烟草—TMV相互作用中质膜NADPH氧化酶的组装

比较 Ｎ Ｎ 烟草（与烟草花叶病毒（ Ｔ Ｍ Ｖ）发生非亲和相互作用）和普通烟草 ３００２ 品种（与 Ｔ Ｍ Ｖ 发生亲和相互作用）在烟草— Ｔ Ｍ Ｖ 的相互作用中质膜 Ｎ Ａ Ｄ Ｐ Ｈ 氧化酶的组装激活、产生活性氧的差异．用两相法制备密闭的正向型质膜（ Ｐ Ｍ）囊泡,以 Ｓ Ｏ Ｄ 敏感的 Ｎ Ａ Ｄ Ｐ Ｈ 依赖的 Ｃｙｔ ｃ 的还原表示 Ｎ Ａ Ｄ Ｐ Ｈ 氧化酶的活性,用人类噬中性白细胞 Ｎ Ａ Ｄ Ｐ Ｈ 氧化酶亚基 ｐ４７ ｐｈｏｘ 的抗体对烟草叶片蛋白进行免疫学检测．结果显示在两种烟草叶片胞质中均存在与 ｐ４７ ｐｈｏｘ 亚基的抗体发生免疫交叉反应的相同分子量的蛋白,该蛋白在 Ｔ Ｍ Ｖ 侵染 Ｎ Ｎ 基因烟草后可向质膜发生转移,且伴随有氧化酶活性的升高．而对于普通烟草则无氧化酶膜组分和酶活性的明显变化．以上结果表明,烟草叶片质膜上存在与哺乳动物 Ｎ Ａ Ｄ Ｐ Ｈ 氧化酶相类似的氧化酶,它的组装和激活可能是烟草— Ｔ Ｍ Ｖ 非亲和相互作用早期活性氧的主要来源．     

THE EFFECTS OF INFECTION WITH TOBACCO MOSAIC VIRUS ON THE PHOTOSYNTHESIS OF TOBACCO LEAVES

The rate of photosynthesis of tobacco leaves infected with the Rothamsted type culture of tobacco mosaic virus was lower than that of comparable healthy tobacco leaves. The lower rate was inferred from Net Assimilation Rates of whole plants and confirmed by direct comparisons of photosynthetic rates of inoculated and healthy leaves. The effect began within 1 hr. of inoculation. It was not caused by an effect of the virus on the stomata, and inactivated virus inoculum did not change the rates. The results indicate either a more rapid movement of virus from the epidermis into the chlorenchyma than has been previously recorded or an effect of virus infection at a site distant from the cells containing virus.     

Fine Structure of Healthy and TMV-Infected Tobacco Cells Grown in Tissue Culture

Three types of tobacco (Nicotiana tabacum cv. Havana 38) callus: 1) healthy stem callus, 2) TMV-infected stem callus, 3) TMV-infected leaf callus; and leaves differentiated from healthy stem callus, and from TMV-infected leaf callus were compared for fine structure. In addition, the fine structure was observed of plastids in cells of leaves differentiated from callus isolated from stem sections of TMV-infected hybrid tobacco plants (N. tabacum cv. Havana 38 ×N. glutinosa) grown under high temperature. The cytoplasmic organelles in tissue cultured cells were similar to those in cells of greenhouse-grown tobacco plants. Except for plastids, TMV infection did not noticeably affect morphologically other cellular organelles in tissue culture cells. In TMV-infected leaf callus, numerous small bodies were seen in plastid-like bodies, while vesicle-like structures were observed in the stroma of plastids in leaves differentiated from callus of hybrid tobacco inoculated with TMV. Morphological variations of mitochondria, such as swelling and vacuolization of the inner matrix, occurred frequently in TMV-infected leaf callus. Needle-like crystalline inclusions or looped inclusions composed of many fine, long filaments were considered TMV particles orientated parallel to each other. The TMV particles were detected in the cytoplasm of tissue culture cells.     

Genotoxicity of Nicotiana tabacum leaves on Helix aspersa

Tobacco farmers are routinely exposed to complex mixtures of inorganic and organic chemicals present in tobacco leaves. In this study, we examined the genotoxicity of tobacco leaves in the snail Helix aspersa as a measure of the risk to human health. DNA damage was evaluated using the micronucleus test and the Comet assay and the concentration of cytochrome P450 enzymes was estimated. Two groups of snails were studied: one fed on tobacco leaves and one fed on lettuce (Lactuca sativa L) leaves (control group). All of the snails received leaves (tobacco and lettuce leaves were the only food provided) and water ad libitum. Hemolymph cells were collected after 0, 24, 48 and 72 h. The Comet assay and micronucleus test showed that exposure to tobacco leaves for different periods of time caused significant DNA damage. Inhibition of cytochrome P450 enzymes occurred only in the tobacco group. Chemical analysis indicated the presence of the alkaloid nicotine, coumarins, saponins, flavonoids and various metals. These results show that tobacco leaves are genotoxic in H. aspersa and inhibit cytochrome P450 activity, probably through the action of the complex chemical mixture present in the plant.     

An arabinoxyloglucan from extracellular polysaccharides of suspension-cultured tobacco cells

An arabinoxyloglucan (AXG) isolated from extracellular polysaccharide of suspension-cultured tobacco cells was investigated by methylation analysis, partial acid hydrolysis and ¹³C NMR spectroscopy. It was found that the AXG is structurally similar to that isolated from the midrib of tobacco leaves.     

Plastid-nuclear complexes in the photosynthesizing cells from their mitosis up to programmed death

Permanent plastid-nuclear complexes (PNCs) exist in tobacco cells from their mitosis up to programmed cell death (PCD). PNCs in senescing cells of tobacco leaves were typical by enclosure of peroxisomes and mitochondria among chloroplasts which were in contact with nucleus. Such a complex position provides simultaneous interaction of these organelles and direct regulation of metabolism and PCD avoiding the cytosol.     

Identification of phloem involved in assimilate loading in leaves by the activity of the galactinol synthase promoter

The definition of "minor" veins in leaves is arbitrary and of uncertain biological significance. Generally, the term refers to the smallest vein classes in the leaf, believed to function in phloem loading. We found that a galactinol synthase promoter, cloned from melon (Cucumis melo), directs expression of the gusA gene to the smallest veins of mature Arabidopsis and cultivated tobacco (Nicotiana tabacum) leaves. This expression pattern is consistent with the role of galactinol synthase in sugar synthesis and phloem loading in cucurbits. The expression pattern in tobacco is especially noteworthy since galactinol is not synthesized in the leaves of this plant. Also, we unexpectedly found that expression in tobacco is limited to two of three companion cells in class-V veins, which are the most extensive in the leaf. Thus, the "minor" vein system is defined and regulated at the genetic level, and there is heterogeneity of response to this system by different companion cells of the same vein.     

类根瘤对烟草叶片中叶绿体及其淀粉粒含量的影响

目的：探讨类根瘤对烟草叶片中的叶绿体及其淀粉粒含量的影响。方法：无菌培养烟草再生植株,其中一部分经过一定浓度的2,4－D与豇豆根瘤菌512快生型突变株菌液诱导处理,另外一部分为对照。运用常规电镜技术制作叶片厚切片,通过显微观察,对两组烟草叶片细胞中的叶绿体及其淀粉粒含量进行对比分析。结果：结瘤植株叶片细胞内的叶绿体含量虽然与对照之间无明显差异,但叶片栅栏组织和海绵组织细胞内淀粉粒的含量却均较对照减少,其中前者减少更为明显（P＜0.05)。结论：结瘤烟草叶片内光合产物的大量同化可能与类根瘤的形成有关。     

Immunochemical Localization of Pathogenesis-Related Proteins Secreted into the Intercellular Spaces of Salicylate-Treated Tobacco Leaves

In tobacco leaves, pathogenesis-related (PR) 1 proteins areabundantly induced by hypersensitive reaction to the infectionwith tobacco mosaic virus (TMV) and by treatment with salicylicacid, and are secreted into the intercellular spaces. To studythe distribution of PR 1 proteins outside of the cells, theimmunogold technique was used with anti-PR 1 antibody. Whensections of salicylate-treated tobacco leaf were reacted withantibody against PR 1a and then with protein A-gold complex,most of the gold label was localized in the intercellular spacesin the region between cells, and a little label was found inthe cytoplasmic matrix and in small electron-dense granulesinside the cells. When salicylate-treated leaves were incubatedwith polygalacturonase and/or cellulase to liberate protoplastsor single cells from the tissue, most of PR 1 proteins weresolubilized far before complete liberation of single cells,suggesting their localization in free spaces or a region susceptableto maceration, such as the secondary cell wall. (Received May 30, 1988; Accepted June 27, 1988)     

The vaccine adjuvant extra domain A from fibronectin retains its proinflammatory properties when expressed in tobacco chloroplasts

We previously showed that recombinant extra domain A from fibronectin (EDA) purified from Escherichia coli was able to bind to toll-like receptor 4 (TLR4) and stimulate production of proinflammatory cytokines by dendritic cells. Because EDA could be used as an adjuvant for vaccine development, we aimed to express it from the tobacco plastome, a promising strategy in molecular farming. To optimize the amount of recombinant EDA (rEDA) in tobacco leaves, different downstream sequences were evaluated as potential fusion tags. Plants generated by tobacco plastid transformation accumulated rEDA at levels up to 2% of the total cellular protein (equivalent to approximately 0.3 mg/g fresh weight) when translationally fused to the first 15 amino acids of green fluorescence protein (GFP). The recombinant adjuvant could be purified from tobacco leaves using a simple procedure, involving ammonium sulfate precipitation and anion exchange chromatography. Purified protein was able to induce production of tumour necrosis factor-α (TNF-α) either by bone marrow-derived dendritic cells or THP-1 monocytes. The rEDA produced in tobacco leaves was also able to induce upregulation of CD54 and CD86 maturation markers on dendritic cells, suggesting that the rEDA retains the proinflammatory properties of the EDA produced in E. coli and thus could be used as an adjuvant in vaccination against infectious agents and cancer. Taken together, these results demonstrate that chloroplasts are an attractive production vehicle for the expression of this protein vaccine adjuvant.     

Studies on the relationship between ploidy level,morphology, the concentration of some phytohormones and the nicotine concentration of haploid and doubled haploid tobacco (Nicotiana tabacum L.) and NICA plants

As compared to doubled haploid plants of the same origin, haploid tobacco plants are characterized by narrow leaves and in these leaves the endogenous concentration of gibberellins was considerably higher than in doubled haploids. This higher GA activity is almost entirely due to elevated levels of polar gibberellins. The same leaf shape as in haploids could be induced by GA₃ sprays to doubled haploids. A similar leaf shape was also observed on tissue culture derived so called NICA plants displaying the morphology of tobacco plants as described by Dudits et al. (1987) from whom the plant material was obtained as a gift. Here, in the leaves of a special strain with narrow lamina again a much higher gibberellin activity was detected than in the leaves of plants of the original tobacco strain. Histochemical determination of the relative DNA content indicated that leaves of NICA were chimaeras containing 1C cells besides cells with higher C values. Obviously, haploidy is somehow related to the endogenous gibberellin activity in tobacco plant material with consequences on the morphological appearance of 1n plants. Comparing some haploid and doubled haploid strains in tissue culture and pot and field experiments in several years apparently the genotype of the plant material is more significant for nicotine concentration than the ploidy level.Abbreviations DW dry weight - FW fresh weight - LSI leaf shape index     

Factors related to iron absorption by enzymically isolated leaf cells

The rate of Fe absorption by cells enzymically isolated from tobacco leaves is correlated with the age of the leaves from which the cells are derived. The cells obtained from younger leaves absorb Fe more rapidly than those from older ones. Ca inhibits Fe and Mn absorption. Fe and Mn are mutually antagonistic in their absorption by leaf cells. Ca enhances the inhibition of Mn absorption by Fe, but reduces the inhibition of Fe absorption by Mn. The affinity constant for Fe absorption by leaf cells is low. The chelate EDDHA (ethylenediamine di(o-hydroxyphenylacetate) competitively inhibits Fe absorption.     

Rapid low temperature-induced stomatal closure occurs in cold-tolerant Commelina communis leaves but not in cold-sensitive tobacco leaves, via a mechanism that involves apoplastic calcium but not abscisic acid

Commelina communis stomata closed within 1 h of transferring intact plants from 27 degrees C to 7 degrees C, whereas tobacco (Nicotiana rustica) stomata did not until the leaves wilted. Abscisic acid (ABA) did not mediate cold-induced C. communis stomatal closure: At low temperatures, bulk leaf ABA did not increase; ABA did not preferentially accumulate in the epidermis; its flux into detached leaves was lower; its release from isolated epidermis was not greater; and stomata in epidermal strips were less sensitive to exogenous ABA. Stomata of both species in epidermal strips on large volumes of cold KCl failed to close unless calcium was supplied. Therefore, the following cannot be triggers for cold-induced stomatal closure in C. communis: direct effects of temperature on guard or epidermal cells, long-distance signals, and effects of temperature on photosynthesis. Low temperature increased stomatal sensitivity to external CaCl(2) by 50% in C. communis but only by 20% in tobacco. C. communis stomata were 300- to 1,000-fold more sensitive to calcium at low temperature than tobacco stomata, but tobacco epidermis only released 13.6-fold more calcium into bathing solutions than C. communis. Stomata in C. communis epidermis incubated on ever-decreasing volumes of cold calcium-free KCl closed on the lowest volume (0.2 cm(3)) because the epidermal apoplast contained enough calcium to mediate closure if this was not over diluted. We propose that the basis of cold-induced stomatal closure exhibited by intact C. communis leaves is increased apoplastic calcium uptake by guard cells. Such responses do not occur in chill-sensitive tobacco leaves.     

THE EFFECT OF INFECTION WITH TOBACCO MOSAIC VIRUS ON THE RESPIRATION OF TOBACCO LEAVES OF VARYING AGES IN THE PERIOD BETWEEN INOCULATION AND SYSTEMIC INFECTION

Leaves of tobacco plants inoculated with tobacco mosaic virus were divided into three groups: ( a ) inoculated leaves; ( b ) younger non-inoculated leaves present at the time of inoculation; ( c ) leaves formed since inoculation. The respiration rate of each group was compared with that of similar leaves from healthy plants. The respiration rate of inoculated leaves was increased by a constant amount for 3 weeks after inoculation, when it decreased. The respiration rate of group ( b ) leaves was not affected at any time, and that of group ( c ) leaves was decreased by 10% when they showed symptoms. The increased respiration in the inoculated leaves occurred too soon to reflect virus formation, and it is suggested that it reflects an initial change in infected cells preparatory to virus synthesis. The subsequent decrease in respiration may be due to the accumulation of virus which does not contribute to the total leaf respiration.     

Absorption of Iron by Enzymically Isolated Leaf Cells

Iron uptake was studied using cells enzymically isolated from green tobacco leaves. Absorption was increased both by light and succinate as probable energy sources. Bicarbonate in the incubation mixture was inhibitory, and citrate also reduced absorption presumably by chelation with the metal. Absorption of iron was temperature sensitive and optimal at 25°C. Temperature coefficients and activation energies suggested that absorption was energy mediated. NaN₃ and DNP inhibited uptake at concentrations of 10-³M and 10^?4M, respectively. The inhibition caused by DNP was not negated by an external supply of ATP. The results suggest that iron absorption is an active metabolic process in cells enzymically isolated from green tobacco leaves. Cells from Fe-chlorotic leaves of PI 54619–5–1 soybean absorbed less iron than those derived from healthy leaves of the same variety, while leaf cells from the variety Hawkeye showed no such differences.     

Molecular characterization of tobacco mitochondrial L-galactono-gamma-lactone dehydrogenase and its expression in Escherichia coli

A cDNA clone encoding L-galactono-gamma-lactone (GAL) dehydrogenase (EC 1.3.2.3) was isolated from tobacco leaves. The cDNA clone contained an open reading frame encoding the protein of 501 amino acids with a calculated molecular mass of 56,926 Da, preceded by a putative mitochondrial targeting signal consisting of 86 amino acid residues. In fact, GAL dehydrogenase was localized in the mitochondria of tobacco cells. The deduced amino acid sequence of the cDNA showed 77 and 82% homology to cauliflower and sweet potato GAL dehydrogenases, respectively. Southern blot analysis showed that tobacco contains one copy of the gene for the enzyme. Northern blot analysis showed that GAL dehydrogenase mRNA (2.0 kb) is expressed in the leaves, stems, and roots in almost equal quantities. We introduced the cDNA clone encoding tobacco GAL dehydrogenase into a pET expression vector to overexpress this protein in Escherichia coli. The partially purified recombinant enzyme was used for comparative studies on the native enzymes from tobacco and other sources; its enzymatic properties were similar to those of other GAL dehydrogenases.     

Characterization of Polypeptides that Accumulate in Cultured Nicotiana tabacum Cells

We characterized the polypeptides that accumulate in photoautotrophicallycultured cells of tobacco. Microsequencing of these polypeptidesaccumulated in large amounts revealed four NH₂-terminal aminoacid sequences that were highly homologous to those of the knownstress proteins, osmotin and chitinase. Further analyses ofour tobacco cell line grown with sucrose in light and in darkness,as well as analyses of newly established cultured cells andregenerating adventitious shoots, clearly showed that all thein vitro cultured cells accumulated these stress proteins. Theaccumulation of these proteins were also observed in old leaves,roots, and leaves infected with Tobacco Mosaic Virus, but notin young healthy leaves. (Received September 27, 1989; Accepted December 4, 1989)     

烘烤过程中外加淀粉类酶对烤烟淀粉降解的影响

为降低烤后烟叶中淀粉含量,研究了烘烤过程中不同外加淀粉类酶对烤烟淀粉降解的影响。结果表明：烘烤过程中,通过外加淀粉类酶来降解烤烟中的淀粉是有效的。烘烤变黄初期,不同外加淀粉类酶烟叶淀粉降解动态基本一致;变黄中期至定色前期,淀粉降解随外加酶量增加而加剧。烤后烟叶淀粉含量随外加酶量增加而减少,水溶性糖和还原糖含量随外加酶量增加而增加。方差分析表明,不同处理烤后烟叶之问淀粉含量存在极显著差异。多重比较结果表明：K326品种适宜的外加酶量为(6 60)U／g;HD的适宜外加酶量为(8 80)U／g。     

Photosynthetic Characteristics and Carbonic Anhydrase Activity in Cells Cultured Photoautotrophically and Mixotrophically and Cells Isolated from Leaves

Cultured green cells of Nicotiana tabacum var. Samsun, Cytisusscoparius Link and Hyoscyamus niger which were grown photoautotrophicallyunder a stream of air enriched with 1% CO₂ or mixotrophicallyin the presence of 3% sucrose and ordinary air showed very lowcarbonic anhydrase activity, which was only 0–9% of thatin the respective intact leaves. The CO₂ compensation pointfor photosynthesis of autotrophically and mixotrophically culturedgreen cells of tobacco was higher than 0.3 mM NaHCO₃ at pH 7.8,but that of the cells isolated from tobacco leaves was lowerthan 0.1 mM NaHCO₃ at pH 7.8. The fact that the cultured cellscannot grow photoautotrophically under ordinary air is due toa high CO₂ compensation point in photosynthesis. The dark respiratoryactivity in both photoautotrophically and mixotrophically culturedtobacco cells was more than 7-fold that in the cells isolatedfrom tobacco leaves. We therefore could not conclude whetherthe high CO₂ compensation point in the cultured cells is dueto the low carbonic anhydrase activity or simply reflects thehigh respiratory activity. (Received July 10, 1980; Accepted November 25, 1980)