Differential regulation of vacuolar H+-ATPase and H+-PPase in Cucumis sativus roots by zinc and nickel

Abstract

Zinc and nickel, as micronutrients, are essential for all organisms. We investigated the effect of 10 and 100 μM Zn and Ni on two tonoplast proton pumps, vacuolar H+-ATPase (V-ATPase) (EC 3.6.3.14) and vacuolar H+-pyrophosphatase (V-PPase) (EC 3.6.1.1), in cucumber roots. ATP-dependent proton transport as well as ATP hydrolysis, catalyzed by V-ATPase, decreased in roots after exposure of plants to both Zn and Ni under all the examined conditions. In contrast, V-PPase activities, measured as PP(i) hydrolysis and PP(i)-driven H+ transport, were stimulated by lower concentration of metals. However, at higher metal concentration, hydrolytic activity of V-PPase remained unchanged, while PP(i)-dependent proton pumping into the tonoplast vesicles was reduced. When heavy metals were introduced into the enzyme reaction medium, both V-ATPase and V-PPase activities were lowered by Zn and Ni in a similar manner. As the gene expression and immunoblot analyses depicted, observed changes in the activity of both tonoplast proton pumps in response to zinc and nickel were not due to the modification in the expression of the CsVHA-A, CsVHA-c and CsVP genes encoding V-ATPase subunit A and c, and V-PPase, respectively, in cucumber roots or in amounts of enzyme proteins. Moreover, Zn as well as Ni ions did not enhance the lipid peroxidation in the root tonoplast fractions. Comparison of ATP and pyrophosphate contents in the control roots and roots treated with heavy metals revealed that Zn and Ni do not affect the ATP amount but reduce the PP(i) level.