Secondary metabolites in grapevine: crosstalk of transcriptional, metabolic and hormonal signals controlling stress defence responses in berries and vegetative organs

Abiotic stresses, such as temperature, heat waves, water limitation, solar radiation and the increase in atmospheric CO concentration, significantly influence the accumulation of secondary metabolites in grapevine berries at different developmental stages, and in vegetative organs. Transcriptional reprogramming, miRNAs, epigenetic marks and hormonal crosstalk regulate the secondary metabolism of berries, mainly the accumulation of phenylpropanoids and of volatile organic compounds (VOCs). Currently, the biological mechanisms that control the plastic response of grapevine cultivars to environmental stress or that occur during berry ripening have been extensively studied in many worlds viticultural areas, in different cultivars and in vines grown under various agronomic managements. A novel frontier in the study of these mechanisms is the involvement of miRNAs whose target transcripts encode enzymes of the flavonoid biosynthetic pathway. Some miRNA-mediated regulatory cascades, post-transcriptionally control key MYB transcription factors, showing, for example, a role in influencing the anthocyanin accumulation in response to UV-B light during berry ripening. DNA methylation profiles partially affect the berry transcriptome plasticity of different grapevine cultivars, contributing to the modulation of berry qualitative traits. Numerous hormones (such as abscisic and jasmomic acids, strigolactones, gibberellins, auxins, cytokynins and ethylene) are involved in triggering the vine response to abiotic and biotic stress factors. Through specific signaling cascades, hormones mediate the accumulation of antioxidants that contribute to the quality of the berry and that intervene in the grapevine defense processes, highlighting that the grapevine response to stressors can be similar in different grapevine organs. The expression of genes responsible for hormone biosynthesis is largely modulated by stress conditions, thus resulting in the numeourous interactions between grapevine and the surrounding environment

New plant breeding technologies towards a more sustainable viticulture

European grapevine cultivars are highly susceptible to many pathogens that are managed through large pesticide use. Nevertheless, the European policies promote pesticide use reduction and new environmentally friendly methods for a more sustainable agriculture. In this framework, grapevine genetic improvement could benefit from New Plant Breeding Technologies. In order to reduce fungal susceptibility, we will produce knock-out plants from embryogenic calli using CRISPR/Cas9 technology. Studies in barley reported the acquisition of powdery mildew resistance by knocking out susceptibility genes belonging to the MLO (Mildew Locus O) family. In this study, our approach takes advantage from CRISPR/Cas9 technology to perform a multiple knockout of MLO genes. Among the 17 VvMLOs reported in grapevine we designed constructs to target VvMLO6 and VvMLO7. Golden Gate assembly was used to produce three different constructs (containing two guideRNAs for each gene) to knocking-out the targets singularly or by producing a double mutant. Usually, the genetic engineering techniques, mediated by A. tumefaciens, involve the insertion of exogenous selectable marker genes. These markers are required for selection of transgenic plants, but they are undesirable to be retained in commercial transgenic plants due to possible toxicity or allergenicity to humans and potential environmental hazard. To overcome these limits, we opted for a \u201cclean\u201d editing strategy developing an inducible excision system. This approach is based on a recombinase technology involving the Cre-loxP system from the P1 bacteriophage under a heat-shock inducible promoter to be activated once the editing event(s) will be confirmed. Obtainment of embryogenic calli is one of the main bottlenecks for application of CRISPR/Cas9: for two seasons, we collected inflorescences from Chardonnay, Glera, Microvine, Pinot Noir, Sangiovese cultivars and two rootstocks, 110 Richter and SO4, cultured and maintained in vitro up to embryo development and then used to perform Agrobacterium tumefaciens GV3101 mediated transformation