Download Immunogold localization of plant surface arabinogalactan

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]
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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).
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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
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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
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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.
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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
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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.
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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
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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
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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-
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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. This
method can be advantageous for the fine immunolocalization studies because fragile thin surface layers and
filamentous structures can easily be altered or damaged
by other preparation techniques, e.g. by critical point drying
conventionally used for SEM (Boyde, 1978) or by immersing
specimens in cooling agent for low temperature SEM
(Ensikat & Barthlott, 1993).
Acknowledgements
J.Š. is grateful to the Alexander von Humboldt Foundation
(Bonn, Germany) for his research fellowship. This work
was supported by AGRAVIS (Bonn, Germany) through
Deutsche Agentur für Raumfahrtangelegenheiten (DARA,
Bonn, Germany) and Ministerium für Wissenschaft und
Forschung (MWF, Düsseldorf, Germany).
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