Detection of a Cytosolic Glutamine Synthetase in Leaves of Nicotiana tabacum L. by Immunocytochemical Methods

Two glutamine synthetase (GS) polypeptides (44 and 39 kD) were immunodetected on western blots of leaf extracts from tobacco (Nicotiana tabacum L.), a plant that has been reported to contain only chloroplast GS in the leaves. By immunocytochemical methods, we confirmed the localization of GS in the cytosol of cells in the vascular tissue and in the chloroplasts of mesophyll cells.     

Subunit Composition of Glutamine Synthetase Isozymes from Root Nodules of Bean (Phaseolus vulgaris L.)

Glutamine synthetase from bean nodules can be separated into two isoforms, GS_n1 and GS_n2. A purification protocol has been developed. It included protamine sulfate precipitation, ammonium sulfate fractionation, anthranilate-affinity chromatography, Dye-Matrex (Orange A) chromatography, and diethylaminoethyl-cellulose ion-exchange chromatography. GS_n1 and GS_n2 have been purified to homogeneity. Subunit structure analysis using two-dimensional polyacrylamide gel electrophoresis revealed that GS_n1 was composed of two different types of subunit polypeptides. They differed in isoelectric points (6.0 and 6.3) but had the same molecular weights (46,000 Daltons). GS_n2 was composed of only one type of subunit polypeptide. It had an isoelectric point of 6.0 and a molecular weight of 46,000 Daltons. It was apparently identical to one of the polypeptides found in GS_n1. Glutamine synthetase holoenzyme consisted of eight subunits. In the nodule there are two different types of glutamine synthetase subunit polypeptides. Random combinations of the polypeptides should generate nine different isozymes. Our electrophoretic analysis revealed that GS_n2 was but one of the isozymes, and GS_n1 was a composite of the other eight. Hence, nodule glutamine synthetase isozymes were homo-octameric as well as hetero-octameric.     

Inhibition of Photosynthesis in Barley with Decreased Levels of Chloroplastic Glutamine Synthetase Activity

Mutant barley plants containing only 8%, 16% or 38% of the wildtype level of glutamine synthetase activity have been isolated.The level of glutamine synthetase activity in the roots of themutant containing only 8% leaf activity was not affected bythis mutation. The plants accumulated high levels of ammoniain leaves exposed to air and although they were able to carryout photosynthetic CO₂fixation normally at low levels of atmosphericO₂, they were unable to maintain wild type rates of CO₂fixationin air. The extent of this inhibition and the extent to whichammonia accumulated in the leaves was dependent on the photonfluence rate intercepted by the plant. When leaves from themutant plant were fed glutamine under non-photorespiratory conditionsfor 40 min before they were transferred to air, the plants exhibitedwild type rates of CO₂ fixation in air but the ammonia contentof the leaves increased to an even higher level. At least inthe short term, therefore, ammonia accumulation was not responsiblefor the dramatic decline in the fixation rate of these mutantsin air. The most probable explanation is that as the supplyof potential amino donors diminished on transfer to air, therewas a restriction on the return of glycerate to the Calvin cyclewithin the chloroplast. Key words: Ammonia toxicity, photorespiration, photosynthesis, GS-deficient barley     

Immunochemical Analysis of Multiple Subunit Polypeptides of Glutamine Synthetase in Methionine Sulfoximine-Sensitive and Tolerant Tobacco Cell Cultures

A methionine sulfoximine (MSX) tolerant cell line of tobacco(Nicotiana tabacum L. cv. Xanthi) cells was selected by culturingthe wild-type cells in suspension media in the presence of MSXat a step-wise increase in its concentration (0.3 to 5 µM).Fifty per cent inhibition of growth occurred at 0.18 µMMSX for the wild-type cells whereas 4.65 µM was requiredfor the tolerant cells. The tolerant cells possessed about 1.5-foldincrease in glutamine synthetase (GS) activity. Kinetic experimentsshowed that an inhibitor constant for MSX was identical betweenGS isolated from these two cell types. Subunit polypeptidesof GS in both cell types were analyzed with an immunoblottingmethod by using polyclonal antibody raised against a chloroplasticGS in spinach. A single polypeptide (41 kDa) was recognizedby the antibody in wild-type cells, whereas two predominantpolypeptides of 41 and 40 kDa were seen in the MSX tolerantcells. When the GS subunit polypeptides in the wild-type cellswere examined with two-dimensional gel electrophoresis, twomajor and four minor polypeptides associating distinct chargeat 41 kDa were detected. The extract from the MSX-tolerant cellshad the same set of polypeptides at 41 kDa and in addition twomajor and some minor spots at 40 kDa. These results indicatethat 1) tobacco GS is consisted of heterogeneous subunit polypeptidesin surface charge and 2) MSX causes formation of additionalmultiple 40 kDa polypeptides which may be related to the tolerantnature of the selected cell line. (Received October 27, 1989; Accepted January 17, 1990)     

Rapid flowering Nicotiana tabacum L.

 Selected Nicotiana tabacum Tobacco Introductions from the Nicotiana Germplasm Collection were crossed, back-crossed, and then selfed. When compared to parents, the new genotypes produce smaller plants with a shortened life-cycle that go from seed to seed in under 11 weeks. Received: 11 December 1998 / Revision accepted: 8 January 1999     

Amino-acid-transport mutant of Nicotiana tabacum L.

The structure of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) has been investigated by tilted-view electron microscopy of negatively stained monolayer crystals and image processing. The structure determined consists of a cylinder of octagonal cross-section with a large central hole. Based on this and other available evidence a model for the arrangement of the large and small subunits is suggested with the eight small subunits arranged equatorially around the core of eight large subunits.Abbreviations LS large subunit - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - SS small subunit     

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)     

Sperm dimorphism in Nicotiana tabacum L.

Sperm cells of tobacco have been intensively studied as examples of isomorphic gametes in which major cellular and organellar parameters remain statistically indistinguishable in the two sperm cells. An examination of sperm cells late in maturation, however, displays that the sperm cell associated with the vegetative nucleus becomes statistically significantly smaller than the other sperm cell in tobacco. If late divergence occurs in the two sperms of other angiosperms, sperm dimorphism may be more prevalent than has previously been assumed and dimorphism may have a major influence on the pattern of double fertilization. Received: 15 December 2000 / Accepted: 4 May 2001     

Antipeptide Antibodies That Can Distinguish Specific Subunit Polypeptides of Glutamine Synthetase from Bean (Phaseolus vulgaris L.)

The amino acid sequences of the β and γ subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (γ_2-9, γ_264-274, and β_264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-β_264-274 antibodies reacted specifically with the β polypeptide and the anti-γ_264-274 and anti-γ_2-9 antibodies reacted specifically with the γ polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.     

烟草的历史

1　吸烟习俗的起源和传播当今遍布于全世界各地的烟草原产于美洲 ,最早栽培并吸食的是当地印第安人。美洲印第安人从什么时候开始吸烟 ,迄今还不很清楚。目前已发现最早的资料是在墨西哥恰帕斯州的帕伦克 ,有在公元 4 32年修建的玛雅文化的古典神庙中的一块浮雕 ,可以看到古代玛雅人在宗教的仪式中有人用管状烟斗来吸烟 ,而且还在喷出烟雾。在美国阿利桑那州的帕布罗城发现公元 6 5 0年前后印第安人居住的洞穴遗址 ,其中有烟叶实物和烟斗 ,烟斗中还存有烟灰。在墨西哥马德雷山上海拔 12 0 0ｍ处的一个山洞中 ,发现一个塞有烟草的空心草管 ,…     

Anther cultures of Nicotiana tabacum L. mutants

Summary The theoretically expected and experimentally observed phenotypic ratios have been compared in populations of haploids derived from chlorophyll mutants of Nicotiana tabacum L. with a known genotypic constitution. The frequencies of mutant genotypes were significantly lower than the expected values, proving the existence of selection in a system of haploid embryoids developing in the anther.The anthers from M₁ plants of a diploidized Nicotiana tabacum haploid cv. Samsun, treated with various concentrations of N-nitroso-N-methylurea and n-butylmethane sulphonate, were cultivated in vitro. The number of anthers which gave rise to haploids (embryogenic anthers) was stimulated by lower concentrations of both the mutagens. The stimulation at the level of M₁ sporophyte is explained by internal genetic heterogeneity induced by adequate mutagen concentration. The average number of haploids per embryogenic anther decreased in all the treatments. The frequency of haploid plants of the mutant phenotypes increased with increasing mutagen concentration.     

Heterogeneity of Glutamine Synthetase Polypeptides in Phaseolus vulgaris L

Glutamine synthetases from roots, nodules, and leaves of Phaseolus vulgaris L. have been purified to homogeneity and their polypeptide composition determined.

The leaf enzyme is composed of six polypeptides. The cytosolic fraction contains two 43,000 dalton polypeptides and the chloroplastic enzyme is formed by four 45,000 dalton polypeptides. Root glutamine synthetase consists only of the same two polypeptides of 43,000 dalton that are present in the leaf enzyme. The nodule enzyme is formed by two polypeptides of 43,000 dalton, one is common to the leaf and root enzyme but the other is specific for N₂-fixing nodule tissue. The two glutamine synthetase forms of the nodule contain a different proportion of the 43,000 dalton polypeptides.

     

Coproporphyrinogenase in tobacco (Nicotiana tabacum L.)

1. Coproporphyrinogenase was extracted and purified from tobacco (Nicotiana tabacum L.). Enzyme activity was mainly located in mitochondria rather than in chloroplasts. The enzyme was purified by differential centrifugation, ammonium sulphate fractionation, calcium phosphate gel adsorption and dialysis. A 69-fold final purification was obtained. 2. An apparent K(m) value of 3.6x10(-5)m was found, the value being largely dependent on the amount of coproporphyrin III recovered after reduction with sodium amalgam to coproporphyrinogen III. Protoporphyrin formation was linear up to 3h and decreased with further incubation. The enzyme activity increased with the concentration of enzyme protein up to 30mug/ml of solution. 3. Enzyme activity was greatly enhanced by increasing Fe(2+) concentrations up to 0.5mm, beyond which inhibition occurred. Co(2+) and Mn(2+) were also found to activate at low concentrations (0.1mm) and inhibit at higher concentrations (5mm). Fe(3+) and Cu(2+), both at 0.1mm, and o-phenanthroline and EDTA, each at 1mm, were found to be inhibitory.     

Steryl Glycoside Formation in Seedlings of Nicotiana tabacum L

Particulate enzyme preparations from tobacco seedlings (Nicotiana tabacum L.) were used in the synthesis of steryl glycoside. The data obtained by measuring cholesterol-4-¹⁴C incorporation generally agree with results obtained with UDP-glucose-¹⁴C. The in vitro reaction was linear for the first 10 minutes and had a pH optimum of 7.0 to 7.4. Addition of ATP activated while UDP-glucose inhibited slightly the reaction. In short term experiments, the percentage disappearance of endogenous and added sterol was about the same.     

Glyphosate Tolerance in Tobacco (Nicotiana tabacum L.)

A glyphosate-tolerant tobacco cell line, Nicotiana tabacum L. Indiana (I7), was selected from the glyphosate-sensitive Wisconsin 38 (W38) line through a single step exposure to the herbicide. Tolerance and growth characteristics of I7 cells were the same for cells maintained for more than 1 year in the presence or absence of glyphosate. Glyphosate tolerance levels were constant through the growth cycle. Tolerance is not due to reduced uptake of glyphosate. Shikimate levels in I7 and W38 cells maintained in glyphosate-free medium were similar, whereas W38 cells accumulated 46 times more shikimate than I7 cells, when cells of both lines were exposed to the herbicide. Glyphosate treatment caused increased levels of aromatic amino acids in W38 cells and slightly lower levels in I7 cells. Specific activities of dehydroquinate synthase, shikimate dehydrogenase, and shikimate kinase were similar in the two cell types, whereas DAHP synthase and EPSP synthase specific activities were elevated in I7 cells. Plants regenerated from I7 cells retained tolerance to glyphosate.     

烤烟种质资源差异性分析

对700多份烤烟种质资源的形态特征、主要性状、抗病性和烟叶化学成分等方面的差异性进行研究和分析,结果表明：烤烟种质资源形态差异主要表现在株型、叶形、叶耳、叶尖、茎叶角度和花序等特征上;主要性状差异表现在株高、打顸株高、叶数、节距、茎围、腰叶的长和宽等性状,其变幅分别为97．0—326．0cm、75.3～133.7cm、8．6—54片、2．6—10．1cm、6.3～13．8cm、41．6-99．0cm、16.6—47．8cm;脚叶、腰叶和项叶的长宽比变幅分别为1．3～3,7、1．5—4．9、1．6～5．8;移栽至现蕾的天数变幅为30-100d;各性状指标的变异系数在7．8％-30．3％之间。不同种质间烟叶化学成分差异较大,变异系数在16．4％-69．2％之间。在702份种质中,抗病、中抗、感病和中感病的种质分别占18．2％、36．0％、6.6％、39．0％,可提供丰富的种质资源供育种选择利用。     

Immunocytochemical Localization of Glutamine Synthetase in Organs of Phaseolus vulgaris L

Glutamine synthetase was localized in nodules, roots, stems, and leaves of red kidney bean (Phaseolus vulgaris L.) by immunocytochemistry. Affinity purified antibodies reactive with glutamine synthetase were prepared using purified nodule-enhanced glutamine synthetase. Immunogold labeling was observed in the cell cytoplasm in each plant organ. In nodules, the labeling was more intense in the infected cells than in the uninfected cells. No labeling was observed in nodule bacteroids, peribacteroid spaces, or in peribacteroid membranes, while previous reports of glutamine synthetase immunolabeling of legume nodules showed labeling in the bacteroid fraction. Significant labeling was observed in nodule proplastids which contained starch granules. Substantial labeling was also observed in leaf chloroplasts. No labeling was observed in other organelles including mitochondria, peroxisomes, and endoplasmic reticulum. Preimmune IgGs did not bind to any structure in the tissues examined.     

Biological effects of ion beams in Nicotiana tabacum L.

The biological effects of ion beams on Nicotiana tabacum L., particularly the induction of chromosome aberrations, were investigated. Dry seeds were exposed to ¹²C⁵⁺, ⁴He²⁺ and ¹H⁺ beams with linear energy transfer (LET) ranging from 1 to 111 keV/μm and irradiated with gamma-rays. Ion beams were more effective in reducing germination and survival of the seeds than gamma-rays. The LD₅₀ for ¹²C⁵⁺ beams, ⁴He²⁺ beams and gamma-rays were 35, 60 and 500 Gy, respectively. The frequencies of mitotic cells with chromosome aberrations, such as chromosome bridges, acentric fragments and lagging chromosomes in the root tip cells of the exposed seeds, increased linearly with increasing doses. Relative biological effectiveness (RBE) values, based on the doses that induced a survival inhibition of 50% and a 10% frequency of aberrant cells, were 14.3–17.5 for the ¹²C⁵⁺ beams, 7.0–8.3 for the ⁴He²⁺ beams and 7.8 for the ¹H⁺ beams. Furthermore, the relative ratios of the chromosome aberration types were significantly different between the ion beam and the gamma-ray regimes: chromosome fragments were more frequent in the former, and chromosome bridges in the latter. Based on these results, we concluded that the repair process of initial lesions induced by ion beams may be different from that induced by low- LET radiation. Received: 29 October 1998 / Accepted in revised form: 25 March 1999