Functional Characterization Of Chalcone Isomerase And Chalcone Synthase From Ferns

Pteridophytes are the most primitive vascular plants,which can be divided into 5 categories,and the most existing category is the Filieophytina.The Filieophytina can be divided into three lineages:Eusporangiates,the Early Leptosporangiates and the Polypodiales.In order to adapt to changes in the environment and cope with the biotic and abiotic stress,an abundance of secondary metabolites are accumulated,including flavonoids,terpenoids and alkaloids.Flavonoids have various functions during the plant growth and development.Meanwhile,flavonoids have been found to be useful as dietary supplements and have many pharmacological activitiesThe biosynthesis of flavonoids is derived from the phenylalanine,which is catalyzed by phenylalanine lyase(PAL),cinnamate 4-hydroxylase(C4H)and 4-coumaryol-CoA ligase(4CL)to form p-coumaryol-CoA.Then one molecule of p-coumaryl CoA and three molecules of malonyl-CoA generate naringenin chalcone through the action of chalcone synthase(CHS).Subsequently,the enzyme chalcone isomerase(CHI)catalyzes the intramolecular and stereospecific cyclization of naringenin chalcone to form(2S)-naringenin.The CHI superfamily comprises four types of CHI-fold proteins.Type ? and ? proteins are the bona fide CHIs with enzymatic activity catalyzing chalcones to yield flavanones.Type ? enzymes isomerize naringenin chalcone to form(2S)-naringenin.Type ? enzymes have broader substrate specificity;besides converting naringenin chalcone,they additionally catalyze isoliquiritigenin to(2S)-liquiritigenin.Previous studies have suggested that the type ? enzymes appear to be legume specific.Besides the bona fide type ? and ?CHIs,additional CHI-fold proteins are also found in Arabidopsis and termed as the type III and type IV CHIs.The type III CHIs have no chalcone cyclization activity but exhibit the fatty acid binding property.Type IV CHI proteins are completely devoid of CHI catalytic activity,but they might function as the enhancer of flavonoid production and flower pigmentation.In our previous studies,we found that the most primitive land plant liverworts and the Selaginella moellendorffii already comprise a complete CHI family genes,and they contain the type II CHI instead of the type I CHI.These findings,in contrast to our current understanding on the evolution of enzymatic CHIs,suggest that emergence of the bona fide type II CHIs is an ancient evolution event prior to the divergence of liverwort lineages.By comparison,the CHI from Selaginella exhibits a relatively low activity to isoliquiritigenin,compared to the liverwort CHIs,poising to a seemingly type I CHI enzyme characteristic.To better understand the function and evolution of CHI fold proteins in ferns,56 putative CHIs were screened from the transcriptome sequence datas of the ferns(52 species).Phylogenetic analysis was performed based on the predicted polypeptide sequences of these 56 putative CHI-fold proteins found in ferns,together with the sequences of previously recognized type II CHIs from liverworts and S.moellendorffii.The outcome of the analysis implied that the evolution of the CHI genes mirrors speciation within the pteridophytes as a whole.However,5 Polypodiales species CHIs appeared to be intermediate between CHIs encoded by S.moellendorffii and species belonging to the Early Leptosporangiates.Sequences alignment indicated that two residues of Medicago sativa CHI(Thr190 and Met191)postulated to determine substrate preference have been replaced by either Thr or Ser and Ile in the pteridophytes,respectively.Accordingly,these pteridophyte CHIs were considered most likely to be type I enzymes.According to the evolution process of ferns,a set of 24 representative pteridophyte CHI genes(from 21 species)were isolated and heterologously expressed in E.coli to characterize their enzymatic properties.The results indicated that 19 of the enzymes were classified as type I CHIs,while the other five exhibited slightly type II CHI activity.Combining the protein homology modeling and site-directed mutagenesis results,the amino acid Phe rather than the residues Thr and Met determined the type II CHI activity of this subgroup of enzymes.The constitutive expression of Lindsaea orbiculata CHI1 and Adiantum capillus-veneris L.CHI1 in the Arabidopsis thaliana tt5 mutant partially rescued the mutant pale yellow phenotype and enhanced the accumulation of both flavonols and total anthocyanins.In this study,we also identified the function of type IV CHI from ferns.By searching the transcriptome databases of Lindsaea orbiculata and Adiantum capillus-veneris L.,two putative CHIL genes and two CHS genes were identified and cloned,then named AcCHIL,LoCHIL,LoCHS1 and LoCHS2,respectively.They were constructed into the prokaryotic expression vector pET32a to induce protein expression.Finally,these two CHSs with different catalytic efficiency were combined with LoCHIL for in vitro enzyme activity.The results showed that CHIL was able to improve the production of naringenin chalcone by limiting the amount of CTAL produced.A yeast two hybrid assay was able to shown that LoCHIL can interact with LoCHIl and LoCHSs,suggesting that CHIL can bind with both CHS and enzymes upstream of CHS to promote flavonoid production.AcCHIL and LoCHIL were transformed into Arabidopsis thaliana chil mutant,respectively.Compared with the chil mutant,the contents of proanthocyanidins and flavonols in the transgenic plants were increased,indicating that AcCHIL and LoCHIL can compensate for the function of CHIL in the mutantIn conclusion,the evidence supports the notion that type ? CHIs evolved into type I CHIs at the time of the emergence of the pteridophytes.Type IV CHIs may act as an anchor for both CHS and CHI proteins;their function is to promote the proper folding and cyclization of polyketide intermediates formed during a CHS-catalyzed reaction,thereby avoiding the formation of derailment products;in so doing,they act to support the accumulation of flavonoids.