The Catalytic Mechanism And Function Of Chrysanthemol Synthase CHS Of Pyrethrum

Pyrethrum(Tanacetum cinerariifolium,syn.Chrysanthemum cinerariifolium)is an ornamental plant of Composite family,and is well-known as a safe and non-toxic natural pesticide of pyrethrins.The supply has not been adequate to the demand.Recent researches highly focus on the extraction of pyrethrins.However,the pyrethrin biosynthasis pathway is unclear to date.Chrysanthemyl diphosphate synthase(CDS)was reported to be the first key synthase invovled in the pyrethrin biosynthesis to catalyse the precusor dimethylallyl diphosphate(DMAPP)into chrysanthemyl diphosphate(CPP).In this work,we cloned a new TcCDS gene and corrected the translation starting position of c DNA region and renamed it as TcCHS.We studied the enzyme function,tissue localization,mechanism of catalytic activity and ecological function of TcCHS.The main results are as followed:1.Identified TcCHS as a bifunctinal enzyme.Here,by mutation and enzyme assay,we showed that the NDXXD catalytic motif of TcCHS catalyzes DMAPP to CPP but also the next step,converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety,leading TcCHS as a bifunctional enzymes with both prenyltransferase and terpene synthase activity.2.Revealed that the TcCHS induced a dual defense system with both COH emission and glycosides accumualtion in trasgenic chrysanthemum.The TcCHS gene was over-expressed in Chrysanthemum morifolium,and resulted in both the emission of volatile chrysanthemol and accumulation of a nonvolatile glycoside derivative,putatively identified as chrysanthemol-malonyl-glucose,with no detrimental phenotypic effects.Dual-choice assays separately assaying the volatile odour and non-volatile components demonstrated independent bioactivity of both components against the cotton aphid(Aphis gossypii).Performance assays showed that the TcCHS plants significantly reduced aphid reproduction,consistent with disturbance of aphid probing activities on these plants as revealed by electropenetrogram(EPG)studies.In open-field trials,aphid population development was very strongly impaired demonstrating the robustness and high impact of the trait.The results suggested that expression of the TcCHS gene induces a dual defense system,with both repellence by chrysanthemol odour and deterrence by its glycoside taste,introducing a promising new option for engineering aphid control into plants.3.Cloned and identifed the trichome specific TcCHS promoter.1128 bp TcCHS promoter was cloned.This sequence included a number of cis-elements which were predicted to be responsive to different hormones,light,and environmental stresses.In order to characterize the promoter,the fragment of TcCHS promoter was fused to reporter gene GFP and transformed into florist’s chrysanthemum‘1581’ The GFP fluorescence in chrysanthemum showed that the TcCHS promoter exclusively expressed in the glandular secreting trichomes(GSTs)of leaf and stem.The main catalytic motif of promoter was also confirmed by transient transformation of deletion fragments of TcCHS promoter to tobacco.The TcCHS promoter transgenic chrysanthemum and pyrethrum plants were further treated by methyl jasmonate(Me JA).The level of TcCHS promoter expression was induced in 12 h and the pyrethrins contents and density of trichome were also increased in pyrethrum after Me JA treatment.The study of TcCHS promoter can be used for trichome-specific metabolic engineering in pyrethrum and other plants.4.Cloned the first farnesol synthase FAS gene expressed in plastid in pyrethrum.Six other homologous genes were cloned from pyrethrum and chrysanthemum when we cloned TcCHS gene.By aligning c DNA sequences,two CHS-like genes and four FDS-like genes could be distinguished in two groups.CHS-like genes shared the high amino acid sequence similarity and expression mode with TcCHS gene in pyrethrum.CHS-like gene was also located in plastid.It prefers to catalyze DMAPP and IPP to produce FOH in vitro and in vivo and also hydrolyze different isopentenyl pyrophosphate to yield related alcohol.We named it as FAS with both prenyltransferase and terpene synthase activity.5.Clarified TcCHS enzyme activity in vitro and in vivo.TcCHS is inactive in vitro while with high activity in E.coli lysate as well as tobacco and chrysanthemum.The GGDS genes were cloned from tobacco and pyrethrum.Bimolecular fluorescence complementation and in vitro enzyme analysis of individual and preincubated TcCHS and GGDS proteins revealed that GGDS can interact with TcCHS and form a heterodimer with enhanced activity to produce product of TcCHS.In conclusion,this study revealed the new bifunction of TcCHS and its insect-resistant molecular ecology and identified the first trichome-specific promoter from pyrethrum and the catalytic mechanism of TcCHS in plants.The study would provide informative theoretical basis for pyrethrins biosynthesis pathway and catalytic mechanism of terpenoid and practical foundation for molecular modified breeding for increasing pyrethrins contents in pyrethrum and enhancing insect resistance in chrysanthemum.