Molecular Cloning And Characterization Of Terpene Synthase 4 In Freesia Hybrida

The constituents of plant volatile organic compounds(VOCs)are extremely complicated,which are mainly composed of volatile terpenoids and their derivatives.In plants,terpene synthases(TPSs)are responsible for the biosynthesis of the various terpene molecules from Geranyl Diphoshate(GPP),Farnesyl Diphoshate(FPP),Geranylgeranyl Diphoshat(GGPP),respectively.Freesia hybrida,with floral scents,pleasent fragrance as well as longer flowering cycle,is an ideal plant species to investigate the biosynthesis and regulation of volatile terpenes.Through in situ TBLASTN searching against transcriptome database established from different Freesia flower tissues,a putative TPS homologous sequence was isolated,which was subsequently subjected to Rapid Amplification cDNA Ends(RACE)analysis to get the full length cDNA,designated as FhTPS4.Sequence analysis showed that FhTPS4 had a complete open reading frame(ORF)of 1701 base pairs encoding a 566 amino acid protein.Bioinformatics analysis showed that the amino acid sequence of FhTPS4 had two conserved domains DDXXD and NSE/DET in the terpene synthase family.Phylogenetic analysis found that FhTPS4 clustered into TPS-g clade.The terpene synthases in this subfamily possess a bifunctional catalytic property which can not only catalyze the synthesis of monoterpene from GPP but also catalyze the formation of sesquiterpene from FPP.In previous studies,TPSs of TPS-g subfamily were mostly found to belocated in chloroplast.Similarly,the subcelllocation of FhTPS4 was in chloroplast confirmed by Arabidopsis protoplast transfection system.In addition,the results of in vitro enzyme assay showed that FhTPS4 could catalyze the formation of linalool and nerolidol from GPP and FPP substrates,respectively,which demonstrated that FhTPS4 possessed bifunctional catalytic properties.In order to further explore the function of FhTPS4 in vivo,the temporal and spatial expression patterns of FhTPS4 were investigated by Real-time PCR and then integrated with the volatile terpenes emission profiles.Based on the correlation between the gene expression and metabolites released as well as TPS catalytic capacities,it can be deduced that FhTPS4 was the predominant gene responsible for the linalool synthesis in ovary and sepals of Freesia hybrida.