SmbHLH53 is relevant to jasmonate signaling and plays dual roles in regulating the genes for enzymes in the pathway for salvianolic acid B biosynthesis in Salvia miltiorrhiza.

Basic helix-loop-helix (bHLH) transcription factors play essential roles in myriad regulatory processes, including secondary metabolism. In this study with Salvia miltiorrhiza, we isolated and characterized SmbHLH53, which encodes a bHLH family member. Expression of this gene was significantly induced by wounding and multiple hormones, including methyl jasmonic acid; transcript levels were highest in the leaves and roots. Phylogenetic analysis indicated that SmbHLH53 clusters withAtbHLH17 and AtbHLH13, two negative regulators of jasmonate (JA) responses, and is localized in the nucleus and cell membrane. Yeast two-hybrid and bimolecular fluorescent complementation assays indicated that SmbHLH53 forms a homodimer as well as a heterodimer with SmbHLH37. It also interacts with both SmJAZs1/3/8 and SmMYC2, the core members of the JA signal pathway. Unexpectedly, we noted that overexpression of SmbHLH53 did not significantly influence the concentrations of rosmarinic acid and salvianolic acid B in transgenic plants. Results from yeast one-hybrid assays showed that SmbHLH53 binds to the promoters of SmTAT1, SmPAL1, and Sm4CL9, the key genes for enzymes in the pathway for phenolic acid synthesis. Assays of transient transcriptional activity demonstrated that SmbHLH53 represses the promoter of SmTAT1 while activating the promoter of Sm4CL9. Thus, the present work revealed that SmbHLH53 may play dual roles in regulating the genes for enzymes in the pathway for Sal B biosynthesis.

Combination of transcriptomic and metabolomic analyses reveals a JAZ repressor in the jasmonate signaling pathway of Salvia miltiorrhiza.

Jasmonates (JAs) are plant-specific key signaling molecules that respond to various stimuli and are involved in the synthesis of secondary metabolites. However, little is known about the JA signal pathway, especially in economically significant medicinal plants. To determine the functions of novel genes that participate in the JA-mediated accumulation of secondary metabolites, we examined the metabolomic and transcriptomic signatures from Salvia miltiorrhiza. For the metabolome, 35 representative metabolites showing significant changes in rates of accumulation were extracted and identified. We also screened out 2131 differentially expressed unigenes, of which 30 were involeved in the phenolic secondary metabolic pathway, while 25 were in the JA biosynthesis and signal pathways. Among several MeJA-induced novel genes, SmJAZ8 was selected for detailed functional analysis. Transgenic plants over-expressing SmJAZ8 exhibited a JA-insensitive phenotype, suggesting that the gene is a transcriptional regulator in the JA signal pathway of S. miltiorrhiza. Furthermore, this transgenic tool revealed that JAZ genes have novel function in the constitutive accumulation of secondary metabolites. Based on these findings, we propose that the combined strategy of transcriptomic and metabolomic analyses is valuable for efficient discovery of novel genes in plants.

Pathway engineering for phenolic acid accumulations in Salvia miltiorrhiza by combinational genetic manipulation.

To produce beneficial phenolic acids for medical and commercial purposes, researchers are interested in improving the normally low levels of salvianolic acid B (Sal B) produced by Salvia miltiorrhiza. Here, we present a strategy of combinational genetic manipulation to enrich the precursors available for Sal B biosynthesis. This approach, involving the lignin pathway, requires simultaneous, ectopic expression of an Arabidopsis Production of Anthocyanin Pigment 1 transcription factor (AtPAP1) plus co-suppression of two endogenous, key enzyme genes: cinnamoyl-CoA reductase (SmCCR) and caffeic acid O-methyltransferase (SmCOMT). Compared with the untransformed control, we achieved a greater accumulation of Sal B (up to 3-fold higher) along with a reduced lignin concentration. This high-Sal B phenotype was stable in roots during vegetative growth and was closely correlated with increased antioxidant capacity for the corresponding plant extracts. Although no outward change in phenotype was apparent, we characterized the molecular phenotype through integrated analysis of transcriptome and metabolome profiling. Our results demonstrated the far-reaching consequences of phenolic pathway perturbations on carbohydrate metabolism, respiration, photo-respiration, and stress responses. This report is the first to describe the production of valuable end products through combinational genetic manipulation in S. miltiorrhiza plants. Our strategy will be effective in efforts to metabolically engineer multi-branch pathway(s), such as the phenylpropanoid pathway, in economically significant medicinal plants.