University of Nevada, Reno Crassulacean acid metabolism in tropical orchids: integrating phylogenetic, ecophysiological and molecular genetic approaches A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry and Molecular Biology by Katia I. Silvera Dr. John C. Cushman/ Dissertation Advisor May 2010 THE GRADUATE SCHOOL We recommend that the dissertation prepared under our supervision by KATIA I. SILVERA entitled Crassulacean Acid Metabolism In Tropical Orchids: Integrating Phylogenetic, Ecophysiological And Molecular Genetic Approaches be accepted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY John C. Cushman, Ph.D., Advisor Jeffrey F. Harper, Ph.D., Committee Member Robert S. Nowak, Ph.D., Committee Member David K.Shintani, Ph.D., Committee Member David W. Zeh, Ph.D., Graduate School Representative Marsha H. Read, Ph. D., Associate Dean, Graduate School May, 2010 i ABSTRACT Crassulacean Acid Metabolism (CAM) is a water-conserving mode of photosynthesis present in approximately 7% of vascular plant species worldwide. CAM photosynthesis minimizes water loss by limiting CO2 uptake from the atmosphere at night, improving the ability to acquire carbon in water and CO2-limited environments. In neotropical orchids, the CAM pathway can be found in up to 50% of species. To better understand the role of CAM in species radiations and the molecular mechanisms of CAM evolution in orchids, we performed carbon stable isotopic composition of leaf samples from 1,102 species native to Panama and Costa Rica, and character state reconstruction and phylogenetic trait analysis of CAM and epiphytism. When ancestral state reconstruction of CAM is overlain onto a phylogeny of orchids, the distribution of photosynthetic pathways shows that C3 photosynthesis is the ancestral state and that CAM has evolved independently several times within the Orchidaceae. Using phylogenetic trait analysis, we found that divergences in photosynthetic pathway and epiphytism are consistently correlated through evolutionary time and are related to the prevalence of CAM epiphytes in lower elevations and abundant species diversification of high elevation epiphytes. The multiple independent evolutionary origins of CAM in orchids suggest that evolution from C3 to weak and strong CAM might involve relatively few genetic changes. In plants performing CAM, phosphoenolpyruvate carboxylase (PEPC) catalyzes the initial fixation of atmospheric CO2 into C4-dicarboxylic acids forming oxaloacetate and inorganic phosphate as a product. PEPC is a ubiquitous enzyme that belongs to a ii multigene family with each gene encoding a function- and tissue-specific isoform of the enzyme. CAM-specific PEPC isoforms might have evolved from ancestral non- photosynthetic C3 isoforms by gene duplication and acquired transcriptional control sequences that mediate increased mRNA expression and leaf-specific or leaf-preferential expression patterns. In order to understand patterns of PEPC family diversification over evolutionary times, PEPC genes families in ten closely-related orchid species from the Subtribe Oncidiinae with a range of photosynthetic pathways from C3-photosynthesis (Oncidium maduroi, Ticoglossum krameri, and Oncidium sotoanum) to weak CAM (Oncidium panamense, Oncidium sphacelatum, Gomesa flexuosa and Rossioglossum insleayi) to strong CAM (Rossioglossum ampliatum, Trichocentrum nanum, and Trichocentrum carthaginense) were characterized. At least three major changes are hypothesized to have occurred during evolution to adapt the CAM progenitor genes for function in CAM plants: 1) CAM isoform genes in orchids have evolved highly expressed mRNA expression patterns; 2) leaf preferential (or specific) expression patterns; and 3) circadian clock control expression patterns. We found that up to five PEPC isoforms are present in orchids, with one putative CAM-specific PEPC isogene with discrete amino acid changes identified in CAM species based on cDNA clone sampling, and an evident shift in PEPC isoform number from 2-3 isoforms in C3 species, to 3-4 isoforms in weak CAM species, to 4-5 isoforms in strong CAM species. Validation of the isotopic analysis and the molecular genetic analysis of PEPC gene family using 24- hour gas exchange showed that weak CAM species exhibit limited amounts of nocturnal CO2 uptake and fixation when compared to strong CAM species. iii To understand the molecular mechanisms responsible for the recruitment of CAM-specific genes, 454 sequencing of cDNA prepared from RNA of the strong CAM species Rossioglossum ampliatum was conducted, and resulted in 189 Mb of DNA sequence, 41,115 contigs, and 100,889 singletons. A NimbleGen microarray constructed and used in a C3 species (Oncidium maduroi), a weak CAM species (Oncidium panamense) and a strong CAM species (Rossioglossum ampliatum), showed that C3 and weak CAM species had average hybridization intensities that diverged from the strong CAM species by 2 and 3 percent, respectively. From 13,566 genes that showed a significant 4.6-fold difference in expression levels from the comparisons between CAM, C3 and weak CAM, 4,520 genes showed a greater than 4.6-fold increase in the ratio of CAM/C3 relative transcript abundance, whereas 3,745 genes showed a greater than 4.6- fold decrease in the ratio of CAM/C3 relative transcript abundance. A maximal increase or decrease in relative transcript abundance of more than 1,000- and 500-fold, respectively, was observed. The results of the microarray analysis will serve as a catalogue of gene expression patterns available for future work aimed at understanding CAM specific expression patterns, and can be used to further understand gene regulation by in-depth analysis of the transcriptional control regions responsible for altered gene expression patterns associated with CAM evolution. Several patterns of CAM evolution have been demonstrated in orchids, thus improving our understanding of the functional significance and evolutionary origins of CAM. The results of this project will aid in understanding photosynthetic plasticity in plants. iv Dedication I dedicate this dissertation to my parents Flor Maria M. de Silvera and Gaspar A. Silvera. They taught me the love for orchids, the patience to cultivate them, and the wisdom to understand them. Beyond all, they gave me the confidence and encouragement to always follow my dreams. I am who I am because of them. v ACKNOWLEDGMENTS I would like to thank my advisor John Cushman for his invaluable guidance throughout these five years. John has been an extraordinary mentor; he gave me the freedom to explore my own ideas, and supported me especially when I was doing research in Riverside and Panama. He always cared about my development as a scientist, he helped me write proposals for funding and he took the time to teach me new techniques. I will always be grateful for your patience, encouragement, and the care you put into reading and editing my writing. I am a much better scientist because of you! I thank the members of my dissertation committee Jeff Harper, Bob Nowak, Dave Shintani and David Zeh for all their helpful advice. They were always willing to meet with me to chat about my project and take the time to work with me on developing new ideas, especially during my qualifying exam. Thank you Klaus Winter for putting me in touch with John Cushman, and for introducing me to the world of CAM. You have been an amazing mentor throughout these years, and have always provided support and guidance while working at STRI in Panama. You have helped me maintain confidence in my research and have always provided me with valuable career advice. Working in your lab is inspiring. I thank the members of the Cushman lab. I have learned so much from each of you! Thank you Becky Albion, you have been an amazing friend and colleague, you are always happy to discuss ideas with me, teach me techniques, and take care of the orchids! You are a wonderful person to have in the lab! Thank you Letty Rodriguez, for working with me in the project and for sharing my passion for orchids. Thank you for always vi being positive and optimistic, even when research was difficult. Thank you JR Tillett for introducing me to so many fun places and people in Reno, and for exchanging music with me. Thank you Patricia Berninsone for opening your home to me and treating me like family! Thanks Leyla and Upul for your friendship. Thanks Miguel Rodriguez for training me when I first arrived to the lab. Thanks Abou, Bahay, Jill, Gadi, Gouthu, Jerome, Lina, Matt, Mark, Mustafa, Sage, Sabine, Sangho, and Tatiana for being such wonderful labmates and friends. Thank you to the supportive staff at UNR, Marianne Davis, Ron Robards, Becky Hess, and Jessica Corey for taking care of my fellowship paperwork every semester. Thanks Tim and Corene for your friendship and for helping me keep things in perspective. Thanks to Tim Close and Ray Fenton for welcoming me in their lab in Riverside. Thank you Mark Whitten for always providing good advice, and for continuing collaborating in orchid projects with me. Thank you Kent Perkins, Kurt Neubig, Norris Williams, Bruce Holst, Jim Solomon, Mireya Correa, and Carmen Galdames for access and assistance with herbarium collections. Thank you Orquideas Tropicales for donating orchids for the project. Thank you Todd Dawson and Stefania Mambelli for isotopic analysis. Thank you Orelis Arosemena for taking care of permits. Special thank you to my parents Flor Maria and Gaspar for your unconditional love and support. To my brothers, and all my family and friends in Panama, who have always cared for me, and taken pride in all my achievements. A very special thank you to my amazing husband Lou Santiago, you are wonderful! Not only are you my husband and best friend, you are also my collaborator! Life with you is exciting and always an adventure. I would have not been able to complete this PhD without you, thank you for your encouragement, unconditional love and support.