The role of LC and FAS in regulating floral meristem and fruit locule number in tomato Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yi-Hsuan Chu, B.S. Graduate Program in Horticulture and Crop Science The Ohio State University 2017 Dissertation Committee Dr. Jyan-Chyun Jang, Dr. Esther van der Knaap, Advisor Dr. Anna Dobritsa Dr. David Mackey Dr. Leah McHale 1 Copyrighted by Yi-Hsuan Chu 2017 2 Abstract In tomato, lc and fas control the variation between the small and bilocular fruits from the wild ancestor (S. pimpinellifolium) and large fruit cultivars (S. lycopersicum var. lycopersicum) with up to ten locules. SlWUS and SlCLV3 are the candidates of lc and fas, respectively. The regulatory balance between these two genes plays a pivotal role in meristem maintenance in Arabidopsis. However, the genetic and molecular mechanisms of SlWUS and SlCLV3 have not been functionally characterized in tomato. Here, we performed a detailed phenotypic analysis of the reproductive organs in tomato near-isogenic lines. The results showed that lc and fas synergistically controlled floral organ and locule number. In addition, results from targeted RNA interference (RNAi) and transgenic complementation of fas clearly demonstrated that SlCLV3 was the gene underlying fas. By using mRNA in situ hybridization and transcriptome profiling, we observed temporal and spatial changes in the expression patterns of these two genes during floral development. Our results indicated that lc was a gain-of-function mutation of SlWUS while fas was a loss-of-function mutation of SlCLV3. We also conducted transcriptome analyses to capture differentially expressed genes (DEGs) responding to single and double mutations of lc and fas across vegetative/reproductive meristems and young flower buds. We adopted a recently developed 3’ Tag RNA-seq method for RNA-seq library preparation and compared its performance with the standard whole mRNA-seq method. Gene Ontology analysis over the DEGs showed enriched functionalities related to meristem/floral development and organ patterning. In addition, co- expression analysis revealed that microtubule motor activity and sterol/brassinosteroids biology might underlie differences between wild and fasciated tomato. In summary, the findings on tomato locule number and meristem control in this dissertation have provided new insights into the mechanisms in tomato fruit development. ii Dedication This dedication is dedicated to my beloved family, my father Chao-Liang Chu, my mother Hui- Chun Sun, my husband Chao-Min Huang and son Morris. iii Acknowledgments I would first like to express my sincere gratitude toward my advisor, Esther van der Kannp, for the tremendous help, advices, encouragement and kind supports along the journey of my doctoral studies. Her passion and curiosity made the lab a wonderful place for scientific discussion and learning, which was always a tremendous source of energy for me. My sincere thanks are extended to my advisor, Dr. JC Jang, for always providing valuable suggestions on my project, improving my presentation and writing skills, accepting me to his lab, and helping me in so many aspects. Without the help from my two talented mentors, this work could never be possible. I am also grateful to my committee members, Dr. Anna Dobritsa, Dr. David Mackey and Dr. Leah McHale for providing valuable comments and supports on my research as well as in all my exams. My appreciation also goes to Dr. Zachary Lippman for offering in situ probes and cooperating in the publication; Dr. Thomas Juenger for offering me the RNA-seq library protocol and primers. I also want to thanks all my lab members and other lab colleagues for numerous help in the lab and also the kind friendship. In particular, I would like to thank Dr. Zejun Huang for collaboration on this project. Thanks also go to Jason Van Houten for offering help in the RNA-seq library preparation, Dr. Shan Wu for teaching me the in situ and CRISPR techniques, Dr. Ying Wang and Dr. Hai- Dong Yu for assistant with RNA-seq materials, Jian Wu for helping with Microtome sectioning, and Dr. Rui Wang for teaching me staining tissues. I also want to acknowledge all my special lab mates, Qi Mu, Dr. Shan Wu, Dr. Liang Sun, Dr. Yangping Wang, Dr. Manohar Chakrabarti, Dr. Hyun Jung Kim, Dr. Eudald Illa, Dr. Neda Keyhaninejad, Dr. Xiaoxi Liu and Nathan Taitano for the friendship, stimulus discussions and helping inside and outside the lab. I am also grateful for Meghan Fisher, Jiheun Cho and Brenda Sanchez Montejo for taking care of my plants. Thanks also go to CCBL members, Fabio Gomez cano, Wilberforce Ouma and Eric Mukundi for providing valuable suggestions in data analysis. I feel extremely fortunate to have worked with these kind and intelligent people. Finally, I want to thank my beloved parents for the endless support, sacrifices throughout my life. Thank you both for giving me strength to chase my dream. Thanks to my husband, Chao-Min for being the best father and husband. Thanks to my son, Morris, who is always the source of happiness. My appreciation to them is more than I can expressed here. Without their support, sacrifice and love, this work would not have been possible. iv Vita 2011……………………………….B.S. Agronomy, National Taiwan University, Taipei, Taiwan 2012 to present…………….......................................Graduate Research Associate, Department of Horticulture and Crop Science, The Ohio State University 2016 and 2017 Spring ………………..…………….Graduate Teaching Associate, Department of Horticulture and Crop Science, The Ohio State University Publications 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. Xu C, Liberatore KL, MacAlister CA, Huang Z, Chu YH, Jiang K, Brooks C, Ogawa-Ohnishi M, Xiong G, Pauly M, Van Eck J. A cascade of arabinosyltransferases controls shoot meristem size in tomato. Nature Genetics. 2015 Jul 1;47(7):784-92. Fields of Study Major Field: Horticulture and Crop Science v Table of Contents Abstract ........................................................................................................................................... ii Dedication ...................................................................................................................................... iii Acknowledgments ......................................................................................................................... iv Vita .................................................................................................................................................. v Table of Contents ........................................................................................................................... vi List of Tables ............................................................................................................................... viii List of Figures ................................................................................................................................ ix Chapter 1. Introduction - The evolution of large cultivated tomatoes from tiny wild relatives ..... 1 Chapter 2. The control of tomato locule number and fruit weight by natural mutant alleles of lc and fas in wild type tomato ........................................................................................................... 16 Abstract ..................................................................................................................................... 16 Introduction ............................................................................................................................... 17 Materials and Methods .............................................................................................................. 19 Results ....................................................................................................................................... 26 Discussion ................................................................................................................................. 31 Chapter 3. Transcriptome analysis of lc and fas controlling tomato fruit development ............... 59 Abstract ..................................................................................................................................... 59 Introduction ............................................................................................................................... 60 vi Materials and Methods .............................................................................................................. 63 Results ....................................................................................................................................... 66 Discussion ................................................................................................................................. 73 Bibliography ................................................................................................................................. 98 Appendix A. Transgene copy number is determined by Southern blot ..................................... 111 Appendix B. Expression of tomato CLE small peptide gene family in floral and inflorescence meristems from whole mRNA-seq analyses ..............................................................................