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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
IN RESPONSE TO LIMITED WATER
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
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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
IN RESPONSE TO LIMITED WATER
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
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)
Ho Chai Ling, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(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
Universiti Putra Malaysia
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
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)
Suhaimi Napis, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)
BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
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