The Cell Membrane of a Novel Rhizobium phaseoli Strain Is the Crucial Target for Aluminium Toxicity and Tolerance

Abstract

Soils with low pH and high aluminium (Al) contamination restrict common bean production, mainly due to adverse effects on rhizobia. We isolated a novel rhizobium strain, B3, from Kenyan soil which is more tolerant to Al stress than the widely used commercial strain CIAT899. B3 was resistant to 50 µM Al and recovered from 100 µM Al stress, while CIAT899 did not. Calcein labeling showed that less Al binds to the B3 membranes and less ATP and mScarlet-1 protein, a cytoplasmic marker, leaked out of B3 than CIAT899 cells in Al-containing media. Expression profiles showed that the primary targets of Al are genes involved in membrane biogenesis, metal ions binding and transport, carbohydrate, and amino acid metabolism and transport. The identified differentially expressed genes suggested that the intracellular γ-aminobutyric acid (GABA), glutathione (GSH), and amino acid levels, as well as the amount of the extracellular exopolysaccharide (EPS), might change during Al stress. Altered EPS levels could also influence biofilm formation. Therefore, these parameters were investigated in more detail. The GABA levels, extracellular EPS production, and biofilm formation increased, while GSH and amino acid level decreased. In conclusion, our comparative analysis identified genes that respond to Al stress in R. phaseoli. It appears that a large portion of the identified genes code for proteins stabilizing the plasma membrane. These genes might be helpful for future studies investigating the molecular basis of Al tolerance and the characterization of candidate rhizobial isolates that perform better in Al-contaminated soils than commercial strains.

Keywords: RNA-Seq; Rhizobium phaseoli; aluminium toxicity; aluminum tolerance; common bean; gene expression.

Figure 1

Percent growth inhibition of isolates B3, S2, S3, and standard R. tropici CIAT899 in media with Al at pH 5.5.

Figure 2

Cell viability approximated by plate count of viable cells after short-time exposure (A) and viability recovery after longtime exposure (B) of isolates B3 and CIAT899 to different Al concentrations. Asterisks represent significance differences between Al-treated CIAT899 and B3 and their respective untreated controls (at 0 µM Al) (one-way ANOVA; * p ≤ 0.05; ** p ≤ 0.001).

Figure 3

The fluorescence signals from Calcein bound to the plasma membranes of Al treated rhizobia cells and untreated control exemplarily quantified with a fluorescence plate reader. Asterisks represent significance differences between fluorescence intensity from CIAT899 and B3 (one-way ANOVA; ** p ≤ 0.001).

Figure 4

Extracellular ATP (A) and extracellular mScarlet-1 (B). Asterisks represent significance differences between luminescence intensity from CIAT899 and B3 (A) and fluorescence intensity between untreated B3 cells with Al-treated cells at various concentrations (one-way ANOVA; * p ≤ 0.05; ** p ≤ 0.001). Values on the y-axis show fold change compared to the untreated cells.

Figure 5

Gene expression analysis of Al-treated B3 cells compared to untreated control cells. A volcano plot (A) shows significantly different (colored) or not significantly different (grey) up-and down-regulated genes in Al-treated cells. (B) Quantitative real-time PCR of the three upregulated genes, highlighted in panel (A). Asterisks in (B) represent significant differences in transcript abundance between Al treated samples and untreated controls (one-way ANOVA; ** p ≤ 0.001).

Figure 6

Subcellular protein localization of the proteins of the DEGs, (A) proteins of upregulated and (B) downregulated genes.

Figure 7

Functional annotation of proteins of DEGs by the cluster of orthologous groups.

Figure 8

Amount of produced exopolysaccharides (A) and biofilm formation (B) by isolate B3 under different concentrations of Al. Asterisks represent the significant differences in the quantity of EPS and biofilm between Al-treated samples and untreated controls (one-way ANOVA; ** p ≤ 0.001).

Figure 9

Expression of GAD1 and GSS genes by RT-qPCR (A), fold change of the amounts of GSH (B), γ-aminobutyric acid (C), and amino acids (D) in Al-treated cells relative to the untreated controls. Asterisks represent significant differences in the expression levels of GAD1 and GSS genes (A), amount of GSH (B), and GABA (C) between Al-treated samples and untreated controls (one-way ANOVA; ** p ≤ 0.001).