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Characterizing the transcriptional regulation of crassulacean acid metabolism in Kalanchoe
AuthorGarcia, Travis Michael
AdvisorCushman, John C
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Due to the agricultural challenges posed by the prospect of a hotter drier climate understanding the molecular basis of plant water-use efficiency is of increasing importance. Species performing crassulacean acid metabolism (CAM) photosynthesis have evolved to be naturally water-use efficient primarily through shifting their carbon uptake to night to minimize water-loss. Relative to C3 and C4 photosynthesis species, CAM plants are enriched for rhythmic circadian clock-dependent regulation of metabolic processes. However, the transcriptional regulation of CAM remains largely uncharacterized. Using Kalanchoe fedtschenkoi, in which CAM develops along a leaf developmental gradient, candidate transcription factors with possible CAM-related functions were identified. The mRNA abundance of these transcription factors increases upon the transition from C3 photosynthesis to CAM and they appear to exhibit a circadian phase-dependent pattern of regulation. To better characterize the transcriptional control circuits underlying CAM, three such of these transcription factors, KfNF-YB3, KfHomeodomain-like, and KfMYB59 were selected for chromatin immunoprecipitation-sequencing (ChIP-seq). However, these experiments failed to identify enriched target genomic loci possibly as a consequence of the unique challenges of adapting experimental protocols designed for model C3 photosynthesis plant species to a succulent plant such as Kalanchoe. Additionally, this work focuses on elucidating the cis-regulatory elements and the trans-acting factors governing the transcriptional control of the phosphoenolpyruvate carboxylase gene (Ppc1) in Kalanchoe. Despite this enzyme’s importance in catalyzing the primary nocturnal fixation of CO2 in CAM species, the complex regulatory mechanisms underlying its expression are not well-studied. We examined the Kalanchoe Ppc1 promoter and identified numerous cis-regulatory elements on the basis of their sequence conservation with known regulatory modules. These individual elements along with two-hundred base pair region segments of the Kalanchoe Ppc1 promoter were used at bait probes in yeast one-hybrid (Y1H) assays. From this analysis, several high-confidence interacting transcriptional regulators were identified including ERF9, ERF106, TCP4, and PIF1. In silico examination of the Ppc1 promoter revealed likely binding sites for these factors based on homology to validated preferred binding sequences in Arabidopsis. The specific transcription factors identified through this work can now serve as the basis for further experiments to confirm interaction with the Ppc1 promoter and elucidate the nature of their regulatory effects. Overall, the work presented in this dissertation attempts to investigate the transcriptional control of crassulacean acid metabolism using the developmental CAM model Kalanchoe.