Resistance to Active Oxygen Toxicity of Transgenic Nicotiana tabacum that Expresses the Gene for Glutathione Reductase from Escherichia coli

A chimeric gene consisting of a gene from Escherichia coli thatencodes glutathione reductase (GR), the 35S promoter of cauliflowermosaic virus and the terminator sequences of the gene for nopalinesynthase, was introduced into tobacco (Nicotiana tabacum SRI)cells via a Ti plasmid vector. Expression of the bacterial genein transformed plants and their descendants was confirmed byimmunochemical analysis. GR activity in leaf extracts variedamong transgenic plants, ranging from about 1.0 to 3.5 timesthe control level. These transgenic plants exhibited lower susceptibilityto paraquat than control plants in terms of the extent of visiblefoliar damage, a result that suggests that GR may play an importantrole in the detoxification of active oxygen in the cytoplasmicmatrix of plant cells. However, the transgenic plants were nomore resistant to ozone than were the controls, both in termsof the extent of visible foliar damage and with respect to photosyntheticactivity. (Received January 28, 1991; Accepted May 9, 1991)     

Enhanced Formation of a Constitutive Sesquiterpenoid in Cultured Cells of a Liverwort, Calypogeia granulata Inoue during Elicitation; Effects of Vanadate

The early cellular responses to elicitation by an abiotic elicitor,vanadate, in suspension-cultured cells of the liverwort Calypogeiagranulata Inoue were investigated. The rate of production of1,4-dimethylazulene, which is a constitutive sesquiterpenoidin intact plants and also in cultured cells of C. granulata,was more than double the control rate after treatment with vanadate.This increase was preceded by doubling of the activity of 3-hydroxy-3-methylglutarylCoA reductase, an enzyme involved in isoprenoid biosynthesis.Activities of superoxide dismutase, ascorbate peroxidase andglutathione reductase increased to 1.5 to 2 times the controlactivities upon treatment with vanadate. Intracellular levelsof glutathione (reduced form) increased to 10 times the controllevel within 24 h after addition of the elicitor. Furthermore,addition of hydrogen peroxide to the culture also enhanced theproduction of 1,4-dimethylazulene to the same extent as vanadate.We propose that treatment with the elicitor resulted in oxidativestress due to generation of active oxygen species which mightact as messengers to activate the biosynthetic pathway to theformation of 1,4-dimethylazulene on the one hand, and to activationof a system for scavenging active oxygen species on the other. (Received June 8, 1992; Accepted January 18, 1993)     

Effects of short-term ozone fumigation on tobacco plants: response of the scavenging system and expression of the glutathione reductase

The role that the constituents of the ascorbate–glutathione cycle play in the mechanism of contrasting ozone sensitivities was examined in mature and old tobacco leaves after acute ozone-fumigation (150 p.p.b., 5 h). Levels of the enzyme activities associated with the detoxifying system were lower in ozone-sensitive Bel W3 control plants than in unfumigated ozone-tolerant Bel B plants. In particular, the endogenous activities of ascorbate peroxidase (APX) and glutathione reductase (GR), and the metabolites ascorbic acid (AA) and reduced glutathione (GSH) were more abundant in Bel B than Bel W3 control plants. These results suggest that the higher tolerance of Bel B to O₃ is associated with a greater initial content of the antioxidant enzymes or metabolites. Only in the mature leaves of the ozone-tolerant Bel B cv. did fumigation trigger activation of APX and, weakly, of dehydroascorbate reductase (DHAR). The activity of these enzymes was significantly lower after ozone treatment in both mature and old leaves of Bel W3 than in control plants. Fumigation had little effect on the ascorbate content. Its main effects on the glutathione pool were that it boosted the oxidized form and lowered the reduced form, particularly in mature Bel W3 leaves. Extractable GR activity remained unchanged in both Bel B and Bel W3 immediately after fumigation, but increased slightly 24 h later, particularly in mature leaves of Bel W3. Exposure to O₃ caused a sharp decline in chloroplastic GR mRNA levels in both cultivars. However, as Western blot analysis failed to detect any major changes in GR protein content at this time, the protein must be highly stable. There is therefore a good correlation between tolerance to O₃ and high endogenous levels of antioxidant metabolites such as AA and GSH in tobacco. In addition, the degree of inducibility of the system discriminates the two cultivars investigated.     

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.     

Effects of Elevated Cytosolic Glutathione Reductase Activity on the Cellular Glutathione Pool and Photosynthesis in Leaves under Normal and Stress Conditions

Tobacco (Nicotiana tabacum var Samsun) was transformed using the bacterial gor gene coding for the enzyme glutathione reductase. Transgenic plants were selected by their kanamycin resistence and expression of the bacterial gor gene. After separation by isoelectric focusing techniques, leaf extracts from transgenic plants having both native and bacterial glutathione reductase activity gave, in addition to the six bands of the native enzyme, two further closely running isoenzymes. These additional bands originating from the expression of the bacterial gor gene were nonchloroplastic. Leaves from transgenic plants had two- to 10-fold higher glutathione reductase activity than non-transgenic controls. The amount of extractable glutathione reductase activity obtained in transgenic plants was dependent on leaf age and the conditions to which leaves were exposed. Both light and exposure to methylviologen increased leaf glutathione reductase activity. Elevated levels of cytosolic glutathione reductase activity in transgenic plants had no effect on the amount or reduction state of the reduced glutathione/oxidized glutathione pool under optimal conditions or oxidative conditions induced by methylviologen. The glutathione pool was unaltered despite the oxidation-dependent loss of CO₂ assimilation and oxidation of enzymes involved in photosynthesis. However, the reduction state of the ascorbate pool was greater in transgenic plants relative to nontransgenic controls following illumination of methylviologen-treated leaf discs. Therefore, we conclude that in the natural state glutathione reductase is present in tobacco at levels above those required for maximal operation of the ascorbate-glutathione pathway.     

Chloroplast development in 4-thiouridine-cultured radish seedlings III. Photochemical activities in isolated plastids

Accumulation of chlorophyll, development of photosystem I andII activities and contents of chloroplastic components wereinvestigated in greening radish seedlings germinated and grownwith 4-thiouridine (4SU). The development of photosystem I activityprior to that of photosystem II was observed also in the 4SU-culturedgreening radish cotyledons in which chlorophyll accumulationwas inhibited up to 60–80% of that of the control. Photochemicalactivities expressed on a plastid protein basis decreased withthe increase of 4SU in the culture medium. In contrast to ferredoxinand ferredoxin-NADP reductase, which were present in significantamounts in the treated cotyledons, chloroplastic cytochromes(f, b₅₅₉ and b₆ decreased in the plastids from 4SU-culturedcotyledons. These results suggest that 4SU interferes in partwith protein synthesis in plastids and thereby with chloroplastdevelopment. (Received December 4, 1979; )     

Cysteine synthase overexpression in tobacco confers tolerance to sulfur-containing environmental pollutants.

Cysteine (Cys) synthase [O-acetyl-L-Ser(thiol)-lyase, EC 4.2.99.8; CSase] is responsible for the final step in biosynthesis of Cys. Transgenic tobacco (Nicotiana tabacum; F(1)) plants with enhanced CSase activities in the cytosol and in the chloroplasts were generated by cross-fertilization of two transformants expressing cytosolic CSase or chloroplastic CSase. The F(1) transgenic plants were highly tolerant to toxic sulfur dioxide and sulfite. Upon fumigation with 0.1 microL L(-1) sulfur dioxide, the Cys and glutathione contents in leaves of F(1) plants were increased significantly, but not in leaves of non-transformed control plants. Furthermore, the leaves of F(1) plants exhibited the increased resistance to paraquat, a herbicide generating active oxygen species.     

Changes in the Activity of the Chloroplastic and Cytosolic Forms of Dihydroxyacetone Phosphate Reductase during Maturation of Leaves

Young or mature rosette leaves from spinach (Spinacia oleracea L.) plants growing in the field, in the greenhouse, or in a growth chamber under a regimen of 8 hours light and 16 hours dark contained 15 to 50 nanomoles per minute per gram wet weight of NADH:dihydroxyacetone phosphate reductase activity. Of this activity, 75 to 87% was the chloroplastic isoform and 25 to 13% was the cytosolic form. When plants were induced to senesce, as measured by stem elongation and flowering, the percentage of the two reductase isoforms in rosette or stem leaves changed to about 12% as the chloroplastic and 88% as the cytosolic isoform. The change in enzyme activity of the rosette leaves occurred within 3 days, before phenotypic changes were observed. Likewise, when plants senesced in continuous darkness, the percentage of chloroplastic to cytosolic reductase changed from 80:20% to 25:75% after 62 hours before changes in total protein or chlorophyll occurred. The ratio of activities did not change in the first 16 hours of darkness or overnight. In each case the change in ratio resulted from about a 75% decrease in activity of the chloroplastic isoform and up to 14-fold increase in cytosolic isoform. In spinach leaves purchased at a local market primarily only the cytosolic isoform remained. When plants were returned to normal day-nights, after 62 hours in continuous darkness, the activity of the chloroplastic isoform increased, but not to control levels after 3 days, while the cytosolic enzyme decreased within 1 day to normal day-night values. Changes in activity were not due to changes during in vitro assays in activation by thioredoxin for the chloroplastic isoform or fructose 2,6-phosphate for the cytosolic isoform.     

拟南芥肌动蛋白解聚因子4基因(AtADF4)在烟草中表达引起植株形态变化

用PCR方法从拟南芥基因组DNA中分离克隆肌动蛋白解聚因子基因（AtADF4）,并进行了序列分析。通过农杆菌介导转化将AtADF4导入烟草,PCR和RT—PCR检测证明AtADF4已整合到烟草基因组中并得到表达。转基因烟草幼苗下胚轴的弯曲度在暗培养与光照培养条件下均比对照的大,暗培养条件下下胚轴弯曲程度较高;下胚轴薄壁细胞壁比对照大,维管束排列不整齐;根毛稀少弯曲,而对照根毛密集且直;转基因烟草开花时间比对照平均延迟了7～8d,花粉萌发时花粉管比对照粗短。     

Drought Induces Oxidative Stress and Enhances the Activities of Antioxidant Enzymes in Growing Rice Seedlings

When rice seedlings grown for 10 and 20 days were subjected to in vitro drought stress of −0.5 and −2.0 MPa for 24 h, an increase in the concentration of superoxide anion (O₂^.−), increased level of lipid peroxidation and a decrease in the concentration of total soluble protein and thiols was observed in stressed seedlings compared to controls. The concentration of H₂O₂ as well as ascorbic acid declined with imposition of drought stress, however glutathione (GSH) concentration declined only under severe drought stress. The activities of total superoxide dismutases (SODs) as well as ascorbate peroxidase (APX) showed consistent increases with increasing levels of drought stress, however catalase activity declined. Mild drought stressed plants had higher guaiacol peroxidase (GPX) and chloroplastic ascorbate peroxidase (c-APX) activity than control grown plants but the activity declined at the higher level of drought stress. The activities of enzymes involved in regeneration of ascorbate i.e. monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) were higher in drought stressed plants compared to controls. Results suggest that drought stress induces oxidative stress in rice plants and that besides SOD, the enzymes of ascorbate-glutathione cycle, which have not been studied in detail earlier under stressful conditions, appear to function as important component of antioxidative defense system under drought stress.     

Expression of arabidopsis cytosolic ascorbate peroxidase gene in response to ozone or sulfur dioxide

The effects of ozone or sulfur dioxide on antioxidant enzymes were investigated in Arabidopsis thaliana. Plants were fumigated with 0.1–0.15 ppm ozone or sulfur dioxide up to about 1 week in an environment-controlled chamber. Both pollutants increased the activities of ascorbate peroxidase and guaiacol per-oxidase in leaves, but had little effect on the activities of superoxide dismutase, catalase, monodehydroascorbate reductase, dehydroascorbate reductase or glutathione reductase. Ozone was more effective than sulfur dioxide in increasing the activities of the peroxidases. Ascorbate peroxidase activity increased 1.8-fold without a lag period during fumigation with 0.1 ppm ozone, while guaiacol peroxidase activity increased 4.4-fold with a 1-day lag. Expression of the APX1 gene encoding cytosolic ascorbate peroxidase was further investigated. Its protein levels in leaves exposed to 0.1 ppm ozone for 4 or 8 days were 1.5-fold higher than in controls. Both ozone and sulfur dioxide elevated APX1 mRNA levels in leaves at 4 and 7 days, whereas at 1 day only ozone was effective. The induction of APX1 mRNA levels by ozone (3.4- to 4.1-fold) was more prominent than that by sulfur dioxide (1.6-to 2.6-fold). The APX1 mRNA level increased by day and decreased by night. Exposure of plants to 0.1 ppm ozone enhanced the APX1 mRNA level within 3 h, which showed a diurnal rhythm similar to that of the control. These results demonstrate that near-ambient concentrations of ozone as well as similar concentrations of sulfur dioxide can induce APX1 gene expression in A. thaliana.Environmental Biology Division     

Enhancement of oxidative stress tolerance in transgenic tobacco plants overproducing Fe-superoxide dismutase in chloroplasts.

A chimeric gene consisting of the coding sequence for chloroplastic Fe superoxide dismutase (FeSOD) from Arabidopsis thaliana, coupled to the chloroplast targeting sequence from the pea ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit, was expressed in Nicotiana tabacum cv Petit Havana SR1. Expression of the transgenic FeSOD protected both the plasmalemma and photosystem II against superoxide generated during illumination of leaf discs impregnated with methyl viologen. By contrast, overproduction of a mitochondrial MnSOD from Nicotiana plumbaginifolia in the chloroplasts of cv SR1 protected only the plasmalemma, but not photosystem II, against methyl viologen (L. Slooten, K. Capiau, W. Van Camp, M. Van Montagu, C. Sybesma, D. Inzé [1995] Plant Physiol 107: 737-750). The difference in effectiveness correlates with different membrane affinities of the transgenic FeSOD and MnSOD. Overproduction of FeSOD does not confer tolerance to H2O2, singlet oxygen, chilling-induced photoinhibition in leaf disc assays, or to salt stress at the whole plant level. In nontransgenic plants, salt stress led to a 2- to 3-fold increase in activity, on a protein basis, of FeSOD, cytosolic and chloroplastic Cu/ZnSOD, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. In FeSOD-overproducing plants under salt stress, the induction of cytosolic and chloroplastic Cu/ZnSOD was suppressed, whereas induction of a water-soluble chloroplastic ascorbate peroxidase isozyme was promoted.     

Effects of ozone and elevated atmospheric carbon dioxide on carbohydrate metabolism of spruce needles. Catabolic and detoxification pathways

We have studied the effects of ozone, carbon dioxide and ozone combined with carbon dioxide fumigations on catabolic and detoxification pathways in spruce ( Picea abies [L.] Karst.) needles. The results obtained showed an increase in the activities of three enzymes involved in the detoxification pathway, superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (AscPOD, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) when trees were exposed to ozone and to ozone‐carbon dioxide treatments. In these two treatments, the fraction of SOD activity due to the chloroplastic isoform was increased (1.5‐fold). In the needles of trees exposed to ozone and to ozone‐carbon dioxide fumigation, an increase in the activities of glucose‐6‐phosphate dehydrogenase (G‐6‐PDH, EC 1.1.1.49) showed that the cell had the capacity to produce more NADPH necessary for the detoxification. Stimulation of other enzymes of catabolic pathways (fumarase [EC 4.2.1.2], phosphofructokinase [PFK, EC 2.7.1.1] and phosphoenolpyruvate carboxylase [PEPC, EC 4.1.1.31]), was also observed making it possible for the cell to provide the reducing power necessary for detoxification as well as energy and carbon skeletons involved in the repair processes.
When carbon dioxide alone was applied, no effects could be detected on these enzyme activities. However, when carbon dioxide was combined with ozone, the effect of ozone on trees was less than that induced by ozone alone, suggesting that elevated atmospheric carbon dioxide concentrations may to some extent protect plants from ozone injury.     

Scavenger Enzyme Activities in Subcellular Fractions of White Clover (Trifolium repens L.) under PEG-induced Water Stress

Scavenger enzyme activities in subcellular fractions under polyethylene glycol (PEG)-induced water stress in white clover (Trifolium repens L.) were studied. Water stress decreased ascorbic acid (AA) content and catalase (CAT) activity and increased the contents of hydrogen peroxide (H₂O₂), thiobarbituric acid reactive substances (TBARS) (measure of lipid peroxidation), and activities of superoxide dismutase (SOD), its various isozymes, ascorbate peroxidase (APOX), and glutathione reductase (GR) in cellular cytosol, chloroplasts, mitochondria, and peroxisomes of Trifolium repens leaves. In both the PEG-treated plants and the control, chloroplastic fractions showed the highest total SOD, APOX, and GR activities, followed by mitochondrial fractions in the case of total SOD and GR activities, whereas cytosolic fractions had the second greatest APOX activity. However, CAT activity was the highest in peroxisomes, followed by the cytosol, mitochondria, and chloroplasts in decreasing order. Although Mn-SOD activity was highest in mitochondrial fractions, residual activity was also observed in cytosolic fractions. Cu/Zn-SOD and Fe-SOD were observed in all subcellular fractions; however, the activities were the highest in chloroplastic fractions for both isoforms. Total Cu/Zn-SOD activity, the sum of activities observed in all fractions, was higher than other SOD isoforms. These results suggest that cytosolic and chloroplastic APOX, chloroplastic and mitochondrial GR, mitochondrial Mn-SOD, cytosolic and chloroplastic Cu/Zn-SOD, and chloroplastic Fe-SOD are the major scavenger enzymes, whereas cellular CAT may play a minor role in scavenging of O₂⁻ and H₂O₂ produced under PEG-induced water stress in Trifolium repens.     

Overexpression of wheat Na+/H+ antiporter TNHX1 and H+-pyrophosphatase TVP1 improve salt- and drought-stress tolerance in Arabidopsis thaliana plants

Transgenic Arabidopsis plants overexpressing the wheat vacuolarNa⁺/H⁺ antiporter TNHX1 and H⁺-PPase TVP1 are much more resistantto high concentrations of NaCl and to water deprivation thanthe wild-type strains. These transgenic plants grow well inthe presence of 200 mM NaCl and also under a water-deprivationregime, while wild-type plants exhibit chlorosis and growthinhibition. Leaf area decreased much more in wild-type thanin transgenic plants subjected to salt or drought stress. Theleaf water potential was less negative for wild-type than fortransgenic plants. This could be due to an enhanced osmoticadjustment in the transgenic plants. Moreover, these transgenicplants accumulate more Na⁺ and K⁺ in their leaf tissue thanthe wild-type plants. The toxic effect of Na⁺ accumulation inthe cytosol is reduced by its sequestration into the vacuole.The rate of water loss under drought or salt stress was higherin wild-type than transgenic plants. Increased vacuolar soluteaccumulation and water retention could confer the phenotypeof salt and drought tolerance of the transgenic plants. Overexpressionof the isolated genes from wheat in Arabidopsis thaliana plantsis worthwhile to elucidate the contribution of these proteinsto the tolerance mechanism to salt and drought. Adopting a similarstrategy could be one way of developing transgenic staple cropswith improved tolerance to these important abiotic stresses. Key words: H⁺-pyrophosphatase, Na⁺/H⁺ antiporter, salt and drought tolerance, sodium sequestration, transgenic Arabidopsis plants