Carotene hydroxylase DcCYP97A3 affects carotenoid metabolic flow and taproot color by influencing the conversion of α-carotene to lutein in carrot

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 <i>DcCYP97A3</i> in carrot remain unclear. Compared to the orange carrot 'Kurodagosun, KRD', the yellow carrot 'Yellowstone, YST' had greater relative transcript levels of <i>DcCYP97A3</i>. DcCYP97A3 was shown to catalyze the β-ring hydroxylation of α-carotene to create zeaxanthin when it was expressed in <i>Escherichia coli</i> accumulating α- and β-carotene. Expression of the <i>DcCYP97A3</i> 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 <i>DcCYP97A3</i> gene, the transcript levels of <i>DcLCYE</i> and <i>DcLCYB1</i> were significantly upregulated and downregulated, respectively. Gene editing of <i>DcCYP97A3</i> in 'YST' resulted in <i>DcCYP97A3</i> knockout mutants with significantly reduced levels of lutein and β-carotene and significantly upregulated transcript levels of <i>DcCHXB2</i> and <i>DcCCD4</i>. These findings advance our knowledge of the molecular mechanisms behind carrot carotenoid metabolism.