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Expression and Functional Analysis of the Nobiletin Biosynthesis-Related www.nature.com/scientificreports OPEN Expression and functional analysis of the nobiletin biosynthesis‑related gene CitOMT in citrus fruit Mao Seoka1,4, Gang Ma1,2,4, Lancui Zhang2, Masaki Yahata1,2, Kazuki Yamawaki1,2, Toshiyuki Kan3 & Masaya Kato1,2* Nobiletin, a polymethoxy favone (PMF), is specifc 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 favedo 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 favones in vitro. Because methylation at the 3′-position of favones 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. Flavonoids are a group of secondary metabolites that include more than 10,000 kinds of derivatives1. According to their structure, favonoids are divided into several subgroups, including favanones, favones, favonols, favanols, isofavones, and anthocyanidins2. Flavonoids are biosynthesized from p-coumaroyl-CoA and three molecules of malonyl-CoA in the phenylpropanoid synthetic pathway3. In favonoid biosynthesis, several modifcation reactions, such as hydroxylation, methylation, glycosylation, and malonylation, occur to produce individual favonoids4. Te position and the number of hydroxyl groups, which are prone to undergo methylation or gly- cosylation, vary greatly among diferent favonoids 5. Methylation of oxygen (O-methylation), nitrogen (N-methylation) and carbon (C-methylation) is a universal process critical to all organisms 6. Te biosynthesis of O-methylated favonoids is catalyzed by O-methyltransferase (OMT) using S-adenosylmethionine (SAM) as a methyl group donor to methylate the favonoid hydroxy groups7. Plant OMT genes are categorized into two types, Types I and II, based on their molecular weight, amino acid sequence and bivalent ion dependency. Type I OMTs (cafeic acid OMT: COMT) are ion-independent enzymes with a molecular weight of 38–43 kDa. COMTs act on a wide range of substrates, such as myoinositol, chal- cones, scoulerine, cafeic acid, 5-hydroxyferulic acid, cafeoyl CoA ester and 5-hydroxyferuloylester8–10. Type II OMTs (cafeoyl-CoA OMT: CCoAOMT) are ion-dependent enzymes involved in the monolignol biosynthesis with the molecular weight of 23–27 kDa. CCoAOMTs act on a narrow range of substrates, which act only on 5-hydroxyferuloyl CoA and cafeoyl CoA 11,12. It is well known that favonoids play important roles in plants, such as inforescence pigments13, signal- ing factors14, antioxidant13,15, antifungal12 and anti-insect16. In addition, favonoids are important for human health due to their various bioactivities. Recently, it was reported that favonoids possess anti-cancer17,18 and 1Graduate School of Integrated Science and Technology, Shizuoka University, 836 Ohya, Suruga, Shizuoka 422-8529, Japan. 2Department of Bioresource Sciences, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga, Shizuoka 422-8529, Japan. 3School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan. 4These authors contributed equally: Mao Seoka and Gang Ma. *email: [email protected] SCIENTIFIC REPORTS | (2020) 10:15288 | https://doi.org/10.1038/s41598-020-72277-z 1 Vol.:(0123456789) www.nature.com/scientificreports/ Satsumamandarin Ponkanmandarin 80 25 9 Totalflavonoid TotalPMF Sinensetin ** 20 ** ** ) 60 6 15 ** DW ** -1 40 g ** 10 ** 3 ** 20 ** (mg 5 0 0 0 ontents 1.5 14 1.4 Tangeretin Nobiletin Heptamethoxyflavone dc 12 1.2 1.2 ** ** 10 1 ** 0.9 8 ** 0.8 ** ** * ** Flavonoi 6 0.6 0.6 ** 4 0.4 0.3 2 0.2 0 0 0 JulOct Dec JulOct Dec JulOct Dec Figure 1. Flavonoid accumulation in the favedos of Satsuma mandarin and Ponkan mandarin during fruit maturation. Te total favonoid content is the sum of identifed favonoids (narirutin, naringin, hesperidin, poncirin, isorhoifolin, rhoifolin, sinensetin, nobiletin, tangeretin and heptamethoxyfavone). Total PMF content is the sum of sinensetin, nobiletin, tangeretin and heptamethoxyfavone. Columns and bars represent the means ± SE (n = 3), respectively. Te signifcant diference between Satsuma mandarin and Ponkan mandarin is indicated by asterisks (*P < 0.05, **P < 0.01). anti-allergy19 activities in in vitro experiments. Moreover, favonoids have been found to exhibit anti-infamma- tory20,21, anti-obesity22, and neuroprotective properties23 in animal models. Nobiletin (3′,4′,5,6,7,8-hexamethoxyfavone), a polymethoxy favone (PMF), is abundant in citrus favedo (Supplementary Fig. S1)24. In recent years, the roles of nobiletin in human health have been investigated exten- sively, and it was suggested that nobiletin was benefcial to human health through its various activities, such as anti-depressant25, anti-allergy26, anti-pigmentation of skin27, and inhibition of heart failure progression28. Cur- rently, the neuroprotective efect of nobiletin has attracted increasing attentions, and nobiletin has been expected to be a potential neuroprotectant for the treatment of cerebral ischemia–reperfusion injury29, Alzheimer’s disease, and Parkinson’s disease 30. Terefore, the development of functional foods or supplements, which are rich in nobiletin, may be of great importance for human health. In nature, nobiletin specifcally accumulates in citrus fruits, and its content varies greatly among diferent citrus species. Nobiletin was markedly accumulates in species of the Acrumen and Aurantium sections, but not in the Fortunella and Poncirus species24. In particular, nobiletin is abundant in the favedos of Acrumen species, such as Dancy tangerine (C. tangerina) and Ponkan mandarin (C. reticulata). Recently, the identifcation and functional characterization of OMTs have been reported in several plants species4,31,32. However, studies on OMTs in citrus fruits are still limited, and the nobiletin biosynthetic pathway in citrus remains to be elucidated. In this study, we investigated favonoid accumulation in two citrus varieties, Satsuma mandarin (Citrus unshiu Marc.), which accumulates a low level of nobiletin, and Ponkan mandarin (Citrus reticulata Blanco), which accumulates a high level of nobiletin. Moreover, a novel O-methyltransferase gene CitOMT was isolated and its functions were investigated in vitro. Te results showed that recombinant protein of CitOMT, which was found to perform methylation the 3′-position hydroxyl groups of favones, was a key gene for the biosynthesis of nobiletin in citrus fruit. Tis study will contribute to the cultivation of high-quality citrus fruits that are rich in nobiletin and the production of supplements and nutrient-rich health foods. Results Flavonoids accumulation in citrus favedo. In this study, the accumulation of favonoids was inves- tigated in the favedos of Satsuma mandarin ‘Miyagawa-wase’ and Ponkan mandarin ‘Ohta Ponkan’ at three development stages: immature stage (July), transition stage (October), and mature stage (December). In the favedos, four favanones (narirutin, naringin, hesperidin, and poncirin), two favones (isorhoifolin and rhoi- folin), and four PMFs (sinensetin, nobiletin, tangeretin, and heptamethoxyfavone) were detected in Satsuma mandarin and Ponkan mandarin. As shown in Fig. 1, total favonoid content decreased in Satsuma mandarin and Ponkan mandarin during fruit maturation. Te total favonoid content in Ponkan mandarin at the mature SCIENTIFIC REPORTS | (2020) 10:15288 | https://doi.org/10.1038/s41598-020-72277-z 2 Vol:.(1234567890) www.nature.com/scientificreports/ stage decreased to approximately one quarter of that at the immature stage. In Satsuma mandarin, the total fa- vonoid content at the mature stage decreased to approximately half of that at the immature stage. In addition, the favonoid composition in the favedos was diferent between Satsuma mandarin and Ponkan mandarin. During fruit maturation, Satsuma mandarin accumulated high levels of favanones, which accounted for more than 85% of the total favonoids, whereas low PMFs, which accounted for less than 6% of the total favonoid content at the mature stage. In contrast, Ponkan mandarin accumulated higher levels of PMFs than Satsuma mandarin during the maturation. At the mature stage, PMFs accounted for more than 53% of the total favonoid content in favedo of Ponkan mandarin. In Ponkan mandarin, the major PMF is nobiletin, followed by sinensetin, tangeretin, and heptamethoxyfavone. During the fruit maturation, the contents of nobiletin, tangeretin, and sinensetin in Pon- kan mandarin were much higher than those in Satsuma mandarin, whereas the content of heptamethoxyfavone in Ponkan mandarin was lower than that in Satsuma mandarin (Fig. 1). The isolation of CitOMT. In this study, we performed a Blast search in the Citrus clementina v.10 genome
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