VlMYB12 and VlDOF1.5 co-target VlCKX6 promoter in response to CPPU promoting fruit set in grape (Vitis vinifera L.)

Grape (Vitis vinifera L.) is an important economic fruit crop widely cultivated worldwide, but serious young fruit set seriously restricts the improvement of yield and quality. The synthetic cytokinin-like plant growth regulator N-(2-chloro-4-pyridinyl)-N'-phenylurea (CPPU) can significantly improve grape fruit set. CPPU treatment reduced endogenous cytokinin levels and resulted in differential expression of the cytokinin oxidase/dehydrogenase 6 (VlCKX6) gene in young fruits. However, the mechanism by which VlCKX6 regulates grape fruit set remains unclear. Here, the molecular mechanism of the VlCKX6 regulating cytokinin levels to regulate grape fruit set was analyzed by studying the function and regulation of VlCKX6 gene. Results showed that VlCKX6 expression was significantly up-regulated in CPPU-treated young grape fruits, with higher expression levels observed in inflorescences and tendrils. GUS activity assay confirmed the promoter activity of VlCKX6, which MeJA and CPPU significantly enhanced its promoter activity, while ABA inhibited its activity. Ectopic overexpression of the VlCKX6 gene in tobacco showed a significant cytokinin deficiency phenotype, including reduced leaf area and decreased cell number. Yeast one-hybrid and dual-luciferase assay verified that VlMYB12 and VlDOF1.5 directly bind to the VlCKX6 promoter. The above results indicate that VlMYB12 and VlDOF1.5 can directly regulate the expression of VlCKX6, modulate the cytokinin level, and thus affect grape fruit set. This study clarifies the role of VlCKX6 in fruit set, and provides a theoretical basis for the mechanism analysis of CPPU-promoted grape fruit set and the directional cultivation of grape varieties with high yield and excellent quality.