A comparative computational analysis of guard cell and mesophyll cell metabolism across C₃ and CAM photosynthesis

Understanding the interlinked metabolism of guard cells (GCs) and mesophyll cells (MCs) and their roles in stomatal movement in C₃ and Crassulacean Acid Metabolism (CAM) is important for designing crop plants with improved water use efficiencies. Although previous studies reported specific metabolic, osmotic, and enzymatic patterns in isolated GC and MC of both C₃ and CAM, and the role of different osmolytes in stomatal opening in combined GC-MC metabolism of C₃, a detailed phase-wise comparison of osmolyte accumulation, metabolism, enzyme activities, and energetics between GC and MC within an integrated GC-MC model for both photosynthetic modes is still lacking. Here, we integrate osmolyte accumulation-dependent stomatal opening, gas exchange, and metabolism in the six-phase diel combined GC-MC metabolic model and simulate cellular metabolism using Flux Balance Analysis. Results capture the phase-specific contribution of diverse osmolytes, including K⁺, Cl^-, NO₃^-, malate^2-, citrate^3-, glucose, fructose, sucrose, etc., on stomatal regulation in C₃ and CAM. It further predicts phase-specific MC-to-GC carbon transfer and reproduces higher activities of C₄-like enzymes, including PEPC, MDH, ME, PEPCK, etc., in C₃ GC relative to MC. It also captures temporal enzymatic patterns in CAM GC and MC and reveals differential fluxes through reactions involved in the production and utilization of ATP and reducing equivalents across the diel cycle. Collectively, these findings provide a quantitative framework for understanding the coordinated metabolic, osmotic, and energetic regulation of GC and MC in C₃ and CAM and identify metabolic processes that may require coordinated regulation for engineering CAM-associated traits into C₃ crops.