Short-term inhibition of legume N2 fixation by nitrate : I. Nitrate effects on nitrate-reductase activities of bacteroids and nodule cytosol

Abstract

The hypothesis of NO2 (-) toxicity as the causative factor of NO3 (-) 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 NO3 (-) 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 NO2 (-) did not occur in nodules. Dissection of nodules into cortical and infected regions, and subsequent NO2 (-) assays in conditions that suppressed enzyme activities, indicated that, in the short-term, bacteroid NR does not generate NO2 (-) in vivo. This is probably because NO3 (-) access is restricted to the nodule cortex. Accumulation of NO2 (-) 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 NO2 (-) is not responsible for the initial NO3 (-)-induced decline of N2ase activity, and that toxic amounts of NO2 (-) only build up in nodules following longer exposures to NO3 (-), when this anion is actively reduced by bacteroid and cytosol enzymes.