Factors Affecting the Enhancement of Oxidative Stress Tolerance in Transgenic Tobacco Overexpressing Manganese Superoxide Dismutase in the Chloroplasts

Two varieties of tobacco (Nicotiana tabacum var PBD6 and var SR1) were used to generate transgenic lines overexpressing Mn-superoxide dismutase (MnSOD) in the chloroplasts. The overexpressed MnSOD suppresses the activity of those SODs (endogenous MnSOD and chloroplastic and cytosolic Cu/ZnSOD) that are prominent in young leaves but disappear largely or completely during aging of the leaves. The transgenic and control plants were grown at different light intensities and were then assayed for oxygen radical stress tolerance in leaf disc assays and for abundance of antioxidant enzymes and substrates in leaves. Transgenic plants had an enhanced resistance to methylviologen (MV), compared with control plants, only after growth at high light intensities. In both varieties the activities of FeSOD, ascorbate peroxidase, dehydroascorbate reductase, and monodehydroascorbate reductase and the concentrations of glutathione and ascorbate (all expressed on a chlorophyll basis) increased with increasing light intensity during growth. Most of these components were correlated with MV tolerance. It is argued that SOD overexpression leads to enhancement of the tolerance to MV-dependent oxidative stress only if one or more of these components is also present at high levels. Furthermore, the results suggest that in var SR1 the overexpressed MnSOD enhances primarily the stromal antioxidant system.     

Increased Tolerance to Mn Deficiency in Transgenic Tobacco Overproducing Superoxide Dismutase

The effect of Mn deficiency on plant growth and activities ofsuperoxide dismutase (SOD) was studied in hydroponically-grownseedlings of transgenic tobacco (Nicotiana tabacum L.) engineeredto overexpress FeSOD in chloroplasts or MnSOD in chloroplastsor mitochondria. In comparison to the non-transgenic parentalline, the activity of MnSOD in the lines overproducing MnSODwas 1.6-fold greater, and the activity of FeSOD in the FeSOD-overproducinglines was 3.2-fold greater, regardless of the Mn treatment (deficientor sufficient). The MnSOD activities decreased due to Mn deficiency,while activities of FeSOD and Cu/ZnSOD remained unaffected 25d after transplanting (DAT). With an increased duration of theMn deficiency stress (45 DAT), FeSOD activity decreased, andthat of MnSOD continued to decrease, while Cu/ZnSOD activitysimultaneously increased. Under Mn sufficiency, non-transgenicparental plants had greater shoot biomass than the transgenics;however, when subjected to Mn deficiency stress, non-transgenicparents suffered a proportionally greater growth reduction thantransgenic lines. Thus, overproduction of MnSOD in chloroplastsmay provide protection from oxidative stress caused by Mn deficiency.Copyright 1999 Annals of Botany Company Manganese deficiency, Nicotiana tabacum, superoxide dismutase (SOD), transgenic tobacco.     

Enhanced sensitivity to oxidative stress in transgenic tobacco plants with decreased glutathione reductase activity leads to a decrease in ascorbate pool and ascorbate redox state

To investigate the possible mechanisms of glutathione reductase (GR) in protecting against oxidative stress, we obtained transgenic tobacco (Nicotiana tabacum) plants with 30–70% decreased GR activity by using a gene encoding tobacco chloroplastic GR for the RNAi construct. We investigated the responses of wild type and transgenic plants to oxidative stress induced by application of methyl viologen in vivo. Analyses of CO₂ assimilation, maximal efficiency of photosystem II photochemistry, leaf bleaching, and oxidative damage to lipids demonstrated that transgenic plants exhibited enhanced sensitivity to oxidative stress. Under oxidative stress, there was a greater decrease in reduced to oxidized glutathione ratio but a greater increase in reduced glutathione in transgenic plants than in wild type plants. In addition, transgenic plants showed a greater decrease in reduced ascorbate and reduced to oxidized ascorbate ratio than wild type plants. However, there were neither differences in the levels of NADP and NADPH and in the total foliar activities of monodehydroascorbate reductase and dehydroascorbate reductase between wild type and transgenic plant. MV treatment induced an increase in the activities of GR, ascorbate peroxidase, superoxide dismutase, and catalase. Furthermore, accumulation of H₂O₂ in chloroplasts was observed in transgenic plants but not in wild type plants. Our results suggest that capacity for regeneration of glutathione by GR plays an important role in protecting against oxidative stress by maintaining ascorbate pool and ascorbate redox state.     

Zinc deficiency enhances ozone toxicity in bush beans (Phaseolus vulgaris L. cv. Saxa)

In zinc-deficient bush beans (Phaseolus vulgaris L. cv. Saxa)ozone sensitivity was enhanced compared to plants sufficientlysupplied with this nutrient. This was correlated with reducedlevels of Cu/ZnSOD activity, but was unrelated to effects ofzinc deficiency on transpiration, rates of ethylene formation,ascorbic acid content or levels of MnSOD and ascorbatedependentperoxidase activities. Thus, these results show that detoxificationof superoxide anions by Cu/ZnSOD is important in plant resistanceto ozone. Additionally, this also indicates the in vivo formationof superoxide anions when plants are exposed to ozone. Key words: Ethylene, ozone, ascorbate peroxidase, superoxide dismutase, zinc deficiency     

Enhanced tolerances of transgenic tobacco plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against methyl viologen-mediated oxidative stress

In order to better understand the role of antioxidant enzymes in plant stress protection mechanisms, transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants were developed that overexpress both superoxide dismutase (SOD) and ascorbate peroxidase (APX) in chloroplasts. These plants were evaluated for protection against methyl viologen (MV, paraquat)‐mediated oxidative damage both in leaf discs and whole plants. Transgenic plants that express either chloroplast‐targeted CuZnSOD (C) or MnSOD (M) and APX (A) were developed (referred to as CA plants and AM plants, respectively). These plant lines were crossed to produce plants that express all three transgenes (CMA plants and AMC plants). These plants had higher total APX and SOD activities than non‐transgenic (NT) plants and exhibit novel APX and SOD isoenzymes not detected in NT plants. As expected, transgenic plants that expressed single SODs showed levels of protection from MV that were only slightly improved compared to NT plants. The expression of either SOD isoform along with APX led to increased protection while expression of both SODs and APX provided the highest levels of protection against membrane damage in leaf discs and visual symptoms in whole plants.     

Effect of hypoxia on photosynthetic activity and antioxidative response in gametophores of <Emphasis Type="Italic">Mnium undulatum</Emphasis>

The effects of hypoxia caused by complete submerging of Mnium undulatum gametophores in water, on their photosynthetic activity and the activity of two antioxidative enzymes: superoxide dismutase (SOD) and catalase (CAT) were investigated. The net photosynthesis was strongly inhibited throughout the experiment, and the strong drop in the maximum quantum yield of the PSII (F_v/F_m) was also observed. Three classes of SOD: MnSOD, FeSOD, Cu/ZnSOD and three isoforms of Cu/ZnSOD were identified. A significant decrease in activity of MnSOD, FeSOD and one Cu/ZnSOD isoform was observed after 24 and 48 h of hypoxia. FeSOD activity decreased already after 1 h of submerging in water and its activity remained at the low level during whole period of the experiment. CAT activity was also strongly inhibited in response to hypoxia stress. The obtained results suggest relationships between photosynthetic activity and antioxidative system in M. undulatum gametophores under oxygen deficiency stress.     

Effect of salinity on antioxidant responses of chickpea seedlings

The changes in the activity of antioxidant enzymes, like superoxide dismutase, ascorbate peroxidase, catalase and glutathione reductase, and growth parameters such as length, fresh and dry weight, proline and H₂O₂ contents, chlorophyll fluorescence (Fv/Fm), quantum yield of PSII and the rate of lipid peroxidation in terms of malondialdehyde in leaf and root tissues of a chickpea cultivar (Cicer arietinum L. cv. Gökçe) under salt treatment were investigated. Plants were subjected to 0.1, 0.2 and 0.5 M NaCl treatments for 2 and 4 days. Compared to controls, salinity resulted in the reduction of length and of the fresh and dry weights of shoot and root tissues. Salinity caused significant (P < 0.05) changes in proline and MDA levels in leaf tissue. In general, a dose-dependent decrease was observed in H₂O₂ content, Fv/Fm and quantum yield of photosynthesis under salt stress. Leaf tissue extracts exhibited three activity bands, of which the higher band was identified as MnSOD and the others as FeSOD and Cu/ZnSOD. A significant enhancement was detected in the activities of Cu/ZnSOD and MnSOD isozymes in both tissues. APX and GR activities exhibited significant increases (P < 0.05) in leaf tissue under all stress treatments, whereas no significant change was observed in root tissue. The activity of CAT was significantly increased under 0.5 M NaCl stress in root tissue, while its activity was decreased in leaf tissue under 0.5 M NaCl stress for 4 days. These results suggest that CAT and SOD activities play an essential protective role against salt stress in chickpea seedlings.     

Superoxide Dismutase Transgenes in Sugarbeets Confer Resistance to Oxidative Agents and the Fungus C. beticola

Sugarbeets carrying superoxide dismutase transgenes were developed in order to investigate the possibility of enhancing their resistance to oxidative stress. Binary T-DNA vectors carrying the chloroplastic and cytosolic superoxide dismutase genes from tomato, were used for Agrobacterium-mediated transformation of sugarbeet petioles. The transgenic plants were subjected to treatments known to cause oxidative stress, such as the herbicide methyl viologen and a natural photosensitizer toxin produced by the fungus Cercospora beticola, namely cercosporin. The transgenic plants exhibited increased tolerance to methyl viologen, to pure cercosporin, as well as to leaf infection with the fungus C. beticola.     

Nitrogen status dependent oxidative stress tolerance conferred by overexpression of MnSOD and FeSOD proteins in Anabaena sp. strain PCC7120

The heterocystous nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120 displayed two superoxide dismutase (SOD) activities, namely FeSOD and MnSOD. Prolonged exposure of Anabaena PCC7120 cells to methyl viologen mediated oxidative stress resulted in loss of both SOD activities and induced cell lysis. The two SOD proteins were individually overexpressed constitutively in Anabaena PCC7120, by genetic manipulation. Under nitrogen-fixing conditions, overexpression of MnSOD (sodA) enhanced oxidative stress tolerance, while FeSOD (sodB) overexpression was detrimental. Under nitrogen supplemented conditions, overexpression of either SOD protein, especially FeSOD, conferred significant tolerance against oxidative stress. The results demonstrate a nitrogen status-dependent protective role of individual superoxide dismutases in Anabaena PCC7120 during oxidative stress.     

Differences in the activity and concentration of elements of the antioxidant system in different layers of Brassica pekinensis head

Differences in the activity of superoxide dismutase, catalase (CAT) and ascorbate peroxidase (APX) as well as in the concentration of ascorbate, tocopherol and hydrogen peroxide (H?O?) were found in leaves from different layers of the Chinese cabbage (Brassica pekinensis (Lour.) Rupr.) head. The youngest chlorophyll-deficient leaves from the most inner layers of the cabbage head were characterized by a high concentration of ascorbate, high activity of iron superoxide dismutase (FeSOD), cooper/zinc superoxide dismutase (Cu/ZnSOD) and a low content of H?O?. On the other hand, activity of CAT, manganese superoxide dismutase (MnSOD) and APX and tocopherol content were highest in chlorophyll-rich leaves from outer parts. The results of this work are interesting from the human nutrition standpoint, as the measured antioxidants have beneficial effects on human health. They can also be utilized to improve storage conditions due to an unequivocal function of antioxidant molecules in maintaining postharvest quality of vegetables.     

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     

Salt stress induces up-regulation of an efficient chloroplast antioxidant system in the salt-tolerant wild tomato species Lycopersicon pennellii but not in the cultivated species

The response of the chloroplastic antioxidant system of the cultivated tomato Lycopersicon esculentum (Lem) and its wild salt-tolerant related species L. pennellii (Lpa) to NaCl stress was studied. An increase in H₂O₂ level and membrane lipid peroxidation was observed in chloroplasts of salt-stressed Lem. In contrast, a decrease in these indicators of oxidative stress characterized chloroplasts of salt-stressed Lpa plants. This differential response of Lem and Lpa to salinity, correlates with the activities of the antioxidative enzymes in their chloroplasts. Increased activities of total superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), glutathione-S-transferase (GST), phospholipid hydroperoxide glutathione peroxidase (PHGPX) and several isoforms of non-specific peroxidases (POD) were found in chloroplasts of salt-treated Lpa plants. In these chloroplasts, in contrast, activity of lipoxygenase (LOX) decreased while in those of salt-stressed Lem it increased. Although total SOD activity slightly increased in chloroplasts of salt-treated Lem plants, differentiation between SOD types revealed that only stromal Cu/ZnSOD activity increased. In contrast, in chloroplasts of salt-treated Lpa plants FeSOD activity increased while Cu/ZnSOD activity remained unchanged. These data indicate that salt-dependent oxidative stress and damage, suffered by Lem chloroplasts, was effectively alleviated in Lpa chloroplasts by the selective up-regulation of a set of antioxidative enzymes. Further support for the above idea was supplied by leaf discs experiments in which pre-exposure of Lpa plants to salt-treatment conferred cross-tolerance to paraquat-induced oxidative stress while increased oxidative damage by paraquat-treatment was found in salt-stressed Lem plants.     

Improved procedure for detection of superoxide dismutase isoforms in potato,Solanum tuberosum L.

Superoxide dismutase (SOD) in-gel activity assay with selective inhibitors (KCN and H₂O₂) is one of the most commonly used methods for identification of SOD isoform types, i.e., FeSOD, MnSOD or Cu/ZnSOD, and evaluation of oxidative stress response in plants. However, there are potential pitfalls that surround this assay, such as problem to detect isoforms with low activity, comigration of SOD isoforms or application of inappropriate inhibitor concentration. We propose an improved method based on the combination of in-gel analysis of SOD activity and native-PAGE immunoblotting for identification of isoforms and determination of SOD isoenzyme activity pattern in potato. Depending on cultivar and growing conditions, one MnSOD, 3 FeSOD and 5–6 Cu/ZnSOD isoforms were identified in potato leaves. The most important qualitative difference between ex vitro- and in vitro-grown plants was the presence of additional FeSOD and Cu/ZnSOD isoforms in plantlets grown in vitro. Compared with results of in-gel activity assay with selective inhibitors, new method allowed accurate identification of comigrating FeSOD and Cu/ZnSOD isoforms and two protein bands of ambiguous identities. Potato SODs were also characterized by SDS-PAGE immunoblotting and single MnSOD (23.6 kDa), three Cu/ZnSOD polypeptides (17.9, 17 and 16.3 kDa) and single FeSOD (25.1 kDa) polypeptide were detected in leaves of four examined cultivars. The difference in the number of FeSOD and Cu/ZnSOD isoforms/polypeptides between native-PAGE and SDS-PAGE immunoblots suggests that SOD proteins may have undergone post-translational modifications affecting protein mobility or existence of isoforms that differ from each other in total protein charge, but not in molecular weight.     

转铜/锌超氧化物歧化酶和抗坏血酸过氧化物酶基因马铃薯的耐氧化和耐盐性研究

高盐等逆境可以加剧植物体内活性氧的产生,进而引起植物细胞死亡。为开发抗逆境作物,以置于氧化诱导型启动子下定位于叶绿体的转铜/锌超氧化物歧化酶（Cu/ZnSOD）和抗坏血酸过氧化物酶基因(APX)马铃薯为材料,研究了其对MV和 NaCl所引起的氧化胁迫的耐受性。结果表明, MV胁迫下,转基因马铃薯叶片膜的相对电导率明显低于对照; NaCl胁迫下,其叶绿素含量高于对照。 在含NaCl 的培养基上,转基因幼苗生根率明显大于对照。另外,NaCl胁迫下转基因马铃薯叶片的SOD和APX酶活性显著高于对照,与其耐盐性的提高相一致。这些研究表明,转入Cu/ZnSOD和APX基因的马铃薯清除活性氧的能力增强,抗逆性得到提高。本实验采用氧化诱导型启动子调控下的SOD和APX两个基因协同作用,使外源基因只有在逆境胁迫时才特异性表达,增强转基因植株的抗逆效果,为培育抗逆经济作物开阔了思路。     

Effects of water stress on antioxidant enzymes of leaves and nodules of transgenic alfalfa overexpressing superoxide dismutases

The antioxidant composition and relative water stress tolerance of nodulated alfalfa plants ( Medicago sativa L. ×  Sinorhizobium meliloti 102F78) of the elite genotype N4 and three derived transgenic lines have been studied in detail. These transgenic lines overproduced, respectively, Mn-containing superoxide dismutase (SOD) in the mitochondria of leaves and nodules, MnSOD in the chloroplasts, and FeSOD in the chloroplasts. In general for all lines, water stress caused moderate decreases in MnSOD and FeSOD activities in both leaves and nodules, but had distinct tissue-dependent effects on the activities of the peroxide-scavenging enzymes. During water stress, with a few exceptions, ascorbate peroxidase and catalase activities increased moderately in leaves but decreased in nodules. At mild water stress, transgenic lines showed, on average, 20% higher photosynthetic activity than the parental line, which suggests a superior tolerance of transgenic plants under these conditions. However, the untransformed and the transgenic plants performed similarly during moderate and severe water stress and recovery with respect to important markers of metabolic activity and of oxidative stress in leaves and nodules. We conclude that the base genotype used for transformation and the background SOD isozymic composition may have a profound effect on the relative tolerance of the transgenic lines to abiotic stress.     

Regulation of superoxide dismutase expression by copper availability

The most abundant copper proteins in green tissues are plastocyanin (PC) in thylakoids and copper/zinc superoxide dismutase (Cu/ZnSOD) of which the major isoforms are found in the cytosol and in the chloroplast stroma. An iron superoxide dismutase (FeSOD) can also be found in the stroma. The expression of superoxide dismutases (SODs) has been studied mainly in the context of abiotic stress. However, the availability of metal cofactors may also determine SOD expression patterns. Indeed, in Arabidopsis thaliana , Cu/ZnSOD enzymes were only expressed when copper was sufficient. This observation was made for plants grown on sucrose-containing tissue culture media and regulation of SOD expression by copper has not been tested for other species. To investigate the effect of copper on SOD expression, we used a hydroponic set-up in which plants grew without any evident stress symptoms. We observed that A. thaliana , Brassica juncea , Lycopersicum lycopersicum , Zea mays and Oryza sativa , downregulated Cu/ZnSOD in response to copper limitation. Under this condition, FeSOD expression was upregulated to replace Cu/ZnSOD in the stroma in all plants except Z. mays , in which FeSOD was not detectable. Copper limitation did not affect PC accumulation in any of the plants except Z. mays . Comparisons of leaf copper contents and SOD expression suggest that Cu/ZnSOD and FeSOD expression levels are good indicators of impending copper deficiency. Plants that downregulate Cu/ZnSOD and upregulate FeSOD under copper limitation can maintain superoxide scavenging and save copper for use in PC, which is essential for photosynthesis.     

Cloned prokaryotic iron superoxide dismutase protects yeast cells against oxidative stress depending on mitochondrial location

Superoxide dismutase (SOD) is considered to be the first line of defense against oxygen toxicity. It exists as a family of three metalloproteins with copper,zinc (Cu,ZnSOD), manganese (MnSOD), and iron (FeSOD) forms. In this work, we have targeted Escherichia coli FeSOD to the mitochondrial intermembrane space (IMS) of yeast cells deficient in mitochondrial MnSOD. Our results show that FeSOD in the IMS increases the growth rate of the cells growing in minimal medium in air but does not protect the MnSOD-deficient yeast cells when exposed to induced oxidative stress. Cloned FeSOD must be targeted to the mitochondrial matrix to protect the cells from both physiological and induced oxidative stress. This confirms that the superoxide radical is mainly generated on the matrix side of the inner mitochondrial membrane of yeast cells, without excluding its potential appearance in the mitochondrial IMS where its elimination by SOD is beneficial to the cells.