EVOLUTIONARY PHYSIOLOGY AND TRANSCRIPTOMICS OF CRASSULACEAN ACID METABOLISM (CAM) IN THE AGAVOIDEAE (ASPARAGACEAE) by CAROLINE HEYDUK (Under the Direction of Jim Leebens-Mack) ABSTRACT Crassulacean acid metabolism (CAM) is a mode of photosynthesis found in ~6% of flowering plants and serves as an adaptation to water-limited habitats. CAM plants open their stomata for gas exchange at night, when transpiration rates are lower, and fix CO2 via an alternative pathway. Carbon is stored as organic acids during the night, then decarboxylated during the day behind closed stomata. CAM results in high levels of CO2 around RuBisCO, the primary carbon-fixing enzyme in all green plants, with minimal water loss. Although CAM occurs in at least 35 separate lineages, its evolutionary trajectory from C3 is unknown. Here we explore the evolutionary patterns of CAM across the Agavoideae, a subfamily of species that includes Agave and Yucca. Anatomical observations paired with character evolution show that species of the Agavoideae may have been preadapted to the CAM syndrome, with many C3 species showing CAM-like morphology. Comparative physiology was explored in more detail in a Yucca hybrid system, where a CAM and C3 species hybridized to form a C3-CAM intermediate. The parents and hybrid offspring were characterized for anatomical and physiological traits and show the hybrid is able to convert from C3 carbon fixation to 100% CAM uptake under periods of drought stress. Finally, the hybrid system in Yucca was used to understand the transcriptional regulation of the CAM pathway; despite lacking any CAM anatomy or physiology, the C3 parental species shows similar gene expression patterns as the CAM species, indicating perhaps an ancestral gene expression pattern that enabled the evolution of CAM in a subset of Yucca species. INDEX WORDS: Crassulacean acid metabolism, physiology, phylogenomics, transcriptomics, Yucca, hybrid EVOLUTIONARY PHYSIOLOGY AND TRANSCRIPTOMICS OF CRASSULACEAN ACID METABOLISM (CAM) IN THE AGAVOIDEAE (ASPARAGACEAE) by CAROLINE HEYDUK BS, University of Wisconsin, 2011 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 2015 © 2015 Caroline Heyduk All Rights Reserved EVOLUTIONARY PHYSIOLOGY AND TRANSCRIPTOMICS OF CRASSULACEAN ACID METABOLISM (CAM) IN THE AGAVOIDEAE (ASPARAGACEAE) by CAROLINE HEYDUK Major Professor: Jim Leebens-Mack Committee: John Burke Katrien Devos Lisa Donovan Chung-Jui Tsai Electronic Version Approved: Suzanne Barbour Dean of the Graduate School The University of Georgia December 2015 ACKNOWLEDGEMENTS Live tissue sampling of Yuccas was assisted by the park rangers that work for the Florida State Parks, South Carolina State Parks, North Carolina State Parks, and the National Park Service. Darren Loomis from the Virginia Park Service went above and beyond to help me find Y. gloriosa samples. Joni Ward at the Desert Botanical Garden assisted with sampling (and re- sampling) their living collection. Additionally, Olle Pellmyr and Jim Leebens-Mack‟s Yucca samples were enormously helpful. For isotopic collections, I thank the Missouri Botanical Garden Herbarium (and Michael McKain), the Desert Botanical Garden Herbarium (Wendy Hodgson), and the Georgia Herbarium (Wendy Zomelefer). Saravanaraj Ayyampalayam assisted with bioinformatics. I had a number of generous funding sources, including the Society for the Study of Evolution, the Society for Comparative and Integrative Biology, the American Society of Plant Taxonomists, the Botanical Society of America, the University of Georgia Graduate School, and NSF. Finally, without the endless support Jim Leebens-Mack, my committee members, and my husband Edward McAssey, none of this work would have been possible. iv TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ........................................................................................................... iv LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix CHAPTER I INTRODUCTION AND LITERATURE REVIEW .....................................................1 The problem with RuBisCO ....................................................................................1 Crassulacean acid metabolism .................................................................................2 Physiology of CAM plants.......................................................................................5 Genetics of CAM plants...........................................................................................6 Study system ............................................................................................................7 References ................................................................................................................9 II EVOLUTION OF CRASSULACEAN ACID METABOLISM IN THE AGAVOIDEAE (ASPARAGACEAE) .........................................................................................15 Abstract ..................................................................................................................16 Introduction ............................................................................................................17 Methods..................................................................................................................20 Results ....................................................................................................................26 Discussion ..............................................................................................................30 References ..............................................................................................................36 v III GAS EXCHANGE AND LEAF ANATOMY OF A C3-CAM HYBRID, YUCCA GLORIOSA (ASPARAGACEAE) .................................................................................................52 Abstract ..................................................................................................................53 Introduction ............................................................................................................54 Methods..................................................................................................................58 Results ....................................................................................................................64 Discussion ..............................................................................................................67 References ..............................................................................................................74 IV TIME-COURSE COMPARATIVE TRANSCRIPTOMICS BETWEEN C3, CAM, AND INTERMEDIATE YUCCA SPECIES ..................................................................................86 Abstract ..................................................................................................................87 Introduction ............................................................................................................88 Methods..................................................................................................................91 Results ....................................................................................................................96 Discussion ............................................................................................................101 References ............................................................................................................106 V CONCLUSION AND DISCUSSION .......................................................................124 CAM in the Agavoideae ......................................................................................124 The CAM continuum ...........................................................................................125 Remaining questions ............................................................................................126 References ............................................................................................................127 APPENDICES A SUPPLEMENTARY FIGURES AND TABLES CH. II ...........................................129 vi B SUPPLEMENTARY FIGURES AND TABLES CH. III .........................................133 C SUPPLEMENTARY FIGURES AND TABLES CH. IV .........................................144 vii LIST OF TABLES Page Table 2.1: Source information for both DNA tissue and for tissue analyzed for δ13C ratios ........42 Table 2.2: Average values (and standard errors) for cross sectional anatomical traits in the Agavoideae ................................................................................................................................45 Table 3.1: Nighttime acid accumulation (DeltaH+, µequivalents g-1) derived from difference in dusk and dawn titratable acidity measurements .............................................................................78 Table 3.2: Mean and standard error for traits in the three species and significance of pairwise comparisons ................................................................................................................................79 Table 4.1: Genotypes of each species used for RNA sequencing ................................................110