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By T-DNA Mutant Analysis and Investigation of Molecular Interactions of Tandem Zinc Finger 1 (TZF1) in Arabidopsis Thaliana Unraveling the Functions of Plant Ran GTPase-Activating Protein (RanGAP) by T-DNA Mutant Analysis and Investigation of Molecular Interactions of Tandem Zinc Finger 1 (TZF1) 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 Thushani Rodrigo-Peiris, M.Eng. Graduate Program in Plant Cellular and Molecular Biology The Ohio State University 2012 Dissertation committee: Professor Iris Meier, Co-advisor Professor Jyan-chyun Jang, Co-advisor Professor Randy Scholl Professor David Mackey Professor Eric Stockinger Copyright by Thushani Rodrigo-Peiris 2012 ABSTRACT RanGAP, the GTPase-Activating Protein (GAP) of the small GTPase Ran, is vital for nucleocytoplasmic trafficking of protein cargo in yeast and animals via the Ran cycle, and mitotic cell division. Arabidopsis thaliana contains two RanGAP paralogs, namely RanGAP1 and RanGAP2. Despite the functional information of RanGAP revealed by extensive studies in yeast and animals, and its high degree of conservation in structure, GAP activity and sub-cellular localization in plants, involvement of RanGAP in plant development is poorly understood. Studies in plants would provide clues on the evolutionary conservation and digression of RanGAP’s roles among eukaryotes, and would also enable insight into its effects on a whole-organism level in a system that undergoes indeterminate development. Here, I have used a genetic approach to generate a series of T-DNA insertion lines in Arabidopsis thaliana that contain decreasing RanGAP levels and increasing severity in vegetative and reproductive phenotypes. The mutants provide valuable experimental material to dissect the functions of plant RanGAP. I have analyzed several of these phenotypes in detail and revealed the importance of Arabidopsis RanGAP in plant genesis, survival and development. The implications of mitotic involvement of RanGAP underlying these phenotypes points to a general conservation of RanGAP functionality in planta, in common with yeast and animals. ii In an independent effort, molecular mechanisms underlying the functions of Tandem Zinc Finger 1 of Arabidopsis thaliana (AtTZF1) were investigated. AtTZF1 is a plant- unique CCCH-type tandem zinc finger (TZF) protein implicated in drought and cold stress tolerance via regulation of gene expression. Studies with the most well studied human TZF; hTTP and AtTZF1 collectively suggest that the functions of AtTZF1 may be mediated by the dynamics in the assembly of stress responsive cytoplasmic mRNA- protein complexes; stress granules (SGs) and P-bodies (PBs), phosphorylation of AtTZF1 by the Mitogen-Activated Protein Kinase (MAPK) pathway and positive regulation of ABA-mediated gene expression in planta. Understanding the mode of function of AtTZF1 particularly in stress tolerance could be useful in developing agronomically important crops. To investigate the molecular mechanisms of AtTZF1 functions that currently remain elusive, assays on protein-protein interaction and AtTZF1 phosphorylation were conducted. Since the assays based on putative candidates were unable to reveal authentic molecular partners for AtTZF1, alternative approches may be used for future investigations. iii Dedicated to my parents, teachers, friends and family iv ACKNOWLEDGEMENTS I wish to express my utmost gratitude to my parents for being my first teachers, my inspiration, and for the unimaginable sacrifices they made on my behalf. Warmest thanks to my husband for being my friend, pillar of support and making me smile through the hardest times over the years. I am grateful to my brother and the rest of my family for being by my side always and unconditionally. I am indebted beyond words to the kindness of the graduate school deans; Drs. Herness, Wallace and Slotnick. It was an honor and a priviledge to have received their guidance, encouragement and support in the time of need to complete my degree. The work of my dissertation would not have been possible if not for the contributions of my advisors; Drs. Meier and Jang, and the dissertation committee; Drs. Scholl, Mackey and Stockinger. Special thanks are due to my collegues and friends in Rightmire Hall and Aronoff Laboratory for sharing their knowledge, ideas and materials, for insightful discussions and experimental help. Particularly I am grateful to my wonderful mentor Dr. Xianfeng Morgan Xu, and my collegues; Ms. Srimathi Bogamuwa, Dr. Nirodhini Siriwardana, Dr. Antje Feller, Dr. Siva Muthuswamy, Dr. Sowmya Venkatakrishnan, Dr. Srilakshmi Makkena, Ms. Jie Qu, Dr. Marcelo Pomeranz, Dr. Gireesha Mohannath, Ms. Veena Patil, Ms. Isabel Casas, Ms. Jamie Jackel, Dr. Ryuta Takeda, Dr. Qiao Zhao, Dr. Priya Raja, Ms. Katja Machemer, Dr. Sumire Fujiwara, Dr. Asuka Itaya and Dr. Kenneth Buckley. Your help, friendship and support over the years had inspired me beyond what you could possibly imagine. v I am thankful to Ms. Horng-Jing (Heather) Wang and Mr. Li Zhang for their unpublished work cited in my dissertation. Assistance with microscopy by Mr. Brian Kemmenoe, Ms. Kathy Walken and Mr. Mike Shade, greenhouse support by Mr. Joe Takayama, technical support by Mr. Scott Hines and Mr. Dave Long, and numerous help by Ms. Melinda Parker, Ms. Laurel Shannon, Ms. Diane Furtney, Dr. Angela White, Dr. Sheila Westendorf, Ms. Karen Kyle and Ms. Lucinda Bolinger are greatly appreciated. Heartfelt thanks are also due to my supervisors at CLSE for constantly providing me a funding line to complete my degree and the exciting opportunities I received in teaching training. I thank all those who were an inspiration, cheered me up, made me laugh, had fun with, got in trouble with, sent me wishes, gave a listening ear, fed me at hungry times, gave me smiles and hugs, and held my hand through all these years. Last but not least, thanks so much to OSU for this valuable and memorable experience. Go bucks! vi VITA 2001 ……………………………………………B.Sc. in Botany University of Colombo, Sri Lanka 2004 ……………………………………………M.Eng. in Biotechnology Osaka University, Japan 2006 – present …………………………………Graduate Teaching/Research Associate Department of Molecular Genetics The Ohio State University PUBLICATIONS RanGAP1 is a continuous marker of the Arabidopsis cell division plane (2008). Xu, X.M., Zhao, Q., Rodrigo-Peiris, T., Brkljacic, J., He, C.S., Muller, S. and Meier, I. Proc Natl Acad Sci USA 105: 18637-18642 RanGAP is required for post-meiotic mitosis in female gametophyte development in Arabidopsis thaliana (2011). Rodrigo-Peiris, T., Xu, X.M., Zhao, Q., Wang, H-J. and Meier, I. J. Exp. Bot. 62(8): 2705-2714 FIELD OF STUDY Plant Cellular and Molecular Biology vii TABLE OF CONTENTS Page Abstract …………………………………………………………………………… ii Dedication ………………………………………………………………………… iv Acknowledgements……………………………………………………………….. v Vita………………………………………………………………………………… vii List of figures………………………………………………………………….…… xiv List of tables……………………………………………………………………….. xvii Chapters: 1. RanGAP is required for post-meiotic mitosis in Arabidopsis thaliana…………. 1 1.1 Abstract………………………………………………………… …………… 2 1.2 Introduction………………………………………………………………….. 3 1.3 Materials and Methods…………………………………………………….. .. 8 1.3.1 Plant material and growth conditions………………………………….. 8 1.3.2 PCR-based genotyping of T-DNA insertion lines……..………………. 8 1.3.3 Antibody preparation…………………………………………………… 9 1.3.4 Floral stage determination……………………………………………… 9 1.3.5 Ovule clearing and differential interference contrast (DIC) optics…….. 9 viii 1.3.6 Aniline blue staining…………………………………………………. .. 10 1.3.7 Cloning…………………………………………………………………. 10 1.3.8 Complementation assay……………………………………………….... 11 1.3.9 β-Glucuronidase assay…………………………………………………. 12 1.4 Results……………………………………………………………………….. 12 1.4.1 RanGAP single knock-out mutants lack observable phenotypes………. 12 1.4.2 RanGAP double knockout mutants rg1-1/rg1-1;rg2-3/rg2-3 were not obtained………………………………………………………………… 13 1.4.3 Transmission of the rg1-1 allele in the rg2-3 background is normal through the male parent but completely blocked through the female parent…… 14 1.4.4 rg1-1;rg2-3 ovules arrest during early megagametogenesis…………… 14 1.4.5 RanGAPs are expressed in the developing embryo sac and pollen……. 16 1.4.6 rg1-1/rg1-1;rg2-3/rg2-3 RanGAP knockout double mutants were rescued by complementation with RanGAP1 and RanGAP2 genomic constructs………………………………………………………………. 16 1.5 Discussion…………………………………………………………………… 17 1.5.1 RanGAP deficiency and lethality………………………………………. 17 1.5.2 RanGAP1 and RanGAP2 are functionally redundant in female gametophyte development……………………………………………………………. 21 1.5.3 Female gametophytes are arrested during a mitotic phase…………….. 21 1.5.4 Embryo sacs are affected more than the pollen………………………... 22 1.5.5 Outcomes of the study and future prospects…………………………… 24 2. Requirement of RanGAP for vegetative development and cell division in Arabidopsis thaliana……………………………………………………………… 44 2.1 Abstract………………………………………………………………………. 45 ix 2.2 Introduction…………………………………………………………………... 47 2.2.1 Ran cycle and the functions of RanGAP in animals and yeast………… 47 2.2.2 RanGAPs in Arabidopsis thaliana …………………………………….. 48 2.2.3 Shoot and root development in Arabidopsis thaliana …………………. 49 2.2.4 Cell division in plant vegetative meristems and the role of sugars…….. 51 2.2.5 Common phenotypes of cell cycle mutants and wild-type plants that were induced for cell division arrest…………………………………………. 54 2.3 Materials and Methods……………………………………………………….. 57 2.3.1 Plant material, growth conditions and constructs……………………….
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