Internal H2O2 Research Articles

Assessing the response lettuce and arugula to MC-LR-contaminated water irrigation: photosynthetic changes and antioxidant defense.

Irrigation of crops with cyanotoxin-contaminated water poses a significant risk to human health. The direct phytotoxic effects of microcystin-LR (MC-LR), one of the most toxic and prevalent microcystin variants in water bodies, can induce physiological stress and hinder crop development and production. This study investigated the impact of environmentally relevant concentrations of MC-LR (1 to 10µg L-1) on photosynthetic parameters and antioxidant response of lettuce (Lactuca sativa L.) and arugula (Eruca sativa L.) following irrigation with contaminated water. During the 15-day experiment, lettuce and arugula were exposed to various concentrations of MC-LR, and their photosynthetic rates, stomatal conductance, leaf tissue transpiration, and intercellular CO2 concentrations were measured using an infrared gas analyzer. These results suggest that the influence of MC-LR on gas exchange in crops is concentration-dependent, with notable disruptions during exposure and recovery tendency during detoxification. Antioxidant response analysis revealed that glutathione S-transferase (GST) and superoxide dismutase (SOD) activities were upregulated during the exposure phase in the presence of MC-LR. However, GST activity decreased during the detoxification phase in both crops, although the effects of the toxin at 10µg L-1 were still evident in arugula. The internal H2O2 concentration in the crops increased after exposure to MC-LR, showing a time- and concentration-dependent pattern, with an increase during the exposure phase (days 1-7) and a decrease during the detoxification phase (days 8-15). Irrigation of lettuce and arugula with MC-LR-contaminated water affected various aspects of the photosynthetic apparatus and antioxidant responses, which could influence the general health and productivity of exposed crops at environmentally relevant microcystin concentrations. Furthermore, investigation of additional vegetable species and long-term MC-LR exposure can be crucial for understanding the extent of contamination risk, detoxification mechanisms, and other parameters affecting these crops.

Hypergolic Ignition by Off-Center Binary Collision of Monoethanolamine-NaBH4 and Hydrogen Peroxide Droplets

Hypergolic ignition of H2O2 and MEA-NaBH4 by off-center collision of their droplets was experimentally studied, focusing on the characteristics and mechanism of droplet mixing, droplet heating and evaporation, and gas-phase ignition. The whole collision ignition process was divided into five stages, which were compared, respectively, with that of head-on collision. Under the condition of a slightly off-center collision (for cases where B < 0.35), H2O2 droplets penetrate MEA-NaBH4 droplets after the collision and coalesce with it, but the internal H2O2 drop inside the MEA-NaBH4 droplet does not form a stable sphere. Instead, it rotates and expands inside the mixed droplet. With B increasing to 0.59, the droplets no longer coalesce after collision but separate away, forming satellite droplets. In such cases, multi-ignition mode is observed. When B increases to a certain extent, specifically, 0.85, a grazing collision is observed such that no mass transfer exists during the interaction of droplets, which leads to ignition failure. A theoretical model quantifying droplet swelling rate was established to calculate the volume change of the droplet. It was found that the swelling can be attributed to the flash boiling of superheated internal H2O2 fluid. Meanwhile, the ignition delay time was found to linearly decrease with B at various Wes until the extent where the chemical reaction takes over control, leading to an almost constant time delay defined as RDT. Additionally, the regime of ignition modes corresponding to different droplet mixing features is summarized in the We-B parametric space.

Mechanistic modelling of solar disinfection (SODIS) kinetics of Escherichia coli, enhanced with H2O2 – part 1: The dark side of peroxide

The present bi-partite work describes the development and validation of a mechanistic kinetic model of SODIS E. coli inactivation, enhanced with H2O2. In this first part, the mechanism of the baseline dark phenomena is modelled. A mechanistic model involving E. coli cellular respiration, inactivation due to HO· and O2·- radicals, and bacterial thermal inactivation, was developed using a series-event model based on the accumulation of damage and cell recovery corrected with the Arrhenius equation for inclusion of the thermal events. The contribution of external H2O2 was included in the internal H2O2 balance, while the balance of extracellular H2O2 considered the consumption caused by its self-decomposition, interactions with cells’ membrane, and organic matter from dead cells. Specifically, the kinetic parameters of the external H2O2 sinks, the oxidation reaction of intracellular Fenton, and bacterial thermal inactivation were independently estimated by model regression from experimental data of E. coli inactivation and H2O2 consumption at different controlled conditions of temperature and initial H2O2 concentration. We complemented the values of the kinetic constants available in the literature with the unknown kinetic parameters estimated from experiemetnal and literature data. The missing kinetic parameters were successfully validated (bacteria error = 4.5%, H2O2 error = 12.9%). This kinetic model helps to understand the intracellular mechanisms and the contributions of each source of inactivation, with the role of radicals’ damage being most important at temperatures below 40 °C, and the thermal inactivation for temperatures above this value.

Tumor Microenvironment-Activated Theranostics Nanozymes for Fluorescence Imaging and Enhanced Chemo-Chemodynamic Therapy of Tumors.

Chemodynamic therapy (CDT) is widely explored for tumor-specific therapy by converting endogenous H2O2 to lethal ·OH to destroy cancer cells. However, ·OH scavenging by glutathione (GSH) and insufficient intratumoral H2O2 levels seriously hinder the application of CDT. Herein, we reported the fabrication of copper ion-doped ZIF-8 loaded with gold nanozymes and doxorubicin hydrochloride (DOX) for the chemotherapy and CDT synergistic treatment of tumors with the assistance of tumor microenvironment (TME)-activated fluorescence imaging. The Cu2+-doped ZIF-8 shell was gradually degraded to release DOX and gold nanoclusters responding to the acidic TME. The fluorescence signal of the tumor region was acquired after the quenched fluorescence of the gold nanoclusters by Cu2+ and DOX by aggregation-induced quenching was turned on because of the interaction of GSH with Cu2+ and the release of free DOX. The Cu2+ ions could deplete the GSH via redox reactions and the generated Cu+ could convert internal H2O2 to ·OH for tumor CDT. The chemotherapeutic effect of DOX was strengthened through drug efflux inhibition and drug sensitivity increase due to the consumption of GSH and ·OH burst. Moreover, DOX could raise the level of H2O2 and augment the effect of CDT. In addition, the fluorescent gold nanoclusters not only served as a peroxidase to convert H2O2 to ·OH but also employed as an oxidase to consume GSH, resulting in the amplification of chemotherapy and CDT. This work presents an approach to construct tumor microenvironment-activated theranostic probes without external stimuli and to achieve the tumor elimination through cascade reactions and synergistic treatment.

Induction of Freezing Tolerance by the Application of Hydrogen Peroxide and Salicylic Acid as Tuber-Dip or Canopy Spraying in Solanum tuberosum L. Plants

Low temperature stress is a current challenge to plants that is associated with climate change. In plants, exposure to extreme temperatures is followed by the accumulation of reactive oxygen species, such as hydrogen peroxide (H2O2), leading to oxidative stress. Salicylic acid (SA) and H2O2 mediate the tolerance responses to stress and have been reported to induce freezing tolerance in potato microplants. The objectives of the present investigation were (1) to evaluate the short- and long-term effects of H2O2 and SA treatments on freezing tolerance in potato (Solanum tuberosum L.) plants grown from tubers and (2) to analyse the relationship between catalase (CAT) activity and H2O2 concentration associated with freezing tolerance responses. We observed the lowest freezing survival rates in 45-day-old potato plants (cv. Granate) compared to younger plants. The two treatments consisted of (1) the tuber-dip (long-term) treatment in which sprouted minitubers were saturated for 1 h in SA 10−5 M or H2O2 1 mM and planted in soil under greenhouse conditions and (2) the crop-spray (short-term) treatment in which plants 5–8 cm high were sprayed twice a week with SA 10−5 M or H2O2 1 mM until 45 days of age. In all treatments, 45-day-old plants were then exposed to − 6 ± 1 °C for 4 h. The survival rate was measured 15 days after freezing. CAT and H2O2 measurements were performed 1 h before and after the freezing treatment. The results showed that SA and H2O2 induced freezing tolerance in both the short- and long-term treatments. Survival was significantly higher in SA- and H2O2-treated plants than in control plants. In both the long- and short-term treatments this higher survival was associated with lower internal H2O2 concentrations after freezing compared with control plants and decreasing oxidative stress. SA and H2O2 induced different levels of CAT activity after freezing compared to that found in the control plants in the long- and the short-term treatments. These results suggest the SA and H2O2 function in independent pathways in terms of their induction of freezing tolerance that depends on the method the treatment was applied, by spraying the canopy or by immersion of the sprouted seed tuber.

Sensitivity of mitochondrial DNA depleted ρ0 cells to H2O2 depends on the plasma membrane status

To clarify the relationship between mitochondrial DNA (mtDNA)-depleted ρ0 cells and the cellular sensitivity to hydrogen peroxide (H2O2), we established HeLa and SAS ρ0 cell lines and investigated their survival rate in H2O2, radical scavenging enzymes, plasma membrane potential status, and chronological change in intracellular H2O2 amount under the existence of extracellular hydrogen peroxide compared with the parental cells. The results revealed that ρ0 cells had higher sensitivity to H2O2 than their parental cells, even though the catalase activity of ρ0 cells was up-regulated, and the membrane potential of the ρ0 cells was lower than their parental cells. Furthermore, the internal H2O2 amount significantly increased only in ρ0 cells after 50 μM H2O2 treatment for 1 h. These results suggest that plasma membrane status of ρ0 cells may cause degradation, and the change could lead to enhanced membrane permeability to H2O2. As a consequence, ρ0 cells have a higher H2O2 sensitivity than the parental cells.

Cylindrospermopsin induced changes in growth, toxin production and antioxidant response of Acutodesmus acuminatus and Microcystis aeruginosa under differing light and nitrogen conditions

Growing evidence suggests that some bioactive metabolites (e.g. cyanotoxins) produced by cyanobacteria have allelopathic potential, due to their inhibitory or stimulatory effects on competing species. Although a number of studies have shown that the cyanotoxin cylindrospermopsin (CYN) has variable effects on phytoplankton species, the impact of changing physicochemical conditions on its allelopathic potential is yet to be investigated. We investigated the physiological response of Microcystis aeruginosa (Cyanobacteria) and Acutodesmus acuminatus (Chlorophyta) to CYN under varying nitrogen and light conditions. At 24h, higher microcystins content of M. aeruginosa was recorded under limited light in the presence of CYN, while at 120h the lower levels of the toxins were observed in the presence of CYN under optimum light. Total MCs concentration was significantly (p<0.05) lowered by CYN after 120h of exposure under limited and optimum nitrogen conditions. On the other hand, there were no significant (p>0.05) changes in total MCs concentrations after exposure to CYN under high nitrogen conditions. As expected, limited light and limited nitrogen conditions resulted in lower cell density of both species, while CYN only significantly (p<0.05) inhibited the growth of M. aeruginosa. Regardless of the light or nitrogen condition, the presence of CYN increased internal H2O2 content of both species, which resulted in significant (p<0.05) changes in antioxidant enzyme (catalase, peroxidase, superoxide dismutase and glutathione S-transferase) activities. The oxidative stress caused by CYN was higher under limited light and limited nitrogen. These results showed that M. aeruginosa and A. acuminatus have variable response to CYN under changing light and nitrogen conditions, and demonstrate that need to consider changes in physicochemical conditions during ecotoxicological and ecophysiological investigations.

CMDH4 encodes a protein that is required for lead tolerance in Arabidopsis

Lead (Pb) is one of the most dangerous soil pollutants with high toxicity to plants and animals. However, the molecular mechanisms behind Pb tolerance are poorly understood. Here we identified a previously unknown cytosol-localized malate dehydrogenase (designated as CMDH4), which is involved in regulating Pb tolerance in Arabidopsis. T-DNA insertional mutants and overexpression lines were used for functional analysis. Lead was externally supplied and phenotypic analysis of these plants was performed. CMDH4 transcription was induced by Pb stress. Loss-of-function of CMDH4 resulted in reduced NAD(P)H level, and Pb hypersensitivity in response to Pb stress. Further analysis revealed that internal H2O2 concentration and cellular damage was elevated in cmdh4 mutants. External supply of NAD(P)H could improve plant tolerance to Pb stress, and recover the sensitivity of cmdh4 loss-of-function mutants to the wild-type level. In addition, the transcription level of PDR12 coding for a Pb2+ pump was significantly reduced in cmdh4 mutants, thereby leading to increased Pb accumulation. To sum up, our data provide evidence that CMDH4 is required for lead tolerance and reduced Pb accumulation in Arabidopsis.

Hydrogen Peroxide Induced Cell Death: The Major Defences Relative Roles and Consequences in E. coli.

We recently developed a mathematical model for predicting reactive oxygen species (ROS) concentration and macromolecules oxidation in vivo. We constructed such a model using Escherichia coli as a model organism and a set of ordinary differential equations. In order to evaluate the major defences relative roles against hydrogen peroxide (H2 O2), we investigated the relative contributions of the various reactions to the dynamic system and searched for approximate analytical solutions for the explicit expression of changes in H2 O2 internal or external concentrations. Although the key actors in cell defence are enzymes and membrane, a detailed analysis shows that their involvement depends on the H2 O2 concentration level. Actually, the impact of the membrane upon the H2 O2 stress felt by the cell is greater when micromolar H2 O2 is present (9-fold less H2 O2 in the cell than out of the cell) than when millimolar H2 O2 is present (about 2-fold less H2 O2 in the cell than out of the cell). The ratio between maximal external H2 O2 and internal H2 O2 concentration also changes, reducing from 8 to 2 while external H2 O2 concentration increases from micromolar to millimolar. This non-linear behaviour mainly occurs because of the switch in the predominant scavenger from Ahp (Alkyl Hydroperoxide Reductase) to Cat (catalase). The phenomenon changes the internal H2 O2 maximal concentration, which surprisingly does not depend on cell density. The external H2 O2 half-life and the cumulative internal H2 O2 exposure do depend upon cell density. Based on these analyses and in order to introduce a concept of dose response relationship for H2 O2-induced cell death, we developed the concepts of “maximal internal H2 O2 concentration” and “cumulative internal H2 O2 concentration” (e.g. the total amount of H2 O2). We predict that cumulative internal H2 O2 concentration is responsible for the H2 O2-mediated death of bacterial cells.

Real-time imaging of hydrogen peroxide dynamics in vegetative and pathogenic hyphae of Fusarium graminearum.

Balanced dynamics of reactive oxygen species in the phytopathogenic fungus Fusarium graminearum play key roles for development and infection. To monitor those dynamics, ratiometric analysis using the novel hydrogen peroxide (H2O2) sensitive fluorescent indicator protein HyPer-2 was established for the first time in phytopathogenic fungi. H2O2 changes the excitation spectrum of HyPer-2 with an excitation maximum at 405 nm for the reduced and 488 nm for the oxidized state, facilitating ratiometric readouts with maximum emission at 516 nm. HyPer-2 analyses were performed using a microtiter fluorometer and confocal laser scanning microscopy (CLSM). Addition of external H2O2 to mycelia caused a steep and transient increase in fluorescence excited at 488 nm. This can be reversed by the addition of the reducing agent dithiothreitol. HyPer-2 in F. graminearum is highly sensitive and specific to H2O2 even in tiny amounts. Hyperosmotic treatment elicited a transient internal H2O2 burst. Hence, HyPer-2 is suitable to monitor the intracellular redox balance. Using CLSM, developmental processes like nuclear division, tip growth, septation, and infection structure development were analyzed. The latter two processes imply marked accumulations of intracellular H2O2. Taken together, HyPer-2 is a valuable and reliable tool for the analysis of environmental conditions, cellular development, and pathogenicity.

A feedback inhibition between nitric oxide and hydrogen peroxide in the heat shock pathway in Arabidopsis seedlings

We even reported that hydrogen peroxide (H2O2) acts upstream of nitric oxide (NO) in the heat shock (HS) pathway in Arabidopsis (Arabidopsis thaliana) seedlings. In this work, we found a strange phenomenon that exogenous application of high concentration of H2O2 could not elevate internal H2O2 level under HS. NO donors sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) intensified this trend, whereas a special NO scavenger, 2-(4-phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxyde (cPTIO) counteracted it, suggesting NO effects on reducing H2O2 accumulation. Western blotting and real-time reverse transcription-polymerase chain reaction demonstrated that NO eliminated H2O2 effects on the DNA-binding activity of HS factors and the accumulation of HS proteins. Subsequent experiments revealed that under HS, SNP and SNAP enhanced the activities of antioxidant enzymes catalase, ascorbate peroxidase and glutathione reductase, whereas c-PTIO inhibited them. Collectively, H2O2-induced NO stimulated the activities of antioxidant enzymes so as to eliminate excessive H2O2 in plant and then inhibit HS factor DNA-binding activity and HS protein accumulation, indicating a feedback inhibition between NO and H2O2 in thermotolerance.

Release of hydrogen peroxide and antioxidants by the coral &amp;lt;i&amp;gt;Stylophora pistillata&amp;lt;/i&amp;gt; to its external &amp;lt;i&amp;gt;milieu&amp;lt;/i&amp;gt;

Abstract. Hydrogen peroxide (H2O2), a common reactive oxygen species, plays multiple roles in coral health and disease. Elevated H2O2 production by the symbiotic algae during stress may result in symbiosis breakdown and bleaching of the coral. We have recently reported that various Red Sea corals release H2O2 and antioxidants to their external milieu, and can influence the H2O2 dynamics in the reef. Here, we present a laboratory characterization of H2O2 and antioxidant activity release kinetics by intact, non-stressed Stylophora pistillata. Experimenting with bleached and non-bleached corals and different stirring speeds, we explored the sources and modes of H2O2 and antioxidant release. Since H2O2 is produced and degraded simultaneously, we developed a methodology for resolving the actual H2O2 concentrations released by the corals. H2O2 and antioxidant activity steadily increased in the water surrounding the coral over short periods of 1–2 h. Over longer periods of 5–7 h, the antioxidant activity kept increasing with time, while H2O2 concentrations were stabilized at ~ 1 μM by 1–3 h, and then gradually declined. Solving for H2O2 release, corals were found to release H2O2 at increasing rates over 2–4 h, and then to slow down and stop by 5–7 h. Stirring was shown to induce the release of H2O2, possibly since the flow reduces the thickness of the diffusive boundary layer of the coral, and thus increases H2O2 mass flux. Antioxidant activity was released at similar rates by bleached and non-bleached corals, suggesting that the antioxidants did not originate from the symbiotic algae. H2O2, however, was not released from bleached corals, implying that the symbiotic algae are the source of the released H2O2. The observed flow-induced H2O2 release may aid corals in removing some of the internal H2O2 produced by their symbiotic algae, and may possibly assist in preventing coral bleaching under conditions of elevated temperature and irradiance.

A dissection of the effects of ethylene, H2O2 and high irradiance on antioxidants and several genes associated with stress and senescence in tobacco leaves

Ethylene and hydrogen peroxide are involved in the modulation of stress responses in plants, but their interrelation is not well understood. This work was designed to find differences between the actions of ethylene and H2O2 on antioxidants and senescence markers. Leaves of Nicotiana tabacum were sprayed with H2O2 or with ethephon (precursor of ethylene). To find the possible modulation of responses to acute abiotic stress, ethephon- and H2O2-sprayed leaves were further subjected to high irradiance (HL). The application of H2O2 strongly stimulated ethylene synthesis (ACC). Ethylene and H2O2, as single factors, stimulated the trolox equivalent antioxidant capacity (TEAC) and the activity of catalase (CAT), in contrast to HL alone (stimulation of nonspecific peroxidases and the total glutathione pool). However, after combined treatments (ethylene+HL and H2O2+HL), the stimulatory action of H2O2 was related to TEAC and CAT activity, while the application of ethylene stimulated the total glutathione pool. Hydrogen peroxide enhanced the expression of the three CAT genes (Cat1, Cat2 and Cat3), in contrast to ethylene (Cat2 and Cat3) and HL (Cat1). In regard to the markers of senescence and pathogenesis the most pronounced difference between the actions of ethylene and H2O2, as single factors, was related to NPR1, whereas when leaf spraying was combined with HL, differences were found at WRKY53 and PR1a. HL reversed the stimulatory effects of H2O2/ethylene-driven enhancements of the expression of several genes (Cat1, Cat2, NPR1, WRKY53). These results show that multiple stressors, as usually encountered by plants in nature, may largely change those expression patterns of genes determined in a single factor analysis. Moreover, the actions of HL (often considered the internal H2O2 trigger) and of exogenous H2O2 on gene expression are clearly different.

Time-dependent alterations in growth, photosynthetic pigments and enzymatic defense systems of submerged Ceratophyllum demersum during exposure to the cyanobacterial neurotoxin anatoxin-a

Recently, aquatic macrophytes have been considered as promising tools for eco-friendly water management with a low running cost. However, only little information is available thus far regarding the metabolic capacity of macrophytes for coping with cyanobacterial toxins (cyanotoxins) in the aquatic environment. Cyanotoxins have become emerging contaminants of great concern due to the high proliferation of cyanobacteria (cyanobacterial bloom) accelerated by eutrophication and climate change. Anatoxin-a, one of the common and major cyanotoxins, is suggested as a high priority water pollutant for regulatory consideration owing to its notoriously rapid mode of action as a neurotoxin. In this study, the time-course metabolic regulation of the submerged macrophyte Ceratophyllum demersum (C. demersum) was investigated during exposure to anatoxin-a at an environmentally relevant concentration (15μg/L). Biotransformation and antioxidative systems in C. demersum responded positively to anatoxin-a through the promoted synthesis of most of the involved enzymes within 8h. Maximum enzyme activities were exhibited after 24 or 48h of exposure to anatoxin-a. However, an apparent decline in enzyme activities was also observed at longer exposure duration (168 and 336h) in company with high steady-state levels of cell internal H2O2, which showed its highest level after 48h. Meanwhile, irreversible inhibitory influence on chlorophyll content (vitality) was noticed, whereas the ratio of carotenoids to total chlorophyll was increased with the increase in exposure duration. Consequently, the reduction in growth (biomass) of C. demersum was observed in sub-chronic exposure to anatoxin-a (8 weeks). Overall results clearly indicate, on the one hand, that anatoxin-a causes negative allelopathic effects on the macrophyte by inducing oxidative stress. On the other hand, the macrophyte might have interactions with anatoxin-a, based on the prompt reaction of its enzymatic defense systems to the toxin. The result obtained from the present study could contribute to the improvement of basic knowledge about the ecological impact of anatoxin-a and the environmental fate of the toxin in the aquatic environment.

Exogenous H2O2 in Phytoplasma-Infected Potato Plants Promotes Antioxidant Activity and Tuber Production Under Drought Conditions

H2O2-mediated symptoms and antioxidant responses were examined in phytoplasma-infected potato plants under drought conditions. In vitro phytoplasma-infected or uninfected plants were transferred to a greenhouse and sprayed twice a week with H2O2. Phytoplasma decreased water potential, catalase (CAT) activity and increased H2O2 content significantly. H2O2 significantly enhanced minitubers weight and starch content in phytoplasma-infected plants. Under drought conditions, minitubers produced by H2O2-sprayed phytoplasma-infected plants had significantly more starch than the controls. Minitubers weight was reduced in both uninfected and infected plants, but H2O2 in the presence of phytoplasma significantly enhanced minitubers weight, CAT activity, and H2O2 content. H2O2 treatment induced higher internal H2O2 concentration, which was associated with positive effects on infected minitubers, such as weight, reduction of number, starch content, sprouting, and tolerance to drought a signal role for H2O2 in lessening symptoms is suggested.

Antioxidant defense properties of Arctic amphipods: comparison between deep-, sublittoral and surface-water species

The toxic reactive oxygen species (ROS), produced naturally in an oxygenated environment, led to the development of antioxidant defenses by aerobic organisms to prevent tissue damage. Polar marine waters are regarded as a strongly prooxidant ecosystem, due to the high dissolved oxygen level and intense UV radiation during summer, while the deep waters are considered refuges against oxidative stress. In order to further elucidate the prooxidant and antioxidant forces associated with cold-water ecosystems and the implications of UV radiation, we sampled three amphipod species living at three different depths in the same water mass of the Arctic Ocean, characterized by low temperature (ca. 0°C), high oxygen level, and high ROS concentration in the surface water. The three species were the deep-sea amphipod Eurythenes gryllus, the benthic sublittoral amphipod Anonyx nugax and the surface-water species Gammarus wilkitzkii inhabiting the ice pack. The total oxyradical scavenging capacity (TOSC) was measured in the cytosolic fraction of the digestive gland and in the cell-free hemolymph of the three amphipod species. A significantly low TOSC toward peroxyl and peroxynitrite radicals (P 0.05) was noted in G. wilkitzkii. These data suggest that A. nugax is highly susceptible to oxidative stress and that G. wilkitzkii is characterized by a mechanism that prevents the diffusion of exogenous ROS through the gills or allows excretion of internal H2O2 through the gills to the environment.

Salt‐induced oxidative stress mediated by activated oxygen species in pea leaf mitochondria

The effect in vivo of salt stress on the activated oxygen metabolism of mitochondria, was studied in leaves from two NaCl‐treated cultivars of Pisum sativum L. with different sensitivity to NaCl. In mitochondria from NaCl‐sensitive plants, salinity brought about a significant decrease of Mn‐SOD (EC 1. 15. 1. 1) Cu, Zn‐SOD I (EC 1. 15. 1. 1) and fumarase (EC 4. 2. 1. 2) activities. Conversely, in salt‐tolerant plants NaCl treatment produced an increase in the mitochondrial Mn‐SOD activity and, to a lesser extent, in fumarase activity. In mitochondria from both salt‐treated cultivars, the internal H2O2 concentration remained unchanged. The NADH‐ and succinate‐dependent generation of O2.−radicals by submitochondrial particles and the lipid peroxidation of mitochondrial membranes, increased as a result of salt treatment, and these changes were higher in NaCl‐sensitive than in NaCl‐tolerant plants. Accordingly, the enhanced rates of superoxide production by mitochondria from salt‐sensitive plants were concomitant with a strong decrease in the mitochondrial Mn‐SOD activity, whereas NaCl‐tolerant plants appear to have a protection mechanism against salt‐induced increased O2.− production by means of the induction of the mitochondrial Mn‐SOD activity. These results indicate that in the subcellular toxicity of NaCl in pea plants, at the level of mitochondria, an oxidative stress mechanism mediated by superoxide radicals is involved, and also imply a function for mitochondrial Mn‐SOD in the molecular mechanisms of plant tolerance to NaCl.