Parallel Changes in H2O2 and Catalase during Thermotolerance Induced by Salicylic Acid or Heat Acclimation in Mustard Seedlings

Spraying mustard (Sinapis alba L.) seedlings with salicylic acid (SA) solutions between 10 and 500 μm significantly improved their tolerance to a subsequent heat shock at 55°C for 1.5 h. The effects of SA were concentration dependent, with higher concentrations failing to induce thermotolerance. The time course of thermotolerance induced by 100 μm SA was similar to that obtained with seedlings acclimated at 45°C for 1 h. We examined the hypothesis that induced thermotolerance involved H₂O₂. Heat shock at 55°C caused a significant increase in endogenous H₂O₂ and reduced catalase activity. A peak in H₂O₂ content was observed within 5 min of either SA treatment or transfer to the 45°C acclimation temperature. Between 2 and 3 h after SA treatment or heat acclimation, both H₂O₂ and catalase activity significantly decreased below control levels. The lowered H₂O₂ content and catalase activity occurred in the period of maximum thermoprotection. It is suggested that thermoprotection obtained either by spraying SA or by heat acclimation may be achieved by a common signal transduction pathway involving an early increase in H₂O₂.     

Heat shock-mediated H2O2 accumulation and protection against Cd toxicity in rice seedlings

Rice (Oryza sativa L.) seedlings stressed with CdCl₂ (0.5 mM or 50 μM) showed typical Cd toxicity (leaf chlorosis, decrease in chlorophyll content, or increase in H₂O₂ and malondialdehyde contents). Rice seedlings pretreated with heat shock at 45°C (HS) for 2 or 3 h were protected against subsequent Cd stress. Rice seedlings pretreated with HS had similar Cd concentration in leaves caused by CdCl₂ as those non-HS. The content of H₂O₂ increased in leaves 1 h after HS exposure. However, APX and GR activities were higher in HS-treated leaves than their respective control, and it occurred after 2 h of HS treatment. Pretreatment of rice seedlings with H₂O₂ under non-HS conditions resulted in an increase in APX, GR, and CAT activities and protected rice seedlings from subsequent Cd stress. HS-induced H₂O₂ production and protection against subsequent Cd stress can be counteracted by imidazole, an inhibitor of NADPH oxidase complex. Results of the present study suggest that early accumulation of H₂O₂ during HS signals the increase in APX and GR activities, which in turn prevents rice seedlings from Cd-caused oxidative damage.     

NADPH oxidase is involved in H2O2-induced differentiation of human promyelocytic leukaemia HL-60 cells

The expression and activity of NADPH oxidase increase when HL‐60 cells are induced into terminally differentiated cells. However, the function of NADPH oxidase in differentiation is not well elucidated. With 150–500 μM H₂O₂ inducing differentiation of HL‐60 cells, we measured phagocytosis of latex beads and investigated cell electrophoresis. Two inhibitors of NADPH oxidase, DPI (diphenyleneiodonium) and APO (apocynin), blocked the differentiation potential of cells induced by 200 μM H₂O₂. However, H₂O₂ stimulated the generation of intracellular superoxide (O₂ ^{? ?}), which decreased in the presence of the two inhibitors. DPI also inhibited H₂O₂‐induced ERK (extracellular‐signal‐regulated kinase) activation, as detected by Western blotting. Furthermore, PD98059, the inhibitor of the ERK pathway, inhibited the differentiation of HL‐60 cells induced by H₂O₂. This shows that H₂O₂ can activate NADPH oxidase, leading to O₂ ^{? ?} production, followed by ERK activation and ultimately resulting in the differentiation of HL‐60 cells. The data indicate that NADPH oxidase is an important cell signal regulating cell differentiation.     

H2O2 mediates nitrate‐induced iron chlorosis by regulating iron homeostasis in rice

The uptake of nitrate by plant roots causes a pH increment in rhizosphere and leads to iron (Fe) deficiency in rice. However, little is known about the mechanism how the nitrate uptake‐induced high rhizosphere pH causes Fe deficiency. Here, we found that rice showed severe leaf chlorosis and large amounts of Fe plaque were aggregated on the root surface and intercellular space outside the exodermis in a form of ferrihydrite under alkaline conditions. In this case, there was significantly decreased Fe concentration in shoots, and the Fe deficiency responsive genes were strongly induced in the roots. The high rhizosphere pH induced excess hydrogen peroxide (H₂O₂) production in the epidermis due to the increasing expression of NADPH‐oxidase respiratory burst oxidase homolog 1, which enhanced root oxidation ability and improved the Fe plaque formation in rhizosphere. Further, the concentrated H₂O₂ regulated the phenylpropanoid metabolism with increased lignin biosynthesis and decreased phenolics secretion, which blocked apoplast Fe mobilization efficiency. These factors coordinately repressed the Fe utilization in rhizosphere and led to Fe deficiency in rice under high pH. In conclusion, our results demonstrate that nitrate uptake‐induced rhizosphere alkalization led to Fe deficiency in rice, through H₂O₂‐dependent manners of root oxidation ability and phenylpropanoid metabolism.     

Biphasic regulation of H2O2 on angiogenesis implicated NADPH oxidase

ROS (reactive oxygen species) take an important signalling role in angiogenesis. Although there are several ways to produce ROS in cells, multicomponent non‐phagocytic NADPH oxidase is an important source of ROS that contribute to angiogenesis. In the present work, we examined the effects of H₂O₂ on angiogenesis including proliferation and migration in HUVECs (human umbilical vein endothelial cells), new vessel formation in chicken embryo CAM (chorioallantoic membrane) and endothelial cell apoptosis, which is closely related to anti‐angiogenesis. Our results showed that H₂O₂ dose‐dependently increased the generation of O₂ ^? (superoxide anion) in HUVECs, which was suppressed by DPI (diphenylene iodonium) and APO (apocynin), two inhibitors of NADPH oxidase. H₂O₂ at low concentrations (10 µM) stimulated cell proliferation and migration, but at higher concentrations, inhibited both. Similarly, H₂O₂ at 4 nmol/cm² strongly induced new vessel formation in CAM, while it suppressed at high concentrations (higher than 4 nmol/cm²). Also, H₂O₂ (200～500 µM) could stimulate apoptosis in HUVECs. All the effects of H₂O₂ on angiogenesis could be suppressed by NADPH oxidase inhibitors, which suggests that NADPH oxidase acts downstream of H₂O₂ to produce O₂ ^? and then to regulate angiogenesis. In summary, our results suggest that H₂O₂ as well as O₂ ^? mediated by NADPH oxidase have biphasic effects on angiogenesis in vitro and in vivo.     

Pretreatment with H(2) O(2) alleviates aluminum-induced oxidative stress in wheat seedlings

Hydrogen peroxide (H₂O₂) is a key reactive oxygen species (ROS) in signal transduction pathways leading to activation of plant defenses against biotic and abiotic stresses. In this study, we investigated the effects of H₂O₂ pretreatment on aluminum (Al) induced antioxidant responses in root tips of two wheat (Triticum aestivum L.) genotypes, Yangmai‐5 (Al‐sensitive) and Jian‐864 (Al‐tolerant). Al increased accumulation of H₂O₂ and O₂^?? leading to more predominant lipid peroxidation, programmed cell death and root elongation inhibition in Yangmai‐5 than in Jian‐864. However, H₂O₂ pretreatment alleviated Al‐induced deleterious effects in both genotypes. Under Al stress, H₂O₂ pretreatment increased the activities of superoxide dismutase, catalase, peroxidase, ascorbate peroxidase and monodehydroascorbate reductase, glutathione reductase and glutathione peroxidase as well as the levels of ascorbate and glutathione more significantly in Yangmai‐5 than in Jian‐864. Furthermore, H₂O₂ pretreatment also increased the total antioxidant capacity evaluated as the 2, 2‐diphenyl‐1‐picrylhydrazyl‐radical scavenging activity and the ferric reducing/antioxidant power more significantly in Yangmai‐5 than in Jian‐864. Therefore, we conclude that H₂O₂ pretreatment improves wheat Al acclimation during subsequent Al exposure by enhancing the antioxidant defense capacity, which prevents ROS accumulation, and that the enhancement is greater in the Al‐sensitive genotype than in the Al‐tolerant genotype.     

Putative role of the malate valve enzyme NADP–malate dehydrogenase in H2O2 signalling in Arabidopsis

In photosynthetic organisms, sudden changes in light intensity perturb the photosynthetic electron flow and lead to an increased production of reactive oxygen species. At the same time, thioredoxins can sense the redox state of the chloroplast. According to our hypothesis, thioredoxins and related thiol reactive molecules downregulate the activity of H₂O₂-detoxifying enzymes, and thereby allow a transient oxidative burst that triggers the expression of H₂O₂ responsive genes. It has been shown recently that upon light stress, catalase activity was reversibly inhibited in Chlamydomonas reinhardtii in correlation with a transient increase in the level of H₂O₂. Here, it is shown that Arabidopsis thaliana mutants lacking the NADP–malate dehydrogenase have lost the reversible inactivation of catalase activity and the increase in H₂O₂ levels when exposed to high light. The mutants were slightly affected in growth and accumulated higher levels of NADPH in the chloroplast than the wild-type. We propose that the malate valve plays an essential role in the regulation of catalase activity and the accumulation of a H₂O₂ signal by transmitting the redox state of the chloroplast to other cell compartments.     

Salinity-induced subcellular accumulation of H2O2 in leaves of rice

The localization of salt-induced H₂O₂ accumulation in the leaves of rice was examined using 3,3-diaminobenzidine and CeCl₃ staining at ultrastructure level. When the 3-week-old rice plants were affected by 100 mM NaCl for 14 days, the swelling of thylakoids and the destruction of thylakoid membranes were observed. H₂O₂ accumulation was also observed in the chloroplast of the leaf treated with NaCl. The electron dense products of 3,3-diaminobenzidine and CeCl₃ were mainly observed especially around the swelling of thylakoids. H₂O₂ accumulation and any ultrastructural changes were not observed in the chloroplasts under dark condition. Furthermore, treatment with ascorbic acid suppressed both H₂O₂ accumulation and the changes in chloroplast ultrastructure. These results suggest that light-induced production of excess H₂O₂ under salinity is responsible for the changes in chloroplast ultrastructure. H₂O₂ accumulation was also observed in the mitochondria, peroxisomes, plasma membrane, and cell walls under light but not dark, suggesting that these organelles are also the source of H₂O₂ and the production is light dependent under salinity.     

Identification of an H2O2 permeable PIP aquaporin in barley and a serine residue promoting H2O2 transport

A barley (Hordeum vulgare) plasma membrane type aquaporin, HvPIP2;5, was identified as an H₂O₂ permeable aquaporin among 21 barley and rice PIPs examined in the heterologous expression system using Saccharomyces cerevisiae. Four TIPs were also detected as H₂O₂‐transporting aquaporins among 15 barley and rice TIPs. Influx of H₂O₂ into yeast cells expressing HvPIP2;5 was determined with a florescent‐dye‐based assay. Indirect immunofluorescence indicated that the expression of HvPIP2;5 protein was ubiquitous in root tissues, and was also weakly observed in leaf epidermal cells and cells in the vascular bundle. Point mutated variants of HvPIP2;5 were generated by the site‐directed mutagenesis. Growth assays of yeast cells expressing these mutated HvPIP2;5 proteins suggested that Ser‐126 in HvPIP2;5 has a large impact on H₂O₂ transport with a minor influence on the HvPIP2;5‐mediated water transport.     

Heat stress-induced H2O2 is required for effective expression of heat shock genes in Arabidopsis

The mechanisms of sensing and signalling of heat and oxidative stresses are not well understood. The central question of this paper is whether in plant cells oxidative stress, in particular H₂O₂, is required for heat stress- and heat shock factor (HSF)-dependent expression of genes. Heat stress increases intracellular accumulation of H₂O₂ in Arabidopsis cell culture. The accumulation was greatly diminished using ascorbate as a scavenger or respectively diphenyleneiodonium chloride (DPI) as an inhibitor of reactive oxygen species production. The mRNA of heat shock protein (HSP) genes, exemplified by Hsp17.6, Hsp18.2, and the two cytosolic ascorbate peroxidase genes Apx1, Apx2, reached similar levels by moderate heat stress (37°C) or by treatment with H₂O₂, butylperoxide and diamide at room temperature. The heat-induced expression levels were significantly reduced in the presence of ascorbate or DPI indicating that H₂O₂ is an essential component in the heat stress signalling pathway. Rapid (15 min) formation of heat shock promoter element (HSE) protein-binding complex of high molecular weight in extracts of heat-stressed or H₂O₂-treated cells and the inability to form this complex after ascorbate treatment suggests that oxidative stress affects gene expression via HSF activation and conversely, that H₂O₂ is involved in HSF activation during the early phase of heat stress. The heat stress induction of a high mobility HSE-binding complex, characteristic for later phase of heat shock response, was blocked by ascorbate and DPI. H₂O₂ was unable to induce this complex suggesting that H₂O₂ is involved only in the early stages of HSF activation. Significant induction of the genes tested after diamid treatment and moderate expression of the sHSP genes in the presence of 50 mM ascorbate at 37°C occurred without activation of HSF, indicating that other mechanisms may be involved in stress signalling. Electronic Supplementary Material Supplementary material is available for this article at http//dx.doi.org/10.1007/s11103-006-0045-4 Roman A. Volkov and Irina I. Panchuk contributed equally     

H2O2 localization in the green alga Micrasterias after salt and osmotic stress by TEM-coupled electron energy loss spectroscopy

Reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂), are constantly generated as by-products of normal metabolic cellular pathways and can be overproduced in response to stress. In this study, we investigated ROS production and localization of H₂O₂ after salt (200 mM KCl) and osmotic (iso-osmotic sorbitol concentration) stress in the unicellular green alga Micrasterias. By means of the dye H₂DCFDA and confocal laser scanning microscopy, most ROS production could be detected in KCl-treated cells when compared to sorbitol-exposed cells and controls. For ultrastructural detection of H₂O₂, CeCl₃, which reacts with H₂O₂ and produces cerium perhydroxide deposits, has been used. Cerium was identified by transmission electron microscopy (TEM)-coupled electron energy loss spectroscopy (EELS) in organelles of KCl- and sorbitol-treated cells and in controls. Statistical measurements of the presence of the cerium M_4,5 edge were performed in mitochondria, chloroplasts, cell walls, and cytoplasmic sites of five individual cells after each treatment. The most pronounced increase in H₂O₂ production was found in chloroplasts of KCl- and sorbitol-treated cells. This shows that the chloroplast reveals the strongest response in H₂O₂ production after stress induction in Micrasterias. Significant elevation of H₂O₂ production also occurred in mitochondria and cytoplasm, whereas H₂O₂ levels remained unchanged or even slightly decreased in cell walls of treated cells. Additionally, TEM micrographs and EELS analyses provided indirect evidence for an increased H₂O₂ production at the plasma membrane of KCl-treated cells, indicating an involvement of the plasma membrane NADPH oxidase in H₂O₂ generation.     

The nuclear transporter SAD2 plays a role in calcium‐ and H2O2‐mediated cell death in Arabidopsis

In response to pathogens, plant cells exhibit a rapid increase in the intracellular calcium concentration and a burst of reactive oxygen species (ROS). The cytosolic increase in Ca²⁺ and the accumulation of ROS are critical for inducing programmed cell death (PCD), but the molecular mechanism is not fully understood. We screened an Arabidopsis mutant, sad2‐5, which harbours a T‐DNA insertion in the 18^th exon of the importin beta‐like gene, SAD2. The H₂O₂‐induced increase in the [Ca²⁺]_cyt of the sad2‐5 mutant was greater than that of the wild type, and the sad2‐5 mutant showed clear cell death phenotypes and abnormal H₂O₂ accumulation under fumonisin‐B1 (FB1) treatment. CaCl₂ could enhance the FB1‐induced cell death of the sad2‐5 mutant, whereas lanthanum chloride (LaCl₃), a broad‐spectrum calcium channel blocker, could restore the FB1‐induced PCD phenotype of sad2‐5. The sad2‐5 fbr11‐1 double mutant exhibited the same FB1‐insensitive phenotype as fbr11‐1, which plays a critical role in novo sphingolipid synthesis, indicating that SAD2 works downstream of FBR11. These results suggest the important role of nuclear transporters in calcium‐ and ROS‐mediated PCD response as well as provide an important theoretical basis for further analysis of the molecular mechanism of SAD2 function in PCD and for improvement of the resistance of crops to adverse environments.     

Relationship between H2O2 and jasmonic acid in pea leaf wounding response

The relationship of H₂O₂ and jasmonic acid (JA) in wound-induced defense response was investigated in the leaves of pea (Pisum sativum L.) plants. The results showed that both wounding and JA treatment led to a significant increase in activities of plasma membrane NADPH oxidase and phenylalanine ammonialyase. However, such an increase was blocked by the pretreatment with plasma membrane NADPH oxidase inhibitors, O ₂ ^? scavengers, or H₂O₂ scavenger, implying that H₂O₂ functions downstream of JA. Furthermore, wounding treatment activated two key enzymes of JA biosynthesis, lipoxygenase and allene oxide synthase, while JA biosynthetic inhibitors impaired the wounding-induced H₂O₂ burst. Thus, it is suggested that H₂O₂ burst depends on JA production in plant wounding response.