Evolutionary Mechanisms and Metabolic Dynamics Required for Crassulacean Acid Metabolism
AuthorGulle Bilgi, Bahay
AdvisorCushman, John C.
Biochemistry and Molecular Biology
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Crassulacean acid metabolism (CAM) is an important photosynthetic adaptation to limited water availability that has evolved independently multiple times in 35 families, including more than 6% of flowering plant species. CAM plants are characterized by nocturnal CO2 fixation, which results in 3- to 6-fold improvement in water use efficiency relative to C4 and C3 species, respectively. CAM predominates among succulent species that are present in arid (e.g., deserts) and semi-arid regions with seasonal or intermittent water availability (e.g., tropical epiphytic habitats). Although the basic biochemistry and physiology of CAM is well characterized, there has been a lack of evidence corroborating the molecular mechanisms responsible for CAM evolution and regulation. In addition, changes in the abundances of key metabolites associated with CAM and the effects of these metabolites on circadian clock regulation need to be investigated to fill the remaining gaps in this developing field. To better understand the molecular mechanism of CAM evolution in orchids and to test the hypothesis that CAM-performing species display greater mRNA expression of CAM-specific isoforms, the glucose-6-phosphate/Pi translocator (GPT) gene family was characterized in C3 photosynthesis-, weak CAM, and strong CAM-performing Oncidiinae species. Findings from this study reveal that CAM evolved from the ancestral C3 photosynthesis by the selective recruitment of Gpt isogenes with circadian clock-controlled and highly increased mRNA expression patterns. The carbonic anhydrase (CA) gene family that supplies bicarbonate for phosphoenolpyruvate carboxylase (PEPC) was also sampled to provide information about the molecular evolution of CAM. The results from this study also suggest that gene duplication events followed by neofunctionalization took place, and that discrete Cah isogenes were selectively recruited to fulfill the CAM-related C4 gene functions. Non-biased, global metabolomics profiling technology, based on GC/MS (Gas Chromatography/Mass Spectrometry) and UHPLC/MS/MS2 (UltraHigh Performance Liquid Chromatography/tandem Mass Spectrometry) platforms, was conducted in order to identify the circadian clock control of metabolic abundances in both wild type and CAM-deficient mutant Mesembryanthemum crystallinum plants. The results presented here show that many compounds in both wild type and CAM-deficient mutant plants exhibit a circadian pattern of expression that is invoked following water-deficit stress. Novel observations from this study include peak accumulation of fumarate at dawn with a circadian pattern. This result validates the metabolic flux through mitochondria in tricarboxylic acid (TCA) cycle components. Also, increased accumulations of soluble sugars, sugar alcohols, nitrogen-rich compounds, and lipids were observed in the CAM-deficient mutant under water-deficit stress conditions. These results suggest that CAM-deficient mutant plants are redirecting excess carbon into alternative pathways to compensate their loss in starch biosynthesis. Accumulations of plant hormone Abscisate (ABA) and antioxidant compounds such as alpha-tocopherol, ascorbate, and glutathione were detected as stress-related responses. The information gained from these studies improved our understanding of the mechanisms responsible for evolutionary origins and circadian regulation of CAM.