Abstract

SUMMARY Sugar content is a pivotal determinant of strawberry fruit quality, influencing flavour perception and consumer acceptance. However, the transcriptional mechanisms regulating sugar metabolism remain largely elusive. Here, we identified FaDOF1.7 and FaDOF1.5 as key regulators of soluble sugar metabolism in strawberry fruit. Comparative transcriptome analysis across different developmental stages between high-sugar cultivar ‘Ningfeng’ and low-sugar cultivar ‘Gama’ identified FaDOF1.7 as a candidate transcription factor associated with sugar content. FaDOF1.7 , a nuclear-localised DOF transcription factor, exhibited high transcription during early fruit development and in stolons. Transient overexpression and virus-induced gene silencing (VIGS) assays demonstrated that FaDOF1.7 promotes the accumulation of fructose, glucose and total soluble sugar by activating three key sugar-metabolic genes, FaSUS1 , FaSUS2 and FaCWINV1 . Moreover, FaDOF1.7 physically interacts with FaDOF1.5 and synergistically enhances their cooperative binding to the promoters of these target genes, thereby activating transcription, as demonstrated by dual-luciferase and β-glucuronidase assays. FaDOF1.5 could also directly bind to the AAAG/CTTT motifs present in the FaSUS1 , FaSUS2 and FaCWINV1 promoters as confirmed by biochemical assays, which contribute to the regulation of soluble sugar accumulation. Stable overexpression of either FaDOF1.5 or FaDOF1.7 in strawberry and tomato fruits reduced sucrose but increased glucose, fructose and total soluble sugars concentrations without affecting fruit ripening or phenotype, highlighting their specific role in sugar metabolism. Altogether, our findings establish FaDOF1.5 and FaDOF1.7 as central transcriptional regulators that coordinate sucrose cleavage and monosaccharide accumulation, providing mechanistic insights into sugar accumulation in strawberry fruit and offering potential targets for metabolic engineering.