Functional Characterization Of Maize Terpene Synthase ZmTPS7,ZmKSL2 And ZmKSL4

Maize(Zea mays L.)is a very important grain crop,but biotic and abiotic stresses lead to the decline in yield and quality.In response to stresses,maize produces many small molecule secondary metabolites.Terpenoids are the largest category,including the phytoalexin zealexin and kauralexin,with effective roles in suppressing the growth of pathogens and the antifeedant activity for insects.Terpenoids are synthesized through the sequential catalytic reactiones from the precursor(FPP/GPP/GGPP)by terpene synthases and the subsequent oxidative modification.Some terpene synthases(TPSs)have been characterized in maize,such as monoterpene synthase ZmTPS26,sesquiterpene synthase ZmTPS6/11 and diterpene synthase ZmKSL5,and so on.But the function of many maize terpene synthase is still unknown.In this study,we investigated the function of three terpene synthases,ZmTPS7,ZmKSL2 and ZmKSL4.By the metabolic engineering recombinant expression in Escherichia Coli and GC-MS detection,the biochemical functions of the three synthase were clarified.In addition,expression patterns of these three genes treated by stresses and phytohormones were analyzed by qRT-PCR.The main results were followed:1.Maize terpene synthase 7(ZmTPS7)was characterized as a ?-cadinol synthase.Here,we characterized ZmTPS7 biochemically through recombinant expression in Escherichia coli and detected that it catalyzed formation of a blend of sesquiterpene and sesquiterpenoid alcohol through GC-MS analysis.Subsequently,the major product was purified and identified as ?-cadinol through nuclear magnetic resonance spectroscopy(NMR)analysis.So that ZmTPS7 was a sesquiterpene synthase,which also was the?-cadinol synthase in maize for the first time.2.Gene expression analysis showed that ZmTPS7 exhibited constitutive and inducible expression patterns in response to biotic and abiotic stress,suggesting involvement in stress response.ZmTPS7 constitutively expressed in aerial tissues while with trace amount of transcript in roots.F.graminearum infection and methyl jasmonate(MeJA)treatment induced gene expression of ZmTPS7,while exogenous ABA treatment dramatically induced the expression of ZmTPS7 in roots.3.The major product of ZmTPS7,?-cadinol was observed to accumulate inducibly in maize leaves by F.graminearum inoculation,which was similar to the gene expression patterns.Meanwhile ?-cadinol was also detected in maize tissues infected by pathogen fungus for the first time.4.Anti-fungal activities of ?-cadinol.Significant inhibitory effects of ?-cadinol were detected on F.graminearum with 50 ?g/mL ?-cadinol in medium.However,the inhibitory concentration was much lower than the concentration of ?-cadinol in the infected maize tissues(-200 ng/g).Such evidence indicated that anti-fungal activity of ?-cadinol was not physiologically relevant,and further investigation is needed to clarify its biological functions in maize.5.ZmKSL2 and ZmKSL4 were diterpene synthase.Each of the two synthases was co-expressed with CPS in E.coli,and enzymatic products were analyzed by GC-MS.The GC-MS analysis results revealed that ZmKSL2 had the ability for using ent-CPP to form ent-isokaurene and ZmKSL4 reacted with ent-CPP to produce one unrecognized diterpene.ZmKSL2 and ZmKSL4 also reacted with syn-CPP to form form syn-pimaradiene and other two diterpenes.6.ZmKSL2 and ZmKSL4 gene expression can be induced.Elevated transcript accumulation of ZmKSL2 and ZmKSL4 were observed in all treatment including leaves infected by F.graminearum or treated by MeJA+ EP or MeJA and roots treated by ABA.The results suggest that the two genes might be involved in stress response,of which ZmKSL2 might involve in the biosynthesis of phytoalexin kauralexin B through catalyzing ent-CPP to form ent-isokaurene.Conclusion:In this study,we identified that ZmTPS7 was ?-cadinol synthase.And?-cadinol was detected in maize tissues infected by pathogen fungus for the first time.ZmKSL2 and ZmKSL4 can react with CPP to form diterpenes.Among these,ZmKSL2 had the ability for using ent-CPP to form ent-isokaurene,which may be involved in kauralexin B biosynthesis.They also catalyze the formation of syn-pimaradiene by syn-CPP and have multifunctional biochemical activities.Those genes were induced by the infection of pathogens and phytohormones treatment,which suggests that they might be involved in stress response.