Effect of polycyclic aromatic hydrocarbons on laccase production by white rot fungus <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> D1

The effect of polycyclic aromatic hydrocarbons (PAHs) on the time course of laccase production by the fungus Pleurotus ostreatus D1 under conditions of submerged cultivation on Kirk’s medium has been studied. It has been shown that phenanthrene, fluoranthene, pyrene, and chrysene actively induce this enzyme, whereas fluorene and anthracene had a smaller effect. Addition of Mn²⁺ ions to cultivation medium elevates the laccase activity twofold and more in the presence of all the studied PAHs. Electrophoresis under nondenaturing conditions demonstrates induction of additional laccase forms by xenobiotics. Ligninolytic peroxidase activities are undetectable under the conditions used.     

Effect of jasmonic acid on the pro-/antioxidant system of wheat coleoptiles as related to hyperthermia tolerance

The effects of treatment with jasmonic acid (JA) of wheat (Triticum aestivum L, cv. Elegia) coleoptiles on the generation of superoxide anion-radical (O ₂ ^·? ), the activity of extracellular peroxidase, enzymatic and non-enzymatic components of the antioxidant system were studied. During the first hour after the start of coleoptile treatment with 1 μM JA, the generation of O ₂ ^·? was enhanced and the extracellular peroxidase was activated. During following 23 h, these effects were gradually reduced. JA-enhanced O ₂ ^·? generation was partially suppressed by coleoptile treatment with the inhibitor of peroxidase salicylhydroxamic acid, the inhibitor of NADPH-oxidase imidazol, and also the calcium chelator EGTA. Under the influence of JA treatment, antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase, and soluble guaiacol peroxidase) in wheat coleoptiles were activated. Treatment with JA improved coleoptile tolerance to damaging heating (10 min at 43°C); it favored the maintenance of the pools of enzymatic and non-enzymatic antioxidants. The inhibitors of NADPH-oxidase and peroxidase, and also calcium chelator reduced a positive JA influence on coleoptile thermotolerance. The role of changes in the pro-/antioxidant balance in plant tissues for the realization of stress-defensive JA effects is discussed.     

Somatic embryogenesis and analysis of peroxidases inPhoenix dactylifera L

To determine some physiological parameters implicated in somatic embryogenesis in date palm (Phoenix dactylifera L.), peroxidases have been studied. Activated charcoal commonly used in date palm tissue culture as an essential antibrowning factor decreased cellular protein contents and peroxidase activities. During the first months of culture, the conventionally used medium (100 mg dm^?3 of 2,4-dichlorophenoxyacetic acid, 3 g dm^?3 charcoal) reduces 2 to 3 and 4 to 6 times protein contents and peroxidase activities, respectively, in comparison with the same one containing only 5 mg dm^?3 of 2,4-D and with or without 150 mg dm^?3 charcoal. In addition, the standard procedure decreased the embryogenic potential which is positively related to the intra- and extracellular (excreted into culture medium) peroxidase activities. In medium with embryogenic calli, extracellular peroxidase activity was three times as high as the activity determined in the same medium with non-embryogenic calli. There were two basic isoforms and four to five acidic bands characterizing the embryogenic calli. It can be suggested that peroxidases play a key role in somatic embryogenesis of date palm and the charcoal used at 3 g dm^?3 constitute a perturbating factor for this process.     

Kir4.1-mediated spatial buffering of K+: Experimental challenges in determination of its temporal and quantitative contribution to K+ clearance in the brain

Neuronal activity results in release of K⁺ into the extracellular space of the central nervous system. If the excess K⁺ is allowed to accumulate, neuronal firing will be compromised by the ensuing neuronal membrane depolarization. The surrounding glial cells are involved in clearing K⁺ from the extracellular space by molecular mechanism(s), the identity of which have been a matter of controversy for over half a century. Kir4.1-mediated spatial buffering of K⁺ has been promoted as a major contributor to K⁺ removal although its quantitative and temporal contribution has remained undefined. We discuss the biophysical and experimental challenges regarding determination of the contribution of Kir4.1 to extracellular K⁺ management during neuronal activity. It is concluded that 1) the geometry of the experimental preparation is crucial for detection of Kir4.1-mediated spatial buffering and 2) Kir4.1 enacts spatial buffering of K⁺ during but not after neuronal activity.     

Changes in the Activity of Antioxidant Enzymes in Wheat Leaves and Roots as a Function of Nitrogen Source and Supply

The activity of enzymes participating in the systems of antioxidant protection was assayed in the second leaf and roots of 21-day-old wheat seedlings (Triticum aestivum L.) grown in a medium with nitrate (NO^– ₃ treatment), ammonium (NH⁺ ₄ treatment), or without nitrogen added (N-deficiency treatment). The activities of superoxide dismutase (SOD), peroxidase, ascorbate peroxidase, glutathione reductase, and catalase in the leaves and roots of the NH⁺ ₄ plants was significantly higher than in the plants grown in the nitrate medium. The activity of SOD decreased and ascorbate peroxidase markedly increased in leaves, whereas the activity of ascorbate peroxidase increased in the roots of N-deficient plants, as compared to the plants grown in nitrate and ammonium. Low-temperature incubation (5°, 12 h) differentially affected the antioxidant activity of the studied plants. Whereas leaf enzyme activities did not change in the NH⁺ ₄ plants, the activities of SOD, peroxidase, ascorbate peroxidase, and catalase markedly increased in the NO^– ₃ plants. In leaves of the N-deficient plant, the activity of SOD decreased; however, the activity of other enzymes increased. In response to temperature decrease, catalase activity increased in the roots of NO^– ₃ and NH⁺ ₄-plants, whereas in the N-deficient plants, the activity of peroxidase increased. Thus, in wheat, both nitrogen form and nitrogen deficiency changed the time-course of antioxidant enzyme activities in response to low temperature.     

A comparative study of the effects of abscisic acid and methyl jasmonate on seedling growth of rice

The effects of abscisic acid (ABA) and methyl jasmonate (MJ) on growth of rice seedlings were compared. The lowest tested concentration of ABA and MJ that inhibited seedling growth was found to be 4.5 and 0.9 µM, respectively. Growth inhibition by ABA is reversible, whereas that by MJ is irreversible. GA₃ was found to be more effective in reversing inhibition of shoot growth by ABA than by MJ. KCl partially relieved MJ-inhibited, but not ABA-inhibited, growth of rice seedlings. The beneficial effect of K⁺ on growth of rice seedlings in MJ medium could not be replaced by Li⁺, Na⁺ or Cs⁺. MJ treatment caused a marked release of K⁺ into the medium. In order to understand whether cell wall-bound peroxidase activity was inversely related to rice seedling growth, effects of ABA and MJ on cell wall-bound peroxidase activity were also examined. Results indicated that both ABA and MJ increased cell wall-bound peroxidase activity in roots and shoots of rice seedlings. Although MJ (4.5 µM) was less effective in inhibiting root growth than ABA (9 µM), MJ was found to increase more cell wall-bound peroxidase activity in roots than ABA.     

Kir4.1-mediated spatial buffering of K+: Experimental challenges in determination of its temporal and quantitative contribution to K+ clearance in the brain

Neuronal activity results in release of K⁺ into the extracellular space of the central nervous system. If the excess K⁺ is allowed to accumulate, neuronal firing will be compromised by the ensuing neuronal membrane depolarization. The surrounding glial cells are involved in clearing K⁺ from the extracellular space by molecular mechanism(s), the identity of which have been a matter of controversy for over half a century. Kir4.1-mediated spatial buffering of K⁺ has been promoted as a major contributor to K⁺ removal although its quantitative and temporal contribution has remained undefined. We discuss the biophysical and experimental challenges regarding determination of the contribution of Kir4.1 to extracellular K⁺ management during neuronal activity. It is concluded that 1) the geometry of the experimental preparation is crucial for detection of Kir4.1-mediated spatial buffering and 2) Kir4.1 enacts spatial buffering of K⁺ during but not after neuronal activity.     

Determination of selected xenobiotics with ferrofluid-modified trypsin

Ferrofluid-modified trypsin has been used for the detection and determination of selected xenobiotics that inhibit trypsin activity. The procedure is useful especially when colored samples or samples containing suspended solid impurities are to be assayed. Ferrofluid-modified trypsin was inhibited by Ag⁺ and Pb²⁺, selected dyes (safranin, thionin), bacitracin and 4-aminobenzamidine. Enzymes immobilized on magnetic particles can form a basis of new automated assay procedures for the determination of xenobiotics.     

Extracellular charge adsorption influences intracellular electrochemical homeostasis in amphibian skeletal muscle

The membrane potential measured by intracellular electrodes, E_m, is the sum of the transmembrane potential difference (E₁) between inner and outer cell membrane surfaces and a smaller potential difference (E₂) between a volume containing fixed charges on or near the outer membrane surface and the bulk extracellular space. This study investigates the influence of E₂ upon transmembrane ion fluxes, and hence cellular electrochemical homeostasis, using an integrative approach that combines computational and experimental methods. First, analytic equations were developed to calculate the influence of charges constrained within a three-dimensional glycocalyceal matrix enveloping the cell membrane outer surface upon local electrical potentials and ion concentrations. Electron microscopy confirmed predictions of these equations that extracellular charge adsorption influences glycocalyceal volume. Second, the novel analytic glycocalyx formulation was incorporated into the charge-difference cellular model of Fraser and Huang to simulate the influence of extracellular fixed charges upon intracellular ionic homeostasis. Experimental measurements of E_m supported the resulting predictions that an increased magnitude of extracellular fixed charge increases net transmembrane ionic leak currents, resulting in either a compensatory increase in Na⁺/K⁺-ATPase activity, or, in cells with reduced Na⁺/K⁺-ATPase activity, a partial dissipation of transmembrane ionic gradients and depolarization of E_m.     

Inhibition of Na(+),K(+)-ATPase activity in hippocampus of rats subjected to acute administration of homocysteine is prevented by vitamins E and C treatment

In the present study we evaluated the effect of acute homocysteine (Hcy) administration on Na⁺,K⁺-ATPase activity, as well as on some parameters of oxidative stress such as total radical-trapping antioxidant potential (TRAP) and on activities of antioxidant enzymes catalase (CAT), superoxide dismutase and glutathione peroxidase in rat hippocampus. Results showed that Hcy significantly decreased TRAP, Na⁺,K⁺-ATPase and CAT activities, without affecting the activities of superoxide dismutase and glutathione peroxidase. We also verified the effect of chronic pretreatment with vitamins E and C on the reduction of TRAP, Na⁺,K⁺-ATPase and CAT activities caused by Hcy. Vitamins E and C per se did not alter these parameters, but prevented the reduction of TRAP, Na⁺,K⁺-ATPase and CAT activities caused by Hcy. Our results indicate that oxidative stress is probably involved in the pathogenesis of homocystinuria and that reduction of Na⁺,K⁺-ATPase activity may be related to the neuronal dysfunction found in homocystinuric patients.     

Oscillations of apoplasmic K+ and H+ activities in Desmodium motorium (Houtt.) Merril. pulvini in relation to the membrane potential of motor cells and leaflet movements

The lateral leaflets of Desmodium motorium exhibit rhythmic upward and downward movements with a period in the minute range. Apoplasmic K⁺ and H⁺ activities were monitored in situ in the abaxial part of the pulvini with ion-selective microelectrodes. An extracellular electric potential was recorded simultaneously. The apoplasmic H⁺ activity of all pulvini exhibiting a regular rhythm of the extracellular electric potential oscillated with the same period between about 10 and 20 mM. The apoplasmic K⁺ activity was high when the membrane potential of the motor cells was depolarized (about 36 mV) and the cells were shrunken. In contrast, the apoplasmic K⁺ activity was low in the swollen state of the motor cells, when the membrane potential was hyperpolarized (about -136 mV). The volatile anesthetic enflurane suppressed reversibly the movement of the leaflets. The same treatment also arrested spontaneous oscillations in the apoplasmic K⁺ activity in the pulvinus. The apoplasmic K⁺ activity oscillated roughly in phase with the K⁺ activity between pH 6.6 and 6.0. Application of white light disturbed the rhythm and increased the extracellular pH. Our results indicate that the physiological mechanism that drives the lateral leaflet movements of Desmodium motorium is closely related to the osmotic motors mediating the leaf movements of Mimosa, Samanea and Phaseolus.Abbreviations E_m membrane potential - E_ex extracellular electric potential - H_ex extracellular H⁺ activity - K_ex extracellular K⁺ activity - R_ex extracellular electrical resistance B. Antkowiak was supported by the Stiftung Volkswagenwerk.     

Fractal analysis and ionic dependence of endocytotic membrane activity of human breast cancer cells

The endocytic membrane activities of two human breast cancer cell lines (MDA-MB-231 and MCF-7) of strong and weak metastatic potential, respectively, were studied in a comparative approach. Uptake of horseradish peroxidase was used to follow endocytosis. Dependence on ionic conditions and voltage-gated sodium channel (VGSC) activity were characterized. Fractal methods were used to analyze quantitative differences in vesicular patterning. Digital quantification showed that MDA-MB-231 cells took up more tracer (i.e., were more endocytic) than MCF-7 cells. For the former, uptake was totally dependent on extracellular Na⁺ and partially dependent on extracellular and intracellular Ca²⁺ and protein kinase activity. Analyzing the generalized fractal dimension (D _q ) and its Legendre transform f(α) revealed that under control conditions, all multifractal parameters determined had values greater for MDA-MB-231 compared with MCF-7 cells, consistent with endocytic/vesicular activity being more developed in the strongly metastatic cells. All fractal parameters studied were sensitive to the VGSC blocker tetrodotoxin (TTX). Some of the parameters had a “simple” dependence on VGSC activity, if present, whereby pretreatment with TTX reduced the values for the MDA-MB-231 cells and eliminated the differences between the two cell lines. For other parameters, however, there was a “complex” dependence on VGSC activity. The possible physical/physiological meaning of the mathematical parameters studied and the nature of involvement of VGSC activity in control of endocytosis/secretion are discussed.     

Small-cell Lung Cancer (Human): Potentiation of Endocytic Membrane Activity by Voltage-gated Na<Superscript>+</Superscript> Channel Expression in Vitro

The possible functional role of voltage-gated Na⁺ channel (VGSC) expression in controlling endocytic membrane activity in human small-cell lung cancer (SCLC) cell lines (H69, H209, H510) was studied using uptake of horseradish peroxidase (HRP). The normal human airway epithelial (16HBE14o) cell line was used in a comparative approach. Uptake of HRP was vesicular, strongly temperature-sensitive and suppressed by cytoskeletal poisons (cytochalasin D and colchicine), consistent with endocytosis. Compared with the normal cells, HRP uptake into SCLC cells was kinetically more efficient, resulting in more than four-fold higher uptake under optimized conditions. Importantly, HRP uptake into SCLC cells was inhibited significantly by the specific VGSC blocker tetrodotoxin, as well as lidocaine and phenytoin. These effects were dose-dependent. None of these drugs had any effect on the uptake into the 16HBE14o cells. Uptake of HRP into SCLC cells was reduced by ∼66% in Na⁺-free medium and was partially (∼30%) dependent on extracellular Ca²⁺. The possibility that the endocytic activity in the H510 SCLC cells involved an endogenous cholinergic system was investigated by testing the effects of carbachol (a cholinergic receptor agonist) and eserine (an inhibitor of acetylcholinesterase). Both drugs inhibited HRP uptake, thereby suggesting that basal cholinergic activity occurred. It is concluded that VGSC upregulation could enhance metastatic cell behavior in SCLC by enhancing endocytic membrane activity.     

Control of the Amiloride-Sensitive Na+ Current in Salivary Duct Cells by Extracellular Sodium

We have previously reported that intralobular salivary duct cells contain an amiloride-sensitive Na⁺ conductance (probably located in the apical membranes). Since the amiloride-sensitive Na⁺ conductances in other tight epithelia have been reported to be controlled by extracellular (luminal) Na⁺, we decided to use whole-cell patch clamp techniques to investigate whether the Na⁺ conductance in salivary duct cells is also regulated by extracellular Na⁺. Using Na⁺-free pipette solutions, we observed that the whole-cell Na⁺ conductance increased when the extracellular Na⁺ was increased, whereas the whole-cell Na⁺ permeability, as defined in the Goldman equation, decreased. The dependency of the whole-cell Na⁺ conductance on extracellular Na⁺ could be described by the Michaelis-Menten equation with a K _m of 47.3 mmol/1 and a maximum conductance (G _max) of 2.18 nS. To investigate whether this saturation of the Na⁺ conductance with increasing extracellular Na⁺ was due to a reduction in channel activity or to saturation of the single-channel current, we used fluctuation analysis of the noise generated during the onset of blockade of the Na⁺ current with 200 μmol/l 6-chloro-3,5-diaminopyrazine-2-carboxamide. Using this technique, we estimated the single channel conductance to be 4 pS when the channel was bathed symmetrically in 150 mmol/l Na⁺ solutions. We found that Na⁺ channel activity, defined as the open probability multiplied by the number of available channels, did not alter with increasing extracellular Na⁺. On the other hand, the single-channel current saturated with increasing extracellular Na⁺ and, consequently, whole-cell Na⁺ permeability declined. In other words, the decline in Na⁺ permeability in salivary duct cells with increasing extracellular Na⁺ concentration is due simply to saturation of the single-channel Na⁺ conductance rather than to inactivation of channel activity. Received: 27 July 1995/Revised: 7 December 1995     

THE EFFECT OF EXTRACELLULAR POTASSIUM CONCENTRATION ON PROTEIN SYNTHESIS IN GUINEA-PIG HIPPOCAMPAL SLICES1,2

Abstract— We have measured the effect of small variations in extracellular potassium concentrations ([K⁺]) upon the incorporation of radioactively labelled amino acid into the protein of the isolated guinea-pig hippocampal slice. The slice is super-perfused with glucose fortified buffer and maintains an ATP concentration of 33–36 nmol/mg protein and incorporates lysine into protein at a rate of 0.82 pmol/(ig protein/h. Within the range of extracellular K⁺ from 1.3 to 8.1 mil the change in the rate of lysine incorporation into protein is proportional to the logarithm of the extracellular K⁺ concentration. Incorporation increases by about 100% over this range. Measurements of the specific activity of the presumed intracellular amino acid pool indicate that the effect of changes in extracellular [K⁺] is to alter the rate of protein synthesis rather than alter the availability of radioactively labelled precursor. Altering extracellular [K⁺] does not affect tissue levels of ATP or creatine phosphate, indicating that the effect on amino acid incorporation does not result from an effect upon energy metabolism. It is suggested that this effect of extracellular [K.⁺] may be a means by which changes in cerebral electrical activity lead to changes in the rate of protein synthesis in brain.     

Dependence of the intracellular concentrations of univalent ions and hydrogenase activity on the salt composition and pH of the medium in the haloalkaliphilic sulfate-reducing bacterium <Emphasis Type="Italic">Desulfonatronum thiodismutans</Emphasis>

It has been shown that the intracellular concentrations of Na⁺, K⁺, and Cl^? ions in Desulfonatronum thiodismutans depend on the extracellular concentration of Na⁺ ions. An increase in the extracellular concentration of Na⁺ results in the accumulation of K⁺ ions in cells, which points to the possibility that these ions perform an osmoprotective function. When the concentration of the NaCl added to the medium was increased to 4%, the concentration gradient of Cl^? ions changed insignificantly. It was found that D. thiodismutans contains two forms of hydrogenase—periplasmic and cytoplasmic. Both enzymes are capable of functioning in solutions with high ionic force; however they exhibit different sensitivities to Na⁺, K⁺, and Li⁺ salts and pH. The enzymes were found to be resistant to high concentrations of Na⁺ and K⁺ chlorides and Na⁺ bicarbonate. The cytoplasmic hydrogenase differed significantly from the periplasmic one in having much higher salt tolerance and lower pH optimum. The activity of these enzymes depended on the nature of both the cationic and anionic components of the salts. For instance, the inhibitory effect of NaCl was less pronounced than that of LiCl, whereas Na⁺ and Li⁺ sulfates inhibited the activity of both hydrogenase types to an equal degree. The highest activity of these enzymes was observed at low Na⁺ concentrations, close to those typical of cells growing at optimal salt concentrations.     

DPNH peroxidase: effector activities of DPN.

At suboptimal H₂O₂ concentrations, DPNH inhibits the peroxidase activity of the flavoprotein, DPNH peroxidase, by converting the enzyme to an unstable intermediate that decays slowly to inactive enzyme. It is postulated that at concentrations of DPNH that are saturating for peroxidase activity, this unstable intermediate is responsible for most of the DPNH oxidation that is supported by alternate electron acceptors, such as O₂ and menadione. DPN⁺ behaves as an activator by reversing the equilibria that lead to the unstable intermediate, thus converting the enzyme to the kinetically active complex that reduces H₂O₂. The data show that DPN⁺ binding will stimulate the peroxidase activity (by lowering the K_m for H₂O₂) and simultaneously lead to strong inhibition of both the rate of enzyme inactivation and the rate at which DPNH is oxidized by alternate electron acceptors.     

Activity-Dependent Adenosine Release May Be Linked to Activation of Na+-K+ ATPase: An In Vitro Rat Study

In the brain, extracellular adenosine increases as a result of neuronal activity. The mechanisms by which this occurs are only incompletely understood. Here we investigate the hypothesis that the Na⁺ influxes associated with neuronal signalling activate the Na⁺-K⁺ ATPase which, by consuming ATP, generates intracellular adenosine that is then released via transporters. By measuring adenosine release directly with microelectrode biosensors, we have demonstrated that AMPA-receptor evoked adenosine release in basal forebrain and cortex depends on extracellular Na⁺. We have simultaneously imaged intracellular Na⁺ and measured adenosine release. The accumulation of intracellular Na⁺ during AMPA receptor activation preceded adenosine release by some 90 s. By removing extracellular Ca²⁺, and thus preventing indiscriminate neuronal activation, we used ouabain to test the role of the Na⁺-K⁺ ATPase in the release of adenosine. Under conditions which caused a Na⁺ influx, brief applications of ouabain increased the accumulation of intracellular Na⁺ but conversely rapidly reduced extracellular adenosine levels. In addition, ouabain greatly reduced the amount of adenosine released during application of AMPA. Our data therefore suggest that activity of the Na⁺-K⁺ ATPase is directly linked to the efflux of adenosine and could provide a universal mechanism that couples adenosine release to neuronal activity. The Na⁺-K⁺ ATPase-dependent adenosine efflux is likely to provide adenosine-mediated activity-dependent negative feedback that will be important in many diverse functional contexts including the regulation of sleep.     

Nickel toxicity and peroxidase activity in seedlings of Triticum aestivum L.

Ni²⁺ toxicity was evaluated in Triticum aestivum L. by its effects on root and shoot length, dry matter production and water content. Over a threshold value of 20 mmol m^?3 Ni²⁺ the degree of toxicity increases as a function of the Ni²⁺ concentration in the medium. Ni²⁺-treated roots show enhanced lipid peroxidation; the higher Ni²⁺ treatment (40mmol m^?3) also increases leakage of K⁺. In roots and shoots, Ni²⁺ enhances both guaiacol and syringaldazine extracellular peroxidase activity. The increase in extracellular peroxidase activity is also associated with an increase in the phenolic contents of roots and shoots. The observed growth inhibition might be partly the result of the effect of Ni²⁺ on cell turgor and cell-wall extensibility. Intracellular soluble peroxidases are also stimulated by Ni²⁺; such effects, independently of the substrate, were detected in extracts of Ni²⁺-treated shoots at a lower Ni²⁺ concentration than in the roots. Intracellular peroxidases might act as scavengers of peroxide radicals produced as a result of nickel toxicity.     

Involvement of extracellular oxidative burst in salicylic acid-induced stomatal closure in Arabidopsis

Salicylic acid (SA), a ubiquitous phenolic phytohormone, is involved in many plant physiological processes including stomatal movement. We analysed SA‐induced stomatal closure, production of reactive oxygen species (ROS) and nitric oxide (NO), cytosolic calcium ion ([Ca²⁺]_cyt) oscillations and inward‐rectifying potassium (K⁺_in) channel activity in Arabidopsis. SA‐induced stomatal closure was inhibited by pre‐treatment with catalase (CAT) and superoxide dismutase (SOD), suggesting the involvement of extracellular ROS. A peroxidase inhibitor, SHAM (salicylhydroxamic acid) completely abolished SA‐induced stomatal closure whereas neither an inhibitor of NADPH oxidase (DPI) nor atrbohD atrbohF mutation impairs SA‐induced stomatal closures. 3,3′‐Diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) stainings demonstrated that SA induced H₂O₂ and O₂^– production. Guard cell ROS accumulation was significantly increased by SA, but that ROS was suppressed by exogenous CAT, SOD and SHAM. NO scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO) suppressed the SA‐induced stomatal closure but did not suppress guard cell ROS accumulation whereas SHAM suppressed SA‐induced NO production. SA failed to induce [Ca²⁺]_cyt oscillations in guard cells whereas K⁺_in channel activity was suppressed by SA. These results indicate that SA induces stomatal closure accompanied with extracellular ROS production mediated by SHAM‐sensitive peroxidase, intracellular ROS accumulation and K⁺_in channel inactivation.