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Dissert 1 Title GENETIC AND BIOCHEMICAL ANALYSIS OF ESSENTIAL ENZYMES IN TRIACYLGLYCEROL SYNTHESIS IN ARABIDOPSIS By KIMBERLY LYNN COTTON A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY WASHINGTON STATE UNIVERSITY Molecular Plant Sciences DECEMBER 2015 © Copyright by KIMBERLY LYNN COTTON, 2015 All Rights Reserved To the Faculty of Washington State University: The members of the Committee appointed to examine the dissertation of KIMBERLY LYNN COTTON find it satisfactory and recommend that it be accepted. ____________________________________________ John A. Browse, Ph.D., Chair _____________________________________________ Michael M. Neff, Ph.D. _____________________________________________ Thomas W. Okita, Ph.D. _____________________________________________ John J. Wyrick, Ph.D. ii ACKNOWLEDGMENTS To my PI John Browse, thank you for your support, guidance and patience over the years. To my committee, John Browse, Michael Neff, Thomas Okita, and John Wyrick, thank you for your insightful discussions and suggestions. To my collaborators Jay Shockey, Anushobha Regmi, Neil Adhikari, John Browse, and Phil Bates, thanks for the great work and great times. To Browse Lab members past and present, thanks for the help, guidance and wonderful memories. To the undergraduate students who have helped me over the years, thank you for all your help in making this project possible; and with a special thank you to Arlene, Tesa, Breana, and “no-longer-undergrads” David and Barbara Cotton for their particular dedication to this project. And finally, a huge thank you to my family and friends who have loved and supported me throughout this venture. Go Cougs! iii GENETIC AND BIOCHEMICAL ANALYSIS OF ESSENTIAL ENZYMES IN TRIACYLGLYCEROL SYNTHESIS IN ARABIDOPSIS Abstract by Kimberly Lynn Cotton, Ph.D. Washington State University December 2015 Chair: John A. Browse Plant oils are used in food, fuel, and feedstocks for many consumer products, and so understanding the process by which they are made and modified will help us to make plant oils more healthy, useful, and sustainable. While some of the genes encoding the ER-localized enzymatic steps to triacylglycerol (TAG) have been well understood and documented, several are still in need of study. The glycerol-3-phosphate acyl transferase (GPAT) enzymatic activity is the first step in the pathway to TAG, and it acylates glycerol 3-phosphate to produce lysophosphatidic acid. GPAT9 (AT5G60620) is conserved across land plants and is homozygous lethal, indicating an essential function. Transcript level in knockdown mutants correlates with GPAT activity and with oil levels, and the protein interacts with other enzymes in the TAG biosynthesis pathway. These data suggest that GPAT9 encodes the main GPAT involved in membrane lipid and TAG synthesis. The phosphatidic acid phosphatase (PAP) step in TAG synthesis is responsible for the hydrolysis of inorganic phosphate from phosphatidic acid and creation of diacylglycerol (DAG). There are 13 putative PAPs in Arabidopsis which are homologous to known PAPs. Most of these are involved in other processes, including the plastidial lipid synthesis pathway and signaling pathways. The Arabidopsis gene LPP β (At4g22550) is expressed in seed tissue, its protein product is localized to the ER, and it encodes PAP activity, indicating that it is a likely candidate for the PAP involved in oil synthesis. At the conclusion of this work, questions remain about the role of iv LPP β in oil synthesis and which genes encode the major enzymes involved in the steps generating phosphatidylcholine and converting it back to DAG; but the main Kennedy Pathway enzymes generating TAG have been identified and characterized. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .................................................................................................................... iii ABSTRACT ...................................................................................................................................... iv LIST OF TABLES ............................................................................................................................ viii LIST OF FIGURES ............................................................................................................................. ix CHAPTER ONE . INTRODUCTION GENERAL INTRODUCTION .....................................................................................................1 REFERENCES .......................................................................................................................12 CHAPTER TWO . INITIAL ASSESSMENT OF POTENTIAL PAP GENES INVOLVED IN OIL SYNTHESIS IN ARABIDOPSIS ............................................................................................................................17 2.1 INTRODUCTION ..............................................................................................................18 2.2 MATERIALS AND METHODS ..........................................................................................25 2.3 RESULTS AND DISCUSSION ............................................................................................30 2.4 CONCLUSION .................................................................................................................41 2.5 REFERENCES .................................................................................................................54 CHAPTER THREE . GENETIC , MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF PUTATIVE PAP GENES ...............................................................................................................................61 3.1 INTRODUCTION ..............................................................................................................62 3.2 MATERIALS AND METHODS ..........................................................................................65 3.3 RESULTS .......................................................................................................................73 3.4 DISCUSSION ..................................................................................................................79 3.5 CONCLUSION .................................................................................................................83 vi 3.6 REFERENCES .................................................................................................................95 CHAPTER FOUR . IDENTIFICATION OF ARABIDOPSIS GPAT9 (A T5G60620) AS AN ESSENTIAL GENE INVOLVED IN TRIACYLGLYCEROL BIOSYNTHESIS .....................................................................99 4.1 INTRODUCTION ............................................................................................................100 4.2 MATERIALS AND METHODS ........................................................................................104 4.3 RESULTS .....................................................................................................................112 4.4 DISCUSSION ................................................................................................................124 4.5 CONCLUSIONS .............................................................................................................131 4.6 REFERENCES ...............................................................................................................148 4.7 SUPPLEMENTAL FIGURES , METHODS , AND REFERENCES ............................................158 4.8 SUPPLEMENTAL REFERENCES .....................................................................................175 CHAPTER FIVE . CONCLUSIONS AND PERSPECTIVES ......................................................................176 REFERENCES .....................................................................................................................185 vii LIST OF TABLES Page CHAPTER 2 1. Literature review and protein information of putative PAPs .........................................45 2. Homologs of Arabidopsis putative PAP proteins in Castor Bean ................................46 3. Homologs of Arabidopsis putative PAP proteins in Soybean ......................................47 4. Homologs of Arabidopsis putative PAPs in seed tissue of oilseeds .............................48 5. Primers used in this work ..............................................................................................53 CHAPTER 3 1. Primers used in this work ...............................................................................................93 CHAPTER 4 1. Non- Mendelian segregation of GPAT9/gpat9-2 under sulfadiazine selection ...........136 2. Transmittance of sulfadiazine resistance from reciprocal crosses … .........................137 3. Ovule viability within GPAT9-2/gpat9-2 half siliques ................................................138 4. Pollen germination and tube growth of GPAT9-2/gapt9-2 within the qrt1 … ...........140 S1. Primers utilized for PCR, RT-PCT, & qPCR ............................................................173 S2. Gene identifiers for GPAT9, LPEAT1, and LPEAT2 sequences ..............................174 viii LIST OF FIGURES Page CHAPTER 1 1. TAG biosynthesis in the eukaryotic pathway in Arabidopsis .......................................11 CHAPTER 2 1. Phylogenetic tree of LPP homologs ..............................................................................42 2. Conserved domains in the PAP2 family of PAPs .........................................................43
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