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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Cucumber (Cucumis sativus L.) is a crop plant being the third most-produced vegetable developed as a new model plant. Heavy metal pollution is a serious global problem that affects crop production. An industrial activity has led to high emissions of Cd into the environment. Plants realize adaptive strategies to diminish the toxic effects of Cd. They can remove excess toxic ions of heavy metals from the cytoplasm to the outside of cells using the metal/proton antiport. The proton gradient needed for the action of the antiporter is generated by the plasma membrane (PM) H+-ATPase (EC 3.6.3.14). We have shown that treatment of cucumber plants with Cd stimulated the diamine oxidase (DAO, EC 1.4.3.6) activity in roots. Under cadmium stress, the PM H+-ATPase activity also increased in cucumber seedlings. The stimulating effect of Cd on the PM H+-ATPase activity and expression of three genes encoding this enzyme (CsHA2, CsHA4, CsHA8) was reduced by aminoguanidine (AG, a DAO inhibitor). Moreover, we have observed that H2O2 produced by DAO promotes the formation of NO in the roots of seedlings. The results presented in this work showed that DAO may be an element of the signal transduction pathway, leading to enhanced PM H+-ATPase activity under cadmium stress.