FeSOD Activity Research Articles

Antioxidative response of Mesembryanthemum crystallinum plants to exogenous SO 2 application

The facultative halophyte, Mesembryanthemum crystallinum shifts its mode of carbon assimilation from the C 3 pathway to crassulacean acid metabolism (CAM) in response to many factors that lead to the generation of reactive oxygen species (ROS) at the cellular level. Reactive oxygen species have been suggested to be involved in the initiation of CAM induction. In our experiment we would like to test whether the space/place of ROS production could play a role in CAM induction. In the present studies we applied exogenously sulphur dioxide to M. crystallinum plants as an oxidative stress factor that accumulates mainly in chloroplasts. On the basis of our data, one can suggest that oxidative stress in the cellular space, mainly in chloroplasts, is not sufficient to induce functional CAM in M. crystallinum plants. The second aim was to evaluate the influence of SO 2 fumigation/sulphite incubation on the activity and mRNA transcript level of SOD isoenzymes, especially FeSOD, that is pointed out as a one of the first indicators correlating with the C 3/CAM transformation. The data indicate that the activity of FeSOD and CuZnSOD isoforms increase under SO 2/sulphite stress, despite of no induction of functional CAM. The increase of the activity level both of these enzymes were not accompanied by the induction of their mRNA transcript levels, suggesting a post-transcriptional regulation of activity of these enzymes. The pattern of FeSOD and CuZnSOD induction suggests that CuZnSOD might take over the role of FeSOD in conditions of severe oxidative stresses. The induction of CuZnSOD is probably due to the action of sulphite per se.

Effect of hypoxia on photosynthetic activity and antioxidative response in gametophores of Mnium undulatum

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 (Fv/Fm) 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.

Differences in the activities of some antioxidant enzymes and in H2O2 content during rhizogenesis and somatic embryogenesis in callus cultures of the ice plant

Callus was obtained from hypocotyls of Mesembryanthemum crystallinum seedlings cultured on two types of medium-germination medium (GM) and callus induction medium (CIM). Following subculture on shoot induction medium SIM1, the callus formed on CIM medium regenerated roots or somatic embryos, while that obtained on GM medium was non-regenerative. The activities of CuZn-superoxidase dismutase (SOD) were comparable in all calli, but the activities of FeSOD and MnSOD varied according to the activity of photosystem II and the regenerative potential of the tissues. Catalase (CAT) activity was related to H2O2 concentration and affected by both the culture conditions and the morphogenic potential of the calli. The possible role of CAT, SODs and H2O2 in the regeneration of M. crystallinum from callus is discussed.

Light-Dependent Oxidative Stress Determines Physiological Leaf Spot Formation in Barley

ABSTRACT We reported previously that physiological leaf spot (PLS) formation in winter and spring barley is dependent on genotype-related oxidative stress under field conditions. In the present study, we searched for factors inducing PLS symptoms in the greenhouse similar to those observed in the field and investigated its relationship to reactive oxygen species (ROS) metabolism. We found that in the greenhouse, oxidative stress induced spring barley cv. Extract, which is sensitive to PLS, to express symptoms similar to those observed in the field. Leaves severely affected by PLS showed significantly lower activities of key enzymes in the Halliwell-Asada cycle such as ascorbate peroxidase, glutathione reductase, dehy-droascorbate reductase, and monodehydroascorbate reductase. The sensitive cultivar also showed lower levels of total superoxide dismutase (SOD) and Cu/Zn-SOD activity but a higher level of chloroplast-specific Fe-SOD activity than that of the insensitive cultivar. Thus, an unbalanced ROS metabolism in chloroplasts may trigger PLS incidence in sensitive cultivars, which is in agreement with the fact that light is essential for the induction of PLS expression under both field and greenhouse conditions. Accordingly, under greenhouse conditions, continuous light stress (7 days), but not light shock treatments, induced PLS similar to that of field conditions in sensitive cv. Extract, but not in resistant cv. Scarlett. Light with a high proportion of energy in the blue wavelength spectrum (350 to 560 nm) was significantly more PLS inductive than light with a pronounced red (photosynthetically active radiation) spectrum (580 to 650 nm). Exposure to ozone did not produce PLS-like symptoms. Furthermore, similar to earlier observations in the field, PLS symptom expression was closely correlated with the accumulation of superoxide (O(2) (-)) detected by both biochemical and histochemical assays. Taken together, these data suggest that PLS in barley is genotype-dependent but its expression appears to be induced by certain environmental stress factors, among which photosyn-thetically active radiation plays a major role.

Copper deficiency induced expression of Fe-superoxide dismutase gene in Matteuccia struthiopteris

Iron-superoxide dismutase (Fe-SOD) activity was not detected in extracts from the leaves of ferns, Equisetum arvense and Matteuccia struthiopteris. To know why ferns lack Fe-SOD activity, the Fe-SOD like gene ( MsFeSOD1) was isolated from M. struthiopteris and its expression was investigated with a focus on the metals Fe and Cu using the prothalli of the fern. The expression of MsFeSOD1 mRNA was induced by a deficiency of Cu, but Fe-SOD activity was not detected. The recombinant protein of MsFeSOD1 produced in E. coli showed Fe-SOD activity. These findings suggest that the fern Fe-SOD like gene was transcriptionally regulated by Cu but an additional mechanism is involved in the formation of an active enzyme.

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.

Pretreatment with Antioxidants Decreases the Effects of Salt Stress on Chloroplast Ultrastructure in Rice Leaf Segments (Oryza sativa L.)

We investigated the kinds of active oxygen species leading to the destruction of chloroplast ultrastructure in salt-stressed rice plants. After the seedlings were grown for 3 wks, leaf segments (5 mm square) were cut from the middle portion of the 5th leaves. Leaf segments were incubated in 200 mM NaCl under dark or light conditions for 24 hr. The chlorophyll content in the leaf segments drastically decreased in light between 12- and 24 hr in 200 mM NaCl, but, no reduction was observed in the dark. In electron microscopic studies, 200 mM NaCl caused swelling of thylakoids and destruction of thylakoid membranes in light. On the other hand, no ultrastructural changes were observed under dark condition. In one experiment, leaf segments were incubated in 200 mM NaCl for 24 hr in light after preincubation with antioxidants for 12 hr in light. Pretreatment with ascorbate and benzoate, which scavenge H2O2 and ˁOH, respectively, effectively suppressed the reduction of chlorophyll content and the destruction of chloroplasts by NaCl in light. However, Tiron and DABCO, which scavenge O2- and 1O2, respectively, could not suppress the effects of salt stress in light. Fe-SOD activity was increased about eight time by salt stress (200 mM NaCl), but, catalase activity was reduced to 69% of the control and ascorbate peroxidase activity was not affected by NaCl. These results suggested that salt-induced injury in chloroplasts is dependent on light, and that H2O2 and ˁOH are responsible for the deleterious effects of salt stress on chlorophyll content and chloroplast ultrastructure.

Pretreatment with a Low Concentration of Methyl Viologen Decreases the Effects of Salt Stress on Chloroplast Ultrastructure in Rice Leaves (Oryza sativa L.)

We investigated the effects of pretreatment with a low concentration of methyl viologen (MV) on the salinity-induced chloroplast degeneration in rice seedlings. The seedlings grown in hydroponic culture containing nutrient solution for 3 wks were treated with 100 nM MV mixed in the hydroponic culture for 3 days, and then with 200 mM NaCl without MV for 3 days. In the plants without MV pretreatment, the chlorophyll content drastically decreased during the NaCl treatment accompanied by swelling of thylakoids and destruction of thylakoid membranes. These damages were alleviated by the pretreatment with MV. The activities of CuZn-SOD and Fe-SOD, which localize in chloroplasts, increased under salt stress in both plants with and without MV pretreatment. In the plants under salt stress without MV pretreatment, ascorbate peroxidase (APX) activity did not differ from that of control. However, in MV-pretreated plants, APX activity under salt stress was about 1.2- to 1.3-fold higher than that of the control. Catalase (CAT) activity in NaCl treated plants was decreased to 52% of the control and the reduction in CAT activity was suppressed by MV pretreatment. These results suggest that MV reduced the damages by salt stress in chloroplasts by increasing APX activity and preventing the decrease in CAT activity.

Location and effects of long-term NaCl stress on superoxide dismutase and ascorbate peroxidase isoenzymes of pea (Pisum sativum cv. Puget) chloroplasts.

The present work describes the intrachloroplast localization and the changes that took place in the thylakoid and stroma-located superoxide dismutases (SOD, EC 1.15.1.1) and ascorbate peroxidases (APX, EC 1.11.1.11), in response to long-term NaCl stress in Pisum sativum L. cv. Puget plants. Native PAGE using high chloroplast protein concentrations pointed to the presence of the two main Fe-SODs, together with CuZn-SODs, both in thylakoids and in the stroma. Western blot and immunogold labelling using the antibodies against chloroplastic Fe-SOD from Nuphar luteum also confirmed the chloroplastic localization of a Fe-SOD. Thylakoidal Fe-SOD activity was induced by a NaCl concentration as low as 70 mM, while CuZn-SOD was induced at 90 mM, although in severe stress conditions (110 mM) both activities were similar to the levels at 90 mM NaCl. NaCl stress also induced stromatic Fe-SOD and CuZn-SOD activities, although these inductions only started at higher NaCl concentration (90 mM) and were significant at 110 mM NaCl. The increase in activity of both Fe-SODs was matched by an increase in Fe-SOD protein. Chloroplastic APX isoenzymes behaved differently in thylakoids and stroma in response to NaCl. A significant increase of stromal APX occurred at 70 mM, whereas the thylakoidal APX activity was significantly and progressively lost in response to NaCl stress (70-110 mM). A significant increase in the H2O2 content of chloroplasts during stress and a reduction in the ascorbate level at 90 mM NaCl also took place, although the oxidized ascorbate pool at the highest NaCl concentration did not show significant changes. These results suggest that the loss of thylakoidal APX may be an important factor in the increase in chloroplastic H2O2, which also results from the increased thylakoid and stroma-located Fe-SOD and CuZn-SOD activities. This H2O2 may be involved in the induction of stromal APX. The up-regulation of the above enzymes in the described stress conditions would contribute to the adaptation of cv. Puget plants to moderate NaCl stress.

Superoxide dismutase activity in callus from the C3-CAM intermediate plant Mesembryanthemum crystallinum

In light-grown callus obtained from M. crystallinum hypocotyls, three classes of superoxide dismutase (SOD): Mn-, Fe- and Cu/ZnSOD were identified. Callus cultured on a medium containing 0.4 M NaCl showed an increase in FeSOD activity on day 4 of the experiment. In contrast, Cu/ZnSOD activity was higher over 16 days of the experiment. Salinity stress induces oxidative stress mainly for the cytosolic SOD form (Cu/ZnSOD). After 16 days of callus culture on salt-containing medium, diurnal malate oscillations, and an increase in NADP-malic enzyme activity were noticed. These results strongly suggest that C3-CAM transition can also be expressed at the cellular level. Therefore, callus tissue could be a useful model, similar to a whole plant, for investigation of mechanisms of stress responses in M. crystallinum.

Effect of Salt Stress on the Superoxide Dismutase Activity in Leaves of Citrus limonum in Different Rootstock-Scion Combinations

The effect of salinity on leaf superoxide dismutase (SOD) activity of lemon trees of different rootstock-scion combinations was studied. In leaves from Citrus limonum cv.Verna scions on Citrus macrophylla and C. reticulata rootstocks, salinity treatment clearly caused a significant depression in both Fe-SOD and Mn-SOD activities and an increase in Cu,Zn-SOD activity. However, in leaves from Citrus limonum on Citrus aurantium rootstook, the reduction observed in the activity values of Fe-SODs and Mn-SODs was not statistically significant. Salt stress also produced a decrease in the content of soluble proteins and chlorophylls. However, this drop was greater in C. limonum leaves on C. macrophylla than for other combinations.

Thermostability of manganese- and iron-superoxide dismutases from Escherichia coli is determined by the characteristic position of a glutamine residue.

The structurally homologous mononuclear iron and manganese superoxide dismutases (FeSOD and MnSOD, respectively) contain a highly conserved glutamine residue in the active site which projects toward the active-site metal centre and participates in an extensive hydrogen bonding network. The position of this residue is different for each SOD isoenzyme (Q69 in FeSOD and Q146 in MnSOD of Escherichia coli). Although site-directed mutant enzymes lacking this glutamine residue (FeSOD[Q69G] and MnSOD[Q146A]) demonstrated a higher degree of selectivity for their respective metal, they showed little or no activity compared with wild types. FeSOD double mutants (FeSOD[Q69G/A141Q]), which mimic the glutamine position in MnSOD, elicited 25% the activity of wild-type FeSOD while the activity of the corresponding MnSOD double mutant (MnSOD[G77Q/Q146A]) increased to 150% (relative to wild-type MnSOD). Both double mutants showed reduced selectivity toward their metal. Differences exhibited in the thermostability of SOD activity was most obvious in the mutants that contained two glutamine residues (FeSOD[A141Q] and MnSOD[G77Q]), where the MnSOD mutant was thermostable and the FeSOD mutant was thermolabile. Significantly, the MnSOD double mutant exhibited a thermal-inactivation profile similar to that of wild-type FeSOD while that of the FeSOD double mutant was similar to wild-type MnSOD. We conclude therefore that the position of this glutamine residue contributes to metal selectivity and is responsible for some of the different physicochemical properties of these SODs, and in particular their characteristic thermostability.

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. x 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.

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.

Influence of light intensity and salt-treatment on mode of photosynthesis and enzymes of the antioxidative response system of Mesembryanthemum crystallinum.

The metabolic switch from C3-photosynthesis to crassulacean acid metabolism (CAM),and the antioxidative response of Mesembryanthemum crystallinum L. plants cultured under severe salt stress and high light intensities, and a combination of both stress conditions, were studied. High light conditions led to a more rapid CAM induction than salinity. The induction time was still shortened when both stress factors were combined. A main pattern observed in CAM plants was a decrease in mitochondrial Mn-superoxide dismutase (SOD) activity during the day. The activities of the chloroplastic Fe-SOD and cytosolic CuZn-SOD were increased due to salt treatment after a lag phase, while catalase activity was decreased. Combination of salt and light stress did not lead to a higher SOD activity as found after application of one stress factor alone, indicating that there is a threshold level of the oxidative stress response. The fact that salt-stressed plants grown under high light conditions showed permanent photoinhibition and lost the ability for nocturnal malate storage after 9 d of treatment indicate serious malfunction of metabolism, leading to accelerated senescence. Comparison of CuZn-SOD activity with CuZn-SOD protein amount, which was determined immunologically, indicates that the activity of the enzyme is at least partially post-translationally regulated.

Physiological causes for decreased productivity under high salinity in Boma, a tetraploid Chloris gayana cultivar. II. Oxidative stress

In tetraploid cultivars of Rhodes grass (Chloris gayana Kunth) productivity decreases significantly under saline conditions. Two closely related clones of cv. Boma (T and S), exhibiting different degrees of salt tolerance, were compared with determine the physiological causes for such decrease. In those clones, salt tolerance was associated with differences in the proportion of dry leaves, salt gland density, Na excretion rate, and oxidative stress damage. The purpose of this paper is to evaluate whether salt tolerance and oxidative stress development in these clones are related to the activity of two antioxidant enzymes. Experiments were conducted in a greenhouse, in winter and summer, in plants treated with 0, 100, or 200 mm NaCl. In the summer, increases in oxidative damage, as determined by malondialdehyde (MDA) concentration, mirrored decreases in CO2 fixation at high salinity, especially in clone S. In clone T, salinity induced higher increases in Fe-SOD (superoxide dismutase) activity in summer-grown plants, and in ascorbate peroxidase (APX) in winter-grown plants. Oxidative stress induced by low paraquat concentration also induced an increase in Fe-SOD in leaf segments of clone T, and APX was less affected in clone T than in clone S. These results suggest that the clones differ in the control of antioxidant enzymes. Nevertheless, leaf death in winter was not related to increases in MDA, indicating that the association between salinity, leaf senescence and oxidatives stress is also influenced by other factors.

Induction of New Isoforms of Superoxide Dismutase and Catalase Enzymes in the Flag Leaf of Wheat during Monocarpic Senescence

Leaf senescence is a programmed cell death phenomenon and involves oxidative stress. Superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT EC 1.11.1.6) activities were studied in the flag leaf of Triticum aestivum cv. Kundan at different stages of grain development. Both SOD and CAT activities showed a decline during monocarpic senescence. Three SOD isozymes were observed in the cytosol, of which one isozyme was observed in the chloroplasts as well. Mitochondria showed the presence of three low abundant SOD isoforms. Inhibitor studies revealed the cytosolic and chloroplastic isoforms to be Cu/Zn SODs. In mitochondria however, two isozymes were MnSOD and one of them appeared to be FeSOD. These isoforms present in the mitochondria increased in activity as senescence progressed. Three isoforms of CAT were observed in peroxisomes which responded differentially during monocarpic senescence. The changes in the kind and pattern of the antioxidant enzymes supported the ordered sequence of events during leaf senescence. This is the first report showing an increase in mitochondrial FeSOD activity during leaf senescence.

Superoxide dismutase activity in the cyanobacterium Microcystis aeruginosa after surface bloom formation.

• The presence of superoxide dismutase (SOD) enzymes and the response of SOD after in vitro induction and decay of a surface bloom are shown in cultures of the cyanobacterium Microcystis aeruginosa. • The SOD enzymes of surface blooms, early degenerate and completely degenerate cultures were assayed by staining for SOD activity, immunoblotting and immunogold labelling. • One band of Mn- and three bands of Fe-SOD were detected in cell extracts. During surface bloom formation, Fe-SOD activity increased fivefold compared with that in control cells; no variation was detected in Mn-SOD activity. However, in early degenerate cultures, Fe-SOD activity decreased to that seen in control cultures, while activity disappeared in completely degenerate cultures. Immunogold labelling showed that Fe-SOD was localized in the cytoplasmic and thylakoid membranes of Microcystis. The extent of labelling paralleled the course of Fe-SOD activity with an increase in particles in surface blooming cells. • The results suggest Fe-SOD increased due to photooxidative stress. However, under prolonged photooxidative stress, high concentrations of active oxygen species could directly, or indirectly, inactivate and degrade Fe-SOD.

Expression studies of superoxide dismutases in nodules and leaves of transgenic alfalfa reveal abundance of iron-containing isozymes, posttranslational regulation, and compensation of isozyme activities.

The composition of antioxidant enzymes, especially superoxide dismutase (SOD), was studied in one nontransgenic and three transgenic lines of nodulated alfalfa plants. Transgenic lines overproduced MnSOD in the mitochondria of nodules and leaves (line 1-10), MnSOD in the chloroplasts (line 4-6), and FeSOD in the chloroplasts (line 10-7). In nodules of line 10-7, the absence of transgene-encoded FeSOD activity was due to a lack of mRNA, whereas in nodules of line 4-6 the absence of transgene-encoded MnSOD activity was due to enzyme inactivation or degradation. Transgenic alfalfa showed a novel compensatory effect in the activities of MnSOD (mitochondrial) and FeSOD (plastidic) in the leaves, which was not caused by changes in the mRNA levels. These findings imply that SOD activity in plant tissues and organelles is regulated, at least partially, at the posttranslational level. All four lines had low CuZnSOD activities and an abundant FeSOD isozyme, especially in nodules, indicating that FeSOD performs important antioxidant functions other than the scavenging of superoxide radicals generated in photosynthesis. This was confirmed by the detection of FeSOD cDNAs and proteins in nodules of other legumes such as cowpea, pea, and soybean. The cDNA encoding alfalfa nodule FeSOD was characterized and the deduced protein found to contain a plastid transit peptide. A comparison of sequences and other properties reveals that there are two types of FeSODs in nodules.

Water stress generates an oxidative stress through the induction of a specific Cu/Zn superoxide dismutase in Lotus corniculatus leaves

Drought brings about different biochemical responses in plants in order to minimize its deleterious effects. Drought induces an oxidative stress in Lotus corniculatus leaves, measured as an increment in lipid membrane peroxidation and in situ detected superoxide. As a result, total superoxide dismutase (SOD: E.C. 1.15.1.1) activity increases after a 4-h drought, when the hydric potential has decreased to −0.77 MPa. Assays with specific inhibitors suggest the presence of MnSOD, Cu/ZnSOD and FeSOD activity in L. corniculatus plants, contribute 60, 30 and 10% of total SOD activity, respectively. The Cu/ZnSOD isoform proved to be the most responsive to drought showing a remarkable increase in its activity as that corresponds to the induction of three different isoenzymes. Expression analysis of SOD isoforms revealed an increase in Cu/ZnSOD transcripts with a maximum accumulation 4 h after drought imposition. The possible role of SOD enzymes as an antioxidant protector system under water stress conditions in L. corniculatus is discussed.