Modulation of reactive oxygen species production during osmotic stress in Arabidopsis thaliana cultured cells: involvement of the plasma membrane Ca2+-ATPase and H+-ATPase.

In Arabidopsis thaliana cells, hypoosmotic treatment initially stimulates Ca2+ influx and inhibits its efflux and, concurrently, promotes a large H2O2 accumulation in the external medium, representative of reactive oxygen species (ROS) production. After the first 10-15 min, Ca2+ influx rate is, however, lowered, and a large rise in Ca2+ efflux, concomitant with a rapid decline in H2O2 level, takes place. The drop of the H2O2 peak, as well as the efflux of Ca2+, are prevented by treatment with submicromolar concentrations of eosin yellow (EY), selectively inhibiting the Ca2+-ATPase of the plasma membrane (PM). Comparable changes of Ca2+ fluxes are also induced by hyperosmotic treatment. However, in this case, the H2O2 level does not rise, but declines below control levels when Ca2+ efflux is activated. Also K+ and H+ net fluxes across the PM and cytoplasmic pH (pH(cyt)) are very differently influenced by the two opposite stresses: strongly decreased by hypoosmotic stress and increased under hyperosmotic treatment. The H2O2 accumulation kinetics, followed as a function of the pH(cyt) changes imposed by modulation of the PM H+-ATPase activity or weak acid treatment, show a close correlation between pH(cyt) and H2O2 formed, a larger amount being produced for changes towards acidic pH values. Overall, these results confirm a relevant role for the PM Ca2+-ATPase in switching off the signal triggering ROS production, and propose a role for the PM H+-ATPase in modulating the development of the oxidative wave through the pH(cyt) changes following the changes of its activity induced by stress conditions.