Genome editing for engineering resilience to climate changes in grapevine

In Arabidopsis stomatal initiation relies on the transcription factor SPEECHLESS, which is positively regulated by AtEPFL9, a small peptide of the Epidermal Patterning Factor family. In grapevine, two EPFL9 paralogs exist, VviEPFL9-1 and VviEPFL9-2 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 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 generated by CRISPR/Cas9. A detailed physiological analysis of epfl9-2 knockout and overexpressing lines under different environmental conditions was also performed. We showed that VviEPFL9-1 is exclusively expressed in leaf primordia, whereas VviEPFL9-2 is also expressed in developing leaves. The differential expression pattern is functionally significant; as previously reported (Clemens et al., 2022), the knockout of VviEPFL9-1 results in reduced stomatal density in very young plants, indicating that its role is limited to early leaf formation. The continuous expression of VviEPFL9-2 during leaf development indicates its significant role in determining stomatal density in fully expanded leaves. An epfl9-2 mutant line with 84% lower stomatal density (SD) than wild type (WT), exhibited a significant improvement in intrinsic water-use efficiency under both well-watered and water-stressed conditions, with little trade- off in photosynthesis and stomatal conductance (gs). When the reduction in SD was closed 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 which may help optimize water use in viticultural practices and mitigate the adverse impacts of climate change on viticulture. Another objective of this study was to ascertain the extent to which the reduced stomatal density could benefit grapevines in terms of resistance to the harmful oomycete Plasmopara viticola, the causal agent of downy mildew, which enter in the plant leaf through the stomata. Leaf disc assays to test Plasmopara viticola pathogenicity were carried out on VviEPFL9-1 and VviEPFL9-2 edited plants of ‘Sugraone’ and ‘Syrah’ and on VviEPFL9-2 overexpressing lines of ‘Sugraone’. The main findings indicate that a reduction in stomatal density is not detrimental to reduce disease severity in grapevine leaves. Moreover, evaluating sporangia density in inoculated leaf tissues, no significant differences were observed among genotypes in three different experiments. Our observation conducted on leaf discs explants suggest that stomatal density may not play a major role in controlling and limiting Plasmopara viticola infection. The role of EPFL9 in organs other than leaf remains to be fully elucidated. Another goal of this study has been to investigate whether VviEPFL9-2 may have a specific biological function in grapevine roots. To this end, epfl9-2 knock-out (KO) lines of the genotype Kober 5BB (Vitis berlandieri x Vitis riparia) were generated using CRISPR/Cas9 technology, and their root morphological traits were evaluated under both well water and water stress conditions, in conjunction with shoot physiological characterization. A transcriptomic analysis was also performed for the root tissues. Finally, grafting experiments were carried out with all possible combinations of WT and epfl9-2 mutants using Syrah as scions and Kober 5BB as rootstock. Phenotypic evaluation in the greenhouse showed that the root system of the mutants was less expanded than that of the wild type, with a significant reduction in the geotropic angle and root length. Interestingly, the secondary roots of the mutants were quantitatively less than those of the WT, but had a larger diameter, similar to that of the primary roots. In addition, grafting showed that the different root phenotype of Kober epfl9-2 KO persisted even in the presence of the unaltered shoot apparatus of the WT scion. Furthermore, RNAseq data from edited Kober roots revealed a strong activation of the lignin pathway compared to WT. Our results demonstrate for the first time that VviEPFL9-2 has a specific role in roots and suggest the existence of a mechanism regulated by EPFL9 that may lead to increased lignin deposition in root tissues. Since lignin is a valuable component of the secondary cell wall that contributes to the improvement of the hydraulic properties of roots under drought, VviEPFL9-2 appeared to be an interesting target for the application of new genomic techniques to make rootstocks better adapted to cope with drier soil environments.