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Identification and Characterization of Genes Involved in Metabolism of N5 Monoene Precursors to N5 Anacardic Acids in the Trichomes of Pelargonium X Hortorum

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Identification and Characterization of Genes Involved in Metabolism of N5 Monoene Precursors to N5 Anacardic Acids in the Trichomes of Pelargonium X Hortorum University of Louisville ThinkIR: The University of Louisville's Institutional Repository Electronic Theses and Dissertations 12-2016 Identification and characterization of genes involved in metabolism of n5 monoene precursors to n5 anacardic acids in the trichomes of Pelargonium x hortorum. Richa A. Singhal Follow this and additional works at: https://ir.library.louisville.edu/etd Part of the Biology Commons, Biotechnology Commons, Molecular Genetics Commons, and the Plant Biology Commons Recommended Citation Singhal, Richa A., "Identification and characterization of genes involved in metabolism of n5 monoene precursors to n5 anacardic acids in the trichomes of Pelargonium x hortorum." (2016). Electronic Theses and Dissertations. Paper 2568. https://doi.org/10.18297/etd/2568 This Doctoral Dissertation is brought to you for free and open access by ThinkIR: The nivU ersity of Louisville's Institutional Repository. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of ThinkIR: The nivU ersity of Louisville's Institutional Repository. This title appears here courtesy of the author, who has retained all other copyrights. For more information, please contact [email protected]. IDENTIFICATION AND CHARACTERIZATION OF GENES INVOLVED IN METABOLISM OF N5 MONOENE PRECURSORS TO N5 ANACARDIC ACIDS IN THE TRICHOMES OF PELARGONIUM × HORTORUM By RICHA A. SINGHAL M.Sc., University of Mumbai, India 2007 B. Sc., University of Mumbai, India 2005 A Dissertation Submitted to the Faculty of the College of Arts and Sciences of the University of Louisville in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy In Biology Department of Biology University of Louisville Louisville, Kentucky December 2016 Copyright 2016 by Richa Singhal © All Rights reserved ii iii IDENTIFICATION AND CHARACTERIZATION OF GENES INVOLVED IN METABOLISM OF N5 MONOENE PRECURSORS TO N5 ANACARDIC ACIDS IN THE TRICHOMES OF PELARGONIUM × HORTORUM By Richa A. Singhal M.Sc., University of Mumbai, India 2007 B. Sc., University of Mumbai, India 2005 A Dissertation Approved on November 21st, 2016 by the following Dissertation Committee: _________________________________________ Dissertation Director Dr. David Schultz _________________________________________ Dr. Michael Perlin _________________________________________ Dr. Joseph Steffen _________________________________________ Dr. Kenneth Palmer _________________________________________ Dr. Pradeep Kachroo ivii DEDICATION This dissertation is dedicated to my family. My mother Mrs. Lalita Singhal, my father Mr. Arunkumar Singhal and my sister Ruma Singhal. I thank them for their unconditional love, support, motivation and prayers. They are the source of my strength and have taught me to never give up and always work hard to achieve my goals. vii i ACKNOWLEDGMENTS I would like to express my heartfelt gratitude towards my mentor Dr. David J. Schultz for his gudiance and patience all these years. I thank him a lot for his insight and expertise which has greatly helped in developing my career as a future scientist and a critical thinker. His support and mentoring has made it possible for me to achieve my academic goals and complete my dissertation. I would also like to thank my committee members, Drs. Michael Perlin, Joseph Steffen, Kenneth Palmer and Pradeep Kachroo for their support, guidance and motivation. I thank all of them for always being available for my scientific queries and helping me develop my skills as a scientist. Their unified inputs along with their encouragement made this dissertation possible. I would especially like to thank Sabine Waigel and Dr. Wolfgang Zacharias from the genomics core facility for allowing me to use their lab facilities and teaching me the nitty gritty of the experiments needed for completion of this degree. I also thank them for providing me with neccesary supplies needed to perform my experiments. Without their support, it wouldn’t have been possible for me to complete the work needed for my dissertation. vi iv I would like to thank Dr. Eric Rouchka for generation of the RNA transcriptome and providing his valuble insights regarding bioinformatics analysis. I would like to thank Dr. Gary Cobbs for analysing HPLC, GC and transgenic data and for teaching me the application of statistical functions. I would especially like to thank the biology department chairman Dr. Ronald Fell for supporting and having faith in my ability to achieve my Ph.D. I would also like to thank Dr. Arnold Karpoff and Dr. Linda Fuselier for mentoring me as a graduate teaching assistant and helping me develop my teaching skills, which will be of great help for my academic career. I would like to thank all the Schultz lab members, Dr. Oliver Starks, Joshua Beck, Susan Hendrick, Ran Tian, Andrew Diddle, Khoi Do and Pratik Thappa for being supportive colleauges and helping my project. I would like to thank my close friends Su San Toh, Jasmit Shah, Jinny Paul, Pooja Schmill, Nisha Solanki, Shambhavi Mishra and Hiral Goel for their constant love and support. I fall short of words to thank my roommate/sister/best friend Sanaya Bamji. Without her constant love, support and motivation I wouldn’t have been able to survive away from my family all these years and stay motivated to achieve my goals. I would also like to thank all of the Soka Gakkai members from the Buddhist organization Soka Gakkai International for their prayers and motivation throughout the course of my Ph.D, particularly Mrs. Hettie Bailey, who has been vii v like a mother and my guide ever since I landed in the United States of America. I want to thank the mentors for my life Dr. Daisaku Ikeda and Dr. Shashishekhar Narayan Sinha for guiding me towards being a good human being, good scientist and maintaing a never give up spirit in life. I would like to thank my previous emplyoers in India, Mr. Rakesh Shah and late Mr. Ravindra Prajapati who supported me for pursuing Ph.D in United States. I would like to thank my extended family members, my uncles Mr. Vasant Salian, Mr. Jayant Salian, Mr. Yatendra Goyal and my cousing sister Garima Goyal for their never ending support during the course of my study at the United States. I finally conclude the acknowledgments by thanking my parents Mrs. Lalita Singhal, Mr. Arunkumar Singhal and my sister Ruma Singhal who are my source of strength and happiness. viviii ABSTRACT IDENTIFICATION AND CHARACTERIZATION OF GENES INVOLVED IN METABOLISM OF N5 MONOENE PRECURSORS TO N5 ANACARDIC ACIDS IN THE TRICHOMES OF PELARGONIUM × HORTORUM Richa Singhal November 21st 2016 Unusual monoenoic fatty acids (UMFA’s) and specialized metabolites called anacardic acids (AnAc) are produced in glandular trichomes of Pelargonium × hortorum (geranium). The UMFA’s, 16:1∆11 and 18:1∆13 are precursors for the synthesis of unsaturated AnAc 22:1n5 and 24:1n5 that contribute to pest resistance in geraniums. UMFAs and their derived AnAc metabolites not only provide a useful biological marker that differentiates the biosynthetic pathway for unusual mononenes from the common fatty acids (i.e. stearic, palmitic, oleic, linoleic and linolenic) but also have industrial, medical and agricultural applications. Fatty acid biosynthesis enzymes like acyl carrier proteins (ACPs); thioesterases (TEs) and β-ketoacyl-ACP synthases (KASs) are required for common fatty acid as well as the UMFA biosynthesis. Based on this, it is hypothesized that the specific isoforms of the fatty acid biosynthesis enzymes are highly expressed in trichomes and are involved specifically in metabolic channeling of UMFAs to anacardic acid synthesis within trichomes of geranium. ix vii This hypothesis is based on the knowledge that there is a novel Δ9 myristoyl- ACP desaturase (MAD) that directs acyl-ACP into UMFA biosynthesis and the products of MAD are correlated with the dominant congeners of AnAc (22:1n5 and 24:1n5). Transcription of MAD as well as production of 16:1∆11 and 18:1∆13 and AnAc 22:1n5 and AnAc 24:1n5 has been found to be highly trichome specific. This dissertation reports the identification of the complete nucleotide and protein sequences of genes for 2 ACPs, 3 FAT-As, 3 FAT-Bs, 4 KAS Is, 1 KAS II and 1 KAS III from a geranium EST database. Quantitative real-time PCR (qRT-PCR) was used to analyze tissue-specific expression patterns of the target genes, which indicated that ACP 1, ACP 2, KAS I-a/b, KAS Ic, FAT-A1, and FAT-A2 are highly expressed in trichomes. To further this research, a de novo RNA and micro-RNA transcriptome was generated from trichomes and bald pedicle of geranium, which helped in identification of several genetic components involved in UMFA synthesis. Bioinformatics analysis of RNA-transcriptome along with qRT-PCR and biochemical assays (HPLC and GC) were used to correlate the effect of temperature (18°C, 23°C and 28°C) on gene expression (ACPs, KASs, FAT-As) and changes in production of 16:1Δ11 and 18:1Δ13 UMFAs and 22:1n5 and 24:1n5 AnAc. Results of this work show that expression of ACP 1, ACP 2, KAS I-c, KAS I-a/b were correlated with changes in UMFAs and AnAc production with temperature, thus indicating their potential role in UMFA metabolism. We also determined that 23°C is an optimal temperature for production of UMFAs viiix and AnAcs as compared to 18°C and 28°C. To determine and verify the function of ACP 1 and ACP 2, we co-expressed these genes in conjunction with a Δ9 myristoyl-ACP (MAD) desaturase in both E. coli and tobacco. E. coli assay results show that expression of ACP 2 with MAD increased the production of UMFAs significantly, thus validating the novel role of ACP 2 in UMFA production. This work, in addition to the generation of a de novo transcriptome, provides a platform for further defining UMFA metabolism within trichomes of geranium. ix xi TABLE OF CONTENTS PAGE DEDICATION………………………………………………………………………….iii ACKNOWLEDGEMENTS…………………………………………………………….iv ABSTRACT…………………………………………………………………………….vii LIST OF TABLES……………………………………………………………………..xiv LIST OF FIGURES……………………………………………………………………xvii CHAPTER PAGE 1.
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