Structure and function of the elongation sink in the stems of higher plants.*

Abstract. Application of an acid aerosol generated from an aqueous HC1 or HNO₃ solution (pH 1-2) to the hypocotyl segment of Vigna sesquipedalis, excised from the elongation zone and abraded with alumina gel, induced rapid elongation growth comparable with that induced by aerosol generated from neutral 1 mol m^?3 1AA aqueous solution. The activity of the first electrogenic ion pump, whose activity is known to be stimulated by IAA aerosol in advance of the increase in growth rate, was little affected by acids. The latent period of the growth response to acids was only 1 min shorter than that to IAA (mean value: 12min), or than the period from the stimulation of the electrogenic ion pump activity by IAA to the beginning of growth acceleration (mean value: 4 min). The growth rate, together with the activities of the first and the second ion pump, was reduced by anoxia in the presence of acid. The acid N₂-sol was ineffective to stimulate the elongation under anoxia. The acid aerosol was ineffective to stimulate the elongation of a non-abraded segment with intact cuticle layer on its surface.     

The role of electrogenic xylem pumps in K+ absorption from the zylem of Vigna unguiculata: the effects of auxin and fusicoccin

Abstract We tested the hypothesis that electrogenic ion pumps, working at the parenchyma symplast/xylem interface of pea hypocotyls, provide the driving force for K⁺ uptake from the xylem. Solutions of known composition were perfused through a hypocotyl segment. The K⁺ activity of the solution flowing out of the xylem (K⁺_out) increased (i.e. K⁺ uptake decreased) when aerobic respiration was inhibited by lack of O₂, and this was preceded by a decrease in V_px (electrical potential difference between parenchyma symplast and xylem). Perfusion with auxin (1AA) and fusicoccin (FC) stimulated the electrogenic activity of the ‘xylem pumps’ (111 and 205% respectively) and stimulated uptake of K ⁺ from the xylem (with 71% and 29% respectively). The close correlation between xylem pump activity and K⁺ uptake corroborated the aforementioned hypothesis. Interestingly, inhibition of pump activity by anoxia was incomplete in the presence of FC. It is thought that FC increases the affinity of the ATP-requiring xylem pump for ATP, thus ensuring that ATP production during fermentation is sufficient to fuel the pump in the absence of O₂.     

Current noise generated by electrogenic ion pumps

Active ion transport by ATP-or light-driven pumps involves a sequence of elementary steps such as binding and release of ions, as well as conformational transitions of the pump protein. At the microscopic level the individual reaction steps occur at random intervals, and therefore the current generated by electrogenic pumps fluctuates around a mean value. In this paper, a theoretical treatment of the electrical noise associated with active ion transport is given. The analysis, which is based on the calculation of the correlation function, yields the spectral intensity S ₁ of current noise as a function of frequency, f. The shape of S _I(f) contains information on the rate constants as well as on the magnitude of the charge displacements occuring during single reaction steps. The contribution of electrogenic pumps to the total voltage noise of the cell may be estimated from S _I(f) and from the frequency-dependent impedance of the cell membrane.     

Xylem perfusion of tap root segments of Plantago maritima: the physiological significance of electrogenic xylem pumps

Abstract A method is described for perfusing xylem vessels in tap root segments of the halophyte P. maritima. Use of excised segments allowed recording of the trans-root potential (TRP) at both ends of a segment. It was shown that there can be a spatial variation of electrogenic ion pump activity along the xylem in one root segment. The pH of perfusion solutions, differing in buffering capacity, was adjusted by the root segment to pH 5.1–5.6 during How through the xylem. This pH range was similar to that of sap produced by root pressure. The K⁺ activity in the outflow solution (K⁺_out) was rather constant at 12–13 mol m^?l3 despite input K⁺ activities ranging from 8 to 20 mol m^?l3. Addition of fusicoccin (10^?l2 mol m^?l3) to the perfusion solution induced a strong acidification of the xylem sap, a decrease in K⁺_out and an increase in Na⁺_out. Inhibition of aerobic respiration through anoxia inhibited electrogenic proton pumping into the xylem and led to an increase in K⁺_out and a decrease in Na⁺_out. It is suggested that transport of K⁺ and Na⁺ to the shoot of the halophyte P. maritima is regulated in the tap root by means of ion exchange between xylem vessels and xylem parenchyma and that this exchange is energized by proton translocating ATPases.     

The electrogenic proton pumping from parenchyma symplast into xylem—direct demonstration by xylem perfusion

Abstract We have devised an experimental system for simultaneous measurement of the activity of the xylem electrogenic ion pump, which is located on the inner cell membrane between the parenchyma symplast (p) and the xylem (x). and pH of the xylem exudate of a hypocotyl segment of Vigna unguiculata under pressurized xylem perfusion. Anoxia caused immediate depolarization of the inner cell membrane followed by alkalization of the xylem exudate several minutes later. Activity of the xylem pump was recovered by reaeration and acidification of the xylem exudate took place. These results indicate that the xylem pump is the respiration-dependent electrogenic proton-pump extruding proton from the parenchyma symplast into the xylem.     

Regulation of plant elongation growth by surface and xylem proton pumps

The roles of plasmalemma electrogenic proton pumps in elongation growth of plant stems are discussed on the basis of growth-electrophysiological studies on hypocotyl segments ofVigna unguiculata. Plant stems usually have two spatially separated electrogenic proton pumps: the surface proton pump which is located on the surface membrane of the symplast and the xylem proton pump, on the cell membrane of the symplast/xylem apoplast boundary. The surface proton pump excretes protons into the surface cell wall layer and causes the loosening of the cell wall. The xylem proton pump excretes protons into the xylem apoplast and drives the uptake of solute and water into the symplastvia secondary and/or tertiary active mechanisms: the proton cotransport system and the apoplast canal system. Both the surface and the xylem proton pumps are active during elongation growth because both the yielding of cell wall loosening and the uptake of water are necessary for continued elongation growth.     

Perfusion rate dependent prostaglandin release from isolated rabbit kidneys

Isolated rabbit kidneys were perfused with 37°C Krebs-Henseleit solution aerated with 95% O₂ + 5% CO₂. Perfusion rate was varied from 1 to 10 ml/min. This was accompanied by parallel changes of perfusion pressure, prostaglandin excretion and release of radioactivity from kidneys with ¹⁴C-arachidonic acid incorporated into the tissue lipid pool. It is suggested that enhancement of perfusion rate raises the intrarenal pressure which increases renal prostaglandin release due to increased substrate availability.     

Trans-Root Electrical Potential in Roots of Plantago media L. as Affected by Hydrostatic Pressure: The Induction of an O2 Deficient Root Core

Excised roots of Plantago media L. were subjected to small hydrostaticpressure gradients. Pressure applied with a displacement pumpeasily extracted gas present in cortical lacunae consequenton the gas saturation deficit of the bathing solution. The temporarypressure treatment resulted in an irreversible decrease in theO₂ diffusion rate through the root tissue and consequently inO₂ deficient root cores. Therefore electrogenic pumps located at the symplast/xylem interface,i.e. in the centre of the root, were inhibited by pressure. The longitudinal distribution of the xylem pumps could be computed:in Plantago media they are located in the basal parts of theroot. The effect of pressure on the electrogenic xylem pumpsdemonstrated here should be taken into account when the processof ion transport from the root to the shoot is studied whilethe root is pressurized. (Received December 5, 1983; Accepted March 26, 1984)     

Review article. Physicochemical aspects of ion relations and pH regulation in plants - a quantitative approach

A quantitative physicochemical approach to ion relations of biological solutions is presented, which applied fundamental laws of physical chemistry to these systems and allows analysis of dependent variables ([H⁺], [OH^-] and the dissociation state of partially dissociated ('weak') ions including carbonate species) in relation to independent variables (concentrations of strong and weak ions, dissociation constants and CO2 partial pressure). Within this concept the influence of strong (fully dissociated) ions is confined to their net unbalanced positive charge which is referred to as SID (strong ion difference). The SID concept is then applied to membrane transport processes and ion relations of xylem and phloem sap: simple transmembrane transport of protons between compartments cannot affect pH on either side of the membrane, because rather small deviations from electrical neutrality results in substantial changes of the membrane potential under natural conditions. Thus the membrane ATPases as electrogenic pumps cannot control the pH of adjacent compartments, but they energize secondary active transmembrane ion transport that results in pH changes. The SID approach is shown to be valid by matching pH values calculated from analysis of xylem and phloem saps with actual measured values. Sensitivity analysis based on the SID approach allows (1) to detect inconsistency in determination of composition in the analysed solutions and (2) quantitatively to analyse the influence of ion export or import and variations of pCO2 on pH and dissociation state of weak acids of complex biological solutions. The SID concept thus allows the evaluation of the contribution of a proposed pH-regulating or pH-affecting mechanism on a quantitative physicochemical basis.Key words: Electrical neutrality, membrane potential, pH regulation, phloem sap, SID, xylem sap.      

Characterization of an anion-permeable channel from sugar beet vacuoles: effect of inhibitors

The vacuole occupies 25-95% of the plant cell volume and plays an essential role in maintaining cytoplasmic homeostasis of nutrients and ions. Recent patch-clamp studies identified ion channels and electrogenic pumps as pathways for the movement of ions and metabolites across the vacuolar membrane (tonoplast). At high cytoplasmic Ca²⁺ (>10^-6 M) and negative potentials (inside the vacuole) non-selective channels of the `slow-vacuolar (SV)-type' were activated resulting in anion release or cation influx. In the present study these vacuolar channels were characterized pharmacologically by ion channel inhibitors. The cation-transport inhibitors Ba<sup>2+</sup>, TEA<sup>+</sup> and amiloride caused only partial and reversible block of the `SV-type'channels, whereas anion-transport inhibitors strongly affected the vacuolar channels. Pyridoxalphosphate and the dimethylaminecarboxylate derivates anthracene-9-carboxylic acid and C 144 reversibly blocked the channels up to 70% and Zncl₂ up to 95%. DIDS and SITS inhibited this channel irreversibly up to 95%. The block developed under a variety of experimental conditions using solutions containing combinations of permanent cations and anions. The DIDS binding site is located on the cytoplasmic surface of the tonoplast, as intravacuolar DIDS did not block the channels. DIDS concentrations in the micromolar range, efficient in blocking 70—80% of the `SV-type' channels did not significantly affect ATP-induced or pyrophosphate-induced proton-pumps. Stilbene derivatives may therefore be useful tools for studies on the substrate binding site on this vacuolar channel and for channel isolation.     

Chloride reabsorption by renal proximal tubules of necturus

Summary Movement of Cl from the lumen ofNecturus proximal tubule into the cells is mediated and dependent on the presence of luminal Na. Intracellular Cl activity was monitored with ion selective microelectrodes. In Cl Ringer's perfused kidneys, cell Cl activity was 24.5±1.1mm, 2 to 3 times higher than that predicted for passive distribution. When luminal NaCl was partially replaced by mannitol (capillaries perfused with Cl Ringer's) cell Cl decreased showing a sigmoidal dependence on luminal NaCl. Peritubular membrane potential was unaltered. Sulfate Ringer's perfusion of the kidneys washed out all cell Cl but did not alter peritubular membrane potential. Chloride did not enter the cell when the tubule lumen was perfused with 100mm KCl, LiCl, or tetramethylammonium Cl. Luminal perfusion of NaCl caused cell Cl to rise rapidly to the same value as the controls in the Cl Ringer's experiments. Perfusion of the tubule lumen with mixtures of NaCl and Na₂SO₄, while the capillaries contained sulfate Ringer's yielded a sigmoidal dependence of cell Cl on luminal NaCl activity. Chloride movement from the lumen into the proximal tubule cells required approximately equal concentrations of Na and Cl. Current clamp experiments indicated that intracellular chloride activity was insensitive to alterations in liminal membrane potential, suggesting that chloride entry was electrically neutral. The transcellular chloride flux was calculated to constitute about one half of the normal chloride reabsorption rate. We conclude that the cell Cl activity is primarily determined by the NaCl concentration in the tubule lumen and that Cl entry across the luminal membrane is mediated.     

Cell physiological aspects of the plasma membrane electrogenic H<Superscript>+</Superscript> pump

This article deals with cell physiological aspects of the plasma membrane electrogenic proton (H⁺) pump and emphasizes the contribution of the giant algal cells of the Characeae in elucidating the mechanism of the pump. First, a history of the development of intracellular perfusion techniques in characean internodal cells is described, including preparation of tonoplast-free cells. Then, an outline of the hypothesis of the electrogenic H⁺ pump proposed by Kitasato is introduced, who prophesied the existence of an electric potential generated by an active H⁺ efflux. Subsequently, a history of finding ATP as the direct energy source of the electrogenic ion pump is presented. Quantitative agreement between the pump current and the ATP-dependent H⁺ efflux supports the notion that the ion carried by the electrogenic ion pump is H⁺. The role of the H⁺ pump in regulation of the cytosolic pH is discussed. Mechanisms of light-induced potential change through photosynthesis-controlled activation of the H⁺ pump are discussed in terms of changes in the levels of adenine nucleotides and in modulation of the K_m value for the ATP of H⁺-ATPase. Recent progress in the molecular mechanism of the blue-light-induced activation of the H⁺-ATPase in guard cells is presented. However, there are cases where H⁺-ATPase activity is inhibited by blue light, indicating the flexibility of the control mechanisms of H⁺-ATPase activity. Finally, modulation of H⁺-pumping or H⁺-ATPase activities in response to environmental factors, such as anoxia, membrane excitation, osmotic and salt stresses, nutrient deficiencies and aluminum toxicity are described. Discussions are presented on the regulation of the electrogenic H⁺ pump.     

Effect of chaotropic anions on the sodium transport by the Na,K-ATPase

The effect of choline iodide, bromide and chloride on the kinetics of the electrogenic sodium transport by the Na,K-ATPase was investigated in a model system of ATPase-containing membrane fragments adsorbed on the lipid bilayer membrane. The kinetic parameters of Na⁺ transport were determined from short circuit currents after fast release of ATP from its caged precursor. The falling phase of the current transients could be fitted by a single exponential with the time constant, τ ₂. Its temperature dependence allowed an estimation of the activation energy of the rate-limiting reaction step, the conformation transition E₁/E₂. Choline iodide and bromide caused a decrease of the activation energy as well as the overall rate of the process expressed as the pre-exponential factor A of the Arrhenius equation. If choline iodide or bromide were present on the cytoplasmic and extracellular sides of the protein, the temperature dependent changes were more pronounced than when present on the cytoplasmic side only. These results can be explained by an effect of the anions on water structure on the extracellular surface of the protein, where a deep access channel connects the ion-binding sites with the solution. Chloride ions also caused a deceleration of the electrogenic transport, however, in contrast to iodide or bromide, they did not affect the activation energy, and were more effective when added on the cytoplasmic side. This effect can be explained by asymmetric screening of the negative surface charges which leads to a transmembrane electric potential that modifies the ion transfer.     

Effects of carbon dioxide on the spatially separate electrogenic ion pumps and the growth rate in the hypocotyl ofVigna sesquipedalis

Carbon dioxide, introduced into the gas phase of the experimental chamber, has distinct effects on two spatially separate membrane potentials and the rate of elongation growth in hypocotyl segments ofVigna sesquipedalis Wight. Both membrane potentials (V _ps andV _px=the electric potential difference between the parenchyma symplast and the surface of the hypocotyl, and that between the parenchyma symplast and the xylem, respectively) hyperpolarized rapidly but transiently at the introduction of CO₂. Prolonged exposure of the hypocotyl to high concentrations of CO₂ (above 10%) caused depolarization of membrane potentials above the level before CO₂ introduction. When CO₂ was replaced with air, the membrane potentials exhibited a distinct depolarization response of transient nature. The growth rate of the hypocotyl segments exhibited similar responses to CO₂ as did the membrane potentials (the increase and the decrease of the growth rate were corresponded to the hyperpolarization and the depolarization, respectively), but these responses always followed the changes of the membrane potentials. The CO₂-induced maximum hyperpolarization ofV _ps and the maximum increase of the growth rate were closely correlated. All these responses were strictly dependent on aerobic metabolism. These results indicate that CO₂ may regulate elongation growth in two ways: by affecting the activity of the electrogenic ion pump via intracellular acidification, and also by acting via apoplastic acidification as a wall-loosening acid.Symbols and abbreviations V _sx electric potential difference between the surface (S) and the xylem (X) of the hypocotyl - V _px electric potential difference between the inside of a parenchyma cell (P) andX - V _ps electric potential difference betweenP andS - V _ps (CO₂, max) the maximum value of CO₂-induced hyperpolarization ofV _ps - GR(CO₂, max) the maximum value of CO₂-induced increase of the growth rate - IAA indole-3-acetic acid     

Role of Cl− in Electrogenic H+ Secretion by Cortical Distal Tubule

The presence of an electrogenic H⁺-ATPase has been described in the late distal tubule, a segment which contains intercalated cells. The present paper studies the electrogenicity of this transport mechanism, which has been demonstrated in turtle bladder and in cortical collecting duct. Transepithelial PD (V _t ) was measured by means of Ling-Gerard microelectrodes in late distal tubule of rat renal cortex during in vivo microperfusion. The tubules were perfused with electrolyte solutions to which 2 × 10⁻⁷ m bafilomycin or 4.6 × 10⁻⁸ m concanamycin were added. No significant increase in lumen-negative V _t upon perfusion with these inhibitors as compared to control, was observed as well as when 10⁻³ m amiloride, 10⁻⁵ m benzamil or 3 mm Ba²⁺ were perfused alone or in combination. The effect of an inhibition of electrogenic H⁺ secretion, i.e., increase in lumen-negative V _t by 2–4 mV, was observed only when Cl⁻ channels were blocked by 10⁻⁵ m 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). This blocker also reduced the rate of bicarbonate reabsorption in this segment from 1.21 ± 0.14 (n= 8) to 0.62 ± 0.03 (8) nmol.cm⁻².sec⁻¹ as determined by stationary microperfusion and pH measurement by ion-exchange resin microelectrodes. These results indicate that: (i) the participation of the vacuolar H⁺ ATPase in the establishment of cortical late distal tubule V _t is minor in physiological conditions, but can be demonstrated after blocking Cl⁻ channels, thus suggesting a shunting effect of this anion; and, (ii) the rate of H⁺ secretion in this segment is reduced by a Cl⁻ channel blocker, supporting coupling of H⁺-ATPase with Cl⁻ transport. Received: 6 July 1996/Revised: 27 December 1996     

Energy coupling for membrane hyperpolarization in Lemna: respiration rate,ATP level and membrane potential at low oxygen concentrations

Respiration rate, ATP content and membrane potential of Lemna have been measured as a function of the concentration of dissolved oxygen. Kinetic analysis showed that within the range from 1 μM to 20 μM O₂, the respiration rate of isolated mitochondria and intact plants was a hyperbolic function of the oxygen concentration. The apparent Michaelis constant (K _m ) for the oxygen of respiration of intact plants (1.15±0.08 μM) is close to that for isolated mitochondria (1.07±0.06 μM), so that diffusion of oxygen within the tissue was obviously not rate-limiting under the applied experimental conditions. The ATP level decreased in parallel with the respiration rate when the oxygen concentration was reduced. In contrast, the hyperpolarization of the membrane potential above the diffusion potential had already decreased at oxygen concentrations where the respiration rate and ATP level remained practically unchanged and was completely abolished at oxygen concentrations above the K _m of respiration. This result is discussed according to the current models for electrogenic pumps. It is concluded that ATP cannot be the fuel for the electrogenic process under investigation.     

Microelectrode characterization of the basolateral membrane of rabbit S3 proximal tubule

Summary The purpose of this study was to characterize the basolateral membrane of the S3 segment of the rabbit proximal tubule using conventional and ion-selective microelectrodes. When compared with results from S1 and S2 segments, S3 cells under control conditions have a more negative basolateral membrane potential (V _bl=–69 mV), a higher relative potassium conductance (t _K=0.6), lower intracellular Na⁺ activity (A _Na=18.4mm), and higher intracellular K⁺ activity (A _K=67.8mm). No evidence for a conductive sodium-dependent or sodium-independent HCO ₃ ^– pathway could be demonstrated. The basolateral Na–K pump is inhibited by 10^–4 m ouabain and bath perfusion with a potassium-free (0-K) solution. 0-K perfusion results inA _Na=64.8mm,A _K=18.5mm, andV _bl=–28 mV. Basolateral potassium channels are blocked by barium and by acidification of the bathing medium. The relative K⁺ conductance, as evaluated by increasing bath K⁺ to 17mm, is dependent upon the restingV _bl in both S2 and S3 cells. In summary, the basolateral membrane of S3 cells contains a pump-leak system with similar properties to S1 and S2 proximal tubule cells. The absence of conductive bicarbonate pathways results in a hyperpolarized cell and larger Na⁺ and K⁺ gradients across the cell borders, which will influence the transport properties and intracellular ion activities in this tubule segment.     

Influence of Ca on the plasma membrane potential and electrogenic uptake of glycine by myeloma cells. Involvement of a Ca-activated K channel

The involvement of Ca²⁺-activated K⁺ channels in the regulation of the plasma membrane potential and electrogenic uptake of glycine in SP 2/0-AG14 lymphocytes was investigated using the potentiometric indicator 3,3′-diethylthiodicarbocyanine iodide. The resting membrane potential was estimated to be −57 ± 6 mV (n = 4), a value similar to that of normal lymphocytes. The magnitude of the membrane potential and the electrogenic uptake of glycine were dependent on the extracellular K⁺ concentration, [K⁺]_o, and were significantly enhanced by exogenous calcium. The apparent V_max of Na⁺-dependent glycine uptake was doubled in the presence of calcium, whereas the K_0.5 was not affected. Ouabain had no influence on the membrane potential under the conditions employed. Additional criteria used to demonstrate the presence of Ca²⁺-activated K⁺ channels included the following: (1) addition of EGTA to calcium supplemented cells elicited a rapid depolarization of the membrane potential that was dependent on [K⁺]_o; (2) the calmodulin antagonist, trifluoperazine, depolarized the membrane potential in a dose-dependent and saturable manner with an IC₅₀ of 9.4 μM; and (3) cells treated with the Ca²⁺-activated K⁺ channel antagonist, quinine, demonstrated an elevated membrane potential and depressed electrogenic glycine uptake. Results from the present study provide evidence for Ca²⁺-activated K⁺ channels in SP 2/0-AG14 lymphocytes, and that their involvement regulates the plasma membrane potential and thereby the electrogenic uptake of Na⁺-dependent amino acids.     

Effects of host-specific toxins on electropotentials of plant cells

Host-specific toxins from Helminthosporium victoriae (HV) and Periconia circinata (PC) caused gradual decreases in the negative electropotentials of single cells of susceptible but not of resistant plants. When tissues were held in a standard nutrient solution, the decrease (depolarization) induced by HV toxin was approximately 50 mv/hr; the decrease induced by PC toxin was even more gradual. Changes in ion efflux were detected before changes in electropotential. In contrast, toxin from H. carbonum caused a rapid but transient increase in negative electropotential of cells. Carbonyl cyanide m-chlorophenylhydrazone, which (like other metabolic inhibitors) blocks electrogenic pumps, caused cell electropotentials to decrease by approximately 50 mv within a few minutes. This suggests that HV and PC toxins do not have direct effects on electrogenic pumps, but do affect passive efflux of ions, or electrically neutral ion exchange systems, across the plasma membrane.     

Adenine-nucleotide levels and metabolism-dependent membrane potential in cells of Nitellopsis obtusa Groves

The relationship between adenine-nucleotide levels and metabolism-dependent membrane potential was studied in cells of Nitellopsis obtusa. Effects of ADP and AMP in the presence of ATP on electrogenic pump activity were measured in the dark, using the continuous perfusion method. Both ADP and AMP acte as competitive inhibitors for ATP, the K_i value for either compound being about 0.4 mM. The role of ADP and AMP as regulating factors for the electrogenic pump was investigated under various metabolic conditions. Application of N₂ gas in the dark caused a significant membrane depolarization amounting to 90 mV, but cytoplasmic streaming and membrane excitability were not affected. Under anoxia, the ATP level decreased from 1.6 to 0.5 mM; ADP increased but only slightly, and AMP increased greatly. However, the time course of changes in the adenine nucleotides was not concurrent with that of the membrane-potential changes, thus, the adenine-nucleotide level changes cannot fully account for the N₂-elicited depolarization. Under light, although the membrane hyperpolarized, no significant changes in the adenine-nucleotide levels were observed. Therefore, the light-induced membrane hyperpolarization cannot be explained solely by changes in adenine-nucleotide levels.Abbreviations APW artificial pond water - E_m membrane potential - R_m membrane resistance