Abscisic-acid-induced turion formation in Spirodela polyrrhiza L. IV. Comparative ion flux characteristics of the turion and the vegetative frond and the effect of ABA during early turion development

Abstract Using the method of compartmental analysis, the ion fluxes and compartment concentrations of Ca²⁺, K⁺ and Cl^- have been compared in the untreated vegetative frond and the abscisic acid (ABA) induced turion of Spirodela polyrrhiza. The ABA-induced turion is characterized by reduced Ca²⁺ exchange across the tonoplast and low vacuolar Ca²⁺ concentration relative to the vegetative frond. In addition the turion exhibits a higher plasmalemma flux with a correspondingly high Ca²⁺ concentration in the cytoplasm. The concentration of K⁺ and Cl^- is much lower in the cytoplasm of the ABA-induced turion than in the vegetative frond with the influx/efflux ratio at both the plasmalemma and the tonoplast being less than 1, a finding exhibited also in dormant storage tissue. Treatment of vegetative fronds with ABA for 18 h resulted in a reduced K⁺ plasmalemma efflux relative to untreated vegetative fronds and a concomitant increase in the cytoplasmic concentration. There was no rapid effect of ABA on Ca²⁺, K⁺ or Cl^- fluxes through either membrane. These results are consistent with the notion that drastic changes in ion fluxes and concentrations in the turion are a secondary consequence of ABA-induced development, possibly due to prior regulation by ABA of enzymes inherent to processes involved in membrane transport.     

Effects of ABA on 86Rb+ fluxes at plasmalemma and tonoplast of stomatal guard cells

Abscisic acid (ABA) induces a transient stimulation of ⁸⁶Rb⁺ from isolated guard cells of Commelina communis L. When ABA is added after 30–50 min of wash-out in the absence of ABA, when tracer is almost entirely vacuolar, its effects on vacuolar release are measured. When ABA is added early in the wash-out (at 2–4 min), when both cytoplasm and vacuole are labelled, the resulting efflux includes both vacuolar and cytoplasmic contributions. Detailed comparison of rates of efflux in the absence of ABA, and in the presence of ABA added early and late in the wash-out, allows the effects of ABA on plasmalemma and tonoplast fluxes to be assessed. Three effects of ABA can be distinguished: these are stimulation of the ⁸⁶Rb⁺ flux from vacuole to cytoplasm (by twofold to 6.7-fold); stimulation of the plasmalemma efflux, by up to twofold, a smaller factor than that of the tonoplast effect and variable between experiments; and a doubling of the half-time for cytoplasmic exchange in ABA, taken to reflect an increase in cytoplasmic ion content as ions flood out of the vacuole. Concentrations of ABA of 0.1–0.2 µM and 1–10 µM are equally effective in the stimulation of plasmalemma efflux, but the effects on tonoplast fluxes are both delayed and reduced at low external concentrations of ABA. It is argued that the delay reflects the need for a threshold internal ABA to be reached before the initiation of vacuolar release, and the reduction reflects the sensitivity of the extent of activation of tonoplast ion channels to concentration of internal ABA. It is likely that the plasmalemma change is mediated by external ABA, and could be the result of the modulation of the stretch-activated channel suggested previously.     

Influence of abscisic acid on unidirectional fluxes and intracellular compartmentation of K+ and Na+ in excised barley root segments

Using compartmental analysis, unidirectional fluxes of K⁺ and Na⁺ and their intracellular compartmentation in excised barley (Hordeum distichon L. cv. Kocher-perle) root segments have been measured during a steady state in the presence or absence of ABA. Almost all flux rates were altered in the presence of external ABA, in particular the xylem transport R’ and the plasmalemma influx Ø_oc (see below) were strongly inhibited in the steady state. At the same time the presence of ABA induced a strong increase in the vacuolar K⁺ and Na⁺ content Q_v and a decrease in the cytoplasmic one (Q_c). Since the fluxes of an ion and its vacuolar or, in particular, cytoplasmic concentrations are interrelated, the ratios of fluxes originating from the cytoplasm and the cytoplasmic ion content were taken into account. On this basis ABA had the following effects: a) the secretion of K⁺ or Na⁺ to the xylem vessels was drastically inhibited; b) the plasmalemma K⁺ or Na⁺ efflux Ø_co was moderately stimulated and c) the tonoplast influx Ø_cv of Na⁺ was stimulated, while the tonoplast influx of K⁺ appeared to be unchanged (the decrease in Ø_cv being due to the decreased cytoplasmic K⁺ content). By a similar argument, also the apparent inhibition of the plasmalemma influx Ø_oc of K⁺ and Na⁺ in the steady state merely is an indirect effect of ABA. It only reflects the strong ABA-induced decrease in the xylem transport, that governs the magnitude of Ø_oc in the steady state. The results are discussed with reference to possible regulatory functions of ABA. In this respect it is suggested that – in particular under conditions of stress – ABA might regulate cellular metabolic processes by changing the cytoplasmic K⁺ level.     

Control of Ion Fluxes in Stomatal Guard Cells

Opening and closing of the stomatal pore is associated with very large changes in K-salt accumulation in stomatal guard cells. This review discusses the ionic relations of guard cells in relation to the general pattern of transport processes in plant cells, in plasmalemma and tonoplast, involving primary active transport of protons, proton-linked secondary active transport, and a number of gated ion channels. The evidence available suggests that the initiation of stomatal opening is regulated through the uptake mechanisms, whereas initiation of stomatal closing is regulated by control of ion efflux at the plasmalemma, and of fluxes to and from the vacuole. In response to a closing signal there are large transient increases in efflux of both Cl^? (or Br^?) and Rb⁺ (K⁺) at the plasmalemma, with also a probable increase in anion flux from vacuole to cytoplasm and decrease in anion flux from cytoplasm to vacuole. A speculative hypothetical sequence of events is discussed, by which the primary response to a closing signal is an increase in Ca²⁺ influx at the plasmalemma, producing depolarisation and increase in cytoplasmic Ca²⁺. The consequent opening of Ca²⁺-sensitive Cl^? channels, and voltage-sensitive K⁺ channels (also Ca²⁺-sensitive?) in the plasmalemma, and of a Ca²⁺-sensitive nonspecific channel in the tonoplast, could produce the flux effects identified by the tracer work; this speculation is also consistent with the Ca²⁺-sensitivity of the response to closing signals and with evidence from patch clamping that such channels exist in at least some plant cells, though not yet all shown in guard cells.     

The effect of CCCP on ion fluxes in the stele and cortex of maize roots

Summary The accumulation of ⁸⁶Rb labelled potassium by isolated stelar and cortical tissues from 7-day-old roots of Zea mays has been compared with the levels accumulated by these tissues in the intact root. Cortical tissues have similar uptake eapacities in these two conditions whereas stelar tissues only exhibit an uptake capacity in the intact root system. The uncoupler carbonylcyanide m-chlorophenylhydrazone caused a considerable decrease in the uptake of potassium by these tissues. In the intact root system it prevented ions from the bathing medium reaching the stelar tissues. The efflux pattern from preloaded isolated stelar and cortical tissues was considerably altered by the inhibitor, a promotion of the efflux occurring in both of these tissues.It is concluded that stelar tissues only accumulated ions when these are supplied through the root symplasm and that the stelar plasmalemma has only a limited uptake capacity per se. Stelar uptake is thus a reflection of vacuolar accumulation across the tonoplast. There is no evidence in the present study of a carrier-mediated active secretion of ions across the stelar plasmalemma. The fact that the efflux was promoted rather than depressed by the uncoupler supports the postulate that a passive leakage is the final stage in the transport of ions across the plant root.     

Control of Volume and Turgor in Stomatal Guard Cells

Water loss from plants is determined by the aperture of stomatal pores in the leaf epidermis, set by the level of vacuolar accumulation of potassium salt, and hence volume and turgor, of a pair of guard cells. Regulation of ion fluxes across the tonoplast, the key to regulation of stomatal aperture, can only be studied by tracer flux measurements. There are two transport systems in the tonoplast. The first is a Ca²⁺-activated channel, inhibited by phenylarsine oxide (PAO), responsible for the release of vacuolar K⁺(Rb⁺) in response to the “drought” hormone, abscisic acid (ABA). This channel is sensitive to pressure, down-regulated at low turgor and up-regulated at high turgor, providing a system for turgor regulation. ABA induces a transient stimulation of vacuolar ion efflux, during which the flux tracks the ion content (volume, turgor), suggesting ABA reduces the set-point of a control system. The second system, which is PAO-insensitive, is responsible for an ion flux from vacuole to cytoplasm associated with inward water flow following a hypo-osmotic transfer. It is suggested that this involves an aquaporin as sensor, and perhaps also as responder; deformation of the aquaporin may render it ion-permeable, or, alternatively, the deformed aquaporin may signal to an associated ion channel, activating it. Treatment with inhibitors of aquaporins, HgCl₂ or silver sulfadiazine, produces a large transient increase in ion release from the vacuole, also PAO-insensitive. It is suggested that this involves the same aquaporin, either rendered directly ion-permeable, or signalling to activate an associated ion channel.     

Changes in Chloride Fluxes and Cytosolic pH Induced by Abscisic Acid in Elodea densa Leaves

The effects of ABA on intracellular pH, net H⁺ extrusion, Cl^? fluxes and E_m values were studied in Elodea densa leaves, and the possible relationships between the ABA-induced changes of cytosolic pH and of Cl^? and H⁺ fluxes were investigated. Cytosolic and vacuolar pH were calculated by the weak acid and weak base distribution method. The data show that, also in this material (a water plant without stomata), ABA induces a decrease in both net H⁺ extrusion and intracellular pH, and strongly inhibits Cl^? efflux. No significant effect of ABA is detectable on E_m values, either at short or long intervals in the presence or absence of K⁺. Cl^? efflux is apparently independent of the activity of the plasmalemma H⁺ pump and of the E_m values. Conversely, it strongly depends on the value of cytosolic pH, a larger efflux occurring for the lower pH values both in the presence and in the absence of ABA. These results indicate that the ABA-induced cytosolic acidification cannot be the cause but, possibly, a consequence of the decrease in Cl^? efflux, and are consistent with the hypothesis of a primary role of ABA in regulating Cl^? efflux, presumably by directly affecting a class of Cl^?-permeable channels.     

Effect of abscisic acid (ABA) on sugar accumulation in the flesh tissue of peach fruit at the start of the maturation stage

Experiments were conducted on¹⁴C-sorbitol, fructose, and glucose uptakeinto flesh discs, and sorbitol efflux from thediscs, with and without ABA application toexamine the effect of abscisic acid (ABA) onsugar accumulation in peach fruit flesh at thestart of the maturation stage in relation tomembrane transport. Total uptake of¹⁴C-sorbitol, fructose, and glucose intoflesh discs was effectively promoted by ABA ata concentration of 10^–5 M. PCMBS(p-chloromercuribenzensulfonicacid)-sensitive uptake, which was considered ascarrier-mediated uptake, of sorbitol into thediscs was clearly stimulated by ABA at10^–5 M, compared with glucose andfructose uptake. Sorbitol efflux from the discsacross the tonoplast was restricted by ABA at10^–5 M. ABA application todeveloping fruit increased sugar accumulationin the fruit. Estimated ABA concentration inthis fruit was approximately 10^–5 M. These results indicate that sugar accumulationin peach fruit flesh is stimulated by ABA at aconcentration of 10^–5 M both invitro and in vivo. ABA stimulatesuptake of sugars, especially sorbitol, into theflesh by enhancing carrier-mediated transportpossibly across both tonoplast and plasmamembrane.     

Two components of auxin transport

The transport of indoleacetic acid-1-¹⁴C out of sunflower stem sections has been analyzed by a compartmental analysis procedure in which the radioactivity moving out of the tissue (log per cent) is plotted against time. The analysis indicates that indoleacetic acid is transported via a fast transport system (t_½ of about 30 minutes) and a slow transport system (t_½ about 10 hours). While we do not know the sources of these two pools, by analogy with ion transport studies, the fast efflux is characteristic of transport from the cytoplasm across the plasmalemma and the slow efflux is characteristic of transport across the tonoplast and thus out of the vacuole. Both components of transport are inhibited by 2,3,5-triiodobenzoic acid.     

Stress-dependent redistribution of abscisic acid (ABA) in Hordeum vulgare L leaves: the role of epidermal ABA metabolism, tonoplastic transport and the cuticle

When ¹⁴C-labelled abscisic acid ([¹⁴C]ABA) was supplied to isolated protoplasts of the barley leaf at pH 6, initial rates of metabolism were about five times higher in epidermal cell protoplasts than in mesophyll cell protoplasts if equal cytosolic volumes were considered. In spite of the fact that epidermal cells make up only about 35% of the total water space in barley leaves, and despite the small cytosolic volume of these cells, in intact leaves all epidermal cells would thus metabolize half as much ABA per unit time as the mesophyll cells (0–27 and 0–51 mmol h^?1 m^?3 leaf water). Therefore, under these conditions epidermal cells seem to be a stronger sink than mesophyll cells for ABA that arrives via the transpiration stream. However, at an apoplastic pH of 7–25, which occurs in stressed leaves, the proportion of total metabolized ABA would be much smaller in epidermal than in mesophyll cells (0–029 and 0–204 mmolh^?l m^?3 leaf water). Our results indicate that under conditions of slightly alkaline apoplastic pH the epidermis may serve as the main source for fast stress-dependent ABA redistribution into the guard cell apoplast. This is partly the result of ABA transport across the epidermal tonoplast, which is dependent on the apoplastic pH and possibly on the cytosolic calcium concentration. The cuticle seems to be of no particular importance in stress-induced apoplastic ABA shifts and cannot be regarded as a significant sink for high ABA concentrations under stress.     

ABA-induced ion efflux in stomatal guard cells: multiple actions of ABA inside and outside the cell

Abscisic acid (ABA) induces a transient stimulation of ⁸⁶Rb⁺ efflux from isolated guard cells of Commelina communis L. The form of the efflux transients produced in suboptimal conditions (low concentrations of ABA and/or high external pH at which ABA will penetrate poorly) has been compared with the full transient. In suboptimal conditions the stimulation of efflux is both delayed and reduced. The pH-dependence of the delay before initiation of the efflux transient suggests that a threshold internal concentration of ABA is required. However in suboptimal conditions even when the threshold internal concentration is reached and a transient is triggered, the degree of stimulation is reduced, an effect which also appears to depend on internal ABA. It is suggested that the differences reflect activation of different numbers of tonoplast ion channels for release of vacuolar ions. By contrast, the same end-state seems to be reached in optimal and suboptimal conditions, but after different times. The relative efflux stimulation during the efflux transient tracks the declining ion content; both the peak and the end of the transient are reached at the same ion content, but at different times. It is suggested that this reflects an ABA-induced change in the set-point of a regulated ion channel which is sensitive to ion content, perhaps a stretch-activated channel. This effect is independent of external concentration in the range 0.1–10 µM, and pH 6 and pH 8 are equally effective, suggesting an external site of action. Thus the results suggest multiple actions of ABA, involving both internal and external receptors. Regulation of both tonoplast ion channels by internal ABA, and of a regulated channel responsive to ion content by external ABA are suggested.     

Further Characterization on the Transport Property of Plasmalemma NADH Oxidation System in Isolated Corn Root Protoplasts

Recent experiments show that exogenous NADH increases the O₂ consumption and uptake of inorganic ions into isolated corn (Zea mays L. Pioneer Hybrid 3320) root protoplasts (Lin 1982, Proc Natl Acad Sci USA 79: 3773-3776). A mild treatment of protoplasts with trypsin released most of the NADH oxidation system from the plasmalemma (Lin 1982 Plant Physiol 70: 326-328). Further studies on this system showed that exogenous NADH (1.5 millimolar) tripled the proton efflux from the protoplasts thus generating a greater electrochemical proton gradient across the plasmalemma. Trypsin also released ubiquinone (11.95 nanomoles per milligrams protein) but not flavin or cytochrome from the system. Kinetic analyses showed that 1.5 millimolar NADH quadrupled V_max of the mechanism I (saturable) component of K⁺ uptake, while K_m was not affected. Diethylstibestrol and vanadate inhibited basal (ATPase-mediated) K⁺ influx and H⁺ efflux, while NADH-stimulated K⁺ uptake was not or only slightly inhibited. p-Chloromercuribenzene-sulfonic acid, N,N′-dicyclohexylcarbodiimide, ethidium bromide, and oligomycin inhibited both ATPase- and NADH-mediated H⁺ and K⁺ fluxes. A combination of 10 millimolar fusicoccin and 1.5 millimolar NADH gave an 11-fold increase of K⁺ influx and a more than 3-fold increase of H⁺ efflux. It is concluded that a plasmalemma ATPase is not involved in the NADH-mediated ion transport mechanism. NADH oxidase is a -SH containing enzyme (protein) and the proton channel is an important element in this transport system. Fusicoccin synergistically stimulates the effect of NADH on K⁺ uptake.     

The permeability of the epidermal cell plasma membrane of barley leaves to abscisic acid

Uptake of ³H-labelled (±)-abscisic acid (ABA) into isolated barley (Hordeum vulgare L.) epidermal cell protoplasts (ECP) was followed over a range of pH values and ABA concentrations. The present results show that ABA uptake is not always linearly correlated with the external concentration of undissociated ABA (ABAH). At pH 7.25, ABA uptake exhibited saturation kinetics with an apparent K _m value of 75 mmol·m^–3 to tal ABA. This saturable transport component was inhibited by pretreating the protoplasts with 1 mol·m^–3 p-chloromercuribenzenesulfonic acid at pH 8.0, conditions that minimized the uptake of this acid sulfhydryl reagent. Moreover, the rate of (±)-[^3]HABA uptake was reduced by addition of 0.1 mol·m^–3 (±)-ABA to 41%, whereas the same concentration of (±)-ABA was approximately half as effective (46% of the inhibitory effect). Thus, it was concluded that only (±)-ABA competes for an ABA carrier that is located in the epidermal cell plasma membrane. The permeability of the epidermal cell plasma membrane was studied by performing a Collander analysis. At pH 6 the overall plasma-membrane permeability of epidermal cells was similar to that of guard cells but was about two times higher than that of mesophyll cells.Abbreviations ABA abscisic acid - ABA^– anion of ABA - ABAH undissociated ABA - 2,4-D 2,4-dichlorophenoxyacetic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - ECP deepidermal cell protoplast - K_r partition coefficient - M_r relative molecular mass - NEM N-ethylmaleimide - PCMBS p-chloromercuriben zenesulfonic acid - P_s permeability coefficient We are grateful to Barbara Dierich for expert technical assistance, to Prof. H. Gimmler (Lehrstuhl für Botanik I, Universität Würzburg, FRG) for helpful discussions and to the Deutsche Forschungsgemeinschaft (SFB 251, TP 3) for financial support.     

Sucrose Efflux from the Maize Scutellum

Sucrose efflux from maize scutellum slices was promoted by high pH and by K+, Na+ or Rb+. Incubation in mannose (which drastically reduces the ATP level) caused high rates of sucrose efflux only when KCl was present at pH 8. The effects of triphenylmethylphosphonium ion (TPMP+, a lipid soluble cation) on sucrose efflux were similar to those of mannose plus KCl. Mannose and TPMP+ caused release of stored sucrose into the cytoplasm, but pH8 and KCl (mannose) or pH 8 (TPMP+) in the bathing solution were necessary for rapid efflux of sucrose. Rb⁺ uptake took place during sucrose efflux. In mannose, rates of Rb⁺ uptake and sucrose efflux were low at pH 5.6 and high at pH 8.0, although the time courses for uptake and efflux were different. It is concluded that sucrose efflux is electrogenic and that it occurs as sucrose-H⁺ symport. A scheme for sucrose transport across plasmalemma and tonoplast is presented.     

Potassium Transport in Corn Roots : III. Perturbation by Exogenous NADH and Ferricyanide

It has recently been reported that plasmalemma electron transport may be involved in the generation of H⁺ gradients and the uptake of ions into root tissue. We report here on the influence of extracellular NADH and ferricyanide on K⁺ (⁸⁶Rb⁺) influx, K⁺ (⁸⁶Rb⁺) efflux, net apparent H⁺ efflux, and O₂ consumption in 2-centimeter corn (Zea mays [A632 × Oh43]) root segments and intact corn roots. In freshly excised root segments, NADH had no effect on O₂ consumption and K⁺ uptake. However, after the root segments were given a 4-hour wash in aerated salt solution, NADH elicited a moderate stimulation in O₂ consumption but caused a dramatic inhibition of K⁺ influx. Moreover, net apparent H⁺ efflux was significantly inhibited following NADH exposure in 4-hour washed root segments.

Exogenous ferricyanide inhibited K⁺ influx in a similar fashion to that caused by NADH, but caused a moderate stimulation of net H⁺ efflux. Additionally, both reagents substantially altered K⁺ efflux at both the plasmalemma and tonoplast.

These complex results do not lend themselves to straightforward interpretation and are in contradiction with previously published results. They suggest that the interaction between cell surface redox reactions and membrane transport are more complex than previously considered. Indeed, more than one electron transport system may operate in the plasmalemma to influence, or regulate, a number of transport functions and other cellular processes. The results presented here suggest that plasmalemma redox reactions may be involved in the regulation of ion uptake and the `wound response' exhibited by corn roots.

     

Regulation of Water Permeability of Vacuolar Symplast

Effect of exogenous ABA and an inhibitor of energy metabolism NaN₃ on water permeability of the desmotubules and tonoplast as the structural elements of vacuolar symplast ensuring water permeability of this transport system was investigated. The methodological approach based on the use of NMR with magnetic field pulse gradient is described in detail. It was shown that ABA affects water permeability of the vacuolar symplast in the root cells of maize (Zea mays L.) seedlings by temporary increase in water permeability of its membrane (tonoplast) and does not modify water permeability of desmotubules. At the same time, the effect of sodium azide is related to the disturbance of water permeability in the latter, and this evidence is corroborated by the additivity in the effects of the two above-mentioned agents on diffusion decay of spin echo produced by vacuolar symplast water molecules. ABA effect was detected only at high exogenous concentrations (10^?4 M). The effect of ABA on water permeability of the tonoplast did not depend on or was weakly related to intracellular concentration of ATP, whereas the open state of desmotubules was ATP-dependent. Observations were made on the role of aquaporins in the ABA influence on tonoplast water permeability and the physiological role of high ABA concentrations.     

Unidirectional sodium fluxes in red beet storage tissue (Beta vulgaris L. cv. Platronde Egyptische): effects of the phytohormones indol-3yl-acetic acid and abscisic acid

Abstract. The influence of indol-3yl-acetic acid (IAA) and abscisic acid (ABA) on the capacities of the cytoplasm and vacuole and their effects on unidirectional sodium fluxes across the plasmalemma and the tonoplast of aged red beet storage tissue was investigated. After loading the tissue in a labelled NaCl solution the efflux of radio-activity was measured in unlabelled NaCl. By means of compartmental analysis the capacities and fluxes were determined and compared with those obtained after loading and elution in the presence of IAA or ABA.
It was established that both IAA and ABA affect sodium transport across the principal cell membranes. Both hormones inhibited the efflux across the plasma-lemma, possibly by affecting a Na⁺ for H⁺ exchanging system. Efflux across the tonoplast was stimulated by IAA and influx across the same membrane was enhanced by ABA. It was suggested that IAA stimulated a proton pump at this level while the influence of ABA remained difficult to explain.     

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     

Turgor pressure changes trigger characteristic changes in the electrical conductance of the tonoplast and the plasmalemma of the marine alga Valonia utricularis

The giant marine alga Valonia utricularis is capable of regulating its turgor pressure in response to changes in the osmotic pressure of the sea water. The turgor pressure response comprises two phases, a fast, exponential phase arising exclusively from water shifting between the vacuole and the external medium (time constant about 10 min) and a second very slow, almost exponential phase adjusting (but not always) the turgor pressure near to the original value by release or uptake of KCl (time constant about 5 h). The changes in the vacuolar membrane potential as well as in the individual conductances of the tonoplast and plasmalemma accompanying turgor pressure regulation were measured by using the vacuolar perfusion assembly (with integrated microelectrodes, pressure transducers and pressure‐regulating valves) as described by Wang et al. (J. Membrane Biology 157, 311–321, 1997). Measurements on pressure‐clamped cells gave strong evidence that the turgor pressure, but not effects related to water flow (i.e. electro‐osmosis or streaming potential) or changes in the internal osmotic pressure and in the osmotic gradients, triggers the cascade of osmotic and electrical events recorded after disturbance of the osmotic equilibrium. The findings definitely exclude the existence of osmosensors as postulated for other plant cells and bacteria. There was also evidence that turgor pressure signals were primarily sensed by ion transporters in the vacuolar membrane because conductance changes were first recorded in the many‐folded tonoplast and then significantly delayed in the plasmalemma independent of the direction of the osmotic challenge. Consistently, turgor pressure up‐regulation (but not down‐regulation) could be inhibited reversibly by external addition of the K⁺ transport inhibitor Ba²⁺ and/or by the Cl^– transport inhibitor 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS). Extensive studies under iso‐, hyper‐ and hypo‐osmotic conditions revealed that K⁺ and Cl^– contribute predominantly to the plasmalemma conductance. Addition of 0.3 mm NaCN showed further that part of the K⁺ and Cl^– transporters depended on ATP. These transporters are apparently up‐regulated upon hyper‐osmotic, but not hypo‐osmotic challenge. These findings explain the strong increase of the K⁺ influx upon lowering turgor pressure and the less pronounced pressure‐dependence of the Cl^– influx of V. utricularis reported in the literature. The data derived from the blockage experiments under hypo‐osmotic conditions were also equally consistent with the experimental findings that the K⁺ efflux is solely passive and progressively increases with increasing turgor pressure due to an increase of the volumetric elastic modulus of the cell wall. However, despite unravelling some of the sequences and other components involved in turgor pressure regulation of V. utricularis the co‐ordination between the ion transporters in the tonoplast and plasmalemma remains unresolved because of the failure to block the tonoplast transporters by addition of Ba²⁺ and DIDS from the vacuolar side.     

Studies on the tonoplast action potential ofNitella flexilis

Summary The origins of the two peaks of the action potential inNitella flexilis were analyzed by inserting two microelectrodes. one into the vacuole and the other into the cytoplasm. It was unequivocally demonstrated that the rapid first peak was generated at the plasmalemma and the slow second peak at the tonoplast. MnCl₂ applied in the external medium abolished the second, tonoplast, peak but not the first, plasmalemma, peak, MnCl₂ also inhibited the cessation of the cytoplasmic streaming accompanying the action potential. CaCl₂ added in MnCl₂-containing medium recovered generation of the tonoplast action potential and the streaming cessation. Since it has been established that the cessation of cytoplasmic streaming on membrane excitation is caused by an increase in cytoplasmic free Ca^2– (Williamson, R.E., Ashley, C.C., 1982.Nature (London) 296:647–651: Tominaga, Y., Shimmen, T., Tazawa, M., 1983,Protoplasma 116:75–77), it is suggested that the tonoplast action potential is also induced by an increase in cytoplasmic Ca²⁺ resulting from the plasmalemma excitation. When vacuolar Cl was replaced with SO ₄ ² by vacuolar perfusion, the polarity of the second, slow peak was reversed from vacuolar positive to vacuolar negative with respect to the cytoplasm, supporting the previous report that the tonoplast action potential is caused by increase in Cl permeability (Kikuyama, M., Tazawa, M., 1976.J. Membrane Biol.29:95–110).