Different responses of tonoplast proton pumps in cucumber roots to cadmium and copper

Cadmium (Cd) and copper (Cu) effects on the two tonoplast proton pumps were compared in cucumber roots. Different alterations of vacuolar H+ transporting ATPase (V-ATPase) (EC 3.6.3.14) and vacuolar H+ transporting pyrophosphatase (V-PPase) (EC 3.6.1.1) activities under heavy metal stress were investigated. ATP-dependent proton transport and ATP hydrolysis increased after exposure of seedlings to Cu, whereas both decreased in plants stressed with Cd. PPi hydrolysis was relatively insensitive to both heavy metals. However, cadmium, but not copper, clearly inhibited PPi-driven H+ transport. Changes in enzyme activities were not due to the metal action on the expression of CsVHA-A, CsVHA-c and CsVP genes encoding V-ATPase subunit A and c, and V-PPase, respectively, in cucumber roots. Moreover, immunoblot analysis using specific antibodies against V-ATPase holoenzyme, phosphoserine and phosphothreonine suggested that the phosphorylation at Ser residue in regulatory subunit B of cucumber V-ATPase was not regulated by metals. Oxidative alterations of membrane lipids were measured as malondialdehyde (MDA) content. Cu ions, in contrast to Cd, visibly enhanced the lipid peroxidation in the root tonoplast fractions. Because ATP and PPi are absolutely required by V-ATPase and V-PPase, respectively, for proton transport, their contents were determined in the control roots and roots treated with cadmium and copper. Both ATP and pyrophosphate amounts decreased under heavy metal stress.