Cytochemical localization of pectinase activity in laticifers ofNerium oleander L.

Summary A method is described for the cytochemical localization of pectinase activity at the ultrastructural level. The procedure involves the use of Benedict's reagent to form an electron-dense copper precipitate when reacted with reducing sugars liberated from exogenously supplied pectin. Using this technique, pectinase activity was examined in the nonarticulated, branched laticifers ofNerium oleander. Electron opaque crystalline deposits indicating the presence of pectolytic enzymes were identified in laticifer central vacuoles. Smaller amounts of reaction product were distributed along the middle lamella between laticifers and adjacent cells. This report represents the first direct evidence for the involvement of pectinase in intrusive growth of nonarticulated laticifers.     

STAINED PECTIN AS SEEN IN THE ELECTRON MICROSCOPE

This paper describes electron microscopic studies on the distribution of pectin within young plant cells. Dark-grown onion roots, from 1 to 3 mm. in length, were used. In order to make the pectic substances selectively dense to electrons, they were first reacted with basic hydroxylamine. This treatment produces pectic hydroxamic acids, which in turn were treated with ferric ion to form insoluble complexes. The tissue was imbedded, sectioned, and then observed by electron microscopy. Dense deposits of iron were found in the region of the middle lamella and in a second area near the surface of the primary wall. Transverse walls of varying maturity were noted. The pectin of the more frequent, immature cross-walls, leads directly into the inner reacting layer of the axillary (longitudinal) wall. The pectin of the more mature transverse walls becomes, on the other hand, intimately associated with the middle lamella pectin of the axillary wall. It is shown that the pectin of the middle lamella represents the hot water-soluble portion of the pectic substance, while the internal layer of the axillary wall and the transverse wall pectin represent the so called residual fraction. Hot versene extraction removes essentially all electron-dense material.     

Pectin esterification is spatially regulated both within cell walls and between developing tissues of root apices

Monoclonal antibodies recognizing un-esterified (JIM5) and methyl-esterified (JIM7) epitopes of pectin have been used to locate these epitopes by indirect immunofluorescence and immunogold electron microscopy in the root apex of carrot (Daucus carota L.). Both antibodies labelled the walls of cells in all tissues of the developing root apex. Immunogold labelling observed at the level of the electron microscope indicated differential location of the pectin epitopes within the cell walls. The un-esterified epitope was located to the inner surface of the primary cell walls adjacent to the plasma membrane, in the middle lamella and abundantly to the outer surface at intercellular spaces. In contrast, the epitope containing methyl-esterified pectin was located evenly throughout the cell wall. In root apices of certain other species the JIM5 and JIM7 epitopes were found to be restricted to distinct tissues of the developing roots. In the root apex of oat (Avena sativa L.), JIM5 was most abundantly reactive with cell walls at the region of intercellular spaces of the cortical cells. JIM7 was reactive with cells of the cortex and the stele. Neither epitope occurred in walls of the epidermal or root-cap cells. These pattern of expression were observed to derive from the very earliest stages of the development of these tissues in the oat root meristem and were maintained in the mature root. In the coleoptile and leaf tissues of oat seedlings, JIM5 labelled all cells abundantly whereas JIM7 was unreactive. Other members of the Gramineae and also the Chenopodiaceae are shown to express similar restricted spatial patterns of distribution of these pectin epitopes in root apices.Abbreviations CDTA 1,2-diaminocyclohexane tetraacetic acid - RG rhamnogalacturonan J.P.K. was supported by the Agricultural and Food Research Council Cell Signalling and Recognition Programme. We thank J. Cooke and N. Stacey for technical assistance, H.A. Schols, Drs. P. Albersheim and A. Darvill for pectic polysaccharides, and Dr. R.R. Selvendran and M. McCann for useful discussions.     

Sub-cellular location of H2O2, peroxidases and pectin epitopes in control and hyperhydric shoots of carnation

In carnation shoots (Dianthus caryophyllus cv. Killer), hyperhydricity was induced in in vitro culture using a low agar concentration. Using transmission electron microscopy, cytochemical techniques and immunolocation of JIM5 and JIM7 pectin epitopes, we followed the sub-cellular modifications of cell walls in relation to peroxidase activity and hydrogen peroxide accumulation during hyperhydricity induction. Peroxidase activity revealed a significant induction of the stomatal and epidermal cells as well as of the intercellular spaces of hyperhydric leaves. Similarly, hydrogen peroxide accumulated in the epidermal cell walls and the intercellular spaces of hyperhydric leaves. Immunolocation of an epitope recognised by the JIM5 antibody revealed the main unesterified nature of the cell walls. Such an epitope was located in the epidermal cell walls as well as in the corners of cell junctions in control leaves. However, hyperhydric leaves showed a total reduction of JIM5 labelling in the corners of cell junctions and a significant reduction of the intercellular spaces and the middle lamella. Highly-methylsterified pectin, recognised by the JIM7 antibody, was present to a slight extent in cell walls in control and hyperhydric leaves. We propose that the altered anatomy observed in hyperhydric carnation leaves could be regulated by the concomitant actions of pectin methyl esterases and free radicals, modifying the structure of the pectin and polysaccharides of the cell walls.     

Antibody localization of extensin in cell walls of carrot storage roots

The accumulation and cross-linking of hydroxyproline-rich glycoproteins (HRGPs) in cell walls of dicotyledonous plants has been correlated with a number of wall-strengthening phenomena. Polyclonal antibodies raised against glycosylated extensin-1, the most abundant HRGP in carrot (Daucus carota L.) cell walls, recognize this antigen on gel and dot blots and on thin sections of epoxy-embedded carrot-root cell walls. Since wall labeling can be largely reduced by preincubating the antibodies with purified extensin-1, most labeling can be attributed to recognition of this antigen. The remaining label may be the result of recognition of extensin-2, a second carrot HRGP, or other wall components (cellulose, hemicellulose and pectin are not recognized). Extensin-1 label was distributed quite uniformly across the cell wall but was absent from the expanded middle lamella at the intersection of three or more cells and was reduced in the narrow middle lamella between two cells. This distribution is essentially the same as that of cellulose. Because of limitations of this labeling technique, it is not possible to construct a complete model of the structure of the cross-linked extensin matrix. Nonetheless, short, linear arrays of gold particles may represent small portions of the extensin matrix or of individual extensin molecules as they are exposed on the surface of sections. These and other results presented here indicate that: a) newly synthesized extensin is added to the wall by intussusception; b) extensin cannot cross the middle lamella separating the walls of adjacent cells; and c) incorporation of extensin is a late event in the development of phloem-parenchyma cell walls in carrot.Abbreviations dE-1 antibodies antibodies raised against deglycosylated extensin 1 - ELISA enzyme-linked immunosorbant assay - gE-1 antibodies antibodies raised against glycosylated extensin 1 - HRGP hydroxyproline-rich glycoprotein - PAGE polyacrylamide gel electrophoresis - RG-1 rhamnogalacturonan I - SDS sodium dodecyl sulfate     

果胶酶的细胞化学定位：马尾松树脂道发生的细胞化学证据

运用薄切片和电镜细胞化学方法观察了马尾松(Pinus massoniana Lamb.)茎皮层树脂道的发育及发育过程中果胶酶的变化。树脂道的发育过程一般可分为4个阶段,即原始细胞阶段、胞间隙形成阶段、腔道扩大阶段和树脂道成熟阶段。在原始细胞阶段,果胶酶的反应产物首先出现在原始细胞膨胀的细胞壁角隅处,然后沿细胞壁中层分布。胞间隙形成后,果胶酶的反应产物分布在细胞壁和胞间隙的交界面。随着胞间隙的扩大,反应产物的密度逐渐降低。当树脂道成熟后,上皮细胞壁上则没有果胶酶的反应产物出现。细胞化学证据表明: 在树脂道的发育过程中,果胶酶降解树脂道原始细胞细胞壁中层,支持树脂道以裂生方式形成。果胶酶的细胞化学定位技术还可用于其他植物中与果胶水解有关的发育过程。     

果胶酶的细胞化学定位:马尾松树脂道发生的细胞化学证据

运用薄切片和电镜细胞化学方法观察了马尾松(Pinus massoniana Lamb.)茎皮层树脂道的发育及发育过程中果胶酶的变化.树脂道的发育过程一般可分为4个阶段,即原始细胞阶段、胞间隙形成阶段、腔道扩大阶段和树脂道成熟阶段.在原始细胞阶段,果胶酶的反应产物首先出现在原始细胞膨胀的细胞壁角隅处,然后沿细胞壁中层分布.胞间隙形成后,果胶酶的反应产物分布在细胞壁和胞间隙的交界面.随着胞间隙的扩大,反应产物的密度逐渐降低.当树脂道成熟后,上皮细胞壁上则没有果胶酶的反应产物出现.细胞化学证据表明:在树脂道的发育过程中,果胶酶降解树脂道原始细胞细胞壁中层,支持树脂道以裂生方式形成.果胶酶的细胞化学定位技术还可用于其他植物中与果胶水解有关的发育过程.     

Use of monoclonal antibodies to detect Mn(II)-peroxidase in birch wood degraded by Phanerochaete chrysosporium

Summary A monoclonal antibody (Mab) produced to purified Mn(II)-peroxidase was visualized on and within cell corners of birch wood degraded by Phanerochaete chrysosporium using colloidal gold immuno-transmission electron microscopy techniques. Labelling of the fungal cell membrane and cell wall was also observed. The same Mab was used to visualize the penetration of extracellular fungal metabolite extracts, infiltrated into previously decayed wood. Binding of antibodies to the lignin-rich cell corner region of the middle lamella in wood decayed by P. chrysosporium was observed in sectioned wood blocks and in wood infiltrated with crude extracellular extracts from P. chrysospirium liquid cultures. When a control monoclonal antiserum, produced to extracellular metabolites of Postia (Poria) placenta and cross-reactive with fungal cellulase, was used in labelling, the cellulose rich region of the wood cell walls were labelled. Labelling in the middle lamella cell corners was only noted in what has been described as nonor poorly lignified cell corner regions. Offprint requests to: G. Daniel     

Cellulose and pectin localization in roots of mycorrhizalAllium porrum: labelling continuity between host cell wall and interfacial material

Two different types of contacts (or interfaces) exist between the plant host and the fungus during the vesicular-arbuscular mycorrhizal symbiosis, depending on whether the fungus is intercellular or intracellular. In the first case, the walls of the partners are in contact, while in the second case the fungal wall is separated from the host cytoplasm by the invaginated host plasmamembrane and by an interfacial material. In order to verify the origin of the interfacial material, affinity techniques which allow identification in situ of cell-wall components, were used. Cellobiohydrolase (CBH I) that binds to cellulose and a monoclonal antibody (JIM 5) that reacts with pectic components were tested on roots ofAllium porrum L. (leek) colonized byGlomus versiforme (Karst.) Berch. Both probes gave a labelling specific for the host cell wall, but each probe labelled over specific and distinct areas. The CBH I-colloidal gold complex heavily labelled the thick epidermal cell walls, whereas JIM 5 only labelled this area weakly. Labelling of the hypodermis was mostly on intercellular material after treatment with JIM 5 and only on the wall when CBH I was used. Suberin bands found on the radial walls were never labelled. Cortical cells were mostly labelled on the middle lamella with JIM 5 and on the wall with CBH I. Gold granules from the two probes were found in interfacial material both near the point where the fungus enters the cell and around the thin hyphae penetrating deep into the cell. The ultrastructural observations demonstrate that cellulose and pectic components have different but complementary distributions in the walls of root cells involved in the mycorrhizal symbiosis. These components show a similar distribution in the interfacial material laid down around the vesicular-arbuscular mycorrhizal fungus indicating that the interfacial material is of host origin.     

An ultrastructural study of the viscin tissue ofPhthirusa pyrifolia (H.B.K.) Eichler (Loranthaceae)

Summary The viscin tissue ofPhthirusa pyrifolia consists at maturity of two different cell types. The first of these (viscin cells) are greatly elongated and have massive secondary walls, the microfibrils of which are arranged strictly transversely, and are soaked with mucilaginous noncellulosic polysaccharides. The second type of cells (vesicular cells) is much broader, with a very extensive vacuolar system and thin primary cell walls only. Viscin cells are capable of being stretched very greatly, the microfibrils of the secondary wall then becoming oriented more or less longitudinally. In nature, this corresponds to the time of attachement to the host surface, and is followed by drying and, possibly, shortening of the viscin cells. Developmental study of the tissue suggests that Golgi bodies are the major organelles participating in polysaccharide production. The actual mucilage seems to originate from three sources: an unorganized substance which accumulates between the plasmalemma and compound middle lamella; spherical vacuoles which seem to be embedded in the central vacuole, some of which are seen open to the outside of the cell; and from the dissolution at maturity of the compound middle lamella. It is suggested that the two types of cells may correspond to the two major functions of viscin, viz., host attachment (viscin cells) and nutrition of the disseminator (vesicular cells).     

Correlation between the distribution of lignin and pectin and distribution of sorbed metal ions (lead and zinc) on coir (Cocos nucifera L.)

Plant fibres are capacious for sorption of metal ions, and can be used in water cleaning. Knowledge about the sorption will help in selection of the fibre and optimisation of its chemical modification, if any. The aim of this paper is to investigate the connection, if any, between the distribution of lignin and pectin and the loading of Pb and Zn on coir (mesocarp fibres from Cocos nucifera L.). The coir consisted mainly of xylem and a fibre sheath. The lignin was evenly distributed in the cell walls of the fibre sheath, but in the xylem, there was no detectable content in the compound middle lamella, and a smaller content of lignin in the secondary walls than in the walls of the fibre sheath. The only detectable content of pectin in the fibre sheath walls was in the middle lamella, cell corners and extracellular matrix, while in the xylem, the pectin was almost evenly distributed in the wall, with a higher concentration in the middle lamella and cell corners. All cell walls facing the lacuna had a high content of pectin. The metal ions were mainly loaded on the xylem and cell walls facing the lacuna, maybe with an additional trend to be loaded on the large fibres. Lead was distributed on and across the whole secondary wall. Zinc was loaded on the secondary walls, but there was no information about the distribution across the wall. If there is a simple correlation between the loading of metal ions and the distribution of lignin or pectin, these investigations point at no correlation with lignin and a positive correlation with pectin. It has to be stressed that these conclusions are made on limited material and are therefore preliminary in nature.     

Immunocytochemistry of pectins in shoot apical meristems: consequences for intercellular adhesion

Summary. The nature of pectins (acidic, methyl-, or acetyl-esterified) in the shoot meristem of Sinapis alba was assessed by immunocytochemistry with the 2F4 monoclonal antibody in light and electron microscopy. This antibody is specific for “egg-boxes” – the polygalacturonic acid conformation induced by calcium as described in Liners et al. (Plant Physiol. 99: 1099–1104, 1992). Hardly any acidic pectin was detected in meristem walls; the pectins were largely methyl-esterified and esterified by acetyl groups and/or other esters. After in situ chemical or enzymatic de-esterification, labeling was distributed over the primary wall and the middle lamella of meristematic cells. Acidic pectin and Ca²⁺-cross-linked homogalacturonans were absent from the pit fields, where plasmodesmata traverse the middle lamella. The type and distribution of pectins are discussed in relation to cellular adhesion between active meristem cells. Correspondence and reprints: Unité de Recherches en Biologie Cellulaire Végétale, Département de Biologie, Facultés Universitaires Notre-Dame de la Paix, rue de Bruxelles 61, 5000 Namur, Belgium.     

Sequential actions of pectinases and cellulases during secretory cavity formation in Citrus fruits

Pectinase and cellulase activities are involved in a number of intercellular space-forming processes in plants. In this study, we combined cytochemistry with ultrastructural analysis to investigate the ontogeny of secretory cavity in fruits of Citrus medica L. var. sarcodactylis (Noot.) Swingle, Citrus reticulata Blanco and Citrus limon (L.) Burm. f. Pectinase activity was first detectable at the initial stage of cavity formation, peaked at the intercellular space-forming stage, and diminished at the following stages. In comparison, no cellulase activity was detected until the early lumen-expanding stage. The cellulase activity increased at the late lumen-expanding stage and culminated at the near-mature stage. In the fruit of C. medica var. sarcodactylis, the distribution of pectinase and cellulase reaction products was restricted to the endoplasmic reticulum (ER), the vesicles derived from ER and the cell wall. We also observed that multivesicular structure containing the pectinase reaction product at the initial stage of cavity formation. Our results suggest that pectinase and cellulase are synthesized on ER and secreted directly into the cell wall through exocytosis of ER-derived vesicles. Our observations are consistent with the notion that the secretory cavity in Citrus fruits is formed through a schizolysigenous process in which pectinase activity is involved in the degradation of the middle lamella, whereas cellulase activity is responsible for the degradation of the cell wall.     

Degenerative changes in the epidermal cell wall of Allium cepa caused by Botrytis allii: Loss of dry mass,pectin, and cellulose in halos

Halos detected using interference microscopy (even- and fringle-field modes with monoand poly-chromatic light) around penetration sites of Botrytis allii in cell walls of normal and protoplast-free outer epidermal tissue of white, yellow, and red onions were alike. Halos in protoplast-free cell walls contained 33% less dry mass than areas of these walls adjacent to halos (quantitative interference miscroscopy with 546 nm light in the even-field mode). Halos were significantly larger in the white onion than in the yellow and red varieties. The loss of cell wall dry mass during the production of halos involved the loss of pectin and cellulose. We infer that this is caused by enzymes released from the pathogen. Cuticle degradation at penetration sites was not observed.     

The leaf internal morphology and ultrastructure of Zostera muelleri irmisch ex aschers. (Zosteraceae): a comparative study of the intertidal and subtidal forms

The leaf anatomy, histochemistry and ultrastructure of the intertidal and subtidal seagrass Zostera muelleri Irmish ex Aschers. from Westernport Bay, Victoria were studied. Unusual anatomical and ultrastructural features are compared with other seagrasses and their functional significance is assessed. Subcuticular cavities are present in the young blade, but not observed in the older blade nor young and old leaf sheath. Wall ingrowths occur in the blade epidermal cells particularly on the inner tangential walls and the lower portions of the radial walls. Plasmodesmata are present between adjacent epidermal cells and between the epidermal and mesophyll cells, suggesting that solutes could transfer between these tissues both symplastically and apoplastically. Each leaf has three longitudinally aligned vascular bundles, each of which comprises a single xylem element isolated from the phloem tissue. The phloem consists of nacreous-walled sieve elements accompanied by phloem parenchyma cells which also process wall ingrowths. The xylem walls are completely hydrolysed and the middle lamella borders directly on the xylem lumen. Leaves have prominent air lacunae bisected transversely by septa at regular intervals along their length. Each septum consists of a file of small parenchyma cells with wall protuberances projecting into intercellular space. There are no major structural differences between the subtidal and intertidal plants, but the former have larger leaves and more leaves per shoot than the latter. In addition, a network of unusual reticulated fungal hyphae is present in the leaf intercellular spaces of the subtidal form and this network may facilitate solute transfer in these plants.     

Loss of pectin is an early event during infection of cocoyam roots by Pythium myriotylum

Cocoyam (Xanthosoma sagittifolium) is an important tuber crop in most tropical zones of Africa and America. In Cameroon, its cultivation is hampered by a soil-borne fungus Pythium myriotylum which is responsible for root rot disease. The mechanism of root colonisation by the fungus has yet to be elucidated. In this study, using microscopical and immunocytochemical methods, we provide a new evidence regarding the mode of action of the fungus and we describe the reaction of the plant to the early stages of fungal invasion. We show that the fungal attack begins with the colonisation of the peripheral and epidermal cells of the root apex. These cells are rapidly lost upon infection, while cortical and stele cells are not. Labelling with the cationic gold, which binds to negatively charged wall polymers such as pectins, is absent in cortical cells and in the interfacial zone of the infected roots while it is abundant in the cell walls of stele cells. A similar pattern of labelling is also found when using the anti-pectin monoclonal antibody JIM5, but not with anti-xyloglucan antibodies. This suggests that early during infection, the fungus causes a significant loss of pectin probably via degradation by hydrolytic enzymes that diffuse and act away from the site of attack. Additional support for pectin loss is the demonstration, via sugar analysis, that a significant decrease in galacturonic acid content occurred in infected root cell walls. In addition, we demonstrate that one of the early reactions of X. sagittifolium to the fungal invasion is the formation of wall appositions that are rich in callose and cellulose.     

Production of Pectinase and Cellulase by Cladosporium cucumerinum with Dissolved Carbohydrates and Isolated Cell Walls of Cucumber as Carbon Sources

The scab fungus Cladosporium cucumerinum can use pectins and polygalacturonic acid as sole sources of carbon. Cellulose and Ca-polygalacturonate are not available carbon sources for the fungus. When growing on sucrose or pectin, pectinase is produced. In these cases the production of cellulase is insignificant. On a mixture of pectin and carboxymethylcellulose also cellulase is produced. Both pectinase and cellulase are released into the culture filtrate when the fungus grows on cell walls without ionic proteins, whereas only cellulase is released when cell walls with ionic proteins are the carbon source. Pectinase produced by the pathogen can bind to isolated cell walls. The bound pectinase can be extracted with 1 M NaCl from cell walls without ionic proteins, but not from cell walls with ionic proteins. A water-extract or 1 M NaCl-extract of cucumber hypocotyls with visible disease symptoms contains cellulase but no pectinase activity. Lack of pectinase activity in the 1 M NaCl-extract may be due to inhibition by a component that could be extracted by NaCl from the cucumber cell walls.     

Differential cell wall degradation byErwinia chrysanthemi in petiole ofSaintpaulia ionantha

Summary Erwinia chrysanthemi is a soft-rot pathogenic enterobacterium that provokes maceration of host plant tissues by producing extracellular cell-wall-degrading enzymes, among which are pectate lyases, pectin methyl esterases, and cellulases. Cell wall degradation in leaves and petiole tissue of infectedSaintpaulia ionantha plants has been investigated in order to define the structural and temporal framework of wall deconstruction. The degradation of major cell wall components, pectins and cellulose, was studied by both classical histochemical techniques (Calcofluor and periodic acid-thiocarbohydrazide-silver proteinate staining) and immunocytochemistry (tissue printing for detection of pectate lyases; monoclonal antibodies JIM5 and JIM7 for detection of pectic substrates). The results show that the mode of progression of the bacteria within the host plant is via the intercellular spaces of the parenchyma leaf and the petiole cortex. Maceration symptoms and secretion of pectate lyases PelA, -D, and -E can be directly correlated to the spread of the bacteria. Wall degradation is very heterogeneous. Loss of reactivity with JIM5 and JIM7 was progressive and/or clearcut. The primary and middle lamella appear to be the most susceptible regions of the wall. The innermost layer of the cell wall frequently resists complete deconstruction. At the wall intersects and around intercellular spaces resistant domains and highly degraded domains occurred simultaneously. All results lead to the hypothesis that both spatial organisation of the wall and accessibility to enzymes are very highly variable according to regions. The use of mutants lacking pectate lyases PelA, -D, -E or -B, -C confirm the important role that PelA, PelD, and PelE play in the rapid degradation of pectins from the host cell walls. In contrast, PelB and PelC seem not essential for degradation of the wall, though they can be detected in leaves infected with wild-type bacteria. With Calcofluor staining, regularly localised cellulose-rich and cellulose-poor domains were observed in pectic-deprived walls.Abbreviations MAb monoclonal antibody - PATAg periodic acid-thiocarbohydrazide-silver proteinate     

Epidermal derivatives as xylem elements and transfer cells: a study of the host-parasite interface in two species of Triphysaria (Scrophulariaceae)

Summary Haustoria ofTriphysaria pusilla andT. versicolor subsp.faucibarbata from a natural habitat were analysed by light and electron microscopy. The keel-shaped edge of the secondary haustorium generally splits the epidermis and cortex of the host root parallel to the root axis, and penetrates to the host vascular tissue. Anticlinally elongated epidermal cells of the haustorium constitute most of the host/parasite interface. Some of these epidermal cells are divided by oblique cell walls. Some of their oblique daughter cells as well as some undivided epidermal cells differentiate into xylem elements. Single epidermal cells occasionally intrude into the vascular tissue of the host and individual host cells can be invaded. The surface area of the plasmalemma in parasitic parenchymatous interface cells is increased by the differentiation of wall labyrinths characteristic of transfer cells and by the development of membrane-lined cytoplasmic tubules or flattened sacs which become embedded in the partly lignified interface cell-wall. Mycorrhizal fungal hyphae enter the xylem bridge in some haustoria. Implications of these observations for the function of the haustorium are discussed.     

A suberized perimedullary reaction zone in Populus balsamifera novel for compartmentalization in trees

 Following artificial inoculation of nonhost Populus balsamifera with Ophiostoma ulmi, structural defensive tissues were formed in the xylem. Among these tissues there was a perimedullary sheath of cells, located adjacent to the invaded xylem, that originated from the dedifferentiation of perimedullary and xylem parenchyma cells. Histochemical tests revealed that this sheath was intensively suberized. A band of lignified cells was frequently detected on both sides of this suberized tissue. The formation of such a tissue at the pith margin represents a new type of anatomical barrier in relation to compartmentalization processes described for trees. Ultrastructural examination showed that the wall of cells forming this zone was generally composed of a compound middle lamella, a suberized secondary wall and a tertiary wall layer. Using colloidal gold conjugated to monoclonal antibodies against pectin and to an exoglucanase for cellulose, only limited labelling was obtained for pectin whereas labelling for cellulose was abundant in the compound middle lamella and the tertiary wall layer. In a few fibres close to this suberized zone, the latter probe also made it possible to distinguish the occasional presence of several alternating wall layers mainly composed of either suberin or cellulose. In Salix sp., another tree species belonging to the Salicaceae, this type of suberized reaction zone was also observed. The new reaction zone is similar in structure and location to a suberized barrier formed in nonwoody carnation (Dianthus caryophyllus) plants in the defense against vascular fungi. Received: 30 July 1996 / Accepted: 7 January 1997