# Unraveling the Biosynthesis of Carvacrol in Different Tissues of _Origanum vulgarae_
## 2 Results
### Identification of VOC-Related Gene Modules Using Modular Organization Analysis  
In Figure S9A, using 16,341 DEGs generated from _O. vulgare_ 'HS', a network heat map plot (interconnectivity plot) of the gene network is displayed, along with the related hierarchical clustering dendrograms and subsequent modules. Using a set of criteria that resulted in improved clusters, we identified nine modules with between 147 and 3,873 DEGs each, colored darkgrey, skyblue3, lightsteelblue1, navajowwhite2, darkgreen, plum1, lightcyan, ivory, and orangered4 (Figure S9B). We also calculated the similarity and clustered nine modules into three subgroups depending on their eigenvalues: (i) the darkgrey, sky  
Figure 4: The expression pattern analysis of genes associated with terpene synthases (TPSs), cytochromes P450s (CYPs), and short-chain dehydrogenase/reductases (SDRs). Heatmap of expression profiles of TPS (**A**), CYP71 (**B**), and SDR (**C**) genes in different tissues of _Origanum vulgare_ ’Hot and Spicy’.  
blue3, and lightestblue1 modules; (ii) the navajowhite2, darkgreen, and plum1 modules; (iii) the lightcyan, ivory, and orangered4 modules. To capture comprehensive transcriptome changes in different tissues of _O. vulgare_ 'HS', we conducted modular organization analysis to classify 16,341 DEGs related to VOCs. The module-VOC relationship revealed that each component was significantly relevant to at least one module (R > 0.5; Figure 5A, Figure 5B). A large portion of carvacrol existed in SEs, BRs, PEs, and LEs, but not in STs, exhibiting a highly positive correlation with orangered4 (R = 0.93), plum1 (R = 0.73), and ivory (R = 0.63) modules. The vital intermediate product of \(\gamma\)-terpinene also presented a similar positive correlation with orangered4 (R = 0.94), plum1 (R = 0.79), and ivory (R = 0.63) modules. STs with a high content of p-cymene exhibited a strongly positive correlation with lightsteelblue (R = 0.77) and navajowhite (R = 0.62) modules (Figure 5B). Additionally, the expression profiles of DEGs in the orangered4 module appeared to be extremely elevated in SEs and STs, and DEGs in the plum1 module exhibited more upregulations in BRs and SEs, while DEGs in the ivory module presented more upregulations in SEs and PEs (Figure 5C).  
With a highly positive correlation between orangered4, plum1, ivory module genes and carvacrol content, we conducted a GO and KEGG term analysis of these module DEGs (Figure S10). As for GO terms, DEGs enriched in'metabolic process' and 'cellular process' showed a high percentage for biological process; DEGs enriched in 'catalytic activity' and  
Figure 5: Modular organization analysis of gene expression in different tissues of _Origanum vulgare_ ‘Hot and Spicy’ associated with volatile organic compounds (VOCs). The clustering tree depicts nine modules of co-expressed genes (**A**). Each of the 16,341 DEGs is represented by a leaf on the tree, and the primary branches of the tree represent modules, which are identified in different colors in the lower panel. The correlations between 21 VOCs and 9 modules (**B**). The expression profiles in different tissues of DEGs in orangered4, plum1, and ivory modules (positive with carvacrol content) (**C**).  
'binding' showed a high percentage for the molecular function; DEGs enriched in 'cell part' and 'cell' showed a high percentage for the cellular component (Figure S10A, Figure S10C, Figure S10E). As for KEGG terms, major DEGs were involved in the metabolism processes of'starch and sucrose metabolism', 'carbon metabolism', 'amino sugar and nucleotide sugar metabolism', 'biosynthesis of amino acids', 'glycolysis/glucoseogenesis', and 'pyruvate metabolism' (Figure S10B, Figure S10D, Figure S10F). Notably, the processes of'starch and sucrose metabolism', 'glycolysis/glucoseogenesis', 'pyruvate metabolism', and 'carbon metabolism' took part in generating G3P and PYR as the precursors of MVA and MEP pathways, and distinct module genes were differently expressed in these processes.