Ascorbate Free Radical Reductase Research Articles

Hyperglycemia enhances the generation of ROS and RNS that impair antioxidant power and cause oxidative damage in human erythrocytes.

Hyperglycemia is a state in which excess glucose circulates in blood. Erythrocytes are in direct contact with this high glucose concentration and are greatly affected by it. We have examined the effect of hyperglycemic condition on isolated human erythrocytes under in vitro conditions. Erythrocytes were incubated with different concentrations of glucose (5, 15, 30, 45 mmol/L) for 24 h, and several biochemical parameters were determined. Treatment with high glucose concentrations increased heme degradation and methemoglobin level, while methemoglobin reductase activity was decreased. A significant increase in protein oxidation and lipid hydroperoxides with a decrease in total sulfhydryl content was seen. This suggested the generation of oxidative stress, which was confirmed by an enhanced production of reactive oxygen and nitrogen species. Hyperglycemia led to a significant decline in the antioxidant power of erythrocytes, lowering their ability to quench free radicals and reduce metal ions to lower oxidation states. The plasma membrane redox system was upregulated, while ascorbate free radical reductase activity was lowered. Glucose exposure inhibited the enzymes of glycolysis and hexose monophosphate shunt. Electron microscopy showed morphological changes resulting in the formation of echinocytes. Thus, the hyperglycemic condition generates reactive species that oxidize proteins, hemoglobin, and lipids; impair the total antioxidant capacity; and alter morphology in human erythrocytes.

Physiological responses of ‘Italia’ grapevines infected with Esca pathogens

Physiological features were examined of a 20-year-old Vitis vinifera ‘Italia’ table grape vineyard cropped in Apulia, Italy. Healthy vines with no foliar symptoms and any indications of wood or berry alterations, vines with natural wood infections by Phaeoacremonium minimum (syn. P. aleophilum) and Phaeomoniella chlamydospora showing brown wood streaking symptoms, and vines naturally infected with P. minimum, P. chlamydospora and Fomitiporia mediterranea with brown wood streaking and white rot symptoms, were surveyed. Bleeding xylem sap, collected at bud-break from healthy vines showed the greatest total ascorbic acid level, while vines with brown wood streaking and white rot had the greatest viscosity coefficient, glutathione concentration, and plant growth regulator activities. Compared to healthy vines, leaves of wood affected vines, sampled during the unfolded leaf, fruit setting, cluster closing and bunch ripening vine growth stages, had reduced fresh and dry weights, total chlorophyll concentrations, and increased leaf surface area. Low ascorbic acid and reduced glutathione concentrations, weak redox state, and moderate levels of dehydroascorbic acid and oxidized glutathione were also detected in these vines. Analyses also detected reduced activities of dehydroascorbate reductase, ascorbate free radical reductase and glutathione reductase in diseased vines. The cell membrane damage, associated with lipid peroxidation, was coupled with high hydrogen peroxide concentrations. These changes could contribute to the cell death of leaves and foliar symptom development. The ascorbate-glutathione cycle supports grapevine susceptibility to Esca complex-associated fungi.

Fluoride enhances generation of reactive oxygen and nitrogen species, oxidizes hemoglobin, lowers antioxidant power and inhibits transmembrane electron transport in isolated human red blood cells

Fluoride is a widespread environmental pollutant that at high levels exerts numerous deleterious effects on human health. The toxic effects of fluoride are a matter of serious concern since many countries have regions of endemic fluorosis. The main source of fluoride exposure for humans is intake of contaminated groundwater. Fluoride is absorbed from the gastrointestinal tract and enters the circulating blood, where the abundant red blood cells (RBC) are an early and major target of fluoride toxicity. Chronic fluoride exposure generates free radicals, reactive species which leads to redox imbalance, cytotoxicity and hematological damage. This study aimed to determine the effect of sodium fluoride (NaF) on human RBC under in vitro conditions. Isolated RBC were incubated with different concentrations of NaF (10–500 µM) for 8 h at 37 °C. Several biochemical parameters were determined in hemolysates or whole cells. Treatment of RBC with NaF enhanced the generation of reactive oxygen and nitrogen species. This increased the oxidation of hemoglobin to yield methemoglobin and oxoferrylhemoglobin, which are inactive in oxygen transport. NaF treatment increased the degradation of heme causing release of free iron from its porphyrin ring. Cellular antioxidant power was significantly decreased in NaF-treated RBC, lowering the metal reducing and free radical quenching ability of cells. The two pathways of glucose metabolism in RBC i.e. glycolysis and hexose monophosphate shunt, were inhibited. NaF also inhibited the plasma membrane redox system, and its associated ascorbate free radical reductase, to disrupt transmembrane electron transport. These results suggest that fluoride generates reactive species that cause extensive oxidative modifications in human RBC.

Zebra chip disease enhances respiration and oxidative stress of potato tubers (Solanum tuberosum L.).

The physiological phenotype of potato tubers afflicted by zebra chip disease is characterized by increased oxidative stress metabolism and upregulation of systems for its mitigation. Starch catabolism and extensive buildup of reducing sugars render potatoes infected with zebra chip (ZC) pathogen (Candidatus Liberibacter solanacearum) unsuitable for fresh market and processing into chips/fries. Here we show that the disease inflicts considerable oxidative stress, which likely constitutes a substantial sink for metabolic energy, resulting in increased respiration rate of afflicted tubers. In contrast to healthy tubers, tissue from diseased tubers had greater ability to reduce 2,3,5-triphenyl-tetrazolium chloride to formazan, indicating enhanced dehydrogenase activity, probable disease-induced changes in cellular redox potential, and increased respiratory activity. The respiration rate of diseased tubers (cv. Atlantic) was 2.4-fold higher than healthy tubers and this correlated with increased activities of glucose-6-phosphate and 6-phosphogluconate dehydrogenases, key enzymes responsible for synthesis of cytosolic reducing equivalents. Wound-induced NADPH oxidase activity was greater for ZC than healthy tubers, but the resulting superoxide was rapidly catabolized by higher superoxide dismutase activity in ZC tubers. Peroxidase, catalase, glutathione reductase and ascorbate free radical reductase activities were also higher in diseased tubers, as was malondialdehyde, a biomarker of peroxidative damage and oxidative stress. Upregulation of the glutathione-ascorbate pathway is a direct response to (and indicator of) oxidative stress, which consumes reducing equivalents (NADPH) to catabolize reactive oxygen species and maintain cellular redox homeostasis. ZC disease substantially altered the oxidative metabolism of tubers, resulting in a physiological phenotype defined by metabolic changes directed toward mitigating oxidative stress. Paradoxically, the increased respiration rate of ZC tubers, which fuels the metabolic pathways responsible for attenuating oxidative stress, likely also contributes to oxidative stress.

Resveratrol Up-Regulates the Erythrocyte Plasma Membrane Redox System and Mitigates Oxidation-Induced Alterations in Erythrocytes During Aging in Humans

Reactive oxygen/nitrogen species (ROS/RNS)-mediated oxidative damage followed by disturbed cellular homeostasis is involved in aging and related consequences. Lipid peroxidation, post-translational modifications of proteins, and an impaired defense system due to increased oxidative stress jeopardize cell fate and functions, resulting in cell senescence. Resveratrol, a natural stilbene, has extensively been reported to elicit a plethora of health-promoting effects. The present study carried out on 97 healthy human subjects (62 males and 35 females) of both sexes provides experimental evidence that resveratrol confers ability to up-regulate the plasma membrane redox system (PMRS) along with ascorbate free radical reductase, a compensatory system operating in the cell to maintain cellular redox state. Furthermore, resveratrol provided significant protection against lipid peroxidation and protein carbonylation and restored the cellular redox homeostasis measured in terms of glutathione (GSH) and sulfhydryl (-SH) group levels during oxidation injury in erythrocytes of different age groups in humans. Findings suggest a possible role of resveratrol in retardation of age-dependent oxidative stress.

Circadian modulation of human erythrocyte plasma membrane redox system by melatonin

BackgroundThe pineal hormone melatonin works for the stabilization of biological rhythms, however, it also modulates several other functions such as cardioprotection, thermoregulation and immunomodulation. Melatonin also shows antioxidant activity. The erythrocyte plasma membrane redox system (PMRS) alongwith ascorbate free radical (AFR) reductase is involved in providing protection against oxidative stress. The present work is an ex vivo study addressing RBC PMRS and AFR reductase activities at two different times of the day. The in vitro modulatory effect of melatonin on PMRS and AFR reductase activities is also reported. Materials and methodsThe study was carried out on 61 healthy donors of both sexes (aged 20–30). Blood samples were collected at two different timings viz., 10:00 a.m. and 10:00 p.m. PMRS and AFR reductase were determined by methods already reported. The concentration-dependent effect of the melatonin was evaluated by incubating the RBCs with the hormone at different doses. ResultsWe present results to show that erythrocyte PMRS and AFR reductase activity are modulated by melatonin, a higher activity (p<0.05) of PMRS and AFR reductase is observed during night when the level of melatonin is high. The effect of in vitro treatment of erythrocytes with melatonin (10−7M to 10−11M final concentration) shows significant changes during day at a melatonin concentration of 10−9M. ConclusionTo the best of our knowledge this study shows for the first time the circadian rhythmicity of erythrocyte PMRS and AFR reductase activities. The modulatory effect of melatonin on PMRS and AFR reductase opens the possibility of melatonin being used in treatment of such physiological and metabolic dysfunctions that involve photic cues in association with oxidative stress.

Upregulation of erythrocyte ascorbate free radical reductase by tea catechins: Correlation with their antioxidant properties

Tea catechins have been shown to possess a variety of health promoting effects, most of these effects have been attributed to their strong antioxidant property. Ascorbate (ASC) is the primary antioxidant in plasma and human erythrocytes play a vital role in regereneration of ASC in blood plasma by reduction of ascorbate free radical (AFR) into ASC by AFR reductase, a transmembrane enzyme. In the present study we report that tea catechins; EGCG, ECG, EGC and EC driven activation of erythrocyte AFR reductase. The efficiency of tea catechins to activate AFR reductase has a significant positive correlation with the antioxidant activities of these compounds, measured by DPPH scavenging and FRAP assays. Since AFR reductase along with other transmembrane oxidoreductases, is reported to act as compensatory/protective mechanism that operates to minimize the extent of oxidative stress, activation of this enzyme by tea catechins may be a mechanism for these compounds to elicit antioxidant effect in vivo.

Altered HBK3 expression affects glutathione and ascorbate metabolism during the early phases of Norway spruce (Picea abies) somatic embryogenesis

Plant homeobox genes play an important role in plant development, including embryogenesis. Recently, the function of a class I homeobox of knox 3 gene, HBK3, has been characterized in the conifer Picea abies (L.) Karst (Norway spruce) [8]. During somatic embryogenesis, expression of HBK3 is required for the proper differentiation of proembryogenic masses into somatic embryos. This transition, fundamental for the overall embryogenic process, is accelerated in sense lines over-expressing HBK3 (HBK3-S) but precluded in antisense lines (HBK3-AS) where the expression of this gene is experimentally reduced. Altered HBK3 expression resulted in major changes of ascorbate and glutathione metabolism. During the initial phases of embryogeny the level of reduced GSH was higher in the HBK3-S lines compared to their control counterpart. An opposite profile was observed for the HBK3-AS lines where the glutathione redox state, i.e. GSH/GSH + GSSG, switched towards its oxidized form, i.e. GSSG. Very similar metabolic fluctuations were also measured for ascorbate, especially during the transition of proembryogenic masses into somatic embryos (7 days into hormone-free medium). At this stage the level of reduced ascorbate (ASC) in the HBK3-AS lines was about 75% lower compare to the untransformed line causing a switch of the ascorbate redox state, i.e. ASC/ASC + DHA + AFR, towards its oxidized forms, i.e. DHA + AFR. Changes in activities of several ascorbate and glutathione redox enzymes, including dehydroascorbate reductase (EC 1.8.5.1), ascorbate free radical reductase (EC 1.6.5.4) and glutathione reductase (GR; EC 1.6.4.2) were responsible for these metabolic differences. Data presented here suggest that HBK3 expression might regulate somatic embryo yield through alterations in glutathione and ascorbate metabolism, which have been previously implicated in controlling embryo development and maturation both in vivo and in vitro.

Ascorbate Recycling by Erythrocytes During Aging in Humans

Erythrocytes play a crucial role in recycling ascorbate in blood plasma. The erythrocyte ascorbate free radical (AFR) reductase is involved in the reduction of AFR to ascorbic acid (ASC) in the plasma. In the present study, we report an age-dependent increase in the activity of erythrocyte AFR reductase in humans that shows a significant positive correlation with the activity of plasma membrane redox system (PMRS). We explain the age-dependent increase in erythrocyte ASC recycling on the basis of a compensatory/protective mechanism that operates to maintain the ASC level in plasma and thereby minimize oxidative stress during aging.

Reduction of ascorbate free radical by the plasma membrane of synaptic terminals from rat brain

Synaptic plasma membranes (SPMV) decrease the steady state ascorbate free radical (AFR) concentration of 1 mM ascorbate in phosphate/EDTA buffer (pH 7), due to AFR recycling by redox coupling between ascorbate and the ubiquinone content of these membranes. In the presence of NADH, but not NADPH, SPMV catalyse a rapid recycling of AFR which further lower the AFR concentration below 0.05 μM. These results correlate with the nearly 10-fold higher NADH oxidase over NADPH oxidase activity of SPMV. SPMV has NADH-dependent coenzyme Q reductase activity. In the presence of ascorbate the stimulation of the NADH oxidase activity of SPMV by coenzyme Q 1 and cytochrome c can be accounted for by the increase of the AFR concentration generated by the redox pairs ascorbate/coenzyme Q 1 and ascorbate/cytochrome c. The NADH:AFR reductase activity makes a major contribution to the NADH oxidase activity of SPMV and decreases the steady-state AFR concentration well below the micromolar concentration range.

Effect of rare earth elements on growth and antioxidant metabolism in Lemna minor L.

Lemna minor is frequently used in bioremediation processes to remove nutrients and contaminants from waste water. In this work the response of L. minor to treatments with lanthanum nitrate and with a mix of several light rare earth elements (REE) nitrates was investigated. Preliminary results indicate that L. minor shows an overall good tolerance to the presence of REE in the media. Toxic effects were observed after prolonged exposition to high concentration of REE. An increase in ascorbate and glutathione content as well as in ascorbate peroxidase, dehy- droascorbate reductase and ascorbate free radical (AFR) reductase activity was observed in treated plants.

Effects of storage temperature on viability, germination and antioxidant metabolism in Ginkgo biloba L. seeds

The behaviour of the Ginkgo biloba L. seeds was studied during storage at 4 and 25 °C. When stored at 25 °C, all the seeds died in 6 months. Cold temperatures preserved seed tissue viability for 1 year but did not preserve their capability to germinate, since such capability decreased after 6 months. A significant increase in lipid peroxidation occurred in the seed both in the embryo and in the endosperm. During storage a progressive deterioration of the endosperm tissues was evident. The two major water soluble antioxidants, ascorbate (ASC) and glutathione (GSH), showed different behaviour in the two conditions of storage and in the two main structures of the seed, the embryo and the endosperm. The ASC content of embryos and endosperms remained quite unchanged in the first 9 months at 4 °C, then increased. At 25 °C a significant decrease in the ASC content in the embryos was evident, whereas it remained more stable in the endosperm. The GSH pool decreased at both storage temperatures in the embryos. As far as the ASC–GSH redox enzymes are concerned, their activities decreased with storage, but changes appeared to be time-dependent more than temperature-dependent, with the exception of the endosperm ascorbate free radical (AFR) reductase (EC 1.6.5.4), the activity of which rapidly decreased at 25 °C. Therefore overall the antioxidant enzymes were scarcely regulated and unable to counteract oxidative stress occurring during the long-term storage.

The Effects of Ascorbate on Root Regeneration in Seedling Cuttings of Tomato

The changes in ascorbate (ASC) and dehydroascorbate (DHA) levels and the activities of ascorbate metabolising enzymes were examined during adventitious root formation in cuttings of tomato (Lycopersicon esculentum Mill. cv. Paw) seedlings. The effects of ASC, DHA and the immediate ascorbate precursor – galactono-γ-lactone (GalL) supplemented to the culture medium on the rooting response, ascorbate content and the activities of the ASC-metabolising enzymes were also investigated. The cuttings treated with abovementioned compounds formed more roots then control plants. However, in contrast to the number of regenerated organs, the elongation of newly formed roots was markedly inhibited. Treatment with auxin (IAA) resulted in a similar phenotype. The inhibitor of auxin polar transport-TIBA (2,3,5-triiodobenzoic acid) effectively blocked rooting. The inhibitory effect of TIBA was reversed by auxin and ASC treatments, while DHA and GalL were ineffective. Both auxin and ASC stimulated cell divisions in an area of pericycle layer of TIBA-treated rooting zones, that enabled cuttings to form roots in the presence of the inhibitor of auxin polar transport. It has been found that the first stages of rooting, preceding the emergence of roots, are accompanied by an increase in endogenous content of ASC with a peak in the 3rd day of rooting. Subsequent stages, when elongation of newly formed roots occurs, are characterised by low level of ASC. The activities of the ascorbate peroxidase (APX), ascorbate oxidase (AOX), ascorbate free radical reductase (AFRR) and dehydroascorbate reductase (DHR) increased in the first 3 days of root formation. The initial period of rooting was also accompanied by the increase of the hydrogen peroxide content and the activities of catalase (CAT) and guaiacol peroxidase (GPX) in the rooting zones. IAA, ASC, DHA as well as Gal stimulated the APX activity, however the rise of the enzyme's activity induced by ASC, DHA and Gal was reversed by TIBA, which was found to inhibit APX. Only exogenous IAA was able to maintain the high level of APX activity in the TIBA-treated cuttings. AOX was strongly affected by ASC and GalL – treatments, its activity increased in the cuttings grown on the media containing ASC in the absence as well as in the presence of TIBA. On the other hand, GalL-dependent stimulation of its activity was suppressed if TIBA was present in a rooting medium.

The effect of osmoticum on ascorbate and glutathione metabolism during white spruce ( Picea glauca) somatic embryo development

Water stress is an important factor which regulates organized development of both zygotic and somatic embryos. Somatic embryos of white spruce were cultured in the presence of polyethylene glycol (PEG), a non-plasmolyzing agent which increases embryo quality and number, and mannitol, a plasmolyzing agent. The effects of these two compounds on both ascorbate and glutathione metabolism were investigated at different stages of embryo development. Compared to control and mannitol-treated embryos, embryos treated with PEG accumulated higher levels of endogenous ascorbate (ASC) in its reduced form, especially during the first half of the maturation period. This increase, also observed in immature seeds, was mainly the result of two different processes: activation of the de novo ASC machinery, and recycling of ASC from ascorbate free radicals (AFR) which was modulated by the activity of ascorbate free radical reductase (AFRR, EC. 1.6.5.4). The activity of this enzyme increased during the early phases of development in both PEG-treated somatic embryos and seeds. Compared to control somatic embryos, mannitol and PEG were shown to change the levels of reduced (GSH) and oxidized glutathione (GSSG). In particular, a constant decline in the GSH/GSSG ratio was observed in the presence of PEG. This pattern was also observed in maturing white spruce seeds. Overall, these data indicate that applications of non-plasmolyzing agents in the culture medium of spruce somatic embryos result in seed-like fluctuations of the ascorbate–glutathione metabolism, which may have a positive effect on embryo yield.

Changes in the ascorbate system in the response of pumpkin ( Cucurbita pepo L.) roots to aluminium stress

The involvement of the ascorbate (AsA) system in the response of pumpkin ( Cucurbita pepo L.) roots to aluminium stress was studied. The treatment of 5-day-old pumpkin seedlings with 50 μM aluminium sulphate resulted in approximately 60% inhibition of root growth within 48–60 h of treatment, while aluminium accumulated in the roots reaching a maximum within 48 h. During the same period, the hydrogen peroxide content of the roots was strongly enhanced. The increased level of hydrogen peroxide was matched by both increased ascorbate peroxidase (APX) (EC 1.11.1.11) activity and ascorbate free radical reductase (AFRR) (EC 1.1.5.4) activity, while dehydroascorbate reductase (DHAR) (EC 1.8.5.1) and glutathione reductase (GR) (EC 1.6.4.2) did not change. The levels of AsA in the roots were also increased by the Al treatment. It was concluded that an oxidative burst is probably involved in the toxicity of Al in pumpkin roots and that plants react to the enhanced production of reactive oxygen species by expressing higher levels of scavenging systems such as the AsA–APX system.

Isolation of ascorbate free radical reductase from rabbit lens soluble fraction

Ascorbate free radical (AFR) reductase with diaphorase activity was isolated from the rabbit lens soluble fraction to characterise some molecular properties of the enzyme. The isolation was accomplished using gel filtration (Sephadex G-75 superfine or Sephacryl S-200 HR), affinity chromatography (Affi-Gel Blue), native isoelectric focusing and two-dimensional gel electrophoresis. A major soluble AFR reductase was found at an isoelectric point of 8·4 and a molecular weight of 31 kDa, and a few minor enzymes were also detected in the range of p I 7·0–8·6. An unknown N-terminal partial amino acid sequence was determined in one peptide fragment prepared from the major enzyme fraction. From the sequence analysis, it is discussed that the lens soluble AFR reductase may differ from NADH-cytochrome b 5 reductase reported to be involved in the membrane-bound AFR reductase activity of mitochondria, microsomes and plasma membrane.

Changes in the ascorbate metabolism of apoplastic and symplastic spaces are associated with cell differentiation.

Ascorbate levels and redox state, as well as the activities of the ascorbate related enzymes, have been analysed both in the apoplastic and symplastic spaces of etiolated pea (Pisum sativum L.) shoots during cellular differentiation. The ascorbate pool and the ascorbate oxidizing enzymes, namely ascorbate oxidase and ascorbate peroxidase, were present in both pea apoplast and symplast, whereas ascorbate free radical reductase and dehydroascorbate reductase were only present in the symplastic fractions. During cell differentiation the ascorbate redox enzymes changed in different ways, since a decrease in ascorbate levels, ascorbate peroxidase and ascorbate free radical reductase occurred from meristematic to differentiated cells, whereas ascorbate oxidase and dehydroascorbate reductase increased. The activity of secretory peroxidases has also been followed in the apoplast of meristematic and differentiating cells. These peroxidases increased their activity during differentiation. This behaviour was accompanied by changes in their isoenzymatic profiles. The analysis of the kinetic characteristics of the different peroxidases present in the apoplast suggests that the presence of ascorbate and ascorbate peroxidase in the cell wall could play a critical role in regulating the wall stiffening process during cell differentiation by interfering with the activity of secretory peroxidases.

Stabilization of Extracellular Ascorbate Mediated by Coenzyme Q Transmembrane Electron Transport

Publisher Summary Ascorbate is a major water-soluble antioxidant found in body fluids of mammals, where it acts as a scavenger of noxious soluble-free radicals or lipid peroxidation-initiating radicals in the aqueous phase, as well as a regenerator of other antioxidants (e.g., vitamin E) in the lipid phase of biological membranes and lipoproteins. Ascorbate oxidation is a two-step process involving the one-electron intermediate ascorbate free radical (AFR) or semidehydroascorbate, and the two-electron, oxidized form dehydroascorbate (DHA). Intracellular reduction of ascorbate has been documented to occur both from AFR and DHA through different enzymatic reactions, with the participation of reduced nicotinamide adenine dinucleotide (NADH) and reduced glutathione (GSH) as electron donors, as well as from ascorbate acting at different cell locations. Among these mechanisms are the AFR reductase found in the chromaffin-vesicle membrane and in the outer mitochondrial membrane, the thioredoxin reductase, and glutaredoxin. The two spectrophotometric assays described in the chapter (i.e., ascorbate measurement at 265 nm and AFR signal at 360 nm), together with the measurement of NADH consumption at 340 nm, are valuable and valid tools to address this issue.

Iron deficiency enhances the levels of ascorbate, glutathione, and related enzymes in sugar beet roots.

The effects of Fe deficiency stress on the levels of ascorbate and glutathione, and on the activities of the enzymes ferric chelate reductase, glutathione reductase (EC 1.6.4.2), ascorbate free-radical reductase (EC 1.6.5.4) and ascorbate peroxidase (EC 1.11.1.11), have been investigated in sugar beet ( Beta vulgaris L.) roots. Plasma membrane vesicles and cytosolic fractions were isolated from the roots of the plants grown in nutrient solutions in the absence or presence of Fe for two weeks. Plants responded to Fe deficiency not only with a 20-fold increase in root ferric chelate reductase activity, but also with moderately increased levels of the general reductants ascorbate (2-fold) and glutathione (1.6-fold). The enzymes of the ascorbate-glutathione cycle in roots were also affected by Fe deficiency. Glutathione reductase activity was enhanced 1.4-fold with Fe deficiency, associated to an increased ratio of reduced to oxidized glutathione, from 3.1 to 5.2. The plasma membrane fraction from iron-deficient roots showed 1.7-fold higher ascorbate free-radical reductase activity, whereas in the cytosolic fraction the enzyme activity was not affected by Fe deficiency. The activity of the cytosolic hemoprotein ascorbate peroxidase decreased approximately by 50% with Fe deprivation. These results show that sugar beet responds to Fe deficiency with metabolic changes affecting components of the ascorbate-glutathione cycle in root cells. This suggests that the ascorbate-glutathione cycle would play certain roles in the general Fe deficiency stress responses in strategy I plants.

Tolerance of kikuyu grass to long term salt stress is associated with induction of antioxidant defences

The response of the antioxidant system to long term (15 d) salt stress (50, 100, 200 mM NaCl) was studied in kikuyu (Pennisetum clandestinum, Hochst.), a perennial grass with a well developed root system (often used as an erosion controlling ground cover) and with high nutritive properties as pasture. The activities of peroxidase, catalase, ascorbate peroxidase, ascorbate-free radical reductase, glutathione reductase and dehydroascorbate reductase enzymes involved in the detoxification from reactive oxygen species were investigated. The growth of kikuyu grass was inhibited at 200 mM NaCl. Salinity affected root length more than leaf length. Lower activities of catalase, ascorbate-free radical reductase, and dehydroascorbate reductase enzymes were observed in stressed plants. The tolerance of kikuyu grass to salt stress (up to 100 mM) appears to be related to up-regulation of ascorbate peroxidase, peroxidase and glutathione reductase enzymes.