Abstract.The activation by abscisic acid (ABA) of current through outward-rectifying K + channels and its dependence on cytoplasmic pH (pHi) was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with multibarrelled and H+-selective microelectrodes to record membrane potentials and pH i during exposures to ABA and the weak acid butyrate. Potassium channel currents were monitored under voltage clamp and, in some experiments, guard cells were loaded with pH buffers by iontophoresis to suppress changes in pH i . Following impalements, stable pH i values ranged between 7.53 and 7.81 (7.67_+ 0.04, n = 17). On adding 20 gM ABA, pHi rose over periods of 5-8 min to values 0.27_+0.03 pH units above the pH i before ABA addition, and declined slowly thereafter. Concurrent voltage-clamp measurements showed a parallel rise in the outward-rectifying K + channel current (IK,out) and, once evoked, both pH i and IK, out responses were unaffected by ABA washout. Acid loads, imposed with external butyrate, abolished the ABA-evoked rise in IK, out. Butyrate concentrations of 10 and 30 mM (pH 0 6.1) caused pH i to fall to values near 7.0 and below, both before and after adding ABA, consistent with a cytoplasmic buffer capacity of 128_+ 12 mM per pH unit (n = 10) near neutrality. Butyrate washout was characterised by an appreciable alkaline overshoot in pH i and concomitant swell in the steady-state conductance of IK,ou t . The rise in pH i and IK,ou t in ABA were also virtually eliminated when guard cells were first loaded with pH buffers to raise the cytoplasmic buffer capacity four-to sixfold; however, buffer loading was without appreciable effect on the ABAevoked inactivation of a second, inward-rectifying class of K + channels (IK,in). The pH i dependence of IK,ou t was (233) 8131 40 consistent with a cooperative binding of at least 2H + (apparent pKa=8.3 ) to achieve a voltage-independent block of the channel. These results establish a causal link previously implicated between cytoplasmic alkalinisation and the activation of IK, out in ABA and, thus, affirm a role for H + in signalling and transport control in plants distinct from its function as a substrate in H+-coupled transport. Additional evidence implicates a coordinate control of IK,in by cytoplasmic-free [Ca 2+] and pH i.

Abscisic acid (ABA) modulates the activities of three major classes of ion channels-inward-and outwardrectifying K+ channels (IK,i and IK,out, respectively) and anion channels-at the guard-cell plasma membrane to achieve a net efflux of osmotica and stomatal closure. Disruption of ABA sensitivity in wilty abil-1 mutants ofArabidopsis and evidence that this gene encodes a protein phosphatase suggest that protein (de-)phosphorylation contributes to guard-cell transport control by ABA. To pinpoint the role of ABIl, the abil-1 dominant mutant allele was stably transformed into Nicotiana benthamiana and its influence on IK,ing, IK,out, and the anion channels was monitored in guard cells under voltage clamp. Compared with guard cells from wild-type and vectortransformed control plants, expression of the abil-l gene was associated with 2-to 6-fold reductions in IK,out and an insensitivity of both hc,in and IK,out to 20 ,uM ABA. In contrast, no differences between control and abil-1 transgenic plants were observed in the anion current or its response to ABA. Parallel measurements of intracellular pH (pHJ) using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) in every case showed a 0.15-to 0.2-pH-unit alkalinization in ABA, demonstrating that the transgene was without effect on the pH; signal that mediates in ABA-evoked K+ channel control. In guard cells from the abil-l transformants, normal sensitivity ofboth K+ channels to and stomatal closure in ABA was recovered in the presence of 100 ,iM H7 and 0.5 ,iM staurosporine, both broad-range protein kinase antagonists. These results demonstrate an aberrant K+ channel behavior-including channel insensitivity to ABAdependent alkalinization of pHr-as a major consequence of abil-l action and implicate ABIl as part of a phosphatase/kinase pathway that modulates the sensitivity of guard-cell K+ channels to ABA-evoked signal cascades.Abscisic acid (ABA) participates in the growth and development of higher plants by controlling seed maturation and dormancy and signals conditions of water stress in vegetative tissues (1, 2). The hormone accumulates in the leaves during drought stress, evoking stomatal closure to reduce transpirational water loss (1). Stomatal closure is achieved through osmotic solute efflux from the guard cells, notably of K+ from the vacuole and cytoplasm across the plasma membrane, and the consequent decline in guard-cell turgor. ABA initiates this flux by modulating three classes of ion channels-inward-and outward-rectifying K+ channels (Iyi,n and I,.out, respectively) and anion channels-events coordinated by at least two independent signal cascades that lead to a rise in cytoplasmic free [Ca2+] ([Ca2+]) and intracellular pH (pHi) (1, 3, 4).Protein phosphorylation has also been implicated from studies of the ABA-insensitive 1 (abil) mutant that interferes with ABA responsiveness inArabidopsis thaliana. Mutations at this locus evoke a range of ABA-related phenotypes, notably aberrant control of stomatal aperture and ...

SummaryTo explore the involvement of a class of seven-transmembrane-span (TTMS) receptors in cellular signalling, a synthetic analogue (mas7) of the amphipathi¢ tetredecapsptide mestoperan was used to mimic hormonal stimulus in guard calls of Vicia faba. The ability for mes7 to substitute for an activated receptor complex was assayed by the effect on guard cell ion channel activities in the absence of any hormonal stimulus. Currents carried by inward-(/K,in) and outward-(/K,out) rectifying potassium channels were determined under voltage clamp conditions before, during, and after exposure to mas7. The dominant effect of mes7 was to inactivate/K, in within 30 sec of application. By contrast, /K,out was largely unaffected under these conditions. The effect of mas7 on IK, in was both concentration-and voltage-dspendent. At any one clamp voltage, mas7 inactivation showed Michenlian beheviour, with a mean/~ of 0.05 _+ 0.02 I~M at -240 mV. Increasing rues7 concentration also shifted the voltage for half-maximal activation of the current negative, with 0.5 IJM mes7 effecting a -13 _+ 2 mV displacement and lengthening the halftime for activation of the current by up to threefold. By contrast, the non-amphipathic analogue of mes7, masCP, had no appreciable effect on the steady-state current or its activation kinetics; nor was the poly-cation polylysine able to substitute for mes7 in its action on the K ÷ channels. Application of the non-hydrolyseble analogue of GDP, GDP-~-S, either by iontophoresis or by diffusion from the microelectrode, effectively blocked mes7-inducad inactivation of/K,in. These, and additional results provide in vivo evidence for the involvement of G-protein-linked 7TMS receptors in the regulation of membrane transport in a higher plant call.