| Bryophytes,which are the earliest advanced plant in the evolution history of plant,underwent the transition of marine to terrestrial plants and suffered the enhanced ultraviolet light and the shortage of water.Bryophytes have evolved some special physiological structures to adapt to the terrestrial environment,including the cuticle of the surface of the bryophytes,which can be used to prevent the loss of water in the body and the invasion of ultraviolet iradiation.The aerial organs of most terrestrial plants are covered by a hydrophobic protective cuticle.The cuticle plays an important role in the process of plant growth and development.The cuticle contains lipid polymers,mostly produced by esterification of aromatic compounds and aliphatic compounds through acyltransferase.Some acyltransferase related to the biosynthesis of the cuticle has been identified in some higher plants.These enzymes belong to the BAHD/HXXXD acyltransferase family.At present,such enzymes have not been studied in bryophytes.In this study,after the preliminary screening of the transcriptome sequencing database of Conocephalum conicum(SRP076966),Conocephalum japonicum(SRP078647)and Marchantia emarginata(SRP078649),three genes annotated as BAHD/HXXXD family were identifid and named as CcHFT,CjHFT and MeHFT,respectively.In addition,each homologous gene was identified from the genomic database of Selaginella moellendorffii and Physcomitrella patens using the sequence of MeHFT as a query and named as SmHFT and PpHFT.MeHFT was found to be localized in the cytoplasm.The enzyme assay indicated that the favorite substrates for CcHFT,CjHFT,MeHFT and SmHFT were feruloyl-CoA and 1-dodecanol.The favorite substrates for PpHFT were caffeoyl-CoA and 16-Hydroxyhexadecanoic acid.In the experiments on the composition analysis of the plant cell wall of C.conicum,C.japonicum,M.emarginata and S.moellendorfii,aromatic compounds are mainly methyl coumarate and methyl ferulate,while Alkane part are mainly 1-hexadecanol and 1-stearyl alcohol.Bryophytes harbor an abundance of secondary metabolites,including flavonoids,lignin,terpenoids,coumarin and bibenzyls.The flavonoids have important biological activity and pharmacological value,such as anti-oxidation,anti-bacterial,anti-inflammatory,anti-cancer,anti-allergic and anti-cytotoxic.The biosynthetic pathway of flavonoids has been studied more clearly in higher plants.The related genes have been cloned and identified in many higher plants.The flavonoid synthase and flavanone-3-hydroxylase are the key oxidase in the flavonoid biosynthesis pathway.The catalytic function and evolutionary relationship of the two need to be elucidated.In previous work,six flavone synthase I(FNS I)genes were cloned and identified in the species of the bryophytes.The enzyme assay indicated that four liverwort FNSIs showed FNSI activity while two showed additional flavanones 2-hydroxylase function.Two F3Hs each from P.patens and S.moellendorffii showed both F3H and FNSI activity.Putative F3Hs from gymnosperms predominantly exhibited F3H activity with minor FNSI activity,while that of angiosperms only showed F3H activity.Sequence alignments and homology modeling of existing genes MpFNSI 2,MeFNSI 2,CjFNSI 2,CcFNSI 1,CjFNSI 1,PaFNSI 1,PpF3H,SmF3H,GbF3H,PsF3H,PrF3H,AtF3H,MtF3H and AtrF3H were performed,and an amino acid site was found as a key amino acid adjacent to the A ring of the substrate naringenin.This key amino acid was Y,M,F and P in the FNSI enzymes in the bryophytes,FNSI/F3H enyzmes in P.patens and S.moellendorffii and F3H enzymes in gymnosperms and angiosperms,respectively.Site-directed mutagenesis experiments demonstrated that the "star site" play an important role in the function and evolution in these enzymes.CjFNSI 1 and CjFNSI2 were ectopic expressed in A.thaliana and the transgenic plant and the wild type were fed with substrate naringinin.The content of apigenin in the transgenic plants overexpressing CjFNSI 1 and CjFNSI 2 were much higher than that in the wild type.The results indicated that CjFNSI 1 and CjFNSI 2 showed the same function as the enzyme assay in vitro. |
