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.
Highlights
Reactive oxygen species (ROS) are biologically important O2 derivatives that are by-products of aerobic metabolism (Köhler et al, 2014)
The whole genome sequence analysis of wild-type RL3841 revealed the presence of a glutathione peroxidase gene and a glutathione reductase encoding gene. gpxA (RL_RS08810, RL1698) is predicted to encode a polypeptide of 182 amino acids with a molecular mass of about 20.02 kDa and a theoretical pI value of 5.51, and gshR (RL_RS13915, RL2694) is predicted to encode a polypeptide of 461 amino acids with a molecular mass of about 50.05 kDa and a theoretical pI value of 5.87
None of the mutant strains presented a substantial reduced growth rate (Supplementary Figure 1). These results suggest neither gpxA gene nor gshR gene has effect on R. leguminosarum growth in acid minimal salts medium (AMS) minimal medium, and contrast with the data from S. meliloti in which glutathione reductase is important for the growth (Tang et al, 2018)
Summary
Reactive oxygen species (ROS) are biologically important O2 derivatives that are by-products of aerobic metabolism (Köhler et al, 2014). The most important ROS include several small chemical compounds such as the superoxide anion, hydroxyl anion (OH−), hydrogen peroxide (H2O2), nitric oxide (NO), and peroxynitrite (ONOO−) (Suvorava and Kojda, 2009). Excessive production of ROS or impaired ROS detoxification causes oxidative damage to lipid, protein, and DNA (Chung et al, 2010). Intracellular antioxidants include enzymes such as superoxide dismutase, catalase, and Gpx, as well as low molecular weight antioxidants like flavonoids, glutathione (GSH) and phenolics (Liu et al, 2014). Glutathione peroxidase plays an important role in scavenging reactive oxygen metabolites and protection of organisms from oxidative damage (Khanyok et al, 1997)
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