Mechanism of Cd and Cu action on the tonoplast proton pumps in cucumber roots

The effect of Cd and Cu on the tonoplast proton pumps, V-ATPase (EC 3.6.3.14) and V-PPase (EC 3.6.1.1) was investigated in cucumber roots subjected to 10 mu M metals for 3 and 6 days. Both hydrolytic and transporting activities of V-ATPase as well as V-PPase increased under copper stress. In contrast, all activities examined were inhibited after the exposure of plants to cadmium. Cd and Cu changed the efficiency of coupling between proton transport and ATP hydrolysis whereas H+/PPi stoichiometry was not modified. Pre-incubation of control tonoplast vesicles with copper caused the stimulation of V-ATPase as well as V-PPase, indicating direct activation by Cu ions. Pre-treatment with cadmium had no significant effect on the activities of both enzymes. The gene expression and western blot analyses showed that observed modifications in enzyme activities were not related to the changes in the transcript levels of genes encoding V-ATPase subunit A and c, and V-PPase or in amounts of enzyme proteins. Moreover, the addition of reduced or oxidized glutathione (GSH and GSSG) to the reaction medium containing tonoplast vesicles isolated from stressed roots did not change the activity level of either enzyme when compared with the controls, suggesting that heavy metal-induced modifications are not simple reversible redox modulations.