A comparative study of glutathione and ascorbate metabolism during germination of Pinus pinea L. seeds

The ascorbate and glutathione systems have been studied during the first stages of germination in orthodox seeds of the gymnosperm Pinus pinea L. (pine). The results indicate that remarkable changes in the content and redox balance of these metabolites occur in both the embryo and endosperm; even if with different patterns for the two redox pairs. Dry seeds are devoid of the ascorbate reduced form (ASC) and contain only dehydroascorbic acid (DHA). By contrast, glutathione is present both in the reduced (GSH) and in the oxidized (GSSG) forms. During imbibition the increase in ASC seems to be mainly caused by the reactivation of its biosynthesis. On the other hand, the GSH rise occurring during the first 24 h seems to be largely due to GSSG reduction, even if GSH biosynthesis is still active in the seeds. The enzymes of the ascorbate--glutathione cycle also change during germination, but in different ways. ASC peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) activities progressively rise both in the embryo and in endosperm. These changes are probably required for counteracting production of reactive oxygen species caused by recovery of oxidative metabolism. The two enzymes involved in the ascorbate recycling, ascorbate free radical (AFR) reductase (EC 1.6.5.4) and DHA reductase (EC 1.8.5.1), show different behaviour: the DHA reductase activity decreases, while that of AFR reductase remains unchanged. The relationship between ascorbate and glutathione metabolism and their relevance in the germination of orthodox seeds are also discussed.     

Changes in ascorbate, glutathione, and related enzyme activities during thidiazuron-induced bud break of apple

The changes of ascorbic acid, dehydroascorbic acid, and glutathione content and related enzyme activities were studied in apple buds during dormancy and thidiazuron-induced bud break. An increase in ascorbic acid, reduced form of glutathione (GSH), total glutathione, total non-protein thiol (NPSH) and non-glutathione thiol (RSH) occurred as a result of induction by thidiazuron during bud break, whereas dehydroascorbic acid and oxidized glutathione (GSSG) decreased during the same period. Thidiazuron also enhanced the ratio of GSH/GSSG, and activities of ascorbate free radical reductase (AFR; EC 1.6.5.4), ascorbate peroxidase (EC 1.11.1.11). dehydroascorbate reductase (DHAR; EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2). The ascorbic acid content and the activities of AFR, ascorbate peroxidase, and DHAR peaked when buds were in the side green or green tip stage just prior to the start of rapid expansion, and declined thereafter. The GSH, NPSH, RSH, ratio of GSH/GSSG, and activities of GR increased steadily during bud development.     

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.     

Antioxidative responses to different altitudes in leaves of alpine plant Polygonum viviparum in summer

Mountain environmental stresses result in increased formation of hydrogen peroxide (H₂O₂) and accumulation of malondialdehyde (MDA) in leaves of Polygonum viviparum. The activities of several antioxidative system enzymes such as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7), glutathione reductase (GR, EC 1.6.4.2), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and the contents of several non-enzymatic antioxidants such as reduced form of ascorbate (ASC), dehydroascorbate (DHA), reduced glutathione (GSH), and oxidized glutathione (GSSG) were investigated in leaves of P. viviparum, which were collected from three altitudes (2,200, 3,200, and 3,900 m) of Tianshan Mountain in China. The activities of these four antioxidative enzymes were accompanied by increases of H₂O₂ levels from 2,200 to 3,200 m. However, the activities of CAT and POD were decreased, whereas the activities of SOD and GR continually increased at 3,900 m. Analyses of isoforms of SOD, CAT, POD, and GR showed that the leaves of P. viviparum exposed different altitude conditions are capable of differentially altering the intensity. Additionally, two new isoforms of SOD were detected at 3900 m. A continual increase in the ASC, ASC to DHA ratio, GSH and GSH/[GSH + GSSG] ratio, and the activity of DHAR were observed in leaves of P. viviparum with the elevation of altitude. These results suggest that the higher contents of ASC, GSH as well as an increase in reduced redox state may be essential to antioxidation processes in the leaves of P. viviparum, whereas antioxidant enzymes system is a cofactor in the processes.     

Oxidative stress responses and shock proteins in the unicellular cyanobacterium Synechococcus R2 (PCC-7942)

Oxidative stress responses were tested in the unicellular cyanobacterium Synechococcus PCC 7942 (R2). Cells were exposed to hydrogen peroxide, cumene hydroperoxide and high light intensities. Activities of ascorbate peroxidase and catalase were correlated with the extent and time-course of oxidative stresses. Ascorbate peroxidase was found to be the major enzyme involved in the removal of hydrogen peroxide under the tested oxidative stresses. Catalase activity was inhibited in cells treated with high H₂O₂ concentrations, and was not induced under photo-oxidative stress. Regeneration of ascorbate in peroxide-treated cells was found to involve mainly monodehydroascorbate reductase and to a lesser extent dehydroascorbate reductase. The induction of the antioxidative enzymes was dependent on light and was inhibited by chloramphenicol. Peroxide treatment was found to induce the synthesis of eight proteins, four of which were also induced by heat shock.Abbreviations ASC ascorbate - DHA dehydroascorbate - MDA monodehydroascorbate - GSH reduced glutathione - GSSG oxidized glutathione - ASC Per ascorbate peroxidase - DHA red. dehydroascorbate reductase - MDA red. monodehydroascorbate reductase - GSSG red. glutathione reductase - HSP heat shock proteins - PSP peroxide shock proteins - C_m chloramphenicol     

The ascorbate system in recalcitrant and orthodox seeds

Recalcitrant seeds of Ginkgo biloba L., Quercus cerris L., Aesculus hippocastanum L. and Cycas revoluta Thunb. are shed by the plant at a high moisture content, contain a large amount of ascorbic acid (ASA) and maintain high ascorbate (ASC) peroxidase (EC 1.11.1.11) activity. Three proteins showing ASC peroxidase activity are present in G. biloba seeds. Conversely, dry orthodox seeds ( Vicia faba L., Avena sativa L., Pinus pinea L.) are completely devoid of ASA and ASC peroxidase. Experimentally induced rapid variations of the water level in both recalcitrant and orthodox seeds do not affect the ASC peroxidase; slow dehydration affects the ASC peroxidase activity moderately in recalcitrant seeds, but provokes a complete loss of germinability. Another peculiar difference between orthodox and recalcitrant seeds concerns the ascorbate recycling enzymes, ascorbate free radical (AFR) reductase (EC 1.6.5.4) and dehydroascorbate (DHA) reductase (EC 1.8.5.1). The DHA reduction capability is low in recalcitrant seeds, but is high in the orthodox ones. In contrast, AFR reductase activity is high in recalcitrant seeds and low in the orthodox ones. Data reported here concerning the ASC system appear to contribute to better understanding the recalcitrance. The presence of three different proteins showing ASC peroxidase activity in the archaic seed-bearing plant G. biloba and its involvement in the spermatophyte evolution is discussed.     

Effect of Selenium on Ascorbate�CGlutathione Metabolism During PEG-induced Water Deficit in Trifolium repens L.

To elucidate the effect of selenium (Se) on the ascorbate?Cglutathione (ASC?CGSH) cycle under drought stress, the activities of antioxidant enzymes and the levels of molecules involved in ASC?CGSH metabolism were studied in Trifolium repens seedlings subjected to polyethylene glycol (PEG)-induced water deficit alone or combined with 5???M Na₂SeO₄. Compared to the control, H₂O₂, thiobarbituric acid reactive substances (TBARS), ascorbate (ASC), dehydroascorbate (DHA), and glutathione disulfide (GSSG) contents increased, whereas a constant content of glutathione (GSH) and decreases in ASC/DHA and GSH/GSSG ratios were observed in the presence of PEG. The activities of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) were upregulated, except for monodehydroascorbate reductase (MDHAR) activity during PEG-induced water deficit. Se application decreased the contents of H₂O₂, TBARS, DHA, and GSSG, increased the levels of GSH and ASC, and inhibited the decreases of ASC/DHA and GSH/GSSG ratios. Although it did not affect APX activity significantly, Se addition improved the activities of MDHAR, DHAR, and GR. Furthermore, GR activity showed the highest increase followed by that of DHAR and MDHAR in decreasing order. These data indicated that fluctuations in ASC?CGSH metabolism resulting from Se may have a positive effect on drought stress mitigation, and the regulation in the ASC?CGSH cycle can be attributed mainly to GR and DHAR in PEG?+?Se-treated T. repens seedlings.     

Hydrogen-peroxide-scavenging systems within pea chloroplasts

The subcellular distribution of ascorbate peroxidase and glutathione reductase (EC 1.6.4.2) in pea leaves was compared with that of organelle markers. Enzyme distribution was found to be similar to that of the chloroplast enzyme NADPH-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). Isolated chloroplasts showed a close correlation between intactness and the percentage of enzyme activity recovered. Chloroplasts of 85% intactness were found to contain a high proportion of leaf dehydroascorbate reductase activity (EC 1.8.5.1), 10% of leaf glutathione and 30% of leaf ascorbate. These results are discussed in relation to the potential role of chloroplast antioxidant systems in plant resistance to environmental and other stress conditions.Abbreviations GSH reduced glutathione - GSSG oxidized glutathione - NADPH-GPD glyceraldehyde-3-phosphate dehydrogenase - SOD superoxide dismutase     

Copper affects the enzymes of the ascorbate-glutathione cycle and its related metabolites in the roots of Phaseolus vulgaris

The involvement of the ascorbate-glutathione cycle in the defence against Cu-induced oxidative stress was studied in the roots of Phaseolus vulgaris L. cv. Limburgse vroege. All the enzymes of this cycle [ascorbate peroxidase (APOD), EC 1.11.1.11; monodehydroascorbate reductase (MDHAR), EC 1.6.5.4; dehydroascorbate reductase (DHAR), EC 1.8.5.1; glutathione reductase (GR), EC 1.6.4.2] were increased, and the total ascorbate and glutathione pools rose after a 15 μ M root Cu treatment. In the first hours after the start of the experiment, the accumulation of dehydroascorbate (DHA), formed as a result of a Cu-mediated direct oxidation of ascorbate (AA), was limited by a non-enzymatic reduction using glutathione (GSH) as the reductant. At 24 h, the enzyme capacities of both DHAR and GR were increased to maintain the redox status of the AA and GSH pools. After 72 h of Cu application, the DHAR capacity was inhibited and MDHAR was responsible for maintaining the AA pool in its reduced form. Although the GR capacity was enhanced after 72 h in the treated plants, the GSSG/GSH ratio was increased. This could be due to direct participation of GSH in the detoxification of Cu through reduction and complexation.     

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 Q₁ and cytochrome c can be accounted for by the increase of the AFR concentration generated by the redox pairs ascorbate/coenzyme Q₁ 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.     

Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography

Glutathione reductase (EC 1.6.4.2) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of polyacrylamide gel electrophoresis on native and SDS-gels. The enzyme has a molecular weight of 145,000 and consists of two subunits of similar size. The pH optimum of spinach glutathione reductase is 8.5–9.0, which is related to the function it performs in the chloroplast stroma. It is specific for oxidised glutathione (GSSG) but shows a low activity with NADH as electron donor. The pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-dependent reduction. The enzyme has a low affinity for reduced glutathione (GSH) and for NADP⁺, but GSH-dependent NADP⁺ reduction is stimulated by addition of dithiothreitol. Spinach glutathione reductase is inhibited on incubation with reagents that react with thiol groups, or with heavymetal ions such as Zn²⁺. GSSG protects the enzyme against inhibition but NADPH does not. Pre-incubation of the enzyme with NADPH decreases its activity, so kinetic studies were performed in which the reaction was initiated by adding NADPH or enzyme. The Km for GSSG was approximately 200 M and that for NADPH was about 3 M. NADP⁺ inhibited the enzyme, assayed in the direction of GSSG reduction, competitively with respect to NADPH and non-competitively with respect to GSSG. In contrast, GSH inhibited non-competitively with respect to both NADPH and GSSG. Illuminated chloroplasts, or chloroplasts kept in the dark, contain equal activities of glutathione reductase. The kinetic properties of the enzyme (listed above) suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions. This prediction was confirmed experimentally. GSH or GSSG play no part in the light-induced activation of chloroplast fructose diphosphatase or NADP⁺-glyceraldehyde-3-phosphate dehydrogenase. We suggest that GSH helps to stabilise chloroplast enzymes and may also play a role in removing H₂O₂. Glucose-6-phosphate dehydrogenase activity may be required in chloroplasts in the dark in order to provide NADPH for glutathione reductase.Abbreviations GSH reduced form of the tripeptide glutathione - GSSG oxidised form of glutathione     

Antioxidants in the apoplast and symplast of beech (Fagus sylvatica L.) leaves: seasonal variations and responses to changing ozone concentrations in air

Concentrations of the antioxidants ascorbate and glutathione were measured in the apoplast of beech (Fagus sylvatica L.) leaves and in leaf tissue. During early leaf development, reduced ascorbate (ASC) was almost absent from the apoplast, whereas levels of oxidized ascorbate (DHA) were high. Less than 20% of the apoplastic ascorbate was reduced. ASC increased towards midsummer, reaching top levels of about 4molm^?3 apoplast volume in July and August. Reduction increased to 60–75% in summer. Neither DHA reductase nor glutathione was detected in the apoplast of beech leaves. Levels of apoplastic ascorbate were compared with ambient concentrations of ozone in air. Statistical analysis indicated a significant interrelation between atmospheric ozone and apoplastic ascorbate. In midsummer of 1993, contents of DHA were increased in the apoplast when ozone concentrations were high. Apoplastic ASC was also positively correlated with ambient ozone concentrations, but with a delay of 3 to 7d. In leaf tissue, levels of ascorbate were between 17 and 21 μmolg^?1 FW in summer. Except for late April and November, more than 95% of the intracellular ascorbate was reduced. Glutathione contents were lowest during the summer. Oxidation was increased in spring and autumn, when apoplastic ascorbate was also largely oxidized. Usually, 80 to 90% of the glutathione was reduced. During the summer, intracellular concentrations of oxidized glutathione (GSSG) were increased, with a delay of about 1d following periods of high ambient ozone concentrations. The transitory accumulation of GSSG may be explained by slow enzymatic regeneration of glutathione.     

Arsenite treatment induces oxidative stress,upregulates antioxidant system,and causes phytochelatin synthesis in rice seedlings

The effects of arsenite treatment on generation of reactive oxygen species, induction of oxidative stress, response of antioxidative system, and synthesis of phytochelatins were investigated in two indica rice (Oryza sativa L.) cvs. Malviya-36 and Pant-12 grown in sand cultures for a period of 5–20 days. Arsenite (As₂O₃; 25 and 50 μM) treatment resulted in increased formation of superoxide anion (O₂^.−), elevated levels of H₂O₂ and thiobarbituric acid reactive substances, showing enhanced lipid peroxidation. An enhanced level of ascorbate (AA) and glutathione (GSH) was observed irrespective of the variation in the level of dehydroascorbate (DHA) and oxidized glutathione (GSSG) which in turn influenced redox ratios AA/DHA and GSH/GSSG. With progressive arsenite treatment, synthesis of total acid soluble thiols and phytochelatins (PC) increased in the seedlings. Among antioxidative enzymes, the activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), total ascorbate peroxidase (APX, EC 1.11.1.11), chloroplastic ascorbate peroxidase, guaiacol peroxidase (EC 1.11.1.7), monodehydroascorbate reductase (EC 1.6.5.4), and glutathione reductase (EC 1.6.4.2) increased in arsenite treated seedlings, while dehyroascorbate reductase (EC 1.8.5.1) activity declined initially during 5–10 days and increased thereafter. Results suggest that arsenite treatment causes oxidative stress in rice seedlings, increases the levels of many enzymatic and non-enzymatic antioxidants, and induces synthesis of thiols and PCs, which may serve as important components in mitigating arsenite-induced oxidative damage.     

Antioxidant defences of the apoplast

Summary The apoplast of barley and oat leaves contained superoxide dismutase (SOD), catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase activities. The activities of these enzymes in the apoplastic extracts were greatly modified 24 h after inoculation with the biotrophic fungal pathogenBlumeria graminis. The quantum efficiency of photosystem II, which is related to photosynthetic electron transport flux, was comparable in inoculated and healthy leaves during this period. Apoplastic soluble acid invertase activity was also modified in inoculated leaves. Inoculation-dependent increases in apoplastic SOD activity were observed in all lines. Major bands of SOD activity, observed in apoplastic protein extracts by activity staining of gels following isoelectric focusing, were similar to those observed in whole leaves but two additional minor bands were found in the apoplastic fraction. The apoplastic extracts contained substantial amounts of dehydroascorbate (DHA) but little or no glutathione (GSH). Biotic stress decreased apoplastic ascorbate and DHA but increased apoplastic GSH in resistant lines. The antioxidant cycle enzymes may function to remove apoplastic H₂O₂ with ascorbate and GSH derived from the cytoplasm. DHA and oxidized glutathione may be reduced in the apoplast or returned to the cytosol for rereduction.Abbreviations AA reduced ascorbate - APX ascorbate peroxidase - DHA dehydroascorbate (oxidised ascorbate) - DHAR dehydroascorbate reductase - G6PDH glucose-6-phosphate dehydrogenase - GSH reduced glutathione - GSSG glutathione disulphide - GR glutathione reductase - MDHA monodehydroascorbate - MDHAR monodehydroascorbate reductase - SOD superoxide dismutase     

Ectopic Expression of Riboflavin-binding Protein Gene TsRfBP Paradoxically Enhances Both Plant Growth and Drought Tolerance in Transgenic Arabidopsis thaliana

Riboflavin is the precursor of the coenzymes flavin monophosphate (FMN) and flavin adenine dinucleotide (FAD), which serve as indispensable redox cofactors in all plants. Numerous data indicate that riboflavin is involved in pathogen resistance but less data are available on abiotic stress tolerance. In this experiment, the overexpression of the riboflavin-binding protein resulted in an enhancement of vegetative growth and net photosynthetic rate, and an acceleration of floral transition in transgenic Arabidopsis thaliana REAT11 (containing less than half the normal levels of free riboflavin, FMN, and FAD) compared to wild-type Col-0 under nonstressed conditions. The effect of drought stress on the antioxidant response of Col-0 and REAT11 was compared, where 20- and 40-day-old grown plants were subjected to 10 % PEG 6000 treatment for 2 days. Stress conditions caused a significant increase in H₂O₂ accumulation, lipid peroxidation, and membrane permeability in Col-0 over that in REAT11. Greater activity levels of superoxide dismutase, ascorbate peroxidase, and glutathione reductase were observed in the leaves of REAT11 compared to those of Col-0. Significant increases in total ascorbate and glutathione content and higher ratios of ASC/DHA: (ASC and DHA are reduced and oxidized ascorbate, respectively) and GSH/GSSG: (GSH and GSSG are reduced and oxidized glutathione, respectively) were observed in the leaves of REAT11 compared to those in Col-0 under drought conditions. In addition, enhancement of free proline and soluble sugar accumulation was observed in REAT11 compared to Col-0 under stress. Our results suggest that a slight deficiency in free riboflavin can paradoxically induce both a higher vegetative growth rate and an enhanced tolerance to drought in transgenic plants. The “stress escape” hypothesis is proposed here to explain this interesting phenomenon.     

Ascorbate-glutathione metabolism during PEG-induced water deficit in <Emphasis Type="Italic">Trifolium repens</Emphasis>

In order to elucidate the response of the ascorbate-glutathione (ASC-GSH) cycle to drought stress, the activities of antioxidant enzymes and the levels of molecules involved in the ASC-GSH metabolism were studied in Trifolium repens L. seedlings subjected to PEG-induced water deficit. Compared to the control, the contents of H₂O₂, thiobarbituric acid reactive substances (TBARS), ascorbate (ASC), dehydroascorbate (DHA), and glutathione disulfide (GSSG) increased in PEG-treated seedlings, whereas the glutathione (GSH) content kept constant during the drought period. Further more, the ASC/DHA and GSH/GSSG ratios decreased in the presence of PEG. Except for that of monodehydroascorbate reductase (MDHAR), the activities of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) were up-regulated during water deficit, and the increases in APX and DHAR activities were much higher than those in GR activity. These data indicate that fluctuations in the ASC-GSH metabolism resulted from PEG treatment may have a positive effect on drought stress mitigation in T. repens.     

Effects of ambient oxygen and of fixed nitrogen on concentrations of glutathione, ascrobate, and associated enzymes in soybean root nodules

Soybean (Glycine max [L.] Merr.) root nodules contain the enzymes of the ascorbate-glutathione cycle for defense against activated forms of oxygen. Nodulated roots of hydroponically grown soybean plants were exposed to atmospheres containing 2, 21, 50, or alternating 21 and 50 kilopascals of O₂. The activities of ascorbate (ASC) peroxidase, monodehydroascorbate (MDHA) reductase, dehydroascorbate (DHA) reductase, and glutathione (GSSG) reductase were higher in nodules exposed to high pO₂. Nodule contents of ascorbate and reduced glutathione were also greater in the high pO₂ treatments. Treatment of nodulated plants with fixed nitrogen (urea) led to concomitant decreases in acetylene reduction activity, in leghemoglobin content, and in activities of ASC peroxidase, DHA reductase, and GSSG reductase. Activity of MDHA reductase and glutathione concentrations in nodules were not affected by treatment with urea. The enzymes of the ascorbate-glutathione cycle were also detected in uninfected soybean roots, although at levels substantially below those in nodules. These observations indicate that the ascorbate-glutathione cycle can adjust to varying physiological conditions in nodules and that there is a key link between N₂ fixation and defenses against activated forms of oxygen.     

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 degrees C. When stored at 25 degrees 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 degrees C, then increased. At 25 degrees 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 degrees C. Therefore overall the antioxidant enzymes were scarcely regulated and unable to counteract oxidative stress occurring during the long-term storage.     

The redox state of the ascorbate-dehydroascorbate pair as a specific sensor of cell division in tobacco BY-2 cells

The effects of ascorbate (ASC) and dehydroascorbate (DHA) on cell proliferation were examined in the tobacco Bright Yellow 2 (TBY-2) cell line to test the hypothesis that the ASC-DHA pair is a specific regulator of cell division. The hypothesis was tested by measuring the levels of ASC and DHA or another general redox pair, glutathione (GSH) and glutathione disulfide (GSSG), during the exponential-growth phase of TBY-2 cells. A peak in ASC, but not GSH, levels coincided with a peak in the mitotic index. Moreover, when the cells were enriched with ascorbate, a stimulation of cell division occurred whereas, when the cells were enriched with DHA, the mitotic index was reduced. In contrast, glutathione did not affect the mitotic-index peak during this exponential-growth phase. The data are consistent in showing that the ASC-DHA pair acts as a specific redox sensor which is part of the mechanism that regulates cell cycle progression in this cell line.     

The redox state of the ascorbate-dehydroascorbate pair as a specific sensor of cell division in tobacco BY-2 cells

Summary The effects of ascorbate (ASC) and dehydroascorbate (DHA) on cell proliferation were examined in the tobacco Bright Yellow 2 (TBY-2) cell line to test the hypothesis that the ASC-DHA pair is a specific regulator of cell division. The hypothesis was tested by measuring the levels of ASC and DHA or another general redox pair, glutathione (GSH) and glutathione disulfide (GSSG), during the exponential-growth phase of TBY-2 cells. A peak in ASC, but not GSH, levels coincided with a peak in the mitotic index. Moreover, when the cells were enriched with ascorbate, a stimulation of cell division occurred whereas, when the cells were enriched with DHA, the mitotic index was reduced. In contrast, glutathione did not affect the mitotic-index peak during this exponential-growth phase. The data are consistent in showing that the ASC-DHA pair acts as a specific redox sensor which is part of the mechanism that regulates cell cycle progression in this cell line.