Paraquat Tolerance of Transgenic Nicotiana tabacum with Enhanced Activities of Glutathione Reductase and Superoxide Dismutase

Transgenic tobacco with enhanced cytosolic activities of glutathionereductase and superoxide dismutase were generated by cross-fertilization.Leaves of the hybrids exhibited further increased toleranceto a O₂^-generating herbicide paraquat than those of their parents.This result indicates the efficiency of manipulating more thanone gene in improving resistance of plants to photooxidativestress. ⁴Present address: Department of Regulation Biology, NationalInstitute for Basic Biology, Myodaiji, Okazaki, 444 Japan     

Enhanced Tolerance to Photooxidative Stress of Transgenic Nicotiana tabacum with High Chloroplastic Glutathione Reductase Activity

A gene from Escherichia coli that encodes glutathione reductasewas connected to a gene for a chloroplastic transit-peptideand the 35S promoter of cauliflower mosaic virus. This chimericgene was introduced into tobacco cells for generation of transgenicplants. Expression of the transgene in leaf cells and accumulationof the product of its translation in chloroplasts of the transgenicplants were confirmed immunochemically. Leaves of some transgenicplants had activities of glutathione reductase that were about3-fold higher than those of control plants. These transgenicplants exhibited lower susceptibility both to paraquat and tosulfur dioxide in the light than the control plants in termsof the extent of visible foliar damage. These results suggestthat the chloroplastic glutathione reductase, a component ofthe system for scavenging active oxygen, plays a role in theresistance of plants to photooxidative stress caused by paraquator sulfur dioxide. (Received June 22, 1992; Accepted November 3, 1992)     

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)     

二氢黄酮还原酶基因的克隆与转基因烟草的获得

以蒺藜苜蓿(Medicagotrunctulacv.5160)幼果总RNA为模板,采用RT-PCR技术克隆到二氢黄酮还原酶(DFR)基因的cDNA序列,所获得的cDNA序列全长1018 bp,具有完整的ORF,编码337个氨基酸。Blast分析表明,该片段与GenBank中注册的DFR基因同源性为99.80%。以植物表达载体pBI121为基础,构建了Ca MV35S启动子驱动的DFR基因植物表达载体pBIDFR;采用直接转化法将pBIDFR导入根癌农杆菌EHA105,用该菌株对普通烟草进行遗传转化获得6株转基因植株。     

The Effects of Methylglyoxal on Glutathione S-Transferase from Nicotiana tabacum

Methylglyoxal (MG) is one of the aldehydes that accumulate in plants under environmental stress. Glutathione S-transferases (GSTs) play important roles, including detoxification, in the stress tolerance systems of plants. To determine the effects of MG, we characterized recombinant GST. MG decreased GST activity and thiol contents with increasing K _m. GST can serve as a target of MG modification, which is suppressed by application of reduced glutathione.     

Development of Nitrate Reductase Activity in Expanding Leaves of Nicotiana tabacum in Relation to the Concentration of Nitrate and Potassium

Up to 80% of the total nitrate reductase activity (NRA) determined in vivo in different parts of vegetative tobacco plant (Nicotiana tabacum) was located in the leaves. The NRA reached a peak when a leaf had expanded to 27% of its final weight and 33% of its final area. Thereafter, with advancing expansion and age of the leaf, the activity declined. This pattern of development of NRA during the ontogenesis of leaves was not influenced by raising the supply of NO₃⁻ from 3 to 6 milliequivalent per cubic decimeter in the substrate solution. The concentration of NO₃⁻ in leaves, stem and root was inversely related to NRA at both NO₃⁻ levels. Raising the supply of K⁺ from 1 to 6 milliequivalent per cubic decimeter at either concentration of NO₃⁻ slowed down the development of NRA in the initial stages of expansion, but promoted it subsequently. The peak of the activity which developed in a leaf of 62% of its final area was higher at the higher supply of K⁺. The higher activity was maintained thereafter in the expanding and in matured and older leaves. It was concluded that NRA and the pattern of its development in expanding leaves is related to the availability of metabolites and their incorporation into enzyme proteins. Both these processes are influenced by: (a) the vertical profile of concentration of K⁺ in the shoot and (b) the concentration of K⁺ in a leaf, which depend upon its supply.     

长喙田菁植物螯合肽合成酶基因在烟草中的表达

以pHANNIBAL及pART27为基础,构建了CaMV35S启动子驱动的长喙田菁(Sesbania rostrata)植物螯合肽合成酶SrPCS1基因植物超量表达载体pAM22,采用电击转化方法将pAM22导入根癌农杆菌EHA105,并用该菌株对烟草进行了转化,通过抗生素筛选、PCR及Northern-blotting检测了目的基因的表达水平,并用ClustalW对SrPCS1进行了进化分析.结果表明:SrPCS1编码233个氨基酸与豆科植物PCSs的同源性较高;得到27株抗性植株,23株PCR阳性植株,Northern-blotting证明得到了超量表达该基因的烟草14株,但基因的表达水平不同.     

Transgenic Nicotiana tabacum and Arabidopsis thaliana plants overexpressing allene oxide synthase

 Allene oxide synthase (AOS), encoded by a single gene in Arabidopsis thaliana (L.) Heynh., catalyzes the first step specific to the octadecanoid pathway. Enzyme activity is very low in control plants, but is upregulated by wounding, octadecanoids, ethylene, salicylate and coronatine (D. Laudert and E.W. Weiler, 1998, Plant J 15: 675–684). In order to study the consequences of constitutive expression of AOS on the level of jasmonates, a complete cDNA encoding the enzyme from A. thaliana was constitutively expressed in both  A. thaliana and tobacco (Nicotiana tabacum L.). Overexpression of AOS did not alter the basal level of jasmonic acid; thus, output of the jasmonate pathway in the unchallenged plant appears to be strictly limited by substrate availability. In wounded plants overexpressing AOS, peak jasmonate levels were 2- to 3-fold higher compared to untransformed plants. More importantly, the transgenic plants reached the maximum jasmonate levels significantly earlier than wounded untransformed control plants. These findings suggest that overexpression of AOS might be a way of controlling defense dynamics in higher plants. Received: 10 February 2000 / Accepted: 11 March 2000     

Somatic Hybridization of Nitrate Reductase Deficient Mutants of Nicotiana tabacum by Protoplast Fusion

Protoplasts were isolated from two mutant cell lines of Nicotiana tabacum L. cv. Gatersleben and fused with the aid of polyethylene glycol. Both mutants lacked nitrate reductase and were thus auxotrophic for reduced nitrogen. The fusion resulted in a high frequency of hybrid cells which were detected by their regained ability to grow in media containing nitrate as sole nitrogen source. Thus, the two mutants were found to complement each other in the hybrids. In control experiments, back mutation and cross-feeding were excluded as possible explanations for the occurrence of cell lines utilizing nitrate. A total of 1061 hybrid lines capable of sustained proliferation were isolated. Some of them were further characterized with respect to nitrate reductase activity, chlorate sensitivity, chromosome number, and shoot formation. The results demonstrate that protoplast fusion can be used for the genetic analysis of cell variants of higher plants and that nitrate reductase-deficient mutants provide efficient selective systems for hybrid cells.     

Expression of a Self-Incompatibility Glycoprotein (S2-Ribonuclease) from Nicotiana alata in Transgenic Nicotiana tabacum

In Nicotiana alata, self-incompatibility is controlled by a single locus, designated the S-locus, with multiple alleles. Stylar products of these alleles are ribonucleases that are secreted mainly in the transmitting tract tissues. N. tabacum plants were transformed with constructs containing the S2-cDNA and genomic S2-sequences from N. alata that were linked to the cauliflower mosaic virus 35S promoter. Unlike other genes controlled by this promoter, the genes were expressed most highly in mature floral organs. This pattern of expression was observed at both the protein and RNA levels. The S2-glycoprotein was detected in the stylar transmitting tract tissues of the transgenic plants. The transgene product was secreted, had ribonuclease activity, and was glycosylated with the correct number of glycan chains. However, the maximum level of S2-glycoprotein in styles of the transgenic plants was approximately 100-fold lower than that found in N. alata styles carrying the S2-allele. Perhaps because of this lower protein level, the plants showed no changes in the incompatibility phenotype.     

过表达谷胱甘肽合成酶基因增强烟草对镉的耐受性

随着工业的发展,土壤污染问题愈发严重,利用基因工程修复土壤技术备受青睐,因此,开发重金属应答中的限速酶基因,将为植物修复重金属污染的土壤提供可应用的基因资源.通过RT-PCR及末端克隆方法获得枸杞谷胱甘肽合成酶 (Lycium chinense,Glutathione synthetase,LcGS>) 基因,采用半定量RT-PCR分析了枸杞LcGS在不同时间镉(Cd)胁迫下表达量的变化,LcGS表达量随着胁迫时间的延长而增强,胁迫9h、12h和24h 后LcGS表达量维持在较高水平.同时构建了植物双元表达载体pCAMBIA2300-LcGS,通过农杆菌介导的方法将LcGS基因转入烟草,PCR证明了LcGS基因成功整合到烟草基因组中,在Cd处理条件下,转基因植株谷胱甘肽 (glutathione,GSH)、植物螯合肽(phytochelatins,PCs)和叶绿素含量比对照组明显高,即转基因植株对重金属的耐逆性比对照组更强,因此,过表达GS植物将是植物修复重金属污染的一个有效策略.     

Increased Salt and Drought Tolerance by D-Ononitol Production in Transgenic Nicotiana tabacum L

A cDNA encoding myo-inositol O-methyltransferase (IMT1) has been transferred into Nicotiana tabacum cultivar SR1. During drought and salt stress, transformants (I5A) accumulated the methylated inositol D-ononitol in amounts exceeding 35 [mu]mol g-1 fresh weight In I5A, photosynthetic CO2 fixation was inhibited less during salt stress and drought, and the plants recovered faster than wild type. One day after rewatering drought-stressed plants, I5A photosynthesis had recovered 75% versus 57% recovery with cultivar SR1 plants. After 2.5 weeks of 250 mM NaCl in hydroponic solution, I5A fixed 4.9 [plus or minus] 1.4 [mu]mol CO2 m-2 s-1, whereas SR1 fixed 2.5 [plus or minus] 0.6 [mu]mol CO2 m-2 s-1. myo-Inositol, the substrate for IMT1, increases in tobacco under stress. Preconditioning of I5A plants in 50 mM NaCl increased D-ononitol amounts and resulted in increased protection when the plants were stressed subsequently with 150 mM NaCl. Pro, Suc, Fru, and Glc showed substantial diurnal fluctuations in amounts, but D-ononitol did not. Plant transformation resulting in stress-inducible, stable solute accumulation appears to provide better protection under drought and salt-stress conditions than strategies using osmotic adjustment by metabolites that are constitutively present.     

Bacillus thuringiensis section sign-Endotoxin Expressed in Transgenic Nicotiana tabacum Provides Resistance to Lepidopteran Insects

The crystal proteins, or §-endotoxins, of Bacillus thuringiensis are specifically lethal to Lepidopteran insects. We utilized a truncated and modified portion of a cloned crystal protein gene to construct a chimeric gene capable of expression in plant cells. Using an Agrobacterium tumefaciens binary vector system, we then transferred the chimeric toxin gene into tobacco (Nicotiana tabacum cv Havana 425) cells and regenerated recombinant plants. One to several copies per cell of the toxin gene are routinely present in the recombinant plants. Hybridization experiments demonstrated that these plants had a new RNA species of the size expected for the truncated toxin mRNA, and a polypeptide having the mobility expected for the truncated toxin was detected by immunoblotting. Significant variation was found in the levels of toxin-specific RNA expression between different recombinants, but the levels of hybridizing RNA in transformants correlated with the level of toxicity demonstrated against Manduca sexta (tobacco hornworm), and other Lepidopteran insects. The recombinant genes were transmitted to progeny and resistance to insects was maintained, thus demonstrating that the introduction of toxin genes into plants may be a practical method of providing protection against certain insect pests.     

Decrease of Nitrate Reductase Activity in Spinach Leaves during a Light-Dark Transition

In leaves of spinach plants (Spinacia oleracea L.) performing CO₂ and NO₃⁻ assimilation, at the time of sudden darkening, which eliminates photosystem I-dependent nitrite reduction, only a minor temporary increase of the leaf nitrite content is observed. Because nitrate reduction does not depend on redox equivalents generated by photosystem I activity, a continuation of nitrate reduction after darkening would result in a large accumulation of nitrite in the leaves within a very short time, which is not observed. Measurements of the extractable nitrate reductase activity from spinach leaves assayed under standard conditions showed that in these leaves the nitrate reductase activity decreased during darkening to 15% of the control value with a half-time of only 2 minutes. Apparently, in these leaves nitrate reductase is very rapidly inactivated at sudden darkness avoiding an accumulation of the toxic nitrite in the cells.     

Expression and Biological Properties of a Novel Methionine Sulfoxide Reductase A in Tobacco (Nicotiana tabacum)

Methionine (Met) residues in proteins/peptides are extremely susceptible to oxidation mediated by reactive oxygen species, resulting in the formation of methionine sulfoxide, which could be inversely reduced back to Met by methionine sulfoxide reductase (MSR). In the present study, an A-type MSR gene, termed NtMSRA4, was isolated from tobacco (Nicotiana tabacum). Sequence analysis of NtMSRA4 amino acid sequence indicated that the gene, encoded a polypeptide with a molecular weight of 21 kDa, possessed the highly conserved motif, ‘GCFWG’ in the N-terminus and ‘KGCNDPIRCY’ motif in the C-terminus respectively. Substrate specific analysis revealed that recombinant NtMSRA4 protein could reduce specifically S-isomer of Dabsyl-MetSO to Dabsyl-Met in vitro using dithiothreitol as an electron donor. Enzymatic properties analysis showed that the temperature of 42 °C and pH 9.0 were optimum for NtMSRA4 activity. The K _m and K _cat values of NtMSRA4 were determined to be 40.04 μM and 0.048 S^?1 in the thioredoxin dependent reduction system. Overexpression of NtMSRA4 in E. coli cells enhanced resistance to H₂O₂ toxicity. Subcellular localization result showed that NtMSRA4 was located in the chloroplast. The expression level of NtMSRA4 was affected differently after exposure to various abiotic stresses.     

Intrinsic GUS Activity in Various Tissues and During Pollen Development of Nicotiana tabacum

The β-glucuronidase (GUS) gene has been widely used as a reporter gene in the study of plant molecular biology and genetic engineering. One of the major reasons leading to the popularity of GUS-fusion system was the belief that there was no detectable intrinsic GUS activity in plant tissues. However, investigators have been troubled by the "false positive" results or "background" activities when GUS assays were performed. In the present experiment, histochemical observations of intrinsic GUS activity in various tissues and during pollen development of tobacco (Nicotiana tabacurn L. ) was carried out using 5-bromo-4- chloro-3-indolyl-β-D-glucuronic acid (X-gluc) as a substrate for overnight incubation of the treated tissues at 37℃. No detectable intrinsic GUS activity was found in seedling root, stem, leaf, anther wall and stigma of different stages, ovule, as well as isolated generative cell and embryo sac. During pollen development, two peaks of intrinsic GUS activity appeared, one, close to the microspore mitosis and the other from the full maturation of pollen lasting to the post-germination pollen tube stage, no or weak activity was found at other pollen developmental stages. GUS was located in the cytoplasm of the pollen. The pH value of staining solution strongly influenced the experimental results. Blue color was visualized at pH 5, even when 20% methanol or 0.2 mmol/L glucaric acid-l-4-1actone (GAL, a specific GUS inhibitor) were added. At pH 7, no detectable reaction was found at all. The aforementioned results indicate that when using tobacco pollen as the target of GUS gene transformation, the assay should be strictly controlled to neutral condition for avoiding false positive resuits.