Reduced stomatal density improves water-use efficiency in grapevine under climate scenarios of decreased water availability.

The grapevine VviEPFL9-2 paralog is specifically expressed during leaf expansion and its knockout provide a phenotype with superior adaptation to environmental stresses via reduced stomatal density. In Arabidopsis stomatal initiation relies on the transcription factor SPEECHLESS, which is positively regulated by AtEPFL9, a peptide of the epidermal patterning factor family. In grapevine, two EPFL9 paralogs exist but despite a structural similarity, their specific function remains unclear. In this study, we investigated their distinct functional roles and the extent to which reduced stomatal density (SD) may be beneficial for grapevine in terms of water use. We combined expression analysis of the two paralogs in untreated and ABA-treated leaves with the functional characterization of the two genes using grapevine epfl9-1 and epfl9-2 mutants. A physiological analysis of epfl9-2 mutants under different environmental conditions was also performed. We showed that VviEPFL9-1 is exclusively expressed in leaf primordia, whereas VviEPFL9-2 plays a predominant role in fine-tuning SD during the leaf expansion. An epfl9-2 mutant line with 84% lower SD than wild type, exhibited a significant improvement in intrinsic water-use efficiency under both well-watered and water-stressed conditions, with little trade-off in photosynthesis. When the reduction in SD was close to 60%, photosynthetic rate and stomatal conductance were comparable to WT. Our results provide compelling evidence that VviEPFL9-2 knockout determines a significant reduction in stomatal density without a major impact on photosynthesis which may help optimize the adverse impacts of climate change on viticulture.