The color diversity of non-purple carrot taproots is mainly affected by carotenoid species and content. Carrot cytochrome P450 carotene β-ring hydroxylase (DcCYP97A3) may influence carotenoid accumulation in carrots; however, the roles of DcCYP97A3 in carrot remain unclear. Compared to the orange carrot ‘Kurodagosun, KRD’, the yellow carrot ‘Yellowstone, YST’ had greater relative transcript levels of DcCYP97A3. DcCYP97A3 was shown to catalyze the β-ring hydroxylation of α-carotene to create zeaxanthin when it was expressed in Escherichia coli accumulating α- and β-carotene. Expression of the DcCYP97A3 of ‘YST’ in DcCYP97A3 functionally deficient orange carrot ‘KRD’ resulted in yellow taproots, decreased α-carotene and β-carotene content, decreased α-/β-carotene ratio, and increased lutein content. In carrots overexpressing the DcCYP97A3 gene, the transcript levels of DcLCYE and DcLCYB1 were significantly upregulated and downregulated, respectively. Gene editing of DcCYP97A3 in ‘YST’ resulted in DcCYP97A3 knockout mutants with significantly reduced levels of lutein and β-carotene and significantly upregulated transcript levels of DcCHXB2 and DcCCD4. These findings advance our knowledge of the molecular mechanisms behind carrot carotenoid metabolism.

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