Co-expression analysis screened out the set of terpene-related genes
To investigate the transcriptomic differences in leaves, stems, and roots of S. rosmarinus , three biological replicates were collected from each organ to ensure accuracy. Raw data underwent filtering, resulting in 7.02-7.10 million 150 bp paired-end reads (Table S13), which were mapped to our assembly at the rate of 67.95% 92.40% (Table S14). Differentially expressed genes (DEGs) were identified using DESeq2 (Varet, Brillet-Gueguen, Coppee, & Dillies, 2016), with a stringent threshold of Log2|FoldChange| >1 and p-value < 0.05. We identified a total of 16,052 DEGs, with 8,833 up-regulated and 7,219 down-regulated genes in the root vs. Leaf comparison, 11,982 DEGs (6,496 up-regulated, 5,486 down-regulated) in the root vs. Stem comparison, and 15,598 DEGs (8,241 up-regulated, 7,357 down-regulated) in the leaf vs. Stem comparison (Figure S14). In addition, 3,475 genes were differentially expressed in all three groups (Figure 3e).
To gain insight into the metabolic processes involved in different organs of S. rosmarinus , KEGG enrichment analysis was performed. The DEGs between roots and leaves were significantly enriched in ”Biosynthesis of other secondary metabolites” and ”Metabolism of terpenoids and polyketides” (p-value< 0.05) (Figure S15, Table S15). While the DEGs between roots and stems were significantly enriched in ”Metabolism of terpenoids and polyketides” and ”Terpenoid backbone biosynthesis” (p-value< 0.05) (Figure S15, Table S15). Notably, the DEGs between stems and leaves were significantly enriched in ”Biosynthesis of other secondary metabolites,” ”Flavonoid biosynthesis,” and ”Phenylpropanoid biosynthesis” (p-value < 0.05) (Figure S15, Table S16). These pathways provided a transcriptomic-level understanding of the metabolic processes in different organs of S. rosmarinus .
To construct the WGCNA co-expression network, we used all DEGs and differential metabolites. By calculating Pearson correlation coefficients (PCCs) between contents of components and gene expression modules (Figure S16, Tables S11, S12), we identified a set of genes associated with monoterpenoids and diterpenoids (PCCs > 0.6). Our results suggested that SrCYP71D8 , SrWRKY2 (with p-value of 0.0315 and 0.0237, respectively) were highly correlated with the accumulation of monoterpene in S. rosmarinus organs. We also screened key genes, including SrHMGR (with p-value located at 0.0019 0.0401) and limonene synthase, which was found to be involved in the biosynthesis process of monoterpene. Moreover, we identified SrCYP81Q32 , and SrMYB1 (with p-values of 0.0004 and 0.0004, respectively) as highly correlated with the accumulation of diterpenoids. Finally, we found that SrGGPPS (with p-values of 0.0004 0.0475) was highly correlated with diterpenoids biosynthesis (Table S21). These findings provide important evidence to support further exploration of terpenoid biosynthesis in S. rosmarinus .