Effects of hyper-osmotic stress on K+ fluxes, H+ extrusion, transmembrane electric potential difference and comparison with the effects of fusicoccin

The stimulation of H+ extrusion by hyper-osmotic stress influx were stimula ted by both MA and FC, (0.2-0.3 M mannitol) in cultured culls of Arabidopsi s thaliana (L.) Heynh. was shown to be associated with an inhibition of Cl- efflux, whereas hypo-osmotic stress, inhibiting H+ extrusion, early and st rongly stimulated Cl- efflux, In this paper, we investigate the contributio n of other factors [K+ transport and transmembrane electric potential diffe rence (E-m)] to the hyper-osmotic-induced activation of the plasma membrane (PM) H+-ATPase. The effects of mannitol (MA) on K+ transport and on E-m we re compared with those of fusicoccin (FC) since the modes of action of osmo tica and of the toxin in stimulating H+-ATPase activity seem to differ at l east in some steps. The changes in H+ extrusion induced by hyper- or hypo-o smotic stress were opposite and could be reversed by the application of the respective opposite stress. The effect of MA on H+ extrusion tvas dependen t on the presence of K+ (or Rb+) similarly to that of FC, while Na+ and Li, which also stimulated the FC effect, were ineffective on that of MA. The MA effect was independent of the anions (Cl-, SO42-, NO3-) accompanying K+. K+ net uptake and K+ Tetraethylammonium (TEA(+)) and Cs+ inhibited both MA - and FC-induced H+ extrusion, suggesting the involvement of K+ channels. M A (0.2 M) induced a strong hyperpolarization of E-m both in the absence and in the presence of K+. The hyperpolarizing effect of MA was also found whe n the cells were already hyperpolarized by FC, and was rapidly reversed by removing the osmoticum from the medium. In the presence of the lipophilic c ation tributylbenzylammonium (TBBA(+)), MA was no longer able to stimulate H+ extrusion, while FC still stimulated it. In cells pretreated with TBBA(), which strongly depolarized E-m, the subsequent addition of FC repolarize d it, while the hyperpolarizing effect of MA was lacking. On the contrary, in cells pretreated with Erythrosine B (EB), E-m was strongly depolarized a nd the following addition of FC did not hyperpolarize it, while the hyperpo larizing effect of MA was still observed. These results suggest that the me chanism of MA in activating H+ extrusion and K+ uptake is different from th at of FC. The rise in net K+ uptake seems to be driven by the activation of some hyperpolarizing system that does not seem to depend on a direct activ ation of PM H+-ATPase, but rather on the inhibition of Cl- efflux induced b y hyper-osmotic stress.