Enhanced antioxidant enzymes are associated with reduced hydrogen peroxide in barley roots under saline stress

Antioxidant enzymes are related to the resistance to various abiotic stresses including salinity. Barley is relatively tolerant to saline stress among crop plants, but little information is available on barley antioxidant enzymes under salinity stress. We investigated temporal and spatial responses of activities and isoform profiles of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), non-specific peroxidase (POX), and glutathione reductase (GR) to saline stress in barley seedlings treated with 200 mM NaCl for 0, 1, 2, 5 days, respectively. In the control plant, hydrogen peroxide content was about 2-fold higher in the root than in the shoot. Under saline stress, hydrogen peroxide content was decreased drastically by 70% at 2 d after NaCl treatment (DAT) in the root. In the leaf, however, the content was remained unchanged by 2 DAT and increased about 14 % at 5 DAT. In general, the activities of antioxidant enzymes were increased in the root and shoot under saline stress. But the increase was more significant and consistent in the root. The activities of SOD, CAT, APX, POX, and GR were increased significantly in the root within 1 DAT, and various elevated levels were maintained by 5 DAT. Among the antioxidant enzymes, CAT activity was increased the most drastically. The significant increase in the activities of SOD, CAT, APX, POX, and GR in the NaCl-stressed barley root was highly correlated with the increased expression of the constitutive isoforms as well as the induced ones. The hydrogen peroxide content in the root.     

Protective role of exogenous spermidine against paraquat toxicity in radish chloroplasts

When radish chloroplasts were pretreated with 1 mM spermidine (Spd) and then exposed to 30 M paraquat (PQ), they improved their tolerance to subsequent PQ-induced oxidative damages. That included the decreases in the contents of chlorophyll, protein, and ascorbate, as well as the increases in malondialdehyde (MDA) and H₂O₂ levels. Analysis of antioxidant enzymes showed that Spd pretreatment effectively prevented the PQ-induced decreases in the total activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX). In contrast, the normally enhanced activities of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) in PQ-treated chloroplasts were reversed by Spd pretreatment In a native gel assay, the Cu/ZnSOD isozyme, which disappeared under the PQ alone treatment, was significantly recovered when tissues were pretreated with Spd. The dominant APX4 isozyme activity, which was preferentially decreased in response to PQ alone treatment, was also strongly reactivated by earlier Spd exposure. Therefore, we suggest that Spd could play a substantial role in protecting the radish chloroplasts from PQ stress. Furthermore, the enhancement of the Cu/ZnSOD and APX4 isozymes by Spd pretreatment seems to be responsible for prevention of the PQ-induced decreases in the total activities of SOD and APX, thereby providing a tolerance to PQ toxicity.     

Changes in activities of antioxidant enzymes and their gene expression during leaf development of sweetpotato

To understand the functions of antioxidant enzymes during leaf development in sweetpotato, we investigated the activities of several antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POX), ascorbate peroxidase (APX) and catalase (CAT). Significant increases were observed in the activities of SOD, POX and APX during the late stage of leaf development, whereas CAT activity increased during the early developmental stage. By RT-PCR analysis, various POX and APX genes showed differential expression patterns during leaf development. Four POX genes swpa3, swpa4, swpa6, swpb4 and one APX gene swAPX1 exhibited high levels of gene expression during the senescence stage of leaf development, but two POX genes, swpa1 and swpa7 were preferentially expressed at both the mature green and the late senescence stages of leaf development. These results indicate that hydrogen peroxide (H₂O₂)-related antioxidant enzymes are differentially regulated in the process of leaf development of sweetpotato.     

Polyamines as antioxidant protectors against paraquat damage in radish (Raphanus sativus L.) cotyledons

Pretreatment of radish cotyledons with polyamines (PAs; especially 1 mM spermidine) significantly improved their tolerance to subsequent 50 μM paraquat (PQ)-induced oxidative damage. Symptoms in the cotyledons, e.g., large accumulations of H₂O₂, and losses of fresh weight, chlorophyll, and proteins, were remarkably alleviated. Likewise, analysis of several enzymes belonging to the Superoxide dismutase (SOD)/ascorbate-glutathione cycle showed that pretreatment with PAs prevented typical PQ-induced declines in the total activities of SOD, ascorbate peroxidase (APX), and glutathione reductase (GR). Dehydroascorbate reductase (DHAR) activity, which normally decreases sharply under prolonged PQ exposure, was also highly maintained by PA treatment. In a native gel assay, two SOD isozymes (FeSOD and Cu/ZnSODI), two APX isozymes (APX1 and APX2), and two GSSG-specific isozymes (GR1 and GR2) proved to be more responsible for PQ tolerance, as manifested by the strong increases in their activities by spermidine (Spd) pretreatment. In addition, experiments with protein synthesis inhibitors (actinomycin D and cycloheximide) indicated that Spd could stimulatede novo synthesis of SOD and APX at the translational level. We can conclude that PAs may function as antioxidant protectors by invoking an efficient SOD/ascorbate-glutathione cycle in radish cotyledons exposed to PQ.     

The activity of antioxidant enzymes in Arabidopsis thaliana exposed to colchicine and H2O2

Studies on the possible interference of colchicine and H2O2 with the activity of some antioxidant enzymes were carried out on Arabidopsis thaliana v. Columbia grown in Murashige and Skooge nutrient medium. Measurements of superoxide dismutase (SOD), guaiacol peroxidase (POX), ascorbate peroxidase (APX) and catalase (CAT) activities were conducted spectrophotometrically. In the presence of colchicine, SOD activity increased, while CAT, APX and POX activities decreased. Inhibitory H2O2 effects on the activity of the enzymes were found. Colchicine pre-treatment resulted in an increase in CAT activity and a further increase in SOD activity in plants treated with H2O2.     

An Enhancing Effect of Exogenous Mannitol on the Antioxidant Enzyme Activities in Roots of Wheat Under Salt Stress

The role of mannitol as an osmoprotectant, a radical scavenger, a stabilizer of protein and membrane structure, and protector of photosynthesis under abiotic stress has already been well described. In this article we show that mannitol applied exogenously to salt-stressed wheat, which normally cannot synthesize mannitol, improved their salt tolerance by enhancing activities of antioxidant enzymes. Wheat seedlings (3 days old) grown in 100 mM mannitol (corresponding to −0.224 MPa) for 24 h were subjected to 100 mM NaCl treatment for 5 days. The effect of exogenously applied mannitol on the salt tolerance of plants in view of growth, lipid peroxidation levels, and activities of antioxidant enzymes in the roots of salt-sensitive wheat (Triticum aestivum L. cv. Kızıltan-91) plants with or without mannitol was studied. Although root growth decreased under salt stress, this effect could be alleviated by mannitol pretreatment. Peroxidase (POX) and ascorbate peroxidase (APX) activities increased, whereas superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) activities decreased in Kızıltan-91 under salt stress. However, activities of antioxidant enzymes such as SOD, POX, CAT, APX, and GR increased with mannitol pretreatment under salt stress. Although root tissue extracts of salt-stressed wheat plants exhibited only nine different SOD isozyme bands of which two were identified as Cu/Zn-SOD and Mn-SOD, mannitol treatment caused the appearance of 11 different SOD activity bands. On the other hand, five different POX isozyme bands were determined in all treatments. Enhanced peroxidation of lipid membranes under salt stress conditions was reduced by pretreatment with mannitol. We suggest that exogenous application of mannitol could alleviate salt-induced oxidative damage by enhancing antioxidant enzyme activities in the roots of salt-sensitive Kızıltan-91.     

Antioxidative defense system in pigeonpea roots under waterlogging stress

Pigeonpea [Cajanus cajan (L.) Millsp.] is a waterlogging-sensitive legume crop. We studied the effect of waterlogging stress on hydrogen peroxide (H₂O₂) content, lipid peroxidation and antioxidant enzyme activities in two pigeonpea genotypes viz., ICPL-84023 (waterlogging resistant) and MAL-18 (waterlogging susceptible). In a pot experiment, waterlogging stress was imposed for 6 days at early vegetative stage (20 days after sowing). Waterlogging treatment significantly increased hydrogen peroxide accumulation and lipid peroxidation, which indicated the extent of oxidative injury posed by stress conditions. Enzyme activities of peroxidase (POX), catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD) and polyphenol oxidase (PPO) increased in pigeonpea roots as a consequence of waterlogged conditions, and all the enzyme activities were significantly higher in waterlogged ICPL-84023 than in MAL-18. POX activity was the maximum immediately after imposing stress, therefore, it was suggested to be involved in early scavenging of H₂O₂, while rest of the enzymes (CAT, APX, SOD and PPO) were more important in late responses to waterlogging. Present study revealed that H₂O₂ content is directly related to lipid peroxidation leading to oxidative damage during waterlogging in pigeonpea. Higher antioxidant potential in ICPL-84023 as evidenced by enhanced POX, CAT, APX, SOD and PPO activities increased capacity for reactive oxygen species (ROS) scavenging and indicated relationship between waterlogging resistance and antioxidant defense system in pigeonpea.     

Investigations on the antioxidative defence responses to NaCl stress in a mangrove, Bruguiera parviflora: Differential regulations of isoforms of some antioxidative enzymes

Two-month-old healthy seedlings of a true mangrove, Bruguiera parviflora, raised from propagules in normal nursery conditions were subjected to varying concentrations of NaCl for 45 d under hydroponic culture conditions to investigate the defence potentials of antioxidative enzymes against NaCl stress imposed oxidative stress. Changes in the activities of the antioxidative enzymes catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POX), glutathione reductase (GR) and superoxide dismutase (SOD) were assayed in leaves to monitor the temporal regulation. Among the oxidative stress triggered chemicals, the level of H₂O₂ was significantly increased while total ascorbate and total glutathione content decreased. The ratio of reduced to oxidized glutathiones, however, increased due to decreased levels of oxidized glutathione in the leaf tissue. Among the five antioxidative enzymes monitored, the APX, POX, GR and SOD specific activities were significantly enhanced at high concentration (400 mM NaCl), while the catalase activities declined, suggesting both up and downregulations of antioxidative enzymes occurred due to NaCl imposed osmotic and ionic stress. Analysis of the stress induced alterations in the isoforms of CAT, APX, POX, GR and SOD revealed differential regulations of the isoforms of these enzymes. In B. parviflora one isoform of each of Mn-SOD and Cu/Zn-SOD while three isoforms of Fe-SOD were observed by activity staining gel. Of these, only Mn-SOD and Fe-SOD2 content was preferentially elevated by NaCl treatment, whereas isoforms of Cu/Zn-SOD, Fe-SOD1 and Fe-SOD3 remained unchanged. Similarly, out of the six isoforms of POX, the POX-1,-2,-3 and -6 were enhanced due to salt stress but the levels of POX-4 and -5 remained same as in control plants suggesting preferential upregulation of selective POX isoforms. Activity staining gel revealed only one prominent band of APX and this band increased with increased salt concentration. Similarly, two isoforms of GR (GR1 and GR2) were visualized on activity staining gel and both these isoforms increased upon salt stress. In this mangrove four CAT-isoforms were identified, among which the prominent CAT-2 isoform level was maximally reduced again suggesting differential downregulation of CAT isoforms by NaCl stress. The results presented in this communication are the first report on the resolutions of isoforms APX, POX and GR out of five antioxidative enzymes studied in the leaf tissue of a true mangrove. The differential changes in the levels of the isoforms due to NaCl stress may be useful as markers for recognizing salt tolerance in mangroves. Further, detailed analysis of the isoforms of these antioxidative enzymes is required for using the various isoforms as salt stress markers. Our results indicate that the overproduction of H₂O₂ by NaCl treatment functions as a signal of salt stress and causes upregulation of APX, POX, GR and deactivations of CAT in B. parviflora. The concentrations of malondialdehyde, a product of lipid peroxidation and lipoxygenase activity remained unchanged in leaves treated with different concentrations of NaCl, which again suggests that the elevated levels of the antioxidant enzymes protect the plants against the activated oxygen species thus avoiding lipid peroxidation during salt stress.     

Effects of paraquat on lipid peroxidation and antioxidant enzymes in aquatic fern <Emphasis Type="Italic">Azolla microphylla</Emphasis>

Paraquat is most extensively used methyl viologen herbicide to control weeds in the rice-Azolla ecosystem. The effects of different paraquat (PQ) dosages on growth, lipid peroxidation, and activity of antioxidant enzymes of Azolla microphylla Kaul. were investigated. The results indicated that Azolla fronds survived only at the concentrations of 2–6 μM PQ. Frond fragmentation and browning occurred after 24 h at 8 μM PQ. At 24 h, the amount of proteins decreased by 48.7 % in Azolla fronds exposed to 10 μM PQ than that in control fronds. The supplementation of 10 μM PQ increased the activities of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), and ascorbate peroxidase (APX) by 2,4-, 1,8-, 3,0-, and 2,2-fold, respectively, as compared with control. The content of PQ and activities of SOD, CAT, GPX, and APX were found to be positively correlated. Our study showed that PQ (2–6 μM) caused ROS overproduction in Azolla fronds, which were scavenged by induced activities of antioxidant enzymes.     

Physiochemical and antioxidant responses of the perennial xerophyte Capparis ovata Desf. to drought

Caper (Capparis ovata Desf.) is a perennial shrub (xerophyte) and drought resistant plant which is well adapted to Mediterranean Ecosystem. In the present study we investigated the plant growth, relative water content (RWC), chlorophyll fluorescence (F_V/F_M), lipid peroxidation (TBA-reactive substances content) as parameters indicative of oxidative stress and antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POX), catalase (CAT) and glutathione reductase (GR) in relation to the tolerance to polyethylene glycol mediated drought stress in C. ovata seedlings. For induction of drought stress, the 35 days seedlings were subjected to PEG 6000 of osmotic potential −0.81 MPa for 14 days. Lipid peroxidation increased in PEG stressed seedlings as compared to non-stressed seedlings of C. ovata during the experimental period. With regard to vegetative growth, PEG treatment caused decrease in shoot fresh and dry weights, RWC and F_V/F_M but decline was more prominent on day 14 of PEG treatment. Total activity of antioxidative enzymes SOD, APX, POX, CAT and GR were investigated in C. ovata seedlings under PEG mediated drought. Induced activities of SOD, CAT and POX enzymes were high and the rate of increment was higher in stressed seedling. APX activity increased on both days of PEG treatment, however, increase in GR activity was highest on day 14 of drought stress. We concluded that increased drought tolerance of C. ovata is correlated with diminishing oxidative injury by functioning of antioxidant system at higher rates under drought stress.