Antioxidant Enzyme Activities during in vitro Morphogenesis of Gladiolus and the Effect of Application of Antioxidants on Plant Regeneration

Activity of antioxidant enzymes was evaluated during somatic embryogenesis and shoot organogenesis from cultured leaf segments of Gladiolus hybridus Hort. The effect of exogenous antioxidants on somatic embryogenesis and shoot organogenesis has also been monitored. Activity of superoxide dismutase (SOD) gradually increased during somatic embryogenesis. while activities of catalase (CAT) and peroxidase (POX) decreased. In contrast, increase in CAT and POX activity and a concomitant decrease in SOD activity were noted during shoot organogenesis. Exogenous application of antioxidants such as glutathione (GSH), α-tocopherol and ascorbate (AA) inhibited somatic embryogenesis but stimulated shoot organogenesis. The frequency of somatic embryogenesis increased with the addition of H₂O₂. However, H₂O₂ inhibited shoot organogenesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro selection and characterization of polyethylene glycol (PEG) tolerant callus lines and regeneration of plantlets from the selected callus lines in sugarcane (Saccharum officinarum L.)

A system for in vitro selection of drought tolerant callus lines in sugarcane was developed. High molecular weight PEG was used as selective agent. Selected callus line grew better than non-selected callus when grown on different concentrations of PEG. The activity of antioxidant enzymes like CAT, POX, APX and SOD were high in selected callus than in non-selected callus. Osmolytes like proline and ascorbic acid were at higher levels in selected callus than in non-selected callus, however at higher concentrations (20–30 %) of PEG, levels of proline and ascorbic acid decreased. The frequency of organogenesis and number of plantlets decreased in selected callus than in non-selected callus. The results can be used for in vitro screening and manipulations of sugarcane for improvement of drought tolerance     

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.     

Reactive oxygen species production and antioxidative defense in pea (Pisum sativum L.) root nodules after short-term aluminum treatment

Pea plants (Pisum sativum L.) were treated with 50???M aluminum chloride at pH 4.5 for 2 or 24?h at room temperature. Following treatment, root nodule Al uptake, the generation of reactive oxygen species (ROS, O ₂ ^?· and H₂O₂), and the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and peroxidase (POX) were investigated. Aluminum accumulation was found chiefly in the apoplast of the nodule cortex, endodermis and meristem, while the formation of peroxide was detected in the nodule cortex, infection threads and bacteroidal tissue. Further, there were increased levels of superoxide in the meristem and bacteroidal tissue. The activity of SOD (EC 1.15.1.1) and POX (EC 1.11.1.7) increased in the Al-treated nodules and the roots of pea plants, whereas CAT (EC 1.11.1.6) activity decreased. The Al absorbed by the nodules induced ROS production. The POX and SOD are important ROS-scavengers in Al-stressed nodules.     

High-frequency vibration improve callus growth via antioxidant enzymes induction in <Emphasis Type="Italic">Hyoscyamus kurdicus</Emphasis>

Effect of high-frequency vibration on growth rate, membrane stability and activities of some antioxidant enzymes were studied in callus tissues of Hyoscyamus kurdicus. Calli initiated from leaf (LE), shoot (SE) and root (RE) explants, and sinusoidal vibrations at 0, 50, 100 and 150 Hz for 30 min were applied on the H. kurdicus calli. Results showed that sinusoidal vibration at 50 and 100 Hz promoted the growth rate as compared to control, and the optimum growth was found at 50 Hz. Sinusoidal vibration increased significantly protein and proline contents and activity of superoxide dismutase (SOD), ascorbate peroxidase (APX) and peroxidase (POX) enzymes, and decreased total carbohydrate, H₂O₂ level and CAT activity as compared to control. Lipid peroxidation also decreased under sinusoidal vibration in all the calli, and the maximum percentage of decrease was observed at 50 Hz. Native polyacrylamide gel electrophoresis indicated different isoform profiles in vibration treated and untreated plants concerning antioxidant enzymes. The responses of different types of calluses were different, and RE callus showed the highest growth, membrane stability and antioxidant enzymes activity as compared to LE and SE calli. These results suggest sinusoidal vibration at optimum frequency could improve callus growth by induction of antioxidative enzymes activity and membrane stability in calli of H. kurdicus.     

Free radical formation and activity of antioxidant enzymes in lupin roots exposed to lead

Inhibition of root growth and modification of root morphology are the most sensitive responses of Lupinus luteus cv. Ventus L. to lead ions - Pb(NO₃)₂. Using electron paramagnetic resonance (EPR), we found that at the lead concentration of 150 mg.L^–1, the level of free radicals remained at control level, whereas at the higher, sublethal concentration of 350 mg.L^–1, they markedly increased. The EPR signal with the g-value at the maximum absorption of 2.0053 implied that the paramagnetic radical is derived from a quinone. The response of antioxidant enzymes, such as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), peroxidase (POX, EC 1.11.1.7) and ascorbate peroxidase (APOX, EC 1.11.1.11), to exogenously applied lead ions was also examined. Enzyme activity was estimated as a function of time and concentration. Native polyacrylamide gel electrophoresis followed by specific staining revealed an increase in the activity of SOD, CAT, POX and APOX coinciding with the time of cultivation. A lead-dependent increase in activities of SOD and POX from root tip extracts was observed, whereas CAT and APOX activities decreased at the higher lead concentrations. These results suggest that at higher lead concentrations, the formation of both free radicals and reactive oxygen species is beyond the capacity of the antioxidant system, which in turn may contribute to the reduced root growth.     

Effects of NaCl on growth, ion accumulation, protein, proline contents and antioxidant enzymes activity in callus cultures of Jatropha curcas

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. The effect of NaCl stress on growth, ion accumulation, contents of protein, proline, and antioxidant enzymes activity in callus cultures of J. curcas was investigated. Exposure of callus to NaCl decreased growth in a concentration dependent manner. NaCl treated callus accumulated Na and declined in K, Ca and Mg contents. Na/K ratio increased steadily as a function of external NaCl treatment. NaCl induced significant differences in quality and quantity of proteins, whereas, proline accumulation remained more or less constant with treatment. NaCl stress enhanced the activity of superoxide dismutase (SOD; E.C. 1.15.1.1) and peroxidase (POX; E.C. 1.11.1.7). Further in the isoenzyme studies, four SOD isoenzymes (SOD 1, 2, 3, and 4) and two POX isoenzymes (POX 1 and 2) were detected with the treatment. NaCl strongly induced activity of SOD 4 isoenzyme in 40, 60, 80 mM and POX 2 isoenzyme in 40 and 80 mM NaCl concentrations. Increase in antioxidant enzymes activity could be a response to cellular damage induced by NaCl. This increase could not stop the deleterious effects of NaCl, but it reduced stress severity and thus allowed cell growth to occur.     

Activity of antioxidant enzyme during <Emphasis Type="Italic">in vitro</Emphasis> organogenesis in <Emphasis Type="Italic">Crocus sativus</Emphasis>

The effect of various hormonal combinations on regeneration of shoots and roots from meristem-derived callus of Crocus sativus L. and activities of antioxidant enzymes have been studied. The most efficient regeneration occurred with 1.0 mg dm⁻³ 1-naphthaleneacetic acid (NAA) + 1.0 mg dm⁻³ thidiazuron and 1.0 mg dm⁻³ NAA + 2.0 mg dm⁻³ kinetin. For sprouting, regenerated shoot were subcultured on Murashige and Skoog medium containing 1.0 mg dm⁻³ NAA + 1.0 mg dm⁻³ benzylaminopurine (BAP). Protein content and superoxide dismutase activity decreased in regenerated shoots and roots and increased in sprouting shoots, while catalase (CAT), peroxidase (POX) and polyphenol oxidase (PPO) activities increased during organogenesis and decreased in sprouting shoots. High CAT and PPO activities were detected in regenerated roots, whereas high POX activity was observed in regenerated shoot.     

Effects of salicylic acid and salinity on apoplastic antioxidant enzymes in two wheat cultivars differing in salt tolerance

The effects of salicylic acid (SA) and salinity on the activity of apoplastic antioxidant enzymes were studied in the leaves of two wheat (Triticum aestivam L.) cultivars: salt-tolerant (Gerek-79) and salt-sensitive (Bezostaya). The leaves of 10-d-old seedlings grown at nutrient solution with 0 (control), 250 or 500 mM NaCl were sprayed with 0.01 or 0.1 mM SA. Then, the activities of catalase (CAT), peroxidase (POX) and superoxide dismutase (SOD) were determined in the fresh leaves obtained from 15-d-old seedlings. The NaCl applications increased CAT and SOD activities in both cultivars, compared to those of untreated control plants. In addition, the NaCl increased POX activity in the salt-tolerant while decreased in the salt-sensitive cultivar. In control plants of the both cultivars, 0.1 mM SA increased CAT activity, while 0.01 mM SA slightly decreased it. SA treatments also stimulated SOD and POX activity in the salt-tolerant cultivar but significantly decreased POX activity and had no effect on SOD activity in the saltsensitive cultivar. Under salinity, the SA treatments significantly inhibited CAT activity, whereas increased POX activity. The increases in POX activity caused by SA were more pronounced in the salt-tolerant than in the salt-sensitive cultivar. SOD activity was increased by 0.01 mM SA in the salt-tolerant while increased by 0.1 mM SA treatment in the salt-sensitive cultivar.     

Antioxidant enzyme activities, malondialdehyde, and total phenolic content of PEG-induced hyperhydric leaves in sugar beet tissue culture

Hyperhydricity is a physiological abnormality that frequently affects shoots that are vegetatively propagated in vitro. In this study, sugar beet (Beta vulgaris L. cv. Felicita) shoot tip explants were cultured on Murashige and Skoog medium supplemented with different concentrations of polyethylene glycol (PEG) 6000. We observed that higher concentrations of PEG 6000 and longer exposure (up to 4 wk) resulted in increasing levels of hyperhydration as well as browning and/or blackening of tissues in culture. A comparison of hyperhydric shoots with controls on the 28th day showed a marked increase in the content of water, phenolics, and malondialdehyde (MDA), which was positively correlated with an increase in the accumulation of PEG 6000. Selected antioxidant enzyme activities, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POX), and polyphenol oxidase (PPO) also increased in hyperhydric shoots, especially at lower concentrations of PEG 6000. Regression analysis indicated that strong linear relationships exist between SOD–APX (R ²?=?0.932), SOD–CAT (R ²?=?0.753), SOD–total phenolic content (R ²?=?0.966), APX–PPO (R ²?=?0.842), APX–total phenolic content (R ²?=?0.904), POX–CAT (R ²?=?0.751), and CAT–total phenolic content (R ²?=?0.806). Despite the correlation between different antioxidant enzymes and between the antioxidant enzymes and antioxidant compounds, was not able to prevent ROS damage in hyperhydric shoots. The negative correlation between SOD–MDA, POX–MDA, CAT–MDA, and MDA–total phenolics also indicated an increase in antioxidant enzyme activities, yet the increase in these antioxidant compound contents did not prevent lipid peroxidation of in vitro propagated beet shoots.     

Induced anti-oxidant activity in soybean alleviates oxidative stress under moderate boron toxicity

The effect of B toxicity on antioxidant responses of soybean (Glycine max) cv. Athow was investigated by growing plants for 43 days at 0.2 (control), 2 and 12 mg B kg^?1. At the end of the treatment period, shoot growth, lipid peroxidation level, the activities of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), and their isoenzymes in leaves were measured. Boron concentration in leaves was significantly increased by the increasing levels of B treatment from 43 to 522 mg kg^?1, and shoot dry matter was depressed at 12 mg B kg^?1. Significant increases in SOD, CAT, and APX activities were determined in leaves under 12 mg B kg^?1; however, GR activities were decreased while POX activity was unchanged. Increased enzymic antioxidant activity arose from a combination of newly formed isoenzymes and activation of existing isoenzymes. By contrast, SOD and GR activities were decreased by 2 mg B kg^?1 concentration as compared to the control groups while POX activity was increased and the activity of CAT did not change. Malondialdehyde content increased under 2 mg B kg^?1 but decreased under 12 mg B kg^?1. These results suggest that higher antioxidant activity observed under 12 than at 2 mg B kg^?1 provided higher free radical-scavenging capacity, and thus a lower level of lipid peroxidation in Athow. While the induction of increased antioxidant activity was related to internal boron levels, the signaling and coordination of responses remain unclear.     

Effects of Co-60 radiation on the activities of three main antioxidant enzymes in Bactrocera dorsalis (Hendel) (Diptera: Tephritidae)

Co-60 radiation has been broadly used for pest management because it results in male sterility and a lack of emergence of unparasitized hosts due to oxidative damage. Insect life is significantly affected by abiotic stress factors, including Co-60 radiation. In the present study, we determined the influence of Co-60 radiation on an important pest in southern China, the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae). We exposed 30-h-old B. dorsalis eggs to Co-60 radiation at doses of (0, 5, 10, 15, 20, 25, 30, 35, and 40?Gy) and determined the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX). CAT and POX activity significantly increased in B. dorsalis in the 1st larvae instar after high-intensity radiation doses (25–40?Gy) and played an important role in the antioxidant response to intensive radiation. The activity of POX in B. dorsalis at the 2nd larval instar sharply decreased after different doses of radiation, except for 10 and 40?Gy. Our results demonstrated that Co-60 radiation affected the activity of antioxidant enzymes and disturbed the physiology of B. dorsalis, especially at early stages (the 1st, 2nd larvae instar). These three antioxidant defense enzymes cooperatively play an important role in protecting B. dorsalis from oxidative damage.     

Changes in antioxidative enzymes activities during Tacitus bellus direct shoot organogenesis

Changes in antioxidative enzymes activities during Tacitus bellus direct shoot organogenesis from leaf explants were examined. During the early stages of shoot organogenesis there was a decrease in superoxide dismutase (SOD) and an increase in catalase (CAT) activity, and later during organogenesis there was an increase in peroxidase (POD) and polyphenol oxidase (PPO) activity. Two highly regulated turning points may be distinguished regarding activities and isoforms of antioxidative enzymes: the initiation of shoot organogenesis and the shoot bud formation. Our data suggest the role of specific CAT, POD, SOD and PPO isoforms in separate processes during T. bellus direct shoot organogenesis.     

Antioxidant responses of citrus red mite, Panonychus citri (McGregor) (Acari: Tetranychidae), exposed to thermal stress

Relatively low or high temperatures are responsible for a variety of physiological stress responses in insects and mites. Induced thermal stress was recently associated with increased reactive oxygen species (ROS) generation, which caused oxidative damage. In this study, we examined the time-related effect of the relatively low (0, 5, 10, and 15 °C) or high (32, 35, 38, and 41 °C) temperatures on the activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), peroxidases (POX), and glutathione-S-transferase (GST), and the total antioxidant capacity (TEAC) of the citrus red mite, Panonychus citri (McGregor). The malondialdehyde (MDA) concentration, as a marker of lipid peroxidation in organisms, was also measured in the citrus red mite under thermal stress conditions. Results showed that SOD and GST activities were significantly increased and play an important role in the process of antioxidant response to thermal stress. Lipid peroxidation levels increased significantly (P < 0.001) and changed in a time-dependent manner. CAT and POX activity, as well as TEAC, did not vary significantly and play a minor role to remove the ROS generation. These results suggest that thermal stress leads to oxidative stress and antioxidant enzymes play an important role in reducing oxidative damage in the citrus red mite.     

Insect-resistant Bt-maize response to the short-term non-target mite-pest infestation and soil drought

Transgenic lepidopteran insect-resistant maize expressing the cry1Ab gene (Bt) and its non-transgenic counterpart at the 12-leaf-stage (V₁₂) were infested by the two-spotted spider mite or dehydrated by cessation of soil watering to check Bt-maize capacity to respond to other stresses than those assured by the presence of Cry protein. Since the antioxidant enzymes are key components of plant defence against biotic and abiotic stresses, the engagement of leaf superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX) in response to 6-day mite feeding and soil drought has been investigated. The reduction of leaf hydration and soluble protein content in the fully expanded 8th leaf was independent of genotype and more pronounced in response to water cessation than mite infestation. Similarly, the changes in enzyme activities depended more on the kind of stress than the presence of the transgene. Water shortage in the soil enhanced the activity of all enzymes, whereas mite feeding decreased the activity of SOD and CAT, and markedly increased POX in the 8th leaf of both cultivars. In mite-infested leaves of the non-transgenic plant, the CAT activity remained unaffected, whereas decreased in leaves of Bt maize due to the hampered activity of CAT-2. In comparison to the control, all enzyme activity in the 10th non-infested leaf of mite-infested non-transgenic maize decreased, whereas it changed in the 10th leaf of Bt maize in the same way as in the 8th mite-infested leaf. The results suggest that SOD, CAT and POX can strongly confer short-term drought-stress response in both maize cultivars, whereas POX is the only responsive enzyme in mite-infested Bt maize.     

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.     

The activity of antioxidant enzymes in Arabidopsis thaliana exposed to colchicine and H2O2

Studies on the possible interference of colchicine and H2O2 with the activity of some antioxidant enzymes were carried out on Arabidopsis thaliana v. Columbia grown in Murashige and Skooge nutrient medium. Measurements of superoxide dismutase (SOD), guaiacol peroxidase (POX), ascorbate peroxidase (APX) and catalase (CAT) activities were conducted spectrophotometrically. In the presence of colchicine, SOD activity increased, while CAT, APX and POX activities decreased. Inhibitory H2O2 effects on the activity of the enzymes were found. Colchicine pre-treatment resulted in an increase in CAT activity and a further increase in SOD activity in plants treated with H2O2.     

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.     

Comparative study on calli from two reed ecotypes under heat stress

The different physiological responses to heat stress in calli from two ecotypes of common reed (Phragmites communis Trin.) plants (dune reed (DR) and swamp reed (SR)) were studied. The relative water content, the relative growth rate, cell viability, membrane permeability (MP), H₂O₂ content, MDA content, proline level, and the activities of enzymes, such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR), and lipoxygenase (LOX) were assayed. Results showed that under heat stress, DR callus could maintain the higher relative growth rate and cell viability than SR callus, while H₂O₂ content, MDA content, and MP in SR callus increased more than in DR callus. The activities of antioxidant enzymes, such as SOD, CAT, POD, APX, and GR in two calli were enhanced by high temperature. However, antioxidant enzymes in DR callus showed the higher thermal stability than those in SR callus. LOX activity increased more in SR callus than in DR callus under heat stress. High temperature markedly elevated proline content in DR callus whereas had no effect on that in SR callus. Taken together, DR callus is more thermotolerant than SR callus, which might be due to the higher activity of antioxidant enzymes and proline level compared with SR callus under heat stress.