Overexpression of Superoxide Dismutase Protects Plants from Oxidative Stress (Induction of Ascorbate Peroxidase in Superoxide Dismutase-Overexpressing Plants)

Photosynthesis of leaf discs from transgenic tobacco plants (Nicotiana tabacum) that express a chimeric gene that encodes chloroplast-localized Cu/Zn superoxide dismutase (SOD+) was protected from oxidative stress caused by exposure to high light intensity and low temperature. Under the same conditions, leaf discs of plants that did not express the pea SOD isoform (SOD-) had substantially lower photosynthetic rates. Young plants of both genotypes were more sensitive to oxidative stress than mature plants, but SOD+ plants retained higher photosynthetic rates than SOD- plants at all developmental stages tested. Not surprisingly, SOD+ plants had approximately 3-fold higher SOD specific activity than SOD- plants. However, SOD+ plants also exhibited a 3- to 4-fold increase in ascorbate peroxidase (APX) specific activity and had a corresponding increase in levels of APX mRNA. Dehydroascorbate reductase and glutathione reductase specific activities were the same in both SOD+ and SOD- plants. These results indicate that transgenic tobacco plants that overexpress pea Cu/Zn SOD II can compensate for the increased levels of SOD with increased expression of the H2O2-scavenging enzyme APX. Therefore, the enhancement of the active oxygen-scavenging system that leads to increased oxidative stress protection in SOD+ plants could result not only from increased SOD levels but from the combined increases in SOD and APX activity.     

Antioxidant responses of pea genotypes to zinc deficiency

The effects of Zn deficiency on antioxidant responses of two pea (Pisum sativum L.) genotypes, a Zn-efficient IPFD-99-13 and Zn-inefficient KPMR-500, grown in sand culture were studied. In the pea genotype KPMR-500, Zn deficiency decreased dry matter yield, tissue Zn concentration, and antioxidant enzyme activities istronger than in the genotype IPFD-99-13. Genotype IPFD-99-13 developed more efficient antioxidant system to scavenge ROS than genotype KPMR-500. Zinc deficiency produced oxidative damage to pea genotypes due to enhanced accumulation of TBARS and H₂O₂ and decreased activities of antioxidant enzymes (Cu/Zn superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX)). In the leaves of IPFD-99-13 genotype, the higher activity of ROS-scavenging enzyme, e.g., SOD, CAT, POD, and glutathione reductase, and antioxidants, such as ascorbate and non-protein thiols, led to the lower accumulation of H₂O₂ and lipid peroxides. These results suggest that, by maintaining an efficient antioxidant defense system, the IPFD-99-13 genotype shows a lower sensivity to Zn deficiency than the KPMR-500 genotype.     

The activities of antioxidant enzymes in response to oxidative stresses and hormones in paraquat-tolerant Rehmannia glutinosa plants

All members of R. glutinosa show the unique characteristic of intrinsic tolerance to paraquat (PQ). Antioxidant enzymes have been proposed to be the primary mechanism of PQ resistance in several plant species. Therefore, the antioxidant enzyme systems of R. glutinosa were evaluated by comparatively analyzing cellular antioxidant enzyme levels, and their responses of oxidative stresses and hormones. The levels of ascorbate peroxidase (APX), glutathione reductase (GR), non-specific peroxidase (POX), and superoxide dismutase (SOD) were 7.3-, 4.9-, 2.7- and 1.6-fold higher in PQ-tolerant R. glutinosa than in PQ-susceptible soybeans. However, the activity of catalase (CAT) was about 12-fold higher in the soybeans. The activities of antioxidant enzymes reduced after PQ treatment in the two species, with the exception of POX and SOD in R. glutinosa, which increased by about 40 %. Interestingly, the activities of APX, SOD and POX in R. glutinosa, relative to those in soybeans, were further increased by 49, 67 and 93 % after PQ treatment. The considerably higher intrinsic levels, and increases in the relative activities of antioxidant enzymes in R. glutinosa under oxidative stress support the possible role of these enzymes in the PQ tolerance of R. glutinosa. However, the relatively lower levels of SOD versus PQ tolerance, and the mixed responses of antioxidant enzymes to stresses and hormones, suggest a possible alternative mechanism(s) for PQ tolerance in R. glutinosa.     

Antioxidant responses of shoots and roots of lentil to NaCl-salinity stress

The effect of salt stress (100 mM and 200 mM NaCl) on antioxidant responses in shoots and roots of 14-day-old lentil (Lens culinaris M.) seedlings was investigated. Salt stress caused a significant decrease in length, wet-dry weight and an increase in proline content of both shoot and root tissues. In leaf tissues, high salinity treatment resulted in a 4.4 fold increase in H₂O₂ content which was accompanied by a significant level of lipid peroxidation and an increase in electrolyte leakage. Root tissues were less affected with respect to these parameters. Leaf tissue extracts exhibited four activity bands, of which two were identified as Cu/Zn-SOD and others as Fe-SOD and Mn-SOD. Fe-SOD activity was missing in root extracts. In both tissues Cu/Zn-SOD activity comprised 70–75% of total SOD activity. Salt stress did not cause a significant increase in total SOD activity of leaf tissues but a significant enhancement (88%) was observed in roots mainly due to an enhancement in Cu/ZnSOD isoforms. Compared to leaf tissues a significantly higher constitutive ascorbate peroxidase (APX) and glutathion reductase (GR) activity was observed in root tissues. Upon salt stress no significant change in the activity of APX, catalase (CAT) and GR was observed in root tissues but a higher APX activity was present when compared to leaf tissues. On the other hand, in leaf tissues, with the exception of CAT, salt stress caused significant enhancement in the activity of other antioxidant enzymes. These results suggested that, root tissues of lentil are protected better from NaCl stress induced oxidative damage due to enhanced total SOD activity together with a higher level of APX activity under salinity stress. To our knowledge this is the first report describing antioxidant enzyme activities in lentil.     

Subcellular distribution of enzymes of the oxidative pentose phosphate pathway in root and leaf tissues

The subcellular distribution of enzymes of the oxidative pentose phosphate pathway was studied in plants. Root and leaf tissues from several species were separated by differential centrifugation into plastidic and cytosolic fractions. In all tissues studied, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were found in both plastidic and cytosolic compartments. In maize and pea root, and spinach and pea leaf, the non-oxidative enzymes of the pentose phosphate pathway (transaldolase, transketolase, ribose 5-phosphate isomerase, ribulose 5-phosphate 3-epimerase) appear to be restricted to the plastid. In tobacco leaf and root, however, the non-oxidative enzymes were found in the cytosolic as well as the plastidic compartments. In the absence of ribose 5-phosphate isomerase and ribulose 5-phosphate 3-epimerase in the cytosol, the product of the oxidative limb of the pathway (ribulose 5-phosphate) must be transported into a compartment capable of utilizing it. Ribulose 5-phosphate was supplied to isolated intact pea root plastids and was shown to be capable of supporting nitrite reduction. The kinetics of ribulose 5-phosphate-driven nitrite reduction in isolated pea root plastids suggested that the metabolite was translocated across the plastid envelope in a carrier-mediated transport process, indicating the presence of a translocator capable of transporting pentose phosphates.Keywords: Pentose phosphate, subcellular, plastid, ribulose 5-phosphate, compartmentation      

Differential response to paraquat induced oxidative stress in two rice cultivars on antioxidants and chlorophyll a fluorescence

The responses of antioxidative system and photosystem II photochemistry of rice (Oryza sativa L.) to paraquat induced oxidative stress were investigated in a chilling-tolerant cultivar Xiangnuo no. 1, and a chilling-susceptible cultivar, IR-50. Electrolyte leakage and malondialdehyde (MDA) content of Xiangnuo no. 1 were little affected by paraquat, but they increased in IR-50. After paraquat treatment, superoxide dismutase (SOD) activity remained high in Xiangnuo no. 1, while it declined in IR-50. Activities of catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) declined with oxidative stress in both cultivars, but Xiangnuo no. 1 had higher GR activity than IR-50. Under paraquat induced oxidative stress, ascorbic acid (AsA) and reduced glutathione (GSH) concentrations remained high in Xiangnuo no. 1, but decreased in IR-50. The results indicated that higher activities of SOD and GR and higher contents of AsA and GSH in Xiangnuo no. 1 under paraquat induced oxidative stress were associated with its tolerance to paraquat, while paraquat induced damage to IR-50 was related to decreased activities of SOD, APX and GR and contents of AsA and GSH. F _v/F _m, Φ _PSII, and qP remained high in Xiangnuo no. 1, while they decreased greatly in IR-50 under paraquat induced oxidative stress.     

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.     

Cadmium effects on mineral nutrition and stress in <Emphasis Type="Italic">Potamogeton crispus</Emphasis>

The mechanisms of plant tolerance to cadmium stress were studied by short-term exposure of Potamogeton crispus L. to various concentrations of Cd ranging from 0 to 0.09 mM. The accumulation of Cd and its influence on nutrient elements, chlorophyll pigments, ultrastructure, proline and MDA contents, and free radical production, as well as the activities of superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR) were investigated. The higher Cd concentration in the medium resulted in a significant enhancement of Cd accumulation. Photosynthetic pigment content decreased and ultrastructural damage to the leaf cells was aggravated with the increase in the Cd concentrations. Disruption of chloroplasts and mitochondria was observed even at the lowest concentration of Cd. Meantime, the rate of O₂^*− generation and the contents of H₂O₂ and MDA significantly increased under Cd stress, suggesting that Cd caused oxidative stress. In addition, the antioxidant system was clearly activated following Cd exposure. SOD and POD activities increased initially and then decreased, while APX and GR activities markedly increased. Simultaneously, mineral nutrition was disturbed. While K, P, Ca, and Cu contents decreased, Na, Fe, and Mn contents increased. Induction of antioxidant enzyme activities in leaves exposed to elevated Cd concentrations may be involved in Cd tolerance of P. crispus.     

Salt and oxidative stress: similar and specific responses and their relation to salt tolerance in Citrus

Salt damage to plants has been attributed to a combination of several factors including mainly osmotic stress and the accumulation of toxic ions. Recent findings in our laboratory showed that phospholipid hydroperoxide glutathione peroxidase (PHGPX), an enzyme active in the cellular antioxidant system, was induced by salt in citrus cells and mainly in roots of plants. Following this observation we studied the two most important enzymes active in elimination of reactive oxygen species, namely, superoxide dismutase (SOD) and ascorbate peroxidase (APX), to determine whether a general oxidative stress is induced by salt. While Cu/Zn-SOD activity and cytosolic APX protein level were similarly induced by salt and methyl viologen, the response of PHGPX and other APX isozymes was either specific to salt or methyl viologen, respectively. Unlike PHGPX, cytosolic APX and Cu/Zn-SOD were not induced by exogenously added abscisic acid. Salt induced a significant increase in SOD activity which was not matched by the subsequent enzyme APX. We suggest that the excess of H₂O₂ interacts with lipids to form hydroperoxides which in turn induce and are removed by PHGPX. Ascorbate peroxidase seems to be a key enzyme in determining salt tolerance in citrus as its constitutive activity in salt-sensitive callus is far below the activity observed in salt-tolerant callus, while the activities of other enzymes involved in the defence against oxidative stress, namely SOD, glutathione reductase and PHGPX, are essentially similar. Received: 10 January 1997 / Accepted: 28 May 1997     

Antioxidant responses to water deficit by drought‐tolerant and ‐sensitive sugarcane varieties

Water deficit is the major yield‐limiting factor of crop plants. The exposure of plants to this abiotic stress can result in oxidative damage due to the overproduction of reactive oxygen species. The aim of this work was to study the antioxidant‐stress response of drought‐tolerant (SP83‐2847 and SP83‐5073) and drought‐sensitive (SP90‐3414 and SP90‐1638) sugarcane varieties to water‐deficit stress, which was imposed by withholding irrigation for 3, 10 and 20 days. The drought‐sensitive varieties exhibited the lowest leaf relative water content and highest lipid peroxidation, hydrogen peroxide (H₂O₂) and proline contents during the progression of the drought‐stress condition. The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPOX) and glutathione reductase (GR) activities changed according to variety and stress intensity. SP83‐2847 exhibited higher CAT and APX activities than the other varieties in the early stage of drought, while the activities of GPOX and GR were the highest in the other varieties at the end of the drought‐stress period. A Cu/Zn SOD isoenzyme was absent at the end of drought period from the SP90‐3414‐sensitive variety. The results indicate that lipid peroxidation and early accumulation of proline may be good biochemical markers of drought sensitivity in sugarcane.     

The spatial patterns of oxidative stress indicators co-locate with early signs of natural senescence in maize leaves

Reactive oxygen species play a crucial role for various physiological and developmental processes in plants. Here, we report a spatial pattern of oxidative stress and antioxidant defence within maize leaf. Localization of hydrogen peroxide in different region of leaf clearly exhibits well-defined increasing pattern of accumulation from the base to the leaf tip. Lipid peroxidation, an index of oxidative damage, also showed a similar pattern-like hydrogen peroxide that is lowest at the base and highest at the leaf tip. NADPH oxidase, an enzyme responsible for superoxide anion generation, showed highest activity in the leaf tip and least in the leaf base regions. Superoxide dismutase (SOD) activity was increased from the base to the leaf tip. Peroxidases, DAB-peroxidase (DAB-POD) and guaiacol-peroxidase (G-POD), catalase (CAT) and glutathione reductase (GR) also showed increases in their activities from the base to the leaf tip. Ascorbate peroxidase (APX), however, showed a reverse trend—highest at the base and least in the leaf tip. The decrease in APX and increases in the activities of other antioxidant enzymes SOD, CAT, DAB-POD, G-POD and GR along with H₂O₂ and lipid peroxidation, ascorbate/dehydroascorbate and non-protein thiol levels from the base to the leaf tip clearly exhibit a spatial pattern prior to the onset of visible signs of senescence in the maize leaf.     

The inductive responses of the antioxidant enzymes by salt stress in the rice (Oryza sativa L.)

To access contributions of inductive responses of the antioxidant enzymes in the resistance to salt stress, activities of the enzymes were determined in the rice (Oryza sativaL. cv. Dongjin) plant. In the leaves of the rice plant, salt stress preferentially enhanced the content of H₂O₂ as well as the activities of the superoxide dismutase (SOD), ascorbate peroxidase (APX), and peroxidase specific to guaiacol, whereas it induced the decrease of catalase activity. On the other hand, salt stress had little effect on the activity levels of glutathione reductase (GR). In order to analyze the changes of antioxidant enzyme isoforms against salt stress, plant extracts were subjected to native PAGE. Leaves of the rice plant had two isoforms of Mn-SOD and five isoforms of Cu/Zn-SOD. Fe-SOD isoform was not observed in the activity gels. Expression of Cu/Zn-1, -2, and Mn-SOD-2 isoforms was preferentially enhanced by salt stress. Seven APX isoforms were presented in the leaves of the rice plants. The intensities of APX-4 to -7 were enhanced by salt stress, whereas those of APX-1 to -3 were minimally in changed response to salt stress. There were seven GR isoforms in the leaves of rice plants. Levels of activity for most GR isoforms did not change in the stressed plants compared to the control plants. On the other hand, the levels of activity for most antioxidant enzymes changed little in the roots of stressed plants compared to the control plants. These results collectively suggest that SOD leads to the overproduction of hydrogen peroxide in the leaves of rice plants subjected to salt stress: The overproduction of hydrogen peroxide functions as the signal of salt stress, which induces the induction of specific APX isoforms but not specific GR isoforms under catalase deactivation.     

Differential reponses to paraquat‐induced oxidative injury in a pea (Pisum sativum) protoplast system

Antioxidant responses to varying degrees of paraquat stress in freshly isolated photosynthesizing pea (Pisum sativum L.) protoplasts from cultivars Progress and Nugget were studied. Leaves of comparable maturity were used for protoplast isolation. Nugget protoplasts were more resistant to paraquat in the micromolar range under our conditions. In Nugget, a non-bleaching paraquat concentration (10 µM) inhibited CO₂-dependent O₂ evolution ca 50% during the first 40 min, remaining at that rate (“coping behavior”) for up to 100 min. In contrast, Progress protoplasts treated with the same concentration of paraquat did not exhibit coping behavior. Antioxidant enzyme activities were unaltered throughout the time course of the experiment in treated protoplasts from Nugget and in chloroplasts isolated from them. Thus, the coping behavior of Nugget protoplasts cannot be attributed to changes in activities of the three antioxidant enzymes tested. Paraquat treatment did not affect antioxidant enzyme activities in Progress protoplasts nor in chloroplasts isolated from them. When higher doses of paraquat were used (12 h, 0.1 mM paraquat), protoplasts from both cultivars were rapidly bleached and total protein decreased to ca 30% of pre-stress levels. Glutathione reductase (GR, EC 1.6.4.2) activity dropped in protoplasts from both cultivars under the severe stress conditions in concert with declines in protein levels. However, superoxide dismutase (SOD, EC 1.15.1.1) activity remained constant over the first 9 h of the time course, increasing to ca 150& of original levels by the final, 12-h time point. The activity of the plastid Cu,Zn isoform, expressed as a percentage of total SOD activity, declined over the time course of the experiment while that of mitochondrial MnSOD appeared to increase. This change in activity of MnSOD correlated with cell decline, therefore, and was not correlated with protection. These data are in agreement with some earlier reports and are compatible with the hypothesis that SOD activity levels increase in response to reactive oxygen species levels, even under conditions leading to cell death.     

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.     

Activation of ascorbate-glutathione cycle inArabidopsis leaves in response to aminotriazole

Aminotriazole(AT)-induced changes in growth, hydrogen peroxide content and activities of H₂O₂-scavenging antioxidant enzymes were investigated in the growing leaves ofArabidopsis plants (Arabidopsis thaliana cv Columbia). Catalase activity of rosette leaves was reduced by 65% with an application of 0.1 mM AT (a herbicide known as a catalase inhibitor), whereas the leaf growth and H₂O₂ content were almost unaffected. However, an approximate 1.6 to 2-fold increase in cytosolic ascorbate peroxidase (APX) activity concomitant with a substantial activation of glutathione reductase (GR) (approx. 22% increase) was observed during leaf growth in the presence of 0.1 mM AT. The activity of cytosolic APX in leaves was also increased by 1.8-fold with an application of exogenous 2 mM paraquat (an inducer of H₂O₂ production in plant cells) in the absence of AT. These results collectively suggest that (a) cytosolic APX and GR operate to activate an ascorbate-glutathione cycle for the removal of H₂O₂ under severe catalase deactivation, and (b) the expression of APX seems to be regulated by a change of the endogenous H₂O₂ level in leaf cells.     

The oxidative stress caused by salinity in two barley cultivars is mitigated by elevated CO2

Changes in antioxidant metabolism because of the effect of salinity stress (0, 80, 160 or 240 m M NaCl) on protective enzyme activities under ambient (350 μmol mol⁻¹) and elevated (700 μmol mol⁻¹) CO₂ concentrations were investigated in two barley cultivars ( Hordeum vulgare L., cvs Alpha and Iranis). Electrolyte leakage, peroxidation, antioxidant enzyme activities [superoxide dismutase (SOD), EC 1.15.1.1; ascorbate peroxidase (APX), EC 1.11.1.11; catalase (CAT), EC 1.11.1.6; dehydroascorbate reductase (DHAR), EC 1.8.5.1; monodehydroascorbate reductase (MDHAR), EC 1.6.5.4; glutathione reductase (GR), EC 1.6.4.2] and their isoenzymatic profiles were determined. Under salinity and ambient CO₂, upregulation of antioxidant enzymes such as SOD, APX, CAT, DHAR and GR occurred. However, this upregulation was not enough to counteract all ROS formation as both ion leakage and lipid peroxidation came into play. The higher constitutive SOD and CAT activities together with a higher contribution of Cu,Zn-SOD 1 detected in Iranis might possibly contribute and make this cultivar more salt-tolerant than Alpha. Elevated CO₂ alone had no effect on the constitutive levels of antioxidant enzymes in Iranis, whereas in Alpha it induced an increase in SOD, CAT and MDHAR together with a decrease of DHAR and GR. Under combined conditions of elevated CO₂ and salinity the oxidative damage recorded was lower, above all in Alpha, together with a lower upregulation of the antioxidant system. So it can be concluded that elevated CO₂ mitigates the oxidative stress caused by salinity, involving lower ROS generation and a better maintenance of redox homeostasis as a consequence of higher assimilation rates and lower photorespiration, being the response dependent on the cultivar analysed.     

Changes in Activities of Antioxidant Enzymes and Their Relationship to Genetic and Paclobutrazol-Induced Chilling Tolerance of Maize Seedlings

The potential role of antioxidant enzymes in protecting maize (Zea mays L.) seedlings from chilling injury was examined by analyzing enzyme activities and isozyme profiles of chilling-susceptible (CO 316) and chilling-tolerant (CO 328) inbreds. Leaf superoxide dismutase (SOD) activity in CO 316 was nearly one-half that of CO 328, in which the high activity was maintained during the chilling and postchilling periods. Activity of glutathione reductase (GR) was much higher in roots than in leaves. CO 328 also possessed a new GR isozyme that was absent in roots of CO 316. Ascorbate peroxidase (APX) activity was considerably lower in leaves of CO 328 than in CO 316, and nearly similar in roots. Paclobutrazol treatment of CO 316 induced several changes in the antioxidant enzyme profiles and enhanced their activities, especially those of SOD and APX, along with the induction of chilling tolerance. These results suggest that increased activities of SOD in leaves and GR in roots of CO 328, as well as SOD and APX in leaves and roots of paclobutrazol-treated CO 316, contribute to their enhanced chilling tolerance.