Altering Carotene Hydroxylase Activity of DcCYP97C1 Affects Carotenoid Flux and Changes Taproot Colour in Carrot

CYP97C1 as a haem-containing cytochrome P450 hydroxylase (P450-type) is important for carotene hydroxylation and xanthophyll biosynthesis. Research about this type of hydroxylase was mainly reported in several model plant species which have no specialized tissues accumulating massive carotenoids. The function of CYP97C1 in the horticultural plant, like carrots, was not fully studied. In this study, we focused on the role of DcCYP97C1 in carotenoid flux and colour formation in carrot. DcCYP97C1 was found highly expressed in the 'turning stage' of carrot taproot. Using stable transformation and CRISPR/Cas9-mediated gene knockout technology, DcCYP97C1 was confirmed the rate-limiting enzyme for lutein biosynthesis and important for taproot colour formation. Overexpression of DcCYP97C1 in an orange carrot KRD (Kurodagosun) resulted in five times overproduction of lutein accompanied by dramatic reduction of carotenes. Knockout of DcCYP97C1 in orange KRD and yellow carrot QTH (Qitouhuang) reduced all kinds of carotenoids including lutein, α-carotene and β-carotene reflecting the key role of DcCYP97C1 for total carotenoid accumulation in taproot 'turning stage'. Our study demonstrated that manipulation of DcCYP97C1 was sufficient to influence carotenoid flux, change carrot colour and for high lutein production. The uncovered role of DcCYP97C1 may be helpful for understanding plant carotenoid metabolism and breeding colourful carrot cultivars.