Red peel regulator1 links Ethylene response factor 25 and β-citraurin biosynthetic genes to regulate ethylene-induced peel reddening in citrus

The reddish apocarotenoid β-citraurin, produced by CAROTENOID CLEAVAGE DIOXYGENASE 4b (CsCCD4b), is responsible for peel reddening in citrus (Citrus spp.). Ethylene induces the characteristic red color of citrus peel, but the underlying molecular mechanism remains largely unclear. Here, we identified Red peel regulator 1 (CsRP1), a trihelix transcriptional activator that regulates ethylene-induced peel reddening by directly binding to a key MYB-binding site in the CsCCD4b promoter, thus activating its transcription. Furthermore, two drought-responsive cis-elements in the CsRP1 promoter are bound by the ethylene-response factor Ethylene response factor 25 (CsERF25). We reconstructed the CsERF25-CsRP1-CsCCD4b transcriptional regulatory cascade through transient expression of CsERF25 and CsRP1 in citrus peel and via stable transformation of citrus calli. In this cascade, CsERF25 expression was induced by ethylene to activate CsRP1 expression, and then CsRP1 directly induced CsCCD4b transcription to catalyze β-citraurin biosynthesis. CsRP1 and CsERF25 also bound to the promoters of other carotenogenic genes and induced their transcription, thereby promoting β-citraurin accumulation. Collectively, our findings reveal a complex regulatory network modulating ethylene-induced citrus peel reddening and provide innovative strategies for improving the nutritional and aesthetic values of citrus and other fruit crops.