Download The Organization Pattern of Root Border

The Organization Pattern of Root Border-Like Cells of
Arabidopsis Is Dependent on Cell Wall
Homogalacturonan1,2[C][W]
Caroline Durand, Maı̈té Vicré-Gibouin, Marie Laure Follet-Gueye, Ludovic Duponchel, Myriam Moreau,
Patrice Lerouge, and Azeddine Driouich*
Laboratoire de Glycobiologie et Matrice Extracellulaire Végétale, Unité Propre de Recherche et
d’Enseignement Supérieur Associé 4358, Institut Fédératif de Recherche Multidisciplinaire sur les Peptides
23, Plate-Forme de Recherche en Imagerie Cellulaire de Haute Normandie, Université de Rouen, 76821 Mont
Saint Aignan, France (C.D., M.V.-G., M.L.F.-G., P.L., A.D.); and UMR CNRS 8516, Laboratoire de
Spectrochimie Infrarouge et Raman, Université de Lille, 59655 Villeneuve d’Ascq Cedex, France (L.D., M.M.)
Border-like cells are released by Arabidopsis (Arabidopsis thaliana) root tips as organized layers of several cells that remain
attached to each other rather than completely detached from each other, as is usually observed in border cells of many species.
Unlike border cells, cell attachment between border-like cells is maintained after their release into the external environment. To
investigate the role of cell wall polysaccharides in the attachment and organization of border-like cells, we have examined their
release in several well-characterized mutants defective in the biosynthesis of xyloglucan, cellulose, or pectin. Our data show
that among all mutants examined, only quasimodo mutants (qua1-1 and qua2-1), which have been characterized as producing
less homogalacturonan, had an altered border-like cell phenotype as compared with the wild type. Border-like cells in both
lines were released as isolated cells separated from each other, with the phenotype being much more pronounced in qua1-1
than in qua2-1. Further analysis of border-like cells in the qua1-1 mutant using immunocytochemistry and a set of anti-cell wall
polysaccharide antibodies showed that the loss of the wild-type phenotype was accompanied by (1) a reduction in
homogalacturonan-JIM5 epitope in the cell wall of border-like cells, confirmed by Fourier transform infrared microspectrometry, and (2) the secretion of an abundant mucilage that is enriched in xylogalacturonan and arabinogalactan-protein
epitopes, in which the cells are trapped in the vicinity of the root tip.
Higher plants rely on their roots to acquire water
and other nutrients in the soil to grow and develop
(Esau, 1977). At the tip of every growing root is a
conical covering consisting of several layers of cells
called the root cap that plays a major role in root
protection and its interaction with the rhizosphere
(Rougier, 1981; Baluška et al., 1996; Barlow, 2003).
Root tips of most plant species produce a large
number of cells programmed to separate from the root
cap and to be released into the external environment
(Hawes et al., 2003). This process occurs through the
action of cell wall-degrading enzymes that solubilize
the interconnections between root cap peripheral cells,
1
This work was supported by the Centre National de la Recherche Scientifique, l’Université de Rouen, and le Conseil Régional de
Haute Normandie.
2
This article is dedicated to the memory of Jean Herbet, who
passed away on February 21, 2009.
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Azeddine Driouich ([email protected]).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.109.136382
causing the cells to separate from each other and from
the root as populations of single cells (Hawes et al.,
2003). Because of their specific position at the interface
between root and soil, these living cells are defined as
root border cells. It has been shown that the number of
these cells per root varies between plant families: from
about 100 (e.g. the Solanaceae family) to several thousands (e.g. 10,000 or more for the Pinaceae; Hawes
et al., 2003). It has also been suggested that species of
the Brassicaceae family including Arabidopsis (Arabidopsis thaliana) do not produce border cells (Hawes
et al., 2003). Indeed, the Arabidopsis root tip does not
produce isolated border cells per se, but it does produce and release cells that remain attached to each
other, forming a block of several cell layers called
border-like cells (Vicré et al., 2005; Fig. 1). This also
occurs in other Brassicaceae species, including rapeseed (Brassica napus), mustard (Brassica juncea), and
Brussels sprout (Brassica oleracea gemmifera), indicating
that such an organization might be specific to this
family (Driouich et al., 2007).
The unique organization pattern of Arabidopsis
border-like cells (e.g. they do not disperse individually) suggests that they might have a specific cell wall
composition and/or structure that makes them resistant to cell wall-hydrolyzing enzymes or that the
enzymes are not present or not functional (Driouich
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Durand et al.
Figure 1. Morphological phenotypes of root tips showing border-like
cells (BLC) of the wild type and cell wall mutants of Arabidopsis. Wildtype Columbia (Col O; A), wild-type Wassilewskija (Ws; B), mur3 (C),
mur2-1 (D), kor1 (E), rsw1 (F), epc1-1 (G), arad1-1 (H), qua1-1 (I), and
qua2-1 (J) are shown. Border-like cells are released from the root tip in
organized cell layers (arrows) in the wild type and in all mutants
examined with the exception of qua1-1 and qua2-1. Note also that
border-like cell organization is similar between Columbia and Wassilewskija. M, Mucilage. Bars = 20 mm (A, B, D–H, and J) or 50 mm (C and I).
et al., 2007). The only information on cell wall composition of Arabidopsis border-like cells was obtained
from immunocytochemical studies, in which it has
been shown that the cell wall of border-like cells is rich
in pectic homogalacturonan and arabinogalactanproteins, two wall polymers believed to be involved
in cell adhesion in plants (Vicré et al., 2005). Based on
this observation, we postulated that pectic polysac-
charides of the cell wall may serve as a glue to cement
border-like cells together, leading to that particular
organization (Vicré et al., 2005).
The cell wall of higher plants comprises mainly
polysaccharides and proteoglycans. Cell wall polysaccharides are assembled into complex macromolecules,
including cellulose, hemicellulose, and pectin. Cellulose forms microfibrils, which constitute an ordered,
fibrous phase, whereas pectin and hemicellulose form
an amorphous matrix phase surrounding the microfibrils (Cosgrove, 1997). Pectins constitute a highly
complex family of cell wall polysaccharides, including homogalacturonan, rhamnogalacturonan I, and
rhamnogalacturonan II. Homogalacturonan domains
consist of a-D-(1/4)-GalUA residues, which can be
methyl esterified, acetylated, and/or substituted with
b-(1/3)-Xyl residues to form xylogalacturonan
(Schols et al., 1995; Willats et al., 2001; Vincken et al.,
2003). Deesterified blocks of homogalacturonan can be
cross-linked by calcium, leading to the formation of a
gel that is believed to be involved in cell adhesion
(Jarvis et al., 2003). Rhamnogalacturonan I consists of
a backbone of up to 100 repeats of the disaccharide
a-(1/4)-GalUA-(1/2)-rhamnose, which carries complex and variable side chains. The rhamnose residues
are commonly substituted with polymeric b-(1/4)linked D-galactosyl residues and/or a-(1/5)-linked
L-arabinosyl residues (Ridley et al., 2001). Rhamnogalacturonan II is a highly complex but conserved molecule consisting of a homogalacturonan-like backbone
substituted with four different side chains containing
specific sugars (O’Neill et al., 2004).
Xyloglucan is the major hemicellulosic polysaccharide of the primary wall of dicotyledonous plants, and
it consists of a b-D-(1/4)-glucan backbone to which
are attached side chains containing xylosyl, galactosylxylosyl, or fucosyl-galactosyl-xylosyl residues. Xyloglucan is the principal polysaccharide that cross-links
the cellulose microfibrils. The xyloglucan-cellulose
network forms a major load-bearing structure that
contributes to the control of cell expansion (Hayashi,
1989; Cosgrove, 1999).
Glycoproteins, such as arabinogalactan-proteins, are
also present in the cell wall matrix (Showalter, 1993;
Seifert and Roberts, 2007). Arabinogalactan-proteins
are highly glycosylated members of the Hyp-rich
glycoprotein superfamily. Many of these glycoproteins, the so-called classical arabinogalactan-proteins,
are anchored to the plasma membrane by a glycosylphosphatidylinositol anchor and have the potential to
bind both cell wall components (Immerzeel et al.,
2006) and cytosolic cortical microtubules (Schultz
et al., 2002; Sardar et al., 2006; Nguema-Ona et al.,
2007). These proteoglycans have been implicated in
many aspects of plant life, including cell expansion,
cell signaling and communication, embryogenesis,
and wound response (Johnson et al., 2003; Seifert
and Roberts, 2007; Driouich and Baskin, 2008).
Although cell-to-cell interaction is a fundamental
feature of plant growth and development, the molec-
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Cell Attachment in Root Border-Like Cells
ular bases of intercellular adhesion and its loss are not
fully understood (Roberts et al., 2002; Jarvis et al., 2003;
Willats et al., 2004). This study aims at investigating
the role of cell wall polysaccharides in cell attachment
and the organization of border-like cells in Arabidopsis. To this end, we took advantage of the recent
characterization of several Arabidopsis mutants affected in the biosynthesis of different classes of cell
wall polysaccharides, including pectin, xyloglucan,
and cellulose. We thus examined the pattern of
border-like cells released by the root tip of selected
Arabidopsis mutants using microscopy and immunocytochemistry. These mutants are (1) quasimodo1-1
(qua1-1) and qua2-1 (Bouton et al., 2002; Mouille
et al., 2007), ectopically parting cells1-1 (epc1-1; Singh
et al., 2005), and arabinan deficient1-1 (arad 1-1; Harholt
et al., 2006), which all have been reported to be
possibly affected in pectin biosynthesis; (2) murus2-1
(mur2-1) and mur3, which make altered xyloglucan
(Vanzin et al., 2002; Madson et al., 2003); and (3)
radially swollen1 (rsw1) and korrigan1 (kor1), which are
affected in cellulose biosynthesis (Arioli et al., 1998;
Nicol et al., 1998).
Our data show that the organization of border-like
cells had a wild-type phenotype in all of the mutants
examined except in qua1-1 and qua2-1. In both of these
mutants, border-like cells had lost the wild-type phenotype, as they were released as single cells separated
from each other. This phenotype was far more pronounced in qua1-1 than in qua2-1. Further analysis of
qua1-1 using immunocytochemistry and Fourier transform infrared microspectrometry showed a substantial
loss of homogalacturonan content in border-like cells.
In addition, border-like cells in the qua1-1 mutant
secreted an abundant mucilage enriched in xylogalacturonan and arabinogalactan-protein epitopes.
RESULTS
Morphological Characterization of Border-Like Cells in
Cell Wall-Altered Mutants of Arabidopsis
A major function of the cell wall is to maintain cellto-cell contact and tissue cohesion (Jarvis et al., 2003).
To investigate the cell wall carbohydrates involved in
border-like cell organization and attachment in Arabidopsis, we examined root cap morphology in mutants
known to be defective in the biosynthesis of different
classes of cell wall polysaccharides, including cellulose, hemicellulose, and pectin (Table I).
We first examined border-like cell organization in
two hemicellulose mutants, mur2-1 and mur3, known
to be deficient in genes encoding for a fucosyltransferase and a galactosyltransferase, two enzymes involved
in xyloglucan biosynthesis (Vanzin et al., 2002;
Madson et al., 2003). As illustrated in Figure 1, no
difference was observed in either border-like cell production or organization between wild-type and mur3
or mur2-1 root tips (Fig. 1, compare A, C, and D).
Similarly, two cellulose-defective mutants, kor1 and
rsw1, did not show any alteration in the overall organization of the same cells (Fig. 1, E and F). Nevertheless, root tips of the mutant rsw1, whose phenotype is
temperature sensitive (Arioli et al., 1998), produced
fewer border-like cells at 30°C than at 20°C.
We next examined border-like cell phenotypes in
mutant lines displaying abnormal pectin content.
Reduced cell adhesion was reported for the epc1-1
mutant, deficient in a gene encoding a putative pectinglycosyltransferase (Singh et al., 2005). Observation of
the root tip in this mutant showed that the organization pattern of border-like cells is similar to that of the
wild type (Fig. 1G). Similarly, the organization of the
cells in the arad1-1 mutant, deficient in a gene encoding an a-1,5-arabinosyltransferase involved in rhamnogalacturonan I biosynthesis (Harholt et al., 2006),
was not altered (Fig. 1H).
In contrast, examination of border-like cells in the
mutant qua1-1, which is deficient in a putative glycosyltransferase involved in homogalacturonan biosynthesis and known to exhibit reduced cell adhesion
(Bouton et al., 2002), revealed a different organization
of border-like cells as compared with the wild type. As
shown in Figure 1I, qua1-1 border-like cells were not
present as organized files of attached cells but were
separated from each other and dispersed individually
in the vicinity of the root tip. Furthermore, these cells
were frequently seen embedded in an abundant layer
Table I. List of Arabidopsis mutants used in this study and description of the root border-like cell organization in each of them
Mutant Name
Hemicellulose
mur2-1
mur3
Cellulose
kor1
rsw1
Pectin
qua1-1
qua2-1
arad1-1
Undefined function
epc1-1
Gene Code
Border-Like Cell Organization
References
At2g03220
At2g20370
Border-like cells organized in files
Border-like cells organized in files
Vanzin et al. (2002)
Madson et al. (2003)
At5g49720
At4g32410
Border-like cells organized in files
Reduced border-like cell formation at 30°C
Nicol et al. (1998)
Arioli et al. (1998)
At3g25140
At1g78240
At2g35100
Isolated border-like cells and thick mucilage
Isolated border-like cells and sometimes thick mucilage
Border-like cells organized in files
Bouton et al. (2002)
Mouille et al. (2007)
Harholt et al. (2006)
At3g55830
Border-like cells organized in files
Singh et al. (2005)
Plant Physiol. Vol. 150, 2009
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Durand et al.
of mucilage (Fig. 1I). Interestingly, careful and daily
examination of border-like cell production during root
growth revealed that the mucilage is secreted concomitantly during border-like cell formation and release
from the root tip. A quite similar but less pronounced
phenotype of border-like cells was also observed in the
qua2-1 mutant (Fig. 1J), which is deficient in a putative
methyltransferase involved in pectin biosynthesis
(Mouille et al., 2007). Separation of the cells in qua2-1
was also accompanied by mucilage secretion, although
less frequently than in the qua1-1 mutant (Fig. 1J).
Together, these observations demonstrate that, unlike
in the wild type and cellulose- and xyloglucan-defective
mutants, root tips of mutants altered in homogalacturonan biosynthesis produced border-like cells that are
separated from each other. This suggests that biosynthesis of homogalacturonan is required for the normal
organization of border-like cells in Arabidopsis.
into the distribution of polysaccharide epitopes at the
surface of border-like cells and in associated mucilage
of the qua1-1 mutant (Table I; Supplemental Data).
Histochemical Staining of Border-Like Cells
To localize pectin epitopes, we immunolabeled root
tips with anti-pectin mAbs, including JIM5, LM5,
LM6, and LM8. Staining with JIM5 was detectable at
the surface of border-like cells, with nearly no difference in localization between the wild type and the
mutant (Fig. 4, A and B). Labeling of the mucilage was
also observed in the qua1-1 line, although very weakly
(Fig. 4B). Immersion immunofluorescence staining,
which allows detection of epitopes at the surface of
the tissue only, did not detect any differences in terms
of intensity of fluorescence between the qua1-1 mutant
and the wild type. However, as reduction in homogalacturonan in the cell walls of qua1-1 was reported to
be particularly associated with the tricellular junction
in suspension-cultured cells (Leboeuf et al., 2005), we
performed the same labeling experiment with JIM5 on
cryofixed and resin-embedded sections of root tips of
the mutant and the wild type (Fig. 4). Under these
conditions, we found a significant loss of JIM5 staining
As the phenotype was more pronounced in qua1-1
than in qua2-1, we focused only on the former for all
of the following histochemical and immunocytochemical analyses. Histochemical staining of root tips with
calcofluor white or ruthenium red showed that borderlike cells are stained with both dyes in wild-type and
qua1-1 plants. In contrast, the mucilage surrounding
the cells of the mutant was only stained with ruthenium red but not with calcofluor white, suggesting
that the mucilage contains acidic polymers such as
pectin but not b-glucans (Fig. 2).
Immunolocalization of Polysaccharide Epitopes in the
Cell Wall of Border-Like Cells and the Secreted Mucilage
of the Homogalacturonan-Defective Mutant
We used immersion immunofluorescence labeling
(Willats et al., 2001; Vicré et al., 2005) to gain insights
Xyloglucan Immunostaining
To investigate the occurrence of xyloglucan, we
probed root tips with the CCRC-M1 monoclonal antibody (mAb). As shown in Figure 3, the antibody
bound strongly to border-like cells produced by wildtype and mutant root tips. In contrast, the secreted
mucilage was not labeled (Fig. 3, C and D). Close
examination of the labeling pattern revealed the presence of labeled filament-like structures at the surface
of border-like cells. These structures can sometimes be
seen peeling off from the surface of the cells.
Pectin Immunostaining
Figure 2. Histochemical staining of root tips showing
border-like cells (BLC) released from the wild type (A
and D) and the qua1-1 mutant (B, C, and E). Roots are
stained with calcofluor white (A–C) or ruthenium red
(D and E). Note the presence of an abundant mucilage (M) surrounding border-like cells of the qua1-1
mutant (E). R, Root apex. Bars = 10 mm (A, C, and D)
or 50 mm (B and E).
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Cell Attachment in Root Border-Like Cells
staining of the cell wall of border-like cells was either
diffuse or not present at all (Fig. 6, B and C). In
contrast, the secreted mucilage showed a very strong
staining in the mutant (Fig. 6, B and C). These observations were confirmed using the mAb LM2 that also
recognizes arabinogalactan-protein epitopes (data not
shown).
Together, the immunolabeling findings demonstrate
that isolated border-like cells produced by the qua1-1
mutant express fewer homogalacturonan epitopes
than the wild type and that the abundant mucilage
they produce is enriched in arabinogalactan-proteins
and xylogalacturonan polysaccharides.
Fourier Transform Infrared Microspectrometry Analysis
of Border-Like Cells
Figure 3. Immunofluorescence labeling at the surface of border-like
cells (BLC) with the mAb CCRC-M1, recognizing xyloglucan, for the
wild type (A and B) and the qua1-1 mutant (C and D). Stained filamentlike structures (arrowheads) are observed at the surface of border-like
cells of the wild type and mutant. Corresponding bright-field microscopy and controls are shown in Supplemental Figures S1 and S5. M,
Mucilage; R, root apex. Bars = 80 mm (A and C) or 8 mm (B and D).
The analysis of Fourier transform infrared data
using principal component analysis revealed two distinct clusters of values for border-like cells, indicating
significant spectral differences between the qua1-1 mutant and the wild type (Fig. 7A). We compared the two
mean second derivative spectra of the qua1-1 mutant
and the wild type in the 920 to 1,780 cm21 region (Fig.
7B). Differences were noticed at the following wave
numbers characteristic of cell wall pectic polysaccharides: 1,740, 1,600, 1,414, 1,243, and 1,018 cm21. The
peak at 1,740 cm21 is specific to carboxylic esters,
probably linked to pectins (Kačuráková et al., 2002);
in cell walls of border-like cells in the qua1-1 mutant
compared with the wild type (Fig. 4, C and D). Such
a decrease in the amount of homogalacturonan in
border-like cells of qua1-1 was further confirmed using
Fourier transform infrared microspectrometry analysis (see below).
Arabinan epitopes recognized by the mAb LM6
were strongly detected in the cell wall of border-like
cells released by the qua1-1 mutant and the wild-type
root tips (Fig. 5, A and B). The mucilage was rarely and
weakly labeled in the qua1-1 mutant (Fig. 5B). The LM5
antibody recognizing galactan epitopes also stained
border-like cells in the wild type and the mutant but
not the mucilage (data not shown).
Labeling with the mAb LM8 specific for xylogalacturonan occurred in the outer surface of border-like
cells as well as in the mucilage from wild-type and
mutant plants (Fig. 5). Interestingly, staining of the
mucilage appeared stronger around isolated cells of
the qua1-1 mutant (Fig. 5, D and E).
Immunostaining of Arabinogalactan-Proteins
We also examined the distribution of epitopes associated with arabinogalactan-proteins in border-like
cells and mucilage using the mAb JIM13 known to
stain these cells in Arabidopsis wild-type plants (Vicré
et al., 2005). As shown in Figure 6A, a uniform staining
of cell wall of border-like cells in the wild type with the
mAb JIM13 was observed. In contrast, in qua1-1,
Figure 4. Immunofluorescence labeling of border-like cells (BLC) with
the mAb JIM5. A and B, Micrographs of fixed root tips for the wild type
(A) and the qua1-1 mutant (B). C and D, Micrographs of fixed/resinembedded root tip sections for the wild type (C) and the qua1-1 mutant
(D). Corresponding bright-field microscopy and controls are shown in
Supplemental Figures S2 and S6. M, Mucilage; R, root apex. Bars = 40
mm (A and B) or 20 mm (C and D).
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Durand et al.
Figure 5. Immunofluorescence labeling of borderlike cells (BLC) with the mAbs LM6 and LM8. A and
B, Micrographs of border-like cells stained with the
mAb LM6 for the wild type (A) and the qua1-1 mutant
(B). C to E, Micrographs of border-like cells stained
with the mAb LM8 for the wild type (C) and the qua1-1
mutant (D and E). Labeling shows that this epitope
associated to xylogalacturonan was particularly present in the thick mucilage (M) surrounding the qua1-1
border-like cells. Corresponding bright-field microscopy and controls are shown in Supplemental Figures
S3 and S6. R, Root apex. Bars = 40 mm (A–C), 16 mm
(D), or 80 mm (E). [See online article for color version
of this figure.]
the peaks at 1,600 and 1,414 cm21 are characteristic of
carboxylate ion stretches for pectin (Marry et al., 2000);
the peak at 1,243 cm21 is assigned to C-O vibrations in
pectic polysaccharides (Kačuráková et al., 2002); and
the peak at 1,018 cm21 corresponds to uronic acid of
pectins (Coimbra et al., 1998).
Interestingly, all of these peaks attributed to pectins
are reduced in the qua1-1 mutant. These results are
consistent with an alteration in GalUA content in the
qua1-1 mutant.
cells was lost. Such a loss of adhesion led to a release of
single border-like cells that secreted abundant mucilage and whose walls contained reduced amounts of
homogalacturonan. These findings indicate that homogalacturonan is involved in the maintenance of
adhesion between border-like cells in Arabidopsis and
show that the loss of cell-to-cell contact is accompanied by secretion of substantial amounts of arabinogalactan-proteins and xylogalacturonan-containing
mucilage, in which the cells are trapped and kept in
the vicinity of the root tip.
DISCUSSION
The goal of this study was to investigate the role of
cell wall polysaccharides in the control of intercellular
attachment of border-like cells that are released by
Arabidopsis root tips as sheets of cells rather than as
single cells. To this end, border-like cells from several
well-defined mutants deficient in the biosynthesis of
cellulose, xyloglucan, or pectin were examined using
microscopy and immunocytochemistry. Among all of
the mutants studied, only the qua1-1 mutant, and to a
lesser extent qua2-1, presented an altered phenotype of
border-like cells in which adhesion between groups of
Homogalacturonan Is Involved in Border-Like Cell
Attachment in Arabidopsis
In almost all plant families, border cells are released
from the root tip as populations of individually separated single cells (Hawes et al., 2003). In pea (Pisum
sativum), for instance, one of the most studied species,
border cells are programmed to separate from each
other upon their release from the root tip. In contrast,
Brassicaceae species, including Arabidopsis, do not
release border cells per se; instead, they release borderlike cells that remain attached together as sheets of
Figure 6. Immunofluorescence labeling of arabinogalactan-proteins recognized by the mAb JIM13 at
the surface of border-like cells (BLC) for the wild type
(A) and the qua1-1 mutant (B and C). Interestingly,
cell walls from the qua1-1 mutant were weakly
labeled compared with the wild type. Note the very
strong labeling of the secreted mucilage (M) in the
qua1-1 mutant. Corresponding bright-field microscopy
and controls are shown in Supplemental Figures S4
and S6. R, Root apex. Bars = 20 mm (A and C) or 40 mm
(B). [See online article for color version of this figure.]
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Cell Attachment in Root Border-Like Cells
Figure 7. Fourier transform infrared microspectrometric analysis of border-like cells. Projection of the
60 preprocessed spectra for the wild type and the
qua1-1 mutant in the principal component analysis
space (A) and mean second derivative absorbance
spectra for the wild type and the qua1-1 mutant (B)
are shown. PC, Principal component. [See online
article for color version of this figure.]
cells rather than being separated from each other
(Vicré et al., 2005). Thus, unlike in pea, cell adhesion
in border-like cells is maintained after their release.
Based on the finding that homogalacturonan epitopes
(recognized by the mAb JIM5) are abundant in the cell
wall of these cells, we postulated that pectin might be
necessary in maintaining their unique organization
(Vicré et al., 2005). This hypothesis was tested in this
study using different available cell wall-defective mutants.
We found that border-like cells could be released
individually in the pectin-defective mutant qua1-1, to a
lesser extent in qua2-1, and not at all in cellulosedefective (kor1 and rsw1) or xyloglucan-defective
(mur2-1 and mur3) mutants or other pectin mutants
studied. Therefore, maintenance of adhesion between
groups of border-like cells in Arabidopsis seems to
depend on QUA genes, mostly on QUA1. It has been
shown that mutation in QUA1 (GAUT8) and QUA2
resulted in dwarfed plant phenotypes and reduced
cell adhesion in the hypocotyl, and the corresponding
gene products were suggested to be involved in
homogalacturonan synthesis (Bouton et al., 2002;
Mouille et al., 2007). The cell walls of qua1-1 and
qua2-1 were shown to have a 25% and 50% reduction,
respectively, in homogalacturonan content (quantification made from 4-week-old whole plants or from the
leaves only) as compared with the wild type (Bouton
et al., 2002; Mouille et al., 2007). Although these
studies have not measured the extent of homogalacturonan reduction in each organ specifically, including
the root cap and border-like cells, they have established a correlation between homogalacturonan content and cell adhesion. They also indicate that even a
partial decrease in this polysaccharide content (25%–
50%) is sufficient to induce a significant loss of cell
adhesion. Therefore, the important loss of cell adhesion observed in border-like cells released by the qua1-1
mutant is possibly related to a loss of homogalacturonan content in these cells. This is supported by (1) the
Fourier transform infrared microspectrometry data,
which revealed a significant alteration in the content of
homogalacturonan in border-like cells of the qua1-1
mutant (Fig. 7B), and (2) the immunolabeling result on
fixed/resin-embedded root tip sections, where staining of border-like cells with JIM5 was significantly
reduced. Thus, the QUA1 gene functions in the production of homogalacturonan by root border-like cells
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Durand et al.
in Arabidopsis and therefore is required for the maintenance of their adhesion and organization.
Further evidence supporting a function for homogalacturonan in maintaining cell adhesion in Arabidopsis border-like cells also arises from previous
studies performed on border cells of pea. Stephenson
and Hawes (1994), using the mAb JIM5 and immunoblotting analysis, showed a substantial increase in the
level of soluble deesterified pectin in the root tip of pea
correlated with border cell production. The amount of
deesterified pectin detected by JIM5 in the watersoluble fraction increased significantly as border cell
separation occurred, whereas the amount of pectin
that was cell wall bound decreased when cell separation began. These data indicate a loss of homogalacturonan concomitant with a loss of border cell
adhesion in pea. Furthermore, loss of cell adhesion
in border cells and their separation from the root cap of
pea was shown to correlate with pectolytic enzyme
activity, such as polygalacturonases responsible for the
hydrolysis of homogalacturonan and pectin methylesterases (Hawes and Lin, 1990). This process seems to
occur via demethylation of pectin followed by the
hydrolysis of polygalacturonic acid by the action of
both enzymes in a way similar to that observed during
fruit ripening (Fischer and Bennett, 1991; Martel and
Giovannoni, 2007). Therefore, we suggest that, unlike
in pea, the hydrolysis of homogalacturonan present in
the cell wall of Arabidopsis border-like cells does not
occur, possibly because the enzymes, if present in the
cell wall of border-like cells, may not be functional.
This is supported by the observation that antisense
inhibition of pectin-methylesterase expression in
transgenic pea resulted in the inhibition of border
cell separation (Wen et al., 1999). In the transformed
plants, border cells remained attached together as
clusters of cells, a phenotype that is somehow similar
to that of border-like cells in Arabidopsis. However, it
remains to be clearly established whether specific
pectin-methylesterase and polygalacturonase genes
are expressed in border-like cells and whether the
corresponding enzymes are active or not (Driouich
et al., 2007).
It is interesting that our findings on border-like cell
separation support the reduced cell adhesion reported
in qua1-1 and qua2-1 (Bouton et al., 2002; Mouille et al.,
2007) but do not support the cell adhesion defect in
hypocotyl tissues reported for the epc1-1 mutant
(Singh et al., 2005). A more recent study on a new
mutant allele of this gene, epc1-2, as well as on epc1-1
itself showed that neither of these mutations affected
cell adhesion (Bown et al., 2007), which was attributed
to differences in growth conditions.
Separation of Border-Like Cells in the qua1-1 Mutant Is
Accompanied by Mucilage Secretion
Localization of rhamnogalacturonan I-containing
epitopes (recognized by the mAbs LM6 and LM5)
suggests that the occurrence of this polysaccharide is
not altered in border-like cells of the qua1-1 mutant.
Thus, the qua1-1 mutation does not seem to affect the
biosynthesis and secretion of rhamnogalacturonan.
The most interesting immunolocalization finding is
that LM8 epitopes associated with xylogalacturonan
were highly expressed in the secreted mucilage of the
qua1-1 mutant. This is consistent with previous reports
indicating that xylogalacturonan was specifically
associated with plant cell separation phenomena
(Willats et al., 2001, 2004), although the functional
relationship between LM8 epitope-containing xylogalacturonan and cell detachment is still not understood.
It is also interesting that distinct spatial differences
were observed in the occurrence of arabinogalactanprotein epitopes detected with the mAbs JIM13 and
LM2 between the wild type and the qua1-1 mutant.
The arabinogalactan-protein epitopes were also particularly abundant in the mucilage, whereas cell walls
of border-like cells in the qua1-1 mutant were less
stained than in the wild type. The disruption of
homogalacturonan biosynthesis and cell adhesion in
border-like cells in the qua1-1 mutant is accompanied
by the production and/or release of other cell wall
components (mainly xylogalacturonan and arabinogalactan-proteins) forming a thick mucilage in which
detached border-like cells were embedded. One explanation for this is that alteration of homogalacturonan synthesis may lead to a significant alteration of
the cell wall structure (e.g. reorganization of wall
polymer interactions), thereby affecting cell wall porosity. Consequently, this would lead to limited retention of xylogalacturonan and arabinogalactan-proteins
in the cell wall and would facilitate their diffusion and
release into the extracellular mucilage. Such a hypothesis has been proposed previously to explain the
increase in the amount of extracellular polymers released in the medium of qua1-1 suspension-cultured
cells compared with wild-type cells (Leboeuf et al.,
2005; Rondeau-Mouro et al., 2008). An alternative
explanation is that the qua1-1 mutant border-like cells
compensate for the lack of homogalacturonan in their
wall by an increase in de novo synthesis and secretion
of xylogalacturonan/arabinogalactan-proteins abundant in mucilage in order to retain nonattached
border-like cells in close proximity to the root tip.
Maintaining border-like cells close to the root cap
either in the wild type (file of attached cells) or in the
qua1-1 mutant (isolated cells entrapped in mucilage)
might be important for the function of border-like
cells, possibly in protecting the root tip. While a protective function of the root cap has been demonstrated
for border cells (Hawes et al., 2000; Gunawardena
et al., 2005), the potential function of border-like cells
in relation to root health has not been assessed.
Border-Like Cells of Arabidopsis as a Novel and Suitable
System for Cell Wall Mutant Screening
Identification of Arabidopsis mutants with altered
structure and synthesis of cell wall polymers has been
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Cell Attachment in Root Border-Like Cells
fundamental in increasing our understanding of the
function of certain wall polysaccharides in relation to
plant development. Nevertheless, selection of cell wall
mutants is a difficult task, and many approaches have
been recently developed for rapidly screening Arabidopsis cell wall mutants. Among other methods, cell
wall mutants have been successfully identified on the
basis of developmental phenotypes (Baskin et al., 1992;
Arioli et al., 1998), Fourier transform infrared microspectrometry analysis (Chen et al., 1998; Mouille et al.,
2003), quantification of monosaccharide composition
by gas chromatography (Reiter et al., 1997), and
matrix-assisted laser desorption ionization-time of flight
spectrometry (Lerouxel et al., 2002). In this study, we
show that based on a direct observation of root borderlike cells of mutants using light microscopy, it was
possible to obtain valuable information on the function
of cell wall molecules. This revealed that homogalacturonan reduction in the qua1-1 mutant leads to peculiar border-like cell phenotypes easily observed with a
light microscope without any staining or further treatment of the roots. Moreover subtle alteration in borderlike cell morphology can also be easily monitored. The
method needs neither staining of the sample nor any
specific treatment apart from immersing the root tip in
water. It is simple, reproducible, and fast. We estimated the overall time from seedling collection to the
final observation of border-like cells to be approximately 4 min. As a consequence, we believe that direct
observation of border-like cells at the light microscope
level is a well-adapted method for fast, easy, and
inexpensive screening for mutants altered in key developmental processes such as cell attachment and
morphology. Therefore, we propose to use it as an
alternative and simple approach for accurate detection
of novel mutants defective in cell wall structure and
function.
MATERIALS AND METHODS
were carefully removed from the root tip using a razor blade. The seedlings
were replaced in petri dishes containing the growing medium. The production
of both mucilage and border-like cells was observed daily for 6 d by placing
the petri dishes on an inverted bright-field microscope stage.
Light Microscopy and Histochemical Staining
Roots were mounted on glass microscope slides in a drop of water and
directly examined for morphological analyses using bright-field illumination.
Staining of cellulose with calcofluor white M2R (Sigma) was performed as
described previously by Andème-Onzighi et al. (2002). Fresh roots were
incubated with the fluorescent probe (1 mg L21) for 30 min in the dark. After
careful washes with distilled water, the roots were observed using a microscope equipped with UV fluorescence (excitation filter, 359 nm; barrier filter,
461 nm). For cytochemical staining of pectins, roots were treated with a
solution of 0.05% (w/v) ruthenium red dye (Sigma) in deionized water for 10
to 15 min, then washed extensively in deionized water. Roots were mounted
as described above and observed using a bright-field microscope. Images
were acquired with a Leica DFC 300 FX camera. For each line, 50 to 60 roots
were observed.
Immunofluorescence Labeling
The anti-pectin mAbs used in this study were JIM5, which recognizes
homogalacturonan (Willats et al., 2000), LM8, which recognizes a xylogalacturonan-associated epitope (Willats et al., 2004), LM5 specific to (1/4)-b-Dgalactan (Jones et al., 1997), and LM6 specific to (1/5)-a-L-arabinan (Willats
et al., 1998). The mAb CCRC-M1, which is specific for fucosylated side chains
of xyloglucan (Puhlmann et al., 1994), was used to label xyloglucan. Arabinogalactan-proteins were stained by two specific mAbs, JIM13 and LM2
(Knox et al., 1991; Smallwood et al., 1996; Yates et al., 1996). The secondary
antibodies used were either fluorescein isothiocyanate (FITC)-conjugated goat
anti-rat (JIM5, LM8, LM5, LM6, JIM13, and LM2) or FITC-conjugated sheep
anti-mouse (CCRC-M1) IgGs (Sigma).
Roots of 15- to 17-d-old seedlings were fixed for 30 min in 4% (w/v)
paraformaldehyde and 1% (v/v) glutaraldehyde in 50 mM PIPES, pH 7, and
1 mM CaCl2 (adapted from Willats et al., 2001). Roots were washed in 50 mM
PIPES, 1 mM CaCl2, pH 7, and incubated for 30 min in a blocking solution of
3% (w/v) low-fat dried milk in phosphate-buffered saline (PBS), pH 7.2. After
being carefully rinsed in PBS containing 0.05% (v/v) Tween 20 (PBST), roots
were incubated overnight at 4°C with the primary antibody (dilution 1:5 in
0.1% [v/v] PBST). After five washes with 0.05% PBST, roots were incubated
with the appropriate secondary antibody (dilution 1:50 in 0.1% PBST) for 2 h at
30°C. Roots were rinsed in 0.05% PBST, mounted in anti-fade solution
(Citifluor AF2; Agar Scientific), and examined using a confocal laser-scanning
microscope (Leica TCS SP2 AOBS; excitation filter, 488 nm; barrier filter, 500–
600 nm). Control experiments were performed by omission of the primary
antibody. An average of 10 to 15 root apices were examined for each antibody.
Plant Material and Growth Conditions
Plant material used was wild-type Arabidopsis (Arabidopsis thaliana ecotypes Columbia and Wassilewskija) and the following mutants: mur2-1 (Vanzin
et al., 2002), mur3 (Madson et al., 2003), rsw1 (Arioli et al., 1998), epc1-1 (Singh
et al., 2005), qua2-1 (Mouille et al., 2007), arad1-1 (Harholt et al., 2006; these
mutants were in the Columbia background), qua1-1 (Bouton et al., 2002), and
kor1 (Nicol et al., 1998; these mutants were in the Wassilewskija background).
Seeds were surface sterilized with 35% (v/v) ethanol for 5 min followed by
immersion in 35% (v/v) bleach for 5 min. After several washes in sterile
distilled water, the seeds were sown on agar-solidified nutrient medium
(Baskin et al., 1992). Growth conditions were identical to those described by
Vicré et al. (2005). Seeds were grown in vertically orientated square petri
dishes in 16-h-day/8-h-night cycles at 24°C for 15 d. The rsw1 mutant was
grown at 24°C for 11 d before being transferred to 30°C for 4 d before
observation as described by Arioli et al. (1998). Mutant seeds were obtained
either directly from the authors who previously described and characterized
the mutant or from the Salk Institute.
Formation of Border-Like Cells and Their Associated
Mucilage in the qua1-1 Mutant
Ten 11-d-old qua1-1 seedlings were aseptically placed into a droplet of
sterile water on autoclaved microscope slides. Border-like cells and mucilage
Fixed Resin-Embedding Root Tip Sections and
Immunofluorescence Labeling
Wild-type and qua1-1 root apices of 15 d (2–3 mm long) were first incubated
in a freezing medium composed of MES buffer (20 mM MES, pH 5.5, 2 mM
CaCl2·2H2O, and 2 mM KCl) containing 200 mM Suc and 10% glycerol. Three to
four roots were placed in gold platelet carriers prefilled with the same freezing
medium and frozen with the EMPACT freezer (Leica Microsystems) as
described previously by Studer et al. (2001).
The root apices were then freeze substituted in anhydrous acetone and
0.5% uranyl acetate at 290°C for 72 h. The temperature was gradually raised
(2°C h21) to 260°C and stabilized during 12 h to 230°C and stabilized again
during 12 h to 215°C. The root apices were washed twice with anhydrous
ethanol for 15 min. Embedding was performed at 215°C in a solution of
ethanol and London Resin White (30%, 50%, and 75% [v/v]) for 8 h each step.
Finally, two infiltrations (24 h each) of pure resin were achieved. Polymerization was realized at 215°C, under UV light, for 48 h.
The polymerized root apices were sectioned with a diamond knife
(Diatome) on an ultramicrotome (UCT; Leica). Semithin sections (300 nm)
were collected on glass well microscope slides coated with poly-L-Lys.
The sections were then incubated, for 5 min, in Tris-buffered saline (50 mM
Tris-HCl, pH 7.4, and 0.9% [w/v] NaCl) containing 0.2% (w/v) bovine serum
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Durand et al.
albumin and 0.01% (v/v) Tween 20, also named 0.01% TBST. After this, they
were incubated for 30 min in a blocking solution of 3% (w/v) low-fat dried
milk in 0.01% TBST. The semithin sections were rinsed with 0.01% TBST and
incubated overnight at 4°C with the primary antibody JIM5 (dilution 1:5 in
0.01% TBST). After five washes with 0.01% TBST, the sections were incubated
with the secondary FITC-conjugated goat anti-rat antibody (dilution 1:50 in
0.01% TBST) for 2 h at 30°C. The sections were rinsed in 0.01% TBST followed
by distilled water washes, mounted in citifluor AF2 and observed in the same
conditions as chemically fixed roots. An average of 10 to 15 root apices were
examined for the wild type and the qua1-1 mutant.
Fourier Transform Infrared Microspectrometry
Sample Preparation and Spectral Acquisition
Fourier transform infrared microspectrometric analysis was carried out
using a Bruker IFS 88 FTIR spectrometer fitted with a Bruker IRSCOPE II
microscope and a MCT detector. For the wild type and the qua1-1 mutant, 15
seedlings were selected. Root tips were placed on a BaF2 window, which
exhibits no interference over the wavelength range chosen for the study (i.e.
600–4,000 cm21). A drop of water was used to release border-like cells. The
sample area to be analyzed was determined prior to the measurement using a
video camera. The focused area was then adjusted to a diameter of 30 mm
using a 363 cassegrain objective in order to analyze a representative number
of border-like cells. The working mode for spectral analysis was transmittance. Absorbance spectra were recorded after 32 scans with a resolution of 2
cm21. Four replicates were made in different parts of border-like cells,
producing a total of 60 spectra for the wild type and the qua1-1 mutant.
Only the 921 to 1,776 cm21 spectral region was kept for the chemometrics
analysis because of its high molecular information content.
Spectral Data Pretreatments and Chemometrics Analysis
All spectral data analyses were computed with Matlab (version 7.1; Math
Works) and the PLS Chemometrics Toolbox (Eigenvector Research). The
spectra were preprocessed in order to obtain representative absorbances for
our samples. The first step was to normalize each spectrum (maximum
absorbance equal to 1) in order to suppress the variance due to the path length
variability between samples. Second derivative absorbance spectra were
obtained in a second step with the well-known algorithm of Savitzky and
Golay (1964). Derivatives were used here to remove uninformative variances
(baseline shift) due to physical scattering effects and to better extract chemical
variances from spectral data. The last step was to apply a statistical tool named
principal component analysis (Massart et al., 1998) on preprocessed spectra.
Such tools are generally used to discover particular structures in multivariate
spectral data sets and more precisely here to determine if different spectral
contributions exist between plant lines.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession numbers 814851 for the MUR2 gene, 816556 for the
MUR3 gene, 835035 for the KOR1 gene, 829376 for the RSW1 gene, 822105 for
the QUA1 gene, 844160 for the QUA2 gene, 818076 for the ARAD1 gene, and
824749 for the EPC1 gene.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Bright-field microscopy corresponding to Figure 3.
Supplemental Figure S2. Bright-field microscopy corresponding to Figure 4.
Supplemental Figure S3. Bright-field microscopy corresponding to Figure 5.
Supplemental Figure S4. Bright-field microscopy corresponding to Figure 6.
Supplemental Figure S5. Controls with omission of primary antibody:
border-like cells were stained with the secondary antibody FITC-conjugated anti-mouse IgG for the wild type (A) and the qua1-1 mutant (B).
Supplemental Figure S6. Controls with omission of primary antibody:
border-like cells were stained with the secondary antibody FITC-conjugated anti-rat IgG for the wild type (A) and the qua1-1 mutant (B).
Supplemental Table S1. Summary of immunolocalization of polysaccharide and arabinogalactan-protein epitopes in cell wall of border-like cells
and secreted mucilage.
ACKNOWLEDGMENTS
We thank Dr. G. Mouille and Prof. H.V. Sheller for providing qua1-1, qua2-1,
and arad1-1 mutant seeds. Thanks are also due to Dr. A. Marchant for his
critical reading of the manuscript and for providing epc1-1 mutant seeds. We
are grateful to Fabrice Richard and Laurence Chevalier for excellent technical
assistance during the preparation of samples for microscopy. Prof. J.C. Mollet,
Prof. A. Staehelin, and M.A. Cannesan are also acknowledged for their
helpful comments on the manuscript and for stimulating discussions on
border-like cells during the course of this work.
Received January 29, 2009; accepted May 12, 2009; published May 15, 2009.
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