POPULUS PHENYLPROPANOID METABOLISM ACROSS BIOLOGICAL SCALES: PHYLOGENOMICS, MOLECULAR PHYSIOLOGY, AND ECOSYSTEM MODELING by LINDSEY KAY TUOMINEN (Under the Direction of Chung-Jui Tsai) ABSTRACT Phenylpropanoid metabolism is a major contributor to plant biotic and abiotic stress responses while also influencing ecosystem-level processes such as nitrogen cycling. Populus in particular is characterized by its rich diversity of phenylpropanoids, varying both qualitatively and quantitatively within and across species. This dissertation develops a cross-scale view of Populus secondary metabolism to consider (1) the genetic basis of metabolic diversity within the genus, (2) influences of phenylpropanoid homeostasis on cellular metabolism, and (3) theoretical ecosystem-level effects of altered Populus phenylpropanoid levels. I first characterized the Populus BAHD acyltransferase family, due to its likely contributions to phenylpropanoid diversity in the taxon, using a phylogenomic approach. The one hundred putative full-length BAHD genes in Populus arose through genome-wide and local duplication events, and possibly through retrotransposition. Correlation of phylogenetic and gene expression data suggests that some recent duplicates have undergone functional divergence. To study the cellular-level effects of phenylpropanoid metabolism in Populus , I analyzed metabolite and gene expression profiles of cell cultures fed phenylpropanoid enzyme inhibitors and/or the elicitor methyl jasmonate. Results suggest that the effects of enzyme inhibitors manifest primarily at the metabolic level, in contrast to the transcriptionally-driven changes under methyl jasmonate elicitation. Links between core phenylpropanoid metabolism and phenylpropanoid derivatives, glycosylation, amino acid metabolism, and the Krebs cycle became evident under metabolic perturbation. To consider possible ecological effects of manipulating phenylpropanoid metabolism in Populus , I developed ecosystem models for probing indirect effects of Populus phenotypes exhibiting increased growth and reduced secondary metabolism. Such phenotypes would be consistent with metabolic engineering goals for trees to be used as biofuels. Initial simulations indicated shifts in biomass of ecosystem components not directly interacting with Populus and generated hypotheses for future field research. The results support the potential of ecological modeling as a research and decision-making tool prior to design and field release of novel tree genotypes. This research advances knowledge of Populus phenylpropanoid metabolism in a coordinated manner across biological scales and subdisciplines, that should be complementary to more traditional, single-discipline oriented research. INDEX WORDS: Populus , secondary metabolism, phenylpropanoids, phylogenomics, metabolic profiling, transcriptomics, plant cell culture, systems ecology, transgenic risk assessment, indirect effects, cross-scale approach. POPULUS PHENYLPROPANOID METABOLISM ACROSS BIOLOGICAL SCALES: PHYLOGENOMICS, MOLECULAR PHYSIOLOGY, AND ECOSYSTEM MODELING by LINDSEY KAY TUOMINEN B.A., Macalester College, 2001 M.S., University of Massachusetts Amherst, 2003 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2012 © 2012 Lindsey Kay Tuominen All Rights Reserved POPULUS PHENYLPROPANOID METABOLISM ACROSS BIOLOGICAL SCALES: PHYLOGENOMICS, MOLECULAR PHYSIOLOGY, AND ECOSYSTEM MODELING by LINDSEY KAY TUOMINEN Major Professor: Chung-Jui Tsai Committee: Jeffrey F.D. Dean Robert O. Teskey James H. Leebens-Mack Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2012 DEDICATION For my parents, who raised me to see that the natural world matters, have allowed me the freedom to choose my own way, and still help me dust myself off every time I realize I’ve dug myself into a hole. For the Blasiaks, who provided me with more encouragement, inspiration, and advice than any doctoral student should ever require, particularly given the circumstances under which I asked for them. Mary will be greatly missed, and her words will not be forgotten. For Gwen, who stuck with me tenaciously from the snowiest winters to the stickiest summers. I know you made sacrifices for my ambition. I still hope to help your own dreams become real in some small way, even if my ability to do so is more limited than we imagined. Last, but not least, for the plants, from whom I have taken much and to whom I have given precious little, except perhaps some alternative pronouns in the places where I can indulge such extravagance. Our species is forever beholden to their kingdom. iv ACKNOWLEDGEMENTS I owe a particular debt of gratitude to my advisor, Dr. Chung-Jui Tsai, who invited me to join her laboratory nine months earlier than I had originally planned to start my doctoral studies. She has been an incredible teacher, providing regular and reliable opportunities to check in, give me feedback, and allow time for questions. She has displayed infinite patience when faced with my own diverse and wandering interests, and she held me back at just the right moment when I was about to walk off a metaphorical cliff, planning to defend a dissertation two-thirds finished. Her generous financial support, supplemented by my own efforts to secure assistantship and travel funding through the Graduate School, helped to sustain my home life through the worst economic downturn in my lifetime. My dissertation committee members, Drs. Leebens-Mack, Dean, and Teskey have each provided helpful research advice along the way. Dr. Leebens-Mack’s comments on an earlier draft of the work presented in Chapter 2 were extremely beneficial and gave me some much- needed confidence that the work as it stood was reasonably competent from the perspective of a molecular phylogeneticist. Dr. Teskey acted as a Capstone Reader for the version of Chapter 4 originally turned in for the Environmental Ethics Graduate Certificate Program, and he has provided me with a wealth of advice for future work to grow that seed into something sturdy enough to withstand the winds of peer review. Dr. Dean provided an array of ideas for new experiments to build on the work in Chapter 3, many of which I failed to fully appreciate until months afterwards. He can rest assured that I’ll be tapping his shoulder for more detailed v editorial advice as the work presented in Chapter 3 is advanced to peer-reviewed publication in the near future. Dr. Scott Harding, the Senior Research Scientist and Co-PI in the Tsai lab, has greatly assisted me by applying his depth of technical expertise and metabolic knowledge both in the laboratory and in the form of substantial editorial advice on the work presented in Chapter 3. Although not officially a member of my advisory committee, he has easily provided me enough help to qualify as an honorary member. I have also benefitted from the advice, collaboration, and expertise of our Assistant Research Scientist, Dr. Chris Frost, and of our postdoctoral researchers, most prominently Drs. Hong-Qiang (Horace) Wang, Batbayar (Baggi) Nyamdari, Ben Babst, Keming Luo, and Yinan Yuan. Our lab managers Hwee Chi (Kate) Tay and Vanessa Michelizzi have been impressively helpful in their detailed knowledge of the logistics of the Tsai lab, and their assistance in scheduling and training undergraduate student workers – along with the direct assistance of the undergraduates themselves -- greatly enhanced my workflow. The Tsai lab computational ninja Ed Johnson has provided cheerful assistance in a wide range of scientific and technical areas of my work relating to bioinformatics and computation, particularly in Chapter 2. Finally, my fellow graduate students have always helped me keep moving when the work was difficult. I particularly thank Raja Payyavula for laying the experimental groundwork for the analyses I carried out in Chapter 3 and for assisting with some early RNAi construct development, Mark Wilson for his ongoing work developing the lab’s custom metabolic profiling database and heroic attempts to translate chemical and biological technical needs into a computational framework, Lei (Eric) Du for applying the SLIM analysis to the microarray data in Chapter 3, Han-Yi Chen for providing refresher training in HPLC-MS and vi introducing me to the peculiarities of data extraction from the Tsai lab instrument, and Michael Bordeaux in the Dean lab for a healthy sense of perspective whenever I was mired in frustration. In a “second life” I have lived outside of the Tsai lab, Dr. Bernie Patten introduced me to the fascinating world of systems ecology. He has been an additional source of inspiration to me throughout my time at UGA, finally confirming for me a suspicion I have long held about the importance of mathematics for biology as a whole and for ecology in particular. Dr. Patten was a Capstone Reader for the work presented in Chapter 4, but his contributions to my thinking in Chapter 3 should not be underestimated. My understanding of metabolic pathways was deepened substantially by my interactions with him and the rest of the faculty, postdoctoral researchers, and graduate students in the Systems Ecology and Engineering group. My doctoral work actually started well over a year before my arrival at UGA, so I also wish to acknowledge the help, inspiration, and advice I received from my committee members at Michigan Technological University prior to