Increase In Ascorbate Peroxidase Activity Research Articles

Determination of anti-oxidant activities in some melon (Cucumis melo L.) varieties and cultivars under salt stress

SummaryThe response of the antioxidant system to salt stress was studied in leaves of seedlings of four Turkish melon (Cucumis melo L.) varieties ‘Besni’, ‘Yuva’, ‘Midyat’ and ‘Semame’ and three melon cultivars ‘Ananas’, ‘Galia C8’ and ‘Galia F1’. The anti-oxidant capability of plants was determined by measuring ascorbate peroxidase (APX) and glutathione reductase (GR) activities together with levels of the anti-oxidant, ascorbic acid (AA). Salt treatment increased APX activities in stress-tolerant ‘Galia C8’ and ‘Galia F1’ and in moderately tolerant ‘Besni’, ‘Midyat’ and ‘Semame’. These increases were greater in salt-tolerant cultivars. On the other hand, salt treatment did not cause a significant increase in APX activity in the salt-sensitive cvs. ‘Yuva’ and ‘Ananas’. GR activity increased in all varieties and cultivars grown under saline conditions compared to control plants. Increases in GR activities were higher in salt-tolerant and moderately-tolerant varieties than in salt-sensitive ones. Likewise, AA contents increased with salt treatment in all varieties and cultivars except the salt-sensitive ‘Ananas’. The data indicate that melon seedlings respond to salt-induced oxidative stress by increasing both their enzymatic and non-enzymatic anti-oxidant defense systems. These increases were most clear in the salt-tolerant cultivars.

Antioxidant defences in olive trees during drought stress: changes in activity of some antioxidant enzymes.

The effects of drought on the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POD), indoleacetate oxidase (IAAox) and polyphenol oxidase (PPO) were studied in 2-year old Olea europaea L. (cv. 'Coratina') plants grown under high temperatures and irradiance levels and gradually subjected to a controlled water deficit. After 20 d without irrigation, mean predawn leaf water potential fell from -0.37 to -5.37 MPa, and decreases in net photosynthesis and transpiration occurred. The activities of SOD, APX, CAT and POD increased in relation to the severity of drought stress in both leaves and roots. In particular, a marked increase in APX activity was found in leaves of plants at severe drought stress. CAT activity increased during severe water deficit conditions in leaves and fine roots. The patterns of POD and IAA oxidase activity ran in parallel and showed increases in relation to the degree of drought. In contrast, PPO activity decreased during the progression of stress in all the tissues studied. The results show that the ability of olive trees to up-regulate the enzymatic antioxidant system might be an important attribute linked to drought tolerance. This could limit cellular damage caused by active oxygen species during water deficit.

Physiological and antioxidant responses of cotton and spurred anoda under interference and mild drought.

The influence of plant interference and a mild drought on gas exchange and oxidative stress was investigated using potted plants of two cotton species (Gossypium hirsutum L. cv. Delta Pine 5415, and Gossypium barbadense L. cv. Pima S-7) and spurred anoda (Anoda cristata L. Schlecht.) of the Malvaceae. Without interference, cotton and spurred anoda had similar net photosynthesis (Pnet) but different pigment profiles. Stomatal conductance (gs) and transpiration rate (E) were greater in spurred anoda than cotton. Net photosynthesis and biomass in cotton were reduced more by spurred anoda interference than by intraspecific interference. With interference, the xanthophyll cycle conversion state and alpha-tocopherol levels increased in cotton, but remained unchanged in spurred anoda. Catalase, ascorbate peroxidase (APX) and glutathione reductase (GR) activities were not influenced by plant interference. Without interference, spurred anoda had lower APX, and similar catalase and GR activities compared with cotton. Mild drought increased APX activity more than 40% in cotton, and 26% in spurred anoda. Upon drought recovery, drought-induced APX activity was still higher in cotton, and GR activity was higher in previously drought-stressed cotton and spurred anoda plants compared with well-watered plants. The greater impact of spurred anoda interference than intraspecific interference on cotton biomass is due mainly to reduced carbon gain in cotton.

Effect of iron on the expression of ascorbate peroxidase in Euglena gracilis

We have investigated the effect of Fe on the induction of ascorbate peroxidase (APX) in Fe-deficient Euglena cells that completely lacked APX activity. Addition of 0.13 mM Fe to Fe-deficient cells caused a two-phase induction of the APX activity: a sharp rapid increase in the first 3 h, followed by a steady gradual increase from 3 through 24 h. Cycloheximide had no effect on the initial increase of activity, but completely inhibited the subsequent increase. Immunoblot analyses using a monoclonal antibody (EAP1) raised against Euglena APX indicated that the former increase is due to activation of APX apo-protein and the latter one is due to de novo synthesis of the enzyme protein. However, Northern analysis revealed that the levels of APX transcripts remained constant, suggesting that the induction of Euglena APX by Fe is regulated at the post-transcriptional stage. The second increase of APX activity was also suppressed by the addition of succinyl acetone, an inhibitor of heme synthesis, suggesting that heme synthesis is essential for the synthesis of APX protein.

Activity of Enzymes Related to H2O2 Generation and Metabolism in Leaf Apoplastic Fraction of Tomato Leaves Infected with Botrytis cinerea

AbstractHydrogen peroxide generation rates of uninfected and infected leaves of two tomato (Lycopersicon esculentum) cultivars showing differential susceptibility to Botrytis cinerea were determined. The superoxide anion, hydroxyl radical, ascorbate contents and changes in NADH peroxidase, superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) activities in the apoplast fraction were analysed. Infected leaves had an increased hydrogen peroxide level. It was greater and generally occurred earlier in plants of the less susceptible cv. Perkoz than in those of the more susceptible cv. Corindo. Induction of nitrotetrazolium blue reducing activity and SOD levels in apoplast were higher in cv. Perkoz 24 h after inoculation. In the controls, NADH peroxidase activity in apoplast was higher in the more susceptible cv. Corindo, but after infection it increased faster and to a higher level in the less susceptible cv. Perkoz. NADH oxidation was inhibited by only 15% by a specific inhibitor DPI (diphenylene‐iodonium) but was completely inhibited by KCN and NaN3. Similar increases in APX activity after 48 h and a small increase in catalase activities were observed in both cultivars soon after infection. These results indicate that resistance of tomato plants to infection by the necrotrophic fungus B. cinerea may result from early stimulation of hydrogen peroxide and superoxide radical generations by NADH peroxidase and SOD in apoplastic space, and they confirm the important role of their enhanced production in apoplastic spaces of plants.

Lead toxicity induces lipid peroxidation and alters the activities of antioxidant enzymes in growing rice plants

When seedlings of two rice ( Oryza sativa L .) cultivars were raised in sand cultures under 500 and 1000 μM Pb(NO 3) 2 in the medium, lengths as well as weights of roots and shoots decreased with increase in Pb concentration. Pb-treated seedlings showed elevated levels of lipid peroxides with a concomitant increase in the activities of the enzymes superoxide dismutase (SOD), guaiacol peroxidase, ascorbate peroxidase and glutathione reductase compared to controls. Though Pb was readily absorbed by growing seedlings, its localization was greater in roots than shoots. The level of Pb accumulation in seedlings was far higher than the supplied one. Seedlings grown for 5–20 days in presence of 1000 μM Pb(NO 3) 2 showed about 21–177% increase in the level of thiobarbituric acid reacting substances (TBARS) in shoots indicating enhanced lipid peroxidation compared to controls. With increase in the level of Pb treatment in situ peroxidases showed more increase in activity than SOD. Under both controls as well as Pb treatments roots maintained higher activity of these enzymes than shoots. About 87–100% increase in SOD activity, 1.2–5.6 times increase in guaiacol peroxidase activity and 1.2–1.9 times increase in ascorbate peroxidase activity was observed in the roots of seedlings grown for 15 days in presence of 1000 μM Pb in the medium. Under similar treatment conditions about 128–196% increase in glutathione reductase activity was recorded in roots and 69–196% increase in shoots compared to control grown seedlings. Pb treatment resulted in a decline in catalase activity in roots whereas in shoots catalase activity increased in seedlings grown at moderately toxic Pb (500 μM) level whereas a highly toxic Pb (1000 μM) level led to a marked inhibition in enzyme activity. Two catalase isoforms were detected in roots and three in shoots of the seedlings. A highly toxic Pb (1000 μM) level led to decrease in the intensity of two pre-existing catalase isoforms in shoots. Results suggest that Pb induces oxidative stress in growing rice plants and that SOD, peroxidases and GR could serve as important components of antioxidative defense mechanism against Pb induced oxidative injury in rice.

Enzymes associated with blackheart development in pineapple fruit

The involvement of browning enzymes, polyphenoloxidase (PPO), peroxidase (POD) and phenylalanine ammonia-lyase (PAL) in blackheart development was investigated in pineapple fruit ( Ananas comosus, Smooth Cayenne) following low temperature storage. An increase in PPO activity was related to the incidence of blackheart symptoms, both temporally and spatially. Fruit maturity significantly affected blackheart susceptibility; immature and over-mature fruits developed less blackheart injury than mature fruit. The effect of maturity on blackheart susceptibility was highly correlated to the response of PPO activity to chilling. POD showed no significant change after chilling. Enhanced PAL activity was observed during chilling at 6, 13 and 18 °C. Chilling (6, 13 and 18 °C) also inhibited the increase of ascorbate peroxidase activity observed in the fruit stored continuously at 25 °C, but had less effect on catalase activity. The results indicate that the development of blackheart symptoms in pineapple fruit results from the disturbance of a number of metabolic processes that occur at sub-ambient temperatures.

Dimethylthiourea, a hydrogen peroxide trap, partially prevents stress effects and ascorbate peroxidase increase in spermidine‐treated maize roots

ABSTRACTInhibition of root growth and accumulation of putrescine caused by exogenous spermidine in roots of maize seedlings (Zea mays L., cv Samodek) were partially prevented by a concomitant treatment with dimethylthiourea (DMTU), that traps H2O2 produced from spermidine by the activity of polyamine oxidase (PAO) in the apoplast. Treatment with spermidine caused a strong increase of ascorbate peroxidase (APX) gene expression, that was induced to a lesser extent by removing spermidine‐generated H2O2 by DMTU. Over‐expression of APX was associated with increased APX activity in spermidine‐treated seedlings whereas the addition of DMTU to spermidine completely prevented spermidine‐induced increase of APX activity. Thus, DMTU permitted the demonstration that exogenous spermidine supplied to maize seedlings causes an oxidative stress and induces APX, a key enzyme of the antioxidant defence mechanism, through H2O2, a spermidine catabolic product.

Dimethylthiourea, a hydrogen peroxide trap, partially prevents stress effects and ascorbate peroxidase increase in spermidine-treated maize roots

Inhibition of root growth and accumulation of putrescine caused by exogenous spermidine in roots of maize seedlings (Zea mays L., cv Samodek) were partially prevented by a concomitant treatment with dimethylthiourea (DMTU), that traps H2O2 produced from spermidine by the activity of polyamine oxidase (PAO) in the apoplast. Treatment with spermidine caused a strong increase of ascorbate peroxidase (APX) gene expression, that was induced to a lesser extent by removing spermidine-generated H2O2 by DMTU. Over-expression of APX was associated with increased APX activity in spermidine-treated seedlings whereas the addition of DMTU to spermidine completely prevented spermidine-induced increase of APX activity. Thus, DMTU permitted the demonstration that exogenous spermidine supplied to maize seedlings causes an oxidative stress and induces APX, a key enzyme of the antioxidant defence mechanism, through H2O2, a spermidine catabolic product.

Effects of Cultural Temperatures on Antioxidant Levels in Leaves of Cattleya and Cymbidium.

Orchids of Brassolaeliocattleya Chian-Tzy Emperor (Cattleya) and Cymbidium Sazanami were cultured at 32/20°C, 30/15°C, and 28/8°C (maximum/minimum temperature) for 2 months. It was found in Cattleya leaves that superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) activities were lower than in Cymbidium leaves, while polyphenol oxidase (PPO) and ascorbate oxidase (AOX) activities were higher. The high cultural temperature enhanced activities in all enzymes in Cattleya and in four enzymes except CAT in Cymbidium, indicating that it greatly affects scavenging enzyme activities compared to the low temperatures which induced only an increase in APX activity in Cattleya and a decrease in CAT activity in Cymbidium. PPO activity increased and AOX decreased in both orchids at the low temperature. Cattleya leaves at both high and low cultural temperatures decreased ascorbic acid, while Cymbidium increased it. Cattleya at the low temperature increased markedly SH-containig substances but the latter at the high temperature decreased these substances. Lipid peroxides increased in Cymbidium leaves under both high and low temperatures, but for Cattleya neither temperature conditions affected lipid peroxides.

Up‐regulation of the ascorbate‐dependent antioxidative system in barley leaves during powdery mildew infection

Abstract The ascorbate-dependent antioxidative system was studied in leaves of barley infected with the barley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh). Increased ascorbate peroxidase (APX) activity was detected upon infection, especially in the compatible interaction. APX activity was determined in epidermal and total leaf tissues. A relatively higher increase in APX activity was found in the epidermis compared to total leaf 72 h after inoculation in the compatible interaction, but the increase was not restricted to the epidermis. Activity assays in native gels and Northern blot hybridization indicated that the increase in APX activity was caused by a cytosolic APX isoform. 'Inverse Northern blot' hybridization results with the cDNA of a cytosolic APX supported the relatively higher increase in epidermal APX activity compared to total leaf activity. In the compatible interaction, monodehydroascorbate reductase (MDHAR) activity increased in temporal and spatial patterns similar to that of APX activity. In contrast to this, dehydroascorbate reductase and glutathione reductase activities either decreased or were unaffected by Bgh infection. The increase in APX and MDHAR activities in the compatible interaction continued until severe infection of the leaves. Thus, an up-regulation of the antioxidative system of the host cells could play a role for maintenance of the biotrophic relationship between Bgh and the barley leaf by preventing proliferating oxidative processes, which would otherwise be harmful to the living plant cell on which the biotrophic powdery mildew fungus depends.

Specific features of the ascorbate/glutathione cycle in cultured protoplasts

Protoplasts isolated from Nicotiana tabacum (L.) leaves were cultured for 6 days in liquid medium and some features of their antioxidant capacity were investigated. Ascorbate exported into the culture medium was oxidized non-enzymically, whereas important modifications of the enzymic scavenging activity were detected inside protoplasts. A new pool of isoenzymes, most of them with cytoplasmic characteristics, ensures the increased ascorbate peroxidase activity. The specific activity of other enzymes involved in the ascorbate/glutathione cycle, such as dehydroascorbate reductase and glutathione reductase, and of glutathione peroxidase were modified, resulting in cultured protoplasts with quantitative differences in antioxidant capacity compared to leaves. The hypothesis presented here suggests that the new scavenging system is related to differences in the compartment-specific accumulation of active oxygen species following protoplast isolation.

Regulation of ascorbate peroxidase activity in dark-grown radish cotyledons by a catalase inhibitor, 3-Amino-1,2,4-Triazole

The present study was performed to see the physiological role of cytosolic ascorbate peroxidase (APX) and its relationship to other enzymes involved in the H2O2 scavenging metabolism, and also to elucidate the regulation of APX expression in dark-grown radish (Raphanus sativus L. cv Taiwang) cotyledons. To do so, 3-amino-l,2,4-triazole (aminotriazole), a known specific inhibitor of catalase, was used to simulate a catalase-deficient phenomenon in cotyledons. Aminotriazole, in very low concetration (10-4 M), inhibited remarkably the development of catalase activity in cotyledons during dark germination. This inhibition of catalase by aminotriazole, however, did not result in any significant changes in the growth response and the H2O2 level of developing cotyledons. In addition, the development of guaiacol peroxidase (GPX) activity was also not significantly affected. Unlike GPX, cytosolic APX activity was induced rapidly and reached a 1.7-fold increase in aminotriazole treated cotyledons at day 7 after germination. However,in vitro incubation of cytosolic APX preparation from cotyledons with aminotriazole did not result in any significant change in activity. One cytosolic APX isozyme (APXa) band involved in this APX activation was predominantly intensified in a native polyacrylamide gel by activity staining assay. This means that this APXa isozyme seems to play a key role in the expression of cytosolic APX activity. On the other hand, 2-day-old control seedlings treated with exogenous 1 mM H2O2 for 1 h showed a significant increase of cytosolic APX acitivity even in the absence of aminotriazole. Also, 2 μM cycloheximide treatment substantially inhibited the increase of APX activity due to aminotriazole. Based on these results, we suggest that a radish cytosolic APX could probably be substituted for catalase in H2O2 removal and that the expression of APX seems to be regulated by a change of endogenous H2O2 level which couples to APX protein synthesis in a translation stage in cotyledons.

Ascorbate peroxidase activity, not the mRNA level, is enhanced in salt‐stressed Raphanus sativus plants

The effect of salt on ascorbate peroxidase (EC 1.11.1.11) activity and mRNA level in radish (Raphanus sativus L. cv. Fakir) was investigated. cDNA encoding a radish ascorbate peroxidase was obtained and sequenced. Sequence data analysis showed that the coding region was well conserved among higher plants. The radish cDNA was used as a probe to measure the steady‐state level of ascorbate peroxidase mRNA in plants subjected to salt stress (100 mM NaCl). The levels of transcripts encoding ascorbate peroxidase were almost identical in control or salt‐grown radish plants in both leaves and roots. The activity of soluble ascorbate peroxidase was enhanced by the salt treatment in both leaves and roots. The salt‐induced increase in ascorbate peroxidase activity requires days to become significant and can be considered as a late response. The increase in activity, but not in mRNA level, suggests that the salt‐induced ascorbate peroxidase expression is probably the consequence of post‐transcriptional events.

Induction of ascorbate peroxidase activity in stressed pine ( Pinus sylvestris L.) needles: a putative role for ethylene

Ascorbate peroxidase (EC 1.11.1.11) was studied in chemically stressed pine ( Pinus sylvestris L.) needles, and the possible role of ethylene in its regulation was evaluated. Ascorbate peroxidase activity was induced both in detached and intact pine needles in response to H 2O 2 and Na 2S 2O 5 treatment through de novo protein synthesis. Both 1-aminocyclopropane-1-carboxylic acid, a direct precursor of ethylene, and ethephon, an artificial ethylene-releasing compound, caused an activation of ascorbate peroxidase which was effectively suppressed by inhibitors of protein synthesis. Chemical stress-dependent increase in ascorbate peroxidase activity was abolished by inhibitors of both ethylene biosynthesis (aminooxyacetic acid) and ethylene action (silver thiosulfate). It appears that both ethylene and H 2O 2 are involved in the control of ascorbate peroxidase induction in stressed pine needles, ethylene being a modulator rather than an inducer of the response.

Evaluation of Paraquat Resistance Mechanisms in Conyza

Experiments were conducted to determine the mechanism(s) of paraquat (1,1′-dimethyl-4, 4′-bipyridinium ion) resistance in a biotype of hairy fleabane ( Conyza bonariensis (L.) Cronq.). Thin-layer chromatographic analysis of leaf extracts indicated that paraquat is not metabolized in either the resistant (R) or the sensitive (S) biotype. Three in vitro studies demonstrated that electron transfer from the photosystem I (PSI) donor ( F A/ F B) to paraquat is similar in both biotypes as was the amount and character of the two F A/ F B iron-sulfur clusters. The relative activities of the stromal enzymes superoxide dismutase, ascorbate peroxidase, and glutathione reductase, which can detoxify paraquat-generated noxious oxygen species, were determined following separation by polyacrylamide gel electrophoresis. Of these enzymes, only an increase in ascorbate peroxidase activity (28%) was observed in stromal extracts of the R (relative to S) biotype. These data indicate that the 100-fold level of paraquat resistance observed in leaves of the R biotype of Conyza is not due to metabolic detoxification, an altered (insensitive) site of action, and/or enhanced activities of stromal enzymes. Paraquat-induced chlorosis (an indicator of sensitivity) was similar in illuminated chloroplast preparations of both biotypes indicating that the resistance factor is located outside of the chloroplast′s envelope. Similar rates of paraquat-induced chlorosis were also observed in illuminated protoplast preparations of both biotypes; however, leaf sections (1 mm width) of the R biotype exhibited a degree of paraquat resistance (81-fold) very similar to that exhibited by whole leaves. These data suggest that resistance is due to a sequestration mechanism that prevents paraquat from diffusing to PSI, the site of paraquat action. The sequestration mechanism appears to require a structurally intact cell wall to be functional.

Molecular cloning and characterization of a gene encoding pea cytosolic ascorbate peroxidase.

A gene encoding cytosolic ascorbate peroxidase (ApxI) from pea (Pisum sativum L.) was isolated and its nucleotide sequence determined. By homologous alignment between the ApxI cDNA (Mittler, R., and Zilinskas, B. (1991) FEBS Lett. 289, 257-259) and the genomic clone, positions of introns and exons were determined. The isolated ApxI gene was found to contain 9 introns, the first of which was located within the 5'-untranslated region of the mRNA. Southern blot analysis of pea genomic DNA suggests that in pea cytosolic ascorbate peroxidase is encoded by a single copy gene. Steady state ApxI transcript levels were found to increase in response to several stresses imposed by drought, heat, and application of ethephon, abscisic acid, and the superoxide-generating agent paraquat. Increases in ascorbate peroxidase activity in response to stresses were less marked than changes observed in transcript levels; cytosolic ascorbate peroxidase protein levels measured by immunoblot analysis remained unchanged.