Identification of a carotenoid cleavage dioxygenase gene TeCCD4a regulating flower color and carotenoid content of marigold

Marigold (Tagetes erecta) serves as both an ornamental and economically significant species, owing to its diverse floral coloration and exceptionally high petal carotenoid content. Carotenoid cleavage dioxygenase (CCD), as the key enzymatic component, mediates the carotenoid degradation process. In this study, we cloned and functionally characterized a CCD4 gene to elucidate its regulatory function in petal color and carotenoid biosynthesis. Our findings revealed that in the deep-orange marigold cultivar ‘Lady’, the expression of the TeCCD4a transcript initially increased before subsequently decreasing, reaching its peak at developmental stage S2. Conversely, in the cream-white cultivar ‘Vanilla’, TeCCD4a expression exhibited a steady rise throughout inflorescence development. Functional characterization revealed that overexpression of TeCCD4a in marigold induced a phenotypic transition of petal coloration from yellow to pale yellow, concomitant with metabolic alterations characterized by elevated accumulation of lycopene and violaxanthin, while reducing zeaxanthin and neoxanthin contents. In marigold transgenic lines, transcript levels of carotenogenic genes TePDS, TeZDS, and TeHYDB were upregulated, while TeLCYB, TeZEP4, and TeCCD4b were downregulated. Furthermore, promoter sequence analysis indicated that TeCCD4a expression may be regulated by multiple factors, as its promoter contains various cis-acting elements including light-responsive elements, stress-responsive elements, and hormone-responsive elements. This study provides a valuable gene resource and establishes a theoretical foundation for improving petal coloration and enhancing carotenoid accumulation in marigold.