Regulation Of Myricetin Biosynthesis By FLSs And F3'5'H In Morella Rubra

Chinese bayberry(Morella rubra Sieb.et Zucc)belongs to Myricaceae family,and is a characteristic fruit crop with important medicinal and economic value in China and other Asian countries.Myricetin with high biological activity has the maximum 3',4',5'-hydroxyl groups at B-ring,and is enriched in M.rubra.It was reported that myricetin was isolated from the bark of Myrica nagi for the first time.However,biosynthesis of kaempferol and quercetin is well studied in plants,whereas information about myricetin biosynthesis is quite limited.Identifying genes involved in myricetin biosynthesis and analyzing the mechanism of myricetin biosynthesis are of great significance to the development and utilization of natural product myricetin,and provide a theoretical basis for the improvement of flavonol biosynthesis.In this paper,Morella containing abundant myricetin was used to explore the biosynthesis mechanism of myricetin by transcriptome analysis,real-time quantitative PCR,prokaryotic recombinant protein,eukaryotic recombinant protein and heterologous transgenic tobacco.The main results are as follows:1.The metabolism of flavonols and anthocyanins in different tissues of M.rubra was analyzed by HPLC.There were four flavonol glycosides and one anthocyanin glycoside in M.rubra,among which myricitrin was detected as the major flavonol,and cyanidin 3-O-glucoside was detected as the major anthocyanin.Higher myricitrin contents were observed in leaves and flowers of M.rubra than that of fruits in both cultivars,and quercetin glycosides accumulated significantly during �Biqi' fruit development,but not in �Dongkui' fruit.Cyanidin 3-O-glucoside did not accumulate in leaves and young fruits of M.rubra,and accumulated significantly in fruit maturity.2.Correlation analysis showed that in different tissues of �Biqi' and �Dongkui' the expression level of MrF3'5'H was well correlated with myricitrin content,the expression level of MrFLS2 gene was well correlated with quercetin glycoside content,and No MrFLS transcript was found to be highly correlated with the accumulation of myricitrin.Interestingly,during the fruit development of both cultivars,the expression level of MrFLS1 was positively correlated with myricitrin content.3.Amino acid sequence analysis showed that both MrFLS1 and MrFLS2 belongs to the2-oxoglutarate dependent dioxygenase family and contained typical 2-oxoglutarate and ferrous dependent dioxygenase signature motifs.Activity assay of recombinant proteins showed that both MrFLS1 and MrFLS2 could convert dihydroflavonol to flavonol,and had some F3 H activity,which could catalyze the hydroxylation of flavanone to dihydroflavonol.For substrate specificity,differences in substrate affinity was rather small between MrFLS1 and MrFLS2 towards catalyzing the three dihydroflavonols,while MrFLS2 exhibited significantly higher turn over rates of dihydroquercetin compared to dihydromyricetin and dihydrokaempferol.Molecular docking technology assisted to verify the affinity of MrFLS1 and MrFLS2 for dihydroflavonol,results showed that both the two FLS proteins from M.rubra have weaker force on dihydromyricetin than dihydroquercetin and dihydrokaempferol.In addition to hydroxylation of flavanones to dihydroflavonols,MrF3 H can also catalyze the oxidation of dihydroflavonol to flavonol.Neither FLS proteins nor F3 H protein from M.rubra can further catalyze the compounds produced itself.Compared with wild-type tobacco,the contents of rutin and kaempferol 3-O-rutinoside in leaves and flowers of overexpressing MrFLS1 or MrFLS2 tobaccos were significantly increased,but the accumulation of myricetin or its related derivatives was not detected.4.MrF3'5'H belongs to CYP75 A subfamily,which contained typical heme binding domain,hydroxylation activity site and substrate recognition site.The activity analysis of recombinant protein in yeast showed that MrF3'5'H could catalyze the hydroxylation of flavanones(naringenin and eriodictyol),dihydroflavonols(dihydrokaempferol and dihydroquercetin)and flavonols(kaempferol and quercetin)at 3' or 5' sites,respectively,and its catalytic activity for naringenin and kaempferol was significantly higher than that of the other four compounds.Compared with wild-type tobacco,the content of myricetin 3-O-rutinoside in leaves and flowers of overexpressing MrF3'5'H tobaccos was significantly increased,indicating that MrF3'5'H is a key factor in myricetin biosynthesis.MrF3'H was a typical F3'H protein that could only catalyze hydroxylation at 3' site but failed to hydroxylation at 5' site.5.The function of FLS proteins from grape,tomato,apple and tobacco was similar to that from Morella,all of them could catalyze the oxidation of dihydroflavonols to flavonols and had some F3 H activity.Interestingly,the catalytic activity of Morella FLS proteins on dihydromyricetin was higher than that of the other four FLS proteins,which may be one of the reasons that myricetin was able to accumulated in M.rubra.Function of plant P450 proteins explored in this paper was different.Although VvF3'5'H showed the highest amino acid identity with that of MrF3'5'H,the preference for kaempferol by F3'5'H from Morella compared to VvF3'5'H enzyme was clear,which may be another key factor that myricetin was enriched in M.rubra.Different with MrF3'H,the other four F3'H enzymes VvF3'H,Sl F3'H,Md F3'H and Nt F3'H all contained some F3'5'H activity.Taken together,our results showed that both MrFLS and MrF3'5'H activity are essential in myricetin biosynthesis,where MrF3'5'H is postulated to be important for driving pathway flux towards the trihydroxylated flavonol by hydroxylating kaempferol without the need of a DHM specific MrFLS in M.rubra.