Spatiotemporal Differences of 24-Epibrassinolide Regulating Anthocyanin and Proanthocyanidin Biosynthesis in Vitis vinifera 'Cabernet Sauvignon'

Brassinosteroids are recognized regulators of anthocyanin and proanthocyanidin biosynthesis in grapevine; however, their spatiotemporal effects remain insufficiently characterized. This study examined the stage-specific impacts of exogenous 24-epibrassinolide and brassinazole on these phenolic compounds in Cabernet Sauvignon. Treatments were applied at fruit set and veraison, with skin and seed tissues collected across six developmental stages. Berry ripening and quality parameters were evaluated, and phenolic profiles were quantified via HPLC. The results revealed that both 24-epibrassinolide and brassinazole significantly influenced grape maturation and phenolic biosynthesis in a timing-dependent manner. Specifically, 24-epibrassinolide application at fruit set increased the content of proanthocyanidins and trihydroxylated subunits, as well as the galloylation percentage, in both skins and seeds, while also altering their composition and subunit architecture. In contrast, veraison-stage treatment reduced these parameters in seeds but promoted them in skins, highlighting a tissue-specific response within the same developmental window. Moreover, compared with fruit-set treatment, 24-epibrassinolide application at veraison more significantly enhanced total anthocyanin content in skins, predominantly through the accumulation of specific monomeric forms. Together, based on two consecutive growing seasons (2022-2023) in Vitis vinifera L. cv. Cabernet Sauvignon, these findings demonstrate that brassinosteroid regulation of anthocyanin and proanthocyanidin biosynthesis in grape berries depends not only on the tissue but also on the phenological stage of application. In conclusion, this study reveals distinct spatiotemporal patterns in the regulation of phenolic biosynthesis by 24-epibrassinolide in grapevine, providing new insights into the hormone-mediated modulation of secondary metabolism and suggesting a potential agronomic strategy for precisely shaping the phenolic profile of wine grapes through stage-targeted brassinosteroid application.