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.

Reassessment of qualitative changes in lignification of transgenic tobacco plants and their impact on cell wall assembly.

In tobacco plants the effect of antisense down-regulation of various genes encoding enzymes of the monolignol biosynthetic pathway resulted in quantitative and qualitative changes in lignin distribution and in diverse alterations of the secondary wall assembly of modified tobacco plants. Total lignin content, composition in syringyl and guaiacyl units, and absolute proportions of condensed and non-condensed substructures occurring in the cell walls, were differentially modified according to the repressed gene. Immunocytochemical characterisation and visualisation of the distribution of condensed and non-condensed lignin substructure epitopes in transmission electron microscopy (TEM) revealed that some transformations entailed profound and specific alterations in the secondary wall biogenesis. Correlation between micro-morphological cell wall alterations and semi-quantitative immuno-analysis of the topochemical distribution of lignin sub-units suggests that the mode of polymerisation of monolignols into non-condensed units, favoured by the microfibril matrix of the secondary wall, plays an important part in the lignified cell wall assembly.

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.

Abiotic and enzymatic degradation of wheat straw cell wall: a biochemical and ultrastructural investigation.

The action of an abiotic lignin oxidant and a diffusible xylanase on wheat straw was studied and characterized at the levels of the molecular structures by chemical analysis and of the cell wall ultrastructure by transmission electron microscopy. While distinct chemical changes in the target polymers were observed when each system was used separately, a combination of the two types of catalysts did not significantly increase either lignin oxidation or hemicellulose hydrolysis. Microscopic observations however revealed that the supramolecular organization of the cell wall polymers was significantly altered. This suggests that the abiotic Mn-oxalate complex and the xylanase cooperate in modifying the cell wall architecture, without noticeably enhancing the degradation of the constitutive polymers.

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.

Chemical analysis and immunolocalisation of lignin and suberin in endodermal and hypodermal/rhizodermal cell walls of developing maize (Zea mays L.) primary roots.

The composition of suberin and lignin in endodermal cell walls (ECWs) and in rhizodermal/hypodermal cell walls (RHCWs) of developing primary maize (Zea mays L.) roots was analysed after depolymerisation of enzymatically isolated cell wall material. Absolute suberin amounts related to root length significantly increased from primary ECWs (Casparian strips) to secondary ECWs (suberin lamella). During further maturation of the endodermis, reaching the final tertiary developmental state characterised by the deposition of lignified secondary cell walls (u-shaped cell wall deposits), suberin amounts remained constant. Absolute amounts of lignin related to root length constantly increased throughout the change from primary to tertiary ECWs. The suberin of Casparian strips contained high amounts of carboxylic and 2-hydroxy acids, and differed substantially from the suberin of secondary and tertiary ECWs, which was dominated by high contents of omega-hydroxycarboxylic and 1,omega-dicarboxylic acids. Furthermore, the chain-length distribution of suberin monomers in primary ECWs ranged from C(16) to C(24), whereas in secondary and tertiary ECWs a shift towards higher chain lengths (C(16) to C(28)) was observed. The lignin composition of Casparian strips (primary ECWs) showed a high syringyl content and was similar to lignin in secondary cell walls of the tertiary ECWs, whereas lignin in secondary ECWs contained higher amounts of p-hydroxyphenyl units. The suberin and lignin compositions of RHCWs rarely changed with increasing root age. However, compared to the suberin in ECWs, where C(16) and C(18) were the most prominent chain lengths, the suberin of RHCWs was dominated by the higher chain lengths (C(24) and C(26)). The composition of RHCW lignin was similar to that of secondary-ECW lignin. Using lignin-specific antibodies, lignin epitopes were indeed found to be located in the Casparian strip. Surprisingly, the mature suberin layers of tertiary ECWs contained comparable amounts of lignin-like epitopes.

Localization of a phosphatidylglycerol/phosphatidylinositol transfer protein in Aspergillus oryzae.

The subcellular localization of the phosphatidylglycerol/phosphatidylinositol transfer protein (PG/PI-TP) of Aspergillus oryzae was investigated using Western blot analysis of the cell protein extracts, a cellular membrane fractionation technique, and transmission electron microscopy. The PG/PI-TP, as detected by Western blot analysis with a specific immune serum, was found to be mainly cytoplasmic and partly associated with intracellular membranes. A fractionation experiment was conducted after homogenization of the filamentous fungus mycelium. The endoplasmic reticulum, Golgi-like vesicles, and the plasma membrane were separated by isopycnic ultracentrifugation on a sucrose gradient, and our data revealed that the immunodetected PG/PI-TP was only associated with the Golgi-like apparatus. All these results were documented by electron microscopy and indicate here for the first time that there exists a specific phospholipid transfer protein in a filamentous fungus that is localized in the cytoplasm and associated with Golgi-like vesicles.

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.

Structural cell-wall proteins in protoxylem development: evidence for a repair process mediated by a glycine-rich protein.

Polyclonal antibodies were used to localize structural cell-wall proteins in differentiating protoxylem elements in etiolated bean and soybean hypocotyls at the light- and electron-microscopic level. A proline-rich protein was localized in the lignified secondary walls, but not in the primary walls of protoxylem elements, which remain unlignified, as shown with lignin-specific antibodies. Secretion of the proline-rich protein was observed during lignification in different cell types. A glycine-rich protein (GRP1.8) was specifically localized in the modified primary walls of mature protoxylem elements and in cell corners between xylem elements and xylem parenchyma cells. The protein was secreted by Golgi bodies both in protoxylem cells after the lignification of their secondary walls and in the surrounding xylem parenchyma cells. The modified primary walls of protoxylem elements were visualized under the light microscope as filaments or sheets staining distinctly with the protein stain Coomassie blue. Electron micrographs of these walls show that they are composed of an amorphous material of moderate electron-density and of polysaccharide microfibrils. These materials form a three-dimensional network, interconnecting the ring- or spiral-shaped secondary wall thickenings of protoxylem elements and xylem parenchyma cells. The results demonstrate that the modified primary walls of protoxylem cells are not simply breakdown products due to partial hydrolysis and passive elongation, as believed until now. Extensive repair processes produces cell walls with unique staining properties. It is concluded that these walls are unusually rich in protein and therefore have special chemical and physical properties.

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.

Use of enzyme-gold complexes for the ultrastructural localization of hemicelluloses in the plant 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.