Isoforms Of Antioxidant Enzymes Research Articles

Upregulation of genes encoding plastidic isoforms of antioxidant enzymes and osmolyte synthesis impart tissue tolerance to salinity stress in bread wheat.

The online version contains supplementary material available at 10.1007/s12298-022-01237-w.

Seed Protection of Solanum lycopersicum with Pythium oligandrum against Alternaria brassicicola and Verticillium albo-atrum.

Pythium oligandrum, strain M1, is a soil oomycete successfully used as a biological control agent (BCA), protecting plants against fungal, yeast, and oomycete pathogens through mycoparasitism and elicitor-dependent plant priming. The not yet described Pythium strains, X42 and 00X48, have shown potential as BCAs given the high activity of their secreted proteases, endoglycosidases, and tryptamine. Here, Solanum lycopersicum L. cv. Micro-Tom seeds were coated with Pythium strains, and seedlings were exposed to fungal pathogens, either Alternaria brassicicola or Verticillium albo-atrum. The effects of both infection and seed-coating on plant metabolism were assessed by determining the activity and isoforms of antioxidant enzymes and endoglycosidases and the content of tryptamine, amino acids, and heat shock proteins. Dual culture competition testing and microscopy analysis confirmed mycoparasitism in all three Pythium strains. In turn, seed treatment significantly increased the total free amino acid content, changing their abundance in both non-infected and infected plants. In response to pathogens, plant Hsp70 and Hsp90 isoform levels also varied among Pythium strains, most likely as a strategy for priming the plant against infection. Overall, our results show in vitro mycoparasitism between Pythium strains and fungal pathogens and in planta involvement of heat shock proteins in priming.

Influence of sodium chloride on the dehydroascorbate reductase activity in Arabidopsis thaliana catalase 2 knokout mutant

Aim. To better understand the mechanisms of abiotic stress resistance in plants, it is important to clarify the role of individual antioxidant enzymes from the same multiproteinic family in the response to stress. It is known that the loss of some isoforms of antioxidant enzymes can be compensated by activation of other enzymes. However, the functional interaction of the ascorbate-glutathione cycle enzymes with catalase under salt stress still remains unexplored. Respectively, we determined the activity of DHAR in knock-out mutants of Arabidopsis thaliana under salt stress. Methods. The DHAR activity was determined in the knock-out line cat2 and in wild-type (WT) Arabidopsis plants after various regimes of treatment with sodium chloride. Results. After treatment with 200 mM sodium chloride in the dark, activation of DHAR was found after 8 hours in WT plants and after 4 hours in the knock-out line cat2. However stress treatment under illumination resulted in significant increase in DHAR activity after 8 hours in both studied lines. In this case, DHAR activity in cat2 was lower than in WT, whereas in non-treated plants or upon stress treatment in the dark no difference between the tested lines was detected. Conclusions. The obtained data indicate that under salt stress conditions, changes in the DHAR activity are included into functional rearrangements of the antioxidant system in cat2 line, which compensate the loss of activity of CAT2 isoenzyme.Keywords: dehydroascorbate reductase, antioxidants, reactive oxygen species (ROS), salt stress, Arabidopsis thaliana

Gene expression and activity of antioxidant enzymes in rice plants, cv. BRS AG, under saline stress.

The rice cultivar (Oryza sativa L.) BRS AG, developed by Embrapa Clima Temperado, is the first cultivar designed for purposes other than human consumption. It may be used in ethanol production and animal feed. Different abiotic stresses negatively affect plant growth. Soil salinity is responsible for a serious reduction in productivity. Therefore, the objective of this study was to evaluate the gene expression and the activity of antioxidant enzymes (SOD, CAT, APX and GR) and identify their functions in controlling ROS levels in rice plants, cultivar BRS AG, after a saline stress period. The plants were grown in vitro with two NaCl concentrations (0 and 136mM), collected at 10, 15 and 20days of cultivation. The results indicated that the activity of the enzymes evaluated promotes protection against oxidative stress. Although, there was an increase of reactive oxygen species, there was no increase in MDA levels. Regarding genes encoding isoforms of antioxidant enzymes, it was observed that OsSOD3-CU/Zn, OsSOD2-Cu/Zn, OsSOD-Cu/Zn, OsSOD4-Cu/Zn, OsSODCc1-Cu/Zn, OsSOD-Fe, OsAPX1, OsCATB and OsGR2 were the most responsive. The increase in the transcription of all genes among evaluated isoforms, except for OsAPX6, which remained stable, contributed to the increase or the maintenance of enzyme activity. Thus, it is possible to infer that the cv. BRS AG has defense mechanisms against salt stress.

Butachlor‐Induced Alterations in Ultrastructure, Antioxidant, and Stress‐Responsive Gene Regulations in Rice Cultivars

Butachlor is an extensively applied herbicide in paddy fields. However, concerns have been raised about its potential adverse effects on the biochemical and physiological processes of non‐target plants. In the present investigation, butachlor‐induced toxic effects in two rice cultivars, Zhejing 88 (ZJ 88) and Xiushui 134 (XS 134), treated with different butachlor treatments were evaluated. A significant decrease in growth rate, pigment concentration, and chlorophyll fluorescence and alternations in chloroplast structures were observed in both rice cultivars at higher treatment levels. The malfunctioning of antioxidant enzymes, the higher uptake of butachlor, and the downregulation of the putative fatty acid elongase gene suggested severe oxidative damage, which was also indicated by enhanced reactive oxygen species (ROS) formation (H2O2, ), lipid peroxidation, and electrolyte leakage and reduced total soluble protein levels in cultivar ZJ 88 than in cultivar XS 134. However, higher proline accumulation and pyrroline‐5‐carboxylate synthase and GABA‐transaminase transcript levels in ZJ 88 suggested their ameliorative role under butachlor‐induced oxidative stress in the sensitive cultivar. In contrast, the stimulated activation of antioxidant enzymes, glutathione, soluble sugars, and phenolics played a vital role in protection against butachlor stress in cultivar XS 134. Thus, butachlor‐induced oxidative stress tolerance in the resistant cultivar (XS 134) is related to the enhanced activation of different isoforms of antioxidant enzymes and a reduced uptake of butachlor compared to the sensitive cultivar (ZJ 88). The present work provides valuable insights into butachlor‐induced toxicity in rice plants.

The content of polyphenolic compounds in the Arabidopsis thaliana knockout mutant Cat2 under salt stress

Aim. In plants, the defense response to abiotic stress includes induction of stress proteins and increase in content of protective metabolites. To date, the role of specific isoforms of antioxidant enzymes in stress responses and their relation to low-molecular weight protective compounds are still not clarified. To study this question the content of polyphenolic compounds (PPC) was evaluated under salt stress in Arabidopsis thaliana wild-type (WT) and in catalase 2 (Cat2) knockout mutant plants. Methods. PPC content in different variants of treatment with sodium chloride was measured. Results. It was shown that under optimal cultivation conditions the content of PPC in leaves of cat2 mutants is higher than in WT leaves. However, cultivation of isolated shoots in nutrient medium resulted in a faster depletion of the PPC pool in the cat2 line. Also, short-term salt stress results in equal depletion of the PPC pool in both, WT and cat2. Conclusions. The increase of PPC content in cat2 leaves is a manifestation of metabolic alterations that aim to compensate the reduced catalase activity.Keywords: multigenic family, knockout mutants, polyphenolic compounds, sodium chloride, Arabidopsis thaliana.

Free prolin content in Arabidopsis thaliana Cat2 and Cat3 knockout mutants under salt stress

Aim. In plants, the enzymatic and non-enzymatic environmental stress resistance mechanisms function in a concerted manner, but the role of specific isoforms of antioxidant enzymes and their relationship to low molecular weight protective compounds is poorly understood. To investigate this question free proline levels were compared under salt stress conditions in wild-type Arabidopsis thaliana and knockout mutants for the catalase genes Cat2 and Cat3. Methods. Free proline content was measured under various treatments of plants with sodium chloride. Results. It was shown that under salt stress conditions free proline increase is impaired in leaves of mutants lacking CAT2 and CAT3 activity. In addition, cat3 knockout line shows a reduced basal level of proline under non-stress conditions. Conclusions. Suppression of response to salt stress points to a possible link between the rearrangement of the antioxidant system in the Cat genes knockouts and the regulation of protective cellular response to salt stress.Key words: Arabidopsis thaliana, knockout mutants, catalase, proline, sodium chloride.

Effects of Cytokinin and Nitrogen on Drought Tolerance of Creeping Bentgrass.

Cytokinin (CK) is a vital plant hormone that controls many aspects of growth and development in plants. Nitrogen (N) is the indispensable macronutrient needed in plants and also one of the most important limiting factors for plant growth. This study was designed to investigate the simultaneous effects of CK and N on the visual turf quality and antioxidant metabolism of drought-stressed creeping bentgrass (Agrostis stolonifera L.). ‘PennA-4’ creeping bentgrass treated with trans-zeatin riboside at three rates of CK concentrations of 0, 10 and 100 μM (designated by CK0, 10, and 100) and two nitrogen rates with 2.5 and 7.5 kg N·ha-1 every 15 days (designated by low and high N) in a complete factorial arrangement was grown under two soil moisture regimes: well-watered and drought stress. Exogenous CK improved turf quality and delayed leaf wilting under drought stress, especially under high N. The grasses treated with CK10 and CK100 had lower O2- production and H2O2 concentration than those without CK treatment. The CK100 treatment enhanced the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and guaiacol peroxidase (POD) by 25%, 22%, 17% and 24%, respectively, relative to CK0. Moreover, the activity changes of the antioxidant enzyme isoforms were more significant under high N condition relative to low N condition. Our results demonstrated the beneficial impacts of CK and N on physiological reactions, especially antioxidant metabolism, and foliar application of CK at 10 or 100 μM plus 7.5 kg ha-1 N biweekly may improve drought stress resistance of creeping bentgrass.

Nitric oxide induces specific isoforms of antioxidant enzymes in soybean leaves subjected to enhanced ultraviolet-B radiation

Antioxidant enzymes play a key role in plant tolerance to different types of stress, including ultraviolet-B (UV-B) radiation. Here we report that nitric oxide (NO) enhances antioxidant enzymes gene expression and increases the activity of specific isoforms protecting against UV-B radiation. Pre-treatments with sodium nitroprussiate (SNP), a NO-donor, prevented lipid peroxidation, ion leakage and H2O2 and superoxide anion accumulation in leaves of UV-B-treated soybean plants. Transcripts levels of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) were significantly induced by SNP. These data correlated with the enhancement of particular antioxidant enzyme isoforms, such as one CAT isoform and two APX isoforms. Moreover, SNP induced the expression of three new isoforms of SOD, identified as Mn-SOD subclass. Further results showed that total activities of SOD, CAT and APX significantly increased by 2.2-, 1.8- and 2.1-fold in SNP-treated plants compared to controls, respectively. The protective effect of SNP against UV-B radiation was negated by addition of the specific NO scavenger cPTIO, indicating that NO released by SNP mediates the enhancement of antioxidant enzymes activities. In conclusion, NO is involved in the signaling pathway that up-regulates specific isoforms of antioxidant enzymes protecting against UV-B-induced oxidative stress.

Effects of UV-B irradiation on isoforms of antioxidant enzymes and their activities in red alga Grateloupia filicina (Rhodophyta)

Macroalgae in a littoral zone are inevitably exposed to UV-B irradiance. We analyzed the effects of UV-B on isoenzyme patterns and activities of superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), and ascorbate peroxidase (APX) of red algae Grateloupia filicina (Lamour.) C. Agardh. The activities of SOD, CAT, and APX changed in response to UV-B in a time- and dose-dependent manner. POX activity increased significantly under all three UV-B treatments. The enzymatic assay showed three distinct bands of SODI (Mn-SOD), SODII (Fe-SOD), and SODIII (CuZn-SOD) under a low (Luv) and medium (Muv) dose of UV-B irradiation, while SODI and SODIII activities decreased significantly when exposed to a high dose of UV-B irradiation (Huv). The activity of POX isoenzymes increased significantly after exposure to UV-B, which is consistent with the total activity. In addition, a clear decrease in activity of CATIV was detected in response to all the three doses of UV treatments. Some bands of APX isoenzyme were also clearly influenced by UV-B irradiation. Correspondingly, the daily growth rate declined under all the three exposure doses, and was especially significant under Muv and Huv treatments. These data suggest that, although the protection mechanisms of antioxidant defense system are partly inducible by UV-B to prevent the damage, G. filicina has incomplete tolerance to higher UV-B irradiation stress.

Oxidative Stress in Experimental Rodent Corneas Infected With Aflatoxigenic and Nonaflatoxigenic Aspergillus flavus

To seek a possible association between aflatoxigenicity and oxidative stress in keratitis caused by Aspergillus flavus in an experimental rodent model. Wistar rats were divided into 3 groups of 6 each. Group 1 served as mock-inoculated controls. Experimental fungal keratitis was induced in group 2 and group 3 rats using aflatoxigenic and nonaflatoxigenic A. flavus conidial suspensions, respectively, and clinical features were scored for 5 days after inoculation. At this time, animals were killed, and test corneas were excised and examined histologically. Expression of IL-1β and TNF-α genes was sought in excised corneas. Levels of malondialdehyde (MDA) and reduced glutathione (GSH) and activities of key antioxidant enzymes were measured in excised corneas and fungal mycelial homogenates. Antioxidant enzyme isoforms were sought in mycelial homogenates by native polyacrylamide gel electrophoresis. Mean levels of MDA and GSH and mean activities of catalase, superoxide dismutase, and glutathione peroxidase were significantly (P < 0.05) higher in a mycelial homogenate of aflatoxigenic A. flavus than in the nonaflatoxigenic mycelial homogenate. Increased numbers of well-stained isoforms were detected in aflatoxigenic mycelial homogenates. Significantly (P < 0.05) higher expression profiles of IL-1β and TNF-α genes, MDA and GSH levels, and antioxidant enzyme activities were noted in group 2 rat corneas than in group 3 rat corneas. Clinical and histological scores suggested a more severe keratitis in group 2 rat corneas than in group 1 and group 3 rat corneas. Aflatoxigenicity is associated with more intense oxidative stress in experimental A. flavus keratitis.

Selenium and spermine alleviate cadmium induced toxicity in the red seaweed Gracilaria dura by regulating antioxidants and DNA methylation

The protective role of exogenously supplied selenium (Se) and polyamines (PAs) such as putrescine (Put) and spermine (Spm) in detoxifying the cadmium (Cd) induced toxicity was studied in the marine red alga Gracilaria dura in laboratory conditions. The Cd exposure (0.4 mM) impede the growth of alga while triggering the reactive oxygen species (ROS viz. O(2)(•-) and H(2)O(2)) generation, inhibition of antioxidant system, and enhancing the lipoxygenase (LOX) activity, malondialdehyde (MDA) level and demethylation of DNA. Additions of Se (50 μM) and/or Spm (1 mM) to the culture medium in contrast to Put, efficiently ameliorated the Cd toxicity by decreasing the accumulation of ROS and MDA contents, while restoring or enhancing the level of enzymatic and nonenzymatic antioxidants and their redox ratio, phycobiliproteins and phytochelatins, over the controls. The isoforms of antioxidant enzymes namely superoxide dismutase (Mn-SOD, ~150 kDa; Fe-SOD ~120 kDa), glutathione peroxidase (GSH-Px, ~120 and 140 kDa), glutathione reductase (GR, ~110 kDa) regulated differentially to Se and/or Spm supplementation. Furthermore, it has also resulted in enhanced levels of endogenous PAs (specially free and bound insoluble Put and Spm) and n-6 PUFAs (C20-3, n-6 and C20-4, n-6). This is for the first time wherein Se and Spm were found to regulate the stabilization of DNA methylation by reducing the events of cytosine demethylation in a mechanism to alleviate the Cd stress in marine alga. The present findings reveal that both Se and Spm play a crucial role in controlling the Cd induced oxidative stress in G. dura.

Toxic Effects of Imidazolium Ionic Liquids on the Green Seaweed Ulva lactuca: Oxidative Stress and DNA Damage

The green credentials of ionic liquids (ILs) are being increasingly questioned due to the growing evidence of their toxicity to aquatic ecosystems, although the mechanisms of toxicity are unknown. This study provides insights into the mechanism of toxicity and biological effects of 1-alkyl-3-methylimidazolium bromide [C(n)mim]Br (n = 4 to 16) on the marine macroalga Ulva lactuca. The cell viability of this alga during IL exposure was found to be negatively correlated to the chain length of the alkyl group. The IL ([C(12)mim]Br) exposure triggers the generation of reactive oxygen species (ROS viz. O(2)(•-), H(2)O(2), and OH(•)), damage of the membrane and DNA, and inhibition of antioxidant systems in the alga. The enhanced production of ROS and lipid peroxidation in the alga subjected to LC(50) concentration for 4 days was largely attributed to lipoxygenase (LOX) activity coupled with the induction of two LOX isoforms (~80 kDa and ~55 kDa). Pretreatment of the algal thallus with enzyme inhibitors such as diphenylene iodonium, sodium azide, cantharidin, and oxadiazoloquinoxalin-1-one, prior to [C(12)mim]Br exposure showed the regulation of ROS by the activation of membrane bound NADPH-oxidase and cytochrome oxidase. The IL exposure resulted in the accumulation of n-3 and n-6 fatty acids at 0.5 LC(50) concentration indicating the induction of desaturase enzymes. Furthermore, antioxidant enzyme activities such as superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) were enhanced by 1.3-2.0-fold, while glutathione peroxidase (GSH-Px) diminished, together with a higher regeneration rate of reduced ascorbate and glutathione. The isoforms of antioxidant enzymes, namely, Mn-SOD (~85 kDa), APX (~125 and 45 kDa), and GR (~135 kDa) regulated differentially to IL exposure. The comet assay performed for the first time for seaweeds revealed the significant induction of DNA damage (>50-70% increase in % tail DNA over control) in alga exposed to ≥ LC(50) concentration.

Differential Responses to Heat Stress in Activities and Isozymes of Four Antioxidant Enzymes for Two Cultivars of Kentucky Bluegrass Contrasting in Heat Tolerance

Understanding antioxidant mechanisms for heat stress is important for improving heat tolerance in cool-season plant species. The objective of this study was to identify antioxidant enzymes associated with cultivar variations in heat tolerance in kentucky bluegrass (Poa pratensis) by comparing heat responses of activity and isoforms of antioxidant enzymes in two cultivars contrasting in heat tolerance. Plants of heat-tolerant ‘Eagleton’ and heat-sensitive ‘Brilliant’ were exposed to 20 °C (control) or 40 °C (heat stress) for 28 days in growth chambers. Chlorophyll (Chl) a content remained unchanged and Chl b content increased in ‘Eagleton’, while both of them decreased in ‘Brilliant’, and by 28 days, ‘Eagleton’ had significantly higher Chl a and b content than ‘Brilliant’. The activities of superoxide dismutase (SOD) were significantly higher in ‘Eagleton’ than in ‘Brilliant’ by 28 days of heat stress. An isozyme SOD2 was induced early during heat stress in ‘Eagleton’, while isozyme SOD3 degraded, to a lesser extent in ‘Eagleton’ than in ‘Brilliant’. Catalase (CAT) activity significantly increased in ‘Brilliant’ but remained constant in ‘Eagleton’, and ‘Brilliant’ had a significantly higher CAT activity and isozyme CAT1 than ‘Eagleton’ during heat stress. Significant increases in ascorbate peroxidase (APX) activities occurred under heat stress, to a greater extent in ‘Eagleton’, whereas isozymes did not exhibit difference between cultivars. Guaiacol-peroxidase (POD) activity declined during heat stress in both cultivars. The intensity of POD isozymes in ‘Brilliant’ remained constant, while ‘Eagleton’ showed a transient increases in POD1 at 7 days of heat stress. Our results indicated that antioxidant defense mechanisms for heat tolerance in kentucky bluegrass could be mainly associated with changes in activity and forms of isozymes of SOD for O2 scavenging and APX activity for H2O2 scavenging under heat stress.

Changes in the pattern of antioxidant enzymes in wheat exposed to water deficit and rewatering

Antioxidant defenses in two wheat cultivars differing in sensitivity to dehydration (YouJian (YJ-24) more sensitive than LongChun (LC-20) were analyzed during water deficit and rewatering. Resistant cultivar (LC-20) showed a higher relative water content than the sensitive cultivar (YJ-24) during the whole period of water withholding. In order to analyze the changes of antioxidant enzymes, native PAGE analysis of protein extract were performed. Wheat leaves had two isoforms of Mn-superoxide dismutase (SOD), two isoforms of Cu/Zn-SOD and one of Fe-SOD. Three catalase (CAT) isoforms were identified in the leaves of wheat. The activities of SOD and CAT isoforms were increased in two cultivars under water deficit. The intensities of SOD and CAT isoforms were slightly lower in LC-20 and increased continuously in YJ-24 after rewatering. Peroxidase (POD) isoforms were significantly increased during the whole dehydration-rehydration period. Three ascorbate peroxidase (APX) isoforms were present in gel. APX-1 and APX-3 were enhanced during water deficit and decreased during rewatering in LC-20. In YJ-24 only the activities of APX-2 were increased under water deficit. Seven isoforms of glutathione reductase (GR) were detected in the native gel. Activities of most of GR isoforms were higher in tolerant (LC-20) than in sensitive cultivar (YJ-24). Different isoforms of GR in two wheat cultivars behaved differently under water deficit and rewatering. These results collectively suggest that water deficit activates the SOD, CAT and ascorbate-glutathione cycle in wheat leaves. The response of enzyme isoforms to drought is not the same for all isoforms of antioxidant enzymes in two wheat cultivars.

Antioxidant Enzyme Isoforms on Gels in Two Poplar Clones Differing in Sensitivity After Exposure to Ozone

The effect of acute ozone (O3) fumigation on isozyme patterns of superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX) in mature (ML) and young leaves (YL) of two poplar clones, contrasting in O3-sensitivity was analysed. Untreated leaves of both the O3-sensitive (O3-S) clone Eridano of Populus deltoides×P. maximowiczii and the O3-resistant (O3-R) clone I-214 of P.×euramericana showed four distinct SOD isoforms with a relative mobility (Rf) of 0.54 (MnSOD), 0.60 (Cu/ZnSOD), 0.65 (unidentified), and 0.71 (Cu/ZnSOD). After O3-fumigation the activity of the SOD isoforms showed only quantitative variations with respect to control plants. In ML of untreated O3-R plants seven POD isoforms (Rf= 0.13, 0.19, 0.34, 0.59, 0.64, 0.70 and 0.75) were found, while in YL one isoform (Rf= 0.34) was undetected. Only three POD isoforms in both ML and YL of untreated O3-S plants were resolved. The electrophoretic pattern of POD in O3-S leaves was greatly modified by acute O3-fumigation with the appearance of new isoforms in both YL and ML and the disappearance of an isoform (Rf= 0.13) in YL. Additionally, O3-exposure induced the appearance of two APX isoforms in YL (Rf= 0.66 and 0.70), and one isoform in ML (Rf= 0.70) of the O3-S clone. By contrast, the activity of the three APX isoformes (Rf= 0.64, 0.70 and 0.76) detected in O3-R leaves showed only quantitative variation with respect to untreated plants. From these data it is concluded that: 1) in these poplar hybrids antioxidant enzyme activity is developmentally regulated and greatly affected by acute O3 stress treatments and 2) the different enzymes activity displayed by the two poplar clones, especially for POD and APX isoformes, could partly explain their distinct O3-sensitivity.

The inductive responses of the antioxidant enzymes by salt stress in the rice ( Oryza sativa L.)

Summary To access contributions of inductive responses of the antioxidant enzymes in the resistance to salt stress, activities of the enzymes were determined in the rice ( Oryza sativa L. cv. Dongjin) plant. In the leaves of the rice plant, salt stress preferentially enhanced the content of H 2 O 2 as well as the activities of the superoxide dismutase (SOD), ascorbate peroxidase (APX), and peroxidase specific to guaiacol, whereas it induced the decrease of catalase activity. On the other hand, salt stress had little effect on the activity levels of glutathione reductase (GR). In order to analyze the changes of antioxidant enzyme isoforms against salt stress, plant extracts were subjected to native PAGE. Leaves of the rice plant had two isoforms of Mn-SOD and five isoforms of Cu/Zn-SOD. Fe-SOD isoform was not observed in the activity gels. Expression of Cu/Zn-1, -2, and Mn-SOD-2 isoforms was preferentially enhanced by salt stress. Seven APX isoforms were presented in the leaves of the rice plants. The intensities of APX-4 to -7 were enhanced by salt stress, whereas those of APX-1 to -3 were minimally in changed response to salt stress. There were seven GR isoforms in the leaves of rice plants. Levels of activity for most GR isoforms did not change in the stressed plants compared to the control plants. On the other hand, the levels of activity for most antioxidant enzymes changed little in the roots of stressed plants compared to the control plants. These results collectively suggest that SOD leads to the overproduction of hydrogen peroxide in the leaves of rice plants subjected to salt stress: The overproduction of hydrogen peroxide functions as the signal of salt stress, which induces the induction of specific APX isoforms but not specific GR isoforms under catalase deactivation.

Chilling stress-induced changes of antioxidant enzymes in the leaves of cucumber: in gel enzyme activity assays

To investigate the antioxidant defense system, chilling stress-induced changes of antioxidant enzymes were examined in the leaves of cucumber ( Cucumis sativus L.). Chilling stress preferentially enhanced the activities of the superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and peroxidase specific to guaiacol, whereas it induced the decrease of catalase activity. In order to analyze the changes of antioxidant enzyme isoforms against chilling stress, foliar extracts were subjected to native PAGE. Leaves of cucumber had four isoforms of Mn-SOD and two isoforms of Cu/Zn-SOD. Fe-SOD isoform was not observed in this plant. Expression of Cu/Zn-SOD and Mn-SOD was preferentially enhanced by chilling stress. Expression of Mn-SOD-2 and -4 was enhanced after 48 h of the poststress period. Five APX isoforms were presented in the leaves of cucumber. The intensities of APX-4 and -5 were enhanced by chilling stress, whereas that of APX-3 was significantly increased in the poststress periods after chilling stress. Gel stained for GR activity revealed six isoforms in the plant. Activation levels for most of GR isoforms were higher in the stressed-plants than the control and poststressed-plants, but that of GR-1 isoform was significantly higher in the poststressed-plants than chilling stressed-plants. These results collectively suggest that chilling stress activates the enzymes of an SOD/ascorbate-glutathione cycle under catalase deactivation in the leaves of cucumber, but the response timing of enzyme isoforms against various environmental stresses is not the same for all isoforms of antioxidant enzymes.