Investigation On Flavonoids Biosynthesis Of Cyclosorus Parasiticus Based On Metabolomics And Transcriptome Analysis

Pteridophytes produce a variety of secondary metabolites including terpenoids and aromatic compounds.These secondary metabolites possess many biological activities and are used as lead compounds for new drug development.The fern species Cyclosorus parasiticus is rich in flavonoids including flavone,flavonone,flavonol and anthocyanin.Flavonoids are widely distributed in the plant kingdom and play a key role in adapting to changes in the environment.Meanwhile,flavonoids show broad bioactivities including anti-inflammatory,anti-fungal,anti-anaphylaxis and anti-tumor.In addition,flavonoids are the main sources of plant pigments.The flavonoids biosynthesis pathway has been extensively studied in seed plants.However,relatively few studies have been performed in pteridophytes.In the present investigation,the metabolomics and transcriptome were used to elucidate the flavonoids biosynthesis and regulation in the fern C.parasiticus.In addition,the selected key genes were further functional and structural characterized.To elucidate the flavonoids biosynthesis in the fern C.parasiticus,two different development stages leaves were chosen for study.The leaves without spores and with brown spores were designated as ’S1’ and ’S2’,respectively.The anthocyanin content of S2 was higher than that of S1.The leaves of S1 and S2 were used for metabolomics and transcriptome analysis.The leaves samples were analyzed using an UPLC-MS/MS system.The results indicated that flavonoids metabolites such as flavones,isoflavones,flavonols,proanthocyanidins,anthocyanins,flavonoid lignans,C-glycosyl flavones and catechin derivatives were detected.PCA and OPLS-DA analysis results showed that metabolites were significantly different between S1 and S2 and the flavonoids content in S2 was much higher than that in S1.In addition,KEGG classification and enrichment results indicated that significantly different metabolites mainly related to flavonoid biosynthesis pathway.To investigate the mechanism underlying the flavonoids accumulation in S2,cDNA libraries were prepared from leaves of S1 and S2 and subjected to RNA-seq analysis.GO functional analysis was performed to characterize all the assembled unigenes,and the results showed that most of the differently expressed genes are related to metabolism.Furthermore,to identify the metabolic pathways that were enriched,a pathway-based analysis was conducted through the KEGG pathway database.The results revealed that the flavonoids and phenylpropanoid biosynthesis pathway were significantly enriched.These results demonstrated that the expression of unigenes which related to flavonoids metabolism was higher in S2 than S1.The transcript levels of these key genes encoding flavonoids biosynthesis enzymes were further confirmed using qRT-PCR.The integrated analysis of transcriptomic and metabolomic data indicated that the differentially expressed unigenes related to flavonoids biosynthesis pathway may involve in regulating the biosynthesis of flavonoids.Meanwhile,2 CHSs were screened from the transcriptomic database of C.parasiticus,named and respectively.The transcript level of CpCHS1 was up-regulated in S2 compared with that of S1 and the expression of CpCHS2 showed no differences between S1 and S2.CpCHS1 and CpCHS2 were cloned from C.parasiticus and heterologous expressed in E.coli.Enzyme activity assay of purified recombinant CpCHSs indicated that CpCHS1 could catalyze cinnamoyl-CoA,p-coumaroyl-CoA and caffeoyl-CoA to form pinocembrin,naringenin and eriodictyol,respectively.However,CpCHS2 was inactive towards all the three substrates.Under the optimum pH and temperature,the reaction kinetics of CpCHS1 was detected.The results revealed that CpCHS1 showed higher catalytic efficiency than those associated with their bryophyta counterparts,but lower than that of angiosperm counterparts.The crystal structures of CpCHS1 in apo form and complex with substrates were solved.To comprehend the structural differences for the evolution of CHS across some major land plant lineages,the structure of CpCHS1 was compared with the structures of CHSs from the angiosperm Arabidopsis thaliana,the monilophyte Equisetum arvense and the bryophyte Physcomitrella patens.CpCHS 1 showed a highly similar conformation and probably shared a similar general catalytic mechanism with angiosperm counterparts.To explore the difference between CpCHS1 and CpCHS2,homology model of CpCHS2 was generated using the modeling server SWISS-MODEL with the CpCHS1 structure as a template.We found that CpCHS2 adopt a very similar conformation to CpCHS 1,except for several amino acid residues located in the substrate binding pocket.These data suggest that the structures of CHSs are highly conserved in the evolution.And the substrate and product specificity of CHSs are caused by subtle differences in the amino acid residues located at the active site.