Cytochrome P450 CitCYP97B modulates carotenoid accumulation diversity by hydroxylating β-cryptoxanthin in Citrus
Yingzi Zhang, Jiajing Jin, Nan Wang, Quan Sun, Di Feng, Shenchao Zhu, Zexin Wang, Shunxin Li, Junli Ye, Lijun Chai, Zongzhou Xie, Xiuxin Deng
Abstract
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.