VvRF2b interacts with VvTOR and influences VvTOR-regulated sugar metabolism in grape

The production of top-quality wines is closely related to the quality of the wine grapes. In wine grapes (Vitis vinifera L., Vv), sugar is a crucial determinant of berry quality, regulated by an interplay of various transcription factors and key kinases. Many transcription factors involved in sugar metabolism remain unexplored. Target of Rapamycin (TOR) is an important protein kinase in plants, recently found to regulate sugar metabolism in grapes. However, transcription factors or other factors involved in this process are rarely reported. Here, we utilized transgenic callus tissues from 'Cabernet Sauvignon' grape fruit engineered via gene overexpression (oe) and CRISPR/Cas9-based gene knockout (ko), and discovered a bZIP transcription factor, VvRF2b, whose knockout resulted in increased accumulation of fructose and sucrose, indicating that VvRF2b is a negative regulator of sugar accumulation. Subcellular localization and transcriptional activation tests showed that VvRF2b is an activator of transcription located both in the nucleus and cell membrane. Analysis of VvRF2b and VvTOR gene levels and sugar contents (glucose, fructose, and sucrose) in 'Cabernet Sauvignon' grape fruits at 30, 70, and 90 days after bloom (DAB) revealed that VvRF2b is expressed more highly during fruit development, while VvTOR is expressed more during the sugar accumulation phase, furthermore, VvTOR gene levels in koVvRF2b transgenic calli increased significantly, suggesting a strong relationship between the knockout of VvRF2b and the overexpression of VvTOR. Additionally, bimolecular fluorescence complementation and luciferase complementation assays demonstrated the interaction between VvRF2b and VvTOR proteins. After knocking out the VvRF2b gene in oeVvTOR calli, it was found that the knockout of VvRF2b promotes VvTOR-regulated sucrose accumulation and enhances the expression of sugar metabolism-related genes regulated by VvTOR. In summary, our results suggest that VvRF2b interacts with VvTOR protein and influences VvTOR-regulated sugar metabolism.