Dry inside: progressive unsaturation within leaves with increasing vapour pressure deficit affects estimation of key leaf gas exchange parameters.

Climate change not only leads to higher air temperatures but also increases the vapour pressure deficit (VPD) of the air. Understanding the direct effect of VPD on leaf gas exchange is crucial for precise modelling of stomatal functioning. We conducted combined leaf gas exchange and online isotope discrimination measurements on four common European tree species across a VPD range of 0.8-3.6 kPa, while maintaining constant temperatures without soil water limitation. In addition to applying the standard assumption of saturated vapour pressure inside leaves (e), we inferred e from oxygen isotope discrimination of CO and water vapour. e desaturated progressively with increasing VPD, consistently across species, resulting in an intercellular relative humidity as low as 0.73 ± 0.11 at the highest tested VPD. Assuming saturation of e overestimated the extent of reductions in stomatal conductance and CO mole fraction inside leaves in response to increasing VPD compared with calculations that accounted for unsaturation. In addition, a significant decrease in mesophyll conductance with increasing VPD only occurred when the unsaturation of e was considered. We suggest that the possibility of unsaturated e should not be overlooked in measurements related to leaf gas exchange and in stomatal models, especially at high VPD.