HALLMARK DISSERTATION Fi

The Roles of Cytokinins and Cytokinin-N-Glucosides in Development and Gene Expression of Arabidopsis thaliana by Howard Tucker Hallmark A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama August 08, 2020 Keywords: Arabidopsis, cytokinin, trans-Zeatin, isopentenyladenine, glucosides, senescence Copyright 2020 by Howard Tucker Hallmark Approved by Aaron M. Rashotte, Chair, Professor of Biological Sciences Leslie R. Goertzen, Associate Professor of Biological Sciences Sang-Wook Park, Assistant Professor of Plant Pathology Haruka Wada, Assistant Professor of Biological Sciences Abstract The phytohormone cytokinin (CK) was first discovered over sixty years ago, and the decades following have produced an enormous amount of data about the various roles CK plays in intracellular signaling, gene expression, and plant development. An often-overlooked characteristic of this phytohormone is that it is not one single molecule; “cytokinin” is a generalized term for dozens of molecules which occur naturally in plants. Much of CK research has used synthetic CKs or only a single form of CK because it has long been believed that only a small subset of CKs display biological activity. Much of this dissertation focuses on CK-N- glucosides (CKNGs), long believed to be inactive forms of CK. Chapter 1 provides a broad overview of CK biology and their functions in plants, with particular focus on the model plant species Arabidopsis thaliana, which the rest of this dissertation work is completed in. Chapter 2 reviews recent advances in understanding of Cytokinin Response Factors, a family of transcription factors involved in response to a variety of environmental signals. Chapter 3 focuses on trans-Zeatin-N-glucosides, revealing that this subclass of CKNGs are capable of delaying leaf senescence and altering gene expression. Chapter 4 focuses on another subclass of CKNGs, isopentenyladenine-N-glucosides, revealing one of these compounds is capable of delaying leaf senescence and mildly altering gene expression in an overlapping manner with isopentenyladenine, a highly active form of CK. Chapter 5 explores potential mechanisms by which trans-Zeatin-N-glucosides delay leaf senescence and suggests they are capable of regulating levels of an active CK, trans-Zeatin. Chapter 6 combines CK measurement data from twelve studies, revealing CKNGs are the most abundant forms of CK in young Arabidopsis thaliana plants. 2 Acknowledgments The process of completing a PhD is an arduous one. There are many people I would like to thank for their personal, professional, and technical assistance. I would first like to thank my Advisor, Dr. Aaron Rashotte, for his guidance, mentorship, and kindness during my undergraduate and graduate work. I must also thank my committee members, Dr. Haruka Wada, Dr. Les Goertzen, and Dr. Sang-Wook Park, for their helpful feedback and assistance over the last few years. I would not have considered pursuing a PhD if it were not for Dr. Robert Locy, who taught me Genomic Biology and gave me my first lessons on the command line. Dr. Paul Zwack and Dr. Erika Keshishian were also advocates of pursuing a graduate degree. I am grateful for their friendship and mentorship. Dr. Chloe Josefson and Rachel Powell, MS, have been great friends and listening ears throughout this journey. I appreciate the efforts of these individuals for getting me into graduate school in the first place and for helping me navigate academia. I could not have completed all of these experiments without some help- though there are many undergraduate researchers who served as incredibly helpful hands, I would like to specifically thank Lesley Turner, Lauren Collins, and Kaki Oberman. Though not an official committee member of mine, I am grateful for the mentorship and guidance of Dr. Courtney Leisner. She has been a great source of advice in the two years since she moved into our “lab neighborhood,” and her presence was especially appreciated during Aaron’s sabbatical in Czech Republic. When I first told my parents that I was thinking of going to grad school, my dad asked how I planned on paying for it. I am grateful that both my mother and father were strongly supportive of me embarking into the odd world of academia, which was foreign to them both. My father worked exceptionally hard to support me and help me earn my Bachelor’s degree, and my mother’s emotional support did not go unnoticed. I am proud to be the first Dr. Hallmark. But based on what I see in some of my nieces and nephews, I won’t be the only doctor in the family for long. I am also incredibly grateful for the support of my sister Sallie Hallmark, one of the most well-composed and successful people I know, whose career advice served me well. Lastly, I cannot begin to express my gratitude for my partner through this and all endeavors, Anthony Hamilton. Your strength and courage through all you faced kept me strong and courageous. I will never be able to surpass your hard-working nature, but you make me work harder, and you taught me to do what I love and to do it well. Thank you for letting me complain and ramble on. Thank you for keeping me together through everything. Thank you for putting your life plans on hold so we could stay together while I finished what I came here for. I dedicate this Dissertation in memory of two family members. First, this is for my paternal grandfather and namesake, Howard Hallmark, who had a grade school education, yet worked his way out of poverty into owning and operating a successful company still in business today. He passed away months before I was born, but I know he would probably be the proudest of anyone over this achievement. Second, for my sister-in-law, Allyson Hamilton, whose sudden passing served as a reminder that life really is too short and that we must cherish those around us. 3 Table of Contents Abstract ......................................................................................................................................... 2 Acknowledgments......................................................................................................................... 3 List of Tables ................................................................................................................................ 5 List of Figures ............................................................................................................................... 6 Chapter 1. Introduction ................................................................................................................ 8 Chapter 2. Cytokinin Response Factors: Responding to more than cytokinin .......................... 46 Chapter 3. trans-Zeatin-N-glucosides have biological activity in Arabidopsis thaliana ........... 68 Chapter 4. Cytokinin isopentenyladenine and its glucoside isopentenyladenine-9-glucoside delay leaf senescence through activation of cytokinin-associated genes .............................. 113 Chapter 5. trans-Zeatin-N-glucoside-delayed senescence involves regulation of trans-Zeatin levels ............................................................................................................................. 152 Chapter 6. Meta-analysis of cytokinin measurements in Arabidopsis thaliana ....................... 170 Chapters 2 and 3 have been previously published, and citation information is listed on each title page. Chapter 4 is in peer review at Plant Direct as of June 29, 2020. 4 List of Tables Table 1 (Chapter 2, Table 1) ....................................................................................................... 67 Table 2 (Chapter 3, Table 1) ....................................................................................................... 79 Table 3 (Chapter 3, Table 2) ....................................................................................................... 82 Table 4 (Chapter 3, Supplemental Table 1) .............................................................................. 110 Table 5 (Chapter 3, Supplemental Table 2) .............................................................................. 111 Table 6 (Chapter 3, Supplemental Table 3) .............................................................................. 112 Table 7 (Chapter 4, Table 1) ..................................................................................................... 141 Table 8 (Chapter 6, Table 1) ..................................................................................................... 181 Table 9 (Chapter 6, Table 2) ..................................................................................................... 183 5 List of Figures Figure 1 (Chapter 1, Figure 1) .................................................................................................... 15 Figure 2 (Chapter 1, Figure 2) .................................................................................................... 23 Figure 3 (Chapter 1, Figure 3) .................................................................................................... 29 Figure 4 (Chapter 2, Figure 1) .................................................................................................... 66 Figure 5 (Chapter 3, Figure 1) .................................................................................................. 101 Figure 6 (Chapter 3, Figure 2) .................................................................................................

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