Rhizobium leguminosarum Glutathione Peroxidase Is Essential for Oxidative Stress Resistance and Efficient Nodulation

Abstract

Glutathione (GSH) plays a key role in regulating the cellular Redox Homeostasis, and appears to be essential for initiation and development of root nodules. Glutathione peroxidase (Gpx) catalyzes the reduction of H₂O₂ and organic hydroperoxides by oxidation of GSH to oxidized GSH (GSSG), which in turn is reduced by glutathione reductase (GR). However, it has not been determined whether the Rhizobium leguminosarum Gpx or GR is required during symbiotic interactions with pea. To characterize the role of glutathione-dependent enzymes in the symbiotic process, single and double mutants were made in gpxA (encoding glutathione peroxidase) and gshR (encoding glutathione reductase) genes. All the mutations did not affect the rhizobial growth, but they increased the sensitivity of R. leguminosarum strains to H₂O₂. Mutant in GpxA had no effect on intracellular GSH levels, but can increase the expression of the catalase genes. The gshR mutant can induce the formation of normal nodules, while the gpxA single and double mutants exhibited a nodulation phenotype coupled to more than 50% reduction in the nitrogen fixation capacity, these defects in nodulation were characterized by the formation of ineffective nodules. In addition, the gpxA and gshR double mutant was severely impaired in rhizosphere colonization and competition. Quantitative proteomics using the TMT labeling method was applied to study the differential expression of proteins in bacteroids isolated from pea root nodules. A total of 27 differentially expressed proteins were identified in these root bacteroids including twenty down-regulated and seven up-regulated proteins. By sorting the down-regulated proteins, eight are transporter proteins, seven are dehydrogenase, deoxygenase, oxidase, and hydrolase. Moreover, three down-regulating proteins are directly involved in nodule process.

Keywords: Rhizobium leguminosarum; antioxidant function; glutathione peroxidase; quantitative proteomics; symbiotic nitrogen fixation.

FIGURE 1

The accumulation of reduced glutathione in R. leguminosarum strains. Data are from three biological samples (±SEM). ^a,bDifferent letters indicates the value is significantly different (two-way ANOVA, P < 0.05).

FIGURE 2

Competition of the wild-type RL3841 (black bars) and the mutants (gray bars) in the pea rhizospheres. The x-axis indicates the initial inoculation ratios, with 1 corresponding to 1 × 10³ CFU. The number of colony-forming bacteria (per plant) from 10 plants are shown. ^a,bValues with different letters are significantly different between mutant and wild-type control (two-way ANOVA, P < 0.05).

FIGURE 3

Structure of pea nodules and bacteroids. Root nodules were induced by RL3841 (A–C), RLgshR (D–F), RLgpxA (G–I), RLgshRgpxA (J–L), RLgpxA(pBBRgpxA) (M–O). Scale bars = 200 μm (A,D,G,J,M), 10 μm (B,E,H,K,N), 1 μm (C,F,I,L,O).