CitERF25 , CitDREB1C , and CitZAT5 orchestrate citrus fruit sugar accumulation via direct and indirect regulation of CitSWEETs

Sugars are essential in plant development and play crucial roles in fruit quality formation. In citrus, three sugars will eventually be exported transporters (SWEETs) have previously been shown to participate in sugar accumulation, but the functions of the rest members and their upstream regulators remain to be elucidated. Here, with 'Youliang' Satsuma mandarin (Citrus unshiu Marc.), we studied expression profiles of 12 CitSWEET members, carried out functional verification, and identified CitSWEET1a as a glucose transporter while CitSWEET4 and CitSWEET11a as fructose transporters. Two APETALA2/ethylene-responsive element-binding factor (AP2/ERF) were identified as positive upstream sugar accumulation regulators, with CitERF25 regulating CitSWEET1a and CitSWEET4, while CitDREB1C (dehydration responsive element-binding 1C) regulating CitSWEET4. Besides, CitDREB1C was also identified as an activator of CitZAT5 (zinc finger of Arabidopsis thaliana 5), a gene previously identified to encode a transcription factor that promotes sugar accumulation. Meanwhile, the interaction between CitERF25 and CitZAT5 enhanced the stimulative effect of CitDREB1C on CitSWEET4 expression. Furthermore, homologous transient overexpression of CitERF25 and CitDREB1C confirmed their essential roles in regulating fruit sugar accumulation. Taken together, it can be concluded that CitERF25 and CitDREB1C play key roles in sugar accumulation in citrus fruit by directly and indirectly stimulating the expression of CitSWEET1a and CitSWEET4. This study provides novel insights into the transcriptional regulatory network governing sugar accumulation in fruit by exerting effects on multiple CitSWEETs.