Nitrate reductase is inhibited in leaves of Triticum aestivum treated with high levels of copper

Abstract

Copper is a potent sulfhydryl reagent which can also catalyse the generation of active oxygen. Since nitrate reductase (EC 1.6.6.1) is an SH‐enzyme sensitive to oxidative environments, the relations among copper, active oxygen species and nitrate reductase (NR) activity are of interest. Foliar segments of wheat (Triticum aestivum cv. Oasis) were floated on CuSO4 solutions (up to 250 μM) for 24 h under continuous light. Copper decreased NR activity before affecting active oxygen generation as estimated by changes in oxidative parameters, including malondialdehyde, K+ leakage and chlorophyll degradation. Cysteine and Na‐benzoate counteracted this decrease, suggesting an oxidative damage of the enzyme in leaves exposed to high copper levels. Copper‐induced NR inactivation was further studied in the partially purified enzyme. Preincubation with CuSO4 inhibited NR. Copper inhibition was reversed by subsequent incubation with EDTA, indicating that the metal bonded to key ‐SH groups of the enzyme. In addition, an ˙OH‐generating system (composed of CuSO4, ascorbate and H2O2) irreversibly decreased the activity of purified NR to a greater extent than copper alone. Our results show that copper affects nitrogen metabolism by diminishing NR activity, involving a direct effect on key SH‐groups and an indirect effect via attack by active oxygen species induced by the metal.