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Journal of Microscopy, Vol. 193, Pt 2, February 1999, pp. 150–157. Received 20 July 1998; accepted 24 September 1998 Immunogold localization of plant surface arabinogalactan-proteins using glycerol liquid substitution and scanning electron microscopy J. ŠAMAJ*‡, H.-J. ENSIKAT,* F. BALUŠKA,* J. P. KNOX,† W. BARTHLOTT* & D. VOLKMANN* *Botanisches Institut, Universität Bonn, Kirschallee 1 and Meckenheimer Allee 170, D-53115 Bonn, Germany †Centre for Plant Biochemistry & Biotechnology, University of Leeds, Leeds LS2 9JT, U.K. ‡Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademická 2, SK-950 07 Nitra, Slovakia Key words. Arabinogalactan-proteins, embryogenic callus, extracellular matrix, immunolocalization, root development, scanning electron microscopy, Zea mays. Summary We have studied the spatial distributions of arabinogalactan-protein (AGP) epitopes on the surface of maize embryogenic calli and roots using monoclonal antibodies JIM4 and MAC207. For this purpose, a new immunogoldscanning electron microscopy (SEM) method was employed which is based on liquid substitution of samples with glycerol. Using this method, we were able to show that the AGP epitopes are distributed along callus and root surfaces and they decorate filamentous structures. In callus cells, the JIM4 epitope was specifically enriched in the outer extracellular layers covering compact clusters of embryogenic meristematic callus cells. In roots, the MAC207 epitope was abundant on the root epidermal surface corresponding to the outer root pellicle, but was only occasionally found on the mucilage layer covering the root cap cells. Silver-enhanced gold particles, indicating AGP epitopes, were often linearly arranged suggesting that AGPs associate with filamentous structures both on the surface of embryogenic calli and root epidermal cells. These results indicate that AGPs are components of the outer extracellular layers and networks that cover the surface of roots and cells undergoing somatic embryogenesis. Introduction Arabinogalactan-proteins (AGPs) form an abundant class of plant cell surface proteoglycans with high water-holding capacity, and inherent stickiness (Chasan, 1994). These molecules are characterized by carbohydrate components Correspondence to: J. Šamaj. Tel: þ 49 228 733350; fax: þ 49 228 732677; email: [email protected] 150 rich in galactose and arabinose, and protein components rich in Hyp, Ala, Ser, Thr and Gly (Nothnagel, 1997). AGPs belong to a family of structural plant cell wall proteins known as Hyp-rich glycoproteins (Kieliszewski & Lamport, 1994). Since the AGP group is very heterogeneous, three main criteria for AGP classification have been proposed by Du et al. (1996): first, the presence of arabinogalactan chains, second, a Hyp-rich protein backbone, and third, the ability to bind b-glucosyl Yariv phenylglycoside (a synthetic phenylazo dye). However, these criteria as a diagnostic test may be too restrictive as some AGPs bind little, if any, Yariv phenylglycoside (Nothnagel, 1997). Biochemical and immunolocalization techniques have indicated that AGPs are associated with plant cell surfaces and they have been found on plasma membranes, within cell walls, and to be secreted by cells in soluble forms (Knox, 1996). Unfortunately, despite two decades of research on AGPs, the exact functions of these plantspecific molecules are still unknown. Nevertheless, several biological roles for AGPs have been proposed including those in water balance regulation and adhesive events in plants (Chasan, 1994), cell proliferation and cell expansion (Schopfer, 1990; Zhu et al., 1993; Serpe & Nothnagel, 1994; Willats & Knox, 1996; Langan & Nothnagel, 1997), and cell signalling in the periplasmic space (Roberts, 1990; Reuzeau & Pont-Lezica, 1995). Exogenous AGPs have been shown to influence somatic embryogenesis (Kreuger & van Holst, 1993, 1995; Egertsdotter & von Arnold, 1995) and have been demonstrated to have cell- and tissue-specific expression in roots (Knox et al., 1991; Smallwood et al., 1994) and flowers (Pennell & Roberts, 1990; Pennell et al., 1991). q 1999 The Royal Microscopical Society I M M U N O G OL D S EM O F P LA N T SUR FAC E A R A B I N O G A LAC TA N - P RO T E I N S 151 This growing body of evidence suggests that cell surface AGPs play an important role in plant morphogenesis (Knox et al., 1989; Pennell et al., 1989; Knox, 1990, 1992, 1996, 1997; Pennell & Roberts, 1990; Nothnagel, 1997). Until now, these molecules have been localized only within organs using tissue sections combined with immunofluorescence and immunogold transmission electron microscopy (e.g. Knox et al., 1991; Pennell et al., 1991, 1992; Knox, 1997). The main aim of this study was to develop a reliable and simple method for the surface localization of AGP epitopes employing silver-enhanced immunogold labelling in combination with scanning electron microscopy. sections were incubated with rat monoclonal antibodies against two different arabinogalactan-proteins: JIM4 (Knox et al., 1989) and MAC207 (Pennell et al., 1989) diluted 1 : 20 in PBS conatining 1% BSA for 1·5 h at 25 8C. After rinsing with PBS containing 1% BSA they were incubated with FITC conjugated goat antirat IgGs (Sigma, St. Louis, MO, U.S.A.) diluted 1 : 20 in the same buffer for 1·5 h at 25 8C. Nuclear DNA was stained with DAPI (1 mg mL¹1) for 2 min. After rinsing in PBS, the stained sections were treated with 0·01% toluidine blue in PBS for 10 min to diminish autofluorescence of the tissues. Sections were finally mounted into antifade mountant containing p-phenylenediamine (Balus̆ka et al., 1992). Materials and methods Immunogold scanning electron microscopy Plant material and tissue culture Grains of maize (Zea mays L., cv. Alarik) obtained from Force Limagrain (Darmstadt, Germany) were soaked and germinated in moist filter paper at 24 8C in darkness. Seedlings with straight primary roots, 30–35 mm long, were selected for immunolabelling. Maize embryogenic callus was induced as described previously (Šamaj et al., 1995) and maintained on MS medium (Murashige & Skoog, 1962) supplemented with 12·7 mM 2,4-dichlorophenoxy acetic acid and 0·088 M sucrose at 24 8C in darkness. Fixation and embedding Apices of primary roots and small clumps of calli were excised and fixed with 4% paraformaldehyde and 0·5% glutaraldehyde in 100 mM phosphate-buffered saline (PBS, pH 7·2) or stabilizing buffer (SB, 50 mM PIPES buffer, 5 mM MgSO4, 5 mM EGTA, pH 7·0) for 1·5 h at 20 8C. After washing in PBS, samples were dehydrated in a graded ethanol series diluted with PBS. Tissue was embedded in Steedman’s wax as described by Balus̆ka et al. (1992). In brief, wax was prepared from PEG 400 distearate and 1-hexadecanol (9 : 1, Aldrich, Milwaukee, WI, U.S.A.). Tissue was embedded at 37 8C and subsequently wax was left to polymerize at room temperature. Indirect immunofluorescence microscopy Tissue sections (7 mm thick) from samples embedded in wax were mounted on slides coated with glycerol albumin (Serva, Heidelberg, Germany) or Biobond (BioCell, Cardiff, U.K.), dewaxed in absolute ethanol, passed through a graded ethanol series and rinsed in SB and PBS containing 5% bovine serum albumin (BSA). Subsequently, the q 1999 The Royal Microscopical Society, Journal of Microscopy, 193, 150–157 Excised callus and root segments were fixed in PBS containing 3·7% formaldehyde and 0·25% glutaraldehyde for 1 h at 25 8C and extensively washed with PBS. Residual aldehydes were blocked with 0·05 M glycine in PBS and proteins were blocked with 5% BSA and 0·1% fish gelatine in PBS, both for 30 min. Thereafter, samples were incubated for 1 h with monoclonal antibodies JIM4 and MAC207 diluted 1 : 50 in PBS containing 1% BSA, and after extensive washing with PBS they were incubated with rabbit antirat IgGs conjugated to 5 nm colloidal gold (BioCell) diluted 1 : 100 in PBS containing 1% BSA for 1 h. After washing with PBS and distilled deionized water (four times, 10 min each), the gold particles were silver enhanced with silver enhancement kit according to the manufacturer’s instructions (BioCell). Following extensive washing in distilled deionized water, samples were continuously substituted with glycerol according to Ensikat & Barthlott (1993) and observed in a Stereoscan S200 SEM (LEO, Cambridge, U.K.) equipped with a detector for backscattered electrons. Both secondary and backscattered electrons were recorded individually or simultaneously as mixed signal. Control callus and root samples were handled as described above except that primary antibody was omitted during incubation. Conventional scanning electron microscopy Samples for conventional SEM using critical point drying were prepared as described elsewhere (Šamaj et al., 1995). Results Immunogold SEM localization of the JIM4 AGP epitope in callus Immunogold SEM revealed that silver-enhanced gold 152 J. ŠA M A J ET AL . Fig. 1. Immunogold SEM images of maize embryogenic callus using JIM4 monoclonal antibody. Larger arrows in a, b and d indicate the same reference point within the same cell. a, b and d. Immunolabelling of embryogenic cells with JIM4 antibody shows distribution of corresponding AGP epitope decorating filamentous structures (larger arrows on a,b,d and smaller arrows on d); a shows a BSE image and b shows a corresponding secondary electron (SE) image to a; d shows a detail of b with labelled filamentous structures (small arrows) c. Control embryogenic callus cells treated without primary antibody show very little nonspecific immunolabelling (BSE image). e. Filamentous nature of surface structure in embryogenic callus after glycerol substitution without immunogold/silver labelling (SE image) f. Cell surface and bridge-like connections between cells (arrows) are densely labelled with JIM4 (BSE image). g. Detail of cell–cell junction (larger arrow) showing accumulation of JIM4 labelling. Silver-enhanced gold particles decorating filamentous structure are indicated by smaller arrows. Scale bars: 2·5 mm for a, b and c; 1·5 mm for d, e and f; and 1 mm for g. q 1999 The Royal Microscopical Society, Journal of Microscopy, 193, 150–157 I M M U N O G OL D S EM O F P LA N T SUR FAC E A R A B I N O G A LAC TA N - P RO T E I N S particles corresponding to the JIM4 epitope occurred preferentially on the surface of embryogenic callus cells organized in compact embryogenic clusters (Fig. 1a,b,d, arrows). When the primary antibody was omitted embryogenic callus cells were not, or only very weakly, labelled, confirming the specificity of the immunogold SEM method (Fig. 1c). The immunogold labelling with subsequent silver enhancement has not altered the surface structure of embryogenic cells compared with control nonlabelled cells substituted with glycerol (Fig. 1e). The JIM4 epitope was especially enriched at cell–cell junctions and bridge-like extracellular connections between adjacent embryogenic cells (Fig. 1f,g, arrows) and embryogenic clumps. Detailed investigations revealed that individual silver-enhanced gold particles corresponding to the JIM4 epitope decorated filamentous 153 structures on the surface of embryogenic cells (Fig. 1b,d, arrows). Immunogold SEM localization of the MAC207 epitope in roots The glycerol substitution method allowed the preservation of fine root surface structure including the mucilage layer covering the root cap cells (Fig. 2a,b, stars). In combination with immunogold labelling, we localized the MAC207 epitope predominantly on the root epidermal surface (Fig. 2b,c) and very occasionally on the surface of root cap mucilage (Fig. 2c, white arrows). Detailed investigations revealed that the MAC207 epitope was arranged in the form of filament-like structures and stripes along the epidermal root surface (Fig. 2c, black arrows and white arrowheads). Fig. 2. Immunogold SEM images of maize roots using MAC207 monoclonal antibody. Stars indicate root cap mucilage. (a) The glycerol substitution method preserves fine mucilages at the root surface including root cap cells (SE image). (b) Dense immunolabelling (white) of the epidermal root surface (BSE image). (c) Detail of the root surface. AGP epitope recognized by MAC207 is arranged in filament-like arrays (black arrows) and stripes (arrowheads) along the epidermal surface (BSE image). Occasionally, weak labelling can be found also on adjacent root cap mucilage (white arrows). Scale bars: 20 mm for a and b, and 5 mm for c. q 1999 The Royal Microscopical Society, Journal of Microscopy, 193, 150–157 154 J. ŠA M A J ET AL . Fig. 3. Immunofluorescence labelling with JIM4 in maize embryogenic callus (a,b) and with MAC207 in maize root (c). a. Outermost extracellular layer (arrowheads) and bridge-like structures (arrow) around embryogenic cell clusters are immunolabelled with JIM4. b. Cell–cell junction domains of cell walls between neighbouring embryogenic cells (arrows) are intensively labelled with JIM4. c. Longitudinal section through the meristematic root zone showing prominent immunolabelling of the root outer pellicle (stars), root epidermis (E) and one outermost layer of the cortex. Scale bars: 5 mm for a, 3 mm for b, and 14 mm for c. Immunofluorescence localization of the JIM4 epitope in callus and the MAC207 epitope in roots Immunofluorescence of tissue sections with JIM4 and MAC207 antibodies supported the results obtained with the immunogold SEM method. In calli, the highest level of immunofluorescence labelling was associated regularly with the distinct outer layer of embryogenic surfaces corresponding to extracellular surface networks covering embryogenic cell clusters (Fig. 3a, arrowheads). The bridge-like structures, directly interconnecting adjacent embryogenic cells, were regularly found to be immunolabelled (Fig. 3a, arrow). The JIM4 epitope was enriched especially at cell wall contacts located close to intercellular spaces (Fig. 3b, arrows). In roots, the MAC207 epitope occurred on the epidermal surface and within the epidermal and cortical cells (Fig. 3c). The immunofluorescence signal was enhanced especially within the outer pellicle of the root epidermis (Fig. 3c, stars). Reliability of glycerol substitution method In preliminary experiments, we tested structural preservation of the mucilage surfaces of calli and roots using critical point drying or glycerol substitution methods. The critical point drying caused an artefactual appearance of mucilage surface layers. This encompassed shrinkage and partial destruction of the surface callus layer accompanied by hole formation (Fig. 4b). Also, the root surface was altered with root cap slime appressed to the root cap cells (Fig. 4d). In contrast, owing to the gentle and continuous replacement of water and absence of drying, the glycerol substitution method allowed an excellent mucilage surface preservation which is closer to native conditions, both in calli and in roots (Fig. 4a,c). Using the latter method, the surfaces appeared smoother, which also helped in the visualization of the silver-enhanced gold particles after immunolabelling. These results unambiguously confirmed the reliability of the glycerol substitution method when used in combination with immunogold labelling. Discussion Immunogold-SEM with conventional preparation of the sample has been used previously for the detection of animal cell-surface antigens such as fibronectins (Trejdosiewicz et al., 1981). When the backscattered electron (BSE) imaging mode was employed in SEM, it noticeably increased the intensity of the signal and allowed the unambiguous identification of the immunogold labelling and corresponding cell morphology in a material-dependent contrast (de Harven et al., 1984; Walther et al., 1984). Moreover, the silver enhancement of gold particles can further improve BSE imaging (Birrell et al., 1986; Scopsi et al., 1986; Herter et al., 1993), especially when small gold particles (1–5 nm), which can bind more antigen epitopes than the large ones (Hodges & Carr, 1990) are used. Importantly, it was shown that glycerol, which has a very low vapour pressure, can be used to infiltrate various biological samples so that they can subsequently be observed in the SEM without drying and coating (Ensikat & Barthlott, 1993; Robards & Wilson, 1996). This simple and versatile glycerol substitution method for SEM was used successfully to preserve immunolabelled actin microfilaments in cut open Chara internodal cells (Reichelt et al., 1995). Here, we have tested the reliability of glycerol substitution for structural preservation of mucilage at plant q 1999 The Royal Microscopical Society, Journal of Microscopy, 193, 150–157 I M M U N O G OL D S EM O F P LA N T SUR FAC E A R A B I N O G A LAC TA N - P RO T E I N S 155 Fig. 4. Comparison of glycerol substitution method (a,c) with conventional SEM (b,d) as applied to maize callus (a,b) and roots (c,d). a. Glycerol substituted callus cells are covered with a continuous extracellular surface layer. b. Critical point drying caused shrinkage and formation of holes (indicated by arrows) in the extracellular surface layer covering embryogenic cells. c. Glycerol substituted roots have smooth surface. d. Roots prepared with critical point drying show shrinkage of mucilage surface (arrow). Mucilage is appressed to root cap cells or removed so that individual root cap cells can be recognized. Root caps are indicated with stars and root epidermis with asterisks on c and d. Scale bars: 2·5 mm for a and b, and 40 mm for c and d. surfaces and we have developed an associated immunogold labelling technique. AGPs are abundant surface proteoglycans with probable morphoregulatory-relevant functions (Knox, 1996; Nothnagel, 1997) and are therefore ideal molecules to be studied on plant surfaces by immuno-SEM methods. Furthermore, the availability of AGP-specific monoclonal antibodies (Knox, 1997) makes them a prime candidate for immunofluorescence and immunogold studies. However, until now, no suitable method has been available for the threedimensional immunolocalization of antigens at plant surfaces. Therefore, we have developed a new immunogold-SEM method for plant surface antigens based on glycerol substitution of the specimen. This method has allowed us to study the spatial surface distributions of the JIM4 and MAC207 AGP epitopes on callus and root. These epitopes appear to occur as components of the filamentous structures covering meristematic cells of maize callus and root epidermis. Moreover, our immunogold SEM results are q 1999 The Royal Microscopical Society, Journal of Microscopy, 193, 150–157 supported by immunofluorescence employing the same antibodies on tissue sections from identical plant material. Previously, the JIM4 epitope has been reported to be developmentally expressed during carrot somatic embryogenesis (Stacey et al., 1990). As we have found the same epitope enriched at the surface of maize embryogenic cultures, our results may indicate a general importance of this AGP epitope during somatic embryogenesis. Using conventional scanning and transmission electron microscopy, the extracellular matrix (ECM) networks have been described during the induction of somatic embryogenesis in several plant species including Coffea (Sondahl et al., 1979), Cichorium (Dubois et al., 1991, 1992), Drosera (Bobák et al., 1995; Šamaj et al., 1995), Papaver (Šamaj et al., 1994; Ovecka et al., 1998) and Pinus (Jásik et al., 1995), as well as during early shoot and root organogenesis of Linum (Šamaj et al., 1997). It has been suggested that they play an important role in plant morphogenesis (Šamaj et al., 1997). Proteinaceous extracellular matrix layers and net- 156 J. ŠA M A J ET AL . works are also structural markers of maize embryogenic callus (Šamaj et al., 1995). The identity of the first molecular components of an ECM network in maize embryogenic tissue cultures have now been identified in this report. For maize roots, it is known that the extracellular matrix covering young columnar epidermal cells is tripartite and that these three distinct layers differ in their chemical composition and structure (Abeysekera & McCully, 1993). The innermost layer was called the cell wall and the two outer layers are the inner and the outer pellicle. Interestingly enough, only the outermost layer (outer pellicle) gives strong positive reactions to staining with Coomassie blue and some lectins. This layer is known to be very strongly coloured by b-glucosyl Yariv phenylglycoside indicating its AGP-based composition (Bacic et al., 1986). In this work, we revealed that the outer pellicle of maize root contains the MAC207 AGP epitope. Detailed observations in this study revealed that silverenhanced particles were linearly arranged, indicating that they decorate filamentous structures both on the callus and on the root surfaces. In this respect, it is important to note that the fibrillar nature of the outermost extracellular layer on the maize root surface has been shown previously by transmission electron microscopy (Abeysekera & McCully, 1993). In conclusion, our new silver-enhanced immunogold SEM method for plant surfaces, based on glycerol substitution, can provide sufficient resolution as well as the necessary structural and antigenic preservation of biological samples to be used for other surface antigens of plant and animal cells. Importantly, samples substituted with glycerol do not need metal coating which could mask smaller gold or silverenhanced gold particles. Thus, gold/silver markers of antigenic sites can be detected with high reliability. 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