Expression of Arabidopsis NDPK2 increases antioxidant enzyme activities and enhances tolerance to multiple environmental stresses in transgenic sweetpotato plants

Transgenic sweetpotato (Ipomoea batatas L. cv. Yulmi) plants expressing the Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) gene under the control of an oxidative stress–inducible peroxidase (SWPA2) promoter (referred to as SN plants) were developed and evaluated for enhanced tolerance of SN plants under various abiotic stress conditions. The level of AtNDPK2 expression and NDPK activity in SN plants following methyl viologen (MV) treatment was positively correlated with the plant’s tolerance to MV. Interestingly, we observed that antioxidant enzyme activities such as peroxidase, ascorbate peroxidase, and catalase increased in MV-treated SN plants. In addition, SN plants showed enhanced tolerance to cold, high salinity, and drought stresses by an increase in the activity of H₂O₂ scavenging enzymes. These results indicate that overexpression of AtNDPK2 in sweetpotato might efficiently modulate oxidative stress from various environmental stresses.     

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.     

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.     

Enhanced tolerance of transgenic potato plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against oxidative stress and high temperature

Oxidative stress is a major damaging factor for plants exposed to environmental stresses. In order to develop transgenic potato plants with enhanced tolerance to environmental stress, the genes of both Cu/Zn superoxide dismutase and ascorbate peroxidase were expressed in chloroplasts under the control of an oxidative stress-inducible SWPA2 promoter (referred to as SSA plants). SSA plants showed enhanced tolerance to 250 μM methyl viologen, and visible damage in SSA plants was one-fourth that of non-transgenic (NT) plants that were almost destroyed. In addition, when SSA plants were treated with a high temperature of 42°C for 20 h, the photosynthetic activity of SSA plants decreased by only 6%, whereas that of NT plants decreased by 29%. These results suggest that the manipulation of the antioxidative mechanism of the chloroplasts may be applied in the development of industrial transgenic crop plants with increased tolerance to multiple environmental stresses.Communicated by I. S. Chung     

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.     

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.     

Enhanced drought tolerance of transgenic rice plants expressing a pea manganese superoxide dismutase

We investigated the role that manganese superoxide dismutase (MnSOD), an important antioxidant enzyme, may play in the drought tolerance of rice. MnSOD from pea (Pisum sativum) under the control of an oxidative stress-inducible SWPA2 promoter was introduced into chloroplasts of rice (Oryza sativa) by Agrobacterium-mediated transformation to develop drought-tolerant rice plants. Functional expression of the pea MnSOD in transgenic rice plants (T1) was revealed under drought stress induced by polyethylene glycol (PEG) 6000. After PEG treatment the transgenic leaf slices showed reduced electrolyte leakage compared to wild type (WT) leaf slices, whether they were exposed to methyl viologen (MV) or not, suggesting that transgenic plants were more resistant to MV- or PEG-induced oxidative stress. Transgenic plants also exhibited less injury, measured by net photosynthetic rate, when treated with PEG. Our data suggest that SOD is a critical component of the ROS scavenging system in plant chloroplasts and that the expression of MnSOD can improve drought tolerance in rice.     

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.     

A novel oxidative stress-inducible peroxidase promoter from sweetpotato: molecular cloning and characterization in transgenic tobacco plants and cultured cells

A strong oxidative stress-inducible peroxidase (POD) promoter was cloned from sweetpotato (Ipomoea batatas) and characterized in transgenic tobacco plants and cultured cells in terms of environmental stress. A POD genomic clone (referred to as SWPA2) consisted of 1824 bp of sequence upstream of the translation start site, two introns (743 bp and 97 bp), and a 1073 bp coding region. SWPA2 had previously been found to encode an anionic POD which was highly expressed in response to oxidative stress. The SWPA2 promoter contained several cis-element sequences implicated in oxidative stress such as GCN-4, AP-1, HSTF, SP-1 reported in animal cells and a plant specific G-box. Employing a transient expression assay in tobacco protoplasts, with five different 5-deletion mutants of the SWPA2 promoter fused to the -glucuronidase (GUS) reporter gene, the 1314 bp mutant deletion mutant showed about 30 times higher GUS expression than the CaMV 35S promoter. The expression of GUS activity in transgenic tobacco plants under the control of the –1314 SWPA2 promoter was strongly induced in response to environmental stresses including hydrogen peroxide, wounding and UV treatment. Furthermore, GUS activity in suspension cultures of transgenic cells derived from transgenic tobacco leaves containing the –1314 bp SWPA2 promoter-GUS fusion was strongly expressed after 15 days of subculture compared to other deletion mutants. We anticipate that the –1314 bp SWPA2 promoter will be biotechnologically useful for the development of transgenic plants with enhanced tolerance to environmental stress and particularly transgenic cell lines engineered to produce key pharmaceutical proteins.     

一种加工型马铃薯品种的农杆菌转化体系研究

大西洋马铃薯是经济价值很高的炸片型加工品种,逆境胁迫下,易产生褐变、空心等问题,影响加工品质。为获取抗逆境胁迫的优质转基因新品种,采用根癌农杆菌介导法,以大西洋马铃薯的茎段为外植体,建立了快速,简便,高效的遗传转化体系。从共培养到转化植株获得只需7-8周,转化频率达80%。结果表明茎段是较好的转化受体,硫代硫酸银可以有效促进不定芽分化并提高再生频率。PCR、Southern杂交分析证明外源基因已经成功整合到马铃薯再生植株的基因组中。该转化体系为大量开发转基因马铃薯植株,进而筛选优质的马铃薯炸片加工型新品种奠定基础。     

High expression of a human lactoferrin in transgenic tobacco cell cultures

Transgenic Nicotiana tabacum cell lines were developed expressing the human lactoferrin gene driven by the oxidative stress-inducible peroxidase (SWPA2) promoter. Western blot analysis showed the accumulation of both the full-length human lactoferrin protein as well as a immuno-reactive truncated fragment. Accumulation of human lactoferrin as monitored by ELISA increased proportionally to cell growth and reached a maximal (up to 4.3% of total soluble proteins) at the stationary phase of growth. Protein extracts from transgenic tobacco cells exhibited antibacterial activity.     

The expression of BrMDHAR gene in chloroplasts and mitochondria enhances tolerance to freezing stress in Arabidopsis thaliana

In chloroplasts and mitochondria, antioxidant mechanisms include the ascorbate-glutathione cycle, and monodehydroascorbate reductase (MDHAR) is important for regeneration of ascorbate (AsA) from monodehydroascorbate (MDHA). To improve detoxification of reactive oxygen species (ROS), we established a construct of the MDHAR gene from Brassica rapa fused to the targeting signal peptides of Pisum sativum glutathione reductase (GR), which was controlled by a stress-inducible SWPA2 promoter, and introduced this expression system into Arabidopsis thaliana. Transgenic (TG) plants overexpressing BrMDHAR targeted to chloroplasts and mitochondria through signal peptides showed an elevated MDHAR activity and an increased ratio of AsA to dehydroascorbate (DHA) when compared to wild-type (WT) plants under a freezing stress. These led to increased photosynthetic parameters, redox homeostasis, and biomass in TG plants when compared to the WT plants. Our results suggest that the overexpression of the BrMDHAR gene targeted to chloroplasts and mitochondria conferred an enhanced tolerance against the freezing stress, and a stress adaptation of dual-targeted BrMDHAR was better than that of single BrMDHAR.     

Stress-inducible expression of barley Hva1 gene in transgenic mulberry displays enhanced tolerance against drought, salinity and cold stress

Coping with different kinds of biotic and abiotic stresses is the foundation of sustainable agriculture. Although conventional breeding and marker-assisted selection are being employed in mulberry (Morus indica L.) to develop better varieties, nonetheless the longer time periods required for these approaches necessitates the use of precise biotechnological approaches for sustainable agriculture. In an attempt to improve stress tolerance of mulberry, an important plant of the sericulture industry, an encoding late embryogenesis abundant gene from barley (HVA1) was introduced into mulberry plants by Agrobacterium-mediated transformation. Transgenic mulberry with barley Hva1 under a constitutive promoter actin1 was shown to enhance drought and salinity tolerance. Here, we report that overexpression of barley Hva1 also confers cold tolerance in transgenic mulberry. Further, barley Hva1 gene under control of a stress-inducible promoter rd29A can effectively negate growth retardation under non-stress conditions and confer stress tolerance in transgenic mulberry. Transgenic lines display normal morphology to enhanced growth and an increased tolerance against drought, salt and cold conditions as measured by free proline, membrane stability index and PSII activity. Protein accumulation was detected under stress conditions confirming inductive expression of HVA1 in transgenics. Investigations to assess stress tolerance of these plants under field conditions revealed an overall better performance than the non-transgenic plants. Enhanced expression of stress responsive genes such as Mi dnaJ and Mi 2-cysperoxidin suggests that Hva1 can regulate downstream genes associated with providing abiotic stress tolerance. The investigation of transgenic lines presented here demonstrates the acquisition of tolerance against drought, salt and cold stress in plants overexpressing barley Hva1, indicating that Arabidopsis rd29A promoter can function in mulberry.