3.5 Iridoids biosynthesis

Iridoids of S. tetraptera comprise loganin, Sinoswertiamatin, sweroside, and gentiopicroside. We measured their concentrations in seven tissues. The content of each iridoid in the seven tissues showed similar variation trends to those reported previously (Yue Liu et al., 2017). Four iridoids had the highest content in CL flowers out of the seven tissues examined, and their contents in these flowers were all obviously higher than those in leaves from the branch on which the CL flowers were growing (termed the ”CL leaf”) (Figure 5a, Table S27). The regulatory pathway of the biosynthesis of iridoids has been reported to consist of the downstream seco-iridoids pathway and the upstream 2-C-methyl-D-erythritol 4- phosphate (MEP) and mevalonate (MVA) pathways (Kellner, Kim, Clavijo, Hamilton, Childs, Vaillancourt, Cepela, Habermann, Steuernagel, Clissold, Mclay, et al., 2015; Yue Liu et al., 2017; Vranová, Coman, & Gruissem, 2013). We identified a total of 56 candidate genes for these two biosynthesis pathways and assessed their expression levels in different tissues for subsequent analyses (Table S28-29). As the primary supplier of intermediate products in the biosynthesis of iridoids, the MEP pathway acts mainly in leaves (Oudin, Courtois, Rideau, & Clastre, 2007; Vranová et al., 2013). However, these iridoid products are always concentrated in the flowers ofSwertia mussotti (Yue Liu et al., 2017) and S. tetraptera . Therefore, the intermediate products of these iridoids may be transported from leaves to flowers in S. tetraptera .
We reconstructed a weighted gene co-expression network for iridoid biosynthesis pathways based on intersections of DEGs containing 8067 genes (618 TFs and 7449 structural genes) for the seven tissues. A total of 12 modules were clustered, and module 8 (blue) was indeed related to leaves, while module 9 (turquoise) was linked to flowers (Figure 5b, Figure S23-24). Many of the previously identified 56 candidate genes for biosynthesis of iridoids were clustered into these two modules. The leaf-related module contained most genes belonging to the MEP pathway (Figure 5b). Most of them showed higher expression levels in leaves than in flowers (Figure 5c, Figure S25-26). However, the candidate genes in the MVA pathway were mainly clustered in the flower-related module and highly expressed in flowers (Figure 5b,5c, Figure S25-26). Similar patterns were also revealed from the correlation analysis between co-expression network modules and the measured iridoid contents (Figure S27). These different clustered networks and differentially expressed genes in the two tissues may suggest the potential genetic basis for the distinct function between leaves and flowers for iridoid production and transport. Furthermore, we found that the genes from the seco-iridoids pathway were distributed in both modules. Of these, two SLS genes were clustered as the hub genes (Figure 5b). They may play an essential role in transporting intermediate products during iridoid biosynthesis between different tissues in S. tetraptera .