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 H 2 O 2 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.

Vacuolar H -ATPase (V-ATPase; EC 3.6.3.14) is the main enzyme responsible for generating a proton gradient across the tonoplast. Under cadmium (Cd) stress conditions, V-ATPase activity is inhibited. In the present work, hydrogen sulfide (H S) and hydrogen peroxide (H O ) cross-talk was analyzed in cucumber (Cucumis sativus L.) seedlings exposed to Cd to explain the role of both signaling molecules in the control of V-ATPase. V-ATPase activity and gene expression as well as H S and H O content and endogenous production were determined in roots of plants treated with 100 μM CdCl and different inhibitors or scavengers. It was found that H S donor improved photosynthetic parameters in Cd-stressed cucumber seedlings. Cd-induced stimulation of H S level was correlated with the increased activities of the H S-generating desulfhydrases. Increased H O and lowered H S contents in roots were able to reduce V-ATPase activities similar to Cd. H O and H S-induced modulations in V-ATPase activities were not closely related to the transcript level of encoding genes, suggesting posttranslational modifications of enzyme protein. On the other hand, exogenous H O raised H S content in root tissues independently from the desulfhydrase activity. Although treatment of control plants with H S significantly stimulated NADPH oxidase activity and gene expression, H S did not affect H O accumulation in roots exposed to Cd. The results suggest the existence of two pathways of H S generation in Cd-stressed cucumber roots. One involves desulfhydrase activity, as was previously demonstrated in different plant species. The other, the desulfhydrase-independent pathway induced by H O /NADPH oxidase, may protect V-ATPase from inhibition by Cd.

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