Characterization of a Caffeic Acid 8-O-Methyltransferase from Citrus and Its Function in Nobiletin Biosynthesis.

Nobiletin (3',4',5,6,7,8-hexamethoxyflavone) is a polymethoxylated flavonoid specifically accumulated in citrus fruit with numerous beneficial effects to human health. In this study, a novel O-methyltransferase (CitOMT2) was isolated from three citrus varieties, Ponkan mandarin (Citrus reticulata Blanco), Nou 6 ("King mandarin" × "Mukaku-kishu"), and Satsuma mandarin (Citrus unshiu Marc.), and its functions were characterized in vitro. The gene expression results showed that CitOMT2 was highly expressed in the two nobiletin abundant varieties of Ponkan mandarin and Nou 6. However, the expression level of CitOMT2 was low in the flavedo of Satsuma mandarin, in which only a small amount of nobiletin was accumulated. Functional analysis suggested that CitOMT2 was a caffeic acid 8-O-methyltransferase, and it catalyzed the O-methylation of 7,8-dihydroxyflavone at 8-OH. As the methylation of flavone at 8-OH was required for nobiletin biosynthesis, the results presented in this study suggested that CitOMT2 was a key gene regulating nobiletin accumulation in citrus fruit.

Blue LED light induces regreening in the flavedo of Valencia orange in vitro.

In the present study, the effects of blue LED light on the regreening of citrus fruit were investigated in an in vitro system of Valencia orange flavedos. The results showed that blue LED light irradiation induced regreening in the flavedos. After four-week culture in vitro, the flavedos exhibited obviously green color in the blue LED light treatment, while the flavedos in the control were still in orange color. During the regreening process, the blue LED light treatment induced chlorophyll accumulation, and substantially altered the carotenoid composition in the flavedos. Compared with the control, the content of 9-cis-violaxanthin was decreased, while the contents of lutein, ß-carotene, and all-trans-violaxanthin were increased by blue LED light. In addition, gene expression results showed that the up-regulation of CitLCYe and down-regulation of CitLCYb2 by blue LED light led to a shift from ß,ß-branch to ß,ε-branch of the carotenoid biosynthetic pathway.

Expression and functional analysis of the nobiletin biosynthesis-related gene CitOMT in citrus fruit.

Nobiletin, a polymethoxy flavone (PMF), is specific to citrus and has been reported to exhibit important health-supporting properties. Nobiletin has six methoxy groups at the 3',4',5,6,7,8-positions, which are catalyzed by O-methyltransferases (OMTs). To date, researches on OMTs in citrus fruit are still limited. In the present study, a novel OMT gene (CitOMT) was isolated from two citrus varieties Satsuma mandarin (Citrus unshiu Marc.) and Ponkan mandarin (Citrus reticulata Blanco), and its function was characterized in vitro. The results showed that the expression of CitOMT in the flavedo of Ponkan mandarin was much higher than that of Satsuma mandarin during maturation, which was consistent with the higher accumulation of nobiletin in Ponkan mandarin. In addition, functional analysis showed that the recombinant protein of CitOMT had methylation activity to transfer a methyl group to 3'-hydroxy group of flavones in vitro. Because methylation at the 3'-position of flavones is vital for the nobiletin biosynthesis, CitOMT may be a key gene responsible for nobiletin biosynthesis in citrus fruit. The results presented in this study will provide new strategies to enhance nobiletin accumulation and improve the nutritional qualities of citrus fruit.