Potassium uptake and export in the resting conditions and in response to the phytohormone abscisic acid (ABA) were examined under voltage clamp in guard cells of Vicia faba L. In 0.1 mM external K+ (with 5 mM Ca2(+)-HEPES, pH 7.4) two distinct transport states could be identified based on the distribution of the free-running membrane voltage (VM) data in conjunction with the respective I-V and G-V relations. One state was dominated by passive diffusion (mean VM = -143 +/- 4 mV), the other (mean VM = -237 +/- 10 mV) exhibited an appreciable background of primary H+ transport activity. In the presence of pump activity the free-running membrane voltage was negative of the respective K+ equilibrium potential (EK+), in 3 and 10 mM external K+. In these cases VM was also negative of the activation voltage for the inward rectifying K+ current, thus creating a strong bias for passive K+ uptake through inward-rectifying K+ channels. In contrast, when pump activity was absent VM was situated positive of EK+ and cells revealed a bias for K+ efflux. Occasionally spontaneous voltage transitions were observed during which cells switched between the two states. Rapid depolarizations were induced in cells with significant pump activity upon adding 10 microM ABA to the medium. These depolarizations activated current through outward-rectifying K+ channels which was further amplified in ABA by a rise in the ensemble channel conductance. Current-voltage characteristics recorded before and during ABA treatments revealed concerted modulations in current passage through at least four distinct transport processes, results directly comparable to one previous study (Blatt, M.R., 1990, Planta 180:445) carried out with guard cells lacking detectable primary pump activity. Comparative analyses of guard cells in each case are consistent with depolarizations resulting from the activation of an inward-going, as yet unidentified current, rather than an ABA-induced fall in H(+)-ATPase output. Also observed in a number of cells was an inward-directed current which activated in ABA over a narrow range of voltages positive of -150 mV; this and additional features of the current suggest that it may reflect the ABA-dependent activation of an anion channel previously characterized in Vicia guard cell protoplasts, but rule out its function as the primary mechanism for initial depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Our understanding of the signalling mechanisms involved in the process of stomatal closure is reviewed. Work has concentrated on the mechanisms by which abscisic acid (ABA) induces changes in speci¢c ion channels at both the plasmalemma and the tonoplast, leading to e¥ux of both K + and anions at both membranes, requiring four essential changes. For each we need to identify the speci¢c channels concerned, and the detailed signalling chains by which each is linked through signalling intermediates to ABA. There are two global changes that are identi¢ed following ABA treatment: an increase in cytoplasmic pH and an increase in cytoplasmic Ca 2+ , although stomata can close without any measurable global increase in cytoplasmic Ca 2+ . There is also evidence for the importance of several protein phosphatases and protein kinases in the regulation of channel activity.At the plasmalemma, loss of K + requires depolarization of the membrane potential into the range at which the outward K + channel is open. ABA-induced activation of a non-speci¢c cation channel, permeable to Ca 2+ , may contribute to the necessary depolarization, together with ABA-induced activation of S-type anion channels in the plasmalemma, which are then responsible for the necessary anion e¥ux. The anion channels are activated by Ca 2+ and by phosphorylation, but the precise mechanism of their activation by ABA is not yet clear. ABA also up-regulates the outward K + current at any given membrane potential; this activation is Ca 2+ -independent and is attributed to the increase in cytoplasmic pH, perhaps through the marked pH-sensitivity of protein phosphatase type 2C.Our understanding of mechanisms at the tonoplast is much less complete. A total of two channels, both Ca 2+ -activated, have been identi¢ed which are capable of K + e¥ux; these are the voltage-independent VK channel speci¢c to K + , and the slow vacuolar (SV) channel which opens only at non-physiological tonoplast potentials (cytoplasm positive). The SV channel is permeable to K + and Ca 2+ , and although it has been argued that it could be responsible for Ca 2+ -induced Ca 2+ release, it now seems likely that it opens only under conditions where Ca 2+ will £ow from cytoplasm to vacuole. Although tracer measurements show unequivocally that ABA does activate e¥ux of Cl À from vacuole to cytoplasm, no vacuolar anion chnnel has yet been identi¢ed.There is clear evidence that ABA activates release of Ca 2+ from internal stores, but the source and trigger for ABA-induced increase in cytoplasmic Ca 2+ are uncertain. The tonoplast and another membrane, probably ER, have IP 3 -sensitive Ca 2+ release channels, and the tonoplast has also cADPRactivated Ca 2+ channels. Their relative contributions to ABA-induced release of Ca 2+ from internal stores remain to be established. There is some evidence for activation of phospholipase C by ABA, by an unknown mechanism; plant phospholipase C may be activated by Ca 2+ rather than by the G-proteins used in many animal cell signalling systems.A further ABA-in...

myo-Inositol hexakisphosphate (InsP6) is the most abundant inositol phosphate in cells, yet it remains the most enigmatic of this class of signaling molecule. InsP6 plays a role in the processes by which the drought stress hormone abscisic acid (ABA) induces stomatal closure, conserving water and ensuring plant survival. Previous work has shown that InsP6 levels in guard cells are elevated in response to ABA, and InsP6 inactivates the plasma membrane inward K ؉ conductance (IK,in) in a cytosolic calciumdependent manner. The use of laser-scanning confocal microscopy in dye-loaded patch-clamped guard cell protoplasts shows that release of InsP 6 from a caged precursor mobilizes calcium. Measurement of calcium (barium) currents ICa in patch-clamped protoplasts in whole cell mode shows that InsP6 has no effect on the calcium-permeable channels in the plasma membrane activated by ABA. The InsP6-mediated inhibition of IK,in can also be observed in the absence of external calcium. Thus the InsP6-induced increase in cytoplasmic calcium does not result from calcium influx but must arise from InsP6-triggered release of calcium from endomembrane stores. Measurements of vacuolar currents in patch-clamped isolated vacuoles in whole-vacuole mode showed that InsP6 activates both the fast and slow conductances of the guard cell vacuole. These data define InsP6 as an endomembrane-acting calciumrelease signal in guard cells; the vacuole may contribute to InsP6-triggered Ca 2؉ release, but other endomembranes may also be involved.