A fucoidan fraction from Ascophyllum nodosum.

A fucoidan fraction was purified from the brown alga Ascophyllum nodosum. The polysaccharide contained L-fucose and sulfate as the only constituents. Combination of methylation analysis, Smith degradation, FTIR and NMR spectroscopy on the native and the de-sulfated polymers demonstrated that the fucoidan consisted of a highly branched core region with primarily alpha-(1-->3)-linked fucosyl residues and a few alpha-(1-->4) linkages. Branch points were at position 2 of the -->3-linked internal residues. The side chains consisted of single and multi-unit fucosyl residues. The combined analytical data suggested also a complex sulfation pattern with substitution principally at position 2 and/or position 4. Such diversity in the structural features of this fucoidan may be of importance for its various biological properties.

In situ analysis of lignins in transgenic tobacco reveals a differential impact of individual transformations on the spatial patterns of lignin deposition at the cellular and subcellular levels.

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.

Simultaneous down-regulation of caffeic/5-hydroxy ferulic acid-O-methyltransferase I and cinnamoyl-coenzyme A reductase in the progeny from a cross between tobacco lines homozygous for each transgene. Consequences for plant development and lignin synthesis.

Inhibition of specific lignin biosynthetic steps by antisense strategy has previously been shown to alter lignin content and/or structure. In this work, homozygous tobacco (Nicotiana tabacum) lines transformed with cinnamoyl-coenzyme A reductase (CCR) or caffeic acid/5-hydroxy ferulic acid-O-methyltransferase I (COMT I) antisense sequences have been crossed and enzyme activities, lignin synthesis, and cell wall structure of the progeny have been analyzed. In single transformed parents, CCR inhibition did not affect COMT I expression, whereas marked increases in CCR activity were observed in COMT I antisense plants, suggesting potential cross talk between some genes of the pathway. In the progeny, both CCR and COMT I activities were shown to be markedly decreased due to the simultaneous repression of the two genes. In these double transformants, the lignin profiles were dependent on the relative extent of down-regulation of each individual enzyme. For the siblings issued from a strongly repressed antisense CCR parent, the lignin patterns mimicked the patterns obtained in single transformants with a reduced CCR activity. In contrast, the specific lignin profile of COMT I repression could not be detected in double transformed siblings. By transmission electron microscopy some cell wall loosening was detected in the antisense CCR parent but not in the antisense COMT I parent. In double transformants, immunolabeling of non-condensed guaiacyl-syringyl units was weaker and revealed changes in epitope distribution that specifically affected vessels. Our results more widely highlight the impact of culture conditions on phenotypes and gene expression of transformed plants.

Ligninolytic characteristics of Pleurotus ostreatus strain F6 and its monokaryotic protoplast derivative P19.

A stable isolate of Pleurotus ostreatus P19 differing in some morphological and physiological characteristics from its parental wild-type strain F6 was obtained via protoplast isolation during the preparation of strains with altered ligninolytic abilities. The isolate is monokaryotic, does not form clamp-connections, and produces much higher activities of enzymes involved in lignin modification (laccase, manganese peroxidase). Cellulase activity was comparable to that of wild-type strain F6, but the xylanase activity was slightly higher in isolate P19. However, this monokaryotic derivative degrades lignin at a slightly lower rate than its parental strain F6. Electron microscopy observations of wood degradation as a function of mycelium growth were performed on three zones of birch wafers delimited according to the distance from the point of inoculation. The different stages of fungal mycelium growth showed differences in the ultrastructural patterns of the decay not only between the strains P19 and F6, but also depending on the distance from the point of inoculation. This suggests a spatio-temporally controlled secretion of enzymes along the hyphae. The enhanced ability of P19 to degrade the condensed forms of lignin in middle lamellae is correlated to its higher laccase activity.

Structure of extracellular polysaccharide produced by lignin-degrading fungus Phlebia radiata in liquid culture.

The extracellular material (EM) produced by the white rot fungus Phlebia radiata cultured in an N-limited liquid medium was studied. Carbohydrate analysis showed maximum concentration of glucose as the major monosaccharide component of EM was found on postinoculation day 9. Beyond day 9 of cultivation the proportion of glucose decreased suggesting that the glucan component of EM had been further metabolized. The analysis of EM at day 9 revealed the presence of the following monosaccharides (in relative %): glucose (62); galactose (16); mannose (13); xylose (4); and fucose (5). The carbohydrate analysis together with the presence of protein in EM corresponds to a mixture of glucan and glycoprotein. Purification by trypsin treatment yielded an enriched glucose-containing extracellular polysaccharide (EPS). Methylation analysis identified EPS as (1-3)-beta-D-glucan highly branched at C-6. The structure of the glucan was confirmed by 13C-NMR spectroscopy. The results suggest that P. radiata's EPS is entangled with a glycoprotein in a complex that makes the extracellular sheath surrounding the hyphae.

Cassia spectabilis DC seed galactomannan: structural, crystallographical and rheological studies.

The seeds of Cassia spectabilis DC (family: Leguminoseae), an Indian fast growing spreading tree, contain about 40% of endosperm and possess the characteristics of becoming a potential source of commercial gum. The purified galactomannan shows Mw 1.1 x 10(6), intrinsic viscosity [eta] 615ml/g with k' = 1.706 x 10(-1), and a mannose to galactose ration of 2.65. The hydrolysis of the fully methylated polysaccharide reveals clearly the expected structure of legume galactomannans. The orthorhombic lattice constants of the hydrated gums are as follows: a = 9.12 A, b = 25.63 A and c = 10.28 A. The results of X-ray fiber studies show that the b dimension of the unit cell is very sensitive to relative humidity (RH), galactose substitution and orientation of the films. The probable space group symmetry of the unit cell is P2(1)2(1)2. Rheological studies of the galactomannan have shown that the transition from semi-dilute to dilute regime occurs at a critical concentration Cc* = 2.75. The slope of the log-log plot of specific viscosity versus C at zero shear rate is 5.87 in the more concentrated regime. The viscoelastic and critical shear rate behavior indicate the characteristics of a coil polymer. The large dependence of the viscosity on the coil overlap parameter is probably due to polymer-polymer interactions and peculiarity of the galactose distribution along the chain. Above 20 g/L concentration, the rheological behavior of the gum is like the one of a weak-gel.

Xylose-rich polysaccharides from the primary walls of embryogenic cell line of Pinus caribaea.

Embryogenic cell lines of Pinus caribaea were isolated from somatic embryogenesis from zygotic embryos. Previous studies showed that the proteins and glycoproteins were characteristic of the embryogenic state. In the present work we were seeking typical feature in the polysaccharide from the cell walls of embryogenic calli at nine days of culture. Sequential extraction with water, ammonium oxalate, dimethyl sulfoxide, sodium borohydride and 4.3 M potassium hydroxide revealed that the extracted polysaccharides contained high proportions of arabinose and significant amounts of xylose. Fractionation of the hydrosoluble polymers on DEAE cellulose afforded a xylose-rich fraction (80% xylose, 24% glucose and lower properties of fucose and mannose). Methylation analysis and 13C-NMR spectra showed that the glycan backbone consisted of beta 1 --> 4 linked xylosyl residues Similar study of the fractions extracted respectively with DMSO and 4.3 M KOH showed the presence of polydisperse glycoxylans but excluded the presence of xyloglucan in significant amount. This could be a characteristic feature of embryogenic cells walls of Pinus caribaea or could be typical of cells grown as calluses. In the various fractions obtained from DEAE cellulose chromatography of the alkaline extract the infrequent occurrence of fucoxylans beside an arabinogalactan showed again the unusual nature of the cell wall polymers of this embryogenic lines, which seems to differ greatly from those found in the primary wall of cells from suspension cultures.

Study of lignification by noninvasive techniques in growing maize internodes. An investigation by Fourier transform infrared cross-polarization-magic angle spinning 13C-nuclear magnetic resonance spectroscopy and immunocytochemical transmission electron microscopy.

Noninvasive techniques were used for the study in situ of lignification in the maturing cell walls of the maize (Zea mays L.) stem. Within the longitudinal axis of a developing internode all of the stages of lignification can be found. The synthesis of the three types of lignins, p-hydroxyphenylpropane (H), guaiacyl (G), and syringyl (S), was investigated in situ by cross-polarization-magic angle spinning 13C-solid-state nuclear magnetic resonance, Fourier transform infrared spectroscopy, and immunocytochemical electron microscopy. The first lignin appearing in the parenchyma is of the G-type preceeding the incorporation of S nuclei in the later stages. However, in vascular bundles, typical absorption bands of S nuclei are visible in the Fourier transform infrared spectra at the earliest stage of lignification. Immunocytochemical determination of the three types of lignin in transmission electron microscopy was possible thanks to the use of antisera prepared against synthetic H, G, and the mixed GS dehydrogenative polymers (K. Ruel, O. Faix, J.P. Joseleau [1994] J Trace Microprobe Tech 12: 247-265). The specificity of the immunological probes demonstrated that there are differences in the relative temporal synthesis of the H, G, and GS lignins in the different tissues undergoing lignification. Considering the intermonomeric linkages predominating in the antigens used for the preparation of the immunological probes, the relative intensities of the labeling obtained provided, for the first time to our knowledge, information about the macromolecular nature of lignins (condensed versus noncondensed) in relation to their ultrastructural localization and development stage.

Changes in wall-bound polysaccharidase activities during the culture cycle of a Rubus fruticosus cell suspension.

Several hydrolytic enzyme activities were detected in the wall of developing cells of Rubus fruticosus in suspension culture. The corresponding substrates of the enzymes are mostly polysaccharide wall constituents, except for chitinase activity. The activities measured when the enzymes were in the free state or wall-bound showed the positive influence of the cell wall micro-environment. Changes in the activities during a cell culture cycle demonstrated that those enzymes acting on xyloglucans behaved differently from the others, and suggest that xyloglucans undergo modifications in vivo over a longer period of time during the exponential growth phase. The same activities were identified in the culture medium. Endo-1,4-beta-D-glucanase activities which depolymerized carboxymethylcellulose (CMC) and xyloglucans (XG) were assayed viscosimetrically. It was found that XG oligosaccharides exhibited an inhibitory effect on the depolymerization of xyloglucans but not on that of CMC. This suggests that true xyloglucanases are present in the culture of Rubus cells.

Structural features and biological activity of xyloglucans from suspension-cultured plant cells.

Different xyloglucan (XG) fractions were isolated from Rubus fruticosus cells cultured in suspension. Sequential extraction showed that two distinct xyloglucans existed in the primary walls. The first could be easily extracted in alkali and the second was tightly associated to cellulose. A third fraction was isolated from the extracellular polysaccharides of the culture medium. The alkali-soluble XG and the extracellular XG showed many structural features in common. By use of an anti-XG polyclonal antibody, electron microscopy examination suggests that the extracellular hemicellulose is progressively released from the wall by a sloughing mechanism. Oligosaccharides prepared from the extracellular XG were purified and their structure examined by FAB-ms technique. When the nonasaccharide was added at low concentrations (10(-5) mg/ml) to the culture medium it was able to elicit several different glycanohydrolase activities associated to the cell wall.

Involvement of an Extracellular Glucan Sheath during Degradation of Populus Wood by Phanerochaete chrysosporium.

Observations by transmission electron microscopy of wood samples of Populus tremula inoculated with the white rot fungus Phanerochaete chrysosporium showed that, at certain stages of their growth cycle, hyphae were encapsulated by a sheath which seems to play an active role in the wood cell wall degradation. Chemical and immunochemical techniques and C nuclear magnetic resonance spectroscopy were applied to demonstrate the beta-1,3-1,6-d-glucan nature of the sheath. Double-staining methods revealed the interaction between the extracellular peroxidases involved in lignin degradation and the glucan mucilage. The glucan was also shown to establish a material junction between the fungus and the wood cell wall. It was concluded that, by means of these interactions, the sheath provides a transient junction between the hyphae and the wood, thus establishing a point of attachment to the site of the degradation. The association of peroxidases to the glucan matrix is in favor of the role of the sheath as a supporting structure. Furthermore, that the sheath was hydrolyzed during the attack demonstrated its active role both in providing the H(2)O(2) necessary to the action of peroxidases and in providing a mode of transport of the fungal enzymes to their substrates at the surface of the wood cell wall.

Use of an alpha-D-glucosidase for the specific cytochemical identification of lateral alpha-D-xylosyl end groups in plant xyloglucans.

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.

Proof of the occurrence of 5,6-O-(1-carboxyethylidene)-D-galactofuranose units in the capsular polysaccharide of Klebsiella K12.

The 13C-n.m.r. spectra of the capsular polysaccharide of Klebsiella K41 and phage-derived oligosaccharides K41-P1 and K41-P2 were compared with spectra from the structurally similar polysaccharide of Klebsiella K12 and oligosaccharides K12-P1 and K12-P2. This led to the conclusion that K41 and K12 contain one and two galactofuranose residues per repeating unit, respectively, and that the terminal, lateral residue in K12 has the 5,6-O-(1-carboxyethylidene)-D-galactofuranose structure rather than that of a 4,6-acetal of D-galactopyranose as originally stated. This is the first reported occurrence in Nature of such a structural unit.

Homologous and heterologous reactions of bacteriophages phi 41 and phi 12 on the capsular polysaccharides from Klebsiella K41 and K12.

The structures of the capsular polysaccharides from Klebsiella K41 and K12 are very similar and differ only in the lateral, terminal group of their respective repeating units. The bacteriophages phi 41 and phi 12 are shown to hydrolyze the same alpha-galactopyranosyl bond in each of the polysaccharides, giving rise to an oligosaccharide characteristic of the starting polysaccharide, irrespective of the phage employed. The presence of the uronic acid function is essential for the phages to be active, but the carboxyl group of the pyruvic acetal in K12 does not appear to play a role in the recognition process.

The structural repeating-unit of the capsular polysaccharide from Klebsiella serotype K48.

Investigation of the structure of the capsular polysaccharide from Klebsiella K48, using methylation analysis, periodate oxidation, Smith degradation, and 1H- and 13C-n.m.r. spectroscopy, indicated the repeating unit to be the pentasaccharide (formula; see text)

Structural investigation of the capsular polysaccharide from Klebsiella K19 by chemical and N.M.R. analyses.

Sugar analysis of the capsular antigen K19 from Klebsiella and of the carboxyl-reduced derivative confirmed its classification into the chemotype containing rhamnose, galactose, glucose, and glucuronic acid residues. Partial acid hydrolysis and phage depolymerization of K19 provided respectively a modified, linear form of the polysaccharide and oligosaccharides of the repeating unit, these were used for the structural elucidation of the original polymer. Methylation analysis, Smith degradation, and 1H- and 13C-n.m.r. spectroscopy of the polysaccharide and derivatives permitted formulation of the following structure for K19: (formula; see text)

Depolymerization of the capsular polysaccharide from Klebsiella K19 by the glycanase associated with particles of Klebsiella bacteriophage luminal diameter 19.

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.

The use of bacteriophage depolymerization in the structural investigation of the capsular polysaccharide from Klebsiella serotype K3.

The structure of the repeating unit of the capsular polysaccharide from Klebsiella serotype K3 has been established from the results of n.m.r. (1H and 13C) spectroscopy and methylation analysis of P1, the pyruvic acetal-bearing pentasaccharide obtained on depolymerization of the polysaccharide with a bacteriophage-borne endogalactosidase, reduced deacetalated P1, and the native polysaccharide. The data permit the assignment of the following structure to the repeating unit: (formula see text)