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TRANSCRlPTIONAL ANALYSIS OF THE

SEVEN IN ABSENTIA GENE FAMILY

IN ARABIDOPSIS

by

MOHAMED NABIL ATTAYA

A THESIS

IN

BIOLOGY

Submitted to the University Honors College ofTexas Tech University

in Partial Fulfillment of the Requirements

for the Degree Designation of

HIGHEST HONORS

May 2006

Randy Dale Allen Chairperson of the Committee

DR. Ri.NDYk ALE ALLEN Professor, Bi ogy

z:;-~ //J/~ ~

DR. GARY . BELL Dean, U · ersity Honors College

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ACKNOWLEDGEMENTS

This research would not have been possible without the support of many

individuals. 1 would like to thank my mentor Dr. Randy Dale Allen whose patience,

guidance, and scholarship serve as a consistent source of inspiration. Moreover, the

completion of this thesis would not be possible without his support, advice, and

invaluable contributions. I would also like to thank Dr. Mohamed Fokar for assisting me

in conducting several experiments.

This thesis is dedicated to my family, especially my parents who have supported

me all these years and have guided me upon the straight path.

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TABLE OF CONTENTS

ACKNOWLEDGMENTS 2

ABSTRACT 5

LIST OF FIGURES 6

LIST OF ABBREVIATIONS 7

CHAPTER

I INTRODUCTION 8

1.1 Protein Degradation 8

1.1.1 Lysosomes 8

1.1.2 Ubiquitin 8

1.1.2.1. Physical properties 9

1.1.2.2. Enzymatic reaction of the ubiquitin pathway 9

1.2 Seven in Absentia regulates eye development in Drosophila 10

1.3 Role of seven in Absentia in mammals 11

1.4 Role of ubiquitination in plant signaling pathways 12

1.5 Role of Seven of Absentia in plants 13

1.6 Investigative purpose 14

II MATERIALS AND METHODS 15

2.1 Plant growth and abiotic treatment 15

2.2 Purification of RNA 15

2.3 Preparation of cDNA 16

2.4 Quantitative PCR 16

2.5 Development of transgenic Arabidopsis plants with promoter GUS fusion 17

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2.6 Histochemical analysis of GUS expression 17

2.7 Microarray data analysis 18

III RESULTS AND DISCUSSION 19

3.1 Transcriptional Profiling of the Sina gene family in Arabidopsis 19

3.1.1 Microarray of tissue specificity of Sina expression 19

3.1.2 Microarray analysis of hormonal sensitivity of Sina expression 20

3.1.3 Microarray analsyis of Sina expression in response to environmental stress...21

3.2 Quantitative PCR analysis of Sina expression in response to environmental stress 22

3.3 GUS expression 25

3.4 Implications and Future Considerations 27

V REFERENCES 28

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ABSTRACT

Specific ubiquitination and subsequent proteolysis of proteins is a fiindamental

stiategy for post-ti-anscriptional regulation of gene expression in eukaryotic cells.

Ubiquitination is a multi-step process that requires at least three types of ubiquitin

transfer proteins. These are ubiquitin activating proteins (El), ubiquitin conjugating

proteins (E2) and ubiquitin ligases (E3). Seven in Absentia (SINA) and Seven in

Absentia Homologs (SIAH) comprise a highly conserved group of proteins that contain a

RING finger domain and ftmction as E3 ubiquitin ligases. SINA was first identified in

Drosophila where it is required for the differentiation of the R7 photoreceptor cells in the

eye. Although ubiquitination of proteins is a common feature of several signaling

pathways in plant cells, our understanding of the functions of plant SiaH genes is

incomplete. The primary goal of this project is to determine the functions of the subset of

six Arabidopsis genes that are most closely related to human SiaH-1. This goal will be

accomplished by answering the following four questions: What are the expression

patterns of the individual members of SiaH gene family and does their expression

correlate with developmental, hormonal or environmental signals?

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LIST OF FIGURES

3.1 Microarray analysis of tissue specificity 19

3.2 Hormonal sensitivity 20

3.3 Induction by environmental stress (microarray) 21

3.4 Induction of SinaT2 by environmental stress (qPCR) 22

3.5 Induction of SinaTSa by environmental stress (qPCR) 23

3.6 Induction of SinaT3b by environmental stiess (qPCR) 23

3.7 Induction of SinaTSc by environmental stress (qPCR) 24

3.8 Induction of SinaT4 by environmental stress (qPCR) 24

3.9 Induction of SinaTS by environmental stress (qPCR) 25

3.10 Localization of GUS activity 26

6 -

LIST OF ABBREVIATIONS

ABA

ARE

AUX/IAA

BL

BRZ

DUB

El

E2

E3

GA

GUS

HECT

PHYL

qPCR

SINA

SIAH

RING

SA

TTK88

Ub

abscisic acid

auxin response factor

auxin/indole-3-acetic acid

brassinolide

brassinozole

de-ubiquitination enzyme

ubiquitin-activating enzyme

ubiquitin-conjugating enzyme

ubiquitin protein ligase

gibberellic acid

6- glucoronidase

homologous to E6-AP C Terminus

phyllopod

quantitative polymerase chain reaction

seven-in-absentia

seven-in-absentia homologs

really interesting new gene

salicylic acid

tramtrak

ubiquitin

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CHAPTER I INTRODUCTION

1.1 Protein Degradation 1.1.1 Lysosomes

An essential constituent of the cellular lifestyle is that of posttranscriptional

modification such as protein degradation. The cells utilize different mechanisms in order

to degrade proteins including the use of endosomes/lysosomes and

ubiquitination/proteasome pathways. Lysosomes contain ~50 hydrolytic enzymes

including many proteases and may maintain a highly acidic pH of ~5. Lysosomes may

degrade substances that the cell internalizes via endocytosis and may recycle intracellular

elements that are enclosed within vacuoles that fuse with the lysosome. Eukaryotes may

also degrade protein via ubiquitination.

1.1.2 Ubiquitin

Ubiquitin (Ub) was fu-st isolated in the thymus and was thought to function as a

thymic hormone (reference). It became clear however that ubiquitin is located in all

eukaryotic tissues and organisms and hence, was given its characteristic name. Ubiquitin

is a highly conserved peptide that has a significant role in protein degradation

(Ciechanover et al. 1980; Wilkinson et al. 1980), chromatin structure (Matsui et al. 1979;

Mueller et al. 1985), the heat-shock response (Finley et al. 1984), and cell surface

receptors (Meyer, et al. 1986; Siegelman et al. 1986). Ubiquitin's conserved amino acid

sequence indicates that it may be essential if not important for several other cellular

functions.

1.1.2.1 Physical properties

Ubiquitin is composed of seventy-six amino acids. The protein has no cysteine or

tryptophan but contains 11 acidic residues and 11 basic residues. Vijay-Kumar et al.

(1985) determined the crystal structure for Ub and showed that it contains two domains

of helical structure, a five-strand beta sheet which composes a hydrophobic core and a

protruding carboxy terminus. The secondary structure of about 90% of the polypeptide

chain is stabilized by hydrogen-bonding. Ub's hydrophobicity and extensive hydrogen

bonding may account for its thermal stability.

1.1.2.2 Enzymatic reaction of the ubiquitin pathway

Ubiquitin exists in a free form or as part of a complex of proteins. Ubiquitin

conjugation to a particular substrate acts as a marking mechanism for selective protein

breakdown. Ubiquitination is a three step process requiring mediator proteins El, E2, and

E3 (Ciechanover et al.l998). The ubiquitin-activating enzyme. El, is responsible for the

ATP-dependent activation of ubiquitin's carboxy terminus and subsequent linkage via a

thioester bond. The ubiquitin-conjugating enzyme E2 accepts activated ubiquitin from El

only to transfer it to the condemned protein via an E3ubiquitin-protein ligase. E3

transfers activated ubiquitin to a Lys e-amino acid of the protein substrate thereby

forming an isopeptide bond (Zachariae et al. 1999). C-terminal residues of more ubiquitin

molecules are linked to lysine 48 of the initial ubiquitin (Aguilar et al. 2003), forming a

polyubiquitin chain. Once a substrate is polyubiquinated, it is then targeted to the

proteasome for degradation (Page et al. 1999). At least four ubiquitin molecules are

required for substrates to be efficiently targeted to the proteasome complex (Thrower et

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al. 2000). It appears that due to direct interaction with the protein substrate, E3's may

have a regulatory role in selecting the protein to be degraded. The extensive number of

E2's (> 20 in the plant Arabidopsis thaliana) however, suggests that they may also

regulate protein substrate specificity. E3 proteins may use one of two catalytic domains;

the HECT (homologous to E6-AP C terminus) or the RING (Really Interesting New

Gene) domains. The number of E3 proteins within the mammalian genome is estimated

to be over 400 (Li et al. 2005).

1.2 Seven in absentia regulates eye development in Drosophila

Seven-in-absentia (SINA) and seven-in-absentia homologs (SIAH) represent a

conserved collection of proteins that contain a RING finger domain and function as E3

ligases (Lorick et al. 1999). SESfA was first identified in Drosophila where it is required

for the differentiation of the R7 photoreceptor cells in the eye. The adult eye of

Drosophila consists of an array of 800 ommatidia, each consisting of eight photoreceptor

cells (R1-R8), eight accessory cells and four lens-secreting cells. Neuronal differentiation

of the R7 photoreceptor requires a RAS/MAPK signaling cascade based on a receptor

tyrosine kinase (sevenless). The sevenless signaling cascade activates transcription

factors and modifies cell cycle regulation (O'Neill et al. 1994). SINA and other

components of the signaling cascade including tramtrack (TTK88), phyllopod (PHYL),

and EBI constitute an interesting post-transcriptional regulatory mechanism (Carthew et

al.; Xiong et al. 1993; Chang et al. 1994).

SINA and PHYL interact with the transcriptional repressor TTK88, leading to

specific ubiquitination of TTK88 and its rapid degradation via the proteasome pathway

10

(Li et al. 1997; Tang, et al. 1997). More recently, EBI was identified an additional factor

in the SINA/PHYL/TTK88 ubiquitination complex (Boulton et al. 2000). EBI contains an

F-box and WD repeats typical of proteins that target protein for ubiquitin-dependent

degradation. Thus, the working model for the regulation of photoreceptor differentiation

in Drosophila suggests that TTK88, which represses the transcription of genes required

for photoreceptor differentiation, is specifically ubiquitinated and degraded in

photoreceptor precursor cells RI, R6 and R7 by interaction with PHYL, EBI and SINA.

The interaction of SINA with ubiquitin conjugating enzyme Dl (UBCDl) further

supports this model (Tang et al. 1997). Interestingly, inactivation of the fat facets gene

leads to the formation of supernumerary R7 cells and the gene product of fat facets is a

protein that cleaves ubiquitin conjugates from proteins (Huang et al. 1995) and may

inhibit degradation of such factors as TTK88 that suppress photoreceptor differentiation

1. 3 Role of seven in absentia in mammals

Mammalian SIAH proteins interact with and mediate the degradation of a number

of regulatory proteins including the netrin 1 receptor DCC (Hu et al.l999; 1997), the

nuclear receptor corepressor N-CoR (Zhang et al. 1998), the motor protein Kid (Germani

et al. 2003), the developmental protein NUMB (Susini et al. 2001), the neural transmitter

protein synaptophysin (Wheeler et al. 2002), and the transcriptional repressor TIEG-1

(Johnsen et al. 2002). Both human SiaHl and SiaH2 were found to interact with a human

homologue of the yeast ubiquitin conjugating protein (Ubc9) (Hu et al. 1997). SiaH can

act as single subunit E3 ubiquitin ligases but it can also function as part of an E3 ligase

complex with other factors. For example, a complex containing SiaH, Skpl, SIP and the

11 -

F-box protein Ebi facilitate the degradation of P-catenin . The N-terminus and RING

domain of SiaH binds E2 proteins (Hu et al. 1999) while the C-terminus interacts with

substrates and cofactors. The ability of SiaH to act both as a single E3 ligase and also to

interact with other factors is unusual and adds considerable flexibility to the cellular role

of SiaH (Conaway et al. 2002).

1.4 Role of ubiquitination in plant signaling pathways

Ubiquitin-dependent proteasomal degradation is essential for plant growth and

development. Regulated protein degradation confers two distinct advantages upon the

regulation of cell signaling. First, proteolysis serves as a dynamic mechanism which may

appropriately manage negative regulation of plant responses to hormonal and

environmental stimuli. Moreover, ubiquitination may occur rapidly, but also may be

transient and quickly reversed by de-ubiquitination enzymes (DUBs) (Vierstra 2003).

These two properties of proteasomal degradation provide the cellular system with

sensitivity as well as selectivity.

Ubiquitin-mediated protein degradation is essential for a variety of signaling

pathways in plants including photomorphogenesis, phytohormone responses, disease

resistance, and self-incompatibility (Hare et al. 2003). For instance, the RING protein

CONSTITUTIVELY PHOTOMORPHOGENIC (COPl) is considered to be a

prototypical E3 ubiquitin ligase. COPl mediates the destabilization of the basic leucine-

zipper transcription factors HYPOCOTYL 5 (HY5) and HY5 homolog (HYH) in

darkness. COPl has never been implicated in the ubiquitination of the two transcription

factors but another protein, COPl interacting protein (CIP8) does promote ubiquitination

of HY5 in vitro (Hardtke et al. 2002). COPl may however directly ubiquitinate LAFl, a

- 1 2 -

MYB-type transcription factor involved in the phyA-mediated photomorphogenic

response.

Ubiquitin-mediated proteolysis is also involved in plant responses to hormones

including auxin, abscisic acid (ABA), gibberillic acid (GA), among others. For example,

SCR™' degrades a subset of the AUXIN/INDOLEACETIC ACID (AUX/IAA) proteins

tiiat repress AUXIN RESPONSE FACTOR (ARE) transcriptional activators. The bZlP

tianscriptional activator ABA-rNSENSlTlVE5 (AB15) accumulates in the cell in the

presence of ABA. ABA's subsequent removal induces the ubiquitination of ABI5.

Moreover, GA responses are repressed by the nuclear protein GA-INSENSITIVE (GAI)

and REPRESSOR of GAI-3 (RGA) in the absence of active GA.

1.5 Role of SEVEN IN ABSENTIA in plants

Although ubiquitination is a pervasive process influencing a broad range of

cellular functions, our understanding of the functions of plant SINA genes is incomplete.

The function of one gene in particular has been partially elucidated. The gene, At5G3360

encodes a protein named SINAT5 that is required for signal transduction in roots (Xie et

al. 2002). SINAT5, a RTNG-motif protein, interacts with and ubiquitinates NO APICAL

MERISTEM/ATAF-1/CUP-SHAPED COTYLEDONLIKE 1 (NACl). Over expression

of SINAT5 in transgenic Arabidopsis plants yields roots that are significantly longer than

wild type. The biological role of the remaining SINA genes has yet to be revealed.

- 1 3 -

1.6 Investigative purpose

The purpose of this project is to identify the fiinctions and biological roles of the

SINA genes in Arabidopsis. Characterizing the biological function of SINA genes will

elucidate the role of targeted ubiquitination in plant signal transduction pathways and

identify important corollaries to animal systems. Moreover this research will instrumental

in provoking an impact upon the agricultural arena by providing novel mechanisms for

genetic manipulation of signaling pathways to optimize crop productivity.

-14

CHAPTER II MATERIALS AND METHODS

2.1 Plant growth and abiotic treatment

Arabidopsis (ecotype Colombia) plants were grown in a chamber at 22°C with

16h / 8h light/dark regime. To determine expression of Sina genes during stress

conditions, three weeks old plants were exposed to the following conditions: ABA, Cold,

hydrogen peroxide, high light, NaCl and heat. Control plants that were not exposed to

any tieatment were also included.

2.2 Purification of RNA

Seeds of wild type (WT) Arabidopsis (ecotype Columbia) were sown on 1/2

Murashige and Skoog salts (MS, Gibco) medium with 0.5 g/L MES (2-(-4morpholino)-

ethane sulphonic acid), 1 ml of B5 vitamins, 1 % sucrose and 0.3% phytagel. Total RNAs

were isolated using the RNAwiz kit protocol (Ambion, cat # 9736). Fresh plant material

was ground in liquid nitrogen into fine powder and then resuspended into 1 ml of

RNAwiz solution and 400 il of chloroform. The mixture was vortexed extensively at

room temperature, and centrifuged at 12,000 x g for 20 min at 4° C. The supernatant was

transferred into another tube, and an equal volume of isopropyl alcohol was added. After

mixing well, the solution was kept at room temperature for 45 min before centrifiigation

for 20 min at 15,000 x g at 4° C. The pellet was washed once with 1 ml of 75% ethanol,

air-dried and then dissolved in 80 |j,l of 0.5% SDS and centrifuged 4,300 x g for 15 min at

4° C. The resulting pellet was allowed to air-dry and then resuspended in 30-50 \i\ of

RNase-free water. To determine the quality of RNA by electrophoresis, 1 |ig of total

15

RNA was run on 1% agarose/formaldehyde denaturing gel and the RNA was stained with

ethidum bromide (EB). The absorbance was measured at 260 nM and 280 nM to check

the purity and the yield of RNA.

2.3 Preparation of cDNA

About four micrgrams of total RNA was used to synthesize the first strand cDNA

using Maloney-Murine leukemia virus reverse transcriptase (MuMLV RT) (Promega, cat

# PRM 1701). Reverse transcription was carried out as follows: Enough DEPC water

was added to 4 |ig of RNA to bring to a final volume of 25 p,l. The sample was heated at

67° C for 10 min in a water bath to rid the RNA of secondary structure. For first strand

synthesis, 10 ^1 of 5X MuMLV buffer, 4 il of dNTPs (2-5 ^iM), and 5.6 il oligo-dT

primer, 1 |xl of Rnasin and 4.4 \i\ of DEPC water to bring up the total volume of 50 il

were added to the microcentrifuge tube. The mixture was mixed well by pipetting and

was incubated at room temperature for 10 min for oligo-dT primer annealing. The

mixture was transferred onto ice for 5 min. To the micro centrifiige tube, 2.5 p,l MuMLV-

RT enzyme was added and the reaction was incubated at 37° C for 1 h. The reaction was

stored at 4° C until use. One microliter of synthesized cDNA was used as a template for

PCR reaction with primers for specific genes designed using the Primer Express (ABI).

2.4 Quantitative PCR

Tissue specific expression of the different Sina genes was analyzed by qPCR in

tissues collected from 3-week-old plants. Real-time PCR was performed using ABI

SYBR Green PCR kit

- 1 6 -

2.5 Development of transgenic Arabidopsis plants with promoter-GUS fusion genes

The Sina promoters were amplified by polymerase chain reaction (PCR) using

specific primers to introduce the restriction sites and these fragments were sub-cloned

into pGEM-T vector. The pBIlOl expression vector is a promoter-less plasmid that

contains a NPTII kanamycin resistant gene driven by a NOS promoter and a P-

glucuronidase (GUS) gene. The target sequence (promoter) was placed upstream of GUS

gene to drive the expression of the reporter gene. The pBI101-5'/na promoter fusions were

introduced into the Agrobacterium strain C58. Transformation of Arabidopsis was

achieved by the floral inoculation method. The primary transformants were selected on

MS medium supplemented with kanamycin.

2.6 Histochemical analysis of GUS expression

Arabidopsis seedlings were placed in 1.5 ml microfuge tubes with 200 |il of

40mg/ml 5-bromo-4-chloro-3-indolyl P-D-glucuronide cyclohexylamine salt (X-gluc)

plus GUS buffer solution. The tissues were infiltrated with staining buffer under vacuum

and incubated ovemight at 37° C. Stained seedlings were then treated with an ethanol,

series (60%, 75%, and 100%) to extract chlorophyll. The stained seedlings were viewed

and photographed using a dissection microscope to detect the expression of the Sina

genes. For the induction of SinaTSc, seedlings were tteated with 200 ^1 of NaCl

ovemight before the staining.

17-

2.7 Microarray data analysis

Expression patterns of the Sina genes in Arabidopsis in various tissues. Data used for the

analysis were downloaded from GENEVESTIGATOR

(https://wvyw.genevestigator.ethz.ch; Zimmermann et al., 2004)

18-

http://wvyw.genevestigator.ethz.ch

CHAPTER III RESULTS AND DISCUSSION

3.1. Transcriptional Profiling of the Sina gene family in Arabidopsis

3.1.1 Microarray of tissue specificity of Sina expression

Analysis of microarray data reveal that the seven in absentia gene family is

differentially expressed (Figure 3.1). SinaT2 shows high expression in leaf and low

expression in root. At3g58040 while SinaTSb is predominately found in pollen and

exhibits low expression in roots. SinaTSc displays a homogenous expression pattern

among all plant tissue types. SinaT4, however is highly expressed in pollen and expressed

at lower levels in rosettes. SinaTS is primarily found within roots.

i

1

18 n

16 -

14 -

pre

ssio

n

o

ro

Rel

ativ

e E

>

O

00

1

4 -

2 -

0 -t"

T

, 1

•

E

1

••

1 I \

E

1 1 1 1 I I < > '

F'igure 3. \. Expression profile of the Sina gene fan differentially expressed.

:c

1

[

1 1 i

• At2g41980-SinaT2

• AI3g61790-SinaT3a

n At3g58040-SinaT3b

D At3g13672-SinaT3c



' V'* ^co^

lily. Data reveal that the genes are

19-

3.1.2 Microarray analysis of hormonal sensitivity of Sina expression

The Seven in absentia gene family exhibits hormonal sensitivity (Figure 3.2).

Plants exposed to specific hormones show changes in expression of Sina genes. Plants

exposed to abscisic acid (ABA) showed a two fold increase in SinaTSc expression.

Brassinolide (BL) exposure led to a 1.3 fold increase in SinaT2 expression. Ethylene

caused a 0.5 fold change decrease in the expression of SinaTS while Indole-3-Acetic acid

(lAA) caused an increase. Salicyclic acid (SA) and methyl jasmonate (MJ) yield a

decrease in SinaTl expression. The brassinosteroid biosynthesis inhibitor Brassinazole

causes a decrease in SinaTSa and SinaTS. Hydrogen peroxide (HP) yields a two fold

decrease in the expression of SinaTSc.

2.5

2 J

1.5 -

1

a. 0.5 O)

D At2g41980-SinaT2

• At3g61790-SinaT3a

nAt3g58040-SinaT3b

nAt3g13672-SinaT3c



L O

o u. -0.5^*

-1 -

-1.5

-2 H

-2.5

<^ T J

O' .v> jnM^—fT—f "" —T f

Treatments

Figure 3. 2 Expression profiles of the Sina gene family after exposure to specific hormones. Plants were exposed to abscisic acid (ABA), brassinolide (BL), ethylene, gibberellic acid (GA3), indole-3-acetic acid (I A A), jasmonate (MJ), Salicyclic acid (SA), Brassinozole (BRZ), and hydrogen peroxide (HP).

-20-

3.1.3 Microarray analsyis of Sina expression in response to envirormiental stress

Plants exposed to different types of environmental stress exhibit unique

expression profiles of the seven in absentia gene family (Figure 3.3). Plants exposed to

osmotic, salt, and age stress show increased expression of SinaTSc. All types of stress led

to a decrease in the expression of SinaT2 except for senescence. Plants exposed to cold

stress showed a decrease in SinaTSb.

1

5 n

4 -

3 -

Ch

ang

e

1 '-

0

-2

I • ^

J L - J . ^ T . ^TW, ^

rj m

Stress Treatments


• At3g61790-SinaT3a

DAt3g58040-SinaT3b

DAt3g13672-SinaT3c



^ ^

s igure 3. 3 Expression profiles of the Sina gene family after plant exposure to different stress treatments.

21

3.2 Quantitative PCR analysis of Sina expression in response to environmental stress

Quantitative polymerase chain reaction (qPCR) was used to confirm and validate

gene expression changes obtained from array analyses. qPCR quantitation corroborated

previous findings for the seven in absentia gene family. Plants exposed to cold stress

show an increased relative expression of SinaT2. High light also induced the expression

of SinaT2. SinaTSa is also highly expressed in plants under cold stress and high light

conditions. Salt stress stimulates the expression SinaTSb. SinaTSc is highly expressed in

response to mannitol, abscisic acid, and salt stress treatments. SinaT4 is highly expressed

in cold stressed plants. SinaTS is highly expressed in plants exposed to high light, cold,

and salt stress.

- 99

SinaTSa

c o

"33 (0

a X Ui 0)

> i

^

^ ^

; # ^ ^ , # -.^^

^^ s<r ^ ^ ^^ .d>

^^ .^^

Figure 3.5 Expression profile of SinaTSa in plants exposed to different stress conditions

SinaTSb 400 n

350 -

300

250

200

c o (0 (0

£ CL X

Ul 0)

I 150 o

100

50 -

0 ± . ^ J" -- ^ ^ ^^ <o

-p^ ^^ / -o-

Figure 3.6 Expression profile of SinaTSb in plants exposed to different stress conditions

-23

SinaT3c

c o "« (0

a X tu a> _> ?

.c> ^^ ^ ' ^^ s^^ or

Figure 3.7 Expression profile of SinaTSc in plants exposed to different stress conditions

SinaT4

(0 M

a.

»

2000

1800

1600

1400

1200

1000

800

600

400

200

0 1 #

^^

W^

^^^ ^^ ^* ^^ #

^ ^ cfi

Figure 3.8 Expression profile of SinaT4 in plants exposed to different stress conditions

24

3.3 GUS expression

The patterns of Sina transcription were confirmed by analysis of the expression of

GUS reporter gene constructs driven by the SinaT2 , SinaTSc. SinaTSa, and SinaTS

promoters (Figure 3.10). The expression of the pSlNAT2: GUS gene construct is weak but

strong in slightly older, flowering plants.pSINATSc:GUS shows in an increase in

expression when induced by salt stress. The expression of the pSINATS: GUS gene

construct is relatively weak and restricted primarily to the cell expansion zone of the

roots. ThepSINATSa:GUS reporter, on the other hand is strongly expressed in shoots

while expression in roots is limited to the vascular cylinder.

25

Figure 3. 10 Localization of GUS activity in representative transgenic Arabidopsis plants that express reporter genes controlled by the SinaT2 (A,B), SinaTSc (C,D), SinaTS (E) and SinaTSa (F) promoters. SinaT2::GUS expression is higher in older (B) than in younger plants (A). SinaTSc::GUS gene construct expression is higher in sah stressed plants (D) than in normal plants (C). While expression from the SinaTS promoter is primarily limited to roots (E), SinaTSa promoter expression is seen throughout the shoot and in the root.

26

3.4 Implications and Future Considerations

Data suggest that the Sina gene family is dynamically regulated in Arabidopsis.

Their active responses to stiess and hormones coupled with a differential tissue

expression pattern suggest that the functions of each Sina studied are diverse and unique.

Moreover, the functions of SlNAs within the plant family will most likely be completely

different than the homologs found in Drosophila, human, or mouse. Albeit, due to the

important development and cell signaling functions SEVEN IN ABSENTIA proteins

have in insects and mammals, the presence of conserved SINA homologs coimotate that

these proteins are important in plants. This project has elucidated a small fraction of the

complex interplay Sina genes may have within the Arabidopsis genome.

Future studies will focus on phenotyping T-DNA knockout mutants for Sina

genes. Specifically, responses to abiotic stress will be evaluated for wild type and T-DNA

mutants. In case of redimdancy double, triple, and quadruple knockouts will be generated.

-27-

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