Brassinosteroids alleviate chilling-induced oxidative damage by enhancing antioxidant defense system in suspension cultured cells of <Emphasis Type="Italic">Chorispora bungeana</Emphasis>

Brassinosteroids (BRs), a class of plant steroid hormones, play a significant role in the amelioration of various biotic and abiotic stresses. In order to further explore and elaborate their roles in plants subjected to chilling stress, suspension cultured cells of Chorispora bungeana with or without 24-epibrassinolide (EBR) application were exposed to 4 and 0°C for 5 days. The EBR treated cells exhibited higher viability after exposure to low temperatures compared with the control. Under chilling stress, reactive oxygen species (ROS) levels and lipid peroxidation were increased in the cultured cells, which were significantly inhibited by EBR application. The activities of antioxidative enzymes such as ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) were increased during chilling treatments, and these increases were more significant in the EBR applied suspension cells. The EBR treatment also greatly enhanced contents of ascorbic acid (AsA) and reduced glutathione (GSH) under chilling stress. From these results, it can be concluded that EBR could play the positive roles in the alleviation of oxidative damage caused by ROS overproduction through enhancing antioxidant defense system, resulting in improving the tolerance of C. bungeana suspension cultures to chilling stress.     

Nitric oxide synthase like activity-dependent nitric oxide production protects against chilling-induced oxidative damage in Chorispora bungeana suspension cultured cells

In the present study, we used suspension cultured cells from Chorispora bungeana Fisch. and C.A. Mey to investigate whether nitric oxide (NO) is involved in the signaling pathway of chilling adaptive responses. Low temperatures at 4 °C or 0 °C induced ion leakage, lipid peroxidation and cell viability suppression, which were dramatically alleviated by exogenous application of NO donor sodium nitroprusside (SNP). The levels of reactive oxygen species (ROS) were obviously reduced, and the activities of antioxidant enzymes such as ascorbate peroxidase (APX, EC 1.11.1.11), catalase (CAT, EC 1.11.1.6), glutathione reductase (GR, EC 1.6.4.2), peroxidase (POD, EC 1.11.1.7) and superoxide dismutase (SOD, EC 1.15.1.1) and the contents of ascorbic acid (AsA) and reduced glutathione (GSH) increased evidently in the presence of SNP under chilling stress. In addition, under low temperature conditions, treatment with NO scavenger PTIO or mammalian NO synthase (NOS) inhibitor l-NAME remarkably aggravated oxidative damage in the suspension cultures compared with that of chilling treatment alone. Moreover, measurements of NOS activity and NO production showed that both NOS activity and endogenous NO content increased markedly under chilling stress. The accumulation of NO was inhibited by l-NAME in chilling-treated cultures, indicating that most NO production under chilling may be generated from NOS-like activity. Collectively, these results suggest that chilling-induced NO accumulation can effectively protect against oxidative injury and that NOS like activity-dependent NO production might act as an antioxidant directly scavengering ROS or operate as a signal activating antioxidant defense under chilling stress, thus conferring an increased tolerance to chilling in C. bungeana suspension cultures.     

Cinnamic acid pretreatment mitigates chilling stress of cucumber leaves through altering antioxidant enzyme activity

To elucidate the physiological mechanism of chilling stress mitigated by cinnamic acid (CA) pretreatment, a cucumber variety (Cucumis sativus cv. Jinchun no. 4) was pretreated with 50 μM CA for 2 d and was then cultivated at two temperatures (15/8 and 25/18 °C) for 1 d. We investigated whether exogenous CA could protect cucumber plantlets from chilling stress (15/8 °C) and examined whether the protective effect was associated with the regulation of antioxidant enzymes and lipid peroxidation. At 2 d, exogenous CA did not influence plant growth, but induced the activities of some antioxidant enzymes, including superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), guaiacol peroxidase (GPX, EC 1.11.1.7), glutathione peroxidase (GSH-Px, EC 1.6.4.2) and ascorbate peroxidase (APX, EC 1.11.1.11) in cucumber leaves, and it also elevated the contents of reduced glutathione (GSH) and ascorbate (AsA). When CA was rinsed and the CA-pretreated seedlings were exposed to different temperatures, the antioxidant activities in leaves at 3 d had undergone additional change. Chilling increased the activities of CAT, GSH-PX, APX, GSH and AsA in leaves, but the combination of CA pretreatment and chilling enhanced the antioxidant activities even more. Moreover, chilling inhibited plant growth and increased the contents of malonaldehyde (MDA), superoxide radical (O₂⁻) and hydrogen peroxide (H₂O₂) in cucumber leaves, and the stress resulted in 87.5% of the second leaves being withered. When CA pretreatment was combined with the chilling stress, we observed alleviated growth inhibition and decreased contents of MDA, H₂O₂ and O₂⁻ in comparison to non-pretreated stressed plants, and found that the withered leaves occurred at a rate of 25.0%. We propose that CA pretreatment increases antioxidant enzyme activities in chilling-stressed leaves and decreases lipid peroxidation to some extent, enhancing the tolerance of cucumber leaves to chilling stress.     

Responses of antioxidant gene, protein and enzymes to salinity stress in two genotypes of perennial ryegrass (Lolium perenne) differing in salt tolerance

Salinity could damage cellular membranes through overproduction of reactive oxygen species (ROS), while antioxidant capacities play a vital role in protecting plants from salinity caused oxidative damages. The objective of this study was to investigate the toxic effect of salt on the antioxidant enzyme activities, isoforms and gene expressions in perennial ryegrass (Lolium perenne L.). Salt-tolerant ‘Quickstart II’ and salt-sensitive ‘DP1′ were subjected to 0 and 250 mM NaCl for 12 d. Salt stress increased the content of lipid peroxidation (MDA), electrolyte leakage (EL) and hydrogen peroxide (H₂O₂), to a greater extent in salt-sensitive genotype. Salt-stressed plant leaves exhibited a greater activity of superoxide dismutase (SOD, EC 1.15.1.1), peroxidase (POD, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11) at 4 d after treatment (DAT), but a lower level of enzyme activity at 8 and 12 d, when compared to the control. Catalase (CAT, EC 1.11.1.6) activity was greater at 4 DAT and thereafter decreased in salt tolerant genotype relative to the control, whereas lower than the control during whole experiment period for salt-sensitive genotype. There were different patterns of five isoforms of SOD, POD and two isoforms of APX between two genotypes. Antioxidant gene expression was positively related to isoenzymatic and total enzymatic activities during 12-d salt-treated leaves of two genotypes, with a relatively higher level in salt-tolerant genotype. Thus, salt tolerance could be related to the constitutive/induced antioxidant gene, leading to more efficient enzyme stimulation and protection in perennial ryegrass.     

Negative regulation of abscisic acid-induced stomatal closure by glutathione in Arabidopsis

We found that glutathione (GSH) is involved in abscisic acid (ABA)-induced stomatal closure. Regulation of ABA signaling by GSH in guard cells was investigated using an Arabidopsis mutant, cad2-1, that is deficient in the first GSH biosynthesis enzyme, γ-glutamylcysteine synthetase, and a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene (CDNB). Glutathione contents in guard cells decreased along with ABA-induced stomatal closure. Decreasing GSH by both the cad2-1 mutation and CDNB treatment enhanced ABA-induced stomatal closure. Glutathione monoethyl ester (GSHmee) restored the GSH level in cad2-1 guard cells and complemented the stomatal phenotype of the mutant. Depletion of GSH did not significantly increase ABA-induced production of reactive oxygen species in guard cells and GSH did not affect either activation of plasma membrane Ca²⁺-permeable channel currents by ABA or oscillation of the cytosolic free Ca²⁺ concentration induced by ABA. These results indicate that GSH negatively modulates a signal component other than ROS production and Ca²⁺ oscillation in ABA signal pathway of Arabidopsis guard cells.     

Responses of antioxidative system to chilling stress in two rice cultivars differing in sensitivity

The responses of antioxidative system of rice to chilling were investigated in a tolerant cultivar, Xiangnuo-1, and a susceptible cultivar, IR-50. The electrolyte leakage and malondialdehyde content of Xiangnuo-1 were little affected by chilling treatment but those of IR-50 increased. Activities of suoperoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase, and ascorbic acid content of Xiangnuo-1 were remained high, while those of IR-50 decreased under chilling. The results indicated that higher activities of defense enzymes and higher content of antioxidant under stress were associated with tolerance to chilling.     

Calcium is involved in the abscisic acid-induced ascorbate peroxidase, superoxide dismutase and chilling resistance in Stylosanthes guianensis

The objective of this work was to test whether Ca²⁺, a second messenger in stress response, is involved in ABA-induced antioxidant enzyme activities in Stylosanthes guianensis. Plants were sprayed with abscisic acid (ABA), calcium channel blocker, LaCl₃, calcium chelator, ethylene glycol-bis(β-amino ethyl ether)-N,N,N′, N′-tetraacetid acid (EGTA), and ABA in combination with LaCl₃ or EGTA. Their effects on superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities and chilling resistance were compared. The results showed that ABA decreased electrolyte leakage and lipid peroxidation but increased maximum photochemical efficiency measured as variable to maximum fluorescence ratio (F_v/F_m) under chilling stress. Treatment with LaCl₃ or EGTA alone and in combination with ABA increased electrolyte leakage and lipid peroxidation, decreased Fv/Fm, suggesting that the block in Ca²⁺ signalling decreased chilling resistance of S. guianensis and the ABA-enhanced chilling resistance. ABA-induced SOD and APX activities were suppressed by LaCl₃ or EGTA. The results suggested that Ca²⁺ is involved in the ABA-enhanced chilling resistance and the ABA-induced SOD and APX activities in S. guianensis.     

Endogenous abscisic acid is involved in methyl jasmonate-induced reactive oxygen species and nitric oxide production but not in cytosolic alkalization in Arabidopsis guard cells

We recently demonstrated that endogenous abscisic acid (ABA) is involved in methyl jasmonate (MeJA)-induced stomatal closure in Arabidopsis thaliana. In this study, we investigated whether endogenous ABA is involved in MeJA-induced reactive oxygen species (ROS) and nitric oxide (NO) production and cytosolic alkalization in guard cells using an ABA-deficient Arabidopsis mutant, aba2-2, and an inhibitor of ABA biosynthesis, fluridon (FLU). The aba2-2 mutation impaired MeJA-induced ROS and NO production. FLU inhibited MeJA-induced ROS production in wild-type guard cells. Pretreatment with 0.1 μM ABA, which does not induce stomatal closure in the wild type, complemented the insensitivity to MeJA of the aba2-2 mutant. However, MeJA induced cytosolic alkalization in both wild-type and aba2-2 guard cells. These results suggest that endogenous ABA is involved in MeJA-induced ROS and NO production but not in MeJA-induced cytosolic alkalization in Arabidopsis guard cells.     

Cross-talks between Ca<Superscript>2+</Superscript>/CaM and H<Subscript>2</Subscript>O<Subscript>2</Subscript> in abscisic acid-induced antioxidant defense in leaves of maize plants exposed to water stress

Here we examined whether Ca²⁺/Calmodulin (CaM) is involved in abscisic acid (ABA)-induced antioxidant defense and the possible relationship between CaM and H₂O₂ in ABA signaling in leaves of maize (Zea mays L.) plants exposed to water stress. An ABA-deficient mutant vp5 and its wild type were used for the experimentation. We found that water stress enhanced significantly the contents of CaM and H₂O₂, and the activities of chloroplastic and cytosolic superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and the gene expressions of the CaM1, cAPX, GR1 and SOD4 in leaves of wild-type maize. However, the increases mentioned above were almost arrested in vp5 plants and in the wild-type plants pretreated with ABA biosynthesis inhibitor tungstate (T), suggesting that ABA is required for water stress-induced H₂O₂ production, the enhancement of CaM content and antioxidant defense. Besides, we showed that the up-regulation of water stress-induced antioxidant defense was almost completely blocked by pretreatment with Ca²⁺ inhibitors, CaM antagonists and reactive oxygen (ROS) manipulators. Moreover, the analysis of time course of CaM and H₂O₂ production under water stress showed that the increase in CaM content preceded that of H₂O₂. These results suggested that Ca²⁺/CaM and H₂O₂ were involved in the ABA-induced antioxidant defense under water stress, and the increases of Ca²⁺/CaM contents triggered H₂O₂ production, which inversely affected the contents of CaM. Thus, a cross-talk between Ca²⁺/CaM and H₂O₂ may play a pivotal role in the ABA signaling.     

Membrane stabilization by abscisic acid under cold aids proline in alleviating chilling injury in maize (Zea mays L.) cultured cells

Previous studies of maize suspension‐cultured cells showed that abscisic acid (ABA) treatment at warm temperatures improved the tolerance of cells to subsequent chilling. In the present study, it is shown that both ABA‐treated and untreated maize cells accumulated proline in response to chilling. However, ABA‐treated cells displayed less lipid peroxidation during chilling, and thus, unlike untreated cells, were able to retain the accumulated proline intracellularly. Proline application experiments indicate that an intracellular proline level higher than 2 µmole (g FW)^?1 prior to chilling was needed to meaningfully reduce chilling‐enhanced lipid peroxidation and significantly improve chilling tolerance. The results suggest that total proline accumulation in ABA‐treated as well as untreated cells during chilling was enough to potentially improve chilling tolerance, but proline leakage rendered the control cells unable to benefit from the endogenous synthesis of proline in relation to the alleviation of chilling injury. Proline participated in chilling tolerance improvement in ABA‐treated maize cells, as evidenced by: (1) the inhibition of proline accumulation by l ‐methionine‐d , l ‐sulphoximine (MSO), an inhibitor of glutamine synthetase, reduced ABA‐improved chilling tolerance, and (2) the addition of glutamine into the medium prevented the MSO‐induced reduction in chilling tolerance. The revised relationship between proline accumulation and membrane stability at cold is discussed in the light of these current findings.     

Extracellular superoxide production associated with secondary root growth following desiccation of Pisum sativum seedlings

The seedling stage is arguably the most vulnerable phase in the plant life cycle, where the young establishing plant is extremely sensitive to environmental stresses such as drought. Here, the production of superoxide (O₂⁻), a molecule involved in stress signaling, was measured in response to desiccation of Pisum sativum L. seedlings. Following desiccation that was sufficient to kill the radicle meristem, viability could be retained by seedlings that grew secondary roots. Upon rehydration, secondary roots formed in a region that had displayed intense extracellular O₂⁻production on desiccation. Treating partially desiccated seedlings with hydrogen peroxide (H₂O₂) prevented viability loss. In summary, reactive oxygen species (ROS) appear to participate in the signaling required for secondary root formation following desiccation stress of P. sativum seedlings.     

Abscisic Acid-induced chilling tolerance in maize suspension-cultured cells

The induction of chilling tolerance by abscisic acid (ABA) in maize (Zea mays L. cv Black Mexican Sweet) suspension cultured cells was examined. Cell viability during exposure to chilling was estimated by triphenyl tetrazolium chloride reduction immediately after chilling and a filter paper growth assay. Both methods yielded comparable results. Chilling tolerance was induced by transferring 5-day-old cultures (late log phase) to a fresh medium containing ABA (10 to 100 micromolar). The greatest chilling tolerance was achieved with ABA at 100 micromolar. Growth of cells was inhibited at this concentration. After a 7-day exposure to 4°C in the dark, the survival of ABA-treated cells (100 micromolar ABA, 28°C for 24 h in the dark) was sevenfold greater than untreated cells. Effective induction of chilling tolerance was first observed when cells were held at 28°C for 6 hours after adding ABA. No tolerance was induced if the culture was chilled at the inception of ABA treatment. Induction of chilling tolerance was inhibited by cycloheximide. These results indicate that ABA is capable of inducing chilling tolerance when ABA-treated cells are incubated at a warm temperature before exposure to chilling, and this induction requires de novo synthesis of proteins.     

Antioxidant status of Lobiger serradifalci and Oxynoe olivacea (Opisthobranchia, Mollusca)

Invasion of the Mediterranean Sea by the two world-wide famous exotic algae species, Caulerpa taxifolia and Caulerpa racemosa, is still a problem and has adverse effects on the Mediterranean sublittoral ecosystem. Biological control studies revealed that the two native Sacoglossans, Oxynoe olivacea and Lobiger serradifalci, may have an effect on the expansion of invasive Caulerpa spp. in the Mediterranean. In the framework of this study, antioxidant enzyme activities, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), lipid peroxidation (LPO) and oxidized glutathione (GSSG) levels, as oxidative stress markers in L. serradifalci and O. olivacea were determined at two different temperature conditions (20 and 27 °C). In both species, SOD, CAT and GSH-Px activities were found to be positively correlated with temperature. The SOD activities in L. serradifalci were higher than those in O. olivacea at both temperatures, whereas the CAT and GSH-Px activities were significantly (p<0.05) higher in O. olivacea, compared to L. serradifalci. As expected, both species showed decreased LPO levels at 27 °C compared to 20 °C. GSSG level at 27 °C in O.olivacea was significantly (p<0.05) higher than that of 20 °C. On the other hand, no statistical (p>0.05) difference in L.serradifalci existed between GSSG levels at two temperatures. But, despite the variations in the antioxidant enzyme activities, there was no significant difference in LPO levels between the species, suggesting that the oxidative consequences of a given environmental condition may vary among different species. Inasmuch as the GSSG levels were in accordance with antioxidant enzyme activities, GSH might have acted as a cofactor of GSH-Px and an individual antioxidant in these sea slugs.     

Protective effects of sulfated chitooligosaccharides against hydrogen peroxide-induced damage in MIN6 cells

Sulfated chitooligosaccharides (COS-S) with different degrees of substitution (DS) were obtained by the chlorosulfuric acid/pyridine method. Protective effects of COS-S against hydrogen peroxide (H₂O₂)-induced damage were investigated in pancreatic β-cells MIN6 cell line. The cell viability, morphology, insulin contents, malondialdehyde (MDA) inhibition, lactate dehydrogenase (LDH) release and the levels of antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidise (GPx) were evaluated under oxidative damage by 150 μM H₂O₂ for 6 h. COS-S did not show any harmful or inhibitory effect on cell growth at concentrations ranging from 0.1 to 0.5 mg/ml. While COS-S could enhance the cell viability, decrease the production of ROS, and reduce the MDA level as well as LDH level in oxidative damaged β-cells by being an antioxidant. The underlining mechanisms of protective effects of COS-S are partly due to the enhancement of antioxidant enzyme activity and inhibition of intracellular ROS production, along with suppressing MIN6 cell apoptosis subsequent to the amelioration of ROS. Moreover, increased DS might contribute to the defense mechanisms against H₂O₂-induced oxidative damage in MIN6 cells. These results indicated that the antioxidant properties of COS-S hold great potential for the oxidative diseases treatment, and the sulfate content of polysaccharides made great role in regulating antioxidant activities.     

Roles of intracellular hydrogen peroxide accumulation in abscisic acid signaling in Arabidopsis guard cells

Reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂), are among the important second messengers in abscisic acid (ABA) signaling in guard cells. In this study, to investigate specific roles of H₂O₂ in ABA signaling in guard cells, we examined the effects of mutations in the guard cell-expressed catalase (CAT) genes, CAT1 and CAT3, and of the CAT inhibitor 3-aminotriazole (AT) on stomatal movement. The cat3 and cat1 cat3 mutations significantly reduced CAT activities, leading to higher basal level of H₂O₂ in guard cells, when assessed by 2′,7′-dichlorodihydrofluorescein, whereas they did not affect stomatal aperture size under non-stressed condition. In addition, AT-treatment at concentrations that abolish CAT activities, showed trivial affect on stomatal aperture size, while basal H₂O₂ level increased extensively. In contrast, cat mutations and AT-treatment potentiated ABA-induced stomatal closure. Inducible ROS production triggered by ABA was observed in these mutants and wild type as well as in AT-treated guard cells. These results suggest that ABA-inducible cytosolic H₂O₂ elevation functions in ABA-induced stomatal closure, while constitutive increase of H₂O₂ do not cause stomatal closure.     

Oxidative Stress in Vivax Malaria

Malaria is still a leading cause of morbidity and mortality. The increase in lipid peroxidation reported in malaria infection and antioxidant status may be a useful marker of oxidative stress during malaria infection. The aim of this study was to investigate the role of antioxidant enzymes against toxic reactive oxygen species in patients infected with Plasmodium vivax and healthy controls. Malondialdehyde levels, superoxide dismutase, and glutathione peroxidase activities were determined in 91 P. vivax patients and compared with 52 controls. Malondialdehyde levels, superoxide dismutase, and glutathione peroxidase activities were 8.07±2.29 nM/ml, 2.69±0.33 U/ml, and 49.6±3.2 U/g Hb in the patient group and 2.72±0.50 nM/ml, 3.71±0.47 U/ml, and 62.3±4.3 U/g Hb in the control group, respectively. Malondialdehyde levels were found statistically significant in patients with vivax malaria higher than in healthy controls (P<0.001). On the other hand, superoxide dismutase and glutathione peroxidase activities were found to be significantly lower in vivax malaria patients than in controls (P<0.05). There was an increase in oxidative stress in vivax malaria. The results suggested that antioxidant defense mechanisms may play an important role in the pathogenesis of P. vivax.     

Antioxidative responses in Vitis vinifera infected by grapevine fanleaf virus

The antioxidative response of grapevine leaves (Vitis vinifera cv. Trebbiano) affected by the presence of grapevine fanleaf virus was studied during the summer of 2010 at three different harvest times (July 1st and 26th, and August 30th). At the first and second harvest, infected leaves showed increases in the concentration of superoxide radical and hydrogen peroxide, the latter increasing for enhanced activity of superoxide dismutase. In contrast, at the last harvest time, increases in the ascorbate pool and ascorbate peroxidase activity maintained hydrogen peroxide to control levels. The glutathione pool was negatively affected as summer progressed, showing a decrease in its total and reduced form amounts. At the same time, increases in the ascorbate pool were observed, making antioxidant defenses of grapevine effective also at the last harvest time. Increases in phenolic acids, and in particular in p-hydroxybenzoic acid, at the first and second harvest might have enhanced the efficiency of the antioxidant system through an interrelation between a peroxidase/phenol/ascorbate system and the NADPH/glutathione/ascorbate cycle. The lack of increase in p-hydroxybenzoic acid at the third harvest could be due instead to the enhanced utilization of this acid for hydrogen peroxide detoxification. With time, grapevine plants lost their capacity to contrast the spread of grapevine fanleaf virus, but acquired a greater ability to counteract pathogen-induced oxidative stress, being endowed with more reduced antioxidant pools.