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Legumes can contribute to emissions of the potent greenhouse gas nitrous oxide (NO) directly by some rhizobia species that are able to denitrify under free-living conditions and in symbiotic association with the plant. In this study, the capacity of Phaseolus vulgaris-Rhizobium etli symbiosis to emit NO in response to nitrate (NO) has been demonstrated for the first time. We found that bacteroidal assimilatory nitrate reductase (NarB) as well as nitrite reductase (NirK) and nitric oxide reductase (cNor) denitrifying enzymes contribute to nitric oxide (NO) and NO formation in nodules. We also show that R. etli NarK is involved in NO extrusion and links NO reduction by NarB in the cytoplasm with NirK and cNor denitrification activities in the periplasm. The knowledge generated in this work will be instrumental for exploring strategies and sustainable practices in agricultural soil management to increase legume crop yield and mitigate greenhouse gas emissions.

Legume-rhizobia symbiotic associations have beneficial effects on food security and nutrition, health and climate change. Hypoxia induced by flooding produces nitric oxide (NO) in nodules from soybean plants cultivated in nitrate-containing soils. As NO is a strong inhibitor of nitrogenase expression and activity, this negatively impacts symbiotic nitrogen fixation in soybean and limits crop production. In Bradyrhizobium diazoefficiens, denitrification is the main process involved in NO formation by soybean flooded nodules. In addition to denitrification, nitrate assimilation is another source of NO in free-living B. diazoefficiens cells and a single domain hemoglobin (Bjgb) has been shown to have a role in NO detoxification during nitrate-dependent growth. However, the involvement of Bjgb in protecting nitrogenase against NO in soybean nodules remains unclear. In this work, we have investigated the effect of inoculation of soybean plants with a bjgb mutant on biological nitrogen fixation. By analyzing the proportion of N in shoots derived from N2-fixation using the 15N isotope dilution technique, we found that plants inoculated with the bjgb mutant strain had higher tolerance to flooding than those inoculated with the parental strain. Similarly, reduction of nitrogenase activity and nifH expression by flooding was less pronounced in bjgb than in WT nodules. These beneficial effects are probably due to the reduction of NO accumulation in bjgb flooded nodules compared to the wild-type nodules. This decrease is caused by an induction of expression and activity of the denitrifying NO reductase enzyme in bjgb bacteroids. As bjgb deficiency promotes NO-tolerance, the negative effect of NO on nitrogenase is partially prevented and thus demonstrates that inoculation of soybean plants with the B. diazoefficiens bjgb mutant confers protection of symbiotic nitrogen fixation during flooding.

Aims: Greenhouse gases are considered as potential atmospheric pollutants, with agriculture being one of the main emission sources. The practice of inoculating soybean seeds with Bradyrhizobium sp. might contribute to nitrous oxide (NO) emissions. We analysed this capacity in five of the most used strains of Bradyrhizobium sp. in South America. Methods and Results: We analysed the denitrification pathway and NO production by Bradyrhizobium japonicum E109 and CPAC15, Bradyrhizobium diazoefficiens CPAC7 and B. elkanii SEMIA 587 and SEMIA 5019, both in free-living conditions and in symbiosis with soybean. The in silico analysis indicated the absence of nosZ genes in B. japonicum and the presence of all denitrification genes in B. diazoefficiens strains, as well as the absence of nirK, norC and nosZ genes in B. elkanii. The in planta analysis confirmed NO production under saprophytic conditions or symbiosis with soybean root nodules. In the case of symbiosis, up to 26.1 and 18.4 times higher in plants inoculated with SEMIA5019 and E109, respectively, than in those inoculated with USDA110. Conclusions: The strains E109, SEMIA 5019, CPAC15 and SEMIA 587 showed the highest NO production both as free-living cells and in symbiotic conditions in comparison with USDA110 and CPAC7, which do have the nosZ gene. Although norC and nosZ could not be identified in silico or in vitro in SEMIA 587 and SEMIA 5019, these strains showed the capacity to produce NO in our experimental conditions. Significance and Impact of the Study: This is the first report to analyse and confirm the incomplete denitrification capacity and NO production in four of the five most used strains of Bradyrhizobium sp. for soybean inoculation in South America.

Aims: Bradyrhizobium diazoefficiens is a soil bacterium capable of establishing nitrogen-fixing symbiosis with soybean plants, an important crop for food production worldwide. This interaction is under the control of several regulatory factors that affect the bacterial efficiency to nodulate legume plants and fix nitrogen within root nodules. To date, the function of the NtrYX two-component system in B. diazoefficiens remains unclear. In this work, we investigated the role of NtrY in the symbiotic interaction of B. diazoefficiens with soybean plants. Methods: We constructed a non-polar mutant of the sensor protein NtrY of the system, and then analysed the symbiosis of this mutant with soybean plants. Results: We found that the ntrY mutant was defective in plant dry weight, nitrogen content and nitrogenase activity. Haem c-staining of cbb3 oxidase components showed a clear reduction in the expression of this terminal oxidase in ntrY bacteroids with respect to wild type bacteroids. Such defect in cbb3 expression correlated with a decreased respiratory capacity of the bacteroids produced by the ntrY mutant. Conclusions: These results suggest that the role of B. diazoefficiens NtrYX in symbiotic nitrogen fixation might be a consequence of its involvement in cbb3 oxidase expression in bacteroids.

Nitrous oxide (NO) is one of the three main biogenic greenhouse gases (GHGs) and agriculture represents close to 30 % of the total NO net emissions. In agricultural soils, NO is emitted by two main microbial processes, nitrification and denitrification, both of which can convert synthetic nitrogen fertilizer into NO. Legume-rhizobia symbiosis could be an effective and environmental-friendly alternative to nitrogen fertilization and hence, to mitigate soil NO emissions. However, legume crops also contribute to NO emissions. A better understanding of the environmental factors involved in the emission of NO from nodules would be instrumental for mitigating the release of this GHG gas. In this work, in vivo NO emissions from nodulated soybean roots in response to nitrate (0, 1, 2 and 4 mM) and flooding have been measured. To investigate the contribution of rhizobial denitrification in NO emission from nodules, plants were inoculated with B. japonicum USDA110 and napA and nosZ denitrification mutants. The results showed that nitrate was essential for NO emissions and its concentration enhanced NO fluxes showing a statistical linear correlation, being the highest NO fluxes obtained with 4 mM nitrate. When inoculated plants grown with 4 mM nitrate were subjected to flooding, a 150- and 830-fold induction of NO emission rates from USDA110 and nosZ nodulated roots, respectively, was observed compared to non-flooded plants, especially during long-term flooding. Under these conditions, NO emissions from detached nodules produced by the napA mutant were significantly lower (p < 0.05) than those produced by the wild-type strain (382 versus 1120 nmol NO h g NFW, respectively). In contrast, nodules from plants inoculated with the nosZ mutant accumulated statistically higher levels of NO compared to wild-type nodules (2522 versus nmol 1120 NO h g NFW, p < 0.05). These results demonstrate that flooding is an important environmental factor for NO emissions from soybean nodules and th

Background The compatible solute trehalose is involved in the osmostress response of Rhizobium etli, the microsymbiont of Phaseolus vulgaris. In this work, we reconstructed trehalose metabolism in R. etli, and investigated its role in cellular adaptation and survival to heat and desiccation stress under free living conditions. Results Besides trehalose as major compatible solute, R. etli CE3 also accumulated glutamate and, if present in the medium, mannitol. Putative genes for trehalose synthesis (otsAB/treS/treZY), uptake (aglEFGK/thuEFGK) and degradation (thuAB/treC) were scattered among the chromosome and plasmids p42a, p42c, p42e, and p42f, and in some instances found redundant. Two copies of the otsA gene, encoding trehalose-6-P-synthase, were located in the chromosome (otsAch) and plasmid p42a (otsAa), and the latter seemed to be acquired by horizontal transfer. High temperature alone did not influence growth of R. etli, but a combination of high temperature and osmotic stress was more deleterious for growth than osmotic stress alone. Although high temperature induced some trehalose synthesis by R. etli, trehalose biosynthesis was mainly triggered by osmotic stress. However, an otsAch mutant, unable to synthesize trehalose in minimal medium, showed impaired growth at high temperature, suggesting that trehalose plays a role in thermoprotection of R. etli. Desiccation tolerance by R. etli wild type cells was dependent of high trehalose production by osmotic pre-conditioned cells. Cells of the mutant strain otsAch showed ca. 3-fold lower survival levels than the wild type strain after drying, and a null viability after 4 days storage. Conclusions Our findings suggest a beneficial effect of osmotic stress in R. etli tolerance to desiccation, and an important role of trehalose on the response of R. etli to high temperature and desiccation stress.