Changes in antioxidant enzyme activities in detached leaves of cucumber exposed to chilling

We studied changes in biochemical and physiological status, level of oxidative damage, and antioxidant enzyme activities in detached leaves of cucumber plants (Cucumis sativus L. cv. Pyunggangnaebyungsamchuk) that were exposed to a low temperature (4°C). Chlorophyll fluorescence (F_v/F_m) declined during the chilling treatment, but was slowly restored after the tissues were returned to 25°C. Likewise, the fluorescence quenching coefficient and relative water content decreased during the stress period, but then increased during recovery. In contrast, we detected a significant rise in protein and hydrogen peroxide contents in the chilled leaves, as well as higher activities for superoxide dismutase, ascorbate peroxidase, peroxidase, and glutathione reductase. However, the level of catalase decreased not only during chilling but also after 24 h of recovery. These results indicate that exposure to low temperatures acts as an oxidative stress. Moreover, we propose that a regulating mechanism exists in the detached cucumber leaves and contains an antioxidant defense system that induces active oxygen species, thereby alleviating the effects of chilling stress within 12 h.     

Cinnamic acid pretreatment mitigates chilling stress of cucumber leaves through altering antioxidant enzyme activity

To elucidate the physiological mechanism of chilling stress mitigated by cinnamic acid (CA) pretreatment, a cucumber variety (Cucumis sativus cv. Jinchun no. 4) was pretreated with 50 μM CA for 2 d and was then cultivated at two temperatures (15/8 and 25/18 °C) for 1 d. We investigated whether exogenous CA could protect cucumber plantlets from chilling stress (15/8 °C) and examined whether the protective effect was associated with the regulation of antioxidant enzymes and lipid peroxidation. At 2 d, exogenous CA did not influence plant growth, but induced the activities of some antioxidant enzymes, including superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), guaiacol peroxidase (GPX, EC 1.11.1.7), glutathione peroxidase (GSH-Px, EC 1.6.4.2) and ascorbate peroxidase (APX, EC 1.11.1.11) in cucumber leaves, and it also elevated the contents of reduced glutathione (GSH) and ascorbate (AsA). When CA was rinsed and the CA-pretreated seedlings were exposed to different temperatures, the antioxidant activities in leaves at 3 d had undergone additional change. Chilling increased the activities of CAT, GSH-PX, APX, GSH and AsA in leaves, but the combination of CA pretreatment and chilling enhanced the antioxidant activities even more. Moreover, chilling inhibited plant growth and increased the contents of malonaldehyde (MDA), superoxide radical (O₂⁻) and hydrogen peroxide (H₂O₂) in cucumber leaves, and the stress resulted in 87.5% of the second leaves being withered. When CA pretreatment was combined with the chilling stress, we observed alleviated growth inhibition and decreased contents of MDA, H₂O₂ and O₂⁻ in comparison to non-pretreated stressed plants, and found that the withered leaves occurred at a rate of 25.0%. We propose that CA pretreatment increases antioxidant enzyme activities in chilling-stressed leaves and decreases lipid peroxidation to some extent, enhancing the tolerance of cucumber leaves to chilling stress.     

Ozone tolerance and antioxidant enzyme activity in soybean cultivars

The current study confirmed earlier conclusions regarding differential ozone (O₃) tolerances of two soybean cultivars, Essex and Forrest, and evaluated antioxidant enzyme activities of these two varieties based on their performance under environmentally relevant, elevated O₃ conditions. The experiment was conducted in open-top chambers in the field during the 1994 and 1995 growing seasons. Exposure of plants to moderately high O₃ levels (62.9 nl l⁻¹ air, 2-year seasonal average) caused chlorophyll loss and increased membrane permeability when compared to control plants grown in charcoal filtered air (24.2 nl l⁻¹ air). The other effects of O₃ treatment were decrease in seed yield, loss of total sulfhydryl groups, reduction of soluble protein content, and increase in guaiacol peroxidase activity in leaves of both cultivars. The O₃-induced increase in guaiacol peroxidase activity was much smaller in cv. Essex leaflets. Cv. Essex had less leaf oxidative damage and smaller reduction in seed yield than cv. Forrest under elevated O₃ conditions. During ozonation, mature leaflets of the more O₃ tolerant cv. Essex had higher levels of glutathione reductase (30%), ascorbate peroxidase (13%), and superoxide dismutase (45%) activity than did mature leaflets of cv. Forrest. Cu,Zn-superoxide dismutase, which represented 95% of total superoxide dismutase activity in the two cultivars, appeared to be increased by O₃ exposure in the leaflets of O₃ tolerant cv. Essex but not in those of cv. Forrest. Cytosolic ascorbate peroxidase activity was also higher in leaflets of cv. Essex than in cv. Forrest regardless of O₃ level. Stromal ascorbate peroxidase and Mn-superoxide dismutase activity did not appear to be involved in the O₃ tolerance of the two soybean cultivars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Radical scavenging activity of tea catechins and their related compounds

(-)-Epigallocatechin gallate was found to be the most effective scavenger among tea catechins for the superoxide anion, hydroxyl radical, and 1,1-diphenyl-3-picrylhydrazyl radical. Examination of the scavenging effects of tea catechins and their glucosides on superoxide anion showed that the presence of at least an ortho-dihydroxyl group in the B ring and a galloyl moiety at the 3 position was important in maintaining the effectiveness of the radical scavenging ability. Stoichiometric factors of tea catechins were estimated to be 2 for (+)-catechin and (-)-epicatechin, 5 for (-)-epigallocatechin, 7 for (-)-epicatechin gallate, and 10 for (-)-epigallocatechin gallate.     

Exogenous nitric oxide promotes waterlogging tolerance as related to the activities of antioxidant enzymes in cucumber seedlings

We investigated the effects of exogenous sodium nitroprusside (SNP), a nitric oxide (NO) donor, on growth of cucumber (Cucumis sativus L., cv. Jinyou No.1) seedlings and antioxidant enzyme activities in cucumber leaves under waterlogging stress. The growth of cucumber seedlings was significantly inhibited when plants were exposed to waterlogging, whereas shoot spraying with SNP significantly alleviated the inhibition of growth from this type of stress: height, fresh and dry weights of the flooded plants increased obviously. Waterlogging also caused the activation of the antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)), the reduction of the chlorophyll content, and the accumulation of MDA and protein in leaves. It was found that SNP treatment further potentiated the antioxidant enzyme activities and maintained the chlorophyll and protein content during the entire water-logging period; however, it reduced the MDA content. Thus, NO protects plants from oxidative damage and promotes growth by activation of antioxidant enzymes in leaves in an extent sufficient for the alleviation of membrane injury. However, exogenous NO had no significant effects on cucumber seedlings growth and antioxidant enzyme activities under nonstress conditions.     

Galactolipase activity but not the level of high-melting-point phosphatidylglycerol is related to chilling tolerance in differentially sensitive Zea mays inbred lines

Galactolipase activity, the level of high-melting-point phosphatidylglycerol (HMP-PG) as well as degradation of lipids during chilling and rewarming were studied in seedlings of maize inbred lines with different chilling responses. In aged chloroplasts of chilling-sensitive (CS) lines, galactolipase activity was considerably higher than that determined in aged chloroplasts isolated from chilling-tolerant (CT) ones. Chilling of seedlings at 5 °C for 6 days induced neither loss of chlorophyll content nor visible changes in the leaves, while a slight decline in total acyl lipid content by about 15.5% and 12.5% in CS and CT lines, respectively, was observed. Among total acyl lipids, only monogalactosyldiacylglycerol (MGDG) levels were decreased significantly upon chilling. Following return to the original growth conditions for 4 days, visible chilling injury in seedlings as well as essential differences in the decrease in total acyl lipids by about 53% and 20% in CS and CT lines, respectively, were found. These changes were accompanied by more extensive degradation of MGDG, digalactosyldiacylglycerol and phosphatidylglycerol in CS than in CT lines. As the levels of HMP-PG in fresh leaves were the same in all four lines of maize, it seems that galactolipase activity and not the level of HMP-PG is related to chilling response in maize. Received: 4 July 1997 / Revision received: 18 September 1997 / Accepted: 3 March 1998     

Endogenous salicylic acid accumulation is required for chilling tolerance in cucumber (Cucumis sativus L.) seedlings

Salicylic acid (SA) is an important plant hormone, and its exogenous application can induce tolerance to multiple environmental stresses in plants. In this study, we examine the potential involvement of endogenous SA in response to chilling in cucumber (Cucumis sativus L.) seedlings. A low temperature of 8 °C induces a moderate increase in endogenous SA levels. Chilling stimulates the enzymatic activities and the expression of genes for phenylalanine ammonia-lyase (PAL) and benzoic acid-2-hydroxylase rather than isochorismate synthase. This indicates that the PAL enzymatic pathway contributes to chilling-induced SA production. Cucumber seedlings pretreated with SA biosynthesis inhibitors accumulate less endogenous SA and suffer more from chilling damage. The expression of cold-responsive genes is also repressed by SA inhibitors. The reduction in stress tolerance and in gene expression can be restored by the exogenous application of SA, confirming the critical roles of SA in chilling responses in cucumber seedlings. Furthermore, the inhibition of SA biosynthesis under chilling stress results in a prolonged and enhanced hydrogen peroxide (H₂O₂) accumulation. The application of exogenous SA and the chemical scavenger of H₂O₂ reduces the excess H₂O₂ and alleviates chilling injury. In contrast, the protective effects of SA are negated by foliar spraying with high concentrations of H₂O₂ and an inhibitor of the antioxidant enzyme. These results suggest that endogenous SA is required in response to chilling stress in cucumber seedlings, by modulating the expression of cold-responsive genes and the precise induction of cellular H₂O₂ levels.     

Reduced citrulline availability by OTC deficiency in mice is related to reduced nitric oxide production

The amino acid arginine is the sole precursor for nitric oxide (NO) synthesis. We recently demonstrated that an acute reduction of circulating arginine does not compromise basal or LPS-inducible NO production in mice. In the present study, we investigated the importance of citrulline availability in ornithine transcarbamoylase-deficient spf(ash) (OTCD) mice on NO production, using stable isotope techniques and C57BL6/J (wild-type) mice controls. Plasma amino acids and tracer-to-tracee ratios were measured by LC-MS. NO production was measured as the in vivo conversion of l-[guanidino-(15)N(2)]arginine to l-[guanidine-(15)N]citrulline; de novo arginine production was measured as conversion of l-[ureido-(13)C-5,5-(2)H(2)]citrulline to l-[guanidino-(13)C-5,5-(2)H(2)]arginine. Protein metabolism was measured using l-[ring-(2)H(5)]phenylalanine and l-[ring-(2)H(2)]tyrosine. OTC deficiency caused a reduction of systemic citrulline concentration and production to 30-50% (P < 0.001), reduced de novo arginine production (P < 0.05), reduced whole-body NO production to 50% (P < 0.005), and increased net protein breakdown by a factor of 2-4 (P < 0.001). NO production was twofold higher in female than in male OTCD mice in agreement with the X-linked location of the OTC gene. In response to LPS treatment (10 mg/kg ip), circulating arginine increased in all groups (P < 0.001), and NO production was no longer affected by the OTC deficiency due to increased net protein breakdown as a source for arginine. Our study shows that reduced citrulline availability is related to reduced basal NO production via reduced de novo arginine production. Under basal conditions this is probably cNOS-mediated NO production. When sufficient arginine is available after LPS stimulated net protein breakdown, NO production is unaffected by OTC deficiency.     

Combined action of antioxidant defense system and osmolytes in chilling shock-induced chilling tolerance in Jatropha curcas seedlings

Low non-freezing temperature is one of the major environmental factors affecting growth, development and geographical distribution of chilling-sensitive plants, Jatropha curcas is considered as a sustainable energy plants with great potential for biodiesel production. In this study, chilling shock at 5 °C followed by recovery at 26 °C for 4 h significantly improved survival percentage of J. curcas seedlings under chilling stress at 1 °C. In addition, chilling shock could obviously enhance the activities of antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR), and the levels of antioxidants ascorbic acid (AsA) and glutathione (GSH), as well as the contents of osmolytes proline and betaine in leaves of seedlings of J. curcas compared with the control without chilling shock. During the process of recovery, GR activity, AsA, GSH, proline and betaine contents sequentially increased, whereas SOD, APX and CAT activities gradually decreased, but they markedly maintained higher activities than those of control. Under chilling stress, activities of SOD, APX, CAT, GR and GPX, and contents of AsA, GSH, proline and betaine, as well as the ratio of the reduced antioxidants to total antioxidants [AsA/(AsA + DHA) and GSH/(GSH + GSSG)] in the shocked and non-shock seedlings all dropped, but shocked seedlings sustained significantly higher antioxidant enzyme activity, antioxidant and osmolyte contents, as well as ratio of reduced antioxidants to total antioxidants from beginning to end compared with control. These results indicated that the chilling shock followed by recovery could improve chilling tolerance of seedlings in J. curcas, and antioxidant enzymes and osmolytes play important role in the acquisition of chilling tolerance.     

Anthocyanins,thiols, and antioxidant scavenging enzymes are involved in Lemna gibba tolerance to arsenic

The influence of arsenic (As) on the growth and the antioxidant system of Lemna gibba L. exposed to five concentrations of As (0.0, 0.25, 0.5, 1.0, and 1.5 mg L^?1) was studied. Although As exposure reduced relative growth rate, L. gibba continued to grow even after the high accumulation of this element after five days of exposure. The concentration of the superoxide anion was unaffected by As, whereas an increase of hydrogen peroxide concentrations was observed with the increasing of As concentration. Increasing concentrations of As also increased the enzyme activity of superoxide dismutase, peroxidase, and glutathione reductase and the total and nonprotein thiols, up to 0.5 mg L^?1, whereas the anthocyanin content increased constantly with As concentration. Catalase and ascorbate peroxidase activities as well as the content of chloroplastic pigments were reduced in plants exposed to all As concentrations. These results support a major role of anthocyanins, nonprotein thiols, and antioxidant scavenging enzymes in L. gibba tolerance to toxic As concentrations.     

ABA application to maize hybrids contrasting for drought tolerance: changes in water parameters and in antioxidant enzyme activity

The objective of this study was to evaluate the effects of abscisic acid (ABA) related to the increase of water-stress tolerance in two drought contrasting maize hybrids: DKB 390 (tolerant) and BRS 1030 (sensitive). The characterization of water status (pre-dawn leaf water potential, Ψ_pd; midday leaf water potential, Ψ_md and stem water potential, Ψ_st) and antioxidant enzyme activity was conducted on greenhouse grown plants. The ABA, hydrogen peroxide (H₂O₂), and malondialdehyde (MDA) contents were also analyzed. Water deficit was imposed for 10 days at the flowering stage and a dosage of 100 μM ABA was applied to plant canopy. Measurements were taken during 10 days after the water recovery. With 5 days of stress, the tolerant hybrid showed lower MDA content, decrease in the water status, and higher activity of the enzymes superoxide dismutase, catalase, ascorbate peroxidase, as well as guaiacol, glutathione reductase, dehydroascorbate reductase, polyphenol oxidase, and l-phenylalanine ammonia-lyase, as compared to the sensitive hybrid. With 10 days of stress, DKB 390 had a decrease in the activity of enzymes whereas BRS 1030 showed a higher activity. In addition, the latter showed greater amounts of H₂O₂ and MDA. ABA application led to a higher tolerance only in DKB 390, due to the increase of water status and the enzymatic activity, mainly the catalase.     

Induction of chilling tolerance in cucumber (Cucumis sativus) seedlings by endogenous and applied ethanol

Five-day-old etiolated cucumber ( Cucumis sativus L.) seedlings cv. Marketmore held at 2°C for 72 h developed chilling injury, resulting in desiccation and collapse of the hypocotyl tissue and eventual plant death. Hypoxia-induced accumulation of ethanol and acetaldehyde led to tolerance of subsequent chilling, as evidenced by continued hypocotyl growth and freedom from injury. Attenuated accumulation of volatiles by applied bisulfite reduced the development of hypoxia-induced chilling tolerance in seedlings. In seedlings held in normoxia cold tolerance was induced by applied ethanol vapors, whereas acetaldehyde had a marginal effect, suggesting that hypoxia-induced cold tolerance may arise from the accumulation and activity of ethanol. Cold tolerance was also induced by exposure of seedlings to volatile anesthetics including n -propanol, n -butanol, chloroform and halothane, suggesting that ethanol activity may result from fluidization of membrane lipids. This view is consistent with results which showed that ethanol activity was not associated with lipid metabolism. However, development of cold tolerance in ethanol-enriched tissues was time dependent, indicating that ethanol activity probably also entails biosynthetic event(s).     

Reduced activity of antioxidant machinery is correlated with suppression of totipotency in plant protoplasts

We previously showed that during protoplast isolation, an oxidative burst occurred and the generation of active oxygen species was differentially mediated in tobacco (Nicotiana tabacum) and grapevine (Vitis vinifera), accompanied by significant quantitative differences (A.K. Papadakis, K.A. Roubelakis-Angelakis [1999] Plant Physiol 127: 197-205). We have now further tested if the expression of totipotency in protoplasts is related to the activity of cellular antioxidant machinery during protoplast culture. Totipotent (T) tobacco protoplasts had 2-fold lower contents of intracellular O2*- and H2O2 and 7-fold lower levels of O2*- and H2O2 in the culture medium, compared with non-totipotent (NT) tobacco protoplasts. Addition of alkaline dimethylsulfoxide, known to generate O2*-, resulted in isolation of tobacco protoplasts with reduced viability and cell division potential during subsequent culture. Active oxygen species levels decreased in tobacco and grapevine protoplasts during culturing, although higher contents of O2*- and H2O2 were still found in NT- compared with T-tobacco protoplasts, after 8 d in culture. In T-tobacco protoplasts, the reduced forms of ascorbate and glutathione predominated, whereas in NT-tobacco and grapevine protoplasts, the oxidized forms predominated. In addition, T-tobacco protoplasts exhibited severalfold lower lipid peroxidation than NT-tobacco and grapevine protoplasts. Furthermore, several antioxidant enzyme activities were increased in T-tobacco protoplasts. Superoxide dismutase activity increased in tobacco, but not in grapevine protoplasts during culturing due to the increased expression of cytoplasmic Cu/Zn-superoxide dismutase. The increase was only sustained in T-tobacco protoplasts for d 8. Together, these results suggest that suppressed expression of totipotency in protoplasts is correlated with reduced activity of the cellular antioxidant machinery.     

Changes in oligosaccharide content and antioxidant enzyme activities in developing bean seeds as related to acquisition of drying tolerance and seed quality.

Seeds of bean (Phaseolus vulgaris cv. Vernel) were collected throughout their development on the plant and dried at 15 degrees C and 75% relative humidity to a final moisture content of about 16% (fresh weight basis) to determine whether the onset of tolerance to this drying condition was related to changes in soluble sugars or the activities of the main antioxidant enzymes, namely superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR). Measurements of soluble sugars and enzyme activities were made after drying the seeds, and drying tolerance was evaluated by the ability of dried seeds to germinate and to produce normal seedlings. Seeds became tolerant to drying at 45 d after anthesis, a time marking physiological maturity. At physiological maturity, the moisture content of seeds was about 50-55% (fresh weight basis) and seed dry matter reached about 190 mg per seed. Seed vigour, evaluated by controlled deterioration and conductivity measurements, continued to increase after seed mass maturity, but decreased when seeds remained thereafter for more than 7 d on the plant. Acquisition of drying tolerance was coincident with an accumulation of raffinose and stachyose. Dried-tolerant seeds were also characterized by a high amount of sucrose, the most abundant sugar, and by a low content of monosaccharides. The (raffinose+stachyose)/sucrose ratio increased during seed filling, reaching a value close to 1 when all the seeds became tolerant to drying, and maintaining this proportion during the final stages of maturation. Acquisition of drying tolerance was also related to a reorientation of the enzymatic antioxidant defence system. Drying-tolerant dried seeds displayed high CAT and GR activities and low SOD and APX activities, while the opposite condition was observed in immature dried seeds. The shift in antioxidant enzymes corresponded to the beginning of the maturation-drying phase. These results suggest that oligosaccharide metabolism and enzymatic antioxidant defences may be involved in acquisition of drying tolerance during bean seed development, but are not related to seed vigour.     

Exogenous catechin increases antioxidant enzyme activity and promotes flooding tolerance in tomato (Solanum lycopersicum L.)

We studied the extent to which catechin applied as a soil drench modifies the effects of soil waterlogging on plant growth, the functioning of the free radical scavenging system and on oxidative stress levels. Forty-day-old tomato plants (Solanum lycopersicum L.) were treated with 0 and 2?mM catechin 48 h prior to 5 d waterlogging followed by a 4 d drainage period. Exogenous catechin increased total fresh and dry weight of flooded plants, reduced membrane damage, maintained chlorophyll concentrations, promoted photosynthesis and increased ATP concentration in the leaves, and raised sucrose synthase and alcohol dehydrogenase activities in the roots. Catechin pre-treatment also reduced hydrogen peroxide and superoxide radical concentration and increased various components of the antioxidative system in leaves. Catechin treatment affected superoxide dismutase and catalase activities in close coordination with ascorbate peroxidases and glutathione reductase. Exogenous catechin can markedly reduce the waterlogging injury in leaves and roots of tomato by enhancing free radical scavenging system sufficiently to lower hydrogen peroxide and superoxide concentrations.     

Arginine decarboxylase is a component activity of the multifunctional enzyme putrescine synthase in cucumber seedlings

A homogenous PreParation of Putrescine synthase, the versatile multifunctional enzyme involved in agmatine →Putrescine conversion inCucumis sativus was found to catalyze enzymatic decarboxylation of arginine also. Similarly, the Purified arginine decarboxylase mediated the comPonent as well as the comPlete set of couPled reactions harboured by Putrescine synthase. Both the enzyme PreParations exhibited identical electroPhoretic and chromatograPhic behaviour and were immunologically indistinguishable. All the enzymic activities are stabilized concurrently by feeding arginine to the intact seedlings. Therefore, it is concluded that the multifunctional Putrescine synthase inCucumis sativus seedlings also harbours arginine decarboxylase activity unlike its counterPart inLathyrus sativus.