Discovery of LEAFY Transcriptional Complex Components Necessary for Flower Formation in Arabidopsis thaliana Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Nirodhini Srimali Siriwardana, B.Sc. Graduate Program in Department of Plant Cellular and Molecular Biology The Ohio State University 2011 Dissertation Committee: Dr. Rebecca Lamb, Advisor Dr. David Somers Dr. Iris Meier Dr. Patrice Hamel Copyright by Nirodhini Srimali Siriwardana 2011 ABSTRACT For successful reproduction, angiosperms must form fertile flowers in the appropriate positions and at the appropriate time. The timing of flower formation is especially important for annual plants, such as the model Arabidopsis thaliana used in this study. Floral fate is controlled by a set of genes termed floral meristem identity genes, the most important of which is the plant-specific transcription factor LEAFY (LFY). LFY‟s function can be divided into two phases, which are both temporally and genetically separable. In the first phase, LFY controls the expression of genes, such as other floral meristem identity factors, that control phyllotaxy and organ number. In the second phase, LFY is necessary for activation of the floral organ identity genes, which are responsible for differentiating the organs of the flower from each other. LFY is a transcription factor that does not have transcriptional activation or repression activities on its own and therefore must act in multiprotein complexes. Region- and stage- specific control of LFY target genes is provided by co-factors. However, until very recently little information was available about other factors that are present in such LFY- containing complexes. This work aims to identify components of LFY transcriptional complexes and characterize their function(s) in flower development. We demonstrate that LFY acts as a homodimer, which is mediated by a domain that is conserved across land ii plant LFY orthologs, suggesting that dimerization is important for the function of the entire LFY family. We report the isolation of putative LFY-interacting proteins and their preliminary characterization. Finally, we present an in depth analysis of one such LFY- interacting protein, FRIEND OF LEAFY1 (FOL1), which is a novel C2H2 zinc finger transcription factor. FOL1 is expressed in inflorescence meristems, floral meristems, male and female gametophytes and in embryos. Based on gain of function and loss of function analyses, FOL1 appears to function in control of meristem size and gametophyte development. We also discuss FOL1‟s physical and genetic interactions with LFY. The conservation of LFY sequence and functions across angiosperms suggests that most if not all of the LFY-interacting proteins identified in Arabidopsis will also function with LFY orthologs in other plants, including important crops. Therefore this work will provide a basis for the development of new strategies to increase agronomical values such as increased yield by manipulating LFY-interacting proteins in economically important crops. iii Dedicated to the most amazing person in my life, my mother (‘amma’). iv ACKNOWLEDGMENTS I could not have done without the help, guidance and critical thinking of my advisor, Dr. Rebecca Lamb. Her positive attitude towards my work and also a friendly atmosphere made my PhD possible. I would like to thank my committee, Drs. David Somers, Iris Meier and Patrice Hamel for their constructive criticism and suggestions, which made my research work, stand strong. Two past lab members Dr. Sachin Teotia and Andrew Moreo initiated this project in the lab and deserves a special note for their work. A special thanks to former graduate student Dr. Srilakshmi Makkena for moral support, for technical help and being there for me as a friend. I would like to thank a former undergraduate, Matthew Tegowski, who helped me tremendously with the work in Chapter 4 and especially for making me laugh in stressful times. I wish to thank undergraduates, Sterling Field, Irene Gentzel and Alyssa Larue for helping me with my research work. My sincere thanks to Matteo Citarelli for the help received with the alignment figures and with computer issues. I am specially thankful to my Aronoff and Rightmire “friends in science”, especially to Sowmya Venkatakrishnan my batch mate, not only for help in research work but also for being there for me every step of the way. Sivaramakrishnan Muthuswamy, Mintu Desai, v Joanna Boruc, Thushani Rodrigo-Peiris for encouraging words. I will never forget my dear friends Rosario Barbieri, Elisa Leyva-Guerrero, Vanessa Falcao and Zoee Gokhale with whom I spent most of my time in my first few years. A special thanks to all the staff members of our department, especially to Rene Hickey, Laurel Shannon and Eduardo Acosta for helping me whole-heartedly whenever I needed help. Also a special thanks to the Biological Sciences green house staff, Joan Leonard and Emily Yoders-Horn for help and support in growing plants. I thank ABRC for supplying us with seeds and Drs. T. Nakagawa, John Lindbo and Keith Slotkin for providing vectors. Special thanks to my dear mother and my sister, without them I couldn‟t have done all this. To my “friends in life” Sharon, Indi, Mekhala, Chamika, Poorani, Shekar, Harshi and Suresh for their encouraging words, laughs and relaxing times which no doubt gave me strength and motivation, especially in the last few months of my PhD. Above all to my love, my husband Ravindra Amunugama for helping me in my most stressful times, in every possible way. Most of all, by loving me and by taking care of our little son. And finally to my happiness, my son Rehan Luc Amunugama for making me the happiest mom on earth, you are the joy of my life! vi VITA 2003................................................................B.Sc. Honors, Plant Biology, University of Colombo, Sri Lanka 2003-2004………………………………….. Teaching Associate, Plant Biology, University of Colombo, Sri Lanka 2004 to present ..............................................Graduate Teaching Associate, Department of Plant Cellular and Molecular Biology, The Ohio State University, U.S.A FIELD OF STUDY Major Field: Plant Cellular and Molecular Biology vii TABLE OF CONTENTS ABSTRACT ........................................................................................................................ ii ACKNOWLEDGMENTS .................................................................................................. v VITA ................................................................................................................................. vii FIELD OF STUDY ........................................................................................................... vii TABLE OF CONTENTS ................................................................................................. viii LIST OF TABLES ............................................................................................................ xii LIST OF FIGURES ......................................................................................................... xiii CHAPTER 1: The Poetry of Reproduction: Flower Formation in Arabidopsis thaliana ............................................................................................................................... 1 1.1 Arabidopsis flower development ............................................................................... 2 1.1.1 Events at the transition to flowering ...................................................................... 2 1.1.1a Converting the VM to IM- the roles of flowering pathways, flowering time regulators and integrators ............................................................................................ 4 1.1.1b FM initiation-the role of the floral meristem identity genes .......................... 10 1.1.1c Flower organ development- the role of organ identity genes. ....................... 14 1.1.2 Maintaining FM identity ...................................................................................... 16 1.2 LEAFY (LFY) ........................................................................................................... 19 1.2.1 Evolution of LFY ................................................................................................. 19 1.2.2 Role of LFY in flowering .................................................................................... 20 1.2.2.1 LFY is involved in precise timing of the floral transition as well as maintaining floral fate. .............................................................................................. 22 viii 1.2.2.2 LFY controls floral homeotic gene expression and is necessary for floral organ identity and differentiation.............................................................................. 24 1.2.3 LFY as a transcription factor ............................................................................... 24 1.2.4 LFY co-regulators ................................................................................................ 25 1.3 Research Objectives .................................................................................................. 28 1.4 Significance of this study .......................................................................................... 30 REFERENCES ................................................................................................................ 40 CHAPTER 2: