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

Carotenoids in plant foods provide health benefits by functioning as provitamin A. One of the vital provitamin A carotenoids, β-cryptoxanthin, is typically plentiful in citrus fruit. However, little is known about the genetic basis of β-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 β-cryptoxanthin. We used data from 81 newly sequenced representative accessions and 69 previously sequenced Citrus cultivars to reveal the genetic basis of β-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 β-ring of β-cryptoxanthin in a heterologous expression system. In planta experiments provided further evidence that CitCYP97B negatively regulates β-cryptoxanthin content. Using the sequenced Citrus accessions, we found that two critical structural cis-element variations contribute to increased expression of CitCYP97B, thereby altering β-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 β-cryptoxanthin-biofortified products.