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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
HASLIZA BINTI HASSAN
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
HASLIZA BINTI HASSAN
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
HASLIZA BINTI HASSAN
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
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)
Mohd Yunus Abd Shukor, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(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
Universiti Putra Malaysia
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
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)
Radzali Muse, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)
Nor Aripin Shamaan, PhD
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
concurrently, submitted for any other degree at Universiti Putra Malaysia or at any
other institution.
________________________
HASLIZA BINTI HASSAN
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