Cytochrome P450s (CYPs) are the largest family of enzymes in plant and play multifarious roles in development and defense but the available information about the CYP superfamily in citrus is very limited. Here we provide a comprehensive genome-wide analysis of the CYP superfamily in Citrus clementina genome, identifying 301 CYP genes grouped into ten clans and 49 families. The characteristics of both gene structures and motif compositions strongly supported the reliability of the phylogenetic relationship. Duplication analysis indicated that tandem duplication was the major driving force of expansion for this superfamily. Promoter analysis revealed numerous cis-acting elements related to various responsiveness. RNA-seq data elucidated their expression patterns in citrus fruit peel both during development and in response to UV-B. Furthermore, we characterize a UV-B-induced CYP gene (Ciclev10019637m, designated CitF3′H) as a flavonoid 3′-hydroxylase for the first time. CitF3′H catalyzed numerous flavonoids and favored naringenin in yeast assays. Virus-induced silencing of CitF3′H in citrus seedlings significantly reduced the levels of 3′-hydroxylated flavonoids and their derivatives. These results together with the endoplasmic reticulum-localization of CitF3′H in plant suggest that this enzyme is responsible for the biosynthesis of 3′-hydroxylated flavonoids in citrus. Taken together, our findings provide extensive information about the CYP superfamily in citrus and contribute to further functional verification.

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