Effects of artificially enhanced levels of ascorbate and glutathione on the enzymes monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase in spinach (Spinacia oleracea)

Effect of high intracellular concentrations of the antioxidants ascorbate and glutathione on the extractable activity of the reducting enzymes dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase were investigated with spinach cells ( Spinacia oleracea ). An elevated ascorbate concentration was obtained by treatment with the ascorbate biosynthesis precursor L-galactono-1,4-lactone (GAL). To increase the intracellular level of glutathione, cells were treated with the 5-oxo-L-proline analog L-2-oxothiazolidin-4-carboxylate (OTC), or with the peroxidative herbicide acifluorfen (sodium 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid). Extractable monodehydroascorbate reductase activity increased in the presence of a high level of ascorbate or glutathione, and enzyme activity was at maximum when cells were treated with acifluorfen + OTC, or acifluorfen + GAL. Extractable dehydroascorbate reductase activity decreased when the intracellular concentration of glutathione was high and non-enzymatic reduction of dehydroascorbate by glutathione was the dominant reaction. Maximal decrease of enzyme activity was found in cells treated with acifluorfen + OTC. Extractable activity of glutathione reductase (GR) increased after treatment of cells with acifluorfen alone, or acifluorfen + OTC, but enzyme activity was unaffected by a high intracellular concentration of glutathione obtained by treatment of cells with OTC alone, or by treatment with acifluorfen + GAL. The degree of GR activation seemed to be controlled by several factors including inhibition by a high concentration of glutathione and possibly oxidative damage to the enzyme. Overall, the enzymes tested in this study, which provide the reduced forms of ascorbate and glutathione, were differently affected by high antioxidant levels.     

Lipid peroxidation in higher plants : the role of glutathione reductase

To study the role of glutathione reductase in lipid peroxidation, bean leaves (Phaseolus vulgaris) cv Fori were treated with the herbicide acifluorfen-sodium (sodium 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid). Acifluorfen is a potent inducer of lipid peroxidation. In beans, decrease of acid-soluble SH-compounds and lipid peroxidation, measured as ethane evolution, were the toxic events after treatment of leaves with acifluorfen. As a primary response to peroxidation, increased production of antioxidants, such as vitamin C and glutathione, was found. This was followed by elevation of glutathione reductase activity. Enhanced activity of the enzyme prevented both further decline of acid-soluble SH-compounds and lipid peroxidation. Increased production of antioxidants and elevated activity of antioxidative enzymes, like glutathione reductase, seem to be a general strategy to limit toxic peroxidation in plants.     

Chloroplast superoxide and hydrogen peroxide scavenging systems from pea leaves: Seasonal variations

Levels of chloroplast antioxidants and enzymes that scavenge oxygen racidals were followed in the leaves of pea plants (Pisum sativum L. cv. Meteor) grown under glasshouse conditions between April 1984 and May 1985. While little variation in pigment levels or superoxide dismutase activity was detected during this period, plants grown in early summer (May–June) contained appreciably higher levels of ascorbate, ascorbate peroxidase and glutathione reductase than plants grown in winter (Dec–Jan.). The role of light intensity in regulating levels of chloroplast antioxidants was examined further using pea plants grown in a constant environment chamber under 100 or 400 μmol m⁻² s ⁻¹ photon flux density. Chloroplasts isolated from plants grown at the higher light intensity contained significantly higher levels of ascorbate, ascorbate peroxidase, glutathione reductase and dehydroascorbate reductase. These data suggest that light intensity may have an important influence on the level and activity of chloroplast antioxidants and oxygen radical scavenger enzymes.     

Antioxidants and Manganese Deficiency in Needles of Norway Spruce (Picea abies L.) Trees

Chlorotic and green needles from Norway spruce (Picea abies L.) trees were sampled in the Calcareous Bavarian Alps in winter. The needles were used for analysis of the mineral and pigment contents, the levels of antioxidants (ascorbate, glutathione), and the activities of protective enzymes (superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate radical reductase, dehydroascorbate reductase, glutathione reductase). In addition, the activities of two respiratory enzymes (glucose-6-phosphate dehydrogenase, NAD-malate dehydrogenase), which might provide the NADPH necessary for functioning of the antioxidative system, were determined. We found that chlorotic needles were severely manganese deficient (3 to 6 micrograms Mn per gram dry weight as compared with up to 190 micrograms Mn per gram dry weight in green needles) but had a similar dry weight to fresh weight ratio, had a similar protein content, and showed no evidence for enhanced lipid peroxidation as compared with green needles. In chlorotic needles, the level of total ascorbate and the activities of superoxide dismutase, monodehydroascorbate radical reductase, NAD-malate dehydrogenase, and glucose-6-phosphate dehydrogenase were significantly increased, whereas the levels of ascorbate peroxidase, dehydroascorbate reductase, glutathione reductase, and glutathione were not affected. The ratio of ascorbate to dehydroascorbate was similar in both green and chlorotic needles. These results suggest that in spruce needles monodehydroascorbate radical reductase is the key enzyme involved in maintaining ascorbate in its reduced state. The reductant necessary for this process may have been supplied at the expense of photosynthate.     

Decrease in Activity of Glutathione Reductase Enhances Paraquat Sensitivity in Transgenic Nicotiana tabacum

Transgenic tobacco (Nicotiana tabacum L. cv SR1) with decreased activity of glutathione reductase exhibited enhanced sensitivity to paraquat in the light as evaluated by chlorophyll destruction and electrolyte leakage from leaf discs. This result indicates the involvement of glutathione reductase in the tolerance of plants to photooxidative stress caused by the herbicide.     

The effect of acute high light and low temperature stresses on the ascorbate–glutathione cycle and superoxide dismutase activity in two Dunaliella salina strains

Dunaliella species accumulate carotenoids and their role in protection against photooxidative stress has been investigated extensively. By contrast, the role of other antioxidants in this alga, has received less attention. Therefore, the components of the ascorbate–glutathione cycle, along with superoxide dismutase (E.C. 1.15.1.1) and peroxidase (E.C. 1.11.1.11) activity were compared in two strains of Dunaliella salina. Strain IR‐1 had two‐fold higher chlorophyll and β‐carotene concentration than Gh‐U. IR‐1 had around four‐fold higher superoxide dismutase, ascorbate peroxidase and pyrogallol peroxidase activities than Gh‐U on a protein basis. Ascorbate and glutathione concentrations and redox state did not differ between strains and there was little difference in the activity of ascorbate–glutathione cycle enzymes (monodehydroascorbate reductase [E.C. 1.6.5.4], dehydroascorbate reductase [E.C. 1.8.5.1] and glutathione reductase [E.C. 1.8.1.7]). The response of these antioxidants to high light and low temperature was assessed by transferring cells from normal growth conditions (28°C, photon flux density of 100 μmol m^?2 s^?1)to 28°C/1200 μmol m^?2 s^?1; 13°C/100 μmol m^?2 s^?1; 13°C/1200 μmol m^?2 s^?1 and 28°C/100 μmol m^?2 s^?1 for 24 h. Low temperature and combined high light‐low temperature decreased chlorophyll and β‐carotene in both strains indicating that these treatments cause photooxidative stress. High light, low temperature and combined high light‐low temperature treatments increased the total ascorbate pool by 10–50% and the total glutathione pool by 20–100% with no consistent effect on their redox state. Activities of ascorbate–glutathione cycle enzymes were not greatly affected but all the treatments increased superoxide dismutase activity. It is concluded that D. salina can partially adjust to photooxidative conditions by increasing superoxide dismutase activity, ascorbate and glutathione.     

Chloroplast-Diphenyl Ether Interactions II

Acifluorfen, a p-nitrodiphenyl ether herbicide, is inhibitory to those photosynthetic functions that require a functioning chloroplast envelope. Functions involving the stroma are also affected. Acifluorfen does not lyse intact spinach chloroplasts, yet does increase the sensitivity of CO₂-dependent O₂ evolution to exogenous inorganic phosphate without directly affecting the function of the phosphate translocator. Acifluorfen penetrates into the chloroplast stroma in a light-independent fashion. Once inside, it causes the inactivation of light and dithiothreitol-activated fructose 1,6-bisphosphatase. Light-activated glyceraldehyde-3-phosphate dehydrogenase (NADP) is also inactivated by acifluorfen.

These data suggest that acifluorfen stimulates a pathway for inactivation of fructose 1,6-bisphosphatase and glyceraldehyde 3-phosphate dehydrogenase (NADP) which uses oxygen as a terminal oxidant and which involves thioredoxin and ferredoxin-thioredoxin reductase.

     

Acifluorfen Enhancement of Cryptochrome-Modulated Sporulation following an Inductive Light Pulse

Acifluorfen enhancement of blue-light mediated phototropism suggested that this diphenyl-ether herbicide augments the light reaction (TY Leong, WR Briggs 1983 Plant Physiol 70: 875-881). The separation of the possible direct interaction of acifluorfen with light reactions from interactions with dark pathways has been elucidated in this paper with Trichoderma harzianum. Acifluorfen at 30 micromolar, given for 5 hours in the growth medium, stimulated the conidiation of Trichoderma in response to blue light without apparently affecting growth. Enhanced conidiation could be elicited by dipping cultures into medium with acifluorfen both before as well as 0.5 hour after inductive blue light. This postphotoinduction stimulation indicates that acifluorfen does not directly augment the effect of light by interacting with cryptochrome(s) in Trichoderma. Instead, acifluorfen most probably interacted with the dark reactions following photoinduction.     

Alterations in the antioxidative system of suspension-cultured soybean cells (Glycine max) induced by oxidative stress

The objectives of this study were the changes of antioxidative key enzyme activities under stress conditions induced by a peroxidizing herbicide using photoheterotrophi-cally grown, suspension-cultured soybean celts ( Glycine max L.). Within two days, 50 to 500 n M oxyfluorfen. a p-nitrodiphenyl ether herbicide, caused up to 100% inhibition of growth, while simultaneously, the chlorophyll was 25% to completely bleached. The major cellular antioxidants ascorbate and glutathione showed different responses. Under stress conditions with more than 250 n M oxyfluorfen, the cellular ascorbate- concentration was halved, whereas dehydroascorbate remained roughly constant. The glutathione content (approximately one-fifth of that of ascorbate in untreated control cells) increased nearly 3-fold in the presence of 250 n M oxyfluorfen. Under this condition, oxidized glutathione was 5 times above the control level. The specific activities of selected enzymes participating in cellular defence, namely ascor-bate peroxidase, glutathione reductase, rnonodehydroascorbate reductase. peroxidase and catalase increased by 40 to 70% with oxyfluorfen concentrations between 50 and 500 n M , while dehydroascorbate reductase showed a significant decrease. Glutathione transferase activity even increased 6-fold under oxyfluorfen stress.     

Changes in Salicylic Acid and Antioxidants during Induced Thermotolerance in Mustard Seedlings

Heat-acclimation or salicylic acid (SA) treatments were previously shown to induce thermotolerance in mustard (Sinapis alba L.) seedlings from 1.5 to 4 h after treatment. In the present study we investigated changes in endogenous SA and antioxidants in relation to induced thermotolerance. Thirty minutes into a 1-h heat-acclimation treatment glucosylated SA had increased 5.5-fold and then declined during the next 6 h. Increases in free SA were smaller (2-fold) but significant. Changes in antioxidants showed the following similarities after either heat-acclimation or SA treatment. The reduced-to-oxidized ascorbate ratio was 5-fold lower than the controls 1 h after treatment but recovered by 2 h. The glutathione pool became slightly more oxidized from 2 h after treatment. Glutathione reductase activity was more than 50% higher during the first 2 h. Activities of dehydroascorbate reductase and monodehydroascorbate reductase decreased by at least 25% during the first 2 h but were 20% to 60% higher than the control levels after 3 to 6 h. One hour after heat acclimation ascorbate peroxidase activity was increased by 30%. Young leaves appeared to be better protected by antioxidant enzymes following heat acclimation than the cotyledons or stem. Changes in endogenous SA and antioxidants may be involved in heat acclimation.     

Composition and Properties of Hydrogen Peroxide Decomposing Systems in Extracellular and Total Extracts from Needles of Norway Spruce (Picea abies L., Karst.)

Hydrogen peroxide (H₂O₂) scavenging systems of spruce (Picea abies) needles were investigated in both extracts obtained from the extracellular space and extracts of total needles. As assessed by the lack of activity of symplastic marker enzymes, the extracellular washing fluid was free from intracellular contaminations. In the extracellular washing fluid ascorbate, glutathione, cysteine, and high specific activities of guaiacol peroxidases were observed. Guaiacol peroxidases in the extracellular washing fluid and needle homogenates had the same catalytic properties, i.e. temperature optimum at 50°C, pH optimum in the range of pH 5 to 6 and low affinity for guaiacol (apparent K_m = 40 millimolar) and H₂O₂ (apparent K_m = 1-3 millimolar). Needle homogenates contained ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, glutathione reductase, and catalase, but not glutathione peroxidase activity. None of these activities was detected in the extracellular washing fluid. Ascorbate and glutathione related enzymes were freeze sensitive; ascorbate peroxidase was labile in the absence of ascorbate. The significance of extracellular antioxidants for the detoxification of injurious oxygen species is discussed.     

Oxidative stress responses and shock proteins in the unicellular cyanobacterium Synechococcus R2 (PCC-7942)

Oxidative stress responses were tested in the unicellular cyanobacterium Synechococcus PCC 7942 (R2). Cells were exposed to hydrogen peroxide, cumene hydroperoxide and high light intensities. Activities of ascorbate peroxidase and catalase were correlated with the extent and time-course of oxidative stresses. Ascorbate peroxidase was found to be the major enzyme involved in the removal of hydrogen peroxide under the tested oxidative stresses. Catalase activity was inhibited in cells treated with high H₂O₂ concentrations, and was not induced under photo-oxidative stress. Regeneration of ascorbate in peroxide-treated cells was found to involve mainly monodehydroascorbate reductase and to a lesser extent dehydroascorbate reductase. The induction of the antioxidative enzymes was dependent on light and was inhibited by chloramphenicol. Peroxide treatment was found to induce the synthesis of eight proteins, four of which were also induced by heat shock.Abbreviations ASC ascorbate - DHA dehydroascorbate - MDA monodehydroascorbate - GSH reduced glutathione - GSSG oxidized glutathione - ASC Per ascorbate peroxidase - DHA red. dehydroascorbate reductase - MDA red. monodehydroascorbate reductase - GSSG red. glutathione reductase - HSP heat shock proteins - PSP peroxide shock proteins - C_m chloramphenicol     

Drought stress induces oxidative stress and the antioxidant defense system in ascorbate-deficient vtc1 mutants of Arabidopsis thaliana

Drought stress has a negative impact on plant cells and results in the generation of reactive oxygen species (ROS). To increase our understanding of the effects of drought stress on antioxidant processes, we investigated the response of the ascorbate-deficient Arabidopsis thaliana vtc1 mutant to drought stress. After drought stress, vtc1 mutants exhibited increases in several oxidative parameters, including H₂O₂ content and the production of thiobarbituric acid reactive substances. Decreases in chlorophyll content and chlorophyll fluorescence parameters were also observed. The vtc1 mutants had higher total glutathione than did wild-type (WT) plants after 48 h of drought stress. A reduced ratio of glutathione/total glutathione and an increased ratio of dehydroascorbate/total ascorbate were observed in the vtc1 mutants compared with the WT plants. In addition, the activities of enzymes that are responsible for ROS scavenging, including superoxide dismutase, catalase, and ascorbate peroxidase, were decreased in the vtc1 mutants compared with the WT plants. Similar reductions in activity in the vtc1 mutant were observed for the enzymes that are responsible for the regeneration of ascorbate and glutathione, including monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase. These results suggest that low intrinsic ascorbate and impaired ascorbate–glutathione cycling in the vtc1 mutant induced a decrease in the reduced form of ascorbate, which enhanced sensitivity to drought stress.     

Biochemical Studies of Paraquat-Tolerant Mutants of the Fern Ceratopteris richardii

Enzymes and metabolites associated with mitigation of paraquat toxicity were compared in two paraquat-tolerant mutants and a sensitive wild-type strain of the fern Ceratopteris richardii Brongn. In 21-day-old gametophytes, the specific activities of superoxide dismutase, catalase, peroxidase, glutathione reductase, dehydroascorbate reductase, and ascorbate peroxidase showed no differences that would explain mutant tolerance. Constitutive levels of ascorbate and glutathione also did not differ significantly in the three strains. An experiment testing the inducibility of paraquat tolerance revealed no change in the dose response of mutant or wild type gametophytes after exposure to sublethal concentrations of the herbicide. Uptake of paraquat by whole gametophytes was also equivalent in mutants and wild type. These data suggest that the physiological basis for tolerance in these mutants, unlike several other tolerant biotypes reported, does not lie in the oxygen radical scavenging system, in an inducible stress response, or in a block to whole-plant uptake.     

Comparison of photosynthesis and antioxidant performance of several Citrus and Fortunella species (Rutaceae) under natural chilling stress

Citrus plants originate from southeastern Asia, in a large area with various climates characterized by a broad range of temperatures. Some species have been diversified in temperate climates, others in subtropical climates. Temperature is assumed to be a key factor in citrus species adaptation and diversification of basic cellular functions. In a field experiment, the tolerance of the three fundamental Citrus species C. medica L., C. reticulata Blanco and C. maxima (Burm.) Merr., and Fortunella japonica (Thunb.) Swing. to photooxidative stress caused by seasonal climatic changes was evaluated on adult trees by measuring net photosynthesis (Pnet), stomatal conductance (Gs), maximum photosynthesis (Pmax) and chlorophyll fluorescence (Fv/Fm). In addition, seasonal changes in oxidative status, antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase) and antioxidant metabolites (ascorbate and glutathione) were monitored. Mandarin and pummelo appeared to be the most tolerant, showing the lowest down-regulation of photosynthetic parameters, and the lowest accumulation of oxidized compounds associated with efficiency of their antioxidant system. Kumquat showed intermediate behaviour, with a large diminution of photosynthetic parameters and marked accumulation of hydrogen peroxide, whereas the malondialdehyde content remained low, with a strong induction of glutathione synthesis. Finally, citron appeared to be the most sensitive genotype with a marked decrease in photosynthetic performance, the largest accumulation of oxidative parameters, insufficient induction of antioxidant enzymes and down-regulation of ascorbate and glutathione synthesis.     

Ascorbate oxidation reduction in Helicoverpa zea as a scavenging system against dietary oxidants

We provide evidence of an important role for ascorbate free radical (AFR) reductase, dehydroascorbate (DHA) reductase, glutathione, and glutathione reductase as components of an oxidant-scavenging system in the midgut of larval Helicoverpa zea. Also, midgut ortho-quinone reductase is a potentially important constituent of the protective system against quinones. The midgut activities of AFR reductase, DHA reductase, glutathione reductase, and ortho-quinone reductase were, respectively, 168, 22.1, 6, and 39.5 nmol/min/mg protein. The relatively high activity of these enzymes in the midgut provides circumstantial evidence for a protective mechanism utilizing ascorbate as an antioxidant and glutathione and/or NADPH as reductants. To our knowledge, the enzymes AFR reductase and DHA reductase have not been reported in insects. The particular relevance of this system to antioxidant protection, and in particular to the detoxication of quinones formed in damaged leaf tissues, is discussed.     

Antioxidant defences of the apoplast

Summary The apoplast of barley and oat leaves contained superoxide dismutase (SOD), catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase activities. The activities of these enzymes in the apoplastic extracts were greatly modified 24 h after inoculation with the biotrophic fungal pathogenBlumeria graminis. The quantum efficiency of photosystem II, which is related to photosynthetic electron transport flux, was comparable in inoculated and healthy leaves during this period. Apoplastic soluble acid invertase activity was also modified in inoculated leaves. Inoculation-dependent increases in apoplastic SOD activity were observed in all lines. Major bands of SOD activity, observed in apoplastic protein extracts by activity staining of gels following isoelectric focusing, were similar to those observed in whole leaves but two additional minor bands were found in the apoplastic fraction. The apoplastic extracts contained substantial amounts of dehydroascorbate (DHA) but little or no glutathione (GSH). Biotic stress decreased apoplastic ascorbate and DHA but increased apoplastic GSH in resistant lines. The antioxidant cycle enzymes may function to remove apoplastic H₂O₂ with ascorbate and GSH derived from the cytoplasm. DHA and oxidized glutathione may be reduced in the apoplast or returned to the cytosol for rereduction.Abbreviations AA reduced ascorbate - APX ascorbate peroxidase - DHA dehydroascorbate (oxidised ascorbate) - DHAR dehydroascorbate reductase - G6PDH glucose-6-phosphate dehydrogenase - GSH reduced glutathione - GSSG glutathione disulphide - GR glutathione reductase - MDHA monodehydroascorbate - MDHAR monodehydroascorbate reductase - SOD superoxide dismutase     

Pre treatment with Bacillus subtilis mitigates drought induced photo-oxidative damages in okra by modulating antioxidant system and photochemical activity

Growth promoting potential of Bacillus subtilis (BS) in drought stressed Abelmoschus esculentus (L.) Moench (okra) was assessed by measuring the chlorophyll stability index (CSI), chlorophyll a (Chl-a) fluorescence, leaf osmotic potential and lipid peroxidation by malondialdehyde content, emission of reactive oxygen species (ROS), osmolyte content and the activity of non-enzyme and enzyme antioxidants. BS treatment significantly increased the leaf osmotic potential, osmolyte production and the activity of non-enzyme and enzyme antioxidants under drought stress. BS treatment mitigated the drought-induced reduction in Chl a fluorescence and CSI. Concomitant increase in total sugar, proline, non-enzyme antioxidants [glutathione and ascorbate] and enzyme antioxidants like superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase and dehydroascorbate reductase modulate the intracellular ROS concentration in okra to resist the stress induced oxidative damage in BS treated plants led to fast recovery and less photodamage.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-00982-8.     

Protective systems against activated oxygen species compared in normal and fully habituated nonorganogenic sugarbeet calluses

Summary The levels of the water-soluble reductants ascorbic acid and glutathione and the activities of the enzymatic antioxidants superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate and dehydroascorbate reductases and glutathione reductase were determined in a fully habituated nonorganogenic sugarbeet callus line (considered a neoplasm) compared with a normal hormone-dependent callus of the same plant. Ascorbic acid was not recovered from either of the two calluses, irrespective of the technique used. Glutathione was titrated at a slightly higher level in the normal callus. Catalase activity was almost nonexistent in the habituated callus. The other enzymes (superoxide dismutase, glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase, and ascorbate peroxidase) were found to have higher activities in the habituated callus. The results are interpreted as a higher protection of the neoplastic habituated cells against oxygen-free radicals and hydroperoxide-dependent oxidations. Such strong scavenging properties of the habituated cell line could explain previous results already reported, namely the stimulation of cell division at the expense of cell differentiation.     

Nitric oxide is involved in the regulation of ascorbate and glutathione metabolism in Agropyron cristatum leaves under water stress

This study investigated the regulation of ascorbate and glutathione metabolism by nitric oxide in Agropyron cristatum leaves under water stress. The activities of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), L-galactono-1,4-lactone dehydrogenase (GalLDH) and γ-glutamylcysteine synthetase (γ-ECS), and the contents of NO, reduced ascorbic acid (AsA), reduced glutathione (GSH), total ascorbate and total glutathione increased under water stress. These increases were suppressed by pretreatments with NO synthesis inhibitors N ^G-nitro-L-arginine methyl ester (L-NAME) and 4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). However, application of L-NAME and cPTIO to plants sufficiently supplied with water did not affect the activities of above mentioned enzymes and the contents of NO and above mentioned antioxidants. Pretreatments with L-NAME and cPTIO increased the malondialdehyde (MDA) content and electrolyte leakage of plants under water stress. Our results suggested that water stress-induced NO is a signal that leads to the upregulation of ascorbate and glutathione metabolism and has important role for acquisition of water stress tolerance.