Monodehydroascorbate Radical Reductase Research Articles

Free radical scavenging capacity and antioxidant enzyme activity in deerberry ( Vaccinium stamineum L.)

Fruit from three genotypes (‘B-76’, ‘B-59’ and ‘SHF-3A’) of deerberry [ Vaccinium stamineum L.] were evaluated for fruit quality, total anthocyanin and phenolic contents, antioxidants, antioxidant capacity, and antioxidant enzyme activity. The fruit soluble solids, titratable acids, total anthocyanins, and total phenolic contents varied with genotypes. Cyanidin 3-galactoside and cyanidin 3-arabinoside were the two predominant anthocyanins. Resveratrol was also found in deerberries. Among the three genotypes, ‘B-76’ had higher amount of anthocyanins, phenolic compounds and resveratrol than ‘B-59’ and ‘SHF-3A’. Deerberries contained potent free radical scavenging activities for 2,2-Di (4- tert-octylphenyl)-1-picrylhydrazyl (DPPH ), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS + ), peroxyl radical (ROO ), superoxide radicals ( O 2 - ), hydrogen peroxide (H 2O 2), hydroxyl radicals ( OH), and singlet oxygen ( 1O 2) radicals and also had high activities of antioxidant enzymes including glutathione-peroxidase (GSH-POD), glutathione reductase (GR), superoxide dismutase (SOD), ascorbate peroxidase (AsA-POD), guaiacol peroxidase (G-POD), monodehydroascorbate radical reductase (MDAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR)] and non-enzyme antioxidants [ascorbic acid (ASA) and reduced glutathione (GSH)]. Antioxidant capacities were highly correlated to antioxidant enzymes activities. Among the three genotypes, ‘B-76’ had the highest level of antioxidants and antioxidant enzyme activity.

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.

Purification and cDNA Cloning of Chloroplastic Monodehydroascorbate Reductase from Spinach

The chloroplastic isoform of monodehydroascorbate (MDA) radical reductase was purified from spinach chloroplasts and leaves. The cDNA of chloroplastic MDA reductase was cloned, and its deduced amino acid sequence, consisting of 497 residues, showed high homology with those of putative organellar MDA reductases deduced from cDNAs of several plants. The amino acid sequence of the amino terminal of the purified enzyme suggested that the chloroplastic enzyme has a transit peptide consisting of 53 residues. A southern blot analysis suggested the occurrence of a gene encoding another isoform homologous to the chloroplastic isoform in spinach. The recombinant enzyme was highly expressed in Eschericia coli using the cDNA, and purified to a homogeneous state with high specific activity. The enzyme properties of the chloroplastic isoform are presented in comparison with those of the cytosolic form.

Crystallization and preliminary crystallographic analysis of monodehydroascorbate radical reductase from cucumber.

Monodehydroascorbate (MDA) radical reductase (EC 1.6.5.4) is an FAD enzyme that catalyzes the univalent reduction of MDA radical to ascorbate using NAD(P)H as an electron donor. The recombinant MDA reductase from cucumber was crystallized using polyethylene glycol 6000 as a precipitant. The crystals belong to space group P2(1), with unit-cell parameters a = 60.8, b = 138.6, c = 61.7 A, beta = 114.5 degrees, and contained two molecules per asymmetric unit. The Matthews coefficient (VM) and the solvent content are 2.46 A3 Da(-1) and 50.0%, respectively. Diffraction data were collected to a resolution of 2.4 A at 100 K using Cu Kalpha radiation with a multi-wire area detector and gave a data set with an overall Rsym of 10.0% and a completeness of 92.5%.

Ascorbate in thylakoid lumen functions as an alternative electron donor to photosystem II and photosystem I

The roles of ascorbate (Asc) in the thylakoid lumen to support photosynthetic electron transport are investigated. Asc can be photooxidized in photosystem (PS) II and PSI. When the water oxidase complex (WOC) was inactivated by acidic pH or by UV-B, Asc was photooxidized in PSII with Asc replacing water as the electron donor. An apparent K m was 2.5 mM. At 20 mM Asc, the electron transport rate reached 50 μmol NADP +-reduced mg Chl −1 h −1. Asc was not oxidized by the PSII reaction center complex having an active WOC, and hence was suggested to function as an emergency donor only when WOC is inactivated. In the presence of 3-(3,4-dichlorophenyl)-1,1 ′-dimethylurea, Asc was photooxidized by PSI, with a lower affinity and an electron transport rate of 70 μmol NADP +-reduced mg Chl −1 h −1 in 50 mM Asc. Thus, Asc can support a PSI-mediated electron flow at a reasonably high rate at Asc concentrations in the physiologically relevant range. During the photooxidation of Asc by PSI, we observed the production of the monodehydroascorbate radical (MDA) that was unscavenged by the exogenously added MDA reductase. Reduction of P700 + by Asc was not affected by the inactivation of plastocyanin with cyanide. These results indicated that Asc was univalently oxidized on the lumenal side of PSI, directly by P700 +. The electron transport Asc → PSI → NADP + did not form a proton gradient across the thylakoid membrane, as determined by 9-aminoacridine fluorescence. Based on these results, we propose that the Asc-dependent cyclic electron flow around PSI, and that through both PSI and PSII can operate when the linear electron transport is partially impaired.

Xanthophyll cycle pigments and water-water cycle in transgenic rice with decreased amounts of ribulose-1,5-bisphosphate carboxylase and the wild-type rice grown under different N levels

Chlorophyll (Chl) fluorescence, gas exchange rates, the amounts of xanthophyll cycle pigments, and the activities of several antioxidant enzymes including superoxide dismutase, ascorbate peroxidase, glutathione reductase, and monodehydroascorbate radical reductase were examined in the leaves of wild-type rice (Oryza sativa L.) and rbcS antisense plants grown under different N levels. The decrease in the CO2 assimilation capacity by the introduction of the rbcS antisense gene and N deficiency was closely related to the decrease in the quantum yield of photo system (PS) II (φPSII) and the enhancement of non-photochemical quenching (NPQ). No differences in the relationships between the electron transport rates from Chl fluorescence and those from gas exchange were found between the wild-type and rbcS antisense plants and there were no differences in the activities of all the antioxidant enzymes per unit of Chl examined. Although a remarkable increase in NPQ was found for the rbcS antisense and N-deficient wild-type plants, the amounts of the total xanthophyll cycle pigments per unit of Chl remained constant in all the plants. NPQ was highly correlated with only the ratio of antheraxanthin plus zeaxanthin to total xanthophyll cycle pigments. In addition, this ratio was negatively correlated with Rubisco content, irrespective of the genotype and N treatment. The results indicate that the low capacity for CO2 assimilation and photorespiration by the introduction of the rbcS antisense gene and N deficiency did not affect the electron flow in the water-water cycle but enhanced the deepoxidation state of the xanthophyll cycle pigments.

Characterisation of antioxidative systems in the ectomycorrhiza-building basidiomycete Paxillus involutus (Bartsch) Fr. and its reaction to cadmium

The effect of different cadmium (Cd) concentrations (5, 50 and 500 microM) on growth, Cd accumulation and antioxidative systems was studied in Paxillus involutus, grown in liquid medium. Cd was rapidly accumulated by P. involutus and resulted in growth inhibition within 24 h. Antioxidative enzymes (superoxide dismutase (SOD), EC 1.15.1.1; catalase (CAT), EC 1.11.1.6; monodehydroascorbate radical reductase (MDAR), EC 1.6.5.4; dehydroascorbate reductase (DAR) glutathione reductase (GR), EC 1.8.1.7 and glutathione-dependent peroxidase (GPx), EC 1.11.1.9) were active in the investigated fungus. Furthermore, high concentrations of glutathione but no ascorbate were detected. Cd exposure resulted in a significant induction of SOD activity. However, activities of enzymes responsible for the detoxification of H2O2 showed no Cd-dependent increase or were only transiently induced (CAT, GPx) and no accumulation of H2O2 was detected. Exposure to low Cd concentrations (5 and 50 microM) caused an increase in GR, while 500 microM Cd led to an inhibition of GR and CAT. Increased glutathione concentrations were observed as a consequence of all Cd treatments. These results suggest that the antioxidative protection of the investigated strain of P. involutus was sufficient to avoid Cd-mediated oxidative stress. It is likely that this strain was able to detoxify high concentrations of Cd by transport of Cd into the vacuole because a high correlation between Cd and sulphur in the vacuole was detected by EDX.

Characterisation of antioxidative systems in the ectomycorrhiza-building basidiomycete Paxillus involutus (Bartsch) Fr. and its reaction to cadmium

The effect of different cadmium (Cd) concentrations (5, 50 and 500 μM) on growth, Cd accumulation and antioxidative systems was studied in Paxillus involutus, grown in liquid medium. Cd was rapidly accumulated by P. involutus and resulted in growth inhibition within 24 h. Antioxidative enzymes (superoxide dismutase (SOD), EC 1.15.1.1; catalase (CAT), EC 1.11.1.6; monodehydroascorbate radical reductase (MDAR), EC 1.6.5.4; dehydroascorbate reductase (DAR) glutathione reductase (GR), EC 1.8.1.7 and glutathione-dependent peroxidase (GPx), EC 1.11.1.9) were active in the investigated fungus. Furthermore, high concentrations of glutathione but no ascorbate were detected. Cd exposure resulted in a significant induction of SOD activity. However, activities of enzymes responsible for the detoxification of H 2O 2 showed no Cd-dependent increase or were only transiently induced (CAT, GPx) and no accumulation of H 2O 2 was detected. Exposure to low Cd concentrations (5 and 50 μM) caused an increase in GR, while 500 μM Cd led to an inhibition of GR and CAT. Increased glutathione concentrations were observed as a consequence of all Cd treatments. These results suggest that the antioxidative protection of the investigated strain of P. involutus was sufficient to avoid Cd-mediated oxidative stress. It is likely that this strain was able to detoxify high concentrations of Cd by transport of Cd into the vacuole because a high correlation between Cd and sulphur in the vacuole was detected by EDX.

Plant phenolic antioxidant and prooxidant activities: phenolics-induced oxidative damage mediated by metals in plants

Plant phenolic compounds such as flavonoids and lignin precursors are important constituents of the human diet. These dietary phytophenolics have been recognized largely as beneficial antioxidants that can scavenge harmful active oxygen species including O 2 −, H 2O 2, OH, and 1O 2. Here we review our current understanding of the antioxidant and prooxidant actions of phenolics in plant cells. In plant systems, phytophenolics can act as antioxidants by donating electrons to guaiacol-type peroxidases (GuPXs) for the detoxification of H 2O 2 produced under stress conditions. As a result of such enzymatic as well as non-enzymatic antioxidant reactions, phenoxyl radicals are formed as the primary oxidized products. Until recently, phenoxyl radicals had been difficult to detect by static electron spin resonance (ESR) because they rapidly change to non-radical products. Application of Zn exerts spin-stabilizing effects on phenoxyl radicals that enables us to analyze the formation and decay kinetics of the radicals. The ESR signals of phenoxyl radicals are eliminated by monodehydroascorbate radical (MDA) reductase, suggesting that phenoxyl radicals, like the ascorbate radical, are enzymatically recycled to parent phenolics. Thus, phenolics in plant cells can form an antioxidant system equivalent to that of ascorbate. In contrast to their antioxidant activity, phytophenolics also have the potential to act as prooxidants under certain conditions. For example, flavonoids and dihydroxycinnamic acids can nick DNA via the production of radicals in the presence of Cu and O 2. Phenoxyl radicals can also initiate lipid peroxidation. Recently, Al, Zn, Ca, Mg and Cd have been found to stimulate phenoxyl radical-induced lipid peroxidation. We discuss the mechanism of phenoxyl radical prooxidant activity in terms of lifetime prolongation by spin-stabilizing agents.

Cadmium and H 2O 2-induced oxidative stress in Populus × canescens roots

Clonal, hydroponically grown poplar plants ( Populus × canescens, a hybrid of Populus tremula × Populus alba) were exposed to Cd or H 2O 2 to find out whether Cd-induced injury was related to the disturbance of the cellular redox control in root tips. Cd exposure resulted in an inhibition of antioxidative enzymes (superoxide dismutase, EC 1.15.1.1; catalase, EC 1.11.1.6; ascorbate peroxidase, EC 1.11.1.11; monodehydroascorbate radical reductase, EC 1.1.5.4; glutathione reductase, EC 1.6.4.2) but had fewer effects on dehydroascorbate reductase (EC 1.8.5.1) activities. Glutathione concentrations decreased, whereas ascorbate remained unaffected by Cd. Five micromoles of Cd were subinjurious in short-term experiments and stimulated root growth. Fifty micromoles of Cd retarded shoot growth faster than root growth, caused a more severe loss in antioxidative capacity than 5 μM Cd and resulted in an accumulation of H 2O 2 in roots. Exposure to H 2O 2 had an effect on antioxidative enzymes similar to that found under the influence of Cd, but caused GSH accumulation, and loss of ascorbate. The present data indicate that both agents acted via the disturbance of the cellular redox control.

Differential temperature dependencies of antioxidative enzymes in two contrasting species: Fagus sylvatica and Coleus blumei

In order to characterise the sensitivity of antioxidative systems to temperature-induced oxidative stress, two species ( Coleus blumei and Fagus sylvatica, L.) representative of environments with contrasting temperature characteristics have been exposed to low or high temperatures of 10 or 35 °C, respectively. Beech leaves were harvested in light and darkness. Coleus leaves were separated into green and white leaf tissue. The thermal dependencies of the activities of protective enzymes and chlorophyll fluorescence over a temperature range from 10 to 35 °C were determined. Ascorbate peroxidase activities were activated at low temperatures in vitro and, thereby, may provide an instantaneous protection against H 2O 2 accumulation which is faster than de novo synthesis. Monodehydroascorbate radical reductase was apparently not involved in short-term acclimation to low or high temperature. After short-term acclimation to low temperature, glutathione reductase and glutathione were more diminished in Coleus than in beech. Both species contained higher concentrations of ascorbate and glutathione at high temperatures than at low temperatures whereas glutathione reductase activity increased. Ascorbate peroxidase activity from Coleus leaves, though detectable under standard assay conditions (25 °C), failed at 35 °C in vitro. The results suggest that the higher temperature susceptibility of Coleus than that of beech was associated with a differential loss in glutathione reductase/glutathione at low temperature and an inhibition of ascorbate peroxidase at high temperature. Since the thermal dependencies of antioxidative enzymes were significantly affected by the preceding environmental conditions, the relative enzymatic activities determined under standard assay conditions may not be representative of enzymatic activities in foliage exposed to varying environmental temperatures.

Dissecting the superoxide dismutase-ascorbate-glutathione-pathway in chloroplasts by metabolic modeling. Computer simulations as a step towards flux analysis.

The present study introduces metabolic modeling as a new tool to analyze the network of redox reactions composing the superoxide dismutase-ascorbate (Asc)-glutathione (GSH) cycle. Based on previously determined concentrations of antioxidants and defense enzymes in chloroplasts, kinetic properties of antioxidative enzymes, and nonenzymatic rate constants of antioxidants with reactive oxygen, models were constructed to simulate oxidative stress and calculate changes in concentrations and fluxes of oxidants and antioxidants. Simulated oxidative stress in chloroplasts did not result in a significant accumulation of O2*- and H2O2 when the supply with reductant was sufficient. Model results suggest that the coupling between Asc- and GSH-related redox systems was weak because monodehydroascorbate radical reductase prevented dehydroascorbate (DHA) formation efficiently. DHA reductase activity was dispensable. Glutathione reductase was mainly required for the recycling of GSH oxidized in nonenzymatic reactions. In the absence of monodehydroascorbate radical reductase and DHA reductase, glutathione reductase and GSH were capable to maintain the Asc pool more than 99% reduced. This suggests that measured DHA/Asc ratios do not reflect a redox balance related to the Asc-GSH-cycle. Decreases in Asc peroxidase resulted in marked H2O2 accumulation without significant effects on the redox balance of Asc/DHA or GSH/GSSG. Simulated loss of SOD resulted in higher H2O2 production rates, thereby affecting all subsequent steps of the Asc-GSH-cycle. In conclusion, modeling approaches contribute to the theoretical understanding of the functioning of antioxidant systems by pointing out questions that need to be validated and provide additional information that is useful to develop breeding strategies for higher stress resistance in plants.

Diurnal fluctuations of antioxidative systems in leaves of field‐grown beech trees (Fagus sylvatica): Responses to light and temperature

To test the hypothesis that antioxidant systems flexibly adjust to short‐term, diurnal fluctuations of ambient environmental conditions, ascorbate‐related systems were studied over several day/night cycles in mature sun‐acclimated leaves of field‐grown beech trees (Fagus sylvatica). Light‐dependent increases in the activities of ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate radical reductase (MDAR, EC 1.1.5.4) and glutathione reductase (GR, EC 1.6.4.2) were not observed. Lowest activities of APX and MDAR were found on hot, sunny days. A strong negative correlation occurred between APX activities and ambient temperatures suggesting that this enzyme was temperature‐ rather than light‐regulated. In contrast to the enzymatic defences, ascorbate levels increased by about 30% under bright sunlight suggesting that protection from excess light is mediated via the adjustment of metabolites. Under these conditions the apparent electron transport rate exceeded the capacity for assimilation and the dehydroascorbate pool increased twofold. Since dehydroascorbate reductase activities were hardly detected, MDAR activities seemed to be the major enzyme to keep ascorbate in its reduced state. However, MDAR appeared to be insufficient to maintain the redox balance of the ascorbate pool under high light intensities in the field.

Chloroplastic monodehydroascorbate radical reductase from spinach,Chloroplastic monodehydroascorbate radical reductase from spinach

Active oxygens are inevitably produced in chloroplats with photosynthesis. In the water-water cycle, ascorbate (AsA) is univalently oxidized to monodehydroascorbate (MDA) radical with scavenging hydrogen peroxide by AsA peroxidase and regenerated via three pathways. MDA radical reductase (MDAR), which contributes to one of these pathways, catalyzes the reduction of MDA radical to AsA using NAD(P)H as the electron donor. Cytosolic MDARs have been purified and characterized, though chloroplastic isoform has never been. We purified MDAR from stromal fraction of intact chloroplasts isolated from spinach. And then we also succeeded to purify chloroplastic isoform from crude extract of spinach leaves, which contained two major MDARs. Its molecular mass was 51 kDa as determined by SDS-PAGE, being 4 kDa larger than that of cytosolic MDAR. Its Km values for NADH and NADPH were 5 µM and 12 µM, respectively. The chloroplastic isoform showed the similar inhibition by SH-modifier and dicumarol to those of cytosolic isoform. The amino acid sequence of the chloroplastic MDAR was determined from the amino termini to 25th residues. The cDNA cloned with oligonucleotide corresponded to the amino-terminal region contained an open reading frame of 1,494 bp length. The open reading frame encoded the region expected to transit peptide consisting of 56 residues. The predicted amino acid sequence of the region expected to mature chloroplastic MDAR contains the FAD- and NAD(P)H-binding domains of flavoproteins and shows partial homology with cytosolic isozymes.

Differential stress responses of antioxidative systems to drought in pendunculate oak (Quercus robur) and maritime pine (Pinus pinaster) grown under high CO2 concentrations

Maritime pine (Pinus pinaster), a drought-avoiding species, contained 2--4-fold lower activities of superoxide dismutase, ascorbate peroxidase, catalase, dehydroascorbate reductase, and glutathione reductase than pendunculate oak (Quercus robur), a drought-tolerant species. The levels of ascorbate, monodehydroascorbate radical reductase activity, and glutathione in pine needles were similar to those in oak leaves. In both species the development of drought stress, characterized by decreasing predawn water potentials, caused gradual reductions in antioxidant protection, increased lipid peroxidation, increased oxidation of ascorbate and glutathione and in pine also significant loss in soluble proteins and carotenoids. These results support the idea that increased drought-tolerance in oak as compared with pine is related to increased biochemical protection at the tissue level. To test the hypothesis that elevated CO(2) ameliorated drought-induced injury, young oak and pine trees acclimated to high CO(2) were subjected to drought stress. Analysis of plots of enzymatic activities and metabolites against predawn water potentials revealed that the drought stress-induced decreases in antioxidant protection and increases in lipid peroxidation were dampened at high CO(2). In pine, protein and pigment degradation were also slowed down. At high CO(2), superoxide dismutase activities increased transiently in drought-stressed trees, but collapsed in pine faster than in oak. These observations suggest that the alleviation of drought-induced injury under elevated CO(2) is related to a higher stability of antioxidative enzymes and an increased responsiveness of SOD to stressful conditions. This ameliorating mechanism existed independently from the effects of elevated CO(2) on plant water relations and is limited within a species-specific metabolic window.

Antioxidative systems, pigment and protein contents in leaves of adult mediterranean oak species (Quercus pubescens and Q. ilex) with lifetime exposure to elevated CO2.

The aim of the present study was to investigate the effects of elevated CO2 on the antioxidative systems and the contents of pigments, soluble protein and lipid peroxidation in leaves of adult oaks, Quercus pubescens and Quercus ilex, grown at naturally enriched CO2 concentrations. For this purpose, a field study was conducted at two CO2 springs in Central Italy. Measurements of the pre-dawn water potentials indicated less drought stress in trees close to CO2 springs than in those grown at ambient CO2 concentrations. Most leaf constituents investigated showed significant variability between sampling dates, species and sites. The foliar contents of protein and chlorophylls were not affected in trees grown close to the CO2 vents compared with those in ambient conditions. Increases in glutathione and other soluble thiols were observed, but these responses might have been caused by a low pollution of the vents with sulphurous gases. At CO2 vents, glutathione reductase was unaffected, and superoxide dismutase activity was significantly diminished, in both species. Generally, the activities of catalase, guaiacol peroxidase and ascorbate peroxidase as well as the sum of dehydroascorbate and ascorbate were decreased in leaves from trees grown in naturally CO2 -enriched environments compared with those grown at ambient CO2 concentrations. The reduction in protective enzymes did not result in increased lipid peroxidation, but increased monodehydroascorbate radical reductase and dehydroascorbate reductase activities found in leaves of Q. pubescens suggest that the smaller pool of ascorbate was subjected to higher turnover rates. These data show that changes in leaf physiology persist, even after lifetime exposure to enhanced atmospheric CO2 . The results suggest that the down-regulation of protective systems, which has also previously been found in young trees or seedlings under controlled exposure to elevated CO2 concentrations, might reflect a realistic response of antioxidative defences in mature trees in a future high-CO2 world.

The FAD-Enzyme Monodehydroascorbate Radical Reductase Mediates Photoproduction of Superoxide Radicals in Spinach Thylakoid Membranes

The photoreduction of dioxygen in spinach thylakoid membranes was enhanced about 10-fold by the FAD-enzyme monodehydroascorbate radical (MDA) reductase at 1 μM. The primary photoreduced product of dioxygen catalyzed by MDA reductase was the superoxide radical, as evidenced by the inhibition of photoreduction of Cyt c by superoxide dismutase. The apparent Km for dioxygen of the MDA reductase-dependent photoreduction of dioxygen was 100 μM, higher by one order of magnitude than that observed with thylakoid membranes only. Glutathione reductase, ferredoxin-NADP+ reductase, and glycolate oxidase also mediated the photoproduction of superoxide radicals in thylakoid membranes at rates similar to those with MDA reductase. Among these flavoenzymes, MDA reductase is the most likely mediator stimulating the photoreduction of dioxygen in chloroplasts; its function in the protection from photoinhibition under excess light is discussed.

Seasonal Fluctuations of Ascorbate-Related Enzymes: Acute and Delayed Effects of Late Frost in Spring on Antioxidative Systems in Needles of Norway Spruce (Picea abies L.)

Seasonal changes of ascorbate peroxidase and monodehydroascorbate radical reductase activities were studied in foliar tissues of Norway spruce {Picea abies L.). In mature needles, APX activities did not show seasonal fluctuations and were similar to those found in resting buds. Monodehydroascorbate radical reductase activity was higher in needles than in buds and higher in winter than in summer. Maximum activities of both enzymes were found before bud break and minimum activities in newly formed needles. When spruce seedlings were exposed to an artifical frost event of — 5°C for one night in spring, ascorbate peroxidase activity declined in young needles before the onset of visible injury but corresponding to a sudden upsurge in lipid peroxidation. After one week, some shoots showed severe symptoms of injury, some were slightly injured and others did not show any visible injury. In lethally injured needles, antioxidative protection (ascorbate peroxidase, monodehydroascorbate radical reductase, glutathione reductase, glutathione, ascorbate, superoxide dismutase) had collapsed. Surviving needles showed a coordinated increase in all components of the antioxidative system suggesting an efficient induction of defense systems. However, enhanced protection was observed only transiently. In fall, needles that had been exposed to frost in spring contained significantly less antioxidants than unstressed needles indicating that unseasonal frost caused memory effects.

Responses of Antioxidative Systems to Drought Stress in Pendunculate Oak and Maritime Pine as Modulated by Elevated CO2.

The aim of the present study was to investigate the effects of an enhanced CO2 concentration alone or in combination with drought stress on antioxidative systems of a deciduous (oak; Quercus robur) and an evergreen (pine; Pinus pinaster) tree species. The seedlings were grown for one season in a greenhouse in tunnels supplied with 350 or 700 [mu]L L-1 CO2. The experiment was repeated in a second year. Antioxidants, protective enzymes, soluble protein, and pigments showed considerable fluctuations in different years. Elevated CO2 caused significant reductions in the activities of superoxide dismutases in both oak and pine. The activities of ascorbate peroxidase and catalase were also reduced in most cases. The activities of dehydroascorbate reductase, monodehydroascorbate radical reductase, glutathione reductase, and guaiacol peroxidase were affected little or not at all by elevated CO2. When the trees were subjected to drought stress by withholding water, the activities of antioxidative enzymes decreased in leaves of pine and oak grown at ambient CO2 and increased in plants grown at elevated CO2 concentrations. The present results suggest that growth in elevated CO2 might reduce oxidative stress to which leaf tissues are normally exposed and enhance metabolic flexibility to encounter increased stress by increases in antioxidative capacity.

Molecular Characterization of Monodehydroascorbate Radical Reductase from Cucumber Highly Expressed in Escherichia coli

Monodehydroascorbate radical (MDA) reductase, an FAD-enzyme, is the first enzyme to be identified whose substrate is an organic radical and catalyzes the reduction of MDA to ascorbate by NAD(P)H. Its cDNA has been cloned from cucumber seedlings (Sano, S., and Asada, K. (1994) Plant Cell Physiol. 35, 425-437), and a plasmid was constructed in the present study that allowed a high level expression in Escherichia coli of the cDNA-encoding MDA reductase using the T7 RNA polymerase expression system. The recombinant MDA reductase was purified to a crystalline state, with a yield of over 20 mg/liter of culture, and it exhibited spectroscopic properties of the FAD similar to those of the enzyme purified from cucumber fruits during redox reactions with NADH and MDA. The red semiquinone of the FAD of MDA reductase was generated by photoreduction. p-Chloromercuribenzoate inhibited the reduction of the enzyme-FAD by NADH, and dicumarol suppressed electron transfer from the reduced enzyme to MDA. The specificity of electron acceptors of the recombinant enzyme appeared to be similar to that of MDA reductase, even though the amino acid sequence encoded by the cDNA was somewhat different from that of the enzyme purified from cucumber fruits. The Km values for NADH and NADPH of the recombinant enzyme indicated a high affinity of the enzyme for NADH. The reaction catalyzed by the enzyme did not exhibit saturation kinetics with MDA up to 3 microM. A second order rate constant for the reduction of the enzyme-FAD with NADH was 1.25 x 10(8) M-1 s-1, as determined by a stopped-flow method, and its value decreased with increases in ionic strength, an indication of the enhanced electrostatic guidance of NADH to the enzyme-FAD.