Plant Physiol. (1998) 116: 1289-1298
Biological Activity of Reducing-End-Derivatized
Oligogalacturonides in Tobacco Tissue Cultures1
Mark D. Spiro2, 3,
Brent L. Ridley3,
Stefan Eberhard,
Keith A. Kates,
Yves Mathieu,
Malcolm A. O'Neill,
Debra Mohnen,
Jean Guern,
Alan Darvill, and
Peter Albersheim*
Complex Carbohydrate Research Center and Department of Biochemistry
and Molecular Biology, The University of Georgia, 220 Riverbend Road,
Athens, Georgia 30602-4712 (B.L.R., S.E., K.A.K., M.A.O., D.M., A.D.,
P.A., M.D.S.); and Institut des Sciences Végétales, Centre
National de la Recherche Scientifique, Bâtiment 22-23,
Avenue de la Terrasse, F-91198 Gif-sur-Yvette cedex, France
(Y.M., J.G.)
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ABSTRACT |
The
biological activity of reducing-end-modified oligogalacturonides was
quantified in four tobacco (Nicotiana tabacum) tissue culture bioassays. The derivatives used were oligogalacturonides with
the C-1 of their reducing end (a) covalently linked to a biotin
hydrazide, (b) covalently linked to tyramine, (c) chemically reduced to
a primary alcohol, or (d) enzymatically oxidized to a carboxylic acid.
These derivatives were tested for their ability to (a) alter
morphogenesis of N. tabacum cv Samsun thin cell-layer explants, (b) elicit extracellular alkalinization by
suspension-cultured cv Samsun cells, (c) elicit extracellular
alkalinization by suspension-cultured N. tabacum cv
Xanthi cells, and (d) elicit H2O2 accumulation
in the cv Xanthi cells. In all four bioassays, each of the derivatives had reduced biological activity compared with the corresponding underivatized oligogalacturonides, demonstrating that the reducing end
is a key element for the recognition of oligogalacturonides in these
systems. However, the degree of reduction in biological activity
depends on the tissue culture system used and on the nature of the
specific reducing-end modification. These results suggest that
oligogalacturonides are perceived differently in each tissue culture
system.
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INTRODUCTION |
Carbohydrates that act as signal molecules in plants
(oligosaccharins) have been isolated from plant and fungal cell wall polysaccharides, from the cell walls of bacterial symbionts of plants,
and from fungal glycopeptides (for review, see Ryan and Farmer, 1991
;
Darvill et al., 1992; Côté and Hahn, 1994). We are
interested in determining the mechanism by which one type of the plant
cell wall-derived oligosaccharins, the oligogalacturonides, elicit
biological responses in plants.
The biological effects elicited in plants by oligosaccharins are
diverse (for review, see Ryan and Farmer, 1991; Darvill et al., 1992;
Côté and Hahn, 1994) but can generally be placed in two
groups: delayed responses and rapid responses. Delayed responses are
usually observed hours or days after oligosaccharin treatment and are
often directly involved in adaptation to environmental conditions,
whereas rapid responses generally occur at the plant cell surface and
are observed within minutes after addition of oligosaccharins. The
delayed responses elicited by oligogalacturonides can be broadly
divided into those in which defense responses are induced and those in
which growth and development are modified. The defense-related
responses, depending on the plant species, include phytoalexin
accumulation (Hahn et al., 1981), lignification of cell walls
(Robertsen, 1986), and proteinase inhibitor accumulation (Bishop et
al., 1984). The responses involving growth and development include
induction of ethylene in tomato fruit (Brecht and Huber, 1988),
inhibition of auxin-induced pea stem elongation (Branca et al., 1988),
and regulation of tobacco (Nicotiana tabacum) TCL explant
morphogenesis (Eberhard et al., 1989). The rapid responses induced by
oligogalacturonides include enhanced protein phosphorylation in tomato
and potato membranes (Farmer et al., 1989), transmembrane ion flux
accompanied by membrane depolarization in tobacco suspension cells
(Mathieu et al., 1991) and carrot protoplasts (Messiaen et al., 1993),
and H2O2 accumulation in
suspension-cultured soybean cells (Apostol et al.,
1989).
Rapid responses elicited by oligosaccharins may act as signal
transduction events in the initiation of the delayed responses; however, this has not been directly demonstrated. Isolation and characterization of oligosaccharin receptors would facilitate the
identification of the oligosaccharin-induced signal-transduction events. High-affinity binding sites for three oligosaccharins, the
oligo-
-glucosides, the oligochitins, and a yeast
N-glycan, have been identified using biologically active,
radiolabeled derivatives (Cosio et al., 1990; Cheong and Hahn, 1991;
Basse et al., 1993; Shibuya et al., 1993; Baureithel et al., 1994). The
specificity of these binding sites has been characterized by measuring
the ability of structural analogs having a range of biological
activities to displace labeled derivatives in competitive binding
studies. A direct correlation between the competitive binding ability
of the analogs and their biological activity has been observed for each
binding site, providing evidence that the sites identified are
physiological receptors of the oligosaccharins. Characterizing the
specificity of binding proteins in this manner requires homogeneous oligosaccharins, labeled oligosaccharin derivatives, and oligosaccharin analogs.
With the ultimate goal of identifying and characterizing
oligogalacturonide receptors, we have quantified the biological
activities of discernibly homogeneous reducing- end-derivatized
oligogalacturonides in four tobacco tissue- culture bioassays. The
oligogalacturonide derivatives assayed include
G13-T, G13-B,
G13-O, and G13-R (Fig. 1). Biotinylated and
125I-labeled tyraminated oligogalacturonides
could be used to detect low-abundance oligogalacturonide-binding
proteins. Unlabeled oligogalacturonides, including reduced and oxidized
oligogalacturonides, could be used to characterize the specificity
of oligogalacturonide-binding proteins.
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MATERIALS AND METHODS |
Purified orange fruit uronic acid oxidase was a gift from Russell
Pressey (U.S. Department of Agriculture, Athens, GA). All reagents were
obtained from Sigma.
Preparation of Homogeneous Oligogalacturonides and Their
Reducing-End Derivatives
In this paper the term oligogalacturonide refers to native or
unmodified, as well as reducing-end-derivatized oligogalacturonides, such as G13-T, G13-B,
G13-R, and G13-O.
Oligogalacturonides, generated by partial
endopolygalacturonase digestion of
polygalacturonic acid, were purified to homogeneity by a three-step
protocol (Spiro et al., 1993) consisting of size-selective
precipitation with ethanol, followed by Q-Sepharose anion-exchange
chromatography, and then semipreparative HPAEC-PAD. All
oligogalacturonides used in this study were homogeneous and
chemically stable at 
20°C, as determined by analytical HPAEC-PAD
and by determination of their Mr by electrospray
MS.
Oligogalacturonides derivatized with tyramine or biotin-x-hydrazide
(6-[biotinoylamino] caproic acid hydrazide) at C-1 of the
reducing-end galacturonic acid were synthesized and purified to
homogeneity as described previously (Spiro et al., 1996; Ridley et al.,
1997).
G13-R was generated by treating the
G13 (5 mg in 1 mL of 1 m
NH4OH) for 16 h at 4°C with
NaBH4 (5 mg). The reaction was terminated by the addition
of 5 volumes of 10% acetic acid in methanol, which destroyed any
remaining NaBH4. This solution was kept for 2 h at
20°C and the resulting precipitate was collected by centrifugation. The supernatant was discarded. The pellet was washed twice with methanol (5 mL) to remove residual salts. The final pellet was dried
under a stream of air at 25°C. The residue was dissolved in water
(0.5 mL) and stored at 20°C.
G13-O was generated by treating
G13 (5 mg) in 10 mm Tris-HCl, pH 8.5 (1 mL), with 3 units of uronic acid oxidase (Pressey, 1993) for 23 h at 37°C. This solution was sterilized by centrifugation through a
0.2-µm nylon filter prior to incubation.
Neither the chemical reduction nor the enzymatic oxidation of
G13 proceeded to completion; therefore,
G13-R and G13-O were individually purified to homogeneity by semipreparative HPAEC-PAD (Spiro et al., 1993). Electrospray MS analysis confirmed that the
expected product had been synthesized.
TCL Bioassay
Tobacco (Nicotiana tabacum cv Samsun) plants were grown
as described by Mohnen et al. (1990). The TCL explant bioassay was performed as described by Eberhard et al. (1989). Briefly, thin strips
of tissue, approximately 1 mm wide and composed of 6 to 10 cell layers,
were cut from surface-sterilized tobacco floral branches. TCL explants,
approximately 10 mm long, were cut from the tissue strips. The explants
were placed in individual wells of 12-well culture dishes (ICN)
containing 2 mL of Linsmaier and Skoog (1965) medium, pH 5.8, supplemented with 167 mm Glc, 3 mm IAA, and 0.3 mm kinetin. The oligogalacturonides to be assayed were
sterilized by filtration through 0.2-µm nylon membrane syringe filters (Nalgene, Rochester, NY). The TCL explants were incubated at
24°C under continuous, cool-white fluorescent light. The overall TCL
explant morphology and the number of flowers formed on each TCL explant
after 23 or 24 d of culture were determined by examination with a
dissecting microscope.
Maintenance of Suspension-Cultured Tobacco Cells
Suspension-cultured N. tabacum cv Samsun cells,
initiated from pith parenchyma callus, were grown in Linsmaier and
Skoog (1965) medium, pH 6.0, supplemented with 4.5 µm
2,4-D and 3% Suc. The cells were cultured under continuous light at
26°C on a rotary shaker at 110 rpm. The PCV obtained after
centrifugation (1000g for 5 min) was used as a measure of
growth. An aliquot of the cell suspension giving an initial PCV of
2.7% was transferred to 100 mL of fresh medium every 7 d. The PCV
after 7 d of culture was between 26 and 33%. The culture of the
N. tabacum cv Samsun cells and the alkalinization bioassays
using these cells were carried out at the Complex Carbohydrate Research
Center (Athens, GA).
Suspension-cultured N. tabacum cv Xanthi cells were grown in
Gamborg B-5 medium, pH 5.7, supplemented with 1 µm 2,4-D,
40 nm kinetin, and 2% Suc. The cells were grown under
continuous light on a rotary shaker at 160 rpm and were subcultured
every 7 d by transferring 10 mL of the culture into 100 mL of
fresh medium. The PCV after 7 d of culture was between 18 and
22%. The culture of the N. tabacum cv Xanthi cells and the
alkalinization and H2O2
accumulation bioassays using these cells were carried out at the Centre
National de la Recherche Scientifique laboratories (Gif-sur-Yvette,
France).
Analysis of Oligogalacturonide-Induced Extracellular Alkalinization
by Suspension-Cultured Tobacco Cells
Aliquots of 7-d-old exponentially growing N. tabacum cv
Samsun suspension-cultured cells (4 mL) in 25-mL Erlenmeyer flasks were
equilibrated for 2 h on a rotary shaker (110 rpm). The pH after
this period was between 4.4 and 4.6. The pH of the oligogalacturonide solutions (50 µL in water) was adjusted to that of the equilibrated cells. The addition of oligogalacturonides to each successive flask was
staggered by 60 s so that pH measurements could be made at fixed
times after sample addition. The extracellular pH was measured
immediately upon addition of the oligogalacturonides and subsequently
at 14-min intervals through at least 56 min using a Micro-Combination
pH electrode (Microelectrodes, Inc., Londonderry, NH) connected to an
Accumet 950 pH meter (Fisher Scientific).
The analysis of oligogalacturonide-induced extracellular alkalinization
by cv Xanthi cells was carried out in the same manner as for the cv
Samsun cells with the following exceptions. Aliquots (3 mL) of the cell
suspensions were placed in 12-mL glass vials, adjusted to 1 mm Mes-Tris (pH 5.2), and equilibrated for 2 h, at
which time the pH was between 5.1 and 5.5. The pH was measured using a
BRV4H glass electrode (Heito, Paris, France) connected to a PSD 11 pH
meter (Heito).
Analysis of Oligogalacturonide-Induced Oxidative Burst
The accumulation of
H2O2 in the growth medium
of suspension-cultured cv Xanthi cells was determined in the same
experiments as the measurement of extracellular alkalinization.
Aliquots (10-40 µL) of the growth medium were removed at intervals
and added to a series of cuvettes containing 8 µm
scopoletin (7-OH-6-methoxycoumarin) and 5 µg of horseradish
peroxidase (P 6782, Sigma) in 2 mL of 50 mm Mes-Tris, pH
5.2. The amount of H2O2
present in the culture medium was determined by the decrease in
fluorescence of scopoletin and compared with the effect of a standard
concentration series of
H2O2 on scopoletin
fluorescence (Root et al., 1975). Fluorescence measurements were made
with a spectrofluorimeter (Photon Technology International, South
Brunswick, NJ) using an excitation wavelength of 350 nm and an emission
wavelength of 460 nm.
| |
RESULTS |
Reducing-End-Derivatized G13 at Submicromolar
Concentrations Alter the Morphogenesis of N. tabacum
cv Samsun TCL Explants
Oligogalacturonides induce flower formation, inhibit root
formation, and reproducibly alter the overall morphology of tobacco TCL
explants cultured on indolebutyric acid and kinetin-containing medium
(Eberhard et al., 1989; Marfà et al., 1991; Bellincampi et al.,
1993). Oligogalacturonides of DP 12 to 14, which have approximately
equal activity, induce the alteration of TCL explant morphogenesis at
submicromolar concentrations, whereas higher concentrations of shorter
and longer oligomers are required to induce an equivalent response. The
mechanism by which oligogalacturonides initiate these responses is
unknown.
We determined the effect of derivatized and underivatized
oligogalacturonides on TCL explants cultured in transition medium containing 3 µm IAA and 0.3 µm kinetin.
A transition medium containing IAA rather than indolebutyric acid
(Mohnen et al., 1990) was used, since the addition of
oligogalacturonides to TCL explants cultured on IAA-containing
transition medium causes a distinct concentration-dependent change in
the overall morphology of the TCL explants. TCL explants, in the
absence of added oligogalacturonides or at low concentrations of
bioactive oligogalacturonides, grow more or less uniformly over their
entire length, resulting in rectangular explants. In the presence of
higher concentrations of bioactive oligogalacturonides, the TCL
explants were significantly inhibited in their growth and had limited
tissue enlargement only at one end, yielding mushroom-shaped explants (Fig. 2). In the absence of
oligogalacturonides, the rectangular explants produced only roots,
whereas they produced roots and occasionally flowers when exposed to
low concentrations of bioactive oligogalacturonides. The
mushroom-shaped explants that resulted from exposure to higher
concentrations of bioactive oligogalacturonides frequently formed
flowers but never roots.
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| Figure 2.
The TCL explants from a representative tobacco
morphogenesis bioassay comparing the activities of G12
(top), G13-B (middle), and G13-T (bottom). The
explants were incubated in growth medium containing the indicated
oligogalacturonide concentrations, with six replicate TCL explants per
concentration. The arrows indicate the concentrations at which
oligogalacturonides induce a distinct change in the overall morphology
of the TCL explants (the morphogenetic switch concentrations).
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The ability of each oligogalacturonide to alter TCL-explant
morphogenesis was tested at nine concentrations and compared with a
similar concentration series of G12. Each
oligogalacturonide was assayed in two independent experiments using six
replicate TCL explants at each concentration.
G12, G13-T,
G13-B, and G13-R each
induced a concentration-dependent increase in the number of flowers
formed on TCL explants. The maximum of the average number of flowers
induced by G12 (1.5 µm, 2.8 flowers/explant) was nearly identical to the maximum of the average
number of flowers induced by the three reducing-end derivatives (2.6 µm, 3.1 flowers/explant). However, as shown in Figure
3 for G12, the
number of flowers induced on TCL explants by a given oligogalacturonide
concentration varied considerably, and thus the flowering data were not
useful for a quantitative comparison of the biological activities of
the various oligogalacturonides.
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| Figure 3.
Plot of the log of G12 concentration
versus the average number of flowers induced per TCL explant. The data
from 12 experiments are shown, and each data point represents the
average number of flowers produced by the six replicate TCL explants
used for each treatment in a given experiment. In the absence of
G12, no flowers were formed. The line represents the
dose-response curve for G12 as determined by least-squares
analysis using log concentration.
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However, the oligogalacturonide concentration at which the overall
morphology of the TCL explants changes from rectangular to
mushroom-shaped (the morphogenetic switch concentration) could be used
to quantify the activities of the oligogalacturonides since it
reproducibly fell within a less-than 4-fold concentration range (Fig.
2). The average morphogenetic switch concentration of
G12 in 12 experiments was 0.058 ± 0.033 µm (Table I). Compared with
G12, the morphogenesis switch concentration for
G13-T was 2.5-fold higher;
G13-B was 3.4-fold higher, and
G13-R was 6.3-fold higher. The difference between
the activity of G12 and each
G13 derivative was statistically significant
(Wilcoxon's Rank-Sum test, confidence level = 98.5%). There was
no discernible difference in the activity of G12
when assayed in the presence of excess free biotin or free tyramine.
These results indicate that derivatization of the reducing end of
G13 results in a reduction in its ability to
alter TCL explant morphogenesis.
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Table I.
Comparison of the ability of
reducing-end-derivatized and underivatized oligogalacturonides to alter
TCL explant morphogenesis
The concentration of each oligogalacturonide that induces a distinct
change in the overall TCL explant morphology is reported as the
morphogenetic switch concentration (as defined in the text). The values
given for each oligogalacturonide are the averages of two independent
bioassays, with the exception of the value for G12, which
is the average of 12 independent experiments (sd = 0.033 µm).
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We compared the morphogenetic switch concentration of various
homogeneously sized biotinylated and underivatized oligogalacturonides to determine whether reducing-end derivatization alters the size requirement for activity. G16-B and
G10-B were both reduced in activity compared with
their corresponding underivatized oligogalacturonides, and the highest
concentration of G8-B tested (2.6 µm) did not induce an alteration in the overall explant
morphology (Table I). G13-B was significantly
more active than longer or shorter biotinylated oligogalacturonides,
which is consistent with the size requirements found using
underivatized oligogalacturonides in this bioassay (Marfà et al.,
1991; Bellincampi et al., 1993).
All Four Derivatives of G13 Were More Than 30-Fold
Reduced in Their Ability to Induce Extracellular Alkalinization by
Suspension-Cultured cv Samsun Cells
Transient membrane depolarization accompanied by rapid
K+ ion efflux and extracellular alkalinization
was induced when suspension-cultured tobacco or carrot cells were
exposed to micromolar concentrations of oligogalacturonides having DPs
of 10 to 15 (Mathieu et al., 1991; Messiaen and Van Cutsem, 1994).
These rapid responses have been hypothesized to act as signaling events
leading to delayed responses, such as the alteration of TCL-explant
morphogenesis (Mathieu et al., 1991; Messiaen and Van Cutsem, 1994). We
have characterized the ability of various sizes of
reducing-end-derivatized and underivatized oligogalacturonides to
induce extracellular alkalinization in cv Samsun suspension
cultures, the same cultivar used for the tobacco morphogenesis
bioassay.
The ability of each oligogalacturonide to induce extracellular
alkalinization by cv Samsun cells was assayed using seven
concentrations of oligogalacturonides in at least two independent
experiments. A standard concentration series of
G12 (Fig. 4) was
assayed in each experiment. Each bioactive oligogalacturonide elicited
a rapid, concentration-dependent, and saturable extracellular
alkalinization response that exhibited three characteristic phases: a
period of rapid alkalinization beginning 30 s after
oligogalacturonide addition, a plateau period during which the pH
remained near its maximum level, and a period of gradual
reacidification during which the pH of the medium returned to
approximately its original value.
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| Figure 4.
Representative time courses of the extracellular
alkalinization by suspension-cultured cv Samsun cells
treated with the indicated concentrations of G12 at time
0.
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The alkalinization response measured 14 min after oligogalacturonide
addition was used to compare the activities of the derivatized and
underivatized oligogalacturonides, since this time consistently fell
within the period of maximum alkalinization. The change in pH was
converted to the R to obtain a linear measure of the
response. Rmax was always found to be
essentially the same between replicates within the same experiment;
however, Rmax varied significantly between
experiments. The alkalinization response elicited by saturating concentrations of each bioactive oligogalacturonide within an experiment was essentially the same between replicates within the same
experiment; however, Rmax varied
significantly between experiments. To correct for variability of
the responses between experiments, the response elicited by each sample
was expressed as the fraction of the Rmax
for that experiment (R/Rmax).
The R/Rmax values were used to
generate dose-response curves (Fig. 5)
and the EC50 were determined from these curves.
G12 and G13 were the most
active of the compounds tested (Fig. 5a) and the difference between
their activities was not statistically significant (paired t
test, level of confidence = 95%); therefore, the data from
G12 and G13 were pooled to
generate a single dose-response curve (EC50 = 0.068 µm). G15 was approximately
5-fold less active (EC50 = 0.36 µm) and G9 was 50-fold less active
(EC50 = 3.4 µm). G3 was inactive over the entire range of
concentrations tested (2-45 µm). These results establish
that the size requirements for oligogalacturonides to elicit
extracellular alkalinization by cv Samsun cells are similar to that
required for alteration of TCL-explant morphogenesis (Marfà et
al., 1991; Bellincampi et al., 1993).
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| Figure 5.
Effect of the DP (a) and reducing-end
derivatization (b) of oligogalacturonides on their ability to induce
extracellular alkalinization by suspension-cultured cv Samsun cells.
The data were plotted as the alkalinization response of each sample
divided by the alkalinization response induced by a saturating
concentration of G12 in that experiment
(R/Rmax). The
R/Rmax values for the various
oligogalacturonides are indicated as follows: G12,  ;
G13,  ; G15,  ; G9,  ;
G13-T,  ; G13-B,  ; G13-O,  ;
and G13-R,  . The data for G12 and
G13 are shown on both plots to aid in comparison. The lines
represent the linear portion of the dose-response curves as determined
by least-squares analysis using log concentration. Separate
dose-response curves were generated using combined data from
G12 and G13 and combined data from the four
reducing-end derivatives of G13 (see text).
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Each of the reducing-end derivatives of G13
elicited a concentration-dependent and saturable extracellular
alkalinization by cv Samsun cells with the same kinetics as
G12 and G13 (Fig. 6, e.g. G13-T),
although they were all approximately 30-fold less active (Fig. 5b).
There was no statistical difference (paired t test, level of
confidence = 95%) between the activities of the four
G13 derivatives in this bioassay. When the data
for all four derivatives were used to generate a single dose-response
curve, an EC50 of 2.2 µm was
obtained, which was approximately 32-fold higher than that of
G12 and G13 (Fig. 5b). The
reduction in the bioactivity of the reducing-end-derivatized
oligogalacturonides was at least 6 times greater in this bioassay
than for any of the G13 derivatives in the
tobacco morphogenesis bioassay. G10-B has an
EC50 of 5 µm, and
G16-B did not induce a measurable response at 2.4 µm, the highest concentration tested. Thus,
G13-B is more active than the shorter or longer
biotinylated oligogalacturonides, which is in accord with the size
requirement for the underivatized oligogalacturonides in this bioassay.
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| Figure 6.
Representative time courses for extracellular
alkalinization by cv Samsun cells induced by the indicated
concentrations of G12 (closed symbols) or G13-T
(open symbols). More than a 30-fold higher concentration of
G13-T, compared with G12, is required to induce
an equivalent alkalinization response.
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The Ability of the Reducing-End-Derivatized Oligogalacturonides to
Induce Extracellular Alkalinization by cv Xanthi Cells
Differs Depending on the Particular Reducing-End Modification
The size requirement for oligogalacturonides to elicit
extracellular alkalinization by cv Samsun cells is similar to that described for cv Xanthi cells (Mathieu et al., 1991). However, the
EC50 for G12 was
approximately 15 times higher for the cv Xanthi cells than for
the cv Samsun cells. Furthermore, with the cv Xanthi cells,
the oligogalacturonide-induced alkalinization and subsequent
reacidification occurred at about half the rate attained with
cv Samsun cells. These differences in the
alkalinization response between the two bioassays may have been due to
differences in the cell culture conditions but were not attributable to
the bioassay conditions, since the differences in sensitivity and kinetics remained when the two cell lines were assayed using the same
conditions (data not shown).
We measured the ability of G13-R and
G13-T to elicit extracellular alkalinization by
suspension-cultured cv Xanthi cells to compare the structural
requirements for activity between the two alkalinization bioassays.
Both G13 derivatives elicited a
concentration-dependent alkalinization response, with kinetics similar
to those elicited by underivatized G13 (Fig.
7, e.g. G13-T).
G13-R induced a saturating response, whereas
G13-T did not, even at the highest concentration tested (8.7 µm). The same method used to determine
EC50s in the cv Samsun alkalinization bioassay
was used for this bioassay. G13 was 1.6-fold more
active than G13-R and 8.7-fold more active than
G13-T, as determined by comparison of their
EC50 values (Table II). Both of these differences are
statistically significant (paired t test, level of
confidence = 99.5%). Thus, the bioactivities of the two
G13 derivatives tested in the cv Xanthi
alkalinization bioassay depend on the specific reducing-end
modification, whereas each of the G13 derivatives
have approximately equal activity in the cv Samsun alkalinization
bioassay.
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| Figure 7.
Representative time courses of extracellular
alkalinization by cv Xanthi cells elicited by G13 and of
G13-T. All treatments were at 8.7 µm.
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Table II.
Comparison of the abilities of
G13, G13-R, and G13-T to elicit
extracellular alkalinization and H2O2
accumulation by suspension-cultured cv Xanthi cells
Each of the oligogalacturonides was tested at five concentrations and
the data were used to calculate the EC50 values.
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The Structural Requirements of the Reducing-End-Derivatized
Oligogalacturonides for Induction of H2O2
Accumulation and for Induction of Extracellular Alkalinization using cv
Xanthi Cells Are Approximately the Same
In the experiments with cv Xanthi described above, in which we
measured extracellular alkalinization, we also measured the ability of
G13, G13-R, and
G13-T to induce the accumulation of extracellular
H2O2.
H2O2 accumulation is of
interest since it is a rapid signaling event involved in the initiation
of defense responses (Tenhaken et al., 1995). G13
and G13-R induced a rapid, concentration-dependent, and saturable accumulation of extracellular H2O2 by cv Xanthi cells. In
contrast, G13-T induced a weak response (Fig.
8); only 20% of the maximum response
induced by G13 was induced at the highest
concentration of G13-T tested (8.7 µm). The kinetics of
H2O2 accumulation and
extracellular alkalinization were at first very similar. However, the
H2O2 concentration returned to its original level in about half the time required for the alkalinization response to return to normal (compare Figs. 7 and 8).
Approximately 1.5-fold higher concentrations were required for
half-maximum induction of
H2O2 accumulation than for
half-maximum induction of extracellular alkalinization in the cv Xanthi
suspension-cultured system. The
H2O2 induction activity of
both derivatives, relative to G13, was similar to
their activity in the extracellular alkalinization bioassays (Table
II). G13 was 1.3-fold more active than
G13-R and in excess of 6-fold more active than
G13-T in the
H2O2 induction assay. These
results indicate that, in the cv Xanthi tissue culture system, the
structural requirements for activity of the reducing-end-derivatized oligogalacturonides are essentially the same for induction of H2O2 accumulation and
extracellular alkalinization.
View larger version (20K):
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| Figure 8.
Representative time courses of
H2O2 accumulation in the growth medium of cv
Xanthi cells elicited by the addition of G13 and G13-T. These measurements were carried out simultaneously
using the same suspension cells as used for the measurement of
extracellular alkalinization (Fig. 7). All treatments were at 8.7 µm.
|
|
| |
DISCUSSION |
Derivatization or modification of the reducing end of
oligogalacturonides significantly reduced their biological activity in
all four tobacco tissue culture bioassays studied. However, the degree
of reduction in activity depends on the specific modification as well
as the tissue-culture system used. Each of the three tissue-culture systems we investigated exhibits a unique capacity to respond to the
different reducing-end-derivatized oligogalacturonides, suggesting that
there is diversity in the way oligogalacturonides are perceived by
these systems. For example, the biological activities of the
reducing-end derivatives of G13 relative to the
activities of the corresponding underivatized oligogalacturonides
(relative activities) of each derivative tested in the cv Samsun
tobacco morphogenesis bioassay (G13-T, 38%;
G13-B, 29%; G13-R, 16%)
are different from their relative activities in the cv Samsun
extracellular alkalinization bioassay, where all four derivatives have
approximately the same relative activity (3%). On the other hand, the
relative activities of the derivatives tested in the cv Xanthi
extracellular alkalinization bioassay (G13-R,
63%; G13-T, 12%) are similar to their relative
activities in the cv Xanthi
H2O2 accumulation bioassay (G13-R, 75%; G13-T,
<17%). The similarity of the results with these two bioassays
suggests that oligogalacturonide induction of extracellular
alkalinization and H2O2
accumulation use signal- transduction pathways with common elements.
It is clear from our results that the reducing-end C-1 of the
oligogalacturonides is a key recognition element in the bioassays tested. In a previous study, the reducing-end C-1 of di- and
trigalacturonides was shown to be required for induction of proteinase
inhibitors in tomato plants (Moloshok et al., 1992). Induction of
tomato proteinase inhibitors likely involves a different receptor than the responses we have investigated here, since the proteinase inhibitors are induced by oligogalacturonides as small as DP 2 (Bishop
et al., 1984). Derivatization of the reducing end of glucan or chitin
oligosaccharins does not affect their biological activity regardless of
the modification used (Sharp et al., 1984a, 1984b; Cheong et al., 1991;
Shibuya et al., 1993; Baureithel et al., 1994). However, the nature of
the modification of the reducing end of a chitosan-derived
octasaccharide does effect its ability to induce phytoalexin
accumulation in pea; the methyl glycoside derivative retains full
activity and the methoxyphenyl glycoside derivative is inactive
(Hadwiger et al., 1994). We have shown that the specific reducing-end
modification differentially reduces the biological activity of
oligogalacturonide derivatives in the tobacco morphogenesis bioassay
and in both of the cv Xanthi suspension-culture bioassays, whereas the
specific modification reduces the activity of the derivatives to an
equal extent in the cv Samsun extracellular alkalinization bioassay.
The nonreducing end of oligogalacturonides can also play a role in the
recognition of biologically active oligogalacturonides by plant cells.
Pectate-lyase-catalyzed introduction of a 4,5-unsaturated bond in the
nonreducing terminal residue of oligogalacturonides and lowered the
most active size from DP 12 to 10 (Davis et al., 1986).
Radiolabeled reducing-end derivatives of an elicitor-active
hepta--glucoside from Phytophthora sojae and
of elicitor-active chitin-derived oligosaccharides have been used to
identify high-affinity, specific binding sites in plant membrane
fractions (Cosio et al., 1990; Cheong and Hahn, 1991; Shibuya et al.,
1993; Baureithel et al., 1994). Each of these derivatives elicits
biological responses (EC50 of 
10 nm) that are indistinguishable from those elicited by their
corresponding underivatized oligosaccharins, indicating that these
derivatives bind to their physiological receptors with high affinity.
Furthermore, the EC50 of these derivatives and a
series of elicitor analogs are closely correlated with the
concentration of each compound required for half-maximum binding to
membrane proteins in vitro, suggesting that the binding proteins
identified are the physiological receptors (Cheong et al., 1991).
We have characterized the bioactivities of G13-T
and G13-B to assess their usefulness for the
identification of oligogalacturonide receptors in the tobacco
tissue-culture system. The G13 derivatives elicit
responses that are qualitatively similar to those elicited by the
underivatized oligogalacturonides, suggesting that in each system the
derivatized oligogalacturonides bind to the physiologically relevant
oligogalacturonide receptor. However, the EC50 of
the labeled G13 derivatives are higher than those
of the underivatized oligogalacturonides, suggesting that the
derivatives have lower affinity for the receptors.
Suspension-cultured cells have been useful for identifying
oligosaccharin-binding proteins because oligosaccharins elicit rapid,
easily measured responses in suspension-cultured cells, and
suspension-cultured cells provide a large amount of homogeneous tissue
for the isolation of membranes (Cosio et al., 1990; Shibuya et al.,
1993; Baureithel et al., 1994). However, the
EC50s for the labeled G13
derivatives in our suspension-culture bioassays are 200- to 900-fold
higher than the EC50 of the labeled
oligosaccharin derivatives that have been used to identify binding
proteins in other systems (Cosio et al., 1990; Cheong and Hahn, 1991;
Shibuya et al., 1993; Baureithel et al., 1994). The high concentrations of G13-T and G13-B that
would be required to identify binding sites in the suspension-culture
systems could result in a high degree of nonspecific binding, which
could be compounded by the polyanionic nature of the
oligogalacturonides. This is likely to make it difficult to detect the
low-abundance, specific, saturable binding that is characteristic of a
receptor protein.
The labeled G13 derivatives have much lower
EC50 in the tobacco morphogenesis bioassay
(150-200 nm) than in the suspension culture bioassays (1.9 to >8.7 µm). The oligogalacturonide concentrations required in the tobacco morphogenesis bioassay are approximately 10-fold higher than the EC50 of the labeled
oligosaccharin derivatives that have been successfully used to identify
binding proteins (Cosio et al., 1990; Cheong and Hahn, 1991; Shibuya et
al., 1993; Baureithel et al., 1994). Thus, the labeled
G13 derivatives could be used to identify
oligogalacturonide-binding proteins in membranes from TCL explants,
although it would be challenging to obtain sufficient tissue from such
small explants (approximately 10 mg each). The labeled derivatives
could also be used to identify clones encoding
oligogalacturonide-binding proteins from a cDNA expression library
constructed with TCL-explant mRNA. Such an approach has been used to
identify membrane-bound receptors (Marullo et al., 1989; Nakayama et
al., 1992).
The differences in the way the three tissue culture systems respond to
the reducing-end-derivatized oligogalacturonides could be due to
different receptors. It is also possible that the
reducing-end-derivatized oligogalacturonides are differentially
degraded or sequestered in the different tissue culture systems. We
have not directly tested this possibility; however, we have found that
suspension-cultured tobacco cells are able to rapidly sequester a
portion of reducing-end-reduced oligogalacturonides and to cleave
the remaining reducing-end-labeled oligogalacturonide (Y. Mathieu, J. Guern, M.D. Spiro, M.A. O'Neill, K. Kates, A.G. Darvill and P. Albersheim, unpublished data). Alternatively, differences in pH, ionic
strength, or ionic composition of the media used in the tissue-culture
systems may affect the binding of oligogalacturonides to their
receptors either directly or indirectly by altering the conformation of
the oligogalacturonides.
Oligogalacturonides of DP 
10 have been hypothesized to form a
multimeric "egg-box" conformation through cooperative
intermolecular binding of Ca2+ ions (Kohn, 1975,
1987; Powell et al., 1982). Monoclonal antibodies have been identified
that are believed to recognize this Ca2+-requiring egg-box
conformation. These antibodies can bind to oligogalacturonides of
DP 9 only in the presence of millimolar Ca2+ (Liners et al., 1992). It is possible that
oligogalacturonide receptors may specifically recognize the egg-box
conformation in a manner similar to these antibodies. Indirect evidence
has been presented that the lower limit of the size requirement for oligogalacturonides to elicit most responses is due to the inability of
oligomers shorter than DP 9 to form the egg box. For example, induction
of ion flux in carrot and tobacco cells and phytoalexin accumulation in
carrot cells requires a minimum DP of 9 or 10 and the presence of
millimolar Ca2+ (Mathieu et al., 1991; Messiaen
et al., 1993; Messiaen and Van Cutsem, 1994). It is possible that the
reduction in biological activity of reducing-end-derivatized
oligogalacturonides reported in this paper is due to a shift in the
equilibrium of the oligogalacturonides away from a physiologically
active conformation, such as the egg box.
| |
FOOTNOTES |
1
This research was supported in part by the U.S.
Department of Energy (DOE)-funded Center for Plant and Microbial
Complex Carbohydrates (grant no. DE-FG05-93-ER20097) and by DOE grant
no. DE-FG02-96ER20221 (to P.A.).
2
Present address: Department of Plant Pathology,
The Pennsylvania State University, University Park, PA 16802.
3
Both of these authors contributed equally to
this manuscript.
*
Corresponding author; e-mail palbersh{at}ccrc.uga.edu; fax
1-706-542-4412.
Received October 15, 1997;
accepted January 20, 1998.
| |
ABBREVIATIONS |
Abbreviations:
DP, degree of polymerization.
EC50, oligogalacturonide concentration that includes a half-maximal response.
G12, dodecagalacturonide.
G13, tridecagalacturonide.
G13-B, biotinylated
tridecagalacturonide.
G13-O, oxidized tridecagalacturonide
(dodecagalacturonide-d-galactaric acid).
G13-R, reduced tridecagalacturonide
(dodecagalacturonide-l-galactonic acid).
G13-T, tyraminated tridecagalacturonide.
HPAEC-PAD, high performance
anion-exchange chromatography with pulsed amperometric detection.
PCV, packed cell volume.
R, change in free proton
concentration.
Rmax, change in free proton
concentration elicited by a saturating concentration of G12
or G13.
TCL, thin cell layer.
| |
LITERATURE CITED |
Apostol I,
Heinstein PF,
Low PS
(1989)
Rapid stimulation of an oxidative burst during elicitation of cultured plant cells. Role in defense and signal transduction.
Plant Physiol
90:
109-116
[Abstract/Free Full Text]
Basse CW,
Fath A,
Boller T
(1993)
High affinity binding of a glycopeptide elicitor to tomato cells and microsomal membranes and displacement by specific glycan suppressors.
J Biol Chem
268:
14724-14731
[Abstract/Free Full Text]
Baureithel K,
Felix G,
Boller T
(1994)
Specific, high affinity binding of chitin fragments to tomato cells and membranes. Competitive inhibition of binding by derivatives of chitin fragments and a nod factor of Rhizobium.
J Biol Chem
269:
17931-17938
[Abstract/Free Full Text]
Bellincampi D,
Salvi G,
De Lorenzo G,
Cervone F,
Marfà V,
Eberhard S,
Darvill A,
Albersheim P
(1993)
Oligogalacturonides inhibit the formation of roots on tobacco explants.
Plant J
4:
207-213
Bishop PD,
Pearce G,
Bryant JE,
Ryan CA
(1984)
Isolation and characterization of the proteinase inhibitor-inducing factor from tomato leaves. Identity and activity of poly- and oligogalacturonide fragments.
J Biol Chem
259:
13172-13177
[Abstract/Free Full Text]
Branca C,
De Lorenzo G,
Cervone F
(1988)
Competitive inhibition of the auxin induced elongation by 
-d-oligogalacturonides in pea stem segments.
Physiol Plant
72:
499-504
Brecht JK,
Huber DJ
(1988)
Products released from enzymatically active cell wall stimulate ethylene production and ripening in preclimacteric tomato (Lycopersicon esculentum Mill.) fruit.
Plant Physiol
88:
1037-1041
[Abstract/Free Full Text]
Cheong J-J,
Birberg W,
Fügedi P,
Pilotti Å,
Garegg PJ,
Hong N,
Ogawa T,
Hahn MG
(1991)
Structure-activity relationships of oligo--glucoside elicitors of phytoalexin accumulation in soybean.
Plant Cell
3:
127-136
[Abstract/Free Full Text]
Cheong J-J,
Hahn MG
(1991)
A specific, high-affinity binding site for the hepta--glucoside elicitor exists in soybean membranes.
Plant Cell
3:
137-147
[Abstract/Free Full Text]
Cosio EG,
Frey T,
Verduyn R,
Van Boom J,
Ebel J
(1990)
High-affinity binding of a synthetic heptaglucoside and fungal glucan phytoalexin elicitors to soybean membranes.
FEBS Lett
271:
223-226
[CrossRef][Medline]
Côté F,
Hahn MG
(1994)
Oligosaccharins: structures and signal transduction.
Plant Mol Biol
26:
1375-1411
Darvill A,
Augur C,
Bergmann C,
Carlson RW,
Cheong J-J,
Eberhard S,
Hahn MG,
Ló,
V-M,
Marfà V,
Meyer B,
and others
(1992)
Oligosaccharins-oligosaccharides that regulate growth, development and defense responses in plants.
Glycobiology
2:
181-198
[Free Full Text]
Davis KR,
Darvill AG,
Albersheim P,
Dell A
(1986)
Host-pathogen interactions. XXX. Characterization of elicitors of phytoalexin accumulation in soybean released from soybean cell walls by endopolygalacturonic acid lyase.
Z Naturforsch
41c:
39-48
Eberhard S,
Doubrava N,
Marfà V,
Mohnen D,
Southwick A,
Darvill A,
Albersheim P
(1989)
Pectic cell wall fragments regulate tobacco thin-cell-layer explant morphogenesis.
Plant Cell
1:
747-755
[Abstract/Free Full Text]
Farmer EE,
Moloshok TD,
Saxton MJ,
Ryan CA
(1989)
In vitro phosphorylation of plant plasma membrane proteins in response to the proteinase inhibitor inducing factor.
Proc Natl Acad Sci USA
86:
1539-1542
[Abstract/Free Full Text]
Hahn MG,
Darvill AG,
Albersheim P
(1981)
Host-pathogen interactions. XIX. The endogenous elicitor, a fragment of a plant cell wall polysaccharide that elicits phytoalexin accumulation in soybeans.
Plant Physiol
68:
1161-1169
[Abstract/Free Full Text]
Hadwiger LA,
Ogawa T,
Kuyama H
(1994)
Chitosan polymer sizes effective in inducing phytoalexin accumulation and fungal suppression are verified with synthetic oligomers.
Mol Plant-Microbe Interact
7:
531-553
[Medline]
Kohn R
(1975)
Ion binding on polyuronates-alginate and pectin.
Pure Appl Chem
42:
371-397
Kohn R
(1987)
Binding of divalent cations to oligomeric fragments of pectin.
Carbohydr Res
160:
343-353
[CrossRef]
Liners F,
Thibault J-F,
Van Cutsem P
(1992)
Influence of the degree of polymerization of oligogalacturonates and of esterification pattern of pectin on their recognition by monoclonal antibodies.
Physiol Plant
99:
1099-1104
Linsmaier EM,
Skoog F
(1965)
Organic growth factor requirement of tobacco tissue cultures.
Physiol Plant
18:
100-127
[CrossRef]
Marfà V,
Gollin DJ,
Eberhard S,
Mohnen D,
Darvill A,
Albersheim P
(1991)
Oligogalacturonides are able to induce flowers to form on tobacco explants.
Plant J
1:
217-225
Marullo S,
Delavier-Klutchko C,
Guillet JG,
Charbit A,
Strosberg AD,
Emorine LJ
(1989)
Expression of human 1 and 2 adrenergic receptors in E. coli as a new tool for ligand screening.
Biotechnology
7:
923-927
Mathieu Y,
Kurkdijan A,
Xia H,
Guern J,
Koller A,
Spiro M,
O'Neill M,
Albersheim P,
Darvill A
(1991)
Membrane responses induced by oligogalacturonides in suspension-cultured tobacco cells.
Plant J
1:
333-343
Messiaen J,
Read ND,
Van Cutsem P,
Trewavas AJ
(1993)
Cell wall oligogalacturonides increase cytosolic free calcium in carrot protoplasts.
J Cell Sci
104:
365-371
[Abstract]
Messiaen J,
Van Cutsem P
(1994)
Pectic signal transduction in carrot cells: membrane, cytosolic and nuclear responses induced by oligogalacturonides.
Plant Cell Physiol
35:
677-689
[Abstract/Free Full Text]
Mohnen D,
Eberhard S,
Marfà V,
Doubrava N,
Toubart P,
Gollin DJ,
Gruber TA,
Nuri W,
Albersheim P,
Darvill A
(1990)
The control of root, vegetative shoot and flower morphogenesis in tobacco thin cell-layer explants (TCLs).
Development
108:
191-201
[Abstract]
Moloshok T,
Pearce G,
Ryan CA
(1992)
Oligouronide signaling of proteinase inhibitor genes in plants: structure-activity relationships of di and trigalacturonic acids and their derivatives.
Arch Biochem Biophys
294:
731-734
[CrossRef][ISI][Medline]
Nakayama N,
Yakota T,
Arai K
(1992)
Use of mammalian cell expression cloning systems to identify genes for cytokines, receptors, and regulatory proteins.
Curr Opin Biotechnol
3:
497-505
[Medline]
Powell DA,
Morris ER,
Gidley MJ,
Rees DA
(1982)
Conformations and interactions of pectins. II. Influence of residue sequence on chain association in calcium pectate gels.
J Mol Biol
155:
517-531
[CrossRef][ISI][Medline]
Pressey R
(1993)
Uronic acid oxidase in orange fruit and other plant tissues.
Phytochemistry
32:
1375-1379
[CrossRef]
Ridley BL,
Spiro MD,
Glushka J,
Albersheim P,
Darvill AG,
Mohnen D
(1997)
A method for biotin labeling of biologically active oligogalacturonides using a chemically stable hydrazide linkage.
Anal Biochem
249:
10-19
[CrossRef][Medline]
Robertsen B
(1986)
Elicitors of the production of lignin-like compounds in cucumber hypocotyls.
Physiol Mol Plant Pathol
28:
137-148
Root RK,
Metcalf J,
Oshino N,
Chance B
(1975)
H2O2 release from human granulocytes during phagocytosis. I. Documentation, quantitation, and some regulating factors.
J Clin Invest
55:
945-955
Ryan CA,
Farmer EE
(1991)
Oligosaccharide signals in plants: a current assessment.
Annu Rev Plant Physiol Plant Mol Biol
42:
651-674
[CrossRef][ISI]
Sharp JK,
McNeil M,
Albersheim P
(1984a)
The primary structures of one elicitor-active and seven elicitor-inactive hexa(-d-glucopyranosyl)-d-glucitols isolated from the mycelial walls of Phytophthora megasperma f. sp. glycinea.
J Biol Chem
259:
11321-11336
[Abstract/Free Full Text]
Sharp JK,
Valent B,
Albersheim P
(1984b)
Purification and partial characterization of a -glucan fragment that elicits phytoalexin accumulation in soybean.
J Biol Chem
259:
11312-11320
[Abstract/Free Full Text]
Shibuya N,
Kaku H,
Kuchitsu K,
Maliarik MJ
(1993)
Identification of a novel high-affinity binding site for N-acetylchitooligosaccharide elicitor in the membrane fraction from suspension-cultured rice cells.
FEBS Lett
329:
75-78
[CrossRef][ISI][Medline]
Spiro MD,
Kates KA,
Koller AL,
O'Neill MA,
Albersheim P,
Darvill AG
(1993)
Purification and characterization of biologically active (1
4)-linked -d-oligogalacturonides after partial digestion of polygalacturonic acid with endopolygalacturonase.
Carbohydr Res
247:
9-20
[CrossRef]
Spiro MD,
Ridley BL,
Glushka J,
Darvill AG,
Albersheim P
(1996)
Synthesis and characterization of tyramine-derivatized (14)-linked -d-oligogalacturonides.
Carbohydr Res
290:
147-157
[Medline]
Tenhaken R,
Levine A,
Brisson LF,
Dixon RA,
Lamb C
(1995)
Function of the oxidative burst in hypersensitive disease resistance.
Proc Natl Acad Sci USA
92:
4158-4163
[Abstract/Free Full Text]
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Abstract
Introduction
