universiti putra malaysia sigit ismawanto fbsb 2013 32 functional

UNIVERSITI PUTRA MALAYSIA

SIGIT ISMAWANTO

FBSB 2013 32

FUNCTIONAL ANALYSIS OF ARABIDOPSIS THALIANA 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID OXIDASE GENE

IN RESPONSE TO LIMITED WATER

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FUNCTIONAL ANALYSIS OF ARABIDOPSIS THALIANA

1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID OXIDASE GENE


By

SIGIT ISMAWANTO

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfillment of the Requirement for the Degree of Master of Science

July 2013

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COPYRIGHT

All material contained within the thesis, including without limitation text, logos,

icons, photographs and all other artwork, is copyright material of Universiti Putra

Malaysia unless otherwise stated. Use may be made of any material contained within

the thesis for non-commercial purpose from the copyright holder. Commercial use of

material may only be made with the express, prior, written permission of Universiti

Putra Malaysia

Copyright © Universiti Putra Malaysia

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia

in fulfillment of the requirement for the degree of Master of Science

FUNCTIONAL ANALYSIS OF ARABIDOPSIS THALIANA

1-AMINOCYCLOPROPANE -1-CARBOXYLIC ACID OXIDASE GENE


By

SIGIT ISMAWANTO

July 2013

Chairman : Assoc. Prof. Dr Mohd Puad Abdullah

Faculty : Biotechnology and Biomolecular Sciences

Prolonged drought conditions pose a serious threat to plant growth and productivity

due to water limitation. To survive, plants adapt by altering various biochemical

pathways leading to physiological changes appropriate for growth under water-

limited condition. One chemical known to be induced during this condition is

ethylene, a plant hormone that affects plant vegetative and reproductive behaviours.

Ethylene is synthesized by a two-step biosynthetic pathway mediated by the enzyme

1-aminocyclopropane-1- carboxylic acid synthase (ACS) and 1-aminocyclopropane-

1-carboxylic acid oxidase (ACO). In plants, both genes are members of separate gene

families. Recent data have shown that members of the ACO and ACS gene families

were affected by limited water condition in Arabidopsis thaliana. The involvement

of the other members of the ACC and ACO gene families is unknown. Therefore, the

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objectives of this study were to determine the ACS and ACO genes responding to

water-limited stress condition in Arabidopsis plants and to functionally analyse the

role of an inducible ACO gene in Arabidopsis plant during development and water-

limited stress. The over-expression construct was generated by using the Gateway

technology and introduced into Arabidopsis by the Agrobacterium-mediated floral

dip method. From nine AtACS genes, only AtACS2 and AtACS6 responded to the

PEG-induced water stress. AtACS2 was induced in roots whereas AtACS6 was down-

regulated in leaves and roots. All six members of the AtACO gene family responded

to PEG-induced water stress with four genes (AtACO3, AtACO4, AtACO5 and

AtACO6) were switched-off in roots whereas three genes (AtACO1, AtACO3 and

AtACO6) were induced in the leaves. Among the inducible AtACO genes, AtACO1

was unique because this gene was not expressed in roots but induced in leaves. Over-

expressing AtACO1 in Arabidopsis plant changed the overall vegetative growth of

the plant, particularly the root system where the root became shorter and lesser.

Interestingly, under PEG-induced water stress the number of lateral roots was

increased. Overall, the transgenic plants responded the same way as wild plants

judging by the biochemical and physiological parameters associated with drought-

stressed plants obtained from the plants. In short, the AtACO1 gene was more likely

involved in plant development particularly the root system.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia

sebagai memenuhi keperluan untuk ijazah Master Sains

FUNGSI ANALISIS GEN 1-AMINOCYCLOPROPANE -1-KARBOSILIK

ASID PADA ARABIDOPSIS THALIANA DI BAWAH KEADAAN

KEKURANGAN AIR

Oleh

SIGIT ISMAWANTO

Julai 2013

Pengerusi : Assoc. Prof. Dr. Mohd Puad Abdullah

Fakulti : Bioteknologi dan Sains Biomolekul

Keadaan kemarau berpanjangan menimbulkan ancaman yang serius kepada

pertumbuhan dan produktiviti tumbuhan akibat kekurangan air. Untuk terus hidup,

tumbuh-tumbuhan menyesuaikan diri dengan mengubah pelbagai tapakjalan

biokimia yang membawa kepada perubahan fisiologi yang sesuai untuk berkembang

di bawah keadaan air yang terhad. Salah satu bahan kimia yang diketahui teraruh

semasa keadaan ini adalah etilena, hormon tumbuhan yang mempengaruhi perlakuan

tumbuhan peringkat vegetatif dan reproduktif. Etilena disintesis oleh tapakjalan

biosintesis dua-langkah yang dimangkinkan oleh enzim 1-aminocyclopropane-1-

carboxylic acid sintase (ACS) dan 1-aminocyclopropane-1- carboxylic acid oksidase

(ACO). Dalam tumbuhan, kedua-dua gen ini adalah ahli famili gen yang berasingan.

Data terkini menunjukkan bahawa ekspresi satu gen daripada famili ACO dan ACS

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terjejas oleh keadaan air yang terhad dalam tumbuhan Arabidopsis thaliana.

Penglibatan ahli-ahli lain dalam famili gen ACC dan ACO tidak diketahui. Oleh itu,

objektif kajian ini adalah untuk menentukan gen ACS dan ACO yang bertindak balas

kepada keadaan air yang terhad dalam tumbuhan Arabidopsis dan untuk

menganalisis peranan gen ACO boleh-aruh dalam tumbuhan tersebut semasa proses

perkembangan dan di bawah keadaan kekurangan air. Binaan gen untuk

pengekspresan-lebih ACO1 telah dibangunkan dengan menggunakan teknologi

Gateway dan diperkenalkan ke dalam Arabidopsis melalui kaedah celupan bunga

berperantara-Agrobacterium. Daripada sembilan gen AtACS, hanya AtACS2 dan

AtACS6 bertindak balas terhadap kekurangan air aruhan-PEG. AtACS2 teraruh dalam

akar manakala ekspresi AtACS6 menurun dalam daun dan akar. Kesemua enam ahli

famili gen AtACO bertindak balas terhadap keadaan kekurangan air aruhan-PEG

dengan ekspresi empat gen (AtACO3, AtACO4, AtACO5 dan AtACO6) dimatikan di

akar manakala tiga gen (AtACO1, AtACO3 dan AtACO6) diaruh atau ditingkatkan

dalam daun. Di antara gen AtACO yang teraruh, AtACO1 adalah unik kerana gen ini

tidak diekspresi di akar tetapi diaruh dalam daun. Pengekspresan secara berlebihan

gen AtACO1 dalam tumbuhan Arabidopsis mengubah keseluruhan pertumbuhan

vegetatif tumbuhan, terutamanya sistem akar di mana ianya menjadi lebih pendek

dan kurang. Menariknya, di bawah keadaan kekurangan air bilangan akar sisi

meningkat. Secara keseluruhannya, tumbuhan transgenik tersebut bertindak balas

dengan cara yang hampir sama seperti tumbuhan liar berdasarkan ukuran parameter

biokimia dan fisiologi berkaitan kemarau yang diperolehi. Secara ringkas, gen

AtACO1 berkemungkinan terlibat dalam perkembangan tumbuhan terutamanya

sistem akar.

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ACKNOWLEDGEMENTS

In the name of Allah, The Most Merciful and The Most compassionate. All praises

and thanks to Allah Almighty for His Mercy and Grace. Sholawat and greetings to

the Prophet, Muhammad S.A.W. With the blessings and Allah’s guidance, I have

completed my research study and preparation of this Master thesis.

First and foremost, I wish to express my deep gratitude to Assoc. Prof. Professor Dr.

Mohd Puad Abdullah, chairman of my supervisory committee, for all his tremendous

support, guidance and advice during the accomplishment of my study. My sincere

thanks are also to my supervisory committee member, Assoc. Prof. Dr. Suhaimi

Napis for his helps, encouragements, suggestions and constructive comments

throughout the period of my study. Greatly appreciate all the help they availed to me

while pursuing my studies.

I would also like to thank to all staff and officer members of faculty of

Biotechnology and Biomolecular Sciences UPM for the assistance. Special

appreciations to all my lab mates for their cooperation, idea and discussion in

completion my lab work and thesis writing, and to all my friends at Universiti Putra

Malaysia, especially to the Indonesian Students Association (PPI-UPM) members

and my house mate at R719 for their friendship during the period of my study.

Special acknowledgement also extended to Sembawa Rubber Research Centre,

Indonesia Rubber Research Institute for supporting me with the scholarship and

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giving me the extension of study period. Without the financial support and extension,

it is impossible for me to complete my study.

Finally my deepest gratitude goes to my parents and all my family members for their

continuous moral support throughout my study.

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I certify that a Thesis Examination Committee has met on 19 July 2013 to conduct

the final examination of Sigit Ismawanto on his thesis entitled “Functional Analysis

of Arabidopsis thaliana 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Gene in

Response to Limited Water” in accordance with the Universities and University

Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A)

106] 15 March 1998. The Committee recommends that the student be awarded the

Master of Science.

Members of the Thesis Examination Committee were as follows:

Janna Ong binti Abdullah, PhD

Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Noor Azmi Shaharuddin, PhD

Senior Lecturer



(Internal Examiner)

Ho Chai Ling, PhD

Associate Professor



(Internal Examiner)

Sreeramanan Subramaniam, PhD

Senior Lecturer

Universiti Sains Malaysia

Malaysia

(External Examiner)

NORITAH OMAR, PhD

Associate Professor and Deputy Dean

School of Graduate Studies


Date: 19 September 2013

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfillment of the requirement for the degree of Master of Science. The

members of the Supervisory Committee were as follows:

Mohd Puad Abdullah, PhD

Associate Professor



(Chairman)

Suhaimi Napis, PhD

Associate Professor



(Member)

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and citations,

which have been duly acknowledged. I also declare that it has not been previously,

and is not concurrently, submitted for any other degree at Universiti Putra Malaysia

or at any other institution.

SIGIT ISMAWANTO

Date: 19 July 2013

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TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRAK iv

ACKNOWLEDGEMENTS vi

APPROVAL viii

DECLARATION x

LIST OF TABLES xv

LIST OF FIGURES xvi

LIST OF ABBREVIATIONS xviii

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 4

2.1 Drought

2.2 Response of plant to water-limited condition

2.2.1 Morphological response

2.2.2 Physiological response

2.2.3 Biochemical response

2.2.4 Molecular response

2.3 Ethylene

2.4 Plant ACC synthase and ACC oxidase genes

2.5 Role of ethylene in plant growth and development

2.5.1 Ethylene and plant growth

2.5.2 Ethylene and Root Formation

2.5.3 Ethylene and fruit ripening

2.5.3 Ethylene and leaf senescence

2.6 Ethylene in plant adaptation to environmental stresses

2.6.1 Ethylene in wounding

2.6.2 Ethylene under water-limited stress

2.6.3 Ethylene and salinity

2.7 Arabidopsis as a model plant for gene functional study

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3 EXPRESSION ANALYSIS OF THE ACC SYNTHASE AND ACC

OXIDASE GENES IN ARABIDOPSIS PLANTS GROWN

UNDER DROUGHT STRESS CONDITION

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3.1. Introduction

3.2. Material and Methods

3.2.1. Plant Material

3.2.2 Chemicals and kits

3.2.3 Growing and maintenance of A. thaliana in hydroponics

3.2.4 Water stress treatment

3.2.5 Determination of relative water content

3.2.6 Determination of proline content

3.2.7 Determination of chlorophyll content

3.2.8 Isolation and purification of total RNA

3.2.9 Reverse trascription-polymerase chain reaction (RT-PC)

assay

3.2.10 Primer design

3.2.11 PCR assay

3.2.12 Construction of phylogenetic tree

3.3. Results and Discussion

3.3.1 PEG-treated Arabidopsis plants demonstrated water-

stressed biochemical symptoms

3.3.2 Expression profiles of ACS and ACO under water-

limited condition

3.3.3 A. thaliana ACS and ACO genes sequences analysis

3.4 Conclusion

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4 FUNCTIONAL ANALYSIS OF THE ACO1 GENE IN

ARABIDOPSIS PLANT

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4.1. Introduction

4.2. Material and Methods

4.2.1 Plasmid and bacterial strains

4.2.2 Specialised kits and reagents

4.2.3 cDNA amplification of AtACO1 gene

4.2.4 Purification of AtACO1 cDNA

4.2.5 Constructing the AtACO1 entry clone

4.2.6 Transformation of AtACO1 entry clone into E. coli

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4.2.7 pENTR-AtACO1 entry clone isolation

4.2.8 Verifying the pENTR-AtACO1entry clone by PCR

4.2.9 Generating the pMDC139-AtACO1 expression vector

4.2.10 Verifying the pMDC139-AtACO1 expression vector

with restriction enzyme digestion

4.2.11 Preparing the A. tumefaciens competent cells

4.2.12 Transforming A. tumefaciens with the expression vector

4.2.13 Verifying pMDC139-AtACO1 in A. tumefaciens cells by

colony PCR

4.2.14 Verifying pMDC139-AtACO1 in A. tumefaciens cells

4.2.15 Preparing A. tumefaciens culture for plant transformation

4.2.16 Transforming A. thaliana by floral dip method

4.2.17 Screening of T1 seeds by hygromycin selection

4.2.18 Verifying T1 transgenic plants by PCR

4.2.19 Segregation analysis of T2 seeds

4.2.20 AtACO1 transgene expression analysis in pMDC139-

AtACO1 by reverse transcription-PCR (RT-PCR)

4.2.21 Growth performance of the transgenic pMDC139-

AtACO1 plant at the seedling stage

4.2.22 Changes in relative water content and proline in

AtACO1-OE plant grown under PEG-induced water

limited stress

4.2.23 Statistical analysis

4.3 Results and Discussion

4.3.1 Construction and transformation of A. tumefaciens with

pMDC139-AtACO1

4.3.2 Transformation of A. thaliana plants

4.3.3 Screening of T1 transformants

4.3.4 Segregation analysis

4.3.5 Expression analysis of the AtACO1 transgene

4.3.6 Effect of AtACO1 overexpression on plant growth

4.3.7 Growth performance of AtACO1-OE plant under

PEG-induced water stress

4.3.8 Effects of AtACO1 overexpression on relative water

content and proline

4.5. Conclusion

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5 SUMMARY, CONCLUSION AND RECOMMENDATIONS

FOR FUTURE RESEARCH

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5.1 Summary and conclusions

5.2 Recommendation for future study

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REFERENCES 102

APPENDICES 121

BIODATA OF STUDENT 129

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universiti putra malaysia sigit ismawanto fbsb 2013 32 functional