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 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.     

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 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)     

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)     

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.     

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.     

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.     

Estimation of Chlorophyll in Tobacco Leaves by Direct Photometry

A simple portable instrument utilizing a cadmium sulphide light-dependentresistor in a bridge circuit, and its use in the in vivo estimationof total chlorophyll content of tobacco leaves, are described.The method has wide applicability in plant physiological andnutritional research, and in agriculture. The relationship betweeninstrument readout and chlorophyll content (calibration curve)was nearly parabolic and was determined largely by the light-scatteringproperties of the leaf. The effects of some environmental andplant parameters on this relationship and on the precision ofthe estimation were analysed. The importance of sampling procedurewas emphasized by a marked heterogeneity of chlorophyll distributionfound within leaves of all ages. A scattered-transmission spectrophotometerwas found to give the same type of calibration curve, and wasused to demonstrate that the ‘reference wavelength’correction used by previous authors has no effect on the shapeof the curve or the precision of the method.     

Analysis of the Proteins in the Leaves of Transgenic Tobacco Plants Expressing Double-Stranded RNA Upon Viral Infection

Generation of transgenic tobacco plants, producing double-stranded RNA with no homology to tobacco genome sequences is reported. The RNA synthesis is mediated by a construct containing an inverted repeat of the pBR322 tetracycline-resistance gene fragment under control of the 35S CaMV promoter. Analysis of the resistance of transgenic plants to the tobacco mosaic virus revealed the changes in the protein spectra of the infected plants. The 25- and 30-kDa proteins found were not detected in the extracts of normal plants. Amino acid sequencing of the 30-kDa peptide with subsequent computer database search revealed the homology of this protein to the hydrolases belonging to the group of plant -glucanases. The role of the novel polypeptides in an increase of the resistance of transgenic plants to TMV, and also the possibility of the regulation of their expression by nonhomologous dsRNA are discussed.