Enhancement of stress tolerance in transgenic tobacco plants overexpressing Chlamydomonas glutathione peroxidase in chloroplasts or cytosol

To evaluate the physiological potential of the defense system against hydroperoxidation of membrane-lipid components caused by environmental stresses in higher plants, we generated transgenic tobacco plants expressing a glutathione peroxidase (GPX)-like protein in the cytosol (TcGPX) or chloroplasts (TpGPX). The activities toward alpha-linolenic acid hydroperoxide in TcGPX and TpGPX plants were 47.5-75.3 and 32.7-42.1 nM min(-1) mg(-1) protein, respectively, while no activity was detected in wild-type plants. The transgenic plants showed increased tolerance to oxidative stress caused by application of methylviologen (MV: 50 microM) under moderate light intensity (200 micro E m(-2) sec(-1)), chilling stress under high light intensity (4 degrees C, 1000 microE m(-2) sec(-1)), or salt stress (250 mM NaCl). Under these stresses, the lipid hydroperoxidation (the production of malondialdehyde (MDA)) of the leaves of TcGPX and TpGPX plants was clearly suppressed compared with that of wild-type plants. Furthermore, the capacity of the photosynthetic and antioxidative systems in the transgenic plants remained higher than those of wild-type plants under chilling or salt stress. These results clearly indicate that a high level of GPX-like protein in tobacco plants functions to remove unsaturated fatty acid hydroperoxides generated in cellular membranes under stress conditions, leading to the maintenance of membrane integrity and increased tolerance to oxidative stress caused by various stress conditions.     

GRP78 Suppresses Lipid Peroxidation and Promotes Cellular Antioxidant Levels in Glial Cells following Hydrogen Peroxide Exposure

Oxidative stress, caused by the over production of reactive oxygen species (ROS), has been shown to contribute to cell damage associated with neurotrauma and neurodegenerative diseases. ROS mediates cell damage either through direct oxidation of lipids, proteins and DNA or by acting as signaling molecules to trigger cellular apoptotic pathways. The 78 kDa glucose-regulated protein (GRP78) is an ER chaperone that has been suggested to protect cells against ROS-induced damage. However, the protective mechanism of GRP78 remains unclear. In this study, we used C6 glioma cells transiently overexpressing GRP78 to investigate the protective effect of GRP78 against oxidative stress (hydrogen peroxide)-induced injury. Our results showed that the overexpression of GRP78 significantly protected cells from ROS-induced cell damage when compared to non-GRP78 overexpressing cells, which was most likely due to GRP78-overexpressing cells having higher levels of glutathione (GSH) and NAD(P)H:quinone oxidoreductase 1 (NQO1), two antioxidants that protect cells against oxidative stress. Although hydrogen peroxide treatment increased lipid peroxidation in non-GRP78 overexpressing cells, this increase was significantly reduced in GRP78-overexpressing cells. Overall, these results indicate that GRP78 plays an important role in protecting glial cells against oxidative stress via regulating the expression of GSH and NQO1.     

Transgenic tobacco plants overexpressing glyoxalase enzymes resist an increase in methylglyoxal and maintain higher reduced glutathione levels under salinity stress

The mechanism behind enhanced salt tolerance conferred by the overexpression of glyoxalase pathway enzymes was studied in transgenic vis-à-vis wild-type (WT) plants. We have recently documented that salinity stress induces higher level accumulation of methylglyoxal (MG), a potent cytotoxin and primary substrate for glyoxalase pathway, in various plant species [Yadav, S.K., Singla-Pareek, S.L., Ray, M., Reddy, M.K. and Sopory, S.K. (2005) MG levels in plants under salinity stress are dependent on glyoxalase I and glutathione. Biochem. Biophys. Res. Commun. 337, 61-67]. The transgenic tobacco plants overexpressing glyoxalase pathway enzymes, resist an increase in the level of MG that increased to over 70% in WT plants under salinity stress. These plants showed enhanced basal activity of various glutathione related antioxidative enzymes that increased further upon salinity stress. These plants suffered minimal salinity stress induced oxidative damage measured in terms of the lipid peroxidation. The reduced glutathione (GSH) content was high in these transgenic plants and also maintained a higher reduced to oxidized glutathione (GSH:GSSG) ratio under salinity. Manipulation of glutathione ratio by exogenous application of GSSG retarded the growth of non-transgenic plants whereas transgenic plants sustained their growth. These results suggest that resisting an increase in MG together with maintaining higher reduced glutathione levels can be efficiently achieved by the overexpression of glyoxalase pathway enzymes towards developing salinity stress tolerant plants.     

Elevated glutathione biosynthetic capacity in the chloroplasts of transgenic tobacco plants paradoxically causes increased oxidative stress

Glutathione (GSH), a major antioxidant in most aerobic organisms, is perceived to be particularly important in plant chloroplasts because it helps to protect the photosynthetic apparatus from oxidative damage. In transgenic tobacco plants overexpressing a chloroplast-targeted gamma-glutamylcysteine synthetase (gamma-ECS), foliar levels of GSH were raised threefold. Paradoxically, increased GSH biosynthetic capacity in the chloroplast resulted in greatly enhanced oxidative stress, which was manifested as light intensity-dependent chlorosis or necrosis. This phenotype was associated with foliar pools of both GSH and gamma-glutamylcysteine (the immediate precursor to GSH) being in a more oxidized state. Further manipulations of both the content and redox state of the foliar thiol pools were achieved using hybrid transgenic plants with enhanced glutathione synthetase or glutathione reductase activity in addition to elevated levels of gamma-ECS. Given the results of these experiments, we suggest that gamma-ECS-transformed plants suffered continuous oxidative damage caused by a failure of the redox-sensing process in the chloroplast.     

Enhanced sensitivity to oxidative stress in transgenic tobacco plants with decreased glutathione reductase activity leads to a decrease in ascorbate pool and ascorbate redox state

To investigate the possible mechanisms of glutathione reductase (GR) in protecting against oxidative stress, we obtained transgenic tobacco (Nicotiana tabacum) plants with 30–70% decreased GR activity by using a gene encoding tobacco chloroplastic GR for the RNAi construct. We investigated the responses of wild type and transgenic plants to oxidative stress induced by application of methyl viologen in vivo. Analyses of CO₂ assimilation, maximal efficiency of photosystem II photochemistry, leaf bleaching, and oxidative damage to lipids demonstrated that transgenic plants exhibited enhanced sensitivity to oxidative stress. Under oxidative stress, there was a greater decrease in reduced to oxidized glutathione ratio but a greater increase in reduced glutathione in transgenic plants than in wild type plants. In addition, transgenic plants showed a greater decrease in reduced ascorbate and reduced to oxidized ascorbate ratio than wild type plants. However, there were neither differences in the levels of NADP and NADPH and in the total foliar activities of monodehydroascorbate reductase and dehydroascorbate reductase between wild type and transgenic plant. MV treatment induced an increase in the activities of GR, ascorbate peroxidase, superoxide dismutase, and catalase. Furthermore, accumulation of H₂O₂ in chloroplasts was observed in transgenic plants but not in wild type plants. Our results suggest that capacity for regeneration of glutathione by GR plays an important role in protecting against oxidative stress by maintaining ascorbate pool and ascorbate redox state.     

Overexpression of wheat mitochondrial uncoupling protein in rice plants confers tolerances to oxidative stresses promoted by exogenous hydrogen peroxide and low temperature

To obtain a better understanding of the function of mitochondrial uncoupling protein (UCP) in higher plants, the wheat gene for mitochondrial uncoupling protein (WhUCP) in rice was overexpressed by Agrobacterium-mediated transformation with a construct containing the WhUCP ORF under control of the 35S promoter. The transgenic rice plants showed a significant increase in tolerance against oxidative stress promoted by exogenous hydrogen peroxide at the seedling stage. The transgenic rice plants overexpressing WhUCP also exhibited greater tolerance against cold stress than did the wild-type plants. These results demonstrated that the mitochondrial UCP in higher plants is positively involved in the pathway for abiotic stress tolerance, probably through a decrease in cellular oxidative damage, and that controlled uncoupling by UCP could be used for improvement of stress tolerance in higher plants.Kenjirou Ozawa and Seiji Murayama are contributed equally to the work     

Salinity-induced glutathione synthesis in Brassica napus

The role of S-assimilation and the biosynthesis of cysteine and glutathione were studied during the response to salt stress of wild-type and salt-tolerant transgenic Brassica napus L. (canola) plants overexpressing a vacuolar Na+/H+ antiporter. A 3-fold increase in cysteine and glutathione content was observed in wild-type plants exposed to salt stress, but not in the transgenic plants. The induction of cysteine and glutathione synthesis during salt stress in the wild-type plants suggests a possible protective mechanism against salt-induced oxidative damage. On the other hand, the salt-tolerant transgenic plants did not show significant changes in either cysteine or glutathione content, confirming the role of vacuolar Na+ accumulation and ion homeostasis in salt tolerance.     

Different Mechanisms of Four Aluminum (Al)-Resistant Transgenes for Al Toxicity in Arabidopsis

We have characterized the mechanism of action of four transgenes (AtBCB [Arabidopsis blue copper-binding protein], parB [tobacco (Nicotiana tabacum) glutathione S-transferase], NtPox [tobacco peroxidase], and NtGDI1 [tobacco GDP dissociation inhibitor]) that independently Al resistance on transgenic Arabidopsis. All four transgenic lines showed lower deposition of callose after Al treatment than the Landsberg erecta ecotype of Arabidopsis, confirming that the four genes function to ameliorate Al toxicity. Influx and efflux experiments of Al ions suggested that the AtBCB gene may suppress Al absorption, whereas expression of the NtGDI1 gene promotes a release of Al in the root tip region of Arabidopsis. The total enzyme activities of glutathione S-transferases or peroxidases in transgenic lines carrying either the parB or NtPox genes were significantly higher than in the Landsberg erecta ecotype of Arabidopsis, and these enzyme activities were maintained at higher levels during Al stress. Furthermore, lipid peroxidation caused by Al stress was repressed in these two transgenic lines, suggesting that overexpression of these two genes diminishes oxidative damage caused by Al stress. Al-treated roots of transgenic plants were also stained by 4',6-diamino-2-phenylindole to monitor cell death caused by Al toxicity. The result suggested that cell death is repressed in the NtPox line. Analysis of F(1) hybrids between the four transgenic lines suggests that more resistant transgenic plants can be constructed by combinations of these four genes.     

Glutathione peroxidase-like protein of Synechocystis PCC 6803 confers tolerance to oxidative and environmental stresses in transgenic Arabidopsis

Glutathione peroxidase (GPX)-like proteins (GPX-1 and GPX-2) of Synechocystis PCC 6803 ( S. PCC 6803) reduce unsaturated fatty acid hydroperoxides using NADPH, but not reduced glutathione (GSH), as an electron donor. Here, we generated transgenic Arabidopsis plants overexpressing S. PCC 6803 GPX-2 in the cytosol (AcGPX2) or chloroplasts (ApGPX2). The activities toward α-linolenic acid hydroperoxide in ApGPX2 and AcGPX2 plants were 6.5–11.5 and 8.2–16.3 nmol min⁻¹ mg protein⁻¹, respectively, while no activity (<0.1 nmol min⁻¹ mg protein⁻¹) was detected in the wild-type plants. Both transgenic lines (AcGPX2 and ApGPX2) showed enhanced tolerance to oxidative damage caused by treatment with H₂O₂ (10 m M ), Fe ions (200 μ M ) or methylviologen (50 μ M ) and environmental stress conditions, such as chilling with high light intensity (4°C, 1000 μmol photons m⁻² s⁻¹), high salinity (100 m M NaCl) or drought. The degree of tolerance of the transgenic plants to all types of stress was correlated with the levels of lipid peroxide suppressed by the overexpression of S. PCC 6803 GPX-2. Under conditions of oxidative stress due to the H₂O₂ treatment, the NADPH/(NADP⁺+ NADPH) ratio in the transgenic plants was lower than that in the wild-type plants. The data reported here indicate that the expression of S. PCC 6803 GPX-2 contributes to the reduction in unsaturated fatty acid hydroperoxides using NADPH in situ under stress conditions in the transgenic plants.     

Overexpression of Loquat Dehydrin Gene <Emphasis Type="Italic">EjDHN1</Emphasis> Promotes Cold Tolerance in Transgenic Tobacco

Dehydrins (DHNs) play vital roles in response to dehydration stress in plants. To examine the contribution of EjDHN to low-temperature stress in loquat (Eriobotrya japonica Lindl.), EjDHN1 was overexpressed in tobacco (Nicotiana tabacum L.). The plant growth of transgenic lines was significantly better than wild type (WT) after 4 d of recovery from cold stress. Cold stress led to membrane lipid peroxidation and reduced photosystem II (PSII) activity in leaves, and these were less severe in transgenic lines. To examine oxidative stress tolerance, the plants were treated with different concentrations of methyl viologen (MV), which inhibited plant growth both in WT and transgenic lines. After exposure to 2.0 μM MV for 10 d, the WT plants had a dramatically lower survival rate. MV treatment in leaf disks confirmed that transgenic lines accumulated less reactive oxygen species (ROS) and suffered less lipid peroxidation. The results suggested that the tolerance of the transgenic plants to cold was increased, and EjDHN1 could protect cells against oxidative damage caused by ROS production under cold stress. It also provided evidences that the enhanced cold tolerance resulted from EjDHN1 overexpression could be partly due to their protective effect on membranes by alleviating oxidative stresses.     

Alleviation of ultraviolet-C-induced oxidative damage through overexpression of cytosolic ascorbate peroxidase

Experiments were conducted to investigate the relationship between ultraviolet (UV) C-induced oxidative damage and the activity of ascorbate peroxidase (APX), using transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana) plants overexpressing cytosolic APX gene (apx1). Transgenic plants having 2.3 fold higher total APX activity, as compared to the wild type plants, showed normal morphological characters. Exposure of 70-day-old plants to fixed intensity UV-C radiation caused an increase in the malondialdehyde (MDA) content in wild type as well as transgenic plants. However, the wild type plants showed significantly higher (p < 0.05) lipid peroxidation as compared to the transgenic plants. Higher proline accumulation was recorded in transgenic plants as compared to the wild type plants, after 24 hours of UV-C exposure. Although the ascorbate content decreased continuously with increasing exposure to UV-C radiation, yet the wild type plants exhibited higher ascorbate levels than the transgenic plants. A marked difference in H₂O₂ content, between the wild type and transgenic plants, was consistently observed up to 20 hours of UV-C exposure. A direct correlation of ascorbate, MDA and H₂O₂ levels was recorded with the extent of oxidative stress, signifying that these could be used as potential bio-marker molecules for oxidative stress. The results clearly demonstrate that overexpression of cytosolic APX can protect tobacco plants from UV-C-induced oxidative damage.     

Glutathione Depletion Due to Copper-Induced Phytochelatin Synthesis Causes Oxidative Stress in Silene cucubalus

The relation between loss of glutathione due to metal-induced phytochelatin synthesis and oxidative stress was studied in the roots of copper-sensitive and tolerant Silene cucubalus (L.) Wib., resistant to 1 and 40 micromolar Cu, respectively. The amount of nonprotein sulfhydryl compounds other than glutathione was taken as a measure of phytochelatins. At a supply of 20 micromolar Cu, which is toxic for sensitive plants only, phytochelatin synthesis and loss of total glutathione were observed only in sensitive plants within 6 h of exposure. When the plants were exposed to a range of copper concentrations for 3 d, a marked production of phytochelatins in sensitive plants was already observed at 0.5 micromolar Cu, whereas the production in tolerant plants was negligible at 40 micromolar or lower. The highest production in tolerant plants was only 40% of that in sensitive plants. In both varieties, the synthesis of phytochelatins was coupled to a loss of glutathione. Copper at toxic concentrations caused oxidative stress, as was evidenced by both the accumulation of lipid peroxidation products and a shift in the glutathione redox couple to a more oxidized state. Depletion of glutathione by pretreatment with buthionine sulfoximine significantly increased the oxidative damage by copper. At a comparably low glutathione level, cadmium had no effect on either lipid peroxidation or the glutathione redox couple in buthionine sulfoximine-treated plants. These results indicate that copper may specifically cause oxidative stress by depletion of the antioxidant glutathione due to phytochelatin synthesis. We conclude that copper tolerance in S. cucubalus does not depend on the production of phytochelatins but is related to the plant's ability to prevent glutathione depletion resulting from copper-induced phytochelatin production, e.g. by restricting its copper uptake.     

Transgenic potato overproducing L-ascorbic acid resisted an increase in methylglyoxal under salinity stress via maintaining higher reduced glutathione level and glyoxalase enzyme activity

Salt-tolerance was studied in transgenic potato. It was conferred by overexpression of ascorbate pathway enzyme (d-galacturonic acid reductase, GalUR). As genetic engineering of the GalUR gene in potato enhances its ascorbic acid content (l-AsA), and subsequently plants suffered minimal oxidative stress-induced damage, we now report on the comprehensive aptness of this engineering approach for enhanced salt tolerance in transgenic potato (Solanum tuberosum L. cv. Taedong Valley). Potatoes overexpressing GalUR grew and tuberized in continuous presence of 200 mM of NaCl. The transgenic plants maintained a higher reduced to oxidized glutathione (GSH:GSSG) ratio together with enhanced activity of glutathione dependent antioxidative and glyoxalase enzymes under salinity stress. The transgenics resisted an increase in methylglyoxal that increased radically in untransformed control plants under salinity stress. This is the first report of genetic engineering of ascorbate pathway gene in maintaining higher level of GSH homeostasis along with higher glyoxalase activity inhibiting the accumulation in methylglyoxal (a potent cytotoxic compound) under salt stress. These results suggested the engineering of ascorbate pathway enzymes as a major step towards developing salinity tolerant crop plants.     

Cloning and Functional Characterization of a Novel Glutathione <Emphasis Type="Italic">S</Emphasis>-Transferase Gene from <Emphasis Type="Italic">Limonium bicolor</Emphasis>

Plant glutathione S-transferases (GSTs) are involved in protecting plants against both diverse biotic and abiotic stresses. In the present study, a novel GST gene (LbGST1) was cloned from Limonium bicolor (Bunge) Kuntze (Plumbaginaceae). To characterize its function in salt tolerance, tobacco lines transformed with LbGST1 were generated. Compared with wild-type (WT) tobacco, transgenic plants overexpressing LbGST1 exhibited both GST and glutathione peroxidase activities. Moreover, superoxide dismutase, peroxidase (POD), and catalase activities in transgenic plants were significantly higher than those in WT plants, particularly when grown under conditions of salt stress. Similarly, levels of proline in transgenic plants were also higher than those in WT plants grown under NaCl stress conditions. Whereas, Malondialdehyde contents in transgenic plants were lower than those in WT plants under NaCl conditions. Furthermore, Na⁺ content in transgenic plants was lower than that in WT plants under these stress conditions. Subcellular localization analysis revealed that the LbGST1 protein was localized in the nucleus. These results suggested that overexpression of LbGST1 gene can affect many physiological processes associated with plant salt tolerance. Therefore, we hypothesize that LbGST1 gene can mediate many physiological pathways that enhance stress resistance in plants.     

Ovexpression of a Vacuolar H<Superscript>+</Superscript>-ATPase c Subunit Gene Mediates Physiological Changes Leading to Enhanced Salt Tolerance in Transgenic Tobacco

H⁺-ATPase subunit c (VHA-c) is involved in the adaptation to environmental stresses, including salt, drought, and heavy metals. However, it remains unclear whether VHA-c can induce a physiological response related to stress tolerance. To investigate this possibility, we generated transgenic tobacco lines overexpressing a V-ATPase subunit c (LbVHA-c1) gene from Limonium bicolor (Bunge) Kuntze. Compared with wild-type (WT) tobacco, superoxide dismutase (SOD) and peroxidase (POD) activities in the transgenic plants were significantly enhanced under salt stress conditions. The level of malondialdehyde (MDA) in the transgenic plants was significantly lower than that in WT plants grown under salt stress conditions. Moreover, the transgenic plants displayed obviously better growth than the WT plants under salt stress. These results suggest that LbVHA-c1 may confer stress tolerance through enhancing POD and SOD activities, and by protecting membranes from damage by decreasing lipid peroxidation under salt stress.     

Involvement of cytosolic ascorbate peroxidase and Cu/Zn-superoxide dismutase for improved tolerance against drought stress

In order to understand the role of cytosolic antioxidant enzymes in drought stress protection, transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants overexpressing cytosolic Cu/Zn-superoxide dismutase (cytsod) (EC 1.15.1.1) or ascorbate peroxidase (cytapx) (EC 1.11.1.1) alone, or in combination, were produced and tested for tolerance against mild water stress. The results showed that the simultaneous overexpression of Cu/Znsod and apx or at least apx in the cytosol of transgenic tobacco plants alleviates, to some extent, the damage produced by water stress conditions. This was correlated with higher water use efficiency and better photosynthetic rates. In general, oxidative stress parameters, such as lipid peroxidation, electrolyte leakage, and H(2)O(2) levels, were higher in non-transformed plants than in transgenic lines, suggesting that, at the least, overexpression of cytapx protects tobacco membranes from water stress. In these conditions, the activity of other antioxidant enzymes was induced in transgenic lines at the subcellular level. Moreover, an increase in the activity of some antioxidant enzymes was also observed in the chloroplast of transgenic plants overexpressing cytsod and/or cytapx. These results suggest the positive influence of cytosolic antioxidant metabolism on the chloroplast and underline the complexity of the regulation network of plant antioxidant defences during drought stress.     

Enhanced tolerance to oxidative stress in transgenic tobacco plants expressing three antioxidant enzymes in chloroplasts

The effect of simultaneous expression of genes encoding three antioxidant enzymes, copper zinc superoxide dismutase (CuZnSOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), and dehydroascorbate (DHA) reductase (DHAR, EC 1.8.5.1), in the chloroplasts of tobacco plants was investigated under oxidative stress conditions. In previous studies, transgenic tobacco plants expressing both CuZnSOD and APX in chloroplast (CA plants), or DHAR in chloroplast showed enhanced tolerance to oxidative stresses, such as paraquat and salt. In this study, in order to develop transgenic plants that were more resistant to oxidative stress, we introduced the gene encoding DHAR into CA transgenic plants. Mature leaves of transgenic plants expressing all three antioxidant genes (CAD plants) had approximately 1.6–2.1 times higher DHAR activity, and higher ratios of reduced ascorbate (AsA) to DHA, and oxidized glutathione (GSSG) to reduced glutathione (GSH) compared to CA plants. CAD plants were more resistant to paraquat-induced stress, exhibiting only 18.1% reduction in membrane damage relative to CA plants. In addition, seedlings of CAD plants had enhanced tolerance to NaCI (100 mM) compared to CA plants. These results indicate that the simultaneous expression of multiple antioxidant enzymes, such as CuZnSOD, APX, and DHAR, in chloroplasts is more effective than single or double expression for developing transgenic plants with enhanced tolerance to multiple environmental stresses.     

Inflammatory response and glutathione peroxidase in a model of stroke

Stroke is one of the leading causes of death in major industrial countries. Many factors contribute to the cellular damage resulting from ischemia/reperfusion (I/R). Experimental data indicate an important role for oxidative stress and the inflammatory cascade during I/R. We are testing the hypothesis that the mechanism of protection against I/R damage observed in transgenic mice overexpressing human antioxidant enzymes (particularly intracellular glutathione peroxidase) involves the modulation of inflammatory response as well as reduced sensitivity of neurons to cytotoxic cytokines. Transgenic animals show significant reduction of expression of chemokines, IL-6, and cell death-inducing ligands as well as corresponding receptors in a focal cerebral I/R model. Reduction of DNA binding activity of consensus and potential AP-1 binding sites in mouse Fas ligand promoter sequence was observed in nuclear extracts from transgenic mice overexpressing intracellular glutathione peroxidase compared with normal animals following I/R. This effect was accompanied by modulation of the c-Jun N-terminal kinase/stress-activated protein kinase pathway. Cultured primary neurons from the transgenic mice demonstrated protection against hypoxia/reoxygenation injury as well as cytotoxicity after TNF-alpha and Fas ligand treatment. These results indicate that glutathione peroxidase-sensitive reactive oxygen species play an important role in regulation of cell death during cerebral I/R by modulating intrinsic neuronal sensitivity as well as brain inflammatory reactions.