Effect of low above-zero temperature on the content of low-molecular antioxidants and activities of antioxidant enzymes in green barley leaves

We studied the effect of low above-zero temperature (2°C) on the content of low-molecular antioxidants (ascorbic acid, glutathione, and carotenoids) and also activities of antioxidant enzymes (ascorbate peroxidase, APO; catalase, CAT; glutathione reductase, GR; and superoxide dismutase, SOD) in green barley (Hordeum vulgare L.) seedlings. Under stress conditions, the content of low-molecular antioxidants, especially that of reduced ascorbate form, increased. Low-temperature stress activated APO, CAT, GR, and SOD. First enzymes responding to the action of stress factor were APO and CAT, i.e., enzymes neutralizing hydrogen peroxide in plant cells, which indicated H₂O₂ active generation at low temperature. Cytoplasmic SOD was more active than its chloroplast isoforms. This indicates that oxidative process initiation under low-temperature stress occurred more active in the cytosol. After termination of stress-factor action, the content of total ascorbate, glutathione, and carotenoids reduced rapidly to the level close to the initial one. During post-stress period, the amount of reduced ascorbate declined as well; however, it remained at the level higher than the initial one. Activities of APO and CAT dropped sharply; activities of GR and SOD reduced gradually. Thus, reduced ascorbate, APO, and CAT play an important role in plant cell defense against above-zero temperatures close to zero; reduced ascorbate, GR, and SOD are especially important during post-stress period.     

Accumulation of hydrogen peroxide and functioning of defense system in overwatered barley seedlings

The effects of overwatering (flooding) on the oxidative potential, the level of low-molecular-weight antioxidants, the content of stress proteins, and activities of antioxidant enzymes in green barley (Hordeum vulgare L.) seedlings were studied. Overwatering retarded barley seedling growth and induced hydrogen peroxide accumulation, a decrease in the total ascorbate content and an increase in the content of reduced glutathione (GSH), but it did not affect the content of oxidized glutathione (GSSG). After the cessation of stress factor action (post-stress period), the content of hydrogen peroxide declined to the initial level, the content of ascorbate reduced still stronger, whereas the content of GSH continued to rise. Under flooding conditions, activities of glutathione reductase (GR) and superoxide dismutase (SOD) increased. After the cessation of stress factor action, activities of these enzymes decreased but remained at rather high levels as compared with control. Activity of catalase (CAT) reduced during stress, whereas activity of ascorbate peroxidase (APX) was not essentially changed. In the post-stress period, CAT activity remained to be low; in contrast, APX activity increased. Barley seedling flooding induced the synthesis of stress proteins, HSP70 and dehydrins (DH). In the post-stress period, the content of stress proteins decreased; however, the content of DH in experimental leaves remained rather high. The results obtained indicate that barley defense system manifested a complex response to overwatering, which may be related to the oxygen shortage under stress conditions and sharp metabolism activation at re-aeration in the post-stress period.     

Effect of zinc on antioxidant response in maize (Zea mays L.) leaves

Maize (Zea mays L. cv kanaujia) plants grown with Zn [10 (control), 0.1 (low) and 20 microM (high)], were investigated for concentration of antioxidants and activities of antioxidative enzymes in leaves. Young leaves of low Zn plants developed whitish-necrotic spots. Leaves of both low and high Zn plants showed decrease in chlorophyll concentration and accumulation of lipid peroxides, ascorbate and dehydroascorbate, associated with a decrease in the activity of ascorbate peroxidase and superoxide dismutase. Low and high Zn, however, showed diverse effect on glutathione reductase. While low Zn increased the activity of glutathione reductase, high Zn decreased its activity. Zinc effect on antioxidative constituents suggested Zn involvement in sustaining the antioxidative defense system in maize leaves.     

Prospects for enhancement of the soluble antioxidants,ascorbate and glutathione

Ascorbic acid (vitamin C) and the tripeptide thiol, glutathione gamma-glutamyl cysteinyl glycine (glutathione) are the major low molecular weight soluble antioxidants in plant cells. The pathway of glutathione biosynthesis is similar in animals and plants while that of ascorbate biosynthesis differs considerably between the two kingdoms. The potential for obtaining substantial constitutive changes in the tissue contents of these antioxidants by manipulation of the biosynthetic enzymes has been demonstrated. Moreover, the concentrations of ascorbate and glutathione are greatly modified in response to a variety of environmental triggers, particularly those that cause increased oxidative stress. It is essential that the signals and associated signal transduction pathways that trigger enhanced antioxidant accumulation are elucidated as these offer an important alternative means of achieving greater nutritional value in edible plant organs.     

Effect of flooding on the activities of some enzymes of activated oxygen metabolism,the levels of antioxidants,and lipid peroxidation in senescing tobacco leaves

Effects of flooding on the activities of some enzymes of activated oxygen metabolism, the levels of antioxidants, and lipid peroxidation in senescing leaves of tobacco were investigated. As judged by the decrease in chlorophyll and protein levels, flooding accelerated the senescence of tobacco leaves. Total peroxide and the lipid peroxidation product, malondialdehyde, increased in both control and flooding-treated leaves with increasing duration of the experiment. Throughout the duration of the experiment, flooded leaves had higher levels of total peroxide and malondialdehyde than did control leaves. Flooding resulted in an increase in peroxidase and ascorbate peroxidase activities and a reduction of superoxide dismutase activity in the senescing leaves. Glycolate oxidase, catalase, and glutathione reductase activities were not affected by flooding. Flooding increased the levels of total ascorbate and dehydroascorbate. Total glutathione, reduced form glutathione, or oxidized glutathione levels in flooded leaves were lower than in control leaves during the first two days of the experiment, but were higher than in control leaves at the later stage of the experiment. Our work suggests that senescence of tobacco induced by flooding may be a consequence of lipid peroxidation possibly controlled by superoxide dismutase activity. Our results also suggest that increased rates of hydrogen peroxide in leaves of flooded plants could lead to increased capacities of the scavenging system of hydrogen peroxide.Abbreviations GSH reduced form glutathione - GSSG oxidized form glutathione - GSSG reductase glutathione reductase - MDA malondialdehyde - SOD superoxide dismutase     

Salt tolerance of barley induced by the root endophyte Piriformospora indica is associated with a strong increase in antioxidants

The root endophytic basidiomycete Piriformospora indica has been shown to increase resistance against biotic stress and tolerance to abiotic stress in many plants. Biochemical mechanisms underlying P. indica-mediated salt tolerance were studied in barley (Hordeum vulgare) with special focus on antioxidants. Physiological markers for salt stress, such as metabolic activity, fatty acid composition, lipid peroxidation, ascorbate concentration and activities of catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase and glutathione reductase enzymes were assessed. Root colonization by P. indica increased plant growth and attenuated the NaCl-induced lipid peroxidation, metabolic heat efflux and fatty acid desaturation in leaves of the salt-sensitive barley cultivar Ingrid. The endophyte significantly elevated the amount of ascorbic acid and increased the activities of antioxidant enzymes in barley roots under salt stress conditions. Likewise, a sustained up-regulation of the antioxidative system was demonstrated in NaCl-treated roots of the salt-tolerant barley cultivar California Mariout, irrespective of plant colonization by P. indica. These findings suggest that antioxidants might play a role in both inherited and endophyte-mediated plant tolerance to salinity.     

Barley leaf antioxidant system under photooxidative stress induced by Rose Bengal

The effects of photooxidative stress induced in green barley (Hordeum vulgare L.) leaves by xanthene dye Rose Bengal (RB) on the content of low-molecular antioxidants and the activity of antioxidant enzymes were studied. During illumination (24 h, 160 mol quanta/(m² s)) of the leaves preincubated in darkness on 10 and 100 M RB, ROS accumulated, and their level increased along with RB concentration and duration of illumination. Under these conditions, the content of reduced ascorbate and reduced glutathione (GSH) increased, the level of -and -tocopherol decreased, and the activity of ascorbate peroxidase, the enzyme participating in H₂O₂ degradation, increased. At the same time, the activity of catalase, also participating in H₂O₂ detoxification, decreased, which may be due to the enzyme inhibition during the photochemical stress. In the illuminated treated leaves, superoxide dismutase (SOD), the enzyme destroying the superoxide anion radicals, was activated. The cytosolic SOD isoform was the first to be activated and chloroplastic isoforms followed. It is supposed that photodynamic processes induced by RB in barley leaves are initiated in the cytosol. The activity of glutathione reductase, participating in glutathione oxidized form reduction, did not change at first and grew only after continuous illumination. Thus, the increase in the GSH level, which we have revealed on the initial stage of photooxidative stress development, was due to its synthesis de novo. In addition, under photooxidative stress induced by the sensitizer RB, the level of tocopherol reduced, whereas the amount of other low-molecular antioxidants increased. The exhaustion of the tocopherol pool, in its turn, could limit the resistance of barley leaves to the photooxidative stress.     

Antioxidant defences of the apoplast

Summary The apoplast of barley and oat leaves contained superoxide dismutase (SOD), catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase activities. The activities of these enzymes in the apoplastic extracts were greatly modified 24 h after inoculation with the biotrophic fungal pathogenBlumeria graminis. The quantum efficiency of photosystem II, which is related to photosynthetic electron transport flux, was comparable in inoculated and healthy leaves during this period. Apoplastic soluble acid invertase activity was also modified in inoculated leaves. Inoculation-dependent increases in apoplastic SOD activity were observed in all lines. Major bands of SOD activity, observed in apoplastic protein extracts by activity staining of gels following isoelectric focusing, were similar to those observed in whole leaves but two additional minor bands were found in the apoplastic fraction. The apoplastic extracts contained substantial amounts of dehydroascorbate (DHA) but little or no glutathione (GSH). Biotic stress decreased apoplastic ascorbate and DHA but increased apoplastic GSH in resistant lines. The antioxidant cycle enzymes may function to remove apoplastic H₂O₂ with ascorbate and GSH derived from the cytoplasm. DHA and oxidized glutathione may be reduced in the apoplast or returned to the cytosol for rereduction.Abbreviations AA reduced ascorbate - APX ascorbate peroxidase - DHA dehydroascorbate (oxidised ascorbate) - DHAR dehydroascorbate reductase - G6PDH glucose-6-phosphate dehydrogenase - GSH reduced glutathione - GSSG glutathione disulphide - GR glutathione reductase - MDHA monodehydroascorbate - MDHAR monodehydroascorbate reductase - SOD superoxide dismutase     

Antioxidative protection in the leaves of dark-senescing intact barley seedlings

In order to elucidate the possibility of in vivo oxidative modification of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39) as a triggering mechanism for its preferential degradation early in senescence, some antioxidant compounds, protective enzymes, H₂O₂ and protein carbonylation levels were studied in the leaves during dark-induced senescence of barley (Hordeum vulgare L. cv. “Obzor”) seedlings. Analyses were performed in extracts as well as in purified chloroplasts. Some weakening of the antioxidative protection was detected during the treatment: diminution in the ascorbate and non-protein SH (mainly glutathione) pools, lower activities of superoxide dismutase, guaiacol and ascorbate peroxidases. However, no accumulation of H₂O₂ was found, lower level of protein carbonylation in darkness was measured and the percentage of reduced ascorbate was maintained high. Data concerning antioxidant compounds in chloroplasts revealed some impairment of the ascorbate and glutathione pools under induced senescence - the level of non-protein thiols declined during early senescence whereas the ascorbate pool was not significantly changed. The percentage of reduced ascorbate remained high in the chloroplasts and the activities of superoxide dismutase and of ascorbate peroxidase were conserved. Taken together the results are not in accordance with the possibility of in vivo oxidative modification of Rubisco in the case of dark-induced senescence. Our data bring some support to the view about redox regulation of Rubisco turnover in senescence through the pool of the low-molecular chloroplastic thiols.     

Age-dependence of the antioxidative system in tobacco with enhanced glutathione reductase activity or senescence-induced production of cytokinins

Tobacco leaves of plants with enhanced glutathione reductase activity (GR46-27, Nicotiana tabacum L. cv. Samsun) or with autoregulated senescence-induced production of cytokinins (P_SAG12-IPT, N. tabacum L. cv. Wisconsin) were studied during the course of leaf development and senescence by measuring photosynthesis, chlorophyll and protein content, the antioxidants ascorbate, glutathione and α -tocopherol as well as the antioxidative enzymes ascorbate peroxidase (APX, EC 1.11.1.11), glutathione reductase (GR, EC 1.6.4.2) and superoxide dismutase (SOD, EC 1.15.1.1). The photosynthetic rate, as well as the chlorophyll and protein content, dropped with increasing leaf age after having reached a maximum at the end of the exponential growth phase. The concentrations of the water-soluble antioxidants ascorbate and glutathione fell continuously with age, whereas the concentration of the lipophilic α -tocopherol increased. The activities of the antioxidative enzymes APX, GR and SOD reached their maximum at the beginning of leaf development, but were reduced in senescing leaves. The age-dependent course of the measured leaf parameters in GR46-27 leaves was similar to the one in wild-type leaves, with the exception of an overall enhanced GR activity. In contrast, in old leaves of P_SAG12-IPT plants, which possess a much higher life span, the chlorophyll and protein content, the photosynthetic rate, the antioxidant concentrations of ascorbate and glutathione as well as the activities of the antioxidative enzymes were higher than in wild-type leaves. The results show that the capacity of the antioxidative system to scavenge radicals is sufficiently balanced with the plant metabolism, and its decline with increasing age is not the cause, but a consequence of senescence and ageing in plants.     

Cold- and light-induced changes of metabolite and antioxidant levels in two high mountain plant species Soldanella alpina and Ranunculus glacialis and a lowland species Pisum sativum

Leaves of the two cold-acclimated alpine plant species Ranunculus glacialis and Soldanella alpina and, for comparison, of the non-acclimated lowland species Pisum sativum were illuminated with high light intensity at low temperature. The light- and cold-induced changes of antioxidants and of the major carbon and phosphate metabolites were analysed to examine which metabolic pathways might be limiting in non-acclimated pea leaves and whether alpine plants are able to circumvent such limitation. During illumination at low temperature pea leaves accumulated high quantities of sucrose, glucose-6-phosphate, fructose-6-phosphate, mannose-6-phosphate and phosphoglycerate (PGA) whereas ATP/ADP-ratios decreased. Although the PGA content also increased in leaves of R. glacialis the other metabolites did not accumulate and ATP/ADP-ratios remained fairly constant in either alpine species. These data indicate a inorganic phosphate (Pi)-limitation in the chloroplasts of pea leaves but not in the alpine species. However, the total phosphate pool and the percentage of free Pi were highest in pea and did not change during illumination in cold. In contrast, free Pi contents declined markedly in R. glacialis leaves, suggesting that Pi is available for metabolism in this species. In S. alpina leaves contents of ascorbate and glutathione doubled in light and cold, while the contents of sugars did not increase. Obviously, S. alpina leaves can use assimilated carbon for ascorbate synthesis, rather than for the synthesis of sugars. A high capacity for ascorbate synthesis might prevent the accumulation of mannose-6-phosphate and Pi-limitation.     

The association of protein synthesis with protochlorophyllide holochrome regeneration in dark-grown barley leaves

A light-stimulated increase in incorporation of radioactive amino acids into protein associated with protochlorophyllide holochrome occurs concomitantly with the regeneration of phototransformable protochlorophyllide in dark-grown barley leaves. This increase in radioactivity and the protochlorophyllide regeneration process are both abolished by incubation of the leaves with inhibitors of cytoplasmic protein synthesis. Prelimiary data implicate protein in the molecular weight range of 45,000–60,000 daltons in this process.     

Carbohydrate Accumulation Affects the Redox State of Ascorbate in Detached Tobacco Leaves

A decreased utilization of NADPH for CO₂ fixation as a result of carbohydrate accumulation in chlorotic leaves is generally believed to be associated with an increase in oxidative stress. Molecular oxygen may serve as an alternative electron acceptor of photosynthesis under these conditions. In order to test this hypothesis mature leaves of tobacco plants (Nicotiana tabacum) were detached, fed with glucose (50 mM) via the petiole, and used to study the effect of carbohydrate accumulation on the pigment content and the components of the superoxide dismutase-ascorbate-glutathione cycle. During a period of five days the concentration of total soluble carbohydrates increased substantially in leaves supplied with glucose in comparison with control leaves supplied only with water. This increase was accompanied by a twofold decrease in the chlorophyll content. In detached water-fed leaves the levels of most of the antioxidative components increased, whereas glucose feeding had no or only little additional effect on the activities of the protective enzymes, but caused a 1.6- and 4-fold increase in the contents of glutathione and ascorbate, respectively. In relation to the total foliar ascorbate pool, the amount of reduced ascorbate increased from about 30 % to 60 % upon feeding with glucose. These results do not support the idea that the accumulation carbohydrates per se cause an increased rate of superoxide production which necessitates increased activities of antioxidative enzymes. It rather appears that glucose-fed leaves have an increased reductive capacity that can be released via glutathione into the ascorbate pool, thereby, regulating the redox state of ascorbate.     

Antioxidative response to cadmium in roots and leaves of tomato plants

Treatment of tomato seedlings (Lycopersicon esculentum Mill. cv. 63/5 F1) with increasing CdCl₂ concentrations in the culture medium resulted in Cd accumulation more important in roots than in leaves. Biomass production was severely inhibited, even at low Cd concentration. Cd reduced chlorophyll content in leaves and enhanced lipid peroxidation. An increase in antioxidative enzyme (superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, glutathione reductase) activities was more pronounced in leaves than in roots, while catalase activity increased only in roots. In addition, changes in isoenzyme composition were observed using the non-denaturing polyacrylamid gel electrophoresis.     

Pathogen-Induced Changes in the Antioxidant Status of the Apoplast in Barley Leaves

Leaves of two barley (Hordeum vulgare L.) isolines, Alg-R, which has the dominant Mla1 allele conferring hypersensitive race-specific resistance to avirulent races of Blumeria graminis, and Alg-S, which has the recessive mla1 allele for susceptibility to attack, were inoculated with B. graminis f. sp. hordei. Total leaf and apoplastic antioxidants were measured 24 h after inoculation when maximum numbers of attacked cells showed hypersensitive death in Alg-R. Cytoplasmic contamination of the apoplastic extracts, judged by the marker enzyme glucose-6-phosphate dehydrogenase, was very low (less than 2%) even in inoculated plants. Dehydroascorbate, glutathione, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase were present in the apoplast. Inoculation had no effect on the total foliar ascorbate pool size or the redox state. The glutathione content of Alg-S leaves and apoplast decreased, whereas that of Alg-R leaves and apoplast increased after pathogen attack, but the redox state was unchanged in both cases. Large increases in foliar catalase activity were observed in Alg-S but not in Alg-R leaves. Pathogen-induced increases in the apoplastic antioxidant enzyme activities were observed. We conclude that sustained oxidation does not occur and that differential strategies of antioxidant response in Alg-S and Alg-R may contribute to pathogen sensitivity.     

Effects of exogenous salicylic acid on manganese toxicity, element contents and antioxidative system in cucumber

The effects of salicylic acid (SA) on manganese (Mn) toxicity in cucumber plants (Cucumis sativus L.) were studied by investigating the symptoms, plant growth, lipid peroxidation, antioxidative enzymes and antioxidants. Excess Mn caused serious chlorosis and inhibited the growth of cucumber plants, and dramatically increased accumulation of Mn in both shoots and roots, furthermore, inhibited the absorption of Ca, Mg and Zn. Addition of SA decreased the transport of Mn from roots to shoots, alleviated the inhibition of Ca, Mg and Zn absorption induced by excess Mn, reduced the toxicity symptoms and promoted the plant growth. The accumulation of reactive oxygen species (ROS) significantly increased in cucumber leaves exposed to excess Mn, and resulted in the lipid peroxidation, which was indicated by accumulated concentration of thiobarbituric acid-reactive substances (TBARS). Addition of SA significantly decreased the level of ROS and lipid peroxidation. Activities of antioxidant enzymes showed different changes, addition of SA inhibited catalase (CAT) and ascorbate peroxidase (APX) activities, while increased activities of superoxide dismutase (SOD), peroxidase (POD), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) in cucumber leaves exposed to excess Mn. As important antioxidants, ascorbate and glutathione contents in cucumber leaves exposed to excess Mn were significantly increased by SA treatment.     

The effect of Botrytis cinerea infection on the antioxidant profile of mitochondria from tomato leaves

Infection of tomato leaves with the necrotrophic fungus Botrytis cinerea resulted in substantial changes in enzymatic and non-enzymatic components of the ascorbate-glutathione cycle as well as in superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione transferase (GST), and l-galactono-gamma-lactone dehydrogenase (GLDH) activities. In the initial phase of the 5 d experiment CuZn SOD was the most rapidly induced isoform (up to 209% of control), whereas later on its activity increase was not concomitant with the constant total SOD enhancement. Starting from the second day B. cinerea infection diminished the mitochondrial antioxidant capacity by decreasing activities of ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) as well as declining ascorbate and glutathione contents. This was accompanied by dehydroascorbate (DHA) and oxidized glutathione (GSSG) accumulation that resulted in ascorbate and glutathione redox ratios decreases. The strongest redox ratio decline of 29% for ascorbate and of 34% for glutathione was found on the 3rd and 2nd days, respectively. Glutathione reductase (GR) induction (185% of control 2 d after inoculation) was insufficient to overcome the decreased antioxidant potential of glutathione. Changes in the ascorbate pool size were closely related to the activity of l-galactono-gamma-lactone dehydrogenase (GLDH). The activities of two glutathione-dependent enzymes: GSH-Px and GST were increased from day 1 to day 4. These results demonstrated that in B. cinerea-tomato interaction mitochondria could be one of the main targets for infection-induced oxidative stress.     

Cadmium-induced oxidative damage and antioxidant responses in Brassica juncea plants

Indian mustard (Brassica juncea L. cv. Vitasso) plants exposed to 10, 30, 50 and 100 μM of Cd for 5 d in hydroponic culture were analysed with reference to the distribution of Cd²⁺, the accumulation of biomass and antioxidants and antioxidative enzymes in leaves. Cd induced a decrease in plant biomass. The maximum accumulation of Cd occurred in roots followed by stems and leaves. Cd induced a decrease in catalase (CAT) and guiacol peroxidase (GPX) activities but an increase in ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activities. Enhancement in dehydroascorbate reductase (DHAR) activity was also at 10 μM Cd. Glutathione reductase (GR) activity showed pronounced stimulation after all treatments, but glutathione S-transferase (GST) and glutathione peroxidase (GPOX) activities decreased. The effectiveness of ascorbate-glutathione cycle (AGC) was determined by the ratio of ascorbate to H₂O₂. This ratio decreased in the Cd-treated leaves which indicated that the cycle was disordered.     

Influences of magnesium deficiency and cerium on antioxidant system of spinach chloroplasts

Magnesium-deficiency conditions applied to spinach cultures caused an oxidative stress status in spinach chloroplast monitored by an increase in reactive oxygen species (ROS) accumulation. The enhancement of lipids peroxide of spinach chloroplast grown in magnesium-deficiency media suggested an oxidative attack that was activated by a reduction of antioxidative defense mechanism measured by analysing the activities of superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, and glutathione reductase, as well as antioxidants such as carotenoids and glutathione content. As the antioxidative response of chloroplast was reduced in spinach grown in magnesium-deficiency media, it caused a significant reduction of spinach plant weight, old leaves turning chlorosis. However, cerium treatment grown in magnesium-deficiency conditions decreased the malondialdehyde and ROS, and increased activities of the antioxidative defense system, and improved spinach growth. Together, the experimental study implied that cerium could partly substitute for magnesium and increase the oxidative stress-resistance of spinach chloroplast grown in magnesium-deficiency conditions, but the mechanisms need further study.