## 2 Results
### DEGs Involved in Terpenoids and Menthol Biosynthesis  
From the transcriptome analysis, 14 DEGs involved in the monotereponoids biosynthetic pathway were identified. These were terpene synthases (TPSs), (\(-\))-isopiperitenone reductases (IPR), menthol dehydrogenase (MR), cytochrome P450 (P450), neomenthol dehydrogenases (NMRs) and alcohol dehydrogenase (ADH). The two TPSs were alpha-pinene/camphene synthase and linalool synthase. We identified 15 DEGs involved in sesquiterpenoids and triterpenoids. Excepted for the Unigene0029907 identified as (3S)-linalool synthase, the other 14 TPSs were dramatically downregulated in the dark but were then upregulated to the same expression level after regaining access to light for 24 h (Figure 6).  
In mint, menthol was the most prevalent chemical compound among the monotereponoids. In our study, orthologous genes encoding enzymes that catalyzed the biosynthesis of (\(-\))-menthol were identified by multiple sequence alignment, using the reference genes of peppermint and spearmint. The nucleotides of these genes were cloned and sequenced in _M. canadensis_ (Supplementary data). The expression profiles of six identified genes, including limonene synthase (LS), (\(-\))-limonene-3-hydroxylase (L3 OH), isopiperitenol dehydrogenase (iPR), isopiperitenone reductase (iPD), pulegone reductase (PR), and neomenthol dehydrogenase (MNR), showed the same patterns: they were significantly downregulated under 24 h of darkness and upregulated after returning to the light for 24 h (Figure 7). Only the MFS gene showed the opposite expression pattern, which was upregulation under darkness and downregulation after removing the covers. Although the GPPS-I, GPPS-s, and MR genes' expression levels were not dramatically altered by light exposure, they were all downregulated as a result of darkness (Figure 7).  
Figure 5: Heatmaps of all DEGs and eight significant expression clusters of all DEGs. (**A**) Heatmap showing the expression profiles of all DEGs between the control and darkness treatments. (**B**) Heatmap showing the expression profiles of all DEGs between the darkness and recovery light treatments. (**C**) The eight expression patterns of DEGs; the cluster names and unigene numbers are shown at the top.  
RT-PCR was used to verify the expression levels of menthol biosynthetic genes (Figure 8). The results indicate that the qRT-PCR expression patterns of 9 genes matched quite well with the RNA-Seq data.  
Figure 6: Heatmaps of the DEGs in the monotereponoid biosynthesis pathway (**A**) and in the sesquiterpenoid and triterpen biosynthesis pathways (**B**). NMR, (+)-neomethol dehydrogenase; P450, cytochrome; MR, (\(-\))-menthol dehydrogenase; TPS, terpene synthase; IPR, (\(-\))-isopiperitenone reductase; ADH, alcohol dehydrogenase.  
Figure 7: Heatmaps of the DEGs in the monotereponoid biosynthesis pathway. GPPS-l, geranyl diphosphate synthase large subunit; GPPS-s, geranyl diphosphate synthase small subunit; LS, (\(-\))-limonene synthase; L3 OH, (\(-\))-limonene-3-hydroxyylase; iPD, (\(-\))-trans-Isopiperitenol dehydrogenase; iPR, (\(-\))-isopiperitenone reductase; iPI, (+)-cis-Isopulegone isomerase; PR, (+)-pulegone reductase; MFS, mentofuran synthase; MR, (\(-\))-menthol dehydrogenase. The green block show the final important compound, menthol; gray blocks show the branches of the final compounds.