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GUARD CELL MOLECULAR RESPONSES TO ELEVATED AND LOW CO2 REVEALED BY METABOLOMICS AND PROTEOMICS By SISI GENG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2016 © 2016 Sisi Geng To my beloved mother, father and husband In memory of my beloved grandfather ACKNOWLEDGMENTS My special thanks goes to my graduate committee: Dr. Sixue Chen, Dr. Kevin Folta, Dr. Julie Maupin-Furlow, and Dr. Harry Klee. Their expertise and achievement inspired and urged me throughout my Ph.D. research. Dr. Sixue Chen, as my supervisor and committee chair was a great role model as a scientist and helped me both in my research project and living. Dr. Bing Yu from Heilongjiang University (a visiting scholar in the Chen lab), Dr. Evaldo de Armas and Dr. Craig Dufresne from Thermo Fisher Scientific, Dr. David Huhman and Dr. Lloyd W. Sumner from Samuel Roberts Noble Foundation, Dr. Hans T. Alborn from United States Department of Agriculture, Dr. Sarah M. Assmann and Dr. Mengmeng Zhu from Pennsylvania State University, and Dr. Zhonglin Mou from Microbiology and Cell Science Department are acknowledged for their help and collaboration in this project. Technical support was provided by the Proteomics and Mass Spectrometry Core at the Interdisciplinary Center for Biotechnology Research, University of Florida. I am also grateful to people who have helped me during my PhD research, especially Dr. Biswapriya Misra, Ning Zhu, Dr. Cecilia Silva-Sanchez, other Chen lab members and all of my friends. My parents, Biao Geng and Wei Zhou, and my husband Dr. Hongbing Liu are specially thanked for their encouragement and emotional support. My study here is funded by China Scholarship Council (CSC), Plant Molecular and Cellular (PMCB) graduate program, and partly by the U.S. National Science Foundation grants NSF-0818051 and NSF-1158000 (to S. Chen). 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ........................................................................................... 10 ABSTRACT ................................................................................................................... 13 CHAPTER 1 INTRODUCTION .................................................................................................... 15 Guard cell CO2 Response ....................................................................................... 15 Short Term Effects of CO2 on Guard Cell Signaling ......................................... 15 Long Term Effects of CO2 on Plant Physiology ................................................ 19 CO2 Effects on Guard Cell Development .......................................................... 22 Guard Cell Metabolism ........................................................................................... 23 Guard Cell Metabolism in General ................................................................... 23 Osmoregulation ................................................................................................ 26 ROS Homeostasis ............................................................................................ 27 Signaling Molecules ......................................................................................... 28 2 JASMONATE-MEDIATED STOMATAL CLOSURE UNDER ELEVATED CO2 REVEALED BY TIME-RESOLVED METABOLOMICS ........................................... 34 Introduction ............................................................................................................. 34 Materials and Methods............................................................................................ 37 Plant Materials .................................................................................................. 37 Preparation of Epidermal Peels for Stomatal Movement and ROS Assays ...... 37 Stomatal ROS Measurement ............................................................................ 38 Large Scale Preparation of Stomata for Metabolomics .................................... 38 Metabolite Extraction ........................................................................................ 39 Metabolite Profiling Using Hyphenated Metabolomics Platforms ..................... 40 Metabolomics Data Processing and Statistical Analysis .................................. 42 Metabolic Pathway Mapping and Enrichment Analyses ................................... 43 Results .................................................................................................................... 43 Elevated CO2 Induced Stomatal Closure and ROS Production ........................ 43 Overview of Temporal Changes in Guard Cell Metabolome under Elevated CO2 ............................................................................................................... 44 Specific Metabolite Changes in Response to Elevated CO2 ............................. 46 Functional Characterization of JA-Mediated CO2 Induced Stomatal Closure ... 48 5 Discussion .............................................................................................................. 49 Changes in Primary Metabolites and Osmolytes in The Course of Stomatal Closure under Elevated CO2 ......................................................................... 49 The Roles of ROS and Flavonoids in CO2-Induced Stomatal Closure ............. 52 Lipids Are Involved in Elevated CO2-Induced Stomatal Movement .................. 53 JA Signaling Mediates High CO2-Induced Stomatal Closure ............................ 54 3 LOW CO2 RESPONSIVE METABOLOME AND PROTEOME IN BRASSICA NAPUS GUARD CELLS ....................................................................................... 111 Introduction ........................................................................................................... 111 Materials and Methods.......................................................................................... 114 Plant Materials ................................................................................................ 114 Preparation of Epidermal Peels for Stomatal Movement Assay ..................... 115 Metabolite Extraction ...................................................................................... 115 Metabolite Profiling ......................................................................................... 117 Metabolomics Data Processing and Statistical Analysis ................................ 119 Metabolic Pathway Mapping and Enrichment Analyses ................................. 119 Protein Extraction and TMT 10-Plex Labeling ................................................ 119 Strong Cation Exchange Fractionation and LC-MS/MS Analysis ................... 121 Protein Identification and Quantification ......................................................... 123 Results .................................................................................................................. 124 Low CO2 Induced Stomatal Opening and Overview of Low CO2 Responsive Guard Cell Metabolism Protein Identification and Quantification ................. 124 Lipid Metabolism under Low CO2 ................................................................... 125 Phytohormone Changes under Low CO2 ....................................................... 126 Proteomic Changes under Low CO2............................................................... 127 Comparison between High and Low CO2 Responsive Metabolites ................ 129 Discussion ............................................................................................................ 130 Primary Metabolism Changes under Low CO2 ............................................... 130 Phytohormone Crosstalk in Guard Cells under Low CO2 Induced Stomatal Opening....................................................................................................... 131 Osmoregulations under Low CO2 ................................................................... 133 Redox Regulation under Low CO2 ................................................................. 134 4 SUMMARY AND PERSPECTIVES ...................................................................... 165 LIST OF REFERENCES ............................................................................................. 168 BIOGRAPHICAL SKETCH .......................................................................................... 188 6 LIST OF TABLES Table page 2-1 MRM period method design .................................................................................... 57 2-2 Information of metabolite detected in Brassica napus guard cells .......................... 67 2-3 Significantly changed metabolites under elevated CO2 ........................................... 80 2-4 Metabolites significantly changed at 3 or more time points or with a fold change over 10 under elevated CO2 ............................................................................... 94 3-1 Fold changes and p values of significantly changed metabolites in low CO2 treated B. napus guard cells ............................................................................
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