SummaryImprovement in crop water-use efficiency (WUE) is a critical priority for regions facing increased drought or diminished groundwater resources. Despite new tools for the manipulation of stomatal development, the engineering of plants with high WUE remains a challenge.We used Arabidopsis epidermal patterning factor (EPF) mutants exhibiting altered stomatal density to test whether WUE could be improved directly by manipulation of the genes controlling stomatal density. Specifically, we tested whether constitutive overexpression of EPF2 reduced stomatal density and maximum stomatal conductance (g w(max) ) sufficiently to increase WUE.We found that a reduction in g w(max) via reduced stomatal density in EPF2-overexpressing plants (EPF2OE) increased both instantaneous and long-term WUE without altering significantly the photosynthetic capacity. Conversely, plants lacking both EPF1 and EPF2 expression (epf1epf2) exhibited higher stomatal density, higher g w(max) and lower instantaneous WUE, as well as lower (but not significantly so) long-term WUE.Targeted genetic modification of stomatal conductance, such as in EPF2OE, is a viable approach for the engineering of higher WUE in crops, particularly in future high-carbon-dioxide (CO 2 ) atmospheres.

SummaryStomatal regulation of plant carbon uptake and water loss under changing environmental conditions was a crucial evolutionary step in the colonization of land by plants. There are currently two conflicting models describing the nature of stomatal regulation across terrestrial vascular plants: the first is characterized by a fundamental mechanistic similarity across all lineages, and the second is characterized by the evolution of major differences in angiosperms compared with more ancient lineages. Specifically, the second model posits that stomata of ferns lack a response to elevated atmospheric CO 2 concentration (c a ) and therefore cannot regulate leaf intercellular CO 2 concentration (c i ).We compared stomatal sensitivity to changes in c a in three distantly related fern species and a representative angiosperm species.Fern and angiosperm stomata responded strongly and similarly to changes in c a . As a result, c i /c a was maintained within narrow limits during c a changes.Our results challenge the model in which stomata of ferns generally lack a response to elevated c a and that angiosperms evolved new dynamic mechanisms for regulating leaf gas exchange that differ fundamentally from ferns. Instead, the results are consistent with a universal stomatal control mechanism that is fundamentally conserved across ferns and angiosperms, and therefore likely all vascular plant divisions.