Search

SUMMARY The deposition of xyloglucans in the incipient wall of Rubus fruticosus cells grown in suspension was examined by transmission electron microscopy. The general distribution of polysaccharides and the formation of a middle lamella was followed along the cell growth period with cytochemical reagents. A predominant morphological change of the cell walls, which are in direct contact with the culture medium, is the reorientation of the superficial cellulose microfibrils which form a loose layer oriented towards the medium. Changes of xyloglucan localization during growth were observed via a polyclonal antibody. When the cells age the loosened superficial microfibrils are progressively depleted in the associated xyloglucans. This phenomenon was accelerated when the nonasaccharide XG9 (XXFG) was added to the culture medium. This is the first observation of an effect of oligosaccharides on the morphology of the plant cell wall at the ultrastructural level.

In nature, wood can be completely degraded by lignolytic fungi. Basidiomycetous white-rot fungi are particularly efficient wood degraders because of their ability to completely mineralize lignin [1,2]. The enzymes produced by white-rot fungi are directed against the two types of macromolecules that make up the walls of wood cells, Polysaccharides and polyphenols.. These enzymes consist of Polysaccharide hydrolases that break down cellulose and hemicelluloses, and of a series of oxidizing enzymes that are able to attack and depolymerize lignins. The latter group consists of a variety of extra-cellular enzymes among which peroxidases — principally lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP) — together with phenol oxidases laccase (Lac), aryl-alcohol oxidase (AAO) and other nonphenol oxidases, contribute to the highly complex mechanisms leading to lignin depolymerization, aromatic rings opening and demethylation [3, 4, 5]. An essential step in the enzymic attack of lignin is the formation of aryl cation radicals originating from both phenolic and nonphenolic aromatic rings [1, 4, 6]. This is the typical mode of action of LiP which uses hydrogen peroxide to generate aryl cation radicals. The other major peroxidase, particularly efficient in lignin oxidation, is manganese peroxidase which oxidizes manganese ions from Mn+2 to Mn+3. These ions that have a short life-time, are stabilized by organic acids, mostly dicarboxylic acids, and are thought to diffuse in the tight lignocellulosic network of the wood cell walls [7]. Another component of the enzyme complex participating in lignin oxidation is laccase, a phenol oxidase which seems now to be rather widespread amont the most efficient wood-degrading white-rot fungi [8].

Using tobacco transgenic lines altered in the monolignol biosynthetic pathway and which differ in their lignin profiles we have evaluated lignin deposition at the cellular and subcellular levels using several microanalytical techniques. Surprisingly, whereas a Cinnamoyl CoA reductase (CCR) down-regulated line with a strong decrease in lignin content exhibited an overall reduction in lignin deposition in the walls of the different xylem cell types, this reduction was selectively targeted to the fibers in a double transformant (down-regulated for both CCR and Cinnamyl alcohol dehydrogenase (CAD)) displaying a similar degree of global lignin content decrease. Fiber and vessel secondary walls of the transgenic tobacco line homozygous for the ccr antisense gene (CCR.H) down-regulated plants were dramatically destructured, particularly in the S2 sublayer, whereas the deposition of lignins in the S1 sublayer was not significantly modified. In contrast, cell wall organization was slightly altered in xylem cells of the double transformant. The relative distribution of non-condensed and condensed units in lignin, evaluated microscopically with specific antibodies, was differentially affected in the transgenics studied and, in a general way, a drop in non-condensed lignin units (beta- 0-4 interunit linkages) was associated with a loss of cohesion and extensive disorganization of the secondary wall. These results demonstrate that lignification is tightly and independently regulated in individual cell types and cell wall sublayers. They also show that down-regulation of specific genes may induce targeted changes in lignin structure and in spatial deposition patterns of the polymer.

Solutions of cellulose diacetate (CDA) from two sources (cotton linters and wood pulp Floranier) were analysed in various solvents by size exclusion chromatography (SEC). Without special precautions, the SEC chromatograms presented three peaks — or prehumps — before the main polymer peak. The first prehump which could be eliminated by ultracentrifugation corresponded to microgels whose sugar composition was determined. These microgels were also investigated by electron microscopy, X-ray and electron diffraction analysis. They corresponded mainly to cellulose triacetate (CTA-II) in the case of CDA from cotton linters and a mixture of CTA-II and xylan diacetate (XDA) in the case of CDA from the wood pulp Floranier. The second and third prehumps could be attributed to ionic effects corresponding to the association of remaining sulfate groups on the CDA molecules with residual calcium. It was found that these ionic effects could be eliminated by the addition of LiBr or LiCl to the elution solvents. This led to chromatograms devoid of prehumps.

alpha-Linked D-xylosyl side chains represent the typical feature common to all xyloglucans not shared by other cell wall polysaccharides. Since no easily available alpha-D-xyloxidase is known, advantage was taken of the conformational and configurational homologies between alpha-D-xylopyranose and alpha-D-glucopyranose to make an alpha-D-glucosidase-gold complex which was able to recognize alpha-D-xylosyl terminal residues of xyloglucans. This marker was used together with alpha-L-fucosidase gold complex for the double labeling on two different structural features of the same macromolecule in plant primary cell wall.

Enzyme-gold complexes have been prepared with an endo beta 1----4 xylanase (EC 3.2.1.8) and a beta 1----4 mannanase (EC 3.2.1.78). The complexes were applied to ultrathin sections of plant cell walls for the ultrastructural localization of xylans in different tissues of a graminea and for the localization of glucomannans in the tracheids of spruce wood. The method proved to be highly specific and gave a very good contrast of the substrate polysaccharides. Used in conjunction with other cytochemical staining the enzyme-gold labelling provided information about the relative distribution of pectic polymers and xylans in primary walls.

Xylose-rich polysaccharide fractions were isolated from cell walls of muskmelon (Cucumis melo L. cv. Cantaloup Charentais) stems. A xylan with a (1 4) linked backbone has been characterized as a component of these fractions. Two xylanases excreted by the fungus pathogen Colletotrichum lagenarium were able to degrade this substrate both after extraction and when it was included in the crude cell walls. The results suggest that the xylanases are involved in the parasitism of this pathogen.

The site of cleavage of the capsular polysaccharide from Klebsiella K19 by the endoglycanase associated with particles of Klebsiella bacteriophage luminal diameter 19 was determined. The specific cleavage of the bond Rhap-(1----2)-Rhap provided a series of oligosaccharides having rhamnose at the reducing end. The enzyme is thus an alpha-rhamnosidase. Structural studies on the oligomers confirmed the sequence of the repeating unit of the polysaccharide from K19. The 1H- and 13C-n.m.r. spectra of the homologous series of oligosaccharides corresponding to one, two, three, and four repeat-units exhibit important differences that denot variation of conformation with chain length. The bacteriophage acted on modified forms of K19 polysaccharide to provide a series of linear oligomers, and emphasized the essential role of the negative charge on the uronic acid in the action of the glycanase.

Five different glucomannan samples were recrystallized from dilute solution. Depending on the experimental conditions, the crystals obtained could be identified as corresponding to the mannan I (anhydrous precipitate of more or less regular lozenge-shaped crystals) or mannan II (hydrated gel-forming pseudo-fibrillar precipitate). High-molecular-weight material, low temperature of crystallization, or a polar crystallization medium favored the mannan II polymorph, whereas a low-molecular weight, a high temperature of crystallization, and a crystallization medium of low polarity yielded the mannan I polymorph. Since the base-plane unit-cell dimensions are fairly constant with respect to variation of glucose, it is likely that isomorphous replacement of mannose by glucose occurs in glucomannan crystallization; the data also indicate that perfection of the glucomannan crystals was reduced in specimens having a high glucose:mannose ratio. The oriented crystallization of glucomannan on cellulose microfibrils was also studied under conditions where the mannan I polymorph was obtained. This gave shish-kebab structures that were characterized.