Involvement of Bradyrhizobium japonicum denitrification in symbiotic nitrogen fixation by soybean plants subjected to flooding

Abstract

Abstract Denitrification by Bradyrhizobium japonicum bacteroids contributes to nitric oxide (NO) production within soybean nodules in response to flooding conditions. However, the physiological relevance of NO production by denitrification in B. japonicum–Glycine max symbiosis is still unclear. In this work, soybean plants were inoculated with B. japonicum strains lacking the nirK or norC genes which encode the copper-containing nitrite reductase and the c-type nitric oxide reductase enzymes, respectively. 14 days flooding increased nodule number of plants inoculated with the WT and norC strains, but not of plants inoculated with the nirK mutant. However, nodule dry weight was not affected by 14 days flooding regardless of the strain used for inoculation. Supporting this observation, individual nodule growth was significantly higher in plants inoculated with nirK than those inoculated with WT or norC after 14 days flooding. Nodule functioning was strongly inhibited by flooding since leghemoglobin content of the nodules induced by any of the strains was significantly decreased after 7 or 14 days flooding compared to control plants. However, this effect was more relevant in nodules of plants inoculated with the WT or norC mutant than in those inoculated with the nirK mutant. Nitrogen fixation was also estimated by analyzing nitrogen content derived from biological nitrogen fixation in shoots, using the 15N isotope dilution technique. By using this approach, we observed that the negative effect of 14 days flooding on nitrogen fixation was more pronounced in plants inoculated with the norC mutant. However, nitrogen fixation of plants inoculated with nirK showed the highest tolerance to 14 days flooding. These findings allowed us to demonstrate the previously proposed hypothesis which suggests that NO formed by copper-containing nitrite reductase in soybean nodules, in response to flooding, has a negative effect on nitrogenase activity. We propose that inoculation of soybeans with a B. japonicum nirK mutant, which does not produce NO from nitrate, increases the tolerance of symbiotic nitrogen fixation to flooding.