Citrus -carotene hydroxylase 2 (BCH2) participates in xanthophyll synthesis by catalyzing the hydroxylation of -carotene and compensates for BCH1 in citrus carotenoid metabolism.

As an essential horticultural crop, has carotenoid diversity, which affects its aesthetic and nutritional values. ,-Xanthophylls are the primary carotenoids accumulated in citrus fruits, and non-heme di-iron carotene hydroxylase (BCH) enzymes are mainly responsible for ,-xanthophyll synthesis. Previous studies have focused on the hydroxylation of , but the role of its paralogous gene in citrus, , remains largely unknown. In this study, we revealed the β-hydroxylation activity of citrus BCH2 (CsBCH2) for the first time through the functional complementation assay using , although CsBCH2 exhibited a lower activity in hydroxylating -carotene into -cryptoxanthin than citrus BCH1 (CsBCH1). Our results showed that overexpression of in citrus callus increased xanthophyll proportion and plastoglobule size with feedback regulation of carotenogenic gene expression. This study revealed the distinct expression patterns and functional characteristics of two paralogous genes, and , and illustrated the backup compensatory role of for in citrus xanthophyll biosynthesis. The independent function of and its cooperative function with in -cryptoxanthin biosynthesis suggested the potential of to be employed for expanding the synthetic biology toolkit in carotenoid engineering.