Cellular damage induced by cadmium and mercury in Medicago sativa

Alfalfa (Medicago sativa) plantlets were exposed to Cd or Hg to study the kinetics of diverse stress indexes. In the so-called beaker-size hydroponic system, plantlets were grown in 30 microM of Cd or Hg for 7 d. Oxidative stress took place and increased over time, a linear response being observed with Cd but not with Hg. To improve the sensitivity of the stress assays used, a micro-assay system, in which seedlings were exposed for 24 h, was developed. Phytotoxicity of metals, quantified as growth inhibition, was observed well before there was any change in the non-protein thiol tissue concentration. When measured with conventional techniques, oxidative stress indexes did not show significant variation. To trace early and small plant responses to Cd and Hg, a microscopic analysis with novel fluorescent dyes, which had not yet been exploited to any significant extent for use in plants, was conducted. These fluorescent probes, which allowed minute cellular responses to 0, 3, 10, and 30 microM of both metals to be visualized in the roots of the alfalfa seedlings, were: (i) 2',7'-dichlorofluorescin diacetate that labels peroxides; (ii) monochlorobimane that stains reduced glutathione/homoglutathione (GSH/hGSH); and (iii) propidium iodide that marks nuclei of dead cells. Oxidative stress and cell death increased after exposure for 6-24 h to Cd and Hg, but labelling of GSH/hGSH decreased acutely. This diminution might be the result of direct interaction of GSH/hGSH with both Cd and Hg, as inferred from an in vitro conjugation assay. Therefore, both Cd and Hg not only compromised severely the cellular redox homeostasis, but also caused cell necrosis. In plants treated with 1 mM L-buthionine sulphoximine, a potent inhibitor of GSH/hGSH synthesis, only the oxidative stress symptoms appeared, indicating that the depletion of the GSH/hGSH pool was not sufficient to promote cell death, and that other phytotoxic mechanisms might be involved.     

Stress Responses of Zea mays to Cadmium and Mercury

A hydroponic experiment was carried out to characterize the oxidative stress responses of maize seedlings (Zea mays L. cv. Dekalb DK604) to cadmium (Cd) and mercury (Hg). Plants were grown hydroponically for 7 days in a nutrient solution supplemented with several concentrations of Cd and Hg: 0.0 (control), 6 or 30 μM. Growth was inhibited by both metals. The effect was more severe in plants exposed to Hg. Oxidative stress was caused by the exposure to the metals, as quantified by malondialdehyde and carbonyl accumulation, by-products of lipid peroxidation and protein oxidation, respectively. The activity of ascorbate peroxidase (APX) and superoxide dismutase (SOD), enzymes involved in the scavenging of reactive oxygen species, were measured upon metal treatment. We found an activation of a cytosolic APX isoform, as identified by using a specific polyclonal antiserum. However, there were negligible changes in SOD activity. Analysis of thiol-peptides revealed that at 6 μM Cd a remarkable increase in root reduced glutathione (GSH) content occurred, and little effect on the relative content of oxidised glutathione (GSSG) was observed. However, at 30 μM Cd and in plants exposed to 6 and 30 μM of Hg, GSH root content either remained stable or decreased significantly, while the proportion of GSSG increased. Moreover, only Cd was able to induce accumulation of phytochelatins at both assayed concentrations. Apparently, Hg was more toxic than Cd, as inferred from the magnitude of the changes found in the physiological parameters tested.     

Lanthanum improves the cadmium tolerance of <Emphasis Type="Italic">Zea mays</Emphasis> seedlings by the regulation of ascorbate and glutathione metabolism

The effect of lanthanum on the metabolism of ascorbate (AsA) and glutathione (GSH) in the leaves of maize seedlings under cadmium stress was investigated. The findings showed that Cd remarkably increased electrolyte leakage (EL), the activities of ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase (MDHAR), glutathione reductase, L-galactono-1,4-lactone dehydrogenase, and γ-glutamylcysteine synthetase, and the content of reduced AsA, reduced GSH, total AsA, total GSH, malondialdehyde (MDA), and Cd, compared with control. However, Cd significantly decreased the dry biomass of roots and shoots. Treatment with La + Cd evidently increased the activities of above enzymes except MDHAR, the content of reduced AsA, reduced GSH, total AsA and total GSH, and the dry biomass of roots and shoots, compared with Cd stress alone. Meanwhile, treatment with La + Cd remarkably decreased EL and the content of Cd and MDA compared with Cd stress alone. Our results suggested that La could be used as a regulator to improve the Cd tolerance of maize for its role in the alleviation of Cd-induced oxidative damage by regulating the metabolism of AsA and GSH.     

Reactive oxygen species, ascorbate–glutathione pool, and enzymes of their metabolism in drought-sensitive and tolerant indica rice (Oryza sativa L.) seedlings subjected to progressing levels of water deficit

Water deficit for rice is a worldwide concern, and to produce drought-tolerant varieties, it is essential to elucidate molecular mechanisms associated with water deficit tolerance. In the present study, we investigated the differential responses of nonenzymatic antioxidants ascorbate (AsA), glutathione (GSH), and their redox pool as well as activity levels of enzymes of ascorbate–glutathione cycle in seedlings of drought-sensitive rice (Oryza sativa L.) cv. Malviya-36 and drought-tolerant cv. Brown Gora subjected to water deficit treatment of ?1.0 and ?2.1 MPa for 24–72 h using PEG-6000 in sand cultures. Water deficit caused increased production of reactive oxygen species such as O₂?^?, H₂O₂, and HO? in the tissues, and the level of production was higher in the sensitive than the tolerant cultivar. Water deficit caused reduction in AsA and GSH and decline in their redox ratios (AsA/DHA and GSH/GSSG) with lesser decline in tolerant than the sensitive seedlings. With progressive level of water deficit, the activities of monodehydroascorbate reductase, dehydroascorbate reductase, ascorbate peroxidase (APX), and glutathione transferase increased in the seedlings of both rice cultivars, but the increased activity levels were higher in the seedlings of drought-tolerant cv. Brown Gora compared to the sensitive cv. Malviya-36. Greater accumulation of proline was observed in stressed seedlings of tolerant than the sensitive cultivar. In-gel activity staining of APX revealed varying numbers of their isoforms and their differential expression in sensitive and tolerant seedlings under water deficit. Results suggest that an enhanced oxidative stress tolerance by a well-coordinated cellular redox state of ascorbate and glutathione in reduced forms and induction of antioxidant defense system by elevated activity levels of enzymes of ascorbate–glutathione cycle is associated with water deficit tolerance in rice.     

Exogenous Selenium Pretreatment Protects Rapeseed Seedlings from Cadmium-Induced Oxidative Stress by Upregulating Antioxidant Defense and Methylglyoxal Detoxification Systems

The protective effect of selenium (Se) on antioxidant defense and methylglyoxal (MG) detoxification systems was investigated in leaves of rapeseed (Brassica napus cv. BINA sharisha 3) seedlings under cadmium (Cd)-induced oxidative stress. Two sets of 11-day-old seedlings were pretreated with both 50 and 100???M Se (Na₂SeO₄, sodium selenate) for 24?h. Two concentrations of CdCl₂ (0.5 and 1.0?mM) were imposed separately or on the Se-pretreated seedlings, which were grown for another 48?h. Cadmium stress at any levels resulted in the substantial increase in malondialdehyde and H₂O₂ levels. The ascorbate (AsA) content of the seedlings decreased significantly upon exposure to Cd stress. The amount of reduced glutathione (GSH) increased only at 0.5?mM CdCl₂, while glutathione disulfide (GSSG) increased at any level of Cd, with concomitant decrease in GSH/GSSG ratio. The activities of ascorbate peroxidase (APX) and glutathione S-transferase (GST) increased significantly with increased concentration of Cd (both at 0.5 and 1.0?mM CdCl₂), while the activities of glutathione reductase (GR) and glutathione peroxidase (GPX) increased only at moderate stress (0.5?mM CdCl₂) and then decreased at 1.0?mM severe stress (1.0?mM CdCl₂). Monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT), glyoxalase I (Gly I), and glyoxalase II (Gly II) activities decreased upon exposure to any levels of Cd. Selenium pretreatment had little effect on the nonenzymatic and enzymatic components of seedlings grown under normal conditions; i.e., they slightly increased the GSH content and the activities of APX, GR, GST, and GPX. On the other hand, Se pretreatment of seedlings under Cd-induced stress showed a synergistic effect; it increased the AsA and GSH contents, the GSH/GSSG ratio, and the activities of APX, MDHAR, DHAR, GR, GPX, CAT, Gly I, and Gly II which ultimately reduced the MDA and H₂O₂ levels. However, in most cases, pretreatment with 50???M Se showed better results compared to pretreatment with 100???M Se. The results indicate that the exogenous application of Se at low concentrations increases the tolerance of plants to Cd-induced oxidative damage by enhancing their antioxidant defense and MG detoxification systems.     

24-Epibrassinolide maintains elevated redox state of AsA and GSH in radish (Raphanus sativus L.) seedlings under zinc stress

Exogenous-applied 24-epibrassinolide (EBR) increased the seedling growth of radish (Raphanus sativus L.) in terms of seedling length, fresh weight and dry weight both in zinc (Zn²⁺)-stressed and unstressed conditions. Moreover, EBR lowered the Zn²⁺ uptake and bioaccumulation. Increased oxidation of ascorbate (AsA) and glutathione (GSH) pools to dehydroascorbate and glutathione disulfide respectively was observed in Zn²⁺-stressed seedlings, a clear indication of oxidative stress. However, exogenous application of EBR to stressed seedlings inhibited the oxidation of ascorbate and glutathione, maintaining redox molecules in reduced form. Under Zn²⁺ stress, enzymatic activities of ascorbate–glutathione cycle such as ascorbate peroxidase, monodehydroascorbate reductase increased but the dehydroascorbate reductase, glutathione reductase decreased. Zn²⁺ stress induced the gamma-glutamylcysteine synthetase, and glutathione-s-transferase activities in radish seedlings were further enhanced with EBR application. Zn²⁺ toxicity decreased the thiol content but, EBR supplementation resulted in restoration of thiol pool. The results of present study clearly demonstrated that external application of EBR modulates the AsA and GSH redox status to combat the oxidative stress of Zn²⁺ in seedlings via the AsA–GSH cycle and glutathione metabolism as an antioxidant defense system.     

Comparative study of alleviating effects of GSH, Se and Zn under combined contamination of cadmium and chromium in rice (Oryza sativa)

A hydroponic experiment was conducted to study the ameliorative effects of separate or combined application of exogenous glutathione (GSH), selenium (Se) and zinc (Zn) upon 20 μM cadmium (Cd) plus 20 μM chromium (Cr) heavy metal stress (HM) in rice seedlings. The results showed that HM caused a marked reduction in seedling height, chlorophyll content (SPAD) and biomass, and activities of catalase (CAT) and ascorbate peroxidase (APX) in leaves and H⁺-ATPase in roots/leaves, but elevated superoxide dismutase (SOD) and guaiacol peroxidase (POD) activities in leaves with elevated malondialdehyde (MDA) accumulation both in leaves and roots over the control. The best mitigation effect was recorded in HM+GSH+Zn and HM+GSH (addition of GSH+Zn and GSH to HM solution), which greatly alleviated HM-induced growth inhibition and oxidative stress. Compared with HM alone, HM+GSH and HM+GSH+Zn markedly reduced Cr uptake and translocation but not affected Cd concentration; improved H⁺-ATPase activity and Fe, Zn, Mn uptake and translocation, and repressed MDA accumulation. Meanwhile exogenous GSH and GSH+Zn counteracted HM-induced response of antioxidant enzymes, via suppressing HM-induced dramatic increase of root/leaf SOD and leaf POD activities, and elevating stress-depressed leaf APX and leaf/root CAT activities.     

黄土高原冰草叶片抗坏血酸和谷胱甘肽合成及循环代谢对干旱胁迫的生理响应

通过盆栽实验, 对干旱胁迫下黄土高原地区冰草(Agropyron cristatum)叶片的抗坏血酸和谷胱甘肽合成及循环代谢相关酶及物质含量进行了研究。结果表明: 冰草可以通过增强叶片的抗坏血酸和谷胱甘肽合成及循环代谢酶: 抗坏血酸过氧化物酶、谷胱甘肽还原酶、脱氢抗坏血酸还原酶、单脱氢抗坏血酸还原酶、L-半乳糖酸-1, 4-内酯脱氢酶和γ-谷氨酰半胱氨酸合成酶活性, 维持植物体内抗坏血酸和谷胱甘肽水平及氧化还原状态, 从而抵御干旱造成的氧化胁迫。但叶片抗坏血酸和谷胱甘肽合成及循环代谢对不同水平干旱胁迫的响应, 随胁迫时间的延长而不同。在胁迫24天以前, 严重干旱下叶片的抗坏血酸和谷胱甘肽合成及循环代谢增强较显著; 在胁迫24天后, 由于该胁迫下植物所遭受的氧化胁迫较为严重, 叶片中上述6种酶的活性均呈降低趋势。而在中度干旱下叶片抗坏血酸和谷胱甘肽合成及循环代谢相关的6种酶在整个胁迫过程中均保持较高的活性。这说明, 冰草能够长时间有效地抵御中度干旱所造成的氧化胁迫, 但只能在一定时间范围内有效地抵御严重干旱所造成的氧化胁迫, 胁迫时间延长则会降低其抵御严重干旱的能力。     

Modulation of cadmium-induced oxidative stress in Ceratophyllum demersum by zinc involves ascorbate-glutathione cycle and glutathione metabolism.

To understand the interaction between Zn, an essential micronutrient and Cd, a non-essential element, Cd-10 microM and Zn supplemented (10, 50, 100, and 200 microM) Cd 10 microM treated Ceratophyllum demersum L. (Coontail), a free floating freshwater macrophyte was chosen for the study. Cadmium at 10 microM concentration decreased thiol content, enhanced oxidation of ascorbate (AsA) and glutathione (GSH) to dehydroascorbate (DHA) and glutathione disulfide (GSSG), respectively, a clear indication of oxidative stress. Zinc supplementation to Cd (10 microM) treated plants effectively restored thiols, inhibited oxidation of AsA and GSH maintaining the redox molecules in reduced form. Cd-10 microM slightly induced ascorbate peroxidase (APX, E.C. 1.11.1.11) but inhibited monodehydroascorbate reductase (MDHAR, E.C. 1.6.5.4), dehydroascorbate reductase (DHAR, E.C. 1.8.5.1) and glutathione reductase (GR, E.C. 1.6.4.2), enzymes of ascorbate-glutathione cycle (AGC). Zn supplementation restored and enhanced the functional activity of all the AGC enzymes (APX, MDHAR, DHAR and GR). Gamma-glutamylcysteine synthetase (gamma-GCS, E.C. 6.3.2.2) was not affected by Cd as well as Zn, but Zn supplements increased glutathione-S-transferase (GST, E.C. 2.5.1.18) activity to a greater extent than Cd and simultaneously restored glutathione peroxidase (GSH-PX, E.C. 1.11.1.9) activity impaired by Cd toxicity. Zn-alone treatments did not change above investigated parameters. These results clearly indicate the protective role of Zn in modulating the redox status of the plant system through the antioxidant pathway AGC and GSH metabolic enzymes for combating Cd induced oxidative stress.     

Reactive oxygen metabolism in mycorrhizal and non-mycorrhizal citrus (Poncirus trifoliata) seedlings subjected to water stress

The effect of the arbuscular mycorrhizal (AM) fungus, Glomus versiforme, on growth and reactive oxygen metabolism of trifoliate orange (Poncirus trifoliata) seedlings was studied in potted plants under well-watered (WW) and water stressed (WS) conditions. Water stress significantly decreased root colonization. Shoot dry weight, plant height and stem diameter were higher in AM than in non-AM seedlings regardless of the water status. Inoculation with G. versiforme increased root dry weight and leaf number per plant of WW seedlings. There was less malondialdehyde (MDA) concentration in leaves and roots of AM seedlings, as well as lower hydrogen peroxide (H(2)O(2)) and superoxide anion radical (O(2)(-)) concentrations in AM roots under WW and WS conditions. AM inoculation did not affect the H(2)O(2) and O(2)(-) concentrations of WW leaves. Whether WS or not, AM symbiosis notably increased the guaiacol peroxidase (G-POD) activity of leaves, glutathione reductase (GR) activity of leaves and ascorbate peroxidase (APX) activity of roots. AM infection also markedly increased the APX activity of WS leaves. Soluble proteins and glutathione (GSH) in leaves and roots and ascorbate (ASC) in leaves were higher in WW AM than in WW non-AM seedlings. AM infection also enhanced the ASC and GSH contents of leaves and roots in WS seedlings. Cross-tolerance might occur in AM plants and be enhanced by AM symbiosis. Our results suggest that the increased concentrations of antioxidant enzymes and non-enzymatic antioxidants found in AM plants may serve to protect the organism against oxidative damage, enhancing drought tolerance.     

Cadmium-induced changes in antioxidative systems,hydrogen peroxide content,and lipid peroxidation in<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

To study the relationship between cadmium (Cd)-induced phytotoxicity and oxidative stress, we grew Cd-sensitive wild-type (WT) and Cd-resistant type (RT) seedlings ofArabidopsis thaliana on MS media containing up to 500 μM CdCl₂. The resistant seedlings showed higher biomasses and lower hydrogen peroxide and lipid peroxidation levels, the latter expressed in terms of malondialdehyde (MDA) production. These results indicate that RT plants experience lower oxidative stress when exposed to Cd. Furthermore, compared with the WT, RT seedlings have significantly higher activities of superoxide dismutase (SOD) and enzymes related to hydrogen peroxide removal, e.g., guaiacol peroxidase (GPX), ascorbate peroxidase (APX), and glutathione reductase (GR). These differential responses suggest that such phytotoxicity could be induced by oxidative stress, and that lower accumulations of hydrogen peroxide confer Cd tolerance in seedlings.     

Cadmium-induced changes in antioxidative systems, hydrogen peroxide content, and differentiation in Scots pine roots.

To investigate whether Cd induces common plant defense pathways or unspecific necrosis, the temporal sequence of physiological reactions, including hydrogen peroxide (H(2)O(2)) production, changes in ascorbate-glutathione-related antioxidant systems, secondary metabolism (peroxidases, phenolics, and lignification), and developmental changes, was characterized in roots of hydroponically grown Scots pine (Pinus sylvestris) seedlings. Cd (50 microM, 6 h) initially increased superoxide dismutase, inhibited the systems involved in H(2)O(2) removal (glutathione/glutathione reductase, catalase [CAT], and ascorbate peroxidase [APX]), and caused H(2)O(2) accumulation. Elongation of the roots was completely inhibited within 12 h. After 24 h, glutathione reductase activities recovered to control levels; APX and CAT were stimulated by factors of 5.5 and 1.5. Cell death was increased. After 48 h, nonspecific peroxidases and lignification were increased, and APX and CAT activities were decreased. Histochemical analysis showed that soluble phenolics accumulated in the cytosol of Cd-treated roots but lignification was confined to newly formed protoxylem elements, which were found in the region of the root tip that normally constitutes the elongation zone. Roots exposed to 5 microM Cd showed less pronounced responses and only a small decrease in the elongation rate. These results suggest that in cells challenged by Cd at concentrations exceeding the detoxification capacity, H(2)O(2) accumulated because of an imbalance of redox systems. This, in turn, may have triggered the developmental program leading to xylogenesis. In conclusion, Cd did not cause necrotic injury in root tips but appeared to expedite differentiation, thus leading to accelerated aging.     

Effect of abscisic acid on active oxygen species, antioxidative defence system and oxidative damage in leaves of maize seedlings.

Leaves of maize (Zea mays L.) seedlings were supplied with different concentrations of abscisic acid (ABA). Its effects on the levels of superoxide radical (O(2)(-)), hydrogen peroxide (H(2)O(2)) and the content of catalytic Fe, the activities of several antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), the contents of several non-enzymatic antioxidants such as ascorbate (ASC), reduced glutathione (GSH), alpha-tocopherol (alpha-TOC) and carotenoid (CAR), and the degrees of the oxidative damage to the membrane lipids and proteins were examined. Treatment with 10 and 100 microM ABA significantly increased the levels of O(2)(-) and H(2)O(2), followed by an increase in activities of SOD, CAT, APX and GR, and the contents of ASC, GSH, alpha-TOC and CAR in a dose- and time-dependent pattern in leaves of maize seedlings. An oxidative damage expressed as lipid peroxidation, protein oxidation, and plasma membrane leakage did not occur except for a slight increase with 100 microM ABA treatment for 24 h. Treatment with 1,000 microM ABA led to a more abundant generation of O(2)(-) and H(2)O(2) and a significant increase in the content of catalytic Fe, which is critical for H(2)O(2)-dependent hydroxyl radical production. The activities of these antioxidative enzymes and the contents of alpha-TOC and CAR were still maintained at a higher level, but no longer further enhanced when compared with the treatment of 100 microM ABA. The contents of ASC and GSH had no changes in leaves treated with 1,000 microM ABA. These results indicate that treatment with low concentrations of ABA (10 to 100 microM) induced an antioxidative defence response against oxidative damage, but a high concentration of ABA (1,000 microM) induced an excessive generation of AOS and led to an oxidative damage in plant cells.     

NaCl胁迫对茄子嫁接幼苗叶片抗坏血酸和谷胱甘肽代谢的影响

以Torvum Vigor为砧木,栽培品种苏崎茄为接穗,研究NaCl胁迫对茄子嫁接苗和自根苗抗氧化物质含量和H2O2产生及清除酶活性的影响.结果表明;(1)NaCl胁迫可诱导叶片中H2O2的产生,导致膜脂过氧化产物丙二醛(MDA)的积累,嫁接苗叶片中H2O2和MDA的含量显著低于自根苗.(2)在NaCl胁迫下,嫁接苗叶片中谷胱甘肽还原酶(GR)和抗坏血酸过氧化物酶(APX)活性上升,自根苗则下降;NaCl胁迫促进了嫁接苗还原型抗坏血酸(AsA)和还原型谷胱甘肽(GSH)的合成,嫁接苗叶片中AsA和GSH的含量显著高于自根苗.由此认为,茄子嫁接苗耐盐性优于自根苗的原因之一在于嫁接苗保持较快的AsA-GSH循环,从而保证GSH和AsA的再生以减轻氧化损伤.     

Manipulation of plant tolerance to herbicides through co-ordinated metabolic engineering of a detoxifying glutathione transferase and thiol cosubstrate

The diphenyl ether herbicide fomesafen can be used selectively in soybean (Glycine max) due to its rapid detoxification by tau class glutathione transferases (GmGSTUs) which preferentially utilize the endogenous thiol homoglutathione (hGSH) as cosubstrate. Soybean cDNAs encoding GmGSTU21, which is highly active in detoxifying fomesafen, and an hGSH synthetase (GmhGS) have been cloned and functionally identified in Escherichia coli. Tobacco plants, which have limited GST activities towards fomesafen and which accumulate glutathione (GSH), rather than hGSH, have been transformed with either GmhGS alone, or a dual construct of GmhGS-GmGSTU21, both under the control of constitutive promoters. Using either construct, the transgenic tobacco accumulated hGSH, with a concomitant increase in GSH content. Segregating T1 plants were analysed for thiol content and GST activity towards fomesafen with GSH and hGSH as cosubstrates, and then scored for photobleaching injury caused by applications of fomesafen. These studies showed that hGSH accumulation alone gave no significant protection against the herbicide and that tolerance was only seen in plants which contained appreciable concentrations of hGSH and GmGSTU21 activity. Tolerance in the dual transformants was associated with the metabolism of radiolabelled fomesafen to inactive hGSH-derived conjugates, while susceptible lines were unable to detoxify the herbicide. These studies confirm the combined importance of specific GSTs and their preferred thiol cosubstrates in conferring herbicide selectivity traits in planta.     

Involvement of hydrogen peroxide in heat shock- and cadmium-induced expression of ascorbate peroxidase and glutathione reductase in leaves of rice seedlings

Hydrogen peroxide (H2O2) is considered a signal molecule inducing cellular stress. Both heat shock (HS) and Cd can increase H2O2 content. We investigated the involvement of H2O2 in HS- and Cd-mediated changes in the expression of ascorbate peroxidase (APX) and glutathione reductase (GR) in leaves of rice seedlings. HS treatment increased the content of H2O2 before it increased activities of APX and GR in rice leaves. Moreover, HS-induced H2O2 production and APX and GR activities could be counteracted by the NADPH oxidase inhibitors dipehenylene iodonium (DPI) and imidazole (IMD). HS-induced OsAPX2 gene expression was associated with HS-induced APX activity but was not regulated by H2O2. Cd-increased H2O2 content and APX and GR activities were lower with than without HS. Cd did not increase the expression of OsAPX and OsGR without HS treatment. Cd increased H2O2 content by Cd before it increased APX and GR activities without HS. Treatment with DPI and IMD effectively inhibited Cd-induced H2O2 production and APX and GR activities. Moreover, the effects of DPI and IMD could be rescued with H2O2 treatment. H2O2 may be involved in the regulation of HS- and Cd-increased APX and GR activities in leaves of rice seedlings.     

Preliminary studies on the involvement of glutathione metabolism and redox status against zinc toxicity in radish seedlings by 28-Homobrassinolide

The effect of exogenous application of 28-Homobrassinolide (HBR) on radish (Raphanus sativus L.) seedlings under zinc (Zn²⁺) stress on glutathione (GSH) production, consumption and changes in redox status was investigated. Zinc toxicity resulted in oxidative burst as evidenced by increased accumulation of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) content. These stress indices were significantly decreased by HBR supplementation. Under Zn²⁺ stress, GSH pool was decreased, while the contribution of oxidized glutathione (GSSG) to total GSH increased (GSSH/GSH ratio), this translated into significant reduction of GSH redox homeostasis. In addition, an increase of phytochelatins (PCs) was observed. In radish seedlings under Zn²⁺ stress, the activities of gamma-glutamylcysteine synthetase (γ-ECS), glutathione synthetase (GS), glutathione peroxidase (GPX), glutathione-S-transferase (GST) and cysteine (Cys) levels increased but the activity of glutathione reductase (GR) decreased. However, application of HBR increased the GSH pool and maintained their redox ratio by increasing the enzyme activities of GSH biosynthesis (γ-ECS and GS) and GSH metabolism (GR, GPX and GST). The results of present study are novel in being the first to demonstrate that exogenous application of HBR modulates the GSH synthesis, metabolism and redox homeostasis to confer resistance against Zn²⁺ induced oxidative stress.     

Ascorbate-glutathione metabolism during PEG-induced water deficit in <Emphasis Type="Italic">Trifolium repens</Emphasis>

In order to elucidate the response of the ascorbate-glutathione (ASC-GSH) cycle to drought stress, the activities of antioxidant enzymes and the levels of molecules involved in the ASC-GSH metabolism were studied in Trifolium repens L. seedlings subjected to PEG-induced water deficit. Compared to the control, the contents of H₂O₂, thiobarbituric acid reactive substances (TBARS), ascorbate (ASC), dehydroascorbate (DHA), and glutathione disulfide (GSSG) increased in PEG-treated seedlings, whereas the glutathione (GSH) content kept constant during the drought period. Further more, the ASC/DHA and GSH/GSSG ratios decreased in the presence of PEG. Except for that of monodehydroascorbate reductase (MDHAR), the activities of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) were up-regulated during water deficit, and the increases in APX and DHAR activities were much higher than those in GR activity. These data indicate that fluctuations in the ASC-GSH metabolism resulted from PEG treatment may have a positive effect on drought stress mitigation in T. repens.     

Accumulation of hydrogen peroxide and functioning of defense system in overwatered barley seedlings

The effects of overwatering (flooding) on the oxidative potential, the level of low-molecular-weight antioxidants, the content of stress proteins, and activities of antioxidant enzymes in green barley (Hordeum vulgare L.) seedlings were studied. Overwatering retarded barley seedling growth and induced hydrogen peroxide accumulation, a decrease in the total ascorbate content and an increase in the content of reduced glutathione (GSH), but it did not affect the content of oxidized glutathione (GSSG). After the cessation of stress factor action (post-stress period), the content of hydrogen peroxide declined to the initial level, the content of ascorbate reduced still stronger, whereas the content of GSH continued to rise. Under flooding conditions, activities of glutathione reductase (GR) and superoxide dismutase (SOD) increased. After the cessation of stress factor action, activities of these enzymes decreased but remained at rather high levels as compared with control. Activity of catalase (CAT) reduced during stress, whereas activity of ascorbate peroxidase (APX) was not essentially changed. In the post-stress period, CAT activity remained to be low; in contrast, APX activity increased. Barley seedling flooding induced the synthesis of stress proteins, HSP70 and dehydrins (DH). In the post-stress period, the content of stress proteins decreased; however, the content of DH in experimental leaves remained rather high. The results obtained indicate that barley defense system manifested a complex response to overwatering, which may be related to the oxygen shortage under stress conditions and sharp metabolism activation at re-aeration in the post-stress period.