INTRODUCTION
The dried roots of Salvia miltiorrhiza Bunge (Danshen) are widely
used in treating inflammation and heart diseases (Dong et al .,
2011; Liu et al ., 2018). S. miltiorrhiza has been
considered as a model medicinal plant due to its important medicinal
value, small genome (~600 Mb), short life cycle,
established
transgenic system, and ease of tissue culture (Ma et al ., 2012;
Xu et al ., 2015). The bioactive compounds ofS.
miltiorrhiza form two main groups: hydrophilic components (SAs), such
as salvianolic acid B (Sal B) and rosmarinic acid (RA), and lipophilic
components (tanshinones, which are a group of diterpenoids), such as
dihydrotanshinone I (DT-I), cryptotanshinone (CT), tanshinone I (T-I)
and tanshinone IIA (T-IIA) (Wang et al .,
2007;
Pei et al ., 2018). The
Chinese
Pharmacopoeia stipulates that the main quality markers of Danshen are
tanshinones and SAs (The State Pharmacopoeia Commission of China, 2015).
The biosynthetic pathways of SAs include phenylpropanoid and
tyrosine-derived pathways (Petersen, 2013; Sun et al. , 2018). Due
to the great medicinal value of tanshinones, the biosynthetic pathways
have been well studied (Ma et al .,
2015; Song et al. , 2015; Zhouet al., 2017). The tanshinones are synthesized from diterpenoids
universal precursor GGPP, which is produced by the mevalonic acid (MVA)
and 2-C-methyl-D-erythritol-4-phosphate (MEP) pathways (Guo et
al. , 2016; Pei et al. , 2018). The key enzyme genes involved in
tanshinones biosyntheses, such as AACT , DXS , CMK ,GGPPS , CPS , KSL1 , and CYP76AH1 have been
characterized in S. miltiorrhiza (Ma et al ., 2012; Xuet al. , 2015). There have been many reports of overexpressing or
RNA interference these biosynthetic genes could regulate the
accumulation of tanshinones (Kai et
al ., 2011; Ma et al ., 2016;
Shi et al ., 2016). Overall, the
supply of these bioactive compounds is limited because of their low
concentrations in the roots of S. miltiorrhiza . And the yields
and quality of S. miltiorrhiza are often affected by unfavorable
environmental conditions. Therefore, research on regulating
secondary
metabolites has become a hot topic. The application of TFs is a
promising approach to improve the efficiency of metabolic engineering in
the plant (Fu et al. , 2018).
TFs play an important role in secondary metabolic engineering. TF can
coordinately regulate the expressions of several genes encoding these
enzymes, therefore guide the metabolic flux towards certain pathways
(Ying et al ., 2019). It is an effective methodology using TFs to
improve the production of secondary metabolites in plants. Several MYBs
have been reported to regulate tanshinones biosynthesis of S.
miltiorrhiza. Overexpression of SmMYB36 promoted tanshinones
accumulation by binding to the promoters of DXR , MCT andGGPPS1 (Ding et al ., 2017). Overexpression ofSmMYB9b also increases tanshinones concentration by stimulating
the MEP pathway (Zhang et al. , 2017). And overexpression ofSmbHLH10 enhanced the tanshinones biosynthesis by binding with
the G-box of DXS2 and CPS5 in S. miltiorrhiza (Xinget al ., 2018). Moreover, SmWRKY1 could positively regulate
tanshinones biosynthesis via target gene DXR in S.
miltiorrhiza (Cao et al. , 2018). But ethylene response factor
(ERF), SmERF115 decreased tanshinones content in S. miltiorrhiza(Sun et al. , 2018). However, the regulatory function of GA
signaling key regulator SmGRASs in tanshinones biosynthesis of S.
miltiorrhiza remains unknown.
GRAS
TF family has been reported playing diverse roles in GA signaling, root
development, light signaling and stress responses (Livne et al. ,
2015; Xu et al. , 2015; Heck et al. , 2016). GRAS has been
found in many plants, such as Arabidopsis , tomato, rice,
grapevine, cotton and Danshen (Tian et al. , 2004; Huang et
al. , 2015; Grimplet et al. , 2016; Bai et al. , 2017; Zhanget al. , 2018). The GRAS family is divided into 13 distinct
subfamilies based on amino acid sequences: DELLA, SCL3, LAS, SCL28,
SCL4/7, SCR, SHR, SCL9 (LISCL), HAM, PAT1, OS4, DLT and OS19 (Zhanget al. , 2018). Among them,
the
SCL3 subfamily has been shown to participate in root cell elongation,
GA/DELLA signaling and stress responses (Hakoshima, 2018). Furthermore,
some evidence has shown that GRAS proteins participated in the
GA-dependent regulatory network and root periderm formation. For
instance, DELLA protein is the repressor of GA and acted as key
regulatory targets in the GA signaling pathway in regulating plant
growth (Murase et al. , 2008; Yoshida et al. , 2014). SCL3
has been reported as a repressor of DELLA. It could positively regulate
the GA signaling pathway and control GA homeostasis inArabidopsis root development (Heo et al. , 2011; Zhanget al. , 2011).
GA is an important phytohormone that plays vital roles in many processes
of plant growth and
metabolism
(Brian, 2010; Du et al. , 2015; Davière and Achard, 2016). For
instance, GA could regulate the flavonol biosynthesis through DELLA to
further promote root growth in Arabidopsis (Tan et al. ,
2019).
And
the GA-mediated control of growth has an interaction with energy
metabolism to coordinates cell wall extension, lipid and secondary
metabolism in Arabidopsis (Ribeiro et al. ,
2012).
Moreover, the biosynthesis pathways of GA and tanshinones are different
branches of the diterpenoid biosynthesis pathway, since they have a
universal precursor GGPP (Xu et al ., 2015). The biosynthesis of
GA from GGPP involves many synthase genes, such as CPS5 ,KS , KAO , GA20ox , GA3ox and GA2ox (Maet al. , 2012; Cui et al. , 2015; Su et al. , 2016).
Thus, crosstalk may occur between GA and tanshinones biosynthesis inS. miltiorrhiza . In our previous report, GA treatment was able to
increase tanshinones accumulation and induce the expressions ofSmGRAS1~5 genes in wild-type hairy roots ofS. miltiorrhiza (Bai et al. , 2018). Therefore, we
speculated that SmGRASs might be involved in the regulation of GA to
tanshinones biosynthesis in S. miltiorrhiza . Although five GRAS
family genes have been cloned in S. miltiorrhiza , however, we
found the SmGRAS5 was the most sensitive gene of them responding
to GA treatment (Bai et al. , 2017). But the roles of SmGRAS5 in
regulating tanshinones biosynthesis through GA signaling are still
unknown.
In this study, we found that overexpressing SmGRAS5 significantly
increased the tanshinones’ content and decreased GA content in S.
miltiorrhiza hairy roots. Y1H, Dual-LUC and EMSA assays indicated that
SmGRAS5 could directly bind to the promoter of SmKSL1 to induce
its expression. And GA treatment improved the tanshinones and GA
accumulation in the SmGRAS5 OE lines. Subsequently, transcriptome
analysis revealed the potential functions of SmGRAS5 in regulating
secondary metabolism. Finally, the molecular mechanism of SmGRAS5
regulated the GA-induced tanshinones biosynthesis was analyzed and
discussed. Our findings revealed a link between SmGRAS and secondary
metabolism and provided important information on GA-mediated secondary
metabolite
biosynthesis in S. miltiorrhiza .