Grapesystress: Use of systems biology and new breeding technology approaches to study water stress-resistant in grapevine plants.

Climate change will severely impact the wine industry, with increased frequency and duration of water stress posing significant threats to grape production in Mediterranean climates. These challenges will be compounded by heightened pest pressure and crop loss. Even though irrigation systems will expand worldwide, they will still not be sufficient to overcome the damage as the main problem is the lack of water. Therefore, the scientific community is working across different fields to address this issue, focusing on improving viticulture techniques and understanding vine physiology under stress conditions. Under this scenario, our duty as plant biotechnologists is to address this specific issue using all the new tools available to us as a community. During this thesis, a systems biology approach was used to analyze public transcriptomic data, creating tissue and cultivar-specific gene expression heat maps and gene co-expression networks related to water deficit. The data obtained from this analysis has been used to create an online tool based on the F.A.I.R. (Findable, Accessible, Interoperable, and Reusable) principles, allowing us to navigate the transcriptomic profile of water stress in grapevines. This tool, called “HydricAtlas” and hosted on the “PlantaeViz” platform, has been used to identify hub nodes (genes) as potential regulators of stress tolerance. These genes have been used to: 1) study markers of susceptibility or resistance associated with drought stress, 2) engineer grapevine genotypes for overexpression or knockout using a CRISPR/Cas9 genome editing approach, and 3) study the molecular pathways involved in the response to hydric stress. In particular, a list of candidate genes has been selected to be functionally characterized by genome editing via knockout by CRISPR-Cas methods based on their function during stress tolerance. During this thesis, we choose seven genes to be edited: VviRAF1, VviGH3.6, VviEM6, VviOPR3, VviExocyt70-02, VviLAX3 and VviNPF7.3. The HydricAtlas was also used to study the Raffinose pathway, an oligosaccharide involved in tolerance to water stress, and the AQUILO transcription factor (TF), an R2R3 MYB TF involved in regulating raffinose synthesis-related genes. In parallel, V. plants expressing the ABACUS biosensor for Abscisic acid (ABA) have been generated to spatially and temporally map ABA levels in grapevine seedlings and fruits in response to various biotic and abiotic stresses, especially during water deficit processes, as well as during plant and fruit development. The primary deliverables of this thesis included the development of an online tool for transcriptome analysis of grapevine under water stress conditions, the production of an over-expressor line for the VviRaf2 enzyme with better tolerance during water stress conditions, the creation of a cisgenic grapevine plant incorporating the VviAquilo gene with the description of the pathways possibly controlled by this transcription factor, the generation of a transgenic line equipped with a biosensor for abscisic acid (ABA), and the initiation of gene editing of susceptible genes identified using the HydriCatlass tool. By coupling all these techniques, we aim to better understand the role of phytohormones, sugars, and osmoprotectants during water stress sensing and adaptation, with the ultimate goal of ensuring sustainable beverage security for future generations.