The Evolution Analysis Of (E)-?-ocimene Synthase In Angiosperms

The plant secondary metabolite plays various ecological roles in protection against the biotic and abiotic stresses.Terpenoid volatile organic compouns produced by plants play crucial roles in processes ranging from plant growth and development to the interactions with their surrounding environment.Terpenoid volatile organic compounds consist of isoprene(C5),monoterpene(C10),and sesquiterpenes(C15).Mulberry is a nutritious and healthful fruit.However,few reports on the mulberry terpenoid volatile organic compouns are published.Moreover,terpene synthase with different activities from the same species tend to be more similar to each other than those with the same activity from different species.The explanation for this is that divergent evolution and occasionally convergent evolution have occurred within the terpene synthases.Although many cases of convergent evolution in the plant secondary metabolite have been reported,little is known for the convergent evolution of terpene synthase family.In the present study,on the basis of the results of bioinformatics and qRT-PCR analyses,we identified putative terpene synthase genes in Morus notabilis.Gas chromatography-mass spectrometry(GC/MS)was used to analyze the terpenoid volatile organic compouns emitted by leaves and root.Combined the genetic,phylogenetic,biochemical analyses and computational chemistry revealed that the origin and evolution of(E)-?-ocimene synthase.The results of this study were as follows:1.Analyses of terpenoid volatile organic compounds emitted by mulberry leaves and rootsSPME-GC/MS was used to analyze the materials in the headspace of both fresh mulberry leaf and root.The results indicated that only ?-pinene was detected.Various terpenoid volatile organic compouns were emitted after methyl jasmonate(MeJA)treatment.In the MeJA-treated leaf,(E)-?-ocimene and linalool were detected.After MeJA treatment,roots emitted mainly 1,8-cineole.There was no difference in terpenoid volatile organic compouns between herbivore-induced and MeJA-treated leaf.2.Identification and sequence analysis of mulberry terpene synthase genesTo identify the terpene synthase genes involved in the producing of mulberry terpenoid volatile organic compounds,we searched the mulberry genomic database(https://www.rosaceae.org/analysis/295).As a result,26 putative mulberry terpene synthase genes were identified.qRT-PCR was then performed to determine which MnTPSs were induced by MeJA treatment.Four putative full-length TPS-b/g genes,MnTPS11,MnTPS12,MnTPS18(Morus018508),and MnTPS25 were induced in the leaves treated by MeJA.In the roots,MnTPSll,MnTPS13,MnTPS18,and MnTPS25 were induced by MeJA treatment.The fusion proteins MnTPS11-EGFP,MnTPS12-EGFP,MnTPS13-EGFP,MnTPS25-EGFP,and MnTPS18-EGFP were targeted to the chloroplasts.3.Functional analyses of MnTPS11,MnTPS12,MnTPS13,MnTPS18,and MnTPS25Cells of E.coli BL21 expressing His-tagged MnTPS11,MnTPS12,MnTPS13,MnTPS18 or MnTPS25 were harvested and lysed.The recombinant MnTPS12 catalyzed the formation of mostly(E)-?-ocimene and a smaller amount of(Z)-?-ocimene.When MnTPS13 was incubated with GPP as the substrate,1,8-cineole as major product.Both MnTPS18 and MnTPS25 catalyzed the synthesis of linalool from with GPP as the substrate.4.Phylogenetic analyses and genomic synteny of(E)-?-ocimene synthaseCombined the phylogenetic reconstructions and genome synteny studies demonstrate that the(E)-?-ocimene synthase appear to have arisen several times in independent lineages during plant evolution.The activity of(E)-?-ocimenesynthase had appeared before the diversification of eudicots.5.Identification amino acid sites be closely related to the convergent evolution of(E)-?-ocimene synthase based on ancestral sequence reconstructionBased on the phylogenetic analyses,ancestral sequence reconstruction,and a structural model,six historical amino acids within a 10 A radius of the active site centre were identified.Four of them(I388-F420-S446-F485)were sufficient to completely convert different terpene synthases into ?-ocimene synthase.6.Obstruction of GPP isomerization produces acyclic monoterpenesIncubation of neryl diphosphate(NPP)with Salvia officinalis 1,8-cineole synthase(SCS)and mutated proteins showed that these four sites obstruct the isomerisation of GPP.QM/MM MD simulations of models of SCS,SCSY420F/I446s,and SCSN338I/Y420F/I446S/L485F with(3R)-linalyl diphosphate((3R)-LPP)suggested that mutations change the configuration of the intermediate to obtain new activities.To summarize all our results,Y420,1446,and a water molecule reduce the space in the catalytic cavity,so that the substrate is precisely positioned to form the ?-terpinyl cation.The mutation F420 could not form H-bonds with the water molecule and the hydrophobicity of Phe may be made it difficult for the water molecule to enter the catalytic cavity.The space in the upper part of the catalytic cavity was increased by the absence of the water molecule and by the smaller side chains of S446.The enlarged active site contour broke the constraint of the intermediate,allowing its conformation to be more flexible to obstruct the isomerisation of GPP.N338I/L485F decreased the distance between the H of C4 and the O2A of PPi,accelerating early termination by deprotonation.The results obtained in the present study will be useful for further studies on the regulation of terpenoid volatile organic compouns formation for eco-friendly breeding projects.Our data provide new perspectives on the evolution of terpene synthases,identify the key residues to control the specificity of engineered terpene synthases and explain the convergent evolution of(E)-?-ocimene synthase at the molecular level.