# Unraveling the Biosynthesis of Carvacrol in Different Tissues of _Origanum vulgarae_
## 2 Results
### RNA-Seq Data Reveal Carvacrol Biosynthesis in Different Tissues of O. vulgare 'HS'
Importantly, \(\gamma\)-terpinene synthase gives rise to \(\gamma\)-terpinene, and other enzymes, such as CYPs and SDRs, are necessarily involved in later steps to confer final arrangements, generating the product of carvacrol [10] (Figure S8; red box). Other monoterpene synthases are implicated in forming various monoterpenes, such as sabinene. Based on the annotated database, we achieved 62 TPS DEGs and analyzed their expression patterns in STs, LEs, BRs, SEs, and PEs (Figure 4A). Of these, 17 SE-specific TPS genes were clustered and presented extremely low expression in STs. Significantly, three \(\gamma\)-terpinene synthase  
Figure 3: Transcript abundance profiles of genes encoding enzymes involved in the mexolonate (MVA) pathway (**A**), the methylerythnitol pathway (**M**EP) pathway (**B**), and the biosynthesis of sesquiterpene skeleton (**C**) in different tissues of _Origanum vulgare_ ‘Hot and Spicy’. genes (A_transcript_9644, A_transcript_7390, A_transcript_11437) exhibited the highest expression in SEs and the lowest expression in STs. An essential class of CYPs is responsible for the oxidation and conjugation of the initial terpene synthase products, and TPS genes are typically found in tandem with the CYP71 gene family in both eudicots and monocots [17]. In the present study, we analyzed 40 CYP71 family genes (including 23 _CYP71 As_, _CYP71 Bs_, and 10 _CYP71 Ds_) in different tissues of \(O\). _vulgare_ 'HS' (Figure 4B). Compared with STs, 26 upregulated CYP genes in SEs contained 16 _CYP71 As_, 3 _CYP71 Bs_, and 7 _CYP71 Ds_; 16 upregulated CYP genes in BRs contained 9 _CYP71 As_, 4 _CYP71 Bs_, and 3 _CYP71 Ds_; 14 upregulated CYP genes in PEs contained 6 _CYP71 As_, 6 _CYP71 Bs_, and 2 _CYP71 Ds_; 12 upregulated CYP genes in LEs contained 8 _CYP71 As_, 3 _CYP71 Bs_, and one _CYP71D_. Simultaneously, 3, 4, 11, and 3 downregulated CYP genes were found in SEs, BRs, PEs, and LEs. Notably, five CYP genes (_CYP71 As_: A_transcript_14387, A_transcript_11561, A_transcript_12504, A_transcript_13769; _CYP71D_: A_transcript_14409) exhibited obviously higher expression in SEs than in other tissues (FPKM > 10). Next, we observed that seven _CYP71 As_ and two _CYP71 Ds_ were most highly expressed in SEs. These results indicate that CYP71 family genes prefer higher transcripts in carvacrol-rich tissues, especially for _CYP71 As_ and _CYP71 Ds_ in _O. vulgare_ 'HS'. A recent study has reported that \(\gamma\)-terpinene was converted to carvacrol by the combined activity of a CYP71D monooxygenase and the short-chain dehydrogenase/reductase (SDR) [10]. The subsequent formation of the aromatic compounds occurs via keto-enol tautomerisms. Based on the annotated database, we achieved 83 SDRs and analyzed their expression patterns in STs, LEs, BRs, SEs, and PEs (Figure 4C). Of these, 19 SE-specific (e.g., A_transcript_11932 and A_transcript_19023), 20 LE-specific (e.g., A_transcript_22742 and A_transcript_23036), and 15 PE-specific (e.g., A_transcript_74343 and A_transcript_31562) SDR genes were clustered and presented extremely low expression in STs.