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.     

Simulating ozone detoxification in the leaf apoplast through the direct reaction with ascorbate

 This paper presents a mathematical model which enables the semi-quantification of ozone (O₃) detoxification, based upon the direct reaction of the pollutant with ascorbate (ASC) located in the aqueous matrix associated with the cell wall (i.e. the apoplast). The model describes the uptake of ozone into the leaf and its direct reaction with ASC, taking into consideration the regeneration of dehydroascorbic acid in the cytosol, the rate of replenishment of cell wall ASC and the distribution of ASC between sub-cellular compartments – based upon the permeability of biomembranes to the neutral species, ascorbic acid and the pH of various sub-cellular compartments. The importance of various physico-chemical characteristics (e.g. stomatal conductance, mesophyll cell wall thickness and tortuosity, chloroplast volume, apoplast pH, ASC:O₃ reaction stoichiometry) in mediating the flux of ozone to the plasmalemma is analysed. Model simulations, supported by experimental observations, suggest that the ASC concentration in the leaf apoplast is high enough to scavenge a significant proportion of the O₃ taken up into the leaf interior, under environmentally relevant conditions. However, there is considerable variation between taxa in the potential degree of protection afforded by apoplastic ASC, emphasizing the need for an improved understanding of the reaction chemistry of O₃ in the cell wall. Received: 13 May 1999 / Accepted: 5 August 1999     

Tolerance to atmospheric ozone in transgenic tobacco over‐expressing glutathione synthetase in plastids

A cross between transgenic tobacco ( Nicotiana tabacum L.) plants which over‐expressed either γ‐glutamylcysteine synthetase (cpGSHI) or glutathione synthetase (cpGSHII) in their chloroplasts was used to compare the consequences of over‐expression of components of the glutathione synthetic pathway on tolerance to atmospheric O₃ or paraquat. A high proportion (50%) of those progeny which carried the cpGSHII transgene alone showed tolerance to atmospheric O₃ but not to paraquat. Progeny of an additional two, independent, self‐pollinated primary transgenic lines, which segregated in a Mendelian fashion for the presence of the cpGSHII transgene and therefore included both transformed and non‐transformed (recessive, wild‐type) plants, were also challenged by fumigation with O₃. Again, in both cases, about 50% of the plants expressing the epGSHII transgene were found to be O₃‐tolerant on the basis of reduced ethylene emissions and increased or unchanged total pigment concentrations. However, this tolerance was not due to specific changes in stomatal densities.     

Oxidative stress responses in transgenic tobacco containing altered levels of glutathione reductase activity

A pea glutathione reductase cDNA was expressed in tobacco. Three classes of construct were used which gave a range of elevated levels of glutathione reductase (GR) activity in the cytosol (GR32), chloroplasts (GR36), or in both chloroplasts and mitochondria (GR46). In some transgenic progeny (T₂) from self-fertilized GR32 and GR36 primary transformants, having approximately twofold elevation of GR activity as compared with recessive siblings, there was an amelioration of the effect on leaf discs of up to 15 µM paraquat. However, lines with similarly elevated levels of GR activity showed no decreased sensitivity to the herbicide. None of the GR32 and GR36 lines was less sensitive to ozone. Conversely, T₂ progeny of GR46 lines, with greater than 4.5-fold elevations of GR activity, showed no reduced sensitivity to paraquat but two out of four of these lines were less sensitive to ozone fumigation. The differential response to stress co-segregated with the presence of the transgene but there was no relationship between the degree of stress response and the level of GR activity. There was an elevation in the total glutathione pool in all lines showing increased GR activity but there was no change in the ratio of oxidized to reduced glutathione. These results demonstrate that the mechanisms of protection against ozone and paraquat are different although both can be mediated by elevated GR activity.     

Transgenic tobacco plants expressing antisense RNA for cytosolic ascorbate peroxidase show increased susceptibility to ozone injury

Ascorbate peroxidase (APX), as a participant in the ascorbate—glutathione cycle, has been suggested to be a particularly important antioxidant enzyme in helping plants survive oxidative stress, but direct evidence for this has not been reported. We demonstrate that expressing antisense RNA comprising 45% of the 3'-coding region of the tobacco cytosolic APX, can reduce significantly both the endogenous APX mRNA levels and the APX catalytic activity in transgenic tobacco plants. Those transgenic plants showing a reduction in both endogenous APX mRNA levels and extractable APX activity display a significant increase in ozone injury following high-level ozone exposure. Lower-level ozone exposure reveals even more drastic differences between the antisense and control plants, suggesting that even a partial loss of APX function in oxidative defence cannot be fully compensated for by other antioxidant measures.     

Overexpression of the acerola (Malpighia glabra) monodehydroascorbate reductase gene in transgenic tobacco plants results in increased ascorbate levels and enhanced tolerance to salt stress

Monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) is a key enzyme of the ascorbate (AsA)-glutathione cycle that maintains reduced pools of AsA and serves as an important antioxidative enzyme. Previously, we have cloned MDHAR cDNA from acerola (Malpighia glabra), a plant that accumulates abundant amount of AsA. In this study, MDHAR cDNA from acerola was introduced into tobacco plants using an Agrobacterium-mediated gene delivery system. Transgenic tobacco plants accumulated greater amounts of AsA and showed higher MDHAR activity than the control plants. Lipid peroxidation and chlorophyll degradation, which were stimulated in control plants, were restrained in transgenic plants subjected to salt stress. These results indicate that overexpression of acerola MDHAR imparts greater tolerance to salt stress.     

Over-expression of ascorbate peroxidase in tobacco chloroplasts enhances the tolerance to salt stress and water deficit

The role of APX (ascorbate peroxidase) in protection against oxidative stress was examined using transgenic tobacco plants. The full length cDNA, coding Arabidopsis thaliana L. APX fused downstream to the chloroplast transit sequence from A. thaliana glutathione reductase, was cloned into appropriate binary vector and mobilized into Agrobacterium tumefaciens C58C2. Leaf discs were infected with the Agrobacterium and cultured on medium supplied with kanamycin. The incorporation of the gene in tobacco genome was confirmed by Southern dot blot hybridization. Transgenic lines were generated, and the line Chl-APX5 shown to have 3.8-fold the level of APX activity in the wild-type plants. The isolated chloroplasts from this line showed higher APX activity. During early investigation, this line showed enhanced tolerance to the active oxygen-generating paraquat and sodium sulphite. The first generation of this line, also, showed enhanced tolerance to salt, PEG and water stresses, as determined by net photosynthesis. The present data indicate that overproducing the cytosolic APX in tobacco chloroplasts reduces the toxicity of H₂O₂.     

The overexpression of an alternative oxidase gene triggers ozone sensitivity in tobacco plants

The alternative oxidase (AOX) of plant mitochondria transfers electrons from the ubiquinione pool to oxygen without energy conservation and prevents the formation of reactive oxygen species (ROS) when the ubiquinone pool is over-reduced. Thus, AOX may be involved in plant acclimation to a number of oxidative stresses. To test this hypothesis, we exposed wild-type (WT) Xanthi tobacco plants as well as Xanthi plants transformed with the Bright Yellow tobacco AOX1a cDNA with enhanced (SN21 and SN29), and decreased (SN10) AOX capacity to an acute ozone (O3) fumigation. As a result of 5 h of O3 exposition (250 nL L(-1)), SN21 and SN29 plants surprisingly showed localized leaf damage, whereas SN10, similarly to WT plants, was undamaged. In keeping with this observation, WT and SN21 plants differed in their response to O3)for the expression profiles of catalase 1 (CAT1), catalase 2 (CAT2), glutathione peroxidase (GPX) and ascorbate peroxidase (APX) genes, and for the activity of these antioxidant enzymes, which were induced in WT. Concomitantly, although ozone induced H2O2 accumulation in WT and in all transgenic lines, only in transgenics with high AOX capacity the H2O2 level in the post-fumigation period was high. The alternative pathway of WT plants was strongly stimulated by O3, whereas in SN21 plants, the respiratory capacity was always high across the treatment. The present results show that, far from exerting a protective role, the overexpression of AOX triggers an increased O3 sensitivity in tobacco plants. We hypothesize that the AOX overexpression results in a decrease of mitochondrial ROS level that in turn alters the defensive mitochondrial to nucleus signalling pathway that activates ROS scavenging systems.     

Effects of exogenous hydrogen sulfide on the redox states of ascorbate and glutathione in maize leaves under salt stress

This study investigated the effects of exogenous hydrogen sulfide (H₂S) on the redox states of ascorbate (AsA) and glutathione (GSH) in maize leaves under NaCl (100 mM) stress. Salt stress increased the activities of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), Γ-glutamylcysteine synthetase (Γ-ECS), and L-galactono-1,4-lactone dehydrogenase (GalLDH), malondialdehyde content and electrolyte leakage, and reduced the ratios of reduced and oxidised forms of AsA (AsA/DHA) and GSH (GSH/GSSG) compared with control. Pretreatment with NaHS (H₂S donor) further enhanced the activities of the above enzymes except MDHAR and ameliorated the decrease in the ratios of AsA/DHA and GSH/GSSG compared with the salt stress alone. Pretreatment with NaHS significantly reduced the malondialdehyde content and electrolyte leakage induced by the salt stress. Pretreatment with NaHS alone did not affect any of the above mentioned parameters compared with the control. Our results suggest that exogenous H2S could maintain the redox states of ascorbate and glutathione by up-regulating the ascorbate and glutathione metabolism and thus play an important role for acquisition of salt stress tolerance in maize.     

cDNA cloning and gene expression of ascorbate oxidase in tobacco

A cDNA clone for ascorbate oxidase (AAO) has been isolated from a cDNA library of tobacco (Nicotiana tabacum) cells. The identity of the amino acid sequence deduced from tobacco AAO cDNA to that from pumpkin AAO cDNA was 68%, which was much lower than the identity (80%) between pumpkin and cucumber AAO. AAO activity in tobacco cells was much lower than that in pumpkin cells, whereas the immunoreactive protein in tobacco cells was more abundant than that in pumpkin cells. We suppose that AAO protein in tobacco cells may be less active than that in pumpkin cells. Genomic Southern blotting suggested that AAO in tobacco was encoded by a single-copy gene. Northern blotting revealed that mRNA of AAO was highly expressed in young and growing tissues of tobacco plant.     

Over-expression of a barley aquaporin increased the shoot/root ratio and raised salt sensitivity in transgenic rice plants

Barley HvPIP2;1 is a plasma membrane aquaporin and its expression was down-regulated after salt stress in barley [Katsuhara et al. (2002) Plant Cell Physiol. 43: 885]. We produced and analyzed transgenic rice plants over-expressing barley HvPIP2;1 in the present study. Over-expression of HvPIP2;1 increased (1) radial hydraulic conductivity of roots (Lp(r)) to 140%, and (2) the mass ratio of shoot to root up to 150%. In these transgenic rice plants under salt stress of 100 mM NaCl, growth reduction was greater than in non-transgenic plants. A decrease in shoot water content (from 79% to 61%) and reduction of root mass or shoot mass (both less than 40% of non-stressed plants) were observed in transgenic plants under salt stress for 2 weeks. These results indicated that over-expression of HvPIP2;1 makes rice plants sensitive to 100 mM NaCl. The possible involvement of aquaporins in salt tolerance is discussed.     

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.     

Expression of Arabidopsis CAX1 in tobacco: altered calcium homeostasis and increased stress sensitivity

Calcium (Ca(2)+) efflux from the cytosol modulates Ca(2+) concentrations in the cytosol, loads Ca(2+) into intracellular compartments, and supplies Ca(2+) to organelles to support biochemical functions. The Ca(2+)/H(+) antiporter CAX1 (for CALCIUM EXCHANGER 1) of Arabidopsis is thought to be a key mediator of these processes. To clarify the regulation of CAX1, we examined CAX1 RNA expression in response to various stimuli. CAX1 was highly expressed in response to exogenous Ca(2+). Transgenic tobacco plants expressing CAX1 displayed symptoms of Ca(2+) deficiencies, including hypersensitivity to ion imbalances, such as increased magnesium and potassium concentrations, and to cold shock, but increasing the Ca(2+) in the media abrogated these sensitivities. Tobacco plants expressing CAX1 also demonstrated increased Ca(2+) accumulation and altered activity of the tonoplast-enriched Ca(2+)/H(+) antiporter. These results emphasize that regulated expression of Ca(2+)/H(+) antiport activity is critical for normal growth and adaptation to certain stresses.     

Expansion of transgenic tobacco protoplasts expressing pumpkin ascorbate oxidase is more rapid than that of wild-type protoplasts

 When pumpkin (Cucurbita spp., cv. Ebisu Nankin) ascorbate oxidase cDNA was introduced into cultured cells of tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow No. 2) by Agrobacterium-mediated transformation, the transgenic cells expressed and secreted the recombinant pumpkin ascorbate oxidase into the culture medium. These transgenic cells showed no morphological difference from wild-type cells. However, in the presence of applied hormones protoplasts prepared from the transgenic cells elongated more rapidly than those of wild-type cells. We propose that ascorbate oxidase may play a key role in the regulation of cell expansion perhaps by controlling transport processes through the plasma membrane, but not by affecting the cell wall. Received: 28 October 1999 / Accepted: 18 January 2000     

Enhanced sensitivity to higher ozone in a pathogen-resistant tobacco cultivar

Investigations of the effects of elevated ozone (O(3)) on the virus-plant system were conducted to inform virus pathogen management strategies better. One susceptible cultivar of tobacco (Nicotiana tabacum L. cv. Yongding) and a resistant cultivar (Nicotiana tabacum L. cv. Vam) to Potato virus Y petiole necrosis strain (PVY(N)) infection were grown in open-top chambers under ambient and elevated O(3) concentrations. Above-ground biomass, foliage chlorophyll, nitrogen and total non-structural carbohydrate (TNCs), soluble protein, total amino acid (TAA) and nicotine content, and peroxidase (POD) activity were measured to estimate the effects of elevated O(3) on the impact of PVY(N) in the two cultivars. Results showed that under ambient O(3), the resistant cultivar possessed greater biomass and a lower C/N ratio after infection than the susceptible cultivar; however, under elevated O(3), the resistant cultivar lost its biomass advantage but maintained a lower C/N ratio. Variation of foliar POD activity could be explained as a resistance cost which was significantly correlated with biomass and C/N ratio of the tobacco cultivar. Chlorophyll content remained steady in the resistant cultivar but decreased significantly in the susceptible cultivar when stressors were applied. Foliar soluble protein and free amino acid content, which were related to resistance cost changes, are also discussed. This study indicated that a virus-resistant tobacco cultivar showed increased sensitivity to elevated O(3) compared to a virus-sensitive cultivar.     

Effects of ozone on wild type and transgenic tobacco

Tocopherol cyclase (TC, encoded by gene VTE1) catalyzes the penultimate step of tocopherol synthesis. In this study we used wild type and transgenic tobacco plants overexpressing VTE1 from Arabidopsis to examine the role of tocopherol in ozone sensitivity. Wild type plants responded to an 4-h exposure to 300 nmol mol⁻¹ ozone by severe leaf necrosis while the transgenic lines exhibited limited injury. Compared with the wild type, VTE1-overexpressing plants had lower increase in hydrogen peroxide, malondialdehyde contents and ion leakage, and lower decrease of net photosynthetic rate 48 h following the ozone exposure. Transgenic plants also better maintained the structural integrity of the photosynthetic apparatus.     

Changes in ascorbate oxidase gene expression and ascorbate levels in cell division and cell elongation in tobacco cells

The biological function of ascorbate oxidase (AAO) was not yet clarified, although it was suggested that AAO may be involved in cell growth. We investigated AAO expression and ascorbate metabolism during non-synchronous, synchronous, and elongation cultures of tobacco BY-2 cells. In non-synchronous culture, AAO mRNA was abundant in logarithmic growth phase. Ascorbate content greatly increased during the growth, whereas dehydroascorbate content was slightly increased. In synchronous division culture, AAO mRNA was detected in all phases, but the levels were quite low in G1 phase. Ascorbate content was high in all phases, whereas dehydroascorbate content was low, especially in G1 phase. In elongation culture, the levels of AAO mRNA increased during elongation of the cells. AAO activity in the culture medium, as well as ascorbate and dehydroascorbate contents in the cells, also increased during the elongation. We propose that AAO expression and production of dehydroascorbate are under the control of the cell cycle and that AAO may function apoplastically as an ascorbate oxidizer in the process of cell elongation.     

The biological functions of glutathione revisited in arabidopsis transgenic plants with altered glutathione levels

A functional analysis of the role of glutathione in protecting plants from environmental stress was undertaken by studying Arabidopsis that had been genetically modified to have altered glutathione levels. The steady-state glutathione concentration in Arabidopsis plants was modified by expressing the cDNA for gamma-glutamyl-cysteine synthetase (GSH1) in both the sense and antisense orientation. The resulting plants had glutathione levels that ranged between 3% and 200% of the level in wild-type plants. Arabidopsis plants with low glutathione levels were hypersensitive to Cd due to the limited capacity of these plants to make phytochelatins. Plants with the lowest levels of reduced glutathione (10% of wild type) were sensitive to as little as 5 microM Cd, whereas those with 50% wild-type levels required higher Cd concentrations to inhibit growth. Elevating glutathione levels did not increase metal resistance. It is interesting that the plants with low glutathione levels were also less able to accumulate anthocyanins supporting a role for glutathione S-transferases for anthocyanin formation or for the vacuolar localization and therefore accumulation of these compounds. Plants with less than 5% of wild-type glutathione levels were smaller and more sensitive to environmental stress but otherwise grew normally.