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

ABSTRACT We investigated how stomatal morphology and physiology control intrinsic leaf water use efficiency ( iWUE ) in grasses. Two C 3 and six C 4 grasses were grown at ambient (400 µl L -1 ) or glacial CO 2 (180 µl L -1 ) and high (1000 µmol m -2 s -1 ) or low light intensity (200 µmol m -2 s -1 ). C 4 grasses tended to have higher iWUE and CO 2 assimilation rates, and lower stomatal conductance (g s ), operational stomatal aperture ( a op ) and guard cell K + influx rate relative to C 3 grasses, while stomatal size (SS) and stomatal density (SD) did not vary according to the photosynthetic type. Overall, iWUE and g s depended most on a op and density of open stomata. In turn, a op 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 C 4 grasses operated with lower g s and a op at ambient CO 2 , they showed a greater potential to open stomata relative to maximal stomatal conductance (g max ), indicating heightened stomatal sensitivity and control. We uncover novel links between a op , g s , iWUE and K + influx amongst grasses and differential K + influx responses of C 4 guard cells to low light, revealing molecular targets for breeding crops with high iWUE . Highlights Across C 3 and six C 4 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.