DYNAMICS, DISTRIBUTION AND DEVELOPMENT OF SPECIALIZED METABOLISM IN GLANDULAR TRICHOME OF TOMATO AND ITS WILD RELATIVES By Zhenzhen Wang A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Biochemistry and Molecular Biology – Doctor of Philosophy 2015 ABSTRACT DYNAMICS, DISTRIBUTION AND DEVELOPMENT OF SPECIALIZED METABOLISM IN GLANDULAR TRICHOME OF TOMATO AND ITS WILD RELATIVES By Zhenzhen Wang Glandular trichomes on the plant surface are specialized epidermal cells which can produce and store a variety of plant specialized metabolites. Specialized phytochemicals produced by trichome are extremely diverse among Solanaceae families and a lot of them have been characterized with the advance of plant omics. Due to their benefits for plant defense system and commercial values, biosynthesis and accumulation of trichome metabolites haven been intensively studied. In this study, analytical chemistry and genetic approaches were integrated to explore the remaining questions about mechanism for the production of specialized metabolites in Solanum trichomes. In particular, this study included three studies addressing the dynamics, distribution and development of specialized metabolism in glandular trichomes from tomato and its relatives. This first study presented the development of analytical methods to profile localized metabolites in biological tissues through chemical imaging, and application of this method to investigations of glandular trichomes. Using this mass spectrometry imaging method, single- cell metabolic profiling and spatially preserved chemical information was achieved in tomato leaf trichomes for several type of trichome specialized metabolites, including acylsugar, flavonoids and terpenes. This work extended imaging mass spectrometry to specialized epidermal cells without using dissection or tissue fixation for the first time. The second study described the isotope labeling methodology for tracing specialized metabolites flux in trichomes. In general, an integrated approach combining whole plant 13 CO2 labeling and data-independent LC-MS quantitative profiling was developed to monitor the metabolic dynamic flux and turnover rate of protective plant specialized metabolites. This analytical strategy is applicable to a wide range of specialized metabolites. The third study applied the isotopic labeling strategy to discover the dynamics of trichome specialized metabolites accumulation and the effect of developmental stage of tomato leaves. Using flux analysis method, we measured fluctuations in labeling enrichment and metabolite accumulation during different tomato leaf developmental stages, and demonstrated that total acylsugar accumulation reaches a steady state at early stage of development. Evidence was generated to support the concept that degradation of undetected metabolic intermediates provides a key regulatory point in the acylsugar metabolic network offers a new viewpoint for engineering plant metabolic networks for high levels of end product acylsugars. This novel regulation mechanism extends our understanding of catabolic activities of specialized metabolites. ACKNOWLEDGMENTS I would like to express the deepest appreciation and thanks to a number of people who have been very supportive during the years of my graduate school and towards finishing this dissertation. First and foremost, a special thanks to my advisor professor A. Daniel Jones for a supportive and patient mentor all the time. Thank you for being available almost 24/7 for any scientific problem we have in the lab, and all the late night and weekend meetings. Thank you for encouraging me on critical thinking and being passionate about my research project all the time. Thank you for truly caring about if the students are happy about their research and course work, with make the mentoring relationship very friendly and memorable. The most important lesson I learn from him is to explore and pursue a career that you feel excited all the time and wish to dedicate yourself with joy and passion. That and only that can make you a cheerful and helpful person to work with. In addition, I admire Dan’s high standard on logic writing and telling an attractive scientific story of my research. Without his encouragement and mentoring, I cannot turn the “impossible” labeling project into “incredible” labeling project; turn the “frustrating” research into “fascinating” research. I consider myself very fortunate to have Dan as my mentor for both scientific career and persona life. I would like to thank my committee member professor Yair Shachar-Hill, professor Gregg Howe, professor Dean DellaPenna and professor Min-Hao Kuo for their guidance and constructive criticism during my entire graduate school research, especially professor Yair Shachar-Hill for providing useful discussions and insights on my labeling project. I would also like to thank our trichome project collaborators from professor Robert Last’s group and professor Cornelius Barry’s group. It is honor to learn plant genetics and molecular biology iv from them and work with them. I also would like to gratefully acknowledge the support the RTSF Mass Spectrometry Core at Michigan State University. My thanks go especially to Lijun Chen and Dr. Scott Smith for being very helpful on suggestion and help for various analyses. Specially, I would like to extend my thanks to close friends at Michigan State University and beyond. I am glad that I have been working with a group of wonderful members from Dan’s lab, especially Dr. Chao Li and Dr. Prabodha Ekanayaka, Chen Zhang, Sujana Pradhan, Xiaoxiao Liu and Afrand Kamali, which made my graduate school life full of memorable happiness. Without their great companion and friendship, graduate school would be difficult and boring. I would also like to make special mentions of Dr. Li Cui, Minyoung So and Dr. Li Zhou. Dr. Jing Ning and Jin Zhang for always being there backup me through the ups and downs of my graduate school life. In the end, I sincerely thank my parents Yan Wang and Zhigang Wang for unconditional love. Their encouragement on pursing better education and a free life drives me this far from my hometown. My deep thanks go to my husband Long Han. I am very grateful for surviving many years of far separation with him and finally graduate from long distance relationship with his coming to the US. His understanding and tolerance through these years make me a confident, optimistic and open-minded person. Thank you for completing me. v TABLE OF CONTENTS LIST OF TABLES.................................................................................................................. viii LIST OF FIGURES ................................................................................................................... ix Chapter 1. Introduction ............................................................................................................... 1 1.1 Importance of plant specialized metabolism ............................................................ 2 1.2 Growing interest and challenges in plant specialized metabolites ........................... 3 1.3 Solanum trichomes as a model system to study plant specialized metabolites ........ 6 1.4 Introduction to acylsugars ........................................................................................ 8 1.5 Analytical strategies for metabolite profiling and flux measurement .................... 10 1.6 Summary of research .............................................................................................. 14 REFERENCES ............................................................................................................. 17 Chapter 2. Chemical Imaging of Trichome Specialized Metabolites using Contact Printing and Laser Desorption/Ionization Mass Spectrometry .............................................................. 26 2.1 Introduction ............................................................................................................ 27 2.2 Materials and methods ............................................................................................ 30 2.2.1 Chemicals ................................................................................................ 30 2.2.2 Plants ....................................................................................................... 31 2.2.3 Sample preparation .................................................................................. 31 2.2.3 LDI imaging ............................................................................................ 33 2.2.4 LC/MS analysis ....................................................................................... 34 2.3 Results and discussion ............................................................................................ 35 2.3.1 Contact printing transfer of trichome metabolites ................................... 35 2.3.2 Whole-leaf imaging using LDI from pencil lead surfaces ...................... 37 2.3.3 Single-trichome imaging ......................................................................... 46 2.3.4 Heterogeneity in localization and quantity of acylsugars in M82 leaf trichomes .......................................................................................................... 51 2.4 Conclusions ............................................................................................................ 54 REFERENCES ............................................................................................................