Uptake of neutral and acidic amino acids by Lemna gibba correlated with the H+-electrochemical gradient at the plasmalemma

Uptake rates of L-alanine, L-serine and L-aspartate and trans-membrane electrical potentials (Δψ) were determined for a pH range in the external medium between 3.5 and 9.0. The proton electrochemical gradients (     ) were calculated from Δψ, pH of the medium, and an assumed cytoplasmic pH of 7.5. At external amino-acid concentrations of 0.1 mol m⁻³, where carrier-mediated uptake dominates total uptake, a linear correlation between uptake rates and     is obtained, which extrapolates to zero uptake at zero     . This corroborates the contention that neutral and acidic amino acids are taken up by Lemna gibba L. by H⁺-cotransport.     

Electrical signalling and cytokinins mediate effects of light and root cutting on ion uptake in intact plants

Nutrient acquisition in the mature root zone is under systemic control by the shoot and the root tip. In maize, exposure of the shoot to light induces short-term (within 1–2 min) effects on net K⁺ and H⁺ transport at the root surface. H⁺ efflux decreased (from −18 to −12 nmol m⁻² s⁻¹) and K⁺ uptake (∼2 nmol m⁻² s⁻¹) reverted to efflux (∼−3 nmol m⁻² s⁻¹). Xylem probing revealed that the trans-root (electrical) potential drop between xylem vessels and an external electrode responded within seconds to a stepwise increase in light intensity; xylem pressure started to decrease after a ∼3 min delay, favouring electrical as opposed to hydraulic signalling. Cutting of maize and barley roots at the base reduced H⁺ efflux and stopped K⁺ influx in low-salt medium; xylem pressure rapidly increased to atmospheric levels. With 100 m m NaCl added to the bath, the pressure jump upon cutting was more dramatic, but fluxes remained unaffected, providing further evidence against hydraulic regulation of ion uptake. Following excision of the apical part of barley roots, influx changed to large efflux (−50 nmol m⁻² s⁻¹). Kinetin (2–4  µ m ), a synthetic cytokinin, reversed this effect. Regulation of ion transport by root-tip-synthesized cytokinins is discussed.     

Cation-stimulated H+ efflux by intact roots of barley

Abstract. Rates of proton extrusion and potassium (⁸⁶Rb) influx by intact roots of barley ( Hordeum vulgare cvs . Fergus, Conquest and Betzes) plants were simultaneously measured in short-term (15min) experiments. The nature and extent of apparent coupling between these ion fluxes was explored by manipulating conditions of temperature, pH and cation composition and concentration during flux determinations. In addition, the influence of salt status upon these fluxes was examined. At low K⁺ concentrations (0.01 to 1 mol m⁻³), H⁺ efflux and K⁺ influx were strongly correlated in both low- and high-K⁺ roots, although K⁺: H⁺ exchange stoichiometries were almost consistently greater than 2:1. At higher concentrations (1 to 5 mol m⁻³), H⁺ efflux was either reduced or remained unchanged while K⁺ influxes increased. In the presence of Na₂SO₄, rates of H⁺ extrusion demonstrated similar cation dependence, although below 10 mol m⁻³ Na₂SO₄, H⁺ fluxes were generally 50% lower than in equivalent concentrations of K₂SO₄. These observations are considered in the context of current hypotheses regarding the mechanisms of k⁺/H⁺ exchange.     

Photoinhibition of photosynthesis in Lemna gibba as induced by the interaction between light and temperature. I. Photosynthesis in vivo

The floating angiosperm Lemna gibba L. was exposed for 2 h to various combinations of photosynthetic photon flux densities and temperature. The extent of photoinhibition of photosynthesis was assayed by measuring the net CO₂ uptake before and after a photoinhibitory treatment, and the time course for photoinhibition was studied. It was found that the maximum quantum yield and the light-saturated rate of CO₂ uptake were affected by the interaction between light and temperature during the photoinhibitory treatment. At a constant photon flux density of 650 μmol m⁻² s⁻¹ the extent of photoinhibition increased with decreasing temperature showing that even a chilling-resistant plant like L. gibba is much more susceptible to photoinhibition at chilling temperatures. About 60% photoinhibition of the quantum yield for CO₂ uptake could be obtained either by a high photon flux density of 1 750 μmol m⁻² s⁻¹ and 25°C or by a moderate photon flux density of 650 μmol m⁻² s⁻¹ and 3°C. The time courses of recovery from 60% photoinhibition produced by either of these two treatments were similar, indicating that the nature of the photoinhibition was intrinsically similar. The extent of photoinhibition was related to the amount of light absorbed in excess to what could be handled by photosynthesis at that temperature. The vital importance of photosynthesis in alleviating photoinhibition is discussed.     

Transmembrane hyperpolarization and increase of K+ uptake in maize roots treated with permeant weak acids

Abstract. Treatment with weak acids (butyrate, isobutyrate, trimethylacetate, DMO) at a concentration of I mol m⁻³ in apical maize root segments induced a rapid, marked hyperpolarization ( ca. 30 mV) of the transmembrane electrical potential, stable for at least 30 min. With butyrate, this effect increased with the increase of butyrate concentration in the medium, reaching a value of ca. 75 mV at a concentration of 5 mol m⁻³.
Both the butyrate uptake and the hyperpolarization were roughly proportional to the pH-regulated, undissociated/dissociated acid ratio in the medium. The butyrate-induced hyperpolarization was reduced progressively, but was still present when K⁺ concentration in the medium was raised from 1 to 10 mol m⁻³.
The hyperpolarization was accompanied by a significant increase of K⁺ uptake, and was almost completely suppressed by the presence of the protonophore carbonylcyanid- p -trichlorometoxy-phenylhydrazone (CCCP) and strongly reduced by erytrosin B, an inhibitor of some animal ATPases and of a K⁺-activated, DCCD- and vanadate-sensitive Mg²⁺-ATPase from plant microsomes. The hyperpolarization effect of butyrate was additive to that of fusicoccin at low, but not at high (5 mol m⁻³), concentrations of the weak acid. These results suggest that the intracellular pH regulates the activity of the electrogenic proton pump at the plasmalemma.     

An electrogenic pump in the xylem parenchyma of barley roots

Analysis of an electrogenic pump in the plasma membrane of xylem-parenchyma protoplasts from barley roots was performed using the patch-clamp technique in the whole-cell configuration. Particularly with regard to understanding xylem loading and unloading, the study of the electrogenic pump from this cell type is important; its functional confirmation was lacking to date. About one-half of the investigated protoplasts displayed current responses with reversal potentials between −80 and −200 mV. The application of fusicoccin, an H⁺-pump stimulator, caused an increase in currents recorded at a membrane potential of 0 mV and a shift of the reversal potential by about −50 mV. Treatment with dicylohexylcarbodiimid, an H⁺-pump inhibitor, resulted in the reduction of the current at 0 mV. The Ca²⁺-pump inhibitor, erythrosin B, showed no effect on current density at 0 mV and on the polarisation of the membrane potential. Enlarging the transmembrane pH gradient by raising the pH of the extracellular solution from 5.8 to 8.8 stimulated the currents. These are strong indications that the electrogenic pump was an H⁺-pump. Neither intracellular pH nor the intracellular Ca²⁺ concentration affected its activity. Simultaneous activity of the electrogenic pump and anion conductances could produce states in which protoplasts exhibited 'intermediate' reversal potentials. It was concluded that the electrogenic pump was not directly involved in the loading of KCl and KNO₃ into the xylem but, in combination with anion channel activities, contributed to the establishment of membrane potentials at which electroneutral salt transport and acid release can proceed.     

Effects of UV-C radiation on extension growth, H+-extrusion and transmembrane electric potential in maize coleoptile segments

The effect of 253.7 nm ultraviolet radiation on elongation growth, medium acidification and changes in electric potential difference between vacuole and external medium in cells of maize ( Zea mays L.) coleoptile segments was investigated. It was found that irradiation with 390, 1170, 3900 and 5 850 J m⁻² UV-C (ultraviolet radiation 253.7 nm) inhibited elongation growth, whereas at 195 J m⁻² stimulation of growth was observed. The administration of IAA (10⁻⁵ M ) to the incubation medium of coleoptile segments partially abolished the inhibitory effect of UV-C. The pH of the incubation medium, measured simultaneously with growth, showed that the exposure of the segments to UV-C caused inhibition of H⁺-extrusion (or stimulation of H⁺ uptake). The presence of IAA (10⁻⁵ M ) in the incubation medium promoted (except after 5850 J m⁻² irradiation) H⁺-extrusion to a level comparable with that produced by IAA in non-irradiated segments. In UV-C irradiated segments the potential difference underwent significant alterations. Irradiation of coleoptile segments with 390 J m⁻² caused a transient depolarization, which was fully reversible within 30 min, while at higher doses depolarization was irreversible. The hyperpolarization of the membrane potential (MP) in cells of maize coleoptile induced by IAA was completely nullified by subsequent irradiation with UV-C. It is suggested that UV-C inhibited IAA-induced growth by a mechanism independent of cell wall acidification.     

The effect of xanthoxylin (2-oxy, 4-6-dimethoxyacetophenone) on potassium-dependent acid: extrusion in wheat and maize root segments

Abstract. Xanthoxylin is a cytotoxic and fungicidal compound with the characteristics of a typical phytoalexin. At a concentration of 0.3 mol m⁻³ it inhibits K⁺-dependent acid extrusion and K⁺ net uptake (or uptake of equivalent alkaline cations such as Rb⁺ and CS⁺) by up to about 80% and hyperpolarizes by about 20% the membrane electrical potential. Its inhibition of the acid extrusion does not depend on altered ion exchange involving the anions in the media, a reduction of the metabolic energy available, or detectable changes in the permeability of the cell membrane to H⁺ ions. The drop in K⁺ net uptake depends on a decrease in the influx of K⁺ into the cell. In functional terms, xanthoxylin is an inhibitor of the K⁺ permeation mechanism and does not appear to interact with the mechanisms creating the electrochemical energy gradient.     

Inhibition of the amino-acid co-transport carrier of Lemna gibba by incorporation ofp-fluorophenylalanine

In Lemna gibba L. a 2 h pretreatment with the amino-acid analogue p -fluorophenylalanine p -FPA inhibited subsequent uptake of L-alanine and reduced the transient depolarization of the plasmalemma caused by L-alanine. Uptake of 3–0-methylglucose was less affected. There was no effect of p -fluorophenylalanine on the resting potential or on the fusicoccin- or light-dependent hyperpolarization of the cells. Furthermore, fusicoccin-stimulation of uptake of L-alanine and 3–0-methylglucose was also unaffected. The results suggest that p -FPA pretreatment selectively acts on the H⁺-amino-acid co-transport carrier, that the H⁺-hexose co-transport carrier is much less affected, and that the proton pump appears to be unaffected by p-FPA.     

Changes in sodium and potassium in Nitellopsis cells treated with transient salt stress

Abstract. Nitellopsis cells grown in fresh water have a relatively low cytoplasmic Na⁺ (11 mol m⁻³) and high cytoplasmic K⁺ (90 mol m⁻³) content. A 30-min treatment with 100 mol m⁻³ external NaCl resulted in a high [Na⁺]_c (90 mol m⁻³) and a low [K⁺]_c (33 mol m⁻³), Subsequent addition of external Ca²⁺ (10 mol m⁻³) prevented Na⁺ influx and then [Na⁺]_c decreased slowly. Changes in [K⁺]_c were opposite to [Na⁺]_c. During the recovery time vacuolar Na⁺ increased, while vacuolar K⁺ decreased. Since all these processes proceeded also under ice-cold conditions, the restoration of original cytoplasmic ion compositions is suggested to be a passive nature. The notion that the passive movement of ions across the tonoplast can act as an effective and economic mechanism of salt tolerance under transient or under mild salt stress conditions is discussed.     

Influence of UV-B radiation on developmental changes, ethylene, CO2 flux and polyamines in cv. Doyenne d'Hiver pear shoots grown in vitro

In vitro shoots of cv. Doyenne ďHiver pear ( Pyrus communis L.) were irradiated under controlled environments for 6 h per day at 5 different levels of biologically effective UV-B radiation (UV-B_BE). UV-B exposure caused a progressive increase in apical necrosis above background levels and stimulated leaf abscission. Shoots grown for 2 weeks at 7. 8 mol m⁻² day ⁻¹ of photosynthetic photon flux (PPF) and treated with 8. 4 or 12. 0 kJ m⁻² day ⁻¹ UV-B_BE produced up to 4 times more ethylene than those given 2. 2 or 5. 1 kJ m⁻² day⁻¹ UV-B_BE or untreated controls. Exposure of shoots to 12 kJ m⁻² day ⁻¹ of UV-B_BE caused an increase in free putreseine content after 4 to 14 days of irradiation. Shoots showed a decrease in CO₂ uptake after 3 days of UV-B: thereafter, they appeared to recover their photosynthetic capacity. Under typical PPF conditions used in micropropagation (90 μmol m⁻² S⁻¹). 8. 4 kJ m⁻² day ⁻¹ of UV-B radiation was injurious to realatively tender tissues of in vitro pear shoots: increasing the level of UV-B_BE to 12 kJ m⁻² day⁻¹ produced even more adverse effects.     

Aluminum enhancement of plant growth in acid rooting media. A case of reciprocal alleviation of toxicity by two toxic cations

The generally rhizotoxic ion Al³⁺ often enhances root growth at low concentrations. The hypothesis that Al³⁺ enhances growth by relieving H⁺ toxicity was tested with wheat seedlings ( Triticum aestivum L.). Growth enhancement by Al³⁺ only occurred under acidic conditions that reduced root elongation. Al³⁺ increased cell membrane electrical polarity and stimulated H⁺ extrusion. Previous investigations have shown that Al³⁺ decreases solute leakage at low _pH and that the alleviation of H⁺ toxicity by cations appears to be a general phenomenon with effectiveness dependent upon charge (C³⁺>C²⁺>C^l+). Alleviation of one cation toxicity by another toxic cation appears to be reciprocal so that Al³⁺ toxicity is relieved by H⁺. It has been argued previously that this latter phenomenon accounts for the apparent toxicity of ALOH²⁺ and Al(OH)⁺₂. Reduction of cell-surface electrical potential by the ameliorative cation may reduce the cell-surface activity of the toxic cation.     

Inhibitory Effect of Omeprazole, a Specific Inhibitor of H+, K+-ATPase, on Spicule Formation in Sea Urchin Embryos and in Cultured Micromere-Derived Cells.

Embryos kept with omeprazole, a specific H⁺, K⁺-ATPase inhibitor, in a period of development between the mesenchyme blastula and the pluteus corresponding stage became abnormal plutei having quite small spicules, somewhat poor pluteus arms and apparently normal archenterons. In micro-mere-derived cells, kept with omeprazole at pH 8.2 in a period between 15 and 40 hr of culture at 20°C, omeprazole strongly inhibited spicule formation but did not block the outgrowth of pseudopodial cables, in which spicule rods were to be formed. These indicate that omeprazole probably exerts no obvious inhibitory effects other than spicule rods formation. Omeprazole-sensitive H⁺, K⁺-ATPase, an H⁺pump, seems to be indispensable for CaCO₃ deposition (formation of spicule rod) in these spicule forming cells. H⁺, produced in overall reaction for CaCO₃ formation: Ca²⁺+ CO₂+H₂O°CaCO₃+2H⁺, is probably released from the cells by this H⁺pump and hence, this reaction tends to go to CaCO₃ production to form spicule rods. Omeprazole, known to become effective following its conversion to a specific inhibitor of H⁺, K⁺-ATPase at acidic pH, is able to inhibit formation of spicule rod at alkaline pH in sea water. This is probably due to an acidification of sea water near the cell surface by H⁺ejection in H⁺, K⁺-ATPase reaction.     

Effect of ultraviolet radiation on accumulation and leakage of 86Rb+ in guard cells of Vicia faba

The effects of UV-C (254 nm), UV-A (365 nm) and broad-band UV (280–380 nm) on guard cells of Vicia faba L. cv. Long Pod were investigated in the presence of white light (450 μmol m⁻² s⁻¹). UV-C (7 μmol m⁻² s⁻¹) was found to cause leakage of ⁸⁶Rb⁺ from guard cells, while UV-A (0.3 μmol m⁻² s⁻¹) stimulated increased uptake in these cells. A relatively small stimulatory effect was observed by broad-band UV (3 μmol m⁻² s⁻¹) during the first 30 min of irradiation with an apparent equilibration of influx and efflux thereafter. Leakage of ⁸⁶Rb⁺ from guard cells continued despite the removal of UV-C and an increase in the amount of white light from 450 to 1500 μmol m⁻² s⁻¹, suggesting that membranes were irreversibly damaged. Irradiation of guard cells with UV-C for 30, 45 and 90 min indicated that these cells began to be affected already by 30 min UV-C irradiation.     

Time dependent K+ currents through plasmalemma of laticifer protoplasts from Hevea brasiliensis

The purpose of our work was to investigate the functioning of K⁺ channels in protoplasts of laticifers of Hevea brasiliensis Muell. Arg., anastomosed into a network devoid of large central vacuoles, after tapping stress. Physiological functions such as proton pump activity and uptake of sucrose (a rubber precursor) were maintained, when the voltage-clamp method was used in vivo to record the whole-cell K⁺ current during the stress response.
A time-dependent inward current was induced in 50 m M KCl and rapidly inactivated (about 100 ms). The activation potential of this inward K⁺ channel was not closely dependent on E_k. This would be coherent with the 'valve model' of Schroeder and Fang (1991, Proc. Natl. Acad. Sci. USA 88: 11583–11587) involving the activation of a H⁺-pump accounting for the K⁺ uptake observed in laticiferous cells under stress. The activation half-time of outward currents was clearly voltage dependent: from about 350 to 60 ms for 125 and 155 mV, respectively. Time-dependent outward current sensitivity to 5 m M BaCl₂ or CaCl₂ or to 5 μ M Erythrosin B showed that the K⁺ channels could be Ca²⁺-dependent. Because of the positive values of the activation potential of the outward current, the possibility opens that an action potential exists, these cells being specialized for stress response.     

Volume changes of Commelina communis guard cell protoplasts in response to K+, light and CO2

The effects of K⁺ concentration, light intensity and CO₂ levels on the volume of Commelina communis L. guard cell protoplasts were studied. Two degrees of swelling response were observed, both dependent on an external supply of K⁺, but not necessarily on the supply of a permeant anion. The presence of K⁺ itself, independent of light or CO₂ level, stimulated swelling at a relatively slow rate. When K⁺, light and low CO₂ conditions were supplied together, the swelling was relatively rapid and of high magnitude. The rapid swelling was specific for K⁺ and Rb⁺ giving a half maximal effect after 2 h at a KCl concentration of about 18 mmol m⁻³. The addition of CaCl₂ at 1 mol m⁻³ inhibited K⁺-dependent swelling under all conditions tested. The response to light and low CO₂ levels by Commelina guard cell protoplasts is thought to reflect a high degree of physiological integrity.     

K+ATP channel opening prevents succinate-dependent H2O2 generation by plant mitochondria

The role of a recently identified K⁺_ATP channel in preventing H₂O₂ formation was examined in isolated pea stem mitochondria. The succinate-dependent H₂O₂ formation was progressively inhibited, when mitochondria were resuspended in media containing increasing concentration of KCl (from 0.05 to 0.15  M ). This inhibition was linked to a partial dissipation of the transmembrane electrical potential (ΔΨ) induced by KCl. Conversely, the malate plus glutamate-dependent H₂O₂ formation was not influenced. The succinate-sustained H₂O₂ generation was also unaffected by nigericin (a H⁺/K⁺ exchanger), but completely prevented by valinomycin (a K⁺ ionophore). In addition, cyclosporin A (a K⁺_ATP channel opener) inhibited this H₂O₂ formation, while ATP (an inhibitor of the channel opening) slightly increased it. The inhibitory effect of ATP was strongly stimulated in the presence of atractylate (an inhibitor of the adenine nucleotide translocase), thus suggesting that the receptor for ATP on the K⁺ channel faces the intermembrane space. Finally, the succinate-dependent H₂O₂ formation was partially prevented by phenylarsine oxide (a thiol oxidant).     

THE COMBINED EFFECTS OF LOW TEMPERATURE AND SO2+NO2 POLLUTION ON THE NEW SEASON'S GROWTH AND WATER RELATIONS OF PICEA SITCHENSIS

Picea sitchensis (Bong.) Carr. seedlings were exposed to SO₂, NO₂ and SO₂+ NO₂ during dormancy in controlled environments, and were taken to night temperatures of 4, 0, −5, −10 and −15 °C in a freezer. Conditions in the freezer were carefully monitored during the low–temperature treatments. In two experiments, different photoenvironments and temperature regimes were imposed prior to the cold treatments, and different effects were observed. In the first, only limited frost hardiness was achieved and night temperatures of −15 °C were lethal. Temperatures of −5 and − 10 °C led to poor survival of lateral buds, particularly in plants exposed to 45 ppb SO₂. The poor bud break in plants exposed to SO₂ and to − 5 °C resulted in a loss of the effectiveness of this temperature as a chill requirement. Pressure-volume analysis showed that the shoots of plants exposed to NO₂ had greater elasticity (lower elastic moduli, e), so that loss of turgor occurred at lower relative water contents. In contrast, a hardening period (2 weeks in night/day temperatures of 3/10 °C and 8 h days at 50 μmol m⁻² s⁻¹ PAR) gave decreased elasticity and lower solute potentials of spruce shoots. In the second experiment, exposure to 30 ppb SO₂ and SO₂+ NO₂ led to slight, but consistent, increases in frost injury to the needles of plants frozen to − 5 and − 10 °C. The results suggest that the main interaction of low temperatures and winter pollutants may be on bud survival rather than on needle damage, but that effects are subtle, only occurring with certain combinations of pollutant dose and cold treatment.     

Influence of some metal chelators and light regimes on bioaccumulation and toxicity of Cd2+ in duckweed (Lemna gibba)

A factorial culture experiment was designed to investigate the influence of light regimes and of some metal chelators on the accumulation of cadmium by Lemna gibba L. The plants were grown in a complete nutrient solution containing Cd²⁺ concentrations ranging from 0 to 27 μ M with or without EDTA, ethylenediamine-N,N'- bis -( o -hydroxyphenylacetic acid) (EDDHA) or salicylic acid. Each experiment was run for eight days in 18 h:6 h light:dark or continuous light. An increase in the Cd²⁺ concentration in plants and a simultaneous drop in accumulation efficiency (ratio of Cd²⁺ concentration in plants to the initial Cd²⁺ concentration in the nutrient solution) with increasing ambient Cd²⁺ levels was best represented by regression power curves. At the lowest Cd²⁺ concentration which caused a significant decrease in the relative growth rate of duckweed, there was a decrease in manganese and zinc and an increase in the iron level in the plants. EDDHA and EDTA protected in some cases against the toxic action of cadmium without preventing its uptake by plants. It was thus observed that 9 μ M or higher levels of Cd²⁺ were toxic to Lemna gibba depending on the chelator and light regime. Duckweed grown in continuous light produced, in general, more dry matter and hence accumulated more cadmium.     

Demonstration of a K+/H+ exchange activity in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

Abstract Washed cells of Rhodopseudomonas sphaeroides forma sp. denitrificans , grown under photodenitrifying conditions, exhibited K⁺ uptake dependent on the transmembrane proton gradient (Δ pH). These cells also acidified the suspension medium in response to K⁺ pulses both aerobically and anaerobically in light and in the dark. The results indicate that the photodenitrifier has a reversible K⁺/H⁺ exchange activity which reflects its role in regulating the intracellular K⁺ concentration, as well as intracellular pH. The acidification of the external medium resulting from K⁺ pulses was inhibited by carbonyl cyanide- m -chlorophenylhydrazone (CCCP) indicating that the antiporter is energy-dependent. Addition of KCl to washed cells depolarized the membrane potential (Δψ) with a concomitant increase in ΔpH, indicating that the K⁺/H⁺ antiporter was electrogenic.