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The Roles of OVATE and Other Elongation Genes in Regulating Proximal-Distal Patterning of Tomato Fruit

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The roles of OVATE and other elongation genes in regulating proximal-distal patterning of tomato fruit Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Shan Wu, M.S. Graduate Program in Horticulture and Crop Science The Ohio State University 2015 Dissertation Committee: Dr. Esther van der Knaap, Advisor Dr. Jyan-chyun Jang Dr. Michelle Jones Dr. Tea Meulia Dr. Eric Stockinger Copyright by Shan Wu 2015 Abstract Domestication of tomato (Solanum lycopersicum) has resulted in a variety of fruit shapes from flat to very elongated. The elongated shape is a common feature that distinguishes many cultivated tomatoes from the undomesticated types, and it is mainly controlled by three loci, ovate, sun and fs8.1. We performed detailed morphological analyses of the reproductive and vegetative organs, which demonstrated that ovate, sun and fs8.1 regulate unique aspects of ovary and fruit elongation and in different temporal manners. The synergistic interaction between sun and ovate or fs8.1 suggested that the pathways involving SUN, OVATE and the gene(s) underlying fs8.1 may converge at a common node. We also conducted a transcriptome comparison analysis between the triple mutant sun/ovate/fs8.1 and wild type by RNA-seq using reproductive meristems and young flower buds. This study revealed changes in the transcription profile possibly caused by the combined effects of the three mutations. To gain insights into the role of OVATE in regulating fruit shape, we searched for its genetic and protein interactors. A synergistic interaction was found between ovate and suppressor of ovate 1 (sov1) in controlling ovary and fruit elongation. Tomato OVATE family protein 20 (SlOFP20) was identified as a candidate gene underlying the sov1 locus. Yeast two-hybrid (Y2H) experiments showed that OVATE interacted with Tonneau1 Recruiting Motif (TRM) proteins, which are a part of a protein complex regulating the formation of preprophase band and organization of cortical microtubule (MT) array. Transient co-expression of OVATE or SlOFP20 with putative MT-associated SlTRMs in N. benthamiana resulted in relocalizations of SlOFPs and SlTRMs. Our findings have shed new light on the roles of OFPs in proximal-distal fruit patterning and provided insights into fundamental aspects of plant organ growth. ii Dedication This dissertation is dedicated to my family. iii Acknowledgments I would like to express my sincere gratitude to my advisor, Dr. Esther van der Knaap, for her mentoring, motivation and patience. Her guidance and encouragement helped me all the time in my research and writing of this dissertation. She sets a good example for me of how to be a scientist with strict standards and great enthusiasm. I would like to thank my study committee members, Dr. JC Jang, Dr. Michelle Jones, Dr. Rebecca Lamb, Dr. Tea Meulia and Dr. Eric Stockinger for providing valuable suggestions and resources to my project and helping me in all the exams. I am also grateful to Dr. Carmen Catala and Dr. Giovannoni for offering me the opportunity to learn RNA-seq library construction, Dr. Martine Pastuglia and Dr. David Bouchez for helpful discussions on the TRMs, and Dr. Sophien Kamoun, Dr. Feng Qu and Dr. Iris Meier for the advices on transient expression assays in tobacco and plasmid construction. I would like to thank all the current and previous lab members for their help and friendship. My special thanks go to Josh Clevenger, Dr. Liang Sun and Dr. Neda Keyhaninejad for their collaboration and insight. I would also like to acknowledge Jason Van Houten, Dr. Hyun Jung Kim, Dr. Manohar Chakrabarti, Dr. Eudald Illa and Dr. Zejun Huang for offering help on this project. My sincere thanks also go to Meghan Fisher, Jiheun Cho and Brenda Sanchez Montejo for taking care of my plants in the greenhouse and field. I also thank my fellow graduate student labmates: Qi Mu, Yi- Hsuan Chu, Yanping Wang, Xiaoxi Liu and Nathan Taitano for the stimulating discussions and for working together late before deadlines. Finally, I thank my family. Words cannot express how grateful I am to my parents and parents-in-law, my beloved husband, Xing, and daughter, Audrey, for always supporting me. iv Vita 2003………………….High School Affiliated to Shanghai Jiao Tong University, Shanghai, China 2007……………………………….B.S. Agonomy, China Agricultural University, Beijing, China 2009……................M.S. Horticulture and Crop Science, The Ohio State University, Wooster, OH 2010 to present ………………………....................... Graduate Reserach Associate, Department of Horticulture and Crop Science, The Ohio State University Publications Wu S, Xiao H, Cabrera A, Meulia T, van der Knaap E. 2011. SUN regulates vegetative and reproductive organ shape by changing cell division patterns. Plant Physiol 157(3):1175-86. Jiang N, Visa S, Wu S and van der Knaap. 2012 Rider transposon insertion and phenotypic change in tomato. Topics in Current Genetics 24: 297–312 Liu N, Wu S, Van Houten J, Wang Y, Ding B, Fei Z, Clarke TH, Reed JW, van der Knaap E. 2014. Down-regulation of AUXIN RESPONSE FACTORS 6 and 8 by microRNA 167 leads to floral development defects and female sterility in tomato. J Exp Bot 65(9): 2507-20. van der Knaap E, Chakrabarti M, Chu YH, Clevenger JP, Illa-Berenguer E, Huang Z, Keyhaninejad N, Mu Q, Sun L, Wang Y, and Wu S. 2014. What lies beyond the eye: The molecular mechanisms regulating tomato fruit weight and shape. Front Plant Sci 5:227. Wu S, Clevenger JP, Sun L, Visa S, Kamiya Y, Jikumaru Y, Blakeslee J and van der Knaap E. 2015. The control of tomato fruit elongation orchestrated by sun, ovate and fs8.1 in a wild relative of tomato. Plant Sci 238:95-104. Fields of Study Major Field: Horticulture and Crop Science v Table of Contents Abstract ............................................................................................................................................ ii Dedication ....................................................................................................................................... iii Acknowledgments........................................................................................................................... iv Vita................................................................................................................................................... v List of Tables .................................................................................................................................. ix List of Figures ................................................................................................................................. xi Chapter 1: The making of an elongated tomato fruit ...................................................................... 1 Chapter 2: The control of tomato fruit elongation orchestrated by sun, ovate and fs8.1 in a wild relative of tomato ............................................................................................................................. 9 Abstract ........................................................................................................................................ 9 Introduction ................................................................................................................................ 10 Materials and Methods ............................................................................................................... 11 Results ........................................................................................................................................ 14 Discussion .................................................................................................................................. 21 Chapter 3: RNA-seq transcriptome analysis of the sun/ovate/fs8.1 NILs during tomato early flower development ....................................................................................................................... 43 Abstract ...................................................................................................................................... 43 vi Introduction ................................................................................................................................ 44 Materials and Methods ............................................................................................................... 46 Results ........................................................................................................................................ 49 Discussion .................................................................................................................................. 55 Chapter 4: The roles of OVATE family proteins in organ patterning and their interaction with TRMs, a group of microtubule modulating proteins ...................................................................... 72 Abstract ...................................................................................................................................... 72 Introduction ................................................................................................................................ 73 Materials and Methods ............................................................................................................... 75 Results ........................................................................................................................................ 81 Discussion
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