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Drought stress is a major constraint to the quality and production of Salvia miltiorrhiza Bunge (Danshen). This study aimed to investigate the posttranslational molecular mechanisms in S. miltiorrhiza leaves in response to drought stress using quantitative phosphoproteomics analysis. S. miltiorrhiza plants were stressed by withholding water for two (moderate drought stress) and four weeks (high drought stress). Leaf samples were prepared with tandem mass tag labeling. Liquid chromatography-tandem mass spectrometry was performed for the quantitative phosphoproteomics. Bioinformatics methods were used to identify the phosphosites and phosphoproteins that had significantly changed phosphorylation levels upon drought stresses. A total of 119 common phosphoproteins were significantly changed by both high and moderate drought stresses. The phosphorylation levels of proteins related to protein processing, photosynthesis, RNA binding, and splicing were significantly changed upon high drought, not moderate drought. Additionally, we identified that the Ser phosphorylation levels of most proteins related to terpene metabolism and RNA splicing were regulated by drought stresses. The Ser and Thr phosphorylation levels of energy metabolism proteins (including FBA2/8, PPC4, and PPCC) and heat shock proteins (including HSP70 and HSP90) were upregulated by drought stresses. Our study showed the posttranscriptional mechanisms in S. miltiorrhiza leaves in response to drought stress.

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Salvia miltiorrhiza Bunge (Danshen) is an important traditional Chinese medicine herb. This study aimed to investigate the drought-responsive metabolic profiling in S. miltiorrhiza using gas chromatography time-of-flight mass spectrometry (GC-TOFMS) analysis. Fifty day-old S. miltiorrhiza seedlings were treated with two (moderate drought, MD) and four weeks (high drought, HD) of withholding water. The S. miltiorrhiza leaf and root samples were prepared for the GC-TOFMS analysis. Differential metabolites with substantial changes in content in S. miltiorrhiza leaf and root were identified using multivariate and univariate statistics. A total of 178 and 157 annotated metabolites were detected in S. miltiorrhiza leaf and root, respectively. Multivariate analysis showed that significantly discriminant metabolites in S. miltiorrhiza leaf by drought were associated with “galactose metabolism” and “citrate cycle”. In addition, the significantly discriminant metabolites in S. miltiorrhiza root were associated with “starch and sucrose metabolism”. Univariate statistics showed that the content of succinic acid, d-glucose, and oxoglutaric acid in S. miltiorrhiza leaf was increased by drought (fold change, FC > 1.5). Allose, d-xylose, melibiose, mannose, sorbitol, quinic acid, sinigrin, and taurine in S. miltiorrhiza root were decreased by drought (FC < 0.67). There were different metabolic profiles between S. miltiorrhiza leaf and root. However, the influence of drought stress on the pharmacological value and accumulation of bioactive constituents in S. miltiorrhiza should be further investigated.

Salvia miltiorrhiza is a traditional Chinese medicinal plant of Labiatae, which has been widely utilized to treat a variety of cardiovascular and cerebrovascular diseases. However, due to the long growth cycle, low content of active ingredients, and serious quality deterioration of S. miltiorrhiza, the use of biotechnology to improve S. miltiorrhiza to meet the growing demand for clinical applications has become a research hotspot. In this study, a novel one-step hairy root regeneration method was developed, which could rapidly obtain hairy roots and regenerated plants with high tanshinone content. By optimizing the parameters of Agrobacterium rhizogenes transformation in S. miltiorrhiza, it was finally established that the explants were infected in Ar.qual (OD600 = 0.6) for 10 min, co-cultured for 3 days, and then screened on the screening medium containing 7.5 mg/l hygromycin, the maximum transformation frequency can reach 73.85%. GFP and PCR detection yielded a total of 9 positive transgenic hairy root lines and 11 positive transgenic regenerated plants. SmGGPPS1 was successfully overexpressed in positive transgenic regenerated plants, according to the results of qRT-PCR. The content of tanshinone IIA and cryptotanshinone were dramatically enhanced in transgenic regenerated plants and hairy roots by Ultra Performance Liquid Chromatography analysis. Based on the Agrobacterium-mediated transformation of S. miltiorrhiza, this study developed a new method for regenerating plants with transgenic hairy roots. This method provides a foundation for the breeding of S. miltiorrhiza and the sustainable development of medicinal plant resources, as well as provides a useful reference for the application of other species.