Soybean Inoculated With One Bradyrhizobium Strain Isolated at Elevated [CO2] Show an Impaired C and N Metabolism When Grown at Ambient [CO2]

Abstract

Soybean (Glycine max L.) future response to elevated [CO₂] has been shown to differ when inoculated with B. japonicum strains isolated at ambient or elevated [CO₂]. Plants, inoculated with three Bradyrhizobium strains isolated at different [CO₂], were grown in chambers at current and elevated [CO₂] (400 vs. 700 ppm). Together with nodule and leaf metabolomic profile, characterization of nodule N-fixation and exchange between organs were tested through ¹⁵N₂-labeling analysis. Soybeans inoculated with SFJ14-36 strain (isolated at elevated [CO₂]) showed a strong metabolic imbalance, at nodule and leaf levels when grown at ambient [CO₂], probably due to an insufficient supply of N by nodules, as shown by ¹⁵N₂-labeling. In nodules, due to shortage of photoassimilate, C may be diverted to aspartic acid instead of malate in order to improve the efficiency of the C source sustaining N₂-fixation. In leaves, photorespiration and respiration were boosted at ambient [CO₂] in plants inoculated with this strain. Additionally, free phytol, antioxidants, and fatty acid content could be indicate induced senescence due to oxidative stress and lack of nitrogen. Therefore, plants inoculated with Bradyrhizobium strain isolated at elevated [CO₂] may have lost their capacity to form effective symbiosis at ambient [CO₂] and that was translated at whole plant level through metabolic impairment.

Keywords: Bradyrhizobium strains; C and N metabolism; N-fixation; elevated [CO2]; metabolomics; nodule; soybean.

FIGURE 1

Effect of [CO₂] on soybean biomass in plants inoculated with three different B. japonicum strains. Nodule, root, stem, and leaf biomass (g DW plant^{– 1}) of soybean plant grown at a[CO₂] (400 ppm) and e[CO₂] (700 ppm) and inoculated with three Bradyrhizobium japonicum strains (USDA110, SFJ4-24, and SFJ14-36). Bars correspond to the mean ± SE of n = 6 of the biomass of each tissue. Results of statistics for total biomass (the sum of nodule, root, leaf, and stem) are shown (two-way ANOVA, P < 0.05). Different letters indicate significant differences (Tukey post hoc test P < 0.05).

FIGURE 2

Effect of [CO₂] on soybean nodule δ¹⁵N in plants inoculated with three different B. japonicum strains. Nodule ¹⁵N isotope labeling (δ¹⁵N, ‰) ± of soybean plants grown at a[CO₂] (400 ppm) and e[CO₂] (700 ppm) and inoculated with three Bradyrhizobium japonicum strains (USDA110, SFJ4-24, and SFJ14-36). Bars correspond to the mean ± SE of n = 3. Results of statistics are shown (two-way ANOVA, P < 0.05). Different letters indicate significant differences (Tukey post hoc test P < 0.05).

FIGURE 3

Effect of [CO₂] on biomass labeled ¹⁵N in three organs of soybean plants inoculated with three different B. japonicum strains. Biomass labeled ¹⁵N (mg ¹⁵N organ^{– 1}) in (A) nodules, (B) roots, and (C) leaves of soybean grown at a[CO₂] (400 ppm) and e[CO₂] (700 ppm) and inoculated with three Bradyrhizobium japonicum strains (USDA110, SFJ4-24, and SFJ14-36). Bars correspond to the mean ± SE of n = 3. Results of statistics are shown (two-way ANOVA, P < 0.05). Different letters indicate significant differences (Tukey post hoc test P < 0.05).

FIGURE 4

Photorespiratory estimations from gas exchange measures. Estimated rate of RuBP Oxygenation (v_o) in soybean leaves grown at a[CO₂] (400 ppm) and e[CO₂] (700 ppm) and inoculated with three Bradyrhizobium japonicum strains (USDA110, SFJ4-24, and SFJ14-36). Bars correspond to the mean ± SE of n = 6. Results of statistics are shown (two-way ANOVA, P < 0.05). Different letters indicate significant differences (Tukey post hoc test P < 0.05).

FIGURE 5

Graphical representation of metabolomic response to different [CO₂] in soybean plants inoculated with three B. japonicum strains. Principal component analysis (PCA) [(A) nodules and (C) leaves] of the different metabolites in soybean plants inoculated with three B. japonicum strains. Scatter plot distribution [(B) nodules and (D) leaves] of the different metabolites in soybean plants inoculated with three B. japonicum strains. Three replicates were examined per strain and CO₂ level.

FIGURE 6

Differentially expressed metabolites involved in [CO₂] responses in function of B. japonicum strain. (A) Hierarchically clustered heat maps of metabolites that were found to be significantly different between Bradyrhizobium strains, [CO₂], and their interaction in nodules. (B) Hierarchically clustered heat maps of metabolites that were found to be significantly different between Bradyrhizobium strains, [CO₂], and their interaction in leaves. Each column represents the average of three replicates. Intensity of red and green indicates increases and decreases relative to the mean respectively, as shown in the color scale.

FIGURE 7

Effects of Bradyrhizobium japonicum strain and [CO₂] on soybean nodule metabolism. Bar charts showing the relative abundance of metabolites in ambient [CO₂] (white) and elevated [CO₂] (black) in soybean plants inoculated with three different Bradyrhizobium japonicum strains. Red, yellow, and blue lightning signs indicate the significant effect of strain, CO₂, and their interaction, respectively. Metabolites in bold indicate metabolites that were found to have significant effect of strain, CO₂, and/or their interaction; italic metabolites indicate metabolites analyzed that were not found to have significant effect of strain, CO₂, and their interaction, and not bold neither italic indicates metabolites not analyzed.

FIGURE 8

Effects of Bradyrhizobium japonicum strain and [CO₂] on soybean leaf metabolism. Bar charts showing the relative abundance of metabolites in ambient [CO₂] (white) and elevated [CO₂] (black) in soybean plants inoculated with three different Bradyrhizobium japonicum strains. Red, yellow, and blue lightning signs indicate the significant effect of strain, CO₂, and their interaction, respectively. Bold metabolites indicate metabolites analyzed that were found to have significant effect of strain, CO₂ and/or their interaction; italic metabolites indicate metabolites analyzed that were not found to have significant effect of strain, CO₂, and their interaction, and not bold neither italic indicates metabolites not analyzed.