Analysis Of Floral Volatiles Biosynthetic Pathways And Functional Characterization Of Mono-tpss Genes From Chimonanthus Praecox

Wintersweet（Chimonanthus praecox L.）is a traditional ornamental plant in China with a pleasant floral scent,and the flowers can be taken as a new source of natural essential oil with important ornamental and economical values.Floral scent is made up of specialized plant metabolites,which can be used as signal substances to attract pollinators as well as protecting themselves from pathogens and resist environmental stress.Floral scent is also an important target character in plant breeding,which plays an important role in determining the aesthetic properties of ornamental plants and cut flowers.The floral scents of wintersweet are rich in monoterpenes and benzenoids.However,its scent profiles markedly differ by genotype with some genotypes having extremely strong fragrances and others have little to none.However,the molecular mechanisms underlying this diversity of fragrance are not clear.Recently genomic,transcriptomic,and proteomic investigations have elucidated the biochemical and molecular basis of the production and emission of floral volatile compounds in many plants.However,due to the lack of genome information,very little is known about the genes involved in terpenoid biosynthesis pathway.In this study,headspace analyses of the aroma chemicals emitted by fully open flower from two C.praecox genotypes,with contrasting phenotypes,were performed.The transcriptome and proteomic data represent excellent source for the identification and functional characterization of floral related terpene synthase genes and transcription factors from C.praecox.This study would provide information theoretical basis for terpenoid biosynthesis and regulation mechanism.The main results as followed:1.To investigate the floral volatile compositions of two different genotype wintersweet（H29 and SW001）,fully open-stage flowers from each genotype were sampled by HS-SPME and detected by gas chromatograph–mass spectrometer（GC-MS）.In total,four categories of volatile compounds were detected in two genotypes,including terpenes（monoterpenes and sesquiterpenes）,benzenoids and phenylpropanoids,aliphatics,and miscellaneous cyclic compounds.A total 36compounds were detected,of which 20 compounds were identified in H29 and 24compounds were identified in SW001.The predominant VOCs identified in H29 were linalool,trans-β-ocimene,benzyl acetate,methyl salicylate,benzyl alcohol,and methyl benzoate,which account for about 89%of the total VOCs in H29.The predominant VOCs identified in SW001 were trans-β-ocimene,allo-ocimene,methyl salicylate,（1S,2S,3R,5S）-（+）-2,3-pinanediol and cis-β-ocimene,which account for about 92%of the total VOCs in SW001.The components with higher aromatic value in scented H29 were linalool（300.92±132.03）,benzyl acetate（66.60±47.47）,methyl salicylate（9.53±4.94）,β-myrcene（6.16±0.80）and limonene（4.72±1.75）.The components with higher aromatic value in unscented SW001 were trans-β-ocimene（157.17±11.42）,allo-ocimene（13.07±0.92）,methyl salicylate（6.21±0.11）,β-myrcene（5.76±1.33）and cis-β-ocimene（5.45±1.09）.2.To understand the molecular basis of polymorphisms in floral scent between the wintersweet genotypes H29 and SW001,the fully open flowers from each genotype were used for high-throughput sequencing and proteome survey.A total of14 443 differentially expressed unigenes and 196 differentially expressed proteins were identified.The credibility of the RNA-Seq and quantitative proteome data were verified by q RT–PCR analyses.In KEGG pathway enrichment analyses,monoterpenoid biosynthesis pathway and terpenoid backbone biosynthesis pathway were significantly enriched.The key DEGs/DEPs involved in terpenoid backbone biosynthesis,monoterpenoid biosynthesis,and benzenoid biosynthesis pathways were identified through bioinformatics analysis,including Cp DXS、Cp GPPS、Cp TPS、Cp PAL、Cp SDRs and Cp BEAT.Using local TBLASTX searches,we identified 16TPSs with significant homology to known TPS enzymes.Among them,only nine transcripts contained full-length open reading frames which were designated Cp TPS1to Cp TPS9.Phylogenetic analyses showed that 5 Cp TPSs belong to mono-TPSs,2Cp TPSs belong to sesqui-TPSs and 2 Cp TPSs belong to di-TPSs.Base on bioinformatics analysis,341 differentially expressed TFs belong to 56 TF families were identified in wintersweet transcriptome.Selection of TFs potentially involved in regulating floral volatile biosynthesis and metabolism,including a promising new option for transcription regulation in wintersweet floral scent.3.In this study,we cloned 5 mono-TPS genes and 2 sesqui-TPSs genes from H29and SW001 genotype wintersweet.The C-terminus of the seven full-length Cp TPSs are two Mg²⁺combined conserved domains（DDXXD and NSE/DTE）,used to chelate divalent metal ions.q RT–PCR analyses were conducted from 5 different genotype wintersweets.The results showed that the expression of Cp TPS1 gene was consistent with that of trans-β-ocimene emission in each genotype and the expression of Cp TPS5gene was consistent with that of linalool emission in each genotype.The subcellular localization results showed that both Cp TPS1 and Cp TPS5 were localized in the plastid.By enzyme assay,we showed that Cp TPS1 catalyzes GPP to produce ocimene,and Cp TPS5 catalyzes GPP to produce linalool but also catalyzes FPP to produce nerolidol.Next the Cp TPS5 gene was over-expressed in tobacco,and resulted in the emission of volatile linalool from the seedling,with no detrimental phenotypic effects.Based on the genome database,1973bp and 1627bp Cp TPS5 gene promoter were cloned from H29 and SW001,respectively.Those sequences included a number of cis-elements which were predicted to be responsive to light,environmental stresses and growth and development.The promoter of Cp TPS5 gene were also predicted including floral scent related regulatory elements.