Analysis of cis-Regulatory Elements of Crassulacean Acid Metabolism (CAM) Genes in Kalanchoe laxiflora

Crassulacean acid metabolism (CAM) reduces 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, which is comprised of a network of transcriptional and posttranscriptional interactions that result in oscillating expression patterns of output genes. To begin to understand the transcriptional regulation of CAM genes under circadian clock control, yeast-one-hybrid (Y1H) experiments are underway that target putative cis-regulatory elements in the promoter region of phosphenolpyruvate carboxylase (PEPC) gene (Ppc1) in Kalanchoe laxiflora.  PEPC is a key CAM enzyme as it catalyzed the primary carboxylation reaction resulting in the nocturnal formation of C₄ acids.  Ppc mRNA expression exhibits an evening-phased transcriptional pattern. Draft genome sequence assemblies for Kalanchoe fedtschenkoi and K. laxiflora were searched for orthologous Ppc genes and candidate CAM orthologs were characterized by mapping RNA-seq data from K. laxiflora leaves performing wither C₃ photosynthesis or CAM. Candidate cis-acting elements for evening-specific mRNA expression and mesophyll expression were identified. Candidate cis-element bait sequences were designed and are being used for Y1H screens. In parallel, Ppc promoter/reporter gene studies will be performed using K. laxiflora mesophyll protoplasts to functionally dissect and validate candidate cis-regulatory elements. Such information will begin to facilitate our understanding of the temporal control of the mRNA expression patterns of key CAM genes that underpin the optimal function of CAM over the diel cycle.