Localization of cell wall polysaccharides in normal and compression wood of radiata pine: relationships with lignification and microfibril orientation

The distribution of noncellulosic polysaccharides in cell walls of tracheids and xylem parenchyma cells in normal and compression wood of Pinus radiata, was examined to determine the relationships with lignification and cellulose microfibril orientation. Using fluorescence microscopy combined with immunocytochemistry, monoclonal antibodies were used to detect xyloglucan (LM15), β(1,4)-galactan (LM5), heteroxylan (LM10 and LM11), and galactoglucomannan (LM21 and LM22). Lignin and crystalline cellulose were localized on the same sections used for immunocytochemistry by autofluorescence and polarized light microscopy, respectively. Changes in the distribution of noncellulosic polysaccharides between normal and compression wood were associated with changes in lignin distribution. Increased lignification of compression wood secondary walls was associated with novel deposition of β(1,4)-galactan and with reduced amounts of xylan and mannan in the outer S2 (S2L) region of tracheids. Xylan and mannan were detected in all lignified xylem cell types (tracheids, ray tracheids, and thick-walled ray parenchyma) but were not detected in unlignified cell types (thin-walled ray parenchyma and resin canal parenchyma). Mannan was absent from the highly lignified compound middle lamella, but xylan occurred throughout the cell walls of tracheids. Using colocalization measurements, we confirmed that polysaccharides containing galactose, mannose, and xylose have consistent correlations with lignification. Low or unsubstituted xylans were localized in cell wall layers characterized by transverse cellulose microfibril orientation in both normal and compression wood tracheids. Our results support the theory that the assembly of wood cell walls, including lignification and microfibril orientation, may be mediated by changes in the amount and distribution of noncellulosic polysaccharides.     

THE COMPOSITION OF THE DEVELOPING PRIMARY WALL IN ONION ROOT TIP CELLS. II. CYTOCHEMICAL LOCALIZATION

Jensen , William A. (U. California, Berkeley.) The composition of the developing primary wall in onion root tip cells. II. Cytochemical localization. Amer. Jour. Bot. 47(4) : 287—295. Illus. 1960.–The composition of the developing cell wall in the first 2 mm. of the onion root tip was studied using a cytochemical technique that permitted the detection of hemicellulose and the noncellulosic polysaccharides as well as the pectic substances and cellulose. The technique is based on the combination of a differential extraction procedure with the periodic acid-Schiff reaction for carbohydrates. The data obtained indicate that the cells of the apical initials are low in all wall substances but that all of the wall materials are present to some extent. Early in cell development, differences appear in the composition of the walls of the various tissues. The cortical cells are relatively high in the noncellulosic polysaccharides and cellulose while relatively low in the pectic substances and hemicellulose. Very early in development the protoderm is similar to the cortex, but differences develop during the radial enlargement of the cells. During this stage the walls of the protodermal cells are low in the noncellulosic polysaccharides and cellulose and high in pectic substances and hemicellulose. As elongation progresses, these differences are lost and the 2 tissues become very similar. The vascular cell walls are low in the noncellulosic polysaccharides and cellulose and are high in pectic substances and hemicellulose early in development. Later, hemicellulose becomes relatively more important. When the cell wall materials are sequentially extracted, no change in the general morphology of the cell occurs until only the noncellulosic polysaccharides and the cellulose remained. When the noncellulosic polysaccharides are then removed, the cells remain intact but are 30% less in diameter. This suggests that while cellulose is of critical importance, the noncellulosic polysaccharides may play a major role in determining the physical characteristics of the wall.     

Cell-Wall Polysaccharides of Developing Flax Plants

Flax (Linum usitatissimum L.) fibers originate from procambial cells of the protophloem and develop in cortical bundles that encircle the vascular cylinder. We determined the polysaccharide composition of the cell walls from various organs of the developing flax plant, from fiber-rich strips peeled from the stem, and from the xylem. Ammonium oxalate-soluble polysaccharides from all tissues contained 5-linked arabinans with low degrees of branching, rhamnogalacturonans, and polygalacturonic acid. The fiber-rich peels contained, in addition, substantial amounts of a buffer-soluble, 4-linked galactan branched at the 0-2 and 0-3 positions with nonreducing terminal-galactosyl units. The cross-linking glycans from all tissues were (fucogalacto)xyloglucan, typical of type-I cell walls, xylans containing (1->)-[beta]-D-xylosyl units branched exclusively at the xylosyl O-2 with t-(4-O-methyl)-glucosyluronic acid units, and (galacto)glucomannans. Tissues containing predominantly primary cell wall contained a larger proportion of xyloglucan. The xylem cells were composed of about 60% 4-xylans, 32% cellulose, and small amounts of pectin and the other cross-linking polysaccharides. The noncellulosic polysaccharides of flax exhibit an uncommonly low degree of branching compared to similar polysaccharides from other flowering plants. Although the relative abundance of the various noncellulosic polysaccharides varies widely among the different cell types, the linkage structure and degree of branching of several of the noncellulosic polysaccharides are invariant.     

Lignin distribution in wood cell walls determined by TEM and backscattered SEM techniques

The lignin distribution in cell walls of spruce and beech wood was determined by high-voltage transmission-electron-microscopy (TEM) in sections stained with potassium permanganate as well as by field-emission-scanning-electron-microscopy (FE-SEM) combined with a back-scattered electron detector on mercurized specimens. The latter is a new technique based on the mercurization of lignin and the concomitant visualization of mercury by back-scattered electron microscopy (BSE). Due to this combination it was possible to obtain a visualized overview of the lignin distribution across the different layers of the cell wall. To our knowledge, this combined method was used the first time to analyse the lignin distribution in cell walls. In agreement with previous work the highest lignin levels were found in the compound middle lamella and the cell corners. Back-scattered FE-SEM allows the lignin distribution in the pit membrane of bordered pits as well as in the various cell wall layers to be shown. In addition, by using TEM as well as SEM we observed that lignin closely follows the cellulose microfibril orientation in the secondary cell wall. From these observations, we conclude that the polymerisation of monolignols is affected by the arrangement of the polysaccharides which constitute the cell wall.     

An Arabidopsis Cell Wall Proteoglycan Consists of Pectin and Arabinoxylan Covalently Linked to an Arabinogalactan Protein

Plant cell walls are comprised largely of the polysaccharides cellulose, hemicellulose, and pectin, along with ∼10% protein and up to 40% lignin. These wall polymers interact covalently and noncovalently to form the functional cell wall. Characterized cross-links in the wall include covalent linkages between wall glycoprotein extensins between rhamnogalacturonan II monomer domains and between polysaccharides and lignin phenolic residues. Here, we show that two isoforms of a purified Arabidopsis thaliana arabinogalactan protein (AGP) encoded by hydroxyproline-rich glycoprotein family protein gene At3g45230 are covalently attached to wall matrix hemicellulosic and pectic polysaccharides, with rhamnogalacturonan I (RG I)/homogalacturonan linked to the rhamnosyl residue in the arabinogalactan (AG) of the AGP and with arabinoxylan attached to either a rhamnosyl residue in the RG I domain or directly to an arabinosyl residue in the AG glycan domain. The existence of this wall structure, named ARABINOXYLAN PECTIN ARABINOGALACTAN PROTEIN1 (APAP1), is contrary to prevailing cell wall models that depict separate protein, pectin, and hemicellulose polysaccharide networks. The modified sugar composition and increased extractability of pectin and xylan immunoreactive epitopes in apap1 mutant aerial biomass support a role for the APAP1 proteoglycan in plant wall architecture and function.     

A Barley xyloglucan xyloglucosyl transferase covalently links xyloglucan, cellulosic substrates, and (1,3;1,4)-beta-D-glucans

Molecular interactions between wall polysaccharides, which include cellulose and a range of noncellulosic polysaccharides such as xyloglucans and (1,3;1,4)-beta-D-glucans, are fundamental to cell wall properties. These interactions have been assumed to be noncovalent in nature in most cases. Here we show that a highly purified barley xyloglucan xyloglucosyl transferase HvXET5 (EC 2.4.1.207), a member of the GH16 group of glycoside hydrolases, catalyzes the in vitro formation of covalent linkages between xyloglucans and cellulosic substrates and between xyloglucans and (1,3;1,4)-beta-D-glucans. The rate of covalent bond formation catalyzed by HvXET5 with hydroxyethylcellulose (HEC) is comparable with that on tamarind xyloglucan, whereas that with (1,3; 1,4)-beta-D-glucan is significant but slower. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analyses showed that oligosaccharides released from the fluorescent HEC:xyloglucan conjugate by a specific (1,4)-beta-D-glucan endohydrolase consisted of xyloglucan substrate with one, two, or three glucosyl residues attached. Ancillary peaks contained hydroxyethyl substituents (m/z 45) and confirmed that the parent material consisted of HEC covalently linked with xyloglucan. Similarly, partial hydrolysis of the (1,3;1,4)-beta-D-glucan:xyloglucan conjugate by a specific (1,3;1,4)-beta-D-glucan endohydrolase revealed the presence of a series of fluorescent oligosaccharides that consisted of the fluorescent xyloglucan acceptor substrate linked covalently with 2-6 glucosyl residues. These findings raise the possibility that xyloglucan endo-transglucosylases could link different polysaccharides in vivo and hence influence cell wall strength, flexibility, and porosity.     

Cell Wall Structure in Cells Adapted to Growth on the Cellulose-Synthesis Inhibitor 2,6-Dichlorobenzonitrile : A Comparison between Two Dicotyledonous Plants and a Graminaceous Monocot

Our previous work (E. Shedletzky, M. Shmuel, D.P. Delmer, D.T.A. Lamport [1990] Plant Physiol 94:980-987) showed that suspension-cultured tomato cells adapted to growth on the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) have a markedly altered cell wall composition, most notably a markedly reduced level of the cellulose-xyloglucan network. This study compares the adaptation to DCB of two cell lines from dicots (tomato [Lycopersicon esculentum] and tobacco [Nicotiana tabacum]) and a Graminaceous monocot (barley [Hordeum bulbosum] endosperm). The difference in wall structures between the dicots and the monocot is reflected in the very different types of wall modifications induced by growth on DCB. The dicots, having reduced levels of cellulose and xyloglucan, possess walls the major integrity of which is provided by Ca²⁺-bridged pectates because protoplasts can be prepared from these cells simply by treatment with divalent cation chelator and a purified endopolygalacturonase. The tensile strength of these walls is considerably less than walls from nonadapted cells, but wall porosity is not altered. In contrast, walls from adapted barley cells contain very little pectic material and normal to elevated levels of noncellulosic polysaccharides compared with walls from nonadapted cells. Surprisingly, they have tensile strengths higher than their nonadapted counterpart, although cellulose levels are reduced by 70%. Evidence is presented that these walls obtain their additional strength by an altered pattern of cross-linking of polymers involving phenolic components. Such cross-linking may also explain the observation that the porosity of these walls is also considerably reduced. Cells of adapted lines of both the dicots and barley are resistant to plasmolysis, suggesting that they possess very strong connections between the wall and the plasma membrane.     

Important new players in secondary wall synthesis

Secondary walls in wood are the most abundant biomass produced by plants. Understanding how plants make wood is not only of interest in basic plant biology but also has important implications for tree biotechnology. Three recent papers report exciting findings regarding a group of novel glycosyltransferases (GTs) involved in secondary wall synthesis. Because little is known about genes involved in the synthesis of wood polysaccharides other than cellulose, the identification of these GTs is a breakthrough in the molecular dissection of wood formation.     

Selection of white-rot fungi for biopulping

Different rates of wood decay and ligninolytic activity were found in wood decayed by various white-rot fungi. Chemical and ultrastructural analyses showed wood decayed by Coriolus versicolor consisted of a nonselective attack on all cell wall components. Lignin degradation was restricted to the cell wall adjacent to hyphae or around the circumference of cell lumina. Decay by Phellinus pini, Phlebia tremellosus, Poria medullapanis and Scytinostroma galactinum was selective for lignin degradation. Secondary walls were void of lignin and middle lamellae were extensively degraded. A diffuse attack on lignin occurred throughout all cell wall layers. Variation in ligninolytic activity was found among strains of Phanerochaete chrysosporium. Differences in weight loss as well as lignin and polysaccharide degradation were also found when wood of different coniferous and deciduous tree species was decayed by various white-rot fungi.     

Structural differences between the lignin-carbohydrate complexes present in wood and in chemical pulps

Lignin-carbohydrate complexes (LCCs) were prepared in quantitative yield from spruce wood and from the corresponding kraft and oxygen-delignified pulps and were separated into different fractions on the basis of their carbohydrate composition. To obtain an understanding of the differences in lignin structure and reactivity within the various LCC fractions, thioacidolysis in combination with gas chromatography was used to quantify the content of beta-O-4 structures in the lignin. Periodate oxidation followed by determination of methanol was used to quantify the phenolic hydroxyl groups. Furthermore, size exclusion chromatography (SEC) of the thioacidolysis fractions was used to monitor any differences between the original molecular size distribution and that after the delignification processes. Characteristic differences between the various LCC fractions were observed, clearly indicating that two different forms of lignin are present in the wood fiber wall. These forms are linked to glucomannan and xylan, respectively. On pulping, the different LCCs have different reactivities. The xylan-linked lignin is to a large extent degraded, whereas the glucomannan-linked lignin undergoes a partial condensation to form more high molecular mass material. The latter seems to be rather unchanged during a subsequent oxygen-delignification stage. On the basis of these findings, a modified arrangement of the fiber wall polymers is suggested.     

Lignin Depletion Enhances the Digestibility of Cellulose in Cultured Xylem Cells

Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from Zinnia elegans . Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulose-binding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis, but realistic enough to capture the natural complexity of lignocellulose in the plant cell wall. Consequently, these cells represent a suitable model for analyzing native lignocellulose degradation.     

Auxin-induced Changes in Avena Coleoptile Cell Wall Composition

Sugar and uronic acid residues were derived from wall polysaccharides of oat (Avena sativa, var. Victory) coleoptiles by means of 2 N trifluoroacetic acid, 72% sulfuric acid, or enzymic hydrolysis. The products of hydrolysis were reduced and acetylated to form alditol acetates which were analyzed using gas chromatography. Time-course studies of auxin-promoted changes in various wall fractions indicate that when exogenous glucose was available, increases in certain wall constituents paralleled increases in length. However, under conditions where exogenous glucose was not available, and where wall synthesis was limited, such correlations with growth were not apparent. Under these latter conditions total wall weight initially increased slightly, then decreased. These changes in weight were the net of increases in cellulose and some noncellulosic constituents and a decrease of over 75% in noncellulosic glucose. When coleoptile sections were preincubated without exogenous glucose for 8 hours to deplete endogenous wall precursors and subsequently treated with auxin, there were no detectable increases in wall weight. There was instead an auxin-promoted decrease in wall weight, and this decrease paralleled a decrease in noncellulosic glucose. There were no significant changes in other wall components. The auxin-promoted decreases in noncellulosic glucose are interpreted as a possible step in the mechanism of growth.     

Screening Wood Decayed by White Rot Fungi for Preferential Lignin Degradation

A screening procedure in which scanning electron microscopy was used indicated that 26 white rot fungi selectively removed lignin from various coniferous and hardwood tree species. Delignified wood from field collections had distinct micromorphological characteristics that were easily differentiated from other types of decay. The middle lamella was degraded, and the cells were separated from one another. Secondary cell wall layers that remained had a fibrillar appearance. Chemical analyses of delignified wood indicated that the cells were composed primarily of cellulose. Only small percentages of lignin and hemicellulose were evident. Delignified wood was not uniformly distributed throughout the decayed wood samples. White-pocket and white-mottled areas of the various decayed wood examined contained delignified cells, but adjacent wood had a nonselective removal of lignin where all cell wall components had been degraded simultaneously. This investigation demonstrates that selective delignification among white rot fungi is more prevalent than previously realized and identifies a large number of fungi for use in studies of preferential lignin degradation.     

Collapsed xylem phenotype of Arabidopsis identifies mutants deficient in cellulose deposition in the secondary cell wall.

Recessive mutations at three loci cause the collapse of mature xylem cells in inflorescence stems of Arabidopsis. These irregular xylem (irx) mutations were identified by screening plants from a mutagenized population by microscopic examination of stem sections. The xylem cell defect was associated with an up to eightfold reduction in the total amount of cellulose in mature inflorescence stems. The amounts of cell wall-associated phenolics and polysaccharides were unaffected by the mutations. Examination of the cell walls by using electron microscopy demonstrated that the decreases in cellulose content of irx lines resulted in an alteration of the spatial organization of cell wall material. This suggests that a normal pattern of cellulose deposition may be required for assembly of lignin or polysaccharides. The reduced cellulose content of the stems also resulted in a decrease in stiffness of the stem material. This is consistent with the irregular xylem phenotype and suggests that the walls of irx plants are not resistant to compressive forces. Because lignin was implicated previously as a major factor in resistance to compressive forces, these results suggest either that cellulose has a direct role in providing resistance to compressive forces or that it is required for the development of normal lignin structure. The irx plants had a slight reduction in growth rate and stature but were otherwise normal in appearance. The mutations should be useful in facilitating the identification of factors that control the synthesis and deposition of cellulose and other cell wall components.     

Mechanical,chemical and X-ray analysis of wood in the two tropical lianas<Emphasis Type="Italic"> Bauhinia guianensis</Emphasis> and<Emphasis Type="Italic"> Condylocarpon guianense</Emphasis>: variations during ontogeny

Mechanical and chemical properties as well as microfibril angles of wood tissues from different ontogenetic stages are determined for the neotropical lianas Bauhinia guianensis and Condylocarpon guianense. The mechanical properties include the elastic moduli under bending and under dynamic torsion. The chemical analyses cover (i) the content of cellulose, lignin and hemicelluloses fractions, (ii) the monomeric composition of the uncondensed lignin, and (iii) the composition of the hemicelluloses with respect to neutral monosaccharides. By comparing the wood properties of these lianas with the corresponding properties of wood from self-supporting deciduous trees, common characteristics and differences are revealed. Additionally, the changes in the lignin and polysaccharides fractions as well as the variations in microfibril orientation that occur during ontogeny of the two liana species are discussed with regard to their implications for the mechanical properties of wood.     

Composition and Structure of Sugarcane Cell Wall Polysaccharides: Implications for Second-Generation Bioethanol Production

The structure and fine structure of leaf and culm cell walls of sugarcane plants were analyzed using a combination of microscopic, chemical, biochemical, and immunological approaches. Fluorescence microscopy revealed that leaves and culm display autofluorescence and lignin distributed differently through different cell types, the former resulting from phenylpropanoids associated with vascular bundles and the latter distributed throughout all cell walls in the tissue sections. Polysaccharides in leaf and culm walls are quite similar, but differ in the proportions of xyloglucan and arabinoxylan in some fractions. In both cases, xyloglucan (XG) and arabinoxylan (AX) are closely associated with cellulose, whereas pectins, mixed-linkage-β-glucan (BG), and less branched xylans are strongly bound to cellulose. Accessibility to hydrolases of cell wall fraction increased after fractionation, suggesting that acetyl and phenolic linkages, as well as polysaccharide–polysaccharide interactions, prevented enzyme action when cell walls are assembled in its native architecture. Differently from other hemicelluloses, BG was shown to be readily accessible to lichenase when in intact walls. These results indicate that wall architecture has important implications for the development of more efficient industrial processes for second-generation bioethanol production. Considering that pretreatments such as steam explosion and alkali may lead to loss of more soluble fractions of the cell walls (BG and pectins), second-generation bioethanol, as currently proposed for sugarcane feedstock, might lead to loss of a substantial proportion of the cell wall polysaccharides, therefore decreasing the potential of sugarcane for bioethanol production in the future.