Phenolic acids and tanshinones are two groups of bioactive ingredients in Salvia miltiorrhiza Bunge. As a heavy metal elicitor, it has been reported that Ag⁺ can induce accumulations of both phenolic acids and tanshinones in S. miltiorrhiza hairy roots. In this study, the effects of Ag⁺ treatment on accumulations of six phenolic acids and four tanshinones in S. miltiorrhiza hairy roots were investigated. To further elucidate the molecular mechanism, expressions of key genes involved in the biosynthesis of these ingredients were also detected. The results showed that although the total phenolic acids content was almost not affected by Ag⁺, accumulations of rosmarinic acid (RA), caffeic acid and ferulic acid were significantly increased, while accumulations of salvianolic acid B (LAB), danshensu (DSU) and cinnamic acid were decreased. We speculate that LAB probably derived from the branch pathway of DSU biosynthesis. Contents of four tanshinones were enhanced by Ag⁺ and their accumulations were more sensitive to Ag⁺ than phenolic acids. Genes in the upstream biosynthetic pathways of these ingredients responded to Ag⁺ earlier than those in the downstream biosynthetic pathways. Ag⁺ probably induced the whole pathways, upregulated gene expressions from the upstream pathways to the downstream pathways, and finally resulted in the enhancement of ingredient production. Compared with phenolic acids, tanshinone production was more sensitive to Ag⁺ treatments. This study will help us understand how secondary metabolism in S. miltiorrhiza responds to elicitors and provide a reference for the improvement of the production of targeted compounds in the near future.

Salvia miltiorrhiza Bunge is a Chinese traditional herb for treating cardiovascular and cerebrovascular diseases, and tanshinones and phenolic acids are the dominated medicinal and secondary metabolism constituents of this plant. WRKY transcription factors (TFs) can function as regulators of secondary metabolites biosynthesis in many plants. However, studies on the WRKY that regulate tanshinones and phenolics biosynthesis are limited. In this study, 69 SmWRKYs were identified in the transcriptome database of S. miltiorrhiza, and phylogenetic analysis indicated that some SmWRKYs had closer genetic relationships with other plant WRKYs, which were involved in secondary metabolism. Hairy roots of S. miltiorrhiza were treated by methyl jasmonate (MeJA) to detect the dynamic change trend of SmWRKY, biosynthetic genes, and medicinal ingredients accumulation. Base on those date, a correlation analysis using Pearson’s correlation coefficient was performed to construct gene-to-metabolite network and identify 9 SmWRKYs (SmWRKY1, 7, 19, 29, 45, 52, 56, 58, and 68), which were most likely to be involved in tanshinones and phenolic acids biosynthesis. Taken together, this study has provided a significant resource that could be used for further research on SmWRKY in S. miltiorrhiza and especially could be used as a cue for further investigating SmWRKY functions in secondary metabolite accumulation.

Salvia miltiorrhiza and S. grandifolia are rich in diterpenoids and have therapeutic effects on cardiovascular diseases. In this study, the spatial distribution of diterpenoids in both species was analyzed by a combination of metabolomics and mass spectrometry imaging techniques. The results indicated that diterpenoids in S. miltiorrhiza were mainly abietane-type norditerpenoid quinones with a furan or dihydrofuran D-ring and were mainly distributed in the periderm of the roots, e.g. cryptotanshinone and tanshinone IIA. The compounds in S. grandifolia were mainly phenolic abietane-type tricyclic diterpenoids with six- or seven-membered C-rings, and were widely distributed in the periderm, phloem, and xylem of the roots, e.g. 11-hydroxy-sugiol, 11,20-dihydroxy-sugiol, and 11,20-dihydroxy-ferruginol. In addition, the leaves of S. grandifolia were rich in tanshinone biosynthesis precursors, such as 11-hydroxy-sugiol, while those of S. miltiorrhiza were rich in phenolic acids. Genes in the upstream pathway of tanshinone biosynthesis were highly expressed in the root of S. grandifolia, and genes in the downstream pathway were highly expressed in the root of S. miltiorrhiza. Here, we describe the specific tissue distributions and mechanisms of diterpenoids in two Salvia species, which will facilitate further investigations of the biosynthesis of diterpenoids in plant synthetic biology.

Salvia miltiorrhiza is a well-known traditional Chinese herb which is used to treat heart disease. Salvia castanea is a substitute product for S. miltiorrhiza in the medicinal field. Previous study has shown that phosphate (Pi) deficiency could promote the accumulation of secondary metabolism in herbs, and it has also developed a strategy for saving Pi resources and increasing the yield of active substances in herbs. In the present study, the hairy roots of S. miltiorrhiza and S. castanea were used to identify the Pi deficiency response mechanisms of these two Salvia species. The results showed that Pi deficiency increased the accumulation of specifically secondary metabolites, such as phenolic acids and tanshinones, which were caused by promoting the expression levels of key enzyme genes. In addition, Pi deficiency promoted the antioxidant activity in these two Salvia species. The data demonstrated that Pi deficiency increased the quality of the medicinal material in the plant. The hairy roots of S. castanea were more adaptive to Pi deficiency than those of S. miltiorrhiza in terms of biomass, secondary metabolism, and antioxidant activity. The results of this study provide insights into breeding herbs that are better adapted to Pi deficiency, which could increase the yield of active ingredients in herbs and save Pi resources.