Deficiency of adenosine kinase activity affects the degree of pectin methyl-esterification in cell walls of Arabidopsis thaliana

Pectin methyl-esterification is catalysed by S-adenosyl-l-methionine (SAM)-dependent methyltransferases. As deficiency in adenosine kinase (ADK; EC 2.7.1.20) activity impairs SAM recycling and utilization, we investigated the relationship between ADK-deficiency and the degree of pectin methyl-esterification in cell walls of Arabidopsis thaliana. The distribution patterns of epitopes associated with methyl-esterified homogalacturonan in leaves and hypocotyls of wild-type (WT) and ADK-deficient plants were examined using immunolocalization and biochemical techniques. JIM5 and LM7 epitopes, characteristic of low esterified pectins, were more irregularly distributed along the cell wall in ADK-deficient plants than in WT cell walls. In addition, epitopes recognized by JIM7, characteristic of pectins with a higher degree of methyl-esterification, were less abundant in ADK-deficient leaves and hypocotyls. Since de-esterified pectins have enhanced adhesion properties, we propose that the higher abundance and the altered distribution of low methyl-esterified pectin in ADK-deficient cell walls lead to the leaf shape abnormalities observed in these plants.     

Distribution of unesterified and esterified pectins in cell walls of pollen tubes of flowering plants

Immunocytochemical localization of polygalacturonic acid (pectin) and methyl-esterified pectin in the walls of pollen tubes of 20 species of flowering plants grown in vitro was investigated by using monoclonal antibodies (MAbs) JIM5 and JIM7 and by means of confocal laser scanning microscopy (CLSM). In general, periodic annular deposits of pectins were found coating the tube wall in species possessing solid styles, and a more uniform pectin sheath in tube walls in species having hollow styles or no styles. We hypothesize that the periodic ring-like structure of the pectin sheath reinforces pollen tubes for passing through the transmitting tract in the style. Esterified pectin which prevents Ca²⁺-induced gelification of pectate is located predominantly at the apex. This implies that pectin esterification is related to tip wall loosening that is required for cell wall expansion during tip growth of pollen tubes. The occurrence of unesterified pectins in other areas of pollen tube walls suggests that de-esterification of pectin following tip expansion leads to a more rigid form of pectin that contributes to the construction of the pollen tube wall.     

Spatial regulation of cell-wall structure in response to heavy metal stress: cadmium-induced alteration of the methyl-esterification pattern of homogalacturonans

Background and Aims

In flax hypocotyls, cadmium-induced reorientation of growth coincides with marked changes in homogalacturonan (HGA) epitopes that were recognized by JIM7 and JIM5 antibodies in the external tangential wall of the epidermis. In the present study, LM7 and 2F4 monoclonal antibodies were used, in addition to JIM5 and JIM7, to extend the investigation on the methyl-esterification pattern of HGA within various domains of the cortical tissues, including the cortical parenchyma where cell cohesion is crucial.

Methods

The PATAg (periodic acid thiocarbohydrazide–silver proteinate) test was applied to ultrathin sections so that the polysaccharides could be visualized and the ultrastructure studied. The monoclonal LM7, JIM5 and JIM7 antibodies that recognize differently methyl-esterified HGA were used. The monoclonal 2F4 antibody that is specific to a particular polygalacturonic acid conformation induced by a given calcium to sodium ratio was also applied. After immunogold labelling, the grids were stained with uranyl-acetate, the samples were observed using a transmission electron microscope and the gold particles were counted.

Key Results

In the presence of cadmium, the increase of LM7 labelling in external tangential wall of the epidermis, together with a decrease of JIM7 labelling, suggested a specific role for randomly partially de-esterified HGA to counteract the radial swelling stress. Enhanced JIM5 and 2F4 labelling in the junctions of the inner tissues indicated that the presence of blockwise de-esterified HGA might oppose cell separation.

Conclusions

The response of the hypocotyl to cadmium stress was to adapt the structure of the wall of cortical tissues by differently modulating the methyl-esterification pattern of HGA in various domains.     

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     

Tissue-related changes in methyl-esterification of pectic polysaccharides in cauliflower (Brassica oleracea L. var. botrytis) stems

Pectic substances are a major component of cell walls in vegetable plants and have an important influence on plant food texture. Cauliflower (Brassica oleracea L. var. botrytis) stem sections at different regions of the mature plant stem have been monitored for tissue-related changes in the native pectic polysaccharides. Chemical analysis detected appreciable differences in the degree of methyl-esterification (ME) of pectic polysaccharides. About 65% of galacturonic acid (GalpA) residues were methyl-esterified in floret tissues. Relative ME showed a basipetal decrease, from 94% in the upper stem to 51% in the lower-stem vascular tissues. The decrease was not related to a basipetal increase in glucuronic acid (GlcpA) residues. The monoclonal antibodies, JIM 5 and JIM 7, produced distinct labelling patterns for the relatively low-methyl-esterified and high-methyl-esterified pectin epitopes, respectively. Labelling was related to cell type and tissue location in the stem. Floret cell walls contained epitopes for both JIM 5 and JIM 7 throughout the wall. Stem vascular tissues labelled more strongly with JIM 5. Whereas pith parenchyma in the upper stem labelled more strongly with JIM 7, in the lower-stem pith parenchyma, JIM 5 labelling predominated. Localization of pectic polysaccharide epitopes in cell walls provides an insight into how structural modifications might relate to the textural and nutritional properties of cell walls. Received: 16 August 1997 / Accepted: 20 December 1997     

Ultrastructural immunolocalization of periodic pectin depositions in the cell wall ofNicotiana tabacum pollen tubes

Summary The monoclonal antibody (MAb) JIM5, marking acidic pectins, was used to localize ultrastructurally pectin molecules in the pollen tube wall ofNicotiana tabacum. Longitudinal sections of LR-White embedded pollen tubes were exposed to antibody treatment; accumulations of pectins were identified by counting the density of the gold particles representing the pectin epitopes along the pollen tube wall. Significant accumulations of gold grains were marked and the distances between them were measured. In many pollen tubes a more or less regular distribution of the accumulations was observed along the tube indicating a periodical deposition of pectin. The distances between the accumulations were 4–6 m. Most of the label was found in the inner part of the outer layer of the bilayered cell wall. These findings correspond to and confirm the earlier observation by our group reporting ring-shaped periodical deposits in pollen tubes after immunofluorescence labelling with the MAb JIM5 under the confocal laser scanning microscope.Abbreviations Ab antibody - MAb monoclonal antibody     

Electrophoretic profiling and immunocytochemical detection of pectins and arabinogalactan proteins in olive pollen during germination and pollen tube growth

Background and Aims

Cell wall pectins and arabinogalactan proteins (AGPs) are important for pollen tube growth. The aim of this work was to study the temporal and spatial dynamics of these compounds in olive pollen during germination.

Methods

Immunoblot profiling analyses combined with confocal and transmission electron microscopy immunocytochemical detection techniques were carried out using four anti-pectin (JIM7, JIM5, LM5 and LM6) and two anti-AGP (JIM13 and JIM14) monoclonal antibodies.

Key Results

Pectin and AGP levels increased during olive pollen in vitro germination. (1 → 4)-β-d-Galactans localized in the cytoplasm of the vegetative cell, the pollen wall and the apertural intine. After the pollen tube emerged, galactans localized in the pollen tube wall, particularly at the tip, and formed a collar-like structure around the germinative aperture. (1 → 5)-α-l-Arabinans were mainly present in the pollen tube cell wall, forming characteristic ring-shaped deposits at regular intervals in the sub-apical zone. As expected, the pollen tube wall was rich in highly esterified pectic compounds at the apex, while the cell wall mainly contained de-esterified pectins in the shank. The wall of the generative cell was specifically labelled with arabinans, highly methyl-esterified homogalacturonans and JIM13 epitopes. In addition, the extracellular material that coated the outer exine layer was rich in arabinans, de-esterified pectins and JIM13 epitopes.

Conclusions

Pectins and AGPs are newly synthesized in the pollen tube during pollen germination. The synthesis and secretion of these compounds are temporally and spatially regulated. Galactans might provide mechanical stability to the pollen tube, reinforcing those regions that are particularly sensitive to tension stress (the pollen tube–pollen grain joint site) and mechanical damage (the tip). Arabinans and AGPs might be important in recognition and adhesion phenomena of the pollen tube and the stylar transmitting cells, as well as the egg and sperm cells.     

Cadmium-induced alterations of the structural features of pectins in flax hypocotyl

In the course of our studies on the putative role of pectins in the control of cell growth, we have investigated the effect of cadmium on their composition, remodelling and distribution within the epidermis and fibre tissues of flax hypocotyl (Linum usitatissimum L.). Cadmium-stressed seedlings showed a significant inhibition of growth whereas the hypocotyl volume did not significantly change, due to the swelling of most tissues. The structural alterations consisted of significant increase of the thickness of all cell walls and the marked collapse of the sub-epidermal layer. The pectic epitopes recognized by the anti-PGA/RGI and JIM5 antibodies increased in the outer parts of the epidermis (external tangential wall and junctions) and fibres (primary wall and junctions). Concomitantly, there was a remarkable decrease of JIM7 antibody labelling and consequently an increase of the ratio JIM5/JIM7. Conversely, the ratio JIM7/JIM5 increased in the wall domains closest to the plasmalemma, which would expel the cadmium ions from the cytoplasm. The hydrolysis of cell walls revealed a cadmium-induced increase of uronic acid in the pectic matrix. Sequential extractions showed a remodelling of both homogalacturonan and rhamnogalacturonan I. In fractions enriched in primary walls, the main part of the pectins became cross-linked and could be extracted only with alkali. In fractions enriched in secondary walls, the homogalacturonan moieties were found more abundantly in the calcium-chelator extract while the rhamnogacturonan level increased in the boiling water extract.     

Involvement of JIM13- and JIM8-Responsive Carbohydrate Epitopes in Early Stages of Cell Wall Formation

Beta vulgaris L.). The spatial and temporal expression of both antigens was studied in suspension cells used as the source-tissue for protoplast isolation, in suspension- and mesophyll-derived protoplasts, and in cells which developed from both types of protoplast. Immunofluorescence and immunocytochemical-electron microscopic methods revealed that labeling was present in the cell walls of most suspension cells and also in the incipients of cell walls synthesized around the protoplasts. This signal became much more intense as rebuilding of the cell wall progressed during culture. Relatively weaker labeling was observed in the cytoplasm, where it was frequently associated with the vacuolar compartment. Signal intensity varied between individual cells of the same population and in successive stages of development, but was always stronger with JIM13 than with JIM8. The role of JIM13-responsive epitope in the development of suspension-derived protoplasts was further studied by its ability to bind antibody added to cultures of different ages. Both JIM8- and JIM13-responsive epitopes were widespread in sugar beet cells of different origin and stage of cell wall synthesis. These epitopes may play an important role in cell wall formation and growth under in vitro conditions. Received 17 August 1998/ Accepted in revised form 13 January 1999     

Distribution of pectic epitopes in cell walls of the sugar beet root

Immunolabelling techniques with antibodies specific to partially methyl-esterified homogalacturonan (JIM5: unesterified residues flanked by methylesterified residues. JIM7: methyl-esterified residues flanked by unesterified residues), a blockwise de-esterified homogalacturonan (2F4), 1,4-galactan (LM5) and 1,5-arabinan (LM6) were used to map the distribution of pectin motifs in cell walls of sugar beet root (Beta vulgaris). PME and alkali treatments of sections were used in conjunction with JIM5-7 and 2F4. The JIM7 epitope was abundant and equally distributed in all cells. In storage parenchyma, the JIM5 epitope was restricted to some cell junctions and the lining of intercellular spaces while in vascular tissues it occurred at cell junctions in some phloem walls and in xylem derivatives. After secondary wall formation, the JIM5 epitope was restricted to inner cell wall regions between secondary thickenings. The 2F4 epitope was not detected without de-esterification treatment. PME treatments prior to the use of 2F4 indicated that HG at cell corners was not acetylated. The LM5 epitope was mainly present in the cambial zone and when present in storage parenchyma, it was restricted to the wall region closest to the plasma membrane. The LM6 epitope was widely distributed throughout primary walls but was more abundant in bundles than in medullar ray tissue and storage parenchyma. These data show that the occurrence of oligosaccharide motifs of pectic polysaccharides are spatially regulated in sugar beet root cell walls and that the spatial patterns vary between cell types suggesting that structural variants of pectic polymers are involved in the modulation of cell wall properties.     

Location of cell-wall components in ectomycorrhizae ofCorylus avellana andTuber magnatum

Summary The cell-wall components in ectomycorrhizae ofCorylus avellana andTuber magnatum have been investigated by using immunocytochemistry and enzyme/lectin-gold techniques. Observations were performed in differentiated regions of hazel roots in the presence and absence of the ectomycorrhizal fungus. The results provided new information on the location of specific components in both the host and the fungal wall. The cellobiohydrolase I (CBH I)-gold complex and the monoclonal antibody (MAb) CCRC-M1 revealed cellulose and xyloglucans, respectively, in the host wall. MAb JIM 5, which detected un-esterified pectins, labelled only the material occurring at the junctions between three cells, while no labelling was found after treatment with MAb JIM 7, which detected methyl-esterified pectins. MAb CCRC-M7, which recognized an arabinosylated -(1,6)-galactan epitope, weakly labelled tissue sections. MAb MAC 266, which detects a carbohydrate epitope on membrane and soluble glycoproteins, labelled the wall domain adjacent to the plasmamembrane. In the presence of the fungus, host walls were swollen and sometimes degraded. The labelling pattern of uninfected tissue was maintained, but abundant distribution of gold granules was found after CBH I and JIM 5 labelling. None of the probes labelled the cementing electron-dense material between the hyphae in the fungal mantle and in the Hartig net. The probes for fungal walls, i.e., wheat germ agglutinin (WGA) and concanavalin A (Con A) and a polyclonal antibody, revealed the presence of chitin, high-mannose side chains of glycoproteins and -1,3-glucans. Con A alone led to a labelling over the triangular electron-dense material, suggesting that this cementing material may contain a fungal wall component.     

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.     

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     

Immunocytochemical localization of pectins in the maturing anther of Allium cepa L.

Distribution of pectins in cell walls of maturing anther of Allium cepa L. was investigated. The monoclonal antibodies against defined epitopes of pectin were used: JIM5 recognizing unesterified pectin and JIM7 recognizing esterified pectin. It has been found that the cell walls of all anther tissues mainly contain esterified pectins. In the somatic tissues only small amounts of unesterified pectins are present in the cell wall junctions and adjacent middle lamellae and in the cell walls of the connective tissue. Thickening of the epiderm cell walls and growth of trabeculae in endothecium are completed through deposition of esterified pectins. In the cell walls of the middle layer and tapetum, unesterified pectins have been found only prior to their disintegration. The primary wall of microsporocytes is made up mainly of esterified pectins. Unesterified pectins occur outside microsporocytes only prior to the callose isolation stage. The presence of esterified pectins has also been detected on the surface of the callose wall surrounding dividing microsporocytes. Lysis of those pectins takes place after microsporogenesis, simultaneously with the lysis of the callosic walls. Before these processes pectins are unesterified. In the sporoderm of pollen grains mainly esterified pectins occur. They have been localized in the intine and aperture. The level of unesterified pectins in the intine is markedly lower.     

Early local differentiation of the cell wall matrix defines the contact sites in lobed mesophyll cells of Zea mays

Background and Aims

The morphogenesis of lobed mesophyll cells (MCs) is highly controlled and coupled with intercellular space formation. Cortical microtubule rings define the number and the position of MC isthmi. This work investigated early events of MC morphogenesis, especially the mechanism defining the position of contacts between MCs. The distributions of plasmodesmata, the hemicelluloses callose and (1 → 3,1 → 4)-β-d-glucans (MLGs) and the pectin epitopes recognized by the 2F4, JIM5, JIM7 and LM6 antibodies were studied in the cell walls of Zea mays MCs.

Methods

Matrix cell wall polysaccharides were immunolocalized in hand-made sections and in sections of material embedded in LR White resin. Callose was also localized using aniline blue in hand-made sections. Plasmodesmata distribution was examined by transmission electron microscopy.

Results

Before reorganization of the dispersed cortical microtubules into microtubule rings, particular bands of the longitudinal MC walls, where the MC contacts will form, locally differentiate by selective (1) deposition of callose and the pectin epitopes recognized by the 2F4, LM6, JIM5 and JIM7 antibodies, (2) degradation of MLGs and (3) formation of secondary plasmodesmata clusterings. This cell wall matrix differentiation persists in cell contacts of mature MCs. Simultaneously, the wall bands between those of future cell contacts differentiate with (1) deposition of local cell wall thickenings including cellulose microfibrils, (2) preferential presence of MLGs, (3) absence of callose and (4) transient presence of the pectins identified by the JIM5 and JIM7 antibodies. The wall areas between cell contacts expand determinately to form the cell isthmi and the cell lobes.

Conclusions

The morphogenesis of lobed MCs is characterized by the early patterned differentiation of two distinct cell wall subdomains, defining the sites of the future MC contacts and of the future MC isthmi respectively. This patterned cell wall differentiation precedes cortical microtubule reorganization and may define microtubule ring disposition.     

Arabinogalactan protein-rich cell walls,paramural deposits and ergastic globules define the hyaline bodies of rhinanthoid Orobanchaceae haustoria

Background and Aims

Parasitic plants obtain nutrients from their hosts through organs called haustoria. The hyaline body is a specialized parenchymatous tissue occupying the central parts of haustoria in many Orobanchaceae species. The structure and functions of hyaline bodies are poorly understood despite their apparent necessity for the proper functioning of haustoria. Reported here is a cell wall-focused immunohistochemical study of the hyaline bodies of three species from the ecologically important clade of rhinanthoid Orobanchaceae.

Methods

Haustoria collected from laboratory-grown and field-collected plants of Rhinanthus minor, Odontites vernus and Melampyrum pratense attached to various hosts were immunolabelled for cell wall matrix glycans and glycoproteins using specific monoclonal antibodies (mAbs).

Key Results

Hyaline body cell wall architecture differed from that of the surrounding parenchyma in all species investigated. Enrichment in arabinogalactan protein (AGP) epitopes labelled with mAbs LM2, JIM8, JIM13, JIM14 and CCRC-M7 was prominent and coincided with reduced labelling of de-esterified homogalacturonan with mAbs JIM5, LM18 and LM19. Furthermore, paramural bodies, intercellular deposits and globular ergastic bodies composed of pectins, xyloglucans, extensins and AGPs were common. In Rhinanthus they were particularly abundant in pairings with legume hosts. Hyaline body cells were not in direct contact with haustorial xylem, which was surrounded by a single layer of paratracheal parenchyma with thickened cell walls abutting the xylem.

Conclusions

The distinctive anatomy and cell wall architecture indicate hyaline body specialization. Altered proportions of AGPs and pectins may affect the mechanical properties of hyaline body cell walls. This and the association with a transfer-like type of paratracheal parenchyma suggest a role in nutrient translocation. Organelle-rich protoplasts and the presence of exceptionally profuse intra- and intercellular wall materials when attached to a nitrogen-fixing host suggest subsequent processing and transient storage of nutrients. AGPs might therefore be implicated in nutrient transfer and metabolism in haustoria.     

Expression of the JIM8 cell wall epitope in carrot somatic embryogenesis

Certain single cells in carrot (Daucus carota L.) suspension cultures react with the monoclonal antibody JIM8, and it has been proposed that these cells represent a transitional stage in somatic embryo formation. Shortly after isolation of the single cells by sieving, up to 80% of the cells react with JIM8. Within 4 d, JIM8 labelling becomes restricted to 1% of the single cells. To obtain evidence for the proposed correlation between expression of the JIM8 cell wall epitope and somatic embryo formation the developmental fate of carrot single cells labelled with JIM8 was determined by cell tracking. The results, obtained by recording 43 000 cells, show that only few JIM8-labelled cells give rise to embryos, and most somatic embryos develop from cells devoid of the JIM8 cell wall epitope. We therefore conclude that the presence of the JIM8 cell wall epitope does not coincide with the ability of single suspension cells to form embryos.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - AGP arabino galactan protein - B5-0 Gamborg's B5 medium - B5-0.2 Gamborg's B5 medium supplemented with 0.2 M 2,4-D - FITC fluoresceïn isothiocyanate - PBS phosphate-buffered saline     

Guard cell wall: immunocytochemical detection of polysaccharide components

The composition of guard cell walls in sugar beet leaves (Beta vulgaris L.) was studied by using histochemical staining and immunocytochemical detection of cell wall antigens. The findings were compared with those in the walls of epidermal and mesophyll cells. Probing of leaf sections with monoclonal antibodies against pectins, terminal fucosyl residues linked alpha-(1-->2) to galactose, beta-(1-->3)-glucans and arabinogalactan-proteins revealed several specific features of guard cells. Pectic epitopes recognized by JIM7 were homogeneously distributed in the wall, whereas pectins recognized by JIM5 were not found in the walls themselves, but were abundant in the cuticular layer. Large amounts of molecules bearing terminal fucose were located predominantly in ventral and lateral guard cell walls. Much smaller amounts were detected in dorsal walls of these cells, as well as in the walls of pavement and mesophyll cells. Conspicuous accumulation of these compounds was observed in the vicinity of the guard cell plasmalemma, whereas labelling was scarce in the areas of the wall adjacent to the cell surface. The presence of callose clearly marked the ventral wall between the recently formed, very young guard cells. Callose also appeared in some mature walls, where it was seen as punctate deposits that probably reflected a specific physiological state of the guard cells. Large amounts of arabinogalactan-proteins were deposited within the cuticle, and smaller amounts of these proteoglycans were also detected in other tissues of the leaf. The histochemical and immunocytochemical structure of the guard cell wall is discussed in the light of its multiple functions, most of which involve changes in cell size and shape.     

The transmitting tissue inBrugmansia suaveolens: immunocytochemical localization of pectin in the style

Summary Two monoclonal antibodies were used to reveal the nature and distribution of pectins in cell walls and in the secretion of the style inBrugmansia (Datura) suaveolens at the light and electron microscope level. The antibodies JIM 5 and JIM 7 distinguish between unesterified and methylesterified pectins. Unesterified pectins occur in the walls of both transmitting tissue and cortex. The high methylesterified pectin is limited to cell walls in the cortex. The intercellular substance contains only unesterified pectins.     

Study of pectin esterase and changes in pectin methylation during normal and abnormal peach ripening

Peach fruit ( Prunus persica cv. Hermosa) were allowed to ripen immediately after harvest or after 30 days of 0°C storage. The fruits lost 75–80% of their firmness after 5 days at 20°C. During ripening after harvest there was a loss of both uronic acid and methyl groups from the cell wall. Cell wall labelling with JIM 7, a monoclonal antibody which recognized pectins with a high degree of methylation, was lower in ripe fruits than in freshly harvested fruits. However, ripe fruit cell walls did not cross-react with JIM 5, which recognizes pectins with low methylation. During storage, de-methylation occurred and in fruit ripened after storage there was little further change in pectin methylation or pectin content in the cell walls. The labelling of stored or stored plus ripened cell walls with JIM 7 was similar, but the cell walls of fruit ripened after storage showed some low cross-reactivity with JIM 5. The in vitro activity and mRNA abundance of pectin esterase (EC 3.1.1.11) was not correlated with the amount of de-esterification as measured chemically or by immuno-labelling in the cell walls. Eighty percent of the fruits which ripened after storage developed a woolly texture. It is suggested that woolliness is due to de-esterification of pectins, not accompanied by depolymerization, which leads to the formation of a gel-like structure in the cell wall.