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.

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Nobiletin (3′,4′,5,6,7, 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.

It was previously reported that the content of β-cryptoxanthin in juice sacs of Satsuma Mandarin was increased by mulching cultivation. In this study, to further clarify the mechanisms of mulching regulating β-cryptoxanthin accumulation, the effects of mulching on the expression of carotenoid metabolic genes were investigated in the juice sacs of Satsuma Mandarin 'Aoshima unshiu', which were planted in two different fields (Fields A and B) of Shimizu-ku, Shizuoka. In the mulching treatment, the β-cryptoxanthin content in the juice sacs and flavedo was 30 and 50% higher than that of the control, respectively. The changes in expression of carotenoid biosynthetic genes by mulching were different between the two fields. In fieldA, the expression of CitZDS was up-regulated, while the expression of CitHYb and CitZEP was down-regulated by mulching treatment in juice sacs. In fieldB, the expression of CitPSY, CitZDS, CitLCYb1, CitLCYb2, CitHYb, CitZEP, CitNCED2, and CitNCED3 in juice sacs was significantly up-regulated by mulching treatment. In particular, the transcript levels of the upstream carotenogenic genes were much higher on mulching treatment in fieldB. These results suggest that the transcription of carotenoid biosynthetic genes in Satsuma Mandarin is induced by mulching treatment. In addition, the high expression of upstream carotenogenic genes (highlevel supply of β-carotene) was considered to be a key mechanism leading to the marked accumulation of β-cryptoxanthin in juice sacs treated by mulching.