Journal of Experimental Botany, Vol. 53, No. 371, pp. 1067-1079,
May 2002
© 2002 Oxford University Press
Original Papers |
Guard cell wall: immunocytochemical detection of polysaccharide components
1Institute of Plant Breeding and Acclimatization, Powsta
ców Wielkopolskich 10, 85-090 Bydgoszcz, Poland
2Department of Biochemistry and Cellular and Molecular Biology of Plants, Experimental Station of Zaidin (CSIC), 18008 Granada, Spain
Received 8 May 2001; Accepted 17 December 2001
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Abstract |
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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
-(1
2) to galactose, ß-(13)-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. Key words: Beta vulgaris L., callose, guard cell wall, pectin, xyloglucan.
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Introduction |
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The leaf epidermis is composed of several types of cells, including pavement cells, guard cells, subsidiary cells, and trichomes, each presenting unique morphological features and each performing well-defined physiological functions. Because each of them is characterized by a common origin from the protodermal layer (Becraft, 1999), the progressive specialization of particular cells within the epidermis provides a suitable model for studies of cell differentiation.
Acquiring guard cell identity involves, among other processes, the formation of specialized cell walls that become especially thick under the outer ledge facing the atmosphere, in the part adjacent to the pore, and on the inner periclinal side that faces the substomatal chamber (Zhao and Sack, 1999). The guard cell walls have been reported to be composed of radially arranged cellulose microfibrils and pectins, and to be covered by a layer of cuticle (Willmer and Fricker, 1996; Becraft, 1999). The details of guard cell wall composition and organization are, unfortunately, not known.
Despite some progress in the identification of genes that contribute to the specialized nature of guard cells (Müller-Röber et al., 1998), little information is available about the genes that might encode the proteins putatively involved in wall structure. Among these, StGCPRP has been shown to encode a protein (Solanum tuberosum guard cell proline-rich protein) with a 26-amino-acid N-terminal sequence, which may serve as the targeting signal for transport into the cell wall. Analysis of the StGCPRP protein revealed that about one-third of the Pro residues may be hydroxylated and several of these glycosylated with Ara residues, thus forming potential cross-linking sites. In addition, the Pro-rich domain of the peptide has a strong positive charge that can result in ionic interaction with highly acidic pectins (Menke et al., 2000). The expression of StGCPRP has been shown to be developmentally regulated. The transcript is especially abundant in all cells of the young leaf and within the youngest leaflets; as the leaf ages, expression is restricted to the epidermis along with the guard cells (Menke et al., 2000).
Guard cells from Nicotiana glauca display high levels of expression of several proteins that are predicted to have signal sequences directing them to the cell wall: NgGRP1 is a glycine-rich protein, NgGPP1 is highly homologous to proline-rich protein from tomato and Arabidopsis thaliana, and NgLTP1 is a lipid transfer protein (Smart et al., 2000). NgLTP1, a member of the family of proteins extractable from the cell wall, has been suggested to play a role in lipid transfer during cuticle deposition (Smart et al., 2000).
Tracing the synthesis of polysaccharides as well as their deposition and linking in the walls is more complicated than is the case for structural wall proteins, because no mRNA template exists for carbohydrate synthesis and assembly. Instead, the necessary information seems to be related to several metabolic events: (1) transport of polysaccharide components from the Golgi apparatus or endoplasmic reticulum to the wall (with the exception of cellulose and callose synthesis), (2) the action of the set of glycosyltransferases involved in binding particular monosaccharides to each other, which results in elongation of the polysaccharide chain, (3) assembly of different polysaccharides in muro, and (4) enzymatic modifications of polysaccharide structures within the wall leading to reorganization and reassembly of wall components (Carpita and Gibeaut, 1993).
Sequencing of cDNAs from guard cell protoplasts of Brassica campestris generated 515 expressed sequence tags, among which only a few were putatively identified as highly homologous to enzymes acting in the cell walls: UTP-glucose glucosyltransferase, ß-fructofuranosidase and peroxidase. None of them, however, was found to be preferentially expressed in guard cells (Kwak et al., 1997).
Because of the limitations in the methods used to investigate the expression of cell wall-related enzymes, detection of polysaccharide epitopes with specific antibodies is, in addition to the analysis of sugar composition and linkage, the method of choice to study structural features of the wall macromolecules and the subcellular compartmentalization of particular components (Knox, 1992, 1996; Pennell and Roberts, 1995). In this paper the presence is reported of identifiable structural epitopes within guard cell walls from leaves of sugar beet seedlings and shoots growing in vitro. The composition was elucidated by immunocytochemical detection of specific polysaccharidic antigens with a series of monoclonal antibodies, including those generated against pectins, terminal fucosyl residues found in xyloglucans and type I rhamnogalacturonans, ß(13) glucans and carbohydrate epitopes of arabinogalactan-proteins (AGPs). The chemical structure in guard cell walls, epidermal and mesophyll cell walls is also compared, and the results are discussed with reference to stomatal functions.
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Materials and methods |
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Plant material
Seeds of the male sterile diploid line 491D of sugar beet (Beta vulgaris L.) from Danisco Seed, Denmark, were used to obtain plants in the field of the Institute for Plant Breeding and Acclimatization in Bydgoszcz, Poland. Tips of generative shoots excised from flowering beets were the explants used to initiate axenic shoot cultures. They were sterilized in 70% ethanol for 30 s and in 5% sodium hypochloride for 20 min. After several washings in sterile distilled water, the explants were placed on modified Murashige and Skoog medium (Murashige and Skoog, 1962) supplemented with 4.4 µM 6-benzylaminopurine (BAP), and the developing shoots were maintained by transferring them onto the same medium containing 0.4 µM BAP and 0.1 µM naphthaleneacetic acid (NAA) every 3–4 weeks. Leaves excised from shoots derived from a single field-growing plant comprised the material studied here.
In addition, line 491D seeds were sown in a greenhouse to obtain seedlings as a source of leaves different from the in vitro cultures.
Fixation and paraffin embedding
The oldest and the youngest leaves were cut off from in vitro shoots 40 d after the last subculture, and from 40-d-old seedlings. All materials were immediately immersed in a fixative composed of 4% paraformaldehyde and 0.25% glutaraldehyde in 0.1 M PBS, pH 7.2. Fixation lasted for 24 h: an initial 3 h period at room temperature under slight vacuum was followed by 21 h at 4 °C. All samples were washed in several changes of 0.1 M PBS and dehydrated in increasing concentrations of ethanol. Then the leaves were passed through graded solutions of xylene in absolute ethanol, immersed in several changes of melted paraffin in xylene (65 °C) and finally embedded by quickly chilling the paraffin blocks in water.
Fixation and embedding in acrylic resin
The leaves were sliced into pieces of several square millimetres and fixed as described for paraffin-embedded samples, except that 0.05 M PIPES buffer, pH 7.2, was used for fixation and post-fixation washings. After the material was dehydrated in a series of ethanols, leaf fragments were infiltrated and embedded in LR Gold resin.
Histochemical detection of ß-glucans
ß-(13) glucans and ß-(14) glucans were stained in vivo on epidermal strips peeled from the abaxial surface of the leaves. Callose was detected by incubating the tissue in 0.1 mM aniline blue in 0.07 M phosphate buffer, pH 8.5, for 3 h, followed by overnight washing in buffer and brief washing in water. Control reactions were performed by incubating the strips in phosphate buffer, followed by washing in water. The fluorescence was observed under a Jenalumar 250 fluorescent microscope (Carl Zeiss, Jena, Germany) with a UV filter (excitation 330–380 nm, barrier filter 410 nm, dichroic mirror 410 nm).
ß-(14)- and ß-(13) glucans were detected by staining the tissues with a 0.1 M solution of Calcofluor white in water, followed by extensive overnight washing. The fluorescence was observed with the UV filter. All photographs were taken with Kodak 400 ASA colour film.
Histochemical detection of AGPs
The presence of AGPs was traced on 10 µm thick paraffin sections of the whole leaf, cut with a Reichert microtome (Austria). Before the leaf tissues were stained, the sections were deparaffined in several changes of xylene and ethanol, then hydrated in ethanols of decreasing concentration, and finally in distilled water. Materials were stained with 300 µM solutions of ß-glucosyl Yariv phenylglycoside (ß-D-Glc)3 in 1% NaCl, which specifically binds and precipitates AGPs. As a control, sections from the same sample were stained with 300 µM solutions of -galactosyl Yariv phenylglycoside (-D-Gal)3, which does not bind AGPs. Both reagents were from Australia Biosupplies (Parkville, Australia). After staining for 72 h at 4 °C, the sections were washed overnight in 1% NaCl to remove excess unbound reagents, then briefly rinsed with distilled water, air-dried, placed in Canada balsam and observed under Jenamed 2 microscope (Carl Zeiss, Jena, Germany).
Immunocytochemical procedures
Semi-thin sections of LR Gold-embedded material (0.5 µm) were cut on an RM 2155 microtome (Leica Microsystems, Nussloch GmbH, Germany) and placed on the surface of glass slides covered with 2% acetone solution of Biobond (BioCell Research Laboratories, UK). Before the immunocytochemical reactions with the panel of antibodies, the sections were blocked with 5% BSA in 0.1 M PBS, pH 7.3, for 36 h at room temperature. Then the following antibodies, diluted with 0.1 M PBS containing 5% BSA, were applied for 12 h to trace the presence and distribution of cell wall antigens: (1) JIM5 and JIM7, diluted 1:3, to detect pectic epitopes, (2) CCRC-M1, diluted 1:5, to detect xyloglucans and rhamnogalacturonan I with terminal fucosyl residues, (3) anti-ß-(13)-glucan, diluted 1:50, to detect callose, (4) LM2 and JIM13, diluted 1:10, to detect carbohydrate epitopes of AGP that contain ß-linked glucuronic acid and GlcpA-ß(13)-D-GalpA-(12)-L-Rha trisaccharide motif, respectively (Yates et al., 1996).
The JIM5, JIM7 and JIM13 antibodies were kindly provided by Dr K Roberts (John Innes Institute, UK); LM2 was provided by Dr P Knox (University of Leeds, UK); and CCRC-M1 by Dr M Hahn (University of Georgia, USA). Anti-ß-(13)-glucan is a commercial antibody (Australia Biosupplies).
All reactions took place at room temperature, and were followed by several 15 min washings in 0.1 M PBS. Then the sections were incubated for 8 h with secondary antibodies (FITC-anti-rat for JIM5, JIM7, LM2, and JIM13, and FITC-anti-mouse for anti-ß-(13)-glucan and CCRC-M1), both diluted 1:20 in 0.1 M PBS containing 0.1% BSA. For CCRC-M1, antibody binding was additionally localized with goat-anti-mouse antibody coupled to 5 nm colloidal gold diluted 1:200 in PBS, followed by a 5 min silver enhancement step according to the manufacturer's instructions (Biocell Research Laboratories, UK). Control reactions were performed by omitting the primary antibody. After overnight washing in several changes of buffer and distilled water, the sections were air-dried, covered with anti-fade solution containing 0.5% p-phenyldiamine in 0.1 M PBS, pH 12.0, mixed with glycerine 1:4, and then observed with a fluorescence microscope equipped with a blue filter (excitation 450 nm, barrier filter 510 nm, dichroic mirror 510 nm). Pictures were taken with Kodak 400 ASA colour film.
Ultrathin sections were obtained with a Reichert Ultracut E ultramicrotome and collected on formvar-coated nickel grids. Immunocytochemical reactions were performed as described for semi-thin sections, except that the blocking step in 5% BSA lasted 2 h, incubation with primary antibodies lasted 3 h, and incubation with secondary antibodies lasted 2 h. The secondary antibodies used to detect the antigens were goat-anti rat coupled with 15 nm colloidal gold diluted 1:50 in buffer supplemented with 0.1% BSA, and goat-anti mouse coupled with 10 nm colloidal gold diluted 1:50 in buffer, also supplemented with 0.1% BSA. The grids were washed in buffer and distilled water, and the sections were briefly stained in a 5% aqueous solution of uranyl acetate and observed under a Zeiss 10C electron microscope (Carl Zeiss, Germany) at 60 kV.
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Results |
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Cells in epidermal strips from sugar beet leaves revealed several morphologically distinct phases of stomatal organization, which corresponded to successive stages of development. These stages were: the primary meristemoid, differentiation of the isodiametric guard mother cell, symmetric mitosis of the guard mother cell, and subsequent phases of growth and differentiation of the guard cell (Fig. 1
). Mature stomatal complexes were composed of two kidney-shaped guard cells surrounded by three or four subsidiary cells of different size, and thus might be classified as anisocytic stomata (Willmer and Fricker, 1996).
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The presence of pectins in the walls of guard cells, pavement cells and mesophyll was traced by the immunocytochemical detection of polymers with JIM7 and JIM5 antibodies. Epitopes recognized by JIM7 were found within the walls of all cell types, and produced a bright green fluorescence throughout the whole leaf section (Fig. 2A
). Labelling with gold-conjugated antibody produced signals in the entire guard cell wall including the thickenings, and resulted in an approximately homogeneous distribution of gold throughout the different wall zones (Fig. 2B, C). Pectins recognized by JIM5 antibody were not seen inside the guard cell walls, but were frequently found at the external surface of the cells facing the environment and the substomatal cavity, as well as between the ventral walls of the two neighbouring cells (Fig. 3A–C). They were also present on the surface of some pavement cells, and were occasionally seen within the mesophyll tissue. In the latter, JIM5-binding polymers appeared as irregular aggregates in some cells and in some wall areas (Fig. 3A). When present, these pectins were distributed in the middle lamella or in the intercellular spaces (not shown).
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The distribution of epitopes containing terminal fucosyl residues, which were detected with the CCRC-M1 antibody, was variable in leaf tissues in terms of both abundance and subcellular location. These compounds proved to be especially abundant in guard cell walls (Fig. 4A
, B), where they showed a distinct spatial pattern and a variable degree of accumulation within the walls surrounding a given cell. The antigen was more abundant in the zone adjacent to the plasma membrane than in the zone closer to the external wall surface (Fig. 4C, D). Moreover, this xyloglucan (rhamnogalacturonan I) epitope was much more abundant in ventral walls and in the lateral walls (Fig. 4D, E) than in the dorsal walls in contact with epidermal cells (Fig. 4F). In longitudinal sections of guard cells, these molecules were found in the central part of the cell, whereas they were relatively scarce at the edges (not shown). The CCRC-M1 antibody also bound to the walls of pavement cells and most mesophyll cells, but the signal was very weak compared to that for the guard cells (Fig. 4A, B).
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The anti-ß-(1
3)-glucan antibody bound most clearly to the wall separating two young guard cells just after the guard mother cells had completed symmetric division (Fig. 5A, C). In this phase of development the walls frequently formed the ‘zig-zag’ line, and the cells themselves were highly vacuolated and already possessed distinctive wall thickenings (Fig. 6A, B). In addition, callose was detected in the walls of some mature guard cells (Fig. 6C), where it frequently showed a punctate distribution (Fig. 5A). Labelling was weak and punctate in epidermal and mesophyll cells (Fig. 5C). Electron microscopic observations showed that this labelling corresponded to plasmodesmatal connections (not shown). Control reactions in epidermal strips (Fig. 5B) and the leaf sections resulted in no labelling.
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Calcofluor staining produced a blue fluorescence in all stomata walls, and the signal was conspicuously stronger in these cells than in the neighbouring pavement cells (Fig. 5D
). Control sections showed no fluorescence (Fig. 5E).
AGPs were detected with histochemical staining with (ß-D-Glc)3 Yariv reagent and an immunocytochemical technique with JIM13 and LM2 monoclonal antibodies, which bind to carbohydrate epitopes of AGPs that contain GlcpA-ß(13)-D-GalpA-(12)-L-Rha trisaccharide motif and ß-linked glucuronic acid, respectively. Both methods revealed the expression of these proteoglycans in almost all mesophyll and epidermal cells, including the guard cells (Fig. 7A, B). AGP epitopes were predominantly located at the plasma membranes or near the membranes, and much weaker fluorescence was observed within the cell walls (Fig. 6B). Very prominent signals were also found in the external cuticular layer covering the epidermis, as indicated by the deep red colour resulting from (ß-D-Glc)3 binding (Fig. 7C) and by the fluorescence signal obtained after incubation with JIM13 and LM2 antibodies (Fig. 7D). Control reactions resulted in a complete lack of staining (Fig. 7E) and no fluorescence signal (Fig. 7F).
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The patterns of distribution of all wall antigens used here were the same in leaves obtained from seedlings and from in vitro shoot cultures. No differences were found between old and young leaves.
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Discussion |
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Guard cells are involved in several processes essential for plant life, and are of particular importance for gas exchange between the plant and the environment, protection against excessive water loss, and protection against pathogen attack. These functions require successive openings and closures of the stomata pore, and are thus related to increases and decreases in cell volume paralleled by marked changes in the shape of the cell wall. The wall of guard cells thus requires a special structure which permits repeated expansion and contraction (Willmer and Fricker, 1996). In addition, these walls must be well adapted to a particularly intense degree of apoplastic transport of ions, water and some organic compounds, and to the rapid interchange of these compounds between the apoplast and symplast (Felle et al., 2000).
Enzymatic treatment of leaf tissue to isolate protoplasts from particular cell types revealed that guard cell walls need much stronger enzyme solutions to be completely digested, compared with their mesophyll counterparts (Boorse and Tallman, 1999; A Majewska-Sawka, A Münster, MI Rodríguez-Garcia, unpublished data). This indicates that the arrangement of the polysaccharide network and the content of certain macromolecules must differ significantly between these two cell types. Although some earlier reports emphasized that differentiation of the guard cells is accompanied by the deposition of radially arranged cellulose microfibrils and by the secretion of pectic polymers (Willmer and Fricker, 1996; Zhao and Sack, 1999), no detailed information on guard cell wall composition and structure is available.
These studies showed that the pectic fraction of the guard cell walls is rich in epitopes recognized by the JIM7 antibody. The structure of these epitopes has not yet been fully characterized, but optimal antibody binding is known to occur when polymers of galacturonic acid are methyl-esterified in a range from about 15–80%, regardless of whether esterification displays a random or blockwise pattern of distribution. Esterification lower than 15% results in significant weakening of binding properties (Willats et al., 2000). On the other hand, optimal binding of epitopes recognized by JIM5 takes place in polymers esterified in a range from 31% to slightly over 40% (Willats et al., 2000).
The presence in guard cell walls of JIM7-recognized epitopes, together with the absence of JIM5-recognized epitopes, led to the assumption that pectins with a level of esterification much greater than 40% were probably being detected.
A high level of esterification makes pectins unable to form the rigid ‘egg-box’ structure through Ca2+ bridges; such epitopes are therefore more flexible than non-esterified or little-esterified epitopes. It has been postulated that the organization of the pectic components controls the porosity and sieving properties of the wall (Fujino and Itoh, 1998), and may thus influence the movement and activity of enzymes for wall metabolism (Carpita and Gibeaut, 1993). The degree of pectin esterification is also related to the strength of the pectin gel; for isolated pectins maximum gel strength was found at about 70% esterification (Davis et al., 1980; McCann and Roberts, 1994). Pectin gels are able to undergo reversible volume changes (swelling), which can be induced by small changes in temperature, type and concentration of salts, pH or electrical field (Carpita and Gibeaut, 1993). Weakly charged pectic polysaccharides display a much more pronounced capacity to swell than do highly charged polymers (Ryden et al., 2000). Pectins are also involved in regulating the water balance within plant cells through their capacity to bind apoplastic water (Zhou et al., 2000). In the light of the aforementioned functions of the guard cell walls, the predominance of epitopes recognized by JIM7 seems logical, since they would facilitate modifications in the pectin matrix required for these processes, while simultaneously making the wall strong and relatively flexible.
The specificity of the JIM5 antibody was recently re-evaluated (Willats et al., 2000). By contrast to the previous report (Knox et al., 1990), it is now assumed that this antibody reacts only very weakly with fully de-esterified pectins, and that optimal binding requires the presence of groups that are methyl-esterified in a range of 31% to slightly over 40%. As for JIM7, the pattern of esterification does not significantly influence binding capacity (Willats et al., 2000). The presence of JIM5 epitopes and the absence of JIM7 epitopes (which show optimum binding to polymers with 15–80% esterification) in the cuticular layer that covers the sugar beet leaf surface, clearly indicates that the differences between these two pectic epitopes involve structural features other than those related to the degree of esterification, possibly the content of continuous stretches of de-esterified residues of galacturonic acid (Willats et al., 2000). The presence of pectins on the external surfaces of guard cell walls is in agreement with the postulated function of this compound in binding the cuticle to the walls of both pavement and guard cells (Becraft, 1999). It is well documented that guard cells tend to have a thick cuticle that covers all surfaces in direct contact with the air, and which thus decreases water vapour diffusion (Willmer and Fricker, 1996).
Molecules bearing terminal fucosyl residues, present in xyloglucans and type I rhamnogalucturonans, accumulate in the guard cell walls of sugar beet, whereas they are scarce in the walls of other cell types. Because CCRC-M1 binding has been shown to be very effective to xyloglucans, and about 50-fold less effective to rhamnogalacturonans I of sycamore (Puhlmann et al., 1994), it seems reasonable to assume that the molecules detected with this antibody in sugar beet cells represent mostly xyloglucan epitopes. Xyloglucans are involved in the formation of a dense macromolecular network with cellulose, and this arrangement of the cell wall components has been found in many types of cell (Carpita and Gibeaut, 1993; Pauly et al., 1999), including epidermal cells of Pisum sativum (Fujino et al., 2000) and Allium cepa (Wilson et al., 2000). Cross-linking of cellulose by xyloglucans is particularly conspicuous in the ventral and lateral walls of sugar beet guard cells, and probably results in decreased stiffness and increased extensibility of these wall zones, as described in other models (Whitney et al., 1999). Cellulose-xyloglucan complexes are typically formed in cells which undergo active changes in volume, and facilitate turgor-mediated cell expansion (Cosgrove, 1999; Wu and Cosgrove, 2000). The guard cells lengthen upon stomatal opening, and the ventral wall extends relatively further than the dorsal wall (Willmer and Fricker, 1996). This would explain the much higher accumulation of xyloglucan molecules in ventral walls compared to dorsal walls.
The capacity of the walls to stretch may be also modulated by the activity of enzymes such as endoglucanases and endotransglycosylases. The activity of the former generates oligosaccharides that may act as signals to modulate further growth (Cosgrove, 1999). In this context, the presence of the -L-Fuc(12)-ß-D-Gal(1) motif (XXFG) in xyloglucan molecules (Puhlmann et al., 1994) might be related to the recently documented role of XXFG as a signalling molecule (Zablackis et al., 1996) responsible for growth inhibition (York et al., 1984; Fry et al., 1993; Dunand et al., 2000). The binding of these oligosaccharides to membrane receptors in blackberry protoplasts was shown to be inhibited by ABA, 2,4-D, GA3 and kinetin in a concentration-dependent manner. Whether XXFG molecules are active in guard cells remains, however, to be determined.
The presence of callose has been shown in guard cell walls in both epidermal strips stained in vivo and in chemically fixed cells. The earliest appearance of callose in the course of stomatal differentiation is related to the formation of the wall separating two very young guard cells. This seems to be a common but temporary feature characterizing all stomata in this phase of development. By analogy with other systems in which callose appears, this phenomenon is probably related to the location of this compound in the cell plate and young primary walls of several cell types (Fulcher et al., 1976; Northcote et al., 1989; Assaad et al., 1997). As far as is known, the presence of ß-(13) glucan in guard cell walls has only been reported in some ferns, including species of Ophioglossum (Peterson et al., 1975), and in the liverwort Marchantia polymorpha (Górska-Brylass, personal communication), but has not been documented in angiosperm species. Later in development, callose is synthesized only in some mature stomata, which indicates that synthesis of this glucan may reflect the physiological state of the cell. The function of this wall compound is unknown, but it may be involved in the response to high turgor and mechanical force during stomatal opening, since callose is a wall-strengthening agent. On the other hand, stomatal closure is known to be induced by ABA; this hormone acts in turn through several intermediates such as hydrogen peroxide and calcium ions (Pei et al., 2000; Zhang et al., 2001). An increase in the concentration of either of these compounds is also involved in the activation of callose synthase (Kauss, 1985; Price, 1990). Refined physiological methods will be required, however, to shed light on these hypotheses.
The results of immunocytochemical studies presented in this paper show clearly that the walls of guard cells from sugar beet leaves show specific differences in composition compared with their neighbouring epidermal and mesophyll cells. The most notable differences are the extremely high accumulation of molecules bearing terminal fucosyl residues and the total absence of pectins recognized by JIM5 in the guard cell walls. Particular features seen during development are the transitory appearance of a callose-rich wall between two recently formed guard cells, and the formation of irregular callose deposits in some mature walls. This unique organization of the guard cell wall seems to be an important prerequisite for stomatal movement and for the transport of water and organic or inorganic compounds.
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Acknowledgements |
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This study was supported by The Polish Committee for Scientific Research (project no. 5 P06A 040 17). The authors thank Dr K Roberts (John Innes Centre, UK) for providing the JIM5, JIM7 and JIM13 antibodies, Dr P Knox (University of Leeds, UK) for the LM2 antibody, and Dr M Hahn (University of Georgia, USA) for the CCRC-M1 antibody. We also thank K Shashok for correcting the English version of the manuscript.
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Footnotes |
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3 To whom correspondence should be addressed. Fax: +48 52 3224454. E-mail: a.majewska{at}ihar.bydgoszcz.pl
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Abbreviations |
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(
-D-Gal)3, -D-galactosyl Yariv phenylglycoside; (ß-D-Glc)3, ß-D-glucosyl Yariv phenylglycoside; AGP, arabinogalactan-protein; BAP, 6-benzylaminopurine; NAA, naphthaleneacetic acid; PBS, phosphate buffer saline; BSA, bovine serum albumin.. |
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