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 Ca(2+) 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, Ca(2+) influx rate is, however, lowered, and a large rise in Ca(2+) efflux, concomitant with a rapid decline in H2O2 level, takes place. The drop of the H2O2 peak, as well as the efflux of Ca(2+), are prevented by treatment with submicromolar concentrations of eosin yellow (EY), selectively inhibiting the Ca(2+)- ATPase of the plasma membrane (PM). Comparable changes of Ca(2+) fluxes are also induced by hyperosmotic treatment. However, in this case, the H2O2 level does not rise, but declines below control levels when Ca(2+) efflux is activated. Also K(+) and H(+) net fluxes across the PM and cytoplasmic pH (pHcyt) 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 pHcyt changes imposed by modulation of the PM H(+)- ATPase activity or weak acid treatment, show a close correlation between pHcyt and H2O2 formed, a larger amount being produced for changes towards acidic pH values. Overall, these results confirm a relevant role for the PM Ca(2+)-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 pHcyt changes following the changes of its activity induced by stress conditions.