The effects of NaCl on antioxidant enzyme activities in callus tissue of salt-tolerant and salt-sensitive cotton cultivars (Gossypium hirsutum L.)

Summary To determine NaCl effects on callus growth and antioxidant activity, callus of a salt-tolerant and a salt-sensitive cultivar of cotton was grown on media amended with 0, 75, and 150 mM NaCl. Callus of the salt-tolerant cultivar, Acala 1517-8 8, grown at 150 mM NaCl, showed significant increases in superoxide dismutase, catalase, ascorbate peroxidase, peroxidase and glutathione reductase activities compared to callus tissue grown at 0 mM NaCl. In contrast, callus tissue of the salt-sensitive cultivar, Deltapine 50, grown at 0, 75, and 150 mM NaCl, showed no difference in the activities of these enzymes. At the 150 mM NaCl treatment, peroxidase was the only antioxidant enzyme from Deltapine 50 with an activity as high as that observed in Acala 1517-88. The NaCl-induced increase in the activity of these enzymes in Acala 1517-88 indicates that callus tissue from the more salt-tolerant cultivar has a higher capacity for scavenging and dismutating superoxide, an increased ability to decompose H₂O₂, and a more active ascorbate-glutathione cycle when grown on media amended with NaCl.     

Cytological changes related with salt tolerance in embryogenic callus of Citrus limon

Electron microscopy observations of salt-tolerant embrogenic calli of Citrus limon [(L.) Burm. f.] showed several changes in cell ultrastructure when compared with control calli. Both types of calli comprised clusters of meristematic cells, but salt-tolerant calli had several structural differences: thick cell walls, ring-shaped mitochondria, an increased content of lipid bodies, microbodies and parallel accumulation of rough endoplasmatic reticulum. These structural features seem to be related with salt tolerance in Citrus limon cells.     

Lipid peroxidation and superoxide dismutase activity in relation to photoinhibition induced by chilling in moderate light

The effect of a chilling stress, at a moderate photon flux density for a few hours, on the peroxidation of membrane lipids and on superoxide dismutase (SOD) activity was compared in leaf slices of chilling-sensitive and chilling-insensitive plants. The aim was to determine if susceptibility to chill-temperature photoinhibition could be related to either damage to membrane lipids by superoxide and-or a decrease in activity of chloroplast SOD. Plants used were Nerium oleander L., grown at 45° C, and Cucumis sativus L., both susceptible to chill-temperature photoinhibition, and N. oleander, grown at 20° C and Spinacia oleracea L., both insensitive to chill-temperature photoinhibition. Lipid peroxidation was assessed by measuring the concentration of malondialdehyde (MDA). Leaf slices from all plants showed a basal level of MDA which decreased by about 15% when the leaf slices were chilled in the light. The level of MDA was not increased by the addition of either KHCO₃ or methyl viologen during chilling but it was increased, up to threefold, by the addition of Rose Bengal, which produces singlet oxygen. Chloroplast SOD activity was assessed in leaf extracts as the cyanide-sensitive production of H₂O₂ in a system which produced superoxide. Activity of SOD was similar in all the plants and was altered little by chilling. The results show that for the plants tested, chilling at a moderate photon flux density for 5 h does not increase the susceptibility of cell membranes to peroxidative damage nor does it decrease the activity of SOD. It was concluded that the susceptibility of chilling-sensitive plants to chill-temperature photoinhibition cannot be explained on the basis of differences in the vulnerability of membrane lipids to damage by superoxide or differences in SOD activity.Abbreviations Chl chlorophyll - MDA malondialdehyde - MV methyl viologen - O ₂ ^- superoxide - 20°-oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - PFD photon flux density - SOD superoxide dismutase Deceased     

Evaluation of NaCl Tolerance in the Callus Cultures of Suaeda nudiflora Moq.

Salt tolerance was studied in the callus cultures of Suaeda nudiflora Moq. a dicotyledonous succulent halophyte. Growth was significantly inhibited at 50, 100, 150 and 200 mM NaCl. Inorganic ions and proline accumulated in response to salinity. Ion accumulation pattern reflected the utilization of Na⁺ as an osmoticum. Na⁺/K⁺ ratio rose steadily as a function of external NaCl concentration. Salt stress enhanced the activity of peroxidase, whereas it decreased activities of superoxide dismutase and catalase. This revised version was published online in July 2006 with corrections to the Cover Date.     

Superoxide dismutase and hydrogen peroxide formation in Campylobacter sputorum subspecies bubulus

Cell-free extracts of Campylobacter sputorum subspecies bubulus contained superoxide dismutase. The enzyme was located in the cytoplasmic fraction and insensitive to cyanide. After centrifuging a cell-free extract at 144000 x g for 1.5 h the total activity in the supernatant fraction was threefold higher than in the crude cell-free extract. The pellet fraction thus obtained was shown to have a lowering effect on superoxide dismutase activities from different sources in the assay method used here. C. sputorum responded to a raised oxygen tension in the culture by an increase in the superoxide dismutase activity. The ability to produce superoxide anion radicals (O₂ ^-·) during oxidation of formate and lactate was demonstrated. Furthermore C. sputorum was found to produce H₂O₂ while oxidizing formate. In experiments in which the reduction of cytochrome c by formate was followed, step-wise kinetics were observed. One of the steady states then obtained was attributed to the oxidizing action of H₂O₂, because it was abolished by the addition of catalase and lengthened by H₂O₂ added in addition to H₂O₂ formed as a product of formate oxidation. An overall reaction for formate oxidation by C. sputorum is discussed.Abbreviations O₂ ^-· superoxide anion radical - NBT p-nitro blue tetrazolium chloride - ABTS 2,2-azino-di-[3-ethylbenzthiazoline sulfonate (6)] - TL-medium tryptose-lactate medium     

Glutathione reductase activity in heterocysts and vegetative cells of the cyanobacterium Nostoc muscorum

Glutathione reductase activity was detected and characterized in heterocysts and vegetative cells of the cyanobacterium Nostoc muscorum. The activity of the enzyme varied between 50 and 150 nmol reduced glutathione· min^-1·mg protein^-1, and the apparent K_m for NADPH was 0.125 and 0.200 mM for heterocysts and vegetative cells, respectively. The enzyme was found to be sensitive to Zn⁺² ions, however, preincubation with oxidized glutathione rendered its resistance to Zn⁺² inhibition. Nostoc muscorum filaments were found to contain 0.6–0.7mM glutathione, and it is suggested that glutathione reductase can regenerate reduced glutathione in both cell types. The combined activity of glutathione reductase and isocitrate dehydrogenase in heterocysts was as high as 18 nmol reduced glutathione·min^-1·mg protein^-1. A relatively high superoxide dismutase activity was found in the two cell types; 34.2 and 64.3 enzyme units·min^-1·mg protein^-1 in heterocysts and vegetative cells, respectively.We suggest that glutathione reductase plays a role in the protection mechanism which removes oxygen radicals in the N₂-fixing cyanobacterium Nostoc muscorum.Abbreviations DTNB 5-5-dithiobis-(2-nitrobenzoic acid) - EDTA ethylenediaminetetra-acetic acid - GR glutathione reductase (EC1.6.4.2) - GSH reduced glutathione - GSSG oxidized glutathione - OPT O-phtaldialdehyde - SOD superoxide dismutase (EC 1.15.1.1)     

The role of salicylic acid and carrot embryogenic callus extracts in somatic embryogenesis of naked oat (Avena nuda)

Enhanced somatic embryogenesis and plant regeneration have been obtained using young leaf bases of naked oat (Avena nuda) as explants by including salicylic acid (SA) and carrot embryogenic callus extracts (CECE) in media. A 5- and 4-fold improvement was achieved in somatic embryogenesis and plant regeneration on the corresponding media supplemented with 0.5 mM SA and CECE as compared to control, respectively. Some physiological and biochemical changes were assayed in both embryogenic callus (EC) and non-embryogenic callus (NEC). The results indicated that superoxide dismutase activity was stimulated and catalases and ascorbate peroxidase activities were inhibited, while the O₂ ^- (superoxide anion) content was reduced and the hydrogen peroxide level was promoted in EC compared with NEC. Reduced malondialdehyde content and relative electrolyte leakage were also detected in EC.     

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.     

Oxygen utilization by Lactobacillus plantarum

Cell-free extracts of Lactobacillus plantarum contain non-proteinaceous compounds which mimic superoxide dismutase activity. Using the test system in which O ₂ ^– is generated by xanthine oxidase, superoxide dismutase activity is found in cell-free extracts, where proteins are removed by precipitation. This activity is strongly decreased after dialysis of cell-free extracts. Superoxide dismutase activity was also investigated by means of pulse radiolysis. Cell-free extracts of Escherichia coli were also investigated as a comparison, which were known to contain superoxide dismutase. With cell-free extracts of both L. plantarum and E. coli the decay of O ₂ ^– was markedly increased. However, the type of reaction of the O ₂ ^– decay was of first order in the presence of E. coli extracts due to superoxide dismutase(s), and of second order in the presence of L. plantarum extracts, indicating that O ₂ ^– elimination is not an enzymic reaction. Mn²⁺ phosphate(s) might be responsible for the observed elimination of O ₂ ^– . The production of O ₂ ^– is not detectable during NADH-, lactate- or pyruvate oxidase reactions in L. plantarum extracts.     

Selection of callus cultures of sugarcane (Saccharum sp.) tolerant to NaCl and their response to salt stress

Stable callus cultures tolerant to NaCl (68 mM) were developed from salt-sensitive sugarcane cultivar CP65-357 by in vitro selection process. The accumulation of both inorganic (Na⁺, Cl⁻ and K⁺) and organic (proline and soluble sugars) solutes was determined in selected and non-selected calli after a NaCl shock in order to evaluate their implication in in vitro salt tolerance of the selected lines. Both salt-tolerant and non-selected calli showed similar relative fresh weight growth in the absence of NaCl. No growth reduction was observed in salt-tolerant calli while a significant reduction about 32% was observed in nonselected ones when both were cultivated on 68 mM NaCl. Accumulation of Na⁺ was similar in both salt-tolerant and non-selected calli in the presence of NaCl. Accumulation of Cl⁻ was lower in NaCl-tolerant than in non-selected calli while proline and soluble sugars were more accumulated in salt-tolerant than in non-selected calli when both were exposed to salt. K⁺ level decreased more severely in non-selected calli than in NaCl-tolerant ones after NaCl shock. The results indicated that K⁺ and Cl⁻ may play a key role in in vitro salt-tolerance in sugarcance cell lines obtained by in vitro selection and that organic solutes could contribute mainly to counteract the negative water potential of the outside medium.     

Production of plants resistant to <Emphasis Type="Italic">Alternaria carthami</Emphasis> via organogenesis and somatic embryogenesis of safflower cv. NARI-6 treated with fungal culture filtrates

The present study describes a system for efficient plant regeneration via organogenesis and somatic embryogenesis of safflower (Carthamus tinctorius L.) cv. NARI-6 in fungal culture filtrates (FCF)-treated cultures. FCF was prepared by culturing Alternaria carthami fungal mycelia in selection medium for host-specific toxin production. Cotyledon explants cultured on callus induction medium with different levels of FCF (10–50%) produced embryogenic callus. In organogenesis, 42.2% microshoots formed directly from embryogenic callus tissues in plant regeneration medium with 40% FCF. Isolated embryogenic callus cultured on embryo induction medium containing 40% FCF induced 50.2% somatic embryogenesis. Embryo germination percentage was decreased from 64.5 to 28 in embryo maturation medium containing 40% FCF. However, nine plantlets from organogenesis and 24 plantlets from somatic embryogenesis were selected as FCF-tolerant. Alternaria carthami fungal spores (5 × 10⁵ spores/ml) sprayed on the leaves of FCF-tolerant plants showed enhanced survival rate over control plants, which plants were more susceptible to fungal attack. The number of leaf spot lesions per leaf was decreased from 3.4 to 0.9 and their lesion length was also reduced from 2.9 to 0.7 mm in organogenic derived FCF-tolerant plants over control. In somatic embryo derived FCF-tolerant plants, the number of lesions was decreased from 3.1 to 0.4 and the lesion size was also reduced to 2.7–0.5 mm when compared to the control. This study also examined antioxidant enzyme activity in FCF-tolerant plants. Catalase (CAT) activity was slightly decreased whereas peroxidase (POD) activity was increased to a maximum of 42% (0.19 μmol min⁻¹ mg⁻¹ protein) from organogenesis and 47% (0.23 μmol min⁻¹ mg⁻¹ protein) from embryogenesis in FCF-tolerant plants. Superoxide dismutase (SOD) activity was also increased to 17% (149 U mg⁻¹ protein) and 19.5% (145 U mg⁻¹ protein) in FCF-tolerant plants derived from organogenesis and somatic embryogenesis when compared with control plants.     

Growth of Chlorella sorokiniana in the presence of sulfite elevates cell content of superoxide dismutase and imparts resistance towards paraquat

1. Growth of Chlorella sorokiniana in the presence of 7.5 mM sulfite, which halved the growth rate while doubling the superoxide dismutase (SOD; EC 1.15.1.1) content per cell, rendered the cells resistant to the toxic effects of 30 M paraquat. 2. While increasing total SOD content, sulfite increased the relative amount of the H₂O₂-resistant manganese-containing SOD. 3. It appears that O₂ ^– may be involved in mediating the toxicity of SO₂ in this green alga.Abbreviations SOD superoxide, dismutase - FeSOD ironcontaining superoxide dismutase - MnSOD manganese-containing superoxide dismutase     

Influence of extremely-low-frequency magnetic field on antioxidative melatonin properties in AT478 murine squamous cell carcinoma culture

Effects of melatonin, extremely-low-frequency magnetic field (ELF-MF), and their combination on AT478 murine squamous cell carcinoma line were studied. Manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (Cu/ZnSOD), and glutathione peroxidase (GSH-Px) were used as markers of cells antioxidative status, and malondialdehyde (MDA) level was used as a marker of lipid peroxidation. After melatonin treatment, antioxidative enzyme activities were increased and MDA level was decreased. Application of ELF-MF on treated cells caused an increase of both superoxide dismutases activity and MDA level, but influence of ELF-MF on GSH-Px activity was negligible. All enzyme activity in culture medium containing melatonin (10⁻³, 10⁻⁴, 10⁻⁵ M) after exposure to ELF-MF were significantly diminished compared to cells treated only with melatonin. Also MDA levels after combined treatment with melatonin and ELF-MF were significantly decreased. Observed changes were statistically significant (p<0.05). These results strongly suggest that ELF-MF attenuates antioxidative actions of melatonin on cellular level.     

Superoxide dismutase in Scenedesmus obliquus

In heterotrophically grown Scenedesmus obliquus, the specific activity of superoxide dismutase (SOD; EC 1.15.1.1) declined when glucose was abundant, increased as it was depleated, and remained steady at a high level when it was absent. Transition to autotrophic growth produced only a small (20% over 5 d) increase in specific activity above the values obtained in dark-grown cells after glucose and starch-reserve depletion. This small, but consistent, increase did, however, parallel a similar increase in photosynthetic capacity. Polyacrylamide-gel electrophoresis showed the existence of nine isoenzymes of SOD. The three major and one of the minor isoenzymes were present in all extracts while three minor isoenzymes were found only in autotrophically grown cells and two only in heterotrophically grown cells. Characterization studies indicated that two of the major isoenzymes are dimeric FeSODs the other is a tetrameric MnSOD, and of the minor isoenzymes, two are dimeric FeSODs and four are dimeric MnSODs.Abbreviation SOD superoxide dismutase     

High-efficiency,microprojectile-mediated cotransformation of sugarcane,using visible or selectable markers

Transient expression of the maize anthocyanin regulatory elements,R andC1, was used to optimise parameters for microprojectile-mediated delivery of DNA into sugarcane embryogenic callus. Osmotic treatment of target tissues and particle acceleration in a high-pressure helium pulse increased the frequency of transient expression to 5–8×10³ cells per bombardment, with minimal tissue damage. An average of 0.34% of transiently expressing cells developed into stably transformed, anthocyanin-pigmented proembryoids which subsequently regenerated into plantlets. However, constitutive expression ofR andC1 proved deleterious, and no anthocyanin-pigmented plant survived beyond 3 cm in height. We also compared selective subculture of callus portions showing luciferase activity with antibiotic selection on medium containing G418 or phosphinothricin, upon bombardment of callus with constructs driving strong expression ofluc, aphA orbar genes. Selective subculture based on luciferase activity enabled recovery of 1.4±0.5 independent transgenic plants per bombardment, compared to 19.8±3.7 independent transgenic plants per bombardment from an optimised G418 selection regimen, and no transformed plants from phosphinothricin selection. Whenluc andaphA on separate plasmids were coprecipitated onto microprojectiles before bombardment, 67–79% of callus lines selected for G418 resistance also showed luciferase activity detectable under a low-light camera. Southern analysis confirmed a very high cotransformation frequency, with variable copy numbers of introduced genes. The high efficiencies of gene transfer, selection and cotransformation in the optimised system, coupled with the simple initiation and regeneration of embryogenic callus, provide an effective tool for practical genetic transformation of sugarcane.     

In vitro selection for salt tolerant lines in Lycopersicon peruvianum

A salt-tolerant callus line of Lycopersicon peruvianum has been obtained by exposing the cells, in suspension cultures and then in callus, to increasing concentrations of NaCl (50–350mM). This selected line grew better than the nonselected line at all levels of NaCl. Moreover, this selected line grew better in media containing salt than in those without it. It retained its tolerance after subculture for 3 passages (3 months) on salt-free medium. The growth of the selected line in mannitol was similar to that of the nonselected line, which suggested that the superiority of the selected line under salt stress was not due to osmotic stress tolerance. The ions SO ₄ ^–– and K⁺ were highly toxic to L. peruvianum root callus, while Na⁺, Mg⁺⁺ and Cl^– were less toxic.     

Effect of oxygen on the cyanobacterium Oscillatoria limnetica

Oscillatoria limnetica grown photoautotrophically under aerobic or anaerobic conditions contained a single superoxide dismutase (SOD) of identical electrophoretic mobility in both cases. Its activity was cyanide resistant and H₂O₂ sensitive, implicating Fe-SOD. The enzyme level was high in aerobically and low in anaerobically growing cells. Anaerobically grown cells were more sensitive than aerobic to photooxidation, as expressed by bleaching of phycocyanin and disintegration of the trichomes.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - SOD superoxide dismutase     

Ethylene and nitric oxide are involved in maintaining ion homeostasis in <Emphasis Type="Italic">Arabidopsis</Emphasis> callus under salt stress

In the present study, the role of ethylene in nitric oxide (NO)-mediated protection by modulating ion homeostasis in Arabidopsis callus under salt stress was investigated. Results showed that the ethylene-insensitive mutant etr1-3 was more sensitive to salt stress than the wild type (WT). Under 100 mM NaCl, etr1-3 callus displayed a greater electrolyte leakage and Na⁺/K⁺ ratio but a lower plasma membrane (PM) H⁺-ATPase activity compared to WT callus. Application of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor) or sodium nitroprusside (SNP, a NO donor) alleviated NaCl-induced injury by maintaining a lower Na⁺/K⁺ ratio and an increased PM H⁺-ATPase activity in WT callus but not in etr1-3 callus. The SNP actions in NaCl stress were attenuated by a specific NO scavenger or an ethylene biosynthesis inhibitor in WT callus. Under 100 mM NaCl, the NO accumulation and ethylene emission appeared at early time, and NO production greatly stimulated ethylene emission in WT callus. In addition, ethylene induced the expression of PM H⁺-ATPase genes under salt stress. The recovery experiment showed that NaCl-induced injury was reversible, as signaled by the similar recovery of Na⁺/K⁺ ratio and PM H⁺-ATPase activity in WT callus. Taken together, the results indicate that ethylene and NO cooperate in stimulating PM H⁺-ATPase activity to modulate ion homeostasis for salt tolerance, and ethylene may be a part of the downstream signal molecular in NO action.     

Manganese superoxide dismutase from a higher plant

A manganese-containing superoxide dismutase (EC 1.15.1.1) was purified to homogeneity from a higher plant for the first time. The enzyme was isolated fromPisum sativum leaf extracts by thermal fractionation, ammonium sulfate salting out, ion-exchange and gel-filtration column chromatography, and preparative polyacrylamide gel electrophoresis. Pure manganese superoxide dismutase had a specific activity of about 3,000 U mg^-1 and was purified 215-fold, with a yield of 1.2 mg enzyme per kg whole leaf. The manganese superoxide dismutase had a molecular weight of 94,000 and contained one g-atom of Mn per mol of enzyme. No iron and copper were detected. Activity reconstitution experiments with the pure enzyme ruled out the possibility of a manganese loss during the purification procedure. The stability of manganese superoxide dismutase at-20°C, 4°C, 25°C, 50°C, and 60°C was studied, and the enzyme was found more labile at high temperatures than bacterial manganese superoxide dismutases and iron superoxide dismutases from an algal and bacterial origin.Abbreviations NBT nitro blue tetrazolium - SOD superoxide dismutase (EC 1.15.1.1)     

Influence of very low doses of ionizing radiation on Synechococcus lividus metabolism during the initial growth phase

Previous results from this laboratory have shown that very low chronic doses of gamma radiation can stimulate proliferation of the Cyanobacterium Synechococcus lividus. This modification of cell proliferation occurred during the first doubling. In this paper, we have compared the metabolism of cells cultivated in a normal environment or under chronic irradiation. Incubation of the cells in a new medium induced a high superoxide dismutase (EC 1.15.1.1, SOD) activity at the 18th hour and a degradation of phycocyanin, thus demonstrating that cells were submitted to a photooxidative stress. This increase in superoxide dismutase activity was followed by concomittant peaks of glutathione reductase (EC 1.6.4.2, GR) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49, G6P-DH) at the 24th hour. Irradiated cultures at a dose of 53.5 mGray/year show an earlier and higher peak of SOD, GR, and G6P-DH. In a second stage, cultures showed an earlier onset of photosynthesis under irradiation, as evidenced by an increase in pigment content and an enhancement of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13, GAP-DH). These results show that the radiostimulation is related to the activation of enzymes protecting against peroxides that were induced under oxidative circumstances and to the activation of a glucose catabolism via the oxidative pentose phosphate pathway.Abbreviations mGy milli-Gray - SOD superoxide dismutase - G6P-DH glucose-6-phosphate dehydrogenase - GAP-DH glycer-aldehyde-3-phosphate dehydrogenase - GSSG oxidized glutathione