DEVELOPMENT OF ENABLING TECHNOLOGIES TO VISUALIZE THE PLANT LIPIDOME Patrick J. Horn, B.Sc. Dissertation Prepared for the Degree of DOCTOR OF PHILOSOPHY UNIVERSITY OF NORTH TEXAS August 2013 APPROVED: Kent D. Chapman, Major Professor Guido F. Verbeck, Committee Member Joel M. Goodman, Committee Member Jyoti Shah, Committee Member Rebecca Dickstein, Committee Member Sam Atkinson, Chair of the Department of Biological Sciences Mark Wardell, Dean of the Toulouse Graduate School Horn, Patrick J. Development of Enabling Technologies to Visualize the Plant Lipidome. Doctor of Philosophy (Biochemistry), August 2013, 192 pp., 8 tables, 47 figures, references, 223 titles. Improvements in mass spectrometry (MS)-based strategies for characterizing the plant lipidome through quantitative and qualitative approaches such as shotgun lipidomics have substantially enhanced our understanding of the structural diversity and functional complexity of plant lipids. However, most of these approaches require chemical extractions that result in the loss of the original spatial context and cellular compartmentation for these compounds. To address this current limitation, several technologies were developed to visualize lipids in situ with detailed chemical information. A subcellular visualization approach, direct organelle MS, was developed for directly sampling and analyzing the triacylglycerol contents within purified lipid droplets (LDs) at the level of a single LD. Sampling of single LDs demonstrated seed lipid droplet-to-droplet variability in triacylglycerol (TAG) composition suggesting that there may be substantial variation in the intracellular packaging process for neutral lipids in plant tissues. A cellular and tissue visualization approach, MS imaging, was implemented and enhanced for visualizing the lipid distributions in oilseeds. In mature cotton seed embryos distributions of storage lipids (TAGs) and their phosphatidylcholine (PCs) precursors were distribution heterogeneous between the cotyledons and embryonic axis raising new questions about extent and regulation of oilseed heterogeneity. Extension of this methodology provides an avenue for understanding metabolism in cellular (perhaps even subcellular) context with substantial metabolic engineering implications. To visualize metabolite distributions, a free and customizable application, Metabolite Imager, was developed providing several tools for spatially-based chemical data analysis. These tools collectively enable new forms of visualizing the plant lipidome and should prove valuable toward addressing additional unanswered biological questions. Copyright 2013 by Patrick J. Horn ii ACKNOWLEDGEMENTS I would like to thank my major professor Dr. Kent Chapman for his advice and support on experimental approaches and execution, writing manuscripts and delivering presentations, and achieving a work-life balance throughout my UNT tenure. My sincere gratitude also goes to members of my graduate committee, Dr. Guido Verbeck, Dr. Jyoti Shah, Dr. Rebecca Dickstein and Dr. Joel Goodman for research guidance and support. Thanks to the UNT graduate school and biology department for financial support through a Doctoral and Dissertation Fellowship as well as a teaching assistant position. I would like to acknowledge those who contributed to this study, mass spectrometry imaging of oilseeds (Dr. Purnima Neogi, Dr. Young-Jin Lee, Andy Korte, Danielle Anderson, Drew Sturtevant, Ebony Love, Dr. Vladimir Shulaev, Dr. Kerstin Strupat and Dr. Mari Conaway), nuclear magnetic resonance (Dr. Ljudmilla Borisjuk and Johannes Fuchs), and lipidomics experiments (Chris James and Dr. Charlene Case). I extend my thanks to members of the Chapman lab past and present, Bikash Adhikari, Yingqi Cai, Dr. Charlene Case, Matt Cotter, Dr. Lionel Faure, Gabe Gonzalez, Chris James, Jantana Kereetaweep, Dr. Aruna Kilaru, Dr. Sang-chul Kim, John Lafin, Clayton Rowe, Dr. Shanmukh Salimath, Drew Sturtevant, Dr. Neal Teaster and Dr. Swati Tripathy. I would also like to thank members of the Shulaev Lab (Dr. Carolina Salazar, Dr. Sarah Holt and Janna Crossley), Verbeck Lab (Dr. William Hofmann and Barbara Walton) and Shah Lab (Vijay Singh and Vamsi Nalam). Last but not least I would not be here without my beautiful, loving and patient wife Ashley as well as wonderful and supporting parents Charles and Jan Horn. iii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ................................................................................................................... iii LIST OF TABLES .............................................................................................................................. viii LIST OF ILLUSTRATIONS................................................................................................................... ix COMPREHENSIVE LIST OF ABBREVIATIONS ................................................................................... xii CHAPTER 1 INTRODUCTION ............................................................................................................ 1 1.1 Lipid Synthesis and Composition in Plants ............................................................. 1 1.2 Lipid Droplets .......................................................................................................... 4 1.3 Lipidomic Analysis in Tissues .................................................................................. 5 1.3.1 Lipidomics in Tissues ................................................................................... 6 1.3.1 Shotgun Lipidomics: High-Resolution, Direct-Infusion Mass Spectrometry ..................................................................................................................... 7 1.3.2 Applications of Shotgun-Based Lipidomics Led to New Insights about Plant Lipid Metabolism and Signaling ....................................................... 10 1.4 Mass Spectrometry Imaging – Localizing Lipids in situ ......................................... 13 1.4.1 Matrix-Assisted Laser Desorption/Ionization ........................................... 14 1.4.2 Desorption Electrospray Ionization (DESI) ................................................ 18 1.4.3 Secondary Ion Mass Spectrometry (SIMS)................................................ 19 1.4.4 Ion Mobility MS (IMMS) ............................................................................ 21 1.5 Figures and Tables ................................................................................................ 23 CHAPTER 2 VISUALIZATION OF LIPID DROPLET COMPOSITION BY DIRECT ORGANELLE MASS SPECTROMETRY ............................................................................................................................ 27 2.1 Abstract ................................................................................................................. 27 2.2 Introduction .......................................................................................................... 28 2.3 Results ................................................................................................................... 31 2.3.1 Direct Organelle Mass Spectrometry .................................................................... 31 2.3.2 Lipid Droplet Characterization .............................................................................. 32 2.4 Discussion.............................................................................................................. 35 iv 2.5 Addendum............................................................................................................. 40 2.5.1 New Insights into Lipid Droplet Composition from DOMS ....................... 40 2.5.2 Chloroplast Galactolipid Analysis .............................................................. 43 2.6 Methods ................................................................................................................ 44 2.6.1 Plant Growth Conditions ........................................................................... 44 2.6.2 Imaging Iipid Droplets In Situ .................................................................... 44 2.6.3 Lipid Droplet Purification .......................................................................... 45 2.6.4 Nanomanipulator Workstation ................................................................. 46 2.6.5 Nanospray Mass Spectrometry ................................................................. 47 2.6.6 Conventional Lipid Extraction ................................................................... 47 2.6.7 Fatty Acid Methyl Ester Preparation and Analysis .................................... 48 2.6.8 Electrospray Mass Spectrometry .............................................................. 48 2.7 Acknowledgements ............................................................................................... 49 2.8 Figures and Tables ................................................................................................ 50 CHAPTER 3 SPATIAL MAPPING OF LIPIDS AT CELLULAR RESOLUTION IN EMBRYOS OF Gossypium hirsutum, L .................................................................................................................................... 62 3.1 Abstract ................................................................................................................. 62 3.2 Introduction .......................................................................................................... 63 3.3 Results ..................................................................................................................