Antioxidant responses to water deficit by drought‐tolerant and ‐sensitive sugarcane varieties

Water deficit is the major yield‐limiting factor of crop plants. The exposure of plants to this abiotic stress can result in oxidative damage due to the overproduction of reactive oxygen species. The aim of this work was to study the antioxidant‐stress response of drought‐tolerant (SP83‐2847 and SP83‐5073) and drought‐sensitive (SP90‐3414 and SP90‐1638) sugarcane varieties to water‐deficit stress, which was imposed by withholding irrigation for 3, 10 and 20 days. The drought‐sensitive varieties exhibited the lowest leaf relative water content and highest lipid peroxidation, hydrogen peroxide (H₂O₂) and proline contents during the progression of the drought‐stress condition. The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPOX) and glutathione reductase (GR) activities changed according to variety and stress intensity. SP83‐2847 exhibited higher CAT and APX activities than the other varieties in the early stage of drought, while the activities of GPOX and GR were the highest in the other varieties at the end of the drought‐stress period. A Cu/Zn SOD isoenzyme was absent at the end of drought period from the SP90‐3414‐sensitive variety. The results indicate that lipid peroxidation and early accumulation of proline may be good biochemical markers of drought sensitivity in sugarcane.     

渗透胁迫对黑麦幼苗活性氧和抗氧化酶活性的影响

用20%聚乙二醇(PEG 6000)研究了渗透胁迫对黑麦(Secale cereale L.)幼苗活性氧(reactive oxygen species, ROS)和主要抗氧化酶—— 超氧化物歧化酶(superoxide dismutase, SOD)、过氧化氢酶(catalase, CAT)、抗坏血酸过氧化物酶(ascorbate peroxidase, APX)和谷胱甘肽还原酶(glutathione reductase, GR)活性的影响。结果表明, 与对照相比, PEG处理明显提高了叶子和根中丙二醛(malondialdehyde, MDA)的含量、ROS的水平和以上4种抗氧化酶的活性。渗透胁迫下,叶子和根中MDA和ROS水平变化的规律基本相似, 但抗氧化酶活性在2种器官中表现不完全相同, 叶子中CAT的活性在对照和处理中无显著差异, 但在根中差异明显, 表明叶子中SOD、APX和GR在植物应答渗透胁迫中起重要作用, 而根中这4种抗氧化酶都参与植物对胁迫的反应。GR活性随PEG处理变化幅度显著高于其它抗氧化酶, 表明GR在黑麦应答渗透胁迫中所起作用可能强于其它抗氧化酶。     

Antioxidative defense system in pigeonpea roots under waterlogging stress

Pigeonpea [Cajanus cajan (L.) Millsp.] is a waterlogging-sensitive legume crop. We studied the effect of waterlogging stress on hydrogen peroxide (H₂O₂) content, lipid peroxidation and antioxidant enzyme activities in two pigeonpea genotypes viz., ICPL-84023 (waterlogging resistant) and MAL-18 (waterlogging susceptible). In a pot experiment, waterlogging stress was imposed for 6 days at early vegetative stage (20 days after sowing). Waterlogging treatment significantly increased hydrogen peroxide accumulation and lipid peroxidation, which indicated the extent of oxidative injury posed by stress conditions. Enzyme activities of peroxidase (POX), catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD) and polyphenol oxidase (PPO) increased in pigeonpea roots as a consequence of waterlogged conditions, and all the enzyme activities were significantly higher in waterlogged ICPL-84023 than in MAL-18. POX activity was the maximum immediately after imposing stress, therefore, it was suggested to be involved in early scavenging of H₂O₂, while rest of the enzymes (CAT, APX, SOD and PPO) were more important in late responses to waterlogging. Present study revealed that H₂O₂ content is directly related to lipid peroxidation leading to oxidative damage during waterlogging in pigeonpea. Higher antioxidant potential in ICPL-84023 as evidenced by enhanced POX, CAT, APX, SOD and PPO activities increased capacity for reactive oxygen species (ROS) scavenging and indicated relationship between waterlogging resistance and antioxidant defense system in pigeonpea.     

Physiochemical and antioxidant responses of the perennial xerophyte Capparis ovata Desf. to drought

Caper (Capparis ovata Desf.) is a perennial shrub (xerophyte) and drought resistant plant which is well adapted to Mediterranean Ecosystem. In the present study we investigated the plant growth, relative water content (RWC), chlorophyll fluorescence (F_V/F_M), lipid peroxidation (TBA-reactive substances content) as parameters indicative of oxidative stress and antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POX), catalase (CAT) and glutathione reductase (GR) in relation to the tolerance to polyethylene glycol mediated drought stress in C. ovata seedlings. For induction of drought stress, the 35 days seedlings were subjected to PEG 6000 of osmotic potential −0.81 MPa for 14 days. Lipid peroxidation increased in PEG stressed seedlings as compared to non-stressed seedlings of C. ovata during the experimental period. With regard to vegetative growth, PEG treatment caused decrease in shoot fresh and dry weights, RWC and F_V/F_M but decline was more prominent on day 14 of PEG treatment. Total activity of antioxidative enzymes SOD, APX, POX, CAT and GR were investigated in C. ovata seedlings under PEG mediated drought. Induced activities of SOD, CAT and POX enzymes were high and the rate of increment was higher in stressed seedling. APX activity increased on both days of PEG treatment, however, increase in GR activity was highest on day 14 of drought stress. We concluded that increased drought tolerance of C. ovata is correlated with diminishing oxidative injury by functioning of antioxidant system at higher rates under drought stress.     

Morphological and Physiological Responses of Cotton (Gossypium hirsutum L.) Plants to Salinity

Salinization usually plays a primary role in soil degradation, which consequently reduces agricultural productivity. In this study, the effects of salinity on growth parameters, ion, chlorophyll, and proline content, photosynthesis, antioxidant enzyme activities, and lipid peroxidation of two cotton cultivars, [CCRI-79 (salt tolerant) and Simian 3 (salt sensitive)], were evaluated. Salinity was investigated at 0 mM, 80 mM, 160 mM, and 240 mM NaCl for 7 days. Salinity induced morphological and physiological changes, including a reduction in the dry weight of leaves and roots, root length, root volume, average root diameter, chlorophyll and proline contents, net photosynthesis and stomatal conductance. In addition, salinity caused ion imbalance in plants as shown by higher Na⁺ and Cl⁻ contents and lower K⁺, Ca²⁺, and Mg²⁺ concentrations. Ion imbalance was more pronounced in CCRI-79 than in Simian3. In the leaves and roots of the salt-tolerant cultivar CCRI-79, increasing levels of salinity increased the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR), but reduced catalase (CAT) activity. The activities of SOD, CAT, APX, and GR in the leaves and roots of CCRI-79 were higher than those in Simian 3. CAT and APX showed the greatest H₂O₂ scavenging activity in both leaves and roots. Moreover, CAT and APX activities in conjunction with SOD seem to play an essential protective role in the scavenging process. These results indicate that CCRI-79 has a more effective protection mechanism and mitigated oxidative stress and lipid peroxidation by maintaining higher antioxidant activities than those in Simian 3. Overall, the chlorophyll a, chlorophyll b, and Chl (a+b) contents, net photosynthetic rate and stomatal conductance, SOD, CAT, APX, and GR activities showed the most significant variation between the two cotton cultivars.     

alpha-Pinene inhibits growth and induces oxidative stress in roots

BACKGROUND AND AIMS: Determining the mode of action of allelochemicals is one of the challenging aspects in allelopathic studies. Recently, allelochemicals have been proposed to cause oxidative stress in target tissue and induce an antioxidant mechanism. alpha-Pinene, one of the common monoterpenoids emitted from several aromatic plants including forest trees, is known for its growth-inhibitory activity. However, its mechanism of action remains unexplored. The aim of the present study was to determine the inhibitory effect of alpha-pinene on root growth and generation of reactive oxygen species, as indicators of oxidative stress and changes in activities of antioxidant enzymes. METHODS: Effects of alpha-pinene on early root growth were studied in five test species, Cassia occidentalis, Amaranthus viridis, Triticum aestivum, Pisum sativum and Cicer arietinum. Electrolyte leakage, lipid peroxidation, hydrogen peroxide generation, proline accumulation, and activities of the enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), catalase (CAT) and glutathione reductase (GR) were studied in roots of C. occidentalis. KEY RESULTS: alpha-Pinene inhibited the radicle growth of all the test species. Exposure of C. occidentalis roots to alpha-pinene enhanced solute leakage, and increased levels of malondialdehyde, proline and hydrogen peroxide, indicating lipid peroxidation and induction of oxidative stress. Activities of the antioxidant enzymes SOD, CAT, GPX, APX and GR were significantly elevated, thereby indicating the enhanced generation of reactive oxygen species (ROS) upon alpha-pinene exposure. Increased levels of scavenging enzymes indicates their induction as a secondary defence mechanism in response to alpha-pinene. CONCLUSIONS: It is concluded that alpha-pinene inhibits early root growth and causes oxidative damage in root tissue through enhanced generation of ROS, as indicated by increased lipid peroxidation, disruption of membrane integrity and elevated antioxidant enzyme levels.     

Antioxidant responses of shoots and roots of lentil to NaCl-salinity stress

The effect of salt stress (100 mM and 200 mM NaCl) on antioxidant responses in shoots and roots of 14-day-old lentil (Lens culinaris M.) seedlings was investigated. Salt stress caused a significant decrease in length, wet-dry weight and an increase in proline content of both shoot and root tissues. In leaf tissues, high salinity treatment resulted in a 4.4 fold increase in H₂O₂ content which was accompanied by a significant level of lipid peroxidation and an increase in electrolyte leakage. Root tissues were less affected with respect to these parameters. Leaf tissue extracts exhibited four activity bands, of which two were identified as Cu/Zn-SOD and others as Fe-SOD and Mn-SOD. Fe-SOD activity was missing in root extracts. In both tissues Cu/Zn-SOD activity comprised 70–75% of total SOD activity. Salt stress did not cause a significant increase in total SOD activity of leaf tissues but a significant enhancement (88%) was observed in roots mainly due to an enhancement in Cu/ZnSOD isoforms. Compared to leaf tissues a significantly higher constitutive ascorbate peroxidase (APX) and glutathion reductase (GR) activity was observed in root tissues. Upon salt stress no significant change in the activity of APX, catalase (CAT) and GR was observed in root tissues but a higher APX activity was present when compared to leaf tissues. On the other hand, in leaf tissues, with the exception of CAT, salt stress caused significant enhancement in the activity of other antioxidant enzymes. These results suggested that, root tissues of lentil are protected better from NaCl stress induced oxidative damage due to enhanced total SOD activity together with a higher level of APX activity under salinity stress. To our knowledge this is the first report describing antioxidant enzyme activities in lentil.     

Effects of Lead on the Activities of Antioxidant Enzymes in Watercress, <Emphasis Type="Italic">Nasturtium officinale</Emphasis> R. Br.

The aim of the present study is to evaluate the oxidative effects of lead with increased concentrations by the determination of antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and ascorbate peroxidase (AP)) and lipid peroxidation levels in the stem and leaves of watercress (Nasturtium officinale R. Br.) which was exposed to lead acetate, Pb (CH₃COOH)₂ regime with concentrations of 0, 50, 100, 200, 250, and 500 mg/L Pb in a hydroponic culture. After 14 days, accumulation of lipid peroxidation in stems and leaves and changes in activity of antioxidant enzymes were determined spectrophotometrically. The maximum accumulation was observed in the highest concentration group. In this group, lipid peroxidation levels were three times higher than the control group in the stem and leaves. The highest induction in SOD and GR activities were determined at 200 mg/L Pb group in stem, whereas CAT and AP activities were higher than other groups at the concentration of 250 and 100 mg/L Pb, respectively. The increase in CAT activity was found to be greater than GR, SOD, and AP activities in stems of watercress under Pb treatment. Both lead accumulation and antioxidant enzyme responses were higher in stems than in leaves. The results of the present study suggested that the induction in antioxidant responses could be occurring as an adaptive mechanism to the oxidative potential of lead accumulation.     

Changes in activities of antioxidant enzymes and their gene expression during leaf development of sweetpotato

To understand the functions of antioxidant enzymes during leaf development in sweetpotato, we investigated the activities of several antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POX), ascorbate peroxidase (APX) and catalase (CAT). Significant increases were observed in the activities of SOD, POX and APX during the late stage of leaf development, whereas CAT activity increased during the early developmental stage. By RT-PCR analysis, various POX and APX genes showed differential expression patterns during leaf development. Four POX genes swpa3, swpa4, swpa6, swpb4 and one APX gene swAPX1 exhibited high levels of gene expression during the senescence stage of leaf development, but two POX genes, swpa1 and swpa7 were preferentially expressed at both the mature green and the late senescence stages of leaf development. These results indicate that hydrogen peroxide (H₂O₂)-related antioxidant enzymes are differentially regulated in the process of leaf development of sweetpotato.     

Postharvest chilling induces oxidative stress response in the dwarf tomato cultivar Micro-Tom

Oxidative stress is involved in the response of Lycopersicon esculentum fruits (cultivar Micro-Tom) to chilling. Changes in activated oxygen scavenging enzymes, superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and glutathione reductase (GR, EC 1.6.4.2) were examined during ripening after postharvest chilling. Also, lipid peroxidation, respiration, and pigment contents were determined. These parameters were affected by chilling, especially the lycopene content and the respiration rate that showed a high value when the fruits were transferred to higher temperatures. CAT activity increased the day after the fruits were re-warmed, while the activity of GR was higher in the chilled than in the non-chilled green fruits. Lipid peroxidation was more evident at the 'pre-chilled' yellow and red fruits. APX and SOD were not affected by previous chilling in ripening fruits. These results indicate that oxidative stress is generated by conservation at 4°C. The antioxidant response of tomato fruit could be mediated by CAT and GR but not by SOD or APX. Moreover, CAT seemed to respond to the increase in the respiration rate.     

Enhanced antioxidant enzymes are associated with reduced hydrogen peroxide in barley roots under saline stress

Antioxidant enzymes are related to the resistance to various abiotic stresses including salinity. Barley is relatively tolerant to saline stress among crop plants, but little information is available on barley antioxidant enzymes under salinity stress. We investigated temporal and spatial responses of activities and isoform profiles of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), non-specific peroxidase (POX), and glutathione reductase (GR) to saline stress in barley seedlings treated with 200 mM NaCl for 0, 1, 2, 5 days, respectively. In the control plant, hydrogen peroxide content was about 2-fold higher in the root than in the shoot. Under saline stress, hydrogen peroxide content was decreased drastically by 70% at 2 d after NaCl treatment (DAT) in the root. In the leaf, however, the content was remained unchanged by 2 DAT and increased about 14 % at 5 DAT. In general, the activities of antioxidant enzymes were increased in the root and shoot under saline stress. But the increase was more significant and consistent in the root. The activities of SOD, CAT, APX, POX, and GR were increased significantly in the root within 1 DAT, and various elevated levels were maintained by 5 DAT. Among the antioxidant enzymes, CAT activity was increased the most drastically. The significant increase in the activities of SOD, CAT, APX, POX, and GR in the NaCl-stressed barley root was highly correlated with the increased expression of the constitutive isoforms as well as the induced ones. The hydrogen peroxide content in the root.     

Antioxidant defense in the leaves of C3 and C4 plants under salinity stress

The effect of salt stress (50, 100 and 150 m M of NaCl) on the activity of superoxide dismutase (SOD, EC. 1.15.1.1), ascorbate peroxidase (APX, EC. 1.11.1.11), glutathione reductase (GR, EC. 1.6.4.2) enzymes and also on the rate of lipid peroxidation in terms of thiobarbituric acid-reactive substances (TBARS) content and photosynthetic capacity in two wheat (C3 plants) and two maize (C4 plants) varieties was studied. In the non-salined control plants, the antioxidant enzymes activities were significantly higher for maize than for wheat. Adding salt to the nutrient solution increased the level of antioxidants in leaves of both maize and wheat. The first substantial response to salinity was found for SOD on the 2nd day, whereas changes occurred for APX on the 4th day and for GR on the 4th/5th day of salt treatment. Although SOD activity increased considerably more in wheat (C3), it never reached as high levels as in maize (C4) grown in the same treatment combinations. The total increase in APX activity was similar for wheat and maize, whereas GR activity was higher in leaves of maize. Lipid peroxidation analyses showed an increase in TBARS contents in both plants' species grown under salinity that corresponded to the damage that occurred in secondary oxidative stress. However, as a result of advanced antioxidant defense in maize, the TBARS quantities did not elevate to as high level as in wheat. Chlorophyll fluorescence measurements revealed a considerable decrease in the efficiency of PS II and electron-transport chain (ETC). Assimilation rate of CO₂ decreased in both plant groups; however, in C4 maize, we observed a much better capacity to preserve the photosynthetic apparatus against overproduction of ROS. Results suggest that efficient antioxidant defense plays an important role in maize, the C₄ plant, resistance to environmental stresses like salinity or drought.     

Potassium starvation induces oxidative stress inSolanum lycopersicumL. roots

The relationship between potassium deficiency and the antioxidative defense system has received little study. The aim of this work was to study the induction of oxidative stress in response to K(+) deficiency and the putative role of antioxidants. The tomato plants were grown in hydroponic systems to determine the role of reactive oxygen species (ROS) in the root response to potassium deprivation. Parameters of oxidative stress (malondialdehyde and hydrogen peroxide (H(2)O(2)) concentration), activities of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR)) and antioxidant molecules (ascorbate (ASC) and glutathione) were investigated. H(2)O(2) was subcellularly located by laser confocal microscopy after potassium starvation in roots. During the first 24h, H(2)O(2) induced the cascade of the cellular response to low potassium, and ROS accumulation was located mainly in epidermal cells in the elongation zone and meristematic cells of the root tip and the epidermal cells of the mature zones of potassium starved roots. The activity of the antioxidative enzymes SOD, peroxidase and APX in potassium deprivation significantly increased, whereas CAT and DHAR activity was significantly depressed in the potassium starvation treatment compared to controls. GR did not show significant differences between control and potassium starvation treatments. Based on these results, we put forward the hypothesis that antioxidant molecule accumulations probably scavenge H(2)O(2) and might be regenerated by the ASC-glutathione cycle enzymes, such as DHAR and GR.     

Cytological and physiological changes in orthodox maize embryos during cryopreservation

Cytological and physiological changes during cryopreservation were studied in maize embryos at 35 days after pollination (DAP). Both dehydration and freezing caused cytological damage, such as plasmolysis, swelled mitochondria, increased heterochromatin, and nuclear shrinkage. Dehydration alone slightly impaired plasma membrane integrity while a drastic increase in electrolyte leakage was observed after freezing of embryos with moisture content above 23%. Damage to cellular ultrastructure and plasmalemma integrity was negatively related to moisture content in unfrozen embryos and positively related in frozen embryos. The pattern of changes in activity of antioxidant enzymes differed from one another during dehydration and/or freezing–thawing treatment. Dehydration increased activity of ascorbate peroxidase (APX) and glutathione reductase (GR) but decreased activity of superoxide dismutase (SOD) and dehydroascorbate reductase (DHAR). Freezing further decreased GR and SOD activity and resulted in extremely low DHAR activity. Embryos at intermediate moisture contents had low catalase (CAT) activity before freezing but highest CAT activity after freeze–thaw. Both dehydration and freezing promoted membrane lipid peroxidation which resulted in an approximately threefold increase at most in the malondialdehyde content in postthaw embryos. Changes in viability of postthaw embryos can be closely related to damage in cellular ultrastructure and plasmalemma integrity but directly related neither to antioxidants nor lipid peroxidation levels.     

Chloroplastic NADPH oxidase-like activity-mediated perpetual hydrogen peroxide generation in the chloroplast induces apoptotic-like death of <Emphasis Type="Italic">Brassica napus</Emphasis> leaf protoplasts

Despite extensive research over the past years, regeneration from protoplasts has been observed in only a limited number of plant species. Protoplasts undergo complex metabolic modification during their isolation. The isolation of protoplasts induces reactive oxygen species (ROS) generation in Brassica napus leaf protoplasts. The present study was conducted to provide new insight into the mechanism of ROS generation in B. napus leaf protoplasts. In vivo localization of H₂O₂ and enzymes involved in H₂O₂ generation and detoxification, molecular antioxidant-ascorbate and its redox state and lipid peroxidation were investigated in the leaf and isolated protoplasts. Incubating leaf strips in the macerating enzyme (ME) for different duration (3, 6, and 12 h) induced accumulation of H₂O₂ and malondialdehyde (lipid peroxidation, an index of membrane damage) in protoplasts. The level of H₂O₂ was highest just after protoplast isolation and subsequently decreased during culture. Superoxide generating NADPH oxidase (NOX)-like activity was enhanced, whereas superoxide dismutase (SOD) and ascorbate peroxidase (APX) decreased in the protoplasts compared to leaves. Diaminobenzidine peroxidase (DAB-POD) activity was also lower in the protoplasts compared to leaves. Total ascorbate content, ascorbate to dehydroascorbate ratio (redox state), were enhanced in the protoplasts compared to leaves. Higher activity of NOX-like enzyme and weakening in the activity of antioxidant enzymes (SOD, APX, and DAB-POD) in protoplasts resulted in excessive accumulation of H₂O₂ in chloroplasts of protoplasts. Chloroplastic NADPH oxidase-like activity mediated perpetual H₂O₂ generation probably induced apoptotic-like cell death of B. napus leaf protoplasts as indicated by parallel DNA laddering and decreased mitochondrial membrane potential.     

2-Benzoxazolinone (BOA) induced oxidative stress, lipid peroxidation and changes in some antioxidant enzyme activities in mung bean (Phaseolus aureus)

2-Benzoxazolinone (BOA), a well-known allelochemical with strong phytotoxicity, is a potential herbicidal candidate. The aim of the present study was to determine whether phytotoxicity of BOA is due to induction of oxidative stress caused by generation of reactive oxygen species (ROS) and the changes in levels of antioxidant enzymes induced in response to BOA. Effect of BOA was studied on electrolyte leakage, lipid peroxidation (LP), hydrogen peroxide (H(2)O(2)) generation, proline (PRO) accumulation, and activities of antioxidant enzymes-superoxide dismutase (SOD, 1.15.1.1), ascorbate peroxidase (APX, 1.11.1.11), guaiacol peroxidase (GPX, 1.11.1.7), catalase (CAT, 1.11.1.6) and glutathione reductase (GR, 1.6.4.2) in Phaseolus aureus (mung bean). BOA significantly enhanced malondialdehyde (MDA) content, a product of LP, in both leaves and roots of mung bean. The amount of H(2)O(2), a product of oxidative stress, and endogenous PRO increased many-fold in response to BOA. Accumulation of PRO, MDA and H(2)O(2) indicates the cellular damage in the target tissue caused by ROS generated by BOA. In response to BOA, there was a significant increase in the activities of scavenging enzymes SOD, APX, GPX, CAT, and GR in root and leaf tissue of mung bean. At 5 mM BOA, GR activity in roots showed a nearly 22-fold increase over that in control. The present study concludes that BOA induces oxidative stress in mung bean through generation of ROS and upregulation of activities of various scavenging enzymes.     

Antioxidant responses of chickpea plants subjected to boron toxicity

This study investigated oxidative stress and the antioxidant response to boron (B) of chickpea cultivars differing in their tolerance to drought. Three‐week‐old chickpea seedlings were subjected to 0.05 (control), 1.6 or 6.4 mm B in the form of boric acid (H₃BO₃) for 7 days. At the end of the treatment period, shoot length, dry weight, chlorophyll fluorescence, B concentration, malondialdehyte content and the antioxidant enzymes superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) were measured. The 1.6 mm B treatment did not cause significant changes in shoot length of cultivars, although shoot length increased in the drought‐tolerant Gökce and decreased in the drought‐sensitive Küsmen after 6.4 mm B treatment. Dry weights of both cultivars decreased with 6.4 mm B treatment. Chlorophyll fluorescence (Fv/Fm) did not change in Gökce at either B level. Nor did it change in Küsmen with 1.6 mm B but Fv/Fm decreased with 6.4 mm B. Boron concentration in the shoots of both cultivars increased significantly with increasing levels of applied B. Significant increases in total SOD activity were observed in shoots of both cultivars given 1.6 and 6.4 mm B. Shoot extracts exhibited five activity bands, two of which were identified as MnSOD and Cu/ZnSOD. In comparison to the control group, all enzyme activities (except APX and SOD) decreased with 1.6 mm B stress. GR activity decreased, while activities of CAT, POX and APX did not change with 6.4 mm B in Küsmen. On the other hand, activities of CAT, APX and SOD increased in Gökce at both B levels. In addition, lipid peroxidation was higher in Küsmen than in Gökce, indicating more damage by B to membrane lipids in the former cultivar. These results suggest that (i) Gökce is tolerant and Küsmen is sensitive to B, and (ii) B tolerance of Gökce might be closely related to increased capacity of the antioxidative system (total SOD, CAT and APX) to scavenge reactive oxygen species and thus suppress lipid peroxidation under B stress. To the best of our knowledge, this is the first report on the antioxidant response of chickpea seedlings to B toxicity.     

Investigations on the antioxidative defence responses to NaCl stress in a mangrove, Bruguiera parviflora: Differential regulations of isoforms of some antioxidative enzymes

Two-month-old healthy seedlings of a true mangrove, Bruguiera parviflora, raised from propagules in normal nursery conditions were subjected to varying concentrations of NaCl for 45 d under hydroponic culture conditions to investigate the defence potentials of antioxidative enzymes against NaCl stress imposed oxidative stress. Changes in the activities of the antioxidative enzymes catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POX), glutathione reductase (GR) and superoxide dismutase (SOD) were assayed in leaves to monitor the temporal regulation. Among the oxidative stress triggered chemicals, the level of H₂O₂ was significantly increased while total ascorbate and total glutathione content decreased. The ratio of reduced to oxidized glutathiones, however, increased due to decreased levels of oxidized glutathione in the leaf tissue. Among the five antioxidative enzymes monitored, the APX, POX, GR and SOD specific activities were significantly enhanced at high concentration (400 mM NaCl), while the catalase activities declined, suggesting both up and downregulations of antioxidative enzymes occurred due to NaCl imposed osmotic and ionic stress. Analysis of the stress induced alterations in the isoforms of CAT, APX, POX, GR and SOD revealed differential regulations of the isoforms of these enzymes. In B. parviflora one isoform of each of Mn-SOD and Cu/Zn-SOD while three isoforms of Fe-SOD were observed by activity staining gel. Of these, only Mn-SOD and Fe-SOD2 content was preferentially elevated by NaCl treatment, whereas isoforms of Cu/Zn-SOD, Fe-SOD1 and Fe-SOD3 remained unchanged. Similarly, out of the six isoforms of POX, the POX-1,-2,-3 and -6 were enhanced due to salt stress but the levels of POX-4 and -5 remained same as in control plants suggesting preferential upregulation of selective POX isoforms. Activity staining gel revealed only one prominent band of APX and this band increased with increased salt concentration. Similarly, two isoforms of GR (GR1 and GR2) were visualized on activity staining gel and both these isoforms increased upon salt stress. In this mangrove four CAT-isoforms were identified, among which the prominent CAT-2 isoform level was maximally reduced again suggesting differential downregulation of CAT isoforms by NaCl stress. The results presented in this communication are the first report on the resolutions of isoforms APX, POX and GR out of five antioxidative enzymes studied in the leaf tissue of a true mangrove. The differential changes in the levels of the isoforms due to NaCl stress may be useful as markers for recognizing salt tolerance in mangroves. Further, detailed analysis of the isoforms of these antioxidative enzymes is required for using the various isoforms as salt stress markers. Our results indicate that the overproduction of H₂O₂ by NaCl treatment functions as a signal of salt stress and causes upregulation of APX, POX, GR and deactivations of CAT in B. parviflora. The concentrations of malondialdehyde, a product of lipid peroxidation and lipoxygenase activity remained unchanged in leaves treated with different concentrations of NaCl, which again suggests that the elevated levels of the antioxidant enzymes protect the plants against the activated oxygen species thus avoiding lipid peroxidation during salt stress.     

Effect of temperature on secondary metabolites production and antioxidant enzyme activities in Eleutherococcus senticosus somatic embryos

Somatic embryos of Eleutherococcus senticosus were exposed at 12, 16, 24 and 30 °C for duration of 45 days in bioreactor. The effects of such treatments on the growth, eleutheroside B, E, E₁, total phenolics, flavonoids, chlorogenic acid concentrations and antioxidant enzymes activities were investigated. The results revealed that low (12 and 18 °C) and high (30 °C) temperature caused significant decrease in fresh weight (FW), dry weight (DW), total phenolics, flavonoids and total eleutheroside accumulation, while low temperature increased eleutheroside E accumulation in somatic embryos. Low temperature significantly increased superoxide dismutase (SOD), catalase (CAT), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) activities whereas a strong increase in ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activity was obtained at 12 °C grown somatic embryos. In contrast, high temperature significantly decreased antioxidant enzymes activities and even guaiacol peroxidase (G-POD) activity also decreased at low temperature in comparison to 24 °C grown embryos. These data suggest that low and high temperature treatment provoked an oxidative stress in E. senticosus embryos, as shown by the increase in lipid peroxidation. The increase in lipid peroxidation was paralleled by a rise in lipoxygenase (LOX) activity and hydrogen peroxide (H₂O₂) content. However, this stress was more prominent at high temperature than low temperature grown embryos. This result suggests that the reduced growth of embryo at 30 °C was concomitant with reduced efficiency of these protective enzymes. On the other hand, increases in antioxidant activities at 12 and 18 °C could also be a response to the cellular damage; however, this increase could not stop the deleterious effects of low temperature, but reduced stress severity thus allowing embryo growth to occur.