## 3 Discussion
In this study, the KEGG pathway enrichment of DEGs indicated that \"Monoterpenoid biosynthesis\" was the most significantly enriched pathway under the treatment of MeJA. 20 DEGs associated with monoterenoid biosynthesis were identified, which consisted of genes encoding TPS (10), IPR (1), MR (1), and NMR (8). Of these, genes encoding IPR, MR, and NMR were involved in menthol and neomenthol biosynthesis and interestingly, the expression levels of the 10 DEGs were almost all up-regulated under MeJA treatment. These results suggest that the biosynthesis of monoterpenes including menthol and neomenthol might be elicited by the MeJA signal in _M. canadensis_. The biosynthesis pathway of menthol has been extensively studied in peppermint and spearmint [2; 3; 4; 5; 6]. In this study, 9 orthologous genes were identified in _M. canadensis_ including 6 new reported genes. However, expression analysis of these 9 genes in CK and MeJA-treated samples indicated that most of the gene expression levels did not change significantly after treatment. qRT-PCR results indicated that _GPPS-s_ and _iPD_ showed a certain degree of up-regulation and _MFS_ was down-regulated. We speculate that only part of the menthol biosynthetic genes responds to the JA signal in _M. canadensis_. The response mechanism of menthol biosynthetic genes to JA signaling needs further study in _M. canadensis_, especially the possible DEGs including _GPPS-s_, _iPD_, and _MFS_. Another explanation might be that different secondary metabolic pathways are controlled by different regulatory modules. For example, in _Medicago truncatula_, genes involved in phenylpropanol biosynthesis were transiently induced after application of MeJA at low concentrations (0.5-5 umol/L), but triterpene biosynthetic genes were induced after 12-24 h at high concentrations (5-500 umol/L) [41]. Many _M. canadensis_ menthol biosynthetic genes might not respond to the MeJA signal under our treatment conditions (200 umol/L, 24 h). Transcriptional regulation is central to plant secondary metabolism. Compared with well-studied metabolic pathways such as flavonoids, the transcriptional regulation of terpene metabolism has been validated in a few studies. Several TFs have been identified from plants including _A. annua_ (AaWRKY1, AaERF1, AaERF2, AabZIP1, and AaMYC2), _Gossypium arboretum_ (GaWRKY1), _Taxus chinensis_ (TaWRKY1), _Hevera brasiliensis_ (EREBP1 and HbWRKY1), and _O. sativa_ (OsTGAP1) that regulate terpene biosynthesis [21; 22; 23; 42; 43; 44; 45; 46; 47; 48]. In _Mentha_ species _M. spicata_, two TFs MsYABBY5 and MsMYB (_M. spicata_ MYB DNA-binding protein) have been reported that negatively regulate monoterpene production [49; 50]. In this study, homologous genes of the two TFs (Unigene0062650, homolog to _MsYABBY5_ and Unigene0030793, homolog to _MsMYB_) were identified in _M. canadensis_. Sequence alignment indicated that both genes had quite high similarities between _M. spicata_ and M. canadensis_ (Supplementary Figure S7). Expression patterns deduced from RPKM values indicated that the expression of the two homologous genes declined to a certain extent under MeJA treatment (52.9-vs.-35.6 for Unigene0062650 and 0.72-vs.-0.38 for Unigene0030793), although the decline was not significantly (two-fold change with FDR <0.05). These results suggest that Unigene0062650 and Unigene0030793 might be MeJA-responsive TFs and similar negative regulation mechanism may also exist in _M. canadensis_.