Edited grapevine knocked-out for VvEPFL9-1 showed reduced stomatal density

Stomatal density and distribution are under the control of small cysteine-rich peptides called epidermal patterning factors (EPFs), highly conserved in a wide range of higher plants. In Arabidopsis and cereals, three members of this family play a key role in the formation of stomata: EPF1, EPF2 and EPFL9. EPF2 and EPF1 prevent stomata formation while EPFL9 is a positive regulator, acting antagonistically to the other two. In this work, CRISPR/Cas9 technology was applied to investigate the role of VvEPFL9-1 gene in grapevine via knockout (KO) mutants. Gene transfer experiments were carried out co-culturing embryogenic calli of a table grape and a rootstock with Agrobacterium tumefaciens carrying the binary vector with a specific sgRNA. Edited lines were phenotypically characterized under well-watered and water-stressed conditions using a full suite of physiological parameters. Vvepfl-1 KO plants showed a reduction in stomatal density compared to the wild type (WT) followed by an improved water-use efficiency both under optimal and water stress conditions. In addition, ad-hoc experiments, carried out to test micrografted combinations of KO and WT lines, showed a moderate gain in water-use efficiency when micrografts were formed by an edited scion or rootstock. Our results highlight the possibility to optimize water-use efficiency by manipulating stomatal density via VvEPFL9-1 knockout and suggests stomatal anatomical features as a promising target for climate change resilient grapevine varieties.