Coordinated stomatal, mesophyll, and biochemical functions in photosynthetic responses to heat and dryness

The intrinsic link between temperature and leaf-to-air vapor pressure difference (Δe) complicates isolation of their individual effects on photosynthesis. Consequently, how CO₂ diffusion changes under heat and high evaporative demand, particularly through mesophyll conductance (g_m) responses, remains poorly understood. The conditions under which biochemical colimitation occurs, meaning Rubisco carboxylation and RuBP regeneration capacities match, are also unclear. To advance understanding of plant responses to climate change, we separated temperature and Δe effects by holding Δe at 1 and 2 kPa while varying leaf temperature (T_leaf) from 20 to 40 °C across five CO₂ levels (150 to 800 μmol mol^-1) in cotton, sunflower, and dwarf bean. Gas exchange and chlorophyll fluorescence measurements showed that g_m responses partly counteract increases in stomatal conductance to CO₂ (g_sc) at high temperatures and declines in g_sc at elevated Δe. Coordination between g_sc and g_m buffers effects of heat and dryness on CO₂ diffusion and stabilizes chloroplast-to-ambient CO₂ ratio (C_c/C_a) across measured T_leaf and Δe ranges. C_c/C_a is more conservative with increasing T_leaf at C_a ≤ 400 μmol mol^-1 than at elevated C_a. Across tested T_leaf and Δe conditions, the transition from Rubisco carboxylation to RuBP regeneration limitation remains near C_a of 400 μmol mol^-1, indicating that biochemical colimitation occurs near current atmospheric CO₂ levels. Our findings reveal that plants alleviate diffusional limitations under heat and dryness through coordinated responses of g_sc and g_m, and maintain biochemical colimitation over broad T_leaf and Δe conditions to efficiently utilize Rubisco carboxylation and RuBP regeneration capacities at near-atmospheric CO₂ levels.