Download Physico-chemical characteristics of cell walls from Arabidopsis

Journal of Experimental Botany, Vol. 55, No. 405, pp. 2087–2097, September 2004
DOI: 10.1093/jxb/erh225 Advance Access publication 30 July, 2004
RESEARCH PAPER
Physico-chemical characteristics of cell walls
from Arabidopsis thaliana microcalli showing
different adhesion strengths
Edouard Leboeuf1,2, Séverine Thoiron2 and Marc Lahaye1,*
1
INRA-Unité de Recherches sur les Polysaccharides, leurs Organisations et Interactions,
BP 71627, F-44316 Nantes, France
2
University of Nantes, Groupe de Physiologie et Pathologie Végétales, Faculté des Sciences et Techniques,
2 rue de la Houssinière, BP 92208, F-44322, Nantes cedex 3, France
Received 17 September 2003; Accepted 8 June 2004
Abstract
Changes in the composition and structure of cell walls
and extracellular polysaccharides (ECP) were studied
during the growth of suspension-cultured Arabidopsis
thaliana microcalli. Three growth phases, namely the cell
division phase, the cell expansion phase, and the stationary phase, were distinguished and associated with
a decreasing cell cluster adhesion strength. Degradation
of the homogalacturonan pectic backbone and of linear
pectic side chains (1,4)-b-D-galactan were observed
concomitantly with the cell expansion and stationary
phases and the decrease in cell adhesion. Also, in the
stationary phase, branched (1,5)-a-L-arabinans were
linearized. The AGP content of the culture medium increased while it decreased in the cell wall during cell
growth and as cell adhesion decreased. These data
suggest that, in addition to homogalacturonan, pectic
side chains and AGP are involved in plant cell development and particularly in cell–cell attachment.
Key words: AGP, Arabidopsis thaliana, (1,5)-a-L-arabinans, cell
adhesion, (1,4)-b-D-galactans, homogalacturonan.
Introduction
Cell adhesion and cell separation are important in the
morphogenesis and development of higher plants (Jarvis
et al., 2003). The adhered cells of young tissues give rise to
more loosely attached cells in mature structures during
abscission, dehiscence, and other development stages inducing cell separation processes. A common feature of all
cell separation processes is that the cell wall is degraded
(Roberts et al., 2002).
Plant cell walls are composed primarily of cellulose
microfibrils, hemicelluloses, pectic polysaccharides, and
small amounts of structural proteins (Carpita and Gibeaut,
1993). Pectins, the major polysaccharides of the middle
lamella and the primary cell wall, are thought to be involved in intercellular attachment. Typically, pectins are
composed of homogalacturonan (HG) and rhamnogalacturonan I (RGI) (Ridley et al., 2001; Vincken et al., 2003). HG
is a polymer of (1,4)-a-linked galacturonic acid residues
partially methyl-esterified and/or acetylated. It can be
further decorated by b-D-xylosyl residues linked to O-3
of galacturonic acid giving xylogalacturonan (XGA) or by
structurally complex sides chains composed of 11 different
types of glycosyl residues giving type II rhamnogalacturonan (RGII) (Ridley et al., 2001; Vincken et al., 2003).
RGI has a backbone of alternating galacturonic acid and
rhamnose residues with 20–80% of the rhamnose residues
substituted with side chains of (1,4)-b-D-galactans, (1,5)a-L-arabinans, or arabinogalactans (Ridley et al., 2001;
Vincken et al., 2003). It has recently been proposed that
HG, XGA, and RGII are other types of side chains of RGI
(Vincken et al., 2003).
Most studies have focused on the role of HG in cell
adhesion, since it is the major component of the middle
lamella which constitutes a cementing structure between
adjacent cells. Indeed, unsubstituted HG forms in vitro
three-dimensional networks through calcium ions-mediated
junction zones and it is thought that it forms similar
interactions in the middle lamella (Jarvis et al., 2003).
However, other unknown covalent linkages are inferred
*To whom correspondence should be addressed. Fax: +33 2 40 67 50 66. E-mail: [email protected]
Journal of Experimental Botany, Vol. 55, No. 405, ª Society for Experimental Biology 2004; all rights reserved
2088 Leboeuf et al.
from studies on the effect of pectin extractions on cell
separations (reviewed by Jarvis et al., 2003). Besides HG,
other macromolecular components and types of interaction
in the primary cell wall are also likely to be involved. These
include galactan, arabinan, and/or arabinogalactan side
chains of RGI (Kikuchi et al., 1996; Iwai et al., 2001;
Wallner and Nevins, 1974), RGII and their borate ester
complexes (Iwai et al., 2002) and, probably, proteins
(Jarvis et al., 2003).
Within the scope of understanding the relationships
between cell wall polymer structures and organizations
with plant cell adhesion, modifications occurring in the cell
wall physico-chemistry and cell cluster fragmentation
during growth of A. thaliana microcalli suspension culture
are reported here.
Materials and methods
Arabidopsis thaliana microcalli suspension cultures
Chlorophyllian Arabidopsis thaliana (Columbia ecotype) microcalli
suspension cultures, cell line T87, were grown according to Axelos
et al. (1992). JPL medium was replaced by Gamborg’s B5 medium
(Sigma-Aldrich, Saint Quentin Fallavier, France).
Determination of cell growth parameters
Cell number was counted on Malassey’s cell after a 16 h enzymatic
maceration at 25 8C of cell aggregates with 0.25% Macerozyme R-10
(Duchefa Biochemie BV, Kalys, Roubaix, France) and 1% Cellulase
Onozuka R-10 (Duchefa Biochemie BV) dissolved in 0.5 M
mannitol. The fresh weight was measured after harvesting cells by
filtration on a filter paper (Macherey-Nagel, Baeckerroot Labo,
Jacou, France) under mild pressure. The dry weight of harvested
cells was determined after 24 h at 60 8C.
Determination of cell adhesion
The cell adhesion of microcalli was determined by the ‘vortexinduced cell separation’ method adapted from Parker and Waldron
(1995). Cell cluster size was measured with a particle size analyser
(Micromeritics, Saturn DigiSizer 5200, Creil, France) before and
after dissociation of cell clusters with a vortex shaker (Top-Mix II,
Fisher Bioblock Scientific, Illkirch, France) for 2, 5, and 8 min at
40 Hz. Absorbance of the cell suspension supernatant between 400
and 800 nm was measured by spectrophotometry after centrifugation
at 1000 g for 10 min.
Preparation of alcohol-insoluble material (AIM), extracellular
medium, and extracellular polymer (ECP)
Cells were harvested by filtration through a 10 lm stainless-steel
mesh. AIM was prepared from about 10 g of cells according to
Bouton et al. (2002).
The cell-free filtrates were freeze-dried and corresponded to the
extracellular medium. The extracellular medium was redissolved in
deionized water (1 g l1) and extracellular polymers were precipitated
by adding ethanol to reach a final concentration of 80%. The precipitate was collected by centrifugation (10 000 g, 10 min), dissolved
in deionized water, dialysed against deionized water, and freeze-dried.
Chemical analyses
Identification and quantification of neutral sugars by gas–liquid
chromotography (GC) was performed after sulphuric acid degradation (Hoebler et al., 1989). Insoluble samples were dispersed in 72%
sulphuric acid for 30 min at 25 8C prior to dilution to 1 M and
hydrolysis (2 h, 100 8C). The soluble samples were directly dispersed
in 1 M sulphuric acid solution and hydrolysed (2 h, 100 8C). For
calibration, standard monosaccharide solutions and inositol as the
internal standard were used.
Starch content was determined by GC according to Englyst and
Cummings (1988).
Uronic acids were quantified by colorimetry using the automated
m-phenylphenol-sulphuric acid method (Thibault, 1979).
The nitrogen content of AIM was performed using the Kjeldahl
method (Miller and Miller, 1948). The amount of protein was
deduced using a conversion factor of 6.25.
The degree of methyl- and acetyl-esterification of pectins was
determined by HPLC following saponification according to Levigne
et al. (2002).
Detection of arabinogalactan-proteins in AIM and ECP
AIM and ECP were suspended at room temperature in 0.15 M NaCl
solution (40 mg ml1 and 10 mg ml1, respectively) for 1 h with
shaking. The preparations were assayed for arabinogalactan-proteins
by the b-glycosyl-Yariv reagent (Biosupplies, Parkville Victoria,
Australia) using the single radial gel diffusion method (van Holst and
Clarke, 1985).
Enzymatic degradations of AIM and ECP
AIM or ECP were degraded by endo-(1,5)-a-L-arabinanase, a-Larabinofuranosidase, endo-(1,4)-b-D-galactanase, endo-polygalacturonase (Megazyme, Bray, Ireland), and b-D-galactosidase (Sigma-Aldrich).
AIM and ECP were suspended (10 mg dry weight in 1.5 ml) in 100 mM
sodium acetate (pH 4) containing 0.02% thimerosal. Enzyme (0.2 U)
was added or not (blank) and the suspensions were incubated for 40 h
for AIM or 16 h for ECP at 30 8C with shaking. Enzyme (0.2 U) was
added again after 24 h of digestion for AIM. After incubation, the
suspensions were centrifuged (5000 g, 10 min) and supernatants were
filtered through 0.45 lm filters (Millex-HV; Millipore, St Quentinen-Yvelines, France) and boiled for 5 min. For ECP, ethanol was added
to the boiled supernatant to reach a final concentration of 80% ethanol
and centrifuged (5000 g, 10 min) to recover enzymatically produced
oligomers.
Uronic acids and neutral sugars contents of the hydrolysates were
measured by colorimetry and GC, respectively.
Nuclear magnetic resonance analysis
13
C-NMR spectra of ECP soluble fraction were recorded on a Bruker
ARX 400 (Bruker, Wissembourg, France) at 100.57 MHz.
ECP were dissolved in water (10 mg ml1) for 16 h at 4 8C. The
solution was centrifuged (5000 g, 10 min) and the supernatant freezedried prior to solubilization in D2O (Sigma-Aldrich). Chemical shifts
were measured from internal acetone assigned to 31.4 ppm.
AIM was extracted by 1% potassium oxalate (1 g in 150 ml) at
25 8C for 1 h with agitation. Residues were recovered by centrifugation (9000 g, 10 min) and supernatants were passed through
a sintered glass filter G4 (5–15 lm porosity). Residues were extracted
twice more as above and the final residues were washed with water.
Starch from the residues was removed by treatment with amyloglucosidase (A7420, Sigma-Aldrich) and residues were dehydrated by
958 ethanol and acetone washes prior to drying at 40 8C. Solid-state
magic angle spinning 13C-NMR spectra of the rehydrated residues
with D2O were recorded using liquid-like conditions on a Bruker
DMX 400 spectrometer operating at carbon frequency of 100.62
MHz (Lahaye et al., 2003).
Water-holding capacity of cell walls
The water-holding capacity of AIM was determined by the centrifugation method adapted from McConnell et al. (1974). About 150 mg
Adhesion strength of Arabidopsis microcalli cells
2089
of AIM were suspended in deionized water (10 ml, 1 h, room
temperature) with shaking before centrifugation at 14 000 g for
20 min. The supernatant was discarded and the pellet drained on
a sintered glass filter G3 (15–40 lm porosity) before weighing. The
weight difference between the wet and dry AIM is ascribed to the
water-holding capacity and expressed as g of water retained g1 of
dry AIM.
Enzyme extraction and assay
Culture medium and cell-surface enzymes were extracted according
to a method adapted from Wallner and Nevins (1974). Cell-surface
enzymes were extracted by shaking cells in cold 1 M sodium citrate
buffer pH 5.4 (1 ml g1) for 1 h.
Enzyme activity was detected by measuring the release of pnitrophenol from b-D-galactoside/a-L-arabinofuranoside substrates
(Sigma–Aldrich) according to Rouau and Odier (1986) or reducing
sugars from polysaccharides (laboratory-prepared sugar beet (1,5)-aL-arabinans, lupin (1,4)-b-D-galactans, and commercial polygalacturonic acid and gum arabic (Sigma–Aldrich)) according to Nelson
(1944). Caseinolytic activity was assayed as described by Tsumaraya
et al. (1990).
HPAEC of extracellular medium
Freeze-dried extracellular media were solubilized in water (10 mg in
1.5 ml) for 16 h at 4 8C. The solutions (20 ll) were injected on
a Dionex analytical Carbopac PA-1 column (Dionex, Sunnyvale, CA
; 43250 mm) equipped with a Carbopac PA-1 guard column
(Dionex; 4350 mm) using alkaline conditions according to Bouton
et al. (2002).
Results
Cell growth and cell adhesion
Although not synchronized, the growth of suspensioncultured Arabidopsis cells (Fig. 1) showed three reproducible phases, namely a cell division phase (days 0–6), a cell
expansion phase (days 6–12) and a stationary phase (after
day 12). During the cell division phase, the cell number
increased about 8-fold with only a 3-fold increase in fresh
weight. The cell expansion phase was characterized by
a 2-fold increase in fresh weight corresponding with water
uptake while the cell number did not change. After 12 d of
culture, cell growth stopped as already described by Axelos
et al. (1992) and probably corresponded with cell senescence (Callard et al., 1996).
The change in cell adhesion at the different growth stages
was observed by cell cluster size measurement before and
after a mild mechanical treatment (vortex shaking) for 2,
5, and 8 min. There was no change in turbidity or in
absorption maxima at 430, 490, and 680 nm for chlorophyll
b, carotenoid, and chlorophyll a absorption, respectively,
demonstrating that the cell integrity was conserved after
vortexing (data not shown). The size distribution of cell
clusters was essentially monomodal and gaussian. The cell
aggregates were grouped into small (<250 lm), middle
(250–500 lm), and big cell clusters (>500 lm).
Before vortexing, the proportion of cell clusters in the
three groups was similar for cells in the division and
expansion stages (Fig. 2). By contrast, cell clusters in the
Fig. 1. Growth curves of Arabidopsis thaliana microcalli suspension
culture and definition of the division, expansion, and stationary growth
phases. Lines are drawn to help reader’s eyes; bars indicate SD (n=5).
stationary phase were more fragmented (Fig. 2). This
fragmentation possibly resulted from the shaking conditions of the culture and indicated that cells in the stationary
phase were less adherent than cells in division or expansion.
After 2, 5 and 8 min of vortexing, cell aggregates in the
expansion phase were richer in small clusters and poorer
in large clusters, than cell aggregates in the division phase
(Fig. 2). Thus, expanding cells are less adherent than
dividing cells. Cell aggregates in the stationary phase were
richer in small clusters and poorer in big clusters than those
of dividing and expanding cells (Fig. 2). These results
suggest that cell adhesion decreases from the cell division
phase to the stationary phase in this Arabidopsis cellculture system.
Cell wall polysaccharides composition and structure
The alcohol-insoluble material (AIM) composition of the
cells at the different growth phases indicated the presence
of uronic acids, glucose, galactose, arabinose, xylose,
fucose, mannose attributed to cell wall polysaccharides,
and to starch (glucose; Fig. 3). It also contained proteins
and uncharacterized compounds (Fig. 3A). The proportion
of cell wall sugars increased during the cell expansion
phase and accounted for about 30–50% of the AIM while
that of starch decreased. Proteins and uncharacterized
material remained constant at about 30% and 15% of dry
weight, respectively.
The relative amount of cell wall sugars in the AIM of
cells in the division, expansion and stationary phases is
shown in Fig. 3B, C, and D. Glucose and uronic acid were
the major sugars (Fig. 3B) while arabinose, galactose, and
xylose represented about 20–30 mol% of all sugars (Fig.
3C, D). Rhamnose, fucose, and mannose were minor sugars
amounting to about 5 mol% of the total sugars. There were
no significant changes in the proportions of uronic acid,
(non-starch) glucose, rhamnose, xylose, fucose, and mannose along the cell culture. By contrast, the proportion of
2090 Leboeuf et al.
Fig. 2. Proportion of dividing, expanding and stationary cell aggregates of size <250 lm, between 250 and 500 lm and >500 lm before (0 min) and after
(2, 5, 8 min) vortex shaking. Results are expressed as a percentage of the total cell cluster population. Bars indicate SD (n=6).
Fig. 3. Chemical composition of the alcohol-insoluble materials from cell aggregates in the dividing, expanding, and stationary phases. (A) Overall dry
weight composition; (B, C, D) molar proportion of wall sugars. (B) Uronic acid and glucose, (C) rhamnose, galactose, and arabinose; (D) fucose, xylose,
and mannose. Bars indicate SD (n=5).
galactose increased about 1.5-fold during the cell division
phase and then gradually decreased to reach a minimum in
the stationary phase. The proportion of arabinose increased
2-fold during the cell division phase, remained stable
during the cell expansion phase, and then decreased 2-fold
in the stationary phase. Expressed on the uronic acid content of the AIM, the pectin degree of methyl-esterification
varied from 30% to 40% and tended to be higher in the
4d
2269
1467
35612
4367
2266
1362
30622
37611
1264
964
1663
3265
74622
666
–
–
7769
1869
–
–
7362
2667
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
663
–
–
–
1765
–
–
–
2464
–
–
5066
–
–
–
52620
–
–
–
53611
–
–
–
2566
–
–
–
1263
–
–
–
964
–
–
–
Ara
Gal
Rha
UA
14 d
14 d
8d
4d
14 d
8d
4d
14 d
8d
14 d
8d
4d
4d
8d
14 d
4d
8d
Polygalacturonase
Arabinanase+
arabinofuranosidase+
galactanase
Galactosidase
Galactanase
Arabinofuranosidase
Arabinanase
Sugars
Results were expressed as weight percentages of invididual sugars content in AIM;6 indicate SD (n=3).
Table 1. Release of arabinose (Ara), galactose (Gal), rhamnose (Rha), and uronic acid (UA) from the alcohol-insoluble material of dividing, expanding, and stationary
cells collected after 4, 8, and 14 d of culture, respectively, by endo-(1,5)-a-L-arabinanase,a -L-arabinofuranosidase, endo-(1,4)-b-D-galactanase, b-galactosidase, and endopolygalacturonase
Adhesion strength of Arabidopsis microcalli cells
2091
cell division phase, while its degree of acetylation increased
from 20% to 30% during the cell division phase and then
gradually decreased during the cell expansion phase to
reach a constant value of 20% in the stationary phase (data
not shown).
To explore the structure of arabinose- and galactosecontaining polysaccharides, alcohol-insoluble material
of cells grown for 4, 8, and 14 d were treated with endo(1,5)-a-L-arabinanase, a-L-arabinofuranosidase, endo-(1,4)b-D-galactanase, and b-D-galactosidase (Table 1). The
endo-a-L-arabinanase released about 2.5-fold more of the
total wall arabinose at day 14 than at days 4 and 8 while the
percentage of total wall galactose released by the endo(1,4)-b-D-galactanase gradually decreased during the cell
culture. The a-L-arabinofuranosidase released about 50%
of total wall arabinose at the three growth phases, but bD-galactosidase released no galactose. In order to test whether
these enzymes could act synergistically on the degradation
of the arabinose- and galactose-containing polysaccharides,
endo-(1,5)-a-L-arabinanase, a-L-arabinofuranosidase, and
endo-(1,4)-b-D-galactanase were also used together (Table
1). The combined enzymes failed to release more galactose
than that released by the endo-(1,4)-b-D-galactanase alone.
By contrast, they increased the amount of arabinose released from cells grown for 4 d and 8 d by about 10%, while
no increase was obtained from cells grown for 14 d.
Altogether, these results indicate that (1,4)-b-D-galactans
are present in the cell wall material and are unlikely to be
ramified. Furthermore, the gradual decrease in galactose
content and the decrease in galactose released by enzymes
as the cell culture progressed to the stationary phase
indicate that pectic galactans side chains are metabolized
and/or diluted by the synthesis of RGI poor in galactan side
chains. Similarly, these results indicate that (1,5)-linked
arabinans are present in these cell walls, but are more
branched in dividing and expanding cells than in nongrowing ones. The decrease in the amount of arabinose in
non-growing cells, the increase in arabinose released by the
endo-(1,5)-a-L-arabinanase alone from these cells, and the
inefficiency of the arabinofuranosidase to release more
arabinose when used in combination with the endoarabinanase suggest that arabinose branches on (1,5)-a-Larabinan are removed as the cell growth stops and/or are
diluted by the synthesis of RGI substituted by few linear
arabinan side chains. The proportion of pectic arabinans
(linear (1,5)-linked a-L-arabinan) and galactans to the total
arabinose and galactose in the wall is low (about 25%, see
Table 1) and suggests the presence of other arabinose/
galactose containing polymers in these cell walls. The
inefficiency of the combined enzymes endo-(1,5)-a-Larabinanase, a-L-arabinofuranosidase, and endo-(1,4)b-D-galactanase in releasing more galactose than
endo-(1,4)-b-D-galactanase alone and the positive reaction
of aqueous suspensions of these different AIM with bglycosyl-Yariv reagent (data not shown) demonstrated the
2092 Leboeuf et al.
presence of arabinogalactan proteins (AGP) in these
materials.
These cell wall preparations were also treated with an
endo-polygalacturonase to follow modifications of the
galacturonan pectic backbone during the different cellgrowth phases. The amount of uronic acid released by the
endo-polygalacturonase was higher from cells grown for
8 d and 14 d than for 4 d (Table 1). As this endopolygalacturonase cleaves blocks of unsubstituted galacturonic acids, the difference in degradation could reflect the
lower methyl- and acetyl-ester substitution of pectins in
expanding and non-growing cells than in dividing cells.
The enzyme released similar amounts of RGI from the cells
at the three growth phases as shown by the close molar
proportions of rhamnose and uronic acids recovered in the
hydrolysate (Table 2). However, the neutral side chains
differ, since the arabinose proportion was higher in the
expanding cells and markedly lower in non-growing cells.
These results agree with wall sugar composition.
To complement the structural analysis of cell wall polysaccharides, the solid-state 13C NMR spectrum was determined with liquid-like magic angle spinning acquisition
conditions (HR-MAS 13C NMR) of the alcohol-insoluble
material from dividing, expanding and non-growing cells
(Fig. 4A). This technique allows the observation of highly
mobile polysaccharides or polysaccharides segments such as
RGI side chains within the cell wall matrix (Foster et al.,
1996; Ha et al., 1996; Rondeau-Mouro et al., 2003). Prior to
observation, the different AIMs were enriched for tightly
linked cell wall materials by oxalate extraction and were then
Table 2. Mole percentage of rhamnose (Rha), arabinose (Ara),
galactose (Gal), and uronic acids (UA) released by endopolygalacturonase from the alcohol-insoluble material of dividing, expanding, and stationary cells collected after 4, 8, and
14 d of culture, respectively;6 indicate SD (n=3)
Sugars
Rha
Ara
Gal
UA
Culture age (d)
4
8
14
4.460.5
12.561.6
8.361.6
74.863.5
5.362.5
18.563.8
9.561.8
66.867.9
6.261.3
9.461.2
7.861.1
76.663.4
Fig. 4. 13C NMR spectra of cell walls (A) and extracellular polysaccharides in the culture medium (B). (A) Solid-state HR-MAS 13C NMR spectra of
oxalate-treated AIM of cells in division, expansion, and stationary phases and spectrum of the empty rotor, and (B) 13C NMR spectra of solutions of
native and a-L-arabinofuranosidase-treated extracellular polysaccharides. Letters correspond to signal in terminal arabinofuranoside (At), (1,4)-linked bD-galactan (G), (1,4)-linked a-D-galacturonan (P), and methyl ester (Me); C-1 to 6 correspond to carbon 1 to 6 in terminally-linked galactose or arabinose
(t-Gal, t-Ara, respectively), 3-, 6-, and 3,6-linked galactose (3-Gal, 6-Gal, 3,6-Gal, respectively); signals referred to as Gal have chemical shifts similar to
those of galactose in Latrix sibirica arabinogalactan (Karacsonyi et al., 1984).
Adhesion strength of Arabidopsis microcalli cells
enzymatically de-starched. The oxalate extract was essentially composed of uronic acids, galactose, arabinose, and
rhamnose that represented 10–30% of the corresponding
total wall sugar (data not shown) which probably corresponded to loosely interacting or ionically linked rhamnogalacturonans and homogalacturonans. The complex
solid-state 13C NMR spectra obtained from the residues
(Fig. 4A) showed major signals attributed to AGP (see below
and Fig. 4B) and pectic polysaccharides (Ryden et al., 1989;
Catoire et al., 1998). Signals for (1,4)-linked b-D-galactan
were observed, but those for (1,5)-linked a-L-arabinan were
not seen, presumably because of overlaps with those of AGP
or losses during preparation of the residues (Fig. 4A). Other
signals at about 20–40 ppm were attributed to proteins, at
171–174 ppm to carboxylic C of pectins (not shown), at
17.0 ppm to the internal hexamethyl benzene reference and
C6 of rhamnose in RGI. The broad signal at 111 ppm was
a contribution from the rotor and/or the probe. Many small
signals could not be assigned, probably resulting in part from
the structural complexity of AGPs and branched pectic side
chains. Modifications in the signals intensity were observed
between the spectra of dividing, expanding, and nongrowing cells. Compared with the signal intensity of the
internal reference, there was an increased contribution of
galacturonan signals in the expanding cell. There was
a decrease in the signals for (1,4)-linked b-D-galactan and
a marked decrease in the signals for terminal arabinofuranosyl residues in the non-growing cell. Because changes
were observed in sugar composition, these modifications
probably resulted from the decrease in the proportion of
some cell wall components rather than a reduction in their
mobility. Signals within the 20–40 ppm range attributed to
proteins were also affected by the growth phases, as their
intensity decreased from dividing to non-growing cells.
Water retention capacity of AIMs
The composition and structural analyses of AIM demonstrated modifications in pectic polysaccharides and AGP
during the cell culture. Pectins are thought to be involved in
the control of cell wall porosity (Willats et al., 2001) while
2093
the function of AGP are still unclear (Showalter, 2001) but
can also play a role through their highly ramified carbohydrate structure on wall porosity and cell wall polymer
interactions. To evaluate the consequence of the modifications of these polysaccharides contents and structure on the
cell wall properties, the water-holding capacity of the
different AIM was measured. The water-holding capacity
increased during cell culture from 5.661.2 at 4 d to
10.261.5 at 8 d, and 13.660.1 at 14 d and suggested an
increase in cell wall porosity.
Extracellular polysaccharides
Given the elimination of arabinose and galactose among the
cell wall polysaccharides during cell culture, the presence
of the corresponding degradation products in the culture
medium was investigated. At day 4, the culture medium
was mainly composed of glucose and mannose, that had
probably originated from sucrose isomerization, while at
days 8 and 14 these sugars were minor compared with the
arabinose, galactose, and uronic acid contents (Table 3).
No monomers and oligomers of arabinose and galactose
were detected in the culture medium by HPAEC. Only
glucose, fructose, and sucrose (peaks 1, 2, and 3, respectively, Fig. 5) were seen and their proportion decreased
during the cell culture in agreement with the GC data. Peak
4 detected at day 8 and, more importantly, at day 14, was
assigned to galacturonic acid.
Extracellular polymers (ECP) were precipitated from the
culture medium by ethanol and their yield on the basis of cell
number (ng cell1) increased during the culture: 0.1760.07
at day 4, 0.3560.02 at day 8, and 0.4060.03 at day 14.
Sugars accounted for 3166, 5162, and 4564% of ECP dry
weight at days 4, 8, and 14, respectively, and were mainly
composed of arabinose, galactose and uronic acids (Table 3).
These sugars amounted to nearly all the arabinose, galactose,
and uronic acids measured in the culture medium (data not
shown) and thus belonged to high molecular weight compounds. ECP was degraded by a-L-arabinofuranosidase
(6762% of total arabinose at day 8 and 6464% at
day 14), but the addition of endo-(1,5)-a-L-arabinanase,
Table 3. Sugar composition (mol%) of the extracellular medium and extracellular polymers of dividing, expanding, and stationary
microcalli recovered after 4, 8, and 14 d of culture, respectively;6 indicate SD (n=3)
Sugarsa
Rha
Fuc
Ara
Xyl
Man
Gal
Glc
UA
a
Extracellular medium
Extracellular polymers
4d
8d
14 d
4d
8d
14 d
0.060.0
0.060.0
0.660.0
0.360.3
10.161.1
0.960.2
85.261.2
2.960.3
1.760.2
1.360.4
15.361.7
3.460.6
5.160.6
15.161.1
12.163.6
45.862.8
1.860.2
1.160.1
17.360.4
3.260.5
4.760.1
16.661.0
7.660.6
47.761.3
1.360.1
1.060.2
15.261.2
6.660.9
4.060.8
19.061.4
15.661.3
34.463.0
1.360.2
0.660.1
14.362.0
2.260.2
2.560.3
14.562.3
5.860.7
55.566.3
1.660.0
0.760.0
17.660.5
1.460.1
2.160.4
17.460.8
4.160.4
52.264.3
Rha, rhamnose; Fuc, fucose; Ara, arabinose; Xyl, xylose; Man, mannose; Gal, galactose; Glc, glucose; UA, uronic acids.
2094 Leboeuf et al.
endo-(1,4)-b-D-galactanase, and b-D-galactosidase to the
arabinofuranosidase had no effect. On solubilization of ECP
for NMR analysis, a uronic acid-rich insoluble material precipitated leaving nearly all the neutral sugars in solution. The
combined solutions of ECP recovered from the 8 d and 14
d cell cultures yielded a 13C NMR spectrum (Fig. 4B) with
major signals attributed to type II arabinogalactans (Gane
et al., 1995; Karacsonyi et al., 1984; Saulnier et al., 1992).
These soluble fractions also yielded a reddish precipitate
with the b-glycosyl-Yariv reagent.
All these results indicated that ECP were made of type II
arabinogalactans belonging to AGPs and partly of homogalacturonan.
Activities of cell wall-degrading enzymes during
cell culture
Together with the structural changes of AIM polysaccharides during the cell growth phases, cell wall and culture
medium enzymatic activities were measured (Table 4).
(1,5)-a-L-Arabinanase, ‘AGPase’ (tested against gum
arabic substrate) and protease activities (tested against
azocasein substate) were not detected. Cell surface bgalactosidase activity was the only enzyme detected at
day 4 and the level of this activity slightly increased during
the cell culture. The culture medium also contained bgalactosidase activity at day 4, but this activity was higher
at days 8 and 14 than at day 4. Cell surface and culture
medium a-arabinofuranosidase activities were very low at
days 4 and 8, but increased at day 14. There was no cell
surface and culture medium (1,4)-b-D-galactanase activity
at day 4. This activity appeared at day 8 and increased at
day 14. Similarly, a low polygalacturonase activity at the
cell surface and in the culture medium appeared at day 8
and markedly increased at day 14 (Table 4).
Discussion
Fig. 5. HPAEC chromatograms of the extracellular culture medium of
Arabidopsis microcalli in division, expansion, and stationary phases.
Peaks labelled Glc, Fru, Suc, and GalA were assigned on the basis of their
retention time to glucose, fructose, sucrose, and galacturonic acid,
respectively.
In the present work, A. thaliana microcalli suspension
culture was assessed as a model to approach the macromolecular basis of cell adhesion. Even though this cell
culture was asynchronous, it presented three reproducible
growth phases enriched in dividing, expanding, and nongrowing cells. These phases were associated with a decrease
in cell cluster adhesion strength, modifications in the cell
wall polysaccharides composition and structure, as well as
in cell wall-degrading enzyme activities. The overall cell
wall sugar composition of these microcalli is similar to that
of A. thaliana seedlings primary wall (Zablackis et al.,
1995; Bouton et al., 2002; Peng et al., 2000). However,
major variations were seen in the galactose and arabinose
contents which peak during the cell division phase and then
decrease at different rates during the cell expansion and
Table 4. Cell wall degrading enzyme activities at the cell surface and in the culture medium of dividing, expanding and stationary
microcalli (4, 8, and 14 d, respectively);6 indicate SD (n=3)
Enzymes
a-L-arabinofuranosidase
b-galactosidase
(1,4)-b-D-galactanase
Polygalacturonase
Cell surface (nkat g1)
Culture medium (nkat ml1)
4d
8d
14 d
4d
8d
14 d
0.0160.01
0.5160.04
0.0060.00
0.0060.00
0.0160.01
0.6060.11
1.3160.52
0.0360.04
0.2560.06
0.6760.07
3.4161.04
2.3160.05
0.0060.00
0.0260.00
0.0060.00
0.0060.00
0.0160.00
0.1060.01
0.1560.12
0.0260.02
0.0560.00
0.1260.03
0.9960.67
0.5760.38
Adhesion strength of Arabidopsis microcalli cells
stationary phases. Part of these sugars was clearly shown to
originate from linear (1,4)-b-D-galactans and branched
(1,5)-a-L-arabinans belonging to RGI side chains. The loss
of these galactans and arabinans was well supported by the
appearance of extracellular (1,4)-b-D-galactanase and arabinofuranosidase activities and led to an increase in waterholding capacity of wall material taken as reflecting an
increase in wall porosity. The decrease in b-D-galactan
during the cell expansion phase agrees with results obtained
on cell suspension cultures of Populus alba (Kakegawa
et al., 2000). The b-D-galactosidase activity observed
throughout the culture cycle and, particularly, peaking
when the cells became less adherent and lost galactose,
agrees with results reported from cell walls of Paul’s Scarlet
Rose cell suspension cultures (Wallner and Nevins, 1974).
All the enzymes reaction products were likely to be rapidly
metabolized because only some galacturonic acid accumulated in the extracellular medium. AGP and HG were the
main accumulating extracellular polymers in agreement
with literature reports (Darjania et al., 2002; Takeuchi and
Komamine, 1978).
The decrease of pectic arabinan and galactan side chains
found in the microcalli culture system used here can be
related to both cell growth and/or adhesion. Willats et al.
(1999) showed that the onset of pectic galactan side chains
in the cell wall of proliferating cells in the carrot root apex
or in carrot calli appeared after the cells were first
conditioned for expansion. Similarly, galactan RGI side
chains were observed at the transition zone near or at the
onset of cell elongation in different tissues of Arabidopsis
seedlings (McCartney et al., 2003). Willats et al. (1999)
also showed that arabinan side chains appeared restricted
to proliferating cells. The different rates of galactose and
arabinose disappearance observed here in Arabidopsis
microcalli cell walls, notably during the elongation phase,
support modulations of RGI neutral side chains composition during cell development. However, the overwhelming
contribution of AGP to the arabinose and galactose contents
and cell asynchrony probably masked fine modifications of
the pectic side chains. In particular, the de-branching of
RGI arabinan observed during the stationary phase of the
microcalli culture would be interesting to follow in planta
with regard to development and cell differentiation.
RGI and their neutral side chains have also been
associated with adhesion and rigidity of primary walls.
The molar ratio of arabinose to galactose in the pectic
fraction of carrot callus cell walls has been correlated with
the average size of cell clusters (Kikuchi et al., 1996) and
probably reflected different cell adhesion. The presence of
galactan side chains have often been related with cell wall
rigidity and with firmer textures of tissues and organs
(McCartney et al., 2000; McCartney and Knox, 2002;
Smith et al., 2002). With regard to arabinan, low levels and
abnormal deposition in cell walls have been found with
large intercellular spaces in the pericarp of the Cnr tomato
2095
mutant (Orfila et al., 2001, 2002) and with loose cells
in Nicotiana plumbaginifolia callus (Iwai et al., 2001).
However, these observations are difficult to interpret
because not only the RGI neutral side chains were affected
but also the galacturonan backbone. In fact, recent mutants
studies suggest that the polyuronan backbone of pectin may
be more important in intercellular attachment than RGI side
chains (Bouton et al., 2002; Iwai et al., 2002; Sorensen
et al., 2000; Skjot et al., 2002; Oomen et al., 2002). The
release of homogalacturonan in the microcalli culture
medium as they became more fragmented supports their
role in adhesion. Since RGI is located in the primary wall
(Jarvis et al., 2003), its role in cell adhesion is likely to be
indirect. It is expected to involve molecular entanglement
of its neutral side chains and ionic interactions of the uronan
backbone with other cell wall and middle lamella polymers.
It is also possible that it be covalently linked, for example,
with xyloglucans (Thompson and Fry, 2000). These linkages and interactions would rigidify the cell wall, anchor
the middle lamella polymers to the primary wall, and control wall porosity, thus affecting the diffusion of enzymes.
The proposition that the middle lamella HG represent
side chains of peculiar RGI (Vincken et al., 2003) remains
to be established, but would fit with the above conception.
However, the bulk of galactose and arabinose affected by
the different growth phase of these A. thaliana microcalli
suspension culture was attributable to AGP. These polymers were found both in the culture medium as previously
reported (Darjania et al., 2002), and tightly bonded in the
cell wall where they are structurally modified according to
the growth phase of the cells. These modifications and their
accumulation in the extracellular medium suggest specific
biological roles (Showalter, 2001), such as on cell growth
(Kreuger and Van Holst, 1993; Toonen et al., 1997).
Nevertheless, like RGI neutral side chains, physicochemical contributions can be expected of the arabinogalactan ramified structure of AGP to the cell wall mechanical
properties and in the control of wall porosity. Furthermore,
being associated or anchored to the plasma membrane
(Showalter, 2001), with b-glycan binding properties
(Baldwin et al., 1993; Pennell et al., 1989; Yariv et al.,
1967), and with the ability to form cross-links (Kjellbom
et al., 1997), AGP are likely to play a role in linking cell
wall macromolecules to the cell membrane, and thus to be
involved in cellular cohesion.
Altogether, these results stress on the complex multimolecular aspect of cell adhesion involving polyuronans,
RGI side chains, and AGP in the whole wall, from the
plasma membrane to the middle lamella. In particular, it is
shown that linearization of RGI arabinan side chains and
modulation of AGP structure are also related to cell growth
and separation processes. However, it is likely that other
cell wall polymers are also involved in cell adhesion, such
as structural wall proteins known to rigidify tissues and to
be able to form complexes with pectins (Cassab, 1998;
2096 Leboeuf et al.
MacDougall et al., 2001). Identification of Arabidopsis
mutants specifically affected in the synthesis, structure, and
location of these different wall polymers will help in
elucidating their function on cell growth and adhesion.
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