University of Missouri, St. Louis IRL @ UMSL Dissertations UMSL Graduate Works 8-6-2015 The Role of Arabidopsis Aminoalcoholphosphotransferase 1 and 2 in Plant Development and Oil Production and Transcriptional Regulation of Seed Oil Accumulation by GLABRA2 Yu Liu University of Missouri-St. Louis, [email protected] Follow this and additional works at: https://irl.umsl.edu/dissertation Part of the Biology Commons Recommended Citation Liu, Yu, "The Role of Arabidopsis Aminoalcoholphosphotransferase 1 and 2 in Plant Development and Oil Production and Transcriptional Regulation of Seed Oil Accumulation by GLABRA2" (2015). Dissertations. 14. https://irl.umsl.edu/dissertation/14 This Dissertation is brought to you for free and open access by the UMSL Graduate Works at IRL @ UMSL. It has been accepted for inclusion in Dissertations by an authorized administrator of IRL @ UMSL. For more information, please contact [email protected]. The Role of Arabidopsis Aminoalcoholphosphotransferase 1 and 2 in Plant Development and Oil Production and Transcriptional Regulation of Seed Oil Accumulation by GLABRA2 Yu Liu University of Missouri-St Louis Department of Biology Program in Cellular and Molecular Biology A Dissertation presented to the Graduate School of Arts and Sciences at the University of Missouri-St Louis in partial fulfillment of the requirements of Doctor of Philosophy in Cell and Molecular Biology August 2015 Advisory Committee Dr. Xuemin Wang Chairperson Dr. Elizabeth Kellogg Dr. Wendy Olivas Dr. Bethany Zolman ABSTRACT Vegetable oils are important commodities as human foods, animal feeds, renewable industrial feedstocks, and biofuels. Understanding how plant lipids are made will facilitate greatly the effort to increase seed oil content and production. Many biochemical and regulation events are involved in seed oil formation, including phospholipid metabolism and transcriptional regulation. In this thesis, I investigated 1) the role of aminoalcoholphosphotransferases (AAPTs) in phospholipid synthesis and plant development, 2) the effects of AAPTs on seed storage lipid production in Arabidopsis and the emerging oil crop Camelina, and 3) the interaction of the lipid mediator phosphatidic acid (PA) with GLABRA2 (GL2), a transcription factor that is a negative regulator in seed oil production. AAPTs are the enzymes catalyzing the last step of the Kennedy pathway to produce phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The Arabidopsis genome contains two AAPTs that I found are indispensable for plant reproduction and vegetative growth, based on the embryonic lethality phenotype of aapt1 aapt2 double knockout (KO) mutant and the retarded growth in AAPT RNA interference (RNAi) plants. Surprisingly, no change in PC level was observed in any of the mutant lines, while PE level decreased drastically in aapt1 KO, aapt1/aapt1 aapt2/AAPT2 and AAPT RNAi lines. The non-Kennedy pathway phospholipids, phosphatidylinositol (PI) and phosphatidylglycerol (PG), also showed mild decrease in AAPT RNAi plants. Detailed analysis revealed extensive lipid species changes. The PC shifted towards 34 carbon (C34) species, while PE species shifted towards C36. The possibility that C34 PC may be exported from chloroplast was not supported by the fatty acid positional analysis. The direct conversion of PI to PC by base-exchange mechanism was also not detected by the co-infiltration of PI and choline in Arabidopsis leaves, but an indirect conversion through lysolipids was suggested by the current results. Radiolabeling of PC and PE by infiltration of 3H- choline and 3H-ethanolamine suggested AAPT1 has more CPT activity than AAPT2 in leaves, while PC is the preferred product of both AAPTs in vivo. These results suggest AAPTs play an important role in plant lipid production and growth, while additional PC producing pathways exist to maintain a stable PC level that is crucial to plants. The fatty acid analysis showed that AAPT mutant lines had decreased oil content and increased long chain fatty acids, while AAPT seed specific overexpression (OE) lines had increased oil content. I also studied the significance of the interaction between PA and the homeodomain transcription factor GLABRA2 (GL2). I found GL2 interacts with PA strongly and specifically. The PA interaction region was mapped to the leucine-zipper region in the DNA-binding domain of GL2. The DNA-binding ability of GL2 was impaired by the interaction with PA. A chromatin immunoprecipitation (ChIP) assay identified possible GL2 targets, which were confirmed by gel mobility shift assay. The seed specific RNAi of GL2 increased oil content, but the mucilage in seed coat was not affected. These results suggest PA negatively regulates GL2, which is a potential master regulator of phospholipids metabolism and oil production. Overall, this work has improved our knowledge of phospholipid metabolism and its regulation by GL2, which will help us in improving lipid/oil production in plants. ACKNOWLEDGEMENT I spent the most important and unforgettable six years at University of Missouri-St. Louis. I have learnt tremendous amount of things as a researcher and as a human being. I am so grateful to meet and interact with the wonderful people here. First and foremost I want to thank my advisor Dr. Xuemin Wang. Without his support and encouragement, I cannot imagine I can complete my Ph. D. study. Dr. Wang gave me countless instructions yet still put his trust on me to complete the majority of my study and research. Dr. Wang inspired me, sometimes forced me to set specific goals and plans about my research, which proved to be invaluable to my development as a scientific researcher. I communicated regularly during writing my manuscript. I received tons of suggestions and improvements from Dr. Wang on the text to make the manuscript significantly better. My thesis committee members, Dr. Bethany Zolman, Dr. Elizabeth Kellogg and Dr. Wendy Olivas gave me great help on the direction of my research project, potential solutions on the difficulties I encountered during my research and proper data presentation. Their review of my dissertation proposal and dissertation are of huge help to the improvement of the content and data arrangement. They deserve my deepest appreciation. I am so thankful to the Department of Biology for accepting me as a Ph.D student. I want to thank all the staffs who are so helpful during my graduate study here. I also have benefited from the current and former members in our lab. They are great friends and provided their help and shared their knowledge with me selflessly. These include: Dr. Geliang Wang, Dr. Liang Guo, Dr. Maoyin Li, Dr. Sangchul Kim, Dr. Yong Zheng, Amanda Tawfall, Brian Fanella, Yuan Su, Xinliang Huang and many intern students. I am grateful to Dr. Ruth Welti and Mary Roth at Kansas State University for their help in lipid profiling and providing phospholipid standards. Finally, I must express my appreciation to my whole family, especially my mother and wife, without their support and sacrifice I could not have finish my project without much distraction. Thank little Adele to give me so much joy. Thank every people I have known during my study and wish them happy and healthy. ABBREVIATIONS AAPT, aminoalcoholphosphotrans- NPC, non-specific phospholipase C ferase OE, overexpression CCT, choline phosphate cytidylyl- PA, phosphatidic acid transferase PAH, phosphatidic acid hydrolase ChIP, chromatin immunoprecipitation PC, phosphatidylcholine CK, choline kinase PDAT, phospholipids: DAG CPT, cholinephosphotransferase acyltransferase DAG, diacylglycerol PE, phosphatidylethanolamine DGAT, DAG acyltransferase PG, phosphatidylglycerol DGDG, digalactosyldiacylglycerol PGP, phosphatidylglycerol-phosphate DGK, DAG kinase PI, phosphatidylinositol EPT, ethanolaminephosphotransferase PL, phospholipids ER, endoplasmic reticulum PLA, phospholipase A FA, Fatty acid PLC, phospholipase C G3P, phosphoglycerol, glycerol-3- PLD, phospholipase D phosphate PS, phosphatidylserine GL, glycolipids PSS, phosphatidylserine synthase GL2, GLABRA 2 qPCR, quantitative real-time PCR KO, knockout TAG, triacylglycerol LPP, lipid phosphate phosphatase WT, wild-type MGDG, monogalactosyldiacylglycerol TABLE OF CONTENTS Chapter 1. Background, goals and objectives ...................................................... 4 Introduction............................................................................................................... 4 Hypotheses and Goals ............................................................................................. 17 References ............................................................................................................... 20 Chapter 2. Role of Aminoalcoholphosphotransferases 1 and 2 in Phospholipid Homeostasis in Arabidopsis ............................................................................... 26 Abstract ................................................................................................................... 27 Introduction............................................................................................................. 27 Results ..................................................................................................................... 30 Disscussion .............................................................................................................. 50 Materials and Methods ........................................................................................... 59 References ..............................................................................................................