ABA activates multiple Ca2+ fluxes in stomatal guard cells, triggering vacuolar K+(Rb+) release

The mechanisms by which abscisic acid (ABA) activates the release of K+(Rb) from the vacuole of stomatal guard cells, a process essential for ABA-ind uced stomatal closure, have been investigated by tracer flux measurements. The form and timing of the ABA-induced efflux transient could be manipulate d by treatments that alter three potential Ca2+ fluxes into the cytoplasm, the influx from the outside and two pathways of internal release, those dep endent on phospholipase C (inhibited by U73122) and cyclic ADP-ribose (inhi bited by nicotinamide). Ba2+, acting as a competitive inhibitor of Ca2+ inf lux but also as an inhibitor of internal release, was an effective inhibito r of the transient. The results suggest that a threshold level of cytoplasm ic Ca2+ is required for the initiation of the minimal efflux transient afte r a lag period and with a low rate of rise. As conditions improve for the g eneration of an efflux transient (higher ABA or reduced Ba2+), a second thr eshold is crossed, generating a transient with zero lag and rapid rate of r ise. This may reflect different Ca2+ levels required for activation of diff erent tonoplast K+ channels. In this state, at high ABA, the transient is i nhibited by removal of external Ca2+, suggesting Ca2+ influx makes a major contribution to increase in cytoplasmic Ca2+. By contrast, at low ABA, the transient is not inhibited by removal of external Ca2+ but is sensitive to either U73122 or nicotinamide, suggesting internal release makes the major contribution, involving both pathways. ABA appears to activate all three pr ocesses, and their relative importance depends on conditions.