Biphasic effect of copper on the ascorbate-glutathione pathway in primary leaves of Phaseolus vulgaris seedlings during the early stages of metal assimilation

Abstract

Copper-imposed oxidative stress and antioxidative defence responses mere investigated in the primary leaves of Phaseolus vulgaris L. plants grown on hydroponics containing 50 muM CuSO4, Copper mainly accumulates in roots; therefore, an increase of the copper content in the leaves was only observed 48 h after the start of the copper supply. Nevertheless, an increase of the thiobarbituric acid reactive metabolites (TBArm) content, an indication of stress, occurred immediately following copper application. Because the ascorbate-glutathione pathway is considered as a major antioxidative defence mechanism, the evolution of the enzymes and the related metabolites involved in this pathway were studied in the primary leaves as a function of plant copper assimilation, The capacities of monodehydroascorbate reductase (EC 1.6.5.4), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2) mere increased before elevated amounts of copper could be detected in the leaves. The early enhancement of glutathione reductase was only temporary. After copper accumulation in the leaves, a second increase of the glutathione reductase capacity and also an increase of the ascorbate peroxidase capacity (EC 1.11.1.11) mere observed. These changes in enzymatic capacity modified the level of the metabolites involved. Increase of the ascorbate pool and maintenance in its reduced form was observed immediately after the start of the treatment. In the beginning of the experiment, the glutathione disulphide/reduced glutathione ratio was higher in the treated plants as compared to the controls. However, towards the end of the experiment, the total glutathione pool, as well as the reduced glutathione content, increased, resulting in a lower ratio value for the treated plants. In conlusion, copper-imposed oxidative stress, as well as the antioxidative defence response in the leaves, appears to be biphasic, An indirect preventive effect on the antioxidative defence system was observed during the first phase before the leaf copper content increased, A root-to-shoot signalling system appears to be involved. Direct oxidation by copper of reduced cell metabolites occurred during the second phase when the leaf copper content was enhanced.