Functional And Evolutionary Investigation Of Flavone Synthase From Liverworts

Flavonoids represent a large group of polyphenolic secondary metablites that are extensively distributed in the plant kingdom.Most of them are important source of pigments.Based on the oxidation state of C-ring and substitution pattern of flavone skeleton,several subgroups were distinguished including flavonones,flavones,dihydroflavonols,flavonols and anthocyanins et al.Flavonoids can help the plants to cope with the biotic and abiotic ecological pressures.For example,flavonoids screen tissues from UV irradiation,help plants to adapt to the conditions of water shortage and protect plants from microorganisms attacking.Flavonoids also play a very important role in nutritional and medicinal application.Although flavonoids are widely distributed in higher plants,they can also be found in liverworts and horsetails.There are different types of flavonoids in plants with different evolutionary status.With the improvement of the status of the plant,the complexity of flavonoids in plants is higher.The biosynthetic pathway of flavonoids in higher plants has been studied extensively,and it was proved that flavonoids were derived from phenylpropane and malonic acid pathway.Four 2-oxoglutarate dependant dioxygenases(20DDs),flavonone 3?-hydroxylase(F3H),flavone synthase I(FNSI),flavonol synthase(FLS)and anthocyanin synthase(ANS),are involved in the flavonoids biosynthesis.Previously,a certain number of FNSIs were characterized from Apiaceae and was suggested locally in ancestral Apiaceae plants through F3H gene multiplication and developed much later than F3H.Recent years,FNSI also has been reported in other plants,including liverworts.F3H is in the key position towards the synthesis of flavonols and anthocyanins during the biosynthesis of flavonoids and is widely distributed in angiosperms,but there are few reports about F3H in lower plants.Morever,according to bioinformatics analysis,there were no evident F3H orthologs in Selaginella and Physcomitrella,although they produce dihydroflavonol-derived metabolites.Thus,the origin and evolution of F3H is an unsolved issue in the biosynthesis of flavonoids.In our laboratory,a flavone synthase called PaFNSI was isolated from Plagiochasma appendiculation.It can catalyze flavanone to 2-hydroxyflavanone and flavone and the alignment result indicated that FNSI in liverworts has higher sequence identity with F3H than FNSI.The above results indicated a probable evolutional connection between liverworts FNSIs and F3Hs.However,due to the lack of genetic informations of bryophytes and pteridophytes,the evolutionary relationships between the two enzymes in primary plants has not been reported before this study.In our study,searching the transcriptome data derived from the five liverwort species Plagiochasma appendiculatum(SRP073827),Marchantia paleacea(SRP078650),Marchantia emarg inata(SRP078649),Conocephalum conicum(SRP076966)and Conocephalum japonicum(SRP078647)using the previously reported PaFNSI(Genbank Accession number KJ439220,Han et al.,2014)as a query,eight cDNAs with homology to PaFNSI were identified.They were designated as PaFNSI 2,PaFNSI 3,MpaFNSI 2,MpaFNSI 3,MeFNSI 2,CcFNSI 1,and CjFNSI 1,2.In addition,when using the Pfam defined 20G-Fe(?)oxygenase superfamily signatures PF03171(http://pfam.xfam.org/)to search the genome sequence of Physcomitrella patens and Selaginella moellendorffii,we identified nine putative F3H genes.Collectively,they were designated as PpF3H,PpF3H-1,PpF3H-2,SmF3H,SmF3H-A,SmF3H-B,SmF3H-C,SmF3H-D and SmF3H-E.F3H and FNSI in plants with different evolutionary status,including F3H genes in gymnosperms(Picea sitchensis,Pinus radiate and Ginkgo biloha)and angiosperms(Amhorella trichopoda,Arabidopsis thaliana and Medicago truncatula)as well as PcFNSI were also investigated in this research.Sequence alignment results indicate that FNSIs from the liverworts all have the corresponding key amino acids that bind to 2-OG and Fe2+.The results of the phylogenetic tree analysis showed that the FNSIs from liverworts and the F3Hs of the P.patens and S.moellendorffii were located between the F3H/FNSI and FLS/ANS branches,together with the PaFNSI studied earlier.We cloned the genes of liverworts,P.patens,S.moellendorffii,A.thaliana and M.truncatula from plants and commercially synthesized the Picea sitchensis,Pinus radiate,Ginkgo biloba and Amborella trichopoda genes by Genwiz.The proteins encoded by thesis genes were expressed as polyhistidine-tagged fusions in E.coli BL21(DE3)and purified for the investigation of their biochemical activity in vitro activity.The results of enzyme activity combined with phylogenetic tree analysis showed that FNSIs are ubiquitous in liverworts and can be classified into two types,one catalyze the desaturation of flavonone and the other showed additional unusual 2-hydroxylation function.Morever,the FNSIs in this study also showed siginficant FLS activity.Our data were in contrast to the common conception which implied that F3Hs emerged earlier than FNSIs.In P.patens and S.moellendorffii,the functional enzymes showed both FNSI and F3H activity.In gymnosperms,the functional enzymes showed significant F3H activity with slight FNSI function.The PcFNSI of Apiceae could catalyze the production of apigenin by naringenin and also exhibited weak FLS activity,which catalyzes the formation of small amounts of kaempferol by dihydrokaempferol.The biochemical function characterization and phylogenetical analysis of enzymes in liverworts,P.patens,S.moellendorffii,gymnosperms and angiosperms indicated that F3Hs were evoluted from FNSIs through the intermediate bifunctional enzymes FNSI/F2H and FNSI/F3H.In order to study the functional domains and key amino acids associated with the formation of 2-position hydroxylated products,the domain exchanging and site-directed mutations were performed on the bifunctional CjFNSI 1 and monofunctional CjFNSI 2 from the C.Japonicum,respectively.It was found that the C-terminus of CjFNSI 1 was a domain that determined the formation of a 2-position hydroxylated product and that single point mutations did not allow a reversible change in the function of CjFNSI 1 and CjFNSI 2.We can not receieve soluble protein from CjFNSI 1 multi-point mutation,indicating that these amino acids work together to ensure the protein expression and they play an important role in the production of 2-hydroxy products.CjFNSI 2 multi-point mutations can not produce 2-hydroxy products.There were no functional F3H being detected in liverworts.