Reactive oxygen species formation and cell death in catalase-deficient tobacco leaf disks exposed to cadmium

The physiological responses of tobacco (Nicotiana tabacum L.) to oxidative stress induced by cadmium were examined with respect to reactive oxygen species (ROS) formation, antioxidant enzymes activities, and cell death appearance in wild-type SR1 and catalase-deficient CAT1AS plants. Leaf disks treated with 100 or 500 µM CdCl₂ increased Evans blue staining and leakage of electrolytes in SR1 or CAT1AS plants, more pronouncedly in the transgenic cultivar, but without evidence of lipid peroxidation in any of the cultivars compared to controls. Cadmium significantly reduced the NADPH oxidase-dependent O ₂ ^? formation in a dose dependent manner in SR1 very strongly at 500 µM (to 5% of the activity in the nontreated SR1 leaf disks). In CAT1AS, the NADPH oxidase activity was constitutively reduced at 50% with respect to that of SR1, but the magnitude of the decay was less prominent in this cultivar, reaching an average of 64% of the C at 21 h, for both Cd concentrations. Hydrogen peroxide formation was only slightly increased in SR1 or CAT1AS leaf disks at 21 h of exposure compared to the respective controls. Cd increased superoxide dismutase activity more than six times at 21 h in CAT1AS, but not in SR1 and reduced catalase activity by 59% at 21 h of treatment only in SR1 plants. Despite that catalase expression was constitutively lower in CATAS1 compared to SR1 nontreated leaf disks, 500 µM CdCl₂ almost doubled it only in CAT1AS at 21 h. The mechanisms underlying Cd-induced cell death were possibly not related exclusively to ROS formation or detoxification in tobacco SR1 or CAT1AS plants.     

Effect of exogenous hydrogen peroxide on enzymatic and nonenzymatic antioxidants in leaves of young pea plants treated with paraquat

The effects of exogenously applied hydrogen peroxide on the antioxidant system of pea plants were investigated. Ten-day-old pea seedlings were sprayed with 2.5 mM H₂O₂ and 24 h later with 0.2 mM PQ. Samples were taken 0, 2 and 5 h after the start of illumination. The protective effect of H₂O₂ was evaluated by monitoring of parameters related to the damage caused by PQ. The treatment with PQ led to a severe leakage of electrolytes from leaf tissues. Malondialdehyde level increased in PQ treated plants, but remained unchanged in H₂O₂ pre-treated ones after 5 h of illumination. Increased catalase and glutathione-S-transferase activity was observed in pea plants treated with H₂O₂ and PQ. Ascorbate peroxidase activity decreased significantly after paraquat application, but pre-treatment with H₂O₂ prevented ascorbate peroxidase inhibition to some extent. Increased guaiacol peroxidase activity was detected after H₂O₂ application. PQ application caused a drastic decline in the levels of thiol-group bearing compounds, reduced glutathione and ascorbate, while the quantity of oxidized glutathione and dehydroascorbate were increased. The results presented on changes in enzymatic and nonenzymatic antioxidants suggest that preliminary H₂O₂ application to pea plants treated with PQ, alleviates the toxic effects of the herbicide.     

Influence of the Herbicide Chlortoluron on Photosynthetic Activity in Transgenic Tobacco Plants

Photosynthetic activity of leaf disks from chlortoluron (2 µmol per plant) treated and non-treated non-transgenic and transgenic (PGF-6) tobacco plants was measured from 1 up to 21 d after treatment under greenhouse conditions. PGF-6 plants, expressing the fused rat cytochrome P4501A1/yeast reductase genes were used. PGF-6 plants were much more chlortoluron-resistant than control plants. In non-transgenic tobacco plants the electron transport flow to PQ pool was strongly inhibited 1 d after treatment with herbicide whereas it was still existing in PGF-6 plants although some reduction was observed. The quantum yield of photosystem 2 (_PS2) which is related to the quantum yield of whole-chain electron transfer was much more inhibited by chlortoluron than the primary PS2 photochemistry, measured by the ratio F_v/F_m. Lower PS2 activity was found for herbicide-treated non-transgenic plants up to the 9^th day. Then it started to increase in both control and PGF-6 plants, but more rapidly in PGF-6 ones, and its values were near to the control level at the 21^st d after chlortoluron treatment.     

Effects of salicylic acid on paraquat tolerance in<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

Foliar spraying ofArabidopsis thaliana (Columbia ecotype) plants with a 1.0-mM salicylic acid (SA) solution significantly improved their tolerance to subsequent paraquat (PQ)-induced oxidative damage. Leaf injuries, including losses of chlorophyll, protein, and fresh weight, were reduced. Our analysis of antioxidant enzymes in the leaves showed that SA pre-treatment effectively retarded rapid decreases in the activities of Superoxide dismutase (SOD), catalase, and ascorbate peroxidase that are normally associated with PQ exposure. In addition, guaiacol peroxidase activity was remarkably increased. In a native gel assay of peroxidase (POD) isozymes, staining activity of the POD1 isozyme, which disappeared in plants exposed only to 10 μM PQ, was significantly recovered by the 1.0-mM SA pre-treatment POD2 isozyme activity was also pronounced in all SA-treated plants compared with the control. A 12-h SA pre-treatment, without subsequent PQ stress, also caused a small increase in the endogenous H₂O₂ content that accompanies the symptoms of mild leaf injuries. This enhanced level occurred in parallel with a slight SOD increase and a catalase decrease. From our results, it can be assumed that, due to the small increase in SOD as well as catalase inactivation via SA pre-treatment, a moderate increase in H₂O₂ levels may occur. In turn, a large induction of guaiacol peroxidase leads to enhanced PQ tolerance inA. thaliana plants.     

An alginate-derived oligosaccharide enhanced wheat tolerance to cadmium stress

Hydroponic experiments were carried out to study the role of alginate-derived oligosaccharides (ADO) in enhancing wheat (Triticum aestivum L.) tolerance to cadmium stress. Data were collected on plant biomass, chlorophyll content, photosynthetic rate, antioxidant enzyme activity and malondialdehyde (MDA) content. Under 100 μM Cd stress, plant growth was significantly inhibited. Shoot length, root length, fresh and dry weight were sharply reduced by 24.21, 34.59, 22.1 and 14.7%, respectively of the control after 10 day of Cd exposure. Superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities were increased and MDA content increased. Wheat seeds were soaked for 5 h in 1,000 mg L⁻¹ ADO solution before cadmium stress. ADO pretreatment alleviated cadmium toxicity symptoms, which were reflected by increasing root and shoot lengths, fresh and dry weight, chlorophyll content and photosynthetic rate (P _n ). Furthermore, ADO pretreatment significantly increased antioxidant enzyme (SOD, CAT and POD) activities and reduced MDA content in leaves and roots. The results indicated that ADO pretreatment partially protected the seedlings from cadmium toxicity during the following growth period.     

Constitutive expression of a fungal glucose oxidase gene in transgenic tobacco confers chilling tolerance through the activation of antioxidative defence system

Scientific evidences in the literature have shown that plants treated exogenously with micromole concentration of hydrogen peroxide (H₂O₂) acquire abiotic stress tolerance potential, without substantial disturbances in the endogenous H₂O₂ pool. In this study, we enhanced the endogenous H₂O₂ content of tobacco (Nicotiana tabaccum L. cv. SR1) plants by the constitutive expression of a glucose oxidase (GO; EC 1.1.3.4) gene of Aspergillus niger and studied their cold tolerance level. Stable integration and expression of GO gene in the transgenic (T₀–T₂) tobacco lines were ascertained by molecular and biochemical tests. Production of functionally competent GO in transgenic plants was confirmed by the elevated levels of H₂O₂ in the transformed tissues. When three homozygous transgenic lines were exposed to different chilling temperatures for 12 h, the electrolyte conductivity was significantly lower in GO-expressing tobacco plants than the control plants; in particular, chilling protection was more prominent at −1°C. In addition, most transgenic lines recovered within a week when returned to normal culture conditions after −1°C–12 h cold stress. However, control plants displayed symptoms of chilling injuries such as necrosis of shoot tip, shoots and leaves, consequently plant death. The protective effect realized in the transgenic plants was comparable to cold-acclimatized wild tobacco. The chilling tolerance of transgenic lines was found associated, at least in part, with elevated levels of total antioxidant content, CAT and APX activities. Based on our findings, we predict that the transgenic expression of GO may be deployed to improve cold tolerance potential of higher plants.     

H2O2 Involvement in Polyamine-Induced Cell Death in Tobacco Leaf Discs

The response of tobacco (Nicotiana tabacum L.) wild-type SR1 leaf discs in terms of reactive oxygen species (ROS) formation and cell death occurrence was evaluated after exposure to the polyamines (PAs) putrescine (Put), spermidine (Spd), and spermine (Spm). Although NADPH oxidase-like enzyme activity was inhibited by all PAs at 3 or 21 h of treatment, H₂O₂ content increased significantly in a time- and concentration-dependent manner, suggesting that H₂O₂ accumulation was linked to the activity of other ROS-generating enzymes. Polyamine oxidase (PAO) activity, which increased markedly upon application of Spd or Spm, is a prime candidate for the increased H₂O₂ accumulation. Except for 0.1 mM Put, which maintained guaiacol peroxidase (GPOX) and catalase (CAT) activities at the same level as the control, the other PA treatments decreased CAT, ascorbate peroxidase, and GPOX activities at 21 h, contributing to the H₂O₂ increase. Esterase activity and Evans blue staining, two cell death parameters, were negatively affected at 3 h of treatment with 1 mM Spd and with both concentrations of Spm, whereas at 21 h there was an increase in cell death with both concentrations of the three PAs, except for 0.1 mM Put, which did not alter those parameters. The expression of the senescence-associated cysteine protease gene CP1 was measured to monitor senescence, a physiological cell death process. Application of all PAs increased the expression of the gene, except for 0.1 mM Put, which decreased its expression at 21 h. This result was in agreement with the prevention of cell death exerted by Put and evidenced by Evans blue staining, esterase activity, and electrolyte release.     

Short-term effect of elevated CO2 concentration and high irradiance on the antioxidant enzymes in bean plants

The effect of short-term exposure to elevated CO₂ concentration and high irradiance on the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidases (GPX) and catalase (CAT), and on the extent of the lipid peroxidation was studied in bean (Phaseolus vulgaris L.) plants. Plants were exposed for 4 d (8 h a day) to irradiance of 100 (LI) or 1000 (HI) μmol m⁻² s⁻¹ at ambient (CA, 350 μmol mol⁻¹) or elevated (CE, 1300 μmol mol⁻¹) CO₂ concentration. Four-day exposure to CE increased the leaf dry mass in HI plants and RuBPC activity and chlorophyll content in LI plants. Total soluble protein content, leaf dry matter and RuBPC activity were higher in HI than in LI plants, although the HI and CE increased the contents of malonyldialdehyde and H₂O₂. Under CA, exposure to HI increased the activity of APX and decreased the total SOD activity. Under CE, HI treatment also activated APX and led to reduction of both, SOD and GPX, enzymes activities. CE considerably reduced the CAT activity at both irradiances, possibly due to suppressed rate of photorespiration under CE conditions.     

Overexpression of bacterial catalase in tomato leaf chloroplasts enhances photo-oxidative stress tolerance

The Escherichia coli gene katE, which is driven by the promoter of the Rubisco small subunit gene of tomato, rbcS3C, was introduced into a tomato (Lycopersicon esculentum Mill.) by Agrobacterium tumefaciens‐mediated transformation. Catalase activity in progeny from transgenic plants was approximately three‐fold higher than that in wild‐type plants. Leaf discs from transgenic plants remained green at 24 h after treatment with 1 µm paraquat under moderate light intensity, whereas leaf discs from wild‐type plants showed severe bleaching after the same treatment. Moreover, ion leakage from transgenic leaf discs was significantly less than that from wild‐type leaf discs at 24 h after treatment with 1 µm paraquat and 10 mm H₂O₂, respectively, under moderate light intensity. To evaluate the efficiency of the E. coli catalase to protect the whole transgenic plant from the oxidative stress, transgenic and wild‐type plants were sprayed with 100 µm paraquat and exposed to high light illumination (800 µmol m^?2 s^?1). After 24 h, the leaves of the transgenic plants were less damaged than the leaves of the wild‐type plants. The catalase activity and the photosynthesis activity (indicated by the F_v/F_m ratio) were less affected by paraquat treatment in leaves of transgenic plants, whereas the activities of the chloroplastic ascorbate peroxidase isoenzymes and the ascorbate content decreased in both lines. In addition, the transgenic plants showed increased tolerance to the oxidative damage (decrease of the CO₂ fixation and photosystem II activity and increase of the lipid peroxidation) caused by drought stress or chilling stress (4 °C) under high light intensity (1000 µmol m^?2 s^?1). These results indicate that the expression of the catalase in chloroplasts has a positive effect on the protection of the transgenic plants from the photo‐oxidative stress invoked by paraquat treatment, drought stress and chilling stress.     

Dye degradation,antimicrobial and larvicidal activity of silver nanoparticles biosynthesized from Cleistanthus collinus

The present study aimed in green synthesis and characterization of silver nanoparticles (AgNPs) using the leaves of Cleistanthus collinus. The NPs showed various absorption peaks between 3402 cm⁻¹ and 1063 cm⁻¹. FTIR spectrum revealed the presence of OH group, alkene, aromatic hydrocarbon, aliphatic fluro compound and aliphatic chloro compounds. Scanning electron microscopic analysis revealed the particle size ranged from 30 to 50 nm. The biosynthesized NPs have potent activity against Shigella dysentriae, Staphylococcus aureus and Bacillus subtilis and the zone of inhibition was 21 ± 1, 20 ± 2, 16 ± 2 mm, respectively. Toxicity of the synthesized NPs was tested on green gram (Vigna radiata) seed at various concentrations (20–100%) and germination was induced by NPs treated seeds. Shoot length and root length was higher in NPs treated plant than control plant (p < 0.01). Elevated level of catalase (CAT) and superoxide dismutase (SOD) and about 13% CAT and 7% SOD activity registered than control. Superoxide dismutase activity of root and shoot varied based on the dosage of AgNPs (p < 0.01). Also, the NPs (1%) showed significant larvicidal activity on Aedes aegypti and 100% mortality was achieved after 24 h treatment. The green synthesized NPs reduced methylene blue and 4-nitrophenol significantly (p < 0.01). The colouration of methylene blue and 4-nitrophenol were considerably reduced after 60 min showed the potential of dye degrading ability.     

Salt and paraquat stress tolerance results from co-expression of the Suaeda salsa glutathione S-transferase and catalase in transgenic rice

GST (Glutathione S-transferase, EC 2.5.1.18) and CAT (Catalase, EC 1.11.1.6) play important roles in oxidative stress resistance. In this study, we transferred both GST and CAT1 of Suaeda salsa into rice (Oryza sativa cv. Zhonghua No.11) by Agrobacterium tumefaciens-mediated transformation under the control of cauliflower mosaic virus (CaMV) 35S promoter, and investigated whether co-expressing the GST and CAT1 in transgenic rice could reduce oxidative damage. Salt and paraquat stresses were applied. The data showed that co-expression of the GST and CAT1 resulted in greater increase of CAT and SOD (Superoxide Dismutase, EC 1.15.1.1) activity in the transgenics compared to non-transgenics following both stress imposition. Whereas the significant increase of GST activity in transgenics only occurred in paraquat stressed plants. While the generation of H₂O₂, Malon dialdehyde and plasma membrane relative electrolyte leakage decreased in the transgenics than in non-transgenics under the same conditions. Moreover, the transgenic rice seedlings showed markedly enhanced tolerance to salt stress compared with non-transgenics upon 200 mM NaCl treatment in greenhouse. The enhancement of the active oxygen-scavenging system that led to increased oxidative stress protection in GST + CAT1-transgenic rice plants could result not only from increased GST and CAT activity but also from the combined increase in SOD activity.     

Association between sheath blight resistance and chitinase activity in transgenic rice plants expressing McCHIT1 from bitter melon

No usable resources with high-level resistance to sheath blight (SB) have yet been found in rice germplasm resources worldwide. Therefore, creating and breeding new disease-resistant rice resources with sheath blight resistance (SBR) are imperative. In this study, we inoculated rice plants with hyphae of the highly pathogenic strain RH-9 of rice SB fungus Rhizoctonia solani to obtain eight stable transgenic rice lines harbouring the chitinase gene (McCHIT1) of bitter melon with good SBR in the T₅ generation. The mean disease index for SB of wild-type plants was 92% and 37–44% in transgenic lines. From 24 h before until 120 h after inoculation with R. solani, chitinase activity in stable transgenic plants with increased SBR was 2.0–5.5 and 1.8–2.7 times that of wild-type plants and plants of a disease-susceptible stable transgenic line, respectively. The correlation between SBR and chitinase activity in McCHIT1-transgenic rice line plants was significant. This work stresses how McCHIT1 from bitter melon can be used to protect rice plants from SB infection.

     

Analysis of transgenic wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) harboring a maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) gene for plastid EF-Tu: segregation pattern,expression and effects of the transgene

We previously reported that transgenic wheat (Triticum aestivum L.) carrying a maize (Zea mays L.) gene (Zmeftu1) for chloroplast protein synthesis elongation factor, EF-Tu, displays reduced thermal aggregation of leaf proteins, reduced injury to photosynthetic membranes (thylakoids), and enhanced rate of CO₂ fixation following exposure to heat stress (18 h at 45°C) [Fu et al. in Plant Mol Biol 68:277–288, 2008]. In the current study, we investigated the segregation pattern and expression of the transgene Zmeftu1 and determined the grain yield of transgenic plants after exposure to a brief heat stress (18 h at 45°C). We also assessed thermal aggregation of soluble leaf proteins in transgenic plants, testing the hypothesis that increased levels of EF-Tu will lead to a non-specific protection of leaf proteins against thermal aggregation. The transgenic wheat displayed a single-gene pattern of segregation of Zmeftu1. Zmeftu1 was expressed, and the transgenic plants synthesized and accumulated three anti-EF-Tu cross-reacting polypeptides of similar molecular mass but different pI, suggesting the possibility of posttranslational modification of this protein. The transgenic plants also showed better grain yield after exposure to heat stress compared with their non-transgenic counterparts. Soluble leaf proteins of various molecular masses displayed lower thermal aggregation in transgenic than in non-transgenic wheat. The results suggest that overexpression of chloroplast EF-Tu can be beneficial to wheat tolerance to heat stress. Moreover, the results also support the hypothesis that EF-Tu contributes to heat tolerance by acting as a molecular chaperone and protecting heat-labile proteins from thermal aggregation in a non-specific manner.     

Expression of Acidothermus cellulolyticus endoglucanase E1 in transgenic tobacco: biochemical characteristics and physiological effects

The expression of the Acidothermus cellulolyticus endoglucanase E1 gene in transgenic tobacco (Nicotiana tabacum) was examined in this study, where E1 coding sequence was transcribed under the control of a leaf specific Rubisco small subunit promoter (tomato RbcS-3C). Targeting the E1 protein to the chloroplast was established using a chloroplast transit peptide of Rubisco small subunit protein (tomato RbcS-2A) and confirmed by immunocytochemistry. The E1 produced in transgenic tobacco plants was found to be biologically active, and to accumulate in leaves at levels of up to 1.35% of total soluble protein. Optimum temperature and pH for E1 enzyme activity in leaf extracts were 81°C and 5.25, respectively. E1 activity remained constant on a gram fresh leaf weight basis, but dramatically increased on a total leaf soluble protein basis as leaves aged, or when leaf discs were dehydrated. E1 protein in old leaves, or after 5h dehydration, was partially degraded although E1 activity remained constant. Transgenic plants exhibited normal growth and developmental characteristics with photosynthetic rates similar to those of untransformed SR1 tobacco plants. Results from these biochemical and physiological analyses suggest that the chloroplast is a suitable cellular compartment for accumulation of the hydrolytic E1 enzyme.     

Selective Feeding of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Hübner) and <Emphasis Type="Italic">Spodoptera litura</Emphasis> (Fabricius) on Meridic Diet with <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Toxins

Laboratory experiments were conducted to evaluate the behavior of Helicoverpa armigera (Hübner) and Spodoptera litura (Fabricius) larvae on meridic diet with different concentrations of Bt spray formulation Delfin or isolated Cry1Ac protein or the foliage and bolls from transgenic cotton, Bollgard hybrid RCH-317 Bt. Both insect species selectively fed on nontreated diet compared with the diet treated with Delfin. While H. armigera exhibited concentration response with Cry1Ac, this protein did not affect S. litura larvae. In general Helicoverpa selected diet with low concentrations (EC₂₀ and EC₅₀ levels) of Cry1Ac compared with higher concentrations of Cry1Ac. In order to develop appropriate management strategies, a thorough understanding of the behavioral mechanisms leading to the responses of insects to the proteins in transgenic varieties is required. Thus, based on results of the insects fed individually on the leaf discs or bolls from transgenic cotton plants alone or under choice situation with meridic diet revealed that H. armigera larvae preferred meridic diet to transgenic leaves or bolls expressing Cry1Ac protein. H. armigera larvae preferred meridic diet to plant material; more than 70% larvae were seen on the meridic diet, and average larval weight gain was in the range of 121.7–130.5 mg. However, in case of S. litura the larvae showed no significant discrimination between meridic diet and the leaf discs. In fact more than 60% larvae preferred leaf discs for feeding, though Cry1Ac expression in leaf discs was in the range of 0.9–2.18 μg/g. Thus differences in behavioral response could potentially impact the level of efficacy of crop cultivars that have been genetically engineered to produce these proteins.     

Overexpressing <Emphasis Type="Italic">SgNCED1</Emphasis> in Tobacco Increases ABA Level,Antioxidant Enzyme Activities,and Stress Tolerance

Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses. 9-cis-epoxycarotenoid dioxygenase (NCED) is the key enzyme of ABA biosynthesis in higher plants. A NCED gene, SgNCED1, was overexpressed in transgenic tobacco plants which resulted in 51–77% more accumulation of ABA in leaves. Transgenic tobacco plants decreased stomatal conductance, transpiration rate, and photosynthetic rate but induced activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate-peroxidase (APX). Hydrogen peroxide (H₂O₂) and nitric oxide (NO) in leaves were also induced in the transgenic plants. Compared to the wild-type control, the transgenic plants improved growth under 0.1 M mannitol-induced drought stress and 0.1 M NaCl-induced salinity stress. It is suggested that the ABA-induced H₂O₂ and NO generation upregulates the stomatal closure and antioxidant enzymes, and therefore increases drought and salinity tolerance in the transgenic plants.     

Effect of <Emphasis Type="Italic">Medicago sativa</Emphasis> ferritin gene on stress tolerance in transgenic grapevine

The effect of Medicago sativa (alfalfa) ferritin gene (MsFer) on abiotic stress tolerance was tested using transgenic Vitis berlandieri × Vitis rupestris cv. ‘Richter 110’ grapevine rootstock lines. Leaf discs from transgenic plants maintained higher photosynthetic activity after NaCl, tert-butyl-hydroperoxide (t-BHP) or paraquat treatment than control ones. These results indicate that the increased production of ferritin significantly improved abiotic stress tolerance in transgenic grapevine plants.