SmGRAS1 and SmGRAS2 Regulate the Biosynthesis of Tanshinones and Phenolic Acids in Salvia miltiorrhiza

Abstract

Salvia miltiorrhiza is one of the most widely used traditional Chinese medicinal plants because of its excellent performance in treating heart diseases. Tanshinones and phenolic acids are two important classes of effective metabolites, and their biosynthesis has attracted widespread interest. Here, we functionally characterized SmGRAS1 and SmGRAS2, two GRAS family transcription factors from S. miltiorrhiza. SmGRAS1/2 were highly expressed in the root periderm, where tanshinones mainly accumulated in S. miltiorrhiza. Overexpression of SmGRAS1/2 upregulated tanshinones accumulation and downregulated GA, phenolic acids contents, and root biomass. However, antisense expression of SmGRAS1/2 reduced the tanshinones accumulation and increased the GA, phenolic acids contents, and root biomass. The expression patterns of biosynthesis genes were consistent with the changes in compounds accumulation. GA treatment increased tanshinones, phenolic acids, and GA contents in the overexpression lines, and restored the root growth inhibited by overexpressing SmGRAS1/2. Subsequently, yeast one-hybrid, dual-luciferase, and electrophoretic mobility shift assays (EMSA) showed SmGRAS1 promoted tanshinones biosynthesis by directly binding to the GARE motif in the SmKSL1 promoter and activating its expression. Yeast two-hybrid assays showed SmGRAS1 interacted physically with SmGRAS2. Taken together, the results revealed that SmGRAS1/2 acted as repressors in root growth and phenolic acids biosynthesis but as positive regulators in tanshinones biosynthesis. Overall, our findings revealed the potential value of SmGRAS1/2 in genetically engineering changes in secondary metabolism.

Transcriptomic analysis reveals the GRAS family genes respond to gibberellin in Salvia miltiorrhiza hairy roots

“…This result also explains a series of composition content changes in the hairy root of S. miltiorrhiza at the transcriptional level after GA treatment, such as the increased tanshinones and phenolic acids contents. In addition, since our previous research found that 3 SmGRAS genes can promote the biosynthesis of tanshinones and inhibit the biosynthesis of GA, we hypothesize that GRAS core regulators of the GA signaling pathway would be involved in the regulation of GA on the secondary metabolism [ 24 , 25 ]. Therefore, we also deeply analyzed the expressions of DEGs in the GA signaling pathway.…”

“…Ten DEGs specific primers were designed by primer 5 (Table S5 ). qRT-PCR was performed on a real-time PCR system (Bio-Rad CFX96, USA), according to our previous methods [ 24 ]. The SmActin gene was used as the endogenous control [ 38 ].…”

“…In addition, GRAS has crucial roles in the GA signaling pathway. Our previous research has shown that 3 SmGRAS genes significantly promote tanshinones biosynthesis, also inhibiting GA biosynthesis in hairy roots of S. miltiorrhiza [ 24 , 25 ] . Therefore, we speculated that SmGRASs might mediate the regulation of secondary metabolism by the GA signaling in S. miltiorrhiza .…”

Identification of peach growth regulating-factors and their expression under UVB

“…To get a better understanding of evolutionary relationships, a neighbor-joining phylogenetic tree was constructed among GRFs from Arabidopsis (9), soybean (22), rice (12) poplar (19), and four Rosaceae species (46). As shown in Fig.…”

“…Supplementary Table S3). In this area we quali cated many other important potential cis-elements, such as GARE motif and P-box (GA responsive element), a CAT-box (cis-acting regulatory element related to meristem expression), AuxRR-core and TGA-element (auxin-responsive element), an RY element (seedspeci c regulation) and a TC-rich repeats (defense and stress responsiveness) [46][47][48][49][50][51]. GARE, P-box, CATbox, AuxRR-core, TGA-element, RY element and TC-rich repeats were distributed within 3, 3, 7, 2, 3, 1 and 5…”