Short-term inhibition of legume N2 fixation by nitrate

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Abstract
The hypothesis of NO 2 − toxicity as the causative factor of NO 3 − inhibition of nitrogenase (N2ase; EC 1.18.6.1) activity has been evaluated using a short-term exposure (3 d) of several legumes. Treatment of plants with 10 mM NO 3 − induced nitrate reductase (NR) from bacteroids (EC 1.7.99.4) and nodule cytosol (EC 1.6.6.1) in most species. Regardless of the levels of both enzymes, significant accumulation of NO 2 − did not occur in nodules. Dissection of nodules into cortical and infected regions, and subsequent NO 2 − assays in conditions that suppressed enzyme activities, indicated that, in the short-term, bacteroid NR does not generate NO 2 − in vivo. This is probably because NO 3 − access is restricted to the nodule cortex. Accumulation of NO 2 − at levels that are damaging for N2ase and leghaemoglobin were only observed when a delay occurred between dissection and assaying of nodules. It is concluded that NO 2 − is not responsible for the initial NO 3 − -induced decline of N2ase activity, and that toxic amounts of NO 2 − only build up in nodules following longer exposures to NO 3 − , when this anion is actively reduced by bacteroid and cytosol enzymes.