Complementary immunolocalization patterns of cell wall hydroxyproline-rich glycoproteins studied with the use of antibodies directed against different carbohydrate epitopes.

Antisera raised against the major hydroxyproline-rich glycoprotein (HRGP) in carrot (Daucus carota L.) taproot, extensin-1, and a minor HRGP, extensin-2, were characterized by western blot analysis, enzyme-linked immunosorbent assay, and periodate oxidation and found to be directed against carbohydrate epitopes shared by both glycoproteins. The anti-extensin-1 antibodies (gE1) target periodate-sensitive epitopes and may recognize the terminal alpha-1,3-arabinoside of extensin-1. The anti-extensin-2 antibodies (gE2) recognize periodate-insensitive epitopes, possibly binding the reducing, internal beta-1,2-arabinosides on the carbohydrate side chains. Despite the cross-reactivity of these antibodies, immunolocalization studies of carrot taproot and green bean (Phaseolus vulgaris L.) leaf tissues reveal a spatial segregation of gE1- and gE2-labeling patterns. The gE1 antibodies bind only to the cellulose-rich region of the cell wall (J.P. Staehelin and L.A. Stafstrom [1988] Planta 174: 321-332), whereas gE2 labeling is restricted to the expanded middle lamella at three cell junctions. Periodate oxidation of nonosmicated, thin-sectioned tissue abolishes gE1 labeling but leads to labeling of the entire cell wall by gE2, presumably as a result of unmasking cryptic epitopes on extensin-1 in the cellulose layer. Purified extensin-2 protein is more efficient than extensin-1 protein at agglutinating avirulent Pseudomonas strains lacking extracellular polysaccharide. Our results indicate that extensin-2 does not form a heterologous HRGP network with extensin-1 and that, in contrast to extensin-1, which appears to serve a structural role, extensin-2 could participate in passive defense responses against phytopathogenic bacteria.     

Cross-linking patterns in salt-extractable extensin from carrot cell walls

Extensins are hydroxyproline-rich glycoproteins (HRGPs) found in the primary cell walls of dicots. Extensin monomers are secreted into the wall and covalently bound to each other, presumably by isodityrosine (IDT) cross-links, to form a rigid matrix. Expression of the extensin matrix is correlated with inhibition of cell elongation during normal development and with increased resistance to virulent pathogens. We have isolated extensin from carrot root tissue (Daucus carota L.) by published techniques and have used gel filtration chromatography to purify fractions enriched in monomers and oligomers. We refer to this protein as “extensin-1” to distinguish it from “extensin-2,” a second extensin-like HRGP from carrot which we will describe later. We prepared extensin-1 for electron microscopy by shadowing it with platinum. Monomers are highly elongated (84 nanometers) and kinked at several sites. Kinks occur at all sites on molecules with nearly equal probability, but do not appear to occur at their ends. The distribution of kinks is similar to that of tyrosine-lysine-tyrosine sequences, which have been shown to be capable of forming intramolecular IDT cross-links, so we suggest that kinks are visible manifestations of intramolecular IDTs. Oligomers likely result from IDT cross-links between monomers, and may be regarded as transient precursors of the fully cross-linked matrix. Nearly 60% of cross-links involve the ends of molecules while the rest are scattered among internal sites. We discuss how the relative positions and proportions of intra- and intermolecular cross-links in extensin-1 may affect the structure, and in turn the function, of the extensin matrix.     

A second extensin-like hydroxyproline-rich glycoprotein from carrot cell walls

The insoluble extensin matrix of dicot cell walls has been studied most fruitfully by examining the salt-extractable precursors to this matrix. Multiple extensin-like hydroxyproline-rich glycoproteins (HRGPs) have been isolated, or their existence inferred, from tomato, potato, bean, soybean, melon, carrot, and other plants. We and others previously have studied a carrot extensin which we call extensin-1. Here we report on the properties of extensin-2, a second salt-extractable hydroxyproline-rich glycoprotein from carrot. Like extensin-1, extensin-2 contains large amounts of hydroxyproline, serine, histidine, and lysine. In contrast, its tyrosine content is only about one-third that of extensin-1. Arabinose and galactose are the most abundant neutral sugars in both proteins, and nearly identical buoyant densities in CsCl suggest a similar proportion of carbohydrate in each. The size of extensin-2 is about half the size of extensin-1 based on: (a) the measured lengths of shadowed molecules (about 40 versus 84 nanometers); (b) the migration of extensin-2 in acid-urea gels relative to monomers, dimers, and trimers of extensin-1; and (c) the Stokes' radii of these molecules as determined by gel filtration chromatography. Electron microscopy of shadowed extensin-2 molecules indicates that they contain kinks, which may indicate the presence of intramolecular isodityrosine cross-links, but intermolecular cross-links, either with other extensin-2 molecules or extensin-1 molecules, are observed rarely if ever.     

Expression of Cell Wall Proteins in Seeds and During Early Seedling Growth of Araucaria araucana is a Response to Wound Stress and is Developmentally Regulated

Araucaria araucana seeds and seedlings respond to wounding after48 h with a 3- to 4-fold increase of hydroxyproline-rich glycoproteins(HRGP) in the cell walls of the embryo and with a 15-fold increasein the cell walls of the megagametophyte. The megagametophytewalls accumulate six times more hydroxyproline per µgof cell wall protein than the embryo in this wound response.Tissue immunoprints of different parts of seeds and seedlingsobtained with polyclonal antibodies raised against HRGP fromcarrot roots or soybean seed coats indicate that the responseis due to an increase in a protein similar to the ones seenin carrot roots or soybean seed coats. Western blots of embryoand megagametophyte cell wall proteins subjected to SDS-PAGEshow three bands that cross-react with these antibodies. Ina native cationic gel system followed by Western blot analysis,only two bands react with these antibodies. Expression of suchproteins in Araucaria araucana seeds seems to be developmentallyregulated and tissue specific, since they are present mainlyin the megagametophyte and the root cap of the embryo. Key words: Araucaria araucana, seeds, seedlings, cell walls, hydroxyproline-rich glycoproteins     

An epitope of rice threonine- and hydroxyproline-rich glycoprotein is common to cell wall and hydrophobic plasma-membrane glycoproteins

A monoclonal antibody, LM1, has been derived that has a high affinity for an epitope of hydroxyproline-rich glycoproteins (HRGPs). In suspension-cultured rice (Oryza sativa L.) cells the epitope is carried by three major proteins with different biochemical properties. The most abundant is the 95-kDa extracellular rice extensin, a threonine- and hydroxyproline-rich glycoprotein (THRGP) occurring in the cell wall and secreted into the medium. This THRGP can be selectively oxidatively cross-linked in the presence of hydrogen peroxide and an endogenous peroxidase with the result that it does not enter a protein gel. A second polypeptide with the LM1 epitope (180 kDa), also occurring in the suspension-cultured cells and medium, is not oxidatively cross-linked. Three further polypeptides (52, 65 and 110 kDa) with the characteristics of hydrophobic proteins of the plasma-membrane also carry the LM1 epitope as determined by immuno-blotting of detergent/aqueous partitions of a plasma-membrane preparation and immuno-fluorescence studies with rice protoplasts. At the rice root apex the LM1 epitope is carried by four glycoproteins and is developmentally regulated. The major locations of the epitope are at the surface of cells associated with the developing protoxylem and metaxylem in the stele, the longitudinal radial walls of epidermal cells and a sheath-like structure at the surface of the root apex.Abbreviations AGP arabinogalactan protein - ELISA enzyme-linked immunosorbent assay - HRGP hydroxyproline-rich glycoprotein - THRGP threonine- and hydroxyproline-rich glycoprotein This work was supported by The Leverhulme Trust. We also acknowledge support from The Royal Society and thank Prof. L.A. Staehelin for the carrot extensin, N. Stacey for the rice cell culture and Dr. J. Keen for protein sequencing.     

Site-specific binding of a nuclear factor to the carrot extensin gene is influenced by both ethylene and wounding

Experiments conducted in vitro using the electrophoretic mobility shift assay have shown that a single region of the extensin gene of carrot (Daucus carota L.) interacts with a protein factor designated Extensin Gene Binding Factor-1 (EGBF-1) present in nuclear extracts obtained from carrot roots. This interaction is sequence-specific as judged by the failure of other plant gene sequences to compete with the extensin gene for EGBF-1 binding. The EGBF-1 activity is organspecific, not being expressed in nuclear extracts obtained from carrot leaves or stems. Both ethylene treatment and wounding of roots are shown to have a controlling influence on the expression of EGBF-1 activity in nuclear extracts. These results demonstrate that at least three distinct signals: ethylene treatment, wounding, and development, are important in determining the activity of EGBF-1 in nuclear extracts, and indicate a role for EGBF-1 in stress-related signal transduction and the regulation of extensin-gene expression.Abbreviations bp base pair(s) - EGBF extensin-gene binding factor - EMSA electrophoretic mobility shift assay - HRGP hydroxyproline-rich glycoprotein - kb kilobase     

Structure and distribution of lignin in primary and secondary cell walls of maize coleoptiles analyzed by chemical and immunological probes

Lignin is an integral constituent of the primary cell walls of the dark-grown maize (Zea mays L.) coleoptile, a juvenile organ that is still in the developmental state of rapid cell extension. Coleoptile lignin was characterized by (i) conversion to lignothiolglycolate derivative, (ii) isolation of polymeric fragments after alkaline hydrolysis, (iii) reactivity to antibodies against dehydrogenative polymers prepared from monolignols, and (iv) identification of thioacidolysis products typical of lignins. Substantial amounts of lignin could be solubilized from the coleoptile cell walls by mild alkali treatments. Thioacidolysis analyses of cell walls from coleoptiles and various mesocotyl tissues demonstrated the presence of guaiacyl-, syringyl- and (traces of)p-hydroxyphenyl units besidesp-coumaric and ferulic acids. There are tissue-specific differences in amount and composition of lignins from different parts of the maize seedling. Electron-microscopic immunogold labeling of epitopes recognized by a specific anti-guaiacyl/syringyl antibody demonstrated the presence of lignin in all cell walls of the 4-d-old coleoptile. The primary walls of parenchyma and epidermis were more weakly labeled than the secondary wall thickenings of tracheary elements. No label was found in middle lamellae and cell corners. Lignin epitopes appeared first in the tracheary elements on day 2 and in the parenchyma on day 3 after sowing. Incubation of coleoptile segments in H₂O₂ increased the amount of extractable lignin and the abundance of lignin epitopes in the parenchyma cell walls. Lignin deposition was temporally and spatially correlated with the appearance of epitopes for prolinerich proteins, but not for hydroxyproline-rich proteins, in the cell walls. The lignin content of coleoptiles was increased by irradiating the seedlings with white or farred light, correlated with the inhibition of elongation growth, while growth promotion by auxin had no effect. It is concluded that wall stiffness, and thus extension growth, of the coleoptile can be controlled by lignification of the primary cell walls. Primary-wall lignin may represent part of an extended polysaccharide-polyphenol network that limits the extensibility of the cell walls.Abbreviations G, S, H guaiacyl, syringyl andp-hydroxyphenyl constituents of lignin - HRGP hydroxyproline-rich glycoprotein - LTGA lignothioglycolic acid - PRP proline-rich protein Dedicated to Professor Benno Parthier on occasion of his 65th birthdayDeceased 7 November 1996     

Immunolocalization of extensin and potato tuber lectin in carrot, tomato and potato

Tissue-specific expression of two members of the cell wall hydroxyproline-rich glycoprotein (HRGP) family, extensin and potato tuber lectin, was examined by immunolocalization at the light microscope level in various organs (leaves, stems, roots, fruit, tuber) of carrot ( Daucus carota cv. Thumbelina), tomato ( Lycopersicon esclentum cv. Pixie Hybrid II), and potato ( Solanum tuberosum cv. Kennebec). Extensin was prominently expressed in vascular tissue, particularly xylem and also phloem, although virtually all cells displayed some degree of staining which varied as a function of the tissue, organ, and plant under study. Antibodies against potato tuber lectin (PTL) displayed a localization pattern similar to that observed for extensin; notably PTL did not stain cambium but did stain epithelial cells lining secretory cavities. These distribution patterns are consistent with a role for extensin, and possibly PTL, in providing mechanical support in tissues subjected to compression or torsional stress imparted by vascular growth, or by similar stress brought about by transport of vascular fluids.     

Purification and Characterization of a Salt-extractable Hydroxyproline-rich Glycoprotein from Aerated Carrot Discs

The salt-extractable hydroxyproline-rich glycoprotein (HRGP) of the cell wall of aerated carrot root discs has been studied by polyacrylamide gel electrophoresis. The predominant proline-labeled protein extractable from the cell wall is rich in hydroxyproline as shown by its specific loss of ³H from proline labeled in position 4 and its shift in electrophoretic mobility after labeling in the presence of an inhibitor of hydroxyproline synthesis. Unlabeled HRGP can be identified by staining gels for carbohydrate. The HRGP has been purified by ion exchange chromatography and CsCl gradient centrifugation. The HRGP consists of about 50% protein and 50% carbohydrate with an overall molecular weight of 86,000. The amino acid composition of the protein portion consists of 50% hydroxyproline, 19% basic amino acids, 12% serine, and 10% tyrosine. This glycoprotein accumulates in a salt-extractable pool in the cell wall beginning between 10 and 20 hours of aeration and may also become incorporated into the nonextractable portion of the cell wall.     

Differential expression of a hydroxyproline-rich cell-wall protein gene in embryonic tissues of Zea mays L.

A hydroxyproline-rich glycoprotein (HRGP) component of the maize cell wall was shown to be present in different organs of the plant by extraction of cell wall proteins and detection by Western blotting and immunocytochemistry. Antibodies raised against the protein or against synthetic peptides designed from the protein sequence immunoprecipitated a proline-rich polypeptide which was synthesized in-vitro from poly(A) ⁺ RNA extracted from different tissues of the plant and from the complete in-vitro-transcribed mRNA. A very low amount of the protein was found in immature embryos. In particular, the protein could not be detected in the scutellum either by Western blotting or by immunocytochemistry. In agreement with this finding, HRGP mRNA was barely detected in the scutellum, in contrast to its accumulation in the embryo axis. Our results indicate the existence of a unique cell wall structure in embryonic tissues from maize as well as a tissuespecific component of the control of maize HRGP gene expression, distinct to others already described such as cell division.Abbreviations HRGP(s) hydroxyproline-richglycoprotein(s) - DAP days after pollination The present work was supported by grants from Plan Nacional de Investigation Cientifica y Técnica (grant BI088-0242) and European Communities (grant BAP-374). L.R.-A. is the recipient of a fellowship from the Plan Nacional de Formación de Personal Investigador.     

Solubilization of covalently bound extensin from capsicum cell walls

Acidified sodium chlorite cleaves isodityrosine and solubilizes covalently bound hydroxyproline-rich material from cell walls. This has been taken as evidence that isodityrosine acts as a cross-link holding the hydroxyproline-rich glycoprotein extensin in the cell wall. However, acidified chlorite was found to cleave peptide bonds in salt-soluble extensin and in bovine serum albumin (BSA). This invalidates the use of conventional acidified chlorite treatment to provide evidence for isodityrosine cross-links. The ratio of BSA:chlorite was important in determining peptidyl cleavage. At a ratio of 0.75:1.00 (mole amino acid residues/mole chlorite), or higher, peptidyl cleavage was not detected. Furthermore, in samples where a low concentration of radioactive extensin was present, BSA substantially protected the peptide bonds of the extensin against peptidyl cleavage during treatment with acidified chlorite, while not preventing the cleavage of isodityrosine. Therefore, acidified sodium chlorite plus BSA was a more specific reagent for the cleavage of isodityrosine than was acidified chlorite alone. This modified treatment solubilized in intact form the `covalently bound' extensin from cell walls of Capsicum frutescens (chili pepper) suspension cultures, providing new evidence compatible with the view that extensin molecules are held in the cell wall by isodityrosine cross-links.     

Purification and Partial Characterization of a Hydroxyproline-Rich Glycoprotein in a Graminaceous Monocot, Zea mays

Graminaceous monocots generally contain low levels of hydroxyproline-rich Glycoproteins (HRGPs). As HRGPs are often at the cell surface, we used the intact cell elution technique (100 millimolar AlCl₃) to isolate soluble surface proteins from Zea mays cell suspension cultures. Further fractionation of the trichloroacetic acid-soluble eluate on the cation exchangers phospho-cellulose and BioRex-70 gave several retarded, hence presumably basic fractions, which also contained hydroxyproline (Hyp). One of these fractions yielded a pure HRGP after a final purification step involving Superose-6 gel filtration. As this HRGP was unusually rich in threonine, (25 mole%) we designated it as a threonine-hydroxyproline-rich glycoprotein (THRGP); it contained about 27% carbohydrate occurring exclusively as arabinosylated Hyp, predominantly as the monosaccharide (15%), and trisaccharide (25%) with 48% Hyp nonglycosylated—a characteristically graminaceous monocot profile. Amino acid analysis confirmed the basic character, and gave a low alanine content. Reaction with Yariv artificial antigen was negative. These characteristics show that the THRGP is not an arabinogalactan protein. On the other hand, antibodies raised against tomato extensin P1 cross-reacted significantly with the THRGP; this cross-reactivity and the above analytical data provide the best evidence to date for the presence of extensin in a graminaceous monocot.     

Spatial organization of the assembly pathways of glycoproteins and complex polysaccharides in the Golgi apparatus of plants

The Golgi apparatus of plant cells is the site of assembly of glycoproteins, proteoglycans, and complex polysaccharides, but little is known about how the different assembly pathways are organized within the Golgi stacks. To study these questions we have employed immunocytochemical techniques and antibodies raised against the hydroxyproline-rich cell wall glycoprotein, extensin, and two types of complex polysaccharides, an acidic pectic polysaccharide known as rhamnogalacturonan I (RG-I), and the neutral hemicellulose, xyloglucan (XG). Our micrographs demonstrate that individual Golgi stacks can process simultaneously glycoproteins and complex polysaccharides. O-linked arabinosylation of the hydroxyproline residues of extensin occurs in cis-cisternae, and glycosylated molecules pass through all cisternae before they are packaged into secretory vesicles in the monensin-sensitive, trans-Golgi network. In contrast, in root tip cortical parenchyma cells, the anti-RG-I and the anti-XG antibodies are shown to bind to complementary subsets of Golgi cisternae, and several lines of indirect evidence suggest that these complex polysaccharides may also exit from different cisternae. Thus, RG-I type polysaccharides appear to be synthesized in cis- and medial cisternae, and have the potential to leave from a monensin-insensitive, medial cisternal compartment. The labeling pattern for XG suggests that it is assembled in trans-Golgi cisternae and departs from the monensin-sensitive trans-Golgi network. This physical separation of the synthesis/secretion pathways of major categories of complex polysaccharides may prevent the synthesis of mixed polysaccharides, and provides a means for producing secretory vesicles that can be targeted to different cell wall domains.     

Structure of the Threonine-Rich Extensin from Zea mays

Chymotryptic digestion of a threonine-rich hydroxyproline-rich glycoprotein (THRGP) purified from the cell surface of a Zea mays cell suspension culture gave a peptide map dominated by the hexadecapeptide TC5: Thr-Hyp-Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys-Pro-Thr-Hyp-Hyp-Thr-Tyr, in which the repetitive motif Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys is homologous with the dominant decamer of P1-type dicot extensins: Ser-Hyp-Hyp-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, modified by a Lys for Hyp substitution at residue 3, a Val-Tyr deletion at residues 8 and 9, and incomplete post-translational modification of proline residues. One of the minor peptides (TC1) contained the 8-residue sequence: Thr-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Tyr corresponding to the C-terminal tail (judging from the recently isolated maize cDNA clone MC56) which is homologous with the major repetitive motif of the `P3' class of dicot extensins. Direct peptide sequencing defined potential glycosylated regions on the THRGP corresponding to clone MC56 and showing that glycosylated and nonglycosylated domains alternate with high regularity. The THRGP is not in the polyproline-II conformation, judging from circular dichroic spectra, but nevertheless is an extended rod, from electron microscopic data. HF-solvolysis of cell walls from maize coleoptile, root, and root tip released deglycosylated THRGP detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots with high titer rabbit polyclonal antibodies raised against the intact THRGP. In a quantitative enzyme-linked immunosorbent assay, these antibodies cross-reacted 20% with tomato P1 extensin, and 18% with anhydrous hydrogen fluoride-deglycosylated P1. These results, together with other previously published data, show that maize THRGP is homologous with the dicot P1 extensins and, as such, is the first extensin isolated from a graminaceous monocot.     

CELL WALL CARBOHYDRATE EPITOPES IN THE GREEN ALGA OEDOGONIUM BHARUCHAE F. MINOR (OEDOGONIALES,CHLOROPHYTA)1

Cell wall changes in vegetative and suffultory cells (SCs) and in oogonial structures from Oedogonium bharuchae N. D. Kamat f. minor Vélez were characterized using monoclonal antibodies against several carbohydrate epitopes. Vegetative cells and SCs develop only a primary cell wall (PCW), whereas mature oogonial cells secrete a second wall, the oogonium cell wall (OCW). Based on histochemical and immunolabeling results, (1→4)‐β‐glucans in the form of crystalline cellulose together with a variable degree of Me‐esterified homogalacturonans (HGs) and hydroxyproline‐rich glycoprotein (HRGP) epitopes were detected in the PCW. The OCW showed arabinosides of the extensin type and low levels of arabinogalactan‐protein (AGP) glycans but lacked cellulose, at least in its crystalline form. Surprisingly, strong colabeling in the cytoplasm of mature oogonia cells with three different antibodies (LM‐5, LM‐6, and CCRC‐M2) was found, suggesting the presence of rhamnogalacturonan I (RG‐I)–like structures. Our results are discussed relating the possible functions of these cell wall epitopes with polysaccharides and O‐glycoproteins during oogonium differentiation. This study represents the first attempt to characterize these two types of cell walls in O. bharuchae, comparing their similarities and differences with those from other green algae and land plants. This work represents a contribution to the understanding of how cell walls have evolved from simple few‐celled to complex multicelled organisms.     

胡萝卜愈伤组织伸展蛋白纯化及某些生化特性的研究

本文报道了胡萝卜愈伤组织伸展蛋白的柱色谱纯化,电泳性质,氨基酸组成及其电镜观察结果。用CM-cellulose柱色谱纯化胡萝卜愈伤组织伸展蛋白时,仅发现有一个组分;它的电泳性质与胡萝卜根产生的第一种类型伸展蛋白相同;它的羟脯氨酸/絲氨酸克分子数比例大约为4/1(羟脯氨酸,42.1mol％;丝氨酸,12.8mol％);此外,在电子显微镜下观察,伸展蛋白具有典型棒状分子结构。     

Location of a cell-wall hydroxyproline-rich glycoprotein,cellulose and β-1,3-glucans in apical and differentiated regions of maize mycorrhizal roots

The cell-wall components of the interface compartment in functioning mycorrhizal roots of maize (Zea mays L. cv. W64A) have been investigated with the use of immunocytochemistry and enzyme/lectin-gold techniques. The distribution of specific cell-wall probes was determined in the apical and differentiated regions of maize roots in the presence and in the absence of the mycorrhizal fungus, Glomus versiforme. Labelling experiments showed that a maize hydroxyproline-rich glycoprotein (HRGP), identified with a specific antibody, was particularly abundant in the apical dividing cells of the root meristem. Cellulose, located with a cellobiohydrolase-gold complex, showed a similar labelling pattern in the walls of both meristematic and differentiated parts of the roots. When the cortex was colonized by the mycorrhizal fungus, the HRGP and cellulose were expressed in two sites: the wall and the interface area created by invagination of the host membrane around the developing fungus. In contrast, in uninfected roots of the same age, they were only present in the inner part of the wall. A specific antibody against -1,3-glucans demonstrated that these glucans were not laid down at the interface between the plant and fungus, while they appeared to be a skeletal component of the fungal wall, together with chitin.Abbreviations CBH I cellobiohydrolase - DAPI 4,6-diamino-2-phenylindole - HRGP hydroxyproline-rich glycoprotein The research was supported by the Italian Murst (40%) grant and by an International Project between Spain and Italy (Azioni Integrate).     

Localization of glucuronoxylans in Japanese beech visualized by immunogold labelling

Summary An antiserum against glucuronoxylans (GXs) has been raised from a mouse. The dot-blot immunoassay and competitive inhibition test indicated that the antibodies could bind specifically to GXs. Therefore, the antiserum was used for immunogold labelling to investigate the localization of GXs in Japanese beech. Labelling of GXs was seen only in the secondary walls of xylem cells, but not in the primary walls or the middle lamella. GXs were evenly distributed in the secondary walls except for the outer part of the outer secondary-wall layer in which they were less abundant. The labelling density in each secondary-wall layer (S₁, S₂, and S₃) increased during cell wall formation. This result strongly suggests that the deposition of GXs occurs in a penetrative way.