Early photosynthetic response of Arabidopsis thaliana to temperature and salt stress conditions

Temperature changes and salt accumulation are among the most common abiotic factors affecting plants in agricultural and natural ecosystems. The different responses of plants to these factors have been widely investigated in previous works. However, detailed mechanism of the early photosynthetic response (first 24 h) has been poorly studied. The aim of the work was to monitor the early response of adult Arabidopsis thaliana plants exposed to different thermal (cold and heat) and salt conditions. Detailed evaluation of the efficiency of photosystem II was done, and the various routes of energy output as well as measurements of the contents of H₂O₂, proline, and photosynthetic pigments at different times during the first 24 h of treatment were examined. The conditions used in the study were those that caused a weak stress with time of exposure. Cold-treated plants showed the most continuous inhibitory effect on photosynthetic activity, with a fast metabolic slowdown (reduced PSII efficiency and decreased pigment contents), although they also demonstrated clear acclimation responses (increased heat dissipation and protein content). Heat-treated plants showed a late but stronger effect on photosynthesis with significantly increased quantum yield of nonregulated energy dissipation (??_NO) and H₂O₂ content at the last measurements. Finally, salt-induced oxidative stress (increased H₂O₂ content), decreased PSII efficiency and pigment content.     

Ascorbate–glutathione pathways mediated by cytokinin regulate H2O2 levels in light-controlled rose bud burst

Rosebush (Rosa “Radrazz”) plants are an excellent model to study light control of bud outgrowth since bud outgrowth only arises in the presence of light and never occurs in darkness. Recently, we demonstrated high levels of hydrogen peroxide (H₂O₂) present in the quiescent axillary buds strongly repress the outgrowth process. In light, the outgrowing process occurred after H₂O₂ scavenging through the promotion of Ascorbic acid–Glutathione (AsA–GSH)-dependent pathways and the continuous decrease in H₂O₂ production. Here we showed Respiratory Burst Oxidase Homologs expression decreased in buds during the outgrowth process in light. In continuous darkness, the same decrease was observed although H₂O₂ remained at high levels in axillary buds, as a consequence of the strong inhibition of AsA–GSH cycle and GSH synthesis preventing the outgrowth process. Cytokinin (CK) application can evoke bud outgrowth in light as well as in continuous darkness. Furthermore, CKs are the initial targets of light in the photocontrol process. We showed CK application to cultured buds in darkness decreases bud H₂O₂ to a level that is similar to that observed in light. Furthermore, this treatment restores GSH levels and engages bud burst. We treated plants with buthionine sulfoximine, an inhibitor of GSH synthesis, to solve the sequence of events involving H₂O₂/GSH metabolisms in the photocontrol process. This treatment prevented bud burst, even in the presence of CK, suggesting the sequence of actions starts with the positive CK effect on GSH that in turn stimulates H₂O₂ scavenging, resulting in initiation of bud outgrowth.

Light-induced bud outgrowth in rosebush results from cytokinin-mediated peroxide scavenging and glutathione metabolism stimulation.     

Xylem parenchyma cells deliver the H<Subscript>2</Subscript>O<Subscript>2</Subscript> necessary for lignification in differentiating xylem vessels

Lignification in Zinnia elegans L. stems is characterized by a burst in the production of H₂O₂, the apparent fate of which is to be used by xylem peroxidases for the polymerization of p-hydroxycinnamyl alcohols into lignins. A search for the sites of H₂O₂ production in the differentiating xylem of Z. elegans stems by the simultaneous use of optical (bright field, polarized light and epi-polarization) and electron-microscope tools revealed that H₂O₂ is produced on the outer-face of the plasma membrane of both differentiating (living) thin-walled xylem cells and particular (non-lignifying) xylem parenchyma cells. From the production sites it diffuses to the differentiating (secondary cell wall-forming) and differentiated lignifying xylem vessels. H₂O₂ diffusion occurs mainly through the continuous cell wall space. Both the experimental data and the theoretical calculations suggest that H₂O₂ diffusion from the sites of production might not limit the rate of xylem cell wall lignification. It can be concluded that H₂O₂ is produced at the plasma membrane in differentiating (living) thin-walled xylem cells and xylem parenchyma cells associated to xylem vessels, and that it diffuses to adjacent secondary lignifying xylem vessels. The results strongly indicate that non-lignifying xylem parenchyma cells are the source of the H₂O₂ necessary for the polymerization of cinnamyl alcohols in the secondary cell wall of lignifying xylem vessels.     

Critical role of poly(ADP‐ribose) polymerase‐1 in modulating the mode of cell death caused by continuous oxidative stress

Continuously generated hydrogen peroxide (H₂O₂) inhibits typical apoptosis and instead initiates a caspase‐independent, apoptosis‐inducing factor (AIF)‐mediated pyknotic cell death. This may be related to H₂O₂‐mediated DNA damage and subsequent ATP depletion, although the exact mechanisms by which the mode of cell death is decided after H₂O₂ exposure are still unclear. Accumulated evidence and our previous data led us to hypothesize that continuously generated H₂O₂, not an H₂O₂ bolus, induces severe DNA damage, signaling poly(ADP‐ribose) polymerase‐1 (PARP‐1) activation, ATP depletion, and eventually caspase‐independent cell death. Results from the present study support that H₂O₂ generated continuously by glucose oxidase causes excessive DNA damage and PARP‐1 activation. Blockage of PARP‐1 by a siRNA transfection or by pharmacological inhibitor resulted in the significant inhibition of ATP depletion, loss of mitochondrial membrane potential, nuclear translocation of AIF and endonuclease G, and eventually conversion to caspase‐dependent apoptosis. Overall, the current study demonstrates the different roles of PARP‐1 inhibition in modulation of cell death according to the method of H₂O₂ exposure, that is, continuous generation versus a direct addition. J. Cell. Biochem. 108: 989–997, 2009. © 2009 Wiley‐Liss, Inc.     

Drought-induced oxidative damage and antioxidant responses in peanut (<Emphasis Type="Italic">Arachis</Emphasis><Emphasis Type="Italic">hypogaea</Emphasis> L.) seedlings

Two cultivars of peanut (Arachis hypogaea L.) which were designated as resistant (Florispan) and sensitive (Gazipasa) according to their growth retardation under drought stress conditions were compared for their oxidative damage and antioxidant responses. Sixteen days-old peanut seedlings were subjected to PEG-6000 solutions of two different osmotic potentials; −0.4 and −0.8 MPa, and various growth parameters, photosystem II activity, changes in malondialdehyde (MDA), hydrogen peroxide (H₂O₂) and proline levels, activities of ascorbate peroxidase (APX), catalase (CAT), peroxidase (POX) and gluthatione reductase (GR) enzymes were determined. Both cultivars exhibited water deficit at −0.8 MPa osmotic potential of PEG-6000 and H₂O₂ levels significantly increased during exposure to −0.4 MPa osmotic potential. However, H₂O₂ levels were under control in both cultivars at exposure to −0.8 MPa osmotic potential. Significant proline accumulation was observed in the tissues of cv. Florispan at −0.8 MPa osmotic potential, whereas proline accumulation did not appear to be an essential part of the protection mechanism against drought in cv. Gazipasa. No significant variation in chlorophyll fluorescence values were detected in neither of the cultivars. Enzyme activity measurements revealed that Gazipasa copes well with lesser magnitudes of drought stress by increasing the activity of mainly APX, and during harsh stress conditions, only APX maintains its activity in the tissues. In cultivar Florispan, GR activity appears to take role in lesser magnitudes of drought stress, whereas CAT and APX activities appear to be very crucial antioxidative defenses during intense stress conditions. The results indicate that, the level of proline and activities of the enzymes CAT and APX are important mechanisms for the maintenance of drought tolerance in peanut plants.     

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.     

Salicylic acid and H2O2 function by independent pathways in the induction of freezing tolerance in potato

The effects of salicylic acid (SA) and hydrogen peroxide (H₂O₂) on freezing tolerance were studied in two potato (Solanum tuberosum) cultivars (Alpha and Atlantic) that differ in cold sensitivity, Alpha being more tolerant to freezing than Atlantic. Lowest freezing survival rates were observed in 4-week-old plants. Freezing treatments consisting of exposure to 6° C for 4 h in the dark were applied 24 h after plants had been transferred from in vitro culture to soil. Catalase activity and H₂O₂ were estimated at the following harvest points: stage (a) 4-week-old in vitro plants treated with either 0.1 mM SA or 5 mM H₂O₂; stage (b) as in (a) but 24 h following transfer to soil prior to freezing treatment; stage (c) as in (b) but measured 15 days after a 4-h freezing treatment. The results show that (1) SA induced freezing tolerance in both cultivars; (2) SA inhibited ascorbate peroxidase activities in both cultivars at all harvest points but inhibited catalase activities in only at stage (a); (3) SA induced H₂O₂ accumulation only in Atlantic at stage (a); (4) H₂O₂ enhanced shoot catalase activities in Atlantic at stages (a) and (b) whereas this treatment had no effect on shoot catalase activities in Alpha; (5) H₂O₂ treatment induced freezing tolerance in Atlantic, even though shoot catalase activities were lower than those of the controls following exposure to freezing temperatures. We conclude that SA does not always lead to H₂O₂ accumulation even though catalase and ascorbate peroxidase activities are decreased as a result of the treatment. Moreover, H₂O₂ accumulation is not always associated with the induction of freezing tolerance, for example at stage (a) where SA-induced tolerance in Alpha was not accompanied by H₂O₂ accumulation. H₂O₂ was able to induce freezing tolerance only in Atlantic, even though H₂O₂ accumulated in both cultivars following this treatment.     

Variations of vinblastine accumulation and redox state affected by exogenous H<Subscript>2</Subscript>O<Subscript>2</Subscript> in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don

Effects of exogenous H₂O₂ application on vinblastine (VBL) and its precursors, vindoline (VIN), catharanthine (CAT) and α-3′,4′-anhydrovinblastine (AVBL), were measured in Catharanthus roseus seedlings in order to explore possible correlation of VBL formation with oxidative stress. VBL accumulation has previously been shown to be regulated by an in vitro H₂O₂-dependent peroxidase (POD)-like synthase. Experimental exposure of plants to different concentrations of H₂O₂ showed that endogenous H₂O₂ and alkaloid concentrations in leaves were positively elevated. The time-course variations of alkaloid concentrations and redox state, reflected by the concentrations of H₂O₂, ascorbic acid (AA), oxidative product of glutathione (GSSG) and POD activity, were significantly altered due to H₂O₂ application. The further correlation analysis between alkaloids and redox status indicated that VBL production was tightly correlated with redox status. These results provide a new link between VBL metabolisms and redox state in C. roseus.     

Nuclear morphology and c‐Jun N‐terminal kinase 1 expression differentiate serum‐starved oxidative stress signalling from hydrogen peroxide‐induced apoptosis in retinal neuronal cell line

Oxidative stress induced by serum starvation and H₂O₂ exposure, both triggers apoptosis in retinal neuronal cell line RGC‐5 (retinal ganglion cell‐5). We have examined whether, despite excess generation of ROS (reactive oxygen species) and apoptosis induction, there is any dissimilarity in nuclear morphology and apoptotic signalling pathway in RGC‐5 under these conditions. Sub‐confluent cells were treated either with H₂O₂ or maintained in SFM (serum‐free medium). ROS level was detected along with nuclear morphology and ultrastructural analysis. Generation of excess intracellular ROS, nuclear localization of Bax and caspase 3 activation along with decrease of cellular viability, confirmed apoptosis induction in RGC‐5 by 72 h serum starvation and 500 M H₂O₂ exposure for 1 h. Nuclear swelling as supported by nuclear cytoplasmic ratio and conspicuous black spots with nuclear remodelling were observed only upon SFM, but not with H₂O₂ treatment. Serum starvation did not alter JNK1 (c‐Jun N‐terminal kinase 1) expression, although nuclear translocation and higher level of pJNK (phospho‐JNK) was evident. Conversely, H₂O₂ exposure blocked the expression and activation of JNK1 to phospho‐JNK as a negligible level of pJNK was present in the cytoplasm. Despite similar ROS generation in both the conditions, difference in nuclear morphology and JNK1 expression leads to the hypothesis that RGC‐5 cells may follow different signalling pathways when challenged with serum starvation and H₂O₂.     

Fluctuating vs. Continuous Exposure to H2O2: The Effects on Mitochondrial Membrane Potential,Intracellular Calcium,and NF-κB in Astroglia

The effects of H₂O₂ are widely studied in cell cultures and other in vitro systems. However, such investigations are performed with the assumption that H₂O₂ concentration is constant, which may not properly reflect in vivo settings, particularly in redox-turbulent microenvironments such as mitochondria. Here we introduced and tested a novel concept of fluctuating oxidative stress. We treated C6 astroglial cells and primary astrocytes with H₂O₂, using three regimes of exposure – continuous, as well as fluctuating at low or high rate, and evaluated mitochondrial membrane potential and other parameters of mitochondrial activity – respiration, reducing capacity, and superoxide production, as well as intracellular ATP, intracellular calcium, and NF-κB activation. When compared to continuous exposure, fluctuating H₂O₂ induced a pronounced hyperpolarization in mitochondria, whereas the activity of electron transport chain appears not to be significantly affected. H₂O₂ provoked a decrease of ATP level and an increase of intracellular calcium concentration, independently of the regime of treatment. However, fluctuating H₂O₂ induced a specific pattern of large-amplitude fluctuations of calcium concentration. An impact on NF-κB activation was observed for high rate fluctuations, whereas continuous and low rate fluctuating oxidative stress did not provoke significant effects. Presented results outline the (patho)physiological relevance of redox fluctuations.     

Hydrogen peroxide pretreatment of roots enhanced oxidative stress response of tomato under cold stress

In the view of physiological role of H₂O₂, we investigated whether exogenous H₂O₂ application would affect short-term cold response of tomato and induce acclimation. Pretreatments were performed by immersing roots into 1 mM H₂O₂ solution for 1 h when transferring seedlings from seedling substrate to soil (acclimated group). Cold stress (3 °C for 16 h) caused significant reduction in relative water content (RWC) of control and non-acclimated (distilled water treated) groups when compared with unstressed plants. H₂O₂ promoted maintenance of relatively higher RWC under stress. Anthocyanin level in leaves of acclimated plants under cold stress was significantly higher than that of unstressed control and non-acclimated plants. Malondialdehyde (MDA) levels demonstrated low temperature induced oxidative damage to control and non-acclimated plants. MDA remained around unstressed conditions in acclimated plants, which demonstrate that H₂O₂ acclimation protected tissues against cold induced lipid peroxidation. H₂O₂ acclimation caused proline accumulation in roots under cold stress. Ascorbate peroxidase (APX) activity in roots of cold stressed and unstressed H₂O₂ acclimated plants increased when compared with control and non-acclimated plants, with highest increase in roots of acclimated plants under cold stress. CAT levels in roots of acclimated plants also increased, whereas levels remained unchanged in unstressed plants. Endogenous H₂O₂ levels significantly increased in roots of control and non-acclimated plants under cold stress. On the other hand, H₂O₂ content in roots of acclimated plants was significantly lower than control and non-acclimated plants under cold stress. The results presented here demonstrated that H₂O₂ significantly enhanced oxidative stress response by elevating the antioxidant status of tomato.     

Propofol Protects Against H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Oxidative Injury in Differentiated PC12 Cells via Inhibition of Ca<Superscript>2+</Superscript>-Dependent NADPH Oxidase

Propofol (2,6-diisopropylphenol) is a widely used general anesthetic with anti-oxidant activities. This study aims to investigate protective capacity of propofol against hydrogen peroxide (H₂O₂)-induced oxidative injury in neural cells and whether the anti-oxidative effects of propofol occur through a mechanism involving the modulation of NADPH oxidase (NOX) in a manner of calcium-dependent. The rat differentiated PC12 cell was subjected to H₂O₂ exposure for 24 h to mimic a neuronal in vitro model of oxidative injury. Our data demonstrated that pretreatment of PC12 cells with propofol significantly reversed the H₂O₂-induced decrease in cell viability, prevented H₂O₂-induced morphological changes, and reduced the ratio of apoptotic cells. We further found that propofol attenuated the accumulation of malondialdehyde (biomarker of oxidative stress), counteracted the overexpression of NOX core subunit gp91^phox (NOX2) as well as the NOX activity following H₂O₂ exposure in PC12 cells. In addition, blocking of L-type Ca²⁺ channels with nimodipine reduced H₂O₂-induced overexpression of NOX2 and caspase-3 activation in PC12 cells. Moreover, NOX inhibitor apocynin alone or plus propofol neither induces a significant downregulation of NOX activity nor increases cell viability compared with propofol alone in the PC12 cells exposed to H₂O₂. These results demonstrate that the protective effects of propofol against oxidative injury in PC12 cells are mediated, at least in part, through inhibition of Ca²⁺-dependent NADPH oxidase.     

Salicylic acid,hydrogen peroxide and calcium-induced saline tolerance associated with endogenous hydrogen peroxide homeostasis in naked oat seedlings

This study investigates the role of salicylic acid (SA), hydrogen peroxide (H₂O₂) and calcium chloride (CaCl₂) singly or in combination, in inducing naked oat plant tolerance to sodium chloride (NaCl). Two-week-old naked oat plants were pretreated with both single and double of 0.5 mM SA, 0.5 mM H₂O₂ and 5 mM CaCl₂ by adding them to the culture solution for 24 h. At the end of the pretreatment, the plants were subjected to 200 mM NaCl exposure for 7 days. Data were collected on plant biomass, H₂O₂ level, antioxidant enzyme activity, non-enzymatic antioxidant content and malondialdehyde (MDA) content. Results showed that exposure to salt significantly inhibited plant growth, and the shoot and root dry weights were reduced 47.5% and 63.4%, and the H₂O₂ levels elevated 5.8 and 2.4 times in comparison with those in the control, respectively. Under the saline stress, the activities of superoxide dismutase (SOD) and catalase (CAT) were induced, but the contents of ascorbic acid (AA) and glutathione (GSH) decreased, and MDA largely accumulated. The various pretreatments efficiently counteracted the salt-caused growth inhibition, especially with H₂O₂ + CaCl₂ the shoot and root dry weights reduced only 9.4% and 24.4% of the non salt-stressed plants. The determination of endogenous H₂O₂ level demonstrated that the pretreatments induced H₂O₂ accumulation, with H₂O₂ + CaCl₂ being most efficient, but the effect was transient. After 7 days of saline stress, the H₂O₂ contents in the pretreated shoots and roots accounted for 23.7–41.8% and 31.7–57.3% of the non-pretreated plants, varying according to the different pretreatments. Under saline stress, SOD and CAT further increased, AA and GSH maintained higher levels and MDA decreased in the pretreated plants compared to the untreated plants. With application of diphenylene iodonium (DPI) during the pretreatment, which inhibited the accumulation of H₂O₂, the ameliorative effect of the pretreatment on salt-caused plant growth inhibition was reduced. However, applied DPI at the immediate end of the pretreatment did not alter its favorable role, indicating a H₂O₂ peak formed at the early time of saline stress might play an important role in regulating plant tolerance to saline stress.     

Characterization of Hydrogen Peroxide Toxicity in Cultured Rat Forebrain Neurons

We investigated the ability of hydrogen peroxide (H₂O₂) to cause apoptotic cell death in cultured rat forebrain neurons and the potential mechanisms by which oxidative stress triggers delayed neuronal death. H₂O₂ (25 M for 5 min) reduced cell viability to 34.5 ± 8.3% of untreated controls 20 h after exposure, and resulted in a significant proportion of neurons which exhibited apoptotic nuclear morphology. Using single cell fluorescence assays, we measured H₂O₂-induced changes in DNA strand breaks, 27 dichlorofluorescin fluorescence, reduced glutathione, intracellular free Ca²⁺, and mitochondrial membrane potential. DNA strand breaks in response to H₂O₂ were not evident immediately following exposure, but were increased 12h and 20h after exposure. Millimolar concentrations of H₂O₂ caused increases in the fluorescence of the oxidant-sensitive fluorescent dye, 27-dichlorofluorescin. H₂O₂ treatment decreased reduced glutathione following 30 minutes of exposure using the fluorescent indicator, 5-chloromethylfluorescein diacetate, and increased intra-neuronal free Ca²⁺ levels in a subpopulation of neurons. Mitochondrial membrane potential, measured by rhodamine 123 localization was unaffected by 25 H₂O₂, while higher concentrations of H₂O₂ (10 or 30 mM) depolarized mitochondria. These studies demonstrate that H₂O₂ is a potent and effective neurotoxin that produces oxidative stress, as well as apoptotic neuronal death     

Effects of hydrogen peroxide on the content of major volatile halogenated compounds in the red alga <Emphasis Type="Italic">Asparagopsis taxiformis</Emphasis> (Bonnemaisoniaceae)

The genus Asparagopsis is a prolific source of halogenated metabolites. Due to its commercial applications, it has been intensively cultivated in southern Portugal. In the present study, we assess if the internal levels of the major halogenated metabolites (bromoform and dibromoacetic acid) in Asparagopsis taxiformis can be increased with hydrogen peroxide (H₂O₂) addition. Previous studies with red algae showed that the production/release of bromoform can be enhanced by exogenously supplying H₂O₂. However, no study has assessed if H₂O₂ supply enhances the content of secondary metabolites within the biomass. This detail is important as the objective of the proposed research is to enhance the content of these valuable metabolites in the produced biomass. Both the activity of the haloperoxidase enzyme and the metabolite content were assessed on short-term and long-term incubation periods to H₂O₂. To determine the susceptibility of A. taxiformis photosynthetic performance to the imposed oxidative stress, the in vivo fluorescence of photosystem II was monitored. A. taxiformis was shown to be physiologically vulnerable to H₂O₂, given the observed decrease of the maximum quantum yield of photosynthesis (F _v/F _m). Contrary to what was expected, the presence of H₂O₂ inhibited the activity of the iodoperoxidase enzyme. Nevertheless, the extracted halogenated metabolites were higher over the first hours of exposure to H₂O₂, decreasing after 48 h. These results are probably related to the prosthetic group of the halogenated enzyme in A. taxiformis and the long-term oxidative stress damage of H₂O₂ exposure. Considering the objective of the proposed research, addition of H₂O₂ to the cultures, prior (3 h) to biomass harvesting, increases the metabolite content.     

Effects of pulsed electric fields on DNA of human lymphocytes

The effects of pulsed electric fields of low frequency (50 Hz) on DNA of human lymphocytes were investigated. The influence of additional external factors, such as hydrogen peroxide (H₂O₂) and γ-irradiation, as well as the repair efficiency in these lymphocytes, was also evaluated. The comet assay, a very sensitive and rapid method for detecting DNA damage at the single cells level was the method used. A significant amount of damage was observed after exposure to the electric fields, compared to the controls. After 2 h incubation at 37^°C, a proportion of damage was repaired. H₂O₂ and γ-irradiation increased the damage to lymphocytes exposed to pulsed electric fields according to the dose used, while the amount of the repair was proportional to the damage.     

Effect of exogenous hydrogen peroxide on enzymatic and nonenzymatic antioxidants in leaves of young pea plants treated with paraquat

The effects of exogenously applied hydrogen peroxide on the antioxidant system of pea plants were investigated. Ten-day-old pea seedlings were sprayed with 2.5 mM H₂O₂ and 24 h later with 0.2 mM PQ. Samples were taken 0, 2 and 5 h after the start of illumination. The protective effect of H₂O₂ was evaluated by monitoring of parameters related to the damage caused by PQ. The treatment with PQ led to a severe leakage of electrolytes from leaf tissues. Malondialdehyde level increased in PQ treated plants, but remained unchanged in H₂O₂ pre-treated ones after 5 h of illumination. Increased catalase and glutathione-S-transferase activity was observed in pea plants treated with H₂O₂ and PQ. Ascorbate peroxidase activity decreased significantly after paraquat application, but pre-treatment with H₂O₂ prevented ascorbate peroxidase inhibition to some extent. Increased guaiacol peroxidase activity was detected after H₂O₂ application. PQ application caused a drastic decline in the levels of thiol-group bearing compounds, reduced glutathione and ascorbate, while the quantity of oxidized glutathione and dehydroascorbate were increased. The results presented on changes in enzymatic and nonenzymatic antioxidants suggest that preliminary H₂O₂ application to pea plants treated with PQ, alleviates the toxic effects of the herbicide.     

Abscisic acid-induced apoplastic H<Subscript>2</Subscript>O<Subscript>2</Subscript> accumulation up-regulates the activities of chloroplastic and cytosolic antioxidant enzymes in maize leaves

The histochemical and cytochemical localization of abscisic acid (ABA)-induced H₂O₂ production in leaves of maize (Zea mays L.) plants were examined, using 3,3-diaminobenzidine (DAB) and CeCl₃ staining, respectively, and the relationship between ABA-induced H₂O₂ production and ABA-induced subcellular activities of antioxidant enzymes was studied. H₂O₂ generated in response to ABA treatment was detected within 0.5 h in major veins of the leaves and maximized at about 2–4 h. In mesophyll and bundle sheath cells, ABA-induced H₂O₂ accumulation was observed only in apoplast, and the greatest accumulation occurred in the walls of mesophyll cells facing large intercellular spaces. Meanwhile, ABA treatment led to a significant increase in the activities of the leaf chloroplastic and cytosolic antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and pretreatment with the NADPH oxidase inhibitor diphenyleneiodonium (DPI), the O ₂ ⁻ scavenger Tiron and the H₂O₂ scavenger dimethylthiourea (DMTU) almost completely arrested the increase in the activities of these antioxidant enzymes. Our results indicate that the accumulation of apoplastic H₂O₂ is involved in the induction of the chloroplastic and cytosolic antioxidant enzymes. Moreover, an oxidative stress induced by paraquat (PQ), which generates O ₂ ⁻ and then H₂O₂ in chloroplasts, also up-regulated the activities of the chloroplastic and cytosolic antioxidant enzymes, and the up-regulation was blocked by the pretreatment with Tiron and DMTU. These data suggest that H₂O₂ produced at a specific cellular site could coordinate the activities of antioxidant enzymes in different subcellular compartments.