Root-to-shoot signalling: apoplastic alkalinization, a general stress response and defence factor in barley (Hordeum vulgare).

We used a noninvasive microprobe technique to record in substomatal cavities of barley leaves the apoplastic pH response to different stress situations. When K+ (or Na+) activity at the roots of intact plants was increased from 1 to 50 mM, the leaf apoplastic pH increased by 0.4 to 0.6 units within 8 to 12 min when stomata were open, and within 15 to 20 min when stomata were closed. This reaction was accompanied by a correlative increase in K+ activity. Addition of 1 microM abscisic acid caused an apoplastic alkalinization of 0.5 to 0.8 units, and low temperatures (4 degrees C) increased pH by 0.2 to 0.3 units. Addition of 100 mM sorbitol or pH changes in the range 4.0 to 7.9 had no effect, ruling out that osmotic potential and/or pH is the carried signal. On detached leaves, the same treatments yielded qualitatively similar results, suggesting that the xylem is the most likely signal path. Following the attack of powdery mildew, the apoplastic pH of barley leaves substantially increases. We demonstrate that in susceptible barley, pretreatment (soil drench) with the resistance-inducing chemical benzo- (1,2,3)thiadiazole-7-carbothioic acid S-methyl ester markedly enhances this pH response. This is consistent with previous finding that apoplastic alkalinization is related to the degree of resistance towards this fungus.

K(+) channel profile and electrical properties of Arabidopsis root hairs.

Ion channels and solute transporters in the plasma membrane of root hairs are proposed to control nutrient uptake, osmoregulation and polar growth. Here we analyzed the molecular components of potassium transport in Arabidopsis root hairs by combining K(+)-selective electrodes, reverse transcription-PCR, and patch-clamp measurements. The two inward rectifiers AKT1 and ATKC1 as well as the outward rectifier GORK dominated the root hair K(+) channel pool. Root hairs of AKT1 and ATKC1 loss-of-function plants completely lack the K(+) uptake channel or exhibited altered properties, respectively. Upon oligochitin-elicitor treatment of root hairs, transient changes in K(+) fluxes and membrane polarization were recorded in wild-type plants, while akt1-1 root hairs showed a reduced amplitude and pronounced delay in the potassium re-uptake process. This indicates that AKT1 and ATKC1 represent essential alpha-subunits of the inward rectifier. Green fluorescent protein (GFP) fluorescence following ballistic bombardment with GORK promoter-GFP constructs as well as analysis of promoter-GUS lines identified this K(+) outward rectifier as a novel ion channel expressed in root hairs. Based on the expression profile and the electrical properties of the root hair plasma membrane we conclude that AKT1-, ATKC- and GORK-mediated potassium transport is essential for osmoregulation and repolarization of the membrane potential in response to elicitors.

Changes in apoplastic pH and membrane potential in leaves in relation to stomatal responses to CO2, malate, abscisic acid or interruption of water supply.

Low CO2 concentrations open CO2-sensitive stomata whereas elevated CO2 levels close them. This CO2 response is maintained in the dark. To elucidate mechanisms underlying the dark CO2 response we introduced pH- and potential-sensitive dyes into the apoplast of leaves. After mounting excised leaves in a gas-exchange chamber, changes in extracellular proton concentration and transmembrane potential differences as well as transpiration and respiration were simultaneously monitored. Upon an increase in CO2 concentration transient changes in apoplastic pH (occasionally brief acidification, but always followed by alkalinization) and in membrane potential (brief hyperpolarization followed by depolarization) accompanied stomatal closure. Alkalinization and depolarization were also observed when leaves were challenged with abscisic acid or when water flow was interrupted. During stomatal opening in response to CO2-free air the apoplastic pH increased while the membrane potential initially depolarized before it transiently hyperpolarized. To examine whether changes in apoplastic malate concentrations represent a closing signal for stomata, malate was fed into the transpiration stream. Although malate caused apoplastic alkalinization and membrane depolarization reminiscent of the effects observed with CO2 and abscisic acid, this dicarboxylate closed the stomata only partially and less effectively than CO2. Apoplastic alkalinization was also observed and stomata closed partially when KCl was fed to the leaves. Respiration increased on feeding of malate or KCl, or while abscisic acid closed the stomate. From these results we conclude that CO2 signals modulate the activity of plasma-membrane ion channels and of plasmalemma H+-ATPases during changes in stomatal aperture. Responses to potassium malate and KCl are not restricted to guard cells and neighbouring cells.

KAT1 is not essential for stomatal opening.

It is generally accepted that K(+) uptake into guard cells via inward-rectifying K(+) channels is required for stomatal opening. To test whether the guard cell K(+) channel KAT1 is essential for stomatal opening, a knockout mutant, KAT1En-1, was isolated from an En-1 mutagenized Arabidopsis thaliana population. Stomatal action and K(+) uptake, however, were not impaired in KAT1-deficient plants. Reverse transcription-PCR experiments with isolated guard cell protoplasts showed that in addition to KAT1, the K(+) channels AKT1, AKT2/3, AtKC1, and KAT2 were expressed in this cell type. In impalement measurements, intact guard cells exhibited inward-rectifying K(+) currents across the plasma membrane of both wild-type and KAT1En-1 plants. This study demonstrates that multiple K(+) channel transcripts exist in guard cells and that KAT1 is not essential for stomatal action.

Dynamics of ionic activities in the apoplast of the sub-stomatal cavity of intact Vicia faba leaves during stomatal closure evoked by ABA and darkness.

Stomatal movement is accomplished by changes in the ionic content within guard cells as well as in the cell wall of the surrounding stomatal pore. In this study, the sub-stomatal apoplastic activities of K+, Cl-, Ca2+ and H+ were continuously monitored by inserting ion-selective micro-electrodes through the open stomata of intact Vicia faba leaves. In light-adapted leaves, the mean activities were 2.59 mM (K+), 1.26 mM (Cl-), 64 microM (Ca2+) and 89 microM (H+). Stomatal closure was investigated through exposure to abscisic acid (ABA), sudden darkness or both. Feeding the leaves with ABA through the cut petiole initially resulted in peaks after 9-10 min, in which Ca2+ and H+ activities transiently decreased, and Cl- and K+ activities transiently increased. Thereafter, Ca2+, H+ and Cl- activities completely recovered, while K+ activity approached an elevated level of around 10 mM within 20 min. Similar responses were observed following sudden darkness, with the difference that Cl- and Ca2+ activities recovered more slowly. Addition of ABA to dark-adapted leaves evoked responses of Cl- and Ca2+ similar to those observed in the light. K+ activity, starting from its elevated level, responded to ABA with a transient increase peaking around 16 mM, but then returned to its dark level. During stomatal closure, membrane potential changes in mesophyll cells showed no correlation with the K+ kinetics in the sub-stomatal cavity. We thus conclude that the increase in K+ activity mainly resulted from K+ release by the guard cells, indicating apoplastic compartmentation. Based on the close correlation between Cl- and Ca2+ changes, we suggest that anion channels are activated by a rise in cytosolic free Ca2+, a process which activates depolarization-activated K+ release channels.

How alfalfa root hairs discriminate between Nod factors and oligochitin elicitors.

Using ion-selective microelectrodes, the problem of how signals coming from symbiotic partners or from potential microbial intruders are distinguished was investigated on root hairs of alfalfa (Medicago sativa). The Nod factor, NodRm-IV(C16:2,S), was used to trigger the symbiotic signal and (GlcNAc)(8) was selected from (GlcNAc)(4-8), to elicit defense-related reactions. To both compounds, root hairs responded with initial transient depolarizations and alkalinizations, which were followed by a hyperpolarization and external acidification in the presence of (GlcNAc)(8). We propose that alfalfa recognizes tetrameric Nod factors and N-acetylchitooligosaccharides (n = 4-8) with separate perception sites: (a) (GlcNAc)(4) and (GlcNAc)(6) reduced the depolarization response to (GlcNAc)(8), but not to NodRm-IV(C16:2, S); and (b) depolarization and external alkalization were enhanced when NodRm-IV(C16:2,S) and (GlcNAc)(8) were added jointly without preincubation. We suggest further that changes in cytosolic pH and Ca(2+) are key events in the transduction, as well as in the discrimination of signals leading to symbiotic responses or defense-related reactions. To (GlcNAc)(8), cells responded with a cytosolic acidification, and they responded to NodRm-IV(C16:2,S) with a sustained alkalinization. When both agents were added jointly, the cytosol first alkalized and then acidified. (GlcNAc)(8) and NodRm-IV(C16:2,S) transiently increased cytosolic Ca(2+) activity, whereby the response to (GlcNAc)(8) exceeded the one to NodRm-IV(C16:2,S) by at least a factor of two.

Genotypical differences in aluminum resistance of maize are expressed in the distal part of the transition zone. Is reduced basipetal auxin flow involved in inhibition of root elongation by aluminum?

Short-term Al treatment (90 microM Al at pH 4.5 for 1 h) of the distal transition zone (DTZ; 1-2 mm from the root tip), which does not contribute significantly to root elongation, inhibited root elongation in the main elongation zone (EZ; 2.5-5 mm from the root tip) to the same extent as treatment of the entire maize (Zea mays) root apex. Application of Al to the EZ had no effect on root elongation. Higher genotypical resistance to Al applied to the entire root apex, and specifically to the DTZ, was expressed by less inhibition of root elongation, Al accumulation, and Al-induced callose formation, primarily in the DTZ. A characteristic pH profile along the surface of the root apex with a maximum of pH 5.3 in the DTZ was demonstrated. Al application induced a substantial flattening of the pH profile moreso in the Al-sensitive than in the Al-resistant cultivar. Application of indole-3-acetic acid to the EZ but not to the meristematic zone significantly alleviated the inhibition of root elongation induced by the application of Al to the DTZ. Basipetal transport of exogenously applied [(3)H]indole-3-acetic acid to the meristematic zone was significantly inhibited by Al application to the DTZ in the Al-sensitive maize cv Lixis. Our results provide evidence that the primary mechanisms of genotypical differences in Al resistance are located within the DTZ, and suggest a signaling pathway in the root apex mediating the Al signal between the DTZ and the EZ through basipetal auxin transport.

Elevation of the cytosolic free [Ca2+] is indispensable for the transduction of the Nod factor signal in alfalfa.

In root hairs of alfalfa (Medicago sativa), the requirement of Ca(2+) for Nod factor signaling has been investigated by means of ion-selective microelectrodes. Measured 50 to 100 microm behind the growing tip, 0.1 microM NodRm-IV(C16:2,S) increased the cytosolic free [Ca2+] by about 0.2 pCa, while the same concentration of chitotetraose, the nonactive glucosamine backbone, had no effect. We demonstrate that NodRm-IV(C16:2,S) still depolarized the plasma membrane at external Ca(2+) concentrations below cytosolic values if the free EGTA concentration remained low (

The Cytosolic Ca2+ Concentration Gradient of Sinapis alba Root Hairs as Revealed by Ca2+-Selective Microelectrode Tests and Fura-Dextran Ratio Imaging.

Using Ca2+-selective microelectrodes and fura 2-dextran ratio imaging, the cytosolic free [Ca2+] was measured in Sinapis alba root hair cells. Both methods yielded comparable results, i.e. values between 158 to 251 nM for the basal [Ca2+] of the cells and an elevated [Ca2+] of 446 to 707 nM in the tip region. The zone of elevated [Ca2+] reaches 40 to 60 [mu]m into the cell and is congruent with the region of inwardly directed Ca2+ net currents measured with an external Ca2+- selective vibrating electrode. The channel-blocker La3+ eliminates these currents, stops growth, and almost completely eliminates the cytosolic [Ca2+] gradient without affecting the basal level of the ion. Growth is also inhibited by pressure-injected dibromo-1,2-bis(o-aminophenoxy)ethane-N,N,N[prime],N[prime]-tetraacetic acid, which causes a decrease in the [Ca2+] in the tip in a concentration-dependent manner. Indole-3-acetic acid, used as a model stimulus, decreases cytosolic free [Ca2+] by 0.2 to 0.3 pCa units in the tip, but only by about 0.1 pCa unit in the shank. Nongrowing root hairs may or may not display a [Ca2+] gradient, but still reversibly respond to external stimuli such as La3+, Ca2+, or indole-3-acetic acid with changes in cytosolic free [Ca2+]. During short time periods, dicyclohexylcarbodiimide inhibition of the plasma membrane H+-ATPase, which stops growth, does not abolish the [Ca2+] gradient, nor does it change significantly the basal [Ca2+] level. We conclude that the cytosolic [Ca2+] gradient and an elevated [Ca2+] in the tip, as in other tip-growing cells, is essential for tip growth in root hairs; however, its presence does not indicate growth under all circumstances. We argue that with respect to Ca2+, tip growth regulation and responses to external signals may not interfere with each other. Finally, we suggest that the combination of the methods applied adds considerably to our understanding of the role of cytosolic free [Ca2+] in signal transduction and cellular growth.

The H+/Cl- Symporter in Root-Hair Cells of Sinapis alba (An Electrophysiological Study Using Ion-Selective Microelectrodes).

In root-hair cells of Sinapis alba, cytosolic pH, cytosolic [Cl-], membrane potential, and membrane resistance have been measured to investigate proton-driven Cl- transport across the plasma membrane. Rapid lowering of the external pH transiently increased cytosolic [Cl-] and acidified the cytoplasm. To an abrupt increase in external [Cl-] the cells reacted with a rapid initial depolarization and a subsequent slower hyperpolarization, which was accompanied by an increase in cytosolic [Cl-] and [H+]. These results are indicative of an nH+/Cl- symport with n > 1. Simultaneous recording of the membrane potential, the proton motive force, cytosolic pH, and cytosolic [Cl-] reveals that kinetically this Cl- transport depends on the pH gradient across the plasma membrane rather than on the membrane potential.

Effect of high pH on the plasma membrane potential and conductance in Elodea densa.

In leaves of Elodea densa the membrane potential measured in light equals the equilibrium potential of H+ on the morphological upper plasma membrane. The apoplastic pH on the upper side of the leaf is as high as 10.5-11.0, which indicates that alkaline pH induces an increased H+ permeability of the plasmalemma. To study this hypothesis in more detail we investigated the changes in membrane potential and conductance in response to alterations in the external pH from 7 (= control) to 9 or 11 under both light and dark conditions. Departing from the control pH 7 condition, in light and in dark the application of pH 9 resulted in a depolarization of the membrane potential to the Nernst potential of H+. In the light but not in the dark, this depolarization was followed by a repolarization to about -160 mV. The change to pH 9 induced, in light as well as in dark, an increase in membrane conductance. The application of pH 11, which caused a momentary hyper- or depolarization depending on the value at the time pH 11 was applied, brought the membrane potential to around -160 mV. The membrane conductance also increased, in comparison to its value at pH 7, as a result of the application of pH 11, irrespective of the light conditions.

The role of the plasma-membrane Ca(2+)-ATPase in Ca (2+) homeostasis in Sinapis alba root hairs.

The regulation of cytosolic Ca(2+) has been investigated in growing root-hair cells of Sinapis alba L. with special emphasis on the role of the plasmamembrane Ca(2+)-ATPase. For this purpose, erythrosin B was used to inhibit the Ca(2+)-ATPase, and the Ca(2+) ionophore A23187 was applied to manipulate cytosolic free [Ca(2+)] which was then measured with Ca(2+)-selective microelectrodes. (i) At 0.01 µM, A23187 had no effect on the membrane potential but enhanced the Ca(2+) permeability of the plasma membrane. Higher concentrations of this ionophore strongly depolarized the cells, also in the presence of cyanide. (ii) Unexpectedly, A23187 first caused a decrease in cytosolic Ca(2+) by 0.2 to 0.3 pCa units and a cytosolic acidification by about 0.5 pH units, (iii) The depletion of cytosolic free Ca(2+) spontaneously reversed and became an increase, a process which strongly depended on the external Ca(2+) concentration, (iv) Upon removal of A23187, the cytosolic free [Ca(2+)] returned to its steady-state level, a process which was inhibited by erythrosin B. We suggest that the first reaction to the intruding Ca(2+) is an activation of Ca(2+) transporters (e.g. ATPases at the endoplasmic reticulum and the plasma membrane) which rapidly remove Ca(2+) from the cytosol. The two observations that after the addition of A23187, (i) Ca(2+) gradients as steep as-600 mV could be maintained and (ii) the cytosolic pH rapidly and immediately decreased without recovery indicate that the Ca(2+)-exporting plasma-membrane ATPase is physiologically connected to the electrochemical pH gradient, and probably works as an nH(+)/Ca(2+)-ATPase. Based on the finding that the Ca(2+)-ATPase inhibitor erythrosin B had no effect on cytosolic Ca(2+), but caused a strong Ca(2+) increase after the addion of A23187 we conclude that these cells, at least in the short term, have enough metabolic energy to balance the loss in transport activity caused by inhibition of the primary Ca(2+)-pump. We further conclude that this ATPase is a major Ca(2+) regulator in stress situations where the cytosolic Ca(2+) has been shifted from its steady-state level, as may be the case during processes of signal transduction.

Auxin-induced H(+)-pump stimulation does not depend on the presence of epidermal cells in corn coleoptiles.

Cell-wall acidification and electrical reactions (depolarization and hyperpolarization) are typical auxin responses in maize (Zea mays L.) coleoptiles. In an attempt to test the role of the outer epidermis in these responses, they have been measured and compared in intact and peeled coleoptile fragments. To exclude interactions between parenchymal and epidermal cells, the coleoptile pieces were completely stripped of their outer epidermis. This preparation was monitored by means of a scanning electron microscope. When externally applied indole-3-acetic acid was tested, we found that neither cell-wall acidification nor the electrical membrane responses depended on the presence of intact epidermal cells.

The electrical response of maize to auxins.

The electrical response of Zea mays coleoptiles and suspension cultured cells to several growth-promoting auxins (IAA, IBA, 2,4-D, 2,4,5-T, 1-NAA) and some of their structural analogues (2,3-D, 2-NAA) has been tested. In coleoptile two typical electrical responses to IAA are observed: an immediated rapid depolarization, and a hyperpolarization following 7-10 minutes after the first external addition of IAA. Of the other tested compounds only 1-NAA significantly depolarized the cells, whereas all auxins as well as the analogues evoked delayed hyperpolarizations. In contrast, the suspension cells were not hyperpolarized by any of the tested compounds, but were strongly depolarized by IAA, 1-NAA, and to a lesser extent by 2-NAA. In these cells IAA and 1-NAA induced inwardly directed currents of positive charge which both saturated around 12 mA/m2. The strong pH-dependence together with the half-maximal currents 0.49 microM IAA and 0.76 microM 1-NAA point to a symport of the anions with at least 2H+. The delayed plasma membrane hyperpolarization is a different response, and seems to be initiated by the protonated auxin species. In accordance with the current literature, it is interpreted as consequence of a stimulated proton extrusion. The finding that all tested compounds evoked a hyperpolarization, makes this response unspecific. It is concluded that a stimulation of proton extrusion is a necessary, but not sufficient step to induce elongation growth.

Proton Gradient Across the Tonoplast of Riccia fluitans as a Result of the Joint Action of Two Electroenzymes.

Using pH-sensitive microelectrodes (in vitro) and acridine orange photometry (in vivo), the actions of the two tonoplast phosphatases, the tp-ATPase and the tp-PPase, were investigated with respect to how effectively they could generate a transtonoplast pH-gradient. Under standard conditions the vacuoles of the aquatic liverwort Riccia fluitans have an in vivo pH of 4.7 to 5.0. In isolated vacuoles a maximal vacuolar pH (pH(v)) of 4.74 +/- 0.1 is generated in the presence of 0.1 millimolar PP(i), but only 4.93 +/- 0.13 in the presence of 2.5 millimolar ATP. Both substrates added together approximate the value for PP(i). Cl(-)-stimulates the H(+)-transport driven by the tp-ATPase, but has no effect on the tp-PPase. The transport activity of the tp-ATPase approximates saturation kinetics (K((1/2)) approximately 0.5 millimolar), whereas transport by the tp-PPase yields an optimum around 0.1 millimolar PP(i). The transtonoplast pH-gradient is dissipated slowly by weak bases, from which a vacuolar buffer capacity of roughly 300 to 400 millimolar/pH(v) unit has been estimated. From the free energy (-11.42 kilojoules per mole) for the hydrolysis of PP(i) under the given experimental conditions, we conclude that the PPase-stoichiometry (transported H(+) per hydrolyzed substrate molecule) must be 1, and that in vivo this enzyme works as a H(+)-pump rather than as a pyrophosphate synthetase.

Ca-selective microelectrodes and their application to plant cells and tissues.

The use of Ca(2+)-selective microelectrodes is difficult because of some basic problems: (a) electrodes with submicron tips may display non-Nernstian slopes; (b) liquid membrane microelectrodes respond only slowly (within seconds) to changes in ion activity; (c) turgid plant cells with tough walls damage the sensitive tip. This article describes concisely recent advances in fabricating Ca(2+)-selective single and double-barreled microelectrodes and their intracellular applications to different plant cell materials. Beveling the electrodes, mixing the sensor components with polyvinylchloride, insulation of the hydrated glass, and stabilization of the tips with inert materials are considered the basic concepts to circumvent most difficulties. It is concluded that the Ca(2+)-electrode can be a useful tool in plant physiology, but in spite of recent progress this technique remains experimentally demanding.

Cytoplasmic free calcium in Riccia fluitans L. and Zea mays L.: Interaction of Ca(2+) and pH?

In cells of Zea mays (root hairs, coleoptiles) and Riccia fluitans (rhizoids, thalli) intracellular Ca(2+) and pH have been measured with double-barrelled microelectrodes. Free Ca(2+) activities of 109-187 nM (Riccia rhizoids), 94-160 nM (Riccia thalli), 145-231 nM (Zea root hairs), 84-143 nM (Zea coleoptiles) were found, and therefore identified as cytoplasmic. In a few cases (Riccia rhizoids), free Ca(2+) was in the lower millimolar range (2.3±0.8 mM). A change in external Ca(2+) from 0.1 to 10 mM caused an initial and short transient increase in cytoplasmic free Ca(2+) which finally levelled off at about 0.2 pCa unit below the control, whereas in the presence of cyanide the Ca(2+) activity returned to the control level. It is suggested that this behaviour is indicative of active cellular Ca(2+) regulation, and since it is energy-dependent, may involve a Ca(2+)-ATPase. Acidification of the cytoplasmic pH and alkalinization of the vacuolar pH lead to a simultaneous increase in cytoplasmic free Ca(2+), while alkalinization of pHc decreased the Ca(2+) activity. Since this is true for such remote organisms as Riccia and Zea, it may be concluded that regulation of cytoplasmic pH and free Ca(2+) are interrelated. It is further concluded that double-barrelled microelectrodes are useful tools for investigations of intracellular ion activities in plant cells.

Auxin causes oscillations of cytosolic free calcium and pH inZea mays coleoptiles.

In epidermal cells of maize (Zea mays L.) coleoptiles, cytosolic pH (pHc), cytosolic free calcium, membrane potential and changes thereof were monitored continuously and simultaneously (pHc/,ψ m, Ca(2+)/ψ m) using double-barrelled ion-sensitive microelectrodes. In the resting cells the cytosolic pH was 7.3-7.5 and the concentration of free calcium was 119±24 nM. One-micromolar indole-3-acetic acid (IAA), added to the external medium at pH 6.0 triggered oscillations inψ m, pHc and free calcium with a period of 20 to 30 min. Acidification of the cytosolic pH increased the cytosolic free calcium. Theψ m oscillations are attributed to changes in activity of the H(+)-extrusion pump at the plasmalemma, triggered off by ΔpH and controlled by pH regulation (pH oscillation). The origin of the pHc and Ca(2+) changes remains unclear, but is possibly caused by auxin-receptor-induced lipid breakdown and subsequent second-messenger formation. It is suggested that the observed cytosolic pH and Ca(2+) changes are intrinsically interrelated, and it is concluded that this onset of regulatory processes through the phytohormone IAA is indicative of calcium and protons mediating early auxin action in maize coleoptiles. It is further concluded that the double-barrelled ion-sensitive microelectrode is an invaluable tool for investigating in-vivo hormone action in plant tissues.

Implications for cytoplasmic pH, protonmotice force, and amino-acid transport across the plasmalemma of Riccia fluitans.

By means of pH-sensitive microelectrodes, cytoplasmic pH has been monitored continuously during amino-acid transport across the plasmalemma of Riccia fluitans rhizoid cells under various experimental conditions. (i) Contrary to the general assumption that import of amino acids (or hexoses) together with protons should lead to cytoplasmic acidification, an alkalinization of 0.1-0.3 pHc units was found for all amino acids tested. Similar alkalinizations were recorded in the presence of hexoses and methylamine. No alkalinization occurred when the substrates were added in the depolarized state or in the presence of cyanide, where the electrogenic H(+)-pump is inhibited. (ii) After acidification of the cytoplasm by means of various concentrations of acetic acid, amino-acid transport is massively altered, although the protonmotive force remained essentially constant. It is suggested that H(+)-cotransport is energetically interconnected with the proton-export pump which is stimulated by the amino-acid-induced depolarization, thus causing proton depletion of the cytoplasm. It is concluded that, in order to investigate H(+)-dependent cotransport processes, the cytoplasmic pH must be measured and be under continuous experimental control; secondly, neither ΔpH nor the protonmotive force across a membrane are reliable quantities for analysing a proton-dependent process.

Vacuolar pH in radish cotyledonal mesophyll cells.

The vacuolar pH in cotyledonal mesophyll cells from radish (Raphanus sativus L. var. sativus) seedlings was determined from vacuoles, isolated from protoplasts through osmotic shock, by means of measurement of vacuole extracts with a pH meter and the "methylamine method", and gave mean pH values of 6.28 and 6.26, respectively. Direct in situ measurements of the vacuolar pH from intact leaf tissue were recorded with pH-sensitive microelectrodes and gave a mean value of 6.0. The results are discussed with respect to possible erroneous pH measurements and the vacuolar location of specific anabolic reactions.