Scavenger Enzyme Activities in Subcellular Fractions of White Clover (Trifolium repens L.) under PEG-induced Water Stress

Scavenger enzyme activities in subcellular fractions under polyethylene glycol (PEG)-induced water stress in white clover (Trifolium repens L.) were studied. Water stress decreased ascorbic acid (AA) content and catalase (CAT) activity and increased the contents of hydrogen peroxide (H₂O₂), thiobarbituric acid reactive substances (TBARS) (measure of lipid peroxidation), and activities of superoxide dismutase (SOD), its various isozymes, ascorbate peroxidase (APOX), and glutathione reductase (GR) in cellular cytosol, chloroplasts, mitochondria, and peroxisomes of Trifolium repens leaves. In both the PEG-treated plants and the control, chloroplastic fractions showed the highest total SOD, APOX, and GR activities, followed by mitochondrial fractions in the case of total SOD and GR activities, whereas cytosolic fractions had the second greatest APOX activity. However, CAT activity was the highest in peroxisomes, followed by the cytosol, mitochondria, and chloroplasts in decreasing order. Although Mn-SOD activity was highest in mitochondrial fractions, residual activity was also observed in cytosolic fractions. Cu/Zn-SOD and Fe-SOD were observed in all subcellular fractions; however, the activities were the highest in chloroplastic fractions for both isoforms. Total Cu/Zn-SOD activity, the sum of activities observed in all fractions, was higher than other SOD isoforms. These results suggest that cytosolic and chloroplastic APOX, chloroplastic and mitochondrial GR, mitochondrial Mn-SOD, cytosolic and chloroplastic Cu/Zn-SOD, and chloroplastic Fe-SOD are the major scavenger enzymes, whereas cellular CAT may play a minor role in scavenging of O₂⁻ and H₂O₂ produced under PEG-induced water stress in Trifolium repens.     

Metabolic bases for differences in sensitivity of two pea cultivars to sulfur dioxide

An oxidative chain reaction of sulfite initiated by the superoxide ion produced in the Mehler reaction has been implicated in the damage of plants exposed to sulfur dioxide. The toxicity of SO₂ may be alleviated by free radical scavenging systems acting to terminate this chain reaction. Hence, the relative sensitivity of plants to SO₂ toxicity could depend on differences in the responses of the levels of antioxidant metabolites and enzymes. The effect of SO₂ exposure on glutathione and ascorbic acid contents, glutathione reductase, and superoxide dismutase activities was assayed in two cultivars (Progress, Nugget) of pea (Pisum sativum L.) in which apparent photosynthesis showed a differential sensitivity to 0.8 microliter per liter SO₂ (R. Alscher, J. L. Bower, W. Zipfel [1987] J Exp Bot 38:99-108). Total and reduced glutathione increased more rapidly and to a greater extent in the insensitive Progress than in the sensitive Nugget, as did glutathione reductase activities. Superoxide dismutase activities increased significantly in Progress, whereas no such change was observed in Nugget as a result of SO₂ exposure. This increase in superoxide dismutase activity was observed at 210 minutes after 0.8 microliter per liter SO₂ concentration had been reached, in marked contrast to the increases in reduced glutathione content and glutathione reductase activity, which were apparent at the 90 minute time point. These data suggest that one basis for the relative insensitivity of the apparent photosynthesis of the pea cultivar Progress to SO₂ is the enhanced response of glutathione reductase, superoxide dismutase activities, and glutathione content.     

Exposure to anoxia of the clam, Chamelea gallina: II: Modulation of superoxide dismutase activity and expression in haemocytes

In order to investigate the influence of anoxic stress on haemocyte immune response, specimens of Chamelea gallina were exposed to 24 and 48 h anoxia. To evaluate recovery capacity, clams were maintained, at the end of the anoxic phase, for 24 h in reoxygenated seawater. In this paper, activity and expression of the antioxidant enzyme superoxide dismutase (SOD) were studied on haemocyte lysate and haemolymph. Reported results have shown that the anoxic stress changed strongly the response of C. gallina blood cells. Indeed, at the end of the anoxic phase in both experiments (24 and 48 h of anoxia exposure), SOD activity in haemocyte lysate decreased significantly with respect to the control, likely because of a decreasing superoxide anion generation in anoxia. Expression analyses were coherent with activity values.In the first experiment (24 h anoxia), reoxygenation determined an increase in activity of both Cu/Zn-SOD and Mn-SOD, but with values that remained significantly lower than those of the controls. It seems that after the applied anoxic stress, 24 h of recovery is not sufficient to restore pre-anoxic conditions. In the second experiment (48 h anoxia), SOD isoforms showed a different response during the recovery of animals. Cu/Zn-SOD activity dropped below the values showed by haemocytes of anoxic bivalves, while Mn-SOD activity values exceeded significantly those of controls. The different haemocyte response could be probably due to a further stress suffered by the clams because of a massive spawning during the reoxygenation phase. Therefore, the high values of activity shown by Mn-SOD during the recovery are likely to be due to the high inducibility of this isoform.In Cu/Zn-SOD expression analyses, two immunoreactive bands were highlighted in both experiments. The former (apparent molecular weight of 16 kDa) corresponds to the expression of SOD1 and the latter (apparent molecular weight of 28-30 kDa) could be attributed to EC-SOD (SOD3), a Cu/Zn-SOD isoform located in extracellular ambient and identified both in vertebrates and invertebrates. The strong SOD3 expression during anoxia exposure and the further spawning stress (second experiment) testified its inducibility in C. gallina haemocytes and haemolymph in response to stressful conditions.     

Characterization of cDNAs encoding CuZn-superoxide dismutases in Scots pine

A Scots pine (Pinus sylvestris L.) cDNA library was screened with two heterologous cDNA probes (P31 and T10) encoding cytosolic and chloroplastic superoxide dismutases (SOD) from tomato. Several positive clones for cytosolic and chloroplastic superoxide dismutases were isolated, subcloned, mapped and sequenced. One of the cDNA clones (PS3) had a full-length open reading frame of 465 bp corresponding to 154 amino acid residues and showed approximately 85% homology with the amino acid sequences of angiosperm cytosolic SOD counterparts. Another cDNA clone (PST13) was incomplete, but encoded a putative protein with 93% homology to pea and tomato chloroplastic superoxide dismutase. The derived amino acid sequence from both cDNA clones matched the corresponding N-terminal amino acid sequence of the purified mature SOD isozymes. Northern blot hybridizations showed that, cytosolic and chloroplastic CuZn-SOD are expressed at different levels in Scots pine organs. Sequence data and Southern blot hybridization confirm that CuZn-SODs in Scots pine belong to a multigene family. The results are discussed in relation to earlier observations of CuZn-SODs in plants.     

Unusual location and characterization of Cu/Zn-containing superoxide dismutase from filamentous fungus Humicola lutea

The present study aims to provide new information about the unusual location of Cu/Zn-superoxide dismutase (Cu/Zn-SOD) in lower eukaryotes such as filamentous fungi. Humicola lutea, a high producer of SOD was used as a model system. Subcellular fractions [cytosol, mitochondrial matrix, and intermembrane space (IMS)] were isolated and tested for purity using activity measurements of typical marker enzymes. Evidence, based on electrophoretic mobility, sensitivity to KCN and H₂O₂ and immunoblot analysis supports the existence of Cu/Zn-SOD in mitochondrial IMS, and the Mn-SOD in the matrix. Enzyme activity is almost equally partitioned between both the compartments, thus suggesting that the intermembrane space could be one of the major sites of exposure to superoxide anion radicals. The mitochondrial Cu/Zn-SOD was purified and compared with the previously published cytosolic enzyme. They have identical molecular mass, cyanide- and H₂O₂-sensitivity, N-terminal amino acid sequence, glycosylation sites and carbohydrate composition. The H. lutea mitochondrial Cu/Zn-SOD is the first identified naturally glycosylated enzyme, isolated from IMS. These findings suggest that the same Cu/Zn-SOD exists in both the mitochondrial IMS and cytosol. Ekaterina Krumova and Alexander Dolashki equally contributed to this work.     

Excess Copper Induces A Cytosolic Cu,Zn-Superoxide Dismutase in Soybean Root

The induction of several proteins in soybean roots in responseto copper was investigated. The major Cu²⁺-induced protein of16 kDa was purified by SDS-PAGE and 2D-PAGE. This Cu²⁺-inducedprotein cross-reacted with antibodies against cytosolic Cu,Zn-SODand the sequence of 40 amino-terminal residues of this proteinwas found to be 70% and 62.5% homologous to the sequences ofcytosolic Cu,Zn-SOD from rice and tomato, respectively. An assayof enzymatic activity revealed that SOD activity of Cu²⁺-treatedsoybean roots was more than twice that in roots of non-treatedcontrol plants. Thus, treatment with copper of soybean roots appears to inducean increase in SOD activity via the synthesis of cytosolic Cu,Zn-SOD.The induction of SOD by such treatment may be the result eitherof a direct effect of copper on the gene for SOD or of an indirecteffect via an increase in levels of O₂^– (Received July 12, 1991; Accepted January 22, 1992)     

鉴别超氧化物歧化酶类型的定位染色法

介绍一种鉴别SOD类型的方法─—聚丙烯酰胺凝胶电泳的定位染色法. 由于不同类型的SOD对抑制剂的表现各异, 电泳后的凝胶经不同的抑制剂处理, 染色, 结果展示在凝胶上, CuZn-SOD酶带在H₂O₂或CN^-的作用下消失, Mn-SOD在CHCl₃-CH₃OH作用下消失, Fe-SOD在H₂O₂或. CHCl₃-CH₃CH₂OH作用下失活, 从酶带消失或存活的情况, 可以判断SOD的类型.     

Cloning and characterization of a cDNA encoding the cytosolic copper/zinc-superoxide dismutase from sweet potato tuberous root

A full-length cDNA clone encoding a putative copper/zinc-superoxide dismutase (SOD) of sweet potato, Ipomoea batatas (L.) Lam. cv Tainong 57, was isolated from a cDNA library constructed in gt10 from tuber root mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 152 amino acid residues. The deduced amino acid sequence showed higher homology (78–86%) with the sequence of the cytosolic SOD than that of the chloroplast SOD from other plant species. The residues required for coordinating copper and zinc are conserved as they are among all reported Cu/Zn-SOD sequences. In addition, it lacks recognizable plastic or mitochondrial targeting sequences. These data suggest that the isolated sweet potato clone encodes a cytosolic Cu/Zn-SOD.     

Effects of Cadmium on Antioxidant Enzyme Activities in Sugar Cane

Sugar cane (Saccharum officinarum L. cv. Copersucar SP80-3280) seedlings were grown in nutrient solution with varying concentrations (0, 2 and 5 mM) of cadmium chloride for 96 h. Leaves were analysed for catalase (CAT), glutathione reductase (GR) and superoxide dismutase (SOD) activities. Although a clear effect of CdCl₂ on plant growth was observed, the activity of SOD was not altered significantly. However, the CAT activity decreased as the concentration of CdCl₂ increased. GR exhibits a significant increase in activity at 2 and 5 mM CdCl₂. CAT and SOD isoenzymes were further characterised by analysis in non-denaturing PAGE. Activity staining for SOD revealed up to seven isoenzymes in untreated control and 2 mM CdCl₂ treated plants, corresponding to Cu/Zn-SOD isoenzymes. At 5 mM CdCl₂, only six Cu/Zn-SOD isoenzymes were observed. No Fe-SOD and Mn-SOD isoenzymes were detected. For CAT, one band of activity was observed.     

Subcellular localization and stress responses of superoxide dismutase isoforms from leaves in the C3-CAM intermediate halophyte Mesembryanthemum crystallinum L.

BSA, bovine serum albumin
CAM, Crassulacean acid metabolism
DTT, dithiothreitol
EDTA, ethylenediaminetetraacetic acid
FPLCfast protein liquid chromatography
HEPES, N-(2-hydroxyethyl)piperazine-?-(ethanesulphonic acid)
ME, β-mercaptoethanol
NBT, nitro blue tetrazolium
PAGE, polyacrylamide gel electrophoresis
SDS, sodium dodecyl sulphate
SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis
Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39)
SOD, superoxide dismutase (EC 1.15.1.1)
TEMED, N,N,?,?-tetramethylethylenediamine
Tris, Tris (hydroxymethyl) aminomethane
Tricine, N-Tris(hydroxymethyl)methylglycine

Treatment of Mesembryanthemum crystallinum for several days with 0·4 kmol m^–3 NaCl in the root medium, in parallel to an increase of the cell sap osmolarity enhances activity of important antioxidative enzymes, such as superoxide dismutases (SODs). M. crystallinum is equipped with three SOD isoforms. These isoforms were identified as Mn-, Fe-, and Cu/Zn-SODs, respectively. Mn-SOD was found in the mitochondrial fraction, Fe-SOD in the chloroplast fraction, and Cu/Zn-SOD is probably localized in the cytosol. The Fe-SOD found in M. crystallinum is the first iron-containing SOD enzyme to be characterized in the plant family Aizoaceae. Salt treatment increases the activity of this isoform earlier than the other SODs. Molecular masses of SOD isoforms were determined as 82, 48 and 34 kDa for Mn-, Fe-, Cu/Zn-SODs, respectively. Native Mn-SOD seems to be a tetramer, while Fe-SOD and Cu/Zn-SOD are dimers. All SOD isoforms show high thermal stability. Mn-SOD is active even after short heating at 90 °C and Fe-SOD at 70 °C. Moreover, high concentrations of β-mercaptoethanol used as a reducing agent did not destroy the function of all isoforms. With the salinity treatment in M. crystallinum, Crassulacean acid metabolism (CAM) is induced. Build-up of large stationary O₂ concentrations in the leaf air spaces is associated with the photosynthetic CO₂ reduction behind closed stomata in phase III of CAM. This illustrates why M. crystallinum may require higher antioxidative activities under NaCl stress and also explains earlier findings that CAM plants are more resistant than C₃ plants to environmental stress as imposed by, for example, SO₂ and O₃.     

Characterization of two copper/zinc superoxide dismutases (Cu/Zn-SODs) from the desert beetle Microdera punctipennis and their activities in protecting E. coli cells against cold

Superoxide dismutases (SODs) are crucial in scavenging reactive oxygen species (ROS); however, studies regarding SOD functions in insects under cold conditions are rare. In this paper, two novel Cu/Zn-SOD genes in the desert beetle Microdera punctipennis, an extracellular copper/zinc SOD (MpecCu/Zn-SOD) and an intracellular copper/zinc SOD (MpicCu/Zn-SOD), were identified and characterized. The results of quantitative real-time PCR showed that MpecCu/Zn-SOD expression was significantly up-regulated by 4 °C exposure for 0.5 h, but MpicCu/Zn-SOD was not. Superoxide anion radical (O₂•^-) content in beetles under 4 °C exposure for 0.5 h showed an initial sharp increase and fluctuated during the cold treatment period, which was consistent with the relative mRNA level of MpecCu/Zn-SOD. The total SOD activity in the beetle was negatively correlated to the O₂•^- content with a correlation coefficient of −0.437. An E. coli system was employed to study the function of each MpCu/Zn-SOD gene. The fusion proteins Trx-His-MpCu/Zn-SODs were over expressed in E. coli BL21 using pET32a vector, and identified by SDS-PAGE and Western blotting. The transformed bacteria BL21(pET32a-MpecCu/Zn-SOD) and BL21(pET32a-MpicCu/Zn-SOD) showed increased cold tolerance to −4 °C as well as increased SOD activity compared to the control BL21(pET32a). The relative conductivity and malondialdehyde content in the two MpCu/Zn-SODs transformed bacteria under −4 °C were significantly lower than the control BL21(pET32a). Furthermore, BL21(pET32a-MpecCu/Zn-SOD) had significantly higher SOD activity and cold tolerance than BL21(pET32a-MpicCu/Zn-SOD) under −4 °C treatment, and had lower conductivity than BL21(pET32a-MpicCu/Zn-SOD). In conclusion, low temperature led to the accumulation of O₂•^- in M. punctipennis, which stimulated the expression of extracellular MpCu/Zn-SOD gene and the increase of total SOD activity. E. coli overexpressing Trx-His-MpCu/Zn-SODs increased resistance to cold treatment-induced oxidative stress. Our findings will be helpful in further study of Cu/Zn-SOD genes in insect cold-tolerance.     

Mitochondria-derived Hydrogen Peroxide Selectively Enhances T Cell Receptor-initiated Signal Transduction

T cell receptor (TCR)-initiated signal transduction is reported to increase production of intracellular reactive oxygen species, such as superoxide (O₂^⨪) and hydrogen peroxide (H₂O₂), as second messengers. Although H₂O₂ can modulate signal transduction by inactivating protein phosphatases, the mechanism and the subcellular localization of intracellular H₂O₂ as a second messenger of the TCR are not known. The antioxidant enzyme superoxide dismutase (SOD) catalyzes the dismutation of highly reactive O₂^⨪ into H₂O₂ and thus acts as an intracellular generator of H₂O₂. As charged O₂^⨪ is unable to diffuse through intracellular membranes, cells express distinct SOD isoforms in the cytosol (Cu,Zn-SOD) and mitochondria (Mn-SOD), where they locally scavenge O₂^⨪ leading to production of H₂O₂. A 2-fold organelle-specific overexpression of either SOD in Jurkat T cell lines increases intracellular production of H₂O₂ but does not alter the levels of intracellular H₂O₂ scavenging enzymes such as catalase, membrane-bound peroxiredoxin1 (Prx1), and cytosolic Prx2. We report that overexpression of Mn-SOD enhances tyrosine phosphorylation of TCR-associated membrane proximal signal transduction molecules Lck, LAT, ZAP70, PLCγ1, and SLP76 within 1 min of TCR cross-linking. This increase in mitochondrial H₂O₂ specifically modulates MAPK signaling through the JNK/cJun pathway, whereas overexpressing Cu,Zn-SOD had no effect on any of these TCR-mediated signaling molecules. As mitochondria translocate to the immunological synapse during TCR activation, we hypothesize this translocation provides the effective concentration of H₂O₂ required to selectively modulate downstream signal transduction pathways.     

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.     

Activities of superoxide dismutase (SOD) isoforms during growth of <Emphasis Type="Italic">Scenedesmus</Emphasis> (chlorophyta) species and strains grown in batch-cultures

Growth of Scenedesmus species and strains, grown for 28 days in mineral BBM medium in batch-cultures, displayed sigmoidal kinetics that comprised a lag, exponential and declining growth phases. Total SOD activity in these autotrophically cultured organisms, which oscillated within 0.6 – 1.4 Umg protein⁻¹, was rather species-specific and only to some extent depended on the growth phase. Contrary, three S. obliquus strains: wild type 276-6, mutant with blocked PS I (strain 56.80) and mutant with blocked PS II (strain 57.80), cultured for 7 days on BBM medium supplemented with bacto-tryptone and yeast extract (BBM+) turned out to be time-dependent and to have several times higher total SOD activity than one obtained for Scenedesmus grown autotrophically. Regardless of the media composition, the phase of growth and studied organism, dominant isoforms of total SOD were together determined Fe- and Mn-SOD. Profiles of SOD isoforms, obtained after PAGE analysis of all autotrophically and exponentially growing organisms, revealed that one Mn-SOD and one Cu/Zn-SOD bands located on gels at the same position whereas location of three bands of Fe-SOD depended on the strain. This suggests the presence of two different groups of Fe-SODs in analyzed organisms. Identical SOD profiles found in two S. armatus strains (276-4a and 276-4d) and S. subspicatus correspond well with their taxonomic position. The SOD profile of S. armatus B1-76 distinctly differed from two other S. armatus strains but was identical to S. microspinal B1-76 and S. quadricauda G-15 despite the fact that there were significant growth rate differences between these three species. SODs profiles of S. acutus 437 and S. obliguus 453 were species-specific. In S. obliquus strains cultured on BBM+ medium, there are four SOD bands: one slightly visible band of Mn-SOD, two intensive bands of Fe-SOD and one band of Cu/Zn-SOD. The above finding suggests that antioxidant response of algae kept in batch-cultures differs according to medium composition and the SOD activity mainly restricted to chloroplasts.     

Enhancement of superoxide dismutase activity in the leaves of white clover (Trifolium repens L.) in response to polyethylene glycol-induced water stress

Effects of polyethylene glycol (PEG)-induced water stress on the activities of total leaf superoxide dismutase (SOD) and chloroplast SOD (including thylakoid-bound SOD and stroma SOD) are described in white clover (Trifolium repens L.) grown in solution culture from rooted cuttings. Both leaf SOD and chloroplast SOD activities were markedly enhanced with increasing concentration of PEG stress, generating osmotic potentials around the roots 0, −0.5, −1.0, −1.5 MPa. The effects increased with time up to 72 h. Chloroplast Fe-containing SOD represented about 30% of the total leaf SOD activity in the control plants and a significant increase in chloroplast SOD activity was found during the stress period. This accounted for about 35.5–71.1% of the total leaf SOD activity. The proportion of chloroplast SOD in total leaf SOD not only increased with the decreasing of osmotic potential, but also increased with incubation time. Furthermore, the increase in thylakoid-bound SOD activity was much higher than that of stroma SOD in chloroplast of plants under water stress. The enhanced chloroplastic SOD activity, especially thylakoid-bound SOD activity, demonstrated in Trifolium repens suggests that Fe-SOD located in chloroplasts play a more important role than cytosolic Cu/Zn-containing SODs in scavenging O₂ ⁻.