FeSOD Activity Research Articles

Protein Pattern and Fe‐Superoxide Dismutase Activity of Bean Plants under Sulphur Dioxide Stress

AbstractProtein synthesis and superoxide dismutase (SODs) activities were analysed in bean plants (Phaseolus vulgaris L., cv. Groffy) exposed to sulphur dioxide (SO2) (30, 60 or 90 nl/l SO2). After long‐term treatments (2, 4 or 7 days), sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of water‐soluble leaf proteins revealed the appearance of new polypeptides corresponding to 34.5 and 31 kDa. Quantitative differences between control and treated plants were observed for subunits of 50, 47, 42, 32, 30 and 28 kDa. In SO2‐fumigated plants, the activity patterns of four SOD‐isoforms, with a relative mobility (Rf) of 0.45 (MnSOD), 0.59 (Cu/ZnSOD), 0.80 (Cu/ZnSOD) and 0.85 (not identified), showed only small quantitative variations in comparison with unexposed plants. In control leaves the FeSOD isoenzyme was not detected, whereas its activity pattern (Rf=0.50) increased to a detectable level always after 2 days of fumigation with 30 nl/l SO2. It is proposed that the rise of FeSOD activity represents the initial activation of the enzymatic antioxidant system to counteract the superoxide anion generated during the radical‐initiated oxidation of SO2 in illuminated chloroplasts.

Differential responses of superoxide dismutase in freezing resistant Solanum curtilobum and freezing sensitive Solanum tuberosum subjected to oxidative and water stress

In photosynthetic tissues superoxide dismutase (SOD) plays an important role by scavenging the superoxide radical whose production is an usual reaction in chloroplast thylakoids. To test the differential response of SOD, two Andean potato species differing in frost resistance, Solanum curtilobum (frost resistant) and Solanum tuberosum (frost sensitive), were subjected to methyl viologen-mediated oxidative stress and polyethylene glycol (PEG)-induced water stress. A significant increment (approximately two-fold) in total SOD and FeSOD activity, which occupied about 50% of the total activity, was found when leaves of S. curtilobum were exposed to water stress. In contrast, the SOD activity in leaves of S. tuberosum remained unchanged. The exposure of leaves of S. curtilobum to oxidative stress increased total SOD and FeSOD activity by 350%. High correlation between SOD activity and the F v/ F m ratio under both PEG induced water stress and MV-mediated oxidative stress was observed. This suggests that SOD can protect PSII from superoxide generated by oxidative and water stress. The higher SOD activity could be an important mechanism to explain why some natives Andean potato like S. curtilobum are more resistant to abiotic stresses than S. tuberosum.

Characterization and mutagenesis of fur gene from Burkholderia pseudomallei

A homolog of the ferric uptake regulator gene ( fur) was isolated from Burkholderia pseudomallei ( Bp) by a reverse genetic technique. Sequencing of a 2.2 kb DNA fragment revealed an open reading frame with extensive homology to bacterial Fur proteins. The cloned gene encodes a 16 kDa protein that cross-reacts with a polyclonal anti- Escherichia coli Fur serum. The transcription start site was determined by the primer extension technique. Expression analysis of fur showed no increased fur mRNA levels in response to various stresses and iron conditions. A positive selection procedure involving the isolation of manganese-resistant mutants was used to isolate mutants that produce altered Fur proteins. Sequencing of a fur mutant revealed a nucleotide change (G to A) converting a conserved amino acid arginine-69 to histidine. The fur missense mutant produced an elevated level of siderophore that could be complemented by a multicopy plasmid carrying the Bp fur. Interestingly, Fur was found to play roles as a positive regulator of FeSOD and peroxidase. The mutant showed a decreased activity of FeSOD and peroxidase, which could be important in its pathogenicity and survival in macrophages.

Acclimation of beans to oxidative stress by treatment with sublethal iron levels

Summary The relationship between Fe 2+ -overloading enhanced antioxidative mechanism and protection from successive oxidative stress in plant cells was studied. The involvement of Fe 2+ in the reduction of superoxide (O 2 − ) and peroxide (H 2 O 2 ) to hydroxyl radical (OH) suggests that excess Fe leads to oxidative stress. An excess of free Fe was induced in Phaseolus vulgaris (cv. Pinto ) plants by soaking the roots in a 900 αmol/L Fe (III)-EDTA solution for 24h. As a result, Fe content in bean leaves increased from 160 mg Fe kg −1 dry weight to 530 mg Fe kg −1 dry weight. Oxidative stress responses were detected by monitoring changes in the activities and contents of a few components of the antioxidative mechanism. The Fe treatment resulted in increased activity of ascorbate peroxidase (APX, EC 1.11.1.11) from 5.3 to 18.2 αmol ascorbate g −1 fresh weight min −1 , increased ascorbate content from 6.6 to 10 mg g −1 fresh weight, and increased catalase (EC 1.11.1.6) and glutathione reductase (GR, EC 1.6.4.2) activities by 380% and 55%, respectively. Fe treatment induced the activity of Fe-SOD (SOD, EC 1.15.11) isozyme, which was not detected in the control bean leaves. The increase in the antioxidative mechanism resulted in acclimation of the Fe treated beans to subsequent methyl viologen treatment relative to control bean plants.

Iron-superoxide dismutase expression in transgenic alfalfa increases winter survival without a detectable increase in photosynthetic oxidative stress tolerance.

To determine whether overexpression of Fe-superoxide (SOD) dismutase would increase superoxide-scavenging capacity and thereby improve the winter survival of transgenic alfalfa (Medicago sativa L.) plants, two genotypes were transformed with the vector pEXSOD10, which contains a cDNA for Arabidopsis Fe-SOD with a chloroplast transit peptide and cauliflower mosaic virus 35S promoter. A novel Fe-SOD was detected by native PAGE in both greenhouse- and field-grown transgenic plants, but activity varied among independent transgenic plants. The increased Fe-SOD activity was associated with increased winter survival over 2 years in field trials, but not with oxidative stress tolerance as measured by resistance of leaves to methyl viologen, a superoxide generator. Total shoot dry matter production over 2 harvest years was not associated with Fe-SOD activity. There was no detectable difference in the pattern of primary freezing injury, as shown by vital staining, nor was there additional accumulation of carbohydrates in field-acclimated roots of the transgenic alfalfa plants. We did not detect any difference in growth of one transgenic plant with high Fe-SOD activity compared with a non-transgenic control. Therefore, the improvement in winter survival did not appear to be a consequence of improved oxidative stress tolerance associated with photosynthesis, nor was it a consequence of a change in primary freezing injury. We suggest that Fe-SOD overexpression reduced secondary injury symptoms and thereby enhanced recovery from stresses experienced during winter.

Low temperature‐induced changes in the distribution of H2O2 and antioxidants between the bundle sheath and mesophyll cells of maize leaves

The distribution of antioxidants between bundle sheath and mesophyll cells of maize leaves was analysed in plants grown at 20 degrees C, 18 degrees C and 15 degrees C. The purity of the isolated bundle sheath and mesophyll fractions was determined using compartment-specific marker enzymes. In plants grown at 15 degrees C, ascorbate peroxidase, CuZn-superoxide dismutase (CuZn-SOD) and monodehydroascorbate reductase activities were increased in the bundle sheath cells, and glutathione reductase, dehydroascorbate reductase and monodehydroascorbate reductase activities were enhanced in the mesophyll cells. SOD was absent from the mesophyll of plants grown at 20 degrees C but an Fe-SOD activity was found in the mesophyll of plants grown at 15 degrees C. Foliar Mn-SOD activities were decreased at 15 degrees C compared to 20 degrees C. Catalase was undetectable in the mesophyll extracts of plants grown at 15 degrees C. Ascorbate and glutathione contents were considerably higher in the mesophyll than the bundle sheath fractions of plants grown at 20 degrees C. The ratios of reduced to oxidized forms of these antioxidants were significantly decreased in the bundle sheath, but increased in the mesophyll of leaves grown at 15 degrees C. Foliar H2O2 accumulated at 15 degrees C compared to 20 degrees C. Most of the foliar H2O2 was localized in the mesophyll tissues at all growth temperatures. The differential distribution of antioxidants between leaf bundle sheath and mesophyll tissues, observed at 20 degrees C, is even more pronounced when plants are grown at 15 degrees C and may contribute to the extreme sensitivity of maize to low temperatures.

Increased Tolerance to Mn Deficiency in Transgenic Tobacco Overproducing Superoxide Dismutase

The effect of Mn deficiency on plant growth and activities of superoxide dismutase (SOD) was studied in hydroponically-grown seedlings of transgenic tobacco (Nicotiana tabacum L.) engineered to overexpress FeSOD in chloroplasts or MnSOD in chloroplasts or mitochondria. In comparison to the non-transgenic parental line, the activity of MnSOD in the lines overproducing MnSOD was 1.6-fold greater, and the activity of FeSOD in the FeSOD-overproducing lines was 3.2-fold greater, regardless of the Mn treatment (deficient or sufficient). The MnSOD activities decreased due to Mn deficiency, while activities of FeSOD and Cu/ZnSOD remained unaffected 25 d after transplanting (DAT). With an increased duration of the Mn deficiency stress (45 DAT), FeSOD activity decreased, and that of MnSOD continued to decrease, while Cu/ZnSOD activity simultaneously increased. Under Mn sufficiency, non-transgenic parental plants had greater shoot biomass than the transgenics; however, when subjected to Mn deficiency stress, non-transgenic parents suffered a proportionally greater growth reduction than transgenic lines. Thus, overproduction of MnSOD in chloroplasts may provide protection from oxidative stress caused by Mn deficiency.

Waterlogging Influences Plant Growth and Activities of Superoxide Dismutases in Narrow-leafed Lupin and Transgenic Tobacco Plants

Summary The effects of waterlogging on plant growth and activities of superoxide dismutase (SOD) forms were studied in seedlings of narrow-leafed lupin ( Lupinus angustifolius L.) and transgenic tobacco ( Nicotiana tabacum L.) plants engineered to overproduce MnSOD or FeSOD in chloroplasts. Waterlogging for 2 days decreased shoot elongation rate by 57% in lupins, and with prolonged waterlogging (4 days) soluble protein concentration declined significantly; however, shoot dry weight was not affected during the whole course of waterlogging stress. After 2 days of recovery (by draining excess water in treatment pots), shoot dry weight was reduced slightly but significantly, while shoot elongation rate and protein concentration continued to decline. The activities of FeSOD and Cu/ZnSOD increased constantly during waterlogging and recovery periods, while those of total SOD and MnSOD decreased initially and increased afterwards. Growth of transgenic tobacco was not affected after 2 days of waterlogging, while non-transgenic parental line showed decreased growth at this stage. Following 2 days of recovery, growth was significantly reduced in all lines, but transgenics suffered proportionally smaller growth reduction than did non-transgenics. This result corresponded with the higher activity of MnSOD or FeSOD in transgenic lines compared to the non-transgenic parent. It is concluded that overproduction of FeSOD or MnSOD in transgenic tobacco enables plants to better tolerate waterlogging-induced oxidative stress and maintain growth rate under stress conditions compared to non-transgenic parent; therefore, engineering lupins for overproduction of FeSOD or MnSOD to increase tolerance to waterlogging stress is warranted.

Drought and salinity differentially influence activities of superoxide dismutases in narrow-leafed lupins

Effects of drought and salinity on growth and activities of superoxide dismutase (SOD) forms were studied in narrow-leafed lupins ( Lupinus angustifolius L.). Shoot dry weight and the elongation rate were depressed after 3 days of drought, with leaf water potential dropping to −1.64 MPa. Activity of total SOD increased by 21%, Cu/ZnSOD by 33% and FeSOD by 50% after 2 days of withholding water; further increases were noted with an increase in severity of drought stress. Two days after resupplying water, leaf water potential and the activity of Cu/ZnSOD returned to the control level, but the FeSOD activity remained high. The activity of MnSOD was unaffected by drought. Root fresh weight and the shoot elongation rate were not affected up to 50 mM NaCl, but were reduced at 100 mM NaCl after 6 days, with leaf water and osmotic potentials being −1.4 and −1.8 MPa, respectively. Concentrations of Na + and Cl − in leaves increased linearly with an increase in NaCl concentration in the growth medium. Salinity stress enhanced activity of Cu/ZnSOD by 145% without influencing the activity of other SOD forms. Drought and salinity differentially influence activity of SOD forms in narrow-leafed lupins, indicating that different mechanisms may be involved in oxidative stress injury caused by drought and salinity.

Response of antioxidant systems and leaf water relations to NaCl stress in pea plants.

A pea (Pisum sativum cv. Puget) cultivar was grown on a medium containing different NaCl concentrations (0-160 mol m-3 ) in order to study the effects of salt stress on leaf water relations and on the activity of antioxidant enzymes. NaCl stress caused a rapid decline in chlorophyll content. Both leaf water (ψl ) and osmotic potentials (ψs ) decreased progressively with the severity of the stress (from 90-160 mol m-3 NaCl) whereas leaf turgor pressure (ψp ) increased in treated plants. Pea leaves contained an iron-containing superoxide dismutase (Fe-SOD) isozyme in chloroplasts alongside a copper-zinc-containing (CuZn-SOD) form (CuZn-SOD II). The lowest NaCl concentration (70 mol m-3 ) had no effect on the activity of these antioxidant enzymes while higher concentrations (110-130 mol m-3 ) enhanced the activity of cytosolic CuZn-SOD I and chloroplastic CuZn-SOD II as well as that of mitochondrial and/or peroxisomal manganese-containing superoxide dismutase (Mn-SOD). These inductions were matched by increases in the activity of ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR). The increased activities coincided with decreased stomatal conductance and were unaffected by the severity of stress except in the case of CuZn-SOD II which fell to control values under the highest stress conditions (140-160 mol m-3 NaCl), when a concomitant increase in chloroplastic Fe-SOD activity was observed. Glutathione reductase (GR) and dehydroascorbate reductase (DHAR) activities were only induced under severe NaCl stress (130-160 mol m-3 ) and were accompanied by losses in the ascorbate and glutathione pools, lower ASC/DHA and GSH/GSSG ratios and increases in GSSG. Electron microscopy showed that the thylakoidal structure of the chloroplasts became disorganized and their starch content decreased in plants treated with 160 mol m-3 NaCl. Overall, the results suggest that salt stress is accompanied by oxidative stress, perhaps at the cell compartment level. The capacity of Puget cultivar to ensure cell turgor and to enhance the activity of enzymes involved in the defence against oxidative stress seems to be important in determining adaptation to moderate NaCl stress conditions. In plants exposed to severe NaCl stress (130-160 mol m-3 ) it seems that such resistance to oxidative stress is overcome, which might contribute to the deleterious effects of salt and significant growth reduction in these conditions.

Molecular cloning and characterization of a cDNA for an iron-superoxide dismutase in rice (Oryza sativa L.).

We have isolated a cDNA encoding Fe-SOD from rice (Oryza sativa L.). The deduced amino acid sequence consists of a polypeptide with 255 amino acids, including a putative transit peptide (40 a.a.) in amino-terminal residues. This sequence is similar to the known plant Fe-SODs but not classified in the group of known Fe-SODs. The metal analysis and SOD assays of the partial purified recombinant protein expressed in E. coli showed that this cDNA encodes an iron-containing SOD. However this SOD activity was not inhibited by the treatment with hydrogen peroxide, which was expected to inhibit known Fe-SOD activity. mRNA of rice Fe-SOD was detected in all vegetative tissues examined, being especially abundant in calli, and strongly increased by light induction. These results suggested that this cDNA encodes rice Fe-SOD, which is apparently distinct from known plant Fe-SODs.

Overproduction of Arabidopsis thaliana FeSOD confers oxidative stress tolerance to transgenic maize.

Transgenic maize (Zea mays L.) plants have been generated by particle gun bombardment that overproduce an Arabidopsis thaliana iron superoxide dismutase (FeSOD). To target this enzyme into chloroplasts, the mature Fesod coding sequence was fused to a chloroplast transit peptide from a pea ribulose-1,5-bisphosphate carboxylase gene. Expression of the chimeric gene was driven by the CaMV 35S promoter. Growth characteristics and in vitro oxidative stress tolerance of transgenic lines grown in control and chilling temperatures were evaluated. The transgenic line with the highest transgenic FeSOD activities had enhanced tolerance toward methyl viologen and had increased growth rates.

Micronutrient deficiency changes activities of superoxide dismutase and ascorbate peroxidase in tobacco plants

The effects of copper (Cu), zinc (Zn), and manganese (Mn) deficiencies on the activities of Superoxide dismutase (SOD) and ascorbate peroxidase (APx) were investigated in seedlings of Nicotiana tabacum cv. Xanthin. After germinating in sand for 7 days, plants were transplanted to solution culture with all nutrients or without Cu, Zn, or Mn. Plants grown without Mn developed visual deficiency symptoms 15 days after transplanting (DAT), and those grown without Zn showed Zn deficiency symptoms 26 DAT. In contrast, plants grown without Cu showed only minor symptoms of Cu deficiency at 33 DAT. When harvested 41 DAT, ‐Mn plants suffered the largest, and ‐Cu the smallest reduction in shoot dry weight compared to plants supplied with all nutrients. Nutrient analysis in leaf tissue showed a considerable decrease in concentration of nutrients not supplied to plants. Total SOD, MnSOD+FeSOD, Cu/Zn SOD, and APx activities declined drastically in ‐Mn and ‐Zn plants, while total SOD, MnSOD+FeSOD and APx activities in ‐Cu plants remained significantly higher than those of control plants. It was concluded that micronutrient deficiencies (Cu, Zn, and Mn) can alter the activities of antioxidative enzymes in tobacco plants depending on the severity and the type of the deficiency stress.

Prokaryotic iron superoxide dismutase replaces cytosolic copper, zinc superoxide dismutase in protecting yeast cells against oxidative stress.

The iron superoxide dismutase (FeSOD) gene of Escherichia coli was cloned in Saccharomyces cerevisiae cells deficient in copper,zinc superoxide dismutase (Cu,ZnSOD). FeSOD replaced Cu,ZnSOD in protecting the yeast cells against oxidative stress. In the recombinant strains the FeSOD gene, which was under the transcriptional control of the yeast phosphoglycerate kinase gene promoter, was functionally expressed at two different levels on episomal and centromeric plasmids. Despite suppression of methionine and lysine auxotrophy, the higher level of FeSOD activity was more beneficial to growth of the mutant yeast cells only when these were exposed to higher levels of oxidative stress induced by paraquat or 100% oxygen. In the presence of paraquat, there was a novel stimulation of FeSOD activity. This was associated with a marked increase in catalase activity, and a decrease in glutathione reductase activity.

Expression ofsodCpandsodBgenes inNicotiana tabacum: effects of light and copper excess

Light and copper are necessary for the biogenesis and normal function of chloroplasts, but are also known to initiate the production of active oxygen species. Tobacco [Nicotiana tabacum L.) contains two plastidlocated superoxide dismutases, Cu/Zn-SOD and Fe-SOD, encoded by the nuclear genes sodCp and sodB, respectively. The expression of both genes is developmentally regulated and influenced by oxidative stress. Here, the effects of light and copper excess on the expression of these two genes were analysed in seedlings and in leaves of non-flowering tobacco plants. The accumulation of both transcripts was induced by light in seedlings and in young leaves of mature plants, but changes in enzymatic activities were only observed in seedlings. Copper excess was induced in two experimental systems. In the first, axenically-grown plants were transferred to the medium containing excess copper and grown for 10 d. Excess copper induced an increase of sodCp mRNA level, but not of enzymatic activity, whereas Fe-SOD activity and sodB transcript level decreased. In contrast, after transfer of hydroponicall y grown plants to a copper excess medium and growth for an additional 3 d, sodB mRNA and enzyme levels increased, whereas the sodCp transcript level was reduced. Changes in the expression levels of both genes were more pronounced in young leaves. The effects of light and copper on the expression of plastid-locat ed SODs are discussed.

Periplasmic copper-zinc superoxide dismutase of Legionella pneumophila: role in stationary-phase survival.

Copper-zinc superoxide dismutases (CuZnSODs) are infrequently found in bacteria although widespread in eukaryotes. Legionella pneumophila, the causative organism of Legionnaires' disease, is one of a small number of bacterial species that contain a CuZnSOD, residing in the periplasm, in addition to an iron SOD (FeSOD) in their cytoplasm. To investigate CuZnSOD function, we purified the enzyme from wild-type L. pneumophila, obtained amino acid sequence data from isolated peptides, cloned and sequenced the gene from a L. pneumophila library, and then constructed and characterized a CuZnSOD null mutant. In contrast to the cytoplasmic FeSOD, the CuZnSOD of L. pneumophila is not essential for viability. However, CuZnSOD is critical for survival during the stationary phase of growth. The CuZnSOD null mutant survived 10(4)- to 10(6)-fold less than wild-type L. pneumophila. In wild-type L. pneumophila, the specific activity of CuZnSOD increased during the transition from exponential to stationary-phase growth while the FeSOD activity was constant. These data support a role of periplasmic CuZnSOD in survival of L. pneumophila during stationary phase. Since L. pneumophila survives extensive periods of dormancy between growth within hosts. CuZnSOD may contribute to the ability of this bacterium to be a pathogen. In exponential phase, wild-type and CuZnSOD null strains grew with comparable doubling times. In cultured HL-60 and THP-1 macrophage-like cell lines and in primary cultures of human monocytes, multiplication of the CuZnSOD null mutant was comparable to that of wild type. This indicated that CuZnSOD is not essential for intracellular growth within macrophages or for killing of macrophages in those systems.

Function and stationary-phase induction of periplasmic copper-zinc superoxide dismutase and catalase/peroxidase in Caulobacter crescentus.

Although cytosolic superoxide dismutases (SODs) are widely distributed among bacteria, only a small number of species contain a periplasmic SOD. One of these is Caulobacter crescentus, which has a copper-zinc SOD (CuZnSOD) in the periplasm and an iron SOD (FeSOD) in the cytosol. The function of periplasmic CuZnSOD was studied by characterizing a mutant of C. crescentus with an insertionally inactivated CuZnSOD gene. Wild-type and mutant strains showed identical tolerance to intracellular superoxide. However, in response to extracellular superoxide, the presence of periplasmic CuZnSOD increased survival by as much as 20-fold. This is the first demonstration that periplasmic SOD defends against external superoxide of environmental origin. This result has implications for those bacterial pathogens that contain a CuZnSOD. C. crescentus was shown to contain a single catalase/peroxidase which, like Escherichia coli KatG catalase/peroxidase, is present in both the periplasmic and cytoplasmic fractions. The growth stage dependence of C. crescentus catalase/peroxidase and SOD activity was studied. Although FeSOD activity was identical in exponential- and stationary-phase cultures, CuZnSOD was induced nearly 4-fold in stationary phase and the catalase/peroxidase was induced nearly 100-fold. Induction of antioxidant enzymes in the periplasm of C. crescentus appears to be an important attribute of the stationary-phase response and may be a useful tool for studying its regulation.

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.

Sensitivity of Superoxide Dismutase Transcript Levels and Activities to Oxidative Stress Is Lower in Mature-Senescent Than in Young Barley Leaves.

Antioxidant enzyme activities are inducible by oxidative stress and decrease during senescence. To determine if the age-dependent decrease of superoxide dismutase (SOD) activities is due to decreased sensitivity to oxidative stress, we have investigated the changes in steady-state levels of transcripts and activities of mitochondrial Mn-SOD (SOD1), chloroplastic Fe-SOD (SOD2), and cytoplasmic Cu-Zn-SOD (SOD3) in young and mature-senescent detached barley (Hordeum vulgare L.) leaves in response to incubation in darkness, growth light (20 W m-2), and photooxidative stress conditions (100 W m-2 with 21 or 100% O2). For a comparison, changes in the mRNA for ribulose bisphosphate carboxylase were also measured. After leaf detachment, the abundance of all three SOD mRNAs increased, then decreased and eventually stabilized after 6 h of incubation. After 20 h of incubation under darkness SOD transcripts decreased in both young and mature-senescent leaves. While under strong photooxidative stress the levels of the three SOD transcripts significantly increased in young leaves; in mature-senescent leaves SOD2 and, to lesser extent, SOD1 and SOD3 transcripts decreased. Generally, SOD activity changes were similar to those of mRNAs. It is proposed that oxidative damage during senescence could be favored by the inability of senescing leaves to modulate the steady-state level of SOD mRNA, and probably those of other antioxidant enzymes, concomitant with the rate of oxyradical formation.

The manganese superoxide dismutase of Escherichia coli K-12 associates with DNA.

Superoxide dismutases (SODs) are vital components in the resistance of aerobic organisms to the toxicity of oxygen. Escherichia coli contains two highly homologous cytoplasmic SODs, a manganese- and an iron-containing enzyme (MnSOD, FeSOD). We previously demonstrated that MnSOD and FeSOD have different physiological functions and that MnSOD is more effective in preventing oxidative damage to DNA. In this report, purified E. coli MnSOD was shown to bind nonspecifically to DNA by electrophoretic mobility shift assay and nitrocellulose-filter binding methodologies. From electrophoretic mobility shift assay, the equilibrium dissociation constants for interaction with a variety of double-stranded and single-stranded oligonucleotides ranged from 1.5 +/- 0.2 to 8.4 +/- 1.3 microM at 20 degrees C. This range of concentrations corresponds to MnSOD concentrations in aerobically grown E. coli. In vivo binding of MnSOD to DNA was supported by colocalization of MnSOD and the E. coli nucleoid in immunoelectron microscopy. Both MnSOD and DNA were inhomogeneously distributed in the cytosol, the concentration of each being higher in the center of the cell and relatively low near the inner membrane. In contrast, there was no evidence for physiologically relevant interaction of FeSOD with DNA. Binding to DNA in vitro was weak, Kd > 40-220 microM, concentrations 7-40 times higher than found in vivo. In addition, the cytoplasmic distribution of FeSOD did not correlate with DNA. FeSOD concentration was higher near the inner membrane and lower in the center of the cytosol. These results demonstrate that E. coli MnSOD associates with DNA in vitro and in vivo. Combined with prior data demonstrating that MnSOD preferentially protects DNA in vivo while an equal enzymatic activity of FeSOD does not (Hopkin, K. A., Papazian, M. A., and Steinman, H. M. (1992) J. Biol. Chem. 267, 24253-24258), our data suggest that E. coli MnSOD acts as a "tethered antioxidant"; association of MnSOD with DNA localizes dismutase activity near a target of oxidative stress and increases protection of DNA from oxidative damage. This model has implications for the therapeutic use of SODs as antioxidants.