CsbHLH6 positively regulates abscisic acid-mediated citrus fruit coloration during postharvest storage

As is well known, postharvest abscisic acid (ABA) treatment of citrus fruits can significantly enhance fruit coloration by promoting the accumulation of carotenoids, however, the underlying molecular mechanism is still unclear. In this study, we further confirmed the effect of ABA treatment in promoting citrus fruit coloration by activating the expression of 4 key carotenoid metabolism genes (CsPSY, CsLCYB, CsHYD, and CsNCED2) during postharvest storage. A novel basic helix-loop-helix (bHLH) transcription factor, CsbHLH6, was identified using the promoters of 3 key ABA-induced carotenoid metabolism genes (CsLCYB, CsHYD, and CsNCED2) by yeast one-hybrid screening. Overexpression of CsbHLH6 enhanced carotenoid accumulation in citrus fruits and calli. Interference with CsbHLH6 expression in citrus calli and fruits indicated that CsbHLH6 was essential for ABA-induced carotenoid biosynthesis and fruit coloration. As a nucleus-localized transcriptional activator, CsbHLH6 directly bound to the promoters of 6 key carotenoid metabolism genes (CsPSY, CsLCYB, CsLCYE, CsHYD, CsZEP, and CsNCED2), subsequently activating their expression in vitro and in vivo. Furthermore, ABA signaling significantly raised CsbHLH6 promoter activity, thus enhancing the regulation of carotenoid accumulation by CsbHLH6. In summary, our study reveals the molecular mechanism by which CsbHLH6 regulates ABA-induced citrus fruit coloration during postharvest storage, which is crucial for improving the postharvest appearance quality of citrus fruits.