转铜/锌超氧化物歧化酶和抗坏血酸过氧化物酶基因马铃薯的耐氧化和耐盐性研究

高盐等逆境可以加剧植物体内活性氧的产生,进而引起植物细胞死亡。为开发抗逆境作物,以置于氧化诱导型启动子下定位于叶绿体的转铜/锌超氧化物歧化酶（Cu/ZnSOD）和抗坏血酸过氧化物酶基因(APX)马铃薯为材料,研究了其对MV和 NaCl所引起的氧化胁迫的耐受性。结果表明, MV胁迫下,转基因马铃薯叶片膜的相对电导率明显低于对照; NaCl胁迫下,其叶绿素含量高于对照。 在含NaCl 的培养基上,转基因幼苗生根率明显大于对照。另外,NaCl胁迫下转基因马铃薯叶片的SOD和APX酶活性显著高于对照,与其耐盐性的提高相一致。这些研究表明,转入Cu/ZnSOD和APX基因的马铃薯清除活性氧的能力增强,抗逆性得到提高。本实验采用氧化诱导型启动子调控下的SOD和APX两个基因协同作用,使外源基因只有在逆境胁迫时才特异性表达,增强转基因植株的抗逆效果,为培育抗逆经济作物开阔了思路。     

Enhanced tolerance of transgenic sweetpotato plants that express both CuZnSOD and APX in chloroplasts to methyl viologen-mediated oxidative stress and chilling

Oxidative stress is one of the major factors causing injury to plants exposed to environmental stress. Transgenic sweetpotato [Ipomoea batatas (L.) Lam. cv. Yulmi] plants with an enhanced tolerance to multiple environmental stresses were developed by expressing the genes of both CuZn superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) under the control of an oxidative stress-inducible SWPA2 promoter in the chloroplasts of sweetpotato plants (referred to as SSA plants). SSA plants were successfully generated by the particle bombardment method and confirmed by PCR analysis. When leaf discs of SSA plants were subjected to 5 μM methyl viologen (MV), they showed approximately 45% less damage than non-transformed (NT) plants. When 200 μM MV was sprayed onto the whole plants, SSA plants showed a significant reduction in visible damage compared to leaves of NT plants, which were almost destroyed. The expression of the introduced CuZnSOD and APX genes in leaves of SSA plants following MV treatment was significantly induced, thereby reflecting increased levels of SOD and APX in the chloroplasts. APX activity in chloroplast fractions isolated from SSA plants was approximately 15-fold higher than that in their counterparts from NT plants. SSA plants treated with a chilling stress consisting of 4°C for 24 h exhibited an attenuated decrease in photosynthetic activity (Fv/Fm) relative to NT plants; furthermore, after 12 h of recovery following chilling, the Fv/Fm of SSA plants almost fully recovered to the initial levels, whereas NT plants remained at a lower level of Fv/Fm activity. These results suggest that SSA plants would be a useful plant crop for commercial cultivation under unfavorable growth conditions. In addition, the manipulation of the antioxidative mechanism in chloroplasts can be applied to the development of various other transgenic crops with an increased tolerance to multiple environmental stresses.     

Overexpression of Superoxide Dismutase Protects Plants from Oxidative Stress (Induction of Ascorbate Peroxidase in Superoxide Dismutase-Overexpressing Plants)

Photosynthesis of leaf discs from transgenic tobacco plants (Nicotiana tabacum) that express a chimeric gene that encodes chloroplast-localized Cu/Zn superoxide dismutase (SOD+) was protected from oxidative stress caused by exposure to high light intensity and low temperature. Under the same conditions, leaf discs of plants that did not express the pea SOD isoform (SOD-) had substantially lower photosynthetic rates. Young plants of both genotypes were more sensitive to oxidative stress than mature plants, but SOD+ plants retained higher photosynthetic rates than SOD- plants at all developmental stages tested. Not surprisingly, SOD+ plants had approximately 3-fold higher SOD specific activity than SOD- plants. However, SOD+ plants also exhibited a 3- to 4-fold increase in ascorbate peroxidase (APX) specific activity and had a corresponding increase in levels of APX mRNA. Dehydroascorbate reductase and glutathione reductase specific activities were the same in both SOD+ and SOD- plants. These results indicate that transgenic tobacco plants that overexpress pea Cu/Zn SOD II can compensate for the increased levels of SOD with increased expression of the H2O2-scavenging enzyme APX. Therefore, the enhancement of the active oxygen-scavenging system that leads to increased oxidative stress protection in SOD+ plants could result not only from increased SOD levels but from the combined increases in SOD and APX activity.     

Enhanced tolerance to methyl viologen‐induced oxidative stress and high temperature in transgenic potato plants overexpressing the CuZnSOD,APX and NDPK2 genes

Oxidative stress is a major threat for plants exposed to various environmental stresses. Previous studies found that transgenic potato plants expressing both copper zinc superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) (referred to as SSA plants), or nucleoside diphosphate kinase 2 (NDPK2) (SN plants), showed enhanced tolerance to methyl viologen (MV)‐induced oxidative stress and high temperature. This study aimed to develop transgenic plants that were more tolerant of oxidative stress by introducing the NDPK2 gene into SSA potato plants under the control of an oxidative stress‐inducible peroxidase (SWPA2) promoter to create SSAN plants. SSAN leaf discs and whole plants showed enhanced tolerance to MV, as compared to SSA, SN or non‐transgenic (NT) plants. SSAN plants sprayed with 400 µM MV exhibited about 53 and 83% less visible damage than did SSA and SN plants, respectively. The expression levels of the CuZnSOD, APX and NDPK2 genes in SSAN plants following MV treatment correlated well with MV tolerance. SOD, APX, NDPK and catalase antioxidant enzyme activities were also increased in MV‐treated SSAN plants. In addition, SSAN plants were more tolerant to high temperature stress at 42°C, exhibiting a 6.2% reduction in photosynthetic activity as compared to plants grown at 25°C. In contrast, the photosynthetic activities of SN and SSA plants decreased by 50 and 18%, respectively. These results indicate that the simultaneous overexpression of CuZnSOD, APX and NDPK2 is more effective than single or double transgene expression for developing plants with enhanced tolerance to various environmental stresses.     

Enhanced stress-tolerance of transgenic tobacco plants expressing a human dehydroascorbate reductase gene

To analyze the physiological role of dehydroascorbate reductase (DHAR, EC 1.8.5.1) catalyzing the reduction of DHA to ascorbate in environmental stress adaptation, T1 transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants expressing a human DHAR gene in chloroplasts were biochemically characterized and tested for responses to various stresses. Fully expanded leaves of transgenic plants had about 2.29 times higher DHAR activity (units/g fresh wt) than non-transgenic (NT) plants. Interestingly, transgenic plants also showed a 1.43 times higher glutathione reductase activity than NT plants. As a result, the ratio of AsA/DHA was changed from 0.21 to 0.48, even though total ascorbate content was not significantly changed. When tobacco leaf discs were subjected to methyl viologen (MV) at 5 mumol/L and hydrogen peroxide (H2O2) at 200 mmol/L, transgenic plants showed about a 40% and 25% reduction in membrane damage relative to NT plants, respectively. Furthermore, transgenic seedlings showed enhanced tolerance to low temperature (15 degrees C) and NaCl (100 mmol/L) compared to NT plants. These results suggest that a human derived DHAR properly works for the protection against oxidative stress in plants.     

Stress-induced expression of choline oxidase in potato plant chloroplasts confers enhanced tolerance to oxidative, salt, and drought stresses

Transgenic potato plants (Solanum tuberosum L. cv. Superior) with the ability to synthesize glycinebetaine (GB) in chloroplasts (referred to as SC plants) were developed via the introduction of the bacterial choline oxidase (codA) gene under the control of an oxidative stress-inducible SWPA2 promoter. SC1 and SC2 plants were selected via the evaluation of methyl viologen (MV)-mediated oxidative stress tolerance, using leaf discs for further characterization. The GB contents in the leaves of SC1 and SC2 plants following MV treatment were found to be 0.9 and 1.43 μmol/g fresh weight by HPLC analysis, respectively. In addition to reduced membrane damage after oxidative stress, the SC plants evidenced enhanced tolerance to NaCl and drought stress on the whole plant level. When the SC plants were subjected to two weeks of 150 mM NaCl stress, the photosynthetic activity of the SC1 and SC2 plants was attenuated by 38 and 27%, respectively, whereas that of non-transgenic (NT) plants was decreased by 58%. Under drought stress conditions, the SC plants maintained higher water contents and accumulated higher levels of vegetative biomass than was observed in the NT plants. These results indicate that stress-induced GB production in the chloroplasts of GB non-accumulating plants may prove useful in the development of industrial transgenic plants with increased tolerance to a variety of environmental stresses for sustainable agriculture applications.     

Overexpression of 2-cysteine peroxiredoxin enhances tolerance to methyl viologen-mediated oxidative stress and high temperature in potato plants

Oxidative stress is one of the major causative factors for injury to plants exposed to environmental stresses. Plants have developed diverse defense mechanisms for scavenging oxidative stress-inducing molecules. The antioxidative enzyme 2-cysteine peroxiredoxin (2-Cys Prx) removes peroxides and protects the photosynthetic membrane from oxidative damage. In this study, transgenic potato (Solanum tuberosum L. cv. Atlantic) expressing At2-Cys Prx under control of the oxidative stress-inducible SWPA2 promoter or enhanced CaMV 35S promoter (referred to as SP and EP plants, respectively) was generated using Agrobacterium-mediated transformation. The transgenic plants were tested for tolerance to stress. Following treatment with 3 μM methyl viologen (MV), leaf discs from SP and EP plants showed approximately 33 and 15% less damage than non-transformed (NT) plants. When 300 μM MV was sprayed onto whole plants, the photosynthetic activity of SP plants decreased by 25%, whereas that of NT plants decreased by 60%. In addition, SP plants showed enhanced tolerance to high temperature at 42 °C. After treatment at high temperature, the photosynthetic activity of SP plants decreased by about 7% compared to plants grown at 25 °C, whereas it declined by 31% in NT plants. These results indicate that transgenic potato can efficiently regulate oxidative stress from various environmental stresses via overexpression of At2-Cys Prx under control of the stress-inducible SWPA2 promoter.     

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.     

Factors Affecting the Enhancement of Oxidative Stress Tolerance in Transgenic Tobacco Overexpressing Manganese Superoxide Dismutase in the Chloroplasts

Two varieties of tobacco (Nicotiana tabacum var PBD6 and var SR1) were used to generate transgenic lines overexpressing Mn-superoxide dismutase (MnSOD) in the chloroplasts. The overexpressed MnSOD suppresses the activity of those SODs (endogenous MnSOD and chloroplastic and cytosolic Cu/ZnSOD) that are prominent in young leaves but disappear largely or completely during aging of the leaves. The transgenic and control plants were grown at different light intensities and were then assayed for oxygen radical stress tolerance in leaf disc assays and for abundance of antioxidant enzymes and substrates in leaves. Transgenic plants had an enhanced resistance to methylviologen (MV), compared with control plants, only after growth at high light intensities. In both varieties the activities of FeSOD, ascorbate peroxidase, dehydroascorbate reductase, and monodehydroascorbate reductase and the concentrations of glutathione and ascorbate (all expressed on a chlorophyll basis) increased with increasing light intensity during growth. Most of these components were correlated with MV tolerance. It is argued that SOD overexpression leads to enhancement of the tolerance to MV-dependent oxidative stress only if one or more of these components is also present at high levels. Furthermore, the results suggest that in var SR1 the overexpressed MnSOD enhances primarily the stromal antioxidant system.     

Response of antioxidative enzymes to plum pox virus in two apricot cultivars

Recent evidence has indicated that activated oxygen species (AOS) may function as molecular signals in the induction of defence genes. In the present work, the response of antioxidative enzymes to the plum pox virus (PPV) was examined in two apricot (Prunus armeniaca L.) cultivars, which behaved differently against PPV infection. In the inoculated resistant cultivar (Goldrich), a decrease in catalase (CAT) as well as an increase in total superoxide dismutase (SOD) and dehydroascorbate reductase (DHAR) activities were observed. Ascorbate peroxidase (APX), glutathione reductase (GR) and monodehydroascorbate reductase (MDHAR) did not change significantly in relation to non-inoculated (control) plants. In the susceptible cultivar (Real Fino), inoculation with PPV brought about a decrease in CAT, SOD and GR, whereas a rise in APX, MDHAR and DHAR activities was found in comparison to non-inoculated (control) plants. Apricot leaves contain only CuZn-SOD isozymes, which responded differently to PPV depending on the cultivar. Goldrich leaves contained 6 SODs and both SOD 1 and SOD 2 increased in the inoculated plants. In leaves from Real Fino, 5 SODs were detected and only SOD 5 was increased in inoculated plants. The different behaviour of SODs (H2O2-generating enzymes) and APX (an H2O2-remover enzyme) in both cultivars suggests an important role for H2O2 in the response to PPV of the resistant cultivar, in which no change in APX activity was observed. This result also points to further studies in order to determine if an alternative H2O2-scavenging mechanism takes place in the resistant apricot cultivar exposed to PPV. On the other hand, the ability of the inoculated resistant cultivar to induce SOD 1 and SOD 2 as well as the important increase of DHAR seems to suggest a relationship between these activities and resistance to PPV. This is the first report about the effect of PPV infection on the antioxidative enzymes of apricot plants. It opens the way for the further studies, which are necessary for a better understanding of the role of antioxidative processes in viral infection by PPV in apricot plants.     

Enhanced tolerance to sulfur dioxide and salt stress of transgenic Chinese cabbage plants expressing both superoxide dismutase and catalase in chloroplasts.

To explore the possibility of overcoming the highly phytotoxic effect of SO(2) and salt stress, we introduced the maize Cu/ZnSOD and/or CAT genes into chloroplasts of Chinese cabbage (Brassica campestris L. ssp. pekinensis cv. Tropical Pride) (referred to as SOD, CAT and SOD+CAT plants). SOD+CAT plants showed enhanced tolerance to 400 ppb SO(2), and visible damage was one-sixth that of wild-type (CK) plants. In addition, when SOD+CAT plants were exposed to a high salt treatment of 200 mM NaCl for 4 weeks, the photosynthetic activity of the plants decreased by only 6%, whereas that of CK plants decreased by 72%. SOD plants had higher total APX and GR activities than CK plants. As expected, SOD plants showed levels of protection from SO(2) and salt stress that were moderately improved compared to CK plants. However, CAT plants showed inhibition of APX activity and provided only limited improvements in plant stress tolerance. Moreover, SOD+CAT plants accumulated more K(+), Ca(2+) and Mg(2+) and less Na(+) in their leaves compared with those of CK plants. These results suggest that the expression of SOD and CAT simultaneously is suitable for the introduction of increased multiple stress protection.     

植物中活性氧的产生及清除机制

环境胁迫使植物细胞中积累大量的活性氧,从而导致蛋白质、膜脂、DNA及其它细胞组分的严重损伤。植物体内有效清除活性氧的保护机制分为酶促和非酶促两类。酶促脱毒系统包括超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GPX)等。非酶类抗氧化剂包括抗坏血酸、谷胱甘肽、甘露醇和类黄酮。利用基因工程策略增加这些物质在植物体内的含量,从而获得耐逆转基因植物已取得一定的进展。     

干旱胁迫下转基因甘薯块根膨大期水分利用效率和生理代谢特征

以转Cu/Zn-SOD和APX基因及其非转基因甘薯进行盆栽试验,在甘薯块根膨大期进行正常供水(田间最大持水量的80%)、中度缺水(田间最大持水量的60%)和重度缺水(田间最大持水量的40%)3种水分处理,分别测定转基因植株和对照植株在薯块膨大期的第20天和第70天的抗氧化酶系统、可溶性糖含量、光合系统之间的差异,以及在不同水分胁迫处理下产量和水分利用效率之间的差异。以此研究外源基因的超表达是否可以提高甘薯的产量及水分利用效率。结果显示:(1)转基因甘薯(TS)的SOD、APX活性以及可溶性糖含量均高于非转基因对照株(NT),但POD活性低于NT;TS和NT植株的APX活性、可溶性糖含量、净光合速率以及蒸腾速率均随干旱胁迫加重呈递减趋势。(2)干旱胁迫70d时,TS和NT植株光合参数均较胁迫20d时降低,且TS和NT间的净光合速率没有明显差异。(3)TS和NT两株系的块根产量在中度胁迫下最高而在重度胁迫下最低,而TS具有较高的块根产量且在重度胁迫下产量降低幅度较小。(4)TS的气孔导度和蒸腾速率显著低于NT,且TS的水分利用效率较NT更高。研究表明,Cu/Zn-SOD和APX基因可以显著增加干旱胁迫下甘薯块根膨大期的SOD、APX活性和可溶性糖含量,提高其水分利用效率,从而减轻干旱胁迫对产量的影响。     

Enhanced photochemical light utilization and decreased chilling-induced photoinhibition of photosystem II in cotton overexpressing genes encoding chloroplast-targeted antioxidant enzymes

The aim of this study was to determine whether increases in stromal superoxide dismutase (SOD; EC 1.15.1.1), ascorbate peroxidase (APX; EC 1.11.1.11) and glutathione reductase (GR; EC 1.6.4.2) via transformation could reduce photosystem (PS) II photoinhibition at low temperature for cotton (Gossypium hirsutum L.) plants and to determine by what mechanism this protection may be realized. During 3-h exposures of lincomycin-treated leaf discs to 10 degrees C and a photon flux density of 500 &mgr;mol m-2 s-1, all transgenic plants exhibited significantly greater PSII activity and O2 evolution than did wild-type plants. Also, the rate constant of PSII photoinactivation was significantly lower for all transgenic plants than for wild-type plants. No significant differences existed between genotypes in non-photochemical quenching of chlorophyll a fluorescence and the regulated component of the thermal dissipation of excitation energy. The relationship between changes in variable to maximum chlorophyll fluorescence (Fv/Fm) and the time-dependent averaged excessive light exposure was similar for all genotypes. This observation excluded the possibility that differences in PSII photodamage were due to improvements in the direct protection of PSII from active oxygen by antioxidant enzyme overproduction. Similar decreases in Fv/Fm during the stress treatment for all genotypes when leaves were pre-treated with 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) suggested that the effect of overproduction involved events downstream of PSII in the electron transfer pathway. Since all transgenic plants exhibited a significantly higher photochemical quenching of chlorophyll fluorescence during the chilling treatment, we concluded that, under the conditions used in this study, the enhancement of the protection of PSII from photodamage by increasing the stromal antioxidant enzyme activity in cotton leaves was due to the maintenance of a higher rate of electron transport and, consequently, a lower reduction state of QA.     

Development of selection marker-free transgenic potato plants with enhanced tolerance to oxidative stress

A binary vector devoid of a plant selection-marker gene (designated as pSSA-F) was constructed to overcome bio-safety concerns about genetically modified plants. This vector carried chloroplast-targeted superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes under the control of an oxidative stress-inducible(SWPA2) promoter, and was utilized to transform potato (Solanum tuberosum L.). Integration of these foreign genes into transgenic plants was primarily performed via PCR with genomic DNA. Twelve marker-free transgenic lines were obtained by inoculating stem explants. The maximum transformation efficiency was 6.25% and averaged 2.2%. Successful integration of the SOD and APX genes rendered transgenic plants tolerant to methyl viologen-mediated oxidative stress at the leaf-disc and whole-plant levels. Our findings suggest that this technique for developing selection marker-free transgenic plants is feasible and can be employed with other crop species.     

转铜/锌超氧化物歧化酶和抗坏血酸过氧化物酶基因甘薯的耐旱性

水分胁迫使转铜／锌超氧化物歧化酶基因（Cu／Zn SOD）和抗坏血酸过氧化物酶基因（APX）甘薯及未转基因甘薯中超氧阴离子（O2^-）、过氧化氢（H2O2）、丙二醛（MDA）含量和细胞膜相对透性增加,在相同条件下以上指标均为转基因甘薯低于未转基因甘薯;而叶片含水量、净光合速率（Pn）和气孔导度（Gs）均下降,SOD和APX酶活性随胁迫程度的加重先增大后减小,胞间CO2浓度（Ci）则先减小后增大,在相同条件下转基因甘薯中以上指标均高于未转基因甘薯。这些结果表明：转入Cu／Zn SOD和APX基因使转基因甘薯清除活性氧的能力增强,在水分胁迫下能保持较高的叶片含水量和Pn,耐旱性得到提高。