Transport of Ascorbic and Dehydroascorbic Acids across Protoplast and Vacuole Membranes Isolated from Barley (Hordeum vulgare L. cv Gerbel) Leaves

Protoplasts, vacuoles, and chloroplasts were isolated from leaves of 8-d-old barley (Hordeum vulgare L. cv Gerbel) seedlings. Transport of ascorbate and dehydroascorbate into protoplasts and vacuoles was investigated. Contents of ascorbic acid, glutathione, and [alpha]-tocopherol and ascorbate peroxidase activity and glutathione reductase activity were analyzed in protoplasts, vacuoles, and chloroplasts. Uptake of ascorbate and dehydroascorbate by protoplasts showed saturation kinetics (Km = 90 [mu]M reduced ascorbic acid, 20 [mu]M dyhydroascorbic acid). Effects of various membrane transport inhibitors suggested that transport was carrier mediated and driven by a proton electrochemical gradient. Translocation of ascorbate and dehydroascorbate into vacuoles did not show saturation kinetics. Neither was it influenced by effectors or by ATP but only by Mg2+, suggesting that translocation did not occur by carrier. Ascorbic acid was predominantly localized in the cytosol. Contents in the chloroplasts and vacuoles were low. The results are consistent with the view that ascorbate is synthesized in the cytosol and released to chloroplasts, apoplast, and vacuole following a concentration gradient. Translocation from the apoplast into the cytosol is against a steep gradient and appears to control the concentration of ascorbic acid in the apoplast. In its function as an antioxidant, ascorbate in the apoplast may be oxidized to dehydroascorbate, which can be efficiently transported back into the cytosol for regeneration to ascorbate.     

Inactivation of Ascorbate Peroxidase in Spinach Chloroplasts on Dark Addition of Hydrogen Peroxide: Its Protection by Ascorbate

Dark addition of hydrogen peroxide to intact spinach chloroplastsresulted in the inactivation of ascorbate peroxidase accompaniedby a decrease in ascorbate contents. This was also the casein reconstituted chloroplasts containing ascorbate, NADP⁺, NAD⁺and ferredoxin. The addition of hydrogen peroxide during light,however, showed little effect on ascorbate contents and ascorbateperoxidase activity in either the intact or reconstituted chloroplasts.In contrast to ascorbate peroxidase, the enzymes participatingin the regeneration of ascorbate in chloroplasts (monodehydroascorbatereductase, dehydroascorbate reductase and glutathione reductase)were not affected by the dark addition of hydrogen peroxide.Ascorbate contents increased again by illumination of the chloroplastsafter the dark addition of hydrogen peroxide. These resultsshow that the inactivation of the hydrogen peroxide scavengingsystem on dark addition of hydrogen peroxide [Anderson et al.(1983) Biochim. Biophys. Acta 724: 69, Asada and Badger (1984)Plant & Cell Physiol. 25: 1169] is caused by the loss ofascorbate peroxidase activity. Ascorbate peroxidase activitywas rapidly lost in ascorbate-depleted medium, and protectedby its electron donors, ascorbate, isoascorbate, guaiacol andpyrogallol, but not by GSH, NAD(P)H and ferredoxin. (Received June 14, 1984; Accepted August 15, 1984)     

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     

Light-dependent reduction of dehydroascorbate and uptake of exogenous ascorbate by spinach chloroplasts

A reconstituted spinach chloroplast system containing thylakoids, stroma and 0.1 mM NADPH supported O₂ evolution in the presence of oxidised glutathione (GSSG). The properties of the reaction were consistent with light-coupled GSSG-reductase activity involving H₂O as eventual electron donor. The reconstituted system also supported dehydroascorbate-dependent O₂ evolution in the presence of 0.6 mM reduced glutathione (GSH) and 0.1 mM NADPH with the concomitant production of ascorbate. The GSSG could replace GSH in which case the production of GSH preceded the accumulation of ascorbate. The data are consistent with the light-dependent reduction of dehydroascorbate using H₂O as eventual electron donor via the sequence H₂O→NADP→GSSG→dehydroascorbate. Approximately 30% of the GSH-dehydrogenase activity of spinach leaf protoplasts is localised in chloroplasts: this could not be attributed to contamination of chloroplasts by activity from the extrachloroplast compartment. Washed intact chloroplasts supported the uptake of ascorbate but the uptake mechanism had a very low affinity for ascorbate (K_m approximately 20 mM). The rate of uptake of ascorbate was less than the rate of light-dependent reduction of dehydroascorbate and too slow to account for the rate of H₂O₂ reduction by washed intact chloroplasts.     

Enhanced tolerance to oxidative stress in transgenic tobacco plants expressing three antioxidant enzymes in chloroplasts

The effect of simultaneous expression of genes encoding three antioxidant enzymes, copper zinc superoxide dismutase (CuZnSOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), and dehydroascorbate (DHA) reductase (DHAR, EC 1.8.5.1), in the chloroplasts of tobacco plants was investigated under oxidative stress conditions. In previous studies, transgenic tobacco plants expressing both CuZnSOD and APX in chloroplast (CA plants), or DHAR in chloroplast showed enhanced tolerance to oxidative stresses, such as paraquat and salt. In this study, in order to develop transgenic plants that were more resistant to oxidative stress, we introduced the gene encoding DHAR into CA transgenic plants. Mature leaves of transgenic plants expressing all three antioxidant genes (CAD plants) had approximately 1.6–2.1 times higher DHAR activity, and higher ratios of reduced ascorbate (AsA) to DHA, and oxidized glutathione (GSSG) to reduced glutathione (GSH) compared to CA plants. CAD plants were more resistant to paraquat-induced stress, exhibiting only 18.1% reduction in membrane damage relative to CA plants. In addition, seedlings of CAD plants had enhanced tolerance to NaCI (100 mM) compared to CA plants. These results indicate that the simultaneous expression of multiple antioxidant enzymes, such as CuZnSOD, APX, and DHAR, in chloroplasts is more effective than single or double expression for developing transgenic plants with enhanced tolerance to multiple environmental stresses.     

Photoinactivation of ascorbate peroxidase in isolated tobacco chloroplasts: Galdieria partita APX maintains the electron flux through the water-water cycle in transplastomic tobacco plants

We evaluated the H2O2-scavenging activity of the water-water cycle (WWC) in illuminated intact chloroplasts isolated from tobacco leaves. Illumination under conditions that limited photosynthesis [red light (>640 nm), 250 micromol photons m(-2) s(-1) in the absence of HCO3-] caused chloroplasts to take up O2 and accumulate H2O2. Concomitant with the O2 uptake, both ascorbate peroxidase (APX) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) lost their activities. However, superoxide dismutase (SOD), monodehydroascorbate radical reductase (MDAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) activities remained unaffected. The extent to which the photosynthetic linear electron flow decreased was small compared with the decline in APX activity. Therefore, the loss of APX activity lowered the electron flux through the WWC, as evidenced by a decrease in relative electron flux through PSII [Phi(PSII)xPFD]. To verify these interpretations, we created a transplastomic tobacco line in which an H2O2-insensitive APX from the red alga, Galdieria partita, was overproduced in the chloroplasts. In intact transplastomic chloroplasts which were illuminated under conditions that limited photosynthesis, neither O2 uptake nor H2O2 accumulation occurred. Furthermore, the electron flux through the WWC and the activity of GAPDH were maintained. The present work is the first report of APX inactivation by endogenous H2O2 in intact chloroplasts.     

Over-expression of chloroplast-targeted Mn superoxide dismutase in cotton (Gossypium hirsutum L., cv. Coker 312) does not alter the reduction of photosynthesis after short exposures to low temperature and high light intensity

Transgenic cotton plants from several independently-transformed lines expressing a chimeric gene encoding a chloroplast-targeted Mn superoxide dismutase (SOD) from tobacco exhibit a three-fold increase in the total leaf SOD activity, strong Mn SOD activity associated with isolated chloroplasts, and a 30% and 20% increase in ascorbate peroxidase and glutathione reductase activities, respectively. The Mn SOD plants did exhibit a slightly enhanced protection against light-mediated, paraquat-induced cellular damage but only at 0.3 µM paraquat. In addition, photosynthetic rates at 10°C and 15°C were similar to those of controls, and the immediate recovery of photosynthesis after a 35-min exposure to 5°C and full sun was only slightly better than that for wild-type plants. The recovery for longer exposure times was comparable for both genotypes as was the deactivation of the H₂O₂-sensitive, Calvin-cycle enzyme, stromal fructose 1,6-bisphosphatase (FBPase). Compared to the controls, Mn SOD plant leaves in full sun prior to chilling stress had a lower activation of FBPase, a higher ratio of oxidized to reduced forms of ascorbate, and a higher total glutathione content. After 35 min at 5°C in full sunlight, total glutathione had risen in control leaves to 88% of the Mn SOD plant values, and oxidized to reduced ascorbate ratios were higher for both genotypes. However, an 80% increase in the ratio of oxidized to reduced glutathione occurred for Mn SOD plant leaves with no change for controls. This increased demand on the ascorbate-glutathione cycle is circumstantial evidence that high Mn SOD activity in the chloroplast leads to increased H₂O₂ pools that could, in some manner, affect photosynthetic recovery after a stress period. We postulate that the pool sizes of reduced ascorbate and glutathione may restrict the ability of the ascorbate-glutathione cycle to compensate for the increased activity of SOD in cotton over-producing mitochondrial Mn SOD in chloroplasts during short-term chilling/high light stress.     

Chloroplast glutathione reductase: Purification and properties

Glutathione reductase was partially purified from isolated pea chloroplasts ( Pisum sativum L. cv. Progress #9). A 1600-fold purification was obtained and the purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)⁻¹ min⁻¹. The enzyme had a native molecular weight of approximately 156 kdalton and consisted of two each of two subunits of about 41 and 42 kdalton. The K_m for oxidized glutathione was 11 μ M and the K_m for NADPH was 1.7 μ M . Enzyme activity was affected by the ionic strength of the assay medium, and maximum activity was observed at an ionic strength of between 60 and 100 m M . The enzyme was inactivated by sulfhydryl modifying reagents and the presence of either oxidized glutathione or NADPH affected the extent of inactivation. Chloroplast glutathione reductase probably serves in the removal of photosynthetically derived H₂O₂ by reducing dehydroascorbate for ascorbate-linked reduction of H₂O₂. Intermediates of this reaction sequence, dehydroascorbate, ascorbate, reduced glutathione, and NADPH had no effect on enzymic activity.     

Ascorbate distribution during hibernation is independent of ascorbate redox state

Distribution of ascorbate into tissues is an essential process in ascorbate antioxidant defense. Hibernating animals are studied as a model of tolerance to ischemia-reperfusion because of their tolerance to fluctuations in blood flow associated with prolonged torpor and periodic arousal episodes. Throughout hibernation, plasma ascorbate concentration ([Asc](p)) repetitively increases during torpor, then falls during periodic arousal bouts. We previously proposed that high [Asc](p) provides a ready source of antioxidant protection for distribution to the central nervous system and peripheral tissues during arousal. Here we tested whether deliberate oxidation of plasma ascorbate by intravenous administration of ascorbate oxidase (AO), prior to arousal, compromised tissue levels of ascorbate or the other water-soluble antioxidants, glutathione (GSH) and urate. Although AO decreased [Asc](p) to below the level of detection during torpor and after arousal, ascorbate oxidation did not decrease post-arousal tissue levels of reduced ascorbate, glutathione, or urate in any tissue examined, except liver. The data imply that ascorbate is taken up equally well into brain and other tissues as either ascorbate or its oxidized product dehydroascorbate, with subsequent intracellular reduction of dehydroascorbate. Lack of effect of ascorbate oxidation on tissue levels of GSH or urate indicates that dehydroascorbate uptake and reduction do not compromise tissue concentrations of these other water-soluble antioxidants. Thus, we show equal availability of reduced and oxidized plasma ascorbate during metabolically demanding thermogenesis and reperfusion associated with arousal from hibernation.     

Nucleoside transport in human erythrocytes. Apparent molecular weight of the nitrobenzylthioinosine-binding complex estimated by radiation-inactivation analysis.

Thiol-treated spinach (Spinacia oleracea) chloroplast fructose bisphosphatase (EC 3.1.3.11) is severely inhibited by H2O2, whereas the freshly purified enzyme is little affected. Dithiothreitol reverses inhibition by H2O2, indicating that essential thiol groups are oxidized during H2O2 inactivation. A new role for the dithiol and thioredoxin systems that are operative in illuminated chloroplasts is proposed.     

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     

Metabolic response to treatment with cold, paraquat, or 3-amino-1,2,4-triazole in leaves of winter wheat.

We treated leaves of winter wheat (Triticum aestivum L.) with cold, paraquat, or 3-amino-1,2,4-triazole and compared the responses. We assayed the activities of glucose-6-phosphate dehydrogenase, catalase, dehydroascorbate reductase and ascorbate free radical reductase and levels of hydrogen peroxide, glucose-6-phosphate, fructose-6-phosphate, ascorbate, dehydroascorbate, reduced and oxidized glutathione. With any of the three treatments, contents of cellular peroxides and hexose phosphates were raised. The content of ascorbate was lowered markedly by paraquat treatment, which produces active oxygen species, whereas such a decrease did not occur in other two treatments. When the plants were treated with 3-amino-1,2,4-triazole, which is a specific inhibitor of catalase, the content of oxidized glutathione increased severalfold. The glucose-6-phosphate dehydrogenase activity increased with all three treatments, but it decreased after glyphosate treatment, which does not stimulate the formation of peroxides. The activities of catalase and dehydroascorbate reductase were increased by the treatment of cold and paraquat, while 3-amino-1,2,4-triazole did not affect the dehydroascorbate reductase activity. The activity of ascorbate free radical reductase increased after treatment by paraquat only.     

Monodehydroascorbate Reductase in Spinach Chloroplasts and Its Participation in Regeneration of Ascorbate for Scavenging Hydrogen Peroxide

The primary reaction product of chloroplast ascorbate peroxidaseactivity was shown to be monodehydroascorbate radical (MDA).MDA reductase (EC 1.6.5.4 [EC] ) was localized in spinach chloroplaststroma. The MDA reductase activity of spinach chloroplasts,using NAD(P)H as electron donor, could account for the regenerationof ascorbate from MDA produced by ascorbate peroxidase activity.In the absence of MDA reductase, MDA disproportionated to ascorbate(AsA) and dehydroascorbate (DHA). The DHA was reduced to AsAby DHA reductase (EC 1.8.5.1 [EC] ) in chloroplasts. Both NADH andNADPH served as the electron donor of partially purified MDAreductase from spinach leaves. (Received September 24, 1983; Accepted January 23, 1984)     

Effects of artificially enhanced levels of ascorbate and glutathione on the enzymes monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase in spinach (Spinacia oleracea)

Effect of high intracellular concentrations of the antioxidants ascorbate and glutathione on the extractable activity of the reducting enzymes dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase were investigated with spinach cells ( Spinacia oleracea ). An elevated ascorbate concentration was obtained by treatment with the ascorbate biosynthesis precursor L-galactono-1,4-lactone (GAL). To increase the intracellular level of glutathione, cells were treated with the 5-oxo-L-proline analog L-2-oxothiazolidin-4-carboxylate (OTC), or with the peroxidative herbicide acifluorfen (sodium 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid). Extractable monodehydroascorbate reductase activity increased in the presence of a high level of ascorbate or glutathione, and enzyme activity was at maximum when cells were treated with acifluorfen + OTC, or acifluorfen + GAL. Extractable dehydroascorbate reductase activity decreased when the intracellular concentration of glutathione was high and non-enzymatic reduction of dehydroascorbate by glutathione was the dominant reaction. Maximal decrease of enzyme activity was found in cells treated with acifluorfen + OTC. Extractable activity of glutathione reductase (GR) increased after treatment of cells with acifluorfen alone, or acifluorfen + OTC, but enzyme activity was unaffected by a high intracellular concentration of glutathione obtained by treatment of cells with OTC alone, or by treatment with acifluorfen + GAL. The degree of GR activation seemed to be controlled by several factors including inhibition by a high concentration of glutathione and possibly oxidative damage to the enzyme. Overall, the enzymes tested in this study, which provide the reduced forms of ascorbate and glutathione, were differently affected by high antioxidant levels.     

18O isotopic 13C NMR shift as proof that bifunctional peptidylglycine alpha-amidating enzyme is a monooxygenase.

The biosynthesis of C-terminal alpha-amidated peptides from their corresponding C-terminal glycine-extended precursors is catalyzed by peptidylglycine alpha-amidating enzyme (alpha-AE) in a reaction that requires copper, ascorbate, and molecular oxygen. Using bifunctional type A rat alpha-AE, we have shown that O2 is the source of the alpha-carbonyl oxygen of pyruvate produced during the amidation of dansyl-Tyr-Val-[alpha-13C]-D-Ala, as demonstrated by the 18O isotopic shift in the 13C NMR spectrum of [alpha-13C]lactate generated from [alpha-13C]pyruvate in the presence of lactate dehydrogenase and NADH. In addition, one-to-one stoichiometries have been determined for glyoxylate formed/dansyl-Tyr-Val-Gly consumed, pyruvate formed/dansyl-Tyr-Val-D-Ala consumed, dansyl-Tyr-Val-NH2 formed/ascorbate oxidized, and dansyl-Tyr-Val-NH2 formed/O2 consumed. Quantitative coupling of NADH oxidation to dansyl-Tyr-Val-NH2 production using Neurospora crassa semidehydroascorbate reductase showed that two one-electron reductions by ascorbate occurred per alpha-AE turnover. The stoichiometry of approximately 1.0 dansyl-Tyr-Val-NH2 produced/ascorbate oxidized observed in the absence of a semidehydroascorbate trap resulted from the disproportionation of two semidehydroascorbate molecules to ascorbate and dehydroascorbate.     

Enhanced sensitivity to oxidative stress in transgenic tobacco plants with decreased glutathione reductase activity leads to a decrease in ascorbate pool and ascorbate redox state

To investigate the possible mechanisms of glutathione reductase (GR) in protecting against oxidative stress, we obtained transgenic tobacco (Nicotiana tabacum) plants with 30–70% decreased GR activity by using a gene encoding tobacco chloroplastic GR for the RNAi construct. We investigated the responses of wild type and transgenic plants to oxidative stress induced by application of methyl viologen in vivo. Analyses of CO₂ assimilation, maximal efficiency of photosystem II photochemistry, leaf bleaching, and oxidative damage to lipids demonstrated that transgenic plants exhibited enhanced sensitivity to oxidative stress. Under oxidative stress, there was a greater decrease in reduced to oxidized glutathione ratio but a greater increase in reduced glutathione in transgenic plants than in wild type plants. In addition, transgenic plants showed a greater decrease in reduced ascorbate and reduced to oxidized ascorbate ratio than wild type plants. However, there were neither differences in the levels of NADP and NADPH and in the total foliar activities of monodehydroascorbate reductase and dehydroascorbate reductase between wild type and transgenic plant. MV treatment induced an increase in the activities of GR, ascorbate peroxidase, superoxide dismutase, and catalase. Furthermore, accumulation of H₂O₂ in chloroplasts was observed in transgenic plants but not in wild type plants. Our results suggest that capacity for regeneration of glutathione by GR plays an important role in protecting against oxidative stress by maintaining ascorbate pool and ascorbate redox state.     

The effect of Botrytis cinerea infection on the antioxidant profile of mitochondria from tomato leaves

Infection of tomato leaves with the necrotrophic fungus Botrytis cinerea resulted in substantial changes in enzymatic and non-enzymatic components of the ascorbate-glutathione cycle as well as in superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione transferase (GST), and l-galactono-gamma-lactone dehydrogenase (GLDH) activities. In the initial phase of the 5 d experiment CuZn SOD was the most rapidly induced isoform (up to 209% of control), whereas later on its activity increase was not concomitant with the constant total SOD enhancement. Starting from the second day B. cinerea infection diminished the mitochondrial antioxidant capacity by decreasing activities of ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) as well as declining ascorbate and glutathione contents. This was accompanied by dehydroascorbate (DHA) and oxidized glutathione (GSSG) accumulation that resulted in ascorbate and glutathione redox ratios decreases. The strongest redox ratio decline of 29% for ascorbate and of 34% for glutathione was found on the 3rd and 2nd days, respectively. Glutathione reductase (GR) induction (185% of control 2 d after inoculation) was insufficient to overcome the decreased antioxidant potential of glutathione. Changes in the ascorbate pool size were closely related to the activity of l-galactono-gamma-lactone dehydrogenase (GLDH). The activities of two glutathione-dependent enzymes: GSH-Px and GST were increased from day 1 to day 4. These results demonstrated that in B. cinerea-tomato interaction mitochondria could be one of the main targets for infection-induced oxidative stress.     

Spermidine application enhances tomato seedling tolerance to salinity-alkalinity stress by modifying chloroplast antioxidant systems

The purpose of this study was to elucidate whether exogenous spermidine (Spd) protection of tomato (Solanum lycopersicum L.) seedlings under salinity-alkalinity stress is associated with antioxidant enzymes in the chloroplast. The effects of exogenous Spd on antioxidant enzyme activity and antioxidant content in the chloroplast were evaluated in seedlings of salt-sensitive ecotype (Zhongza 9) grown in a 75 mM salinity-alkalinity solution, with or without 0.25 mM Spd foliar spraying. Results showed that salinity-alkalinity stress increased MDA content, superoxide anion O₂^?- generation rate, superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR) activities and ratio of AsA/DHA and reduced contents of ascorbate (AsA), dehydroascorbate (DHA), AsA+DHA, glutathione (GSH), oxidized glutathione (GSSG), GSH+GSSG, dehydroascorbate reductase (DHAR) activity and ratio of GSH/GSSG in chloroplasts. The exogenous Spd application combined with salinity-alkalinity stress decreased the O₂^?- generation rate and MDA content compared to salinity-alkalinity stress alone. The exogenous Spd also increased AsA-GSH cycle components and increased all antioxidant enzyme activities in most cases. Therefore, exogenous Spd alleviates salinity-alkalinity stress damage using antioxidant enzymes and non-enzymatic systems in chloroplasts.     

Action of calcium ions on spinach (Spinacia oleracea) chloroplast fructose bisphosphatase and other enzymes of the Calvin cycle.

Thiol-treated spinach (Spinacia oleracea) chloroplast fructose bisphosphatase is powerfully inhibited by Ca2+ non-competitively with respect to its substrate, fructose 1,6-bisphosphate. 500 microM-Ca2+ causes virtually complete inhibition and the Ki is 40 microM. Severe inhibition of sedoheptulose bisphosphatase is also caused by Ca2+. A role for Ca2+ in regulation of the Calvin cycle in spinach chloroplasts is proposed.     

Alterations in the antioxidative system of suspension-cultured soybean cells (Glycine max) induced by oxidative stress

The objectives of this study were the changes of antioxidative key enzyme activities under stress conditions induced by a peroxidizing herbicide using photoheterotrophi-cally grown, suspension-cultured soybean celts ( Glycine max L.). Within two days, 50 to 500 n M oxyfluorfen. a p-nitrodiphenyl ether herbicide, caused up to 100% inhibition of growth, while simultaneously, the chlorophyll was 25% to completely bleached. The major cellular antioxidants ascorbate and glutathione showed different responses. Under stress conditions with more than 250 n M oxyfluorfen, the cellular ascorbate- concentration was halved, whereas dehydroascorbate remained roughly constant. The glutathione content (approximately one-fifth of that of ascorbate in untreated control cells) increased nearly 3-fold in the presence of 250 n M oxyfluorfen. Under this condition, oxidized glutathione was 5 times above the control level. The specific activities of selected enzymes participating in cellular defence, namely ascor-bate peroxidase, glutathione reductase, rnonodehydroascorbate reductase. peroxidase and catalase increased by 40 to 70% with oxyfluorfen concentrations between 50 and 500 n M , while dehydroascorbate reductase showed a significant decrease. Glutathione transferase activity even increased 6-fold under oxyfluorfen stress.