Functional Characterization Of CrOMT1 And CrOMT2 Genes Involved In The Biosynthesis Of Polymethoxylated Flavones In Citrus

Polymethoxylated flavones(PMFs),which accumulate almost exclusively in Citrus has gained much attention due to their extent bioactivity.Methoxy groups on PMFs are catalyzed by Omethyltransferase(OMT).Characterization of OMT genes involved in PMF biosynthesis is of great importance in the investigation of PMF biosynthesis pathway in citrus.‘Ougan'(Citrus reticulata CV.Suavissima)fruit is rich of PMFs,and is used in this paper for the identification and functional characterization of OMT genes involved in PMF biosynthesis.The main results are as follows:1.A comprehensive study regarding OMT gene family was conducted,including phylogenetic evolution,gene structure,protein structure and gene expression pattern of OMT gene family members,based on Citrus clementina genome and transcriptome.A total of 64 OMT genes were identified from C.clementina genome and could be divided into two subfamilies according to phylogenetic analysis at the protein level,i.e.,caffeic acid O-methyltransferase(COMT)(57)and caffeoyl Co A Omethyltransferase(CCo AOMT)(7).The gene structures,protein domains and motifs of the two subfamilies showed obvious difference,while the OMT members with close relationship displayed a similar pattern.OMT gene family members showed different expression patterns in different development stages of citrus fruit and exposure of fruit to UV-B radiation based on the transcriptome data.2.Identification and metabolic pattern of the flavonoid component in the peel of ‘Ougan' are performed using HPLC and LC-MS/MS.A total of nine flavonoids were identified from citrus peel,including six flavanone glycosides and three PMFs.The content of flavanone glycosides declined both in the flavedo and albedo,while PMFs specifically accumulated in the flavedo and showed a trend from rising to declining during developmental stages of ‘Ougan' fruit.Exposure of fruit to UV-B treatment had no significant effect on the accumulation of flavanone glycosides in the peel,while it enhanced the accumulation of PMFs.3.The transcript level of CrOMT1 was positively correlated with PMF accumulation by both tissue and developmental stages of ‘Ougan' fruit and in response to UV-B irradiation.CrOMT1(belonging to CCo AOMT subfamily)was the first candidate gene associated with PMF content in citrus.Recombinant CrOMT1 could catalyze a wide range of flavonoids with neighboring hydroxy moieties in vitro,and showed the highest catalytic efficiency for flavones,The enzyme favored 5,6,7,8,3',4'-hexahydroxy flavones(PMF-type)analogs with methylation at positions 6,8 and 3',although the catalytic activity toward 3' was relatively lower.These preferences(especially the 6-and 8-Omethylation)corresponded precisely with the essential methylation sites for the naturally occurring PMFs.Transient overexpression analysis in citrus peel showed that overexpression of CrOMT1 enhanced the accumulation of PMFs.These results together indicated that CrOMT1 was involved in the PMF biosynthesis in citrus.4.Sequence homology analysis revealed that the amino acid sequence of CrOMT2(belonging to COMT subfamily)was highly consistent(identity 73.35%)with that of flavonol 3'-Omethyltransferase(At OMT1)from Arabidopsis thaliana.The transcript level of CrOMT2 was correlated with PMF content in different developmental stages and tissues of ‘Ougan' fruit,while UVB treatment had no significant effect on its transcript level.Recombinant CrOMT2 could accept a wide array of flavonoids with neighboring hydroxy moieties in vitro and favored the 3' position of PMFtype substrate analogs.Notably,it also displayed methylation at 7 position of PMF-type substrate analogs with relatively lower activity.Transient overexpression of CrOMT2 in citrus peel increased the content of PMFs.Taken together,CrOMT2 was another gene involved in PMF biosynthesis in citrus.