Wheat catalase expressed in transgenic rice can improve tolerance against low temperature stress

We examined whether the expression of wheat catalase (EC 1.11.1.6) cDNA in transgenic rice ( Oryza sativa L.) could enhance tolerance against low temperature injury. Transgenic rice plants expressing wheat CAT protein showed an increase of activities in leaves at 25°C, 2- to 5-fold that in non-transgenic rice. At 5°C, catalase activities were about 4–15 times higher than those in non-transgenic rice were. A comparison of damage observed in leaves as they withered due to chilling at 5°C showed that transgenic rice displayed an increased capability to resist low temperature stress. The exposure of these plants to low temperature at 5°C for 8 days resulted in decreased catalase activities in leaves at 25°C, but the transgenic plants indicated 4 times higher residual catalase activities than those of non-transgenic ones. The concentration of H₂O₂ in leaves was kept lower in transgenic rice than that of the control plants during the 8 days chilling. These results suggest that the improved tolerance against low temperature stress in genetically engineered rice plants be attributed to the effective detoxification of H₂O₂ by the enhanced catalase activities.     

Is hydrogen peroxide a second messenger of salicylic acid in systemic acquired resistance?

Elevated levels of salicylic acid (SA) are required for the induction of systemic acquired resistance (SAR) in plants. Recently, a salicylic acid-binding protein (SABP) isolated from tobacco was shown to have catalase activity. Based on this finding elevated levels of hydrogen peroxide (H₂O₂) were postulated to act as a second messenger of SA in the SAR signal transduction pathway. A series of experiments have been carried out to clarify the role of H₂O₂ in SAR-signaling. No increase of H₂O₂ was found during the onset of SAR. Induction of the SAR gene, PR-1, by H₂O₂ and H₂O₂-inducing chemicals is strongly suppressed in transgenic tobacco plants that express the bacterial salicylate hydroxylase gene, indicating that H₂O₂ induction of SAR genes is dependent on SA accumulation. Following treatment of plants with increasing concentrations of H₂O₂, a dose-dependent accumulation of total SA species was found, suggesting that H₂O₂ may induce PR-1 gene expression through SA accumulation. While the results do not support a role for H₂O₂ in SAR signaling, it is suggested that SA inhibition of catalase activity may be important in tissues undergoing a hypersensitive response.     

The oxidative stress response in Enterococcus faecalis: relationship between H2O2 tolerance and H2O2 stress proteins

The hydrogen peroxide (H₂O₂) stress response in Enterococcus faecalis ATCC19433 was investigated. A 2·4 mmol l⁻¹ H₂O₂ pretreatment conferred protection against a lethal concentration (45 mmol l⁻¹) of this agent. The relatively high concentrations of H₂O₂ used for adaptation and challenge treatments in Ent. faecalis emphasised the strong resistance towards oxidative stress in this species. Various stresses (NaCl, heat, ethanol, acidity and alkalinity) induced weak or strong H₂O₂ cross-protection. This paper describes the involvement of protein synthesis in the active response to lethal dose of H₂O₂, in addition to the impressive enhancement of synthesis of five H₂O₂ stress proteins. Combined results suggest that these proteins might play an important role in the H₂O₂ tolerance response.     

Glutathione peroxidase-like protein of Synechocystis PCC 6803 confers tolerance to oxidative and environmental stresses in transgenic Arabidopsis

Glutathione peroxidase (GPX)-like proteins (GPX-1 and GPX-2) of Synechocystis PCC 6803 ( S. PCC 6803) reduce unsaturated fatty acid hydroperoxides using NADPH, but not reduced glutathione (GSH), as an electron donor. Here, we generated transgenic Arabidopsis plants overexpressing S. PCC 6803 GPX-2 in the cytosol (AcGPX2) or chloroplasts (ApGPX2). The activities toward α-linolenic acid hydroperoxide in ApGPX2 and AcGPX2 plants were 6.5–11.5 and 8.2–16.3 nmol min⁻¹ mg protein⁻¹, respectively, while no activity (<0.1 nmol min⁻¹ mg protein⁻¹) was detected in the wild-type plants. Both transgenic lines (AcGPX2 and ApGPX2) showed enhanced tolerance to oxidative damage caused by treatment with H₂O₂ (10 m M ), Fe ions (200 μ M ) or methylviologen (50 μ M ) and environmental stress conditions, such as chilling with high light intensity (4°C, 1000 μmol photons m⁻² s⁻¹), high salinity (100 m M NaCl) or drought. The degree of tolerance of the transgenic plants to all types of stress was correlated with the levels of lipid peroxide suppressed by the overexpression of S. PCC 6803 GPX-2. Under conditions of oxidative stress due to the H₂O₂ treatment, the NADPH/(NADP⁺+ NADPH) ratio in the transgenic plants was lower than that in the wild-type plants. The data reported here indicate that the expression of S. PCC 6803 GPX-2 contributes to the reduction in unsaturated fatty acid hydroperoxides using NADPH in situ under stress conditions in the transgenic plants.     

Properties and functions of glutathione reductase in plants

The assay and in vitro characterization of glutathione reductase (EC 1.6.4.2) is discussed. In vivo the H₂O₂-scavenging system in chloroplasts is the best documented role of reduced glutathione and glutathione reductase in plants. Similarly, redaction of H₂O₂, outside of the chloroplasts, requires glutathione and glutathione reductase; but the pathway, in terms of intermediates, is controversial. The notion that biological stress frequently causes cellular oxidation has lead to the suggestion that glutathione and glutathione reductase may play a role in stress resistance or tolerance mechanisms. The changes in glutathione reductase levels in response to low temperature, oxidative stress and drought are discussed.     

Responses of lactic acid bacteria to oxygen

Abstract A small number of flavoprotein oxidase enzymes are responsible for the direct interaction of lactic acid bacteria (LAB) with oxygen; hydrogen peroxide or water are produced in these reactions. In some cultures exposed to oxygen, hydrogen peroxide accumulates to inhibitory levels.
Through these oxidase enzymes and NADH peroxidase, O₂ and H₂O₂ can accept electrons from sugar metabolism, and thus have a sparing effect on the use of metabolic intermediates, such as pyruvate or acetaldehyde, as electron acceptors. Consequently, sugar metabolism in aerated cultures of LAB can be substantially different from that in unaerated cultures. Energy and biomass yields, end-products of sugar metabolism and the range of substrates which can be metabolised are affected.
Lactic acid bacteria exhibit an inducible oxidative stress response when exposed to sublethal levels of H₂O₂. This response protects them if they are subsequently exposed to lethal concentrations of H₂O₂. The effect appears to be related to other stress responses such as heat-shock and is similar, in some but not all respects, to that previously reported for enteric bacteria.     

Changes in catalase activity and hydrogen peroxide concentration in plants in response to low temperature

Taxicity of oxygen species such as free radicals and H₂O₂ has been invoked to explain a number of degradative processes in plants, most involving photo-oxidation. Since catalase is a major protectant against accumulation and toxicity of H₂O₂, we examined alterations in catalase activity in several plant species ( Pisum sativum L. cv. Greenfeast, Vigna radiata (L.) R. Wilcz, Cucumis sativus L. cv. Heinz Pickling, and Passiflora spp.) during chilling, and compared this change to change in H₂O₂ content. Catalase activity was reduced in a range of chilling sensitive and tolerant species by exposure to low temperature. This reduction in catalase activity correlated better with the onset of visible symptoms than with the treatment itself. Visible injury in turn was dependent on light and temperature differences. Hydrogen peroxide concentrations invariably decreased with low temperatures.
Reduction in catalase activity therefore does not necessarily imply accumulation of H₂O₂ to damaging levels. The absence of a clear inverse relationship between catalase activity and H₂O₂ concentration suggests the continued activity of other reactions that remove H₂O₂ and these may be important in the tolerance of plants to oxidative attack. Loss of catalase activity may result from the inability of damaged peroxisomal membranes to transport catalase precursors into the peroxisome.     

Detection of hydrogen peroxide produced by meat lactic starter cultures

Twelve strains of meat lactic starter cultures (Pediococcus spp. and Lactobacillus plantarum) were found to produce hydrogen peroxide in vitro. The (cumulative) amounts of H₂O₂ produced were measured through the peroxidative action of catalase on H₂O₂ and oxidation of added formate to CO₂ by the H₂O₂-catalase complex formed. There was a problem in building a calibration curve for converting values of formate oxidation into amounts of H₂O₂, either by adding H₂O₂ directly to the assay mixture or having it produced via a glucose-glucose oxidase system.     

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.     

Oxidative burst and expression of germin/oxo genes during wounding of ryegrass leaf blades: comparison with senescence of leaf sheaths

Two bursts of H₂O₂ production have been detected by in situ 3,3'-diaminobenzidine (DAB) staining after cutting of Lolium perenne L. leaf blades. The first burst, which occurred immediately after wounding was inhibited by Na-diethydithiocarbamate (DIECA), a Cu/Zn–superoxide dismutase (SOD) inhibitor. The second burst, which was initiated several hours later, coincided with the induction of oxalate oxidase (G-OXO) activity detected in vitro or visualized in situ by the α-chloronaphtol assay. Four genes encoding G-OXO have been identified from cDNA obtained from wounded L. perenne L . leaf blades. Comparison of protein sequences revealed more than 91% homology in the coding region between G-OXOs of the true cereals and G-OXOs of ryegrass, which is a Gramineae belonging to the tribe of Festucaceae. The wound-dependent increase of G-OXO activity in floated cut leaf blades was the result of differential induction of the four g-oxo genes. The involvement of G-OXOs in wound-induced H₂O₂ production coincided with the presence in leaf tissues of oxalate throughout the period of increase of G-OXO synthesis. Moreover, expression of g-oxo genes was enhanced by an exogenous supply of H₂O₂ or methyljasmonate (MeJa). Expression of the four g-oxo genes was also induced after in planta stinging of leaf blades. The pattern of their expression in planta was identical to that occuring in senescing leaf sheaths. These results emphasize the importance of G-OXOs in H₂O₂ production in oxalate-producing plant species such as ryegrass. G-OXOs might be crucial during critical events in the life of plants such as cutting and senescence by initiating H₂O₂-mediated defences against pathogens and foraging animals.     

Hydrogen peroxide does not function downstream of salicylic acid in the induction of PR protein expression

The roles of salicylic acid (SA) and H₂O₂ in the induction of PR proteins in tobacco have been examined. Studies were conducted on wild-type tobacco and plants engineered to express a bacterial salicylate hydroxylase capable of metabolizing SA to catechol (SH-L plants). Wild-type and PR-1a—GUS-transformed plants express PR-1a following challenge with Pseudomonas syringae pathovar syringae , SA or 2,6-dichloro-isonicotinic acid (INA). In contrast, SH-L plants failed to respond to SA but did express PR-1a following INA treatment. H₂O₂ and the irreversible catalase inhibitor 3-amino-1,2,4-triazole (3-AT) were found to be weak inducers of PR-1a expression (relative to SA) in wild-type tobacco but were unable to induce PR-1a in SH-L plants, suggesting that the action of these compounds depends upon the accumulation of SA. A model has been proposed suggesting that SA binds to and inhibits a catalase inducing an increase in H₂O₂ leading to PR protein expression. Catalase activity has been measured in tobacco and no significant changes in activity following infection with P. syringae pv. syringae were detected. Furthermore, inhibition of catalase activity in vitro in plant extracts requires pre-incubation and only occurs at SA concentrations above 250 µM. Leaf disks pre-incubated with 1 mM SA do accumulate SA to these levels and PR-1a is efficiently induced but there is no apparent inhibition of catalase activity. It is also shown that a SA-responsive gene, PR-1a, and a H₂O₂-sensitive gene, AoPR-1, are both relatively insensitive to 3-AT suggesting that induction of these genes is unlikely to be due entirely to inhibition of an endogenous catalase.     

Drought acclimation confers oxidative stress tolerance by inducing co-ordinated antioxidant defense at cellular and subcellular level in leaves of wheat seedlings

Wheat ( Triticum aestivum L.) seedlings of a drought-resistant cv. C306 were subjected to severe water deficit directly or through stress cycles of increasing intensity with intermittent recovery periods (drought acclimation). The antioxidant defense in terms of redox metabolites and enzymes in leaf cells, chloroplasts, and mitochondria was examined in relation to ROS-induced membrane damage. Drought-acclimated seedlings modulated growth by maintaining favorable turgor potential and RWC and were able to limit H₂O₂ accumulation and membrane damage as compared with non-acclimated plants during severe water stress conditions. This was due to systematic upregulation of H₂O₂-metabolizing enzymes especially ascorbate peroxidase (APX, EC 1.11.1.11) and by maintaining ascorbate–glutathione redox pool in acclimated plants. By contrast, failure in the induction of APX and ascorbate–glutathione cycle enzymes makes the chloroplast susceptible to oxidative stress in non-acclimated plants. Non-acclimated plants protected the leaf mitochondria from oxidative stress by upregulating superoxide dismutase (SOD, EC 1.15.1.1), APX, and glutathione reductase (GR, EC 1.6.4.2) activities. Rewatering led to rapid enhancement in all the antioxidant defense components in non-acclimated plants, which suggested that the excess levels of H₂O₂ during severe water stress conditions might have inhibited or downregulated the antioxidant enzymes. Hence, drought acclimation conferred enhanced oxidative stress tolerance by well-co-ordinated induction of antioxidant defense both at the chloroplast and at the mitochondrial level.     

A stress-induced oxidative burst in Eucheuma platycladum (Rhodophyta)

A hurst of hydrogen peroxide has been found in the red macroalga Eucheuma platycladwn Schmitz as a response to mechanical stress. After exposure of pieces of thalli (2 cm) broken from the plant and stirred with a magnetic bar an oxidative burst was registered, as measured by luminol dependent chemiluminescence (LDC). The burst was totally inhibited by cataluse (EC 1.11.1.6). showing the generation of H_2:O_2; Ten g of seaweed in 300 ml sea water caused a maximal medium concentration of LDC corresponding to 7 u .M H₂O_2; The burst decayed after about 30 min. The decay is probably caused by increased catalase aciivity of the sea water. due to leakage of catalasc from the seaweed. Addition of NaN₃ caused a dramatic increase in LDC. probably due to inhibition of catalase. Similar bursts of active oxygen, involving active oxygen species such as O₂, H₂O₂ and OH. have been reported as pan of the hypcrsensitive reaction in some higher plants, e.g. tobacco. potato and soybean. Exposure of plants or cell suspension cultures to some pathogenic bacteria, fungi, inorganic elicitors or physical damage causes an oxidalive burst that is often followed by necrosis. The production ot active oxygen is thought to he a first defence against invading pathogens. We assume that the oxidative burst from E. platycladum is of a defensive nature, providing a protection against grazers and pathogenic organisms. To our knowledge this is the first repoil of an oxidalive burst from seaweeds.     

Role of hydrogen peroxide and different classes of antioxidants in the regulation of catalase and glutathione S-transferase gene expression in maize (Zea mays L.)

The role of hydrogen peroxide (H₂O₂) and various antioxidants in the regulation of expression of the three Cat and Gst1 genes of maize ( Zea mays L.) has been investigated. Low concentrations of H₂O₂ appeared to inhibit Cat1 , Cat3 , and Gst1 gene expression, while higher doses strongly induced these genes. Time course experiments indicated that high concentrations of H₂O₂ induced Cat1 , Cat2 , and Gst1 gene expression to higher levels, and in less time, than lower H₂O₂ concentrations. Induction of Cat3 was superimposed on the circadian regulation of the gene. These results demonstrate a direct signaling action of H₂O₂ in the regulation of antioxidant gene responses in maize.The effects of the antioxidant compounds N-acetylcysteine, pyrrolidine dithiocarbamate, hydroquinone, and the electrophile antioxidant responsive element (ARE)-inducer β -naphthoflavone were quite different and specific for each gene/compound/concentration combination examined. The response of each gene to each antioxidant compound tested was unique, suggesting that the ability of these compounds to affect expression of the maize Cat and Gst1 genes may not be the result of a common (antioxidant) mode of action. A putative regulatory ARE motif involved in the regulation of antioxidant and oxidative stress gene responses in mammalian systems is present in the promoter of all three maize catalase genes and we tested its ability to interact with nuclear extracts prepared from 10 days post-imbibition senescing scutella. Protein-DNA interactions in the ARE motif and the U2 snRNA homologous regions of the Cat1 promoter were observed, suggesting that ARE may play a role in the high induction of Cat1 in a tissue which, due to senescence, is under oxidative stress.     

Hydrogen Peroxide Induces a Long-Lasting Inhibition of the Ca2+-Dependent Glutamate Release in Cerebrocortical Synaptosomes Without Interfering with Cytosolic Ca2+

Abstract: We studied the action of H₂O₂ on the exocytosis of glutamate by cerebrocortical synaptosomes. The treatment of synaptosomes with H₂O₂ (50–150 µ M ) for a few minutes results in a long-lasting depression of the Ca²⁺-dependent exocytosis of glutamate, induced by KCl or by the K⁺-channel inhibitor 4-aminopyridine. The energy state of synaptosomes, as judged by the level of phosphocreatine and the ATP/ADP ratio, was not affected by H₂O₂, although a transient decrease was observed after the treatment. H₂O₂ did not promote peroxidation, as judged by the formation of malondialdehyde. In indo-1-loaded synaptosomes, the treatment with H₂O₂ did not modify significantly the KCl-induced increase of [Ca²⁺]_i. H₂O₂ inhibited exocytosis also when the latter was induced by increasing [Ca²⁺]_i with the Ca²⁺ ionophore ionomycin. The effects of H₂O₂ were unchanged in the presence of superoxide dismutase and the presence of the Fe³⁺ chelator deferoxamine. These results appear to indicate that H₂O₂, apparently without damaging the synaptosomes, induces a long-lasting inhibition of the exocytosis of glutamate by acting directly on the exocytotic process.