Induction of Resistance in Tobacco Leaves to Tobacco Mosaic Virus by Dodecylbenzenesulfonate

Application of dodecylbenzenesulfonate (DBS) to half leaves of Nicotiana tabacum cv. Xanthi nc. before TMV inoculation resulted in a marked decrease in lesion number and size as well as in virus content of the lesions in the untreated half leaves. Systemic induction of resistance in untreated leaves of the plants was not detected.     

Synthesis of Stress Proteins in Tobacco Leaves

Pathogenesis-related proteins (PR proteins), which are knownto be induced in tobacco leaves in response to infection withtobacco mosaic virus (TMV), were isolated by a simple procedureinvolving ammonium sulfate fractionation and preparative gelelectrophoresis. A rabbit antibody to one of the purified PRproteins, designated as PR1a, also reacted with two other PRproteins, designated as PR1b and PR1c in double immunodiffusiontests. Quantitative analysis of these proteins using rocketimmunoelectrophoresis with the antibody showed that they wereinduced not only by infection with TMV but also by mechanicalinjury and osmotic stress at 20?C, although not at 30?C. Basedon these findings, we propose that these proteins be called"stress proteins" rather than "pathogenesis-related proteins." (Received October 23, 1984; Accepted January 18, 1985)     

Induction of UDP-Glucose:Salicylic Acid Glucosyltransferase Activity in Tobacco Mosaic Virus-Inoculated Tobacco (Nicotiana tabacum) Leaves

Salicylic acid (SA) is a putative signal that activates plant resistance to pathogens. SA levels increase systemically following the hypersensitive response produced by tobacco mosaic virus (TMV) inoculation of tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaves. The SA increase in the inoculated leaf coincided with the appearance of a [beta]-glucosidase-hydrolyzable SA conjugate identified as [beta]-O-D-glucosylsalicylic acid (GSA). SA and GSA accumulation in the TMV-inoculated leaf paralleled the increase in the activity of a UDP-glucose:salicylic acid 3-O-glucosyltransferase (EC 2.4.1.35) ([beta]-GTase) capable of converting SA to GSA. Healthy tissues had constitutive [beta]-GTase activity of 0.076 milliunits g-1 fresh weight. This activity started to increase 48 h after TMV inoculation, reaching its maximum (6.7-fold induction over the basal levels) 72 h after TMV inoculation. No significant GSA or elevated [beta]-Gtase activity could be detected in the healthy leaf immediately above the TMV-inoculated leaf. The effect of TMV inoculation on the [beta]-GTase and GSA accumulation could be duplicated by infiltrating tobacco leaf discs with SA at the levels naturally produced in TMV-inoculated leaves (2.7-27.0 [mu]g g-1 fresh weight). Pretreatment of leaf discs with the protein synthesis inhibitor cycloheximide inhibited the induction of [beta]-GTase by SA and prevented the formation of GSA. Of 12 analogs of SA tested, only 2,6-dihydroxybenzoic acid induced [beta]-GTase activity.     

Localization of Pathogenesis-Related Proteins in the Epidermis and Intercellular Spaces of Tobacco Leaves after Their Induction by Potassium Salicylate or Tobacco Mosaic Virus Infection

The localization of pathogenesis-related (PR) proteins inducedin tobacco leaves by treatment with potassium salicylate ora hypersensitive response to tobacco mosaic virus (TMV) infectionwas studied using immunochemical methods. Total PR protein levelsincreased with time after these treatments. The proportion ofPR proteins in the intercellular spaces to the total contentin the leaf discs rapidly rose in the later stage of the treatmentto about 75% on the 9th day after salicylate treatment and tomore than 80% on the 6th to 9th day after TMV inoculation. After5 days of salicylate treatment, the amounts of PR proteins inthe peeled leaf epidermis were two fold those in the mesophylltissue. Only five percent or less of the total PR proteins inthe epidermal and mesophyll tissues of salicylate-treated leaveswere detected in the isolated epidermal and mesophyll protoplasts.The sugar content in highly purified PR la, lb and lc was lessthan one mole of monosaccharide per mole of each protein. Theseresults show that the PR proteins are non-glycoproteins secretedinto the intercellular spaces. (Received January 16, 1987; Accepted July 14, 1987)     

Accumulation of Pathogenesis-Related Proteins in Tobacco Leaves Irradiated with UV-B

Nicotiana tabacum L. (cv. Petit Havana SR1) were grown under ultraviolet-B (UV-B, 290–320 nm) irradiation, and soluble proteins were extracted from the leaves. Two-dimensional electrophoresis revealed that a minimum of 12 polypeptides were induced by UV-B. Polypeptides which were so abundant as to be detectable by Coomassie brilliant blue staining were then subjected to N-terminal amino acid sequence analyses. Two of the polypeptides were identified as a 23 kDa protein of PS II and 6 as a pathogenesis-related protein 5 (PR-5). Immunoblotting demonstrated that other PR proteins, PR-1 and PR-3 were also induced by UV-B. Salicylic acid (SA), which is an important component of signal transduction that leads to the expression of PR proteins and exhibition of acquired resistance to pathogens, increased in response to exposure to UV-B. In addition, the activity of phenylalanine ammonialyase, which catalyzes the synthesis from phenylalanine of trans-cinnamic acid, the endogenous precursor of SA, was transiently increased by UV-B irradiation. These results suggest that UV-B activates the signal transduction pathway, which is a common step in pathogen infection. Received 8 May 2000/ Accepted in revised form 29 August 2000     

Purification and Properties of Cytokinin-Binding Proteins from Tobacco Leaves

A cytokinin-binding protein complex was purified 700-fold fromleaves of tobacco (Nicotiana sylvestris). The purification procedureconsisted of four chromatographic steps on columns of DEAE-cellulose,Mono Q, Phenyl Superose and Superose 12, respectively. The purifiedcytokinin-binding protein complex behaved as a 130-kDa globularprotein on gel filtration. This complex contains two proteinspecies whose molecular masses are estimated to be 57 kDa and36 kDa. Binding to benzyl[8-¹⁴C]adenine was inhibited by adenine,ATP, zeatin and cAMP but not by indoleacetic acid. Scatchardanalysis indicated the existence of at least two cytokinin-bindingsites in the purified complex. The dissociation constant forthe high-affinity site was 2.1 10^-5 M. (Received October 19, 1992; Accepted February 27, 1993)     

Induction and Secretion of Pathogenesis-Related Proteins by Salicylate or Plant Hormones in Tobacco Suspension Cultures

Pathogenesis-related proteins (PR proteins), that are inducedin tobacco leaves in hypersensitive response to infection withtobacco mosaic virus (TMV) or by treatment with chemicals, werefound to be also inducible in a dedifferentiated system, tobaccosuspension culture. Quantitative determination of these proteinsusing anti PR 1a IgG showed that their increase started at aboutthe end of cell growth period and that their production couldbe enhanced by the addition of potassium salicylate, Eosin Yellowishand plant hormones (GA3, IAA and 2,4-D). The production dependedon the concentration of the chemical inducer and the cell lineused. In BY-2 cell line, PR proteins amounted to 12 µgat day 5 and then increased exponentially with time, reaching280 µg or 70 µg per g fr wt of cells at day 9 withor without the addition of 25 µM potassium salicylate.More than 90% of the induced PR proteins was found in the mediumand less than 10% in the cells at day 9. Peroxidase activityin the medium was constant throughout the experiment althoughtotal activity in the flask increased with cell growth, indicatingthat PR proteins are actively secreted into the medium. (Received November 12, 1986; Accepted March 6, 1987)     

Induction of Salicylic Acid {beta}-Glucosidase in Tobacco Leaves by Exogenous Salicylic Acid

Salicylic acid (SA) has been proposed to be an endogenous signalfor systemic acquired resistance to infection by pathogens inplants. In general, most SA is found in an inactive form asSA ß-glucoside (SAG). SAG seems to be a storage formof SA from which bioactive SA can be generated. Recent reportsindicate that ß-glucosidase might be involved in regulatingthe signaling activity of phytohormones. Therefore, it seemslikely that SA ß-glucosidase, the enzyme that hydrolyzesSAG to yield free SA, might also play an important role by regulatingthe level of free SA. Since hydrolysis of SAG seems to occurin intercellular spaces, we attempted to isolate SA ß-glucosidaseactivity from the intercellular spaces of SA-treated tobaccoleaves, where we found considerable amounts of the enzymaticactivity. Furthermore, increased levels of SA and SA ß-glucosidaseactivity were found in the leaves after treatment with exogenousSA. The role of SA ß-glucosidase in plant defensesystems is discussed. (Received November 15, 1994; Accepted January 20, 1995)     

Cd、Pb及其复合污染对烤烟叶片生理生化及细胞亚显微结构的影响

本研究以水培的烤烟给予不同浓度的Cd、Pb及其复合物处理10d后的烟叶为材料,分析了烟叶过氧化氢酶、硝酸还原酶的活性变化,测试了烟叶可溶性糖含量的变化情况,通过透射电子显微镜观察到了Cd和Pb对烟叶叶肉细胞亚显微结构的改变,特别是对叶绿体、线粒体和细胞核结构的损伤情况进行了详细观察。并探讨其毒害机理。研究结果表明：1)烟叶过氧化氢酶的活性剧烈地被Cd抑制;而随着Pb浓度的增加,其活性则表现为先增加后减弱的变化。2)Cd对硝酸还原酶活性的影响表现为先刺激增强,当Cd浓度超过50mg·L-1后,Cd剧烈地抑制其活性,当Cd浓度为200mg·L-1时,其活性几乎为零;Pb抑制烟叶硝酸还原酶的活性,仅在1000mg·L-1时出现一个低于正常活性的抗性峰。3)烟叶可溶性糖含量对Cd、Pb及其复合污染非常敏感,在较低浓度的污染处理时,其含量就明显下降,烟叶可溶性糖含量的变化可作为监测Cd、Pb污染的指标。4)Cd对烟叶叶肉细胞亚显微结构具有较强的损伤诱变作用,对细胞核、叶绿体和线粒体造成不可逆转的伤害,破坏了细胞正常生理活动所需的结构基础。电镜观察表明Cd严重地破坏细胞的膜结构。这可能是由于Cd离子与蛋白质结合而使蛋白质变性,从而使得以蛋白质为重要组成成份之一的膜的结构改变,功能丧失。5)在细胞膜的外面可以看到大量的Pb沉积粒,细胞膜可以阻止部分Pb进入原生质体内部,但在细胞质和叶绿体中仍可看到Pb沉积粒。Pb同样的损伤叶绿体、线粒体、细胞核的亚显微结构。     

烤烟叶片衰老期氨气挥发特征及其生理调控研究

以烤烟品种K326为试验材料,利用氨气收集装置测定烟叶的氨气挥发量,并利用谷氨酰胺合成酶(GS)抑制剂(Glufosinate)处理叶片和质外体提取等方法,研究了叶片氨气挥发及其与氮代谢相关生理指标的关系。结果表明:(1)随着叶片的衰老,氨气挥发量在叶龄70d时最大(10.96μg.m-2.h-1),与衰老初期(叶龄40d)相比增加了2.15倍;质外体NH4+浓度和pH、氨气补偿点逐渐上升,GS和硝酸还原酶(NR)活性下降,谷氨酸脱氢酶(GDH)活性升高,可溶性蛋白和总氮降解,叶片NH4+浓度升高。(2)GS抑制剂处理后,叶片组织NH4+浓度和氨气补偿点升高,氨气挥发量增大,与对照相比差异显著。(3)氨气挥发量与质外体NH4+浓度、质外体pH和氨气补偿点呈极显著或显著正相关,与GS活性呈显著负相关,与GDH活性呈显著正相关,与叶片组织NH4+浓度等其他指标相关性不显著。研究认为,烤烟叶片衰老期间氨挥发量大幅上升,挥发量的大小受气孔氨气补偿点、GS和GDH活性的直接调控,以及其他氮素代谢相关指标的间接调控,其中GS起主导作用。     

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)     

Changes in the Proteins of Bean Leaves Infected With Tobacco Necrosis or Alfalfa Mosaic Viruses

Acidic extracts from TNV and AMV infected “Saxa” bean leaves were electrophoretically examined for protein content. In native conditions of resolution (PAGE) at least three protein bands (PS_1–3) not present in the control were found. In denaturing conditions (SDS–PAGE) at least one (PS_a), but often two or three such proteins (PS_{b, c}) were found in the same extracts. Chromatographic resolution of proteins on Sephadex G-100 column resulted in partial purification of the PS-proteins. Additional, not known before, slow-migrating protein (PS₀) induced by hypersensitive viral infection was discovered in some of the eluted fractions. Both PS_0–3 and PS_a–c proteins were present in the same fractions. This fact suggests their similarity in molecular weights and/or shapes.     

Sunflower (Helianthus annuus L.) Pathogenesis-Related Proteins (Induction by Aspirin (Acetylsalicylic Acid) and Characterization)

Sunflower leaf discs floated on a solution containing aspirin (acetylsalicylic acid) produced a set of new proteins extractable at pH 5.2 and excreted into the intercellular space. More than 80% of the proteins found in the intercellular fluids of induced leaf discs have been identified as pathogenesis-related (PR) proteins by their immunological relationship with tobacco PR proteins. Members of the four major classes of PR proteins have been characterized. Sunflower PR proteins of type 1 (PR1) and of type 3 (PR3) were found to have acidic isoelectric points, whereas the induced PR protein of type 2 (PR2) had a basic isoelectric point. Members of the type 5 PR proteins (PR5), known in tobacco as thaumatin-like proteins, showed a more complex pattern. Multiple sunflower PR5 isomers of similar molecular weight but of different isoelectric points were excreted from the cells in response to the aspirin treatment. PR2 and PR3 proteins were found at very low basal levels in untreated leaves, whereas PR1 and PR5 proteins could not be detected at all in the same extracts. Glucanase and chitinase activities were always associated with PR2 and PR3 proteins in partially purified sunflower extracts. All of these data indicate that, in response to aspirin treatment, sunflower plants produce a complete set of PR proteins characterized by an apparently exclusively extracellular localization.     

Biphasic Temporal and Spatial Induction Patterns of Defense-Related mRNAs and Proteins in Fungus-Infected Parsley Leaves

Previous experiments using in situ RNA hybridization have shown that the mRNAs encoding phenylalanine ammonia-lyase, 4-coumarate:coenzyme A ligase, and pathogenesis-related protein 1 accumulated transiently around fungal infection sites in parsley (Petroselinum crispum) leaf buds. These studies have now been extended by (a) analyzing different stages of the infection process and (b) monitoring the timing of appearance and the spatial distribution of the proteins as well as the corresponding mRNAs. An early and short period of mRNA induction throughout a large portion of the infected leaf was followed by a longer period, during which the mRNA levels remained high in a more localized area around the site of fungal penetration with sharp borders toward the surrounding tissue. This biphasic pattern of mRNA accumulation was followed after some delay by the same pattern of protein accumulation.