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

Abstract

As an essential horticultural crop, Citrus has carotenoid diversity, which affects its aesthetic and nutritional values. β, β-Xanthophylls are the primary carotenoids accumulated in citrus fruits, and non-heme di-iron carotene hydroxylase (BCH) enzymes are mainly responsible for β, β-xanthophyll synthesis. Previous studies have focused on the hydroxylation of BCH1, but the role of its paralogous gene in citrus, BCH2, 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 Escherichia coli, although CsBCH2 exhibited a lower activity in hydroxylating β-carotene into β-cryptoxanthin than citrus BCH1 (CsBCH1). Our results showed that overexpression of CsBCH2 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, CsBCH1 and CsBCH2, and illustrated the backup compensatory role of CsBCH2 for CsBCH1 in citrus xanthophyll biosynthesis. The independent function of CsBCH2 and its cooperative function with CsBCH1 in β-cryptoxanthin biosynthesis suggested the potential of CsBCH2 to be employed for expanding the synthetic biology toolkit in carotenoid engineering.

Acknowledgements

The authors are very grateful to Prof. Norihiko Misawa for providing the pACCAR16ΔcrtX plasmid and grateful to Prof. Robert M. Larkin and Prof. Pengwei Wang for providing expression vectors. We thank Prof. Ping Liu for language improvement. This research was supported by the National Natural Science Foundation of China (No. 31930095 and 32172527) and the Modern Agro-industry Technology Research System (CARS-26).

Author contributions

X.X.D. supervised the research; Y.Z.Z. and X.X.D. designed the experiments; Y.Z.Z. performed the experiments with contributions from J.J.J.; S.C.Z. and Z.Z.X. provided the plant materials. Y.Z.Z. and X.X.D. wrote the manuscript; Q.S., Y.Z., and J.L.Y. provided critical comments on manuscript editing.

Data availability

All relevant data are included in the paper and its supplementary files.

Conflict of interest

The authors declare no competing interests.

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