Crassulacean Acid Metabolism (CAM) Ppc Promoter and Transcription Factor Analysis in Kalanchoe Species

Crassulacean acid metabolism (CAM) decreases daytime transpirational water loss and improves water-use efficiency by shifting part or all atmospheric CO₂ fixation to the nighttime. This temporal shift in atmospheric gas exchange is presumably controlled by a complex transcriptional network orchestrated by the core circadian clock. To understand the transcriptional regulation of CAM-specific genes under circadian clock control, yeast-one-hybrid (Y1H) experiments were performed with target putative cis-regulatory elements within the promoter region of the phosphenolpyruvate carboxylase (PEPC) gene (Ppc1) in Kalanchoe laxiflora. PEPC is a key CAM enzyme that catalyzes the primary carboxylation reaction resulting in the nocturnal formation of C₄ acids. Ppc mRNA exhibits an evening-phased pattern of transcriptional expression. By mapping RNA-seq data from K. laxiflora, CAM Ppc orthologs were characterized from draft genome sequence assemblies for both diploid Kalanchoe fedtschenkoi and tetraploid Kalanchoe laxiflora. Candidate cis-regulatory elements for evening-specific mRNA expression and mesophyll expression were used to design five bait sequences for Y1H screening. Nine candidate transcription factors that bound to the bait sequences were selected for more detailed evaluation and the identification of their cognate cis-elements by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and verification by Electrophoretic Mobility Shift Assays (EMSA). In parallel, deletion series of Ppc promoter/reporter genes were stably transformed into diploid K. laxiflora to dissect the in vivo function of cis-regulatory regions within the promoter. Such information will facilitate our understanding of the temporal mRNA expression patterns of key CAM genes that underpin the function of CAM over the diel cycle.