Short-term inhibition of legume N2 fixation by nitrate

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Abstract
A comparison was made of changes in nitrogenase (N2ase; EC 1.18.6.1) activity, oxygen diffusion resistance and NO 3 − metabolism in symbioses ofPhaseolus vulgaris L. andVigna radiata (L.) Wilczek during a 3-d exposure to 10 mM NO 3 − . Bacteroids fromPhaseolus nodules lacked nitrate reductase (NR;EC 1.7.99.4) but those fromVigna nodules had elevated amounts of the enzyme. The nodule cytosol of both species contained assimilatory NR (EC 1.6.6.1). Both symbioses showed a C2H2-induced decline in N2ase activity, the extent of which remained constant with NO 3 − exposure forPhaseolus but became greater forVigna. Nitrate application for 3 d reduced maximum (pre-decline) rates of C2H2-reduction activity by 83% and 36% inPhaseolus andVigna, respectively. Nitrogenase-linked respiration (NLR) closely paralleled N2ase activity as the carbon costs of N2ase were not significantly altered by NO 3 − . The relationship between NLR and increases in external O2 concentration from 21 to 60% was used to characterize the oxygen diffusion resistance (R) of nodules from both species. In absolute terms the minimum R ofPhaseolus nodules increased with NO 3 − , whereas the ability to adjust this R in response to O2 was lost after 2d. ForVigna nodules the increase in minimum R was much smaller and the adjustment ability was retained for the 3-d period of NO 3 − exposure. Bacteroids ofVigna and the cytosol of both species contained NR prior to NO 3 − exposure, and activities increased 1.5- to 2-fold during the treatment period. Despite this, NO 2 − was not detected in nodules ofPhaseolus, and showed only a very small accumulation in the cytosol ofVigna nodules. It is proposed that nodules have a two-stage response to applied NO 3 − . In the first stage NO 3 − is restricted to the nodule cortex and causes a reversible increase in R. In the second stage NO 3 − may enter the infected region and toxic amounts of NO 2 − can be generated in nodules having high bacteroid andor cytosol NR activities. This NO 2 − can irreversibly damage the nodules and accelerate their senescence.