Tobacco Cell Wall Research Articles

Pectin's polygalacturonate single sugar chain helix visualized in Pt/C replicas

Pectic polysaccarides are 34% by weight of tobacco cell wall materials, more than any other cell wall component (cellulose, 18.7%; hemicellulose, 11.4%; protein, 21.6%; lignin, 4.1%). During cell growth, the cell wall is loosened by acidification which then enables it to elongate by its own turgor pressure. It has been suggested that the primary action of acidification is the loosening of a calcium pectate gel which at 34% by weight must permeate the wall and bind the cell wall together. This same work suggests that minimum Ca2+binding occurs at pH 3 and lower and maximum Ca2+ binding occurs at pH 6 and that Ca2+ binding capacity is lost by treatment with polygalacturonase. If the capacity for acid-induced wall loosening Is switched on, which is dependent on protein not carbohydrate synthesis, then the rate of wall loosening is proportional to the pH between 5.7 and 4.5.

Inhibition of the hypersensitive response in tobacco by pectate lyase

Tobacco leaves pretreated with low levels of an Erwinia chrysanthemi pectate lyase isozyme did not express the hypersensitive response which normally follows inoculation with Pseudomonas syringae pv. pisi . The enzyme was obtained from the periplasmic shock fluids of an Escherichia coli strain containing a cloned pectate lyase gene ( pelC ) from E. chrysanthemi . Crude shock fluids or homogenous pectate lyase purified from the fluids were effective in preventing the hypersensitive response in tobacco leaves; shock fluids from E. coli lacking the pelC gene and buffer treatments were ineffective. Prevention of the hypersensitive response by pectate lyase was light-dependent and required an induction period of approximately 1·5 h prior to challenge with P. syringae pv. pisi . Pretreatment of tobacco leaves with a purified isozyme preparation containing 0·03 units (umol product min −1 at pH 6·0) of activity per millilitre and less than 3 ng protein ml −1 caused suppression of the multiplication of both compatible P. syringae pv. labaci ) and incompatible ( P. syringae pvs. pisi and syringae ) bacteria. Pectate lyase pretreatment also inhibited the production of disease symptoms by P. syringae pv. labaci . Heat-stable products released by pectate lyase from isolated tobacco cell walls were similarly able to suppress the hypersensitive response in tobacco leaves, suggesting that the protection phenomenon may be mediated by galacturonic acid-containing oligosaccharides.

Binding of Pseudomonas solanacearum fimbriae to tobacco leaf cell walls and its inhibition by bacterial extracellular polysaccharides

Fimbriae may mediate attachment of plant pathogenic bacteria to host cell walls, and this is thought to be involved in the elicitation of resistant (hypersensitive) responses. Fimbrial extracts and purified fimbriae of the avirulent B1 strain of Pseudomonas solanacearum agglutinated isolated tobacco cell wall fragments, erythrocytes and polystyrene latex spheres. In all three assays, agglutination was inhibited by purified extracellular polysaccharides from the virulent K60 strain of P. solanacearum, Rhizobium meliloti, and Agrobacterium tumefaciens. Bovine serum albumin, fetuin and conalbumin were also strong inhibitors of agglutination. Pretreatment of tobacco cell -walls with P. solanacearum extracellular polysaccharide greatly reduced their agglutination by Bl fimbriae. This inhibition was not reversed by sugars, indicating that specific carbohydrate receptors on the tobacco cell wall or the erythrocyte surface may not be involved in agglutination. It is proposed that the fimbriae bind to these surfaces via nonspecific interactions. Results are discussed in relation to (a) the possible involvement of fimbriae in immobilization of avirulent bacteria in vivo, and (b) the role of extracellular polysaccharide in preventing the attachment of virulent strains by virtue of its interaction with the tobacco cell wall.

Surface components involved in bacterial pathogen-plant host recognition.

During their initial association with plant hosts, pathogenic bacteria interact with plant cell walls. The results of this interaction appear to determine whether bacterial multiplication will take place. With one group of bacterial plant pathogens (e.g. Agrobacterium tumefaciens), attachment to the host surface appears essential for pathogenesis. With another group (e.g. Pseudomonas solanacearum), only those strains that do not attach to the host cell wall are able to multiply in the intercellular spaces. Attachment of many incompatible strains to tobacco mesophyll cell walls leads to a rapid hypersensitive response (HR) and a drastic reduction in bacterial multiplication. Our working hypothesis is that these differences in host response to strains of P. solanacearum are the result of a recognition response in which surface components of both host and pathogen play important roles. Our approach is based on the use of spontaneous or transposon (Tn5)-generated mutants of strains K60 (virulent) and B1 (avirulent) that differ in surface components and in their ability to attach to host cells and to induce the HR. A study of the surface components of bacterial and tobacco cell walls has led to the tentative conclusion that bacterial lipopolysaccharide (LPS) and plant hydroxyproline-rich glycoproteins mediate initial attachment, apparently as a result of charge-charge interaction. This initial attachment is reversed by high salt concentrations during the first 15 min, but not thereafter. Firm attachment appears to depend on hydrophobic interactions mediated by bacterial pili. At the normal ionic strength of intercellular fluids, extracellular polysaccharide (EPS) appears to inhibit only the pili-mediated attachment. Several HR- mutants of strain B1 have been obtained by Tn5 insertion, but they remain avirulent on tobacco. We are examining the EPS, LPS and pili production, and the attachment characteristics of these strains.

Ultrastructural Changes of Tobacco Cell Walls Following Sequential Extraction

Abstract The role of calcium in plants has been the subject of research for many years. Calcium has been postulated to cover a wide variety of functions which have major and minor influences on the plant's metabolism. Calcium interaction with pectin has been postulated as a major source of cell wall stability, however, no direct measurements of this interaction have been made. In this study, a sequential extraction method has been utilized to fractionate the various forms of calcium present in cured bright and Burley tobacco. The extraction method uses water, potassium chloride, lanthanum chloride and hydrochloric acid with emphasis on the lanthanum chloride extraction which appears to preferentially replace the structural calcium. Extraction data in conjunction with light microscopy (LM) and transmission electron microscopy (TEM) data have been used to predict the role of structural calcium in the cell wall. Oxalate and calcium analyses have been made at each of the extraction steps for bright tobacco. It is shown that the major portion of calcium extracted is not associated with oxalate except for the hydrochloric acid step. The data show that approximately 20 % of the calcium is structurally related and that calcium oxalate utilizes a maximum of another 20 % of the total calcium. The remaining 60 % is non-structural and non-oxalate and is probably inorganic salts and salts of organic acids. Combining these two techniques provides a means of obtaining quantitative information that either technique used independently could not provide. The techniques used in this study are applicable to the investigation of other plant types and may be useful in furthering the general knowledge of the role of calcium in plant materials.

Lipopolysaccharide-Defective Mutants of the Wilt Pathogen Pseudomonas solanacearum

Lipopolysaccharide (LPS)-defective mutants of Pseudomonas solanacearum were used to test the hypothesis that differences in LPS structure are associated with the ability or inability of different strains to induce a hypersensitive response (HR) in tobacco. To obtain these mutants, LPS-specific bacteriophage of P. solanacearum were isolated and used to select phage-resistant mutants of the virulent, non-HR-inducing strain K60. The LPS of 24 of these mutants was purified and compared with that of K60 and its HR-inducing variant, B1. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, LPS from K60 and other smooth strains separated into many evenly spaced bands that migrated slowly, whereas LPS from B1 and most phage-resistant strains separated into one to three bands that migrated rapidly. Carbohydrate analysis showed that the LPS of the phage-resistant strains lacked O-antigen sugars (rhamnose, xylose, and N-acetylglucosamine) and could be grouped into (i) those that had all core sugars (rhamnose, glucose, heptose, and 2-keto-3-deoxyoctonate), (ii) those that had no core rhamnose, and (iii) those that lacked all core sugars except for 2-keto-3-deoxyoctonate. The LPS composition of 10 of the rough, phage-resistant mutants was similar to that of the HR-inducing strain, B1, yet none of them induced the HR. Only 2 of 13 mutant strains tested caused wilting of tobacco, and these had rough LPS but produced large amounts of extracellular polysaccharide, unlike most LPS-defective mutants. The evidence did not support the hypothesis that the initial interaction between rough LPS and tobacco cell walls is the determining factor in HR initiation.

Neutral Sugar Analysis of Tobacco Cell Wall Fractions

AbstractA method for the determination of the neutral sugar content of tobacco cell wall fractions has been developed. The seven neutral sugars - rhamnose, fucose, arabinose, xylose, mannose, glucose and galactose - determined in the analysis represent the usual glycosyl residues found in plant cell wall polysaccharides. In this procedure, the structural polysaccharides are acid hydrolysed into their constituent monosaccharides. The monosaccharides are then reduced with sodium borohydride to the corresponding alditols. The alditols are converted to the acetates with acetic anhydride. The alditol acetates are quantitatively measured by capillary gas chromatography with flame ionization detection. Both 72 % H

A hydroxyproline-rich bacterial agglutinin from potato: its localization by immunofluorescence

Potato tubers (cv. Katahdin) contain a hydroxyproline-rich glycoprotein (HPRG) that agglutinates certain avirulent strains of the bacterial wilt pathogen, Pseudomonas solanacearum. This and similar agglutinins are thought to play an important rôle in the immobilization of incompatible bacteria in potato and tobacco tissues. The agglutinin from potato tubers was purified by ion exchange chromatography. Antisera to the intact or deglycosylated agglutinin were obtained from New Zealand white rabbits after multiple intradermal and intramuscular injections. Immunoglobulins were precipitated with (NH4)2SO4 and antibodies specific for the agglutinin were purified by affinity chromatography. Frozen sections of petiole or leaf tissue from tobacco and potato were treated firstly with sheep normal immunoglobulin and then with either anti-agglutinin antibodies or normal rabbit immunoglobulin for 20 min. The sections were rinsed and then treated with fluorescein isothiocyanate-conjugated sheep anti-rabbit immunoglobulin. When the sections were examined by fluorescence microscopy, it was determined that anti-agglutinin antibodies bound only to the cell walls, particularly those of parenchyma. Fluorescence was also evident on the cell walls of tobacco and potato xylem vessels, epidermis, and collenchyma. Control sections treated with normal rabbit immunoglobulin did not bind the labelled anti-rabbit immunoglobulin. Cell walls in tissue sections from non-solanaceous plants such as soybean, corn, or begonia, treated in the same manner, were also stained by the labelled antibodies. Antibodies to both intact and deglycosylated potato agglutinin bound to these plant cell walls, indicating that the receptors are proteins with antigenic determinants which are similar to those of proteins from potato or tobacco cell walls. Such proteins (HPRGs) are common components of plant cell walls and may play a rôle in immobilizing bacteria that gain access to the intercellular spaces.

Role of Peroxidase in Lignification of Tobacco Cells

The two peroxidase isoenzyme groups (G(I) and G(III)) localized in the cell walls of tobacco (Nicotiana tabacum L.) tissues were compared with respect to their capacity for NADH-dependent H(2)O(2) formation. Peroxidases of the G(III) group are slightly more active than those of the G(I) group when both are assayed under optimal conditions. This difference is probably not of major regulatory importance. NADH-dependent formation of H(2)O(2) required the presence of Mn(2+) and a phenol as cofactors. The addition of H(2)O(2) to the reaction mixture accelerated subsequent NADH-dependent H(2)O(2) formation. In the presence of both cofactors or Mn(2+) alone, catalase oxidized NADH. However, if the cofactors were absent or if only dichlorophenol was present, catalase inhibited NADH oxidation. No H(2)O(2) accumulation occurred in the presence of catalase. Superoxide dismutase inhibited NADH oxidation quite significantly indicating the involvement of the superoxide radical in the peroxidase reaction. These results are interpreted to mean that the reactions whereby tobacco cell wall peroxidases catalyze NADH-dependent H(2)O(2) formation are similar to those proposed for horseradish peroxidase (Halliwell 1978 Planta 140: 81-88).

Adsorption of pseudomonads to tobacco cell walls and its significance to bacterium—host interactions

Cells of Pseudomonas fluorescens, a saprophyte, P. pisi, a pea pathogen, and P. tabaci, a tobacco pathogen, were introduced into the intercellular spaces of tobacco leaf tissue and subsequently recovered by centrifugation. The percentage recoveries of P. pisi and P. tabaci cells were respectively at least 10·8 and 4·1 times that of P. fluorescens cells. These results indicate that the efficiency of adsorption to tobacco cell walls is greatest for P. fluorescens and least for P. pisi. Thus the induction of the hypersensitive reaction in tobacco by P. pisi does not require efficient adsorption of bacterial cells to tobacco cell walls. Scanning electron micrographs showed that the tobacco cell wall beneath P. fluorescens Gells was eroded within 5 h of inoculation. Adsorption of bacterial cells to tobacco cell walls may facilitate this erosion.

Hydroxyproline arabinosides from suspension-cultured tobacco cell wall.

Hydroxyproline tri- and tetra-arabinosides were isolated from suspension-cultured tobacco cell wall preparation by Ba(OH)2 treatment followed by gel filtration and ion-exchange chromatography. Methylation analysis showed that they consisted of terminal, 1,2-linked and 1,3-linked arabinofuranosyl residues in molar ratios of 1: 2: 0 and 1:2: 1, respectively. A part of trans-4-hydroxy-l-proline in the arabinosides was revealed to have been isomerized into cis-4-hydroxy-d-proline during the alkaline hydrolysis.