Plant Cell Physiol.

43(1): 21–26 (2002)

JSPP © 2002

Action of Xyloglucan Hydrolase within the Native Cell Wall Architecture and
Its Effect on Cell Wall Extensibility in Azuki Bean Epicotyls
Tomomi Kaku 1, Akira Tabuchi 1, 2, Kazuyuki Wakabayashi, Seiichiro Kamisaka 3 and Takayuki Hoson 4
Department of Biological Sciences, Graduate School of Science, Osaka City University, Sugimoto, Sumiyoshi-ku, Osaka, 558-8585 Japan

Xyloglucan hydrolase (XGH) has recently been puri-
fied from the cell wall of azuki bean (Vigna angularis Ohwi
et Ohashi) epicotyls as a new type of xyloglucan-degrading
enzyme [Tabuchi et al. (2001) Plant Cell Physiol. 42: 154].
In the present study, the effects of XGH on the mechanical
properties of the cell wall and on the level and the molecu-
lar size of xyloglucans within the native wall architecture
were examined in azuki bean epicotyls. When the epider-
mal tissue strips from the growing regions of azuki bean
epicotyls were incubated with XGH, the mechanical exten-
sibility of the cell wall dramatically increased. XGH exoge-
nously applied to cell wall materials (homogenates) or epi-
dermal tissue strips decreased the amount of xyloglucans
via the solubilization of the polysaccharides. Also, XGH
substantially decreased the molecular mass of xyloglucans
in both materials. These results indicate that XGH is capa-
ble of hydrolyzing xyloglucans within the native cell wall
architecture and thereby increasing the cell wall extensibil-
ity in azuki bean epicotyls.
Key words: Azuki bean (Vigna angularis) — Cell wall exten-
sibility — Epicotyl — Plant growth — Xyloglucan hydrolase.
Abbreviations: EXGT, endo-xyloglucan transferase; XET,
xyloglucan endotransglycosylase; XGH, xyloglucan hydrolase.
Introduction
The capacity of the cell wall to extend (cell wall extensi-
bility) has been regarded as an important factor determining the
rate of plant cell growth (Taiz 1984, Cosgrove 1986, Masuda
1990). Biochemical modifications of the cell wall, such as
changes in the chemical structures and quantities of cell wall
polysaccharides, have been considered to be involved in the
regulation of wall extensibility (Taiz 1984, Masuda 1990,
Carpita and Gibeaut 1993). The primary cell walls of growing
tissues are mainly composed of cellulose microfibrils and
matrix polysaccharides (hemicellulose and pectin). In the cell
walls of dicotyledonous plants, xyloglucans are the major
hemicellulosic polysaccharides and account for as much as 20–
1
These authors contributed equally to this work.
2
Present address: Research Institute for Bioresources, Okayama University, Chuo, Kurashiki, 710-0046 Japan.
3
Present address: Department of Biology, Faculty of Science, Toyama University, Gofuku, Toyama, 930-8555 Japan.
4
Corresponding author: E-mail, hoson@sci.osaka-cu.ac.jp; Fax, +81-6-6605-2577.
;
25% of the constituents of the primary cell walls (Hayashi
1989). Also, xyloglucans bind tightly to cellulose microfibrils
via hydrogen bonds and serve as cross-links between cellulose
microfibrils (Fry 1989, Hayashi 1989). Therefore, xyloglucan
molecules probably give rigidity to the cell wall. Indeed, dur-
ing auxin-induced cell wall loosening and cell elongation of
stem segments of dicotyledons, auxin stimulates the breakdown
of xyloglucans (Hayashi 1989, Masuda 1990, Hoson 1991).
The results that xyloglucan-binding lectins and antibodies
raised against xyloglucan oligosaccharides suppress auxin-
induced cell elongation and cell wall loosening as well as the
breakdown of xyloglucans, support the view that the break-
down of xyloglucans is an essential process for auxin-induced
cell wall loosening responsible for cell elongation in stems of
dicotyledons (Hoson 1991, Hoson 1993). Also, xyloglucan
metabolism is greatly modified during growth regulation by
environmental signals such as light (Miyamoto et al. 1997) and
gravity (Soga et al. 1999, Soga et al. 2000).
Xyloglucans are actively metabolized in the cell wall apo-
plast by the action of wall enzymes. Endo-1,4-b-glucanases
and endo-xyloglucan transferases (EXGTs, also called xyloglu-
can endotransglycosylases, XETs) have been supposed to be
involved in the breakdown and modification of xyloglucan
molecules. Endo-1,4-b-glucanases are hydrolases capable of
cleaving 1,4-b-glucosyl linkages in several glucans, including
xyloglucans, and have also been termed cellulase because of
their potential hydrolytic activity towards cellulose (Wong et
al. 1977, Hayashi and Maclachlan 1984). This enzyme is asso-
ciated with cell wall dissolution during tissue softening and
abscission (Lashbrook et al. 1994). EXGTs cleave xyloglucan
polymers endolytically and then rejoin one of the newly gener-
ated free ends with another xyloglucan chain (Fry et al. 1992,
Nishitani and Tominaga 1992, Nishitani 1997). The physiologi-
cal role of EXGTs in plant growth processes has not been clari-
fied, because EXGTs do not appear to cause extension of iso-
lated cell walls in vitro (McQueen-Mason et al. 1993).
Conventional EXGTs lack hydrolase activity and it is likely
that they are involved in the molecular grafting or the modifi-
cation rather than the breakdown of xyloglucans in the cell wall.
Recently, a new type of EXGT showing hydrolytic activity
towards xyloglucans was purified from the cell wall of azuki
bean epicotyls (Tabuchi et al. 2001). This enzyme, termed

21
22 Xyloglucan hydrolase induces wall loosening

Fig. 1 The amount of polysaccharides in XGH-treated cell wall materials. The wall homogenates of azuki bean epicotyls were incubated in
XGH solution, and then fractionated into four fractions. Total sugar content in each fraction was determined by the phenol-sulfuric acid method.
Xyloglucan content in the hemicellulose II fraction was determined by the iodine staining method. XG, xyloglucan. Data are means ± SE of
results from three independent samples.

xyloglucan hydrolase (XGH), hydrolyzes xyloglucans in an

endo-type manner up to 5 kDa fragments, without showing any
endotransferase activity. Partial amino acid sequences of XGH
shared an identity with the EXGT family (Tabuchi et al. 2001),
and XGH appears to belong to group 3 or a new independent
group in the classification by Campbell and Braam (1999). XGH
is believed to be involved in xyloglucan breakdown necessary
for the wall loosening. However, such a mode of action was
demonstrated by the analysis of their actions on isolated
xyloglucans, and the catalytic properties of XGH on xyloglucans
within the intact cell wall architecture have not been studied yet.
Also, whether this enzyme can influence the mechanical proper-
ties of the cell wall has not been examined. The present study
was carried out to clarify these points using purified XGH. The
results indicated that XGH increased the cell wall extensibility
of epidermal tissues of azuki bean epicotyls by hydrolyzing
xyloglucans within the native cell wall architecture.

Results

Effects of XGH on levels and molecular mass of xyloglucans in

Fig. 2 The amount of sugars released from XGH-treated cell walls.
The polymers released during enzyme treatment were hydrolyzed and
their sugar compositions were determined by gas-liquid chromatogra-
phy. Rha, rhamnose; Fuc, fucose; Ara, arabinose; Xyl, xylose; Gal,
galactose; Glc, glucose. Data are means ± SE of results from three
independent samples.
the cell wall material
The cell wall materials (homogenates) prepared from the
growing regions of dark-grown azuki bean epicotyls were
treated with purified XGH. Treated cell wall material was frac-
tionated into the pectin, hemicellulose I, hemicellulose II, and
cellulose fractions, and the amount of polysaccharides in each
fraction was determined (Fig. 1). The treatment with XGH
decreased the amount of hemicellulose II by about 10%,
whereas it did not affect the amounts of pectin, hemicellulose I,
or cellulose.
Xyloglucans are the principal polysaccharides in the
 Xyloglucan hydrolase induces wall loosening 23

Table 1 The mechanical properties of the cell wall of XGH-treated epidermal tissues
Extensibility (mm g–1) Minimum stress-relaxation time (ms) Relaxation rate (%)
Control 3.8±0.2 35.1±0.6 5.9±0.1
XGH 19.0±0.7* 31.7±0.4* 6.1±0.1
The mechanical extensibility, the minimum stress-relaxation time, and the relaxation rate of the cell wall of
the isolated epidermal tissue strips were measured with a tensile tester. Data are means ± SE (n = 30). Aster-
isks denote a significant difference from the denatured control at 5% level.

hemicellulose II fraction of dicotyledons. Therefore, the
amount of xyloglucans in this fraction was determined by the
iodine staining method (Fig. 1). In azuki bean epicotyl cell
walls, xyloglucans accounted for about 75% of the total
polysaccharides in the hemicellulose II fraction. XGH
decreased the amount of xyloglucans by 15% during incuba-
tion, which corresponded to the decrease in total polysaccha-
rides in the hemicellulose II fraction. These results suggest that
XGH degraded xyloglucans and induced their solubilization.
To ascertain this possibility, the sugar composition of the mate-
rials released from the cell wall during incubation was ana-
lyzed by gas-liquid chromatography (Fig. 2). Released materi-
als were composed of rhamnose, fucose, arabinose, xylose,
galactose, and glucose. XGH caused increases in the amounts
of fucose, xylose, and glucose, which are major components of
xyloglucan molecules. However, XGH did not influence the
amounts of other sugars. The result supports the view that
XGH promoted the solubilization of xyloglucans and caused a
decrease in their levels in the cell wall materials.
To examine the effect of XGH on the molecular size of
xyloglucans, the extracted hemicellulose II polysaccharides were
separated with a gel filtration column on an HPLC system (Fig.
3). The elution pattern of xyloglucans extracted from XGH-
treated cell wall materials greatly shifted towards the low molec-
ular mass regions. The calculated weight-average molecular
mass of xyloglucans extracted from XGH-treated cell wall mate-
rials was remarkably lower than that from denatured control.
Levels and molecular mass of xyloglucans in the cell wall of
XGH-treated epidermal tissues
The epidermal tissue strips were homogenized and frac-
tionated into the pectin, hemicellulose I, hemicellulose II, and
cellulose fractions. The changes in the amounts of polysaccha-
rides in each fraction due to enzyme treatment were examined.
XGH decreased the amount in the hemicellulose II fraction
(Fig. 4). Such a decrease was completely due to the decrease in
xyloglucan levels. In addition, XGH slightly decreased the
amount of cellulose.
Xyloglucans in XGH-treated epidermal tissues eluted in
the low molecular mass regions as compared with those in con-
trol tissues on a gel filtration column (Fig. 5). Therefore, XGH
clearly decreased the weight-average molecular mass of
xyloglucans.
Discussion
The reaction of an enzyme depends on the surrounding
environment, such as pH and the presence of other molecules.
Thus, the mode of action of the cell wall enzymes, demon-

The mechanical properties of the cell wall of XGH-treated

epidermal tissues
The effects of purified XGH on the mechanical properties
of cell walls were examined in azuki bean epicotyls. Because
auxin is believed to induce elongation growth of stems by caus-
ing cell wall loosening in the epidermal tissues (Tanimoto and
Masuda 1971, Kutschera and Briggs 1987), and because the
loosening of the epidermal cell walls is considered to be due to
xyloglucan breakdown in dicotyledons (Wakabayashi et al.
1991, Hoson et al. 1993), the epidermal tissue strips isolated Fig. 3 Molecular mass distributions of xyloglucans extracted from
from the epicotyls were treated with the enzyme. XGH greatly XGH-treated cell wall materials. Polysaccharides in the hemicellulose
increased (up to five times) the cell wall extensibility of the II fraction were separated with a gel filtration column on an HPLC sys-
tem. Xyloglucans were detected by the iodine staining method. The elu-
epidermal tissues (Table 1). This result indicates that XGH is
tion positions of molecular mass standards and the void volume (V0)
able to increase the capacity of the cell wall to extend. XGH are shown at the top. Each elution profile is the mean of three inde-
also decreased the minimum stress-relaxation time, but the pendent samples. The weight-average molecular mass of xyloglucans
relaxation rate did not change significantly. was calculated from the elution profiles and shown with SE (n = 3).
24 Xyloglucan hydrolase induces wall loosening

Fig. 4 The amount of cell wall polysaccharides in XGH-treated epidermal tissues of epicotyls. The isolated epidermal tissue strips of azuki bean
epicotyls were incubated in XGH solution, and then homogenized and the wall materials were fractionated into four fractions. Total sugar content
in each fraction was determined by the phenol-sulfuric acid method. Xyloglucan content in the hemicellulose II fraction was determined by the
iodine staining method. XG, xyloglucan. Data are means ± SE of results from three independent samples.

strated by the analysis of their actions on isolated substrates, epidermal tissue strips of azuki bean epicotyls (Table 1). The
does not always reflect their behavior in the actual cell wall wall extensibility of the treated strips was five times that of the
architecture (Hoson 1993). Using isolated xyloglucans, XGH denatured control. XGH also decreased the minimum stress-
was shown to have xyloglucan-specific hydrolase activity relaxation time (Table 1). This parameter is another good meas-
(Tabuchi et al. 2001). However, the nature of this enzyme’s ure of the degree of cell wall loosening and seems to represent
action in the cell wall in situ had not been clarified. The present the viscous nature of the cell wall (Yamamoto et al. 1970). The
study clearly showed that XGH hydrolyzed xyloglucans in an decrease in the minimum stress-relaxation time means that
endo-type manner within the intact cell wall architecture as in XGH decreases the viscosity of the cell wall, which is sup-
vitro. In the cell wall materials prepared from the growing posed to be caused by the decrease in molecular size of their
regions of azuki bean epicotyls, XGH substantially decreased
the amount and the molecular mass of xyloglucans (Fig. 1, 3).
Also, XGH enhanced the release of fucose, xylose, and glu-
cose, which are major components of xyloglucan molecules,
from the cell wall (Fig. 2), indicating that the solubilization of
xyloglucans underlies such changes.
XGH was also capable of hydrolyzing xyloglucans when
applied to the epidermal tissue strips of azuki bean epicotyls
(Fig. 4, 5). The result indicates that XGH molecules can pass
through the pores in the cell wall, reach the proper substrates,
and then react with them in a manner as in vitro. The diameter
of the cell wall pores has been estimated as 8 to 10 nm, which
limits the diffusion of proteins with a size of 100 kDa and
above (Hoson 1993). Because the molecular size of XGH is
33 kDa (Tabuchi et al. 2001), the enzyme appears to possess
enough mobility within the cell wall architecture. In the epider-
mal tissue strips, XGH induced a slight decrease in the amount
of cellulose, in addition to that in xyloglucans (Fig. 4). There Fig. 5 Molecular mass distributions of xyloglucans extracted from
would be a possibility that the breakdown of xyloglucans indi- XGH-treated epidermal tissues of epicotyls. Conditions of HPLC-gel
filtration chromatography are as shown in Fig. 3. Each elution profile
rectly influences the stability of cellulose microfibrils.
is the mean of three independent samples. The weight-average molecu-
The most striking result of the present study is that XGH lar mass of xyloglucans was calculated from the elution profiles and
greatly increased the cell wall extensibility, when applied to the shown with SE (n = 3).
 Xyloglucan hydrolase induces wall loosening
constituents (Masuda and Yamamoto 1985, Sakurai 1991).
Thus, the decrease in the molecular mass of xyloglucans by
XGH corresponds well with the changes in the mechanical
properties of the cell wall.
The present study indicates that XGH is capable of hydro-
lyzing xyloglucans within the intact cell wall architecture of
azuki bean epicotyls as in vitro, thereby increasing cell wall
extensibility. Plant cell walls contain another group of wall-
loosening proteins, termed expansins (Cosgrove 2000).
Expansins have the capacity to promote extension of isolated
cell walls in vitro, without showing a glycanolytic or EXGT
activity (McQueen-Mason et al. 1992, McQueen-Mason et al.
1993). The exact mechanism of expansin action is still
unknown, but it has been suggested that expansins disrupt
hydrogen bonds between cellulose microfibrils and hemicellu-
loses (McQueen-Mason and Cosgrove 1995). Expansins are
active on composites containing xyloglucans, indicating that
cellulose–xyloglucan complex is the site of expansin action
(Whitney et al. 2000). It has also been suggested that expansins
could increase the accessibility of xyloglucans to cell wall
enzymes such as XGH. Therefore, the interactions between
XGH and expansins may be important in the regulation of the
mechanical properties of the cell wall. The cloning of XGH
gene and the analysis of its expression pattern under various
growth conditions will elucidate further the role of the enzyme
in the regulation of plant cell growth.
Materials and Methods
Plant material
Seeds of azuki bean (Vigna angularis Ohwi et Ohashi cv. Takara)
were soaked in running tap water at 30°C for 24 h and they were
allowed to germinate and were grown on gauze in a plastic dish filled
with water at 25°C in the dark. After 5 d, seedlings which had 40–
60 mm long epicotyls were selected, and 10 mm segments were
excised from the region 5–15 mm below the epicotyl top. For prepara-
tion of the epidermal strips, the epidermal tissues were peeled from the
excised segments with a razor and forceps. The whole segments and
the isolated epidermal tissue strips were immediately boiled in metha-
nol at 80°C for 10 min and stored in fresh methanol at 4°C.
Enzyme treatment
XGH was purified from the cell wall of azuki bean epicotyls by
the method of Tabuchi et al. (2001). For the treatment of the cell wall
materials (homogenates), the methanol-fixed epicotyl segments were
rehydrated and then homogenized in ice-cold deionized water with a
mortar and a pestle. The homogenate was poured onto polypropylene
mesh (32 mm) and washed successively with acetone, a mixture of
methanol and chloroform (1 : 1, v/v), ethanol, and ice-cold deionized
water. The cell wall materials obtained from 30 segments (ca. 8.5 mg
cell wall equivalents) were suspended in 1.6 ml of 0.2 M sodium phos-
phate buffer (pH 5.0, optimal pH of XGH, Tabuchi et al. 2001), con-
taining 2 mg of purified XGH, and then incubated for 48 h at 37°C. As
a control, the enzyme denatured by heating at 100°C for 5 min was
used. After the incubation, the reaction was terminated by boiling.
Treated cell wall materials were precipitated by centrifugation and the
supernatant was collected as the released sugar fraction.
For the treatment of the epidermal tissue strips, the methanol-
25
fixed epidermal strips were rehydrated with deionized water. The
rehydrated-epidermal strips obtained from 15 segments (ca. 1.8 mg cell
wall equivalents) were suspended in 0.8 ml of 0.2 M sodium phosphate
buffer (pH 5.0) containing 2 mg of purified XGH, and then incubated
for 48 h at 37°C. After the enzyme treatment, the epidermal strips were
transferred to the methanol solution, boiled at 80°C for 10 min to termi-
nate the reaction, and then stored in fresh methanol at 4°C.
Fractionation of the cell wall polysaccharides
Fractionation of cell wall polysaccharides was conducted as pre-
viously reported (Nishitani and Masuda 1979). The enzyme-treated cell
wall materials were treated with 2 units ml–1 a-amylase (from porcine
pancreas, Sigma, St. Louis, MO, U.S.A.) in 50 mM sodium acetate
buffer (pH 6.5) at 37°C for 3 h and then washed with deionized water.
After the amylase treatment, pectic substances were extracted from the
wall materials three times (15 min each) with 50 mM EDTA at 95°C.
Then hemicellulose was successively extracted three times (8 h each)
with 4% (w/v) KOH and 24% (w/v) KOH containing 0.02% NaBH4 at
25°C. The fractions extracted with 4% and 24% KOH were designated
as hemicellulose I and hemicellulose II, respectively. Hemicellulose I
and II fractions were neutralized with acetic acid and then dialyzed
against deionized water. The alkali-insoluble fraction (cellulose frac-
tion) was washed successively with 0.03 M acetic acid and ethanol, and
then dried at 40°C. The dried cellulose fraction was dissolved in 72%
sulfuric acid at 25°C for 1 h, and then diluted with 29-fold volume of
deionized water. Total sugar content in each fraction was determined by
the phenol-sulfuric acid method (Dubois et al. 1956). The amount of
xyloglucans in the hemicellulose II fraction was determined by the
iodine staining method (Kooiman 1960).
The enzyme-treated epidermal tissue strips were rehydrated and
then homogenized in deionized water with a mortar and a pestle. The
homogenate was washed successively with acetone, a mixture of meth-
anol and chloroform (1 : 1, v/v), ethanol, and deionized water. After
the washing, the cell wall materials were treated with a-amylase and
then fractionated as described above.
Determining the sugar composition of released polymers
The released sugar fraction obtained during the treatment of the
cell wall materials with XGH was dried at 50°C under a stream of fil-
tered air. The dried materials were hydrolyzed with 2 M trifluoroace-
tic acid at 121°C for 1 h. The sugar composition of the hydrolyzed
sample was determined by a gas-liquid chromatography according to
the method of Albersheim et al. (1967).
Determining the molecular mass of xyloglucans
The hemicellulose II polysaccharides were lyophilized, and then
samples were dissolved in 1.0 ml of 50 mM potassium-phosphate
buffer (pH 7.2), and a portion of the solution (0.2 ml) was injected into
the gel permeation column (TSK-GEL 5000 PW; Tosoh Co., Ltd.,
Tokyo, Japan) in an HPLC system (LC-6A, Shimadzu Co., Kyoto,
Japan). The sample was eluted with 50 mM potassium-phosphate
buffer (pH 7.2) at a flow rate of 1 ml min–1. Fractions were collected
with a fraction collector (model-203, Gilson, Middleton, WI, U.S.A.)
at 0.5 min intervals. The xyloglucan content in each fraction was
determined by the iodine staining method. The weight-average molec-
ular mass of xyloglucans was calculated according to the following
formula:
Weight-average molecular mass = S × Mi · Wi / S × Wi
where Wi is the xyloglucan content of ith fraction and Mi is the molec-
ular mass of ith fraction estimated from the calibration curve (Nishi-
tani and Masuda 1981). Dextrans of 10, 40, 70, 120 and 500 kDa
26 Xyloglucan hydrolase induces wall loosening
(Sigma) were used as molecular mass markers.
Measuring the mechanical properties of the cell wall
Before the mechanical properties of the cell wall were measured,
the enzyme-treated epidermal tissue strips were rehydrated for 3 h at
4°C with several changes of deionized water. The mechanical proper-
ties of the cell wall were measured with a tensile tester (Tensilon
RTM-25, Toyo Baldwin, Tokyo, Japan). The epidermal tissue strips
were fixed between two clamps (the distance between the clamps was
5 mm) and stretched by lowering the bottom clamp at a speed of
20 mm min–1 until a stress of 10 g was produced. The cell wall exten-
sibility (strain stress–1) was calculated based on the slope of the load-
extension curve obtained between 8 and 10 g. The minimum stress-
relaxation time and the relaxation rate were calculated as reported by
Yamamoto et al. (1970).
Acknowledgments
We thank Ryuji Mori and Mizue Saiki for their technical assist-
ance in the measurement of mechanical properties of the cell wall. The
present study was supported in part by Grants-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science and
Technology, a Grant for Basic Research in Space Station Utilization
from the Institute of Space and Astronautical Science and a Grant for
Ground Research for Space Utilization from Japan Space Forum.
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(Received April 25, 2001; Accepted October 15, 2001)