# Unraveling the Biosynthesis of Carvacrol in Different Tissues of _Origanum vulgarae_
## 3 Discussion
### WCENA of Genes Modules Related to Carvacrol Biosynthesis  
The systems technique for data mining using WCENA was exceptionally efficient in discovering information about modules shared by subsets of samples and connected to phenotypic characteristics; it is extremely adept at exposing trait-specific modules and important genes for biology sciences [31]. In this work, we used 16,341 DEGs derived from different tissues of _O. vulgare_ 'HS' and clustered nine modules based on expression profiles. Frequently, the amount of specialized metabolites generated by a plant is strongly associated with the density of glandular trichomes present on the surface of the epidermis, and the higher EO content in oregano may be attributable to the significantly greater abundance of glandular trichomes [12; 13]. Therefore, we not only observed gene modules positively correlated with carvacrol metabolite (orangered4, plum1, ivory), but also focused on the PGT-related gene modules of orangered4, plum1, and lightcyan (positive correlations) and darkgrey and skyblue3 (negative correlations). Moreover, SEs had the highest amount of PGTs, followed by BRs, PEs, LEs, and STs in _O. vulgare_ 'HS', which agreed with the VOC content trend in tissues, especially for carvacrol. Different _Solanum_ species accumulate different contents of terpenoids due to the shape and metabolic capacity of glandular trichomes [32; 33]. Measurements of PGT sizes in some Lamiaceae plants with leaf surface view were made through the diameter of the head along with the above subcuticular space [34; 35]. Thus, we measured the diameter of PGTs and found that SEs showed the biggest size of PGTs, followed by BRs, LEs, PEs, and STs, similar to tissue-dependent VOC contents. Our work confirms that the development of PGTs is highly positively correlated with carvacrol biosynthesis with tissue-dependence in _O. vulgare_ 'HS'.  
Progresses have been made in confirming six TF families related to the regulation of terpenoid metabolism, such as AP2/ERFs, bHLHs, MYBs, NACs, WRKYS, and bZIIPs [36]. Our WGCNA results indicated that modules positively correlated with carvacrol and PGT density/size contained _ZIPs_, _bHLHs_, _MYBs_, _WRKYs_, _NACs_, and _AP2/ERF-ERFs_, and negative modules presented _bHLHs_, _WRKYs_, _ZIPs_, and _AP2/ERF-ERFs_. For instance, _CitAP2.10_ and _CitERF71_ regulates valencene and (E)-geraniol biosynthesis in citrus, _MsMYB_ inhibits monoterpene synthesis in spearmint, and _GaWRKY1_ upregulates sesquiterpene phytoalexin accumulation in cotton [37; 38; 39; 40]. The mutation in the _NAC_ binding region of TPS gene promoters affects monoterpene contents in two kiwifruit species, while overexpression of _OsbZIP79_ reduces phytoalexin accumulation in rice [41; 42]. As reported, a set of TFs act as positive regulators of trichome formation, and a set of TFs act as negative regulators [43]. The WGCNA modules exhibited positive or negative correlations with PGT density/size, which implies positive or negative roles in regulating PGT development, which in turn affects carvacrol biosynthesis in _O. vulgare_ 'HS'.