Effect of hydrogen peroxide on nitrate reductase activity in detached oat leaves in darkness

Abstract

Nitrate reductase (NR, EC 1.6.6.1) is sensitive to O2 concentration, and therefore it was of interest to study the action of H2O2, a normal substance in plant metabolism, on NR activity in segments of 7‐, 14‐ and 17‐day‐old leaves of oat (Avena sativa L. ev. Suregrain). After 4 h of treatment in the dark, H2O2 decreased NR activity as measured with the in vivo assay. The effect was stronger in 14‐ and 17‐ than in 7‐day‐old leaves. Vacuum infiltration of cysteine did not prevent this decrease. When NR was determined with the in vitro assay, H2O2 did not seem to affect the activity after the 4 h treatment. but NR decreased when crude extracts prepared from untreated 14‐day‐old leaves were incubated directly with H2O2. This effect was prevented by addition of cysteine, ascorbate or reduced glutathione to the extracts. In order to study the possibility that low activity of the system for defense against oxidations could account for the age‐dependent response of NR to H2O2 in the in vivo test, activities of catalase, ascorbate peroxidase and glutathione reductase were measured during leaf development and after a 4‐h treatment with H2O2 in the dark. No clear correlation was found between the activities of those enzymes and changes in in vivo NR activity caused by H2O2. The results suggest that H2O2 might affect NR both directly by oxidizing SH‐groups and indirectly by decreasing reductant availability as a result of NADH oxidation. The age‐dependent response of NR to H2O2 treatment could also be explained in terms of decreased NADH availability in the tissues due to decreased NADH synthesis and/or increased degradation.