Investigation of Plant Specialized Metabolism (Secondary Metabolism) Using Metabolomic and Proteomic Approaches Item Type text; Electronic Dissertation Authors Xie, Zhengzhi Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 25/09/2021 17:46:43 Link to Item http://hdl.handle.net/10150/195218 INVESTIGATION OF PLANT SPECIALIZED METABOLISM (SECONDARY METABOLISM) USING METABOLOMIC AND PROTEOMIC APPROACHES By Zhengzhi Xie A Dissertation Submitted to the Faculty of the DEPARTMENT OF PHARMACEUTICAL SCIENCES In Partial Fulfillment of the Requirements For the Degree DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 2007 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Zhengzhi Xie entitled Investigation of plant specialized metabolism (secondary metabolism) using metabolomic and proteomic approaches and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy ____________________________________________________________Date: 5/30/07 David R. Gang ____________________________________________________________Date: 5/30/07 Myron K. Jacobson ____________________________________________________________Date: 5/30/07 Danzhou Yang Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. _____________________________________________________________Date: 5/30/07 Dissertation Director: David R. Gang 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: Zhengzhi Xie _ 4 ACKNOWLEDGEMENTS I would like to thank all the people who have provided their kind help to me. I thank Dr. David R. Gang and Dr. Barbara N. Timmermann for their support and guidance for my research. I am truly grateful for the wonderful opportunity they gave me to work on this interesting project, their outstanding teaching skills stimulating my critical scientific thinking, and for their patience when they helps me improve my communication skills. I would like to thank Dr. Myron K. Jacobson, Dr. Victor J. Hruby, and Dr. Danzhou Yang for their encouragements, and their helpful and constructive suggestions which have been critical for both my Ph.D. study and my future career. I am thankful to all the members of Dr. Gang’s laboratory for their friendship and the enjoyable working environment they created. I would like to thank Dr. Jeremy Kapteyn for his help, collaboration and instructive discussion on the sample preparation, and data analysis of the proteomic experiments. I would like to thank Dr. Xiao-Qiang Ma and Hyun Jo Koo for their help, collaboration and instructive discussion on the experimental design, sample preparation, and data collection on the metabolomic experiments. I would like to thank Brenda L. Jackson and Eric McDowell for their assistance in instrumental operation and instructive discussion on computer programming. I would like to thank former members of Dr. Gang’s laboratory, Dr. Hongliang Jiang and Dr. Maria del Carmen Ramirez-Ahumada for their kind help and advice on my project. Finally, I would like to thank my parents Mr. and Mrs. Shuyuan Xie, my wife Hong Gao and my sister Zhenghui Xie for their love, support, strong confidence in my scientific abilities, and giving me the strength and courage to complete the doctoral degree and pursue my scientific career. This project is supported by the National Institutes of Health NCCAM/ODS Research Training Grant # 3P50AT000474-03S1 to B.N.T., NSF Plant Genome Program Grant DBI-0227618 to D.R.G., and NSF Metabolic Biochemistry Program Grant MCB- 0210170 to D.R.G. 5 TABLE OF CONTENTS LIST OF FIGURES………………………………………………………………….… 8 LIST OF TABLES………………………………………………………………..…… 10 LIST OF ABBREVIATIONS…………………………………………...…………….. 11 ABSTRACT………………………………………………………………………..….. 15 CHAPTER 1: INTRODUCTION…………………………………………………….. 17 1.1 Brief introduction of plant specialized metabolism...………………………...….. 17 1.1.1 The phenylpropanoid pathway and the terpenoid pathway ………….….. 17 1.1.2 Regulation of plant specialized metabolism and variation of herbal product quality………………………………………………… 21 1.2 Review: plant specialized metabolism research using proteomic and metabolomic approaches……………………………………………………. 23 1.2.1 Approaches of proteomics………………………………………………. 23 1.2.2 Proteomic research on herbal plant specialized metabolism…………….. 28 1.2.3 Introduction of metabolomics……………………………………………. 30 1.2.4 Metabolomic research on herbal plant specialized metabolism…………. 34 1.3 Review: Basil glandular trichome ...……………………………………………... 37 1.3.1 Basil produces phenylpropanoids and terpenoids in glandular trichome…………………………………………………………………. 37 1.3.2 Basil glandular trichome as a model system of plant specialized metabolism research…………………………………………. 39 1.4 Review: turmeric………………………………………………………………… 45 1.4.1 History, chemistry, and biological activities of turmeric……..…………. 45 1.4.2 Biosynthesis of diarylheptanoids in turmeric……………………………. 54 1.4.3 Effects of agricultural factors on yield and quality of turmeric rhizome……………………………………………………….56 CHAPTER 2: MATERIALS AND METHODS………………………………………. 59 2.1 Materials…………………………………………………………………………. 59 2.1.1 General materials………………………………………………………… 59 2.1.2 Growth of basil and leaf sample collection……………………………… 59 2.1.3 Growth of turmeric and rhizome sample collection……………………... 60 2.2 Equipment and data analysis software…………………………………………… 61 2.2.1 Equipment……………………………………………………………….. 61 2.2.2 Computer languages and software tools for data processing and analysis……………………………………………………………… 62 2.3 Methods………………………………………………………………………….. 63 2.3.1 GC-MS analysis of basil leaves………………………………………….. 63 2.3.2 Isolation of peltate trichome glands from basil young leaves………….… 64 6 TABLE OF CONTENTS – continued 2.3.3 Protein purification and fractionation from the basil glandular trichomes………………………………………………………. 65 2.3.4 Proteomic analysis using GeLC-MS/MS………………………………… 66 2.3.5 Proteomic analysis using MudPIT……………………………………….. 67 2.3.6 Database searching and data processing for protein identification and TSC calculation……………………………………….. 68 2.3.7 Data processing for protein functional categorization…………………… 69 2.3.8 Searching for PTMs using X!tandem……………………………………. 70 2.3.9 Sample preparation for metabolomic analysis of turmeric rhizomes……. 71 2.3.10 GC-MS analysis of turmeric rhizomes…………………………………... 72 2.3.11 LC-MS and LC-MS/MS analysis of turmeric rhizomes…………………. 73 2.3.12 LC-PDA analysis of turmeric rhizomes……………….…………………. 75 2.3.13 Data analysis of the targeted analysis…….……………………………… 76 2.3.14 Data analysis of the non-targeted analysis…….…….…………………… 76 CHAPTER 3: RESULTS………………………………………………………………. 78 3.1 Protein expression and modification in basil glandular trichomes..…..…………. 78 3.1.1 Profiles of volatile metabolites in basil lines ……..…………………...… 79 3.1.2 Protein identification using shotgun proteomic approaches…………...… 79 3.1.3 Protein functional categorization based on gene ontology (GO) controlled vocabulary……………………………………………… 84 3.1.4 Protein and mRNA levels of enzymes related to the biosynthesis of phenylpropanoids and terpenoids……………………..… 89 3.1.5 Identification of protein post-translational modifications (PTMs)…………………………………………………………………… 92 3.2 Metabolomic investigation of turmeric rhizome …………………………………101 3.2.1 Targeted analysis of three major curcuminoids .……………..………….. 102 3.2.2 Non-targeted analysis using LC-MS and GC-MS…...………...……...…. 107 3.2.3 Correlation between the abundance patterns of metabolites: co-regulated metabolite modules………………………..…. 110 3.2.4 Distribution of Pearson correlation coefficient between the abundance patterns of metabolites……………………….…. 111 3.3 Identification of diarylheptanoids in turmeric rhizome using Tandem-MS-ASC (Assisted Spectra Comparison)……………………….. 126 3.3.1 Development of Tandem-MS-ASC (Assisted Spectra Comparison)……………………………………………………………… 127 3.3.2 Identification of diarylheptanoids in turmeric rhizome based on their LC-MS/MS……………………………………………….. 133 CHAPTER 4: DISCUSSION…………………………………………………………... 141 4.1 Discussions of proteomic results…………………………………………………... 141 7 TABLE OF CONTENTS – continued 4.1.1 Shotgun approaches as high-throughput tools for plant proteomics research…………………………………………….. 141 4.1.2 Differential expression of specific enzymes controls chemical