Synthesis and Accumulation of Nitrite in Soybean Nodules Supplied with Nitrate

Abstract

It has been known for over a century that combined nitrogen inhibits the growth of legume nodules, and, in recent decades, it has been shown that combined nitrogen also inhibits N2 fixation activity of nodules (6,7,9,13,17,22). While there has been some interest in the effect of ammonium (7, 9), most of the emphasis has been on the effect of nitrate, which is the most common form of nitrogen absorbed by plant roots growing in cultivated soil. The mechanism by which nodule growth and nitrogenase activity are adversely affected by nitrate is still not clear.One of the simplest explanations for the effect of nitrate on nodule growth and nitrogenase activity states that some product of nitrate metabolism is inhibitory. Nitrite is a logical candidate for such an inhibitory role because (a) it is a potent inhibitor of nitrogenase activity when mixed with the purified enzyme (24) (b) it deoxygenates and oxidizes leghemoglobin in vitro resulting in the formation of ferric leghemoglobin ( 18); and (c) it (ie. nitrous acid) reacts spontaneously with primary and secondary amines.There have been very few reports demonstrating the actual presence of nitrite in nodules on plants supplied with nitrate.Rigaud has reported 140 to 780 nmole NO2/g fresh weight of soybean nodules 6 to 24 h after supplying plants with 7 mM N03 (17). More recently, Manhart and Wong have reported high NO2 concentrations in lupine and cowpea nodules supplied with very high (15 mM) nitrate (13). They also showed that the amount of NO2-accumulated in nodules was positively correlated with NR2 activity in bacteroids (13). In both of these studies, the main interest was in exploring the inhibition of nitrogenase activity by nitrate over periods of 1 to 5 d.After the discovery of NR in Rhizobium japonicum bacteroids (5) it was generally assumed that the synthesis of nitrite in nodules occurs in the bacteroids. However, Gibson and Pagan showed that growth and N2-fixing activity of Macroptilium atropurpureum and Trifolium subterraneum nodules formed by Rhizobia lacking NR were still depressed when plants were supplied with nitrate (6). This observation has recently been extended to Vigna unguiculata and Lupinus angustifolius nodules (13). Coupled with the fact that nitrite was not detected in these nodules (6, 13), these observations make it less likely that the generation of nitrite in nodules may be the mechanism by which nitrate inhibits nodule growth and activity. The work reported here was focused on the effect of nitrate on soybean nodule growth. The objectives were to (a) Beeson, were planted in silica sand. Variations in nitrate concentration in nutrient solutions were provided by balancing nitrate and chloride. Details on nutrient solution composition and plant growth conditions have been published (22).Plants for experiments 1 and 2 were grown outdoors during the summer months without alteration of the natural photoperiod.

Effect of Water Stress on the Reduction of Nitrate and Nitrite by Soybean Nodules

“…It has been reported that cytosol of the nodule also contained NRA (12,13). The results in this study confirmed this finding.…”

“…Although the majority of absorbed N03-was reduced in the aerial portion of bean plant, NO3-reduction was also detectable in symbiotic nodules (1,2,4,8,13 (7), Russell et al (9), and Ishizawa (4) suggested that the reduction of NO3-in soybean nodules could also be assimilatory, and, thus contributed to the nitrogen economy ofthe bean plant.…”

“…The results show that nitrate reduction occurred both in the cytosol and bacteroids of water-stressed soybean nodules. The reduction of nitrate functioned at different physiological modes in these two fractions.Soybean plant can utilize both soil NO3-and atmospheric N2.Although the majority of absorbed N03-was reduced in the aerial portion of bean plant, NO3-reduction was also detectable in symbiotic nodules (1,2,4,8,13 (7), Russell et al (9), and Ishizawa (4) suggested that the reduction of NO3-in soybean nodules could also be assimilatory, and, thus contributed to the nitrogen economy ofthe bean plant.The NO2-, produced as a result of NO-reduction, is further reduced by NiR2. Daniel and Appleby (2) were unable to detect NiRA in bacteroids of soybean nodules, even though enzyme activity could be found in Rhizobium japonicum grown anaerobically.…”

Nitrate and Nitrite Reduction in Relation to Nitrogenase Activity in Soybean Nodules and Rhizobium japonicum Bacteroids

“…It is the nodule cytosol NR that most likely accounts for the residual NR activity shown by nodules initiated by the NR-strains in Table I. The occurrence of NR, but not NiR, has previously been shown in nodule cytosols of peas (2) and soybeans (22). The activity of NiR assayed in vitro was several times the corresponding activity of NR (Table II) and, therefore, in principle NR but not NiR should be considered as the ratelimiting step in the sequence of reactions involved in NO-metabolism in nodules.…”

“…Nitrite accumulation within the nodules has been shown in NO3--treated plants of several legumes including soybeans (15,22). NO2-is a known inhibitor of several aspects of N2 fixation (12,19,21,24).…”