Rapid dehydration drives a nondiffusional drop in C3 photosynthesis that aligns with phosphate limitation.

Drought is an abnormally prolonged water deficit posing major challenges to plants. Stomatal closure has long been considered the primary factor limiting photosynthesis during the early stages of drought. However, emerging evidence suggests that nonstomatal limitation may also arise, particularly under rapid dehydration. We hypothesised that under rapid dehydration, photosynthesis is constrained by an impeded supply of CO2 to the chloroplasts. To test this, we conducted an innovative experiment inducing rapid but controlled dehydration in hydroponically grown wheat and sunflower. We measured water vapour and CO2 exchange in real time, along with their isotopic compositions and Chl fluorescence. Our results revealed a decrease in CO2 levels in the substomatal cavity at the onset of drying, followed by a reduction in assimilation rate due to both stomatal and nonstomatal limitations. The nonstomatal component was attributed to a combination of diffusional limitation, linked to reduced mesophyll conductance, and a biochemical limitation. Our analyses indicate that the biochemical limitation is primarily due to a reduction in ATP production, rather than a diminished capacity for Rubisco carboxylation. This is likely attributable to a phosphate shortage, which aligns with an observed reversal in the oxygen sensitivity of CO2 assimilation.