CIRCADIAN CLOCK ASSOCIATED 1 modulates drought-induced acid and sugar accumulation in citrus fruit

Citrus fruit accumulates acid at the early stage of fruit enlargement and sugar at the fruit maturity stage. Drought stress during these stages accelerates their accumulation. Here, we reported a citrus circadian clock gene, CIRCADIAN CLOCK ASSOCIATED 1 (CsCCA1), which exhibits circadian rhythmicity and impacts citrate accumulation in citrus fruit under drought stress, even naturally working in cultivars with high or low fruit citrate levels. Further, it was found to participate in fruit sugar accumulation. Overexpression of CsCCA1 in citrus juice sacs, calli, leaves, and tomato fruits was associated with an increase in the concentration of citrate, sucrose, fructose, and glucose, whereas the silenced lines showed the opposite effect. The higher concentrations of citrate and sugar enhanced the drought tolerance of citrus callus. We also found that CsCCA1 positively regulates the bHLH transcription factor ANTHOCYANIN 1 (AN1), which plays a role in citrate transport to the vacuole, and sucrose transporter 1 (SUT1), which facilitates the entry of sucrose into cells. This regulation occurs through the binding of CsCCA1 to the evening element sequences in their promoters. This indicates the circadian clock's role in coordinating drought signal with the accumulation of citrate and sugar, providing novel insights into the formation of citrus fruit quality.