Utilization of nitrate by bacteroids of Bradyrhizobium japonicum in the soybean root nodule

Abstract

Bacteroids of Bradyrhizobium japonicum strain CB1809, unlike CC705, do not have a high level of constitutive nitrate reductase (NR; EC 1.7.99.4) in the soybean (Glycine max. Merr.) nodule. Ex planta both strains have a high activity of NR when cultured on 5 mM nitrate at 2% O2 (v/v). Nitrite reductase (NiR) was active in cultured cells of bradyrhizobia, but activity with succinate as electron donor was not detected in freshly-isolated bacteroids. A low activity was measured with reduced methyl viologen. When bacteroids of CC705 were incubated with nitrate there was a rapid production of nitrite which resulted in repression of NR. Subsequently when NiR was induced, nitrite was utilized and NR activity recovered. Nitrate reductase was induced in bacteroids of strain CB1809 when they were incubated in-vitro with nitrate or nitrite. Increase in NR activity was prevented by rifampicin (10 μg· ml(-1)) or chloramphenicol (50 μg·ml(-1)). Nitrite-reductase activity in bacteroids of strain CB1809 was induced in parallel with NR. When nitrate was supplied to soybeans nodulated with strain CC705, nitrite was detected in nodule extracts prepared in aqueous media and it accumulated during storage (1°C) and on further incubation at 25°C. Nitrite was not detected in nodule extracts prepared in ethanol. Thus nitrite accumulation in nodule tissue appears to occur only after maceration and although bacteroids of some strains of B. japonicum have a high level of a constitutive NR, they do not appear to reduce nitrate in the nodule because this anion does not gain access to the bacteroid zone. Soybeans nodulated with strains CC705 and CB1809 were equally sensitive to nitrate inhibition of N2 fixation.

Role of Oxygen in the Limitation and Inhibition of Nitrogenase Activity and Respiration Rate in Individual Soybean Nodules

“…Also, values of Oi measured at TNAPeak were lower than those measured previously in minimally disturbed soybean nodules (King and Layzell, 1991). However, the TNA, k values were equal to, or greater than, most TNA values reported in the literature for nodulated roots of soybeans (5.1-54 kmol g-' dry weight of nodule h-') (Ralston and Imsande, 1982;van Kessel and Bums, 1983;van Berkum et al, 1985;Bethlenfalvay et al, 1987;Giannakis et al, 1988;Imsande, 1988;Kanayama et al, 1990;Kohl et al, 1991). This observation is consistent with the suggestion that many of the standard methods that have been used for assay of nitrogenase activity inhibit the process being measured (Minchin et al, 1983(Minchin et al, , 1986, presumably due to a reduction in Oi and the rate of nitrogenase-linked respiration.…”

Nitrate transport pathway into soybean nodules traced by tungstate and15NO3-

“…Smith and Smith (1986) produced evidence that NOa-reaches the infected region of nodules, and that it is reduced to N20 by bacteroid enzymes. On the other hand Sprent et al (1987) suggested that NOa-could not penetrate into the infected region, and Giannakis et al (1988) concluded that NO2-accumulation in soybean nodules is an artifact resulting from the mixing of infected and cortical regions. Becana et al (1989) attributed these discrepancies to the differences in the experimental conditions used in terms of NO3-concentration, treatment duration, or plant-microbe species.…”

Effect of Nitrate on Nodulation and Nitrogen Fixation of Soybean

“…Kanayama and Yamamoto proposed that NO formed from NO 3 -binds to Lb to make nitrosylleghemoglobin and defect the O 2 binding activity (Kanayama & Yamamoto, 1990). On the other hand, Giannakis et al (1988) suggested that nitrate metabolism does not occur in symbiotic region of soybean nodule, even when a dissimilatory NR is expressed, because of restricted access of nitrate. Leghemoglobin (Lb) plays a crucial role in N 2 fixation of leguminous nodules by facilitating O 2 supply to the bacteroids.…”