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.

Scientific RepoRtS | (2020) 10:15288 | https://doi.org/10.1038/s41598-020-72277-z www.nature.com/scientificreports/ stage decreased to approximately one quarter of that at the immature stage. In Satsuma mandarin, the total flavonoid content at the mature stage decreased to approximately half of that at the immature stage. In addition, the flavonoid composition in the flavedos was different between Satsuma mandarin and Ponkan mandarin. During fruit maturation, Satsuma mandarin accumulated high levels of flavanones, which accounted for more than 85% of the total flavonoids, whereas low PMFs, which accounted for less than 6% of the total flavonoid 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 flavonoid content in flavedo of Ponkan mandarin. In Ponkan mandarin, the major PMF is nobiletin, followed by sinensetin, tangeretin, and heptamethoxyflavone. During the fruit maturation, the contents of nobiletin, tangeretin, and sinensetin in Ponkan mandarin were much higher than those in Satsuma mandarin, whereas the content of heptamethoxyflavone 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 databases (https ://www.phyto zome.net/) using the sequence of ROMT-9 as a query, which has been reported to have strict specificity for the 3′-hydroxy group of flavonoids 33 . One OMT gene (Ciclev10020814m.g) was identified in the citrus genome database. We isolated the full-length CDS of OMT (CitOMT) from Satsuma mandarin (LC516612) and Ponkan mandarin (LC616611) using the primers designed according to the sequences obtained from the citrus genome database (Supplementary Table S1 . 2a). CitOMT showed conserved motifs (Motif I-V), which may be involved in interactions with the cofactor SAM 5,34 . The amino acid residues (His-270, Glu-298 and Glu-330 in CitOMT) are known as the catalytic residues in MsIOMT 6 . A phylogenetic tree of OMTs was constructed using Phylogeny. fr (https ://www.phylo geny.fr/simpl e_phylo geny.cgi), and we found that CitOMT was categorized within the plant COMTs (Fig. 2b).

Expression of flavonoid biosynthetic genes in citrus flavedo.
In this study, the expression of flavonoid metabolic genes (CitCHS1, CitCHS2, CitCHI, CitFNS, CitF3′H, and CitF6H), as well as CitOMT was investigated in the flavedos of Satsuma mandarin and Ponkan mandarin during fruit maturation. Sets of TaqMan probes and primers were designed based on the common sequences of the two varieties using Primer Express software (Supplementary Table S2). In Satsuma mandarin, the expression of CitCHS1 and CitCHS2, which are related to the biosynthesis of chalcone, decreased to a low level at the transition stage, and then increased rapidly during maturation. In Ponkan mandarin, the expression of CitCHS2 decreased at the transition stage, whereas the expression of CitCHS1 increased slightly during fruit maturation. The expression levels of CitCHS1 and CitCHS2 in Satsuma mandarin was 3 times and 2.2 times higher than those in Ponkan mandarin at the mature stage, respectively. The expression levels of CitCHI, CitFNS, CitF3′H, CitF6H, and CitOMT increased gradually in the two citrus varieties during maturation. In Ponkan mandarin, the expression levels of CitFNS, CitF6H, and CitOMT were much higher than those in Satsuma mandarin during fruit maturation (Fig. 3).

Enzyme activity of CitOMT in vitro.
In order to investigate the function of CitOMT in citrus fruit, the cDNA of CitOMT was cloned into a pGEX-6P-1 vector, and the recombinant plasmid was transformed into E. coli strain XL1-Blue Competent Cells. Recombinant protein of CitOMT was obtained and affinity purified using PreScission Protease. The recombinant protein of CitOMT was detected as a single band by SDS-PAGE. The molecular weight of CitOMT was estimated 40.0 kDa, which was consistent with the estimated amino acid molecular weight.
To further confirm the role of CitOMT, the reaction product of 3′,4′-dihydroxyflavone was analyzed by Direct Analysis in Real Time (DART) MS on positive mode. Mass spectrometry showed that the O-methylated product of 3′,4′-dihydroxyflavone (Peak P2 at 18.9 min, Fig. 4d) had a parent ion peak [M + H] + at an m/z of 269.081, and its formula was calculated as C 16 H 13 O 4 . The results of mass spectrometry analysis suggested that the O-methylated product of 3′,4′-dihydroxyflavone (P2) was a mono-methylated flavone (Fig. 4e). In addition, because the hydroxy group on its 4′-position cannot be methylated by CitOMT (Table 1), it was indicated that CitOMT may function to methylate the 3′-hydroxy group of flavones in vitro.

Discussion
Citrus plants are a rich source of flavonoids, and the accumulation of flavonoids is closely related to the appearance, taste, as well as the nutritional values of the fruit. The major flavonoids accumulated in citrus fruit are divided into two groups, flavanone glycosides, such as naringin, hesperidin, neo-hesperidin, and PMFs, such as, nobiletin, sinensetin, and tangeretin 35 . It is well known that the accumulation of flavonoids in citrus fruit depends on several factors, including the genetic origin, maturity stage, and the different parts of the fruit (flavedo, albedo, seeds, Supplementary Figure S1). Moreover, the composition of flavonoids varies greatly among different citrus species 24,36,37 . In the present study, the accumulation of flavonoids was investigated in two citrus varieties, Satsuma mandarin and Ponkan mandarin. The results showed that there were significant differences in flavonoid composition between Satsuma mandarin and Ponkan mandarin (Fig. 1). In Satsuma mandarin, high amounts of flavanones were accumulated, while the contents of PMFs were extremely low, which accounted for less than 6% of the total flavonoid in the flavedo of mature fruit. In Ponkan mandarin, in contrast, high levels of PMFs were accumulated in the flavedo. In the flavedo of mature fruit, the PMFs contents in Ponkan mandarin accounted for more than 53% of total flavonoid. In Ponkan mandarin, four kinds of PMFs, sinensetin, nobiletin, tangeretin and heptamethoxyflavone, were detected, and among them nobiletin was found to be the major PMF accumulated in the flavedo, followed by sinensetin, tangeretin, heptamethoxyflavone. In the mature fruit, the nobiletin content in Ponkan mandarin was approximately 13 times higher than that in Satsuma mandarin. In the study of Zohra et al., the accumulation of nobiletin and tangeretin was investigated in 11 citrus cultivars. The results revealed that there was a significant correlation between the accumulation of nobiletin and tangeretin in the flavedos of citrus fruit, and nobiletin tended to accumulate at higher level than tangeretin in the flavedos of the 11 citrus cultivars 38 .
To date, although flavonoid accumulation has been extensively reported in different citrus cultivars, the molecular mechanism regulating the biosynthesis of PMFs, especially nobiletin, in citrus fruit is still unclear. In the present study, to further elucidate the high accumulation of nobiletin in Ponkan mandarin, the expression of flavonoid biosynthetic genes (CitCHS1, CitCHS2, CitCHI, CitFNS, CitF3′H, CitF6H, and CitOMT) was investigated (Fig. 3). The results showed that the expression levels of genes that are responsible for PMF biosynthesis  OMTs that transfer the methyl group of SAM to the hydroxyl group of flavonoids are key enzymes for PMF biosynthesis. Plant OMTs are a large gene family, which are categorized into two types, COMT and CCoAOMT, according to their molecular weight and bivalent ion dependency. In plants, numerous OMT genes have been identified, and their functions have been extensively investigated in various plant species, such as Arabidopsis 39 , barley 40 , mango 41 , rice 42 , tomato 43 , and sweet basil 44 . In citrus, it was reported that 58 OMT genes existed and were unevenly distributed on the nine chromosomes of Citrus sinensis. Among them, 27 OMTs were predicted to be involved in the O-methylation of flavonoids from the DGE and qRT-PCR analysis 45 . To date, two OMTs, CdFOMT5 and CrOMT1, have been isolated and their functions were characterized in citrus fruit. Recombinant proteins of CdFOMT5 and CrOMT1 exhibited high substrate specificity and regioselectivity. Recombinant CdFOMT5 demonstrated methylation activity for the 3-,5-,6-, and 7-hydroxyl groups of flavones in vitro 31 . Different from CdFOMT5, CrOMT1 is a CCoAOMT-like enzyme, and it had a strong preference for flavones with highest catalytic efficiency at the 6-and 8-hydroxyl groups of flavones in vitro 46 . In the present study, we isolated a novel OMT gene (CitOMT) from Satsuma mandarin and Ponkan mandarin, using the sequence of ROMT-9 as a query, which has been reported to have strict specificity for the 3′-hydroxy group of flavonoids 33 . In the phylogenetic analysis, it was shown that CitOMT was clustered into COMT, which is independent of a cation and known to have the enzymic activity for flavonoids [8][9][10] (Fig. 2). In addition, multiple sequence alignment of CitOMT with other plant OMTs suggested that the amino acid sequence of CitOMT had the same conserved sequences including SAM binding residues and catalytic residues as other plants OMTs 5,6,34 , which indicates that CitOMT may possess O-methyltransferase activity with flavonoids in citrus fruit (Fig. 2).
Sequence analysis showed that CitOMT shared 53.8% and 23% identity with CdFOMT5 and CrOMT1 at the amino acid level, respectively. The relatively low identity levels indicated that the functions of CitOMT may be different from CdFOMT5 and CrOMT1 in citrus fruit. In the present study, to investigate the roles of CitOMT in citrus fruit, the cDNA of CitOMT was isolated from Ponkan mandarin, and cloned into a pGEX-6P-1 vector. A single band of the recombinant CitOMT protein was detected at approximately 40.0 kDa by SDS-PAGE. Functional analysis showed that the recombinant protein of CitOMT methylated two flavones (3′,4′,5,7-tetrahydroxyflavone and 3′,4′-dihydroxyflavone), whereas it had no activity with flavanones (3′-hydroxyflavanone, 4′-hydroxyflavanone, naringenin, hesperidin) and isoflavone (daidzein) in vitro. To further confirm the methylation position of CitOMT in flavones, the substrates, 4′-hydroxyflavone, 7-hydroxyflavone, 7,8-dihydroxyflavone, were also tested in vitro assays, and no new product was detected. These results suggested that CitOMT cannot methylate flavones at positions 4′, 7, or 8 in vitro. In addition, the O-methylated product of 3′,4′-dihydroxyflavone  Fig. 4d) was analyzed by DART MS, and the results showed that the O-methylated product of 3′,4′-dihydroxyflavone (P2) was a mono-methylated flavone (Fig. 4e). Because the hydroxy group on its 4′-position cannot be methylated by CitOMT (Table 1), it was indicated that CitOMT might have the function to methylate the 3′-hydroxy group of flavones in vitro.
In conclusion, the roles of a novel OMT gene (CitOMT) in nobiletin biosynthesis was investigated in two citrus varieties, Satsuma mandarin, which accumulates a low level of nobiletin, and Ponkan mandarin, which accumulates a high level of nobiletin. The results showed that the expression level of CitOMT in the flavedo of Ponkan mandarin was much higher than that in Satsuma mandarin during fruit maturation. In addition, functional analysis suggested that CitOMT was a key gene responsible for nobiletin biosynthesis in citrus fruit. Recombinant protein of CitOMT had methylation activity to transfer a methyl group to the 3′-hydroxy group of flavones in vitro, which is vital for nobiletin biosynthesis. The results presented in this study may contribute to elucidating the mechanism of nobiletin biosynthesis in the flavedo of Ponkan mandarin, which will provide strategies to improve flavonoid accumulation in citrus fruit.

Methods
Plant materials. Satsuma mandarin 'Miyagawa-wase' (C. unshiu Marc.) and Ponkan mandarin 'Ohta Ponkan' (C. reticulata Blanco) were grown at the Center for Education and Research in Field Sciences (Shizuoka, Japan). Flavedo was separated from sampled fruits, frozen in liquid nitrogen immediately, and kept at − 80 °C until use.
Flavonoid analysis. Powdered flavedos were freeze dried. Portions (20 mg) were extracted by homogenization and ultrasonicating in 400 mL of DMSO:methanol (1:1, v/v) at room temperature. After centrifugation at 21,500 × g for 10 min, the supernatant was collected, and the remaining residue was extracted twice with 300 μL of the same solvent. In total, 1 mL of supernatant was filtered through a membrane filter, TORAST Disc (hole diameter: 0.22 μm, SHIMADZU GLC, Japan).  www.nature.com/scientificreports/ The high-performance liquid chromatography (HPLC) system consisted of a pump, autosampler, photodiode array detector, column oven (JASCO, Japan), and a YMC-UltraHT Pro C 18 column (100 × 3.0 mm i.d. S-2 μm, 12 nm; YMC, Japan). The detector was monitored at 274, 310, 324, 338, and 362 nm. A two-solvent gradient system of 1% phosphoric acid (A) and acetonitrile-methanol (1:1, v/v) (B) was used. The gradient program consisted of three periods: (1) 0-33 min, 78% A, (2) 33-47.5 min, 16% A, (3) 47.5-75 min, 78% A. The flow rate was 0.6 mL min −1 , the column was operated at 44 °C, and the sample injection volume was 10 μL. Standard flavonoids (eriocitrin, narirutin, naringin, hesperidin, rhoifolin, isorhoifolin, diosmin, sinensetin, nobiletin, tangeretin and heptamethoxyflavone) were purchased in FUJIFILM Wako Pure Chemical Corporation (Japan). The flavonoids were identified by comparing their retention times and UV spectra with those of authentic standards stored in a data processor. The concentration of each flavonoid was calculated from the integrated peak area of the sample and the corresponding standard. Each sample was replicated three times, and mean values and standard error were calculated. Gene expression. Total RNA was extracted from the flavedo of Satsuma mandarin and Ponkan mandarin according to the method described by Ikoma et al. 47 . The total RNA was purified using a RNeasy Mini Kit (Qiagen, Germany) and treated with DNase (Takara, Japan) digestion on the column. The cDNA was synthesized from 600 ng of purified RNA and a random hexamer primer at 37 °C for 60 min using TaqMan Reverse Transcription Regents (Applied Biosystems, USA).

CitOMT
Real-time PCR was performed to investigate the expression of CitCHS1, CitCHS2, CitCHI, CitFNS, CitF3′H, CitF6H, and CitOMT. TaqMan probes and sets of primers were designed based on the common sequences with Primer Express software (Supplementary Table S2 Table S3). The cDNA fragments of CitOMT-coding gene were digested by BamHI and XhoI and purified using a GFX PCR DNA and Gel Band Purification Kit (GE Healthcare, Japan). The purified DNA fragment was ligated into the expression vector pGEX-6P-1 (Amersham Bioscience, UK), which had been digested with the same restriction enzymes. The constructed plasmid was transformed into E. coli strain XL1-Blue Competent Cells (Agilent Technology, Japan). For protein expression, 2 mL of overnight culture of the transformants harboring the gene of CitOMT was used to inoculate a 200 mL culture in a 2 × YT medium (5 g yeast extract, 8 g bacto-tryptone, and 2.5 g NaCl) to OD 600 0.8 at 37 °C with shaking. The expression and purification of recombinant protein of CitOMT were carried out using the method described by Kato et al. 48 . The expression of protein was induced by the addition of isopropyl-β-D-thiogalactoside (100 μM) at 27 °C for 17 h. The E. coli cells were collected by centrifugation at 3,300 × g for 10 min, and then resuspended in 20 mL of 1 × PBS stock solution of a GST Bulk Kit (GE Healthcare, Japan) containing 5 mM DTT. Suspensions containing the E. coli cells were lysed by sonication, and then 1% (v/v) of Triton X-100 was added and shaken on ice for 30 min. After centrifugation at 3,300 × g for 90 min, recombinant protein of CitOMT bonded to Glutathione Sepharose 4B (GE Healthcare) was washed twice with wash buffer [1 × PBS stock solution, 5 mM DTT, and 1% Triton X-100 (v/v)] and equilibrated twice with cleavage buffer [50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, and 0.05% Triton X-100 (v/v)]. The recombinant protein was released using PreScission Protease (GE Healthcare, Japan) in cleavage buffer at 4 °C for 16 h. The recombinant protein was analyzed by SDS-PAGE using a 12.5% (v/v) polyacrylamide gel and WIDE-VIEW Prestained Protein Size Marker (Wako, Japan) using PhastSystem (Amersham Bioscience, US).