ABA activates multiple Ca(2+) 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-induced stomatal closure, have been investigated by tracer flux measurements. The form and timing of the ABA-induced efflux transient could be manipulated by treatments that alter three potential Ca(2+) fluxes into the cytoplasm, the influx from the outside and two pathways of internal release, those dependent on phospholipase C (inhibited by ) and cyclic ADP-ribose (inhibited by nicotinamide). Ba(2+), acting as a competitive inhibitor of Ca(2+) influx but also as an inhibitor of internal release, was an effective inhibitor of the transient. The results suggest that a threshold level of cytoplasmic Ca(2+) is required for the initiation of the minimal efflux transient after a lag period and with a low rate of rise. As conditions improve for the generation of an efflux transient (higher ABA or reduced Ba(2+)), a second threshold is crossed, generating a transient with zero lag and rapid rate of rise. This may reflect different Ca(2+) levels required for activation of different tonoplast K(+) channels. In this state, at high ABA, the transient is inhibited by removal of external Ca(2+), suggesting Ca(2+) influx makes a major contribution to increase in cytoplasmic Ca(2+). By contrast, at low ABA, the transient is not inhibited by removal of external Ca(2+) but is sensitive to either or nicotinamide, suggesting internal release makes the major contribution, involving both pathways. ABA appears to activate all three processes, and their relative importance depends on conditions.

Inositol hexakisphosphate is a physiological signal regulating the K+-inward rectifying conductance in guard cells.

(RS)-2-cis, 4-trans-abscisic acid (ABA), a naturally occurring plant stress hormone, elicited rapid agonist-specific changes in myo-inositol hexakisphosphate (InsP(6)) measured in intact guard cells of Solanum tuberosum (n = 5); these changes were not reproduced by (RS)-2-trans, 4-trans-abscisic acid, an inactive stereoisomer of ABA (n = 4). The electrophysiological effects of InsP(6) were assessed on both S. tuberosum (n = 14) and Vicia faba (n = 6) guard cell protoplasts. In both species, submicromolar concentrations of InsP(6), delivered through the patch electrode, mimicked the inhibitory effects of ABA and internal calcium (Ca(i)(2+)) on the inward rectifying K(+) current, I(K,in), in a dose-dependent manner. Steady state block of I(K,in) by InsP(6) was reached much more quickly in Vicia (3 min at approximately 1 microM) than Solanum (20-30 min). The effects of InsP(6) on I(K,in) were specific to the myo-inositol isomer and were not elicited by other conformers of InsP(6) (e.g., scyllo- or neo-). Chelation of Ca(2+) by inclusion of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or EGTA in the patch pipette together with InsP(6) prevented the inhibition of I(K,in), suggesting that the effect is Ca(2+) dependent. InsP(6) was approximately 100-fold more potent than Ins(1,4,5)P(3) in modulating I(K,in). Thus ABA increases InsP(6) in guard cells, and InsP(6) is a potent Ca(2+)-dependent inhibitor of I(K,in). Taken together, these results suggest that InsP(6) may play a major role in the physiological response of guard cells to ABA.

Signal transduction and ion channels in guard cells.

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 specific ion channels at both the plasmalemma and the tonoplast, leading to efflux of both K+ and anions at both membranes, requiring four essential changes. For each we need to identify the specific channels concerned, and the detailed signalling chains by which each is linked through signalling intermediates to ABA. There are two global changes that are identified following ABA treatment: an increase in cytoplasmic pH and an increase in cytoplasmic Ca2+, although stomata can close without any measurable global increase in cytoplasmic Ca2+. 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-specific cation channel, permeable to Ca2+, 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 efflux. The anion channels are activated by Ca2+ 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 identified which are capable of K+ efflux; these are the voltage-independent VK channel specific 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 Ca2+, and although it has been argued that it could be responsible for Ca(2+)-induced Ca2+ release, it now seems likely that it opens only under conditions where Ca2+ will flow from cytoplasm to vacuole. Although tracer measurements show unequivocally that ABA does activate efflux of Cl- from vacuole to cytoplasm, no vacuolar anion channel has yet been identified. There is clear evidence that ABA activates release of Ca2+ from internal stores, but the source and trigger for ABA-induced increase in cytoplasmic Ca2+ are uncertain. The tonoplast and another membrane, probably ER, have IP3-sensitive Ca2+ release channels, and the tonoplast has also cADPR-activated Ca2+ channels. Their relative contributions to ABA-induced release of Ca2+ 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 Ca2+ rather than by the G-proteins used in many animal cell signalling systems. A further ABA-induced channel modulation is the inhibition of the inward K+ channel, which is not essential for closing but will prevent opening. It is suggested that this is mediated through the Ca(2+)-activated protein phosphatase, calcineurin. The question of Ca(2+)-independent stomatal closure remains controversial. At the plasmalemma the stimulation of K+ efflux is Ca(2+)-independent and, at least in Arabidopsis, activation of anion efflux by ABA may also be Ca(2+)-independent. But there are no indications of Ca(2+)-independent mechanisms for K+ efflux at the tonoplast, and the appropriate anion channel at the tonoplast is still to be found. There is also evidence that ABA interferes with a control system in the guard cell, resetting its set-point to lower contents, suggesting that stretch-activated channels also feature in the regulation of guard cell ion channels, perhaps through interactions with cytoskeletal proteins. (ABSTRACT TRUN

Signalling in guard cells and regulation of ion channel activity.

A review is presented of the properties of ion channels in plasmalemma and tonoplast of stomatal guard cells, their regulation, with particular reference to Ca(2+) and protein phosphorylation/dephosphorylation, and of the evidence for ABA-induced changes in specific ion channels, with an attempt to identify the signalling chains involved in each such change. A key question is whether a local increase in Ca(2+), close to cell membranes and capable of triggering Ca(2+)-dependent changes in a variety of ion channels, is a universal feature of the ABA-reponse. If this is so, then there exist Ca(2+)-coupled mechanisms for most of the observed changes, including inhibition of the inward K(+) channel and activation of the slow anion channel in the plasmalemma, and activation of two channels in the tonoplast, the K(+)-selective (VK) channel and the slow vacuolar (SV) channel, initiating efflux of both anions and cations from the vacuole. The detailed signalling chains are not complete, and the role of protein phosphorylation/dephosphorylation is not clearly defined, nor linked to ABA. Control of the outward K(+) channel is Ca(2+)-independent; its activation by ABA may be mediated by cytoplasmic alkalinization, but the role of protein dephosphorylation in the signalling chain has still to be clarified. If Ca(2+) is not available as second messenger, then the signalling chains involved have hardly begun to be understood. Detailed comparison of the efflux transients in different conditions provides evidence that ABA changes the 'set-point' of a stretch-activated channel, initiating loss of vacuolar K(+). The inclusion of Ba(2+) in the bathing solution has effects similar to those of reduced ABA concentration, a delay in initiating the vacuolar transient, and a slower rise to a reduced peak height. It is suggested that this could be the result of inhibition of the process of Ca(2+) release from internal stores, by blocking a charge-balancing K(+) flux.

Role of calcium in the modulation of Vicia guard cell potassium channels by abscisic acid: a patch-clamp study.

There is evidence for a role of increased cytoplasmic Ca2+ in the stomatal closure induced by abscisic acid (ABA), but two points of controversy remain the subject of vigorous debate--the universality of Ca2+ as a component of the signaling chain, and the source of the increased Ca2+, whether influx across the plasmalemma, or release from internal stores. We have addressed these questions by patch-clamp studies on guard cell protoplasts of Vicia faba, assessing the effects of ABA in the presence and absence of external Ca2+, and of internal Ca2+ buffers to control levels of cytoplasmic Ca2+. We show that ABA-induced reduction of the K+ inward rectifier can occur in the absence of external Ca2+, but is abolished when Ca2+ buffers are present inside the cell. Thus, some minimum level of cytoplasmic Ca2+ is a necessary component of the signaling chain by which ABA decreases the K+ inward rectifier in stomatal guard cells, thus preventing stomatal opening. Release of Ca2+ from internal stores is capable of mediating the response, in the absence of any Ca2+ influx from the extracellular medium. The work also shows that enhancement of the K+ outward rectifier by ABA is Ca2+ independent, and that other signaling mechanisms must be involved. A role for internal pH, as suggested by H.R. Irving, C.A. Gehring and R.W. Parish (Proc. Natl. Acad. Sci. USA 89:1790-1794, 1990) and M.R. Blatt (J. Gen. Physiol. 99:615-644, 1992), is an attractive working hypothesis.

Ca2+ and cell signalling in guard cells.

Abscisic acid (ABA)-induced stomatal closure involves two different signalling chains, only one of which is Ca(2+)-dependent. ABA induces deactivation of the inward K+ channel and activation of an inward 'background' current, changes also produced by high cytoplasmic Ca2+ or injection of inositol 1,4,5-trisphosphate. It is argued that ABA produces local increases in Ca2+, which are obligatory for the response, even where global increases are not observed with present methodology. Deactivation of the inward K+ channel is abolished in the presence of internal Ca2+ chelator, but not by external Ca2+ chelator, arguing for release from internal stores. ABA-induced turnover in the polyphosphoinositide cycle occurs within 30 s, and may precede the electrical changes. Activation of the outward K+ channel is Ca(2+)-independent; changes in cytoplasmic pH, of unknown origin, may be responsible.

Membrane transport in stomatal guard cells: the importance of voltage control.

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)

Raising the intracellular level of inositol 1,4,5-trisphosphate changes plasma membrane ion transport in characean algae.

Inositol 1,4,5-trisphosphate (InsP3) was introduced into the cytoplasm of characean algae in two different ways: (i) by iontophoretic injection into cytoplasm-enriched fragments from Chara and (ii) by adding InsP3 to the permeabilization medium of locally permeabilized cells of Nitella. In both systems this operation induced a depolarization of the membrane potential, ranging from a few mV to sequences of action potentials. The effect of InsP3 on locally permeabilized Nitella cells was abolished when InsP3 was added together with 30 mM EGTA. When inositol 1,4-bisphosphate or myo-inositol were substituted for InsP3 in this system, there was no change in the membrane potential. On the other hand, increasing the free Ca2+ concentration in the permeabilization medium induced, in a similar fashion to InsP3, action potentials. Similarities between InsP3 and Ca2+ action were also observed upon injection into Chara fragments. Both injections increased an inward current. In the first few seconds after injection the current/voltage characteristics of the InsP3-induced current resembled those of the Ca2(+)-sensitive current. Subsequently, differences between the InsP3- and Ca2(+)-induced phenomena became apparent in that the InsP3-induced current continued to increase while the Ca2(+)-induced current declined, returning to the resting level. Our results suggest that these plant cells contain an InsP3 sensitive system that, under experimental conditions, is able to affect membrane transport via an increase in cytoplasmic free Ca2+.

Cytoplasmic calcium affects the gating of potassium channels in the plasma membrane ofChara corallina: a whole-cell study using calcium-channel effectors.

The action of a wide range of drugs effective on Ca(2+) channels in animal tissues has been measured on Ca(2+) channels open during the action potential of the giant-celled green alga,Chara corallina. Of the organic effectors used, only the 1,4-dihydropyridines were found to inhibit reversibly Ca(2+) influx, including, unexpectedly, Bay K 8644 and both isomers of 202-791. Methoxyverapamil (D-600), diltiazem, and the diphenylbutylpiperidines, fluspirilene and pimozide were found not to affect the Ca(2+) influx. Conversely, bepridil greatly and irreversibly stimulated Ca(2+) influx, and with time, stopped cytoplasmic streaming (which is sensitive to increases in cytoplasmic Ca(2+)). By apparently altering the cytoplasmic Ca(2+) levels with various drugs, it was found that (with the exception of the inorganic cation, La(3+)) treatments likely to lead to an increase in cytoplasmic Ca(2+) levels caused an increase in the rate of closure of the K(+) channels. Similarly, treatments likely to lead to a decrease in cytoplasmic Ca(2+) decreased the rate of K(+) channel closure. The main effect of bepridil on the K(+) channels was to increase the rate of voltage-dependent channel closure. The same effect was obtained upon increasing the external concentration of Ca(2+), but it is likely that this was due to effects on the external face of the K(+) channel. Addition of any of the 1,4-dihydropyridines had the opposite effect on the K(+) channels, slowing the rate of channel closure. They sometimes also reduced K(+) conductance, but this could well be a direct effect on the K(+) channel; high concentrations (50 to 100 µM) of bepridil also reduced K(+) conductance. No effect of photon irradiance or of abscisic acid could be consistently shown on the K(+) channels. These results indicate a control of the gating of K(+) channels by cytoplasmic Ca(2+), with increased free Ca(2+) levels leading to an increased rate of K(+)-channel closure. As well as inhibiting Ca(2+) channels, it is suggested that La(3+) acts on a Ca(2+)-binding site of the K(+) channel, mimicking the effect of Ca(2+) and increasing the rate of channel closure.

Calcium influx at the plasmalemma of isolated guard cells of Commelina communis : Effects of abscisic acid.

The influx of (45)Ca into isolated guard cells of Commelina communis L. has been measured, using short uptake times, and washing in ice-cold La(3+)-containing solutions to remove extracellular tracer after the loading period. Over 0.5-4 min the uptake was linear with time, through the origin. Over 20-200µM external Ca(2+) the influx measured with 10-20 mM external KCl was in the range 0.3-2.3 pmol·cm(-2)·s(-1) (on the basis of estimated guard-cell area); with only 1 mM KCl externally the (45)Ca influx was significantly reduced, in the range 0.3-1.1 pmol·cm(-2)·s(-1) for external Ca(2+) of 50-100 µM. The results indicate that the Ca-channel is voltage-sensitive, opening with depolarisation. No consistent effect of the addition of abscisic acid could be found. In different experiments, on the addition of 0.1 mM abscisic acid the Ca(2+) influx was sometimes stimulated by 28-79%, was sometimes unaffected, and was sometimes inhibited by 16-29%. The results rule out a long-lasting stimulation of (45)Ca influx by ABA, but they do not rule out a transient stimulation followed by inhibition, perphaps as a consequence of down-regulation of Ca(2+) influx by increasing cytoplasmic Ca(2+). The hypothesis that ABA may act via an action on Ca(2+) influx, increasing cytoplasmic Ca(2+), with consequent effects on voltage-dependent and Ca(2+)-dependent ion channels in both plasmalemma and tonoplast, is neither proved nor disproved by these results.

Calcium influx at the plasmalemma of Chara corallina.

Influx of (45)Ca into internodal cells of Chara corallina has been measured, using short uptake times, and a wash in ice-cold La(3+)-containing pondwater after the labelling period to overcome the difficulty of distinguishing extracellular tracer from that in the cell. Over 5-15 min the uptake was linear with time, through the origin. The basal influx from 0.1 mM Ca(2+) externally was 0.25-0.5 pmol·cm(-2)·s(-1), but some batches of cells showed higher fluxes. The influx was markedly stimulated by depolarisation in pondwater containing 20 mM K(+). In cells in which the control flux was less than about 0.5 pmol·cm(-2)·s(-1) there was no effect of 50 µM nifedipine. In cells in which the control flux was greater than about 0.5 pmol·cm(-2)·s(-1) (whether by natural variability, pretreatment, or by depolarisation in 20 mM K(+)), the flux was reduced by 50 µM nifedipine to a value in the range 0.25-0.59 pmol·cm(-2)·s(-1). It is suggested that two types of Ca-channel are probably involved, both opening on depolarisation, but only one sensitive to nifedipine. The flux was inhibited by 10 µM BAY K 8644, which in animal cells more commonly opens Ca-channels. The apparent influx measured over long uptake times was much reduced, and the kinetics indicated filling a pool of apparent size about 1.45 nmol·cm(-2) with a halftime of about 38 min, probably representing cytoplasmic stores. It is argued that in spite of the very small pool of (free+bound) cytoplasmic Ca(2+) the measured influx is a reasonable estimate of the influx at the plasmalemma.

Sodium efflux from perfused giant algal cells.

Internodal cells of the giant alga Chara corallina were perfused internally to replace the native cytoplasm, tonoplast and vacuole with artificial cytoplasm. Sodium efflux from perfused cells, measured by including (22)Na in the perfusion media, was increased by increasing the internal sodium concentration and by decreasing the external pH, and was inhibited by external application of the renal diuretic amiloride. The sodium efflux was markedly ATP-dependent, with a 50-fold decrease in efflux observed after perfusion with media lacking ATP. Efflux in the presence of ATP was reduced by 33% by inclusion of 10 µM N,N'-dicyclohexylcarbodiimide in the perfusion medium. The membrane potential of the perfused cells approximated that of intact cells from the same culture. It is suggested that sodium efflux in perfused Chara cells proceeds via a secondary antiporter with protons, regulated by ATP in a catalytic role and with the proton motive force acting as the energy source.

Salt tolerance in Aster tripolium L. II. Ionic regulation.

Measurements of tissue ion contents (Na, K and Cl) were carried out at frequent intervals on plants of Aster tripolium L. grown at a range of salinities for 36 d. Aster tripolium behaved as a typical halophyte showing high levels of inorganic ion accumulation even at low salinities. As salinity increased Na replaced K to a large extent in the shoot but root K was unaffected up to 500 mol m-3 external NaCl. Shoot (Na + K) concentration on a tissue water basis was maintained constant in all treatments throughout the experiment, whereas shoot (Na + K) on a dry weight basis showed marked fluctuations in some treatments. An increase in (Na + K) per gram dry weight was, however, accompanied by a parallel increase in fresh weight: dry weight (FW : DW) ratio. Transport of (Na + K) to the shoot per unit root weight changed during the experiment in the manner expected, given the observed changes in shoot relative growth rate and FW : DW to result in a constant shoot (Na + K) concentration on a water basis. Chloride was the major balancing anion in the shoot at high salinity, but never accounted for more than 38% of the (Na + K) found in the root tissue. At all salinities (Na + K) salts accounted for the majority of the measured shoot sap osmotic potential. The interactions between salinity, growth, ion transport and osmotic adjustment are discussed.

Salt tolerance in Aster tripolium L. I. The effect of salinity on growth.

A study of the growth of the maritime halophyte Aster tripolium L. has been carried out over a range of salinity treatments. The regression approach to growth analysis using frequent small harvests has been used to allow 'continuous' measurement of growth over a period of 36 d. Salinity was applied with the major ions present in ratios typical of those found in seawater. Growth was inhibited in terms of both dry weight production and leaf expansion at salinity levels equivalent to 0.625 strength sea water (full culture solution 300) and above, with the greatest effect being seen in terms of leaf area. Aster tripolium did not show increased succulence at high salinity, leaf fresh weight to dry weight ratio in fact declined, whilst leaf fresh weight per unit area remained constant. It should be noted that the plants exhibit low growth rates due to the low light intensity used.