Shi Qiaofang, Liu Yiting, Wang Ruxin, Zhang Haimeng, Jing Pengwei, Yu Yihe
College of Horticulture and Plant Protection, Henan University of Science and Technology, Luoyang, 471023, Henan Province, China; Henan Provincial Engineering Research Center on Characteristic Berry Germplasm Innovation & Utilization, Luoyang, 471023, Henan, China.
College of Horticulture and Plant Protection, Henan University of Science and Technology, Luoyang, 471023, Henan Province, China; Henan Provincial Engineering Research Center on Characteristic Berry Germplasm Innovation & Utilization, Luoyang, 471023, Henan, China.
Plant Physiol Biochem. 2025 Aug 19;229(Pt A):110409. doi: 10.1016/j.plaphy.2025.110409.
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.
葡萄（Vitis vinifera L.）是一种重要的经济水果作物，在全球广泛种植，但严重的幼果坐果率严重限制了产量和品质的提高。合成的细胞分裂素类植物生长调节剂N-(2-氯-4-吡啶基)-N'-苯基脲（CPPU）能显著提高葡萄的坐果率。CPPU处理降低了内源细胞分裂素水平，并导致幼果中细胞分裂素氧化酶/脱氢酶6（VlCKX6）基因的差异表达。然而，VlCKX6调控葡萄坐果的机制尚不清楚。在此，通过研究VlCKX6基因的功能和调控，分析了VlCKX6调控细胞分裂素水平以调控葡萄坐果的分子机制。结果表明，在CPPU处理的葡萄幼果中，VlCKX6表达显著上调，在花序和卷须中观察到更高的表达水平。GUS活性分析证实了VlCKX6的启动子活性，茉莉酸甲酯（MeJA）和CPPU显著增强了其启动子活性，而脱落酸（ABA）抑制了其活性。VlCKX6基因在烟草中的异位过表达表现出明显的细胞分裂素缺乏表型，包括叶面积减小和细胞数量减少。酵母单杂交和双荧光素酶分析验证了VlMYB12和VlDOF1.5直接结合到VlCKX6启动子上。上述结果表明，VlMYB12和VlDOF1.5可以直接调控VlCKX6的表达，调节细胞分裂素水平，从而影响葡萄坐果。本研究阐明了VlCKX6在坐果中的作用，为CPPU促进葡萄坐果的机制分析以及高产优质葡萄品种的定向培育提供了理论依据。
