Identification,Molecular Cloning And Functional Characterization Of Mep Pathway Genes (DXS,GPPS) From Wintersweet Flower

The scent of a flower is one of the key factors in determining its economical and ornamental values.Wintersweet(Chimonanthus praecox L.)is a perennial deciduous shrub with a distinctive fragrance and unique flowering time in winter.The floral scent of wintersweet is predominantly a combination of volatile terpenoids(monoterpenes and sesquiterpenes)and benzenoids.Volatile monoterpenes biosynthesis mainly occurs in the plastid localized methylerythritol phosphate(MEP)pathway.So far,less information is known about the key genes that could be involved in regulating the monoterpene biosynthesis in wintersweet.In the current study,geranyl diphosphate synthase(GPPS)and deoxy-D-xylulose-5-phosphate synthase(DXS)genes that belong to the MEP pathway were identified and characterized in wintersweet.Main findings of the current study are the following.1.In the first experiment,5 full-length GPPS/geranylgeranyl diphosphate synthases(GGPPS)genes were identified from the wintersweet transcriptome database.Sequence analysis revealed that the isolated c DNAs have high protein sequence similarity with the other plants GPPS and GGPPS genes.The phylogenetic analysis classified these c DNAs into four distinct clades,representing heterodimeric GPPS small subunits(SSU1 and SSU2),homodimeric GPPS,and GGPPS.The Cp GPPS.SSU1,Cp GPPS.SSU2,Cp GGPPS1,and Cp GGPPS2 proteins contain the conserved Cxxx C motif that is required for physical interaction between GPPS.SSU and GPPS.LSU/GGPPS-like genes.Moreover,homodimeric Cp GPPS,Cp GGPPS1,and Cp GGPPS2 proteins encode two DDX_(2-4)D essential for catalytic function.While the protein sequence of both Cp GPPS.SSU1 and Cp GPPS.SSU2 lack this motif.Temporal expression analysis revealed that all genes have the highest transcript level at the full-open flower stage.Moreover,Cp GPPS.SSU1 and Cp GGPPS1 were predominantly expressed in petal and flower,whereas Cp GPPS.SSU2,Cp GPPS,and Cp GGPPS2 showed a constitutive expression.The subcellular localization assay identified the chloroplast localization of SSUs and GGPPSs proteins,and the yeast two-hybrid assay showed that both Cp GPPS.SSU1 and Cp GPPS.SSU2 can interact with the GGPPS-like proteins.Moreover,overexpression of Cp GPPS.SSU1 slightly increased the linalool content in tobacco transgenic lines as compared to wild type SR1 plants and transgenic plants showed leaf chlorosis phenotype in T₀and T₁seedlings;while in the case of Cp GPPS.SSU2,no alteration was found in volatile content(monoterpene)between transgenic lines and wild type plants.2.In another experiment,we found 5 DXS annotated transcripts from the transcriptome database of wintersweet flower.Of them,two DXS transcripts had a full-length open reading frame and shared high sequence similarity with the DXS from other plants.Both DXS transcripts have the TPP binding and DRAG domains.Moreover,the phylogenetic analysis revealed that both Cp DXS2A and Cp DXS2B belongs to clade 2 of DXS genes.Spatial expression analysis showed that both Cp DXS2A and Cp DXS2B had high expression in petal and flower parts.Moreover,temporal expression analysis of both genes showed that Cp DXS2A and Cp DXS2B had the maximum transcript level at partial open flower stage and full open flower stage,respectively.The subcellular localization assay identified that the DXS proteins are localized in the chloroplast.Additionally,in bacterial genetic complementation assay,both Cp DXS2A and Cp DXS2B produced coloured colonies due to synthesis of?-carotene that exhibits to encode functional protein in wintersweet.Taken together,this study revealed that a heterodimeric GPPS could be involved in monoterpene biosynthesis with possibly Cp GPPS.SSU1 playing a regulatory role.Moreover,both Cp DXS2A and Cp DXS2B genes could be important in regulating the metabolic flux of the MEP pathway by anticipating the availability of precursor for monoterpene biosynthesis in wintersweet flower.