Salicylic acid increases tolerance to oxidative stress induced by hydrogen peroxide accumulation in leaves of cadmium-exposed flax (Linum usitatissimum L.)

The aim of this study is to investigate the impacts of exogenous salicylic acid (SA) pretreatments on hydrogen peroxide (H₂O₂) accumulation, protein oxidation, and H₂O₂-scavenging enzymes in leaves of Cd-treated flax seedlings. Cd-enhanced H₂O₂ levels were related to increased activities of guaiacol peroxidase (POX, EC 1.11.1.7) and ascorbate peroxidase (APX, EC 1.11.1.11), and were independent of changes in catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC 1.15.1.1) activities. In control flax seedlings, exogenous SA pretreatments inhibited the activity of CAT, resulted in an enhanced production of H₂O₂ suggesting that SA requires H₂O₂ to initiate an oxidative stress. However, although leaves of Cd-free flax seedlings pretreated with SA accumulated in vivo H₂O₂ by 1.2-fold compared with leaves of Cd-only exposed ones; the damage to growth and proteins after the exposure to Cd was significantly less, indicating that SA can regulate the Cd-induced oxidative stress. Moreover, the Cd-treated seedlings primed with SA exhibited a higher level of total antioxidant capacities and increased activities of H₂O₂-detoxifying enzymes.     

The antioxidant action of N-acetylcysteine: Its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid

N-acetylcysteine has been widely used as an antioxidant in vivo and in vitro. Its reaction with four oxidant species has therefore been examined. N-acetylcysteine is a powerful scavenger of hypochlorous acid (H---OCl); low concentrations are able to protect ₁-antiproteinase against inactivation by HOCl. N-acetylcysteine also reacts with hydroxyl radical with a rate constant of 1.36 × 10¹⁰ M⁻¹s⁻¹, as determined by pulse radiolysis. It also reacts slowly with H₂O₂, but no reaction of N-acetylcysteine with superoxide (O₂⁻) could be detected within the limits of our assay procedures.     

Abscisic acid-induced hydrogen peroxide is required for anthocyanin accumulation in leaves of rice seedlings

The role of hydrogen peroxide (H(2)O(2)) in abscisic acid (ABA)-induced anthocyanin accumulation in detached and intact leaves of rice seedlings was investigated. Treatment with ABA resulted in an accumulation of anthocyanins in detached rice leaves. Dimethylthiourea, a chemical trap for H(2)O(2), was observed to be effective in inhibiting ABA-induced accumulation of anthocyanins. Inhibitors of NADPH oxidase (diphenyleneiodonium chloride and imidazole), phosphatidylinositol 3-kinase (wortmannin and LY 294002), and a donor of nitric oxide (N-tert-butyl-alpha-phenylnitrone), which have previously been shown to prevent ABA-induced H(2)O(2) accumulation in detached rice leaves, inhibited ABA-induced anthocyanin increase. Exogenous application of H(2)O(2), however, was found to increase the anthocyanin content of detached rice leaves. In terms of H(2)O(2) accumulation, intact (attached) leaves of rice seedlings of cultivar Taichung Native 1 (TN1) are ABA sensitive and those of cultivar Tainung 67 (TNG67) are ABA insensitive. Upon treatment with ABA, H(2)O(2) and anthocyanins accumulated in leaves of TN1 seedlings but not in leaves of TNG67. Our results, obtained from detached and intact leaves of rice seedlings, suggest that H(2)O(2) is involved in ABA-induced anthocyanin accumulation in this species.     

Feasibility of simultaneous measurement of cytosolic calcium and hydrogen peroxide in vascular smooth muscle cells

Interplay between calcium ions (Ca²⁺) and reactive oxygen species (ROS) delicately controls diverse pathophysiological functions of vascular smooth muscle cells (VSMCs). However, details of the Ca²⁺ and ROS signaling network have been hindered by the absence of a method for dual measurement of Ca²⁺ and ROS. Here, a real-time monitoring system for Ca²⁺ and ROS was established using a genetically encoded hydrogen peroxide indicator, HyPer, and a ratiometric Ca²⁺ indicator, fura-2. For the simultaneous detection of fura-2 and HyPer signals, 540 nm emission filter and 500 nm∼ dichroic beamsplitter were combined with conventional exciters. The wide excitation spectrum of HyPer resulted in marginal cross-contamination with fura-2 signal. However, physiological Ca²⁺ transient and hydrogen peroxide were practically measurable in HyPer-expressing, fura-2-loaded VSMCs. Indeed, distinct Ca²⁺ and ROS signals could be successfully detected in serotonin-stimulated VSMCs. The system established in this study is applicable to studies of crosstalk between Ca²⁺ and ROS. [BMB Reports 2013; 46(12): 600-605]     

Effect of carbon source on the production of oxalic acid and hydrogen peroxide by brown-rot fungus Poria placenta

Oxalic acid and hydrogen peroxide have been suggested to be essential in the degradation of wood carbohydrates by brown-rot fungi. The production of oxalic acid, hydrogen peroxide and endo-β-1,4-glucanase activity by the brown-rot fungus Poria placenta was studied on crystalline cellulose, amorphous cellulose and glucose media. Oxalic acid and hydrogen peroxide by P. placenta were clearly produced on culture media containing either crystalline or amorphous cellulose. Oxalic acid and hydrogen peroxide were formed simultaneously and highest amounts of oxalic acid (1.0 g l⁻¹) and hydrogen peroxide (39.5 μM) were obtained on amorphous cellulose after 3 weeks cultivation. On glucose medium the amounts were low. The endoglucanase activity was observed to increase during the cultivation and was most pronounced on glucose medium and thus indicated the constitutive characteristics of the brown-rot cellulases.     

Implications of water stress-induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings

Vigna cutjang Endl. cv. Pusa Barsati seedlings, subjected to increasing degrees of water stress (−0.5, −1.0, −1,5 MPa), produced an approximately proportional increase in glycolate oxidase activity, hydrogen peroxide (H₂O₂) and proline content but a decrease in catalase activity, ascorbic acid and protein content. Leaf water potential (leaf ψ) and relative water content (RWC) were also lowered with increasing stress. Pretreatment with l -cysteine and reduced glutathione (10-3 M) decreased glycolate oxidase activity, H₂O₂ content, ascorbic acid oxidase activity, proline content and also slightly improved the water status of leaves stressed (−1.0 MPa) for 2 days. Pretreatment of non-stressed seedlings with these antioxidants had little or no effect. These studies indicate that treatment with antioxidants makes the plant tolerant against water stress by modulating the endogenous levels of H₂O₂ and ascorbic acid in stressed tissue.     

Role of thiocyanate, bromide and hypobromous acid in hydrogen peroxide-induced apoptosis

We have previously reported that H₂O₂-induced apoptosis in HL-60 human leukemia cells takes place in the presence of chloride, requires myeloperoxidase (MPO), and occurs through oxidative reactions involving hypochlorous acid and chloramines. We now report that when chloride is replaced by the pseudohalide thiocyanate, there is little or no H₂O₂-induced apoptosis. Furthermore, thiocyanate inhibits H₂O₂-induced apoptosis when chloride is present at physiological concentrations, and this occurs at thiocyanate concentrations that are present in human serum and saliva. In contrast, bromide can substitute for chloride in H₂O₂-induced apoptosis, but results in a lower percent of the cells induced into apoptosis. Hypobromous acid is likely a short-lived intermediate in this H₂O₂/MPO/bromide apoptosis, and reagent hypobromous acid and bromamines induce apoptosis in HL-60 cells. We conclude that the physiologic concentrations of thiocyanate found in human plasma could modulate the cytototoxicity of H₂O₂ and its resulting highly toxic MPO-generated hypochlorous acid by competing with chloride for MPO. Furthermore, the oxidative products of the reaction of thiocyanate with MPO are relatively innocuous for human leukemic cells in culture. In contrast, bromide can support H₂O₂/MPO/halide apoptosis, but is less potent than chloride and it has no effect in the presence of physiological levels of chloride.     

Soybean MAPK, GMK1 Is dually regulated by phosphatidic acid and hydrogen peroxide and translocated to nucleus during salt stress

Mitogen-activated protein kinase (MAPK) is activated by various biotic and abiotic stresses. Salt stress induces two well-characterized MAPK activating signaling molecules, phosphatidic acid (PA) via phospholipase D and phospholipase C, and reactive oxygen species (ROS) via nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase. In our previous study, the activity of soybean MAPK, GMK1 was strongly induced within 5 min of 300 mM NaCl treatment and this early activity was regulated by PA. In this study, we focused on the regulation of GMK1 at the later stage of the salt stress, because its activity was strongly persistent for up to 30 min. H₂O₂ activated GMK1 even in the presence of PA generation inhibitors, but GMK1 activity was greatly decreased in the presence of diphenyleneiodonium, an inhibitor of NADPH-oxidase after 5 min of the treatment. On the contrary, the n-butanol and neomycin reduced GMK1 activity within 5 min of the treatment. Thus, GMK1 activity may be sustained by H₂O₂ 10 min after the treatment. Further, GMK1 was translocated into the nucleus 60 min after NaCl treatment. In the relationship between GMK1 and ROS generation, ROS generation was reduced by SB202190, a MAPK inhibitor, but was increased in protoplast overexpressing TESD-GMKK1. However, these effects were occurred at prolonged time of NaCl treatment. These data suggest that GMK1 indirectly regulates ROS generation. Taken together, we propose that soybean GMK1 is dually regulated by PA and H₂O₂ at a time dependant manner and translocated to the nucleus by the salt stress signal.     

Benzoic acid may act as the functional group in salicylic acid and derivatives in the induction of multiple stress tolerance in plants

Benzoic acid, sulfosalicylic acid and methyl salicylic acid wereevaluated for their regulatory role in inducing multiple stress tolerance inbean (Phaseolus vulgaris cv Brown Beauty) and tomato(Lycopersicum esculentum cv Romano) plants. All threemolecules were effective in inducing tolerance to heat, drought and chillingstress similar to that reported previously for salicylic and acetylsalicylicacids. Benzoic acid is effective at lower concentrations than salicylic acid orits derivatives. The benzoic acid structural portion is common to all fivemolecules and is the most likely basic functional molecular structure impartingstress tolerance in plants.     

Supercritical carbon dioxide and hydrogen peroxide cause mild changes in spore structures associated with high killing rate of Bacillus anthracis

The present work examines chemical and structural response in B. anthracis spores killed by a mixture of supercritical carbon dioxide (SCCO₂) and hydrogen peroxide (H₂O₂). Deactivation of 6-log of B. anthracis spores by SCCO₂ + H₂O₂ was demonstrated, but changes in structure were observed in only a small portion of spores. Results from phase contrast microscopy proved that this treatment is mild and does not trigger germination-like changes. TEM imaging revealed mild damage in a portion of spores while the majority remained intact. Dipicolinic acid (DPA) analysis showed that < 10% of the DPA was released from the spore core into the external milieu, further demonstrating only modest damage to the spores. Confocal fluorescent microscopy, assessing uptake of DNA-binding dyes, directly demonstrated compromise of the permeability barrier. However, the magnitude of uptake was small compared to spores that had been autoclaved. This work suggests that SCCO₂ + H₂O₂ is quite mild compared to other sterilization methods, which has major implications in its application. These results provide some insight on the possible interactions between spores and the SCCO₂ + H₂O₂ sterilization process.     

Antioxidant action of sodium diethyldithiocarbamate: Reaction with hydrogen peroxide and superoxide radical

The oxidation of sodium diethyldithiocarbamate (DDC) by hydrogen peroxide or superoxide radicals has been investigated. Hydrogen peroxide oxidizes DDC, leading to the formation of a hydrated form of disulfiram, a dimer of DDC having a disulfide group. In equimolar conditions, the overall process appears as a first-order reaction (k = 0.025±0.005 s⁻¹), the first step being a second-order reaction (k = 5.0±0.1mol⁻¹.1. s⁻¹). No radical intermediate was observed in this process. In the presence of an excess of any of the reagents, the hydrated form of disulfiram transforms into different products corresponding to the fixation of oxygen by sulfur atoms or replacement of C = S group by ketone function, in the presence of an excess of hydrogen peroxide. Superoxide anions (produced by steady-state ⁶⁰Co γ-radiolysis) oxidize DDC, yielding similar products to those obtained with hydrogen peroxide with a maximum oxidation G-value of 0.3 μmol.J⁻¹. The rate constant k(O₂^·− + DDC) is equal to 900 mol⁻¹. 1. s⁻¹.     

Salicylic acid induced physiological and biochemical changes in wheat seedlings under water stress

Salicylic acid (SA) is an important signal molecule modulating plantresponses to stress. It is recently reported to induce multiple stresstolerancein plants including drought. An experiment was, therefore, conducted toascertain the effect of salicylic acid on the growth and metabolic profile ofwheat seedlings under water stress. Irrespective of the SA concentration(1–3 mM) and water stress, SA treated plants showed, ingeneral, a higher moisture content, dry mass, carboxylase activity of Rubisco,superoxide dismutase (SOD) activity and total chlorophyll compared to those ofuntreated seedlings. SA treatment, under water stress, protected nitratereductase (NR) activity and maintained, especially at 3 mM SAconcentration, the protein and nitrogen content of leaves compared to watersufficient seedlings. Results signify the role of SA in regulating the droughtresponse of plants and suggest that SA could be used as a potential growthregulator, for improving plant growth under water stress.     

Cadmium effect on hydrogen peroxide, gluthatione and phytochelatins levels in potato tuber

Short-term treatment of potato tuber (Solanum tuberosum L.) dises with CdCl₂ (1mM) induced an oxidative stress, manifested by higher levels of H₂O₂, and activated the synthesis of phytochclatins ((γ-Glu-Cys)_n-Gly): PC₂, PC₃ and PC₄. If in the tissues with a lower GSH level, the oxidative stress was induced by treatment with 3-aminotriazol (AT), or with AT and H₂O₂, the elevation of H₂O₂ and GSH levels and then some accumulation of thiols, including PC₂, PC₃ and PC₄, were observed. However, this increase of PC concentration was considerably lower when compared with the effects brought about by Cd⁺² treatment. If such a procedure of evoking subsequent moderate oxidative stress in tissues preceded Cd-treatment, a marked limitation of PC synthesis was observed. The presented results support the role of H₂O₂ as the second messenger in activating GSH synthesis and thus suggest a possibility of redox type regulation mechanism of PCs synthesis.     

An assessment of proposed mechanisms for sensing hydrogen peroxide in mammalian systems

Despite much recent interest in the biochemistry of reactive oxygen species, the mechanisms by which hydrogen peroxide (H2O2) functions in mammalian cells remain poorly defined. Proposed mechanisms for sensing H2O2 in mammalian cells include inactivation of protein tyrosine phosphatases and dual specificity phosphatases as well as inactivation of peroxiredoxins. In this critical review, proteins proposed to serve as sensors for H2O2 in mammals will be compared to peroxidases, catalases, and the bacterial H2O2 sensor OxyR for their ability to react with H2O2, in the context of our current knowledge concerning the concentrations of H2O2 present in cells.     

Induction of baroresistance by hydrogen peroxide, ethanol and cold-shock in Saccharomyces cerevisiae

The acquisition of tolerance to high hydrostatic pressure of 220 MPa (HHP) in response to a 0.4 mM hydrogen peroxide, 6% ethanol and cold-shock (10 degrees C) pretreatment for different lengths of times was studied in the yeast Saccharomyces cerevisiae. The protection conferred by these different treatments was similar ( approximately 3 log cycles) and time-dependent. Analysis of the induction of the most pressure up-regulated genes under these conditions was investigated by RT-PCR. Our results revealed that the cell stress response to HHP shares common features with hydrogen peroxide and ethanol stresses, but differs in some way to cold-shock.     

Salicylic acid and nitric oxide alleviate osmotic stress in wheat (Triticum aestivum L.) seedlings

Salicylic acid (SA) and nitric oxide (NO) are reported to alleviate the damaging effects of stress in plants rather similarly when applied at appropriate low concentrations. An experiment was therefore conducted to study the impact of SA, sodium nitroprusside (SNP; as NO donor), and methylene blue (MB; as a guanylate cyclase inhibitor) on wheat seedling performance under osmotic stress. Osmotic stress significantly reduced shoot fresh weight (SFW), chlorophyll contents (Chla, Chlb, total Chl), and membrane stability index (MSI) and also increased malondialdehyde (MDA) level, lipoxygenase (LOX) activity, and hydrogen peroxide production. Moreover, enzymatic antioxidant activities including superoxide dismutase, guaiacol peroxidase, and glutathione reductase activity were enhanced under osmotic stress. On the contrary, SA or SNP pretreatment reduced the damaging effects of osmotic stress by further enhancing the antioxidant activities that led to increased SFW, Chl, and MSI and reduced MDA level and LOX activity. However, pretreatment of plants with MB reversed or reduced the protective effects of SA and SNP suggesting that the protective effects were likely attributed to NO signaling. Therefore, NO may act as downstream of SA signaling in reduction of induced oxidative damage in wheat seedlings.     

Differential distribution of ascorbic acid,peroxidase activity,and hydrogen peroxide along the root axis in Allium cepa L. and its possible relationship with cell growth and differentiation

Summary.  In this paper we show an asymmetrical distribution of apoplastic and symplastic ascorbic acid content, peroxidase activities and hydrogen peroxide along the root axis in Allium cepa L. For most of these metabolites, a marked gradient from the root apex to the onion base was observed and was different for apoplastic and symplastic compartments. In total homogenates, ascorbic acid content was higher in the zones closer to the apex and decreased towards the root base. However, an opposite pattern was observed in the apoplastic fraction. Peroxidase activities with guaiacol, ferulic acid, ascorbic acid, and coniferyl alcohol were also different depending on the evaluated zone and the fraction used (apoplastic or symplastic). In general, each activity had a specific and unique pattern. Immunodetection of peroxidase proteins in Western blots using anti-horseradish peroxidase and anti-ascorbate peroxidase antibodies revealed different bands at the different zones of the root. Hydrogen peroxide was detected by electron microscopy and was mainly found in cell walls of epidermis (or rhizodermis), meristem, and elongating cells. The number of cell walls showing hydrogen peroxide decreased dramatically towards the root base. The results suggest that the different zones of the root show specific requirements for ascorbic acid and hydrogen peroxide. Also, each fragment of the root seems to express specific peroxidase proteins. Different processes that take place at every part of the root, as cell proliferation and elongation near the root apex and gradual lignification and differentiation towards the root base are the key to explain the results. Received May 10, 2002; accepted September 20, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Edificio C-6, Campus de Rabanales, Universidad de Córdoba, 14014 Córdoba, Spain.