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Sabharwal-Dissertation-2016 Copyright by Tanya Sabharwal 2016 The Dissertation Committee for Tanya Sabharwal Certifies that this is the approved version of the following dissertation: Rapid remodeling of lipids and transcriptome in the diatom Phaeodactylum tricornutum in response to defense related decadienal Committee: Mona C. Mehdy, Supervisor K Sathasivan, Co-Supervisor Enamul Huq Stanley J. Roux, Jr Edward C. Theriot Rapid remodeling of lipids and transcriptome in the diatom Phaeodactylum tricornutum in response to defense related decadienal by Tanya Sabharwal, B.Tech; M.S Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin May 2016 Dedication To my Dad and Mom, who have always believed in me and kept me strong Acknowledgements I will always be grateful to my advisor Dr. Mona Mehdy whom I wrote an email back in 2008 when I was pursuing my Masters in Chicago, for giving me a chance to volunteer in her lab (while visiting family in Austin during summers) and from there on it has been a long journey and she had taught me so much. She introduced me to Dr. K Sathasivan (Dr Sata) and I was lucky to get hired initially as a research scientist intern on Dr. Sata’s DARPA project and started learning about the world of algae before formally getting admitted to school as a PhD student. I am thankful to Dr. Mehdy and Dr. Sata for accepting to be my advisors, for all their guidance, support, patience and invaluable advice during my work here as a doctoral student. They always encouraged me to do good and honest research, and be an independent scientist and a good technical writer. They are both wonderful people at heart who genuinely care about people, society and have always given me valuable advice both in and outside of research that has immensely helped me grow both professionally and personally. I would also like to thank my nice committee members Dr. Stanely Roux, Dr. Ed Theriot and Dr. Enamul Huq for agreeing to be on my committee and for their valuable suggestions during our committee research meetings. I would like to sincerely thank Dr. Susan Weintraub, Kevin Hakala and Sam Pardo at UT Health Science Center, San Antonio and thanks to Dr. Ruth Welti and Mary Roth at Kansas State Lipidomics center, Manhattan for their help with lipid work. My honest thanks to Dr. Schonna Manning for her valuable discussions and suggestions on algae lipid biology. It was a big help to have her on campus. My thanks to UTEX culture collection and its staff for being helpful and friendly. I would also like to thank on campus facilities such as Greenhouse, ICMB core, GSAF and CCBB and people associated with them for help in my project in one way or other. v I would like to thank all the former and current undergraduates and graduate students of Mehdy lab and Sathasivan lab, Hali Fechtmeyer, Emily Beck, Shahima Islam, Andrew Ngo, Shyam Ramachandran, Sangwa Park, Drew Wallen and Elborz Safarzadeh for their help in many ways during my time in the lab. Thanks to wonderful friends I made here at university and my friends outside university for their support and friendship, which helped in keeping a good work life balance. Last and most importantly would like to thank my biggest support my family, my Mom and Dad, my husband Ravi, my brother Karan, my family-in-law and my love, my daughter Amaira for all their patience, love and support. I thank ‘God’ for blessing my life with such people. Thank you all! vi Rapid remodeling of lipids and transcriptome in the diatom Phaeodactylum tricornutum in response to defense related decadienal Tanya Sabharwal, Ph.D. The University of Texas at Austin, 2016 Supervisor: Mona C. Mehdy Co-Supervisor: K. Sathasivan Abstract Diatoms rapidly release extracellular oxylipins (oxygenated lipids) including polyunsaturated aldehydes (PUAs) upon cell damage, as a defense response to inhibit reproduction in many herbivores. PUAs are synthesized from membrane phospholipids. In diatoms, PUAs rapidly increase intracellular Ca2+ and nitric oxide (NO) levels and affect gene expression and physiology. Treatment of the marine diatom Phaeodacylum tricornutum, with the PUA, decadienal (DD, 10 µM) altered lipid compositions. In cells treated for 3 hr, nine out of ten measured saturated and unsaturated fatty acids declined (range of 0.5-0.7 fold of solvent control levels). At 6 hr, most fatty acid species 14:0, 18:0, 20:5 remained at lower levels while 18:1 and 18:2 increased (1.12 fold and 1.46 fold respectively). Phospholipids in DD treated cells at 3 hr showed decline in PG1 (0.69 fold) and PS1 (0.36 fold) whereas PE1 increased (1.79 fold). At 6 hr, PI1 (0.79 fold), PS1 (0.28 fold) and LPG1 (0.56 fold) declined whereas PC1 (1.21 fold) and PE1 (2.7 fold) increased. There were no effects on abundant chloroplast glycolipids (DGDG/MGDG)1. Molecular 1 PG= phosphotidylglycerol; PS= phosphotidylserine; PE= phosphatidylethanolamine; PI= phosphotidylinositol; PS=phosphotidylserine; LPG= lyso phosphotidylglycerol; PC= phosphotidylcholine; DGDG= digalactosyl diacylglycerol; MGDG= monogalactosyl diacylglycerol vii species composition of phospholipids showed a greater decline in polyunsaturated fatty acids than monounsaturated fatty acids and saturated fatty acids, which indicates a shift towards saturated fatty acids that may protect membranes from oxidative stress. C-28 brassicasterol declined 0.86 fold at 3hr whereas there was no change at 6hr. Non-polar lipids increased by 1.16 fold and 1.38 fold at 3hr and 6hr. Decreased membrane permeability in DD treated cells was suggested by reduced uptake of cytological dyes. To our knowledge, this is the first report documenting novel rapid (within 6 hr) phospholipid changes in response to PUA suggesting early membrane lipid remodeling to aid in adaptation to PUA stress in algae. Transcriptome analysis at 3hr and 6hr of DD treatment indicated differential regulation of a subset of genes in fatty acid metabolism pathway in co-ordination with genes in multiple metabolic pathways. This suggests that rapid lipid remodeling may be regulated at transcriptome levels to help adapt to decadienal stress. viii Table of Contents List of Tables ........................................................................................................ xii List of Figures ...................................................................................................... xiv Chapter 1: Introduction ...........................................................................................1 Stress in diatoms and adaptive strategies ...............................................2 Effect of abiotic stress on changes in lipids in algae .............................8 Effect of mechanical wounding stress on lipids in algae .....................11 Phaeodactylum tricornutum: Choice of organism under study for this project .........................................................................................14 Lipid composition and known lipid biosynthesis pathways in P. tricornutum ........................................................................15 Regulation of various stresses at transcriptome levels in P. tricornutum ........................................................................18 Broad aims of this project ....................................................................19 Hypothesis............................................................................................20 Significance of this study .....................................................................20 Chapter 2: Defense-related decadienal elicits changes in fatty acid and lipid class composition in the diatom Phaeodactylum tricornutum ...............................30 Abstract ................................................................................................30 Introduction ..........................................................................................31 Materials and methods .........................................................................34 Strain and culture conditions ......................................................34 Chemicals ....................................................................................34 Growth assay ...............................................................................35 Viability assay .............................................................................35 Measuring NO production in cells ..............................................35 FAME preparation ......................................................................36 GC/MS Analyses ........................................................................36 ESI-MS/MS analysis of lipid molecular species ........................37 Sterol Extraction and GC-MS Analysis ......................................38 ix Measuring neutral lipids in P. tricornutum cells by Bodipy .......39 PUA analysis ...............................................................................39 Membrane permeability assays ...................................................40 Results and Discussion ........................................................................41 Growth and viability assay to determine effect of DD on cells ..41 DD dose and time dependent NO production in the cells ...........41 Effect of DD on changes in fatty acid levels in P. tricornutum ..42 ESI-MS/MS analysis of various lipid classes in DMSO solvent and DD treated P. tricornutum cells .........................................45
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