Metabolomic Analysis of Alfalfa (Medicago sativa L.) Root-Symbiotic Rhizobia Responses under Alkali Stress

Abstract

Alkaline salts (e.g., NaHCO₃ and Na₂CO₃) causes more severe morphological and physiological damage to plants than neutral salts (e.g., NaCl and Na₂SO₄) due to differences in pH. The mechanism by which plants respond to alkali stress is not fully understood, especially in plants having symbotic relationships such as alfalfa (Medicago sativa L.). Therefore, a study was designed to evaluate the metabolic response of the root-nodule symbiosis in alfalfa under alkali stress using comparative metabolomics. Rhizobium-nodulized (RI group) and non-nodulized (NI group) alfalfa roots were treated with 200 mmol/L NaHCO₃ and, roots samples were analyzed for malondialdehydyde (MDA), proline, glutathione (GSH), superoxide dismutase (SOD), and peroxidase (POD) content. Additionally, metabolite profiling was conducted using gas chromatography combined with time-of-flight mass spectrometry (GC/TOF-MS). Phenotypically, the RI alfalfa exhibited a greater resistance to alkali stress than the NI plants examined. Physiological analysis and metabolic profiling revealed that RI plants accumulated more antioxidants (SOD, POD, GSH), osmolytes (sugar, glycols, proline), organic acids (succinic acid, fumaric acid, and alpha-ketoglutaric acid), and metabolites that are involved in nitrogen fixation. Our pairwise metabolomics comparisons revealed that RI alfalfa plants exhibited a distinct metabolic profile associated with alkali putative tolerance relative to NI alfalfa plants. Data provide new information about the relationship between non-nodulized, rhizobium-nodulized alfalfa and alkali resistance.

Keywords: GC-TOF/MS; alfalfa; alkali stress; metabolomics; symbiotic rhizobium.

Figure 1

Phenotype of alfalfa plants with or without rhizobium inoculation before and after NaHCO₃ treatment. (A) Untreated, (B) 4 d after alkali stress, (C) 6 d after alkali stress, and (D) 8 d after alkali stress. The left two pots in each image have inoculated RI group plants and the right two are non-inoculated NI plants.

Figure 2

Changes in MDA (A) and proline (B) and GSH (E) content and in the activities of SOD (C), and POD (D) in the roots of alfalfa plants with (RI group) and without (NI group) rhizobium inoculation under alkali stress. Values are means of six replicates. Standard error bars annotated by different lower-case letters in the same stress type indicate significant differences at p < 0.05 according to Duncan's method.

Figure 3

Principal component analysis (PCA) score plots of metabolic profiles in alfalfa roots under the alkali stress. (A) PCA score plot for NI control (black), NI alkali-treated (blue) samples, (B) PCA score plot for RI control (red) and RI alkali-treated (yellow) samples, (C) PCA score plot for NI control (black) and RI control (red) samples, (D) PCA score plot for NI alkali-treated (blue) and RI alkali-treated (yellow) samples.

Figure 4

Partial least squares-discriminate analysis (PLS-DA) score plots of metabolic profiles in alfalfa roots under the alkali stress. (A) PLS-DA score plot for NI control (black), NI alkali-treated (blue) samples, (B) PLS-DA score plot for RI control (red) and RI alkali-treated (yellow) samples, (C) PLS-DA score plot for NI control (black) and RI control (red) samples, (D) PLS-DA score plot for NI alkali-treated (blue) and RI alkali-treated (yellow) samples.

Figure 5

(A) Effects of alkali stress on the metabolomes of NI. (B) Heat map analysis combined with hierarchical cluster analysis of A and A1. (C) Effects of alkali stress on the metabolomes of RI. (D) Heat map analysis combined with hierarchical cluster analysis of B and B1. A, NI control; A1, NI 6 d after alkali stress; B, RI control; B1, RI 6 d after alkali stress.

Figure 6

(A) Effects of metabolomes in NI and RI alfalfa. (B) Heat map analysis combined with hierarchical cluster analysis of A and B. (C) Effects of alkali stress on the metabolomes of NI and RI. (D) Heat map analysis combined with hierarchical cluster analysis of A1 and B1. A, NI control; B, RI control; A1, NI 6 d after alkali stress; B1, RI 6 d after alkali stress.

Figure 7

(A) NI Control and NI 6 days after alkali stress pathway enrichment (B) RI Control and RI 6 days after alkali stress pathway enrichment (C) NI Control and RI Control pathway enrichment (D) RI 6 d after alkali stress and NI 6 d after alkali stress. Pathway enrichment, the abscissa pathway impact represents the influencing factor of the path topological analysis, and the ordinate −log (P) represents the P-value of the pathway enrichment analysis (negative logarithm). At the same time, the bubble size indicates the influence factor of topological analysis, the bigger the bubble is, the bigger the impact factor is. The color of the bubbles indicates the P-value (negative logarithm) of the enrichment analysis. The darker the color, the larger the value of −log (P), the more significant the enrichment.

Figure 8

Metabolic changes involved in the primary pathways of alfalfa roots under alkali treatment. The significantly up- and down-regulated (VIP > 1, P < 0.05) metabolites were indicated in red and green, respectively. NI alkali-treated/NI Control (A1/A); RI alkali-treated/RI Control (B1/B), RI Control/NI Control (B/A), RI alkali-treated/NI alkali-treated (B1/A1).