Expression and functional analysis of citrus carotene hydroxylases: unravelling the xanthophyll biosynthesis in citrus fruits. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Ma, Gang; Zhang, Lancui; Yungyuen, Witchulada; Tsukamoto, Issei; Iijima, Natsumi; Oikawa, Michiru; Yamawaki, Kazuki; Yahata, Masaki; Kato, Masaya
• Is Part Of: BMC plant biology. Volume 16:Issue 1(2016)
• Page Start: 1
• Page End: 12
• Publication Date: 2016-12
• Subjects: β-Cryptoxanthin -- Flavedo -- Juice sacs -- Lutein -- Satsuma mandarin; Plant molecular biology -- Periodicals; Botany -- Periodicals; 580.5
• DOI: 10.1186/s12870-016-0840-2
• Languages: English
• ISSNs: 1471-2229

Abstract

Abstract Background Xanthophylls are oxygenated carotenoids and fulfill critical roles in plant growth and development. In plants, two different types of carotene hydroxylases, non-heme di-iron and heme-containing cytochrome P450, were reported to be involved in the biosynthesis of xanthophyll. Citrus fruits accumulate a high amount of xanthophylls, especially β, β-xanthophylls. To date, however, the roles of carotene hydroxylases in regulating xanthophyll content and composition have not been elucidated. Results In the present study, the roles of four carotene hydroxylase genes (CitHYb, CitCYP97A, CitCYP97B, andCitCYP97C ) in the biosynthesis of xanthophyll in citrus fruits were investigated. Phylogenetic analysis showed that the four citrus carotene hydroxylases presented in four distinct clusters which have been identified in higher plants. CitHYb was a non-heme di-iron carotene hydroxylase, while CitCYP97A, CitCYP97B, and CitCYP97C were heme-containing cytochrome P450-type carotene hydroxylases. Gene expression results showed that the expression ofCitHYb increased in the flavedo and juice sacs during the ripening process, which was well consistent with the accumulation of β, β-xanthophyll in citrus fruits. The expression ofCitCYP97A andCitCYP97C increased with a peak in November, which might lead to an increase of lutein in the juice sacs during the ripening process. The expression level ofCitCYP97B was much lower than that ofCitHYb, CitCYP97A, andCitCYP97C in the juice sacs during the ripening process. Functional analysis showed that the CitHYb was able to catalyze the hydroxylation of the β-rings of β-carotene and α-carotene inEscherichia coli BL21 (DE3) cells. Meanwhile, when CitHYb was co-expressed with CitCYP97C, α-carotene was hydroxylated on the β-ring and ε-ring sequentially to produce lutein. Conclusions CitHYb was a key gene for β, β-xanthophyll biosynthesis in citrus fruits.CitCYP97C functioned as an ε-ring hydroxylase to produce lutein using zeinoxanthin as a substrate. The results will contribute to elucidating xanthophyll biosynthesis in citrus fruits, and provide new strategies to improve the nutritional and commercial qualities of citrus fruits.