Dehydroascorbic Acid Reductase Research Articles

Involvement of Nitric Oxide and Melatonin Enhances Cadmium Resistance of Tomato Seedlings through Regulation of the Ascorbate-Glutathione Cycle and ROS Metabolism.

Melatonin (MT) and nitric oxide (NO) act as signaling molecules that can enhance cadmium (Cd) stress resistance in plants. However, little information is available about the relationship between MT and NO during seedling growth under Cd stress. We hypothesize that NO may be involved in how MT responds to Cd stress during seedling growth. The aim of this study is to evaluate the relationship and mechanism of response. The results indicate that different concentrations of Cd inhibit the growth of tomato seedlings. Exogenous MT or NO promotes seedling growth under Cd stress, with a maximal biological response at 100 μM MT or NO. The promotive effects of MT-induced seedling growth under Cd stress are suppressed by NO scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO), suggesting that NO may be involved in MT-induced seedling growth under Cd stress. MT or NO decreases the content of hydrogen peroxide (H2O2), malonaldehyde (MDA), dehydroascorbic acid (DHA), and oxidized glutathione (GSSG); improves the content of ascorbic acid (AsA) and glutathione (GSH) and the ratios of AsA/DHA and GSH/GSSG; and enhances the activities of glutathione reductase (GR), monodehydroascorbic acid reductase (MDHAR), dehydroascorbic acid reductase (DHAR), ascorbic acid oxidase (AAO), and ascorbate peroxidase (APX) to alleviate oxidative damage. Moreover, the expression of genes associated with the ascorbate-glutathione (AsA-GSH) cycle and reactive oxygen species (ROS) are up-regulated by MT or NO under Cd conditions, including AAO, AAOH, APX1, APX6, DHAR1, DHAR2, MDHAR, and GR. However, NO scavenger cPTIO reverses the positive effects regulated by MT. The results indicate that MT-mediated NO enhances Cd tolerance by regulating AsA-GSH cycle and ROS metabolism.

2-hydroxy-3-phenylpropanoic acid suppressed the growth of Alternaria alternata through damaging cell membrane integrity and modulating reactive oxygen species metabolism

Black spot rot caused by Alternaria alternata is one of the major postharvest disease of apple fruit during logistic. This study evaluated in vitro inhibitory effect of 2-hydroxy-3-phenylpropanoic acid (PLA) at various concentrations on A. alternata and the possible mechanisms involved in its action. Results showed that different concentrations of PLA inhibited conidia germination and mycelial growth of A. alternata in vitro, and 1.0 g L−1 was the lowest effective concentration to suppress A. alternata growth. Moreover, PLA significantly reduced relative conductivity and increased malondialdehyde and soluble protein contents. PLA also increased H2O2 and dehydroascorbic acid contents, but reduced ascorbic acid content. Additionally, PLA treatment inhibited catalase, ascorbate peroxidase, monodehydroascorbate acid reductase, dehydroascorbic acid reductase and glutathione reductase activities, whereas promoted superoxide dismutase activity. All these findings suggest that the possible mechanisms involved in the inhibitory effect of PLA on A. alternata included damaging the cell membrane integrity to cause electrolyte leakage and destroying reactive oxygen species balance.

Gaseous ozone regulates reactive oxygen species metabolism and ascorbate–glutathione cycle to delay the senescence of fresh-cut red pitaya (Selenicereus undatus) fruit

Mechanical damage during processing can accelerate the senescence and deterioration of fresh-cut red pitaya (Selenicereus undatus), thereby affecting its nutritional quality and commodity value. In this study, the effects of a low-concentration and long-term (10 μL L−1 for 60 min) gaseous ozone treatment on the quality, reactive oxygen species (ROS) metabolism, and enzyme and non-enzyme antioxidant activities of fresh-cut red pitaya fruit were investigated. The results showed that ozone treatment delayed the decline in fruit firmness, total soluble solids, and titratable acid content. Ozone treatment also maintained a higher free radical scavenging capacity and lower membrane lipid peroxidation than the control treatment. It prevented the rapid increase in ROS level by improving the activity of antioxidant enzymes, including superoxide dismutase, peroxidase, and catalase. Additionally, the treatment activated the ascorbate–glutathione cycle. Compared with the control group, ozone treatment maintained the activities of ascorbic acid peroxidase, dehydroascorbic acid reductase, monodehydroascorbic acid reductase, and glutathione reductase. Although the contents of ascorbic acid (AsA) and dehydroascorbic acid (DHA) decreased within 10 days of storage, the AsA/DHA and glutathione/oxidized glutathione ratios in the ozone-treated group were higher than those in the control group from day 0 to day 6. Thus, ozone treatment regulated ROS metabolism, enhanced the ROS-scavenging system, maintained the redox balance of fresh-cut red pitaya, and delayed senescence.

Exogenous Hemin alleviated cadmium stress in maize (Zea mays L.) by enhancing leaf photosynthesis, AsA-GSH cycle and polyamine metabolism.

Cadmium (Cd) stress is one of the principal abiotic stresses that inhibit maize growth. The research was to explore (hemin chloride) Hemin (100 μmol L−1) on photosynthesis, ascorbic acid (AsA)-glutathione (GSH) cycle system, and polyamine metabolism of maize under Cd stress (85 mg L−1) using nutrient solution hydroponics, with Tiannong 9 (Cd tolerant) and Fenghe 6 (Cd sensitive) as experimental materials. The results showed that Hemin can increase leaf photosynthetic pigment content and ameliorate the ratio of Chlorophyll a/chlorophyll b (Chla/Chlb) under Cd stress. The values of ribose 1, 5-diphosphate carboxylase/oxygenase (RuBPcase) and phosphoenolpyruvate carboxylase (PEPCase), and total xanthophyll cycle pool [(violoxanthin (V), antiflavin (A) and zeaxanthin (Z)] increased, which enhancing xanthophyll cycle (DEPS) de-epoxidation, and alleviating stomatal and non-stomatal limitation of leaf photosynthesis. Hemin significantly increased net photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (Tr), photochemical quenching coefficient (qP), PSII maximum photochemical efficiency (Fv/Fm), and electron transfer rate (ETR), which contributed to the improvement of the PSII photosynthetic system. Compared with Cd stress, Hemin can reduce thiobartolic acid reactant (TBARS) content, superoxide anion radical (O2−) production rate, hydrogen peroxide (H2O2) accumulation, and the extent of electrolyte leakage (EL); decreased the level of malondialdehyde (MDA) content and increased the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT); slowed the decrease in dehydroascorbic acid reductase (DHAR) and monodehydroascorbate reductase (MDHAR) activity and the increase in glutathione reductase (GR) and ascorbate peroxidase (APX) activity in leaves; promoted the increase in AsA and GSH content, decreased dehydroascorbic acid (DHA) and oxidized glutathione (GSSG), and increased AsA/DHA and GSH/GSSG ratios under Cd stress. Hemin promoted the increase of conjugated and bound polyamine content, and the conversion process speed of free putrescine (Put) to free spermine (Spm) and spermidine (Spd) in maize; decreased polyamine oxidase (PAO) activity and increased diamine oxidase (DAO), arginine decarboxylase (ADC), ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) enzyme activities in leaves under Cd stress.

Beneficial effects of abscisic acid and melatonin in overcoming drought stress in cotton (Gossypium hirsutum L.).

Pot experiments were performed to study the effects of abscisic acid (ABA) and melatonin (MT) on cotton drought tolerance and to explore their combined effects. ABA or MT spraying promoted water status and antioxidant capacity of drought-stressed leaves, which was conducive to scavenge ROS, finally increasing lint yield. However, the mitigation mechanisms of ABA and MT on drought were not identical, which were mainly manifested as: (1) ABA increased the relative water content (RWC) of drought-stressed leaves via, reducing water loss, but MT increased it via, promoting water uptake efficiency; (2) for enzymatic antioxidant system, ABA and MT might modulate different kinds of superoxide dismutase to catalyze the reduction of O2 - under drought; and (3) for ascorbic acid (AsA)-glutathione (GSH) cycle, MT increased the glutathione reductase activity in drought-stressed leaves, but ABA did not. ABA + MT spraying led to higher leaf RWC and total antioxidant capacity than single hormone under drought, leading to a lower H2 O2 level. For the enzymatic antioxidant system, single hormone treatment affected Cu/ZnSOD or MnSOD expression, but ABA + MT upregulated both genes in drought-stressed leaves. Hormones combined application also had higher CAT expression than single hormone. For AsA-GSH cycle, ABA + MT had higher dehydroascorbic acid reductase activity than single hormone, resulting in higher AsA content. Moreover, hormones combined application caused higher ascorbate peroxidase activity than single hormone, suggesting that their combination synergistically improved the ability of AsA to eliminate ROS. All these confirmed that ABA plus MT had synergistic effects on improving crop drought resistance.

Changes in Antioxidative Compounds and Enzymes in Small-Leaved Linden (Tilia cordata Mill.) in Response to Mistletoe (Viscum album L.) Infestation.

Mistletoe infestation leads to a decrease in the growth of woody plants, their longevity, and partial or complete drying of the top, as well as premature death. Various environmental stress factors, both abiotic and biotic, stimulate the formation of reactive oxygen species and the development of oxidative stress in plant tissues. This study aimed to investigate the effect of mistletoe (Viscum album L.) infestation on the response of the antioxidative defense system in leaves of small-leaved linden (Tilia cordata Mill.). Leaves from infested trees were taken from branches (i) without mistletoe, (ii) with 1–2 mistletoe bushes (low degree of infestation), and (iii) with 5–7 mistletoe bushes (high degree of infestation). The relative water content and the chlorophyll a and b contents in leaves from linden branches affected by mistletoe were significantly lower than those in leaves from non-infested trees and from host-tree branches with no mistletoe. At the same time, leaves from branches with low and high degrees of infestation had significantly higher electrolyte leakage, malondialdehyde and hydrogen peroxide content, oxidized forms of ascorbic acid (dehydroascorbic and 2,3-diketogulonic acids), and oxidized glutathione. The results of principal component analysis show that the development of oxidative stress was accompanied by an increase in proline content and in superoxide dismutase, ascorbate peroxidase, glutathione peroxidase, and glutathione reductase activity. Several biochemical parameters (proline, ascorbic acid, dehydroascorbic acid, glutathione, glutathione peroxidase, ascorbate peroxidase, and dehydroascorbate reductase) were found to be altered in leaves from host-tree branches with no mistletoe. This result indicates that the mistletoe infestation of trees not only causes local changes in the locations of hemiparasite attachment, but also affects the redox metabolism in leaves from other parts of the infested tree.

LED Light Quality of Continuous Light before Harvest Affects Growth and AsA Metabolism of Hydroponic Lettuce Grown under Increasing Doses of Nitrogen.

To study the effects of light quality of continuous light before harvest on the growth and ascorbic acid (AsA) metabolism of lettuce (Lactuca sativa L.) grown under relative high nitrogen level, lettuce plants grown under different nitrogen levels (8, 10 and 12 mmol·L−1) were subjected to continuous light with different red: blue light ratios (2R:1B and 4R:1B) before harvest. The results showed that the shoot fresh weight of lettuce under 12 mmol·L−1 nitrogen level was significantly higher than that under other treatments. There were no significant differences in shoot dry weight, root fresh weight, root dry weight, soluble sugar content, nitrate content and AsA content in leaves among the treatments at different nitrogen levels. The content of AsA in leaves was significantly higher than that in petioles before and after continuous light. Under the same nitrogen level, the fresh weight of lettuce under continuous light quality 4R:1B was significantly higher than that under other treatments. The content of AsA in lettuce leaves increased in different degrees after continuous light before harvest. High yield and AsA content could be obtained by 72 h continuous light with red and blue light 4R:1B at 12 mmol·L−1 nitrogen level. After continuous light, the content of AsA increased significantly due to the increase of the ratio of red light and nitrogen level, which increased the activities of L-galactono-1,4-lactone dehydrogenase (GalLDH) and dehydroascorbic acid reductase (DHAR) involved in AsA synthesis and in the recycling of DHAR to AsA respectively.

Effects of magnetic treatment on the ascorbate–glutathione cycle and endogenous hormone levels in Populus × euramericana ‘Neva’ under cadmium stress

Cadmium (Cd) contamination in soil has become a serious worldwide environmental and health problem. Cd is easily taken up by plants and translocated to aboveground tissues. A pot experiment was carried out to explore the role of the ascorbate–glutathione (AsA–GSH) cycle and endogenous hormones in enhancing Cd tolerance and promoting translocation of Cd in one-year-old seedlings of Populus × euramericana ‘Neva’. The antioxidant substances ascorbic acid (AsA), dehydroascorbic acid (DHA), glutathione (GSH), and oxidized glutathione (GSSG); the activities of the antioxidant enzymes ascorbic acid peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), and dehydroascorbic acid reductase (DHAR); the levels of the endogenous hormones indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellin (GA3), and zeatin riboside (ZR); and the contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2) were investigated after 30 days of irrigation with half-Hoagland solution containing 0 or 100 μmol·L–1 Cd(NO3)2 under magnetic treatment (MT) or nonmagnetic treatment (NMT). The results were as follows. (i) Compared with NMT, MT increased the AsA levels in roots compared with those in leaves under Cd stress, whereas it increased the DHA levels in the leaves but decreased the DHA levels in the roots. The GSH and GSSG levels both increased by 8%–151% under MT. (ii) MT inhibited the APX activity in blades and roots, and a similar effect was observed on the foliar activities of GR and MDHAR, with a decrease of 8%–50%; however, MT increased the activation of DHAR in the blades and GR in the roots. In addition, compared with NMT, MT increased the activities of GR, MDHAR, and DHAR by 19%–285% in Populus (poplar). (iii) With the exogenous addition of Cd, the Cd accumulation and biological transport coefficient of Cd from roots to leaves (S/R) were enhanced in poplar, accompanied by increased levels of H2O2 and MDA due to MT. (iv) The levels of IAA, ABA, GA, and ZR were inhibited by 19%–95% in the leaves following MT. In contrast, the levels of these endogenous hormones were increased by 18%–203% in the roots following MT. (v) MT improved the seedling growth of poplar, with an increase of 0.4%–90%, compared with that of the NMT. The ground diameter and number of root tips showed the greatest increases, with average ratios of 29% and 87%, respectively. These results suggested a large increase in AsA and decreases in poplar antioxidant enzymes, especially in the leaves, with a high GSH level. In this review, we concluded that the antioxidant substance GSH plays an important role in the AsA–GSH cycle following exposure to a magnetic field under Cd stress. Additionally, the roots play a major role in eliminating oxygen free radicals by regulating the levels and ratios of various endogenous hormones. Moreover, magnetization could alleviate Cd-induced oxidative stress by stimulating MDHAR, DHAR, and GR activities; enhance the defense capability of the AsA–GSH cycle; and maintain normal physiological metabolism in poplar.

Foliar application of 5-aminolevulinic acid (ALA) alleviates NaCl stress in cucumber (Cucumis sativus L.) seedlings through the enhancement of ascorbate-glutathione cycle

The natural plant growth regulator 5-aminolevulinic acid (ALA) promotes plant growth. The present study assessed the effects of exogenously applied ALA (25 mg L−1) on the growth and H2O2 scavenging system (ascorbate-glutathione cycle, AsA-GSH cycle) under NaCl stress (50 mmol L−1) in cucumber (Cucumis sativus L.) seedlings. Growth of cucumber seedlings was significantly inhibited by NaCl stress. Moreover, NaCl caused accumulation of H2O2 and malonaldehyde (MDA) as well as dehydroascorbic acid (DHA) and oxidized glutathione (GSSG) in cucumber leaves. However, the application of ALA reversed the adverse effect of NaCl on growth of the cucumber seedlings by increasing the shoots and roots biomass. The application of ALA also increased the contents of AsA and GSH of the seedlings under moderate salt stress. The ratios of reduced/oxidized antioxidant, such as AsA/DHA and GSH/GSSG, in seedlings under NaCl treatment were also improved by ALA, indicating that the regeneration efficiency of antioxidants could be promoted by exogenous ALA. In addition, exogenous ALA also promoted the activities of enzymes involved in the AsA-GSH cycle, including ascorbic acid oxidase (AAO), ascorbate peroxidase (APX), monodehydroascorbic acid reductase (MDHAR), dehydroascorbic acid reductase (DHAR) and glutathione reductase (GR). These results suggested that ALA alleviate the damages caused by NaCl through the enhancement of the AsA-GSH pathway in cucumber seedlings.

Amelioration of cold-induced oxidative stress by exogenous 24-epibrassinolide treatment in grapevine seedlings: Toward regulating the ascorbate–glutathione cycle

Ascorbate-glutathione (AsA-GSH) cycle plays a key role in tolerance of plant under biotic or abiotic stress. However, little is known regarding the role of brassinolide in AsA-GSH cycle in grape under chilling stress. In this study, a wine grape cultivar, Cabernet Sauvignon (Vitis vinifera), was subjected to chilling stress for different time points after treated by 24-Epibrassinolide (EBR). The result showed EBR plays a positive role in amelioration of leaf morphology and root growth under chilling stress. In terms of physiological aspect, EBR treatment reduce the O2− and H2O2 contents than that in the control. This is primarily related to the improved antioxidant system and the regulation of EBR to AsA-GSH cycle. There was an obvious increase in superoxide dismutase (SOD) activities in EBR treatment compared with in the control, and similar result also was observed in level of AsA, and AsA/DHA. Alleviation of EBR to chilling-stressed injury was also reflected in glutathione (GSH) and GSH/GSSG, as high levels of these parameters were observed in EBR treatment. Furthermore, ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbic acid reductase (DHAR) and monodehydroascorbate reductase (MDHAR) also showed elevated activities in the EBR treatment. Interestingly, the regulation of EBR to these components in AsA-GSH cycle showed a difference in time course, suggesting that the cycle might be affected by EBR at earlier and middle term of chilling stress. Our work will provide the information for understanding the role of EBR in AsA-GSH cycle.

Novel Glutaredoxin Activity of the Yeast Prion Protein Ure2 Reveals a Native-like Dimer within Fibrils

Ure2 is the protein determinant of the Saccharomyces cerevisiae prion [URE3]. Ure2 has structural similarity to glutathione transferases, protects cells against heavy metal and oxidant toxicity in vivo, and shows glutathione-dependent peroxidase activity in vitro. Here we report that Ure2 (which has no cysteine residues) also shows thiol-disulfide oxidoreductase activity similar to that of glutaredoxin enzymes. This demonstrates that disulfide reductase activity can be independent of the classical glutaredoxin CXXC/CXXS motif or indeed an intrinsic catalytic cysteine residue. The kinetics of the glutaredoxin activity of Ure2 showed positive cooperativity for the substrate glutathione in both the soluble native state and in amyloid-like fibrils, indicating native-like dimeric structure within Ure2 fibrils. Characterization of the glutaredoxin activity of Ure2 sheds light on its ability to protect yeast from heavy metal ion and oxidant toxicity and suggests a role in reversible protein glutathionylation signal transduction. Observation of allosteric enzyme behavior within amyloid-like Ure2 fibrils not only provides insight into the molecular structure of the fibrils but also has implications for the mechanism of [URE3] prion formation.

Somatic embryogenesis in saffron (Crocus sativus L.). Histological differentiation and implication of some components of the antioxidant enzymatic system

The ontogenetic developmental stages of saffron somatic embryogenesis have been studied and characterized using light microscopy and the biochemical determination of the antioxidant enzymatic system. The embryogenic callus underwent internal segmented divisions with the formation of globular embryos that were attached to the callus surface by a broad multicellular structure. Further development of the embryoids was characterized by the emergence of a shoot apical meristem and cotyledon (monopolar stage) with the subsequent differentiation of a minicorm at the basal part of the somatic embryo (dipolar stage). During the morphological differentiation of the somatic embryos changes in the antioxidant enzymatic system with increased superoxide dismutase (SOD) and catalase (CAT) activities were detected at the initial stages of somatic embryogenesis. The isoforms of SOD, including two Mn-SODs and four Cu, Zn-SODs, were also detected. Although all the isoforms were expressed during the successive stages of somatic embryogenesis, an increase in Mn-SODs and a decrease in Cu, Zn-SODs during the last two stages was observed. Significant changes were also detected in the antioxidant activities ascorbate peroxidase, dehydroascorbic acid reductase and glutathione reductase.

Ascorbic acid recycling in human erythrocytes is induced by smoking in vivo

Tobacco smoke contains large numbers of radicals that burden the antioxidant defense and, thus, lower plasma antioxidants, in particular vitamin C or ascorbic acid, is commonly observed among smokers. Ascorbic acid recycling describes the process in which ascorbic acid is oxidized to dehydroascorbic acid by various pathways and subsequently reduced back to ascorbic acid intracellularly, e.g., in erythrocytes, thereby preserving the ascorbic acid pool. In humans who are unable to synthesize ascorbic acid, and in smokers in particular, who are prone to oxidation, this process must be very efficient and of great importance. It has previously been reported that isolated erythrocytes subjected to tobacco smoke in vitro had significantly lower ascorbic acid recycling as compared to controls. In contrast to these findings, we now report that freshly isolated erythrocytes from long-term smokers (n = 39) display a significantly increased rate of ascorbic acid recycling in vivo as compared to those isolated from nonsmokers (n = 31; p < .0001). Preliminary data suggests that the increase results from induction of dehydroascorbic acid reductase activity rather than from differences in energy status, glutathione content, or altered transport capacity. The induction of ascorbic acid recycling as a potential adaptation mechanism of the antioxidant defense to oxidative insults is discussed.

Ascorbic acid recycling in human erythrocytes is induced by smoking in vivo

Tobacco smoke contains large numbers of radicals that burden the antioxidant defense and, thus, lower plasma antioxidants, in particular vitamin C or ascorbic acid, is commonly observed among smokers. Ascorbic acid recycling describes the process in which ascorbic acid is oxidized to dehydroascorbic acid by various pathways and subsequently reduced back to ascorbic acid intracellularly, e.g., in erythrocytes, thereby preserving the ascorbic acid pool. In humans who are unable to synthesize ascorbic acid, and in smokers in particular, who are prone to oxidation, this process must be very efficient and of great importance. It has previously been reported that isolated erythrocytes subjected to tobacco smoke in vitro had significantly lower ascorbic acid recycling as compared to controls. In contrast to these findings, we now report that freshly isolated erythrocytes from long-term smokers ( n = 39) display a significantly increased rate of ascorbic acid recycling in vivo as compared to those isolated from nonsmokers ( n = 31; p < .0001). Preliminary data suggests that the increase results from induction of dehydroascorbic acid reductase activity rather than from differences in energy status, glutathione content, or altered transport capacity. The induction of ascorbic acid recycling as a potential adaptation mechanism of the antioxidant defense to oxidative insults is discussed.

Ebselen Has Dehydroascorbate Reductase and Thioltransferase-like Activities

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a seleno-organic compound, has been reported to mimic glutathione peroxidase (GPX). Since bovine erythrocyte GPX showed dehydroascorbic acid (DHA) reductase and thioltransferase (TTase) activities, ebselen was also examined for DHA reductase and TTase-like activities. Evidence is reported that, in the presence of GSH, ebselen catalyzed the in vitro reduction of DHA to L-ascorbic acid in a dose-dependent manner. Using S-sulfocysteine and GSH as co-substrates, ebselen catalyzed the in vitro formation of glutathione disulfide in a dose-dependent manner, thereby acting as a TTase mimic. 1-Chloro-2,4-dinitrobezene (CDNB), a co-substrate with GSH for glutathione S-transferase, was used to measure rates of adduct formation with ebselen pretreated with GSH and compared with GSH alone. The reaction rate was proportional to ebselen, and ebselen was about 250 times more reactive than GSH on an equimolar basis. The DHA reductase and TTase-like activities, in addition to the powerful nucleophilic reactivity of ebselen selenol, may contribute to ebselen's significant anti-inflammatory and anti-oxidative properties in vivo.

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.

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.

Antioxidant defense systems of two lipidopteran insect cell lines

Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines were found to contain unique assemblages of antioxidant enzymes. Specifically, the Sf-9 insect cell line contained Manganese and Copper-Zinc superoxide dismutase (MnSOD and CuZnSOD) for reducing the superoxide radical (O2•−) to hydrogen peroxide (H2O2) and ascorbate peroxidase (APOX) for reducing the resulting H2O2 to H2O. Approximately one third of the total SOD activity was found to be MnSOD. The Tn-5B1-4 cells were also found to contain MnSOD (∼ two thirds of the total SOD activity), CuZnSOD and APOX activities. However, the Tn-5B1-4 cell line, in contrast to the Sf-9 cell line, contained catalase (CAT) activity for reducing H2O2 to H2O. Both the Sf-9 and Tn-5B1-4 cell lines contained glutathione reductase and dehydroascorbic acid reductase activities for regenerating the reduced forms of glutathione and ascorbic acid, respectively. In addition, both cell lines contained glutathione S-transferase peroxidase activity towards hydroperoxides other than H2O2. Finally, neither cell line contains the glutathione peroxidase activity that is ubiquitous in mammalian cells.

Enzymes of the ascorbate biosynthesis and ascorbate-glutathione cycle in cultured cells of tobacco Bright Yellow 2

The ascorbate (ASC) and glutathione (GSH) metabolisms were studied in cultured Nicotiana tabacum cv. Bright Yellow 2 (TBY-2) cells. TBY-2 cells were found to be endowed with L-galactono-γ-lactone dehydrogenase (GLDH) (EC 1.3.2.3), an enzyme that converts L-galactono-γ-lactone into ASC. Cellular fractionation of TBY-2 protoplasts indicated that this enzyme is exclusively localised in mitochondria and associated to the membrane fractions. During the growth cycle of TBY-2 cell culture, GLDH transiently increased, reaching the maximum value on the third day of culture, at the beginning of the exponential phase, when the cell proliferative activity was also higher. Similar behaviour has been observed for ASC and GSH contents. The activities of ascorbate peroxidase (APX) (EC 1.11.1.11), ascorbate-free radical reductase (AFRR) (EC 1.6.5.4), dehydroascorbic acid reductase (DHAR) (EC 1.8.5.1) and glutathione reductase (GR) (EC 1.6.4.2) also transiently raised. However, the scale of the increases varied being about 4-fold for APX and AFRR, 2-fold for DHAR and more than 11-fold for GR. The behaviour of the ASC and GSH recycling enzymes allowed TBY-2 cells to maintain both dehydroascorbic acid and glutathione disulphide at low levels, even under conditions of high ASC and GSH utilisation. The relationship between the ASC and GSH metabolisms during the growth cycle of TBY-2 cell suspension cultures is also discussed.

Dehydroascorbic acid uptake in a human keratinocyte cell line (HaCaT) is glutathione-independent

Vitamin C plays an important role in neutralizing toxic free radicals formed during oxidative metabolism or UV exposure of human skin. This study was performed to investigate the mechanisms that regulate the homoeostasis of vitamin C in HaCaT cells by identifying the events involved in the transport and in the reduction of dehydroascorbic acid. Dehydroascorbic acid accumulated to a greater extent and faster compared with ascorbic acid; its transport appeared to be mediated by hexose transporters and was entirely distinct from ascorbic acid transport. Dehydroascorbate reductase activity was unaffected by glutathione depletion, although it was sensitive to thiol protein reagents. These observations, as well as the subcellular distribution of this enzymic activity and the cofactor specificity, indicate that thioredoxin reductase and lipoamide dehydrogenase play an important role in this reduction process. HaCaT cells were able to enhance their dehydroascorbic acid reductase activity in response to oxidative stress.