## 2 Results
### Identification and Analysis of Differentially Expressed TFs under MeJA Treatment  
To further understand the transcriptional regulation of MeJA-treated _M. canadensis_, differentially expressed TFs were identified between CK and MeJA-treated samples. As a result, 1535 TFs belonged to 55 families were identified by annotating in PlantTFDB database. bHLH (basic/helix-loop-helix), ERF (ethylene-responsive factor), MYB_related (myeloblastosis DNA-binding related protein), C2H2 (C2H2 zinc-finger protein), MYB (myeloblastosis DNA-binding protein), WRKY (WRKY-type DNA binding protein), C3H (Cys3 His zinc finger protein), NAC (NAC domain protein), bZIP (basic region/leucine zipper motif), and GRAS (Gibberellin-insensitive (GAI), Repressor of GA (RGA), and SCARECROW (SCR) protein) families were the top 10 largest TF families (Supplementary Table S3). Of the 153 TFs, 157 belonged to 32 families were differentially expressed. WRKY, bHLH, ERF, MYB, MYB_related, NAC, bZIP, C2H2, HD-ZIP (Homeodomain-leucine zipper protein), and Trihelix families were the top 10 families that had the largest number of DEGs. Each of these 10 families had more than 5 DEGs and the WRKY and bHLH family were the largest differentially expressed families that both contained 21 DEGs (Figure 5 and Supplementary Table S3). Comparing the expression levels of these DEGs, 102 of the 157 DEGs were up-regulated and the other 55 were down-regulated. Considering the family distribution of the up-regulated and down-regulated DEGs, 19 families showed that the up-regulated unigenes were more abundant than the down-regulated ones, especially in the WRKY family, 19 unigenes were up-regulated and only 2 were down-regulated. On the contrary, there were 11 families that contained more down-regulated unigenes than up-regulated and 2 families contained the same number of up-regulated unigenes and down-regulated (Figure 5). Identification and Expression Verification of Monoterpenoids and Menthol Biosynthetic Genes under MeJA Treatment  
In _M. canadensis_, monoterenes are the major constituents of essential oils. In this study, the enrichment results of the KEGG database indicated that \"Monoterpenoid biosynthesis\" was one of the most significantly enriched pathways under MeJA treatment. 20 DEGs associated with monoterenoid biosynthesis were identified, which consisted of genes encoding 10 terpene synthases (TPSs), 1 (\(-\))-isopiperitenone reductase (IPR), 1 menthol dehydrogenase (MR), and 8 neomenthol dehydrogenases (NMRs). _TPS_ is a multi-gene family that is widespread in plants, whose diversity and substrate complexity lead to the wide variety of terpenoids [32]. In this study, 10 differentially expressed _TPSs_ were identified under MeJA treatment, 8 of which were up-regulated and the other 2 were down-regulated. The remaining 10 DEGs in \"Monoterpenoid biosynthesis\" were menthol/neomenthol biosynthesis related genes. Interestingly, the expression levels of the 10 DEGs were almost all up-regulated except for an _NMR_ (Unigene0051701) under MeJA treatment (Figure 6). Figure 5: Statistics of differentially expressed TFs under MeJA treatment. Menthol is the main constituent of monoterponoids in _M. canadensis_, which is synthesized by a series of enzymatic reactions in the peltate glandular trichomes on the aerial surfaces of the plant. The biosynthetic pathway and enzyme catalysis mechanism of menthol has been extensively studied in peppermint and spearmint, two other species of genus _Mentha_[2; 3; 4; 5; 6]. Using the reference genes of peppermint and spearmint as queries, 9 orthologous genes were identified in _M. canadensis_ (Supplementary Figure S6), including the previously reported _GPPS-I, GPPS-s_, and _LS_[15]. The other 6 genes that catalyze the biosynthesis of (\(-\))-Menthol from (\(-\))-Limonene were identified in _M. canadensis_ for the first time. Considering the transcriptional levels of the 9 genes, although their RPKMs showed a certain level of increase after MeJA treatment, they did not reach significant levels above controls.