Short-term inhibition of legume N2 fixation by nitrate

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.