Effect of different oxygen availability on the nitrate reductase activity in Cucumis sativus roots

The effect of different oxygen availability on the nitrate reductase (NR) activity was studied in cucumber roots from cucumber seedlings grown hydroponically. NR activity measured in the presence of Mg2+ (actual NR activity) as well as NR activity measured with EDTA (maximum NR activity) increased distinctly after 30 min of root incubation in a medium flushed with N2 (anaerobic conditions). In contrast, aeration of roots decreased both enzyme activities. Such inactivation of NR was rapidly reversed after transferring the roots to anaerobic conditions. An air-induced decrease of the actual enzyme activity was prevented by staurosporin, a protein kinase inhibitor, whereas microcistin LR, an inhibitor of protein phosphatases, completely eliminated the reactivation of NR actual activity under limited oxygen availability. An increase of the NR actual activity in roots incubated in a nitrogen-flushed buffer was correlated with a lower content of ATP in root tissues. These data suggest that reversible protein phosphorylation is involved in the regulation of NR activity under limited oxygen. On the other hand, feeding roots with inhibitors of protein kinases as well as phosphatases did not affect the maximal activity of NR indicating that other modification(s) of enzyme activity could also function in cucumber roots. Since the changes in the expression level of gene encoding nitrate reductase (CsNR) under different oxygen availability were not correlated with the enzyme activity, the transcription level of oxygen action was excluded. On the other hand, it was demonstrated that oxygen-induced alteration of NR was dependent on the ratio of oxidized/reduced pyridine nucleotides in tissues. In aerobic conditions, when maximal NR activity was inhibited, a drop of the NAD(P)H level was also observed. These data point to hysteretic modifications of NR protein induced by NAD(P)H as the target of reversible and rapid changes in maximal enzyme activity under different oxygen availability.