Functional Identification Of Wheat Sesquiterpene Synthase TaPS And Barley Diterpene Synthase HvKSL4

Wheat(Triticum aestivum L.)and barley(Hordeum vulgare L.)are the main food crops and the main source of human caloric intake.These plants will produce terpenoids to repel pests or attract predators or parasitoids of insect pests.These volatile terpenes,including the monoterpene linalool,sesquiterpene caryophyllene,limonene and bergamotene,are involved in the indirect defense of plants.In addition,terpenoids can also play a direct defensive role as phytoalexins to improve plant resistance.Although some terpene synthases have been identified in wheat and barley,there are still a large number of unidentified terpene synthases involved in plant defense responses.In this study,the functional characterization of wheat sesquiterpene synthase TaPS and barley diterpene synthase HvKSL4,as commented in the database,was performed by screening published data and consulting related literature.Firstly,the microbial metabolic engineering technology was applied to recombinant expression in Escherichia coli.Then,the biochemical functions of these two enzymes were determined by GC-MS,mass spectrogram comparison and NMR analysis.Meanwhile,based on the analysis of the physical properties of the products and the gene expression pattern of TaPS in response to stress,the role of TaPS in the indirect defense of plants was studied,and the following results were achieved:1.This study demonstrated that TaPS is a sesquiterpene synthase.GC-MS detection showed that TaPS could catalyze trans-FPP to produce sesquiterpenoid β-patchoulene.Therefore,the terpene synthase was named TaPS.2.Based on analysis of the gene expression pattern of TaPS,it was found that TaPS exhibited low constitutive expression in wheat leaves,stems and roots.In addition,the expression of TaPS under various stressors revealed that Alamethicin treatment,F.graminearum spore inoculation and herbivory with beet armyworm larvae feeding all significantly induced the expression of TaPS gene in wheat leaves,which indicated that it was mainly involved in the response to biotic stress.3.Based on gene expression analysis,alamethicin was the powerful elicitor to induce TaPS gene expression,which then was used to treat wheat leaves for terpene analysis by GC-MS.β-patchoulene was clearly detected in treated wheat tissues,this for the first time,but not in controls,the results were consistent with previous gene expression patterns.At the same time,alamethicin is usually used to simulate insect herbivory on plants,so we speculate that TaPS may be involved in the process of insect resistance.4.Arabidopsis overexpressing TaPS produced β-patchoulene to repel S.exigua larvae.TaPS gene was transformed into Arabidopsis,and we detected β-patchoulene in transgenic Arabidopsis through GC-MS in a headspace sampling method,but not in wild-type Arabidopsis.Furthermore,S.exigua larvae preferred WT over transgenic leaves,indicating repellent activity of β-patchoulene produced by TaPS overexpression in Arabidopsis.Finally,the potential toxicity of β-patchoulene on larvae was also explored and no significant growth inhibition was observed when larvae fed transgenic Arabidopsis.5.Exploration on the catalytic mechanism of TaPS.Based on the results of homology modeling and docking,the author found that the residues I392 and C393 of the gene were closely localized to the final product β-patchoulene.By comparing with the similar sesquiterpene synthase,we mutated I392 and C393 of TaPS to the equivalent C and G of Patchoulol synthase,respectively.Some hydroxylated sesquiterpenoids were produced by TaPS mutants.The results showed that these two amino acids in TaPS are related to the protonation and stability of intermediate and product formation,play an important role in the catalysis of TaPS.6.HvKSL4,a diterpene synthase cloned from barley,was co-expressed with GGPP synthase and CPP synthase in E.coli.GC-MS detection showed that HvKSL4 react with syn-CPP to generate multiple diterpenoid compounds,three of which were identified as syn-stemar-13-ene,syn-stemod-13(17)-ene and syn-stemod-12-ene.These three compounds participate in the formation of diterpenoid phytoalexin.In addition,HvKSL4 can also catalyze normal-CPP to generate a single diterpene compound with a molecular weight of 272.The product was identified as a novel diterpene by separating,purifying and NMR analysis.Conclusions: Here we identified TaPS from wheat as the β-patchoulene synthase with inducible gene expression.Especially the simulation of insect herbivory on wheat leaves by Alamethicin strongly induces the expression of the TaPS gene,andβ-patchoulene was first identified in wheat.Moreover,overexpression of Arabidopsis could release β-patchoulene to repel S.exigua larvae,indicating that TaPS is involved in the indirect defense response of plants.The diterpene synthase HvKSL4 in barley has a variety of functions,which can catalyze the formation of a series of diterpenoids from CPP with different configurations.These diterpenes are modified by downstream oxidation to produce diterpenoid phytoalexins with biological activity and participate in plant direct defense response.These results further elucidate the biosynthetic pathway of terpenoids in wheat crops,and provide theoretical basis for the combination of direct and indirect defense responses in the cultivation of resistant varieties to control pests and diseases.