| Flavonoids are important secondary metabolites found in many plant species.These metabolites have a variety of pharmacological activities and play an important role in plant growth and development,and participate in plant resistance to abiotic stress.Flavone synthaseⅠs(FNS Ⅰs)convert flavanones to flavones.Flavanone 3-hydroxylases(F3Hs)catalyze the oxygenation of flavanones C-3 to form dihydroflavonols,which lead to the formation of flavonols and anthocyanins,respectively.FNS Ⅰs and F3Hs belong to the 2-oxoglutarate/Fe(Ⅱ)-dependent dioxygenases.F3Hs are mainly characterized in angiosperms.Our previouS Ⅰnvestigation revealed that the FNS Ⅰ and the bifunctional enzyme FNS Ⅰ/F2H emerged in liverworts and moss FNS Ⅰ has weak F3H function.Further evolution led to the emergence of F3H with a minor level of FNS Ⅰ activity in gymnosperm species.These results suggested that the canonical F3H of angiosperms probably evolved from liverworts FNS Ⅰthrough FNS Ⅰ/F3H bifunctional enzymes.As important intermediate plants in the evolution from liverworts to seed plants,ferns have more common flavonols and anthocyanins than liverworts,but there are no reports on FNS Ⅰs and F3Hs.Therefore,mining FNS Ⅰs and F3Hs from ferns will help to analyze their characteristics,and further clarify the relationship between FNS Ⅰs and F3HS Ⅰn plants.Psilotum nudum,the only plant of the Psilotaceae in China,belongs to the basal group of ferns and is rich in flavonoids.One putative 2ODD sequence annotated as flavanone 3β-hydroxylase was identified from transcriptomic sequences of P.nudum.This gene was designated as Pn2ODD.Phylogenetic analysis was performed in the MEGA v5.0 based on the polypeptide sequences of the 2ODDs from other plants.The results implied that the Pn2ODD was located at the base of gymnosperms F3H and angiosperms F3H.Multiple sequence alignment revealed that the Pn2ODD showed higher identity with Ginkgo biloba F3H and the key amino acid residue related to FNS Ⅰ and F3H activity was the same as that of seed plants F3H,but different from Tyr of liverworts FNS Ⅰ.The enzymatic analysis results showed that Pn2ODD showed the activities of FNS Ⅰ and F3H that could convert naringenin to apigenin and dihydrokaempferol.Therefore,it was renamed PnFNS Ⅰ/F3H.The PnFNS Ⅰ/F3H had a broad substrate specificity and catalyzed the flavanones to generate the corresponding flavones.According to the analysis of the evolutionary relationship between FNS Ⅰs and F3Hs in the early stage of the laboratory,site-directed mutagenesis was performed based on the homology modeling with AtANS(2brt)as a template.The enzymatic activity of the PnFNSⅠ/F3H-P228Y and PnFNS Ⅰ/F3H were further analyzed.The results showed that the function of PnFNS Ⅰ/F3H-P228Y was shifted from F3H to F2H with the remaining FNS Ⅰ activity.It indicated that there may be an evolutionary relationship between FNS Ⅰ/F2H and FNS Ⅰ/F3H.To verify the activity of PnFNS Ⅰ/F3H in plants,the gene encoding PnFNS Ⅰ/F3H was transformed into the Arabidopsis thaliana tt6 and dmr6 mutant.Flavonoids profiles were analyzed and the results showed that the contents of flavones,flavonols,and anthocyanins increased in PnFNS Ⅰ/F3H-tt6 transgenic plants.The flavone apigenin and flavonol quercetin in PnFNS Ⅰ/F3H-dmr6 transgenic plants were also increased.According to those results,we found that the flavones in two transgenic plants increased more significantly than flavonols,which proved that PnFNS Ⅰ/F3H had FNS Ⅰ activity and weak F3H activity in plants.This is the first time that FNS Ⅰ/F3H enzyme was discovered in P.nudum.The activity of PnFNS Ⅰ/F3H-P228Y was transformed from FNS Ⅰ/F3H to FNS Ⅰ/F2H by site-directed mutation.PnFNS Ⅰ/F3H can increase the content of flavonoids in Arabidopsis.This study is helpful to further clarify the relationship between angiosperms F3Hs and liverworts FNS Ⅰs and provides a candidate gene for the synthetic biology of flavonoids. |
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