Transport of [2-14C]jasmonic acid from leaves to roots mimics wound-induced changes in endogenous jasmonic acid pools in Nicotiana sylvestris

Jasmonic acid (JA) is part of a long-distance signal-transduction pathway that effects increases in de-novo nicotine synthesis in the roots of Nicotiana sylvestris Speg et Comes (Solanaceae) after leaf wounding. Elevated nicotine synthesis increases whole-plant nicotine pools and makes plants more resistant to herbivores. Leaf wounding rapidly increases JA pools in damaged leaves, and after a 90-min delay, root JA pools also increase. The systemic response in the roots could result from either: (i) the direct transport of JA from wounded leaves, or (ii) JA synthesis or its release from conjugates in roots in response to a second, systemic signal. We synthesized [2-¹⁴C]JA, and applied it to a single leaf in a quantity (189 μg) known to elicit both a whole-plant nicotine and root JA response equivalent to that found in plants subjected to leaf wounding. We quantified radioactive material in JA, and in metabolites both more and less polar than JA, from treated and untreated leaves and roots of plants in eight harvests after JA application. [2-¹⁴C]Jasmonic acid was transported from treated leaves to roots at rates and in quantities equivalent to the wound-induced changes in endogenous JA pools. The [2-¹⁴C]JA that had been transported to the roots declined at the same rate as endogenous JA pools in the roots of plants after leaf wounding. Most of the labeled material applied to leaves was metabolized or otherwise immobilized at the application site, and the levels of [2-¹⁴C]JA in untreated leaves did not increase over time. We measured the free JA pools before and after four different hydrolytic extractions of root and shoot tissues to estimate the size of the potential JA conjugate pools, and found them to be 10% or less of the free JA pool. We conclude that the direct transport of wound-induced JA from leaves to roots can account for the systemic increase in root JA pools after leaf wounding, and that metabolism into less polar structures determines the duration of this systemic increase. However, the conclusive falsification of this hypothesis will require the suppression of all other signalling pathways which could have shoot-to-root transport kinetics similar to that of endogenous JA. Received: 14 April 1997 / Accepted: 9 June 1997     

Growth, membrane potential and endogenous ion currents of willow (Salix viminalis) roots are all affected by abscisic acid and spermine

Growth measurements of hormone-treated roots from willow cuttings were combined with electrophysiological recordings to study hormone-induced changes in membrane potential and in endogenous ion currents. The mean growth rate of roots was 10 ± 2 μm min^?1 in regular nutrient solution. It increased to 13 ± 2 μm min⁺¹ after application of spermine and decreased to 0.07 ± 0.01 μm min^?1 after treatment with abscisic acid (ABA). Transient depolarizations were elicited in root cortex cells by spermine, while ABA caused a transient hyperpolarization. All changes in membrane potential were accompanied by transient responses of the endogenous current. These responses suggest that first anions, then cations leave the root during spermine-induced depolarizations. From the changes of the endogenous current an apparent efflux of anions (presumably Cl^?) and cations (presumably K⁺) of 200 to 700 pmol cm^?2 per depolarization was calculated. To further investigate a possible relation between endogenous ion currents, growth and the growth regulators ABA and spermine, long-lasting extracellular vibrating-probe measurements were performed. Control roots showed an inward current of about 1.5 μA cm^?2 at the apical elongation zone and an outward current with a maximum density of 1.3 μA cm^?2 at the central and basal elongation zone. The addition of ABA and spermine (final concentration 0.1 mM) to the bathing medium affected the endogenous current in opposite ways: ABA caused a reduction of inward and outward current, while spermine stimulated both. Since protons are a major component of the endogenous current, and sucrose can be taken up by root cells from the apoplast via symport with H⁺, a role of the endogenous current in growth regulation is indicated.     

Quantification, correlations and manipulations of wound-induced changes in jasmonic acid and nicotine in Nicotiana sylvestris

Jasmonic acid (JA) is thought to be part of a signal-transduction pathway which dramatically increases de-novo nicotine synthesis in the roots and increases whole-plant (WP) nicotine pools in response to the wounding of the leaves in Nicotiana sylvestrisSpegazzini and Comes (Solanaceae). We report the synthesis of a doubly labeled JA ([1, 2-¹³C]JA) and use it as an internal standard to quantify by gas chromatography-mass spectrometry the changes in root and shoot JA pools in plants subjected to differing amounts of standardized leaf wounding. Wounding increased JA pools 10-fold locally in damaged leaves within 90 min and systemically in the roots (3.5-fold) 180 min after wounding. If JA functions as an intermediary between stimulus and response, quantitative relationships among the stimulus, JA, and the response should exist. To examine these relationships, we varied the number of punctures in four leaves and quantified both the resulting JA in damaged leaves after 90 min and the resulting WP nicotine concentration after 5 d. We found statistically significant, positive relationships among number of leaf punctures, endogenous JA, and WP nicotine accumulation. We used two inhibitors of wound-induced nicotine production, methyl salicylate and indole-3-acetic acid, to manipulate the relationships between wound-induced changes in JA and WP nicotine accumulation. Since wounding and the response to wounding occur in widely separated tissues, we applied inhibitors to different plant parts to examine their effects on the local and systemic components of this response. In all experiments, inhibition of the wound-induced increase in leaf JA 90 min after wounding was associated with the inhibition of the nicotine response 5 d after wounding. We conclude that wound-induced increases in leaf JA are an important component of this long-distance signal-transduction pathway. Received: 24 April 1996 / Accepted: 18 July 1996     

Differential Expression of Two Soybean (Glycine max L.) Proline-Rich Protein Genes after Wounding

We have investigated the wound-induced expression of two members of the soybean (Glycine max L.) proline-rich cell wall protein gene family and show that SbPRP1 and SbPRP2 exhibit unique patterns of expression after physical damage. SbPRP1 mRNA can be detected in the hook of soybean seedlings within 2 h after wounding and is present at high levels in the hook and elongating hypocotyl 20 h after wounding. In contrast, SbPRP2 mRNA increases transiently and rapidly throughout the soybean seedling after wounding. SbPRP2 is also induced by wounding in soybean leaves, but the pattern of mRNA accumulation in leaves is distinct from that seen in seedlings and reaches high levels of expression 20 h after physical damage. SbPRP2 mRNA levels were also found to increase in the mature hypocotyl and roots of seedlings in response to treatment with 10 [mu]M indoleacetic acid and naphthalene-1-acetic acid. These data indicate that the wound-induced expression of PRPs in soybean is tissue specific and that the regulation of these genes after physical damage may operate through different signal transduction pathways.     

Utilization of the Vibrating Probe and Ion-Selective Microelectrode Techniques to Investigate Electrophysiological Responses to Wounding in Pea Roots

This paper examines the ionic composition of wound-induced electricalcurrents in higher plant tissue, using two non-injurious electrophysiologicaltechniques. By simultaneous recording of K⁺, H⁺ , and Ca²⁺ ionfluxes with extracellular ion-selective microelectrodes, wehave determined that a Ca²⁺ influx (2.4 µA cm^–2),a small H⁺ influx (0.17 µA cm^–2) and a large K⁺efflux (16 µA cm^–2) occur immediately after woundingin roots of Pisum sativum L. var. Greenfeast. Using an extracellularvibrating probe at the wound site, net ion currents of 26 µAcm^–2 were measured 5 min after wounding. In a more concentratedbathing medium (1/4 rather than 1/16 strength Hoagland's solution),net ion currents of 59 µA cm^–2 were measured, andthese would appear to be the largest extracellular currentsthat have been measured in plants. We made a quantitative comparisonof the summed ion fluxes with the net ion currents and thisrevealed that ion fluxes, in addition to those measured here,occur after wounding. Key words: Wounding, ion flux, electric current, calcium, potassium     

Rapid Uptake of Aluminum into Cells of Intact Soybean Root Tips (A Microanalytical Study Using Secondary Ion Mass Spectrometry)

A wide range of physiological disorders has been reported within the first few hours of exposing intact plant roots to moderate levels of Al3+. Past microanalytic studies, largely limited to electron probe x-ray microanalysis, have been unable to detect intracellular Al in this time frame. This has led to the suggestion that Al exerts its effect solely from extracellular or remote tissue sites. Here, freeze-dried cryosections (10 [mu]m thick) collected from the soybean (Glycine max) primary root tip (0.3-0.8 mm from the apex) were analyzed using secondary ion mass spectrometry (SIMS). The high sensitivity of SIMS for Al permitted the first direct evidence of early entry of Al into root cells. Al was found in cells of the root tip after a 30-min exposure of intact roots to 38 [mu]M Al3+. The accumulation of Al was greatest in the first 30 [mu]m, i.e. two to three cell layers, but elevated Al levels extended at least 150 [mu]m inward from the root edge. Intracellular Al concentrations at the root periphery were estimated to be about 70 nmol g-1 fresh weight. After 18 h of exposure, Al was evident throughout the root cross-section, although the rate of accumulation had slowed considerably from that during the initial 30 min. These results are consistent with the hypothesis that early effects of Al toxicity at the root apex, such as those on cell division, cell extension, or nutrient transport, involve the direct intervention of Al on cell function.     

Inward-Rectifying K+ Channels in Root Hairs of Wheat (A Mechanism for Aluminum-Sensitive Low-Affinity K+ Uptake and Membrane Potential Control)

K+ is the most abundant cation in cells of higher plants, and it plays vital roles in plant growth and development. Extensive studies on the kinetics of K+ uptake in roots have shown that K+ uptake is mediated by at least two transport mechanisms, one with a high and one with a low affinity for K+. However, the precise molecular mechanisms of K+ uptake from soils into root epidermal cells remain unknown. In the present study we have pursued the biophysical identification and characterization of mechanisms of K+ uptake into single root hairs of wheat (Triticum aestivum L.), since root hairs constitute an important site of nutrient uptake from the soil. These patch-clamp studies showed activation of a large inward current carried by K+ ions into root hairs at membrane potentials more negative than -75 mV. This K+ influx current was mediated by hyperpolarization-activated K+-selective ion channels, with a selectivity sequence for monovalent cations of K+ > Rb+ [almost equal to] NH4+ >> Na+ [almost equal to] Li+ > Cs+. Kinetic analysis of K+ channel currents yielded an apparent K+ equilibrium dissociation constant (Km) of [almost equal to]8.8 mM, which closely correlates to the major component of low-affinity K+ uptake. These channels did not inactivate during prolonged stimulation and would thus enable long-term K+ uptake driven by the plasma membrane proton-extruding pump. Aluminum, which is known to inhibit cation uptake at the root epidermis, blocked these inward-rectifying K+ channels with half-maximal current inhibition at [almost equal to]8 [mu]M free Al3+. Aluminum block of K+ channels at these Al3+ concentrations correlates closely to Al3+ phytotoxicity. It is concluded that inward-rectifying K+ channels in root hairs can function as both a physiologically important mechanism for low-affinity K+ uptake and as regulators of membrane potential. The identification of this mechanism is a major step toward a detailed molecular characterization of the multiple components involved in K+ uptake, transport, and membrane potential control in root epidermal cells.     

Wound-induced expression of a tobacco peroxidase is not enhanced by ethephon and suppressed by methyl jasmonate and coronatine

In tobacco plants, wounding induces production of a set of defense-related proteins such as basic pathogenesis-related (PR) proteins and proteinase inhibitors (PIs) via the jasmonate/ethylene pathway. Although class III plant peroxidase (POX) is also wound-inducible, the regulatory mechanism for its wound-induced expression is not fully understood. Here, we describe that a tobacco POX gene (tpoxN1), which is constitutively expressed in roots, is induced locally 30 min after wounding and then systemically in tobacco plants. Infection of necrotizing virus also induced tpoxN1 gene. The wound-induced expression was not enhanced by known wound-signal compounds such as methyl jasmonate (MeJA) and ethephon in contrast to other wound-inducible genes such as basic PR-1 and PI-II genes. And treatment with MeJA and coronatine, biological analogs of jasmonate, rather suppressed the tpoxN1 expression. Salicylic acid, an antagonist of jasmonate-based wound signaling, did not suppress the wound-induced expression of tpoxN1. Only spermine, which is reported as an endogenous inducer for acidic PR genes in tobacco mosaic virus-infected tobacco leaves, could induce tpoxN1 gene expression. These results suggest that wound-induced expression of the tpoxN1 gene is regulated differently from that of the basic PR and PI-II genes.     

Evidence for G-Protein Regulation of Inward K+ Channel Current in Guard Cells of Fava Bean

Recent reports have shown that GTP-binding proteins (G-proteins) are present in plants but have given limited indication as to their site of action. G-proteins in animal cells transduce extracellular signals into intracellular or membrane-mediated events, including the regulation of ion channels. Using whole-cell patch clamp, we provide evidence that a G-protein in guard cells of fava bean regulates the magnitude (and not the kinetics) of inward current through K+-selective ion channels in the plasma membrane. GDP[beta]S (100 to 500 [mu]M) increases inward K+ current, whereas GTP[gamma]S (500 [mu]M) has the opposite effect. The control nucleotides ADP[beta]S and ATP[gamma]S (500 [mu]M) do not affect K+ current. Reduction of inward current by GTP[gamma]S is eliminated in the presence of the Ca2+ chelator, BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N[prime],N[prime],-tetraacetic acid) (5 mM). When applied intracellularly, the G-protein regulators, cholera toxin and pertussis toxin, both decrease inward K+ current. The entry of K+ (and anions) into guard cells increases their turgor, opening stomatal pores in the leaf epidermis that allow gas exchange with the environment. Our data suggest the involvement of a G-protein in the inhibition of K+ uptake and stomatal opening. Changes in stomatal aperture, vital to both photosynthesis and plant water status, reflect guard-cell responsiveness to a variety of known environmental signals. The results presented here indicate that, in plants as well as animals, ion channel regulation by environmental stimuli may be mediated by G-proteins.     

Relationship between incurrent flow, calcium ion concentration and membrane potential

A dependence of the inward current across the cell membrane giant neurones at garden snail was investigated under voltage champ. It has been concluded that there are two components of the inward current: a calcium-dependent and I0. The latter current probably was carried by sodium ions. The inward Ca current (ICa) is then given as a function [Ca]2+ by: formula (see text) : KCa is a dissociation constant of the sites in outer part channel independent of membrane voltage. The experimental data are interpreted by two barrier membrane model bases of absolute reaction rate Eyring's theory.     

Maize Root Phytase (Purification,Characterization, and Localization of Enzyme Activity and Its Putative Substrate)

Three phytase (EC 3.1.3.26) isoforms from the roots of 8-d-old maize (Zea mays L. var Consul) seedlings were separated from phosphatases and purified to near homogeneity. The molecular mass of the native protein was 71 kD, and the isoelectric points of the three isoforms were pH 5.0, 4.9, and 4.8. Each of the three isoforms consisted of two subunits with a molecular mass of 38 kD. The temperature and pH optima (40[deg]C, pH 5.0) of these three isoforms, as well as the apparent Michaelis constants for sodium inositol hexakisphosphate (phytate) (43, 25, and 24 [mu]M) as determined by the release of inorganic phosphate, were only slightly different. Phytate concentrations higher than 300 [mu]M were inhibitory to all three isoforms. In contrast, the dephosphorylation of 4-nitrophenyl phosphate was not inhibited by any substrate concentration, but the Michaelis constants for this substrate were considerably higher (137-157 [mu]M). Hydrolysis of phytate by the phytase isoforms is a nonrandom reaction. D/L-Inositol-1,2,3,4,5- pentakisphosphate was identified as the first and D/L-inositol-1,2,5,6-tetrakisphosphate as the second intermediate in phytate hydrolysis. Phytase activity was localized in root slices. Although phosphatase activity was present in the stele and the cortex of the primary root, phytase activity was confined to the endodermis. Phytate was identified as the putative native substrate in maize roots (45 [mu]g P g-1 dry matter). It was readily labeled upon supplying [32P]phosphate to the roots.     

Effects of Acetyl-Coenzyme A Carboxylase Inhibitors on Root Cell Transmembrane Electric Potentials in Graminicide-Tolerant and -Susceptible Corn (Zea mays L.)

Herbicidal activity of aryloxyphenoxypropionate and cyclohexanedione herbicides (graminicides) has been proposed to involve two mechanisms: inhibition of acetyl-coenzyme A carboxylase (ACCase) and depolarization of cell membrane potential. We examined the effect of aryloxyphenoxypropionates (diclofop and haloxyfop) and cyclohexanediones (sethoxydim and clethodim) on root cortical cell membrane potential of graminicide-susceptible and -tolerant corn (Zea mays L.) lines. The graminicide-tolerant corn line contained a herbicide-insensitive form of ACCase. The effect of the herbicides on membrane potential was similar in both corn lines. At a concentration of 50 [mu]M, the cyclohexanediones had little or no effect on the membrane potential of root cells. At pH 6, 50 [mu]M diclofop, but not haloxyfop, depolarized membrane potential, whereas both herbicides (50 [mu]M) dramatically depolarized membrane potential at pH 5. Repolarization of membrane potential after removal of haloxyfop and diclofop from the treatment solution was incomplete at pH 5. However, at pH 6 nearly complete repolarization of membrane potential occurred after removal of diclofop. In graminicide-susceptible corn, root growth was significantly inhibited by a 24-h exposure to 1 [mu]M haloxyfop or sethoxydim, but cell membrane potential was unaffected. In gramincide-tolerant corn, sethoxydim treatment (1 [mu]M, 48 h) had no effect on root growth, whereas haloxyfop (1 [mu]M, 48 h) inhibited root growth by 78%. However, membrane potential was the same in roots treated with 1 [mu]M haloxyfop or sethoxydim. The results of this study indicate that graminicide tolerance in the corn line used in this investigation is not related to an altered response at the cell membrane level as has been demonstrated with other resistant species.     

Cation fluxes cause plasma membrane depolarization involved in beta-glucan elicitor-signaling in soybean roots

Inducible and specific ion fluxes on plasma membranes represent very early events during elicitation of plant cells. The hierarchy of such ion fluxes involved is still unknown. The effect of Phytophthora sojae-derived beta-glucan elicitors on the plasma membrane potential as well as on surface K+, Ca2+, and H+ fluxes has been investigated on soybean roots using ion-selective microelectrodes. Beta-Glucans with different degrees of polymerization transiently depolarized the plasma membrane. The elicitor concentration necessary for half-maximal depolarization closely resembled the corresponding binding affinities of soybean root membranes toward the respective beta-glucans. Upon repeated elicitor treatment, the root cells responded partially refractory, suggesting a complex responsiveness of the system. Within the root hair space, characteristic decreasing K(+)- and Ca(2+)-free concentrations were induced by the elicitors, probably causing depolarization through the influx of positive charges. Whereas K+ fluxes were inverted after passing the K+ equilibrium (Nernst-) potential, Ca2+ influx continued. No anion fluxes sufficient to account for charge compensation were observed under the same experimental conditions. K+ and Ca2+ fluxes as well as depolarization were inhibited by 100 microM or less of the Ca2+ antagonist La3+. Contrasting other systems, in soybean the main cause for elicitor-induced plasma membrane depolarization is the activation of cation instead of anion fluxes.     

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.     

Identification of a Transport Mechanism for NH4+ in the Symbiosome Membrane of Pea Root Nodules

Symbiosome membrane vesicles, facing bacteroid-side-out, were purified from pea (Pisum sativum L.) root nodules and used to study NH4+ transport across the membrane by recording vesicle uptake of the NH4+ analog [14C]methylamine (MA). Membrane potentials ([delta][psi]) were imposed on the vesicles using K+ concentration gradients and valinomycin, and the size of the imposed [delta][psi] was determined by measuring vesicle uptake of [14C]tetraphenylphosphonium. Vesicle uptake of MA was driven by a negative [delta][psi] and was stimulated by a low extravesicular pH. Protonophore-induced collapse of the pH gradient indicated that uptake of MA was not related to the presence of a pH gradient. The MA-uptake mechanism appeared to have a large capacity for transport, and saturation was not observed at MA concentrations in the range of 25 [mu]M to 150 mM. MA uptake could be inhibited by NH4+, which indicates that NH4+ and MA compete for the same uptake mechanism. The observed fluxes suggest that voltage-driven channels are operating in the symbiosome membrane and that these are capable of transporting NH4+ at high rates from the bacteroid side of the membrane to the plant cytosol. The pH of the symbiosome space is likely to be involved in regulation of the flux.     

Correlation between Root-Generated Ionic Currents, pH, Fusicoccin, Indoleacetic Acid, and Growth of the Primary Root of Zea mays

Correlations between root-generated ionic currents, extracellular pH, indoleacetic acid, fusicoccin, and growth were investigated. Current consistently entered the meristematic and elongating tissues of intact growing roots of Zea mays cv Golden Bantam. Mature root regions generated the outward limb of the current loop. Ion-substitution and pH-profile experiments suggested that the bulk of the ionic current was carried by H⁺. Calcium ions did not carry current, but calcium may regulate the proton circulation since the proton current density was slightly larger in calcium-depleted media. Increased root elongation at low pH was associated with increased current density and an extended zone of inward current. Conversely decreased elongation at high pH was associated with a reduced current density and a more restricted zone of inward current. The effect of the fungal toxin fusicoccin was to increase the current density of the inward limb of the ion current and to increase root extension. Concentrations of indoleacetic acid that reduced root growth, also reduced the density of the inward current and shortened the inward current zone. The results emphasize the point that roots are electrically contiguous over many millimeters and that the electrophysiology of root growth is best studied in intact root systems.     

Electrogenic transport properties of growing Arabidopsis root hairs : the plasma membrane proton pump and potassium channels

Ion transport, measured using double-barreled micropipettes to obtain current-voltage relations, was examined in Arabidopsis thaliana root hairs that continued tip growth and cytoplasmic streaming after impalement with the micropipette. To do this required in situ measurements with no handling of the seedlings to avoid wounding responses, and conditions allowing good resolution microscopy in tandem with the electrophysiological measurements. Two ion transport processes were demonstrated. One was a tetraethylammonium-sensitive potassium ion current, inward at hyperpolarized potentials and outward at depolarized potentials. The addition of tetraethylammonium (a potassium channel blocker) caused the potential to hyperpolarize, indicating the presence of a net inward potassium current through the ion channels at the resting potential. The potassium influx was sufficient to “drive” cellular expansion based upon growth rates. Indeed, tetraethylammonium caused transient inhibition of tip growth. The other electrogenic process was the plasma membrane proton pump, measured by indirect inhibition with cyanide or direct inhibition by vanadate. The proton pump was the dominant contribution to the resting potential, with a very high current density of about 250 microamperes per square centimeter (seen only in young growing root hairs). The membrane potential generated by the proton pump presumably drives the potassium influx required for cellular expansion. The pump appears to be a constant current source over the voltage range −200 to 0 millivolts. With this system, it is now possible to study the physiology of a higher plant cell in dynamic living state using a broad range of cell biological and electrophysiological techniques.     

Ionic components of electric current at rat corneal wounds

Background

Endogenous electric fields and currents occur naturally at wounds and are a strong signal guiding cell migration into the wound to promote healing. Many cells involved in wound healing respond to small physiological electric fields in vitro. It has long been assumed that wound electric fields are produced by passive ion leakage from damaged tissue. Could these fields be actively maintained and regulated as an active wound response? What are the molecular, ionic and cellular mechanisms underlying the wound electric currents?

Methodology/Principal Findings

Using rat cornea wounds as a model, we measured the dynamic timecourses of individual ion fluxes with ion-selective probes. We also examined chloride channel expression before and after wounding. After wounding, Ca²⁺ efflux increased steadily whereas K⁺ showed an initial large efflux which rapidly decreased. Surprisingly, Na⁺ flux at wounds was inward. A most significant observation was a persistent large influx of Cl⁻, which had a time course similar to the net wound electric currents we have measured previously. Fixation of the tissues abolished ion fluxes. Pharmacological agents which stimulate ion transport significantly increased flux of Cl⁻, Na⁺ and K⁺. Injury to the cornea caused significant changes in distribution and expression of Cl⁻ channel CLC2.

Conclusions/Significance

These data suggest that the outward electric currents occurring naturally at corneal wounds are carried mainly by a large influx of chloride ions, and in part by effluxes of calcium and potassium ions. Ca²⁺ and Cl⁻ fluxes appear to be mainly actively regulated, while K⁺ flux appears to be largely due to leakage. The dynamic changes of electric currents and specific ion fluxes after wounding suggest that electrical signaling is an active response to injury and offers potential novel approaches to modulate wound healing, for example eye-drops targeting ion transport to aid in the challenging management of non-healing corneal ulcers.     

Investigation of the Apparent Induction of Nitrate Uptake in Barley (Hordeum vulgare L.) Using NO3--Selective Microelectrodes (Modulation of Coarse Regulation of NO3- Uptake by Exogenous Application of Downstream Metabolites in the NO3- Assimilatory Pathway)

The influence of a 12-h pretreatment with either NO3-, NH4+, glutamine, or glutamate (300 [mu]M) on the apparent induction of NO3- uptake was investigated. Net fluxes of NO3- into roots of intact, 7-d-old barley (Hordeum vulgare L. cv Prato) seedlings in solution culture were estimated from ion activity gradients measured with NO3--selective microelectrodes in the unstirred layer of solution immediately external to the root surface. Control plants, pretreated with nitrogen-free nutrient solution, exhibited a sigmoidal increase in net NO3- uptake, reaching a maximum rate between 8 and 9 h after first exposure to NO3-. Plants pretreated with NH4+ or Glu exhibited a delay of several hours in the initiation of the induction process after they had been exposed to NO3-. In Gln-pretreated plants, however, responses ranged from no delay of the induction process to delays comparable to those observed following NH4+ or Glu pretreatments. Only treatment with NO3-resulted in the induction of NO3- uptake, whereas pretreatments with NH4+, Gln, or Glu tended to delay induction of NO3- uptake upon subsequent exposure to NO3-.     

Ionic movements mediated by monensin in frog skeletal muscle.

Monensin-mediated ionic movements were studied in frog skeletal muscle. The ionophore, which forms electrically neutral complexes with monovalent cations, induced dose dependent fluxes of Na+, K+ and H+ in and out of the fibers. Monensin concentrations ([MON]) ranged from 2 to 40 microM. In the presence of normal Ringer's solution the following maximum ionic exchanges were generated by monensin (in pmol cm-2 s-1): (1) Nai+/Nao+ 112, (2) Nai+/Ho+ 30.7, (3) Ki+/Nao+ 14.2 (4) Hi+/Nao+ 49. The maximum net fluxes produced by these exchanges (i.e. for [MON] = infinity) are (in pmol cm-2 s-1): Na+ (inward) 32.5, K+ (outward) 14.2, H+ (outward) 18.3. The last one appears to be largely offset by a passive (monensin-independent) H+ influx down an inwardly directed electrochemical gradient promoted by pH reduction of the T-tubular lumen content as a consequence of the monensin-mediated net H+ efflux. Maximum unidirectional cationic fluxes mediated by monensin amounted to 206 pmol cm-2 s-1 and had the following composition: influx: 85% Na+ and 15% H+; efflux: 69% Na+, 7% K+, 24% H+.