DcLcyB1 and DcLcyB2, two lycopene β-cyclases with partial functional overlap modulate carotene profiles in carrot roots via distinct catalytic properties

In plant, lycopene β-cyclase is a crucial enzyme in carotenoid metabolic pathway, which can induce color alteration. Carrots (Daucus carota) possess two genes encoding lycopene β-cyclase, namely DcLcyB1 and DcLcyB2. Little is known regarding the functional disparities between these two proteins in regulating carrot carotenoid accumulation. We found that the expression level of DcLcyB2 was higher than that of DcLcyB1 in carrot roots. Enzyme reaction in E. coli demonstrated that both two DcLcyB proteins were capable of cycloconverting lycopene to β-carotene, but DcLcyB2 tended to have a higher preference for monocyclic carotene substrates, resulting in more α-carotene production. After the DcLcyBs were separately overexpressed in red carrots, the roots turned yellow, accompanied by the reduction of lycopene and β-carotene content and the entire carotenoid metabolism flowed downstream towards xanthophylls. The expression levels of DcCHXE, DcCYP97A3, DcCHXB1 and DcCHXB2 in the DcLcyB-OE lines raised sharply. After gene editing of DcLcyB1/2, the α-/β-carotene ratio changed conspicuously, particularly in the dclcyb2 mutants, where α-carotene content dropped sharply, while β-carotene remained high. The expression levels of most structural genes in carotenoid pathway responded dynamically. Our results enriched the understanding of functionally redundant but differentiated roles of two DcLcyB isoenzymes in carrot root coloring.