Cytochrome P450 CitCYP97B modulates carotenoid accumulation diversity by hydroxylating ß-cryptoxanthin in Citrus.

Carotenoids in plant foods provide health benefits by functioning as provitamin A. One of the vital provitamin A carotenoids, ß-cryptoxanthin, is typically plentiful in citrus fruit. However, little is known about the genetic basis of ß-cryptoxanthin accumulation in citrus. Here, we performed a widely targeted metabolomic analysis of 65 major carotenoids and carotenoid derivatives to characterize carotenoid accumulation in Citrus and determine the taxonomic profile of ß-cryptoxanthin. We used data from 81 newly sequenced representative accessions and 69 previously sequenced Citrus cultivars to reveal the genetic basis of ß-cryptoxanthin accumulation through a genome-wide association study. We identified a causal gene, CitCYP97B, which encodes a cytochrome P450 protein whose substrate and metabolic pathways in land plants were undetermined. We subsequently demonstrated that CitCYP97B functions as a novel monooxygenase that specifically hydroxylates the ß-ring of ß-cryptoxanthin in a heterologous expression system. In planta experiments provided further evidence that CitCYP97B negatively regulates ß-cryptoxanthin content. Using the sequenced Citrus accessions, we found that two critical structural cis-element variations contribute to increased expression of CitCYP97B, thereby altering ß-cryptoxanthin accumulation in fruit. Hybridization/introgression appear to have contributed to the prevalence of two cis-element variations in different Citrus types during citrus evolution. Overall, these findings extend our understanding of the regulation and diversity of carotenoid metabolism in fruit crops and provide a genetic target for production of ß-cryptoxanthin-biofortified products.

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

As an essential horticultural crop, Citrus has carotenoid diversity, which affects its aesthetic and nutritional values. ß,ß-Xanthophylls are the primary carotenoids accumulated in citrus fruits, and non-heme di-iron carotene hydroxylase (BCH) enzymes are mainly responsible for ß,ß-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 ß-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 ß-carotene into ß-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 ß-cryptoxanthin biosynthesis suggested the potential of CsBCH2 to be employed for expanding the synthetic biology toolkit in carotenoid engineering.