Plasma membrane enriched vesicles were isolated from 5 week old Col 0, pks5 1, j3 1, and j3 2 plants treated with or without 250 mM NaCl for 3 or 6 d, and the ATP hydrolytic and H transport activities of the H ATPase were measured. Assays in the presence of specific H ATPase inhibitors demonstrated that the vesicles were enriched in plasma membranes and that they were transport competent . Unless indicated, all PM H ATPase activity assays shown monitor the H transport activity of the H ATPase. Without NaCl treatment, there was no significant difference in PM H ATPase activity between Col 0 and the j3 mutants . After plants were treated with NaCl, PM H ATPase activity increased significantly in all plants. However, the increase in activity was significantly lower in the vesicles isolated from j3 mutant plants compared with activity in Col 0 . Consistent with previous results , PM H ATPase activity in pks5 1 mutant plants was higher than activity in Col 0 . When we increased the time of treatment to 6 d, PM H ATPase activity increased further relative to activity after the 3 d treatment. The increase in Col 0 was 44%, but only 11% in pks5 1, 14% in j3 1, and 19% in j3 2 mutants .
These results suggest that loss of either PKS5 or J3 in Arabidopsis alters regulation of PM H ATPase activity during salt stress. The difference in PM H ATPase activity between Col 0 and j3 increased to 32% and 31% after 6 d of treatment compared with 14% and 16% after 3 d of treatment. Consistent with what we have shown previously, the pH optimum for PM H ATPase activity in pks5 Sorafenib PDGFR inhibitor selleck chemicals 1 shifted from 6.5 to 7.0, while for j3 mutants, the optimum stayed at 6.5, the same as for Col 0, but with lower activity . Addition of recombinant PKS5 protein to plasma membrane vesicles isolated from the pks5 1 mutant rescuedPMH ATPase activity to Col 0 levels . To determine if addition of J3 protein in the assay also affects PM H ATPase activity, the J3 coding region was cloned into the pGEX 6p 1 vector to generate a translational fusion to glutathione S transferase . The resulting plasmid was transferred into E. coli BL21 , and the J3 protein was purified and added to PM H ATPase assays.
In the presence of 250 ng mL J3, PM H ATPase activity increased in vesicles isolated from Col 0 and the j3 mutant . As a control, GST or boiled J3 protein was added to assays and did not have any effect on PM H ATPase activity . When we monitored the level ofPMH ATPase protein in response to NaCl treatment with immunoblot analysis using PM H ATPase antibodies, we did not observe Quercetin significant differences in protein levels among Col 0, pks5 1, j3 1, and j3 2 plants . Addition of J3 protein to membranes isolated from pks5 1 had no significant effect on activity . These data indicate that J3 is a novel regulator of PM H ATPase activity and that this regulation likely takes place via mediation of PKS5 activity.