Intrinsic water use efficiency depends on stomatal aperture rather than stomatal density in C3 and C4 grasses grown at glacial CO2 and low light

We investigated how stomatal morphology and physiology control intrinsic leaf water use efficiency (iWUE) in grasses. Two C3 and six C4 grasses were grown at ambient (400 {micro}l L-1) or glacial CO2 (180 {micro}l L-1) and high (1000 {micro}mol m-2 s-1) or low light intensity (200 {micro}mol m-2 s-1). C4 grasses tended to have higher iWUE and CO2 assimilation rates, and lower stomatal conductance (gs), operational stomatal aperture (aop) and guard cell K+ influx rate relative to C3 grasses, while stomatal size (SS) and stomatal density (SD) did not vary according to the photosynthetic type. Overall, iWUE and gs depended most on aop and density of open stomata. In turn, aop correlated with K+ influx, stomatal opening speed on transition to high light and SS. Species with higher SD had smaller and faster-opening stomata. Although C4 grasses operated with lower gs and aop at ambient CO2, they showed a greater potential to open stomata relative to maximal stomatal conductance (gmax), indicating heightened stomatal sensitivity and control. We uncover novel links between aop, gs, iWUE and K+ influx amongst grasses and differential K+ influx responses of C4 guard cells to low light, revealing molecular targets for breeding crops with high iWUE. HighlightsAcross C3 and six C4 grasses, intrinsic water use efficiency was strongly associated with stomatal conductance, operational stomatal aperture, guard cell K+ influx and stomatal opening speed on transition to high light.