Action potentials and variation potentials in sunflower: An analysis of their relationships and distinguishing characteristics

Sunflower plants ( Helianthus annuus L.) were given an electrical stimulus to the stem or a heat (flame)‐wound to a single leaf or a cotyledon. The resulting electrical activity was monitored with extracellular electrodes. An electrical stimulus applied to the stem frequently evoked an action potential (AP), but never a variation potential (VP). In contrast, a heat‐wound applied to a leaf virtually always elicited a VP, which was often accompanied by one or more superimposed spikes (putative APs). The kinetic parameters of the AP and the VP were investigated. The AP appears to propagate without decrement in velocity or magnitude, whereas the VP parameters decrease significantly with distance. The heat stimulus triggered rapid alterations in stem elongation/contraction, which preceded changes in electrical potential, indicating the transmission of a hydraulic signal. Light‐off and light‐on stimuli evoked negative‐ and positive‐going changes in extracellular electrical potential, respectively, corresponding to de‐ and hyper‐polarization of the plasma membrane. Membrane depolarization (extracellularly manifested as a VP) evoked by both the light‐off and heat‐wounding stimuli was able to trigger one or more APs. We interpret these results to suggest that APs are "genuine" electrical signals involving voltage‐gated ion channels or pumps, which can be evoked directly by electrical stimulation or indirectly by changes in membrane potential occurring during the VP or after the light‐off stimulus. In contrast, VPs appear to be a local (non‐transmissible) electrical consequence of the passage of a rapidly transmitted hydraulic signal in the xylem, presumably acting on mechanosensitive ion channels or pumps in adjacent living cells.     

Time-Resolved Analysis of Signals Involved in Systemic Induction of Pin2 Gene Expression

The systemic induction of proteinase inhibitor genes in tomato plants is mediated either by electrical signals, hydraulic signals or chemical messengers. In the present study we analyzed the effects of mechanical wounding, heat treatment and electrical current application on wild-type tomato plants (Lycopersicon esculentum Mill, cv Moneymaker) and ABA-deficient mutants of tomato (sitiens). Kinetic studies revealed that systemic Pin2 gene expression could be slightly induced by the fast transient membrane potential change which left the damaged leaf within 30–60s after wounding. Moreover, a signal leaving the damaged tissue between 2 and 4 minutes after wounding was responsible for a significant amplification of Pin2 gene expression. This signal could either be a decrease in turgor pressure, which occurred 3–4min after treatment, or a slow electrical transient. In addition, mechanical wounding and electrical current seem to involve ABA to induce changes in membrane potential and to promote Pin2 gene expression. In contrast, heat triggers fast and slow electrical transients leading to an induction of Pin2 gene expression within the plant independently of ABA. Turgor pressure, in turn, is presumably adjusted in relation to ionic movements across the membrane, elucidated by membrane potential recordings. In conclusion, wound-induced changes in membrane potential seem to be dependent on the endogenous level of ABA. These shifts in membrane potentials, in turn, are involved in regulation of turgor pressure within the plant.     

Surface potentials and hydraulic signals in wheat leaves following localized wounding by heat

Abstract. Localized burning of a leaf causes a rapid change in apoplastic electrical potential throughout the shoot of wheat seedlings ('variation potential'). It also causes marked increases in turgor pressure in epidermal cells of adjoining leaves. These turgor increases indicate rapid propagation throughout the seedling, of a hydraulic pressure wave from the site of wounding. Evidence is presented that this pressure wave is caused by relief of xylem tension, by water released from damaged cells in the wounded region. It is demonstrated that, in the absence of wounding, pressure waves imposed at the tip of one leaf can travel to neighbouring leaves, and can there induce change in apoplastic electrical potential similar to a 'variation potential'. This indicates that the hydraulic event produced by wounding is the signal responsible for systemic induction of the 'variation potential'. This signal has been termed 'Ricca's factor'. It is suggested that arrival of the hydraulic wave alters leaf water potential and thereby induces stomatal activity. Leaf surface potential may be dominated by electrogenic ion pumping or flux at stomatal cells, and the 'variation potential' may therefore be a reflection of stomatal activity induced by the hydraulic signal.     

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.      

Simultaneous recording of xylem pressure and trans-root potential in roots of intact glycophytes using a novel xylem pressure probe technique

The radial electrical potential difference between the root xylem and the bathing solution, i.e. the so-called trans-root potential, was measured in intact maize and wheat plants using a xylem pressure probe into which an Ag/AgCl electrode was incorporated. Besides other advantages (e.g. detection and removal of tip clogging; determination of the radial root resistance), the novel probe allowed placement of the electrode precisely in a single xylem vessel as indicated by the reading of sub-atmospheric or negative pressure values upon penetration. The trans-root potentials were of the order of 0 to – 70 mV and + 40 to – 20 mV for 2- to 3-week-old maize and wheat plants, respectively. Osmotic experiments performed on maize demonstrated that addition of 100 mM mannitol to the solution resulted in a decrease of xylem pressure associated with a slow, but continuous depolarization. The depolarization was reversible upon removal of the mannitol. For wheat plants it could be shown that the oscillations of the xylem pressure described recently by Schneider et al. (1997, Plant, Cell and Environment 20, 221–229) were accompanied by (rectangular, saw-tooth and/or U-shaped) oscillations in the trans-root potential (but not by corresponding changes of the membrane potential of the cortical cells measured simultaneously with conventional microelectrodes). Increase of the light intensity (up to 550 μmol m^–2 s^–1) resulted in a drop of the xylem pressure in wheat, whereas the trans-root potential showed a biphasic response: first hyperpolarization (by about 10 mV) was observed, followed by depolarization (by up to about + 40 mV). Similar light-induced biphasic (but often less pronounced) changes in the trans-root potential were also recorded for maize plants. Most interestingly, the response of the trans-root potential was always faster (by about 1–3 min) than the response of the xylem pressure upon illumination, suggesting that changes in the transpiration rate are reflected very quickly in the electrical properties of the root tissue. The impact of this and other findings on long-distance transport of solutes and water as well as on long-distance signalling is discussed.     

Surface dynamics of voltage-gated ion channels

Neurons encode information in fast changes of the membrane potential, and thus electrical membrane properties are critically important for the integration and processing of synaptic inputs by a neuron. These electrical properties are largely determined by ion channels embedded in the membrane. The distribution of most ion channels in the membrane is not spatially uniform: they undergo activity-driven changes in the range of minutes to days. Even in the range of milliseconds, the composition and topology of ion channels are not static but engage in highly dynamic processes including stochastic or activity-dependent transient association of the pore-forming and auxiliary subunits, lateral diffusion, as well as clustering of different channels. In this review we briefly discuss the potential impact of mobile sodium, calcium and potassium ion channels and the functional significance of this for individual neurons and neuronal networks.     

The cyclic nucleotide gated channel of olfactory receptor neurons

Olfactory transduction proceeds through a G-protein coupled cascade that produces the ubiquitous second messenger cyclic AMP. The cyclic AMP causes a chance in membrane potential by acting directly on an ion channel that allows cations to flow into the cell. This ion channel is one of a new family of ion channels that are activated by intracellular cyclic nucleotides. However, even though they are activated by binding a ligand their amino acid structure shows that they share a common ancestry with voltage activated channels, especially voltage dependent Ca²⁺ channels. In olfactory neurons these channels perform a critical role in the transduction of chemical information in the environment into changes in membrane electrical properties that are transmitted to higher order processing centers in the brain.     

Age-dependent changes of photosynthetic responses induced by electrical signals in wheat seedlings

Electrical signals in plants, namely, the action potential (AP) and variation potential (VP) alter the activity of many processes, including photosynthesis. The functional responses induced by electrical signals vary in direction and amplitude, which might be determined by variable conditions of plants prior to stimulation, by the development stage in particular. In this work, the parameters of VP-induced photosynthetic responses were analyzed at various stages of wheat seedling development. Local wounding of the second leaf in wheat plants induced the propagation of VP and altered the activity of photosynthesis at a distance from the wound location. The amplitude of VP was enlarged when the seedling age increased from 11 to 18 days. The VP-induced photosynthetic response changed with age both qualitatively and quantitatively. The amplitude of VP-induced changes in CO₂ assimilation and nonphotochemical quenching (NPQ) increased with age, which might be due to the increase in VP amplitude and associated changes in Ca²⁺ and H⁺ concentrations. The quantum yield of photosystem II photoreaction was subject to age-dependent changes: the photochemical quantum yield (γ(PSII)) was found to increase after VP in young leaves, whereas the decline in γ(PSII) was observed after the VP propagation in mature leaves. The results may explain the diversity of photosynthetic responses caused by the electrical signals.     

Bicarbonate-induced alkalinization of the xylem sap in intact maize seedlings as measured in situ with a novel xylem pH probe

In higher plants the pH of the xylem sap plays an important role in drought signaling, growth regulation, and plant nutrition. However, the interpretation of the data is very controversial. The main reason for this is that the xylem pH in intact plants was not directly accessible hitherto. We present here a novel, minimally-invasive probe based on the xylem pressure-potential probe (used for measuring directly xylem pressure and the electrical potential between root xylem sap and medium). Single-tipped, double-barreled capillaries were used, one barrel served as H(+)-selective electrode, whereas pressure and electrical potential were recorded by the other one. Upon insertion of the probe into the root xylem of maize (Zea mays) seedlings, pH values ranging between about 4.2 and 4.9 were monitored when the roots were immersed in standard nutrient solution. The pH did not respond to changes in light irradiation (up to 300 micromol m(-2) s(-1)), but increased upon exposure of the root to 5 or 20 mm bicarbonate in the bath solution. Changes in pH could also be recorded in transpiring plants when the root was cut below the insertion point of the probe and placed in media with different pH. The data support the hypothesis of Mengel ([1994] Plant Soil 165: 275-283) that upon external supply with bicarbonate Fe is immobilized in the leaf apoplast via changes in xylem pH.     

Stomatal conductance and xylem sap properties of aspen (Populus tremuloides) in response to low soil temperature

The mechanisms regulating stomatal response following exposure to low (5°C) soil temperature were investigated in aspen ( Populus tremuloides Michx.) seedlings. Low soil temperature reduced stomatal conductance within 4 h, but did not alter shoot xylem pressure potential within 24 h. The xylem sap composition was altered and its pH increased from 6.5 to 7.1 within the initial 4 h of the low temperature treatment. However, the increase in abscisic acid (ABA) concentration in xylem sap was observed later, after 8 h of treatment. These changes were accompanied by a reduction in the electrical conductivity and an increase in the osmotic potential of the xylem sap. The timing of physiological responses to low soil temperature suggests that the rapid pH change of the xylem sap and accompanying changes in ion concentration were the initial factors which triggered stomatal closure in low temperature-treated seedlings, and that the role of the more slowly accumulating ABA was likely to reinforce the stomatal closure. When leaf discs were exposed to xylem sap extracted from low soil temperature-treated plants, stomatal aperture was negatively correlated with ABA and positively correlated with K⁺ concentrations of the xylem sap. The stomatal opening in the leaf discs linearly increased in response to exogenous KCl concentrations when K⁺ concentrations were in the similar range to those detected in the xylem sap. The lowest concentration of exogenous ABA to induce stomatal closure was several-fold higher compared with the concentration present in the xylem sap.     

Induction and ionic basis of slow wave potentials in seedlings of Pisum sativum L.

Slow wave potentials (SWPs) are transient depolarizations which propagate substantial distances from their point of origin. They were induced in the epidermal cells of pea epicotyls by injurious methods such as root excision and heat treatment, as well as by externally applied defined steps in xylem pressure (Px) in the absence of wounding. The common principle of induction was a rapid increase in Px. Such a stimulus appeared under natural conditions after (i) bending of the epicotyl, (ii) wounding of the epidermis, (iii) rewatering of dehydrated roots, and (iv) embolism. The induced depolarization was not associated with a change in cell input resistance. This result and the ineffectiveness of ion channel blockers point to H(+)-pumps rather than ion channels as the ionic basis of the SWP. Stimuli such as excision, heat treatment and pressure steps, which generate SWPs, caused a transient increase in the fluorescence intensity of epicotyls loaded with the pH-indicator DM-NERF, a 2',7'-dimethyl derivative of rhodol, but not of those loaded with the pH indicator 2',7'bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Matching kinetics of depolarization and pH response identify a transient inactivation of proton pumps in the plasma membrane as the causal mechanism of the SWP. Feeding pump inhibitors to the cut surface of excised epicotyls failed to chemically simulate a SWP; cyanide, azide and 2,4-dinitrophenol caused sustained, local depolarizations which did not propagate. Of all tested substances, only sodium cholate caused a transient and propagating depolarization whose arrival in the growing region of the epicotyl coincided with a transient growth rate reduction.     

Ion Channels in the Xylem Parenchyma of Barley Roots (A Procedure to Isolate Protoplasts from This Tissue and a Patch-Clamp Exploration of Salt Passageways into Xylem Vessels

To identify mechanisms for the simultaneous release of anions and cations into the xylem sap in roots, we investigated voltage-dependent ion conductances in the plasmalemma of xylem parenchyma cells. We applied the patch-clamp technique to protoplasts isolated from the xylem parenchyma by differential enzymic digestion of steles of barley roots (Hordeum vulgare L. cv Apex). In the whole-cell configuration, three types of cation-selective rectifiers could be identified: (a) one activated at membrane potentials above about -50 mV; (b) a second type of outward current appeared at membrane potentials above +20 to +40 mV; (c) below a membrane potential of approximately -110 mV, an inward rectifier could be distinguished. In addition, an anion-specific conductance manifested itself in single-channel activity in a voltage range extending from about -100 to +30 mV, with remarkably slow gating. In excised patches, K+ channels activated at hyperpolarization as well as at depolarization. We suggest that salt is released from the xylem parenchyma into the xylem apoplast by simultaneous flow of cations and anions through channels, following electrochemical gradients set up by the ion uptake processes in the cortex and, possibly, the release and reabsorption of ions on their way to the xylem.     

Fluorescence imaging of electrically stimulated cells

Designing high-throughput screens for voltage-gated ion channels has been a tremendous challenge for the pharmaceutical industry because channel activity is dependent on the transmembrane voltage gradient, a stimulus unlike ligand binding to G-protein-coupled receptors or ligand-gated ion channels. To achieve an acceptable throughput, assays to screen for voltage-gated ion channel modulators that are employed today rely on pharmacological intervention to activate these channels. These interventions can introduce artifacts. Ideally, a high-throughput screen should not compromise physiological relevance. Hence, a more appropriate method would activate voltage-gated ion channels by altering plasma membrane potential directly, via electrical stimulation, while simultaneously recording the operation of the channel in populations of cells. The authors present preliminary results obtained from a device that is designed to supply precise and reproducible electrical stimuli to populations of cells. Changes in voltage-gated ion channel activity were monitored using a digital fluorescent microscope. The prototype electric field stimulation (EFS) device provided real-time analysis of cellular responsiveness to physiological and pharmacological stimuli. Voltage stimuli applied to SK-N-SH neuroblastoma cells cultured on the EFS device evoked membrane potential changes that were dependent on activation of voltage-gated sodium channels. Data obtained using digital fluorescence microscopy suggests suitability of this system for HTS.     

Electrical correlates of secretion in endocrine and exocrine cells

Many types of secretory cells including neurons and cells of endocrine and exocrine glands show changes in electrical potential and resistance when secretion is stimulated. These electrical correlates result from the movement of ions across the cell membrane through specific ion-selective channels. In neurons and certain endocrine cells (such as pancreatic beta cells and certain cells of the anterior pituitary), these channels are voltage dependent and open transiently upon depolarization leading to action potentials. Thus some endocrine cells are electrically excitable, a property previously held to occur only in nerve and muscle. In other nonexcitable endocrine and exocrine cells (such as the pancreas and parotid), ion channels are responsive to either occupancy of specific membrane receptors or changes in intracellular metabolites and second messengers. Ion fluxes through these latter channels also lead to changes in the electrical potential and resistance, but these changes are generally more sustained and action potentials are not seen. The entry of Ca2+ through both voltage-dependent and voltage-independent ion channels plays a major role in the activation of secretion via exocytosis.     

Electrical signals in higher plants: Mechanisms of generation and propagation

Local stimulation induces generation and propagation of electric signals in higher plants. Noninvasive stimulus induces an action potential and damaging influences lead to the variation potential. The mechanism of the generation of an action potential is rather complex in nature and is associated with both activation of ion channels (Ca²⁺, Cl^–, and K⁺) and transient change in the activity of the plasma membrane H⁺-ATPase. Generation of the variation potential, the duration of which is considerably longer than that of the action potential, is based on transient inactivation of the electrogenic pump; however, passive ion fluxes also contribute to such process, which causes qualitative similarity of the mechanisms of action potential and variation potential generation. Propagation of electrical signals mainly occurs in conducting bundles; thus, transfer of an action potential is associated with vascular parenchyma and sieve elements, while the variation potential is connected to the xylem vessels. The mechanism of the distribution the action potential is similar to nerve impulse transmission, while generation of the variation potential is induced by transfer of a chemical substance, whose propagation is accelerated by a hydraulic wave.     

Electrical potential oscillations – movement relations in circumnutating sunflower stem and effect of ion channel and proton pump inhibitors on circumnutation

The physiological control and molecular mechanism of circumnutation (CN) has not yet been fully understood. To gain information on the CN mechanism, the relationship between the changes of electrical potential and movement in the circumnutating sunflower stem and effect of ion channels and proton pump inhibitors on CN parameters were evaluated. Long‐term electrophysiological measurements and injection of solutions of ion channel inhibitors (ICI) into sunflower stem with the simultaneous time‐lapse recording of the movement were made. The oscillations of electrical potential (OEP) – movement relations – consist of cells depolarization on the deflected side of the stem and, at this same time, cells hyperpolarization on the opposite side of the stem. The delay of organ movement in relation to electrical changes of approximately 28 min (22% of the period) may indicate that the ionic fluxes causing the OEP are the primary phenomenon. The biggest decrease of CN period was observed after injection of proton pump (approximately 26%) and cation channel (approximately 25%) inhibitors, while length and amplitude were reduced mainly by calcium channel inhibitors (approximately 67%). Existence of OEP only in circumnutating part of sunflower stem and reduction of CN parameters and OEP amplitude after application of ICI prove that the CN cellular mechanism is associated with transmembrane ion transport.     

Control of Sodium Transport in Sunflower Roots

Electrochemical potential differences (driving forces) for sodiumdistributed between the outside solution and the exuding sapof water-culture-grown sunflower plants (Helianthus annuius)have been determined. The results indicated that sodium wasmoving from the outside solution to the xylem against the electrochemicalpotential gradient at external concentrations below approximately0.30 mM Na. At higher external concentrations sodium appearedto be actively excluded from the xylem. An electrical potential difference between the exuding sap andthe external solution of approximately 30 mV was observed. Itwas unaffected by the external sodium concentration. Use ofa short-circuiting technique indicated that the trans-root potentialresides at the plasmalemma of the cortical cells. Driving forces on sodium distributed between the external solutionand the root and between the xylem sap and the root were calculated.They indicated that the root is able to accumulate sodium activelyboth from the external solution and the xylem sap. It is concludedthat sodium transport to the xylem in this species is controlledby the balance of these two opposing forces.