Fusicoccin-induced catalase inhibitor is produced independently of H+-ATPase activation and behaves as an organic acid

The phytotoxin fusicoccin (FC) was found to induce an increase in apoplastic H₂O₂ content in Arabidopsis thaliana cells, apparently linked to the presence of an as yet unidentified catalase inhibitor detectable even in the external medium of FC‐treated cells. This study, aimed to further characterize the inhibitor's features, shows that (1) FC‐induced H₂O₂ accumulation increases as a function of FC concentration and correlates to the amount of inhibitor released at apoplastic level. The pattern of H⁺ efflux, conversely, does not fit with that of these two parameters, suggesting that neither the production nor the release of the catalase inhibitor is linked to the main role of FC in activating the plasma membrane (PM) H⁺‐ATPase; (2) treatment with 10 µM erythrosin B (EB) early and totally inhibits net H⁺ and K⁺ fluxes across the PM, indicative of the H⁺ pump activity; nevertheless, also in these conditions a huge FC‐induced H₂O₂ accumulation occurs, confirming that this effect is not related to the FC‐induced PM H⁺‐ATPase activation; (3) the inhibitor's release increases with time in all conditions tested and is markedly affected by extracellular pH (a higher pH value being associated to a larger efflux), in agreement with a weak acid release; and (4) the inhibitor can be almost completely recovered in a CH₂Cl₂‐soluble fraction extracted from the incubation medium by sequential acid–base partitioning which contains nearly all of the organic acids released. These final results strongly suggest that the metabolite responsible for the FC‐induced catalase inhibition belongs to the organic acid class.     

Effects of aphids Myzus persicae on the changes of Ca2+ and H2O2 flux and enzyme activities in tobacco

In order to understand the continuous defense reactions of host plants against insect attack, a tobacco variety G140 was infested by tobacco aphid Myzus persicae for 2 h to 5 d. The changes of transmembrane ionic fluxes (Ca²⁺) and hydrogen peroxide (H₂O₂) were detected by the technique of noninvasive micro-test and their relationship was further studied. It was found that H₂O₂ accumulation depended on Ca²⁺ influx. Ca²⁺ flux exhibited a strong influx at all infestation periods by aphids, while H₂O₂ showed an efflux behavior. The slight variation tendency of Ca²⁺ influx and H₂O₂ efflux was consistent. The activities of the corresponding defense proteins, peroxidase (POD) and catalase (CAT) enzyme, were enhanced to respond to the insect attacks, much higher than those tobacco in control. The Ca²⁺ influx and H₂O₂ efflux, as well as the activities of POD and CAT enzymes, were increased in a long period of aphid feeding. It indicated that a continuous physiological response of tobacco to aphid infestation could be initiated and lasted for a long time.     

Potential role of Noxes in the protection of mucosae: H2O2 as abacterial repellent

Duox proteins are members of the NADPH oxidase (Nox) family and are responsible for hydrogen peroxide (H₂O₂) production by various tissue types including bronchial and intestinal mucosae. The antimicrobial killing role of H₂O₂ in leukocytes and macrophages is generally considered as the paradigm of its function. We investigated here the positive role of H₂O₂ in the prevention of cellular invasion by Salmonella. We show that H₂O₂, under conditions that preserved bacterial growth, has a repellent effect on Salmonella motility on agar plates. In addition, H₂O₂ produced by PCCl3, a rat thyroid cell line, reduces bacterial invasion of the cells by around 40%. To test whether the observed phenotype is attributable to H₂O₂ production, we constructed a CHO stable cell line expressing Duox2 protein at the cell surface (CHO-D2). The transfected cells produce a high amount of H₂O₂. Upon infection with Salmonella, the invasion of CHO-D2 cells was reduced by up to 60%. In both PCCl3 and CHO expressing Duox2 cells, normal invasion was restored upon incubation with catalase. Our data suggest that H₂O₂ at reduced concentrations acts as a repellent for bacteria, keeping them away from cells, a situation that could naturally prevent mucosal cells infection in vivo.     

Inactivation of human pathogens and spoilage bacteria on the surface and internalized within fresh produce by using a combination of ultraviolet light and hydrogen peroxide

Aims: To evaluate the efficacy of ultraviolet (UV) light (254 nm) combined with hydrogen peroxide (H₂O₂) to inactivate bacteria on and within fresh produce. Methods and Results: The produce was steep inoculated in bacterial cell suspension followed by vacuum infiltration. The inoculated samples were sprayed with H₂O₂ under constant UV illumination. The log count reduction (LCR) of Salmonella on and within lettuce was dependent on the H₂O₂ concentration, temperature and treatment time with UV intensity being less significant. By using the optimized parameters (1·5% H₂O₂ at 50°C, UV dose of 37·8 mJ cm^?2), the surface Salmonella were reduced by 4·12 ± 0·45 and internal counts by 2·84 ± 0·34 log CFU, which was significantly higher compared with H₂O₂ or UV alone. Higher LCR of Escherichia coli O157:H7, Pectobacterium carotovora, Pseudomonas fluorescens and Salmonella were achieved on leafy vegetables compared with produce, such as cauliflower. In all cases, the surface LCR were significantly higher compared with the samples treated with 200 ppm hypochlorite. UV–H₂O₂‐treated lettuce did not develop brown discolouration during storage but growth of residual survivors occurred with samples held at 25°C. Conclusions: UV–H₂O₂ reduce the bacterial populations on and within fresh produce without affecting the shelf‐life stability. Significance of the Study: UV–H₂O₂ represent an alternative to hypochlorite washes to decontaminate fresh produce.     

Mass spectrometry-based quantitative proteomic analysis of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Enteritidis protein expression upon exposure to hydrogen peroxide

Background  

Salmonella enterica, a common food-borne bacterial pathogen, is believed to change its protein expression profile in the presence of different environmental stress such as that caused by the exposure to hydrogen peroxide (H₂O₂), which can be generated by phagocytes during infection and represents an important antibacterial mechanism of host cells. Among Salmonella proteins, the effectors of Salmonella pathogenicity island 1 and 2 (SPI-1 and SPI-2) are of particular interest since they are expressed during host infection in vivo and are important for invasion of epithelial cells and for replication in organs during systemic infection, respectively. However, the expression profiles of these proteins upon exposure to H₂O₂ or to host cells in vivo during the established phase of systemic infection have not been extensively studied.     

Selection of Vaginal H2O2-Generating Lactobacillus Species for Probiotic Use

Lactobacilli are believed to contribute to the control of the vaginal microflora by different mechanisms such as production of antagonistic substances like lactic acid, bacteriocins, and H₂O₂. This paper describes the selection of H₂O₂-generating lactobacilli among 35 hydrophobic isolates from the human vagina. Lactobacillus crispatus F117, which generated the highest H₂O₂ level, was chosen to study: (a) the kinetics of H₂O₂ production considering different culture conditions, and (b) the effect of this metabolite on the growth of urogenital tract pathogens. The levels of H₂O₂ in L. crispatus supernatant increased during its growth and were maximum at the early stationary phase (3.29 mmol H₂O₂L⁻¹) under aerated conditions (agitated cultures). In nonagitated cultures there were no detectable levels of H₂O₂. L. crispatus F117 spent supernatant inhibited Staphylococcus aureus growth in plaque assay. Inhibition was due to H₂O₂ since catalase treatment of the supernatant suppressed inhibition. In mixed cultures performed with L. crispatus and S. aureus a significant decrease in pathogen growth was observed. The inhibitory effect depended on the initial inoculum of S. aureus. Further evaluation of the properties of L. crispatus F117 will be performed to consider its inclusion in a probiotic for local use in the vaginal tract. Received: 17 November 1998 / Accepted: 17 December 1998     

Nitric oxide and hydrogen peroxide are important signals mediating the allelopathic response of Arabidopsis to p‐hydroxybenzoic acid

Both nitric oxide (NO) and hydrogen peroxide (H₂O₂) are important signals that mediate plant response to environmental stimulation. Their role in plants' allelopathic interactions has also been reported, but the underlying mechanism remains little understood. p‐Hydroxybenzoic acid (pHBA) has been proposed to be an allelopathic chemical. Here, we found that pHBA at 0.4 mM efficiently suppressed Arabidopsis growth. Meanwhile, pHBA rapidly induced the accumulation of NO and H₂O₂, where such effect could be reversed by NO or H₂O₂ metabolism inhibitors or scavengers. Also, pHBA‐induced NO and H₂O₂ could be compromised in NO synthesis mutants noa1, nia1 and nia2, or H₂O₂ metabolism mutant rbohD/F, but suppressing NO accumulation with a NO synthesis inhibitor or using NO synthesis‐related mutants did not reduce pHBA‐induced H₂O₂ accumulation. Furthermore, we found that the effect of pHBA on allelopathic inhibition of growth was aggravated in NO/H₂O₂ metabolism‐related mutants or reducing NO/H₂O₂ by different inhibitors, whereas the addition of an NO/H₂O₂ donor could partly relieve the inhibitory effect of pHBA on the growth of wild type. However, adding only an NO donor, but not low concentration of H₂O₂ as the donor, could relieve the inhibitory effect of pHBA on root growth in NO metabolism mutants. On the basis of these results, we propose that both NO and H₂O₂ are important signals that mediate Arabidopsis response to the allelopathic chemical pHBA, where during this process H₂O₂ may work upstream of the NO signal.     

Formation of myeloperoxidase compound II during aerobic stimulation of rat neutrophils

We have made simultaneous spectrophotometric and O₂ measurements on suspensions of rat neutrophils during activation of the respiratory burst. Under aerobic conditions an absorption increase attributable to myeloperoxidase compound II was observed in parallel with the rapid phase of O₂ uptake. Identification of this compound was confirmed by analysis of a spectrum obtained with purified myeloperoxidase and H₂O₂. Whereas a second addition of stimulus did not increase O₂ uptake any further, a second phase of myeloperoxidase release and compound II formation was observed. These results suggest thatin vivo myeloperoxidase reacts with H₂O₂ generatedvia the respiratory burst to form compound II under conditions in which the chlorination reaction would be the expected major pathway.Abbreviations FMLP N-formylmethionylleucyl phenylalanine - MPO²⁺.H₂O₂ Myeloperoxidase compound II - MPO³⁺.H₂O₂ Myeloperoxidase compound I - {ei275-1} superoxide     

Metabolism of hydrogen peroxide in isolated hepatocytes: Relative contributions of catalase and glutathione peroxidase in decomposition of endogenously generated H2O2

The compartmentation of hydrogen peroxide catabolism was studied in isolated hepatocytes. Hydrogen peroxide generation in the peroxisomal compartment was stimulated by addition of glycolate and in the endoplasmic reticular compartment (cytosolic compartment) by ethylmorphine. The rate of catabolism by catalase was estimated from the concentration of methanol required to decrease the steady-state concentration of catalase Compound I to the half-maximal value. The rate of catabolism by glutathione peroxidase was assessed in a semiquantitative manner by the rate of GSSG efflux. The relationship of GSSG efflux to catalase-dependent metabolism of H₂O₂ in the presence of increasing concentrations of glycolate was sigmoidal. This indicates that the function of glutathione peroxidase is small relative to that of catalase at low rates of H₂O₂ production in the peroxisomal fraction, but that the contribution of the former system increases as the peroxisomal H₂O₂ production rate is enhanced, and suggests that the accumulation of a steady-state concentration of H₂O₂ in the nanomolar range in the peroxisomes is sufficient to allow diffusion of H₂O₂ into the cytosol. Following pretreatment of animals with aminotriazole to inhibit catalase, glycolate caused GSSG release at rates nearly double those in control cells. This indicates that even incomplete inhibition of catalase in cells can result in enhanced release of H₂O₂ into the cytosol and demonstrates the relationship of GSSG release to H₂O₂ production under these conditions. An estimate of the rate of H₂O₂ diffusion to catalase during ethylmorphine metabolism was made from the steady-state level of Compound I and measured formate concentrations. This rate increased threefold as the rate of GSH loss increased from 1 to 2 nmol/10⁶ cells per min, indicating that as the rate of H₂O₂ production in the endoplasmic reticulum becomes maximally stimulated in the presence of ethylmorphine, the rate of H₂O₂ metabolism by catalase becomes larger. A comparison of ethylmorphine-stimulated rates of GSSG efflux from cells of control and aminotriazole-treated rats shows that, unlike experiments with glycolate, no difference in the rate of efflux is observed. These results support the conclusion that in hepatocytes catalase has a relatively minor role in catabolism of H₂O₂ at low rates of H₂O₂ generation in the endoplasmic reticulum, but that the catalase function increases as the rate of H₂O₂ production is enhanced.     

Melatonin delays ABA-induced leaf senescence via H2O2-dependent calcium signalling

Precocious leaf senescence can reduce crop yield and quality by limiting the growth stage. Melatonin has been shown to delay leaf senescence; however, the underlying mechanism remains obscure. Here, we show that melatonin offsets abscisic acid (ABA) to protect photosystem II and delay the senescence of attached old leaves under the light. Melatonin induced H₂O₂ accumulation accompanied by an upregulation of melon respiratory burst oxidase homolog D (CmRBOHD) under ABA-induced stress. Both melatonin and H₂O₂ induced the accumulation of cytoplasmic-free Ca²⁺ ([Ca²⁺]_cyt) in response to ABA, while blocking of Ca²⁺ influx channels attenuated melatonin- and H₂O₂-induced ABA tolerance. CmRBOHD overexpression induced [Ca²⁺]_cyt accumulation and delayed leaf senescence, whereas deletion of Arabidopsis AtRBOHD, a homologous gene of CmRBOHD, compromised the melatonin-induced [Ca²⁺]_cyt accumulation and delay of leaf senescence in Arabidopsis under ABA stress. Furthermore, melatonin, H₂O₂ and Ca²⁺ attenuated ABA-induced K⁺ efflux and subsequent cell death. CmRBOHD overexpression and AtRBOHD deletion alleviated and aggravated the ABA-induced K⁺ efflux, respectively. Taken together, our study unveils a new mechanism by which melatonin offsets ABA action to delay leaf senescence via RBOHD-dependent H₂O₂ production that triggers [Ca²⁺]_cyt accumulation and subsequently inhibits K⁺ efflux and delays cell death/leaf senescence in response to ABA.     

H2O2 and cytosolic Ca2+ signals triggered by the PM H+‐coupled transport system mediate K+/Na+ homeostasis in NaCl‐stressed Populus euphratica cells

Using confocal microscopy, X‐ray microanalysis and the scanning ion‐selective electrode technique, we investigated the signalling of H₂O₂, cytosolic Ca²⁺ ([Ca²⁺]_cyt) and the PM H⁺‐coupled transport system in K⁺/Na⁺ homeostasis control in NaCl‐stressed calluses of Populus euphratica. An obvious Na⁺/H⁺ antiport was seen in salinized cells; however, NaCl stress caused a net K⁺ efflux, because of the salt‐induced membrane depolarization. H₂O₂ levels, regulated upwards by salinity, contributed to ionic homeostasis, because H₂O₂ restrictions by DPI or DMTU caused enhanced K⁺ efflux and decreased Na⁺/H⁺ antiport activity. NaCl induced a net Ca²⁺ influx and a subsequent rise of [Ca²⁺]_cyt, which is involved in H₂O₂‐mediated K⁺/Na⁺ homeostasis in salinized P. euphratica cells. When callus cells were pretreated with inhibitors of the Na⁺/H⁺ antiport system, the NaCl‐induced elevation of H₂O₂ and [Ca²⁺]_cyt was correspondingly restricted, leading to a greater K⁺ efflux and a more pronounced reduction in Na⁺/H⁺ antiport activity. Results suggest that the PM H⁺‐coupled transport system mediates H⁺ translocation and triggers the stress signalling of H₂O₂ and Ca²⁺, which results in a K⁺/Na⁺ homeostasis via mediations of K⁺ channels and the Na⁺/H⁺ antiport system in the PM of NaCl‐stressed cells. Accordingly, a salt stress signalling pathway of P. euphratica cells is proposed.     

A dynamic model for photosynthesis by an aquatic plant, Egeria densa

Abstract This paper describes a dynamic model for photosynthesis by an aquatic plant, Egeria densa. The model takes into account an HCO^?₃ pump, high diffusion resistances and PEP carboxylase, and develops a set of differential equations to form the time-dependent solutions for photosynthesis. The predicted changes in pH, [CO₂]_aq and total inorganic carbon are compared with experimental data and the model is found to describe the data. The model is then used to examine the effect of O₂ on photosynthesis under these conditions, and shows that the increase in internal CO₂ concentration due to the recycling of photorespiratory CO₂ directly stimulates gross CO₂ fixation and can more than compensate for the O₂ inhibition of gross photosynthesis. The importance of the HCO^?₃ pump in O₂ inhibition is also examined. The CO₂ compensation point (where inorganic carbon influx and efflux are equal) is examined and the importance of the HCO^?₃ pump and PEP carboxylase in reducing the compensation concentration is discussed. The model was developed in order to study the photosynthesis of an aquatic weed, which will be reported in a later paper.     

The effect of polar auxin transport on adventitious branches formation in Gracilaria lichenoides in vitro

Seaweed tissue culture (STC) is an important micropropagation tool that has been applied for strain improvement, micropropagation and genetic engineering. Because the mechanisms associated with STC are poorly understood, its application to these organisms lags far behind that of tissue culture propagation of higher plants. Auxin, calcium (Ca²⁺) and hydrogen peroxide (H₂O₂) fluxes all play key roles during plant growth and development. In this study, we therefore measured indole‐3‐acetic acid, Ca²⁺ and H₂O₂ fluxes of Gracilaria lichenoides explants during adventitious branches (ABs) formation for the first time using noninvasive micro‐test technology. We confirmed that polar auxin transport (PAT) also occurs in the marine red alga G. lichenoides. We additionally found that N‐1‐naphthylphthalamic acid may suppress auxin efflux via ABCB1 transporters and then inhibit ABs formation from the apical region of G. lichenoides segments. The involvement of Ca²⁺ and H₂O₂ fluxes in PAT‐mediated AB formation in G. lichenoides was also investigated. We propose that complex feedback among Ca²⁺, H₂O₂ and auxin signaling and response systems may occur during ABs polar formation in G. lichenoides explants, similar to that in higher plants. Our results provide innovative insights that should aid future elucidation of mechanisms operative during STC.     

A New γ-Dihydropyrone from Streptomyces sp. as a Microtubule Association Inhibitor toward Pronuclear Fusion in Sea Urchin Eggs

A conidia suspension of Magnaporthe grisea carried elicitor activity that induced the expression of defense-related genes and the production of H₂O₂ in suspension-cultured rice cells. The levels of H₂O₂ produced were dependent on fungal isolates and were correlated with the catalase activity in the supernatant fraction of each conidia suspension, not with gene-for-gene interactions.     

Structure-activity relationships of 2-aryl-1H-indole inhibitors of the NorA efflux pump in Staphylococcus aureus

The synthesis of 22 2-aryl-1H-indoles, including 12 new compounds, has been achieved via Pd- or Rh-mediated methodologies, or selective electrophilic substitution. All three methods were based on elaborations from simple indole precursors. SAR studies on these indoles and 2-phenyl-1H-indole in Staphylococcus aureus as NorA efflux pump inhibitors indicated 5-nitro-2-(3-methoxycarbonyl)phenyl-1H-indole was a slightly more potent inhibitor than the lead INF55. A promising new antibacterial lead compound against S. aureus (2-phenyl-1H-indol-5-yl)-methanol, was also found.     

Functional roles of superoxide and hydrogen peroxide generated by mitochondrial DNA mutation in regulating tumorigenicity of HepG2 cells

Mitochondria are a major source of reactive oxygen species (ROS). Recent studies have estimated that mitochondrial DNA mutations inducing the overproduction of ROS are associated with human cancer. However, a substantial challenge in elucidating their diverse roles in regulating tumorigenesis is the lack of methods for probing ROS in living systems with molecular specificity. In this study, we reported the application of two fluorescent probes, 2‐chloro‐1,3‐dibenzothiazolinecyclohexene and naphthofluorescein disulfonate, which showed high selectivity for superoxide (O₂^•−) and hydrogen peroxide (H₂O₂). They were capable of detecting and visualizing O₂^•− and H₂O₂ overproduction caused by a mutation in the gene encoding nicotinamide adenine dinucleotide dehydrogenase subunit 6 (ND6) in HepG2 cells. The levels of O₂^•− and H₂O₂ in mitochondria isolated from HepG2 cells were found to be 0·63 ± 0·07 and 1·13 ± 0·05 μM, respectively. Using assays of tumorigenesis in mouse models, we found that treatment of the mice with different ROS scavengers suppressed tumour growth. These findings suggested that ROS generated by ND6 gene mutation do play an important role in regulating tumorigenesis and H₂O₂ may be a key modulator. Copyright © 2011 John Wiley & Sons, Ltd.     

Cross-talks between Ca<Superscript>2+</Superscript>/CaM and H<Subscript>2</Subscript>O<Subscript>2</Subscript> in abscisic acid-induced antioxidant defense in leaves of maize plants exposed to water stress

Here we examined whether Ca²⁺/Calmodulin (CaM) is involved in abscisic acid (ABA)-induced antioxidant defense and the possible relationship between CaM and H₂O₂ in ABA signaling in leaves of maize (Zea mays L.) plants exposed to water stress. An ABA-deficient mutant vp5 and its wild type were used for the experimentation. We found that water stress enhanced significantly the contents of CaM and H₂O₂, and the activities of chloroplastic and cytosolic superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and the gene expressions of the CaM1, cAPX, GR1 and SOD4 in leaves of wild-type maize. However, the increases mentioned above were almost arrested in vp5 plants and in the wild-type plants pretreated with ABA biosynthesis inhibitor tungstate (T), suggesting that ABA is required for water stress-induced H₂O₂ production, the enhancement of CaM content and antioxidant defense. Besides, we showed that the up-regulation of water stress-induced antioxidant defense was almost completely blocked by pretreatment with Ca²⁺ inhibitors, CaM antagonists and reactive oxygen (ROS) manipulators. Moreover, the analysis of time course of CaM and H₂O₂ production under water stress showed that the increase in CaM content preceded that of H₂O₂. These results suggested that Ca²⁺/CaM and H₂O₂ were involved in the ABA-induced antioxidant defense under water stress, and the increases of Ca²⁺/CaM contents triggered H₂O₂ production, which inversely affected the contents of CaM. Thus, a cross-talk between Ca²⁺/CaM and H₂O₂ may play a pivotal role in the ABA signaling.     

Oxidative stress impairs autophagic flux in prion protein-deficient hippocampal cells

We previously reported that autophagy is upregulated in Prnp-deficient (Prnp^0/0) hippocampal neuronal cells in comparison to cellular prion protein (PrP^C)-expressing (Prnp^+/+) control cells under conditions of serum deprivation. In this study, we determined whether a protective mechanism of PrP^C is associated with autophagy using Prnp^0/0 hippocampal neuronal cells under hydrogen peroxide (H₂O₂)-induced oxidative stress. We found that Prnp^0/0 cells were more susceptible to oxidative stress than Prnp^+/+ cells in a dose- and time-dependent manner. In addition, we observed enhanced autophagy by immunoblotting, which detected the conversion of microtubule-associated protein 1 light chain 3 β (LC3B)-I to LC3B-II, and we observed increased punctate LC3B immunostaining in H₂O₂-treated Prnp^0/0 cells compared with H₂O₂-treated control cells. Interestingly, this enhanced autophagy was due to impaired autophagic flux in the H₂O₂-treated Prnp^0/0 cells, while the H₂O₂-treated Prnp^+/+ cells showed enhanced autophagic flux. Furthermore, caspase-dependent and independent apoptosis was observed when both cell lines were exposed to H₂O₂. Moreover, the inhibition of autophagosome formation by Atg7 siRNA revealed that increased autophagic flux in Prnp^+/+ cells contributes to the prosurvival effect of autophagy against H₂O₂ cytotoxicity. Taken together, our results provide the first experimental evidence that the deficiency of PrP^C may impair autophagic flux via H₂O₂-induced oxidative stress.     

Chloroplast-generated ROS dominate NaCl- induced K+ efflux in wheat leaf mesophyll

Mesophyll K⁺ retention ability has been recently reported as an important component of salinity stress tolerance in wheat. In order to investigate the role of ROS in regulating NaCl^-induced K⁺ efflux in wheat leaf mesophyll, a series of pharmacological experiments was conducted using MV (methyl viologen, superoxide radical inducer), DPI (an inhibitor of NADPH oxidase), H₂O₂ (to mimic apoplastic ROS), and EGCG ((−)-Epigallocatechin gallate, ROS scavenger). Mesophyll pre-treatment with 10 μM MV resulted in a significantly higher NaCl^-induced K⁺ efflux in leaf mesophyll, while 50 μM EGCG pre-treatment alleviated K⁺ leakage under salt stress. No significant change in NaCl^-induced K⁺ efflux in leaf mesophyll was found in specimens pre-treated by H₂O₂ and DPI, compared with the control. The highest NaCl^-induced H⁺ efflux in leaf mesophyll was also found in samples pre-treated with MV, suggesting a futile cycle between increased H⁺-ATPase activity and ROS-induced K⁺ leak. Overall, it is suggested that, under saline stress, K⁺ efflux from wheat mesophyll is mediated predominantly by non-selective cation channels (NSCC) regulated by ROS produced in chloroplasts, at least in bread wheat.