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Identifying Molecular Functions of Heliotropic Motor Tissue Through Proteomic Analysis of Soybean Pulvini

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Identifying Molecular Functions of Heliotropic Motor Tissue Through Proteomic Analysis of Soybean Pulvini ABSTRACT Title of Document: IDENTIFYING MOLECULAR FUNCTIONS OF HELIOTROPIC MOTOR TISSUE THROUGH PROTEOMIC ANALYSIS OF SOYBEAN PULVINI Hakme Lee, M.S., 2012 Directed By: Professor Joseph Sullivan, Plant Sciences and Landscape Architecture Heliotropic and nyctinastic leaf movement are mediated in soybean through turgor changes in the motor cells of the pulvinus, located at the base of the leaves. While some elements of the signaling pathways have been studied, a broad-scale protein identification has not yet been reported. In my research pulvini proteins were extracted in light- and dark-harvested soybean using the TCA/acetone method and identified by LC-MS/MS. Gene ontology analysis revealed proteins involved in proton transport were enriched in the soybean pulvinus proteome compared to a background soybean proteome. Proteins more highly expressed in the light were mostly stress response proteins, while under-expressed proteins were categorized as energy proteins. Further investigations using more sensitive extraction protocols and a multitude of sample times will build on these initial results to provide a thorough examination of heliotropic mechanisms at the molecular level. IDENTIFYING MOLECULAR FUNCTIONS OF HELIOTROPIC MOTOR TISSUE THROUGH PROTEOMIC ANALYSIS OF SOYBEAN PULVINI By Hakme Lee Thesis submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Master of Science 2012 Advisory Committee: Professor Joseph Sullivan, Chair Associate Professor Irwin Forseth Adjunct Assistant Professor Savithiry Natarajan © Copyright by Hakme Lee 2012 Dedication I would like to thank my parents and my siblings for loving me just as I am. ii Acknowledgements I wish to thank Joseph Sullivan for advising me on my research for the past two and a half years, Savi Natarajan for research guidance, and post-docs/technicians Xi Chen, Farooq Khan, and Adelbary Prince for protocol assistance. Special thanks to Irv Forseth for participating in my thesis committee, Wes Garrett for mass spectrometry work, Bill Kenworthy for donating seeds, and Shaun Faulkner and Sydney Wallace for their assistance in growing the plants in the greenhouse. This research was made possible through a Cooperative Agreement between University of Maryland, College Park, and USDA Beltsville Agricultural Research Center (BARC). iii Table of Contents Dedication ..................................................................................................................... ii Acknowledgements ...................................................................................................... iii Table of Contents ......................................................................................................... iv List of Tables ............................................................................................................... vi List of Figures ............................................................................................................. vii Chapter 1: Introduction and Literature Review ............................................................ 1 Heliotropism overview.............................................................................................. 1 Introduction ........................................................................................................... 1 Benefits of heliotropism ........................................................................................ 1 Modulating factors in heliotropism ....................................................................... 2 Heliotropism and implications for agriculture ...................................................... 4 Pulvinar Structure and Mechanisms ......................................................................... 4 Histology ............................................................................................................... 4 Basic mechanism .................................................................................................. 5 Ion channels .......................................................................................................... 6 Induction factors for ion movement ...................................................................... 7 Other molecules implied in turgor change ............................................................ 8 Proteins involved in heliotropism ......................................................................... 9 Proteomics............................................................................................................... 10 History of Protein Analysis ................................................................................. 10 Study objectives .................................................................................................. 12 Chapter 2: Proteomic analysis of the Pulvinus, a Heliotropic Tissue, in Glycine max13 Abstract ................................................................................................................... 13 Introduction ............................................................................................................. 14 Experimental ........................................................................................................... 16 Plant Material ...................................................................................................... 16 Protein Extraction ............................................................................................... 16 2D gel electrophoresis......................................................................................... 17 In-gel digestion ................................................................................................... 18 Mass spectrometry .............................................................................................. 19 Data analysis ....................................................................................................... 19 Results and Discussion ........................................................................................... 20 Protein Extraction, Identification, and Functional Analysis ............................... 20 Proton transport ................................................................................................... 28 Malate metabolism .............................................................................................. 29 Cytoskeletal processes ........................................................................................ 30 Ion transporters and Aquaporins ......................................................................... 30 Pulvinus proteome compared to guard cells ....................................................... 31 Conclusion .............................................................................................................. 33 Chapter 3: Differentially Expressed Proteins of Soybean (Glycine max) Pulvinus in Light and Dark Conditions.......................................................................................... 35 Abstract ................................................................................................................... 35 iv Introduction ............................................................................................................. 36 Experimental ........................................................................................................... 38 2-D PAGE ........................................................................................................... 38 Results ..................................................................................................................... 39 Discussion ............................................................................................................... 42 Overview ............................................................................................................. 42 Pulvinus proteins more highly expressed in the light include stress response proteins ................................................................................................................ 44 The increased expression of beta amylase in the light may reflect greater sucrose demand by the pulvinus ...................................................................................... 45 Proteins with increased expression in the dark may be a result of multiple factors ............................................................................................................................. 46 Rubisco activase expression levels are possibly a result of circadian rhythms .. 46 Other explanations for diminished photosynthetic and carbon-fixing proteins in the light ............................................................................................................... 47 Conclusion .............................................................................................................. 49 Chapter 4: Conclusions ............................................................................................... 50 Overview ................................................................................................................. 50 A note on methodology ........................................................................................... 52 New directions ........................................................................................................ 53 Bibliography ..............................................................................................................
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