Ethylene activation of carotenoid biosynthesis by a novel transcription factor CsERF061.

Carotenoids are indispensable for both human health and plant survival. Citrus, known for being rich in carotenoids, is a vital source of dietary carotenoids in the human diet. Ethylene can accelerate carotenoid accumulation in citrus, but the underlying transcriptional regulation mechanisms remain unclear. Chromoplast-specific lycopene β-cyclase (LCYb2) is a critical carotenogenic enzyme, which controls the massive accumulation of downstream carotenoids, especially provitamin A carotenoids in citrus, but its regulatory metabolism is largely unknown. Here a group I ethylene response factor CsERF061 was identified in citrus by yeast one-hybrid screen with the promoter of LCYb2. The expression of CsERF061 was induced by ethylene. Transcript and protein levels of CsERF061 were increased during fruit development and coloration. CsERF061 is a nucleus-localized transcriptional activator, which directly binds to the promoter of LCYb2 and activates its expression. Additionally, overexpression of CsERF061 in citrus calli and tomato fruits enhanced carotenoid accumulation by increasing the expression of key carotenoid pathway genes, increased the number of chromoplasts needed to sequester the elevated levels of carotenoids, and also accompanied by the alteration of abscisic acid and gibberellin contents. Electrophoretic mobility shift assay and dual-luciferase assay verified that CsERF061 activates the promoters of the other nine key carotenoid pathway genes, including PSY1, PDS, CRTISO, LCYb1, BCH, ZEP, NCED3, CCD1, and CCD4, revealing the multitargeted regulation of CsERF061. Collectively, our findings decipher a novel regulatory network of carotenoid enhancement by CsERF061, induced by ethylene, which is useful for manipulating the carotenoid accumulation in citrus, as well as other plants.