Differential extractability of calcium and pectic substances in different wall regions of epicotyl cells in young flax plants.

We applied the simultaneous use of a subtractive method and two imaging techniques (secondary ion mass spectrometry and electron microscopy after PATAg staining) to correlate the distribution of Ca2+ to pectic substances in cell walls of young flax plants. The calcium images were compared with the structural electron microscopy images. This suggests that the linkage of the pectic substances within the wall is mainly by calcium bridges in the intercellular junctions of most types of cells under study (epidermis, subepidermis, fiber layer, and endodermis) and in the outer part (close to the cuticle) of the wall of the epidermal cells. In the primary walls of the various types of cells under study and in the inner part (close to the cytoplasm) of the wall of the epidermal cells, the linkage of the pectic substances would be mainly by covalent bonds. In the middle lamellae of the various cells, and in the intercellular junctions within the cortical parenchyma, both types of linkages apparently coexist. The mechanism of "ionic condensation" may provide an interpretation for the chemical status of the Ca2+ ions which are associated with the pectic components solubilized in boiling water, and which do not seem to contribute to the linkage of these components within the wall.     

Distribution of pectin and arabinogalactan protein epitopes during organogenesis from androgenic callus of wheat

The distribution of several arabinogalactan protein and pectic epitopes were studied during organogenesis in androgenic callus of wheat. In cell wall of mature and degenerating parenchyma cells, the arabinogalactan epitopes JIM4, JIM14, JIM16 or LM2 were expressed differently according to the cells location. LM2 was observed also in meristematic cells of regenerated shoot buds and leaves. Anti-pectin JIM7 labelled the wall of meristematic cells but fluorescence was strongest in outer walls of surface cells of callus and shoot buds coated by extracellular matrix surface network (ECMSN). During leaves growth the ECMSN disappeared, and JIM7 fluorescence decreased. JIM5 epitope was abundant in the cell walls lining the intercellular spaces of callus parenchyma and in tricellular junctions within regenerated buds and leaves.     

Micro-heterogeneity of pectins and calcium distribution in the epidermal and cortical parenchyma cell walls of flax hypocotyl

Summary Pectic polysaccharides are major components of the plant cell wall matrix and are known to perform many important functions for the plant. In the course of our studies on the putative role of pectic polysaccharides in the control of cell elongation, we have examined the distribution of polygalacturonans in the epidermal and cortical parenchyma cell walls of flax seedling hypocotyls. Pectic components have been detected with (1) the nickel (Ni²⁺) staining method to visualize polygalacturonates, (2) monoclonal antibodies specific to low (JIM5) and highly methylesterified (JIM7) pectins and (3) a combination of subtractive treatment and PATAg (periodic acid-thiocarbohydrazide-silver proteinate) staining. In parallel, calcium (Ca²⁺) distribution has been imaged using SIMS microscopy (secondary ion mass spectrometry) on cryo-prepared samples and TEM (transmission electron microscopy) after precipitation of calcium with potassium pyroantimonate. Our results show that, at the tissular level, polygalacturonans are mainly located in the epidermal cell walls, as revealed by the Ni²⁺ staining and immunofluorescence microscopy with JIM5 and JIM7 antibodies. In parallel, Ca²⁺ distribution points to a higher content of this cation in the epidermal walls compared to cortical parenchyma walls. At the ultrastructural level, immunogold labeling with JIM5 and JIM7 antibodies shows a differential distribution of pectic polysaccharides within cell walls of both tissues. The acidic polygalacturonans (recognized by JIM5) held through calcium bridges are mainly found in the outer part of the external wall of epidermal cells. In contrast, the labeling of methylesterified pectins with JIM7 is slightly higher in the inner part than in the outer part of the wall. In the cortical parenchyma cells, acidic pectins are restricted to the cell junctions and the wall areas in contact with the air-spaces, whereas methylesterified pectins are evenly distributed all over the wall. In addition, the pyroantimonate precipitation method reveals a clear difference in the Ca²⁺ distribution in the epidermal wall, suggesting that this cation is more tightly bound to acidic pectins in the outer part than in the inner part of that wall. Our findings show that the distribution of pectic polysaccharides and the nature of their linkages differ not only between tissues, but also within a single wall of a given cell in flax hypocotyls. The differential distribution of pectins and Ca²⁺ in the external epidermal wall suggests a specific control of the demethylation of pectins and a central role for Ca²⁺ in this regulation.Abbreviations Cdta diamino-1,2-cyclohexane tetra-acetic acid - PATAg periodic acid-thiocarbohydrazide-silver proteinate - PGA polygalacturonic acid - PME pectin methylesterase - RG I rhamnogalacturonan I - SIMS secondary ion mass spectrometry - TEM transmission electron microscopy     

Building flax fibres: more than one brick in the walls

The objective of the review is to provide fundamental knowledge on the chemical composition and structural characteristics of flax fibres. These are long and multinucleate cells without septum or partition (average length 2–5 cm) and have a secondary wall of very large thickness (5–15 μm). Fibres are gathered in bundles of one to three dozen cells that encircle the vascular cylinder. The bundle cohesion is insured by pectins, accumulating in the primary wall and cell junctions. In contrast, lignin, which is present in very low amount, does not seem to play a major role in bundle cohesion. At maturity, secondary wall is characterised by (i) a high level of cellulose with microfibrils locked into an almost axial direction and (ii) 5–15% non-cellulosic polysaccharides (NCPs). The chemical composition of NCPs depends on growth stage, indicating important cell wall remodelling, fibre position and variety. Despite the large disparity of the results reported in the literature, galactose appears to be the predominant sugar of NCPs, and β-1-4-galactan together with rhamnogalacturonan of type I (RG-I) and polygalacturonic acid (PGA) become, with fibre maturity, the most abundant tightly bound NCPs. Glycine-rich proteins (GRPs) and arabinogalactan-proteins (AGPs), also present in flax fibres, are both characterised by appreciable levels of glycine and acidic amino acid and are deficient in hydroxyproline, and may contribute to the cross-linking of pectins. (Galacto)glucomanans/glucans rather than xylans consist of cross-linking polymers in fibre secondary wall. A model is proposed where cellulose microfibrils, tethered by cross-linking (galacto)glucomanans/glucans, are embedded in a pectic matrix.     

The morphology and physiological properties of nereid giant fibres

• 1.1. The three sets of giant fibres in the nerve cord of Nereis virens are connected by both electrotonic and chemically transmitting junctions.
• 2.2. The paired laterals and paramedials are connected to their partners by electronic junctions. The laterals are also electrically coupled to the median giant fibre.
• 3.3. The laterals are connected to the paramedials by an excitatory chemical synapse, while in the anterior segments the paramedials provide an inhibitory input to the median giant fibre.
• 4.4. Afferent input to the giant fibres through the segmental nerves two and four is excitatory, except that to the median fibre in the caudal segments.
• 5.5. There is no evidence of the segmental origin of the lateral giant fibres, either in the form of macrosynapses or segmental cell bodies.
• 6.6. The median giant fibre originates from two groups of cell bodies in the sub-oesophageal ganglia.

     

Tricellulin constitutes a novel barrier at tricellular contacts of epithelial cells

For epithelia to function as barriers, the intercellular space must be sealed. Sealing two adjacent cells at bicellular tight junctions (bTJs) is well described with the discovery of the claudins. Yet, there are still barrier weak points at tricellular contacts, where three cells join together. In this study, we identify tricellulin, the first integral membrane protein that is concentrated at the vertically oriented TJ strands of tricellular contacts. When tricellulin expression was suppressed with RNA interference, the epithelial barrier was compromised, and tricellular contacts and bTJs were disorganized. These findings indicate the critical function of tricellulin for formation of the epithelial barrier.     

Loss of occludin affects tricellular localization of tricellulin

The tricellular tight junction (tTJ) forms at the convergence of bicellular tight junctions (bTJs) where three epithelial cells meet in polarized epithelia, and it is required for the maintenance of the transepithelial barrier. Tricellulin is a four transmembrane domain protein recently identified as the first marker of tTJ, but little is known about how tricellulin is localized at tTJs. As for the molecular mechanism of association of tricellulin with tight junctions (TJs), we found that tricellulin was incorporated into claudin-based TJs independently of binding to zona occludens-1. Unexpectedly, exogenous expression of tricellulin increased cross-links of TJ strands in the plasma membrane. As for the molecular mechanisms for localization of tricellulin at tricellular junctions, we found that knockdown of occludin caused mislocalization of tricellulin to bTJs, implying that occludin supports tricellular localization of tricellulin by excluding tricellulin from bTJs.     

The polysaccharide structure of potato cell walls: Chemical fractionation

Cell walls of potato tubers were fractionated by successive extraction with various reagents. A slightly degraded pectic fraction with 77% galacturonic acid was extracted in hot, oxalate-citrate buffer at pH 4. A further, major pectic fraction with 38% galacturonic acid was extracted in cold 0.1 M Na₂CO₃ with little apparent degradation. These two pectic fractions together made up 52% of the cell wall. Most of the oxalate-citrate fraction could alternatively be extracted with cold acetate-N,N,N-tetracetic acid (CDTA) buffer, a non-degradative extractant which nevertheless removed essentially all the calcium ions. This fraction was therefore probably held only by calcium binding, and the remainder of the pectins by covalent bonds. Electrophoresis showed that both pectic fractions contained a range of molecular types differing in composition, with a high arabinose: galactose ratio as well as much galacturonic acid in the most extractable fractions. From methylation data, the main side-chains were 1,4-linked galactans and 1,5-linked arabinans, with smaller quantities of covalently attached xyloglucan. Extraction with NaOH-borate removed a small hemicellulose fraction and some cellulose. The main hemicelluloses were apparently a galactoxyloglucan, a mannan or glucomannan and an arabinogalactan.Abbreviations GLC gas-liquid chromatography - MS mass spectrometry - V₀ void volume - MW weight-average molecular weight - DMSO dimethylsulphoxide - EDTA ethylenediamine tetraacetic acid - TFA trifluoroacetic acid - CDTA N,N,N-tetraacetic acid     

Changes in non-cellulosic cell-wall polysaccharides during the growth of carrot cells in suspension cultures

The cell-wall composition of carrot (Daucus carota L.) cells has been studied during their growth in suspension culture. Pectic and hemicellulosic polymers were fractionated according to molecular size by a Sepharose 4B column. Polyuronides in the pectic fraction were resolved into high- and low-molecular-weight components. The low-molecular-weight polyuronides were relatively free of neutral sugars and showed a marked increase during the growth of the cell wall. Hemicellulosic polysaccharides were of disperse molecular size. As cell expansion proceeded, the contents of glucose and xylose in the high-molecular-weight region increased while those in the low-molecular-weight fraction decreased. Removal of auxin from the medium apparently caused degradation of high-molecular-weight polymers in both the pectic and hemicellulosic fractions.     

c‐Jun N‐terminal kinase is largely involved in the regulation of tricellular tight junctions via tricellulin in human pancreatic duct epithelial cells

Tricellulin (TRIC) is a tight junction protein at tricellular contacts where three epithelial cells meet, and it is required for the maintenance of the epithelial barrier. To investigate whether TRIC is regulated via a c‐Jun N‐terminal kinase (JNK) pathway, human pancreatic HPAC cells, highly expressed at tricellular contacts, were exposed to various stimuli such as the JNK activators anisomycin and 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), and the proinflammatory cytokines IL‐1β, TNFα, and IL‐1α. TRIC expression and the barrier function were moderated by treatment with the JNK activator anisomycin, and suppressed not only by inhibitors of JNK and PKC but also by siRNAs of TRIC. TRIC expression was induced by treatment with the PKC activator TPA and proinflammatory cytokines IL‐1β, TNFα, and IL‐1α, whereas the changes were inhibited by a JNK inhibitor. Furthermore, in normal human pancreatic duct epithelial cells using hTERT‐transfected primary cultured cells, the responses of TRIC expression to the various stimuli were similar to those in HPAC cells. TRIC expression in tricellular tight junctions is strongly regulated together with the barrier function via the JNK transduction pathway. These findings suggest that JNK may be involved in the regulation of tricellular tight junctions including TRIC expression and the barrier function during normal remodeling of epithelial cells, and prevent disruption of the epithelial barrier in inflammation and other disorders in pancreatic duct epithelial cells. J. Cell. Physiol. 225: 720–733, 2010. © 2010 Wiley‐Liss, Inc.     

The ultrastructure of the human fetal pineal gland

Summary The innervation of the pineal gland, the cell junctions in this organ and junctions between ependymal cells in the pineal recess were investigated in 27 human fetuses (crown-rump length 30–190 mm).Free nerve boutons containing clear and a few dense core vesicles were present in the pineal parenchyma and in the perivascular spaces. The boutons did not make synaptic contacts with the pinealocytes. No evidence for the presence of noradrenaline in the vesicles of nerve boutons was found.Gap junctions, intermediate-like junctions and desmosomes were frequently seen between the pinealocytes. Ruthenium red was used in three fetuses as an extracellular marker.The continuous endothelial cells surrounding the capillary lumen were connected by tight junctions. This indicates the presence of a blood-brain barrier.Tight junctions were present between the ependymal cells in the pineal recess. These junctions constitute an extracellular barrier between the pineal and the cerebrospinal fluid. Acknowledgements: The author wishes to thank Inger Ægidius and Jb Machen for their technical, Ruth Fatum for her linguistic and Karsten Bundgaard for his photographical assistance     

Incorporation of D-[U-14C]Glucose in the Cell Wall of Linum Plantlets during the First Steps of Growth

Flax plantlets, cultivated from day 3 in liquid medium and undercontinuous light showed linear growth. Electron microscopy observationsshowed that treatment of the cell walls with cdta-Na₂ clearedout the middle lamella and the cell junctions, whereas boilingwater extracted pectic polysaccharides from the primary cellwall in each tissue (epidermis, cortical parenchyma and phloem). Pulse-chase experiments showed that there was during the growthof flax plantlets a continuous redistribution of radioactivityin all parts of the cell walls: 1) from pectins to hemicellulosesand even to the cellulosic residues. 2) from oligomers to polymers.3) from neutral to acidic polymers in the core of the middlelamella. 4) from acidic to neutral pectins in the primary cellwalls. The elongation zone which was restricted to a small zoneback from the tip, involved strong synthesis of neutral pectinsin all the cell walls. Conversely, the redistribution of radioactivitywas related mainly to the plant cell maturation, and especiallyto the acidification of the cell wall. Demethylation of someneutral pectins occurred in the middle lamella whereas stronglyacidic pectins were synthetized in the primary cell wall. (Received October 1, 1990; Accepted April 9, 1991)     

Shigella targets epithelial tricellular junctions and uses a noncanonical clathrin-dependent endocytic pathway to spread between cells

Bacteria move between cells in the epithelium using a sequential pseudopodium-mediated process but the underlying mechanisms remain unclear. We show that during cell-to-cell movement, Shigella-containing pseudopodia target epithelial tricellular junctions, the contact point where three epithelial cells meet. The bacteria-containing pseudopodia were engulfed by neighboring cells only in the presence of tricellulin, a protein essential for tricellular junction integrity. Shigella cell-to-cell spread, but not pseudopodium protrusion, also depended on phosphoinositide 3-kinase, clathrin, Epsin-1, and Dynamin-2, which localized beneath the plasma membrane of the engulfing cell. Depleting tricellulin, Epsin-1, clathrin, or Dynamin-2 expression reduced Shigella cell-to-cell spread, whereas AP-2, Dab2, and Eps15 were not critical for this process. Our findings highlight a mechanism for Shigella dissemination into neighboring cells via targeting of tricellular junctions and a noncanonical clathrin-dependent endocytic pathway.     

Immunocytochemical localization of cell wall polysaccharides in the root tip ofAvena sativa

Summary Two polyclonal antisera, anti-xyloglucan (anti-XG) and anti-polygalacturonic acid/rhamnogalacturonan I (anti-PGA/RG-I), which recognize, respectively, noncellulosic -(14)-D-glucan containing polysaccharides and the unesterified forms of the acidic pectic polysaccharide polygalacturonic acid/rhamnogalacturonan I, were used to localize epitopes recognized by the two antisera in the root tip of oat (Avena sativa). Immunoblot analysis shows that epitopes recognized by the anti-XG antibodies are present in both the mixed linkage -(13)-(14)-D-glucans (MG) and in xyloglucan (XG). Immunogold electron microscopy shows that the cell walls of meristematic, cortical, epidermal, columella, and peripheral cells contain significant amounts of such epitopes. In contrast, the molecules that carry these MG/XG epitopes appear to be sparse in the expanded middle lamella of meristematic cells, but dense in the expanded middle lamella of peripheral root cap cells. This finding suggests that the porosity of the middle lamella is altered in peripheral root cap cells to facilitate mucilage secretion. In contrast, few PGA/RG-I epitopes were detected in any cell walls of any of the cell types examined. Double immunogold labeling experiments revealed an intriguing localization pattern of MG/XG and of PGA/RG-I epitopes in the peripheral mucilage-secreting cells of the root cap. Whereas MG/XG epitopes were abundant in the cell wall, they were sparse in both the secreted mucilage and in intracellular secretory vesicles. In marked contrast, PGA/RG-I epitopes were detected at high density in intracellular secretory vesicles, but unexpectedly, were quite sparse in both the cell wall and in the mucilage. These immunolabeling patterns are consistent with the hypotheses that the synthesis and secretion of particular -D-glucans is subject to both activation and down-regulation during cell development and differentiation and that post-secretory alterations of pectic polysaccharides, such as enzymatic release of RG-I-type mucilage molecules from PGA/RG-I precursors, may occur in the peripheral cell walls of the oat root cap.Abbreviations MG mixed linkage -(13)-(14)-D-glucan - PGA/RG-I polygalacturonic acid/rhamnogalacturonan I - SEPS sycamore extracellular polysaccharides - TGN trans Golgi network - XG xyloglucan     

Composition of the cell walls of different asparagus (Asparagus officinalis) tissues

Portions of stems from the base of asparagus spears (Asparagus officinalis L. cv. Connovor Collossus) were dissected to give the following tissues: (1) pith, which was free of vascular bundles, (2) two surrounding layers, parenchyma and fibre I and II (PFI and PFII), containing parenchyma and vascular bundles, (3) sclerenchyma sheath, (4) epidermis and sub-epidermal layers and (5) asparagus vascular fibre (AVF). The alcohol-insoluble residues (AIRs) from these tissues were shown to be free of starch. They were analysed for moisture and protein, and the component sugars were released by two hydrolytic procedures, which helped to distinguish the sugars from non-cellulosic polysaccharides and cellulose. The AIRs from pith and epidermal tissues were relatively low in xylose, but were rich in cellulosic glucose, and sugars associated with pectic polysaccharides such as galacturonic acid, galactose and arabinose. Their major component polysaccharides (in decreasing amounts) were inferred to be pectic polysaccharides, cellulose, and hemicelluloses. AIR from sclerenchyma was rich in glucose and xylose, suggesting the presence of much cellulose and (acidic) xylans. The AIRs of PFI, PFII and AVF contained significant amounts of xylose in addition tn other sugars, and the major polysaccharides inferred to be present were pectic polysaccharides, cellulose and hemicelluloses, a significant proportion of which may be acidic xylans. Methylation analysis of the AIRs confirmed the above inferences. The bulk of the glucosyl residues were (1–4)-linked, and there were small but significant amounts of (1–4, 6)-linked glucosyl residues (the linkage characteristic of xyloglucans) in all the preparations. The presence of (1–4)-linked galactosyl, (1–5)-linked arabinosyl, terminal galactosyl, terminal arabinosyl, (1–2)- and (1–2, 4)-linked rhamnosyl residues in all the AIRs except that from sclerenchyma, confirmed the presence of significant levels of pectic polysaccharides in all the parenchyma tissues. All the preparations containing vascular tissues contained significant amounts of (1–4)-linked xylosyl residues, probably derived from acidic xylans. Even in the AIR of pith, a significant amount of (1–4)-linked xylosyl residues were detected. This may be due to the ability of these cells and the parenchyma cells associated with the vascular bundles, to undergo lignification in mature asparagus plants.     

γ-Tubulin-like molecules in the mouse duodenal epithelium

A mouse monoclonal antibody (G9, Horio et al. in Cell Motil Cytoskel 44:284–295, 1999) that was raised against the γ-tubulin from a fission yeast, Schizosaccharomyces pombe, showed a unique staining in the mouse small intestine. Similar to another anti-γ-tubulin antibody that is commercially available, G9 showed typical dot-like staining corresponding to the microtubule-organizing center in the free cells of the epithelium and the connective tissue under it. In addition, G9 stained the cell–cell contacts in the epithelium. This stained region was not bicellular but tricellular junctions of the enterocytes. This staining was unique to G9 and was diminished on the sample of the mouse small intestine, which had lost most of its filamentous microtubules through the preparation process. The tricellular junction is thought to be the weakest point of the epithelial barrier, and no other junctional structures have been identified except for the central sealing elements extending from the tight junctions between the two cells. Our results suggest the existence of a new molecule underlying the tricellular junctions, which may relate to γ-tubulin and the microtubules.     

The ultrastructure of lymphatic valves in the adult rabbit lung

Summary An electron microscopic investigation has revealed that the pulmonary lymphatic valves of adult rabbits are not simple duplicatures of the lymphatic vessel wall. They consist of an uninterrupted central connective tissue core, covered on both sides with a single layer of flattened endothelial cells. Near their insertion in the lymphatic vessel wall, the connective tissue core reveals a distinct thickening being composed mainly of collagen bundles. In the other parts it contains mainly elastic fibers and fine filaments, enclosing also some rather peculiar connective tissue cells. Nervous and muscular elements were not observed. The endothelium is continuous and exhibits no open junctions. The valvular basement membrane is better developed than in lymphatic capillaries. The endothelial cells contain numerous cytoplasmic filaments which might be endowed with contractile properties. The nuclei of the endothelial and the connective tissue cells are irregularly spaced and frequently clustered near the free edge of the valve.These ultrastructural features suggest that the function of the lymphatic valves is mainly passive. They are firmly inserted in the lymphatic vessel wall by collagen fibers and their moving parts are slender and elastic. Their endothelium appears relatively impermeable and is firmly attached to the subjacent connective tissue.This study has been supported by a grant from The Council for Tobacco Research—U.S.A.. We thank Professor Robert C. Rosan (Saint Louis University—U.S.A.) for expert advice, R. Janssens for technical, G. Pison and St. Ons for photographic and N. Tyberghien for secretarial assistance.     

Changes in the composition of cotton fibre cell walls during development

Purified cell walls, prepared from cotton fibres (Gossypium arboreum L.) at different growth stages, were subjected to successive extractions to give pectic, hemicellulosic, and -cellulosic fractions. The protein content and sugars obtained after hydrolysis of the total cell walls and of the various fractions were quantitatively estimated. The amount of protein in the fibre cell walls from one ovule reached a maximum value at the end of the elongation growth, decreased, and then reached a second maximum at the end of the secondary wall deposition. The absolute amounts of fucose, galactose, mannose, rhamnose, arabinose, uronic acid, and non-cellulosic glucose residues all reached a maximum at the end of the primary wall formation or at the beginning of the secondary wall formation. Only the absolute amounts of xylose and of the cellulosic glucose residues increased until the end of the fibre development. Most conspicuous was the decrease in the absolute amounts of non-cellulosic glucose and of arabinose residues during the secondary wall formation, possibly indicating a turnover of at least some of the hemicellulosic wall material.Abbreviations DPA days post anthesis - TLC thin layer chromatography - SDS sodium dodecyl sulphate     

Fractionation and Partial Characterization of Pectic Polysaccharides in Cell Walls from Liverwort (Marchantia polymorph) Cell Cultures

Konno, H., Yamasalu, Y. and Katoh, K. 1987. Fractionation andpartial characterization of pectic polysaccharides in cell wallsfrom liverwort (Marchantia polymorpha) cell cultures.—Jexp. Bot. 38: 711–722. Pectic polysaccharides were extracted from the starch-free cellwall preparation of cell suspension cultures of Marchantia polymorpha.The polysaccharides were fractionated by DEAE-Sephadex A-50ion-exchange chromatography yielding the five fractions, andthe degree of polymerization and glycosyl composition determinedfor each fraction. The neutral rich and acidic pectic polymerswere depolymerized by purified endoglucanase (l,4-ß-D-glucan4-glucanohydrolase, E.C. 3.2.1.4 [EC] .) and endopolygalacturonase(poly-l,4--Dgalacturonide glycanohydrolase, E.C. 3.2.1.15 [EC] ),respectively. The degraded pectic fractions were fractionatedby gel filtration chromatography on Bio-Gel A-5m and Bio-GelP-2, and glycosyl composition determined for each fraction.The results indicate that pectic polysaccharides contain glucose-richpolymer, rhamnogalacturonan and homogalacturonan in a ratioof 1:4:0–6. In addition, pectic polysaccharides were releasedas five pectic fragments from the cell walls by purified endopectatelyase (poly-l,4--D-galacturonide lyase, E.C. 4.2.2.2 [EC] ). Basedon the analysis of glycosyl composition of each fragment, thepectic polysaccharides of Marchantia cell walls are characterized Key words: Cell suspension culture, cell wall, liverwort, Marchantia polymorpha, pectic polysaccharides