title: Expression and functional analysis of citrus carotene hydroxylases: unravelling the xanthophyll biosynthesis in citrus fruits.
abstract: 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.
  
  
  
  In the present study, the roles of four carotene hydroxylase genes (CitHYb, CitCYP97A, CitCYP97B, and CitCYP97C) 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 of CitHYb 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 of CitCYP97A and CitCYP97C 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 of CitCYP97B was much lower than that of CitHYb, CitCYP97A, and CitCYP97C 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 in Escherichia 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.
  
  
  
  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.
authors.affiliation: Department of Biological and Environmental Sciences, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka, Suruga, 422-8529, Japan.
authors.forename: Gang
authors.initials: G
authors.lastname: Ma
authors.affiliation: Department of Biological and Environmental Sciences, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka, Suruga, 422-8529, Japan.
authors.forename: Lancui
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Biological and Environmental Sciences, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka, Suruga, 422-8529, Japan.|The United Graduate school of Agricultural Science, Gifu University (Shizuoka University), Yanagido, Gifu, 501-1193, Japan.
authors.forename: Witchulada
authors.initials: W
authors.lastname: Yungyuen
authors.affiliation: Department of Biological and Environmental Sciences, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka, Suruga, 422-8529, Japan.
authors.forename: Issei
authors.initials: I
authors.lastname: Tsukamoto
authors.affiliation: Department of Biological and Environmental Sciences, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka, Suruga, 422-8529, Japan.
authors.forename: Natsumi
authors.initials: N
authors.lastname: Iijima
authors.affiliation: Department of Biological and Environmental Sciences, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka, Suruga, 422-8529, Japan.
authors.forename: Michiru
authors.initials: M
authors.lastname: Oikawa
authors.affiliation: Department of Biological and Environmental Sciences, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka, Suruga, 422-8529, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Yamawaki
authors.affiliation: Department of Biological and Environmental Sciences, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka, Suruga, 422-8529, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Yahata
authors.affiliation: Department of Biological and Environmental Sciences, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka, Suruga, 422-8529, Japan. amkato@ipc.shizuoka.ac.jp.
authors.forename: Masaya
authors.identifier: 0000-0002-6574-8031
authors.initials: M
authors.lastname: Kato
mesh_terms: D002957:Citrus / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005638:Fruit / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism*; D024341:Xanthophylls / Q000096:biosynthesis*
chemical_list: D010940:Plant Proteins; D024341:Xanthophylls; D006899:Mixed Function Oxygenases
keywords: Flavedo; Juice sacs; Lutein; Satsuma mandarin; β-Cryptoxanthin