and the left border of the T-DNA, followed by sequencing of the

genomic DNA/T-DNA junction, confirmed the exact insertion site and

allowed the isolation of a homozygous line for the insertion named

scl30a-1 (Fig. 3.2B). RT-PCR analysis of SCL30a expression in

scl30a-1 plants using primers annealing upstream of the insertion site

revealed transcript levels comparable to the wild type, but no


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expression was detected when primers flanking or annealing

downstream of the T-DNA segment were used (Fig. 3.2C).

Consistent with the location of the T-DNA insertion, no splice variants

were detected in the mutant. Thus, scl30a-1 expresses a single

SCL30a truncated transcript (22% of the full-length coding sequence)

that lacks the sequence corresponding to the entire RS domain as

well as most of the RRM and is hence expected to encode a

nonfunctional SR protein. These results strongly suggest that the

scl30a-1 allele is a true loss-of-function mutant.

In order to gain insight into the biological role(s) of the SCL30a

SR protein, we carried out a detailed phenotypical analysis of the

scl30a-1 mutant. Despite ubiquitous SCL30a expression in vegetative

tissues, no evident mutant phenotypes were observed in adult plants

grown under greenhouse conditions. Given the marked induction of

the SCL30a gene during seed imbibition and germination (see Figure

3.1), we next paid particular attention to embryonic tissues and early

developmental stages. Strikingly, mature scl30a-1 seeds were found

to display a significant reduction in size, with dry and imbibed seeds

being respectively 12% and 14% smaller than seeds of wild-type

plants (Fig. 3.3A). However, maturing seeds collected from

developing scl30a-1 siliques exhibited no significant differences in

size when compared to the wild type (data not shown), suggesting

that SCL30a specifically controls the latest stages of seed maturation.

Interestingly, the scl30a-1 mutant also exhibited enhanced

seed dormancy. As shown in Figure 3.3B, upon incubation for 7 d in

darkness, germination of freshly-harvested, non-stratified scl30-1

mutant seeds was only about one third of that of wild-type seeds. As

a control for equal seed viability, we used stratified seeds germinated


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Figure 3.3. Seed and germination phenotypes of the scl30a-1 mutant. (A) Representative images of wild-type (Col-0) and mutant (scl30a-1) dry seeds (scale bars = 1.5 mm), and quantification of the area of dry and imbibed Col-0 (white bars) and scl30a-1 (black bars) seeds (means ± SE, n = 50). (B) Germination of freshly-harvested Col-0 (white bars) and scl30a-1 (black bars) seeds scored upon either stratification and 7 d of incubation in light or 7 d of incubation in darkness (means ± SE, n = 3). (C) Germination rates of Col-0 (open circles) and scl30a-1 (closed circles) stratified seeds under control conditions (means ± SE, n = 3). (D) Germination of Col-0 (open circles) and scl30a-1 (closed circles) stratified seeds in the presence of different ABA or NaCl (scored 5 d after stratification) or mannitol (scored 3 d after stratification) concentrations (means ± SE; n = 3). Asterisks indicate statistically significant differences from the wild type according to Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.001).


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in light (Fig. 3.3B). In fact, despite their hyperdormancy and apart

from a slight delay in the first 24 h, scl30a-1 seeds germinated as well

as wild-type seeds under optimal conditions (Fig. 3.3C). Early

seedling development, measured as cotyledon greening and

expansion, was also unaffected in the mutant (data not shown).

Nevertheless, although selective light conditions did not affect seed

germination, the scl30a-1 mutation conferred a significantly enhanced

response to far-red, red and blue light in the repression of hypocotyl

elongation, with no clear phenotypic alterations being observed under

darkness (Fig. 3.4A).

The seed-specific phenotypes of the scl30a-1 mutant

prompted us to analyze its response to the phytohormone ABA, which

is essential for seed maturation, dormancy and germination in

Arabidopsis (reviewed in Finkelstein et al., 2008; Holdsworth et al.,

2008). Indeed, the ABA dose-response curve depicted in Figure 3.3D

shows that scl30a-1 displays hypersensitivity to ABA, with seeds of

the mutant germinating less than half of wild-type seeds at

concentrations of the hormone as low as 1 µM. We then sought to

examine scl30a-1 seed germination under different abiotic stress

conditions. As with exogenously applied ABA, the germination rates

of mutant seeds were markedly reduced under high salinity stress

imposed by NaCl concentrations above 100 mM (Fig. 3.3D).

Similarly, the osmotic stress induced by high mannitol concentrations

inhibited the germination of scl30a-1 seeds more than that of wild-

type seeds (Fig. 3.3D). Seeds from the scl30a-1 mutant also showed

oversensitivity to the KCl salt during germination, but were unaffected

by sugars (data not shown).


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Figure 3.4. Hypocotyl elongation of SCL30a loss-of-function and overexpression lines under selective light conditions. (A) Hypocotyl lengths of Col-0 (white bars) and scl30a-1 (black bars) seedlings as well as of the phyA (orange bars), phyB (red bars) and cry1/2 (blue bars) mutants after 4 d under darkness or two different fluence rates of far-red, red or blue light irradiation (means ± SE, n = 40). (B) Hypocotyl lengths of seedlings of Col-0 (white bars), scl30a-1 (black bars) and two SCL30a overexpression lines, OX1 and OX2 (grey bars), after 4 d under darkness or 1 µmol.m-2.s-1 of far-red, red or blue light irradiation (means ± SE, n = 40). Asterisks indicate statistically significant differences from the wild type according to Student’s t-test (***P < 0.001).

To verify that the identified seed and germination phenotypes

were indeed due to disruption of the gene encoding the SCL30a

protein, we transformed the scl30a-1 mutant with either a DNA

fragment spanning the SCL30a gene, which included the same

promoter sequence used in the reporter gene experiments, or the

SCL30a.1 coding sequence under the control of the 35S promoter.


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Three independent transgenic lines expressing each of these

constructs (Fig. 3.5A) were isolated and characterized. As shown in

Figure 3.5B, all but one of these lines exhibited complementation of

Figure 3.5. Complementation of the scl30a-1 mutant phenotypes. (A) RT-PCR analysis of SCL30a transcript levels in seedlings of the wild-type (Col-0), mutant (scl30a-1) and six complementation lines expressing either the SCL30a genomic fragment under the control of the endogenous promoter (C1, C2, C3) or the SCL30a.1 coding sequence under the control of the 35S promoter (C4, C5, C6) in the scl30a-1 mutant background. The location of the F2 and R1 primers used is shown in Figure 3.2A. Ubiquitin 10 (UBQ10) was used as a loading control. (B) Size (expressed as area in mm2) of imbibed Col-0 (white bars), scl30a-1 (black bars) and C1-C6 complementation line (grey bars) seeds (means ± SE, n = 50). (C) Germination, scored 3 d after stratification, of Col-0 (white bars), scl30a-1 (black bars) and C1-C6 complementation line (grey bars) seeds under control conditions or in the presence of 1 mM ABA, 150 mM NaCl or 375 mM mannitol (means ± SE; n = 3). Different letters indicate statistically significant differences between genotypes under each condition according to Student’s t-test (P < 0.05).


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the seed size phenotype. Moreover, the enhanced sensitivity to ABA,

NaCl and mannitol during germination was reverted in all six

transgenic lines (Fig. 3.5C), confirming that loss of SCL30a function

is responsible for the observed mutant phenotypes. Importantly, the

cDNA encoding the full-length SCL30a.1 protein was able to

complement all phenotypes as efficiently as the SCL30a genomic

fragment, suggesting that the two PTC-containing splice variants,

SCL30a.2 and SCL30a.3, are either not translated or encode proteins

with no function, at least in the seed-specific and germination traits

analyzed.

These findings show that the SCL30a SR protein plays an in vivo role

in embryonic tissues, affecting seed size and dormancy. Furthermore,

although not essential for seed germination per se, SCL30a affects

the ability of seeds to germinate under unfavorable environmental

conditions such as salt and osmotic stress.

3.3.3. SCL30a is a novel component of the ABA pathway

To investigate whether the salt and osmotic stress phenotypes

of the scl30a-1 mutant are mediated by the ABA phytohormone, we

first performed the same germination assays in the presence of

fluridone, an inhibitor of ABA biosynthesis (Moore and Smith, 1984;

Ullah et al., 2002; Lin et al., 2007). As seen in Figure 3.6A, and

consistent with the well established role of ABA as a key mediator of

drought and salt stress responses, addition of 1 µM fluridone notably

relieved the inhibition imposed by high NaCl or mannitol

concentrations on the germination of wild-type seeds. Most

importantly, the presence of fluridone rescued both the scl30a-1 salt


129

and osmotic stress phenotypes, with the two genotypes germinating

at similar rates in NaCl or mannitol (Fig. 3.6A). These results show

that the mutant’s stress germination phenotypes depend on the

endogenous production of ABA and indicate that loss of SCL30a

function results in either impaired biosynthesis or sensing/signaling of

the hormone.

To conclusively establish the involvement of SCL30a in the

ABA pathway, we next turned to epistatic analysis and assessed the

genetic interaction between SCL30a and ABA2, encoding a cytosolic

short-chain reductase involved in the conversion of xanthoxin to ABA-

aldehyde during ABA biosynthesis (Schwartz et al., 1997), or ABI4,

which encodes an AP2-type transcription factor involved in ABA

signal transduction (Finkelstein et al., 1998). To this end, scl30a-1

was independently crossed with the ABA-deficient aba2-1 and ABA-

insensitive abi4-101 mutant alleles to generate the corresponding

homozygous double mutants. Interestingly, we found that the ABA

single mutants exhibit enhanced SCL30a expression (Fig. 3.6B),

indicating that both ABA2 and ABI4 are implicated in the

transcriptional repression of this SR gene. Furthermore, as seen in

the dose-response curves depicted in Figure 3.6C, seeds from the

scl30a-1aba2-1 and scl30a-1abi4-101 double mutants behaved as

those of the corresponding single ABA mutants when germinated

under salt or osmotic stress conditions. This indicates that the role of

SCL30a in salt and osmotic stress responses during germination fully

relies on functional ABA2 and ABI4 genes.

During the seed germination assays, we noticed that the

aba2-1 mutant produced larger seeds than the wild type. This

prompted us to test whether the smaller seed size of scl30a-1 (see

Figure 3.3A) was also linked to the ABA pathway. The size of wild-


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Figure 3.6. ABA dependence of the scl30a-1 mutant phenotypes. (A) Germination of Col-0 (white bars) and scl30a-1 (black bars) seeds under control conditions or in the presence of 200 mM NaCl (scored 5 d after stratification) or 450 mM mannitol (scored 3 d after stratification) supplemented or not with 1 µM fluridone (means ± SE, n = 3). (B) RT-PCR analysis of SCL30a transcript levels in Col-0, scl30a-1, aba2-1, abi4-101, scl30a-1aba2-1 and scl30a-1abi4-101 7-d old seedlings. The location of the F1 and R1 primers used is shown in Figure 2A. Ubiquitin 10 (UBQ10) was used as a loading control. (C) Germination of Col-0, scl30a-1, aba2-1, abi4-101, scl30a-1aba2-1, and scl30a-1abi4-101 seeds under different NaCl (scored 4 d after stratification)


131

or mannitol (scored 2 d after stratification) concentrations (means ± SE, n = 3). (D) Size (expressed as area in mm2) of imbibed Col-0, scl30a-1, aba2-1, scl30a-1aba2-1, abi4-101 and scl30a-1aba4-101 seeds (means ± SE, n = 50). (E) Germination of freshly-harvested Col-0, scl30a-1, aba2-1, abi4-101, scl30a-1aba2-1, and scl30a-1abi4-101 seeds scored upon either stratification and 7 d of incubation in light or 7 d of incubation in darkness (means ± SE, n = 3). Asterisks indicate statistically significant differences between the scl30a-1 mutant and the wild type or the double mutants and the corresponding ABA single mutant according to Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.001).

-type, mutant and double mutant imbibed seeds was measured, and

as aba2-1 seeds, the scl30a-1aba2-1 seeds were larger than the wild

type (Fig. 3.6D). abi4-101 seeds were found to be similar in size to

the wild type (Fig. 3.6D). Interestingly, the ABI4 mutation in the

scl30a-1 background abolished the smaller size of scl30a-1 seeds,

restoring it to wild-type values (Fig. 3.6D). Thus, as observed for the

germination phenotypes, the altered seed size of scl30a-1 is ABA

dependent.

Since ABA is known to regulate seed dormancy (reviewed in

Finkelstein et al., 2008 and Holdsworth et al., 2008), we also

investigated a potential link between the higher dormancy of scl30a-1

(see Figure 3.3B) and this phytohormone. As expected, mutant

aba2-1 seeds were markedly non-dormant and the same was

observed for scl30a-1aba2-1 (Fig. 3.6E). The scl30a-1aba4-101

double mutant displayed similar dormancy levels to the wild type and

abi4-101, an abi4 allele unaffected in seed dormancy. These results

clearly indicate that the enhanced dormancy of scl30a-1 seeds is also

dependent on a functional ABA pathway.

ABA also plays important roles during later stages of plant

development, particularly in the regulation of stomatal aperture and


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thus in the control of leaf water loss under drought stress conditions

(reviewed in Cutler et al., 2010 and Hubbard et al., 2010). Although

no growth defects were observed in scl30a-1 adult plants, we asked

whether the SCL30a SR protein also plays an ABA-dependent role in

stomata and measured the transpiration rates of detached rosette

leaves from the wild type and the scl30a-1 mutant, including the ABA-

related mutants as controls (Fig. 3.7). Impairment of SCL30a

expression appeared to increase slightly the rate of water loss from

leaves when compared to the wild type (Fig. 3.7). Given the ABA

hypersensitive responses of scl30a-1 during germination (Fig. 3.3D),

Figure 3.7. Leaf water loss in SCL30a loss- and gain-of-function lines and in ABA-related mutants. Water loss of Col-0, scl30a-1, aba2-1, abi4-101, scl30a-1aba2-1, scl30a-1abi4-101 and SCL30a.1OX1 5-week old detached rosette leaves (n = 2). Results are representative of three independent experiments.

and given that ABA triggers stomatal closure and represses opening

(reviewed in Cutler et al., 2010 and Hubbard et al., 2010), we would

expected reduced leaf transpiration rates in the mutant. The

hypothesis that SCL30a regulates the ABA pathway differently in


133

embryonic and vegetative tissues should not be discarded, but we

nevertheless considered that the water loss changes observed for the

scl30a-1 mutant were insufficient to infer an essential activity of the

SCL30a SR protein in stomatal movements.

3.3.4. SCL30a-overexpressing plants produce larger seeds

exhibiting abiotic stress tolerance during germination

Given that the SCL30a.1 transcript alone was able to

complement the mutant seed size and stress-germination phenotypes

(see Figure 3.5), we next expressed this splice variant in the Col-0

background for SCL30a gain of function studies. Two lines effectively

overexpressing SCL30a were selected and named SCL30a.1OX1

and SCL30a.1OX2 (Fig. 3.8A).

Importantly, both transgenic Arabidopsis lines overexpressing

the SCL30a.1 isoform produced significantly larger seeds than the

wild type (Fig. 3.8B), but seed dormancy appeared unaltered (Fig.

3.8C) as well as the response to selective light conditions during

hypocotyl elongation (see Figure 3.4B). Remarkably, when seed

germination under different stress conditions was assessed for the

two SCL30a.1-overexpressing lines, both displayed clear insensitivity

to high ABA, salt and mannitol concentrations (Fig. 3.8D). Therefore,

the SCL30a SR protein confers tolerance to these different abiotic

stresses during seed germination. Interestingly, consistent with the

increased transpiration rates found for scl30a-1, overexpression of

SCL30a may lead to a slight reduction in water loss rates of detached

adult leaves when compared to the wild type (see Figure 3.7).


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However, the weak effect observed supports the notion that the SR

protein is not essential for stomatal movements.

Figure 3.8. Phenotypes conferred by gain of SCL30a function. (A) RT-PCR analysis of SCL30a transcript levels in seedlings of the wild-type (Col-0), mutant (scl30a-1) and two SCL30a overexpression lines (SCL30a.1OX1 and SCL30a.1OX2). The location of the F2 and R1 primers used is shown in Figure 2A. Ubiquitin 10 (UBQ10) was used as a loading control. (B) Size (expressed as area in mm2) of imbibed Col-0 (white bars), scl30a-1 (black bars) and SCL30a.1OX1 or SCL30a.1OX2 (grey bars) seeds (means ± SE, n = 50). (C) Germination of freshly-harvested Col-0 (white bars), scl30a-1 (black bars) and SCL30a.1OX1 or SCL30a.1OX2 (grey bars) seeds scored upon either stratification and 7 d of incubation in light or 7 d of incubation in darkness (means ± SE, n = 3). (D) Germination of Col-0 (white bars), scl30a-1 (black bars) and SCL30a.1OX1 or SCL30a.1OX2 (grey bars) under control conditions, 3 µM ABA or 200 mM NaCl (scored 5 d after stratification) or 450 mM mannitol (scored 3 d after stratification). Bars represent means ± SE, n = 3. Asterisks indicate significant differences from the wild type according to Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.001).


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3.3.5. SCL30a regulates ABA signaling under salt stress

Taken together, the results gathered so far in this study

indicate that the SCL30a SR protein modulates the ABA pathway to

regulate a myriad of seed traits including size, dormancy and

germination under salt and osmotic stress. To narrow down this SR

protein’s role in the ABA pathway, and dissect whether it acts at the

level of biosynthesis of the hormone or solely in its signal

transduction, we determined the endogenous ABA levels in wild-type,

scl30a-1 and SCL30a.1OX1 seeds germinated under control

conditions or high salinity stress. We chose the latter condition as

plant responses to salt and drought stress are closely related, with

overlapping underlying mechanisms (reviewed in Tuteja, 2007 and

Cutler et al., 2010). Moreover, salt stress can be experimentally

imposed more easily and precisely in the laboratory. We selected the

highest salt concentration used in the dose response curves (see

Figure 3.3D), 200 mM NaCl, and harvested seeds at the onset of

germination to ensure all samples were at similar developmental

stages.

As expected, high salinity increased endogenous ABA levels

in wild-type seeds (Table 3.1), with the same being observed in

scl30a-1 and SCL30a.1OX1 seeds. The ABA-deficient aba2-1 mutant

was included as negative control, and indeed its ABA content was

unaltered by high salinity stress. Importantly, the ratio between the

levels of ABA in seeds grown under both control and salt conditions

was similar in Col-0, scl30a-1 and SCL30a.1OX1, indicating that

these genotypes synthesize the same amounts of the phytohormone.

It is therefore unlikely that SCL30a acts on ABA biosynthesis, being

rather involved in signal transduction of the hormone in response to


136

high salinity stress during seed germination. This conclusion is

consistent with the scl30a-1 and SCL30a.1OX1 responses to

exogenously applied ABA during germination (see Figures 3.3D and

3.8D), which indicate altered sensing/signaling of the phytohormone.

Table 3.1. Effect of loss or gain of SCL30a function on endogenous ABA levels. ABA levels (means ± SE) in ng/g fresh weight of Col-0, scl30a-1, SCL30a.1OX1 and aba2-1 seeds at the onset of germination in the presence of 0 or 200 mM NaCl. Letters indicate significant differences between genotypes among each condition and asterisks significant differences for each genotype between control and salt conditions, according to Student’s t-test (p<0.01).

Genotype Control NaCl Ratio

NaCl/Control

Col-0 81.77 ± 3.53 103.97 ± 4.31 1.27 ± 0.11

scl30a-1 90.76 ± 4.62 129.10 ± 5.07 1.42 ± 0.13

SCL30a.1OX1 92.85 ± 3.90 122.82 ± 4.15 1.32 ± 0.10

aba2-1 127.79 ± 1.91 126.89 ± 1.40 0.99 ± 0.03

3.3.6. SCL30a affects the expression and splicing pattern of

several Arabidopsis genes

To gain insight into the molecular mechanisms governing the

role of the SCL30a RNA-binding protein in seed maturation and

germination under stress, we next conducted a global transcriptome

analysis of wild-type, scl30a-1 mutant and SCL30a.1 overexpressing

seeds by next generation sequencing, in order to identify its

downstream targets.

The deep-sequencing approach allows obtaining the reads for

each nucleotide within a specific transcript, a number that is a direct

a

**

**

**


137

result of the expression level of that particular mRNA. Furthermore,

comparing the reads for each genotype within specific regions allows

detection of different splice junctions and therefore distinct splice

variants among the various genotypes. Since we identified SCL30a

as an important regulator of salt responses during seed germination,

the RNA used in the deep-sequencing analysis was extracted from

seeds of the three genotypes plated on high levels of salt (200mM

NaCl) and collected at the onset of germination, ensuring that the

three genotypes were at similar developmental stages.

Based on the hypothesis that disruption and overexpression of

SCL30a expression would result in altered expression and/or splicing

profiles of several genes, we first examined the total gene expression

levels obtained from the deep-sequencing analysis and isolated the

genes whose expression was found to be significantly induced in the

mutant and downregulated in the overexpression line when compared

to the wild type, or vice-versa. Using this approach, we obtained a

final list of 8 genes, shown in Table 3.2. The ninth gene shown in this

Table is the SCL30a gene itself, supporting the reliability of the data.

We then used the same rationale to identify genes whose

splicing was altered in both the T-DNA insertion and the

overexpression lines when compared to the wild type. In this case,

two comparisons were performed. Firstly, we identified genes with

unique splice junctions in all three genotypes, and obtained a list with

50 loci, depicted in Table 3.3. Secondly, we identified genes with

increased expression of a particular splice variant, over the total gene

expression, in the mutant and decreased in the overexpression line,

when compared to the wild type, or vice-versa. Table 3.4 lists the 30

genes isolated using this approach. Appendix I compiles the results

obtained using the analytical methods employed in Tables 3.2, 3.3


138

and 3.4, but comparing only the mutant or the overexpressor to the

wild type.

Table 3.2. Genes whose expression is oppositely regulated in the scl30a-1 mutant (KO) and the SCL30a.1OX1 line (OX). The fold change is the average obtained from two replicates.

Average Fold Change Locus ID Gene Description Cufflinks

ID KO OX

At5g09400 KUP7 Potassium transmembrane transporter

XLOC_ 024708 7.443 -3.998

At4g19150 T18B16.120 Ankyrin repeat family

XLOC_ 019710 5.725 -1.002

At1g47395 Unknown protein

XLOC_ 001949 2.063 -1.382

At3g07130 PAP15

Purple acid phosphatase, phytase activity; phosphorus reserves mobilization

XLOC_ 014540 1.293 -1.052

At5g16050 GRF5

General regulatory factor, 14-3-3 gene family member; response to cadmium ion

XLOC_ 025089 1.250 -1.021

At2g35240 T4C15.9 Plastid developmental protein

XLOC_ 010421 1.102 -1.128

At1g45233 THO5 Transport of mRNA precursors

XLOC_ 001929 -1.042 30.198

At3g58590 F14P22.180 Pentatricopeptide repeat

XLOC_ 013875 -1.372 11.690

At3g13570 SCL30a 30kD SC35-like splicing factor

XLOC_ 014877 -10.236 3.043

Finally, the comparisons performed in Tables 3.2, 3.3 and 3.4

were merged, i.e., we analyzed the genes that showed a significantly


139

altered total expression level in the mutant and unique splice

junctions or altered splice variant expression ratios in the

overexpression line, or vice-versa. Table 3.5 lists the 25 genes

isolated following this approach. It is interesting to note that, in

relation to alternative splicing changes, all cases identified here only

show altered splice variant expression ratios, never displaying unique

splice junctions. Interestingly, the SCL30a gene was listed in Table

3.5, suggesting that overexpression of SCL30a.1 or disruption of its

function to residual levels interfere with splicing of its own pre-mRNA.

The approaches used to analyze the deep-sequencing data

allowed the identification of 104 genes whose expression and/or

splicing pattern are controlled by the SCL30a SR protein, with a few

genes being retrieved in more than one of the different analyses

performed. The SCL30a endogenous targets under salt stress appear

to fulfill different molecular functions and to be associated with a

variety of biological processes (Fig. 3.9). Regarding molecular

function (Fig. 3.9A), the most represented group accounts for one

third of the genes retrieved and is related to RNA and DNA

processing. Other major groups relate to signal transduction and

binding to proteins or other molecules. The less represented classes

are generally related to the regulation of protein activity and to the

progression of the cell cycle. As for biological processes (Fig. 3.9B),

we found that a fifth of the SCL30a targets play roles in stress

responses. In addition to the 15% of genes associated to general

signaling, almost 40% of the retrieved targets are involved in stimuli

sensing, signaling and the activation of stress responses. Other

biological groups listed relate to general metabolism and to the cell

cycle, but also, to a lesser extent, to light responses, organ size

regulation or to genes specifically expressed in pollen.


140

Table 3.3. Genes with altered splice junctions (SJs) in all three genotypes. Wild type, WT; scl30a-1, KO; and SCL30a.1OX1, OX.

# unique SJs (% total) Locus ID Gene Description Cufflinks ID

Total # of SJs WT KO OX

At3g17050 K14A17.12 Transposable element XLOC_012273 23 7 (30) 6 (26) 3 (13)

At5g45490 MFC19.16 ATP hydrolase XLOC_022973 8 2 (25) 1 (13) 3 (38)

At5g59660 MTH12.10 Serine-threonine protein kinase XLOC_023731 8 2 (25) 2 (25) 3 (38)

At5g53500 MNC6.4 Transducin/ WD40 repeat-like XLOC_026549 4 1 (25) 1 (25) 1 (25)

At3g23450 MLM24.18 Unknown protein XLOC_012659 55 12 (22) 12 (22) 18 (33)

At5g52110 CCB2 Cytochrome b6f complex assembly XLOC_026475 11 2 (18) 1 (9) 1 (9)

At1g63130 F16M19.5 siRNA; targets At1g62930 XLOC_002698 6 1 (17) 2 (33) 1 (17)

At5g14830 T9L3.130 Transposable element XLOC_021750 13 2 (15) 2 (15) 3 (23)

At1g11100 T19D16.2 Helicase activity XLOC_004378 7 1 (14) 1 (14) 3 (43)

At2g26350 PEX10 Protein import into the peroxisome;

embryogenesis and dormancy XLOC_009987 21 3 (14) 2 (10) 1 (5)

At2g45880 BAM7 Beta-amylase; cellulose synthesis XLOC_011010 14 2 (14) 1 (7) 1 (7)

At1g08010 GATA11 Transcription factor XLOC_004197 8 1 (13) 1 (13) 1 (13)

At4g05330 AGD13 Regulator of GTPase activity XLOC_019161 17 2 (12) 9 (53) 2 (12)

At3g12670 EMB2742 Embryo development ending in seed

dormancy; CTP synthase XLOC_014817 30 3 (10) 1 (3) 1 (3)


141

At4g12870 T20K18.220 Gamma interferon responsive

lysosomal thiol reductase XLOC_017225 10 1 (10) 1 (10) 1 (10)

At5g25380 CYCA2;1 Cyclin-dependent protein kinase

regulator activity XLOC_022351 10 1 (10) 5 (50) 2 (20)

At3g59330 F25L23.190 Unknown protein XLOC_016439 41 4 (10) 2 (5) 1 (2)

At5g01950 T20L15.220 Serine-threonine protein kinase XLOC_024249 33 3 (9) 1 (3) 3 (9)

At4g09970 T5L19.100 Unknown protein XLOC_017083 12 1 (8) 1 (8) 2 (17)

At1g32940 SBT3.5 Serine-type endopeptidase XLOC_001773 12 1 (8) 1 (8) 1 (8)

At5g40270 MSN9.18 Phospho hydrolase XLOC_025866 26 2 (8) 6 (23) 2 (8)

At1g51310 F11M15.16 tRNA methyl transferase XLOC_005723 13 1 (8) 1 (8) 1 (8)

At2g14520 T13P21.10 Unknown protein XLOC_009492 13 1 (8) 1 (8) 1 (8)

At2g33980 NUDT22 Hydrolase activity XLOC_010361 13 1 (8) 1 (8) 2 (15)

At4g23910 T32A16.80 Unknown protein XLOC_017811 27 2 (7) 2 (7) 1 (4)

At4g27830 BGLU10 Beta-glucosidase XLOC_020129 27 2 (7) 1 (4) 1 (4)

At3g02030 F1C9.19 Acyltransferase XLOC_011225 14 1 (7) 1 (7) 1 (7)

At4g33945 ARM repeat superfamily XLOC_018507 15 1 (7) 1 (7) 1 (7)

At1g77890 F28K19.10 DNA-directed RNA polymerase II XLOC_003560 16 1 (6) 1 (6) 1 (6)

At5g43990 SUVR2 Histone methyl transferase XLOC_022907 16 1 (6) 2 (13) 2 (13)

At1g50500 HIT1 Golgi to ER transport; response to heat

and osmotic stress XLOC_005704 35 2 (6) 2 (6) 3 (9)

At3g57060 F24I3.140 Chromosome condensation XLOC_016327 35 2 (6) 3 (9) 3 (9)


142

At4g25070 F24A6.14 Unknown protein XLOC_017906 18 1 (6) 1 (6) 1 (6)

At5g48310 K23F3.3 Unknown protein XLOC_026274 18 1 (6) 7 (39) 1 (6)

At1g12790 F13K23.4 RuvA domain 2-like XLOC_004460 19 1 (5) 1 (5) 1 (5)

At2g06005 FIP1 Frigida interacting protein; flowering

time XLOC_007266 19 1 (5) 1 (5) 1 (5)

At3g08000 F17A17.34 RNA binding XLOC_014592 19 1 (5) 1 (5) 1 (5)

At2g20290 XIG Myosin complex XLOC_009692 44 2 (5) 1 (2) 1 (2)

At2g48060 T9J23.21 Unknown protein XLOC_011101 23 1 (4) 1 (4) 1 (4)

At1g16800 F17F16.1 ATP hydrolase; RNA binding XLOC_004662 23 1 (4) 7 (30) 1 (4)

At1g30420 MRP12 ATPase activity coupled to

transmembrane transport XLOC_001646 48 2 (4) 3 (6) 2 (4)

At5g46470 RPS6 ATP binding; defense response XLOC_023025 24 1 (4) 2 (8) 1 (4)

At3g13065 SRF4 Serine-threonine protein kinase XLOC_011995 25 1 (4) 1 (4) 1 (8)

At4g27050 F10M23.390 F-box domain, cyclin-like XLOC_020065 25 1 (4) 1 (4) 2 (8)

At4g15233 ABCG42 PDR ABC-type XLOC_017362 26 1 (4) 1 (4) 3 (12)

At3g14150 MAG2.11 Aldolase; oxidation-reduction process XLOC_014917 28 1 (4) 1 (4) 1 (4)

At2g39090 APC7 Anaphase-promoting complex XLOC_010635 32 1 (3) 1 (3) 1 (3)

At1g77600 T5M16.19 ARM repeat superfamily XLOC_006854 35 1 (3) 2 (6) 3 (9)

At5g21326 Ca2+ serine-threonine protein kinase XLOC_022143 39 1 (3) 2 (5) 2 (5)

At1g02080 T7I23.15 Transcription regulator XLOC_000063 55 1 (2) 1 (2) 1 (2)


143

Table 3.4. Genes whose transcript expression ratios are significantly different in all three genotypes. Wild type, WT; scl30a-1, KO; and SCL30a.1OX1, OX. The relative transcript level is the average obtained from two replicates.

Cufflinks ID % of relative transcript level (difference to the WT) Locus ID Gene Description

Gene Transcript KO WT OX

At5g09820.2 MYH9.3 Plastid-lipid associated XLOC_ 024729

TCONS_ 00082689

94.8 (63.3) 31.4 0.4 (-31.0)

At5g20410.1 MGD2 Monogalactosyldiacylglycerol synthase; response to phosphate starvation

XLOC_ 022082

TCONS_ 00074537

93.7 (44.6) 49.1 25.7 (-23.4)

At5g65300.1 MNA5.2 Unknown protein XLOC_ 027226

TCONS_ 00076766

89.4 (37.9) 51.5 6.5 (-45.0)

At4g26690.1 SHV3 Phosphodiesterase; cell wall cellulose accumulation and pectin linking

XLOC_ 020033

TCONS_ 00057545

73.5 (34.6) 38.8 6.7 (-32.2)

At1g05380.2 T25N20.3 Regulation of transcription XLOC_ 000249

TCONS_ 00014801

78.8 (31.1) 47.8 26.9 (-20.9)

At2g40340.1 DREB2C Transcription factor; response to ABA, drought

XLOC_ 010714

TCONS_ 00026106

88.6 (28.9) 59.7 38.8 (-21.0)

At3g59530.2 LAP3 Calcium-dependent phosphotriesterase superfamily; strictosidine synthase

XLOC_ 013954

TCONS_ 00041871

65.1 (25.4) 39.7 3.3 (-36.3)

At4g24040.1 TRE1 Trehalase XLOC_ 017818

TCONS_ 00052512

68.7 (25.4) 43.3 21.9 (-21.4)

At5g67030.1 ABA1 First step of ABA synthesis XLOC_ 024172

TCONS_ 00072339

93.8 (24.7) 69.1 18.3 (-50.7)

At1g76890.2 GT2 Regulation of transcription XLOC_ 006818

TCONS_ 00014563

63.9 (24.5) 39.4 19.3 (-20.2)


144

At3g18040.2 MPK9 Similar to MAP kinases; ABA signaling XLOC_ 012336

TCONS_ 00048402

54.9 (24.1) 30.8 4.5 (-26.3)

At4g16470.1 DL4260C Tetratricopeptide repeat (TPR)-like superfamily

XLOC_ 019566

TCONS_ 00060915

68.1 (23.6) 44.5 12.2 (-32.3)

At1g26840.1 ORC6 DNA binding; inititation of DNA replication XLOC_ 001465

TCONS_ 00001503

97.9 (22.7) 75.3 28.6 (-46.7)

At2g23348.1 Unknown protein XLOC_ 009840

TCONS_ 00033042

79.4 (21.9) 57.4 26.8 (-30.7)

At5g57740.1 XBAT32 Ubiquitin ligase; negative regulation of ethylene synthesis

XLOC_ 026786

TCONS_ 00073741

11.6 (-20.4) 32.0 55.2 (23.2)

At1g07870.1 F24B9.4 Serine-threonine protein kinase XLOC_ 004192

TCONS_ 00004163

24.5 (-21.1) 45.6 79.6 (34.0)

At2g20650.1 F23N11.3 RING/U-box superfamily XLOC_ 009714

TCONS_ 00027954

13.7 (-21.9) 35.6 65.6 (30.1)

At5g52290.1 SHOC1 Similar to XPF endonucleases; meiotic recombination

XLOC_ 026480

TCONS_ 00069694

44.1 (-22.6) 66.7 96.5 (29.7)

At1g50240.2 FU Serine-threonine protein kinase; cytokinesis

XLOC_ 002145

TCONS_ 00018358

8.8 (-23.3) 32.2 66.1 (34.0)

At1g05380.2 T25N20.3 Regulation of transcription XLOC_ 000249

TCONS_ 00000261

20.0 (-24.8) 44.8 68.1 (23.3)

At1g20780.1 SAUL1 Ubiquitin ligase; negative regulation of ABA synthesis

XLOC_ 001206

TCONS_ 00017995

6.4 (-24.9) 31.3 58.6 (27.3)

At4g14965.1 MAPR4 Membrane-associated progesterone binding protein

XLOC_ 017349

TCONS_ 00052042

13.7 (-25.6) 39.3 94.6 (55.4)

At3g56250.1 F18O21.210

Unknown protein XLOC_ 013738

TCONS_ 00041762

23.6 (-26.1) 49.7 75.5 (25.8)

At1g61100.1 F11P17.17 Disease resistance XLOC_ 006075

TCONS_ 00006165

5.2 (-27.3) 32.4 64.8 (32.4)


145

At5g26010.1 T1N24.8 PP2C family XLOC_ 025579

TCONS_ 00068843

21.5 (-27.6) 49.1 83.5 (34.3)

At4g13650.1 F18A5.40 Pentatricopeptide repeat (PPR) superfamily

XLOC_ 019402

TCONS_ 00054138

16.5 (-28.1) 44.6 71.4 (26.8)

At3g18040.1 MPK9 Similar to MAP kinases; ABA signaling XLOC_ 012336

TCONS_ 00035931

8.5 (-29.1) 37.6 93.2 (55.5)

At5g06710.1 HAT14 Regulation of transcription XLOC_ 024552

TCONS_ 00067783

10.9 (-36.0) 46.9 73.3 (26.4)

At5g65300.1 MNA5.2 Unknown protein XLOC_ 027226

TCONS_ 00073962

10.6 (-37.9) 48.5 93.5 (45.0)

At5g09820.2 MYH9.3 Plastid-lipid associated XLOC_ 024729

TCONS_ 00082690

1.2 (-66.0) 67.3 98.7 (31.4)

Table 3.5. Genes up- or down-regulated at least two fold in the KO (scl30a-1) and showing significant changes in transcript expression ratios between the OX (SCL30a.1OX1) and the WT (wild type), or vice-versa. The fold change and the relative transcript level are averages obtained from two replicates.

Average Fold

Change Cufflinks ID

% of relative transcript level

(difference to the WT)

Locus ID Gene Description

KO VS WT Gene Transcript WT OX

At5g38720 MKD10.4 Unknown protein 4.938 XLOC_ 022640

1. TCONS_ 00065824 35.7 56.4 (20.7)


146

1. TCONS_ 00072399 82.1 29.8 (-52.3)

At5g02800 F9G14.110

Protein serine/threonine kinase activity 2.163 XLOC_

024307 1. TCONS_ 00078207 6.8 66.3 (59.5)

2. TCONS_ 00056739 6.8 32.5 (25.7)

At4g35560 DAW1 Transducin/WD40 repeat-like superfamily; pollen sperm cell differentiation

2.069 XLOC_ 018600

2. TCONS_ 00062103 48.7 22.8 (-25.9)

2. TCONS_ 00056662 5.0 32.9 (27.9)

At4g33925 SSN2 Suppressor of SNI transcription factor; immune responses and homologous recombination

-2.107 XLOC_ 018506

2. TCONS_ 00060498 40.7 9.5 (-31.2)

At4g32560 L23H3.40 Paramyosin-related -2.174 XLOC_

018401 3. TCONS_ 00056616 11.7 35.5 (23.8)

At5g36790 F5H8.7 Haloacid dehalogenase-like hydrolase -2.450 XLOC_

025755 3. TCONS_ 00083023 47.9 21.5 (-26.3)

At3g14510 MOA2.15 Polyprenyl synthetase -2.840 XLOC_ 014936

1. TCONS_ 00038523 37.5 2.8 (-34.7)

1. TCONS_ 00052509 96.1 63.1 (-33.0)

At4g24010 CSLG1 Cellulose synthase like -3.517 XLOC_ 017815 1. TCONS_

00058446 2.8 31.1 (28.3)

At4g15760 MO1 Similar to monooxygenases known to degrade salicylic acid -3.542 XLOC_

019521 2. TCONS_ 00057243 11.5 40.0 (28.5)


147

2. TCONS_ 00059145 28.1 48.4 (20.3)

1. TCONS_ 00063751 60.4 11.6 (-48.8)

At5g24670 TAD3/

EMB2820

Unknown protein -4.935 XLOC_ 025532

2. TCONS_ 00068784 6.1 37.0 (30.9)

1. TCONS_ 00038466 9.2 36.0 (26.8)

At3g13570 SCL30a 30kD SC35-like splicing factor -10.236 XLOC_ 014877 1. TCONS_

00045020 49.6 21.7 (-27.9)

1. TCONS_ 00037393 0.4 40.0 (39.6)

At3g57870 SCE1 SUMO conjugating enzyme; responses to ABA -23.303 XLOC_

013838 1. TCONS_ 00044614 99.5 43.0 (-56.5)

Locus ID Gene Description Average

Fold Change

Cufflinks ID % of relative

transcript level (difference to the

WT) OX VS

WT Gene Transcript WT KO

At3g51640 T18N14.20 Unknown protein 6.052 XLOC_

016056 2. TCONS_ 00043134 63.2 91.6 (28.4)

1. TCONS_ 00022616 67.7 42.9 (-24.8)

At2g07728 T5E7.16 Unknown protein 5.075 XLOC_ 007305 1. TCONS_

00030691 32.3 57.1 (24.8)


148

1. TCONS_ 00003276 67.4 18.3 (-49.1)

At1g73340 T9L24.44 Cytochrome P450 superfamily 4.265 XLOC_ 003287 1. TCONS_

00003278 32.6 81.7 (49.1)

1. TCONS_ 00054963 57.9 13.9 (-44.1)

At4g29080 PAP2/ IAA27

Phytochrome-associated; auxin responsive; regulation of transcription and translation

3.796 XLOC_ 020198

1. TCONS_ 00057611 11.6 76.2 (64.6)

1. TCONS_ 00045020 49.6 11.9 (-37.7)

At3g13570 SCL30a 30kD SC35-like splicing factor 3.043 XLOC_ 014877 1. TCONS_

00045021 6.0 39.6 (33.6)

1. TCONS_ 00033484 24.3 87.7 (63.4)

At2g01530 MLP329/ZCE2

MLP-like; copper ion binding and response to biotic stimulus 2.966 XLOC_

007112 1. TCONS_ 00033485 75.7 12.3 (-63.4)

1. TCONS_ 00069694 66.7 44.1 (-22.6)

At5g52290 SHOC1 Similar to XPF endonucleases; related to meiotic recombination 2.683 XLOC_

026480 1. TCONS_ 00079034 33.3 55.9 (22.6)

1. TCONS_ 00031565 8.8 49.7 (40.9)

At2g21280 GC1/SULA Plastid-targeted 2.455 XLOC_

009751 1. TCONS_ 00033000 34.8 5.1 (-29.7)


149

1. TCONS_ 00001503 75.3 97.9 (22.7)

At1g26840 ORC6 Origin recognition complex subunit; initiation of DNA replication

2.197 XLOC_ 001465

1. TCONS_ 00012047 24.7 2.1 (-22.7)

1. TCONS_ 00022623 40.8 4.1 (-36.7)

At2g07680 ABCC13/MRP11

MRP subfamily; ATPase activity, coupled to transmembrane movement of substances

2.125 XLOC_ 007309

1. TCONS_ 00030698 20.2 52.1 (31.9)

2. TCONS_ 00068784 6.1 61.1 (55.0)

At5g24670 TAD3/

EMB2820

Unknown protein -3.938 XLOC_ 025532 2. TCONS_

00080903 42.7 10.9 (-31.7)

1. TCONS_ 00066339 25.7 3.0 (-22.7)

At5g48830 K24G6.16 Unknown protein -6.328 XLOC_

023146 1. TCONS_ 00071764 74.3 97.0 (22.7)

1. TCONS_ 00037393 0.4 66.4 (66.0)

At3g57870 SCE1 SUMO conjugating enzyme; responses to ABA -45.618 XLOC_

013838 1. TCONS_ 00044614 99.5 19.0 (-80.5)


150

Figure 3.9. Classification of the SCL30a targets in seeds germinating under salt stress according to their (A) molecular function and (B) involvement in biological processes. The numbers indicate the relative contribution of each subgroup (%) to the total set of SCL30a targets.


151

Altogether, these results substantiate a function for SCL30a in

regulating gene expression and particularly pre-mRNA splicing. The

deep-sequencing approach also allowed detecting novel splice

variants not previously annotated. Finally, from the 104 genes

isolated it is likely that only some are effectively direct targets of the

SR protein, while others will result from an indirect effect of the

altered SCL30a levels in the genotypes examined. Future work will

address the identification of direct splicing targets of the SCL30a SR

protein and their validation as functional targets.

3.3.7. The SCL33 and SCL30a duplicated gene pair displays

similar expression patterns in Arabidopsis

The structurally-conserved protein domains within SR protein

subfamilies result in high degrees of similarity among several splicing

factors. Moreover, from the 18 SR proteins in Arabidopsis, 12 have

duplicated pairs among the family (Kalyna and Barta, 2004). Given

these two observations, we were interested in studying possible

functional redundancies between SCL30a and other proteins of the

SCL subfamily. By BLAST analysis, and as presented in Table 3.6,

we found the SCL33 SR protein to be the closest member to SCL30a,

showing the highest levels of identity and similarity among the SCL

subfamily. This result was expected as the two genes likely arose

from a genome duplication event (Kalyna and Barta, 2004).

To start assessing potential functional redundancy between

these two SR proteins, we first examined the SCL33 expression

pattern. RT-PCR analyses of tissue-specific SCL33 expression (Fig.

3.10A) indicated that it is similar to that of SCL30a (see Figure 3.1A),


152

except during the earliest stages of seedling development, where

SCL33 expression was undetectable.

Table 3.6. BLAST homology between SR members of the SCL plant-specific subfamily. Score hits obtained by BLAST search (% identity / % similarity).

SCL SCL33 SCL30a SCL30 SCL28

SCL33

SCL30a 75/82

SCL30 57/76 58/76

SCL28 57/72 56/73 54/73

Using the GUS reporter gene assay, we found the SCL33

promoter to be active in various Arabidopsis tissues (Fig. 3.10B-I),

again similarly to what we observed for the SCL30a gene (see Figure

3.1B-N). Nevertheless, a difference in the activity of the two

promoters was visible in leaves: as seen in Figure 3.10C, the SCL33

promoter appears to be highly active in stomata, a feature that was

not observed for the SCL30a promoter. Finally, although SCL33

expression was not detected during the earliest developmental stages

by RT-PCR, the analysis with the GUS reporter points to SCL33

promoter activity in embryonic tissues and germinating seeds (Fig.

3.10G-I), as also observed for SCL30a.

Importantly, we were able to isolate a homozygous T-DNA

insertion line in SCL33 (Figs. 3.11A and B), scl33-1, in which

expression of the full-length SCL33 transcript was abolished (Fig.

3.11C). This mutant line was then crossed with the scl30a-1 mutant,

allowing the isolation of a double mutant homozygous line for both

SCL30a and SCL33 (Fig. 3.11D). Preliminary phenotypical analyses


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Figure 3.10. SCL33 expression pattern and promoter activity in Arabidopsis. (A) RT-PCR analysis of SCL33 transcript levels in vegetative tissues of wild-type (Col-0) plants. The cyclophilin (ROC1) gene is shown as a loading control. (B) to (I) Histochemical observation of GUS activity in transgenic Col-0 plants carrying the SCL33 promoter-GUS fusion construct. ProSCL33:GUS expression in 3-week old seedlings (B), the leaf epidermis (C), the primary root tip (D), a lateral root (E), mature and immature flowers (F) the embryo (G) and testa (H) from imbibed mature seeds, and seeds germinated for 2-3 d (I). Bar = 100 µM.

indicate that the scl33-1 single mutant is not impaired in germination

under control conditions, or in the presence of exogenous ABA, high

salinity or osmotic stress. Characterization of the scl30a-1scl33-1

double mutant is still to be initiated.


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Figure 3.11. SCL33 gene structure and isolation of the scl33-1 loss-of-function mutant. (A) Schematic representation of the SCL33 gene showing the site of insertion and orientation of the T-DNA in the scl33-1 mutant (boxes indicate exons with UTRs in grey, lines between boxes represent introns, and arrows indicate the location of SCL33- and T-DNA-specific primers). (B) PCR-based genotyping of an scl33-1 homozygous line. The location of the F2, R2 and LBc primers is shown in (A). (C) RT-PCR analysis of SCL33 transcript levels in wild-type (Col-0) and mutant (scl33-1) 5-d old seedlings using primers flanking the T-DNA insertion. The location of the F2 and R3 primers is shown in (A). The ubiquitin 10 (UBQ10) gene was used as a loading control. (D) PCR-based genotyping of a scl30a-1scl33-1 double mutant homozygous line. The location of the SCL33 F2 and R2, SCL30a F3 and R2, and the LBc primers is shown in (A) and in Figure 3.2A.

3.4. Discussion

The Arabidopsis SR protein family of essential splicing

regulators comprises 18 members (Barta et al., 2010), but few

functional studies have addressed their roles during plant growth and

development or in stress responses. The work presented here

demonstrates that SCL30a, belonging to plant-specific SCL-subfamily


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of SR proteins, is a negative regulator of the ABA pathway and

controls splicing of several genes in Arabidopsis.

Previous studies on Arabidopsis SR proteins have mainly

addressed protein-protein interactions and in vivo subcellular

localization. The Arabidopsis SCL33 and the SR-related SR45 factor

interact with the splicesosome U1-70K snRNP (Golovkin and Reddy,

1999). RS2Z33 interacts with several members of the SC35-like

(SCL) subfamily, including SCL30a (Lopato et al., 2002). In general,

SR proteins are found in nuclear speckles (Lorkovic et al., 2004;

Tillemans et al., 2005), shuttling to the nucleoplasm in dynamic

movements correlating with transcriptional spots and chromatin

organization (Fang et al., 2004; Tillemans et al., 2006; Lorkovic et al.,

2008). On the other hand, the two reported functional studies on

Arabidopsis SR proteins showed that increased SR30 and RS2Z33

levels cause altered splicing patterns of other SR genes and general

growth abnormalities (Lopato et al., 1999; Kalyna et al., 2003). By

characterizing the first loss-of-function mutant of an SR protein gene,

we demonstrate that SCL30a negatively regulates ABA signaling

during repression of germination under high salinity and osmotic

stress, relying also on the ABA pathway to control two seed-specific

traits, size and dormancy (Fig 3.12). Overexpression of the full-length

SCL30a protein reverts the mutant phenotypes and produces larger

seeds, which are also less sensitive to salt and osmotic signals during

germination.

The plant hormone ABA regulates numerous processes such

as seed maturation and germination, lateral root development,

transition from the vegetative to the reproductive phase, and abiotic

and biotic stress responses (reviewed in Cutler et al., 2010 and

Hubbard et al., 2010). ABA cell-surface receptors were recently


156

uncovered (Miyazono et al., 2009; Park et al., 2009) and many

genetic components acting on this hormone signaling pathway have

been identified, the large majority of which is associated with

transcriptional regulation (reviewed in Cutler et al., 2010).

Figure 3.12. SCL30a negatively regulates the ABA pathway in embryonic tissues and during early seedling development. Biological roles identified for the Arabidopsis SCL30a protein. Green and red lines indicate inductive and repressive signals, respectively. Blue dashed lines represent hypotheses still requiring experimental validation.

In seeds challenged with drought and/or high salinity stress

signals, the endogenous levels of the hormone increase and lead to a

delay, or ultimately a total arrest, of germination. Disruption of

SCL30a expression results in a hypersensitive response to these

stress conditions, in contrast to the overexpression of the SR protein,

which accelerates germination when compared to the wild type.

Blocking ABA synthesis, by exogenously applying fluridone, and

epistatic analyses between SCL30a and the ABA biosynthesis-related

gene, ABA2, and the ABA signalling gene, ABI4, showed that

SCL30a acts on the ABA pathway to negatively control the inhibition

of germination under drought and salt stress. As the three genotypes


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with different SCL30a expression revealed similar fold changes in

ABA levels in response to stress, the germination phenotypes do not

appear to result from different ABA endogenous contents and,

therefore, SCL30a likely acts on the ABA pathway to repress

signaling of the hormone instead of downregulating its levels.

Furthermore, the increased SCL30a expression in ABA-related

mutants suggests that ABA may in turn regulate SCL30a levels,

controlling the activity of the SR protein via a negative feedback

mechanism. Interestingly, SCL30a is not essential for germination per

se, as mutant and overexpressor seeds show ABA levels and

germination rates similar to the wild type under control conditions.

During seed development, ABA plays essential roles in

maturation and establishment of primary dormancy (reviewed in

Finkelstein et al., 2008 and Holdsworth et al., 2008). ABA

accumulates at the end of seed maturation to prevent early

germination and lowers to basal levels during after-ripening, allowing

the initiation of germination upon sensing of adequate external

conditions. In addition to ABA, many genetic components and

different hormones, such as gibberellins, cytokinins or ethylene, have

been implicated in the control of seed dormancy, but our epistatic

analyses demonstrate that induction of the dormant state by SCL30a

requires an intact ABA pathway. Measurement of endogenous ABA

levels was only performed in mature seeds. Although the wild type,

the slc30a-1 mutant and an SCL30a overexpression line contained

similar levels of the hormone in mature seeds at the onset of

germination under control conditions, which could suggest that

SCL30a acts at the level of ABA signaling to control seed maturation,

it cannot be excluded that SCL30a acts via different mechanisms in

dormant and in mature seeds. We also report a role for ABI4 in the


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control of dormancy. It is likely that the absence of altered dormancy

levels in abi4 single alleles results from functional redundant activities

of other ABI-related transcription factors.

Control of seed size promotes efficient seed dispersal and,

together with controlled size dormancy, is fundamental in ensuring

species survival. Indeed, considerable efforts have been directed

towards the selection of larger seeds as a means of improving crop

yields (Shomura, 2008). SCL30a appears to enhance seed size by

repressing the ABA pathway. Interestingly, we also report here a

novel phenotype for the aba2-1 mutant, which displays larger seeds

than the wild type. Although the abi4-101 mutant does not exhibit

aberrant seed size, the ABI4 transcription factor is also required for

proper control of seed size by SCL30a. The absence of seed size

phenotypes in the abi4-101 background may result from the activity of

redundant genes compensating for the loss of the ABI4 transcription

factor activity, similarly to what was suggested above for the

dormancy phenotype. The impact of ABA in seed size regulation is

less known than its role in abiotic stress responses or seed dormancy

control. One study has reported that the ubiquitin receptor DA1 gene

is induced by ABA and disruption of its expression results in aberrant

seed and organ sizes as well as reduced sensitivity to ABA (Li et al.,

2008). However, DA1 seems to act independently of ABI4 and ABI5,

two fundamental transcription factors transducing ABA signaling (Li et

al., 2008). The final size of a seed depends on the differential

contribution of imprinted parental and maternal factors inherited upon

fertilization. Increased paternal genome dosage delays endosperm

development, increasing its size, as well as the final seed size. In

contrast, higher maternal genome dosage delays cellularization of the

peripheral endosperm and hypertrophy of the chalazal endosperm,


159

resulting in smaller seeds (Scott et al., 1998). Future work should

investigate the influence of the parental genomes on the size of

scl30a-1 mutant seeds, through reciprocal crosses of the wild type

and the mutant line.

Intriguingly, we also uncovered a role for SCL30a in the

repression of hypocotyl elongation by restricted light conditions during

early seedling development. It would be interesting to examine

whether the SR protein also acts on the ABA pathway to repress

photomorphogenesis. Although the potential function of this hormone

in light responses has been poorly addressed, ABA has been shown

to play a role in these responses in young seedlings. For example,

transgenic Arabidopsis lines overexpressing the ABA signaling

transcription factor ABI5 have been reported to exhibit, similarly to the

scl30a-1 mutant, exaggerated responses to red, far-red and blue light

(Chen et al., 2008). Interestingly, links between light responses and

the control of seed size have also been uncovered. A gain-of-function

allele in the short hypocotyl under blue 1 (SHB1) displays longer

hypocotyls under blue, far-red and red light, whereas its knockout

exhibits shorter hypocotyls under blue light (Kang and Ni, 2006). The

mutant also produces smaller seeds than the wild type, in contrast to

the larger seeds of overexpression line, and no obvious phenotypes

during adult growth (Zhou et al, 2009). Nevertheless, it is proposed

that SHB1 controls seed size and light responses via independent

pathways (Zhou et al, 2009).

The Arabidopsis SCL30a was found to be ubiquitously

expressed in vegetative tissues throughout development and to

generate, under our experimental conditions, three splice variants by

alternative splicing. These observations are consistent with previous

data showing SCL30a expression in different Arabidopsis tissues


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(Palusa et al., 2007), and the production of multiple alternative

transcripts (Palusa et al., 2007; Zhang et al., 2011), although to date

only one has been annotated (www.arabidopsis.org). In light of the

SCL30a ubiquitous expression but seed- and early seedling-specific

activity, we have also addressed the hypothesis of functional

redundancy between the SCL30a splicing factor and other

Arabidopsis SR proteins. Among the SCL-subfamily members, the

SCL33 protein is the closest to SCL30a (Kalyna and Barta, 2004) and

hence we initiated its characterization. In general, SCL33 exhibited a

similar expression pattern to that observed for SCL30a, except for the

GUS coloration in stomata, where an intense signal was visualized for

SCL33 but not for SCL30a. The crucial role of ABA in regulating

stomatal aperture (reviewed in Cutler et al., 2010 and Hubbard et al.,

2010) and the fact that the abolishment or enhanced expression of

SCL30a did not substantially interfere with the rates of water loss

from leaves suggest that future work should aim at investigating

whether SCL33 compensates for the loss of SCL30a activity in

leaves.

We have shown that the SCL30a transcript encoding the full-

length protein is sufficient to rescue the impaired seed size, dormancy

and germination under drought and salt stress displayed by the

scl30a-1 mutant. The other two splice variants contain PTCs, a

feature that has been identified in almost all members of the

Arabidopsis SR gene family undergoing alternative splicing (Palusa et

al., 2007). Although the presence of PTCs suggests degradation by

NMD, only roughly half of these PTC-containing transcripts seem to

be indeed targeted to degradation by this mRNA quality control

mechanism (Palusa and Reddy, 2010). Future work on the two

alternative SCL30a splice variants will be required to establish


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whether the inclusion of PTCs leads to their degradation via NMD or

results in the expression of truncated isoforms containing portions of

the RRM domain. Alternative splicing of an SR protein gene could

provide a means of rapidly adapting its expression levels upon a

specific condition, and putatively existent truncated protein forms

could compete with the full-length splicing factor in binding (a) specific

pre-mRNA target(s), thus influencing the splicing reaction.

The SCL30a expression and splicing pattern is unaffected by

exogenous application of ABA, salt or mannitol (this work, Palusa et

al., 2007 and Zhang et al., 2011). This suggests that, under these

conditions, SCL30a activity is not regulated at the

transcriptional/posttranscriptional levels, but rather at the

posttranslational level. The activity of SR proteins is well known to be

regulated by phosphorylation of the RS domain (Graveley, 2000; de

la Fuente van Bentem et al., 2006; Tillemans et al., 2006) and

therefore the phosphorylation status of SCL30a, under control and

stress conditions or at different developmental time points, should be

investigated in the future. In addition, members of the SCL-subfamily

of SR proteins possess, besides the single RRM and RS domains, an

N-terminal extension rich in arginine, proline, serine, glycine and

tyrosine residues (Barta et al., 2010), which is also a candidate for

posttranslational modifications. Indeed, the floral initiator Shk1 kinase

binding protein 1/protein arginine methyltransferase 5 (SKB1/PRMT5)

suppresses expression of the flowering locus C (FLC) through histone

H4R3 symmetric dimethylation, promoting flowering, but also

methylates the U6 small ribonucleoprotein-specific Sm-like protein

LSM4 (Zhang et al., 2011), which modulates its activity during splicing

(Deng et al., 2010; Sanchez et al., 2010; Zhang et al., 2011). In

addition, mutations in SKB1 result in hypersensitivity to salt and


162

severe splicing defects (Deng et al., 2010; Sanchez et al., 2010;

Zhang et al., 2011). These recent reports shed light on the regulation

of spliceosome-related proteins, which can include SR proteins,

strengthening the notion that the regulation of pre-mRNA splicing is

tightly related to transcription, chromatin remodelling and epigenetic

markers (Anastasiadou et al., 2011; Zhang et al., 2011). Interestingly,

the ABA pathway is also known to regulate several biological

processes, including seed maturation and abiotic stress responses,

through epigenetic processes (reviewed in Chinnusamy et al., 2008),

supporting the notion that potential modifications of arginine residues

at the N-terminal domain of SCL30a should be investigated.

A global analysis on the transcriptome of three lines with

different SCL30a expression levels showed that this SR protein is

involved in regulating splicing. Under high salinity conditions, a large

majority of the SCL30a targets are associated to RNA or DNA

metabolism and are involved in biological processes such as seed

development or responses to stress signals, including to ABA.

Interestingly, from the few studies in the literature reporting the

functional characterization of individual alternative splicing events in

Arabidopsis, a considerable number has shown the activity of the

resulting splice isoforms in seeds and/or responses to ABA. For

instance, the two splice forms of the transcription factor encoded by

the phytochrome interacting factor 6 (PIF6) gene have distinct roles in

the control of seed dormancy and in regulating responses to specific

light conditions (Penfield et al., 2010). Alternative splicing of ABI3 is

developmentally-regulated and generates two isoforms which act as

transcription factors and compete for repressing and inducing

germination after seed maturation (Sugliani et al., 2010). Another

example is the SR45 gene, which encodes a splicing factor that


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generates two transcripts by alternative splicing (Ali et al., 2007).

While the longer isoform is active in flowers, the second regulates

root growth (Zhang and Mount, 2009). Interestingly, both protein

isoforms are active in responses to ABA and sugars during early

seedling development (Carvalho et al., 2010).

Based on a three-step approach, future work should aim at

validating, within the genes found by deep-sequencing, direct targets

of the SCL30a protein. The rationale of this validation will firstly

involve the selection of the most relevant genes whose molecular

function or associated biological process can further help in

pinpointing the role of SCL30a in the ABA pathway. A good

candidate, for instance, is the DREB2C gene (Table 3.4), which

encodes a transcription factor involved in drought responses and

whose expression is induced by salt and mannitol (Lee et al., 2010).

Overexpression of the sole DREB2C splice variant annotated has

been reported to result in hypersensitivity to ABA during germination

an to increased water loss at adult stages (Lee et al., 2010).

Interestingly, our deep-sequencing results allowed identifying a novel

alternative splicing event in this gene, and a higher relative

expression ratio for the referred transcript in the scl30a-1 mutant,

whilst lower in the SCL30a.1OX1 line, when compared to the wild

type. Another potential candidate is SAUL1/PUB44 (Table 3.4), which

has only one transcript annotated, encoding an E3 ligase acting on

the ABA pathway during germination upon stress but also at later

stages of growth, such as in adult leaves (Salt et al., 2011). On the

other hand, the PEX10 gene (Table 3.3) has no alternative splicing

events annotated and encodes a protein known to be active in protein

import into the peroxisome and to regulate embryogenesis (Sparkes

et al., 2003). The sumo conjugating enzyme 1 (SCE1) encoding gene


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is also among the SCL30a targets (Table 3.5). SCE1, whose

alternative splicing has not been reported, presumably plays a role

during seed development and the establishment of dormancy, having

been reported to regulate ABA responses in roots (Lois et al., 2003).

Surprisingly, the ABA1 gene, a component of ABA biosynthesis

(Koornneef et al., 1982), which has two splice forms annotated, was

also retrieved from the deep-sequencing analysis (Table 3.4), and

despite our assumption that SCL30a does not directly interfere with

the levels of the ABA hormone, it may be worthwhile to include ABA1

in the validation tests.

Following selection of the most relevant candidates, validation

by RT-PCR of their altered splicing/expression profiles in the three

genotypes with varying SCL30a levels should be carried out. Lastly, it

will be important to confirm direct RNA-protein interactions via RNA

immunoprecipitation assays. The validation by semi-quantitative RT-

PCR may not be effective when detecting small differences in

expression or altered splice junctions that result in transcripts with

very similar length. However, it may provide evidence of considerably

altered splicing ratios or relative gene expression levels. On the other

hand, while quantification by real time RT-PCR may be more

accurate, it may sometimes be unable to provide information on

alternative splicing changes, depending on the structure of the gene’s

splice variants in question.


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3.5. Materials and Methods

3.5.1. Plant materials and growth conditions

The Arabidopsis thaliana Columbia (Col-0) ecotype was used

in this study. Seeds were surface-sterilized for 10 minutes in 50%

(v/v) bleach and 0.07% (v/v) Triton X-100 under continuous shaking,

washed 3 times with sterile water and stratified for 3 d at 4ºC in the

dark. Seeds were then plated on media containing 1 x Murashige and

Skoog (MS) salts (Duchefa Biochemie), 2.5 mM MES (pH adjusted to

5.7 with 1M KOH), 0.5 mM myo-inositol and 0.8% (w/v) agar and

transferred to a growth chamber under long day (16 h light; 90

µmol.m-2.s-1 white light) conditions, at 60% RH and 22ºC (light period)

and 18ºC (dark period). For adult growth, individual plants were

transferred to pots after 2 to 3 weeks.

The T-DNA insertion lines, SALK_041849 and SALK_058566,

in SCL30a (At3g13570) and in SCL33 (At1g55310), respectively,

were obtained from the SALK collection (http://signal.salk.edu). While

the first line is sensitive to kanamycin, the latter is resistant to this

antibiotic. Table 3.7 lists the primers used both for confirmation of the

T-DNA insertion site and for genotyping of the isolated scl30a-1 and

scl33-1 mutants. The aba2-1 (Leon-Kloosterziel et al., 1996) and

abi4-101 (Laby et al., 2000) mutants were isolated previously, as well

as phyA-211 (Nagatani et al., 1993), phyB-9 (Reed at al., 1993) and

cry1/cry2 (Ahmad et al., 2002).

The scl30a-1 mutant was crossed with aba2-1 and abi4-101,

containing point mutations, and double mutants isolated using

primers listed in Table 3.7. The LB primer was used for genotyping of

the scl30a-1 allele, whereas both the aba2-1 and abi4-101 alleles


166

were amplified and sequenced. The scl30a-1 mutant was

backcrossed twice with the wild type (Col-0).

3.5.2. Generation of transgenic plants

Genomic DNA was extracted from wild-type Col-0 1-week old

seedlings using a sodium acetate/isopropanol-based protocol. For

GUS reporter studies, a 2206-bp sequence, finishing immediately

upstream of the SCL30a start codon, was amplified using primers 5’-

AGACTTACTAGTTTCTGTAAATTACATAGCCAAAG-3’ (introducing

a SpeI restriction site) and 5’-

AGAGTTCCGCGGTGTGTCTAGACTTTAATATCTATATC-3’ (SacII

restriction site) and subcloned into the pGEM vector. The eGFP-GUS

segment was isolated from the pKGWFS7 vector, using SacII and

NcoI, and cloned at the 3’ of the SCL30a promoter sequence in

pGEM. The promoter-eGFP-GUS segment was then transferred from

pGEM (SpeI / NcoI) into the final pKGWFS7 vector, by substitution of

the CmR-ccdB-eGFP-GUS original cassette. For the SCL33

promoter, the same procedure was followed and primers 5’-

ATCCATACTAGTTGTAGATACCAACAGCTTGACTTG-3’

(introducing a SpeI restriction site) and 5´-

GTACTTCCGCGGGAGTCAAGCTTCAATCTCTCTATC-3’ (SacII

restriction site) were used to clone a 1184-bp sequence finishing

immediately upstream of the SCL33 start codon.

For SCL30a promoter-gene cloning, primers 5’-

AAGACTTCATGATTCTGTAAATTACATAGCCAAAG-3’ and 5’-

TCTGTCTCGAGTTTTCTGCAATTTTTTATTTTATTTC-3’ (BspHI and

XhoI restriction sites, respectively) were used to amplify the first 1649


167

bp of the same sequence used for the GUS reporter construct. This

fragment was cloned into the pBA vector, replacing the 35S promoter.

Primers 5’-TCTGTCTCGAGAACCCTAATGACCCGTTCGC-3’ and 5’-

TCAGAGGCGCGCCACTTGTCTCAGGGTTTGGATTTTG-3’ (XhoI

and AscI restriction sites, respectively) were used to amplify the rest

of the sequence – a 3062-bp sequence starting immediately after the

1649-bp sequence cloned first and finishing 200 bp downstream of

the 3’ UTR. This fragment was then cloned into the pBA plasmid,

after the first cloned segment.

Total RNA was extracted from wild-type Col-0 1-week old

seedlings and first-strand cDNA synthesized using M-MLV Reverse

Trasncriptase (Promega) and a poly-T primer. Primers 5’-

ACAGAGGCGCGCCATGAGAGGAAGGAGCTACAC-3’ and 5’-

AGACATTAATTAATCACTGGCTTGGAGAACGGTC-3’ (AscI and

PacI restriction sites, respectively) were used to amplify the

SCL30a.1 coding sequence and clone it into the pBA vector under the

control of the 35S promoter. Similarly, primers 5’-

ACAGAGGCGCGCCATGAGAGGAAGGAGCTACAC-3’ and 5’-

AAGAGAACGCGTAACATAAACTGGCTTGGAGAAC-3’ (AscI and

MluI restriction sites, respectively) were used to clone the same

sequence into HApBA.

All constructs were transformed into the Agrobacterium

tumefaciens strain EHA105 for floral dip transformation (Clough and

Bent, 1998) of Col-0 wild-type plants with the ProSCL30a:GUS-

pKGWFS7, ProSCL33:GUS-pKGWFS7 and 35S:SCL30a.1-HApBA

constructs or of the scl30a-1 mutant with the ProSCL30a:SCL30a-

pBA and 35S:SCL30a.1-pBA constructs. T1 transformants were

selected on kanamycin (pKGWFS7 vector) or BASTA (pBA

backbone), grown to maturity and selfed. T2 generation plants were


168

used for GUS-reporter fusion and gene expression analyses of pBA-

transformed plants (see Table 3.7 for primers used). Phenotypical

analyses were conducted at the T3 generation.

3.5.3. Gene expression analyses

Total RNA was extracted from the wild-type (Col-0) at different

stages of plant development using either the Tri Reagent (T924;

Sigma-Aldrich), following the manufacturer’s protocol, or the

innuPREP Plant RNA Kit (analytikjena) for extraction from dry,

imbibed and up to 5 days-germinated seeds. RNA samples were then

treated with DNAse I (Promega) and purified by phenol-chloroform

extraction. cDNA was synthesized as described above. Paq5000

DNA polymerase (Stratagene) was used for expression analysis of

SCL30a, SCL33 and the housekeeping genes cyclophilin (ROC1) and

ubiquitin10 (UBQ10). The primers used are listed in Table 3.7. The

number of PCR cycles in a linear range of amplification for semi-

quantitative analysis was determined for each gene (see Table 3.7).

PCR products were separated and visualized in 1.5% agarose gels,

stained with RedSafe (Intron Biotechnology).

For GUS reporter gene analysis, a total of six independent

kanamycin-resistant lines expressing the ProSCL30a:GUS-

pKGWFS7 construct in the wild-type background were selected at the

T1 generation, grown to maturity and selfed. For the ProSCL33:GUS-

pKGWFS7 construct, two lines were isolated. GUS coloration was

assessed at the T2 generation using the X-glucoro (CHA salt,

A1113,0100, AppliChem) substrate. Seeds, seedlings or tissues from

adult plants were incubated in GUS buffer (1 mg.ml-1 X-glucoro, 25


169

mM sodium phosphate buffer, 2 mM ferrocyanide, 2 mM ferricyanide,

0.1 % (v/v) Triton X-100, 1 mM EDTA) overnight at 37°C. Samples

were then washed once with 100 mM sodium phosphate buffer (28.85

mM Na2HPO4, 21.15 mM NaH2PO4), incubated for 1 h in acetic

acid/ethanol (3:1) at room temperature, washed twice with 100 mM

sodium phosphate buffer and blue coloration from the GUS reporter

gene assessed.

To identify SCL30a splice variants, total RNA was extracted

from Col-0 wild-type 1 week-old seedlings and cDNA synthesized as

described above. SCL30a was amplified using the SCL30a primers

F1 and R1 listed in Table 3.7, and the resulting PCR product was

cloned into pGEM. E. coli TOP10 cells were transformed with the

ligation product and a colony-PCR was performed on 50 positive

clones. PCR products were run on agarose gels and colonies

selected based on the size differences of the amplified bands.

Plasmid DNA was extracted from the selected colonies and the

corresponding SCL30a-pGEM sequenced.

3.5.4. Phenotypical analyses

Plants of different genotypes were sown and grown to maturity

simultaneously under the same conditions. For dormancy assays,

seeds from freshly matured siliques were immediately collected from

the tree and directly used for phenotypical analysis. For germination

assays, fully mature siliques from dehydrated plants of the same age

were collected and stored in the dark at room temperature, for at least

one week before phenotypical analysis.


170

Seed size was assessed at different stages of seed

development, by collecting seeds from developing and mature

siliques or by observation of mature seeds collected from dehydrated

plants. The area of dry and imbibed mature seeds was measured

using the ImageJ software (http://rsbweb.nih.gov/ij) and around 50

seeds of each genotype were used in each experiment.

For germination assays, seeds were surface-sterilized and

stratified as described above and plated on MS media supplemented

or not with ABA (mixed isomers, A1049; Sigma), NaCl or mannitol,

with or without addition of fluridone (45511, Fluka Analytical). In each

assay, 70-100 seeds per genotype per plate and three plates per

genotype were used. Petri dishes were then transferred to long-day

condition growth chambers and germination scored by measuring the

percentage of emerged radicles from the seed coat over the total

number of seeds.

For dormancy assays, seeds were surface-sterilized but not

stratified, plated on MS media, and immediately transferred to dark.

Control seeds were stratified, plated and grown under long-day

conditions. The percentage of germination was assessed 7 d after

seeds were sown.

For light response assays, seeds were surface-sterilized,

stratified and plated on MS media. Around 40 seeds were used per

genotype in each assay. Petri dishes were transferred to dark, red,

far-red, or blue light conditions. After 4 d, hypocotyl length was

measured using the ImageJ software.


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Table 3.7. Sequences of the primers used for PCR and RT-PCR analyses.

Name Gene ID Primer name Primer sequence # cycles

F1 5’-TTCCCCTGTGTTTT TCTTCG-3’ 30

R1 5’-CTTTGGCTCCTTGC TTGTTC-3’ 28/30

F2 5’-ATGAGAGGAAGGA GCTACAC-3’ 28/30

R2 5’-CCTAAAGTGACTC GAAGAGGG-3’ 35*

F3 5’-TCTCTTGGTTCGCA ACTTACG-3’ 35*

R3 5’-CTTGCCTGCAATCA TGACGTAAG-3’ 30

SCL30a At3g13570

F4 5’-CTTCCTAGGGATTA CTATAC-3’ 30

F1 5’-CTCCGTCGTTCCTC ACCACCG-3’ 28

R1 5’-GTTCCCCACATGTT CCATAG-3’ 28

F2 5’-TCGGGATAGAAGA CGTACTCC-3’ 35*

R2 5’-ATCACTGGCTTGGT GAACGG-3’ 35*

SCL33 At1g55310

R3 5’-TATGCTTCTTCTAG GGCTGG-3’ 28

rocF 5’-GTCTGATAGAGATC TCACGT-3’ 25

ROC1 At4g38740

rocR 5’-AATCGGCAACAAC AACAGGC-3’ 25

ubqF 5’-GATCTTTGCCGGA AAACAATTGG-3’ 23

UBQ10 At4g05320

ubqR 5’-TAGAAAGAAAGAG ATAACAGG-3’ 23

T-DNA segment LBc1 5’-CAAACAGGATTTT CGCCTGCTGGGG-3’ 35*

* number of cycles used for PCR-based genotyping of the scl30a-1 and scl33-1 insertion lines.


172

3.5.5. Determination of ABA content

Seeds of Col-0, scl30a-1, SCL30.1OX1 and aba2-1 were

sown on 200 mM NaCl and collected at the onset of germination (2 d

after transfer to light) to ensure samples were at the same

developmental stage. Six replicas were performed per genotype, and

within each replica around 300 seeds were used. Collected seeds

were frozen in liquid nitrogen, grounded and homogenized in 1 mL of

ABA-extraction buffer (10 mg.L-1 butylated hydroxytoluene, 20 mL.L-1

acetic acid, 90% methanol). Samples were incubated in the ABA-

extraction buffer overnight at 4°C under continuous shaking, and the

supernatant collected and evaporated to dryness using a speed-vac.

ABA levels were determined using the Phytodetek-ABA-kit (AGDIA

Inc.), following the manufacturer’s protocol. Mixed ABA isomers

(mixed isomers, A1049; Sigma) were used as a standard.

3.5.6. Deep-sequencing

Seeds of Col-0, scl30a-1 and SCL30.1OX1 were sown on 200

mM NaCl and collected at the onset of germination (2 d after transfer

to light) to avoid the effect of differences in developmental stage. Two

replicas were performed per genotype and harvested seeds (300 per

replica) frozen and grounded in liquid nitrogen. Total RNA was

extracted using the innuPREP Plant RNA Kit (analytikjena), digested

with DNAse I and purified with phenol-chloroform, as described

above. The quality of the total RNA was evaluated using the 2100

Bioanalyzer RNA Nano Chip (Agilent Technologies).


173

3.5.6.1 Mapping reads to the Arabidopsis genome

Release 10 of the Arabidopsis genome was downloaded from

The Arabidopsis Information Resource (TAIR) website

(www.arabidopsis.org) and formatted for use as a reference

sequence database using the bowtie-build tool included in the Bowtie

0.12.5 read mapping package (Langmead et al., 2009). Each of the

six libraries was then mapped to the Arabidopsis genome using

Tophat 1.0.14 (Trapnell et al., 2009), which both runs the Bowtie

alignment algorithm and predicts splice junctions between exons,

using the default values for all parameters.

3.5.6.2 Transcript prediction and expression level estimation

The read mapping data for the six libraries was then further

analyzed using Cufflinks 0.8.3 (Trapnell et al., 2010), which groups

the splice junctions identified by Tophat into putative transcripts.

Cufflinks estimates the relative expression levels for each transcript in

fragments per kilobase of exon per million fragments mapped

(FPKM), a unit of expression that is normalized for both transcript

length and library size. Gene loci are also identified as regions

containing one or more overlapping transcripts, and total expression

levels for each gene are calculated as well. For each library, this

analysis produced a description of the structure of all transcripts in

General Transcript Format (GTF) and tabular listings of expression

levels for both genes and individual transcripts.


174

3.5.6.3. Comparison with TAIR annotation and testing for

differential expression

The six lists of predicted transcripts, along with a reference set

of Arabidopsis transcript annotation from TAIR, were then fed into the

program "Cuffcompare" which is part of the Cufflinks package.

Cuffcompare produced a non-redundant listing of transcripts found in

the supplied lists, labelled the predicted genes and transcripts using a

unified naming scheme, and identified their closest match among the

TAIR reference set.

Two statistical analysis programs were used to identify genes

and transcripts showing significant differences in expression levels

between the wild type (WT), the overexpressor (OX), and the

knockout (KO) genotypes: Gene Set Enrichment Analysis (GSEA)

(Subramanian et al., 2005) and Significance Analysis of Microarrays

(SAM) (Tusher et al., 2001). For GSEA, the six samples were divided

into three classes with two replicates each, the transcripts were

grouped by the Gene Ontology annotation of their TAIR matches, and

each transcript’s estimated FPKM values were used in lieu of spot

intensity values. For SAM, two analyses were performed, the first

comparing the OX and WT, and the second comparing the KO and

WT. In both analyses, the data type was set to two class unpaired,

the minimum fold change was 2, and a value of k=5 was used in the

K-Nearest Neighbor missing data imputation algorithm. In order to

obtain 150-200 significant genes in each analysis, the delta value was

adjusted, resulting in a value of 0.8855 being used for the OX/WT

analysis, and 1.7755 for the KO/WT.


175

3.5.6.4. Splice junction analysis

In order to identify differences in mRNA splicing patterns, we

compiled lists of the splice junctions found within each gene in each

of the three genotypes. A junction was only added to the gene’s list

for that genotype if it was found in both replicates. Then, for each

gene, the junctions found in only one genotype were identified. The

total number of splice junctions in each gene was derived from the list

of all transcripts derived from that gene that appeared in both

replicates of at least one genotype.

3.5.6.5 Transcript expression ratio analysis

For each gene, its total expression level in a given genotype

was defined as the sum of the FPKM values for all transcripts

expressed in that genotype. Each transcript’s fraction of the total

expression was then calculated by dividing its FPKM in that genotype

by the gene’s total expression level. A gene was then defined as

having a significant difference in expression ratios in all three

genotypes if it had at least one transcript expressed in all three

genotypes whose fraction of the total expression was at least 20

percentage points higher in the mutant or the overexpressor than in

the wild type, and at least 20 percentage points lower than the wild

type in the other genotypes.


176

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CHAPTER 4 CONCLUDING REMARKS AND FUTURE PERSPECTIVES

Chapter 4 – Concluding remarks and future perspectives

187

4.1. Introduction

Plant growth and development are regulated by a complex

combination of several signaling pathways that integrate internal and

environmental cues. The plastic modulation of these pathways allows

correct responses to different signaling molecules, ensuring

maintenance of plant growth under favorable conditions or survival

when the environment is adverse. Gene expression and its regulation

have major impacts on the control of the signaling pathways that

dictate overall plant growth and development. Alternative splicing

allows the generation of proteomic diversity and functional

complexity, being likely to provide an important molecular tool for

optimal plant growth and development as well as for the response to

external cues.

The most recent estimate indicates that 42% of the

Arabidopsis genes undergo alternative splicing (Filichkin et al., 2010),

and large-scale approaches have proven useful in revealing different

splicing patterns at particular developmental time points or upon

exposure to specific stress conditions (Iida et al., 2004, Tanabe et al.,

2006; Palusa et al., 2007; Filichkin et al., 2010). However, there is still

a striking and evident lack of functional studies clearly demonstrating

the biological role and relevance of alternative splicing in plants. This

is in huge contrast with studies in humans, where 95% of the genes

have been shown to undergo alternative splicing (Pan et al., 2008;

Wang et al., 2008), and where it has been documented that this

mechanism of gene expression regulation plays important roles

during development, having a major impact in many diseases

(reviewed in Kalsotra and Cooper, 2011), such as cancer (Karni et al.,

2007) or Alzheimer’s (Twine et al., 2011).


188

The overall goal of the present work was to gain further insight

into the biological meaning of alternative splicing in plants, using

Arabidopsis thaliana as the model organism. As referred by Kazan

(2003), studies on alternative splicing in plants should focus on four

main subjects: identifying alternative splicing patterns by large-scale

approaches, reporting developmental- and stress-specific cues that

influence splicing, characterizing the splicing reaction while dissecting

the methods of splice site selection, and analyzing individual

alternative splicing events by functionally characterizing the produced

splice variants. Almost ten years later, the first two issues have since

then been widely studied while the other two were poorly addressed.

In agreement with this, a two-step approach was designed and put

forward in the presented thesis. Firstly, as described in Chapter 2, an

Arabidopsis gene undergoing alternative splicing was functionally

analyzed and the physiological relevance of its two splice forms

studied. Secondly, as presented in Chapter 3, the in vivo role of an

important plant-specific splicing factor, belonging to the Arabidopsis

SR protein family that plays key roles in the regulation of alternative

splicing, was investigated. Moreover, in both approaches, efforts to

examine potential functional redundancies between duplicated gene

pairs were also initiated.

4.2. Alternative Splicing of the XBAT35 RING E3 Ligase

The selected case study of an alternatively-spliced pre-mRNA

was that of the XBAT35 gene, from the Arabidopsis XBAT family of

RING E3 ligases. E3 ligases provide specificity to the ubiquitination

pathway, a fundamental process for protein turnover by the 26S

proteasome and the maintenance of cellular homeostasis during


189

several biological processes. We were able to characterize two splice

variants generated by the XBAT35 gene, which are both ubiquitously

expressed under the conditions tested. An exon skipping event in the

XBAT35 pre-mRNA excludes a nuclear localization signal from the

shorter variant and targets the encoded protein to the cytoplasm,

whereas the constitutively-spliced transcript produces a nuclear

protein. In plants, very few reports exist in which alternative splicing is

found to control protein targeting to different subcellular

compartments, and therefore the data presented in Chapter 2

provides valuable new information on the functional significance of

alternative splicing in Arabidopsis. Going deeper into the relevance of

the two XBAT35 isoforms, we first tested their ability to efficiently

function as E3 ligases and indeed concluded that both have the

capacity to mediate autoubiquitination activity in vitro.

When we investigated the regulation of the XBAT35

expression pattern by external stimuli, we found that transcript levels

were affected by temperature and sugars and, similarly to the

developmental and photoperiod-specific expression patterns, the

transcript encoding the nuclear form was always observed at slightly

higher levels than the shorter one. We were therefore unable to

identify any specific developmental stage or condition in which one of

the XBAT35 splice variants was effectively more prominent than the

other, a possibility that we first hypothesized as a valuable guide in

dissecting the biological meaning of this particular alternative splicing

event. Moreover, and despite ubiquitous expression of both

alternative transcripts, the different loss-of-function lines we

generated for XBAT35 displayed no striking defects during normal

growth under standard greenhouse conditions, nor did they respond

differently to heat and cold stress or glucose exposure during the

early steps of seedling development. Two hypotheses were raised to


190

explain this absence of phenotypes: either we were looking at

conditions under which XBAT35 was inactive, or other genes with

redundant activity were masking the loss of XBAT35 function. The

sole member of the XBAT family previously characterized is XBAT32,

which has been shown to regulate lateral root development by

modulating the levels of the ethylene phytohormone (Nodzon et al.,

2004; Prasad et al., 2010). Based on this hint, we analyzed the

behavior of the XBAT35 loss-of-function lines in response to an

ethylene precursor during early development of etiolated seedlings.

This allowed uncovering a role for the XBAT35 E3 ligase in the

control of apical hook curvature by the referred hormone.

Interestingly, XBAT35 does not seem to act in the regulation of

hypocotyl elongation nor in root development, the other two aspects

that, together with apical hook exaggeration, are components of the

so-called triple response observed in dark-grown seedlings. The fact

that XBAT35 plays no role in root development had also been

described in a previous study (Prasad et al., 2010).

To examine the contribution of the two XBAT35 isoforms to

apical hook exaggeration in response to an ethylene precursor, each

splice variant was expressed in a XBAT35 mutant background and

independent transgenic lines for each splice form were analyzed.

Both isoforms revealed to be active under this growth condition, being

able to rescue the mutant’s apical hook curvature defect. In face of

this finding, it is difficult to understand the relevance of the XBAT35

alternative splicing event. However, the cytoplasmic isoform appears

to confer a stronger response than the nuclear one, with several

complementation lines expressing the shorter splice variant exhibiting

an overexpression phenotype, as seen by a reduced apical hook

curvature in response to ethylene when compared to the wild type.

This suggests that both proteins differentially contribute to ethylene


191

signaling during apical hook formation. Still, further experimental work

is required in order to dissect the molecular rationale underlying these

observations. Given their different intracellular localization, it is

possible that both XBAT35 isoforms target different sets of proteins,

but one can also hypothesize that as both proteins are likely able to

bind the same substrates (the protein-protein interaction domain is

unchanged in the two isoforms), it is possible that (a) putative

XBAT35 target(s) is found at higher levels in the cytoplasm.

A first attempt to identify proteolytic substrates of this E3

ligase was undertaken by means of a yeast two-hybrid assay.

Although future biochemical analyses to verify the isolated targets

must be performed, it seems clear that the yeast two-hybrid approach

did not uncover any protein that only interacts with one of the

XBAT35 isoforms. This is consistent with the functional activity of the

two XBAT35 proteins in a same physiological process, but did not

provide further clues in the elucidation of the biological relevance of

the studied alternative splicing event.

Among the five members of the Arabidopsis XBAT family of

E3 ligases, XBAT34 is the closest member to XBAT35, being likely

that both genes evolved from a gene duplication event (Kalyna and

Barta, 2004). The hypothesis of overlapping protein functions

between XBAT34 and XBAT35 was also considered in Chapter 2.

Interestingly this issue has been previously addressed for XBAT32,

XBAT34 and XBAT35 but only in what concerns root growth (Prasad

et al., 2010). Indeed, challenging single mutant lines with ethylene

revealed a role for XBAT32 in root growth but not for the other two

genes (Prasad et al., 2010). Aiming at further characterizing the

XBAT34 gene, we first looked at its expression pattern throughout

Arabidopsis development and found it to be strikingly similar to that of

XBAT35. Nevertheless, the expression pattern of XBAT34 was not as


192

extensively analyzed as for XBAT35, and future work should test the

effect of additional external cues on the XBAT34 gene expression

pattern. Interestingly, no alternative splice forms have been predicted

or identified for XBAT34.

To gain further insight into the in vivo role of XBAT34, we tried

isolating loss-of-function lines for this gene, also in an attempt to

generate double mutants for the XBAT34/XBAT35 duplicated gene

pair. Analysis of the latter mutants would allow establishing potential

functional redundancy between the two XBAT members, which would

in turn explain the very restricted phenotype found for the XBAT35

loss-of-function lines. However, from the insertion lines tested for

XBAT34 none revealed a significant reduction in gene expression

levels when compared to the wild type. We were therefore unable to

examine plant behavior in the absence of XBAT34 function or

simultaneous loss of XBAT34 and XBAT35 expression. Nevertheless,

clarifying this issue should be again attempted in future work. Other

T-DNA insertion lines in XBAT34 must be screened or silencing lines

directed at effectively targeting both XBAT genes could be generated.

Moreover, the effects of independent expression of the two XBAT35

splice variants could also be analyzed in the XBAT34/XBAT35 double

mutant background. E3 ligases have the ability to ubiquitinate

themselves or other ligases, so the possibility that XBAT34 targets

one of the XBAT35 isoforms should not be discarded.

Despite the fact that over 40% of the Arabidopsis genome is

now estimated to undergo alternative splicing (Filichkin et al., 2010),

the information on the biological significance of this

posttranscriptional regulatory mechanism is surprisingly scarce.

Chapter 2 provides several lines of evidence supporting functional

relevance for a specific alternative splicing event. We show that

alternative splicing of the XBAT35 RNA determines dual targeting of


193

two functional ubiquitin E3 ligase isoforms, which may differentially

contribute to apical hook curvature control. Two main issues should

be addressed in future work. Further testing of the XBAT35 protein

substrate candidates will allow investigating the specificity of each of

the E3 ligase isoforms, thus dissecting the precise biological

significance of this alternative splicing event. To this end, biochemical

assays such as in vitro co-immunoprecipitation and ubiquitination

assays should be conducted, starting with the XBAT35 targets

isolated from the yeast two-hybrid screen. It would be interesting to

include XBAT34 as a substrate and to test XBAT35 as a target for its

duplicated gene pair product. Finally, it is expected that the

generation of a XBAT34/XBAT35 double mutant line and its

subsequent phenotypical analysis will provide invaluable information

on the functional redundancy of these close homologs.

4.3. Biological Roles of the Plant-Specific SCL30a SR

Protein

SR proteins are essential splicing factors that recruit core

spliceosome components and help in the precise selection of splice

sites, thus playing central roles in alternative splicing. Members of the

SR protein family exist in higher number in plants than in metazoans

and, interestingly, more than half of the Arabidopsis SR proteins are

specific to plants (Barta et al., 2010). However, the role of SR

proteins in plants has been poorly addressed. Different research

groups have attempted but failed to develop a plant in vitro splicing

assay, possibly due to the use of inappropriate pre-mRNA substrates

not recognized by plant splicing factors. On the other hand, no loss-

of-function mutants in any of these splicing factors had been reported


194

in plants, hindering the functional analysis of the SR protein family in

plants.

Chapter 3 reports the functional analysis of a plant-specific SR

protein and the characterization of the first loss-of-function line for a

member of the Arabidopsis family of these splicing factors, shedding

light on its biological roles. We show that SCL30a controls seed-

specific traits and responses to adverse external conditions during

germination, such as osmotic and salt stress, by negatively regulating

the abscisic acid (ABA) signaling pathway. Moreover, SCL30a is also

involved in regulating light responses, at least during the early stages

of seedling development.

The SCL30a gene undergoes alternative splicing, with Reddy

and coworkers (Palusa et al., 2007) reporting the identification of

seven SCL30a splice variants, although only five were actually

sequenced. In young Arabidopsis seedlings, we were only able to

detect expression of three SCL30a splice variants, at approximately

constant relative levels, one of which has not been previously

reported. The two splice variants that do not encode the full-length

protein contain premature stop codons, thus either representing

strong candidates for nonsense-mediated decay (Palusa and Reddy,

2010) or producing truncated isoforms. As the SCL30a transcript

encoding the full-length protein complemented all of the scl30a-1

phenotypes tested, it is likely that the other splice variants play a

minor or no role at all in the overall activity of this splicing factor.

Moreover, it is interesting to note that the splice variant encoding the

full-length protein confers overexpression phenotypes during

germination under stress and regarding seed size but not in what

relates to seed dormancy. One possible explanation is that the

SCL30a truncated forms compete with the full-length SR protein to

bind a specific RNA target during particular environmental or


195

developmental conditions, potentially affecting its spatial

conformation. The two putative truncated forms include the N-terminal

end rich in arginine, proline, serine, glycine and tyrosine residues and

portions of the RNA recognition motif (Barta et al., 2010). It is

therefore conceivable that they are capable of binding RNA

molecules and/or be responsive to potential protein modifications at

the N-terminal extension. Future work should consider the functional

analysis of the truncated SCL30a forms, eventually starting by

addressing their functional activity when individually expressed in the

SCL30a loss-of-function background or in parallel with the full-length

protein. Since SCL30a has been reported to be able to interact with

itself (Lopato et al., 2002), it may also be useful to test this interaction

via co-immunoprecipitation assays upon different stress cues.

SCL30a gene expression was found to be induced during

seed imbibition and germination under control conditions, but not

when germinating seeds were challenged with ABA, salt or mannitol,

three conditions under which we have identified a role for SCL30a.

These observations have also been reported by two other research

groups (Palusa et al., 2007; Zhang et al., 2011). The fact that the

SCL30a splicing pattern is unchanged in the large majority of the

tested conditions indicates that the analysis of the expression and

splicing pattern of a particular gene may not always be useful in

gaining insight into its function. Moreover, it suggests a role for non-

transcriptional regulatory mechanisms in the control of SCL30a

activity. This hypothesis is supported by the fact that the activity of SR

proteins is known to be regulated at the posttranslational level,

especially by phosphorylation (Lorkovic et al., 2004; Tripathi et al.,

2010). Therefore, future work should aim at investigating the SCL30a

phosphorylation levels at different developmental time points and

under the most relevant stress conditions. In addition, it may also be


196

useful to identify potential kinases and phosphatases regulating

SCL30a activity, in order to dissect molecular components acting with

this splicing factor to control the activity of their downstream gene

targets in response to specific cell-surface signals. Moreover, since

phosphorylation also affects SR protein dynamics, it will be interesting

to examine the subcellular localization of SCL30a upon the

exogenous application of different compounds that interfere with

protein phosphorylation levels. The mobility of SCL30a could also be

analyzed under the presence of other external stimuli, such as those

that lead to phenotypes in the SCL30a mutant during seed

germination. In addition, it would be interesting to investigate whether

the referred stimuli disturb the nuclear speckle localization, which has

been reported for SCL30a (Lorkovic et al., 2008).

Besides phosphorylation, recent reports have referred to

additional levels of posttranslational regulation that should hence be

further explored. For instance, the Arabidopsis U6 small nuclear

ribonucleoprotein Sm-like 4 has been shown to be methylated at

arginine residues upon salt stress, with disruption of expression of the

corresponding gene resulting in hypersensitivity to high salinity and in

defects in splicing of hundreds of transcripts (Zhang et al., 2011).

Therefore, it could be of major interest to investigate the degree of

methylation at the arginine residues present in the N-terminal end of

the SCL30a SR protein. Interestingly, among the SR protein family in

Arabidopsis, only the SCL-subfamily contains the referred additional

residues at the N-terminal (Barta et al., 2010), suggesting that the

additional three members of this subgroup may also be subjected to

this regulatory mechanism, which could be a means of conferring

functional specificity.

In contrast to the fact that loss of SCL30a function only affects

embryonic tissues and early seedling development, analysis of the


197

SCL30a gene expression pattern in Arabidopsis revealed ubiquitous

expression throughout development, suggesting that members from

the same subfamily of SR proteins exert redundant functions. We

therefore initiated the functional characterization of SCL33, the

SCL30a closest homolog and its duplicated pair (Kalyna and Barta,

2004). Future work focusing on the detailed analysis of this SR

protein gene pair, including single and double mutant phenotypical

characterization, should conclusively establish the existence of

functional redundancy between these two SR genes. The

confirmation of this hypothesis could also open the way to future

research on other SR members, as it appears that two thirds of the

SR protein family in Arabidopsis possess duplicated gene pairs

(Kalyna and Barta, 2004).

Each SR protein likely targets different RNA molecules in a

same tissue, also depending on the tissue, developmental window

and/or exposure to particular stress conditions. Moreover, splicing of

each target may be regulated by different SR proteins. As the

SCL30a protein controls different aspects of Arabidopsis early

development and stress responses, we decided to perform a global

transcriptome analysis on seeds germinating under high salinity

levels, a condition of wide interest to the research community, given

the pressing issue of crop production improvement. The deep-

sequencing analysis allowed identifying new splice variants in several

genes, including in some that were not previously predicted

(www.arabidopsis.org) to undergo alternative splicing. This

observation highlights the fact that the most recent estimate on the

number of Arabidopsis genes undergoing alternative splicing

(Filichkin et al., 2010) is still likely to represent an underestimate.

Finally, the use of three genotypes with varying SCL30a

expression levels proved pivotal in detecting dozens of genes whose


198

splicing is targeted by the SR protein. We were thus able to show that

the SCL30a protein is indeed functional in this posttranscriptional

regulatory mechanism. In the future, it will be necessary to

investigate, within the identified genes, which ones encode the

functional targets active in the uncovered phenotypes. This will

increase the chances of finding direct SCL30a targets that will

actually link the biological activity of this splicing factor to the control

of a particular splicing event. The most promising candidates should

therefore be the subject of RNA immunoprecipitation assays and

functional analyses in planta. Finally, regarding the physiological

meaning of alternative splicing of SR protein genes, it would also be

interesting to perform RNA immunoprecipitation assays with the full-

length protein in both the mutant and wild-type backgrounds, and

assess whether the truncated SCL30a isoforms interfere with binding

of the splicing factor to the pre-mRNA target.

4.4. Conclusions

To conclude, the work presented in this thesis provides a

significant contribution to the elucidation of the biological relevance of

alternative splicing in higher plants. Using a two-fold approach, we

have addressed the significance of a particular splicing event in

Arabidopsis and investigated the in vivo roles of a plant-specific

splicing factor involved in the regulation of alternative splicing. The

XBAT35 gene undergoes exon skipping, the least common type of

alternative splicing in Arabidopsis, which leads to exclusion of a

nuclear localization signal in one of its encoded isoforms, thus

determining subcellular localization. Furthermore, the two isoforms

appear to differentially contribute to the ethylene-mediated apical


199

hook exaggeration in etiolated seedlings. Regarding the SCL30a

protein, we show its function as a splicing regulator and, by reporting

the first loss-of-function mutant in a plant SR protein splicing factor,

we demonstrate that its full activity is required for seed maturation

and germination under salt and osmotic stress, as well as in seedling

development under restricted light conditions, implying a role for

alternative splicing in the control of different aspects of plant early

development and responses to stress. Research in coming years is

expected to reveal the larger extent of the impact that alternative

splicing exerts in overall plant growth and development. It is

nevertheless clear that this is a highly complex research field, still

lacking extensive knowledge. In this regard, useful guiding clues and

different experimental approaches to be considered in the future are

suggested.

4.5. References


Filichkin, S.A., Priest, H.D., Givan, S.A., Shen, R., Bryant, D.W., Fox, S.E., Wong, W.-K., and Mockler, T.C. (2010). Genome wide-mapping of alternative splicing in Arabidopsis thaliana. Genome Res 20, 45-58.

Iida, K., Seki, M., Sakurai, T., Satou, M., Akiyama, K., Toyoda, T., Konagaya, A., and Shinozaki, K. (2004) Genome-wide analysis of alternative pre-mRNA splicing in Arabidopsis thaliana based on full-length cDNA sequences. Nucleic Acids Res 32, 5096-5103.


200

Kalsotra, A., and Cooper, T.A. (2011). Functional consequences of developmentally regulated alternative splicing. Nat Rev Genet 12, 715-729.


Karni, R., de Stanchina, E., Lowe, S.W., Sinha, R., Mu, D., and Krainer, A.R. (2007). The gene encoding the splicing factor SF2/ASF is a proto-oncogene. Nat Struct Mol Biol 14, 185-193.

Kazan, K. (2003). Alternative splicing and proteome diversity in plant: the tip of the iceberg has just emerged. Trends Plant Sci 8, 468-471.

Lopato, S., Forstner, C., Kalyna, M., Hilscher, J., Langhammer, U., Indrapichate, K., Lorkovic, Z.J., and Barta, A. (2002). Network of interactions of a novel plant-specific Arg/Ser-rich protein, AtRSZ33, with SC35-like splicing factors. J Biol Chem 277, 39989-39998.

Lorkovic, Z.J., Lopato, S., Pexa, M., Lehner, R., and Barta, A. (2004). Interactions of Arabidopsis RS domain containing cyclophilins with SR proteins and U1 and U11 small nuclear ribonucleoprotein-specific proteins suggest their involvement on pre-mRNA splicing. J Biol Chem 32, 33890-33898.

Lorkovic, Z.L., Hilscher, J., and Barta, A. (2008). Co-localisation studies of Arabidopsis SR splicing factors reveal different types of speckles in plant cell nuclei. Exp Cell Res 314, 3175-3186.

Nodzon, L.A., Xu, W.-H., Wang, Y., Pi, L.-Y., Chakrabarty, P.K., and Song, W.-Y. (2004). The ubiquitin ligase XBAT32 regulates lateral root development in Arabidopsis. Plant J 40, 996-1006.



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Pan, Q., Shai, O., Lee, L.J., Frey, B.J., and Blencowe, B.J. (2008). Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet 40, 1413-1415.

Prasad, M.E., Schofield, A., Lyzenga, W., Liu, H., and Stone, S.L. (2010). Arabidopsis RING E3 ligase XBAT32 regulates lateral root production through its role in ethylene biosynthesis. Plant Physiol 153, 1587-1596.

Tanabe, N., Yoshimura, K., Kimura, A., Yabuta, Y., and Shigeoka, S. (2006). Differential expression of alternatively spliced mRNAs of Arabidopsis SR protein homologs, atSR30 and atSR45a, in response to environmental stress. Plant Cell Physiol 48, 1036-1049.

Tripathi, V., Ellis, J.D., Shen, Z., Song, D.Y., Pan, Q., Watt, A. T., Freier, S.M., Bennett, C.F., Sharma, A., Bubulva, P.A., Blencowe, B.J., Prasanth, S.G., and Prasanth, K.V. (2010). The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation. Mol Cell 24, 925-938.

Twine, N.A., Janitz, K., Wilkins, M.R., and Janitz, M. (2011). Whole transcriptome sequencing reveals gene expression and splicing differences in brain regions affected by Alzheimer’s disease. PLoS One 6, e16266.

Wang, E.T., Sandberg, R., Luo, S., Khrebtukova, I., Zhang, L., Mayr, C., Kingsmore, S.F., Schroth, G.P., and Burge, C.B. (2008). Alternative isoform regulation in human tissue transcriptomes. Nature 456, 470-476.

Zhang, Z., Zhang, S., Zhang, Y., Wang, X., Li, D., Li, Q., Yeu, M., Li, Q, Zhang, Y., Xu, Y., Xue, Y., Chong, K., and Bao, S. (2011). Arabidopsis floral initiator SKB1 confers high salt tolerance by regulating transcription and pre-mRNA splicing through altering histone H4R3 and small nuclear ribonucleoprotein LSM4 methylation. Plant Cell 23, 396-411.

APPENDIX I

Appendix I

205

Table A. Genes whose total expression is up- or down-regulated in the scl30a-1 mutant.

Locus ID Gene Description Cufflinks ID Fold Change (> 2)

AT2G23020 tRNA-Met-1 pre-tRNA; translAtional elongAtion XLOC_009825 82.756 AT4G39510 CYP96A12 Cytochrome P450, family 96, subfamily A XLOC_020832 24.125 AT1G55830 F20N2.20 Unknown protein XLOC_005921 12.554 AT1G69828 Defensin-like (DEFL) family XLOC_003092 12.317 AT5G16290 VAT1 AcetolactAte synthase activity; amino acid biosynthesis XLOC_025109 10.784 AT5G27980 F15F15.50 Seed mAturAtion XLOC_025668 9.096 AT5G64450 T12B11.4 PutAtive endonuclease or glycosyl hydrolase XLOC_024012 8.711 AT5G06350 MHF15.13 ARM repeAt superfamily XLOC_024526 8.688 AT1G02310 MAN1 Glycosyl hydrolase; carbohydrAte mAtabolism XLOC_003841 7.657 AT5G09400 KUP7 Potassium transmembrane transporter XLOC_024708 7.443 AT4G19150 T18B16.120 Ankyrin repeAt family XLOC_019710 5.725 AT5G38720 MKD10.4 Unknown protein XLOC_022640 4.938 AT5G24830 F6A4.40 TetrAtricopeptide repeAt (TPR)-like superfamily XLOC_025540 4.681 AT1G16960 F17F16.18 Ubiquitin domain-containing protein XLOC_000980 4.586 AT5G35935 Transposable element XLOC_025745 4.190 AT5G43130 TAF4 Transcription inititAtion XLOC_022871 4.075 AT4G05494 Unknown protein XLOC_019165 4.007

AT1G09090 ATRBOHB NADPH-oxidase; seed after-ripening and superoxide production in germinAting seeds XLOC_000514 3.493

AT5G53190 SWEET3 Nodulin MtN3 family; sugar transmembrane transporter XLOC_026525 3.438

AT3G57870 SCE1/EMB1637

SUMO ligase; embryo development ending in seed dormancy XLOC_016374 3.409

AT5G60160 F15L12.1 Zn-dependent exopeptidases superfamily XLOC_023760 3.146 AT1G07620 ATOBGM GTPase activity XLOC_004165 2.986

AT1G64625 Serine/threonine-protein kinase WNK (With No Lysine)-relAted; regulAtion of transcription XLOC_002787 2.919

AT5G13650 SVR3 Suppressor of VariegAtion 3; GTPase activity XLOC_024946 2.797

Appendix I

206

AT2G43660 F18O19.23 CarbohydrAte-binding X8 domain superfamily XLOC_010891 2.754 AT5G60860 RABA1F GTP binding and mediAted signal transduction XLOC_023795 2.676

AT2G19330 PIRL6 Plant Intracellular Ras-group-relAted LRR; signal transduction XLOC_007583 2.595

AT5G41760 K16L22.3 Nucleotide-sugar transmembrane transporter XLOC_025942 2.578 AT5G66760 SDH1-1 Mitochondrial succinAte dehydrogenase XLOC_027304 2.540

AT5G26880 AGL26 DNA-dependent RNA methyltransferase activity; root specific XLOC_025614 2.504

AT5G40010 AATP1 ATPase-in-seed development; seed and silique development XLOC_025859 2.425

AT5G02170 T7H20.220 Transmembrane amino acid transporter XLOC_020975 2.421

AT5G56290 PEX5/EMB2790

Peroxisomal targeting signal type 1 receptor; embryo defective XLOC_026696 2.384

AT5G16550 MQK4.30 Unknown protein XLOC_021847 2.379

AT4G16144 AMSH3 Protein desumoylAtion; intracellular trafficking and vacuole biogenesis XLOC_019546 2.378

AT2G33490 F4P9.26 Hydroxyproline-rich glycoprotein family XLOC_008343 2.377 AT5G17530 K10A8.10 Phosphoglucosamine mutase family XLOC_025195 2.359

AT1G79500 ATKDSA1 3-deoxy-8-phosphooctulonAte (KDOP - cell wall component) synthase activity XLOC_007088 2.346

AT2G16870 F12A24.5 ATPase; disease resistance (TIR-NBS-LRR class) family XLOC_009555 2.266

AT4G04930 DES-1-LIKE Sphingolipid delta4-desAturase; sphingolipid biosynthesis; expressed in floral tissues XLOC_016972 2.188

AT1G72570 F28P22.24 Integrase-type DNA-binding; transcriptional regulAtion and organ morphogenesis XLOC_003245 2.175

AT5G02800 F9G14.110 Protein serine/threonine kinase activity XLOC_024307 2.163

AT4G35560 DAW1 Transducin/WD40 repeAt-like superfamily; pollen sperm cell differentiAtion XLOC_018600 2.069

AT1G47395 Unknown protein XLOC_001949 2.063 AT2G25740 F3N11.21 ATP-dependent protease La (LON) domain XLOC_007926 2.056 AT3G46840 T6H20.130 Serine-type endopeptidase activity; subtilase family XLOC_016686 2.009

Appendix I

207

Locus ID Gene Description Cufflinks ID Fold Change (< -2)

AT3G16240 AQP1/DELTA-TIP1

Delta tonoplast intrinsic protein; wAter and ammonium channel XLOC_012222 -2.021

AT5G19220 ADG2/APL1 ADP-glucose pyrophosphorylase large subunit; starch biosynthesis XLOC_025277 -2.049

AT4G16690 MES16 Methyl esterase XLOC_019588 -2.061 AT5G06120 K16F4.8 ARM repeAt; involved in intracellular protein transport XLOC_021222 -2.089 AT3G49130 F2K15.1 Suppressor-of-White-Apricot; RNA-binding XLOC_013324 -2.092

AT4G33925 SSN2 Suppressor of SNI transcription factor; involved in plant immune responses and homologous recombinAtion XLOC_018506 -2.107

AT5G09390 T5E8.190 CD2-binding XLOC_024705 -2.110 AT1G57580 T8L23.5 F-box domain XLOC_002478 -2.127

AT3G60140 BGLU30/DIN2 Glycoside hydrolase; response to light and to sucrose XLOC_013986 -2.133

AT5G23380 T32G24.1 Unknown protein XLOC_022249 -2.133

AT3G13525 60560.SNORNA00001 snoRNA; rRNA modificAtion XLOC_012044 -2.174

AT4G32560 L23H3.40 Paramyosin-relAted XLOC_018401 -2.174 AT3G13400 SKS13 SKU5 similar; oxidoreductase activity XLOC_012033 -2.180 AT2G33830 T1B8.13 Dormancy/auxin associAted XLOC_010356 -2.208 AT3G54890 LHCA1 Photosystem I light harvesting antenna complex XLOC_016234 -2.265 AT1G31330 PSAF Photosystem I subunit F XLOC_005304 -2.268 AT3G01940 F1C9.28 Unknown protein XLOC_014212 -2.270 AT3G50820 OEC33 Photosystem II oxygen evolving complex subunit XLOC_016014 -2.305 AT3G54060 F24B22.20 Unknown protein XLOC_016188 -2.309 AT3G27400 K1G2.22 Pectin lyase-like; endomembrane system XLOC_012911 -2.310 AT1G04660 T1G11.8 Glycine-rich; endomembrane system XLOC_003981 -2.326 AT5G22580 MQJ16.12 Stress responsive A/B Barrel Domain XLOC_022199 -2.395 AT5G36790 F5H8.7 Haloacid dehalogenase-like hydrolase XLOC_025755 -2.450 AT1G53480 MRD1 Mto 1 responding down XLOC_005815 -2.521

Appendix I

208

AT4G28740 F16A16.150 Unknown protein XLOC_018139 -2.563

AT2G05520 GRP3 Glycine-rich; response to ABA, salicylic acid, ethylene and dessicAtion XLOC_007250 -2.585

AT5G05965 Unknown protein XLOC_024488 -2.609

AT5G63910 FCLY Farnesylcysteine lyase; involved in farnesyl diphosphAte metabolic process and ABA signaling XLOC_023969 -2.619

AT4G23670 F9D16.140 Polyketide cyclase/dehydrase and lipid transport; response to salt stress, cadmium ion and bacteria XLOC_019901 -2.641

AT1G08380 PSAO Photosystem I subunit O XLOC_004221 -2.651 AT3G01345 O-glycosyl coumpounds hydrolase activity XLOC_011176 -2.656

AT1G76100 PETE1 Plastocyanin; involved in electron transport under copper-limiting conditions XLOC_006780 -2.761

AT3G07620 MLP3.7 Exostosin XLOC_014575 -2.832 AT3G14510 MOA2.15 Polyprenyl synthetase XLOC_014936 -2.840 AT1G14380 IQD28 IQ-domain; calmodulin binding XLOC_004535 -2.854 AT5G54900 RBP45A RNA-binding protein XLOC_026630 -2.987 AT2G43520 TI2 Trypsin defensin-like inhibitor; defense responses XLOC_008931 -2.998

AT2G34870 MEE26 MAternal effect embryo arrest; embryo development ending in seed dormancy XLOC_008420 -3.042

AT3G61470 LHCA2 Photosystem I light harvesting antenna complex XLOC_014060 -3.234 AT1G20840 TMT1 Tonoplast monosaccharide transporter; response to fructose XLOC_004855 -3.249

AT5G48485 DIR1 PutAtive apoplastic lipid transfer; involved in systemic acquired resistance XLOC_026282 -3.284

AT3G46780 PTAC16 Plastid transcriptionally active XLOC_013200 -3.405 AT4G24010 CSLG1 Cellulose synthase like XLOC_017815 -3.517 AT4G15760 MO1 Similar to monooxygenases known to degrade salicylic acid XLOC_019521 -3.542 AT1G32060 PRK Phosphoribulokinase; response to cold and to bacteria XLOC_001731 -3.725 AT1G49750 F14J22.4 Leucine-rich repeAt XLOC_005667 -4.311 AT5G18820 EMB3007 Embryo development ending in seed dormancy XLOC_025262 -4.502

AT3G20470 GRP5 Glycine-rich, cell wall constituent; response to ABA and salicylic acid XLOC_015270 -4.907

Appendix I

209

AT5G24670 TAD3/EMB2820 tRNA adenosine deaminase; embryo defective XLOC_025532 -4.935

AT2G05380 GRP3S Glycine-rich protein 3 XLOC_007249 -6.961 AT2G15680 F9O13.23 Calcium-binding EF-hand XLOC_007419 -7.437 AT5G56020 MDA7.6 Got1/Sft2-like; vesicle-mediAted transport XLOC_026687 -9.853 AT3G13570 SCL30a 30kD SC35-like splicing factor XLOC_014877 -10.236 AT5G33395 Transposable element XLOC_022495 -17.284 AT5G36210 MAB16.20 Alpha/beta-hydrolase XLOC_022556 -18.779 AT5G45780 MRA19.22 Leucine-rich repeAt protein kinase XLOC_022993 -20.100

AT3G46230 HSP17.4 Class I small heAt-shock protein; expressed in mAturing seeds XLOC_015820 -21.736

AT3G57860 OSD1 Omission of second division; control of entry into the second meiotic division XLOC_013838 -23.303

AT5G66550 K1F13.22 Maf-like XLOC_024144 -112.395

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Table B. Genes whose total expression is up- or down-regulated in the SCL30a.1OX1 line.

Locus ID Gene Description Cufflinks ID Fold Change (> 2)

AT5G62790 DXR/PDE129 1-deoxy-d-xylulose 5-phosphAte reductoisomerase; isoprenoid biosynthesis; pigment-defective embryo XLOC_023910 112.980

AT1G24490 ALB4 Homologue of Alb3/Oxa1/YidC; chloroplast organizAtion XLOC_001380 44.890

AT1G03640 50828.TRNA-PHE-1 pre-tRNA XLOC_003920 43.194

AT1G45233 THO5 Transport of mRNA precursors XLOC_001929 30.198 AT4G14980 DL3531C Cysteine/Histidine-rich C1 domain; signal transduction XLOC_019478 29.744 AT5G16260 ELF9 RNA binding protein; regulAtion of flower development XLOC_025102 27.664

AT2G32920 PDI9 Disulfide isomerase-like, within the thioredoxin superfamily; response to endoplasmic reticulum stress XLOC_010302 18.649

AT3G44006 Unknown protein XLOC_013073 15.009 AT5G61140

MAF19.14 ATP-dependent helicase; 30% identity with MER3/RCK XLOC_026983 14.523

AT5G40770 PHB3 Prohibitin; cell division; response to salt stress, to nitric

oxide, to auxin XLOC_025902 12.609

AT3G58590 F14P22.180 PentAtricopeptide repeAt XLOC_013875 11.690

AT1G76770 F28O16.14 HSP20-like chaperone XLOC_007085 11.526

AT3G04900 T9J14.15 Heavy metal transport/detoxificAtion XLOC_014398 9.183

AT5G17430 BBM Baby boom AP2-domain containing transcription factor; expressed in embryos and lAteral root primordium, involved in organ morphogenesis

XLOC_021914 8.973

AT1G20400 F5M15.25 Unknown protein XLOC_004831 8.758 AT5G14710 T9L3.10 Proteasome assembly chaperone XLOC_021742 6.631 AT5G24350 K16H17.4 Unknown protein XLOC_025518 6.647

AT2G46455 OxaA/YidC-like; involved into protein insertion into membrane XLOC_011035 6.071

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AT3G51640 T18N14.20 Unknown protein XLOC_016056 6.052

AT1G55870 AHG2/ATPARN

ABA-hypersensitive germinAtion; poly(A)-specific ribonuclease XLOC_002424 6.033

AT1G15190 F9L1.13 Fasciclin-like arabinogalactan; endomembrane system XLOC_000894 5.555 AT4G32175 PNAS-3 relAted; endomembrane system XLOC_018366 5.347 AT5G12050 F14F18.220 Unknown protein; circadian rhythm XLOC_021580 5.199

AT1G11290 CRR22 PentAtricopeptide repeAt containing the DYW motif endonuclease; multiple plastid transcripts editing XLOC_004387 5.163

AT2G07728 T5E7.16 Unknown protein XLOC_007305 5.075 AT5G55470 NHX3 Sodium proton exchanger XLOC_023487 4.914 AT5G16340 MQK4.6 AMP-dependent synthetase and ligase XLOC_021834 4.902 AT5G23955 Transposable element XLOC_022285 4.893 AT3G13320 CAX2 Low affinity calcium antiporter XLOC_012019 4.570 AT5G41970 MJC20.7 Metal-dependent protein hydrolase XLOC_025954 4.472

AT1G66520 PDE194 Hydroxymethyl-, formyl-, relAted transferase activity; purine ribonucleotide biosynthesis XLOC_006325 4.445

AT4G37400 CYP81F3 Cytochrome P450 superfamily XLOC_018730 4.425 AT1G73340 T9L24.44 Cytochrome P450 superfamily XLOC_003287 4.265 AT3G57620 F15B8.190 Glyoxal oxidase-relAted; endomembrane system XLOC_013824 4.252 AT4G18460 F28J12.130 D-Tyr-tRNA(Tyr) deacylase family XLOC_019671 4.172

AT4G27030 FAD4 PalmitAte desAturase; unsAturAted fAtty acid biosynthetic process XLOC_018024 3.817

AT4G00390 F5I10.6 DNA-binding storekeeper protein-relAted transcriptional regulAtor XLOC_018939 3.806

AT4G29080 PAP2/IAA27 Phytochrome-associAted; auxin responsive; regulAtion of transcription and translAtion XLOC_020198 3.796

AT3G15250 K7L4.5 Unknown protein XLOC_014977 3.793 AT5G23830 MRO11.13 MD-2-relAted lipid recognition domain-containing protein XLOC_022277 3.645 AT5G57320 VLN5 Villin, putAtive; actin binding, cytoskeleton organizAtion XLOC_023605 3.605 AT1G21100 IGMT1 O-methyltransferase activity XLOC_004870 3.573

AT5G65420 CYCD4;1 D-type cyclin; upregulAted during germinAtion and involved in stomAtal cell lineage proliferAtion in the hypocotyl XLOC_024074 3.434

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AT2G20495 Serine-threonine protein kinase XLOC_007668 3.361 AT1G60270 BGLU6 CAtion binding, hydrolase activity XLOC_006033 3.289 AT2G07042 Noncoding RNA XLOC_009413 3.277

AT1G22670 T22J18.16 Protease-associAted RING/U-box zinc finger family; endomembrane system XLOC_004935 3.213

AT2G43040 NPG1 Calmodulin-binding; specifically expressed in pollen, required for pollen development XLOC_010857 3.108

AT3G13570 SCL30a 30kD SC35-like splicing factor XLOC_014877 3.043

AT2G01530 MLP329/ZCE2 MLP-like; copper ion binding and response to biotic stimulus XLOC_007112 2.966

AT5G55130 CNX5/SIR1 PutAtive molybdopterin synthase sulphurylase XLOC_023465 2.910

AT3G57870 SCE1/EMB1637

SUMO ligase; embryo development ending in seed dormancy XLOC_016374 2.894

AT5G57110 ACA8 Autoinhibited Ca2+ -ATPase; localized to the plasma membrane XLOC_023575 2.780

AT5G62020 HSFB2A HeAt stress transcription factor family XLOC_023855 2.739 AT1G50440 F11F12.20 RING/FYVE/PHD zinc finger superfamily XLOC_002161 2.715 AT1G52450 F6D8.33 Ubiquitin carboxyl-terminal hydrolase-relAted protein XLOC_005767 2.685

AT5G52290 SHOC1 Similar to XPF endonucleases; relAted to meiotic recombinAtion XLOC_026480 2.683

AT5G66760 SDH1-1 Mitochondrial succinAte dehydrogenase XLOC_027304 2.680 AT5G48960 K19E20.8 HAD-superfamily hydrolase; 5'-nucleotidase activity XLOC_026305 2.589 AT3G56740 T8M16.70 Ubiquitin-associAted; endomembrane system XLOC_013761 2.589

AT4G03080 BSL1 BRI1 suppressor 1-like; protein serine/threonine phosphAtase activity XLOC_019082 2.582

AT1G08620 PKDM7D DNA binding transcription factor XLOC_000481 2.541 AT5G18420 F20L16.140 Unknown protein XLOC_021974 2.482 AT2G21280 GC1/SULA Plastid-targeted XLOC_009751 2.455 AT5G41760 K16L22.3 Nucleotide-sugar transporter family; endomembrane system XLOC_025942 2.451 AT5G03905 Iron-sulphur cluster biosynthesis family XLOC_021082 2.435

AT3G52130 F4F15.240 Bifunctional inhibitor/lipid-transfer protein/seed storage 2S albumin superfamily XLOC_016086 2.371

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AT5G04260 WCRKC2 Chloroplast stroma-localized thioredoxin; cell redox homeostasis XLOC_024388 2.358

AT3G25610 T5M7.10 ATPase activity; relAted to transmembrane ion movement and phospholipid transport XLOC_015502 2.351

AT3G10360 PUM4 Pumilio; regulAtion of mRNA stability and translAtion XLOC_014690 2.333 AT4G02655 Unknown protein XLOC_016861 2.300 AT5G48440 MJE7.7 FAD-dependent oxidoreductase family XLOC_023122 2.298

AT5G60520 MUF9.15 LAte embryogenesis abundant protein-relAted; endomembrane system XLOC_026947 2.231

AT1G26840 ORC6 Origin recognition complex subunit; initiAtion of DNA replicAtion XLOC_001465 2.197

AT5G08600 MAH20.16 U3 ribonucleoprotein family; rRNA processing XLOC_024673 2.150 AT1G17360 F28G4.18 COP1-interacting protein-relAted XLOC_001006 2.145 AT1G70430 F17O7.3 Protein serine/threonine kinase XLOC_003118 2.141 AT5G62890 MQB2.190 Xanthine/uracil permease family XLOC_027081 2.129

AT3G15340 PPI2 Similar to PPI1, which interacts with the plasma membrane H+ ATPase AHA1; regulAtion of proton transport XLOC_014981 2.128

AT5G44750 REV1 DNA polymerase activity; response to DNA damage stimulus XLOC_026096 2.126

AT5G56290 PEX5/EMB2790

Peroxisomal targeting signal type 1 receptor; embryo defective XLOC_026698 2.126

AT2G07680 ABCC13/MRP11

MRP subfamily; ATPase activity, coupled to transmembrane movement of substances XLOC_007309 2.125

AT5G10840 T30N20.110 Golgi apparAtus, endomembrane system XLOC_021496 2.125

AT3G04850 T9J14.20 Tesmin/TSO1-like CXC domain-containing protein; DNA binding transcription factor activity XLOC_014395 2.100

AT5G02690 F9G14.2 Unknown protein; endomembrane system XLOC_021004 2.085 A1G49940 F2J10.17 RelAted to tetrAtricopeptide repeAt-like superfamily XLOC_005675 2.064

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Locus ID Gene Description Cufflinks ID Fold Change (< -2)

AT1G07710 F24B9.19 Ankyrin repeAt family XLOC_004181 -2.012

AT3G44300 NIT2 Indole-3-acetonitrile nitrilase; involved in IAA biosynthesis; response to bacterium and cadmium ion XLOC_013082 -2.024

AT5G12250 TUB6 Beta-tubulin; structural constituent of cytoskeleton; response to cold treAtment XLOC_021595 -2.041

AT3G10300 F14P13.10 Calcium-binding EF-hand family XLOC_011803 -2.107

AT1G19376 52195.SNORNA00002 snoRNA; rRNa modificAtion XLOC_001135 -2.168

AT5G05965 Unknown protein XLOC_024488 -2.197 AT1G17810 F2H15.4 Beta-tonoplast intrinsic protein; wAter channel activity XLOC_001049 -2.235 AT3G29647 T13J10.8 Transposable element XLOC_016673 -2.252

AT4G33925 SSN2 DNA binding transcription factor activity; involved in plant immune response and homologous recombinAtion XLOC_018506 -2.292

AT2G32190 F22D22.6 Unknown protein XLOC_008287 2.490

AT4G28530 NAC074 NAC domain containing protein; DNA binding transcription factor activity; multicellular organism development XLOC_020163 -2.626

AT3G07620 MLP3.7 Exostosin family; endomembrane system XLOC_014575 -2.760 AT5G16940 F2K13.90 Carbon-sulfur lyase XLOC_025145 -2.936

AT1G47540 F16N3.19 Scorpion toxin-like knottin superfamily; ion channel inhibitor activity, defense response XLOC_005554 -3.075

AT2G19750 F6F22.22 Ribosomal protein S30 family XLOC_007612 -3.082 AT5G23955 Transposable element XLOC_025500 -3.701 AT3G53120 VPS37-1 Modifier of rudimentary XLOC_016136 -3.728

AT5G57480 MUA2.5 P-loop containing nucleoside triphosphAte hydrolases superfamily XLOC_026769 -3.777

AT1G20840 TMT1 Tonoplast monosaccharide transporter XLOC_004855 -3.787

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AT5G24670 TAD3/EMB2820 Unknown protein XLOC_025532 -3.938

AT5G08380 AGAL1 Alpha-galactosidase XLOC_021365 -4.015

AT1G74550 CYP98A9 Cytochrome P450; oxidAtion-reduction process; spermidine metabolic process XLOC_003361 -4.251

AT1G29071 SNOR105 C/D box type of snoRNA; rRNAs or snRNAs modificAtion; role in ribosome biogenesis XLOC_005188 -4.378

AT4G03070 AOP1 Possible 2-oxoglutarAte-dependent dioxygenase; glucosinolAte biosynthesis XLOC_016893 -4.657

AT5G57110 ACA8 Autoinhibited Ca2+ -ATPase; localized to the plasma membrane XLOC_023581 -4.907

AT3G52605 Potential nAtural antisense gene, overlapping with At3g52610 XLOC_016111 -5.345

AT3G27170 CLC-B Chloride channel XLOC_012886 -5.400 AT2G33793 Unknown protein XLOC_008358 -6.245 AT5G48830 K24G6.16 Unknown protein XLOC_023146 -6.328 AT5G33395 Transposable element XLOC_022495 -8.130

AT5G61590 K11J9.4 Ethylene response factor subfamily B-3 of ERF/AP2 transcription factor family; response to wAter deprivAtion XLOC_027011 -8.591

AT1G11300 T28P6.6 Protein serine/threonine kinase XLOC_000675 -8.885 AT3G23480 MEE5.2 Cyclopropane-fAtty-acyl-phospholipid synthase XLOC_012661 -10.984 AT4G11000 T22B4.2 Ankyrin repeAt family XLOC_017137 -13.559 AT5G66550 K1F13.22 Maf-like XLOC_024144 -15.347 AT3G17620 MKP6.18 F-box domain, cyclin-like XLOC_015107 -17.026 AT3G01345 O-glycosyl compounds hydrolase XLOC_011176 -39.702

AT3G57860 OSD1 Omission of second division; control of entry into the second meiotic division XLOC_013838 -45.618

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Table C. Genes with unique splice junctions (SJs) in the wild type (WT) and the scl30a-1 mutant (KO).

Locus ID # unique SJs (% total)

Cufflinks ID Total # of SJsWT KO

AT1G60680 XLOC_006049 5 4 (80) 1 (20)AT1G72420 XLOC_003227 2 1 (50) 1 (50)AT2G02680 XLOC_007156 2 1 (50) 1 (50)AT2G42840 XLOC_008877 2 1 (50) 1 (50)AT4G05440 XLOC_019165 2 1 (38) 1 (50)AT5G60030 XLOC_026919 8 3 (38) 2 (25)AT1G09450 XLOC_000537 14 5 (36) 2 (14)AT2G23348 XLOC_009840 3 1 (33) 1 (33)AT3G29644 XLOC_015677 6 2 (33) 1 (17)AT1G12070 XLOC_004427 7 2 (29) 1 (14)AT2G17970 XLOC_009601 11 3 (27) 2 (18)AT2G27380 XLOC_008039 20 5 (25) 7 (35)AT5G51700 XLOC_026462 5 1 (20) 1 (20)AT2G28940 XLOC_010126 6 1 (17) 1 (17)AT2G02800 XLOC_009299 7 1 (14) 1 (14)AT5G19770 XLOC_025304 7 1 (14) 1 (14)AT2G07180 XLOC_009421 8 1 (13) 1 (13)AT3G61010 XLOC_016505 16 2 (13) 1 (6)AT1G06820 XLOC_004125 17 2 (12) 1 (6)AT4G11310 XLOC_017153 9 1 (11) 1 (11)AT1G26530 XLOC_001446 10 1 (10) 1 (10)AT1G28281 XLOC_001543 10 1 (10) 4 (40)AT2G24765 XLOC_007858 10 1 (10) 1 (10)AT3G26680 XLOC_012858 10 1 (10) 2 (20)AT4G05450 XLOC_017000 10 1 (10) 2 (20)AT4G17310 XLOC_019618 10 1 (10) 2 (20)AT4G27490 XLOC_018059 10 1 (10) 2 (20)

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AT5G22720 XLOC_025438 10 1 (10) 1 (10)AT3G46200 XLOC_013180 11 1 (9) 1 (9)AT4G14920 XLOC_019474 11 1 (9) 1 (9)AT5G06350 XLOC_021245 22 2 (9) 1 (5)AT5G20020 XLOC_022063 11 1 (9) 1 (9)AT3G51880 XLOC_016074 12 1 (8) 3 (25)AT3G62080 XLOC_014097 12 1 (8) 1 (8)AT4G20350 XLOC_017634 24 2 (8) 3 (13)AT4G31790 XLOC_018335 12 1 (8) 1 (8)AT5G19300 XLOC_022029 12 1 (8) 1 (8)AT5G64010 XLOC_027143 12 1 (8) 2 (17)AT1G02305 XLOC_000068 13 1 (8) 1 (8)AT1G07670 XLOC_004169 13 1 (8) 2 (15)AT3G01540 XLOC_014199 13 1 (8) 2 (15)AT5G07940 XLOC_021332 13 1 (8) 1 (8)AT1G53510 XLOC_005817 14 1 (7) 1 (7)AT2G42780 XLOC_008873 14 1 (7) 1 (7)AT5G17890 XLOC_021938 14 1 (7) 1 (7)AT5G03300 XLOC_021042 29 2 (7) 1 (3)AT1G71240 XLOC_003166 15 1 (7) 3 (20)AT5G10240 XLOC_024747 15 1 (7) 1 (7)AT5G13710 XLOC_024954 15 1 (7) 1 (7)AT5G57990 XLOC_026798 15 1 (7) 1 (7)AT1G76030 XLOC_003436 16 1 (6) 1 (6)AT2G23420 XLOC_007806 16 1 (6) 1 (6)AT2G27350 XLOC_010048 16 1 (6) 2 (13)AT3G52050 XLOC_013476 18 1 (6) 1 (6)AT2G24420 XLOC_007849 19 1 (5) 2 (11)AT3G12590 XLOC_014814 19 1 (5) 1 (5)AT1G28100 XLOC_001533 20 1 (5) 1 (5)AT1G06590 XLOC_004113 22 1 (5) 1 (5)AT4G21150 XLOC_017655 22 1 (5) 1 (5)

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AT2G14120 XLOC_009486 24 1 (4) 1 (4)AT4G09680 XLOC_017073 24 1 (4) 1 (4)AT5G35560 XLOC_025727 24 1 (4) 2 (8)AT5G56360 XLOC_026705 24 1 (4) 1 (4)AT1G71810 XLOC_006555 25 1 (4) 3 (12)AT2G29200 XLOC_010133 25 1 (4) 1 (4)AT2G35110 XLOC_010416 25 1 (4) 1 (4)AT5G07810 XLOC_024610 28 1 (4) 1 (4)AT1G50140 XLOC_005688 30 1 (3) 1 (3)AT4G02400 XLOC_016833 30 1 (3) 1 (3)AT4G21090 XLOC_019775 30 1 (3) 2 (7)AT2G13370 XLOC_009456 33 1 (3) 1 (3)AT5G08620 XLOC_021390 37 1 (3) 5 (14)

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Table D. Genes with unique splice junctions (SJs) in the wild type (WT) and the SCL30a.1OX1 line (OX). Locus ID # unique SJs (% total)

Cufflinks ID Total # of SJs

WT OXAT1G26771 XLOC_005086 2 1 (50) 1 (50)AT1G68940 XLOC_006425 2 1 (50) 1 (50)AT2G17770 XLOC_007521 2 1 (50) 1 (50)AT2G38240 XLOC_010591 4 2 (50) 1 (25)AT5G01180 XLOC_024209 2 1 (50) 1 (50)AT5G66760 XLOC_027303 2 1 (50) 1 (50)AT4G00760 XLOC_018961 16 6 (38) 1 (6)AT1G15160 XLOC_000892 6 2 (33) 1 (17)AT2G04395 XLOC_009360 3 1 (33) 1 (33)AT3G06390 XLOC_014487 3 1 (33) 1 (33)AT3G22780 XLOC_012594 3 1 (33) 1 (33)AT5G42350 XLOC_022823 3 1 (33) 1 (33)AT5G42360 XLOC_025972 3 1 (33) 1 (33)AT5G50950 XLOC_023250 9 3 (33) 1 (11)AT3G14690 XLOC_014941 7 2 (29) 3 (43)AT2G05812 XLOC_007259 4 1 (25) 2 (50)AT3G29770 XLOC_013008 4 1 (25) 3 (75)AT2G45500 XLOC_010996 13 3 (23) 1 (8)AT1G22960 XLOC_004951 5 1 (20) 1 (10)AT1G63420 XLOC_002715 10 2 (20) 1 (10)AT1G70430 XLOC_003118 15 3 (20) 6 (40)AT5G04170 XLOC_021103 5 1 (20) 1 (20)AT5G23410 XLOC_025472 5 1 (20) 1 (20)AT5G65070 XLOC_024056 5 1 (20) 1 (20)AT4G29360 XLOC_020210 6 1 (17) 1 (17)AT5G66050 XLOC_027266 12 2 (17) 1 (8)AT1G64860 XLOC_002815 13 2 (15) 2 (15)

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AT5G04130 XLOC_024379 40 1 (3) 2 (5)AT2G31960 XLOC_008267 48 1 (2) 1 (2)

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Table E. Genes whose transcript expression ratios are significantly different in the wild type (WT) and the scl30a-1 mutant (KO). Cut-off line: 50%.

Cufflinks ID Relative transcript level Difference to the WT Locus ID

Gene Transcript WT KO (KO - WT)AT4G35640.1 XLOC_020605 TCONS_00055384 2.0% 86.6% 84.6%AT4G23990.1 XLOC_017815 TCONS_00058446 2.8% 85.9% 83.1%AT5G24170.1 XLOC_025505 TCONS_00080895 0.8% 81.8% 81.0%AT5G05610.2 XLOC_024470 TCONS_00080547 0.7% 80.6% 79.9%AT3G57910.1 XLOC_013842 TCONS_00037397 17.8% 96.9% 79.1%AT1G15400.1 XLOC_004599 TCONS_00004610 2.2% 79.9% 77.7%AT1G77090.1 XLOC_006833 TCONS_00006962 18.7% 95.2% 76.5%AT3G21215.1 XLOC_012544 TCONS_00041129 17.2% 92.7% 75.5%AT5G01630.1 XLOC_024234 TCONS_00080476 7.0% 82.5% 75.5%AT4G17360.1 XLOC_019620 TCONS_00059192 13.8% 89.1% 75.3%AT3G14540.1 XLOC_014938 TCONS_00045048 16.3% 91.3% 75.1%AT1G60090.1 XLOC_006028 TCONS_00014189 14.4% 88.3% 74.0%AT3G11530.1 XLOC_011886 TCONS_00035510 2.3% 76.0% 73.7%AT5G15070.1 XLOC_025042 TCONS_00075824 21.4% 94.5% 73.1%AT4G09840.2 XLOC_019239 TCONS_00053949 14.5% 87.4% 72.9%AT3G43700.1 XLOC_013066 TCONS_00041415 9.8% 79.3% 69.5%AT1G31770.1 XLOC_001716 TCONS_00001771 11.4% 80.8% 69.3%AT4G17180.1 XLOC_017484 TCONS_00052171 18.0% 86.9% 68.9%AT2G47015.1 XLOC_009164 TCONS_00024467 15.9% 84.7% 68.8%AT2G26470.1 XLOC_007972 TCONS_00023300 20.7% 89.4% 68.7%AT1G56340.1 XLOC_002462 TCONS_00012479 25.6% 94.0% 68.4%AT3G09060.1 XLOC_014623 TCONS_00047128 6.8% 75.2% 68.4%AT2G25140.1 XLOC_007874 TCONS_00030887 19.2% 87.5% 68.4%AT5G44600.1 XLOC_022936 TCONS_00066109 1.3% 68.4% 67.0%AT3G24340.1 XLOC_015457 TCONS_00039064 17.1% 83.7% 66.6%AT2G19800.1 XLOC_007614 TCONS_00032273 1.1% 67.4% 66.4%

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AT5G54570.1 XLOC_026610 TCONS_00079073 2.3% 68.4% 66.0%AT3G57860.1 XLOC_013838 TCONS_00037393 0.4% 66.4% 66.0%AT5G55560.1 XLOC_026665 TCONS_00076537 21.2% 86.7% 65.4%AT2G29340.1 XLOC_008115 TCONS_00023445 4.3% 69.7% 65.3%AT2G17442.4 XLOC_009576 TCONS_00029880 20.0% 85.1% 65.1%AT5G16180.1 XLOC_021818 TCONS_00081776 6.6% 71.2% 64.6%AT4G29080.1 XLOC_020198 TCONS_00057611 11.6% 76.2% 64.6%AT4G28450.1 XLOC_018122 TCONS_00060362 14.8% 79.4% 64.5%AT4G31150.1 XLOC_020337 TCONS_00057677 14.3% 78.7% 64.4%AT1G65390.1 XLOC_002844 TCONS_00015791 13.3% 77.6% 64.3%AT2G30790.1 XLOC_010193 TCONS_00030153 2.3% 66.2% 63.9%AT1G63110.3 XLOC_002703 TCONS_00018531 28.8% 92.4% 63.6%AT2G01530.1 XLOC_007112 TCONS_00033484 24.3% 87.7% 63.4%AT5G09820.2 XLOC_024729 TCONS_00082689 31.4% 94.8% 63.3%AT5G16230.1 XLOC_021824 TCONS_00074392 5.8% 68.8% 63.1%AT5G63900.1 XLOC_023968 TCONS_00075377 3.8% 66.6% 62.8%AT5G48310.2 XLOC_026274 TCONS_00081148 12.9% 75.1% 62.2%AT5G27290.2 XLOC_025624 TCONS_00076120 8.4% 70.2% 61.8%AT3G15520.1 XLOC_012166 TCONS_00050064 11.0% 72.2% 61.2%AT2G06025.1 XLOC_007268 TCONS_00026650 2.8% 63.8% 61.0%AT3G19210.2 XLOC_015205 TCONS_00045168 8.8% 69.7% 61.0%AT2G25964.1 XLOC_009965 TCONS_00025302 32.6% 93.5% 60.9%AT5G55900.1 XLOC_023509 TCONS_00066739 16.1% 76.8% 60.7%AT2G26920.1 XLOC_008003 TCONS_00023336 25.1% 85.8% 60.6%AT1G56670.1 XLOC_002475 TCONS_00015640 27.2% 87.9% 60.6%AT2G30440.1 XLOC_008172 TCONS_00023495 10.1% 70.2% 60.1%AT1G53090.2 XLOC_002276 TCONS_00002319 25.2% 85.2% 60.0%AT2G25350.1 XLOC_007891 TCONS_00032366 5.0% 65.0% 60.0%AT4G14700.1 XLOC_017331 TCONS_00058236 13.1% 72.8% 59.7%AT1G07650.1 XLOC_004167 TCONS_00009636 6.6% 66.1% 59.5%AT3G11960.2 XLOC_011912 TCONS_00040787 27.3% 86.7% 59.4%AT4G15110.1 XLOC_019488 TCONS_00063744 19.9% 79.2% 59.4%

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AT1G77765.3 XLOC_003555 TCONS_00016072 3.6% 55.2% 51.7%AT1G13610.2 XLOC_004499 TCONS_00004504 9.5% 61.0% 51.6%AT3G53330.1 XLOC_013548 TCONS_00037099 20.3% 71.8% 51.6%AT4G33465.1 XLOC_018475 TCONS_00053171 38.3% 89.8% 51.5%AT1G13330.1 XLOC_000778 TCONS_00000768 30.5% 82.0% 51.5% AT5G46390.2 XLOC_023020 TCONS_00080072 5.7% 57.2% 51.4% AT1G54330.1 XLOC_005859 TCONS_00014073 36.8% 88.2% 51.4% AT1G04250.1 XLOC_000187 TCONS_00014768 19.5% 70.8% 51.3% AT5G25790.1 XLOC_025572 TCONS_00078693 20.6% 71.8% 51.2% AT2G17030.1 XLOC_007462 TCONS_00022764 4.6% 55.7% 51.1% AT2G48080.1 XLOC_011103 TCONS_00032103 41.0% 92.1% 51.1% AT5G46180.1 XLOC_023005 TCONS_00071671 41.3% 92.4% 51.1% AT5G58790.1 XLOC_023689 TCONS_00066898 1.2% 52.3% 51.1% AT1G13580.3 XLOC_004498 TCONS_00013439 4.3% 55.0% 50.7% AT2G29650.3 XLOC_010148 TCONS_00025521 12.5% 63.0% 50.4% AT3G14500.1 XLOC_012109 TCONS_00035701 18.7% 69.1% 50.4% AT1G35612.1 XLOC_001846 TCONS_00008360 17.1% 67.4% 50.3% AT1G50240.2 XLOC_002145 TCONS_00015498 14.8% 65.0% 50.2% AT1G08520.1 XLOC_004231 TCONS_00021383 6.8% 56.9% 50.1% AT5G51180.2 XLOC_026417 TCONS_00069642 4.2% 54.2% 50.1% AT5G60410.5 XLOC_023774 TCONS_00082370 50.7% 0.7% -50.0% AT5G14610.2 XLOC_021733 TCONS_00081750 63.8% 13.8% -50.1% AT1G35612.1 XLOC_001846 TCONS_00001924 82.9% 32.6% -50.3% AT4G03410.2 XLOC_016917 TCONS_00055826 73.7% 23.3% -50.4% AT1G04490.2 XLOC_003971 TCONS_00016246 62.8% 12.2% -50.6% AT3G61440.1 XLOC_016541 TCONS_00040231 61.2% 10.4% -50.8% AT3G50840.1 XLOC_016017 TCONS_00051204 56.5% 5.6% -50.9% AT5G46180.1 XLOC_023005 TCONS_00077686 58.7% 7.6% -51.1% AT5G51810.1 XLOC_026465 TCONS_00069677 63.1% 12.0% -51.1% AT2G48080.1 XLOC_011103 TCONS_00034855 59.0% 7.9% -51.1% AT4G09510.2 XLOC_019224 TCONS_00053934 80.1% 28.9% -51.1% AT1G77080.5 XLOC_003513 TCONS_00003482 57.1% 5.9% -51.2%

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AT1G04250.1 XLOC_000187 TCONS_00000200 80.5% 29.2% -51.3% AT3G60330.2 XLOC_013998 TCONS_00050594 78.8% 27.4% -51.3% AT1G54330.1 XLOC_005859 TCONS_00010633 63.2% 11.8% -51.4% AT3G05850.1 XLOC_014455 TCONS_00037998 63.9% 12.5% -51.4% AT1G13330.1 XLOC_000778 TCONS_00020317 69.5% 18.0% -51.5% AT4G33470.1 XLOC_018475 TCONS_00053172 61.7% 10.2% -51.5% AT1G66730.1 XLOC_002915 TCONS_00008934 60.5% 8.9% -51.6% AT5G63910.1 XLOC_023968 TCONS_00075378 83.1% 31.5% -51.6% AT2G23950.1 XLOC_009873 TCONS_00034414 64.3% 12.6% -51.6% AT1G77765.3 XLOC_003555 TCONS_00018791 96.4% 44.8% -51.7% AT1G76710.2 XLOC_003485 TCONS_00003466 84.3% 32.6% -51.7% AT3G25070.1 XLOC_015488 TCONS_00051045 71.3% 19.4% -51.9% AT5G35670.1 XLOC_022529 TCONS_00065723 62.3% 10.2% -52.1% AT5G62640.1 XLOC_023894 TCONS_00080357 90.1% 38.0% -52.1% AT5G22220.2 XLOC_022173 TCONS_00079751 55.3% 3.1% -52.2% AT3G17420.1 XLOC_015097 TCONS_00049272 59.1% 6.8% -52.2% AT5G67390.1 XLOC_027338 TCONS_00070497 78.9% 26.6% -52.3% AT5G45730.1 XLOC_022988 TCONS_00066167 60.9% 8.6% -52.3% AT2G15480.1 XLOC_007413 TCONS_00033594 54.2% 1.8% -52.4% AT5G09290.1 XLOC_021404 TCONS_00077053 82.3% 29.6% -52.7% AT3G20080.3 XLOC_015254 TCONS_00050983 87.0% 34.1% -52.9% AT3G43700.1 XLOC_013066 TCONS_00050327 56.2% 3.2% -53.0% AT1G10840.2 XLOC_000642 TCONS_00017816 73.9% 20.7% -53.2% AT4G31770.1 XLOC_018332 TCONS_00053018 93.0% 39.9% -53.2% AT5G19840.2 XLOC_022058 TCONS_00071167 59.5% 5.9% -53.6% AT3G57680.1 XLOC_013830 TCONS_00048847 77.0% 23.3% -53.7% AT2G25850.4 XLOC_009955 TCONS_00034459 66.8% 13.1% -53.7% AT2G29970.1 XLOC_008144 TCONS_00023467 75.6% 21.9% -53.7% AT1G72510.1 XLOC_003235 TCONS_00009118 91.7% 38.0% -53.7% AT5G24670.2 XLOC_025531 TCONS_00080902 87.6% 33.6% -53.9% AT1G65910.1 XLOC_006304 TCONS_00010917 79.0% 24.8% -54.2% AT1G78620.1 XLOC_003596 TCONS_00003571 73.7% 19.1% -54.5%

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AT4G23990.1 XLOC_017815 TCONS_00052509 96.1% 7.5% -88.5%

Table F. Genes whose transcript expression ratios are significantly different in the wild type (WT) and the SCL30a.1OX1 line (OX). Cut-off line: 50%.

Cufflinks ID Relative transcript level DifferenceLocus IDGene Transcript WT OX (OX - WT)

AT2G15880.1 XLOC_007422 TCONS_00033597 8.8% 97.4% 88.6%AT5G27290.2 XLOC_025624 TCONS_00076120 8.4% 94.2% 85.9%AT2G44120.2 XLOC_008966 TCONS_00024291 10.3% 95.5% 85.2%AT2G06025.1 XLOC_007268 TCONS_00026650 2.8% 87.6% 84.8%AT2G45840.1 XLOC_009094 TCONS_00024394 6.2% 90.7% 84.5%AT3G58850.1 XLOC_016417 TCONS_00040092 8.2% 89.5% 81.3%AT5G24170.1 XLOC_025505 TCONS_00080895 0.8% 79.5% 78.7%AT4G28450.1 XLOC_018122 TCONS_00060362 14.8% 93.5% 78.7%AT5G12240.1 XLOC_021594 TCONS_00064813 7.4% 85.4% 78.0%AT2G42860.1 XLOC_008879 TCONS_00031224 15.4% 92.3% 76.8%AT3G04680.1 XLOC_011425 TCONS_00035103 18.6% 94.2% 75.7%AT3G14990.1 XLOC_014956 TCONS_00050886 4.1% 78.5% 74.4%AT3G26935.1 XLOC_012873 TCONS_00041308 9.6% 82.8% 73.2%AT3G50685.1 XLOC_013391 TCONS_00036942 12.1% 84.5% 72.4%AT4G17360.1 XLOC_019620 TCONS_00059192 13.8% 85.8% 71.9%AT2G46455.1 XLOC_011035 TCONS_00033443 10.7% 82.3% 71.6%AT1G11905.1 XLOC_000709 TCONS_00000681 2.7% 74.1% 71.4%AT1G20830.1 XLOC_001210 TCONS_00001215 8.0% 78.8% 70.7%AT3G62910.1 XLOC_016621 TCONS_00045832 4.9% 75.6% 70.7%AT4G09840.2 XLOC_019239 TCONS_00053949 14.5% 84.4% 69.9%AT1G73960.1 XLOC_003319 TCONS_00021086 15.1% 84.6% 69.5%AT1G56340.1 XLOC_002462 TCONS_00012479 25.6% 94.9% 69.3%

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AT5G53110.1 XLOC_023352 TCONS_00071878 33.0% 86.6% 53.6%AT1G34010.1 XLOC_001804 TCONS_00008327 4.1% 57.6% 53.5%AT1G76890.2 XLOC_006818 TCONS_00011236 6.9% 60.3% 53.4%AT3G21730.2 XLOC_015335 TCONS_00038948 13.8% 67.1% 53.3%AT5G48030.1 XLOC_026264 TCONS_00076383 13.5% 66.6% 53.1%AT1G65040.1 XLOC_002831 TCONS_00008887 11.6% 64.6% 52.9%AT1G76020.1 XLOC_006776 TCONS_00011213 24.0% 76.8% 52.8%AT4G20310.3 XLOC_017630 TCONS_00058356 3.9% 56.6% 52.7%AT3G50620.1 XLOC_013388 TCONS_00044458 35.0% 87.4% 52.4%AT3G23175.1 XLOC_015388 TCONS_00039001 33.1% 85.6% 52.4%AT1G17280.1 XLOC_004692 TCONS_00009964 12.7% 65.1% 52.4%AT5G16790.1 XLOC_025139 TCONS_00068371 23.0% 75.5% 52.4% AT3G17050.1 XLOC_012273 TCONS_00050094 25.8% 78.1% 52.4% AT2G32700.5 XLOC_008309 TCONS_00023622 7.5% 59.9% 52.4% AT4G23940.1 XLOC_017813 TCONS_00058441 2.8% 55.1% 52.3% AT1G11170.2 XLOC_000667 TCONS_00000649 11.1% 63.3% 52.2% AT1G12620.1 XLOC_004453 TCONS_00004450 35.0% 87.1% 52.0% AT1G73220.1 XLOC_003285 TCONS_00003272 19.4% 71.4% 52.0% AT2G25850.4 XLOC_009955 TCONS_00031646 9.6% 61.6% 52.0% AT4G35040.1 XLOC_018574 TCONS_00056720 29.3% 81.2% 51.9% AT3G52110.1 XLOC_016084 TCONS_00043158 9.5% 61.3% 51.8% AT2G01730.1 XLOC_007123 TCONS_00026556 12.5% 64.2% 51.7% AT2G20290.1 XLOC_009692 TCONS_00025022 8.8% 60.4% 51.5% AT4G33180.1 XLOC_018448 TCONS_00053151 16.6% 68.2% 51.5% AT3G24340.1 XLOC_015457 TCONS_00039064 17.1% 68.6% 51.5% AT3G14540.1 XLOC_014938 TCONS_00045048 16.3% 67.7% 51.5% AT3G59580.2 XLOC_013960 TCONS_00037501 13.3% 64.7% 51.4% AT1G79890.1 XLOC_003678 TCONS_00016124 9.6% 60.8% 51.2% AT5G20300.1 XLOC_025344 TCONS_00068569 35.2% 86.3% 51.1% AT3G04850.1 XLOC_014395 TCONS_00037934 24.5% 75.4% 50.9% AT1G76670.1 XLOC_003478 TCONS_00016036 12.7% 63.6% 50.9% AT2G47000.1 XLOC_011055 TCONS_00033453 19.0% 69.9% 50.9%

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AT1G12370.2 XLOC_004443 TCONS_00004441 17.9% 68.6% 50.8% AT3G15520.1 XLOC_012166 TCONS_00050064 11.0% 61.7% 50.8% AT5G67030.1 XLOC_024172 TCONS_00067395 30.9% 81.7% 50.7% AT5G01760.1 XLOC_020947 TCONS_00076871 5.8% 56.4% 50.7% AT2G38170.3 XLOC_010584 TCONS_00025971 6.7% 57.3% 50.5% AT1G11330.2 XLOC_000677 TCONS_00020285 63.9% 13.9% -50.0% AT4G11230.1 XLOC_019300 TCONS_00057107 71.7% 21.5% -50.3% AT5G67030.1 XLOC_024172 TCONS_00072339 69.1% 18.3% -50.7% AT5G03250.1 XLOC_021041 TCONS_00076914 57.1% 6.4% -50.7% AT4G25520.1 XLOC_019980 TCONS_00059357 86.8% 36.0% -50.8% AT3G04850.1 XLOC_014395 TCONS_00048996 75.5% 24.6% -50.9% AT1G63110.1 XLOC_002702 TCONS_00008801 79.7% 28.6% -51.1% AT2G02540.1 XLOC_007151 TCONS_00028847 71.1% 20.0% -51.1% AT5G20300.3 XLOC_025344 TCONS_00082866 64.8% 13.7% -51.1% AT4G09970.2 XLOC_017083 TCONS_00063038 63.1% 11.9% -51.2% AT1G55810.3 XLOC_002419 TCONS_00020822 52.6% 1.2% -51.4% AT3G24340.1 XLOC_015457 TCONS_00049392 82.9% 31.4% -51.5% AT3G20090.1 XLOC_012469 TCONS_00036069 74.6% 22.9% -51.7% AT4G21070.1 XLOC_017652 TCONS_00063200 61.3% 9.5% -51.8% AT5G02470.1 XLOC_024281 TCONS_00072392 74.6% 22.6% -52.0% AT1G12620.1 XLOC_004453 TCONS_00021460 65.0% 12.9% -52.0% AT4G25610.1 XLOC_017939 TCONS_00060278 71.5% 19.3% -52.2% AT5G02800.1 XLOC_024307 TCONS_00072399 82.1% 29.8% -52.3% AT5G38780.1 XLOC_022644 TCONS_00077564 89.4% 37.1% -52.3% AT5G16790.1 XLOC_025139 TCONS_00082802 77.0% 24.5% -52.4% AT3G23175.1 XLOC_015388 TCONS_00049375 66.9% 14.4% -52.4% AT3G50620.1 XLOC_013388 TCONS_00044457 65.0% 12.6% -52.4% AT5G64130.1 XLOC_023990 TCONS_00067212 59.2% 6.5% -52.6% AT4G32130.1 XLOC_018360 TCONS_00058666 65.1% 12.5% -52.7% AT4G20310.3 XLOC_017630 TCONS_00052323 96.1% 43.4% -52.7% AT3G58720.2 XLOC_016411 TCONS_00049714 75.4% 22.7% -52.7% AT5G51980.2 XLOC_026469 TCONS_00076466 84.1% 31.3% -52.8%

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AT3G24820.1 XLOC_012769 TCONS_00046444 77.9% 10.7% -67.2% AT1G22910.3 XLOC_001312 TCONS_00015176 74.6% 6.9% -67.7% AT1G20830.1 XLOC_001210 TCONS_00011917 87.9% 20.1% -67.7% AT1G33415.2 XLOC_001787 TCONS_00018243 79.9% 12.1% -67.8% AT1G50430.2 XLOC_002154 TCONS_00012362 83.1% 15.3% -67.9% AT5G40270.1 XLOC_025866 TCONS_00076226 72.0% 4.0% -68.0% AT1G56340.1 XLOC_002462 TCONS_00002524 72.5% 4.2% -68.3% AT4G14730.1 XLOC_017334 TCONS_00058238 91.5% 22.8% -68.7% AT1G63855.3 XLOC_006215 TCONS_00014300 85.1% 16.3% -68.8% AT5G54570.1 XLOC_026610 TCONS_00083267 78.3% 9.3% -69.0% AT1G73960.1 XLOC_003319 TCONS_00015991 84.9% 15.4% -69.5% AT4G09840.2 XLOC_019239 TCONS_00062336 85.5% 15.6% -69.9% AT5G05610.1 XLOC_024470 TCONS_00067695 86.7% 16.7% -70.0% AT3G26935.1 XLOC_012873 TCONS_00036477 85.5% 14.9% -70.6% AT1G11905.1 XLOC_000709 TCONS_00011722 97.3% 25.9% -71.4% AT1G77810.2 XLOC_006867 TCONS_00014591 75.9% 4.2% -71.6% AT2G36835.1 XLOC_008511 TCONS_00029439 81.9% 10.1% -71.8% AT4G17360.1 XLOC_019620 TCONS_00054380 86.2% 14.2% -71.9% AT3G08720.2 XLOC_014606 TCONS_00044904 88.3% 16.1% -72.2% AT3G50685.1 XLOC_013391 TCONS_00036943 87.9% 15.5% -72.4% AT4G23950.2 XLOC_017813 TCONS_00052507 73.3% 0.7% -72.7% AT3G04680.1 XLOC_011425 TCONS_00040531 81.4% 5.8% -75.7% AT2G42860.1 XLOC_008879 TCONS_00031223 84.6% 7.7% -76.8% AT1G52260.1 XLOC_002240 TCONS_00012392 82.6% 5.5% -77.1% AT5G12240.1 XLOC_021594 TCONS_00079567 92.6% 14.6% -78.0% AT4G28450.1 XLOC_018122 TCONS_00052778 85.2% 6.5% -78.7% AT3G58850.1 XLOC_016417 TCONS_00047983 91.8% 10.5% -81.3% AT2G45840.1 XLOC_009094 TCONS_00032766 93.8% 9.3% -84.5% AT2G44120.2 XLOC_008966 TCONS_00027554 89.7% 4.5% -85.2% AT5G27290.1 XLOC_025624 TCONS_00080947 91.6% 5.8% -85.9% AT2G15880.1 XLOC_007422 TCONS_00022725 91.2% 2.6% -88.6%

Appendix I

Documents

Financial support from Fundação para a Ciência e Thesis.pdf · Firstly, in a case-study approach, the functional relevance of an alternative splicing event in an E3 ubiquitin ligase