SmGRAS5 promotes tanshinones biosynthesis in S. miltiorrhiza
To further explore the roles of SmGRAS5 in the biosynthesis of tanshinones, the transgenic hairy roots lines were generated through overexpression and antisense expression approaches. The positive transgenic hairy roots were identified by PCR (Fig. S3). Control hairy roots were developed using ATCC15834 without plasmids. Three independent OE and three AE lines were randomly selected for functional analysis (G5O14, G5O17, G5O20 and G5A11, G5A16, G5A21). Collectively, the gene expression levels of SmGRAS5 were 18- to 50-fold higher in the OE lines than in the control but decreased by 70%–85% in the AE lines (Fig. 3A).
The phenotypes of control and SmGRAS5 transgenic hairy roots are shown in Figure S4. The SmGRAS5 OE lines showed redder and smaller compared with the control. The HPLC analysis showed that the contents of four tanshinones (DT-I, CT, T-I, T-IIA) were all significantly increased in the SmGRAS5 OE lines (Fig. 3B). Tanshinones’ contents were the highest in the G5O14 lines, reaching 5-fold (DT-I), 10.85-fold (CT), 2.94-fold (T-I), and 3.77-fold (T-IIA) of the control lines, respectively. In contrast, the contents of four tanshinones were reduced in the SmGRAS5 AE lines, especially T-I (Fig. 3C). The results showed that SmGRAS5 promoted the accumulation of tanshinones.
To further confirm these results, we then analyzed the expression levels of key genes in the biosynthetic pathways of tanshinones (Fig. 3D). As expected, the expressions of the genes whose promoters contained the GA response motif were consistent with HPLC results. Expressions of most of the key genes in the tanshinones biosynthetic pathway were upregulated in the SmGRAS 5 OE lines. The expressions of downstream genes of the tanshinones biosynthetic pathway, such as GGPPS1 ,CPS1 and KSL1 , were indeed significantly upregulated in the SmGRAS 5 OE lines. In contrast, expressions of most of the tanshinones biosynthetic pathway genes were decreased in the AE lines, especially AACT2 , HDS , CMK and KSL1 . These data showed that SmGRAS5 could regulate the biosynthesis of tanshinones through regulating the expressions of key biosynthesis genes. Collectively, our findings indicated that SmGRAS5 acted as a positive regulator of tanshinones biosynthesis in S. miltiorrhiza hairy roots.