Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2012 Chemical characterization of hydrocarbons and transcriptome profiling to elucidate pathway(s) of hydrocarbon biosynthesis in maize, pea, Botryococcus braunii and Emiliania huxleyi Wenmin Qin Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biochemistry Commons Recommended Citation Qin, Wenmin, "Chemical characterization of hydrocarbons and transcriptome profiling to elucidate pathway(s) of hydrocarbon biosynthesis in maize, pea, Botryococcus braunii and Emiliania huxleyi" (2012). Graduate Theses and Dissertations. 12905. https://lib.dr.iastate.edu/etd/12905 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Chemical characterization of hydrocarbons and transcriptome profiling to elucidate pathway(s) of hydrocarbon biosynthesis in maize, pea, Botryococcus braunii and Emiliania huxleyi by Wenmin Qin A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Biochemistry Program of Study Committee: Basil J. Nikolau, Major Professor Alan Dispirito Jacqueline Shanks Martin Spalding Marna Yandeau-Nelson Iowa State University Ames, Iowa 2012 Copy right © Wenmin Qin, 2012. All rights reserved. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS v CHAPTER 1. OVERVIEW 1 Introduction 1 Hydrocarbon Biosynthesis 2 Hydrocarbons in Maize Silk 8 Hydrocarbons in Pea Epidermis 11 Hydrocarbons in Botryococcus braunii 13 Hydrocarbons in Emiliania huxleyi 15 Research Goals 19 Dissertation Organization 21 References 23 CHAPTER 2. THE NATURAL VARIATION IN HYDROCARBON ACCUMULATION IN POLLEN AND SILKS FROM A DIVERSE SET OF MAIZE INBREDS 38 Abstract 38 Introduction 38 Results and Discussion 41 Conclusion 52 Experimental 53 References 55 Tables and Figures 59 CHAPTER 3. INHERITANCE PATTERNS OF HYDROCARBON ACCUMULATION EXHIBIT DIFFERENTIAL HETEROTIC EFFECTS IN POLLEN AND SILKS OF MAIZE HYBRIDS 70 Abstract 70 Introduction 71 Materials and Methods 73 Results 76 Discussion 83 References 86 Figures and Tables 93 CHAPTER 4. COMPARATIVE TRANSCRIPTOME PROFILING OF SILKS FROM MAIZE INBRED B73 FOR THE IDENTIFICATION OF GENES INVOLVED IN THE BIOSYNTHESIS OF SURFACE LIPIDS 101 Abstract 101 Introduction 102 Experimental 105 iii Results 111 Discussion 125 References 128 Table and Figures 132 CHAPTER 5. DIFFERENT SPATIAL AND TEMPORAL ACCUMULATION OF SURFACE LIPIDS ON LEAFLETS BUT NOT ON STIPULES IN AN ARG MUTANT OF PISUM SATIVUM 154 Abstract 154 Introduction 155 Results 157 Discussion 162 Experimental Procedures 167 References 170 Table and Figures 175 CHAPTER 6. HYDROCARBON ACCUMULATION IN IN BOTRYOCOCCUS BRAUNII EXPOSED TO DIFFERENT AVAILABILITIES OF CARBON, NITROGEN AND PHOSPHATE 184 Abstract 184 Introduction 185 Materials and Methods 187 Results 191 Discussion 195 References 199 Tables and Figures 203 CHAPTER 7. PHYSIOLOGICAL REGULATION OF LONG-CHAIN ALKENE-ACCUMULATION BY THE AVAILABILITY OF CARBON, NITROGEN AND PHOSPHATE NUTRIENTS IN EMILIANIA HUXLEYI 213 Abstract 213 Introduction 219 Materials and Methods 222 Results 225 Discussion 231 References 236 Tables and Figures 240 CHAPTER 8. GENERAL CONCLUSIONS 262 APPENDIX A. RNA PREPARATIONS FOR ILLUMINA SEQUENCING FROM MAIZE, PEA, BOTRYOCOCCUS BRAUNII AND EMILIANIA HUXLEYI: iv APPLIED TO SAMPLES ISOLATED FROM CONDITIONS LEADING TO HYPER- AND HYPO-ACCUMULATION OF HYDROCARBONS 276 APPENDIX B. AMINO ACID AND TOTAL METABOLITE ANALYSIS OF ENCASED AND EMERGED SILKS OF MAIZE 282 v ACKNOWLEDGEMENTS I would never have been able to finish my dissertation without the guidance of my committee members, help from my lab members, collaborators and friends, and support from my family. I would like to express my deepest gratitude to my advisor, Dr. Basil Nikolau, for his excellent guidance, caring, patience, and providing me with an excellent atmosphere and financial support for doing research. I would like to thank Dr. Marna Yandeau-Nelson for guiding my research for the past several years and helping me to develop my background in maize physiology and biochemistry. I also would like to thank Dr. Nikolau and Dr. Yandeau-Nelson for their help in the preparation of this dissertation. Dr. Jacqueline Shanks was enthusiastic and always provide insight to my work and available to answer questions in our biweekly hydrocarbon group meeting. I would like to thank other committee members, Dr. Alan Dispirtio and Dr. Martin Spalding and my former committee members, Dr. Tom Bobik, Dr. Volker Brendel and Dr. Reuben Peters for serving on my Program of Study committee. I would like to thank Dr. Ann Perera and Dr. Zhihong Song teach me how to use GC-MS and identify all the metabolites in all my samples and analyze data generated by GC-MS. Dr. Wolfe Gordon from Univesity of California-Chico teaches me how to grow Emiliania huxleyi, which needs special nutrients and growth conditions. I would like to thank Dr. Laura Jarboe for letting me use the Bio-Rad qPCR machine in her lab and Dr. Kumar vi Babu Kautharapu for helping me start with the qPCR experiments. Also, I would like to thank our lab manager, Dr. Libuse Brachova for maintaining the lab and make all the experiments possible, Sam Condon, an undergraduate student in our lab for collecting and preparing maize samples, extracting hydrocarbons, preparing media for algae and cell counting of Emiliania huxleyi and all other lab members for their suggestion about the my research, for their jokes, like a seasoning of life here and their support. I would also like to thank my parents, especially my mom, Suzhi Liu, two younger sisters and one younger brother, who always support me and encourage me with their best wishes. Finally, I would like to thank my husband, Wenzhi Lan, who was always there cheering me up and stood by me through the good times and bad. Our daughter, Lisa Lan, wipes away my stress and gives me strength when I feel tired and weak. 1 CHAPTER 1: OVERVIEW INTRODUCTION Hydrocarbons are compounds comprised of only carbon and hydrogen. Based on their structures, hydrocarbons can be classified into four different types: 1) saturated hydrocarbons (alkanes); 2) unsaturated hydrocarbons with at least one double (alkene) or triple (alkyne) bond between carbon atoms; 3) cycloalkanes containing one or more carbon rings; and 4) aromatic hydrocarbons having one or more aromatic rings. Simple, linear hydrocarbons such as alkanes and alkenes occur at low levels but are widely distributed in the biosphere. For example, hydrocarbons are found in some bacteria (Han and Calvin 1969a, b; Park 2005) and algae (Dennis and Kolattukudy 1991b; Rieley et al. 1998a) as well as in the cuticles of insects (Endler et al. 2004; Samuels et al. 2008b; Wackett et al. 2007) and plants (Samuels et al. 2008b). Hydrocarbons mainly exist in surface lipids of plants and insects and they serve as a protective barrier against water loss and pathogen attack (Kunst and Samuels 2003b; Kunst and Samuels 2009; PostBeittenmiller 1996; Samuels et al. 2008b). Because aliphatic hydrocarbons are the predominant components of petroleum-based fuels such as gasoline, diesel, and jet fuel, biological-derived hydrocarbons are ideal replacement of transportation fuels (Fortman et al. 2008; Jetter and Kunst 2 2008; Regalbuto 2009) and thus are potential alternative biorenewable fuel for petroleum (Jetter and Kunst 2008). Hydrocarbon biosynthesis Hydrocarbons are thought to be synthesized from very long chain fatty acid (VLCFA) precursors (Tornabene 1982). However, the biological synthesis of hydrocarbons from VLCFAs is poorly understood. Four different hypotheses for the mechanism(s) of hydrocarbon biosynthesis have been proposed (Beller et al. 2010; Frias et al. 2010; Park 2005; Schirmer et al. 2010; Sukovich et al. 2010a; Sukovich et al. 2010b; Templier et al. 1991a, b) (Fig1). Figure 1. Potential hydrocarbon biosynthesis pathways The majority of the linear hydrocarbons that are found in biological systems are of odd-number of carbon atoms. Because most fatty acids are assembled by the condensation of 2-carbon units (acetate), theoretically therefore, odd-numbered hydrocarbons (chain length 2n-1) can be derived 3 from even-numbered fatty acid (2n) by either the loss of a single carbon atom or by the addition of a single carbon. The theory that hydrocarbon biosynthesis occurs via the loss of one carbon is supported by the finding that across many diverse biological systems odd-numbered hydrocarbons (2n-1) occur together with their corresponding even-numbered fatty acid (2n) precursors (Chibnall and Pipe 1934; Largeau et al. 1980a; Major and Blomquist 1978). This theory was further supported by radio-labeling studies in which either the carboxyl group or alkyl chain of a fatty acid was radio-labeled, and it was demonstrated that the labeled carboxyl carbon is lost in the reaction, whereas labeled carbons within the alkyl chain are retained (Cassagne and Lessire 1974; Khan and Kolattukudy 1974; Kolattukudy et al. 1972). However, the mechanism by which the single carbon atom is lost during hydrocarbon biosynthesis is still poorly understood. Four different hydrocarbon biosynthetic mechanisms have been proposed,