UNIVERSITI PUTRA MALAYSIA. PROTEOMIC PROFILES OF FLORAL AND LEAF TISSUES OF Michelia alba DC. HASLIZA BINTI HASSAN

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1 UNIVERSITI PUTRA MALAYSIA PROTEOMIC PROFILES OF FLORAL AND LEAF TISSUES OF Michelia alba DC. HASLIZA BINTI HASSAN FBSB

2 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 i

3 Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirement for the degree of Master of Science Chairman Faculty PROTEOMIC PROFILES OF FLORAL AND LEAF TISSUES OF Michelia alba DC. By HASLIZA BINTI HASSAN October 2012 : Associate Professor Mohd Puad Abdullah, PhD : 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 ii

4 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 iii

5 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 fragrancerelated 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. iv

6 Abstrak thesis yang dikemukakan kepada senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains Pengerusi Fakulti PEMPROFILAN PROTEOMIK PADA TISU BUNGA DAN DAUN Michelia alba DC. Oleh HASLIZA BINTI HASSAN Oktober 2012 : Professor Madya Mohd Puad Abdullah, PhD : 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. v

7 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. Prasyarat 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. vi

8 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. vii

9 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.: 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, viii

10 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. ix

11 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 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: x

12 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: xi

13 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 xii

14 TABLES OF CONTENTS Page ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS CHAPTER 1 INTRODUCTION 1 2 LITERATURE REVIEW Michelia alba Biology of Michelia alba Studies on Michelia alba Volatile oils Enzymes Fragrance chemistry in plants Biosynthesis of monoterpenoid compounds Biosynthesis of phenylpropanoids fragrances Biosynthesis of fatty acid derivatives compounds Fragrance producing plants Specific organs for fragrance production Fragrance compounds Fragrant plants Clarkia breweri Other fragrant plants In planta studies of floral scent Molecular biology approach Biochemical approach Scent engineering Improvement of plant defense Metabolic engineering of floral volatiles Improvement of aroma quality of fruits, vegetables and 29 herbs ii v viii x xii xvi xvii xx xiii

15 2.6 Profiling of proteins Plant protein extraction Two-Dimensional Gel Electrophoresis Peptide Mass Fingerprinting 32 3 MATERIALS AND METHODS Materials Chemicals and Reagents Plant materials Methods Experimental Design Protein Extraction Bis-Tris/acetone extraction method Tris/acetone extraction method TCA/acetone extraction method Determination of protein content Sodium Dodecyl Sulfate Polyacrylamide Gel 41 Electrophoresis (SDS-PAGE) Preparation of Stock Solutions Methodology of SDS-PAGE Two-Dimensional Gel Electrophoresis First Dimension: Isoelectric Focusing (IEF) Solution and buffers preparation Casting and Running the IEF gel Second Dimension: Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Visualization of Electrophoresis Gels Image Analysis of 2D gels using Melanie version software Excision of Spots for Sequencing Service Database mining for protein identification Statistical analysis 58 xiv

16 4 RESULTS AND DISCUSSION Optimization of protein extraction for proteome profiling of the 59 flower and leaf of M. alba Effect of different extraction buffers methods on total 60 protein content Effect of different types of extraction buffers on SDS- 62 PAGE banding patterns D-electrophoretic patterns of the proteomes 64 extracted by different protocols 4.2 Profiling of proteomes at different developmental stages of 68 M. alba Total protein content of flower and leaf tissues at different 68 developmental stages Reproducibility of 2D gel electrophoresis Overall between 2D gels reproducibility Specific reproducibility between protein spots 77 in the 2-DGE Spot detection and matching in 2D gels Among different stages of flower Among different stages of leaf DGE profiles of the leaf and flower proteomes 83 of M. alba at different developmental stages Differentially-expressed proteins during flower 89 and leaf developments 4.3 Identities of protein spots based on peptide mass fingerprinting Classification of identified proteins Putative fragrance-related proteins of M. alba aminocyclopropane-1-carboxylate (ACC) 103 Synthase S-adenosylmethionine decarboxylase Guaiadiene synthase Acyl carrier protein 3, chloroplast precursor Putative caffeate o-methyltransferase SUMMARY, GENERAL CONCLUSION AND 117 RECOMMENDATION FOR FUTURE RESEARCH 5.1 SUMMARY GENERAL CONCLUSION RECOMMENDATION FOR FUTURE RESEARCH 119 REFERENCES 121 APPENDICES 131 BIODATA OF STUDENT 137 LIST OF PUBLICATIONS 138 xv

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