Characterization of the Transcriptional Regulation of Crassulacean Acid Metabolism (CAM) in Kalanchoe laxiflora mesophyll Tissue

Increasing bioenergy crop production will be vital to meet energy demands in the future.  However, about 40% of the world’s surface area is too arid for biomass production.  Engineering biofuel crops for growth in these environments could greatly increase biomass yields.  Introducing crassulacean acid metabolism (CAM) into these crops might confer the improved water-use efficiency required for biomass production in semi-arid lands.  CAM is strongly reliant on rhythmic circadian clock-dependent mRNA regulation.  Using the CAM models Kalanchoe and Mesembryanthemum crystallinum, several putative CAM-related transcription factors were identified.  These transcription factors’ mRNA expression profiles fluctuate in a circadian rhythm distinct from that of C₃ photosynthesis and become highly induced upon transition from C₃ 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, Isolation of Nuclei Tagged in Specific Cell Types (INTACT) will be employed to purify mesophyll nuclei from diploid Kalanchoe laxiflora expressing epitope-tagged transcription factors of interest.  INTACT utilizes mesophyll-specific nuclear biotin labeling followed by affinity isolation of biotinylated nuclei.  Chromatin immunoprecipitation followed by sequencing (ChIP-seq) will then be conducted to identify transcription factor cis-regulatory genomic targets.  Complementary approaches consisting of Y1H screening and transient co-transfection assays will be employed to validate ChIP-seq results and determine the functional roles of these transcription factors. Through these experiments, insights into the transcriptional control of CAM will be obtained to inform its engineering into bioenergy crops.