Characterization of the Transcriptional Regulation of Crassulacean Acid Metabolism (CAM) in Kalanchoe fedtschenkoi Mesophyll Tissue

Increasing bioenergy crop production is vital to meet future renewable energy demands. Much of the world, however, is too arid for sustainable biomass production. Engineering biofuel crops for growth in these environments could greatly increase potential biomass production areas. Introducing crassulacean acid metabolism (CAM) into these crops might confer the improved water-use efficiency required for growth in arid lands. CAM relies upon rhythmic circadian clock-dependent mRNA regulation. Using Kalanchoe, in which CAM develops along a leaf developmental gradient, and Mesembryanthemum crystallinum, a facultative CAM model, candidate transcription factors with CAM-related functions were identified and confirmed as nuclear localized in Kalanchoe. These transcription factors’ mRNA expression profiles fluctuate in a circadian rhythm distinct from that observed in C₃ photosynthesis and increase in abundance during the transition from C₃ photosynthesis to CAM. To elucidate the temporal regulation of CAM in mesophyll tissue as an initial step in engineering CAM into C₃ crops, target genes of these transcription factors will be identified. To accomplish this, INTACT (Isolation of Nuclei Tagged in Specific Cell Types) is being employed to purify mesophyll nuclei from transformed lines of Kalanchoe fedtschenkoi that express epitope-tagged transcription factors of interest. INTACT utilizes mesophyll-specific nuclear envelope biotin labeling followed by affinity isolation of biotinylated nuclei. Chromatin harboring tagged transcription factors will be immunoprecipitated from purified mesophyll nuclei and chromatin immunoprecipitation-sequencing (ChIP-seq) conducted to identify transcription factor cis-regulatory genomic targets. Through these experiments, insight into the transcriptional regulation of CAM will be obtained to better inform its engineering into bioenergy crops.