The Ascorbate System in Two Bryophytes: Brachythecium velutinum and Marchantia polymorpha

The ascorbate system, one of the major antioxidant systems, has been studied in two bryophytes; a moss, Brachythecium velutinum (Hedw.) B., S. & G., and a liverwort, Marchantia polymorpha L. The moss and liverwort gametophytes contain ascorbate both in the reduced and oxidized form; utilize ascorbate in removing hydrogen peroxide by means of ascorbate peroxidase and reconvert to ascorbate its oxidation products by means of dehydroascorbate reductase and monodehydroascorbate reductase. Ascorbate oxidase activity was measured in the cytosolic fraction suggesting a localization of the enzyme different from more evolved organisms. The ascorbate content was maintained in the moss after drought stress while it declines in the liverwort, which seems more sensitive to water stress. Since ascorbate recycling is more efficient in the moss than in the liverwort, this seems to suggest a correlation between efficiency of ascorbate recycling and water stress tolerance. This revised version was published online in July 2006 with corrections to the Cover Date.     

Changes in the antioxidative systems in mitochondria during ripening of pepper fruits

The presence of enzymes of the ascorbate–glutathione cycle was studied in mitochondria purified from green and red pepper (Capsicum annuum L.) fruits. All four enzymes, ascorbate peroxidase (APX; EC 1.11.1.11), monodehydroascorbate reductase (MDHAR; EC 1.6.5.4), dehydroascorbate reductase (DHAR; EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2) were present in the isolated mitochondria of both fruit ripening stages. The activity of the reductive ascorbate–glutathione cycle enzymes (MDHAR, GR and DHAR) was higher in mitochondria isolated from green than from red fruits, while APX and the antioxidative enzyme superoxide dismutase (SOD; EC 1.15.1.1) were higher in the red fruits. The levels of ascorbate and L-galactono-γ-lactone dehydrogenase (GLDH; EC 1.3.2.3) activity were found to be similar in the mitochondria of both fruits. The higher APX and Mn-SOD specific activities in mitochondria from red fruits might play a role in avoiding the accumulation of any activated oxygen species generated in these mitochondria, and suggests an active role for these enzymes during ripening.     

Composition and Properties of Hydrogen Peroxide Decomposing Systems in Extracellular and Total Extracts from Needles of Norway Spruce (Picea abies L., Karst.)

Hydrogen peroxide (H₂O₂) scavenging systems of spruce (Picea abies) needles were investigated in both extracts obtained from the extracellular space and extracts of total needles. As assessed by the lack of activity of symplastic marker enzymes, the extracellular washing fluid was free from intracellular contaminations. In the extracellular washing fluid ascorbate, glutathione, cysteine, and high specific activities of guaiacol peroxidases were observed. Guaiacol peroxidases in the extracellular washing fluid and needle homogenates had the same catalytic properties, i.e. temperature optimum at 50°C, pH optimum in the range of pH 5 to 6 and low affinity for guaiacol (apparent K_m = 40 millimolar) and H₂O₂ (apparent K_m = 1-3 millimolar). Needle homogenates contained ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, glutathione reductase, and catalase, but not glutathione peroxidase activity. None of these activities was detected in the extracellular washing fluid. Ascorbate and glutathione related enzymes were freeze sensitive; ascorbate peroxidase was labile in the absence of ascorbate. The significance of extracellular antioxidants for the detoxification of injurious oxygen species is discussed.     

Purification and characterization of pea cytosolic ascorbate peroxidase

The cytosolic isoform of ascorbate peroxidase was purified to homogeneity from 14-day-old pea (Pisum sativum L.) shoots. The enzyme is a homodimer with molecular weight of 57,500, composed of two subunits with molecular weight of 29,500. Spectral analysis and inhibitor studies were consistent with the presence of a heme moiety. When compared with ascorbate peroxidase activity derived from ruptured intact chloroplasts, the purified enzyme was found to have a higher stability, a broader pH optimum for activity, and the capacity to utilize alternate electron donors. Unlike classical plant peroxidases, the cytosolic ascorbate peroxidase had a very high preference for ascorbate as an electron donor and was specifically inhibited by p-chloromercurisulfonic acid and hydroxyurea. Antibodies raised against the cytosolic ascorbate peroxidase from pea did not cross-react with either protein extracts obtained from intact pea chloroplasts or horseradish peroxidase. The amino acid sequence of the N-terminal region of the purified enzyme was determined. Little homology was observed among pea cytosolic ascorbate peroxidase, the tea chloroplastic ascorbate peroxidase, and horseradish peroxidase; homology was, however, found with chloroplastic ascorbate peroxidase isolated from spinach leaves.     

Expression of arabidopsis cytosolic ascorbate peroxidase gene in response to ozone or sulfur dioxide

The effects of ozone or sulfur dioxide on antioxidant enzymes were investigated in Arabidopsis thaliana. Plants were fumigated with 0.1–0.15 ppm ozone or sulfur dioxide up to about 1 week in an environment-controlled chamber. Both pollutants increased the activities of ascorbate peroxidase and guaiacol per-oxidase in leaves, but had little effect on the activities of superoxide dismutase, catalase, monodehydroascorbate reductase, dehydroascorbate reductase or glutathione reductase. Ozone was more effective than sulfur dioxide in increasing the activities of the peroxidases. Ascorbate peroxidase activity increased 1.8-fold without a lag period during fumigation with 0.1 ppm ozone, while guaiacol peroxidase activity increased 4.4-fold with a 1-day lag. Expression of the APX1 gene encoding cytosolic ascorbate peroxidase was further investigated. Its protein levels in leaves exposed to 0.1 ppm ozone for 4 or 8 days were 1.5-fold higher than in controls. Both ozone and sulfur dioxide elevated APX1 mRNA levels in leaves at 4 and 7 days, whereas at 1 day only ozone was effective. The induction of APX1 mRNA levels by ozone (3.4- to 4.1-fold) was more prominent than that by sulfur dioxide (1.6-to 2.6-fold). The APX1 mRNA level increased by day and decreased by night. Exposure of plants to 0.1 ppm ozone enhanced the APX1 mRNA level within 3 h, which showed a diurnal rhythm similar to that of the control. These results demonstrate that near-ambient concentrations of ozone as well as similar concentrations of sulfur dioxide can induce APX1 gene expression in A. thaliana.Environmental Biology Division     

Hydroperoxide metabolism in cyanobacteria

The enzymes involved in antioxidative activity and the cellular content of the antioxidants glutathione and ascorbate in the cyanobacteria Nostoc muscorum 7119 and Synechococcus 6311 have been examined for their roles in hydroperoxide removal. High activities of ascorbate peroxidase and catalase were found in vegetative cells of both species and in the heterocysts of N. muscorum. The affinity of ascorbate peroxidase for H2O2 was 15- to 25-fold higher than that of catalase. Increased activity of ascorbate peroxidase was observed in N. muscorum when H2O2 production was enhanced by photorespiration. Catalase activity was decreased in dilute cultures whereas ascorbate peroxidase activity increased. Ascorbate peroxidase activity also increased when the CO2 concentration was reduced. Ascorbate peroxidase appears to be a key enzyme in a cascade of reactions regenerating antioxidants. Dehydroascorbate reductase was found to regenerate ascorbate, and glutathione reductase recycled glutathione. In vegetative cells glutathione was present in high amounts (2-4 mM) whereas the ascorbate content was almost 100-fold lower (20-100 microM). Glutathione peroxidase was not detected in either cyanobacterium. It is concluded from the high activity of ascorbate peroxidase activity and the levels of antioxidants found that this enzyme can effectively remove low concentrations of peroxides. Catalase may remove H2O2 produced under photooxidative conditions where the peroxide concentration is higher.     

Ascorbate oxidase of Cucumis melo L. var. reticulatus: purification, characterization and antibody production

Ascorbate oxidase activity and ascorbic acid content were followedduring the development of muskmelon (Cucumis melo L. var. reticulatus)fruits. The enzyme was highly expressed in ovaries and veryyoung fruit tissues, followed by a decrease in 10- and 20-d-oldfruits and an increase in 30- and 35-d-old fruits which coincidedwith early events of fruit ripening. Ascorbic acid content wasnegatively correlated with ascorbate oxidase activity. The enzymewas purified to homogeneity following ion exchange, affinityand gel filtration chromatographic trials. The purified enzymewas a glycoprotein of molecular weight 137 000 composed of twosubunits of molecular weight 68000, and formed by six isoenzymeswith isoelectric points in the range of pH 7.7 to 8.3. Its electronparamagnetic resonance and optical spectra were in agreementwith other copper proteins and the enzyme contained eight copperatoms per dimeric molecule. The K_m of the enzyme for ascorbicacid was 50 µM. Ascorbate oxidase activity was inhibitedby azide and by EDTA, two inhibitors of copper proteins. Optimalconditions for enzyme activity was pH 5.5, and a temperatureof 37 C. Polyclonal antibodies were produced against the purifiedprotein and immunoprecipitated ascorbate oxidase activity. Key words: Cucumis melo, muskmelon, ascorbate oxidase, fruit ripening     

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 H₂O₂ 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.     

Ascorbate-dependent hydrogen peroxide detoxification and ascorbate regeneration during germination of a highly productive maize hybrid: Evidence of an improved detoxification mechanism against reactive oxygen species

Ascorbate content and the activities of some key enzymes involved in the detoxification from reactive oxygen species were investigated in germinated embryos of two Zea mays L. inbred lines (B73 and Mo17) and of their heterotic F1 hybrid (B73×Mo17). The F1 hybrid showed a higher ascorbate biosynthetic capability owing to a higher activity of l -galactono- Γ -lactone dehydrogenase (EC 1.6.5.4), the last enzyme in ascorbate biosynthesis. Ascorbate peroxidase (EC 1.11.1.11), ascorbate free radical reductase (EC 1.6.5.4) and dehydroascorbate reductase (EC 1.8.5.1) activities were much higher in the F1 hybrid than in either inbred line, whereas catalase (EC 1.11.1.6) activity was similar in the three genotypes. Native polyacrylamide gel electrophoresis (PAGE) analysis showed three forms of cytosolic ascorbate peroxidase, both in parental lines and in the F1 hybrid. On the other hand, a complex pattern of proteins with dehydroascorbate reductase activity was observed, with the hybrid combining the different dehydroascorbate-reducing proteins expressed by the inbred lines. The possible involvement of the enzymes of the ascorbate system in the phenomenon of hybrid vigour is discussed.     

Purification, properties, and distribution of ascorbate peroxidase in legume root nodules

All aerobic biological systems, including N₂-fixing root nodules, are subject to O₂ toxicity that results from the formation of reactive intermediates such as H₂O₂ and free radicals of O₂. H₂O₂ may be removed from root nodules in a series of enzymic reactions involving ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. We confirm here the presence of these enzymes in root nodules from nine species of legumes and from Alnus rubra. Ascorbate peroxidase from soybean nodules was purified to near homogeneity. This enzyme was found to be a hemeprotein with a molecular weight of 30,000 as determined by sodium dodecyl sulfate gel electrophoresis. KCN, NaN₃, CO, and C₂H₂ were potent inhibitors of activity. Nonphysiological reductants such as guaiacol, o-dianisidine, and pyrogallol functioned as substrates for the enzyme. No activity was detected with NAD(P)H, reduced glutathione, or urate. Ascorbate peroxidation did not follow Michaelis-Menten kinetics. The substrate concentration which resulted in a reaction rate of ½ V_max was 70 micromolar for ascorbate and 3 micromolar for H₂O₂. The high affinity of ascorbate peroxidase for H₂O₂ indicates that this enzyme, rather than catalase, is responsible for most H₂O₂ removal outside of peroxisomes in root nodules.     

Salt and oxidative stress: similar and specific responses and their relation to salt tolerance in Citrus

Salt damage to plants has been attributed to a combination of several factors including mainly osmotic stress and the accumulation of toxic ions. Recent findings in our laboratory showed that phospholipid hydroperoxide glutathione peroxidase (PHGPX), an enzyme active in the cellular antioxidant system, was induced by salt in citrus cells and mainly in roots of plants. Following this observation we studied the two most important enzymes active in elimination of reactive oxygen species, namely, superoxide dismutase (SOD) and ascorbate peroxidase (APX), to determine whether a general oxidative stress is induced by salt. While Cu/Zn-SOD activity and cytosolic APX protein level were similarly induced by salt and methyl viologen, the response of PHGPX and other APX isozymes was either specific to salt or methyl viologen, respectively. Unlike PHGPX, cytosolic APX and Cu/Zn-SOD were not induced by exogenously added abscisic acid. Salt induced a significant increase in SOD activity which was not matched by the subsequent enzyme APX. We suggest that the excess of H₂O₂ interacts with lipids to form hydroperoxides which in turn induce and are removed by PHGPX. Ascorbate peroxidase seems to be a key enzyme in determining salt tolerance in citrus as its constitutive activity in salt-sensitive callus is far below the activity observed in salt-tolerant callus, while the activities of other enzymes involved in the defence against oxidative stress, namely SOD, glutathione reductase and PHGPX, are essentially similar. Received: 10 January 1997 / Accepted: 28 May 1997     

Ascorbate and glutathione metabolism in embryo axes and cotyledons of germinating lupine seeds

Changes in ascorbate and glutathione contents and the activities and isoenzyme patterns of enzymes of the ascorbate-glutathione cycle were investigated in embryo axes and cotyledons of germinating lupine (Lupinus luteus L.) seeds. Ascorbate content was not significantly affected over the initial 12 h of imbibition in embryo axes, but afterwards increased, with the most rapid accumulation coinciding with radicle emergence. A somewhat similar trend was observed for glutathione with significant increase in embryo axes shortly before radicle protrusion followed by decline in the next hours. In cotyledons the ascorbate pool rose gradually during germination but the amount of glutathione showed fluctuations during a whole germination period. The activity of ascorbate peroxidase (APX) rose progressively in embryo axes, while activities of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) showed transient increase during germination. New isoforms of APX and GR were synthesized, suggesting that they play a relevant role during germination. All analyzed enzymes were already present in dry seeds which allowed them to be active immediately after imbibition.     

Increased antioxidative capacity in maize calli during and after oxidative stress induced by a long lead treatment

Maize (Zea mays L., cv. Samodek) callus cultures were exposed for long period (22 months) to lead (0.5 mM lead chloride) and lead content, oxidative damage and antioxidative response were evaluated at different steps. Inductively coupled plasma (ICP) emission spectroscopy analysis showed that lead entered the cells and it accumulated, but its internal concentration was maintained 10-fold less than the external one. Increase of both polyamine and lipid peroxide content indicated that cells underwent a stress condition due to an oxidative attack, counteracted by an increase of antioxidative defence enzyme activities, ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2). After 10 months, from the start of the lead treatment, a stock of calli was transferred for 6 months in a lead-deprived medium and then re-exposed to lead for a further 6 months. Analysis indicated that lead-deprived calli maintained high levels of APX and GR activities, suggesting that, over the experimental time–course, cells with high APX and GR activity were selected and allowed to enrich the cultures. These cultures, after a new lead treatment, showed a lower oxidative damage compared to continuously lead-treated calli.     

Delayed wheat flag leaf senescence due to removal of spikelets is associated with increased activities of leaf antioxidant enzymes,reduced glutathione/oxidized glutathione ratio and oxidative damage to mitochondrial proteins

Removal of reproductive ‘sink’ i.e. spikelets from wheat at anthesis delays the rate of flag leaf senescence. In this work, the antioxidant defense was studied in the flag leaf of Triticum aestivum cv. Kalyansona plants showing normal (S + plants) and delayed senescence via removal of spikelets (S? plants). This was done by measurement of metabolites and activities of enzymes such as superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase. S? plants had higher reduced glutathione/oxidized glutathione (GSH/GSSG) ratio and antioxidant enzyme activities than the control plants and the differences were apparent from 21 days after anthesis (DAA). The removal of the reproductive sink led to an increased antioxidant defense which may be contributing towards the delayed flag leaf senescence in wheat. Chloroplasts and mitochondria, important sources of ROS, were isolated at two stages representing early (7 DAA) and late (21 DAA) senescence. Oxidative damage to proteins was studied in these organelles in relation to SOD and APX. Mitochondria had higher levels of damaged proteins than chloroplasts at 7 DAA in both S+ and S? plants. Higher damage was related to the lower antioxidant enzyme levels of SOD and APX in mitochondria as compared to chloroplasts.     

Cytosolic Ascorbate Peroxidase in Seedlings and Leaves of Maize (Zea mays)

Ascorbate peroxidase (APX) was purified to homogeneity frommaize (Zea mays L. cv.) coleoptiles. APX was a monomer witha molecular mass of 28 kDa, as determined by gel nitration andSDS-polyacrylamide gel electrophoresis. It contained one protohememoiety per molecule, with the oxidized form giving a Soret peakat 403 nm with small peaks at 502 and 638 nm, and the reducedform giving peaks at 435 and 556 nm. The enzyme was not inactivatedby depletion of ascorbate. Cell fractionation and immunohistochemicalstudies using polyclonal antibodies raised against maize APXrevealed that the enzyme was not located in the chloroplastsof green leaves. It was abundant in the cytoplasm but not inthe vacuoles of cells in the coleoptile, mesocotyl and youngleaves of seedlings. In mature green leaves, small amounts ofthe enzyme were distributed in vascular systems, in particularin the companion cells. The N-terminal amino acid sequence ofmaize APX exhibited high homology to pea cytosolic APX, spinachAPX and Arabidopsis APX, but not to APX from tea chloroplasts. (Received February 15, 1993; Accepted May 6, 1993)     

Modeling and phylogenetic analysis of cytosolic ascorbate peroxidase (OsAPX1) from rice reveal signature motifs that may play a role in stress tolerance

Ascorbate peroxidase (APX) is a crucial, haeme-containing enzyme of the ascorbate glutathione cycle that detoxifies reactive oxygen species in plants by catalyzing the conversion of hydrogen peroxide to water using ascorbate as a specific electron donor. Different APX isoforms are present in discrete subcellular compartments in rice and their expression is stress regulated. We revealed the homology model of OsAPX1 protein using the crystal structure of soybean GmAPX1 (PDB ID: 2XIF) as template by Modeller 9.12. The resultant OsAPX1 model structure was refined by PROCHECK, ProSA, Verify3D and RMSD that indicated the model structure is reliable with 83 % amino acid sequence identity with template, RMSD (1.4 Å), Verify3D (86.06 %), Zscores (-8.44) and Ramachandran plot analysis showed that conformations for 94.6% of amino acid residues are within the most favoured regions. Investigation revealed two conserved signatures for haeme ligand binding and peroxidase activity in the alpha helical region that may play a significant role during stress.     

Cadmium and copper induction of oxidative stress and antioxidative response in tomato (<Emphasis Type="Italic">Solanum lycopersicon</Emphasis>) leaves

We compare cadmium and copper induced oxidative stress in tomato leaves and the antioxidative enzyme response during a time course of 96 h. Plants were subjected to 25 μM of CdCl₂ or CuSO₄ and malondialdehyde (MDA) level and activity of guaiacol peroxidase, superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase were determined. The results showed that there was an early increase in the MDA level and in the guaiacol peroxidase activity more pronounced with copper exposure during almost all the time course of the experiment. The activity of superoxide dismutase and catalase was induced very early after cadmium and copper treatment, reached a maximal value after 12 h and then declined but it remained always slightly higher than the control at the end of the experiment. Ascorbate peroxidase activity pathway was similar to superoxide dismutase or catalase with a maximal activity after 48 h of heavy metal exposure. Induction of glutathione reductase activity observed only under copper exposure is maintained during almost all the experimental time. The antioxidative activity developed by tomato leaves is more induced by copper treatment. This can be related to the ability of this metal to induce more than cadmium an accumulation of reactive oxygen species (ROS) at the cellular level. Decline in the antioxidative enzymes activity at the end of the experiment can be a consequence of cadmium- and copper-inducing a further ROS formation that might affect enzymes activity.     

Opposing roles for superoxide and nitric oxide in the NaCl stress-induced upregulation of antioxidant enzyme activity in cotton callus tissue

The roles of superoxide and NO in the NaCl-induced upregulation on antioxidant enzyme activity were investigated in NaCl-tolerant cotton calli. Both NaCl and paraquat treatments resulted in significant increases in superoxide production. The activities of ascorbate peroxidase (APX), catalase, glutathione reductase (GR), and peroxidase also increased significantly within 2 h after applying the stress. Pre-treatment with the superoxide scavenger, N-acetyl l-cysteine (NAC), completely removed the superoxide and inhibited the upregulation of antioxidant enzyme activity in the tissue treated with either NaCl or paraquat. NaCl stress also resulted in a significant increase in the NO level. Experiments were also carried out to measure antioxidant enzyme activity in cotton calli exposed to NO, the NO producer sodium nitroprusside (SNP), and the NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO) under different salt stress conditions. The direct addition of NO gas produced no change in the activities of catalase and GR and caused a significant decrease in APX activity when compared to the controls. When the calli was treated with SNP in the absence of NaCl stress, APX and GR activities decreased significantly and catalase activity was only slightly higher than the control. Treatment with SNP in the presence of NaCl stress resulted in a significant decrease in APX activity, and GR and APX activities were not significantly different from those observed in the NaCl treatment alone. In the presence of PTIO, the activities of all three enzymes increased in the presence or absence of NaCl stress. These results suggest that reactive oxygen species (ROS) such as superoxide radicals may serve as signal transduction molecules to switch “on” the early NaCl-induced up-regulation of antioxidant enzyme activity, while NO may play a role in switching “off” the response after other mechanisms in the cascade of events responsible for NaCl tolerance have been activated.