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.