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UNIVERSITI PUTRA MALAYSIA

HASLIZA BINTI HASSAN

FBSB 2012 38

PROTEOMIC PROFILES OF FLORAL AND LEAF TISSUES OF Michelia alba DC.

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PROTEOMIC PROFILES OF FLORAL AND LEAF TISSUES OF

Michelia alba DC.

By


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

in Fulfillment of the Requirements for the Master of Science

October 2012

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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

PROTEOMIC PROFILES OF FLORAL AND LEAF TISSUES OF

Michelia alba DC.

By


October 2012

Chairman : Associate Professor Mohd Puad Abdullah, PhD

Faculty : Biotechnology and Biomolecular Sciences

Michelia alba DC is a well known fragrant plant that is rich in high quality essential

oils of economic importance especially for the perfumery industry. At biochemical

level, the exact metabolic pathway that is responsible for the generation of the

essential oils in M. alba and the regulatory mechanism is poorly understood.

Therefore, profiling of proteomes from the organs that are involved in the formation

of the oils is an approach to obtain the potential candidate proteins related to the oils

production. This study was carried out as an attempt to identify differentially

expressed proteins during the development of fragrant-producing organs by

optimizing the methods of protein extraction and profiling the proteomes at different

developmental stages. The objectives of this study were i) to optimize the protein

extraction protocols for profiling of M. alba proteomes by two-dimensional gel

electrophoresis (2-DGE), ii) to optimize the reproducibility of two-dimensional

polyacrylamide gel electrophoresis (2-DGE) in flower and leaf tissues of M. alba, iii)

to profile the proteomes of the leaf and flower of M. alba using 2-DGE at different

developmental stages of the organs, iv) to find trends and correlation of the

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proteomic profile in term of regulation of unique proteins and v) to identify uniquely

proteins expressed for fragrance production throughout developmental stages.

Two-dimensional gel electrophoresis (2-DGE) is currently one of the methods that

can offer a relatively good separation power for proteomics analysis. In this study,

traditional 2-DGE was used to profile the proteomes of the leaf and flower organs of

M. alba at different developmental stages. A prerequisite for a reproducible, high

resolution of proteomic protein separation on the 2-DGE, is the availability of protein

extraction protocol that is optimized for the plant especially for M. alba as a woody

plant. Three different protein extraction methods were evaluated for their abilities to

extract high amounts of soluble protein, to produce high resolution of protein

separation of the crude extract on 1D and 2D gels, and to eliminate maximum

amounts of non-protein contaminants from the extract. The protocol based on the use

of Bis-Tris/acetone was the best protein extraction protocol for M. alba based on the

criteria above and was significantly highest in total protein content in both flower

and leaf tissues (p<0.05).

Using this protein extraction protocol, the proteomes of different developmental

stages of M. alba leaf and flower were successfully resolved on 2-DGE. The

numbers of protein spots and their expression levels were monitored during the

development of the organs. Generally the numbers of protein spots increased during

the development of the organs. During the development of M. alba flower, the

numbers of protein spots reached its peak when the flower was about to open, but

then decreased once the flower bloomed. Profiling of the leaf and flower proteomes

from different developmental stages generated 80 to 210 spots, of which some

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showed differentially-expressed patterns. Five protein spots from the flower

proteome and eight from the leaf were successfully identified based on peptide mass

fingerprinting method. These proteins are categorized into three major classes

according to their functions: primary metabolism, developmental and fragrance-

related proteins. Five of these are fragrance-related proteins namely 1-

aminocyclopropane-1-carboxylic acid synthase, S-adenosylmethionine

decarboxylase, guaiadiene synthase, acyl carrier protein 3 and caffeate o-

methyltransferase. The findings of the study provide some fundamental information

for a more comprehensive approach to analyze and then explain the mechanism

involved in fragrance biosynthesis and its regulation in this plant.

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Abstrak thesis yang dikemukakan kepada senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains

PEMPROFILAN PROTEOMIK PADA TISU BUNGA DAN DAUN

Michelia alba DC.

Oleh


Oktober 2012

Pengerusi : Professor Madya Mohd Puad Abdullah, PhD

Fakulti : Bioteknologi dan Sains Biomolekul

Michelia alba DC merupakan tumbuhan wangian yang terkenal dan kaya dengan

minyak perlu yang berkualiti tinggi dan berkepentingan dari segi ekonomi

terutamanya kepada industri wangian. Salah satu daripada komponen utama minyak

perlu tersebut ialah monoterpena. Dalam tumbuhan, terpena disintesis dalam tapak

jalan metabolik yang kompleks yang melibatkan kedua-dua metabolisma utama dan

sekunder. Pada peringkat biokimia, tapak jalan metabolik yang tepat yang

bertanggungjawab dalam penghasilan minyak perlu dalam M. alba dan mekanisma

pengawalaturannya masih lagi kurang difahami. Oleh itu, pemprofilan menyeluruh

ke atas proteome dari organ-organ yang terlibat dalam pembentukan minyak perlu ini

merupakan salah satu pendekatan untuk memperoleh protein-protein yang berkaitan

dan berpotensi dalam penghasilan minyak perlu tersebut. Secara amnya, kajian ini

dijalankan untuk mengenalpasti protein yang berbeza pengekspresan semasa

peringkat perkembangan melalui pengenalpastian kaedah pengekstrakan protein yang

optimum dan pemprofilan proteome pada peringkat perkembangan yang berbeza.

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Objektif kajian ini adalah untuk i) mengoptimumkan kaedah pengekstrakan protein

untuk pemprofilan proteome M. alba dengan menggunakan elektroforesis gel dua

dimensi (2-DGE), ii) mengoptimumkan kebolehulangan 2-DGE dalam tisu bunga

dan daun M. alba, iii) memprofil proteome bunga dan daun M. alba pada peringkat

perkembangan organ yang berbeza dengan menggunakan 2-DGE, iv) mengenalpasti

arah aliran dan hubungkait profil proteomik dari sudut pengawalaturan protein yang

unik dan v) mengenalpasti protein yang diekspress secara unik untuk penghasilan

wangian semasa peringkat perkembangan.

Elektroforesis gel dua dimensi (2-DGE) adalah salah satu kaedah yang dapat

menghasilkan pemisahan yang baik untuk analisis pengekspresan protein. Dalam

kajian ini, kaedah 2-DGE yang tradisional telah digunakan untuk memprofil protein

pada organ daun dan bunga M. alba di peringkat perkembangan yang berbeza. Pra-

syarat untuk mendapatkan kebolehulangan dan resolusi yang tinggi dalam kaedah

proteomik pemisahan protein 2-DGE adalah pengekstrakan protein tumbuhan yang

dioptimumkan terutamanya tumbuhan berkayu seperti M. alba. Tiga kaedah

pengekstrakan protein yang berbeza telah dinilai keupayaannya iaitu dari segi

pengekstrakan jumlah protein larut yang tinggi, penghasilan pemisahan protein

resolusi tinggi bagi ekstrak mentah pada gel 1D dan 2D dan dapat menyingkirkan

pencemar bukan protein daripada ekstrak protein tersebut dengan maksimum.

Protokol yang melibatkan penggunaan Bis-Tris/aseton merupakan kaedah

pengekstrakan protein yang terbaik bagi M. alba berdasarkan ciri-ciri tersebut dan

secara signifikannya (p<0.05) mempunyai kandungan protein yang paling tinggi

dalam tisu bunga dan daun.

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Dengan menggunakan protokol pengekstrakan ini, proteome pada peringkat

perkembangan yang berbeza dalam daun dan bunga M. alba telah berjaya diperoleh

melalui kaedah 2-DGE. Bilangan bintik protein dan tahap pengekspresannya telah

dipantau semasa peringkat perkembangan organ-organ tersebut. Secara umumnya,

bilangan bintik protein bertambah secara progresif semasa peringkat perkembangan

organ-organ tersebut. Semasa perkembangan bunga M. alba, bilangan bintik protein

adalah paling tinggi semasa bunga mula berkembang namun berkurangan setelah

bunga berkembang penuh. Pemprofilan proteome untuk daun dan bunga daripada

peringkat perkembangan yang berbeza menghasilkan 80 hingga 210 bintik yang

menunjukkan pola pengekspresan yang berbeza, yang mana sebahagiannya telah

berjaya dijujuk. Lima bintik protein daripada proteome bunga dan lapan daripada

daun telah berjaya dikenalpasti identitinya dengan menggunakan kaedah

pengenalpastian cap jari jisim peptida (PMF). Protein-protein ini dapat dikategorikan

kepada tiga kelas utama mengikut fungsi masing-masing: metabolisme utama,

perkembangan dan protein yang berkaitan dengan wangian. Lima daripada protein

yang berkaitan dengan wangian ialah 1-aminocyclopropane-1-karboksilik asid

synthase, S-adenosylmethionine dekarboksilase, guaiadiene synthase, acyl carrier

protein 3 dan caffeate o-metiltransferase. Penemuan daripada kajian ini menyediakan

maklumat asas untuk pendekatan analisa yang lebih komprehensif dan dapat

menerangkan mekanisme yang terlibat dalam biosintesis wangian dan

pengawalaturannya dalam tumbuhan ini.

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ACKNOWLEDGEMENTS

First of all I would like to thank Allah S.W.T the Almighty for His blessings and

wisdom to enable me to complete this thesis.

I would like to take this opportunity to express my deep appreciation and gratitude to

the chairman of my supervisory committee, Assoc. Prof. Dr. Mohd. Puad Abdullah

for his invaluable advice, excellence guidance and contribution in making the

completion of this thesis a success. I am also sincere thanks to my co-supervisors of

my supervisory committee, Assoc. Prof. Dr. Radzali Muse and Prof. Dr. Nor Aripin

Shamaan for their support, assistance, friendliness and as well as their suggestion

throughout this research.

I wish to thank Ministry of Science, Environment and Innovation (MOSTI) for the

IRPA grant (Grant No.: 09-03-03-004 BTK/TD/007) and the National Science

Fellowship (NSF) awarded. I would also to thank Universiti Putra Malaysia for the

laboratory and libraries provided throughout the whole project.

Special thanks to all staff of Faculty Biotechnology and Biomolecular Sciences at

Universiti Putra Malaysia (UPM), especially staffs of Natural Product Laboratory,

Toxicology Laboratory, Enzymology Laboratory, Microbiology Laboratory and

Biotech 2 administration office who had contributed directly or indirectly towards

the success of this research.

I also wish to express my sincere appreciation and gratitude to Dr. Mohd Rosni

Sulaiman, Natarajan Perumal, Tham Lik Gin, Ram Chandra Basnyat, Rafidah,

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Azzreena and Farida Haryani for sharing their knowledge, friendship, ideas, as well

as their life experiences during the course of this project.

Lastly but not least, my heartfelt gratitude goes to my beloved husband, parents and

family for their love, supports and helps throughout the project.

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I certify that an Examination Committee has met on 4 October 2012 to conduct the

final examination of Hasliza Binti Hassan on her Master of Science thesis entitled

“Proteomic Profiles of The Floral and Leaf Tissues of Michelia alba DC.” in

accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and

Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee

recommends that the student be awarded the degree of Master of Science.

Members of the Examination Committee were as follows:

Noorjahan Banu Mohamed Alitheen, PhD

Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Adam Leow Thean Chor, PhD

Lecturer



(Internal Examiner)

Mohd Yunus Abd Shukor, PhD

Associate Professor



(Internal Examiner)

Salmijah Surif, PhD

Professor

Pusat Pengajian Sains Sekitaran & Sumber Alam

Fakulti Sains dan Teknologi

Universiti Kebangsaan Malaysia

Malaysia

(External Examiner)

________________________________

SEOW HENG FONG, PhD

Professor and Deputy Dean

School of Graduate Studies


Date:

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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 Supervisory Committee were as follows:

Mohd Puad Abdullah, PhD

Associate Professor



(Chairman)

Radzali Muse, PhD

Associate Professor



(Member)

Nor Aripin Shamaan, PhD

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

concurrently, submitted for any other degree at Universiti Putra Malaysia or at any

other institution.

________________________


Date: 4 OCTOBER 2012

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TABLES OF CONTENTS

Page

ABSTRACT ii

ABSTRAK v

ACKNOWLEDGEMENTS viii

APPROVAL x

DECLARATION xii

LIST OF TABLES xvi

LIST OF FIGURES xvii

LIST OF ABBREVIATIONS xx

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 5

2.1 Michelia alba 5

2.1.1 Biology of Michelia alba 6

2.1.2 Studies on Michelia alba 7

2.1.2.1 Volatile oils 7

2.1.2.2 Enzymes 8

2.2 Fragrance chemistry in plants 9

2.2.1 Biosynthesis of monoterpenoid compounds 9

2.2.2 Biosynthesis of phenylpropanoids fragrances 12

2.2.3 Biosynthesis of fatty acid derivatives compounds 14

2.3 Fragrance producing plants 16

2.3.1 Specific organs for fragrance production 16

2.3.2 Fragrance compounds 18

2.3.3 Fragrant plants 21

2.3.3.1 Clarkia breweri 21

2.3.3.2 Other fragrant plants 22

2.4 In planta studies of floral scent 23

2.4.1 Molecular biology approach 23

2.4.2 Biochemical approach 25

2.5 Scent engineering 27

2.5.1 Improvement of plant defense 27

2.5.2 Metabolic engineering of floral volatiles 28

2.5.3 Improvement of aroma quality of fruits, vegetables and 29

herbs

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2.6 Profiling of proteins 30

2.6.1 Plant protein extraction 30

2.6.2 Two-Dimensional Gel Electrophoresis 32

2.6.3 Peptide Mass Fingerprinting 32

3 MATERIALS AND METHODS 34

3.1 Materials 34

3.1.1 Chemicals and Reagents 34

3.1.2 Plant materials 34

3.2 Methods 37

3.2.1 Experimental Design 37

3.2.2 Protein Extraction 38

3.2.2.1 Bis-Tris/acetone extraction method 38

3.2.2.2 Tris/acetone extraction method 39

3.2.2.3 TCA/acetone extraction method 39

3.2.3 Determination of protein content 40

3.2.4 Sodium Dodecyl Sulfate Polyacrylamide Gel 41

Electrophoresis (SDS-PAGE)

3.2.4.1 Preparation of Stock Solutions 41

3.2.4.2 Methodology of SDS-PAGE 43

3.2.5 Two-Dimensional Gel Electrophoresis 45

3.2.5.1 First Dimension: Isoelectric Focusing (IEF) 45

3.2.5.1.1 Solution and buffers preparation 45

3.2.5.1.2 Casting and Running the IEF gel 48

3.2.5.2 Second Dimension: Sodium Dodecyl

Sulfate-Polyacrylamide Gel Electrophoresis 50

3.2.6 Visualization of Electrophoresis Gels 52

3.2.7 Image Analysis of 2D gels using Melanie version 7.0 53

software

3.2.8 Excision of Spots for Sequencing Service 56

3.2.9 Database mining for protein identification 56

3.2.10 Statistical analysis 58

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4 RESULTS AND DISCUSSION 59

4.1 Optimization of protein extraction for proteome profiling of the 59

flower and leaf of M. alba

4.1.1 Effect of different extraction buffers methods on total 60

protein content

4.1.2 Effect of different types of extraction buffers on SDS- 62

PAGE banding patterns

4.1.3 2D-electrophoretic patterns of the proteomes 64

extracted by different protocols

4.2 Profiling of proteomes at different developmental stages of 68

M. alba

4.2.1 Total protein content of flower and leaf tissues at different 68

developmental stages

4.2.2 Reproducibility of 2D gel electrophoresis 71

4.2.2.1 Overall between 2D gels reproducibility 71

4.2.2.2 Specific reproducibility between protein spots 77

in the 2-DGE

4.2.3 Spot detection and matching in 2D gels 78

4.2.3.1 Among different stages of flower 78

4.2.3.2 Among different stages of leaf 80

4.2.4 2-DGE profiles of the leaf and flower proteomes 83

of M. alba at different developmental stages

4.2.5 Differentially-expressed proteins during flower 89

and leaf developments

4.3 Identities of protein spots based on peptide mass fingerprinting 96

4.3.1 Classification of identified proteins 102

4.3.2 Putative fragrance-related proteins of M. alba 103

4.3.2.1 1-aminocyclopropane-1-carboxylate (ACC) 103

Synthase

4.3.2.2 S-adenosylmethionine decarboxylase 106

4.3.2.3 Guaiadiene synthase 109

4.3.2.4 Acyl carrier protein 3, chloroplast precursor 111

4.3.2.5 Putative caffeate o-methyltransferase 113

5 SUMMARY, GENERAL CONCLUSION AND 117

RECOMMENDATION FOR FUTURE RESEARCH

5.1 SUMMARY 117

5.2 GENERAL CONCLUSION 118

5.3 RECOMMENDATION FOR FUTURE RESEARCH 119

REFERENCES 121

APPENDICES 131

BIODATA OF STUDENT 137

LIST OF PUBLICATIONS 138

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