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 .