VviRafS5 is a raffinose synthase involved in cold acclimation in grapevine woody tissues

The accumulation of raffinose family oligosaccharides (RFOs) is a hallmark of plant response to different abiotic stresses, including cold. The synthesis of galactinol, by galactinol synthases (GolS), and raffinose, by raffinose synthases (RafS), are fundamental for stress-induced accumulation of RFOs, but the role of these enzymes in the cold response of grapevine (Vitis vinifera L.) woody tissues is still unclear. To address this gap in the literature, 1-year-lignified grapevine canes were incubated at 4°C for 7 and 14days and tissues were analyzed for sugar content and gene expression. Results showed that, in parallel to starch breakdown, there was an increase in soluble sugars, including sucrose, glucose, fructose, raffinose, and stachyose. Remarkably, abscisic acid (ABA) levels increased during cold acclimation, which correlated with the increased expression of the key ABA-synthesis genes VviNCED2 and VviNCED3. Expression analysis of the VviGolS and VviRafS family allowed the identification of VviRafS5 as a key player in grapevine cold response. The overexpression of VviRafS5 in Saccharomyces cerevisiae allowed the biochemical characterization of the encoded protein as a raffinose synthase with a size of ~87kDa. In grapevine cultured cells, VviRafS5 was upregulated by cold and ABA but not by heat and salt stresses. Our results suggest that ABA accumulation in woody tissues during cold acclimation upregulates VivRafS5 leading to raffinose synthesis.

The work was supported by National Funds by FCT— Portuguese Foundation for Science and Technology, under the strategic program UIDB/04050/2020. The work was also supported by FCT and European Funds (FEDER/POCI/COMPETE2020) through the research projects MitiVineDrought (PTDC/BIAFBT/30341/2017 and POCI 01-0145-FEDER-030341), BerryPlastid (PTDC/BIA-FBT/28165/2017 and POCI-01-0145-FEDER-028165), and AgrifoodXXI (NORTE-01-0145-FEDER-000041). AS and HN were supported by FCT grants SFRH/BD/135782/2018 and SFRH/BPD/115518/2016, respectively. This work also benefited from the networking activities within COST ACTION CA17111 INTEGRAPE and Collaborative Laboratory “CoLab ADVID Vines & Wines.”

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