Molecular changes in Mesembryanthemum crystallinum guard cells underlying the C₃ to CAM transition

KEY MESSAGE: The timing and transcriptomic changes during the C₃ to CAM transition of common ice plant support the notion that guard cells themselves can shift from C₃ to CAM. Crassulacean acid metabolism (CAM) is a specialized type of photosynthesis: stomata close during the day, enhancing water conservation, and open at night, allowing CO₂ uptake. Mesembryanthemum crystallinum (common ice plant) is a facultative CAM species that can shift from C₃ photosynthesis to CAM under salt or drought stresses. However, the molecular mechanisms underlying the stress induced transition from C₃ to CAM remain unknown. Here we determined the transition time from C₃ to CAM in M. crystallinum under salt stress. In parallel, single-cell-type transcriptomic profiling by 3'-mRNA sequencing was conducted in isolated stomatal guard cells to determine the molecular changes in this key cell type during the transition. In total, 495 transcripts showed differential expression between control and salt-treated samples during the transition, including 285 known guard cell genes, seven CAM-related genes, 18 transcription factors, and 185 other genes previously not found to be expressed in guard cells. PEPC1 and PPCK1, which encode key enzymes of CAM photosynthesis, were up-regulated in guard cells after seven days of salt treatment, indicating that guard cells themselves can shift from C₃ to CAM. This study provides important information towards introducing CAM stomatal behavior into C₃ crops to enhance water use efficiency.