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Text of My Dissertation Abstract COKER, JEFFREY SCOTT. The systemic response to fire damage in tomato plants: A case study in the development of methods for gene expression analysis using sequence data. (Under the direction of Dr. Eric Davies.) Fire is a natural component of most terrestrial ecosystems and can act as a local wound stimulus to plants. The ultimate goal of this work was to characterize the array of transcripts which systemically accumulate in plants after fire damage. Before this could be accomplished, substantial development of methods for gene expression analysis using sequence data was necessary. This involved developing methods for identifying contamination in DNA sequence data (Chapter 2), identifying over 78,000 false sequences in GenBank and several thousand more in the indica rice genome (Chapter 2), developing a novel method for identifying housekeeping controls using sequence data (Chapter 3), performing relative expression analyses for 127 potential housekeeping control transcripts (Chapter 3), and characterizing 23 transcripts which encode all 13 subunits of vacuolar H+- ATPases in tomato plants (Chapter 4). A subtractive cDNA library served as a starting point to identify and characterize 9 novel tomato transcripts systemically up-regulated in leaves in the first hour after a distant leaf is flame wounded (Chapters 5). Real-time RT-PCR using leaf RNA isolated at different times after flaming showed that the most common pattern of transcript accumulation was an increase within 30 to 60 minutes, followed by a return to basal levels within 3 hours. Expression analyses also showed that most up-regulated transcripts were already present in unwounded tissues. A total of 46 different transcripts were identified from the subtractive cDNA library (Chapters 6). Compared with the entire tomato transcriptome, these 46 transcripts are very highly conserved in plants. The vast majority fell into 5 classes: enzymes of general metabolism; protein synthesis, modification, and transport; transcription; membrane transport; and photosynthesis and respiration. At least half of the transcripts have been previously associated with wounding or stress, suggesting that the systemic response to fire damage has components similar to those of other wound and stress responses. On the other hand, 30% of transcripts were associated with photosynthesis and respiration, suggesting that part of the response to fire damage is notably different from other wound and stress responses. Conclusions and future directions are included in Chapter 7. THE SYSTEMIC RESPONSE TO FIRE DAMAGE IN TOMATO PLANTS: A CASE STUDY IN THE DEVELOPMENT OF METHODS FOR GENE EXPRESSION ANALYSIS USING SEQUENCE DATA by JEFFREY SCOTT COKER A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy DEPARTMENT OF BOTANY Raleigh 2004 APPROVED BY: ___________________________ ____________________________ Dr. Judy Thomas Dr. Jack Wheatley Advisory committee member Advisory committee member ___________________________ ____________________________ Dr. Dominique Robertson Dr. Chris Brown Advisory committee member Advisory committee member ___________________________ Dr. Eric Davies Chair of Advisory Committee Dedication The dissertation of Jeffrey S. Coker, which completes the Degree of Doctor of Philosophy, is dedicated to the educators of Plymouth, North Carolina. Leafie Bryant Carolyn Watlington Julia Towe Joyce Hardison Rita Rhodes Donna Whitfield Frances Callander A. Willingham Ann Bland Robert Moore Doris Downing Kevin Cutler Ruth Pharr Leroy Bland Beth Thompson Kathy Stanfield Shirley Thomas Kerry Koeppl Sally Woolard Donald Rote Glenda Smith Pam Benson Bea Waters Becky Brown Judy Wynn Robert Cody Ms. Wilkins Alma Phifer Senya Norman Frances Jones Roxanna Brown Marian Floyd Judy Bragg Ed Clark Mary Kay Bradshaw Geraldine Rodgers Janet Swain Charlene Evans Joyce Hardison Susan Owens Dianne Staten Hector Palacios Donald Hassell Susie Jakeman Louis Spencer Victor Davis Marty Alligood Patrick Parr Mr. and Mrs. Sermons Michelle Stewart Julius Walker Robert Cody ii Biography Jeffrey Scott Coker was born the son of Jerry and Debra Coker in the small town of Plymouth, North Carolina. His interest in plants is probably due to a family of gardeners, pulp and paper engineers, and wood-workers, as well as a community where farms, forests, ball fields, and swamps are plentiful. Jeffrey attended Davidson College, where he studied biology and ancient Greek and Roman civilizations, and played baseball. After graduation, he worked for one year at the Helen Paesler School in Raleigh, NC, teaching high school biology, chemistry, and calculus, as well as middle school science/math. It was during this year that he found a passion for teaching science and decided to pursue it at the college level. Jeffrey entered graduate school at N.C. State University in 1999 as an RA/TA in the Botany Department, where he taught laboratories in Botany and Biotechnology, and co- taught a new Whole Plant Physiology course. He earned a M.Ed. in Science Education in the spring of 2001, and formally became a Ph.D. student in Botany (under Dr. Eric Davies) shortly thereafter. He has been recognized for his teaching at N.C. State by receiving the CALS Outstanding Teaching Assistant Award, the Martha Sue Sebastian Memorial Award for Excellence in Teaching, a GSA Outstanding Teaching Award, an Alcoa Teaching Fellowship, and a NACTA Graduate Student Teaching Award. Student researchers under his supervision have been recognized locally and nationally for their work. While in Raleigh, Jeffrey met a wonderful girl named Beth, and they were married on December 20, 2003, in Greenville, N.C. Beginning in August of 2004, Jeffrey will be an Assistant Professor in the Biology Department at Elon University. He looks forward to a successful career in teaching and research, and to spending many happy years with Beth. iii Acknowledgements There are many people who have supported me over the last five years in various capacities. My committee members have been extremely supportive, and for that I am most grateful. Dr. Eric Davies has been an outstanding research advisor in every sense. His openness to new ideas, support of my work, willingness to integrate scientific and educational pursuits, careful review of manuscripts, daily friendliness, and general guidance have all been invaluable. Perhaps the most distinct impression Eric has left on me is the amount of effort he spends helping to advance the lives and careers of his students and colleagues. I cannot think of a more admirable quality. We have had many conversations about how many students do not fully appreciate a teacher or mentor until years later. Let me assure you that I am fully aware of what an outstanding advisor I have had. Dr. Judy Thomas has been an excellent mentor and friend, and was an instrumental part of my success in graduate school. She believed in me when others were skeptical, and set me on the right path more times than I can count. Dr. Chris Brown has been a role model for me in terms of professionalism, teaching, and the leadership of research and teaching collaborations. He introduced me to concepts of Space Biology which changed the way I look at my own discipline. I credit Dr. Niki Robertson with shaping my earliest thoughts about biotechnology, and value her thoughts very highly. Her enthusiastic and insightful approaches to science and life are contagious among her students. Dr. Jack Wheatley’s presence on my committee is especially meaningful because he represents good teaching and educational scholarship. I am thankful for his guidance, patience, and insightful reviews of my teaching. iv A number of people worked alongside me in the laboratory, and provided daily assistance for which I am thankful. Dr. Raul Salinas was especially helpful and patient. Most of my “co-workers” were high school and undergraduate student researchers who always made the lab a more enjoyable place. In particular, I am thankful to have worked with Derek Jones regarding vacuolar ATPases and enjoyed both his enthusiasm and friendship. Other student researchers included Katie Grant, Jessica Staley, Holly Cline, Ryan Parks, Ashwynn Stanger, John Pollard, and Turqouise Ross. Dr. Gerald Van Dyke has been an invaluable teaching mentor and friend. His excitement about teaching and commitment to students have inspired me to seek excellence in the classroom. My time at N.C. State would not have been the same without the friendship and conversation of Dr. Isaac Bruck. I am also thankful for the Botany administrative staff, especially Sue Vitello and Vicki Lemaster, who dealt with many issues on my behalf. I am blessed with a loving family which has provided support in many forms. Mom, Dad, Grandmother, Chris, Eric, Laura, Sheila, Mike, Josh, and Debbie have all played important roles in my life. On at least two occasions, family members (Eric and Mom) helped me to overcome significant research difficulties. Finally, I could not dream of having a more supportive wife. Beth has been at my side through virtually every step of my dissertation research. She has assisted me in the field, in the laboratory, and in the classroom. She has read my papers, inspected tables and figures, listened to whole lectures just so I could practice and, perhaps most importantly, encouraged me to work long hours when deadlines approached or I became really excited about something (which happens frequently). She
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