Download Activation of cell wall-associated peroxidase

Journal of Experimental Botany, Vol. 47, No. 305, pp. 1897-1904, December 1996
Journal of
Experimental
Botany
Activation of cell wall-associated peroxidase isoenzymes
in pea epicotyls by a xyloglucan-derived nonasaccharide
Hildegard Maria Warneck, Thomas Haug and Harms Ulrich Seitz1
Botanisches Institut, University of TObingen, Auf der Morgensteiie 1, 72076 TObingen, Germany
Received 14 March 1996; Accepted 12 July 1996
Abstract
The cell wall-derived xyloglucan nonasaccharide XXFG
was found to increase the extractable activity of distinct cationic cell wall-associated peroxidase isozyme
groups isolated from etiolated pea epicotyls.
Peroxidase activation occurred in the first 10 h of
incubation with the nonasaccharide in the pea epicotyl
bioassay. At the same time varying concentrations of
XXFG caused growth inhibition up to 35%. Neither the
increase of peroxidase activity nor the growth inhibition was restricted to a certain XXFG concentration.
The increase in peroxidase activity was not just an
oligosaccharide effect in general. The corresponding
heptasaccharide XXXG neither inhibited growth nor
increased peroxidase activity. The isozymes extracted
from pea epicotyls were additionally separated by
cation-exchange chromatography and submitted to
isoelectric focusing. With one exception, all of the
ionically-bound, cell wall-associated peroxidases present in pea epicotyls were cationic or slightly anionic.
It is proposed that the growth inhibition caused by
XXFG is at least in part the result of peroxidasecatalysed cell wall tightening induced by the
nonasaccharide.
Key words: XXFG, growth inhibition, cell wall-associated
peroxidases, cell wall tightening, pea epicotyls.
Introduction
Xyloglucan oligosaccharides play an important role in
plant growth and development. The xyloglucan nonasaccharide XXFG (nomenclature—Fry et al., 1993) has been
found to influence several processes in plant growth.
Nanomolar concentrations inhibited 2,4-D-induced
1
(York et al., 1984; McDougall and Fry, 1988; Emmerling
and Seitz, 1990), NAA-induced (Warneck, 1994), protoninduced (Lorences et al., 1990), and gibberellic acidinduced elongation growth as well as endogenous growth
(Warneck and Seitz, 1993). The growth-inhibiting effects
were dependent on the fucosyl-galactosyl side-chain. The
corresponding heptasaccharide XXXG lacking this sidechain caused no growth inhibition. To date there is no
information on the mechanism after XXFG-application
leading to growth inhibition.
Since the inhibitory effect of XXFG is independent of
the phytohormone applied to the system, the nonasaccharide seems to influence those processes which lead to
elongation-growth in general. Due to the low XGO
concentrations being most effective a direct interaction
with the wall components seems to be unlikely. The
specific structural requirements for the growth-inhibiting
activity of XXFG suggest a specific receptor for XXFG
and related fucose-containing XGOs (McDougall and
Fry, 1988).
The involvement of peroxidases in growth processes
was suggested (Gaspar et al., 1982; Penel et al., 1992)
several years ago and various attempts have been made
to explain their participation in a pathway of reactions
controlling cell wall rigidification (Srivastava and van
Huystee, 1973; Ricard and Job, 1974; Gaspar et al.,
1985). The role of peroxidases in plant growth was shown
indirectly by the development of the enzyme along the
growth gradient of mung bean hypocotyls being correlated with a loss in plasticity (Goldberg et al., 1986a, b,
1987). Zheng and van Huystee (1992) demonstrated a
direct link between growth inhibition and peroxidase
activities in peanut hypocotyls by the addition of either
antibodies of individual peroxidases or purified isoenzymes to the culture medium. Furthermore, peroxidases
are thought to regulate growth by catalysing oxidative
To whom the correspondence should be addressed. Fax: +49 7071 293387.
Abbreviations: PRX, peroxidases; XGOs, xyloglucan oligosaccharides; XXFG, xyloglucan nonasaccharide; XXXG, xyloglucan heptasaccharide.
© Oxford University Press 1996
1898
Warneck et al.
Peroxidase extraction
coupling of phenols, for example, the formation of
Peroxidase extraction was modified from the methods of
diphenyl bridges (Fry, 1983, 1984; Cooper and Varner,
Masuda et al. (1983) and Sato et al. (1993). After determination
1984; Epstein and Lamport, 1984), which leads to a crossof fresh weight, 20 pea epicotyl segments of each treatment in
linking of polysaccharides and glycoproteins in the cell
the bioassay were homogenized in liquid nitrogen with mortar
wall (Fry, 1986; Zheng and van Huystee, 1991a; Hartley
and pestle, suspended in 5 ml TRIS/HC1 buffer (0.1 M, pH 7.0)
and Jones, 1975). This process is proposed to lead to cell
and homogenized again by ultrasonication (3 x 30 s, 70 W). In
order to isolate the cell wall fraction together with the ionicallywall tightening and thus to a loss of plasticity of the wall.
bound peroxidases, the suspension was centrifuged at 1000 g
Valero et al. (1991) showed that the activity of cell wallfor 10 min. The supernatant contained the intracellular
associated peroxidases in epicotyls of Cicer arietinum was peroxidase fraction. The pellet was resuspended in 5 ml 0.1 M
related to cell wall tightening.
TRIS/HC1 (pH 7.0) and centrifuged again at 6000 g for 10
min. The supernatant was discarded and the pellet washed
Since the presence of certain peroxidases, very often
twice in 0.1 M TR1S/HC1 (pH 7.0). Cell walls were resuspended
cationic isozymes, has been positively correlated with a
in 2 ml 0.1 M TRIS/HC1 (pH 7.0) containing 0.2 M CaCI2 and
number of physiological processes such as cessation of
stirred for 2 h at 4°C to extract ionically-bound, cell wallgrowth, it was hypothesized that growth inhibition caused
associated peroxidases. The extraction was terminated by
centrifugation (18000 g, 20 min). The pellet was resuspended
by the nonasaccharide XXFG was possibly the result of
and centrifuged again. The resulting supernatants were comperoxidase-catalysed processes in the cell wall. During
bined and contained the cell wall-associated peroxidases isolated
auxin-stimulated elongation growth a variety of xyloat pH 7.0. In some experiments peroxidases were isolated by
glucan fragments are released by cellulase-catalysed
incubation with 0.1 M acetate (pH 4.5) containing 0.2 M CaCl 2 .
degradation of wall xyloglucans. The resulting biologicThese fractions contained the cell wall-associated peroxidases
isolated at pH 4.5.
ally active fragments are likely to act in the apoplast or
at the plasma membrane. Interest was, therefore, focused
All enzyme extracts were concentrated by ultrafiltration
(Centricon-10, Amicon, Inc., Beverly, MA, USA).
on ionically-bound, cell wall-associated peroxidases. The
The protein concentration was determined according to
influence of the cell wall-derived xyloglucan oligosaccharBradford (1976).
ides XXFG and XXXG on cell wall-associated peroxidase
isozymes was investigated in the pea epicotyl bioassay.
Separation of isoenzymes by non-denaturing gel electrophoresis
Growth inhibition was examined at the same time. The
Peroxidase isoenzymes were separated by discontinuous polydata support the hypothesis that cell wall-associated
acrylamide gel electrophoresis (separating gel 7.5%, pH 4.3;
stacking gel 3.75%, pH 6.8) under non-denaturing conditions,
peroxidase isozymes are activated by XXFG and might
modified from the methods of Reisfeld et al. (1962) and Hames
be involved in XXFG-induced growth inhibition.
Materials and methods
Cell cultures and preparation of xyloglucan-derived
oligosaccharides
Suspension-cultured carrot cells (Daucus carota L.) were maintained as previously described (Seitz et al., 1985). Xyloglucanderived oligosaccharides (XXXG and XXFG) were prepared
from the hemicellulosic fraction of cell walls of suspensioncultured Daucus carota cells by the controlled action of cellulase
in vitro. Purification and identification was carried out as
previously described (Emmerling and Seitz, 1990; Warneck and
Seitz, 1993).
Pea epicotyl bioassay
The pea epicotyl bioassay was carried out as previously
described (Warneck and Seitz, 1993), but in a slightly modified
version. These experiments were performed with 6-7-d-old
etiolated pea epicotyls, varying in length from 3.5-6 cm, but
possessing a partly developed second internode (up to 1.3 cm).
After length determination the epicotyls were incubated between
1 h and 10 h with or without varying concentrations of the
XGOs (final concentration 1 0 ~ n - 1 0 ~ 7 M ) in the absence of
exogenously applied phytohormones. After incubation, the final
length was determined again and a 1.5 cm long segment excised
directly below the plumular hook for peroxidase extraction.
The segments were frozen in liquid nitrogen and stored at 18 °C.
and Rickwood (1981). According to their affinity for ionexchange chromatography material, peroxidases are classified
as anionic or cationic isozymes (Nessel and Mader, 1977).
Anionic peroxidases are separated by using a basic pH-value of
the separating gel; cationic isozymes are separated at an acidic
pH. Cathodic separations were performed at pH 4.3 and led to
a separation of cationic isoenzymes. Anodic separations were
carried out at pH 8.8, leading to a separation of anionic
isoenzymes. Samples for electrophoresis (22.5 fil) contained
enzyme extracts (with equal amounts of protein, between 5 and
10 pg as indicated, in 0.1 M TRIS/HC1 buffer, containing 0.2 M
CaCl 2 (pH 7.0) and 4-fold concentrated sample buffer (7.5 ^1)
with 10% glycerol, 1-fold concentrated stacking buffer (375 mM
HOAc, 60 mM K.OH, pH 4.3) and 0.002% phenosafranin as
the tracking dye. A volume of 30 /J was applied to each slot.
The gels were run in a water-cooled (4°C) 'Mighty Small'
system (Pharmacia, Uppsala, Sweden) for 4 h at 9 raA per gel
with HOAc-buffer (140mM, containing 342 mM 0-alanine)
at pH4.5.
Staining for peroxidases
After gel electrophoresis peroxidase activity was detected by
staining with o-dianisidine in the presence of H 2 O 2 (Stegemann
el al., 1983). Peroxidases convert o-dianisidine into a waterinsoluble brown dye. The gels were equilibrated for 5 min in a
solution containing 30 mg o-dianisidine dissolved in 18 ml
methanol and 48 ml 0.25 M NaH 2 PO 4 (pH 5.8). The reaction
was then initiated by the addition of 120 ^1 H 2 O 2 (245 /*M).
Photographs were taken after 30 min. In some experiments,
o-dianisidine was replaced by guaiacol, and the peroxidases
were stained according to Siegel and Galston (1967).
XXFG-induced activation of pea peroxidase
SDS-PAGE
SDS-was conducted according to Laemmli (1970) using a 5%
stacking and a 10% separating gel. Proteins were silver stained
according to Ansorge (1985).
Preparation of crude extracts for ion-exchange
chromotography
Pea epicotyl segments (1.5 cm length) from 6-7-d-old etiolated
pea seedlings with a partly developed second internode were
excised directly below the plumular hook and homogenized as
described above. Cell walls were also prepared as described
above with the exception that the TRIS/HC1 buffer used
throughout the extraction was replaced by Mcllvain buffer
(10 mM citrate, 20 mM Na2HPO4, pH 5.8). Ionically-bound
peroxidase isoenzymes were isolated by incubation with 3 M
LiCl in Mcllvain buffer (pH 5.8) for 2 h at 4°C. The insoluble
material was removed from the crude protein extract by
centrifugation (18000 g, 10 min). The volume was reduced by
ultrafiltration (model 52, Amicon, Witten, FRG; Diaflo PM10).
Desalting and buffer exchange (20 mM Na-acetate, pH 5.0) was
carried out by using PD-10 columns (Sephadex G 25 M,
Pharmacia, Uppsala, Sweden).
Ion-exchange chromatography
The crude protein fraction was applied to a cation-exchange
CM-Sepharose 'Fast-Flow column' (2.6x16 cm) equilibrated
with 20 mM sodium acetate buffer (pH 5.0), at a flow rate of
1.5 ml min"1 (Pharmacia Biotech with a GradiFrac system and
HiLoad-pump P-50, Uppsala, Sweden). Bound proteins were
eluted by applying a linear gradient from 20 to 600 mM
Na-acetate buffer (pH 5.0). Fractions of 8 ml were collected
and monitored at 280 nm (Monitor UV-1, Pharmacia Biotech,
Uppsala, Sweden). The fractions were assayed for peroxidase
activity with guaiacol and H2O2 according to Siegel and Galston
(1967). Fractions corresponding to the major cationic peroxidases were pooled, concentrated by ultrafiltration (Centricon-10,
Amicon, Beverly, MA, USA) and submitted to non-denaturing
gel electrophoresis suitable for the separation of cationic
peroxidases as well as to isoelectric focusing.
inhibition by XXFG was observed (Warneck and Seitz,
1993). Since quantitative assays for peroxidases do not
distinguish between individual isozymes, peroxidases were
separated by non-denaturing gel electrophoresis. In these
experiments there was a focus on cationic isozymes
because anionic isozymes were not detectable even when
separated under basic conditions. Using a gel system with
an acidic pH-value it was possible to separate five different
cationic isozyme groups named Cl to C5 (Fig. 1). This
isozyme pattern was typical of cell wall-associated peroxidases and was independent of the pH-value (4.5 or 7.0)
of the buffer used. Intracellular peroxidases showed a
different pattern of isozymes compared to the cell wallassociated peroxidases, but lacked the C4 and C5 isozyme
groups when separated at an acidic pH. The incubation
with the nonasaccharide XXFG caused an increase in
peroxidase activity of distinct isoenzyme groups between
1 h and 10 h. Samples extracted from pea epicotyls
incubated with different concentrations of the nonasaccharide showed a clear increase of the isozyme groups C4
and C5, with either one or both of them being activated
(Fig. 1). At the same time XXFG, inhibited endogenous
growth up to 26% (Fig. 2). The increase in peroxidase
activity as well as XXFG-induced growth inhibition was
not restricted to a certain concentration but varied
between the experiments. In some experiments all the
XXFG concentrations caused a clear activation of distinct
isoenzyme groups compared to the control independent
of the incubation time (Fig. 3). In order to prove that
the increase in enzyme activity of the isozyme groups C4
C.PRX
Isoelectric focusing
Ultrathin-layer isoelectric focusing was performed in 200 ^m
polyacrylamide gels (pH gradient 2-11) according to Radola
(1980) using a flat-bed apparatus (Pharmacia-LKB, Freiburg,
FRG). Peroxidase activity was detected by staining with
diaminobenzidine and benzidine according to Gebhardt el al.
(1982). Commercially available horseradish peroxidase from
Sigma (Deisenhofen, FRG) was used as a standard.
C1
C2
C3
XXFG
•vniit
C4
MUttM
Statistical significance
Each experiment consisted of 18-20 epicotyls per oligosaccharide concentration and control. Each experiment was repeated
at least three times on separate days. The elongation of the
XXFG-treated epicotyls was significantly different from that
of the control by Student's Mest. (a) />^0.001; (b)
0.001 <,P<,0.005; (c) 0.005^^^0.01; (d) 0.01 ^P^O.025; (e)
0.025 ^ P < 0.05; (f) P^O.OS; (a-e = significantly different; f=
not significantly different).
Results
Peroxidases were extracted from a 1.5 cm long segment
excised directly below the plumular hook. In this region
maximum elongation growth as well as maximum growth
1899
C5
-3
_fc
c
0
-3
-fc
e
0
s
r-
^
—
r
l l
CD
CD
=
o->
11
CD
2:
ON
CD
5=
?^
I
CD
Fig. 1. Cathodic separation of ionically-bound, cell wall-associated
cationic isoperoxidases under non-denaturing conditions. Before peroxidase-extraction pea epicotyls were treated with varying concentrations
of XXFG ( 1 0 " " , 10"', 10" 7 M) for 7 h. The controls were incubated
without XXFG. Peroxidases were isolated from the growth region of
etiolated pea epicotyls. Each lane contains peroxidase-isozymes isolated
from 20 epicotyls and was loaded with 8 fig protein. Peroxidase activity
was visualized by staining with o-dianisidine in the presence of H 2 O 2 .
1900
Warneck et al.
C.PRX
30
•
C2
20
i
C3
o
en
XXFG
1 111
I! !!
C1
3?
XXXG
C4
10
g
-5
_t
CZ
O
0.01
1.0
100
concentration of nonasaccharide [nM]
Fig. 2. Inhibition of endogenous growth of etiolated pea epicotyls by
XXFG. The data show the growth inhibition caused by the nonasaccharide in the same experiment as shown in Fig 1. Inhibition of growth by
the nonasaccharide (%) is related to the control incubated in the
absence of XXFG. Growth inhibition was calculated as previously
described (Warneck and Seitz, 1993). Each value is the mean of 38—40
epicotyls; a-f indicate the significance of difference from the control.
C.PRX
x:
r-
2:
o
1
1
O
O
2:
?f
I
O
o
c
0
21
1
0
0
2:
21
1
T
0
Fig. 4. Influence of the xyloglucan oligosacchandes XXXG and XXFG
on the activity of lonically-bound, cell wall-associated peroxidases of
etiolated pea epicotyls. Pea epicotyls were treated for 6 h with varying
concentrations (10" u , 10"', 10"7 M) of the oligosacchandes in the
pea epicotyl bioassay. The controls were incubated without XGOs.
Each lane contains isoperoxidases isolated from 18-20 epicotyls and
was loaded with 9.8 ^g protein. The cathodic separation was earned
out under non-denaturing conditions.
C4
30
XXFG
XXXG
a? 20
10
Fig. 3. Cathodic separation of ionically-bound, cell wall-associated
cationic peroxidase isozymes isolated from the growth region of
etiolated pea epicotyls. This figure shows only the C4 isozyme. Before
peroxidase-extraction pea epicotyls were treated with varying concentrations (ltT 11 , 10"9, 10"7 M) of XXFG for 3, 5 and 7 h. Each lane
contains peroxidase-isozymes extracted from 18-20 epicotyls and was
loaded with 18 ^g protein. Peroxidase activity was visualized by staining
with o-dianisidine in the presence of H2O2.
and C5 was not just the result of an oligosaccharide effect
in general, but a specific reaction to XXFG, the influence
of the corresponding heptasaccharide XXXG with that
of XXFG on peroxidase activity was compared. XXXG
lacks the fucosyl-galactosyl side-chain which is responsible for the growth-inhibiting effect of the XGOs.
Peroxidase activity was extracted after 6 h of incubation
with varying concentrations of the oligosacchandes. All
concentrations of XXXG tested (KT 11 , 10"9, 10"7 M)
failed to cause any distinct increase of enzyme activity
(Fig. 4) whereas 10~7 M and 10~9 M concentrations of
XXFG clearly enhanced the activity of the isozyme group
C5. C4 was activated by treatment with 10 ~7 M XXFG
-10
I
Y///S1
f
f
f
0.01 1.0
100
0.01 1.0
100
concentration of ollgosaccharlde [nM]
Fig. 5. Influence of XXXG and XXFG on endogenous growth of
etiolated pea epicotyls after 6h of incubation. The data show the
influence of the XGOs on elongation growth in the same experiment as
shown in Fig. 4. Inhibition of growth (%) is related to the control
incubated in the absence of XGOs. Each value is the mean of 18-20
epicotyls; a-f indicate the significance of difference from the control.
and slightly activated by 10" n M XXXG. At the same
time the heptasaccharide showed no growth inhibition
(Fig. 5). XXFG inhibited endogenous growth up to 30%
at 10"11 M.
XXFG-induced activation of pea peroxidase
1901
9
In the same experiment 10 M XXFG inhibited
growth up to 20% after 9 h (data not shown). Peroxidase
activity had already decreased by that time. Epicotyls
treated with 10"" M XXFG still showed growth inhibition of 34% while peroxidase activity stayed at the level
of the controls. From these experiments it is concluded
that the activation of certain peroxidase isozymes takes
place some time before growth inhibition occurrs and
slowly decreases afterwards. This would imply that 10" n
M XXFG had caused an activation of the isozyme groups
C4 and C5 earlier and that the activity had already
decreased after 6 h resulting in a clear growth inhibition.
In some experiments a slight activation of the C4 or
C5 isozyme group was observed in one of the controls or
even XXXG-treated samples. Interestingly, elongation
growth of these samples was also reduced compared with
parallel samples treated identically. Since the endogenous
concentration of XXFG in the epicotyls is unknown it
could be supposed that the slight activation which sometimes occurrs in non-treated samples is due to the endogenous concentration of XXFG in the epicotyls. The
endogenous XXFG concentration might also be the
reason for variations in the most effective concentration
of XXFG as well as variations in the percentage of
growth inhibition caused by the nonasaccharide throughout the experiments. When peroxidases of XXFG-treated
samples were separated by SDS-PAGE, no additional
protein bands compared to the controls appeared (data
not shown), even though XXFG had caused a clear
activation of the C4 and C5 isozyme groups when separated by non-denaturing gel electrophoresis. In order to
characterize the XXFG-activated isozymes in more detail,
the crude extract of cell wall-associated peroxidases
extracted with high salt concentrations, e.g. LiCl (3 M),
was separated by cation-exchange chromatography equilibrated with 20 mM Na-acetate and bound proteins
eluted with a linear Na-acetate gradient. Five peaks of
activity were obtained by assaying for peroxidase activity
using guaiacol as a substrate (Fig. 6).
The elution profile was similar to the one Valero et al.
(1991) obtained from cell wall-associated peroxidases of
Cicer arietinum epicotyls. In the experiments, the amount
of two fractions designated as Px-1 and Px-2 was correlated with the age of the epicotyls. Px-2 showed an inverse
relationship to their growth capacity. The shoulder of
Px-1 eluted at the same concentration as peak 3 containing
the isozyme groups C4 and C5 that were increased by
XXFG-treatment.
The fractions under the peaks were pooled, concentrated and submitted to non-denaturing gel electrophoresis under acidic conditions. The isozyme group C4 which
was activated by XXFG eluted in peak 3, with a slight
contamination in peak 4 (Fig. 7). The isozyme group C5
was also present in peak 3, but the activity in this
preparation was too low to be visible in Fig. 7. The five
80 fractions
Fig. 6. Elution profile of CM-sepharose ion-exchange chromatography
of the lomcally-bound cell wall proteins from 6-d-old etiolated pea
epicotyls. Proteins were eluted by a linear gradient from 20 to 600 mM
Na-acetate buffer (pH 5.0). Proteins were monitored at 280 nm (O).
Peroxidase activity was measured with guaiacol as the substrate (A).
C.PRX
C1
C2
C3
crude 1 2
extract
4
5
wmm
1* Ik i
II
U
C5
3
i
Fig. 7. Cathodic separation under non-denaturing conditions of the five
peroxidase activity peaks obtained by ion-exchange chromatography
(Fig. 6). Lane 1: crude extract before chromatography; lane 2: aliquot
of peak 1; lane 3: aliquot of peak 2; lane 4: aliquot of peak 3; lane 5:
aliquot of peak 4; lane 6: aliquot of peak 5. In lanes 1-6 the peroxidase
activity loaded was between 1.5 and 2.0 A A min"1 in 50 ^1 using
guaiacol as a substrate.
peaks were additionally separated by isoelectric focusing.
All the five peaks obtained by cation-exchange chromatography contained mainly cationic isozymes (Fig. 8) with
a pi between 9.5 and 7.0, which confirmed the results of
the non-denaturing gels as well as a group of slightly
anionic isozymes with a pi between 7.0 and 6.0. The
crude extract contained a slight amount of one strongly
anionic isoenzyme with a pi of 3.0, which was lost by
cation-exchange chromatography. The pis of the isozymes
in peaks 1 and 2 were all located between pH 8.0 and
8.7. The pis of the isozymes in peak 3 were located at
pH 9.5, 8.5, 8.0, and between pH 8.0 and 6.5. The pis of
the isozymes in peak 4 were located around pH 9.0,
1902
Warneck et al.
X
X
1 2
3
4
at
anode
cathode
Fig. 8. Ultrathin-layer isoelectnc focusing (pH 2-11). Lanes 1-5 were
loaded with an aliquot of the peaks containing peroxidase activity after
ion-exchange chromatography (Fig. 6). Lanes 1-6 were loaded with
peroxidase activity between 1.5 and 2.0 AA min"1 in 50 jA using
guaiacol as the substrate. 1' peak 1; 2- peak 2; 3: peak 3; 4: peak 4; 5:
peak 5; 6: crude extract before ion-exchange chromatography; 7:
commercial horseradish peroxidase.
pH 8.0 and between pH 7.0 and 6.0. The isozymes in
peak 5 were located between pH 6.5 and 5.5 showing a
slightly anionic character. From the IEF data it was not
possible to asign each isozyme to one of the five isozyme
groups obtained on the non-denaturing gels. The data
imply that each isozyme group consists of several different
peroxidase-isozymes.
Discussion
The xyloglucan-derived nonasaccharide XXFG inhibits
elongation growth in general (Warneck and Seitz, 1993)
independent of the phytohormone applied to the system.
The role of peroxidases in the control of plant growth
includes the regulation of the indole-3-acetic acid (IAA)
levels through oxidative catabolism as well as their participation in the loss of plasticity of the cell walls through
the phenolic cross-linking of cell wall polymers (Biggs
and Fry, 1987) such as via isodityrosine or diferulate.
The cross-linking of wall polymers results in an irreversible tightening which leads to resistance against turgordriven expansion.
Since peroxidases are very often induced during plant
pathogen interactions (Gaspar et al., 1982; Flott et al.,
1989) it should be mentioned that the biologically active
XXFG does not show any elicitor activity (Warneck,
1994).
It has been shown that varying concentrations of
XXFG increased the extractable activity of distinct peroxidase isozymes in the first 10 h of incubation. At the same
time growth was significantly inhibited. In some experiments the increase in peroxidase activity was independent
of the nonasaccharide concentration applied and the
incubation time. Variations in the most effective concentrations of the nonasaccharide are probably due to the
short incubation times and the unknown concentrations
of endogenous XXFG in each epicotyl. Normally, growth
inhibition in the pea epicotyl bioassay is assayed after
24 h of incubation (Warneck and Seitz, 1993). Shorter
incubation times lead to greater variations in the growth
rate which normally disappear within 24 h. The reason
for carrying out these short incubation times was that
XXFG-induced growth inhibition was already achieved
after 3-5 h. In these experiments peroxidase activity had
increased by this time. Similar observations were made
in suspension cultures of carrot (Warneck, 1994).
Incubation of carrot cells with nanomolar concentrations
of XXFG caused an increase in activity of certain cell
wall-associated peroxidase isozyme groups up to 10 h.
Unfortunately, elongation growth and its inhibition
cannot readily be measured in these cell cultures. The
postulated involvement of peroxidase-catalysed tightening
in XXFG-induced growth inhibition implicates an activation of peroxidases being correlated with the early appearance of growth inhibition. The role of peroxidases in
growth has been shown directly (Zheng and van Huystee,
1992) or indirectly (Goldberg et al, 1986a, b, 1987) by
the development of the enzyme activity along the growth
gradient of hypocotyls. Valero et al. (1991) examined
changes in the occurrence of cell wall-associated peroxidases in epicotyls of Cicer arietinum. The activity of two
isolated fractions designated as Px-1 and Px-2 was correlated with the age of the epicotyls. The activity of Px-1
decreased when the epicotyl age advanced showing a
slightly increasing shoulder. Peroxidase activity of Px-1
was clearly increased at pH-values lower than 5.0. The
elution profile was similar to the one obtained from pea
epicotyls. The shoulder of Px-1 eluted at the same concentration as peak 3 containing the isozyme groups C4 and
C5 that were activated by XXFG. Further characterization of these isozymes concerning the pH-optimum would
be of great interest. The higher activity of the fraction
Px-1 at low pH-values might thus be due to processes
initiating the end of elongation growth. It is suggested
that different isozymes might control successive steps in
the process of cell wall tightening.
All the ionically-bound, cell wall-associated peroxidases
isolated from pea epicotyls belonged to the group of
cationic or slightly anionic isozymes as proved by nondenaturing gel electrophoresis and IEF. Only one strongly
anionic isozyme with a pi of 3.0 was present in the cell
wall of pea. In peanut cationic peroxidase is the predomin-
XXFG-induced activation of pea peroxidase
ant isozyme in the cell wall (Zheng and van Huystee,
1991a, b) or the medium (Hu et al., 1989), although
anionic isozymes were described (van Huystee, 1990;
Zheng and van Huystee, 1991a). The cationic peroxidase
isolated from the medium of cell cultures of peanut had
a high homology with ionically and covalently-bound cell
wall peroxidase (Zheng and van Huystee, 19916). Both
cationic and anionic ionically-bound isozymes were found
in the cell walls of lupin (Ferrer et al., 1992; Ros Barcelo
et al., 1987, 1988) and of mung bean (Goldberg et al.,
1986a, b, 1987). Siegel and Galston (1967) examined the
patterns of isoperoxidases in different organs of tall and
dwarf pea cultivars. Interestingly, the pattern for cationic
peroxidases in shoots was similar to the one in our system.
They found anionic isozymes in extracts of shoots, but
they were not classified as cell wall-located enzymes.
Many attempts have been made to define clear functions
for anionic or cationic peroxidase isozymes (Gaspar et al.,
1985). Cationic isozymes were found to be more effective
in IAA-oxidation (van den Berg et al., 1983) which leads
to a clear alteration in the concentration of IAA and the
phytohormone level in general (Trewavas, 1991). Anionic
isozymes have been suggested to be involved in crosslinking of cell wall polymers and in the lignification
process (Gaspar et al., 1985) although cationic peroxidases have been described in this process as well (Zheng
and van Huystee, 1991a; van Huystee and Zheng, 1993).
In peanut, both the cationic and the anionic isozymes
were able to oxidize tyrosine (Zheng and van Huystee,
1991a), but with different pH-optima, although the cationic isozyme is the major peroxidase in the cell wall of
peanut (Hu et al., 1989). Additionally in experiments on
the oxidation of ferulic acid, antibodies against the cationic isozyme were more inhibitory (van Huystee and
Zheng, 1993).
In summary this work supports the hypothesis that
peroxidase-catalysed processes in the cell wall are involved
in XXFG-induced growth inhibition. It is further hypothesized that the activity of XXFG-activated isozymes
interferes with cell elongation and growth processes.
Acknowledgements
This work was supported by the European Communities'
Biotechnology Programme as part of the Project of
Technological Priority, 1993-1996. We wish to thank Professor
SC Fry for valuble discussion. We are grateful to Mrs I Freitag
for performing the isoelectric focusing. We thank Professor
Feucht and his coworkers for discussing the IEF data.
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