Differential hydroxylation efficiency of the two non-heme carotene hydroxylases: DcBCH1, rather than DcBCH2, plays a major role in carrot taproot

Abstract

Carotene hydroxylase plays an important role in catalyzing the hydroxylation of carotene to xanthopylls, including two types: non-heme carotene hydroxylase (BCH type) and heme-containing cytochrome P450 hydroxylase (P450 type). Two BCH-encoding genes were annotated in the carrot genome. However, the role of BCHs and whether there are functional interactions between the duplicated BCHs in carrot remains unclear. In this study, two BCH encoding genes, <i>DcBCH1</i> and <i>DcBCH2</i>, were cloned from carrot. The relative expression level of <i>DcBCH1</i> was much higher than that of <i>DcBCH2</i> in carrot taproots with different carotene accumulation levels. Overexpression of <i>DcBCH1</i> in 'KRD' (high carotene accumulated) carrot changed the taproot color from orange to yellow, accompanied by substantial reductions in α-carotene and β-carotene. There was no obvious change in taproot color between transgenic 'KRD' carrot overexpressing <i>DcBCH2</i> and control carrot. Simultaneously, the content of α-carotene in the taproot of <i>DcBCH2-</i>overexpressing carrot decreased, but the content of β-carotene did not change significantly in comparison with control carrot. Using the CRISPR/Cas9 system to knock out <i>DcBCH1</i> in 'KRD' carrot lightened the taproot color from orange to pink-orange; the content of α-carotene in the taproot increased slightly, while the β-carotene content was still significantly decreased, compared with control carrot. In <i>DcBCH1</i>-knockout carrot, the transcript level of <i>DcBCH2</i> was significantly increased. These results indicated that in carrot taproot, DcBCH1 played the main function of BCH enzyme, which could hydroxylate α-carotene and β-carotene; DcBCH1 and DcBCH2 had functional redundancy, and these two DcBCHs could partially compensate for each other.