Modulation by cytosolic components of proton pump activities in plasma membrane and tonoplast from Cucumis sativus roots during salt stress.

Abstract

The effect of NaCl on the plasma membrane and tonoplast ATPases measured as the hydrolytic and H(+)-pumping activity was studied. Treatment of cucumber seedlings with salt increased the membrane-bound ATPases of the plasma membrane as well as the tonoplast. In both types of membranes the stimulation of ATP-hydrolysis was much higher than the stimulation of H(+)-transport suggesting that the salt- treatment of plants partially uncoupled the membrane proton pumps. It was shown that the soluble fraction obtained from the unstressed or NaCl-stressed roots stimulated the ATPase activities in both membranes isolated from unstressed plants. A stimulatory effect of the soluble fraction on the proton pump activities was considerably enhanced in the salt conditions indicating the presence of a salt-inducible factor (s) in the soluble fraction, which could rapidly modulate the membrane-bound ATPases. Staurosporine, a specific protein kinase inhibitor, totally abolished the stimulatory action of the soluble fractions on the membrane proton pumps, whereas okadaic acid, a phosphatase inhibitor, had no effect. Inclusion of calcium in the mixture of membranes and the soluble fraction from unstressed roots elevated the ATPase activities to the levels determined with the soluble fraction isolated from NaCl-stressed roots. Cation chelators (EGTA), as well as calmodulin antagonist (W7) cancelled the stimulatory effect of calcium ions. The above results strongly suggest the involvement of specific calcium-calmodulin-dependent protein kinases in the activation of the membrane ATPases under salt-stress conditions.