Plant Cell Physiol. 43(4): 411–418 (2002)
JSPP © 2002
Changes in the Sugar Composition and Molecular Mass Distribution of
Matrix Polysaccharides during Cotton Fiber Development
Hayato Tokumoto 1, Kazuyuki Wakabayashi 1, Seiichiro Kamisaka 2 and Takayuki Hoson 1, 3
1
2
Department of Biological Sciences, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka, 558-8585 Japan
Department of Biology, Faculty of Science, Toyama University, Gofuku, Toyama, 930-8555 Japan
;
Cotton (Gossypium herbaceum L.) fiber development
consists of a fiber elongation stage (up to 20 d post-anthesis)
and a subsequent cell wall thickening stage. Cell wall analysis revealed that the extractable matrix (pectic and hemicellulosic) polysaccharides accounted for 30–50% of total
sugar content in the fiber elongation stage but less than 3%
in the cell wall thickening stage. By contrast, cellulose
increased dramatically after the fiber elongation ceased.
The amounts of extractable xyloglucans and arabinose- and
galactose-containing polymers per seed increased in the
early fiber elongation stage and decreased thereafter. The
amounts of extractable acidic polymers and non-cellulosic
>-glucans (mainly composed of >-1,3-glucans) increased in
parallel with fiber elongation and then decreased. The
molecular masses of extractable non-cellulosic >-glucans,
and arabinose- and galactose-containing polymers
decreased during both fiber elongation and cell wall thickening stages. The molecular mass of extractable xyloglucans also decreased during the fiber elongation stage, but
this decrease ceased during the cell wall thickening stage.
Conversely, the molecular size of acidic polymers in the
extractable pectic fraction increased during both stages.
Thus, not only the amounts but also the molecular size of
the extractable matrix polysaccharides showed substantial
changes during cotton fiber development.
2001). Basra and Malik (1984) divided cotton fiber development into four stages: (i) initiation, (ii) elongation, (iii) secondary wall thickening, and (iv) maturation. The initiation stage
starts at anthesis, followed by the fiber elongation stage. Fiber
cells elongate up to 30 mm within 3 weeks after anthesis (primary wall stage). The subsequent secondary wall growth (3–6
weeks post-anthesis) is marked by the deposition of a thick
wall.
The amount of cellulose per seed increases dramatically
during the secondary wall thickening and maturation stages and
cell walls of mature fibers are composed mostly of cellulose.
By contrast, the primary cell walls of fibers have been reported
to contain 35–50% cellulose (Huwyler et al. 1979). Therefore,
the matrix polysaccharides are dominant constituents in cell
walls of growing cotton fibers. The active turnover of matrix
polysaccharides has been considered to be associated with
plant cell growth (Sakurai 1991). Specific biochemical modifications, such as changes in the quantities and molecular masses
of the matrix polysaccharides are likely to be involved in the
regulation of the mechanical properties of the cell wall.
Researchers have studied the compositions of matrix polysaccharides in growing fibers and found that acidic polymers
(galacturonans), >-glucans (>-1,3-glucans) and xyloglucans
showed significant quantitative changes (Huwyler et al. 1979,
Maltby et al. 1979, Shimizu et al. 1997). The amount of
xyloglucans and acidic polymers per seed increased during the
elongation stage and then decreased after cessation of elongation (Huwyler et al. 1979, Hayashi and Delmer 1988, Shimizu
et al. 1997) while the amount of >-1,3-glucans in cotton fibers
was low during the fiber elongation stage (primary wall synthesis) and rose at approximately the onset of secondary wall synthesis (Maltby et al. 1979). These results indicate that substantial synthesis and degradation of the matrix polysaccharides is
apparent in developing cotton fibers.
The molecular masses of matrix polysaccharides, in addition to their amounts, contribute to determining the viscosity of
cell walls. The molecular masses of matrix polysaccharides in
cell walls of rapidly growing tissues, such as auxin-treated
stem segments, are lower than in those of slowly growing ones
(Nishitani and Masuda 1981, Wakabayashi et al. 1991, Hoson
et al. 1991). Timpa and Triplett (1993) reported the molecular
mass distribution of whole cotton fiber cell walls solubilized
with dimethyl acetamide and lithium chloride. On the other
Key words: Cell wall — Cotton fiber — Elongation — Matrix
polysaccharides — Molecular mass — Gossypium herbaceum.
Abbreviations: Ara, arabinose; DPA, days post-anthesis; Fuc,
fucose; Gal, galactose; Glc, glucose; GLC, gas-liquid chromatography; HC-I, hemicellulose I; HC-II, hemicellulose II; Man, mannose;
NS, neutral sugar; Rha, rhamnose; TS, total sugars; UA, uronic acid;
Xyl, xylose.
Introduction
Fiber cells are an excellent system for studying the mechanisms controlling cell differentiation and cell elongation
(Zhong et al. 2001). Cotton fibers, which differentiate from single epidermal cells of developing cotton seeds without cell
division, are attractive materials in this sense (Kim and Triplett
3
Corresponding author: E-mail, [email protected]; Fax, +81-66605-2577.
411
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Cell walls in growing cotton fibers
Fig. 1 Growth of cotton fibers. Length (open circles) and dry weight
(closed circles) of cotton fibers at different growth stages were measured. Data are means ± SE (n = 30 and 3 for determining fiber length
and dry weight, respectively). DPA, days post-anthesis.
hand, Gokani and Thaker (2000) measured the amounts of low
and high molecular mass xyloglucans, extracted with 1 M and
4 M KOH, respectively, in cotton fiber cell walls. However, the
changes in the molecular size of each wall polysaccharide during cotton fiber development have not yet been described. In
the present study, we measured not only the amounts but also
the molecular masses of the pectic and hemicellulosic polysaccharides in developing cotton fibers.
Results and Discussion
Quantifying cell wall polysaccharides
The length of cotton fibers increased almost linearly up to
20 d post-anthesis (DPA) and reached a final length of ca.
30 mm (Fig. 1). By contrast, the dry weight increased slowly
during the first 20 d and rapidly thereafter (Fig. 1). We
obtained similar results on fiber growth in repeated experiments (in 1997 and 1998). Thus, development of cotton fibers
consists of the fiber elongation stage up to 20 DPA and the following cell wall thickening stage.
The cell walls of cotton fibers were fractionated into the
pectic, the hemicellulose I (HC-I), the hemicellulose II (HC-II),
and the cellulose fractions (Fig. 2). The uronic acid (UA) component of the pectic and the HC-I fractions made up about 60–
70% and 10–35%, respectively, of the total sugars (TS) in those
fractions, while the HC-II fraction contained negligible
amounts of UA (less than 1% of TS). The amount of xyloglucans in the HC-II fractions as determined by the iodine method,
was similar to that of the TS, suggesting that xyloglucans are
the major components of this fraction. The amounts of extractable pectic and hemicellulosic polysaccharides (including
acidic polymers) per seed increased during the fiber elongation
period, and in particular, xyloglucans in the HC-II fraction
increased in the early fiber elongation stage. The amounts of
these polysaccharides then decreased during the cell wall thickening stage. Conversely, the amount of cellulose increased continuously during fiber development, and that increase was
especially dramatic after the cessation of fiber elongation (Fig.
2). The matrix polysaccharides accounted for 30–50% of total
cell wall sugars in the fiber elongation stage but less than 3% in
the cell wall thickening stage.
Fig. 3 shows the changes in the amounts of neutral sugars
(NSs) per seed in the pectic, the HC-I, and the HC-II fractions.
Glucose (Glc) was the major NS constituent in all fractions and
accounted for 50–70% of the total extractable NS in these fractions. It has been shown that >-1,3-glucans are major polysaccharides in the matrix fraction of growing cotton fiber cell
walls and such glucans are easily extracted from fiber cell
walls with hot water and hot dimethyl sulfoxide (Maltby et al.
1979). In the present study the levels of Glc in the pectic and
the HC-I fractions were reduced to less than 10% by treatment
with >-1,3-glucanase (mollusk laminarinase) (data not shown).
Therefore, most of the Glc residues in the pectic and the HC-I
fractions may be components of >-1,3-glucans. The amounts of
Glc in the pectic and the HC-I fractions increased in parallel
with fiber elongation and then decreased during the cell wall
thickening stage. In addition to Glc, the pectic and HC-I fractions contained substantial amounts of arabinose (Ara), galactose (Gal) and rhamnose (Rha). Ara and Gal in both fractions
increased in the early fiber elongation stage and then decreased
before the cessation of fiber elongation. Rha in the pectic fraction also increased in the early fiber elongation stage and then
decreased gradually.
The HC-II fraction contained xylose (Xyl), fucose (Fuc),
Gal, Ara and mannose (Man) residues in addition to Glc.
Xyloglucan molecules in dicotyledons are known to be composed of Glc, Xyl, Fuc, Gal and Ara residues (Darvill et al.
1980, Hayashi 1989). The amounts of Xyl and Fuc increased in
the early fiber elongation stage and then decreased gradually.
The pattern of change in the total amount of Xyl, Fuc and Glc
residues was comparable with that of the xyloglucan content
determined by the iodine method (Fig. 2). Ara and Gal also
increased in the early fiber elongation stage and then decreased
rapidly. The changes in the Ara and Gal levels in the HC-II
fraction were almost consistent with those in the pectic and
HC-I fractions. The amount of Man in the HC-II fraction
Cell walls in growing cotton fibers
413
Fig. 2 Amounts of sugars in the pectic, the HC-I, the HC-II and the cellulose fractions of cotton fiber cell walls. Total sugars (TS, open circles)
and uronic acid (UA, open squares) content of each fraction was determined by the phenol-sulfuric acid method and the carbazole method, respectively, and expressed on the basis of one seed. Xyloglucan (XG, closed squares) content of the HC-II fraction was determined by the iodine
method. Data are means ± SE of results from three independent samples.
Fig. 3 Amounts of neutral sugars in the cell wall fractions of cotton fibers. The amounts of NSs in the pectic (upper panel), the HC-I (middle
panel) and the HC-II (lower panel) fractions were determined by GLC, and expressed on the basis of one seed. Data are means ± SE of results
from three independent samples.
increased during the elongation stage and then decreased gradually. These results suggest that xyloglucans are the major
components of the HC-II fraction and that in this fraction polymers containing Ara, Gal and Man residues are present as
minor components, as shown in stem cell walls of dicotyledons (Nishitani and Masuda 1981, Sakurai et al. 1987).
The analysis of the sugar composition of the cellulosic
fraction showed that this fraction contained about 3% Gal and
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Cell walls in growing cotton fibers
Fig. 4 Elution profiles of the pectic components in cell walls of cotton fibers. The pectic substances extracted from cell walls of 12, 17 and 28
DPA cotton fibers were subjected to gel-permeation chromatography on an HPLC system, and the elute was collected at a rate of 0.5 ml per fraction. The amounts of NS components and UA in each column fraction were determined by GLC and by the carbazole method, respectively, and
expressed on the basis of one seed. Numbers in parentheses are the weight-average molecular mass (in kDa) of each sugar residue calculated from
the elution profile. Data of UA are means ± SE of results from three independent samples. Three independent samples were combined for analyzing molecular mass distributions of NS components. Vertical bars at the top denote the elution positions of authentic dextrans with molecular
masses in kDa, glucose (Glc) and the void volume (V0).
Fig. 5 Elution profiles of the HC-I components in cell walls of cotton fibers. The materials were prepared and the conditions were the same for
gel-permeation chromatography as in Fig. 4. Numbers in parentheses are the weight-average molecular mass (in kDa) of each sugar residue calculated from the elution profile. Data of UA are means ± SE of results from three independent samples. Three independent samples were combined
for analyzing molecular mass distributions of NS components. Vertical bars at the top denote the elution positions of authentic dextrans with
molecular masses in kDa, glucose (Glc) and the void volume (V0).
3% Ara residues in addition to Glc at 14 DPA. The amount of
Ara decreased during fiber elongation, leaving less than 3%
Gal as the only detectable component other than Glc residues
after 21 DPA. These results suggest that most of the matrix
polysaccharides, other than those containing Gal and Ara, were
effectively extracted from the fiber cell walls by the sequential
extraction with hot EDTA, and weak and strong alkali solutions.
Molecular mass of the matrix polysaccharides
Changes in the molecular mass distribution of polysaccharides in the extractable pectic, HC-I, and HC-II fractions dur-
Cell walls in growing cotton fibers
415
Fig. 6 Elution profiles of the HC-II components in cell walls of cotton fibers. The materials were prepared and the conditions were the same for
gel-permeation chromatography as in Fig. 4. Numbers in parentheses are the weight-average molecular mass (in kDa) of each sugar residue calculated from the elution profile. Three independent samples were combined for analyzing molecular mass distributions of NS components. Vertical
bars at the top denote the elution positions of authentic dextrans with molecular masses in kDa, glucose (Glc) and the void volume (V0).
ing fiber development were analyzed by assaying NS and UA
residues in fractions from a gel filtration column on an HPLC
system (Fig. 4, 5, 6). In the fiber elongation stage, the extractable pectic and HC-I fractions contained considerable amounts
of UA (Fig. 2). UA residues in the pectic fraction obtained
from the 12 DPA fibers eluted mainly in the low molecular
mass regions, around fraction 20 and the elution pattern shifted
toward the high molecular mass regions from 17 to 28 DPA
(Fig. 4). Thus, the molecular mass of UA-containing polymers
in the pectic fraction increased during development. In addition to UA, the molecular mass of Rha-containing polymers in
the pectic fraction also increased from 17 to 28 DPA (Fig. 4).
Since Rha is a constituent of rhamnogalacturonans (Darvill et
al. 1980), this result suggests that the molecular mass of Rhacontaining galacturonans in the pectic fraction increased during fiber development, particularly in the wall thickening stage.
By contrast, UA residues in the HC-I fraction showed a
bimodal molecular mass distribution and the average molecular mass of UA-containing polymers decreased during development (Fig. 5).
Ara, Gal and Rha in the pectic and HC-I fractions showed
a bimodal molecular mass distribution: high molecular mass
regions around fraction 10, and low molecular mass regions
around fractions 20–25 (Fig. 4, 5). In both fractions Ara, Gal
and Rha showed similar elution patterns and the molecular
masses of polymers containing these residues decreased during
the development stages, except for Rha in the pectic fraction at
28 DPA. In a preliminary experiment we tried to separate pectic polysaccharides into neutral and acidic polymers using an
anion-exchange column (DEAE-Sephadex A-25) and examine
the molecular mass distribution of both polysaccharides independently. The flow-through fraction from the column was
composed of Glc (higher than 99% of total sugar content of the
fraction) and did not contain UA residues (data not shown).
The elution pattern, on the gel filtration column, of polymers in
the flow-through fraction was almost consistent with that of
Glc residue shown in Fig. 4. On the other hand, the polysaccharides bound to the column were not eluted even with solutions
containing higher concentration of NaCl (up to 3 M NaCl),
indicating a strong binding to the anion-exchange resin as compared with those obtained from stem cell walls (Sakurai et al.
1987). Mort et al. (1993) showed that the esterification values
for pectins of cotton fiber cell walls were low as compared with
those of other plant cell walls. The low esterification level of
acidic polymers in cotton fibers may cause the strong binding
to the anion-exchange resin. The observations that the flowthrough fraction from the column did not contain Gal and Rha
residues and that less than 10% of applied Ara was recovered
in the fraction (data not shown), suggest that Ara- and Galcontaining polysaccharides in the pectic fraction are probably
attached to acidic polymers, such as rhamnogalacturonans
(Darvill et al. 1980). The amount of Ara eluting in the high
molecular mass regions in the pectic fraction at 12 DPA was
substantially greater than that at 17 and 28 DPA (Fig. 4). It has
previously been shown that the pectic fraction obtained from
cell walls of carrot embryogenic callus contained a large
amount of high molecular mass arabinans, but such arabinans
decreased in cell walls of non-embryogenic callus (Kikuchi et
al. 1996). Therefore, it is possible that cotton fiber cell walls
obtained in the early fiber elongation stage contain high molecular mass Ara-containing polysaccharides (arabinans) which
may be linked to acidic polymers. These Ara-containing
polysaccharides in cotton fiber cell walls were depolymerized
during development.
Glc in the pectic and HC-I fractions eluted in the intermediate molecular mass regions around fractions 10–20 as a single peak (Fig. 4, 5). The molecular masses of Glc in the HC-I
fraction were about four times those in the pectic fraction dur-
416
Cell walls in growing cotton fibers
to, or slightly lower than, that of xyloglucans from stems of
dicotyledons, such as squash hypocotyls (Wakabayashi et al.
1991), azuki bean epicotyls (Hoson et al. 1991), and outer and
inner tissues of pea epicotyls (Miyamoto et al. 1997). The
molecular mass of xyloglucans in cotton fiber cell walls
decreased from 12 to 17 DPA and then slightly increased from
17 to 28 DPA. Such a change showed a good correlation with
the alteration of molecular masses of Glc, Xyl and Fuc residues in this fraction during fiber development (Fig. 6).
Fig. 7 Elution profiles of xyloglucans in the HC-II fractions in cell
walls of cotton fibers. The materials were prepared and the conditions
were the same for gel-permeation chromatography as in Fig. 4. The
amount of xyloglucans in each column fraction was determined by the
iodine method. Numbers in parentheses are the weight-average molecular mass (in kDa) calculated from the elution profile of xyloglucans.
Data are means ± SE of results from three independent samples. Vertical bars at the top denote the elution positions of authentic dextrans
with molecular masses in kDa, glucose (Glc) and the void volume
(V0).
ing the development stages, suggesting that cotton fiber cell
walls contain at least two populations of non-cellulosic >glucans: one containing polymers with a high molecular mass
(1–2 MDa) and the other with an intermediate molecular mass
(nearly 500 kDa) polymers. This difference in the molecular
mass may be the cause of the different extractability of these
polymers observed in the present study. Although the total
amounts of Glc in the pectic and HC-I fractions increased during fiber elongation, the average molecular mass decreased.
In the HC-II fraction, Glc, Gal, Xyl, Ara and Fuc residues
eluted mainly in the intermediate molecular mass regions,
while Man eluted in the low molecular mass regions (Fig. 6).
The molecular masses of polymers containing Man and Ara
decreased throughout fiber development, while those containing Glc, Xyl, Gal and Fuc decreased from 12 to 17 DPA and
then increased slightly or remained constant during the subsequent stages.
The molecular mass distribution of xyloglucans in the
HC-II fraction, as determined by the iodine method, is shown
in Fig. 7. The molecular mass of xyloglucans in cotton fiber
cell walls was about 400–600 kDa. This value is comparable
Cell wall changes and fiber development
In the present study we found that the amounts and the
molecular sizes of the extractable matrix polysaccharides
changed during cotton fiber development. Since cotton fiber
elongation appears to be initially achieved by cell wall loosening (Ruan et al. 2001), some of such changes may contribute to
cell elongation. In this context, the dynamics of xyloglucans are
noteworthy. Xyloglucans, the predominant hemicellulosic
polysaccharides in cell walls of dicotyledons, are attached to
cellulose microfibrils by hydrogen bonds and thus form a
xyloglucan-cellulose network (Hayashi 1989, Fujino et al.
2000). Modification of this network may play a role in cell wall
loosening in stems of dicotyledons (Sakurai 1991). In fact, it has
been clearly demonstrated that the regulation of xyloglucan
breakdown is involved in auxin-induced elongation (Hoson et
al. 1991) and plant response to the environmental signals such
as light and gravity (Miyamoto et al. 1997, Soga et al. 1999,
Soga et al. 2000). The rapid increase in the amounts of extractable xyloglucans in the early stage of fiber elongation and their
molecular mass downshift during the elongation stage (Fig. 2,
7) suggest that a massive turnover of xyloglucans contributes to
cell wall loosening, leading to the rapid elongation of cotton fibers. In a preliminary experiment we detected xyloglucandegrading activity in the cell wall of cotton fibers. Whether such
an activity is attributed to xyloglucan endo-1,4->-glucanases
(Shimizu et al. 1997) or to endoxyloglucan transferases (Nishitani 1997), especially those with xyloglucan hydrolase activities (Tabuchi et al. 1997, Tabuchi et al. 2001), is an unsolved
problem. Recently, the analysis of growth mutants in Arabidopsis has shown that cellulose synthesis is required for normal cell
elongation (Arioli et al. 1998, Sato et al. 2001). Thus, the harmonious formation and turnover of the xyloglucan-cellulose
network may also be involved in cotton fiber elongation.
In the present study the amounts of extractable Ara- and
Gal-containing polysaccharides also increased rapidly in the
early stage of fiber elongation and then decreased, and their
molecular mass decreased during the fiber elongation stage
(Fig. 3, 4, 5). The transient increase in (arabino)galactan content during the maximum growth rate of stem tissues was also
observed in intact squash hypocotyls (Sakurai et al. 1987) and
azuki bean epicotyls (Nishitani and Masuda 1979). Auxin stimulated the turnover of (arabino)galactans when it induced cell
elongation of stem segments of dicotyledons (Seara et al.
1988). Therefore, the synthesis and turnover of Ara- and Gal-
Cell walls in growing cotton fibers
containing polysaccharides, in addition to that of xyloglucans,
may be associated with cotton fiber elongation.
Cell walls of elongating cotton fibers contained large
amounts of acidic polymers (galacturonans) and non-cellulosic
>-glucans (>-1,3-glucans); and, their extractable amounts
increased as fibers grew, and then decreased after elongation
ceased (Fig. 2, 3). Vaughn and Turley (1999) suggested that
changes in the abundance and structure of acidic polymers in
cotton fibers occurred in the primary cell walls, particularly in
the outer layer. It has been shown that the acidic polymer
network, instead of the xyloglucan-cellulose network, contributed to maintaining cell shape of the 2,6-dichlorobenzonitrileadapted cultured tomato and tobacco cells (Shedletzky et al.
1992). Thus, production of acidic polymers may contribute to
extending and fixating cell walls in elongating cotton fibers. In
addition to the amount, the increase in the molecular mass of
acidic polymers in the extractable pectic fraction (Fig. 4) also
seems to be associated with the maintenance of cell wall
strength during fiber development. In a preliminary experiment we found that the molecular size of acidic polymers also
increased in vitro, suggesting the direct involvement of certain
wall enzymes in the process. The mechanism and the significance of the increase in the molecular size of acidic polymers
are under investigation.
Materials and Methods
Plant materials
Seeds of cotton (Gossypium herbaceum L. cv. Shisomen) were
sown in the middle of May 1997 and 1998 at an experimental field
adjacent to the university campus. On the day of flowering, each individual flower was tagged, and then at various developmental stages (9,
12, 14, 17, 21, 28 and 35 DPA), healthy bolls were harvested. The
pericarp was removed immediately after harvest and the ovules were
frozen in liquid nitrogen and stored at –30°C.
Measuring the length and dry mass of cotton fibers
Before measuring fiber length, we fixed frozen ovules for 10 min
in boiling methanol, and then rehydrated them for 30 min at room temperature. The length of the fibers was measured using a scale. After
measurement, fibers were carefully detached from the ovule using forceps and dried at 60°C for 2 d. The dry mass of fibers was measured.
Twenty ovules from five bolls were used for measuring the length and
dry mass of fibers.
Fractionation of cell wall polysaccharides
Fibers were separated from the ovules which had been fixed in
boiling methanol and then rehydrated. Cell wall polysaccharides were
extracted from the fibers and fractionated by the method of Sakurai et
al. (1987) and Wakabayashi et al. (1991) with slight modification.
Rehydrated fibers were homogenized in ice-cold water with a mortar
and pestle, washed with water, acetone and a methanol : chloroform
mixture (1 : 1, v/v) and then treated with 2 units ml–1 porcine pancreatic a-amylase (EC 3.2.1.1, type I-A, Sigma, St. Louis, MO, U.S.A.)
in 50 mM sodium acetate buffer (pH 6.5) at 37°C for 3 h. After the
amylase treatment, pectic substances were extracted from the cell wall
materials three times (15 min each) with 50 mM EDTA (pH 6.8) at
95°C. The acidic polysaccharides in the pectic fraction are degraded in
417
mild alkali at the site where a sugar is glycosidically linked to O-4 of
an esterified uronic acid unit, known as b-elimination (Fry 1988).
However, neither shortening nor extending the extraction period with
EDTA influenced the molecular mass distribution of the pectic
polysaccharides, suggesting that the effect of b-elimination can be
ignored under the present experimental conditions. The hemicellulose
was then successively extracted from the cell wall materials three
times (12 h each) with 4% (w/v) KOH and three times (12 h each) with
24% (w/v) KOH containing 0.02% NaBH4 at room temperature. The
fractions extracted with 4% and 24% KOH were designated as the HCI and the HC-II, respectively. The HC-I and the HC-II fractions were
neutralized with acetic acid. The pectic fraction and the neutralized
HC-I and HC-II fractions were dialyzed in cellulose tubing (18/32,
molecular weight cut-off: 12,000–14,000, Wako Pure Chemical, Ltd.,
Osaka) against water. At the dialysis step, 80–90% of applied wall
polysaccharides were recovered. The alkali-insoluble fraction (cellulose fraction) was washed successively with 0.03 M acetic acid and
ethanol, and dried at 40°C. The cellulose fraction was dissolved in
72% (v/v) sulfuric acid for 1 h at room temperature, and then diluted
with a 29-fold volume of water.
TS content was determined by the phenol-sulfuric acid method
(Dubois et al. 1956), and UA content by the carbazole method (Galambos 1967). The amounts of xyloglucans were determined by the iodine
staining method (Kooiman 1960, Nishitani and Masuda 1981) using
xyloglucans of azuki bean epicotyls as a standard. The dialyzed pectic, HC-I and HC-II fractions were lyophilized, and then hydrolyzed
with 2 M trifluoroacetic acid for 1 h at 121°C. After the trifluoroacetic
acid had been removed by evaporation under a stream of air at 50°C,
the sugars were reduced with NaBH4 in 1 M ammonia, and acetylated
with acetic anhydride for 3 h at 121°C. The amounts of the acetylated
alditoles derived from NSs were analyzed by gas-liquid chromatography (GLC) according to the method of Albersheim et al. (1967). In
addition, portions of the lyophilized sample of the pectic and the HC-I
fractions were incubated with 200 mg ml–1 of b-1,3-glucanase (laminarinase from mollusk; Sigma) in 20 mM sodium acetate buffer (pH 5.5)
for 4 h at 37°C (Kakimoto and Shibaoka 1992). The reaction mixtures
were dialyzed in cellulose tubing as described above, and the levels of
Glc in the dialyzed solutions were determined by GLC as described
above. To determine the sugar composition of the cellulose fraction,
we incubated the samples that had been dissolved in 72% sulfuric acid
and diluted with a 29-fold volume of water for 1 h at 121°C, and then
neutralized with barium hydroxide and barium carbonate. After the
neutralized solution had been dried, the sugars in the dried materials
were reduced, acetylated and then analyzed by GLC as described above.
Gel-permeation chromatography of pectic and hemicellulosic
polysaccharides
Dialyzed pectic, HC-I and HC-II fractions 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 (0.3–0.4 ml) was separated on a TSK-GEL 5000PW column (Tosoh Co., Ltd., Tokyo) on an
HPLC system (LC-6A, Shimadzu Co., Kyoto) equipped with a refractive index detector (RID-6A, Shimadzu Co.). 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. TS, UA, and
xyloglucan content in each column fraction was determined by the
phenol-sulfuric acid method, the carbazole method, and the iodine
method, respectively. The amounts of NS in each column fraction were
determined by GLC as described above. The weight-average molecular mass of polysaccharides was calculated from the equation proposed by Nishitani and Masuda (1981). Dextrans (Sigma) of 10, 40,
70, 120, 500 kDa and Glc were used as molecular mass markers.
418
Cell walls in growing cotton fibers
Acknowledgments
We thank Professor Emeritus Y. Masuda, Osaka City University,
and Professor Emeritus K. Nishio, Mukogawa Women’s University,
for invaluable suggestions and discussions.
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(Received September 25, 2001; Accepted February 1, 2002)