Western University Scholarship@Western Electronic Thesis and Dissertation Repository 6-28-2017 12:00 AM GmMYB176 Interactome and Regulation of Isoflavonoid Biosynthesis in Soybean Arun Kumaran Anguraj Vadivel The University of Western Ontario Supervisor Dr. Sangeeta Dhaubhadel The University of Western Ontario Joint Supervisor Dr. Mark Bernards The University of Western Ontario Graduate Program in Biology A thesis submitted in partial fulfillment of the equirr ements for the degree in Doctor of Philosophy © Arun Kumaran Anguraj Vadivel 2017 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Molecular Biology Commons, and the Plant Biology Commons Recommended Citation Anguraj Vadivel, Arun Kumaran, "GmMYB176 Interactome and Regulation of Isoflavonoid Biosynthesis in Soybean" (2017). Electronic Thesis and Dissertation Repository. 4639. https://ir.lib.uwo.ca/etd/4639 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. i Abstract MYB transcription factors are one of the largest transcription factor families characterized in plants. They are classified into four types: R1 MYB, R2R3 MYB, R3 MYB and R4 MYB. GmMYB176 is an R1MYB transcription factor that regulates Chalcone synthase (CHS8) gene expression and isoflavonoid biosynthesis in soybean. Silencing of GmMYB176 suppressed the expression of the GmCHS8 gene and reduced the accumulation of isoflavonoids in soybean hairy roots. However, overexpression of GmMYB176 does not alter either GmCHS8 gene expression or isoflavonoid levels suggesting that GmMYB176 alone is not sufficient for GmCHS8 gene regulation. I hypothesized that GmMYB176 acts cooperatively with another factor(s) for the regulation of GmCHS8 gene expression and it may also regulate other isoflavonoid biosynthetic genes in soybean. The objective of this research was to identify the GmMYB176 interactome for GmCHS8 gene regulation and elucidate the role of GmMYB176 in isoflavonoid biosynthesis in soybean. GmMYB176 interacting proteins were identified using two translational fusion baits (GmMYB176-YFP and YFP-GmMYB176) by co-immunoprecipitation, followed by liquid chromatography-tandem mass spectrometry. The interaction of selected candidates with GmMYB176 was validated in planta and their DNA binding activities determined. GmMYB176 may form a transcriptional complex with Gm04bZIP and/or Gm05bZIP for the regulation of GmCHS8 gene expression. RNA-seq and metabolomics analyses of soybean hairy roots in which GmMYB176 was either silenced or over-expressed revealed that GmMYB176 regulates multiple genes in the isoflavonoid biosynthesis pathway, affecting the production of metabolites in phenylpropanoid pathway such as phenylalanine, liquiritigenin, daidzin, genistin, glycitein, and glyceollin. The knowledge and information generated on the role of GmMYB176 in the regulation of isoflavonoid biosynthesis will allow genetic manipulation of isoflavonoid level in soybean and/or introduce isoflavonoid pathway in non-legumes. Keywords Co-immunoprecipitation, LC-MS/MS, MYB, protein-protein interaction, transcriptomics, metabolomics, isoflavonoid, phenylpropanoid, secondary metabolism, transcriptional complex, gene regulation, gene families, soybean ii Acknowledgements I dedicate this thesis to my non-biological father, Mr. N. Thangavel. Thanks as well to my parents Mr. Anguraj Vadivel and Mrs. Thilagavathy, my sister, Mrs. Priyadharshini for their support in me pursue a research in Canada, far away from home. I also thank my cousins Ms. Kamalapriya and Mr. Suryakumar for their support. I would like to thank my adopted family at Agriculture and Agri-Food Canada (AAFC), London. Everyone in the Dhaubhadel lab (past and present), Mehran, Hemanta, Kishor, Pravesh, Arjun, Caroline, Xuyan, Shaomin, Serina, Amelia, Jaeju, Kevin, Kelsey, Smarth, and Nishat – thank you for your support and encouragement. I would also like to thank Ling, for her lab technical advice and life advice. Moreover, I would like to thank all my friends in other labs (in AAFC) FM lab, CD lab, IS lab, AH lab, RM lab, RA lab, YC lab (and so many) for lighting up my days and for all those memories in last five years. I also thank my friends, Aagam, Milan, Preetam and Rohan from dept of chemistry, biochemistry, and geology. I must thank the individuals who have been essential to the present research: my advisory committee, Dr. Jim Karagiannis and Dr. Frederic Marsolais for their input and guidance; I also thank my co-supervisor, Dr. Mark Bernards for his insights and his invaluable advice for my research and in general. I would like to thank Dr. Justin Renaud for his help in analyzing metabolite samples and teaching me a lot about metabolomics. I also thank London Regional Proteomics Center, UWO for their help in analyzing my protein samples. Most importantly, I must thank my supervisor, Dr. Sangeeta Dhaubhadel for giving me the opportunity to join her lab and for her guidance, support, and encouragement in science. iii Table of Contents Abstract ................................................................................................................................. i Acknowledgements ............................................................................................................ ii Table of Contents .............................................................................................................. iii List of Tables .................................................................................................................... vii List of Figures .................................................................................................................. viii List of Appendices ............................................................................................................... x List of Abbreviations .......................................................................................................... xi 1 GENERAL INTRODUCTION ...................................................................................... 1 1.1 Soybean ................................................................................................................... 1 1.1.1 Soybean genome .......................................................................................... 1 1.2 Phenylpropanoid pathway ....................................................................................... 2 1.3 Isoflavonoids ........................................................................................................... 2 1.3.1 Role of isoflavonoids in human health and nutrition .................................. 4 1.3.2 Role of isoflavonoids in plants .................................................................... 4 1.4 Isoflavonoid biosynthesis in soybean ...................................................................... 5 1.4.1 Overview of the pathway ............................................................................. 5 1.4.2 Regulation and accumulation of isoflavonoids in soybean tissues ............. 9 1.5 MYB transcription factors and transcriptional complexes .................................... 11 1.6 GmMYB176: the regulator of isoflavonoid biosynthesis in soybean ................... 14 1.7 Objectives of the study .......................................................................................... 17 1.8 Literature cited ....................................................................................................... 18 2 IDENTIFICATION OF SOYBEAN CHS GENE FAMILY MEMBERS ................... 28 2.1 Introduction ........................................................................................................... 28 2.2 Materials and methods ........................................................................................... 30 iv 2.2.1 In silico and phylogenetic analysis ............................................................ 30 2.2.2 Generation of a heat map ........................................................................... 30 2.3 Results ................................................................................................................... 31 2.3.1 The GmCHS family contains 14 putative members .................................. 31 2.3.2 GmCHS transcript accumulation vary in soybean tissues ......................... 32 2.4 Discussion .............................................................................................................. 40 2.4.1 Soybean genome contains 14 putative GmCHS genes .............................. 40 2.4.2 GmCHSs display tissue-specific expression in soybean............................ 42 2.5 Conclusion ............................................................................................................. 43 2.6 Literature cited ....................................................................................................... 44 3 GmMYB176 INTERACTOME: KEY FACTORS IN ISOFLAVONOID BIOSYNTHESIS IN SOYBEAN .............................................................................................................. 47 3.1 Introduction ........................................................................................................... 47 3.2 Materials and methods ........................................................................................... 49 3.2.1 Plant