Glycobiology vol. 20 no. 6 pp. 775–786, 2010
doi:10.1093/glycob/cwq034
Advance Access publication on March 3, 2010
Chitosan oligosaccharides modulate the supramolecular
conformation and the biological activity of
oligogalacturonides in Arabidopsis
2
2
Juan-Carlos Cabrera , Aurélien Boland ,
2
2,3
Pierre Cambier , Patrick Frettinger ,
1,2
and Pierre Van Cutsem
2
Unité de Recherche en Biologie Cellulaire Végétale, Facultés Universitaires
Notre-Dame de la Paix, rue de Bruxelles 61, B-5000 Namur, Belgium
Received on December 18, 2009; revised on February 26, 2010; accepted on
February 27, 2010
Plant cell walls undergo remodeling during growth and development and are the first target of many invading
pathogens. Acidic pectin (homogalacturonans) binds calcium and forms chain dimers called egg boxes and even
multimers at higher calcium ion concentrations. Chitosan,
the deacetylated form of chitin produced by fungi when invading plant tissues, is a cationic polymer that can interact
with negatively charged pectin. The interaction between
chitosan oligomers (COS) and pectic egg boxes was investigated using 2F4, a monoclonal antibody specific for
calcium-associated dimers of pectin. Depending on the size
of the pectic molecules, the COS to pectin ratio, the degree
of polymerization and the degree of acetylation of COS in
the mixture, the calcium-induced egg box conformation of
oligogalacturonides (OGA) was strongly stabilized or destroyed. The biological activity of COS-stabilized egg
boxes was assayed on Arabidopsis cell suspensions. COS–
OGA egg boxes strongly enhanced extracellular alkalinization and decreased potassium fluxes compared to control
COS and OGA alone. Furthermore, OGA rescued Arabidopsis from cell death induced by higher concentrations of
deacetylated COS. The stabilized COS–OGA egg boxes
could constitute a combined emergency signal that informs
plant cells on both cell wall degradation and pathogen
presence, triggering a much stronger response than individual components alone.
Keywords: Arabidopsis /chitooligosaccharides/egg box /
elicitation /oligogalacturonides
Introduction
Pectin constitutes a dense molecular network that accounts for
much of the physico-chemical properties of the primary plant
1
To whom correspondence should be addressed: Tel: +32-(0)-8172-4414;
e-mail: [email protected]
3
Current address: Unité Mixte de Recherches INRA-CNRS-Université de
Bourgogne, Plante-Microbe Environnement, INRA, 17 Rue Sully, BP
86510, 21065 Dijon cedex, France
cell wall. Cross-linking of the pectic molecules by Ca2+ ions
according to the so-called “egg-box” model (Grant et al.
1973) plays an important role in the properties of the pectin
network which strongly affects the physiological significance
of the pectic polysaccharides in plant cell walls.
Upon plant invasion, most pathogens secrete pectolytic enzymes that degrade wall pectin into bioactive alpha-1,4-linked
oligogalacturonides (OGA).These OGA function as elicitors of
defense responses and regulators of developmental responses
in plants (Aldington and Fry 1993; Cote and Hahn 1994;
Van Cutsem and Messiaen 1994; Ridley et al. 2001). The structural requirements for OGA with degree of polymerization
(DP) ≥ 9 to adopt the calcium-induced egg box conformation
determine most of their bioactivity (Mathieu et al. 1991; Messiaen et al. 1993; Messiaen and Van Cutsem 1993a, 1993b,
1994, 1999). This egg box conformation can be modulated
by polycations such as the polyamines spermidine and spermine that prevent polygalacturonic acid (PGA) from
adopting the Ca 2+ -induced supramolecular conformation,
block the transduction of the pectic signal and consequently
their elicitor activity (Messiaen and Van Cutsem 1999).
No pectin receptor has been described up to now, but
WAK1, a transmembrane receptor-like wall-associated kinase,
contains a cytoplasmic Ser/Thr kinase domain and an extracellular domain in contact with the pectin fraction of the cell
wall. The extracellular domain of WAK1 from Arabidopsis
thaliana binds to PGA, OGA and pectins extracted from A.
thaliana cells (Decreux and Messiaen 2005; Decreux et al.
2006).
Recently, we reported that OGA dimerization through Ca2+
bridges increases with the age of the solution (Cabrera et al.
2008). We call this process egg box maturation and it consists
of a progressive increase in length of the junction sequences
between two calcium-associated chains that slide along each
other in order to form a maximum number of calcium bridges
and dimer ends. We could show that maturation of egg boxes
induced a significant increase of both their binding to the extracellular domain of WAK1 and their bioactivity as inducer of
extracellular alkalinization of A. thaliana suspension cells.
The cell wall of fungi and several oomycetes contains
chitin (Werner et al. 2002, Kamoun 2003). Plants attacked
by fungi respond by secreting pathogenesis-related proteins
among which several chitinases. N-Acetylchitooligosaccharides and chitosan oligosaccharides, its de-N-acetylated
derivative, have been shown to elicit defense responses in
various plants (Hahn 1996). Chitin fragments bind to rice
CEBiP, a transmembrane protein with two extracellular
LysM motifs that lacks an intracellular signaling domain
© The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]
775
J-C Cabrera et al.
(Kaku et al. 2006). CERK1, a LysM RK may act with CEBiP to transduce chitin signaling (Miya et al. 2007).
Chitosan oligosaccharides that result from chitin deacetylation and hydrolysis elicit defense responses mostly in
dicots (Shibuya and Minami 2001). The bioactivity of polycationic chitosans has been associated to their interaction
with negatively charged phospholipids (Kauss et al. 1989).
However, both deacetylated (Vasconsuelo et al. 2003) and
acetylated (Wan et al. 2004) elicitors seem to activate a
common mitogen-activated protein kinase cascade. The oligosaccharide concentration needed in plant bioassays also
seems to differ between elicitors and depends on the plant
model used. The chitosan concentrations required are usually
much higher than those necessary for chitin oligosaccharides
to elicit similar responses (Yamaguchi et al. 2000).
Polycationic chitosans can interact in solution with polyanionic pectins to form polyelectrolyte complexes, coacervates,
networks and multilayers. These attractive interactions between
polymers have been extensively studied (Marudova et al. 2004;
Murata et al. 2005; Chou et al. 2006) and used in technological
applications (Meshali and Gabr 1993; Hoagland 1996; Macleod
et al. 1999; Jang et al. 2003). However, the interactions between
chitosan oligosaccharides and oligopectic egg boxes have never
been explored.
In this study, we investigate the effect of chitooligosaccharides on the egg box conformation of poly- and
oligogalacturonans and the consequences of their interaction
on the early responses and viability of challenged A. thaliana
cells in suspension.
Results
Oligosaccharides preparation and characterization
Two defined OGA fractions of low DPs (mainly up to six) and
high DPs (containing hepta- and octa-oligomers until heptadeca-α-(1,4)-D-oligogalacturonides) were obtained by enzymatic
hydrolysis of PGA and isolated by selective precipitation.
Two sets of deacetylated chitooligomers of low DPs (up to
six) and high DPs (between five and nine) were obtained by
acid and enzymatic hydrolysis of chitosan, respectively. Acetylated chitosan oligomers (COS) were prepared by chemical
reacetylation. All COS sets were characterized by matrix-assisted laser desorption ionization time-of-flight (MALDITOF) mass spectrometry (Cabrera and Van Cutsem 2005).
Since the degree of acetylation (DA) is an average value and
reacetylated COS are polydisperse in terms of acetylated residues, we use the terms “deacetylated” COS for essentially fully
deacetylated chitooligomers; “partially acetylated” COS for
chitooligomers with DA between 15 and 25 and “highly acetylated” COS for chitooligomers with DA ∼50%. Chitin
oligosaccharides had DA higher than 60.
Enzyme-linked immunosorbent assay (ELISA) procedure
The conformational changes of the pectic molecules induced
by calcium ions have been traditionally studied using equilibrium dialysis, circular dichroism (CD) (Morris et al. 1982;
Powell et al. 1982) and IR spectroscopy (Jarvis and Apperley
1995). In general, these methods require relatively high concentration of the pectic oligo/polysaccharides. A valuable
alternative is the ELISA procedure described by Liners et al.
776
(1989), in which Ca2+-induced dimers of pectin can be quantitatively detected using the 2F4 antibody that specifically
recognizes this conformation. Any modification of the pectic
egg box conformation can thus be detected by the antibody.
To study the interaction between COS and pectic egg boxes,
we used the monoclonal antibody (MoAb) 2F4 that specifically
recognizes Ca2+-induced pectin dimers. The first experiments
were performed using an immunoprecipitation followed by an
ELISA test of the remaining 2F4 in the supernatant as described by Liners et al. (1992). In this procedure, the MoAb
is mixed and preincubated with the assayed compounds
(OGA plus COS), and after centrifugation, the supernatant
containing unreacted 2F4 MoAbs but also unreacted COS
are transferred to PGA-coated microwells and bound 2F4
MoAbs detected by a horseradish peroxidase (HRP)-labeled
secondary antibody.
Before using this procedure, we carried out a control test in
which COS were directly added to PGA-coated microwells
(Figure 1A). We observed that fully deacetylated high DP
COS inhibited binding of 2F4 MoAbs to PGA adsorbed in
the microwells. Clearly, COS bound the immobilized, negatively charged PGA, thereby preventing access of the 2F4
antibody to its epitope. This ruled out the use of this test to
study the interaction between COS and egg boxes in solution.
Another immunoprecipitation and ELISA test was then set
up in which PGA coating of the microwells was substituted
for GaMIg coating, a goat antimouse immunoglobulin to capture the unreacted 2F4 IgG1 from the supernatant. In this
GaMIg ELISA test, preincubation of 2F4 MoAbs with increasing COS concentrations did not interfere with the subsequent
capture of 2F4 MoAbs by the antimouse immunoglobulin
(Figure 1B), confirming that COS did not interact with 2F4.
A calibration curve for low and high DP OGA and PGA detection by 2F4 MoAbs is presented in Supplemental Figure 1.
This test allowed quantification of PGA and OGA egg boxes
over at least four orders of magnitude. As already known
(Liners et al. 1992) and confirmed by CD (Cabrera et al.
2008), low DP OGA were not able to adopt the egg box conformation recognized by the 2F4 antibodies, even at high
concentration. High DP OGA and PGA, dimerized in presence
of suitable calcium–sodium ratios, were specifically bound by
the 2F4 antibodies, and the absorbance of the inhibition ELISA
test was inversely proportional to the concentration of pectin
molecules present in the immunoprecipitation solution. The regression lines of Supplemental Figure 1 were used to
determine the relative egg box concentrations in the immunoprecipitation — ELISA assays. Alginates (Supplemental
Figure 1), methylesterified high DP OGA and methylesterified
PGA were not recognized by the 2F4. In presence of ethylenediaminetetraacetic acid, a calcium chelator, no egg box could
be detected whatever the pectin tested (data not shown). This
allowed using this immunoprecipitation — GaMIg ELISA test
as a specific quantitative measurement of calcium-induced egg
box dimers of pectin molecules.
Chitooligosaccharides interact with Ca2+-induced dimers of
pectic molecules
PGA or OGA in Ca/Na ELISA buffer were incubated with partially acetylated COS, and the concentration of pectic egg box
dimers in the mixture was determined using the immunoprecip-
COS modulate OGA egg box bioactivity
2,5
A
Absorbance at 450 nm
2,0
1,5
1,0
0,5
0,0
0
1
3
5
7
10
COS concentration
20
30
40
30
40
(mg.L-1)
1,5
B
Absorbance at 450 nm
1,2
0,9
0,6
0,3
0,0
0
1
3
5
7
10
20
COS concentration (mg.L-1)
Fig. 1. Effect of completely deacetylated chitosan oligosaccharides on
detection of 2F4 MoAbs in ELISA tests. (A) Inhibition by COS of 2F4 MoAb
binding to pectin. PGA-coated microwells were preincubated with increasing
concentrations of deacetylated high DP COS, washed, incubated with the 2F4
MoAbs and the binding of the antibodies revealed by a secondary HRP-SAM
antibody. (B) Absence of inhibition by COS of 2F4 binding to trapper antibody
(immunoprecipitation — GaMIg ELISA). The 2F4 MoAbs were incubated
with different concentrations of deacetylated high DP COS, centrifuged and the
supernatant transferred to GaMIg-coated microwells. After washing, bound
2F4 MoAbs were detected by a secondary HRP-SAM antibody. The mean
values (n = 3) are presented with the standard errors.
itation — GaMIg ELISA procedure (Figure 2). Concentrations
were calculated using calibration curves (Supplemental
Figure 1) and expressed as percentages of controls without
COS. In presence of partially acetylated COS, a modification
in the recognition of the Ca2+-induced PGA conformation detected by the 2F4 MoAbs was observed (Figure 2A). When
partially acetylated high DP COS were present at a COS/
PGA molar monomer unit ratio about 0.5, the detected amount
of pectic egg box dimers was more than seven times higher than
that of PGA alone. Low DP COS showed a similar although
smaller effect at higher COS/PGA molar monomer unit ratio.
For both long and short COS fractions, increasing the COS/
PGA molar monomer unit ratio finally completely suppressed
PGA recognition by the 2F4 antibody. Glucosamine monomers
Fig. 2. Effect of partially acetylated chitosan oligosaccharides on egg box
dimer formation by polygalacturonides and high DP OGA. Egg boxes were
detected by 2F4 MoAbs in an immunoprecipitation — GaMIg ELISA test,
their concentration determined with the help of calibration curves
(Supplemental Figure 1) and expressed as percentages of egg boxes formed by
control PGA or OGA without COS. Whatever their size, the partially acetylated
COS induced a pronounced stabilization of the egg box dimers up to an
optimum COS/PGA or COS/OGA ratio. Excess COS concentrations destroyed
the egg box conformation, depending on COS length (low and high DPs) and
pectin size (PGA or OGA). (A) PGA (0.2 mg L−1) egg box detection in
presence of increasing concentrations of partially acetylated COS of low (○)
and high DP (•). (B) High DP OGA (4.0 mg L−1) egg box detection in
presence of increasing concentrations of partially acetylated COS of low (○)
and high DP (•).
at any concentration did not interfere with PGA recognition by
the 2F4 antibody (not shown).
The same partially acetylated COS had similar effects on the
egg box conformation of OGA with DP > 8 (Figure 2B). However, the stabilization of the egg box conformation by COS
occurred at lower molar monomer unit ratios for OGA than
for PGA. Whatever the COS to OGA ratio, no egg boxes were
detected in low DP OGA and methylesterified high DP OGA
solutions when they were either alone or mixed with COS (data
not shown). Polycations such as histone, poly-L-lysine and
polyethyleneimine (PEI) readily destroyed the egg boxes (Sup777
J-C Cabrera et al.
Alkalinization (R/RCOS, DA~0 %)
200
a
A
b
150
b
c
100
50
d
0
DA ~15 DA ~0
Low DP
DA ~25 DA ~35 DA ~50
High DP
120
a
80
b
60
40
+
K efflux (R/RCOS DA~0 %)
B
100
20
c
c
c
0
DA ~15
Low DP
Fig. 3. Effect of the degree of acetylation of chitosan oligosaccharides on egg
box dimer formation by polygalacturonides and high DP OGA. Egg boxes
were detected by 2F4 MoAbs in an immunoprecipitation — GaMIg ELISA
test, their concentration determined with the help of calibration curves
(Supplemental Figure 1) and expressed as percentages of egg boxes formed by
control PGA or OGA without COS. Only partially acetylated COS induced a
pronounced stabilization of the egg box dimers up to an optimum COS/PGA or
COS/OGA ratio beyond which egg boxes were destabilized. Largely acetylated
COS (○) did not interfere with PGA and OGA at all while completely
deacetylated COS (▴) destroyed the egg boxes. Only partially acetylated COS
(•) could stabilize the egg boxes. (A) PGA (0.2 mg L-1) egg box detection in
presence of increasing concentrations of high DP COS deacetylated (▴),
partially acetylated (•) and highly acetylated (○). (B) High DP OGA
(4.0 mg L-1) egg box detection in presence of increasing concentrations of high
DP COS deacetylated (▴), partially acetylated (•) and highly acetylated (○).
plemental Figure 2) at concentrations at least 10 times lower in
the case of OGA as compared to PGA.
The specific effect of COS acetylation on the egg box conformation of PGA and OGA was studied using high DP COS
with average DAs of 0%, 15%–25% and 50% (Figure 3). In
agreement with Figure 2, the pectic egg boxes were best recognized by the 2F4 antibody in presence of partially acetylated
COS. Preincubation of the pectic molecules with highly acetylated COS (i.e. chitin oligomers) did not modify the egg box
concentration detected by the 2F4 MoAbs, while deacetylated
778
DA ~0
DA ~25 DA ~35 DA ~50
High DP
Fig. 4. Effect of size and acetylation of chitosan oligosaccharides on medium
alkalinization and K+ efflux in Arabidopsis cell suspensions. Washed cells
were resuspended in 20 mg L-1 COS solution for 30 min. Values of the control
solutions without elicitor were subtracted from all data, and the response (R)
induced by each treatment was expressed as a fraction of RCOS DA ∼0, the
response with COS fragments of DA ∼0. Data reflect the mean of at least two
independent experiments. Different letters among treatments indicate
significant differences (P ≤ 0.05). (A) Medium alkalinization. High but not
low DP COS induced medium alkalinization. (B) Potassium efflux. Only
deacetylated or partially acetylated high DP COS could induce potassium
efflux. The pattern of medium alkalinization response differs from K+ efflux,
suggesting two separate perception and transduction pathways.
COS completely suppressed egg box dimer formation above a
molar monomer unit ratio of 0.5 in the case of PGA, which is
consistent with Figure 1A. High molar monomer unit ratios of
partially acetylated high DP COS also progressively abolished
the egg box stabilization. The ratio required to dismantle the
egg boxes increased with the DA of the chitooligosaccharide
assayed (data not shown). Partially acetylated COS did not induce alginates to form any egg boxes detectable by the 2F4
MoAbs (data not shown).
To summarize, addition of COS either stabilized or destroyed
the calcium-induced egg box conformation of the pectic substances, depending on the DA and the DP of COS and on the
ratio between COS and pectic molecules in the mixture.
COS modulate OGA egg box bioactivity
OGA inhibit Arabidopsis cell death induced by COS
Cabrera et al. (2006) showed that deacetylated high DP COS at
concentrations higher than 100 mg L−1 induce cell death of
Alkalinization (R/R OGA, %)
300
A
250
200
150
100
-1
50
COS + OGA (40 mg.L )
COS
0
0
20
40
60
80
COS (mg.L-1)
1400
B
COS + OGA (40 mg.L-1)
COS
1200
+
K efflux (R/R OGA, %)
COS–OGA interaction modifies early responses of A. thaliana
cell suspensions to both elicitors
The biological relevance of the interaction between COS and
OGA was studied by measuring the induction of early membrane responses by both oligosaccharides alone and their
combinations in A. thaliana suspension cells.
Medium alkalinization and K+ efflux are two of the earliest
responses of suspension-cultured cells to elicitors. High DP
OGA in calcium-induced egg box conformation have been
shown to increase significantly both cellular responses. A rapid
and transient alkalinization reaches a plateau at 20 µg mL−1
OGA while K+ efflux is also dose dependent, but no plateau
is observed within the OGA concentration range tested (0–
50 µg mL−1). While K+ efflux is not affected by OGA egg
box maturation, the extracellular alkalinization increases significantly (Cabrera et al. 2008).
Depending on size and DA, COS induced a strong alkalinization of the extracellular medium and K+ efflux out of A.
thaliana suspension cells (Figure 4). High DP COS with a
DA ∼25 was the most potent elicitor of alkalinization
(Figure 4A) while fully deacetylated COS induced the strongest K+ efflux. No K+ efflux could be detected when the DA
of COS was higher than 25% (Figure 4B). In presence of low
DP COS, neither alkalinization nor K+ efflux was detected.
These early membrane responses were then used to test the
bioactivity of mixtures of both OGA and partially acetylated
high DP COS that formed strongly stabilized egg boxes. First,
the effect of the COS/OGA molar monomer unit ratio on the
bioactivity was tested (Figure 5). In this experiment, OGA concentration was 40 mg L −1 and COS concentration was
increased. While increasing COS concentration alone monotonously increased both medium alkalinization and K+ efflux,
the COS + OGA mixture had contrasting effects. When COS
fragments were present at concentrations up to nearly twice the
OGA concentration, they clearly increased medium alkalinization over what was observed with COS alone (Figure 5A). K+
efflux on the other hand was always lower in mixtures when
COS concentration was higher than or equal to OGA concentration. When COS concentration in the mixture was lower
than that of OGA (i.e. 40 mg L−1), K+ efflux was not really
different from the treatment with OGA alone (Figure 5B).
Highest medium alkalinization and lowest K+ efflux occurred
with a mixture of 40 mg L−1 OGA and 30 mg L−1 COS.
Arabidopsis cell suspensions were then treated with this optimum COS–OGA mixture, and the time course of medium
alkalinization and K+ efflux was evaluated (Figure 6). After
2 h, the alkalinization induced by the mixture was higher than
the simple additive effects of COS and OGA alone
(Figure 6A). In absence of the ionic conditions required to induce egg box dimers (Figure 6C), such an effect of the mixture
on alkalinization could not be observed. While COS-treated
cells exhibited high K+ efflux, the presence of OGA in combination with COS completely abolished the effect of COS
(Figure 6B). The high DP OGA had the same effect on K+ efflux induced by COS in absence of the appropriate Ca/Na ionic
solution (Figure 6D).
1000
800
600
400
200
0
0
20
40
60
80
COS (mg.L-1)
Fig. 5. Effect of partially acetylated chitosan oligosaccharides with or without
high DP OGA on alkalinization and potassium efflux in Arabidopsis cell
suspensions. Cells were treated in presence or absence of 40 mg L-1 high DP
OGA in Ca/Na bioassay solution for 30 min. Values of the control solutions
without elicitor were subtracted from all data, and the response induced by each
treatment was expressed as the percentage of ROGA, the response elicited by
40 mg L-1 high DP OGA alone. Data are the mean of three replicates of at least
two independent experiments. (A) Medium alkalinization. Increasing COS
concentration in presence of OGA finally neutralized the effect of OGA on
medium alkalinization. (B) Potassium efflux. The presence of OGA nearly
suppressed potassium efflux until COS were more concentrated than OGA.
Arabidopsis cell suspensions within 24 h after treatment and
that acetylation of these chitosan oligosaccharides suppressed
or decreased their toxicity. In Figure 7A, the viability of suspension cells was evaluated 24 h after treatment with acetylated and
deacetylated high DP COS at 300 mg L−1 alone or in combination with OGA. Acetylated high DP COS, monogalacturonic
acid and the pectic molecules alone did not affect cell viability.
Deacetylated high DP COS (DA ∼0, 300 mg L−1) killed the
cells but their combination with low or high DP OGA significantly prevented cell death, probably because OGA titrated
out polycationic COS, preventing them from reaching their
cell targets. Cell viability decreased with increasing concentrations of deacetylated high DP COS but was rescued in
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J-C Cabrera et al.
Without Ca/Na
With Ca/Na
Alkalinization (pH units)
C
A
0,6
COS
COS + OGA
OGA
0,4
0,2
COS
COS + OGA
OGA
0,0
2,0
D
B
COS
COS + OGA
OGA
K+ efflux (mM)
1,5
COS
COS + OGA
OGA
1,0
0,5
0,0
0
20
40
60
80
100
120
Time (min)
140 0
20
40
60
80
100
120
140
Time (min)
Fig. 6. Time–response curves of extracellular alkalinization and K+ efflux in Arabidopsis cell suspensions. Cell suspensions were treated with partially acetylated
high DP COS (30 mg L−1), high DP OGA (40 mg L−1) or their mixture in presence (A and B) or absence (C and D) of Ca/Na bioassay buffer. Values of the control
solutions without elicitors were subtracted from all data. Data are representative of the means of five independent experiments. (A) Medium alkalinization induced
by the elicitors in presence of calcium. The alkalinization reached no plateau in presence of the COS–OGA mix. (B) Potassium efflux caused by the elicitors in
presence of calcium. The addition of OGA to COS reduced the K+ efflux down to the level of OGA alone. (C) Medium alkalinization induced by the elicitors in
absence of calcium. OGA-containing treatments induced less medium alkalinization in absence of calcium while COS-treated cells induced similar or even higher
medium alkalinization. (D) Potassium efflux caused by the elicitors in absence of calcium. Potassium efflux was strongly reduced in absence of calcium in the
medium.
presence of OGA (Figure 7B): higher COS concentrations
were needed to kill the cells when OGA were present.
Discussion
Since chitosans are polycations and pectins are polyanions, we
tested the interaction between OGA and COS oligomers to see
whether chitosan fragments could modulate the elicitating
properties of pectic oligomers.
COS–pectic egg box interaction
Partly acetylated COS (DA 15–25%) strongly increased the formation of egg box dimers of PGA and of high DP OGA up to an
optimum that was dependent on the size of both COS and pectic
molecules (Figure 2) while glucosamine monomer had no effect
(not shown). Since COS did not interfere with 2F4 MoAbs
(Figure 1B), this means that COS first stabilized the egg box
780
conformation while higher COS concentrations destroyed it,
probably by displacing Ca2+ ions from the pectic anions. The
higher the DP of COS, the lower the concentration needed to
observe the stabilizing effect. Similarly, the higher stability of
PGA dimers also required more COS for altering their conformation (Figure 2A) as compared to OGA (Figure 2B).
COS acetylation
Highly cationic COS (DA ∼0%) readily destroyed the pectic
egg boxes (Figure 3) as did other polycations such as polyamines (11), histones, poly-L-lysine and PEI (Supplemental
Figure 2). Only partially acetylated COS (DA ∼25) stabilized
the egg box structures while even a substantial concentration of
weakly charged COS (DA 50%) could not hinder pectic egg
box formation.
At the usual cell wall pH of 5.5, completely deacetylated
chitosans bear a positive charge on each glucosamine resi-
COS modulate OGA egg box bioactivity
Cell viability (% of control)
0
A
20
40
60
80
100
120
a
Control
a
COS DA~50
c
COS DA~0
d
COS DA~0 + MonoGalA
b
COS DA~0 + OGA Low DP
ab
COS DA~0 + OGA High DP
a
MonoGalA
a
a
OGA Low DP
OGA High DP
B
120
COS
COS + OGA ( 100 mg.L-1)
COS + OGA ( 200 mg.L-1)
Cell viability
(% of control)
100
80
60
40
20
0
0
100
200
300
COS (mg.
L-1)
400
500
Fig. 7. Cell viability of Arabidopsis cell suspensions measured 24 h after treatment with chitosan oligosaccharides, OGA and their combination. (A) Effect of
acetylated COS (DA ∼50), deacetylated COS (DA ∼0) (300 mg L−1) and combinations of deacetylated COS (300 mg L−1) with monogalacturonic acid and OGA of
low and high DPs (200 mg L−1). OGA rescued cells from cell death caused by deacetylated chitosan oligosaccharides. Different letters among treatments indicate
significant differences (P ≤ 0.05). (B) Cell viability after treatment with increasing concentrations of deacetylated chitooligomers (DA ∼0%) alone or in
combination with high DP OGA. Increasing OGA concentrations rescued more cells from death caused by deacetylated COS. Data reflect the means of at least three
independent experiments.
due and form single helicoidal stiff chains stretched by
electrostatic repulsions. In this zigzag structure, chitosan
chains take a 2-fold helical conformation (Okuyama et al.
1997) which means that chitosans have positive charges alternating on each side of the chain at 1.03 nm between
neighboring charges on the same side of the chain (Sikorski et al. 2005), exactly the same way homogalacturonans
have negative charges alternating at a similar distance on
each side of its 2-fold zigzag structure. Positive charges
of chitosans can then interact with identically spaced negative charges of the pectin chains to form OGA–COS
complexes (Figure 8A) and the longer the chains the higher
the binding affinity.
Totally deacetylated chitosans, like any other polycation, are
strongly attracted by polyanionic pectin and could completely
displace calcium ions from within pectin egg box dimers
(Figure 8A). The resulting pectin–COS complexes are not rec-
ognized by the 2F4 antibodies. Because of cooperativity of egg
box formation, longer pectic chains bind calcium ions more
strongly, and consequently higher COS concentrations are required to dismantle egg box dimers.
Partly acetylated oligochitosans bearing fewer positive
charges per unit length could not displace calcium ions so easily from within egg box dimers for steric and coulombic
reasons but they stabilized the association. A model for partly
acetylated COS– OGA interaction can b e proposed
(Figure 8B). Two oligopectate chains associate cooperatively
through calcium ions to form an egg box dimer, leaving external charges free to bind monovalent cations in solution. Due to
hydrogen bonding, the two chains are not coplanar but form an
angle with each other (Braccini and Perez 2001). This geometry could allow one partly acetylated COS to bind to the
external faces of the egg box without being able to displace
the calcium ions from within this egg box. The 2F4 antibody
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J-C Cabrera et al.
Fig. 8. Model for the interaction of chitosan oligosaccharides with OGA. (A) Schematic representation of OGA egg box disruption by fully deacetylated COS.
Polycationic COS interact with identically spaced negative charges of OGA, replacing calcium ions and dismantling pectic egg boxes. (B) Schematic representation
of OGA egg box stabilization by interaction with partly acetylated COS. Side view (left) represents pectin egg box dimer with at least five calcium ions that bind
both chains together. The angle between OGA chains was drawn after a computer simulation by Braccini and Perez (2001). A positively charged, partly acetylated
chitosan molecule binds external negative charges of calcium-bound pectin while higher COS concentrations could dislodge calcium from the complex. The
chitosan molecule being shorter than the OGA, the extremities of the egg box remain accessible. Top view (right) schematically represents the location of charges on
the oligomers. The 2F4 antibody could still bind the COS-stabilized egg box dimer on the calcium-binding side of the complex.
would still bind the complex on the calcium-binding side of
pectin chains.
The geometry of this ternary Ca2+–COS–OGA complex also involves kinetic considerations. The partly and irregularly
acetylated COS do not bind as strongly and stably with pectin
as completely deacetylated chitosans and could therefore have
more rapid kinetics of binding and dissociating. Partially
acetylated COS bind probably only transiently to the “outside” charged groups of pectic dimers, competing with
782
monovalent cations, but nevertheless strongly enough to increase the effective concentration of egg boxes for interaction
with antibody or cells.
Effect of COS–OGA egg boxes on Arabidopsis cells
Beside production of an oxidative burst, suspension-cultured
plant cells challenged by elicitors undergo perturbations of
their cellular ionic balance with K+ and Cl− efflux, Ca2+ influx,
COS modulate OGA egg box bioactivity
external alkalinization and cellular acidification. These early
events are associated with changes in plasma membrane properties that play the role of a switch between the elicitor
recognition and the pathogen defense-signaling pathways
(Hagendoorn et al. 1991; Amborabe et al. 2003). Partly acetylated COS combined with OGA egg boxes induced a
prolonged and sustained alkalinization of the culture medium
well above levels attained by either COS or OGA alone
(Figure 6A). This synergy was dependent on the COS/OGA
ratio (Figure 5A) in a similar way as partly acetylated COSstabilized OGA egg boxes in ELISA tests (Figure 2B). In absence of calcium, this synergistic effect was not observed
(Figure 6C). It is therefore very tempting to interpret this
stronger alkalinization as the result of partly acetylated COS
binding and stabilizing OGA egg boxes that in turn triggered
more intense signaling.
K+ efflux and H+ influx are not coupled
K+ efflux in plant cells induces water loss and cell shrinkage
commonly observed during cell death involved in the hypersensitive response (Garcia-Brugger et al. 2006). Completely
deacetylated chitosans that induced the strongest K+ efflux
(Figure 4B) also killed Arabidopsis cells while reacetylation
of COS suppressed both K+ efflux and cell death (Figure 7),
but not medium alkalinization (Figure 4). Adding OGA to
partially acetylated COS increased medium alkalinization
and decreased K+ efflux (Figure 6A and B) in a calcium-dependent manner. The COS/OGA ratio that induced maximal
alkalinization for minimal K + efflux was found to be
30 mg L−1 COS for 40 mg L−1 OGA (Figure 5). This ratio
is higher than the optimum detected by the ELISA test of
Figure 2B and could be explained by binding of a fraction
of COS to the cell wall pectin.
The toxicity of deacetylated COS can be suppressed by either
OGA addition or COS reacetylation (Figure 7). We explain this
cell rescue by a simple titration of COS positive charges by the
pectic anions or by chemical neutralization of amine groups by
acetyl.
Biological implications
Several fungi start producing chitin deacetylase when they establish intimate contact with the tissue of their host plants
(Hadwiger and Beckman 1980; Siegrist and Kauss 1990;
Deising and Siegrist 1995), and deacetylated chitosans but
not chitin are present on the surface of their infection structures
growing within the invaded plant tissues (El Gueddari et al.
2002). Depolymerization of deacetylated chitosan can be carried out, at least in vitro, by a plethora of enzymes including
lipases, glucanases, cellulases, hemicellulases and pectinases
(Cabrera and Van Cutsem 2005, Kim and Rajapakse 2005).
How are COS of different DAs differentially perceived by
plant cells? Considering that suspension cells respond to COS
within minutes, the perception mechanism must be located in
the plasma membrane. LysM RLK1 that binds completely acetylated COS (chitin oligomers) is essential for chitin signaling in
plants and is involved in chitin-mediated innate immunity in
plants (Wan et al. 2008). However, concerning COS of DA
<35, to our knowledge, no receptor has ever been described,
but it is often considered that the polycationic nature of chitosan
may lead to membrane disturbance through its interaction with
negatively charged phospholipids (Kauss et al. 1989; Shibuya
and Minami 2001; Zuppini et al. 2004).
Taking together the results reported here, a tentative picture
describing biological implications of COS–pectin egg box interaction can be drawn: strongly cationic COS target anionic
pectin of the plant cell wall. This is in agreement with an
earlier report in which chitosan is shown to displace calcium
ions from isolated cell walls of Glycine max suspension cultures (Young and Kauss 1983). The disruption of calciumbound pectin of the plant cell wall by deacetylated chitosan
could then be perceived and interpreted by plant cells as a
distress signal commanding the strongest possible response
including cell death.
On the other hand, the presence of free OGA in the egg
box conformation is sensed by plant cells (Van Cutsem and
Messiaen 1994). This constitutes a warning signal indicating a
depolymerization of the cell wall that could be due to the presence of a pathogen but also to a normal turnover of the cell
wall such as during xylem differentiation or organ abscission.
The simultaneous presence of partly acetylated COS that
strongly stabilize OGA egg boxes would dramatically reinforce this warning signal, informing the cell upon active
invasion by a fungal pathogen. However, increasing concentrations of even partly acetylated COS would finally displace
calcium from any pectin and profoundly affect cell wall integrity: COS would titrate all egg boxes out and even single chain
pectin of the walls would start binding excess COS. This could
be detected by cell wall integrity sensors (Hematy, Cherck and
Somerville 2009), triggering a strong concentration-dependent
increase of K+ efflux. High concentrations of COS, whether
partly acetylated or not, alone or in presence of OGA finally
kill plant cells.
In other words, COS-stabilized OGA egg boxes could represent an emergency signal for plant cells while excess COS
could be a hypersensitivity signal in the vicinity of extensive
fungal penetration.
Materials and methods
Chemicals
Chitin from lobster was supplied by Mario Muñoz Pharmaceutical Laboratories (Hacendado 1, Ciudad Habana, Cuba) and
used to prepare chitosan under heterogeneous conditions following the methodology described by Cabrera et al. (2000).
PGA (sodium salt, Sigma, p-3850), histone from calf thymus
(H9250) and poly-L-lysine hydrobromide (P-1399 mol wt
150,000–300,000) were from Sigma-Aldrich (St Louis, MO).
Preparation and characterization of oligosaccharides
Chitooligosaccharides (COS) were obtained and characterized
as previously described (Cabrera et al. 2006). High DP COS
were reacetylated as described by Hirano and Yamaguchi
(1976). All COS samples were analyzed by MALDI-TOF mass
spectrometry (Cabrera and Van Cutsem 2005).
OGA were obtained by enzymatic hydrolysis of PGA, and
the pectic oligosaccharides were selectively fractionated in
low DP and high DP OGA and characterized by high pH anion-exchange chromatography with pulsed amperometric
detection analysis as described by Cabrera (2000).
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J-C Cabrera et al.
Preparation of the mixtures of pectic molecules and
chitooligosaccharides
PGA, OGA and COS stock solutions (1–10 mg/mL) were prepared in Milli-Q grade water. PGA and OGA stock solutions
were diluted to the working concentration in 50 mM acetate
buffer pH 5.7 containing 0.5 mM Ca2+ and 150 mM final
Na+ concentration. The PGA or OGA–COS mixtures were
prepared by adding COS to the pectic solutions followed
by vortexing. The ratio between COS and pectic molecules
was expressed as molar monomer unit ratio (COS/OGA or
COS/PGA).
Egg box detection by ELISA
PGA Coating of Microwells. Fifty microliters of poly-L-lysine
hydrobromide (0.05 mg mL−1 in deionized water) was dispensed into wells of NUNC High Binding Capacity
microplates (MAXISORP) and incubated for 1 h at room temperature. Identical volumes of a 200 mg L−1 PGA sample in
50 mM acetate buffer pH 5.7 containing 0.5 mM Ca2+ and
150 mM Na+ (Ca/Na ELISA buffer) were dispensed and left
overnight at 4°C. Nonspecific binding was blocked by incubating the wells for 2 h at 37°C with 250 μL of 30 mg mL−1
ELISA-grade bovine serum albumin (3% BSA) prepared in
the same buffer.
Incubation with COS. After removal of the excess albumin,
50 μL of high DP fully deacetylated COS solutions was added
to the wells and incubated for 30 min. The microplates were
washed with the Ca/Na buffer before incubation with 50 μL
of 2F4 MoAbs diluted 354 times in the Ca/Na buffer for
60 min at 37°C.
Washing and Detection. The microplates were washed eight
times with the Ca/Na buffer containing 0.1% Tween 20 before
addition of 50 μL of HRP-labeled sheep antimouse (SAM) immunoglobulin (1:5000 in 50 mM acetate buffer containing Ca/
Na solution) for 60 min at 37°C. After a second washing cycle,
the binding of the antibodies was revealed by incubation for
20 min in dark at room temperature with 100 μL of enhanced
K-blue TMB substrate (Neogen Corporation, Lexington, KY).
The process was stopped by addition of 50 μL of 1 N HCl. The
absorbance of the solution was measured after 15 min with a
Titertek multiscan microplate reader at 405 nm.
Immunoprecipitation — GaMIg ELISA procedure
Microwells Coating with Antimouse Immunoglobulin. Fifty
microliters of goat antimouse immunoglobulin (GaMIg whole
molecule, Sigma Chemicals M 5899 (Sigma-Aldrich, St Louis,
MO), 0.05 mg mL−1 in 50 mM carbonate buffer pH 9.5) was
dispensed into NUNC High Binding Capacity microplates
(MAXISORP) and left overnight at 4°C. Nonspecific binding
was blocked by incubating the wells for 2 h at 37°C with
250 μL BSA (30 mg mL-1) prepared in Ca/Na buffer. After removal of the BSA buffer, supernatants of PGA/OGA–COS–
antibody mixtures (see next paragraph) were added to the wells.
Incubation with antibodies, washing and detection were performed as described in the section Egg box detection by ELISA.
Incubation of the Mixtures of Pectic Molecules and COS
with the 2F4 Antibodies. A hundred microliters of the mixture of PGA or OGA and COS was incubated with 100 μL
2F4 MoAbs diluted 177 times in Ca/Na buffer for 30 min at
784
25°C. Controls consisted in PGA or OGA without COS and
in buffer. These PGA/OGA–COS–antibody mixtures were
then centrifuged for 10 min at 7500 × g before dispensing
the supernatants in GaMIg-coated microwells and incubating
for 60 min at 37°C. The washing and detection steps were
carried out as described in the section Egg box detection by
ELISA. The concentrations of pectic dimers detected were calculated as percentages of the concentrations detected in control
solutions without added chitosan oligosaccharides.
Bioassay
Suspension-cultured cells derived from leaves of A. thaliana
strain L-MM1 ecotype Landsberg erecta were grown in Murashige and Skoog medium (4.43 g L−1) with sucrose (30 g L−1),
0.5 mg L−1 naphthalene acetic acid, 0.05 mg L−1 kinetin,
pH 5.7. Cultures were maintained under a 16/8 h light/dark
photoperiod, at 25°C, on a rotary shaker at 100 rpm. Cells were
diluted 10-fold in fresh medium every 7 days.
Alkalinization and K+ efflux assay
Seven-day-old cells were filtrated on Miracloth (Calbiochem,
Merck, Darmstadt, Germany), washed and equilibrated in a
K+-free medium containing 10 mM sucrose, 0.5 mM Ca2+,
50 mM Na+ and 0.5 mM 2-(N-morpholino)-ethanesulfonic acid
adjusted to pH 5.7 with Tris-(hydroxymethyl)-aminomethane
(Ca/Na bioassay solution) for 30 min at room temperature with
gentle shaking. This washing was repeated four times at 30min intervals. After the last washing, the cells were incubated
in the same Ca/Na bioassay solution for an additional 120 min
before use.
OGA, COS and their combination were dissolved and preequilibrated for 1 h in the Ca/Na bioassay solution. Fivemilliliter aliquots of washed cell (100 mg fresh weight mL−1)
were placed in glass vials, and the incubation medium was
changed for equal volumes of assayed solutions using a Pasteur
pipette and agitated on a rotatory shaker at 150 rpm. The extracellular pH and K+ concentrations were determined in aliquots
of the incubation medium obtained by rapid filtration of the
cells through Miracloth. pH was monitored with a Hamilton
Biotrode pH electrode, and K+ concentrations were determined
in 1 N HCl using an atomic absorption spectrophotometer
(PU 9200X, Pye-Unicam, Cambridge, UK).
Cell viability assay
Seven-day-old cells were treated with OGA, COS and their
combination for 24 h. Cell viability was determined using
2,3,5-triphenyltetrazolium chloride (TTC) viability assay (Dixon 1985). Five milliliters of the treated cell suspension was
centrifuged for 5 min at 100 × g and 4°C to collect the cells. Cells
were incubated with 2 mL of 0.05 M KH2PO4 at pH 8.0 containing 0.6% TTC for 15 min in the dark at 20°C. This mixture
was centrifuged at 4000 rpm for 1 min, and the water-insoluble
red formazan accumulated by living cells was extracted by
heating at 60°C for 15 min with 3 mL 95% (v/v) ethanol.
The absorbance of ethanol solution was measured at 485 nm.
Statistical analysis
Statistical analysis of data was performed using STATISTICA 7
software. Data were subjected to one-way analysis of variance
COS modulate OGA egg box bioactivity
and Duncan honestly significant difference for comparison of
means. Unless stated otherwise, means ± standard error
are reported.
Funding
This work was supported by Région Wallonne, Belgium (Elite
Internationale fellowship to J.-C.C.).
Conflict of interest statement
None declared.
Abbreviations
BSA, bovine serum albumin; CD, circular dichroism; COS,
chitosan oligomers; DA, degree of acetylation; DP, degree of polymerization; ELISA, enzyme-linked immunosorbent assay;
GaMIg, goat antimouse immunoglobulin; HRP-SAM, horseradish peroxidase-labeled sheep antimouse; MALDI-TOF, matrixassisted laser desorption ionization time-of-flight; MoAbs,
monoclonal antibodies; OGA, oligogalacturonides; PEI, polyethyleneimine; PGA, polygalacturonic acid; TTC, 2,3,5triphenyltetrazolium chloride; WAK, wall-associated kinases.
Supplementary data
Supplementary data for this article is available online at
http://glycob.oxfordjournals.org/.
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