The effect of heavy metals on the modification of plasma membrane H⁺-ATPase (EC 3.6.3.14) activity in cucumber roots was studied. In plants stressed for 2 h with 10 μM or 100 μM Cd, Cu or Ni the hydrolytic as well as the transporting activity of H⁺-ATPase in the plasma membranes of root cells was decreased. Transcript levels of Cucumis sativus plasma membrane H⁺-ATPase in roots treated with 10 μM Cd, Cu, or Ni as well as with 100 μM Cu or Ni were similar to the control, indicating that the action of metals did not involve the gene expression level. Only in roots exposed to 100 μM Cd was the level of plasma membrane H⁺-ATPase mRNA markedly decreased. The inhibition of the plasma membrane proton pump caused by 100 μM Cd, Cu and Ni was partially diminished in the presence of cantharidin, a specific inhibitor of protein phosphatases. Western blot analysis with the antibody against phosphothreonine confirmed that decreased activity of plasma membrane H⁺-ATPase under heavy metals resulted from dephosphorylation of the enzyme protein. Taken together, these data strongly indicated that alteration of the enzyme under heavy metal stresses was mainly due to the post-translational modification of its proteins in short-term experiments.

The effect of heavy metals on plasma membrane (PM) H⁺-ATPase (EC 3.6.3.14) activity in cucumber (Cucumis sativus) roots was studied. The aim of this work was to explain the mechanism of modification of the PM H⁺-ATPase activity in plants subjected to heavy metals. Plants were treated with 10 μM Cd or Cu for 6 d. After 3 d exposure to the heavy metals, some of the plants were transferred to control conditions for a further 3 d (3/3 plants). The activity of PM H⁺-ATPase was found to be increased in plants treated with heavy metals. The highest activity measured as proton transport was observed in 3/3 plants. Estimation of transcript levels of C. sativus PM H⁺-ATPase in roots indicated that the action of Cd, but not Cu, affected the gene expression level. Transcript levels of C. sativus PM H⁺-ATPase (CsHA2, CsHA3, CsHA4, CsHA8, and CsHA9) genes increased in roots treated with Cd. Moreover, Western blot analysis with antibody against phosphothreonine and 14-3-3 protein indicated that increased activity of PM H⁺-ATPase under heavy-metal stress resulted from phosphorylation of the enzyme. It was found that Cu markedly increased the activity of catalase and ascorbate peroxidase and reduced the level of H₂O₂ in cucumber roots. In contrast, Cd did not affect these parameters. These results indicate that Cd and Cu can, in different ways, lead to modification of PM H⁺-ATPase activity. Additionally, it was observed that treatment of plants with heavy metals led to an increased level of heat-shock proteins in the tissues. This suggests that the plants had started adaptive processes to survive adverse conditions, and increased PM H⁺-ATPase activity could further enhance the repair processes in heavy-metal-stressed plants.