Untargeted LC-MS/MS-Based Metabolomic Profiling for the Edible and Medicinal Plant Salvia miltiorrhiza Under Different Levels of Cadmium Stress

Abstract

Salvia miltiorrhiza, a medicinal and edible plant, has been extensively applied to treat cardiovascular diseases and chronic hepatitis. Cadmium (Cd) affects the quality of S. miltiorrhiza, posing serious threats to human health. To reveal the metabolic mechanisms of S. miltiorrhiza's resistance to Cd stress, metabolite changes in S. miltiorrhiza roots treated with 0 (CK), 25 (T1), 50 (T2) and 100 (T3) mg kg^-1 Cd by liquid chromatography coupled to mass spectrometry (LC-MS/MS) were investigated. A total of 305 metabolites were identified, and most of them were amino acids, organic acids and fatty acids, which contributed to the discrimination of CK from the Cd-treated groups. Among them, S. miltiorrhiza mainly upregulated o-tyrosine, chorismate and eudesmic acid in resistance to 25 mg kg^-1 Cd; DL-tryptophan, L-aspartic acid, L-proline and chorismite in resistance to 50 mg kg^-1 Cd; and L-proline, L-serine, L-histidine, eudesmic acid, and rosmarinic acid in resistance to 100 mg kg^-1 Cd. It mainly downregulated unsaturated fatty acids (e.g., oleic acid, linoleic acid) in resistance to 25, 50, and 100 mg kg^-1 Cd and upregulated saturated fatty acids (especially stearic acid) in resistance to 100 mg kg^-1 Cd. Biosynthesis of unsaturated fatty acids, isoquinoline alkaloid, betalain, aminoacyl-tRNA, and tyrosine metabolism were the significantly enriched metabolic pathways and the most important pathways involved in the Cd resistance of S. miltiorrhiza. These data elucidated the crucial metabolic mechanisms involved in S. miltiorrhiza Cd resistance and the crucial metabolites that could be used to improve resistance to Cd stress in medicinal plant breeding.

Keywords: Cd treatment; LC-MS/MS; Salvia miltiorrhiza; discriminating metabolites; metabolic pathways.

Figure 1

Variation in the total metabolites in S. miltiorrhiza roots under different levels of Cd stress. (A,B) represent changes in the total number of upregulated and downregulated metabolites in S. miltiorrhiza roots with different levels of Cd treatment, respectively; different colors in (A,B) represent different sources of upregulated metabolites and downregulated metabolites, respectively; (C), Total relative amino acid, organic acid, fatty acid, ketones, sugar, amide and others content in S. miltiorrhiza roots; Different lowercase letters on the columns of the same metabolite indicate significant differences within each treatment in the roots (p < 0.05); vertical bars above the columns indicate the standard error of each mean (mean ± SE).

Figure 2

Score plots of principal component analysis (PCA) for metabolomic data from S. miltiorrhiza roots under different levels of Cd stress. PC1, the first principal component; PC2, the second principal component. The ellipse indicates Hotelling's T2 (95%); (A–C) stand for score plots PCA of metabolite data obtained from LC–MS/MS in S. miltiorrhiza roots of T1 (25 mg/kg Cd group) and CK (control group), of T2 (50 mg/kg Cd group) and CK, and of T3 (100 mg/kg Cd group) and CK, respectively.

Figure 3

Score plots of orthogonal projections to latent structures discriminant analysis (OPLS-DA) of metabolite data in S. miltiorrhiza roots. (A–C) stand for score plots OPLS-DA of metabolite data obtained from LC–MS/MS in S. miltiorrhiza roots of T1 (25 mg/kg Cd group) and CK (control group), of T2 (50 mg/kg Cd group) and CK, and of T3 (100 mg/kg Cd group) and CK, respectively.

Figure 4

Variation in the discriminating metabolites in S. miltiorrhiza roots under different levels of Cd stress. (A,B) stand for overlap of the upregulated and downregulated discriminating metabolites of S. miltiorrhiza roots in response to different Cd treatments, respectively; different colors in (A,B) represent different sources of upregulated discriminating metabolites, and downregulated discriminating metabolites, respectively; (C), Heatmap analysis of amino acids ((Li et al.), fatty acids (b), esters (c), organic acids (d), ketones (e), sugars (f) and others (g) of the discriminating metabolites in S. miltiorrhiza roots between the control and Cd stressed groups; T1, T2 and T3 stand for discriminating metabolites in roots between the control and 25 mg kg⁻¹ Cd group (T1), 50 mg kg⁻¹ group (T2), and 100 mg kg⁻¹ Cd group (T3), respectively; Log2(FC), an estimate of the log2-transformed ratio of the relative content of metabolites in S. miltiorrhiza roots of the Cd treated group to that of the control group. The colors indicate the log2 transform of the ratios for the relative content of metabolites between the control and Cd treated groups in S. miltiorrhiza roots, ranging from black (low level) to red (high level). The gray ellipses stand for not discriminating metabolites.

Figure 5

Pathway of discriminating metabolites in S. miltiorrhiza roots between the control and Cd-stressed groups. (A–C) represent pathways of discriminating metabolites between CK and T1 (A), T2 (B), and T3 (C), respectively; the size and color of the bubble represent the number hits and -log10(p) values for each pathway from pathway analysis; (D), overlap of metabolic pathways discriminating metabolites of S. miltiorrhiza roots based on KEGG in response to different levels of Cd stress; different colors represent different sources of metabolic pathways for discriminating metabolites.

Figure 6

Metabolic map of S. miltiorrhiza roots based on LC–MS/MS with different levels of Cd stress. Metabolisms in orange were involved in isoquinoline alkaloid biosynthesis, betalain biosynthesis, and tyrosine metabolism, and purple and green were involved in aminoacyl-tRNA biosynthesis and unsaturated fatty acids biosynthesis, respectively; G3P, PEP, L-Ser, SA, EU, L-Phe, L-Try, RA, L-Asp, L-His, L-Pro, L-Arg and L-Asn represent glucose-3-phosphate, phosphoenolpyruvic acid, L-serine, shikimic acid, eudesmic acid, L-phenylalanine, L-tyrosine, rosmarinic acid, L-aspartic acid, L-histidine, L-proline, L-arginine, and L-asparagine, respectively; Log2(FC), an estimate of the log2-transformed ratio of the relative content of metabolites in S. miltiorrhiza roots of the Cd treated group to that of the control group.