## 2 Results
### Assessment of Correlations between Terenoid Content and Gene Expression  
To better understand the regulation of volatile oil biosynthesis, transport, and accumulation over time in _A. argyi_ leaves, we conducted further analyses of the genes associated with terpenoid backbone biosynthesis and modification. Eleven of these genes (from Figure 6) were maximally expressed in June, when these leaf samples exhibited peak volatile oil levels. Pearson correlation analyses revealed these genes to be significantly correlated with the terpenoid content in these leaf samples (Figure 7). In particular, the expression of HMGR1 from the MAV pathway was significantly correlated with 1,8-Cineole, (1R)-4-methyl-1-propan, \(\gamma-\)Tepinene, Sabinene, Thujone, and \(\beta\)-Selinene contents, while MDD1 and MDD2 were correlated with \(\alpha\)-Terpineol levels. The HDR1 and HDR3 genes of the MEP pathway were also significantly correlated with O-Cymene, 1,8-Cineole, and \(\beta\)--Farmesene content, while HDS7 was significantly correlated with the \(\gamma\)-Terpinene, Sabinene, and \(\beta\)-Selinene levels. In addition, TPS2 was significantly positively correlated with the levels of (1R)-4-methyl-1-propan contents, and TPS5 was significantly positively correlated with the levels of the O-Cymene, Thujone, and \(\beta\)--Farmesene contents, respectively. These results identified six key genes: one 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR1), one (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS7), two 1-deoxy-D-xylulose-5-phosphate reductioisomerase (HDR1, HDR7), and two terpene synthase (TPS2,TPS5) genes that were significantly correlated with the levels of monoterpenes or sesquiterpenpenes in the _A. argyi_ samples. The structural diversity of the terpenoids is related to the generation of diverse terpene skeletons by TPS [19], and these latter two enzymes are likely to regulate monoterpene and sesquiterpene distributions over the course of _A. argyi_ leaf development. RT-qPCR results revealed that these six gene expression profiles were generally consistent with the FPKM values (Figure 8).  
Figure 6: Schematic overview of the putative _A. argyi_ terpenoid biosynthetic pathway, with corresponding gene expression levels. (**A**) The predicted pathway of _A. argyi_ terpenoid biosynthesis, with the numbers in brackets denoting the number of enzymes in each family. (**B**) A clustered heatmap demonstrating the log-2-transformed FPKM expression levels of 77 terpenoid backbone genes and 7 TPS genes in _A. argyi_ samples collected during the indicated months.  
Figure 8: Expression analyses of selected genes (from Figure 7) using both qRT-PCR and RNA-Seq (FC of FPKM) approaches. Relative expression levels were estimated from the threshold of the PCR cycle, by the delta–delta Ct method. The error bars indicate the standard errors of three biological replicates.  
Figure 7: Pearson correlation bubble chart corresponding to gene expression patterns and terpenoid contents in _A. argyi_ samples collected at four time points. The size of circles corresponds to correlation coefficient (R) values, and colors indicate whether a correlation is negative or positive.