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Plant Physiol. (1998) 116: 1013-1021
Xyloglucan Octasaccharide XXLGol Derived from the Seeds of
Hymenaea courbaril Acts as a Signaling
Molecule1
Carem Vargas-Rechia2,
Fany Reicher,
Maria Rita
Sierakowski,
Alain Heyraud,
Hugues Driguez, and
Yvette Liénart*
Department of Biochemistry, Universidad Federal do Paranã, BP
19046, 81531-970, Curitiba, Paranã, Brazil (F.R.); Department of
Chemistry, Universidad Federal do Paranã, BP 19081, 81531-990,
Curitiba, Paranã, Brazil (M.R.S.); and Centre de Recherches sur
les Macromolécules Végétales-Centre National de la
Recherche Scientifique, BP 53, 38041 Grenoble cedex 9, France
(affiliated with the Joseph Fourier University, Grenoble; C.V.-R.,
A.H., H.D., Y.L.)
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ABSTRACT |
Treatment
of the xyloglucan isolated from the seeds of Hymenaea
courbaril with Humicola insolens
endo-1,4- -d-glucanase I produced xyloglucan
oligosaccharides, which were then isolated and characterized. The two
most abundant compounds were the heptasaccharide (XXXG) and the
octasaccharide (XXLG), which were examined by reference to the
biological activity of other structurally related xyloglucan compounds.
The reduced oligomer (XXLGol) was shown to promote growth of wheat
(Triticum aestivum) coleoptiles independently of the
presence of 2,4-dichlorophenoxyacetic acid (2,4-D). In the presence of
2,4-D, XXLGol at nanomolar concentrations increased the auxin-induced
response. It was found that XXLGol is a signaling molecule, since it
has the ability to induce, at nanomolar concentrations, a rapid
increase in an  -l-fucosidase response in suspended cells or protoplasts of Rubus fruticosus L. and to modulate
2,4-D or gibberellic acid-induced -l-fucosidase.
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INTRODUCTION |
Xyloglucan is a major hemicellulosic polysaccharide in the primary
cell walls of dicotyledons and nongraminaceus monocotyledons and is
also present as a storage polysaccharide in the seeds of many
dicotyledons (McNeil et al., 1984 ; Reid, 1985; Lima et al., 1993; York
et al., 1993; Braccini et al., 1995). The heterogeneity of xyloglucans
results from differences in their molecular mass, distribution, and
levels of substituted xylosyl units with galactosyl and fucosyl
residues. Thus, the fucosyl residue has been found to be (1 2)-linked
to the -galactosyl residue in suspended cells of
Rosa sp. (McDougall and Fry, 1988) and sycamore (Stephen,
1983) or when xyloglucans were isolated from the stems and the roots of
etiolated pea (Pisum sativum L.) plants (Guillén
et al., 1995). This residue is unusual in seed xyloglucans (Siddiqui
and Wood, 1977; Guidley et al., 1991).
The metabolism of xyloglucans in cellulose microfibril networks is
important for cell wall expansion. Support for this idea comes from
alterations to xyloglucan that contribute to wall extensibility. Endo-1,4--d-glucanases, xyloglucanase,
-l-fucosidase, and/or endo-type transferases such as
xyloglucan endotransglycosylase are involved in the auxin- or
acid-promoted breakdown of xyloglucans (Hayashi et al., 1984;
Hetherington and Fry, 1993). Furthermore, xyloglucan-derived oligomers
exhibited signaling effects. The Fuc-rich xyloglucan from suspension
cultures of Rosa sp. digested by Trichoderma
viride cellulase resulted in the formation of a nonasaccharide-containing terminal fucosyl residue (XXFG; Fry et al.,
1993a). This oligosaccharide acts as an anti-auxin 2,4-D growth promotor in etiolated pea stems (McDougall and Fry, 1988) and
was also able to inhibit GA3 induction of pea
segments (Yang et al., 1996), stimulate glycan-synthase activities, and
increase the viability of protoplasts (Emmerling and Seitz, 1990).
Substitution of the XXXG core with one or two Gal residues to give XXLG
and XLLG resulted in growth promotion and in the in vitro stimulation of cellulase (McDougall and Fry, 1990).
Hymenaea courbaril (Leguminosae) is a species that occurs
abundantly throughout Brazilian forests from the northeast to the south, and the seeds contain 40 to 45% xyloglucan, the structure of
which consists of a cellulosic-type (14)-linked
-d-glucan main chain and side chains containing
-d-xylopyranosyl and
-d-galactopyranosyl-(12)--d-xylopyranosyl residues, each (16)-linked to the main chain (Lima et al., 1993, 1995). Here, the ability of endoglucanase I of Humicola
insolens to cleave the (14)-linked -d-glucosyl
residues of xyloglucan from H. courbaril seeds was carried
out. The released oligomers (such as XXLG and XXXG) were then examined
under the reduced form in terms of their biological activity with other
structurally related xyloglucan compounds. The auxin activity of XXLGol
in vivo was investigated using wheat coleoptiles. Parallel to this, a
system of Rubus fruticosus L. protoplasts was
used to study the signaling effect of the induction of
-l-fucosidase activity.
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MATERIALS AND METHODS |
Chemicals
The Humicola insolens endoglucanase I (121 endocellulase units mg 1) was a gift of Dr.
Schülein (Novo Nordisk, Bagsvaerd, Denmark) and was obtained as
described by Armand et al. (1997). Caylase 345 (cellulase) and Caylase
M3 (pectinase) were purchased from Cayla (Toulouse, France).
-l-Fuc-(12)--d-Gal-(14)--d-Glc (2 -fucosyl-lactose) was from Oxford GlycoSystems (Coger, Paris, France). XXFGol, prepared from Rubus fruticosus L. suspended
cells as described by Joseleau et al. (1992), was a gift of Dr.
Gérard Chambat (Centre de Recherches sur les Macromolécules
Végétales-Centre National de la Recherche Scientifique,
Grenoble, France). The trisaccharide methyl
-l-Fuc-(12)--d-Gal-(12)--d-Xyl
side chain of the xyloglucan molecule was prepared as described by Lopez et al. (1994) and was a gift of Dr. Fernandez-Mayoralas (Instituto de Quimica Organica General, Consejo Superior de
Investigaciones Científicas, Madrid, Spain). The
oligogalacturonides of DP ranging from 12 to 15 and maltopentaose were
a gift of Dr. Alain Heyraud (Centre de Recherches sur les
Macromolécules Végétales-Centre National de la
Recherche Scientifique). Potassium benzyl penicillin, cycloheximide,
actinomycin D, and pnitrophenyl
-l-fucopyranoside were from Sigma. BioGelP2 was from
Bio-Rad.
Materials
Suspensions of R. fruticosus L. cultures, originally
derived from cambial explants from twigs, were grown as described by Hustache et al. (1975). Hymenaea courbaril seeds were
collected in July 1995, at the campus of the University of São
Paulo, Ribeirão Preto, Brazil. Seeds of wheat (Triticum
aestivum var Festival) were purchased from EARL Benoist (Airvault,
France).
Preparation of XXXGol and XXLGol
Preparation of XXXGol and XXLGol was as follows: Purified
xyloglucan from H. courbaril seeds was obtained according to
the method of Lima et al. (1995). A sample (500 mg) was incubated (96 h, 37°C) in water (100 mL) containing
endo-1,4--d-glucanase type I (5 mg) from H. insolens (EC 3.2.1.4; 121 endocellulase units/mg). Aliquots (0.25 mL) were removed from the incubated sample at 0, 24, 48, 72, and
96 h, and the reducing sugars were determined. The enzymatic
reaction was stopped by heating at 100°C for 5 min, insoluble
material was removed by centrifugation, and the supernatant was
concentrated and lyophilized. Aliquots of the
oligosaccharide mixture (50 mg, 1 mL) were filtered on a cellulose nitrate membrane (0.45 µm) and applied to a column (1.5 × 210 cm; 60°C) of BioGel-P2 (400 mesh). Elution with water was at a flow
rate of 0.5 mL min1, controlled by a
peristaltic pump (Milton-Roy, Rochester, NY). Eluted oligosaccharides
were monitored with a differential refractometer (model R403,
Waters).
Oligosaccharides were reduced as described by York et al. (1993): they
were first dissolved (5 mg mL1) in water
containing NaBH4 (5 mg
mL1; 3 h). Excess of borohydride was
decomposed with glacial acetic acid, and residual borate was removed by
coevaporation with methanol. The resulting oligoglycosyl
alditols (5 mg per injection) were filtered on a cellulose nitrate
membrane (0.3 µm), desalted, and separated by reverse-phase
chromatography on a Nucleosil C-18 semipreparative column (25 × 0.46 cm) eluted with 7% aqueous methanol as the mobile phase at a flow
rate of 2 mL min1; eluted oligosaccharides were
detected by monitoring the refractive index.
NMR Spectroscopy
NMR spectra were recorded with a spectrometer (model AC300,
Bruker, Billerica, MA). The alditol samples were dissolved in D2O (2 mg mL1
[1H] and 10 mg mL1
[13C]). Chemical shifts are reported as  relative to internal acetone as 2.04 (1H) and
29.8 (13C) with respect to the signals for
tetramethylsilane at 333 K (1H) and 303 K
(13C).
FAB-MS Spectrometry
Low-mass resolution measurements were performed on a quadripole
mass spectrometer (model R.10.10C, Nermag, Rveil-Malmaison, France)
using a glycerol matrix and FAB(+) mode.
Wheat Coleoptile Growth Biossays
Seeds were grown in the dark and 3-d-old wheat seedings with the
first internode measuring 15 mm were selected. These samples were
incubated in sterile, plastic Petri dishes (15-20 seeds per dish) in
two independent replicate sets containing 15 mL of fresh medium (1%
[w/v] Suc, 5 mm
KH2PO4 [pH 6.1] and
0.02% potassium benzyl penicillin) with or without effector. The
bioassay was started by the addition of 2,4-D (1 µm)
and/or XXLGol (0.5, 1, 10, 50, and 100 nm), XXFGol (0.5, 1, 10, and 50 nm), 2-fucosyl-lactose (1, 10, and 100 nm), and the coleoptile length was measured at intervals up
to 62 h. Controls were concomitantly run in the described medium
without the addition of 2,4-D or oligosaccharide. The variability in
elongation of the 15 to 20 stem segments in a single dish was measured
with a Student's t test, and data with P < 0.05 were analyzed. Data points of kinetic curves are each means ± se of 30 to 40 determinations (Fig.
1).
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| Figure 1.
Effect of the xyloglucan oligomers XXLGol (a) and
XXFGol (b) and of 2-fucosyl-lactose (c) on the straight growth of
wheat coleoptiles (A) and on the elongation promoted by 2,4-D (B). Two independent experiments, each conducted from 15 to 20 coleoptile segments treated or not with 2,4-D (1 µm) for 62 h
were monitored and the mean increments of length ( L) were
plotted. In A each value of a curve is the average of 30 to 40 experiments, and vertical bars represent ses. Data are
presented as L versus log sugar concentration; L is the
additional increase in length between 10 and 20 h. The absolute
increase in length of controls (sets in the absence of 2,4-D or
oligosaccharides) exhibited the mean value of 3.87 mm. This value was
the reference (L = 0). In B data are presented as mean
percentages of inhibition or activation of 2,4-D-stimulated growth,
calculated as percentages of activation or inhibition = L(2,4-D + oligosaccharide)  (L[2,4-D])/(L[2,4-D]) L(control) × 100%, where
L(2,4-D) is the mean final length of segments treated with 2,4-D, L(control) is
the mean final length of segments incubated without 2,4-D, and
L(2,4-D + oligosaccharide) is the mean final
length of segments treated with 2,4-D plus the oligosaccharide. Plotted
data represent the means ± se.
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Preparation of Cells and Protoplasts from R. fruticosus
L.
Cells in the exponential growth phase (15-18 d after
subculturing) were collected by centrifugation at 4000g for
5 min, washed with Heller's medium, and resuspended in 50 mm sodium-citrate buffer, pH 5.9, containing 2% Suc, 4 mm EGTA, 3 mm MgCl2, and 0.06 mm CaCl2. For protoplast
isolation, 18- to 20-d-old cells (40 g fresh weight) were incubated
overnight at room temperature in 300 mL of the growth medium, pH 5.9, supplemented with 0.56 m mannitol and 0.25% (w/v)
cellulase and 0.01% (w/v) pectinase. The released protoplasts were
filtered through a 100-µm nylon mesh washed twice with the incubation
medium with no cell wall-degrading enzymes before being pelleted at
500g for 5 min. They were resuspended in 25 mm
Bis-Tris-HCl buffer, pH 4.8, containing 0.56 m
mannitol, 0.06 m Suc, 1 mm KCl, 1 mm CaCl2, and 6% (w/v) Ficoll 400, and were then centrifuged at 500g for 5 min, and finally
washed with Bis-Tris-HCl buffer without Ficoll. Protoplast yields
ranged from 70 to 85% of the initial number of treated cells.
Bioassays
Protoplasts (2 × 106; or cells) of
R. fruticosus were suspended under stirring in 25 mL of
buffer (25 mm Bis-Tris-HCl, pH 4.8, containing 0.56 m mannitol, 0.06 m Suc, 1 mm KCl,
and 1 mm CaCl2) in the presence or
absence of: oligo-, polysaccharide inducer up to 100 nm;
2,4-D or GA3 (10 nm); cycloheximide
(1 µm); or actinomycin D (1 µg
mL1). The concentration of oligomers is
expressed as molarity, and the molar concentration of the polymer is
reported by reference to XXLG repeating units. Protoplasts (or cells)
were harvested at various intervals by centrifugation at
300g for 8 min at 4°C, before being subjected to enzyme
extraction. The viability of protoplasts was controlled using Evan's
blue indicator.
-l-Fucosidase Assays
Enzymes were extracted in 50 mm Tris-HCl, pH 7.2, containing 1 m NaCl by homogenizing the protoplasts on ice
with a polytron at full speed, 15 times for 45 s. The extracts
were dialyzed and concentrated using ultrafiltration units equipped
with a molecular mass cutoff value of a 10-kD membrane (Ultrafree,
Millipore, Bedford, MA). -l-Fucosidase (EC 3.2.1.51) was
assayed as follows: p-nitrophenyl -l-fucopyranoside (2 mg mL1) was
incubated at 40°C for 0 to 90 min with crude enzyme extract (1 µg
of protein based on colorimetric determination; Bradford, 1976) in 200 µL of 0.1 m sodium acetate buffer, pH 5.0. The enzyme reaction was quenched with 100 µL of 0.1 m
Na2CO3,
and -l-fucosidase activity was
determined by measuring the A410 of the
p-nitrophenolate ion according to the method of Lee and
Zeikus (1993).
For each oligosaccharide tested, enzyme assays were performed from
three to four replications from three independent elicitation sets, and
kinetic curves were drawn and fitted with the second-order polynomial
regressions. The velocity of enzyme activities was calculated from
regression equations using Excel software, and enzyme activation is
expressed as R, which is the ratio of the slopes of the
fitted curves obtained from treated protoplasts versus controls. The
treated protoplasts were elicited by one oligosaccharide and/or one
hormone. The controls were the protoplasts suspended in buffer without
any effector. Blanks (without enzyme or without substrate) were carried
out for each sample.
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RESULTS |
Preparation and Characterization of Oligosaccharides Derived from
Xyloglucan of H. courbaril Seeds
The enzymatic hydrolysis of the xyloglucan isolated from H. courbaril seeds was carried out using
endo-1,4--d-glucanase I from H. insolens
(Armand et al., 1997). The water-soluble oligosaccharides (450 mg) were
partially fractionated according to their molecular weights by
BioGel-P2 chromatography. Higher-molecular-weight oligosaccharide components represented 50% of eluted material and were not analyzed further. Fractions corresponding to oligomers of DP 5 to 8 were pooled
and converted to their corresponding alditol derivatives. The mixture
was further fractionated by reverse-phase chromatography. Only the two
major oligosaccharides (4 and 42 mg), with retention times of 35 and
41.26 min, respectively, were isolated and characterized.
The oligosaccharides were identified by their
1H-NMR spectra as XXXGol and XXLGol. The shape
and chemical shift of their 1H-NMR signals were
in agreement with those already published (York et al., 1993),
considering the differences in experimental conditions. Our NMR spectra
were recorded on a spectrometer (model AC300, Bruker, Wissembourg,
France) at 333 K at a concentration of 10 to 20 mg
mL1; York et al. (1993) used a Bruker 500 at
298 K and a concentration of 1 to 10 mg mL1. In
the 1H-NMR spectrum of XXLGol, H-1 signals of
-Xylp units were a doublet ( 5.03, J 4.0 Hz) and a
triplet of two superimposed signals centered at 4.81, each with J
4.0 Hz. The H-1 signal of the -Galp unit was detected as
a doublet ( 4.50, J 10 Hz). The H-1 signal of the
-Glcp gave a broad doublet ( 4.40, J 8 Hz, 3 units).
The 13C-NMR spectrum of the octasaccharide XXLGol
was consistent with the structure (York et al., 1993; Guillén et
al., 1995), with C-1 signals at 105.5 (-Galp); 103.8, 103.6, and 103.3 (each -Glcp); 99.9 (2 × -Xylp) and 99.3 (-Xylp); and
-Glcp 4-O-substituted resonances at 70.5, 80.3, 80.7, and 80.9. Its positive-ion FAB-MS indicated a molecular
weight at 1226 (i.e. GalXyl3Glc3Gol; [M + H]+ at m/z 1227; [M + Na]+ at m/z 1249). Because of the rupture of
glycosidic linkages, the ions at m/z 1085, 1065, 983, 791, and 645 were
also assigned.
XXXGol was also characterized on the basis of its
1H-NMR and FAB-MS (Guillén et al., 1995).
The H-1 region has neither the signal at .5.0, which corresponds to
Xyl substituted at O-2 by Gal, nor that of
-Galp. Its positive-ion FAB-MS indicated a molecular weight at 1064 (i.e.
Xyl3Glc3Gol; [M + Na]+ at m/z 1087).
Growth Induction by Xyloglucan Oligosaccharides and
2-Fucosyl-Lactose
The coleoptiles incubated in Petri dishes showed no significant
variability in elongation. Therefore, all of the samples subjected to a
particular treatment on a given day were viewed as a single population,
despite the fact that they were distributed between distinct dishes.
The ability of the xyloglucan oligosaccharides XXLGol and XXFGol and of
2-fucosyl-lactose to interact with coleoptile growth, induced or not
with 2,4-D, were bioassayed during 62 h. The oligosaccharides were
used here at the narrow nanomolar concentration range up to 100 nm, which can promote enzyme-activation responses as
detailed below. The trisaccharide was chosen to test the importance of
Xyl in the activity by reference to the 2-fucosyl-lactose. The growth
rates between 10 and 20 h of incubation were first investigated as
a function of experimental time (not shown) and then expressed as L
versus log sugar concentration (Fig. 1A) or as a percentage of growth
promotion (percentage of activation) or growth inhibition (percentage
of inhibition) by 2,4-D (Fig. 1B).
The results without 2,4-D (Fig. 1A) clearly indicate that XXLGol at a
concentration ranging from 0.5 to 100 nm
exhibited growth stimulation (curve a), that
XXFGol was not active (curve b), and that 2-fucosyl-lactose showed an
inhibiting effect (curve c). In the presence of 2,4-D used at 1 µm (Fig. 1B), XXLGol increased the auxin-induced
response (curve a), whereas XXFGol (curve b) and 2-fucosyl-lactose
(curve c) showed anti-auxin activity. It was observed that XXFGol,
which was less active than 2-fucosyl-lactose, had anti-auxin activity
at low concentrations but exhibited growth-restoring activity at high
concentrations.
Xyloglucosyl Oligomers as Inducers of
-l-Fucosidase Activity
R. fruticosus protoplasts were incubated for 15 min in
the presence of sugar inducers at concentrations up to 100 nm. The inducers were the xyloglucan polymer from H. courbaril seeds (Lima et al., 1995), the derived oligosaccharides
XXLGol, XXXGol, and XXFGol obtained from R. fruticosus
xyloglucan, and the trisaccharide methyl
-l-Fuc(12)--d-Gal(12)--d-Xyl.
It was verified that protoplast viability was not affected by the
treatment and remained identical to the control protoplasts (90%)
throughout the experiments.
Dose-response curves for -l-fucosidase activation are
shown in Figure 2. With all of the
inducers used up to 100 nm, e.g. the oligomers XXLGol,
XXFGol, and XXXGol (curves a, b, and d, respectively), the
trisaccharide (curve e), and the xyloglucan polymer (curve f),
bell-shaped curves were obtained. The highest activities, with maximum
R values of approximately 3, were observed for XXXGol,
XXFGol, and XXLGol at 0.1, 1, and 5 nm, respectively. The
trisaccharide and the polymer with maximal R values of 2 only exhibited higher optimal concentrations (10 and 50 nm,
respectively).
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| Figure 2.
Dose-response curves for
-l-fucosidase response induced by the xyloglucan
oligomers XXLGoL (a), XXFGol (b), and XXXGol (d), the trisaccharide
methyl
-l-Fuc-(12)--d-Gal(12)--d-Xyl
(e), and the polymer (f). R. fruticosus protoplasts
(2 × 106) in 25 mL of buffer were incubated for 15 min in the presence of inducer up to 100 nm. Each curve was
obtained by least-squares regression of data from three to four
replications carried out from three independent inducer sets.
R is reported as the rate of
-l-fucosidase activity of treated over control
protoplasts.
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Kinetic measurements of -l-fucosidase activation in
R. fruticosus protoplasts and/or cells in the presence of
the inducer used at its optimal concentration and shown in Figure
3 were carried out. We verified that
protoplast (or cell) viability was not affected by the treatment and
remained as high as in control protoplasts (90%) or control cells
(95%) throughout the experiments. When the protoplasts were challenged
with XXLGol, XXFGol, XXXGol, and trisaccharide (Fig. 3, curves a, b, d,
and e, respectively), the detected responses were biphasic and
triphasic with respect to their kinetics. The oscillation of the early
responses of protoplasts peaked after 10 to 20 min and 45 min for the
oligomers; the response to the synthetic trisaccharide was delayed,
since it peaked at 30 min and 6 h. When the inducers were compared
at their respective maximal R values, the order of their
effectiveness between 10 and 30 min was XXFGol > XXXGol
approximately trisaccharide > XXLGol, with respective
R values of 5.8, 4.1, and 3. After 45 min, the order was
XXXGol > XXLGol > XXFGol, with R values of 6.5, 4.7, and 1.8, respectively.
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| Figure 3.
Time course for -l-fucosidase
activation in R. fruticosus protoplasts challenged with
the sugar inducers XXLGol (a), XXFGol (b), and XXXGol (d) and the
trisaccharide methyl
-1-Fuc-(12)--d-Gal-(12)--d-Xyl (e). Protoplasts (2 × 106) in 25 mL of
buffer were challenged with inducer used at the optimal concentration
(0.1 nm XXXGol, 1 nm XXFGol, 5 nm
XXLGol, and 10 nm trisaccharide). Each curve was obtained
by least-squares regression of data from three to four replications
carried out from three independent inducer sets. R is
reported as the rate of -l-fucosidase activity of
treated over control protoplasts. Control sets were run without
addition of 2,4-D or oligosaccharides.
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It is significant that only 10 min was required for XXXGol to trigger a
response, as opposed to 15 and 20 min for XXLGol and XXFGol,
respectively, and 30 min for the trisaccharide. Incubation for a longer
duration of up to 96 h resulted in a large response increase;
R values of 6 and 8 were found from XXXGol and XXLGol, respectively (not shown). In the presence of inhibitors of
transcription (1 µm cycloheximide) or of translation (1 µg mL1 actinomycin D), the short responses of
up to 45 min were maintained, whereas the long-term treatment resulted
in a markedly attenuated response (40 or 50% inhibition at 96 h
being detected from the XXLGol inducer). The cell-suspension cultures
monitored instead of protoplasts gave rise to plateau responses, and
the enzyme activation was largely attenuated, since the highest
detected maximal R values were only 1.2 and 3.5 (not shown).
Oligosaccharides structurally unrelated to xyloglucan, such as
oligogalacturonides of 10 to 15 DP and maltopentose, were also used as
potential inducers but failed to promote any response in protoplasts or
in cells.
Effects of Xyloglucosyl Oligomers on
2,4-D-Induced -l-Fucosidase Response
Induction of -1-fucosidase activity (R
value of 1.5) occurred when 2,4-D was used with R. fruticosus protoplasts for 15 min at 10 nm. The
oligomers XXFGol, XXLGol, XXXGol, and the trisaccharide methyl
-l-Fuc-(12)--d-Gal-(12)--d-Xyl
up to 100 nm were assayed for their effect on the
auxin-stimulated -l-fucosidase. One of our aims was to
determine which glycosyl residues were required for modifying the
-l-fucosidase response. Dose-response curves in the
presence of 10 nm 2,4-D indicated that XXFGol and
trisaccharide inducers exhibited a similar behavior (Fig.
4A, curves b and e, respectively). The
absence of a fucosyl residue in XXLGol resulted in the promotion of
enzyme activation, with an R value of about 6 (Fig. 4B,
curve a), whereas XXXGol, which lacks the 2-fucosyl Gal side chain,
was not active (Fig. 4B, curve d). The data shown in Figure 4C are the
percentages of modulation of 2,4-D-induced -l-fucosidase. They clearly show that the terminal
galactosyl residue of XXLGol is the structural feature required to
promote an -l-fucosidase response (curve a) and that the
lack of the galactosyl residue led to a compound with inhibitory
activity only (curve d). The presence of the fucosyl residue attached
to the 2-position of the galactosyl unit common to XXFGol (curve b) and
the trisaccharide methyl
-l-Fuc-(12)--d-Gal-(12)--d-Xyl (curve e) caused either inhibition or activation depending on the sugar
concentration, the trisaccharide being the more potent inhibitor of
these two sugars.
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| Figure 4.
Effects of XXLGol (a), XXFGol (b), and XXXGol (d)
and the trisaccharide methyl
-l-Fuc(12)--d-Gal(12)--d-Xyl
(e) on 2,4-D-stimulated -l-fucosidase in R. fruticosus protoplasts. The results are expressed as the
R value in A and B and as the mean percentage of
activation (inhibition) of 2,4-D-stimulated response in C. Protoplasts
(2 × 106) in 25 mL of buffer were incubated for 15 min with sugar inducer up to 100 nm in the presence of 10 nm 2,4-D. Each curve was obtained by least-squares
regression of data from three to four replications carried out
from three independent inducer sets. The induced response given as R is reported as the rate of
-l-fucosidase activity of treated
protoplasts over controls. The mean percentage of activation (inhibition) is calculated as
R(2,4-D + oligosaccharide) (R[2,4-D])/(R[2,4-D]) R(control) × 100, where
R(2,4-D + oligosaccharide) and
R(2,4-D) are R values in
protoplasts incubated with sugar inducer and 2,4-D and with
2,4-D, respectively, and R(control) is the
R value in protoplasts suspended in buffer without the
addition of 2,4-D or oligosaccharides.
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Effects of Xyloglucosyl Oligomers on GA3
-l-Fucosidase Activation
GA3 acting for 15 min at 10 nm
was also found to promote -l-fucosidase activation in
R. fruticosus protoplasts (R value of about 1.5).
The dose-response curves resulting from the presence of
GA3 (Fig. 5, A and
B) and the oligomers showed that XXXGol (curve d) and XXFGol (curve b)
were highly effective in the enhancement of enzyme activation, with
R values of 7 and 6, respectively. XXLGol (curve a) was less
active (R value of 2 only) and the xyloglucosyl side chain
of methyl
-l-Fuc-(12)--d-Gal-(12)--d-Xyl
did not promote an increase in the -l-fucosidase
response (curve e). The data shown in Figure 5C, presented as
percentages of activation of
GA3-induced--l-fucosidase, clearly
reveal that the presence of two xylosyl residues attached at final and
penultimate Glc units are needed for the biological activity (curves d
and b), but the galactosyl residue at position 2 antagonized the
inducing effect of xylosyl residue, since XXLGol was poorly effective
(curve a). It is significant that the presence of a terminal fucosyl residue in XXFGol (curve b) could only partially restore the activity, but this restoration required a feature mimicking the XXXGol backbone, since the trisaccharide was not active (curve e).
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| Figure 5.
Effects of XXLGol (a), XXFGol (b), XXXGol (d) and
the trisaccharide methyl
-l-Fuc-(12)--d-Gal-(12)--d-Xyl
(e) on GA3-stimulated -l-fucosidase in
R. fruticosus protoplasts. The results are expressed as
the R value in A and B and as the mean percentage of
activation of GA3-stimulated response in C. Protoplasts
(2 × 106) in 25 mL of buffer were incubated for 15 min with sugar up to 100 nm in the presence of 10 nm GA3. Each curve was obtained by least-squares regression of data from three to four replications carried out from three independent inducer sets. R is
reported as the rate of -1-fucosidase activity of
treated protoplasts over controls. The mean percentage of activation is
calculated as = R(GA3 + oligosaccharide) (R[GA3])/(R[GA3]) R(control) × 100, where
R(GA3 + oligosaccharide) and
R(GA3) are R values in
protoplasts incubated with sugar and GA3 and with
GA3, respectively, and
R(control) is the R value in
protoplasts suspended in buffer without the addition of 2,4-D or
oligosaccharides.
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DISCUSSION |
Sugar-signaling molecules of fungal or plant origin, which are
produced by acidic or enzymatic hydrolysis of cell wall polysaccharides or glycoproteins, induce plant-defense responses and/or exhibit effects
on growth and development (Aldington and Fry, 1993). In relation to the
xyloglucan oligomers, nanomolar or micromolar concentrations with
specific structural elements initiate characteristic physiological or
biochemical responses. Thus, the structural features required for XXFG
to exhibit an anti-auxin effect have been extensively studied (York et
al., 1984; McDougall and Fry, 1988, 1989a, 1989b, 1990; Augur et al.,
1992). Optimal anti-auxin activity is observed in a nanomolar range of
XXFG (but not of XXLG or XXXG). XXFG is as effective as the reduced
form XXFGol according to Augur et al. (1992). This effect is mimicked
only by the related -l-Fuc unit containing xyloglucosyl
oligomers and by the 2-fucosyl-lactose. It has also been postulated
that the activity of XXFG may be the result of two opposing effects: at
lower concentrations, the Fuc-dependent-anti-auxin effect predominates,
whereas at higher concentrations the Fuc-independent, growth-promoting effect is expressed.
These results suggest the presence of specific recognition systems for
xyloglucan oligomers in plants. It is worth noting that when XLLG,
XXXG, and XXFG promoted the elongation of pea stem segments in the
absence of 2,4-D, the detected effect differed in several important
respects from the growth-inhibiting effect reported above. Indeed, the
optimal concentration for growth promotion is approximately micromolar,
the fucosyl residue is not required, and some of the
Xyl3 Glc4 backbone is
required. This prompted Fry et al. (1993a) to suggest that the
oligosaccharides exert their effect by acting as substrates of
xyloglucan endo-transglycosylase.
We have used the xyloglucan from the seeds of H. courbaril
as a source of signaling molecules. The monosaccharide composition of
this xyloglucan is Glc, Xyl, and Gal in a ratio of 50:35:13, and its
structure was determined by methylation, periodate oxidation, and
13C-NMR spectroscopy (Lima et al., 1993, 1995) to
be similar to that of the well-characterized xyloglucan from the seeds
of Tamarindus indica (Guidley et al., 1991). The
polysaccharide was treated with H. insolens
endo-1,4--d-glucanase I. Because it belongs to the
glycosyl hydrolase family 7, the enzyme is a retaining endoglucanase
that needs as a substrate an unsubstituted glucosyl unit in its
subsite-1 (Armand et al., 1997), as do most of the endoglucanases used
for xyloglucan degradation (Vincken et al., 1995). The enzyme
preparation was free of galactosidase and xylosidase, since no
monosaccharide was detected on elution with BioGel-P2. The
octasaccharide, representing the basic structure of the xyloglucan, was
readily obtained, as well as a minor heptasaccharide component. The
resulting hepta- and octasaccharide were isolated by size-exclusion chromatography and then by HPLC; the corresponding alditols were characterized by their NMR and FAB-MS spectra. The data are in agreement with those previously published (York et al., 1993; Guillén et al., 1995).
XXLGol and XXXGol were used as signaling molecules. Purified XXLGol was
shown to promote, at a nanomolar concentration range, the growth of
wheat coleoptiles independently of the presence of 2,4-D, as described
previously for micromolar xyloglucan in pea stem segments (McDougall
and Fry, 1990). In the presence of 2,4-D, nanomolar concentrations of
XXLGol increased the auxin-induced response, but 2-fucosyl-lactose and
XXFGol, to a lesser extent, can exhibit the anti-auxin effect,
depending on the concentration used. Therefore, the data obtained for
wheat coleoptiles confirmed that the anti-auxin activity depends on the
presence of the -l-fucopyranosyl -(12)-d-galactopyranosyl -(1) side chain of the
xyloglucan.
The data reported for suspensions of R. fruticosus
protoplasts (or cells) revealed that xyloglucan oligomers exhibited
signaling molecules by their ability to increase
-l-fucosidase activity and to modulate 2,4-D- or
GA3-induced -l-fucosidase. Here
the auxin and/or the anti-auxin activity of xyloglucosyl oligomers in
biological systems is totally different from that previously reported,
and the GA3-induced response confirmed the
results obtained by Warneck and Seitz (1993). The biological responses
mainly depended on parameters such as the structural features of the
xyloglucosyl oligomers and side chain, the chemical structure of the
hormone, and the sugar concentration.
It was interesting that in the presence of 2,4-D the terminal
-d-galactosyl residue was the determinant for biological
activity: XXLGol contains the residue promoting an auxin
-l-fucosidase response, and the lack of the galactosyl
residue in XXXG led to inhibitory activity. The galactosyl residue can
be antagonized by a Fuc molecule attached to the 2 position of the
galactosyl unit, as is also the case with XXFGol and synthetic
trisaccharide. In the presence of GA3, the
xylosyl residue(s) attached in the final or/and penultimate position is
required for biological activity. They could be antagonized by the
galactosyl residue at position 2, since XXLGol is not active. It was
worth noting that the terminal fucosyl of the XXFGol side chain (but
not the Fuc unit in a trimeric structure) revealed antagonistic effects
toward galactosyl residues and the same order of effectiveness of
XXFGol and XXXGol.
The protoplast responses had a very rapid and transient nature,
suggesting an early signal transduction cascade. This was especially
true in the case -l-fucosidase induction, which started within a few minutes after the addition of xyloglucan signals. The
elicitor treatment up to 180 min did not change the total amount of
extractable protein but affected the specific enzyme activity. The
induction of such responses did not require the presence of cell walls,
and it was fully maintained in the presence of inhibitors of
transcription or translation. These results, which strongly suggest the
evidence of receptor molecules for xyloglucan at plasma membranes,
contribute to a more general application of the receptor hypothesis.
Extensive research on signaling sugars has revealed a sequence of
biochemical events, including transcription and translation of specific
genes, resulting in induction de novo synthesis of enzymes, but the
initial process of signal perception and transduction has still not
been elucidated (Côté and Hahn, 1994). The presence of
high-affinity binding sites, putative receptors for sugars (hepta
-glucoside [Cosio et al., 1992],
N-acetylchitooligosaccharide [Shibuya et al., 1993], and
the trisaccharide determinant of H-type 1 human determinant
[Liénart et al., 1992]), have been reported in plants, and a
binding protein for a -glucan elicitor of Phytophthora megasperma origin has been cloned (Umemoto et al., 1997). However, the initial process of perception and transduction of signaling oligosaccharides remains to be elucidated.
The present study confirmed that xyloglucan oligomers can have
hormone-dependent effects in a system other than pea-stem bioassays. This had been previously reported from data on the anti-auxin activity
in carrot protoplasts (Emmerling and Seitz, 1990) or on the ability of
FG to control the morphogenesis in cultured wheat embryos (Pavlova et
al., 1992). In addition, the effect of elongation of XXXG and XXFG was
recently correlated with the viscoelastic properties of pea shoots
(Cutillas-Iturralde and Lorences, 1997). McDougall and Fry (1990) and
Augur et al. (1995) speculated that cellulase and
-l-fucosidase activities participate in the regulation
of plant growth by controlling both the hydrolysis of xyloglucan and
the concentration of fucosylated oligomers.
This is consistent with our experiments showing that the treatment of
R. fruticosus protoplasts by xyloglucosyl oligomers in the
presence or absence of hormone greatly modulated the
-l-fucosidase activity within a few minutes of
application. When -l-fucosidase was assayed in plants
(Farkas et al., 1991; Augur et al., 1995; Hoson et al., 1995), the
enzyme was able to hydrolyze terminal fucosyl residues from XXFG but
failed to cleave p-nitrophenyl -l-fucopyranoside. In contrast, we observed that the
enzyme in R. fruticosus hydrolyzes the artificial
substrate, as do the mammalian -l-fucosidases (Johnson
and Alhadeff, 1991). Research is now in progress to explain these
observations.
| |
FOOTNOTES |
1
Financial support was provided by Conselho
Nacional de Desenvolvimento Científico e Tecnológico
(Brazil).
2
Permanent address: Department of Chemistry and
Physical, Faculdade de Ciêncas Farmacêuticas de
Ribeirão Preto-Universidade de São Paulo, 14040-903,
Ribeirão Preto, São Paolo, Brazil.
*
Corresponding author; e-mail liénart{at}cermav.cnrs.fr; fax
33-4-76-54-72-03.
Received August 18, 1997;
accepted November 17, 1997.
| |
ABBREVIATIONS |
Abbreviations:
DP, degree of polymerization.
FAB, fast-atom
bombardment.
XXXG, XXLG, and XXFG, the xyloglucan-derived hepta-,
octa-, and nonasaccharide, respectively (see Fry et al., 1993b) .
XXXGol, XXLGol, and XXFGol, the reduced oligomers of XXXG, XXLG, and
XXFG, respectively.
| |
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Abstract
Introduction