Effects of in vivo treatment with abscisic acid and/or cytokinin on activities of vacuolar H⁺ pumps of tonoplast-enriched membrane vesicles prepared from barley roots

We investigated the effects of in vivo treatment (1 day) of barley roots with abscisic acid (ABA) and/or a cytokinin (6-benzyladenine; BA) on the ATP- and PP_i-dependent H⁺ transport activities of tonoplast-enriched membrane vesicles prepared from the roots. Treatment with ABA significantly increased the two H⁺ transport activities. By contrast, treatment with BA significantly decreased PP_i-dependent H⁺ transport activity, while the change in ATP-dependent H⁺ transport activity was small. Increases in the two H⁺ transport activities caused by treatment with ABA were suppressed during treatment with ABA and BA. Changes in the NO^--inhibitable ATPase activity and the Na⁺-inhibitable PP_iase activity of membrane vesicles after treatment of roots with phytohormone(s) (ABA, BA, ABA + BA) were similar to changes in the ATP- and PP_i dependent H⁺ transport activities of the membrane vesicles, respectively. Immunoblot analysis with antibodies raised against the functional catalytic subunits of the vacuolar H⁺ pumps (H⁺- ATPase and H⁺-PP_iase) of mung bean revealed that only the level of the functional catalytic subunit of the H⁺-PP_iase of the membrane vesicles was significantly increased by treatment with ABA alone and in combination with BA. These results suggest that treatment with ABA has a stimulatory effect on the activities of the two H⁺ pumps of the vacuolar membrane of barley roots, with increase in the level of the catalytic subunit of the H⁺-PP_iase, and that treatment with BA has an inhibitory effect on the two H⁺ pump activities of the vacuolar membrane without changes in the levels of the catalytic subunits of either H⁺ pump, with the limitation that treatment with BA has an inhibitory effect only when the activity of the H⁺-ATPase has been increased by treatment with ABA.