Effects of salt stress on H +-ATPase activity of plasma membrane-enriched vesicles isolated from sunflower roots

Abstract

It has previously been shown that mild and severe salt-stress increased both ATP- and PP i-dependent H + transport and induced a Na +/H + exchange activity in tonoplast vesicles from sunflower seedling roots. In this study, the vanadate-sensitive H +-ATPase activity was characterized in plasma membrane-enriched vesicles isolated by discontinuous sucrose gradient centrifugation from sunflower ( Helianthus annuus L.) roots exposed for 3 days to 0, 75 or 150 mM NaCl. Blots immunoassayed with an Arabidopsis thaliana plasma membrane ATPase polyclonal antibody revealed the existence of a band of about 100 kDa, which was highly enriched in the 32/43% sucrose interface. ATP hydrolyzing activity in this fraction was mostly inhibited by vanadate, and scarcely by azide, oligomycin, nitrate and molybdate, indicating that it was essentially enriched in plasma membrane vesicles. Properties of vanadate-sensitive ATPase activity, such as inhibitor sensitivity, pH optimum, substrate specificity, ion effects and kinetic data were consistent with those of other plasma membrane ATPases. Although the pH profile, ion dependence, apparent K m and the amount of antigenic protein were unaffected by salt-stress, the V max of the vanadate-sensitive ATPase activity (measured in the presence of detergent) was reduced as a function of salt treatments. Likewise, while mild and severe salt-stress reduced the basal ATP hydrolyzing activity (measured in the absence of detergent) about a 35 and 55%, respectively, the ATP-dependent H + transport activity sensitive to vanadate remained unchanged. As a consequence, the ratio of H + pumping to the basal phosphohydrolase activity was proportionally increased. These results and those previously reported at the sunflower tonoplast suggest that the activity of H + pumps under salt stress can form part of a tolerance mechanism in sunflower roots, which could regulate the ion fluxes (Na + and Cl −) across the tonoplast and plasma membrane.