Involvement of plasma membrane H⁺-ATPase in diamide-induced extracellular alkalization by roots from pea seedlings

The plasma membrane H⁺-ATPase can be considered as a redox-dependent enzyme, because diamide-mediated inhibition of its hydrolytic and transport activities is accompanied by alkalization of the rhizosphere and retardation of root growth. Plasma membranes were isolated from roots of etiolated pea seedlings treated in the presence of an oxidant-diamide and an inhibitor of redox-sensitive protein phosphatase-phenylarsine oxide. Hydrolytic and proton transport activities of H⁺-ATPase were determined. The effects of diamide appeared in inhibition of both ATP hydrolysis and the proton transport. However, root treatment with phenylarsine oxide only slightly reduced V_max, but did not affect ATP-dependent proton transport. The thiol groups of cysteines in the proteins can act as molecular targets for both compounds. However, treatment of isolated membranes with diamide or dithiothreitol did not have any effect on the H⁺ transport. It can be assumed that water-soluble diamide acts indirectly and its effects are not associated with oxidation of H⁺-ATPase cysteines. Therefore, plasmalemma was subjected to PEGylation-process where reduced cysteines available for PEG maleimide (5 kDa) were alkylated. Detection of such cysteines was carried out by Western blot analysis with anti-ATPase antibodies. It was found that shifts in the apparent molecular weight were detected only for denaturated proteins. These data suggest that available thiols are not localized on the enzyme surfaces. BN-PAGE analysis showed that the molecular weights of the ATPase complexes are almost identical in all samples. Therefore, oligomerization is probably not the reason for the inhibition of ATPase activity. Roots treated with these inhibitors in vivo exhibited stunted growth; however, a strong alkaline zone around the roots was formed only in the presence of diamide. Involvement of H⁺-ATPase redox regulation in this process is discussed.