DIFFERENTIAL EXPRESSION AND REGULATION OF SUCROSE TRANSPORTERS IN RICE (Orzya sativa L. cv Nipponbare) DURING

ENVIRONMENTAL STRESS CONDITIONS

A THESIS

submitted in fulfilment of the requirements for the degree of

DOCTOR OF PHILOSOPHY (PhD) BIOCHEMISTRY

DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY UNIVERSITY OF FORT HARE

By Omodele IBRAHEEM

SUPERVISOR: Prof. G. Bradley CO SUPERVISOR: Prof. C.E.J.Botha

March, 2011

II

ABSTRACT

Plant productivity is greatly affected by environmental stresses such as drought, salinity and

insect herbivory. Plants respond and adapt to these stresses by exhibiting physiological as well as

biochemical changes at the cellular and molecular levels in order to survive. Expression of a variety

of genes which encode numerous membrane transporters have been demonstrated to be induced by

these stresses in a variety of plants. The nutritional status of plants is controlled by these

transporters, which are regulated by the transcription of the corresponding genes.

In spite of these adverse stress effects on agricultural yield, only a few studies have focused

on gene transcriptional and translational regulation of membrane transporters during environmental

stress situations. Rice, like other plants, contains a number of sucrose transporters encoded by a

family of genes. However, detailed knowledge of their roles, localization and regulation during

environmental stress conditions is lacking.

Bioinformatic tools were used to identify putative cis-acting regulatory elements that may

be involved in the regulation of rice and Arabidopsis thaliana sucrose transporters. The possible

cis-acting regulatory elements were predicted by scanning genomic sequences 1.5 kbp upstream of

the sucrose transporter genes translational start sites, using Plant CARE, PLACE and Genomatix

Matinspector professional data bases. Several cis-acting regulatory elements that are associated

with plant development, plant hormonal regulation and stress response were identified, and were

present in varying frequencies within the 1.5 kbp of 5′ regulatory region. The putative cis-acting

regulatory elements that possibly are involved in the expression and regulation of sucrose

transporter gene families in rice and Arabidopsis thaliana during cellular development or

environmental stress conditions were identified as: A-box, RY, CAT, Pyrimidine-box, Sucrose-box,

ABRE, ARF, ERE, GARE, Me-JA, ARE, DRE, GA-motif, GATA, GT-1, MYC, MYB, W-box,

and I-box.

Expression analysis was used to elucidate the role of rice (Oryza sativa L. cv Nipponbare)

sucrose transporter (OsSUT) genes during drought and salinity treatments of three week old rice

plants ( at four leaf stage) over a 10 days. Among the five rice OsSUT genes identified, only

OsSUT2 was observed to be progressively up-regulated during drought and salinity treatments,

while OsSUT1, OsSUT4 and OsSUT5 were expressed at low levels, and OsSUT3 showed no

detectable transcript expression. Sucrose transport will be essential to meet the cellular energy

demands and also for osmoprotectant activities during drought and salinity stresses. It therefore

indicates that OsSUT2 which facilitates transport of sucrose from photosynthetic cells will be

III

essential for rice plants to cope with drought and salinity stresses, and cultivars with a higher

OsSUT2 expression should be able to tolerate these environmental stresses better.

The role of OsSUT in assimilate transport during rusty plum aphids (Hysteroneura setariae;

Thomas) infestation on the leaves of three week old rice (Orzya sativa L. cv Nipponbare) cultivar

plants, over a time-course of 1 to 10 days of treatments, was also examined by combination of gene

expression and β-glucuronidase (GUS) reporter gene analysis.

Real Time PCR analysis of the five OsSUT genes revealed that the expression of OsSUT1

was progressively up-regulated during the course of aphid infestation. OsSUT2 and OsSUT4

expression were comparatively low in both the control and treated plants. OsSUT5 showed no clear

difference in transcript expression in both control and treated plants, while no detectable transcript

expression of OsSUT3 could be found. The up-regulation of OsSUT1 gene was verified at protein

level by western blot analysis in both the control and treated plants. OsSUT1 protein expression

was found to increase with time during aphid infestation. A similar trend was noticeable in the

control plants, however at a lower expression level. These demonstrate that the cellular expression

of OsSUT1is regulated by both developmental and environmental factors.

OsSUT1-promoter:::GUS reporter gene expression was observed within the vascular

parenchyma and/or companion cells associated with phloem sieve elements of the large and small

bundles in the phloem tissues of the flag leaf blade regions where feeding aphids were confined,

which progressively increased with time of infestation. It is suggested that OsSUT1 may primarily

play an essential role in phloem transport of assimilate to wounded tissues from adjacent health

tissues or may be involved in the retrieval of assimilate back into the phloem to minimize loss

caused by the infestation. Some OsSUT1-promoter:::GUS expression was also found in the

metaxylem at 10 days after infestation, which could signify a recovery system in which sucrose lost

into the xylem as a result of aphids feeding are retrieved back into the phloem through the vascular

parenchyma. This was supported by the exposure of cut ends of matured OsSUT1-promoter:::GUS

rice plant leaf to 2% sucrose solution. OsSUT1-promoter:::GUS expression was observed within the

protoxylem, xylem and phloem parenchyma tissues. This indicates that sucrose translocating within

the xylem tissues are retrieved into the phloem via the OsSUT1 localized within the parenchyma

tissues.

In conclusion, the differential expression and regulation of rice (Orzya sativa L. cv

Nipponbare) sucrose transporters as reported here suggest that OsSUT2 and OsSUT1 were

constitutively expressed compared to other rice sucrose transporters during drought and salinity,

and rusty plum aphids (Hysteroneura setariae; Thomas) infestation stresses respectively. Thus, the

expression and regulation of the sucrose transporters could be related to the physiological and

IV

nutritional requirements of the cells during plant developmental or environmental stress state that

allows their differential expression.

V

DECLARATION

I, Omodele IBRAHEEM do hereby declear that this thesis entitled “Differential expression

and regulation of sucrose transporters in rice (Orzya sativa L. cv Nipponbare) during environmental

stress conditions” submitted for the award of the degree PhD (Biochemistry) at the University of

Fort Hare, is my own original work and has not been submitted for any degree or examination at

any other university. I further declear that all sources of my information have been quoted as

indicated in the text and/or reference list.

Omodele IBRAHEEM

VI

DEDICATION

This work is dedicated to him that controls the beginning and the end of all things, the Almighty God, and to my family.

VII

ACKNOWLEDGEMENTS

To God be the glory for been my alpha and omega, given me the strength and wisdom to

start and to finish this race. His name is praised forever. Amen.

I greatly appreciated the invaluable love, help and advice given to me by my supervisor,

Prof. Graeme Bradley. I will forever be grateful for the opportunity he gave me to join his research

group for my doctoral study and for the bank of knowledge which he had impacted unto me from

his years of scholastic and intellectual endeavour. I appreciate you sir and I say a very big thank

you.

I’m also grateful for the love, support and accommodation given to me by my co-supervisor,

Prof C.E.J. Botha, for the inestimable instructions that he dropped onto my ears from his well of

wisdom and of course the absolute free access to his well-stocked and equipped laboratory. Thank

you so much sir.

I’m very grateful for the assistance and accommodation given to me by Prof. Saartjie Roux

and Dr. Gill Dealtry of Department of Biochemistry & Microbiology, Nelson Mandela

Metropolitan University, South Africa, while working in their Laboratory. Thank you so much, God

bless you two.

I greatly appreciate the assistance and attention of Mr Mahboob Jimoh throughout the

course of my work. Thank you so much, God will reward you. Amen

I acknowledge the Plant Stress Response Group members; both present and past. They have

been a source of encouragement to me at all times. I pray God reward them abundantly. Amen

I acknowledge the efforts of Drs O.A. Aiyegoro and E.O Igbinosa, Department of

Biochemistry and Microbiology, University of Fort Hare, for introducing me to Prof. Graeme

Bradley and processing my admission. God will reward both of you. Amen

I also will like to thank the academic members of Department of Biochemistry and

Microbiology, University of Fort Hare, and all the postgraduate students. Thank you all.

Special thanks to Mrs Leane Smith; Manager, School of Environmental and Biological

Sciences and Mrs Abimbola Badmus of the Phytomedicine Group, University of Fort Hare, for their

motherly attention and assistance at all times. God bless you and families. Amen.

VIII

In will like to thank Govan Mbeki Research and Development Centre (GMRDC) University

of Fort Hare, for their unflinching financial support; the award of a three years bursary to me and

funding of our research. Thank you very much.

I will also like to thank Bells University of Technology for granting me a study leave.

Thank you.

I will like to use this medium to express my sincere appreciation to Dr E. Obuotor and Dr

(Mrs) A. Kuku, both of Department of Biochemistry Obafemi Awolowo University, Nigeria, for the

fatherly and motherly role they played while preparing to come to South Africa. God in his

abundant greatness will reward both of you. Amen.

Thanks Rapheal, though we seldomly communicate while I was here, but am sure your kind

heart is always with me. God bless you and your family.

I’m also thanking the entire staff members of Department of Biochemistry, Obafemi

Awolowo University, Nigeria, for their impact in one way or the other. Surely, it was the

background training which I have gained from them all that carries me to this day. Most

importantly is Prof. Adeyinka Afolayan. Thank you sir, will forever remember and be grateful to

you.

Special thanks to Dr. R. Furbank, CSIRO Plant Industry, Canberra, ACT2601, Australia for

providing us seeds of rice (Orzya sativa L. cv Nipponbare) as well as the OsSUT1 promoter::GUS

transgenic rice seeds.

I thank Lloyd E. Adams, Professor Emeritus (deceased) and Department of Entomology,

The Pennsylvania State University for allowing me to use their aphid diagram in this thesis (Figure

5.1: Characteristic feature of aphids). Thank you.

With genuineness of heart and deep sense of appreciation, I acknowledge the efforts of my

parents, Chief and Mrs R.A. Ibraheem. From my day one on earth to this day, they have been my

mirrors and gold. My prayer is for them to live long, see my good works and eat the fruits thereof

with good days of life in Jesus name. Amen.

My three delectable and gorgeous sisters: Omosalewa, Omowunmi, and Omobosinuola are

treasure in my course of life. They have all made great contribution in my life, God bless and

multiply you all in Jesus name. Amen.

Finally, all glory and adoration be to Almighty God for being with me throughout the course

of my study.

IX

TABLE OF CONTENTS

Page Number

ABSTRACT……………………………………………………………………………. II

DECLARATION……………………………………………………………………….. V

DEDICATION…………………………………………………………………………. VI

ACKNOWLEDGEMENTS……………………………………………………………… VII

TABLE OF CONTENTS…………………………………………………………………. IX

LIST OF FIGURES………………………………………………………………………. XV

LIST OF TABLES……………………………………………………………………….. XVII

LIST OF ABBREVIATIONS……………………………………………………………. XVIII

BRIEF CHAPTER SYNOPSIS………………………………………………………….. XXI

CHAPTER 1: LITERATURE REVIEW……………………………………………… 1

1.1 Introduction…………………………………………………………………….. 2

1.2 Structural Analysis……………………………………………………………… 5

1.3 Expressional Analysis…………………………………………………………… 11

1.4 Mechanism of Action…………………………………………………………….. 12

1.5 Substrate Specificity……………………………………………………………… 14

1.6 Regulation of Action…………………………………………………………….... 17

1.7 References…………………………………………………………………………. 20

CHAPTER 2: RESEARCH MOTIVATION AND HYPOTHESIS………………….. 26

2.1 Introduction………………………………………………………………………. 27

2.2 Problem Statements………………………………………………………………. 27

2.2.1 Drought and Salinity……………………………………………………… 27

2.2.2 Aphid Infestation…………………………………………………………… 27

X

2.3 Hypotheses………………………………………………………………………… 28

2.4 Aim …………………………………………………………………………………. 28

2.5 Specific Objectives………………………………………………………………….. 28

2.6 Significance of Research……………………………………………………………. 29

2.7 References…………………………………………………………………………… 30

CHAPTER 3: A COMPARATIVE IN-SILICO ANALYSIS OF CIS-ACTING REGULATORY ELEMENTS IN PROMOTER REGIONS OF SUCROSE TRANSPORTER GENE FAMILIES IN RICE (Oryza sativa JAPONICA) AND ARABIDOPSIS THALIANA…………………… 32

3.1 Introduction………………………………………………………………………… 33

3.2 Promoter Prediction……………………………………………………………….. 34

3.3 Core Promoter Elements…………………………………………………………... 36

3.4 Identification of Cis-Acting Regulatory Elements and Composite

Transcription Factor Modules…………………………………………………….. 37

3.5 Stress Induced/Regulated Genes…………………………………………………... 37

3.6 Specific Objective…………………………………………………………………… 38

3.7 Method……………………………………………………………………………….. 39

3.7.1 Identification of the 5́UTR of Sucrose Transporter Genes………………… 39

3.7.2 Cis-Acting Regulatory Elements Analysis…………………………………. 39

3.8 Results………………………………………………………………………………. 40

3.9 Discussion…………………………………………………………………………… 55

3.9.1 Cellular Development Associated Cis-Acting Regulatory Elements……..…. 57

3.9.2 Hormonal Regulation Associated Cis-Acting Regulatory Elements…………. 59

3.9.3 Stress Response Associated Cis-Acting Regulatory Elements…..…………… 62

3.10 Conclusion………………………………………………………………………….. 67

3.11 References…………………………………………………………………………… 68

XI

CHAPTER 4: EFFECT OF DROUGHT AND SALINITY ON THE EXPRESSIONAL LEVELS OF SUCROSE TRANSPORTERS IN RICE (Oryza sativa L. cv NIPPONBARE). ………………………. 83

4.1 Introduction………………………………………………………………………. 84

4.2 Specific Objective………………………………………………………………….. 85

4.3 Method……………………………………………………………………………... 86

4.3.1 Cultivation of Plant Materials and Treatment ……………………………… 86

4.3.1.1 Cultivation of rice (Orzya sativa L. cv Nipponbare) at optimum

conditions…………………………………………………………… 86

4.3.1.2 Treatment of rice (Orzya Sativa L. cv Nipponbare) ……………….. 86

4.3.1.2.1 Drought treatment………………………………………………….. 86

4.3.1.2.2 Salinity treatment…………………………………………………… 87

4.3.2 Analysis of Differentially Expressed Sucrose Transporter………………….. 88

4.3.2.1 Isolation of total RNA from controls and treated rice plants……...… 88

4.3.2.2 Amplification of sucrose transporters (SUT) genes by reverse

transcriptase polymerase chain reaction (RT-PCR) ……………........ 89

4.3.2.3 Relative quantification of amplified SUT genes by Real-Time

PCR…………………………………………………………………… 90

4.4 Results………………………………………………………………………………….. 91

4.4.1 Effect of Treatment on Rice Plants…………………………………………… 91

4.4.2 Total RNA Extraction and OsSUTs’ Primer Pairs Confirmation…………… 92

4.4.3 Quantitative Real-Time PCR………………………………………………… 93

4.5 Discussion……………………………………………………………………………. 96

4.6 Conclusion…………………………………………………………………………… 98

4.7 References…………………………………………………………………………… 99

XII

CHAPTER 5: EFFECT OF RUSTY PLUM (Hysteroneura setariae; Thomas) APHIDS

INFESTATION ON THE EXPRESSIONAL LEVELS OF SUCROSE

TRANSPORTERS IN RICE (Oryza sativa L. cv NIPPONBARE)………. 102

5.1 Introduction…………………………………………………………………………… 103

5.2 Specific Objective……………………………………………………………………… 105

5.3 Method………………………………………………………………………………..... 106

5.3.1 Cultivation of Plant Materials and Treatment ………………………………..... 106

5.3.1.1 Cultivation of rice (Orzya Sativa L. cv Nipponbare) at optimum

conditions……………………………………………………………….. 106

5.3.1.2 Maintenance of rusty plum aphids (Hysteroneura setariae; Thomas)

Colonies…. ……………………………………………………………... 107

5.3.1.3 Aphids infestation on rice (Orzya Sativa L. cv Nipponbare) ……............ 107

5.3.2 Analysis of Differentially Expressed Sucrose Transporter Genes …………….... 108

5.3.2.1 Isolation of total RNA from controls and treated rice plants……..…....... 108

5.3.2.2 Amplification of sucrose transporters (SUT) genes by reverse

transcriptase polymerase chain reaction (RT-PCR) …………....……...... 108

5.3.2.3 Relative quantification of amplified SUT genes by Real-Time

PCR…………………………………………………………………........ 109

5.3.2.4 Isolation of total proteins from control and treated rice plants….............. 109

5.3.2.5 Monitoring the expression levels of OsSUT1 proteins by western

blot analysis and its relative quantification…………………………… 110

5.3.3 Analysis of OsSUT1:::GUS Activity Localization............................................. 111

5.3.3.1 Construction of OsSUT1:::GUS gene and transformation into rice

plant …................................................................................................... 111

5.3.3.2 Cultivation of OsSUT1:::GUS transgenic rice plants and aphid

treatment …........................................................................................... 112

XIII

5.3.3.3 Exposure of cut ends of OsSUT1:::GUS transgenic rice plant to 2%

sucrose solution……………………………….….................................. 112

5.3.3.4 Staining of transgenic rice for GUS expression ….................................. 112

5.4 Results…………………………………………………………………………………. 113

5.4.1 Total RNA Extraction and OsSUTs’ Primer Pairs Confirmation……………… 113

5.4.2 Quantitative Real-Time PCR…………………………………………………… 114

5.4.3 Western Blot Analysis and Relative Quantification of OsSUT1 Protein………. 116

5.4.4 OsSUT1:::GUS Activity Localization ………………………………………… 118

5.5 Discussion……………………………………………………………………………… 121

5.6 Conclusion…………………………………………………………………………….. 124

5.7 References……………………………………………………………………………… 125

CHAPTER 6: FUTURE PROSPECT…………………………………….……………....... 130

LIST OF ARTICLES EXTRACTED FROM THESIS……………………………………… 134

APPENDICES ………………………………………………………………………………… 136 Appendix A RNA Quantification Protocol…………………………………………… 137

Appendix B OsSUTs cDNA Sequences……………………………………………… 137

Appendix C RC/DC Protein Assay Protocol. ………………………………………… 143

Appendix D OsSUTs Protein Sequences…………………………………………….. 144

Appendix E Sodium Docecyl Sulphate Polyacrylamide Gel Electrophoresis

(SDSPAGE) Analysis…..……………………………………………….. 146

Appendix F 1% Agarose Gel Electrophoresis of Total RNA Extracts from Control and Stressed Rice Plants ……………………………………….. 148

Appendix G Melt Curve Analysis of OsSUTs’ Genes………………………………… 149

XIV

Appendix H Clustal W Multiple Sequence Alignment of OsSUT2, AtSUT4 and

HvSUT2…………………………………………………………..……. 150

Appendix I Copyright of Aphid Diagram Used in Thesis.……………………..….. 151

XV

LIST OF FIGURES

CHAPTER 1 Page Number

Figure 1.1 The path of sucrose from source to sink. …………………………………. 4

Figure 1.2 Phylogenetic tree for 48 confirmed sucrose transporter protein sequences from publicly accessible databases……………….………………………… 6

Figure 1.3 Two dimensional models of sucrose transporters for the different

subgroups. …………………………………………………………………… 10

CHAPTER 3

Figure 3.1 Map of the promoter regions of rice (Oryza sativa Japonica cultiva-group) sucrose transporter gene family………………………………………………... 44

Figure 3.2 Map of the promoter regions of Arabidopsis thaliana sucrose transporter gene family…………………………………………………………………….. 45

Figure 3.3 Illustration of the frequencies of the cis-acting regulatory elements identified in the promoter sequences of rice (Oryza sativa Japonica cultiva-group) sucrose transporter gene family……………………………………………………….. 48

Figure 3.4 Illustration of the frequencies of the cis-acting regulatory elements identified

in the promoter sequences of Arabidopsis thaliana sucrose transporter gene

family………………………………………………………………………… 49

Figure 3.5 Representation of the summation of cis-acting regulatory elements associated

with cellular development, Plant hormone, abiotic and biotic stresses in each

rice (Oryza sativa Japonica cultiva-group) and Arabidopsis thaliana sucrose

transporter gene families……………………………………………………… 57

CHAPTER 4

Figure 4.1 Greenhouse set-up of rice plants for drought experiment…………………… 87

Figure 4.2 Greenhouse set-up of rice plants for salinity experiment…………………… 88

Figure 4.3 Effect of drought and salinity on two week old rice plants, at 10 days

post treatment………………………………………………………………… 91

Figure 4.4 Representative sample of 1% agarose gel electrophoresis of total RNA extracts of (A) drought experiment (B) salinity experiment………..…………………. 92

Figure 4.5 Confirmation of RT-PCR amplification bythe various primer pairs on 1% agarose gel...…………………………………………………………………… 92

XVI

Figure 4.6 OsSUT1 melt curve analysis; a representative sample …..…………………. 93

Figure 4.7 Quantitative PCR analysis of OsSUT1, OsSUT2, OsSUT4 and OsSUT5 genes during drought treatment of rice (Orzya sativa L. cv Nipponbare) cultivar plants.………………………………………………………………... 94

Figure 4.8 Quantitative PCR analysis of OsSUT1, OsSUT2, OsSUT4 and OsSUT5 genes during salinity treatment of rice (Orzya sativa L. cv Nipponbare) cultivar plants. …………………………………………………….……….... 95

CHAPTER 5

Figure 5.1 Characteristic features of aphids ……….…………………………………….. 104

Figure 5.2 Rusty plum aphid (Hysteroneura setariae)………………………………….. 105

Figure 5.7 (A) Rusty plum aphids (Hysteroneura setariae; Thomas) maintained in insect

cage………………………………………...……………………………..... 107

(B) Rusty plum aphids (Hysteroneura setariae; Thomas) on rice leaf………… 107

Figure 5.4 Conviron set-up of rice plants for Rusty plum aphids (Hysteroneura setariae;

Thomas) infestation experiment……………………………………………… 108

Figure 5.5 Representation of OsSUT1:::GUS gene fusion construct…..………………… 111

Figure 5.6 Representative sample of 1% agarose gel electrophoresis of total RNA extracts….…...................................................................................................... 113

Figure 5.7 Confirmation of RT-PCR amplification by the various primer pairs on 1% agarose gel ……………………………………………………………………. 114

Figure 5.8 OsSUT1 melt curve analysis; a representative sample ….……………………. 114

Figure 5.9 Quantitative PCR analysis of OsSUT1, OsSUT2 and OsSUT4 genes during Rusty plum aphids (Hysteroneura setariae; Thomas) infestation on rice (Orzya sativa L. cv Nipponbare) cultivar plants …..………………………….. 115

Figure 5.10: Western blotting analysis and relative quantification of OsSUT1protein in rice (Orzya sativa L. cv Nipponbare) cultivar plants. ………………………… 117

Figure 5.11: (A – I) Cross-sections through the mid portions of mature of rice leaf blade tissues, counterstained with ImaGene Green(TM) for the cellular localization of OsSUT1- Promoter:::GUS expression during aphid infestation…………… 119

Figure 5.12 : ( A-B) Show the effect of exposure of the cut ends of mature leaves to a 2% sucrose solution…………………………………………………………… 120

XVII

LIST OF TABLES

CHAPTER 1 Page Number

Table 1.1 Sucrose transporters affinity for sucrose ………………………………... 16

CHAPTER 3

Table 3.1 The BLASTN result of rice (Oryza sativa Japonica cultiva-group) and

Arabidopsis sucrose transporter gene families. …………………………… 41

Table 3.2: Location of the mRNA and CDS sequences of rice (Oryza sativa Japonica cultiva-group) and Arabidopsis thaliana sucrose transporter gene families as they appear in the different chromosomes, indicating the positions of all exons and introns…………………………………………………………….. 42

Table 3.3 Putative cis-acting regulatory elements identified in the promoter regions

of rice (Oryza sativa Japonica cultiva-group) and Arabidopsis thaliana sucrose transporter gene families……………………………………………. 46

Table 3.4 Percentage composition of cis-acting regulatory elements per 300 base pair

up-stream of 5́UTR of rice (Oryza Sativa) and Arabidopsis thaliana sucrose transporter genes……………………………………………………………….. 52

Table 3.5 The clustal-W multiple sequence alignment of rice (Oryza sativa Japonica

cultiva-group) and Arabidopsis thaliana sucrose transporter gene families.…… 53 Table 3.6: The clustal-W multiple sequence alignment of rice (Oryza sativa Japonica

cultiva-group) sucrose transporter protein sequences …………………………… 54 Table 3.7: The clustal-W multiple sequence alignment of Arabidopsis thaliana sucrose

transporter protein sequences ……………………………………………………. 54

CHAPTER 4

Table 4.1 Rice sucrose transporter primer pairs………………………………………….. 89

XVIII

LIST OF ABBREVIATIONS

ABA Abscisic acid

APS Ammonium persulphate

AU Arbitrary Unit

BLAST Basic Local Alignment Search Tool

Bp Base Pairs

CC Companion Cell

cDNA Complementary Deoxyribonucleic Acid

ESTs Expressed Sequence Tags

ET Ethylene

GUS β-Glucuronidase

HR Hypersensitive Response

IPG Immobilised pH Gradient

JA Jasmonic Acid

kDa Kilodalton

Km Michaelis Constant

mA Milliampere

MC Mesophyll Cell

mRNA Messenger Ribonucleic Acid

NCBI National Centre For Biotechnology Information

OA Okadaic Acid

PCR Polymerase Chain Reaction

PD Plasmodesmata

PEG Polyethylene Glycol

XIX

pI Isoelectric Point

PMF Proton Motive Force

PPU Pore Plasmodesmata Unit

PR Pathogenesis Related Proteins

R gene Resistance Gene

RNA Pol II Ribonucleic acid Polymerase II

ROS Reactive Oxygen Species

RT-PCR Reverse Transcriptase Polymerase Chain Reaction

SA Salicylic Acid

SAR Systemic Acquired Resistance

SC Sink Cell

SDS Sodium Dodecyl Sulphate

SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis

SE Sieve Elements

SUC Sucrose Carrier

SUT Sucrose Transporter

TEMED N,N,N’N’ tetramethylethylenediamine

TFBs Transcription Factor Binding Site

TFs Transcription Factor

TRIS Tris Hydroxyl Methyl Amino Methane

TSS Transcription Start Site

ULD Unloading Domain

Vac Vacuole

X-Gluc 5-Bromo-4-Chloro-3-Indolyl-β-D-Glucuronide

XX

∆ψ Electrical Gradient

∆H pH Gradient

5′-UTR 5′-Untranslated Region

XXI

BRIEF CHAPTER

SYNOPSIS

XXII

CHAPTER 1:

This chapter discusses a concise but comprehensive literature review of sucrose transporters

(SUT’s) in plants in general. The structural and expressional analysis, mechanism of action,

substrate specificities and regulation of SUT’s action are discussed.

CHAPTER 2:

The overall rationale and motivation for the present study are discussed in this chapter,

followed by the hypothesis and specific objectives which this study seeks to actualize.

CHAPTER 3:

Discussed here is an inclusive comparative analysis of cis-acting regulatory elements

present within the 5́- untranslated region (5΄-UTR) of the sucrose transporter gene families in rice

(Oryza sativa Japonica cultivar - group) and Arabidopsis thaliana, using available bioinformatics

tools. The cis-acting regulatory elements were predicted by scanning 1500 base pairs upstream of

the translational start sites (ATG), using Plant CARE, PLACE and Genomatix Matinspector

professional data bases. It is assumed that these cis-acting regulatory elements may serve as target

binding sites for functional promoters for the expression and regulation of these genes.

CHAPTER 4:

Drought and salinity stresses are two major adverse abiotic stresses that pose a great

challenge to survive of plants, most especially commercial crops such as rice. In this chapter, RT-

PCR and Real- Time quantitative analyses were carried out to determine the differential expression

of individual OsSUT genes, induced by these stress treatments on 3 week 4 leaf rice (Orzya sativa

L. cv Nipponbare) plants.

CHAPTER 5:

Among plant insect pests, aphids (Hemiptera, Aphidoidae) have been reported to cause

significant damage to valuable economic cereal crops including rice, wheat, barley and oats. This

XXIII

chapter is focused on RT-PCR and Real-Time quantitative analyses of the differential expression of

individual OsSUT genes, in relation to rusty plum aphid (Hysteroneura setariae; Thomas)

infestation on 3 week 4 leaf rice (Orzya sativa L. cv Nipponbare) plants. Western blotting analysis

was used to confirm that the up-regulated OsSUT1 gene is translated into full functional protein.

Furthermore, β-glucuronidase (GUS) gene expressional analysis was used to confirm in-vivo, the

up-regulated OsSUT1 gene expression during rusty plum aphid (Hysteroneura setariae; Thomas)

infestation on 3 week, 4 leaf rice (Orzya sativa L. cv Nipponbare) plants, and also to determine the

sub-cellular localization of expression during this stress period.

CHAPTER 6:

This chapter briefly discusses future research that should be carried out in order to provide

further insight into the role of SUTs during environmental stress conditions, thus providing

information that may aid in general crop improvement.