## 4 Discussion  
Terpenoids play important roles in plant defense [22]. Terpene volatiles were involved in defense against pests and/or disease pathogens in many plants including maize and rice [42; 43; 44]. However, terpenoid metabolism in wheat has not been studied thoroughly. Recently, characterization of wheat FPP synthases suggested existence of sesquiterpene biosynthesis in wheat with potential involvement in anti-herbivory response [34]. We searched the wheat genomic sequence released on Phytozone and found a number of genes with annotation of terpene synthase. Here, we identified TaPS from wheat as the \(\beta\)-patchoulene synthase. To the best of our knowledge, TaPS is the first sesquiterpene synthase characterized in wheat. Alamethicin is usually used to simulate insect herbivory on plants [45]. _TaPS_ was induced drastically by the alamethicin treatment in wheat, and it also elicited \(\beta\)-patchoulene accumulation. _TaPS_ gene expression and its enzymatic product accumulation responded to alamethicin elicitation,  
Figure 5: Homology modeling of TaPS and site-directed mutagenesis analysis. (**A**) Catalytic cavity of TaPS was predicted based on homology modeling with docking of the final product \(\beta\)-patchoulene (red). Two key aspartates in the conserved DDX motif of terpene synthases were shown in blue. The two unique residues in TaPS, I392 and C393 were shown in yellow and green, respectively. The predicted distances between I392 or C393 and C2 of \(\beta\)-patchoulene were labeled as well (3.3 Å and 3.8 Å, respectively); (**B**) amino acid alignment of TaPS and patchoul synthase (PatTPS177). The unique I392 and C393 were indicated by the red line. The full alignment was shown in Figure S4; (**C**) GC-MS chromatograms of TaPS:I392C and C393G with FPP as the substrate. The new products were labeled as peak 1–10 and their mass spectra were listed in Figure S5. The authentic (\(-\))-patchoul was injected for comparison. suggesting involvement in the anti-herbivory response. This was confirmed by the inducible gene expression of TaPS in response to beet armyworm larvae feeding on wheat leaves. Furthermore, Arabidopsis overexpressing _TaPS_ produced \(\beta\)-patchoulene that acted as the repellent against insect feeding, which also demonstrated _TaPS_ involvement in the anti-herbivory response. We further tested the potential toxicity of \(\beta\)-patchoulene to beet armyworm larvae. When the transgenic Arabidopsis leaves were provided as the sole food, larvae did not have other choices and fed on these leaves emitting the disliked \(\beta\)-patchoulene, and slight lower weight gain was observed in comparison with those feeding on WT (Figure S3), indicating no direct toxicity of \(\beta\)-patchoulene on these larvae. In Arabidopsis, semivolatile diterpene, rhizathalene deterred fungus gnat larvae infestation in roots [46]. With artificial diet feeding, fungus gnat larvae consumed less diet containing different concentrations of rhizathalene than the control [46]. In this study, two tested TaPS transgenic lines emitted similar amount of \(\beta\)-patchoulene, which might not reach the toxic concentration. Artificial diet with higher concentration of \(\beta\)-patchoulene should be used for anti-herbivory assays in future studies. Further investigation of such anti-herbivory assays also should be conducted with \(\beta\)-patchoulene spraying on Arabidopsis WT leaves to exclude the potential bad taste of overexpressed TaPS protein in transgenic plants. A number of volatiles have been identified to defend insect attack through multiple ways in plants. Myrcene, \(\beta\)-caryophyllene and \(\beta\)-farnesene are emitted by plants upon insect damage and acted as the attractors of herbivore enemies [47]. Volatiles also play as the signal molecules to prime defense in neighbor plants [48]. Here we only investigated the antifeedent function of \(\beta\)-patchoulene in Arabidopsis, further study is needed to explore its other potential functions in wheat.