Plant Physiol. (1990) 93, 396-402
Received for publication August 22, 1989
and in revised form January 10, 1990
0032-0889/90/93/0396/07/$01 .00/0
Enzymic Analysis of Feruloylated Arabinoxylans (Feraxan)
Derived from Zea mays Cell Walls1
111. Structural Changes in the Feraxan during Coleoptile Elongation
Kazuhiko Nishitani2 and Donald J. Nevins*
Department of Vegetable Crops, University of California, Davis, California 95616
ABSTRACT
In Poaceae cell walls, feraxan3 and (l-*3),(l--4)-#-D-glucans constitute the major components of the cell wall matrix
polymers (2, 11, 24). Recent studies have implicated feruloylated polymers as a means to control mechanical properties
of the cell wall (4, 7-9, 20, 21). However, little is known about
the structural changes which occur in feraxan during growth
and maturation of plant tissues. This is due, in part, to a lack
of suitable enzymes capable of specific and quantitative dissociation of feraxan components from the wall matrix. Classical alkaline extraction procedures do not afford release of
native feraxan fragments, in part, because ester linkages,
which link ferulic acid to arabinoxylans, are cleaved (1 1).
Recently, we (17) purified two enzyme systems capable of
specifically and quantitatively dissociating feraxan and 3-Dglucan fragments from maize cell wall. Upon analysis of
feraxan fragments of the maize coleoptile, we were able to
identify certain changes in the structural features of matrix
polymers during coleoptile elongation. This paper describes
the results.
Changes in structural features of feraxan (feruloylated arabinoxylans) in cell walls during development of maize (Zea mays
L.) coleoptiles were investigated by analysis of fragments released by feraxanase, a specific enzyme purified from Bacillus
subtilis. The following pattems were identified: (a) The total
quantity of carbohydrate dissociated from a given dry weight of
cell wall by feraxanase remained rather constant throughout the
initial 10 days of coleoptile development. However, during the
same period the proportion of ferulic acid in the fraction increased
12-fold. The absolute amount of ferulic acid per coleoptile also
increased rapidly during this developmental phase. (b) Fragments
dissociated by the enzyme were resolved into feruloylated and
nonferuloylated components by reversed phase chromatography.
While the quantity of feruloylated fractions represented a small
portion of the total arabinoxylan during the phase of maximum
coleoptile elongation (days 2-4) this component increased in
abundance to reach a plateau (after 8-10 days). In contrast,
nonferuloylated fractions were most abundant during the stage
of maximum elongation but declined to a constant level by day 6.
(c) Glycosidic linkage analysis of carbohydrate reveals that substitution of the xylan backbone of feraxan by arabinosyl residues
decreased during coleoptile growth. We conclude that significant
incorporation of ferulic acid occurs and/or more feruloyated domains are added to the arabinoxylan during development. This
augmentation in phenolic acids is accompanied by a concerted
displacement of arabinosyl residues and/or a reduction in the
incorporation of regions enriched in arabinosyl sidechains.
MATERIALS AND METHODS
Growth Measurement
Caryopses of maize (Zea mays L. hybrid B73 x Mo17),
after being soaked for 12 h in running tap water, were sown
in moistened vermiculite and grown at 25°C in darkness.
Coleoptiles were harvested and the leaves removed. The mean
lengths (n = 30) and the total number of coleoptiles recovered
were recorded. Coleoptile tissue was immediately heated in
boiling 80% ethanol for 5 min and stored in 95% ethanol.
Morphological changes in plant cell walls, including those
processes leading to extension and differentiation, are considered, at least in part, to be an expression of adjustments in
the mechanical properties of the cell wall. In an attempt to
understand the chemical basis for mechanical properties,
changes in chemical structure of wall matrix polysaccharides
during hormone induced elongation of excised plant parts
have been studied in detail (5, 12, 15, 16, 22). However, the
chemical basis for mediation of rheological properties of the
cell wall remains unresolved.
Preparation of Coleoptile Sections
Coleoptiles, after heat treatment in 80% ethanol, were
washed successively with organic solvents and 3 M LiCl, then
subjected to a-amylase digestion and dried according to the
procedure described previously (17). Cell wall dry weight per
coleoptile was calculated by dividing total dry weight of the
recovered coleoptile preparation by the total number of coleoptiles in the sample.
' Supported, in part, by National Science Foundation Grant DMB8505901.
2
Permanent address: Dr. Kazuhiko Nishitani, Department of Biology, College of Liberal Arts and Sciences, Kagoshima University,
Enzyme Digestion
Feraxanase was prepared as described previously (17). Fifty
mg of the coleoptile wall preparation was treated with 5 mL
'Abbreviation: feraxan, feruloylated arabinoxylans.
Korimoto, Kagoshima 890, Japan.
396
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FERAXAN STRUCTURAL CHANGES DURING COLEOPTILE ELONGATION
(5 ,g protein) of feraxanase in 20 mm Na-phosphate buffer
(pH 6.8) containing 0.1% sodium azide. The incubation
mixture was maintained at 37°C for 24 h. After incubation,
the supernatant was filtered through ACRO LC 13 disposable
filters (pore size 0.45 um, Gelman Sciences, Inc., MI), then
the sample was heated at 90°C for 5 min to inactivate the
feraxanase (F-fraction).
Glucanase was also prepared as described previously (17).
Fifty mg of the wall preparation was incubated with 5 mL of
Bacillus subtilis (1--3),(l-*4)-f3-D-glucan-4-glucanohydrolase
(10 ,ug of protein in 20 mm Na-phosphate buffer [pH 6.8],
containing 0.1 S% sodium azide). The incubation mixture was
maintained at 37°C. After 24 h the mixture was filtered with
the aid of an ACRO membrane and heated in boiling water
for 5 min. The soluble phase of the glucanase digest yielded
the G-fraction.
Fractionation of Feraxanase Products
One mL of the F-fraction was adjusted to pH 3.2 by the
addition of 30 ,uL of glacial acetic acid. The acidified fraction
was applied to a Sep-Pak C18 cartridge prewashed with 15
mL of ethanol, 20 mL of water, and 10 mL of 0.05% acetic
acid solution (pH 3.2). The cartridge was eluted successively
with 10 mL of a 0.05% acetic acid solution, 5 mL of a 10%
ethanol solution containing 0.05% acetic acid, and 5 mL of a
60% ethanol solution containing 0.05% acetic acid. Fractions
F-A, F-B, and F-C were obtained (18). F-A and F-B fractions
were free of phenolic acids, whereas F-C contained ferulic
acid and other phenolics.
Portions of these fractions were assayed for total carbohydrates, uronic acids, phenolic acids, and neutral sugar composition. The remainder of each of the fractions was neutralized by addition of 1 M sodium acetate solution, evaporated,
redissolved in water, desalted on a Bio Gel P-2 column, and
finally lyophilized. The lyophilized oligomers were subjected
to glycosidic linkage analysis by permethylation followed by
gas chromatography (17).
Oligosaccharide composition of the G fraction was analyzed
by HPLC on an Amnex HPX-42A column (7.8 x 300 mm)
(17).
Gel Permeation Chromatography
The F-B and F-C fractions derived from 4- and 10-d-old
coleoptiles were resolved by chromatography on a Bio Gel P10 (200-400 mesh) column (1.5 x 83 cm) and a Bio Gel A
0.5 m (200-400 mesh) column (1.7 x 83 cm), respectively.
Fractions were eluted with 50 mm Na-acetate buffer (pH 5.4).
The total sugar content in the eluate was determined by the
phenol sulfuric acid method (6). Total phenolics in the eluate
were estimated by the measurement of UV absorption at 324
nm and the absorbance values were converted to ferulic acid
equivalents. HPLC was used for the specific determination of
ferulic acid (18).
RESULTS
Coleoptile length, cell wall dry weight, and the growth rate
are shown as a function of age in Figure 1. An enhanced
growth rate and net deposition of cell wall substances prevailed from d 1 through 3 and d 2 through 6, respectively.
After d 6 both processes diminished and coleoptile development stabilized.
cm
E
E
E
c
0)
0)
3
0
._
r.
2
I
I_
I
I
I
I
I
2
4
B
Analyses for Carbohydrates and Ferulic Acid in Enzyme
Digests
Total sugar content was determined by the phenol-sulfuric
acid method (6) and the values converted to xylose equivalents. Total uronic acid content was determined by the
method of Blumenkrantz and Asboe-Hansen (1), and the
values were converted to glucuronic acid equivalents. Neutral
sugar composition was determined by GLC on a SP 2330
glass WCOT capillary column as described previously (17).
Glycosidic linkage arrangements were determined by permethylation followed by acetylation according to Hakomori (10)
with modification (17, 23). The glycosidic derivatives were
subjected to analysis by a GLC equipped with a DB 225 fused
silica capillary column (0.25 mm x 30 m). Individual, partially methylated alditol acetates were identified by GLC-MS
(17).
Ferulic acid content of cell wall preparations and F-C
fractions was determined by HPLC on Biosil ODS-5S column
(4 x 250 mm) after saponification by 0.5 M NaOH. The
procedure was described previously (18).
397
0
1
0
0
0
6
Days
I
aI
8
10
Figure 1. Growth of maize coleoptiles. Coleoptiles were excised
from seedlings maintained at 250C in darkness. A, The mean accumulated coleoptile length (0) and the total cell wall dry weight of
individual coleoptiles (0) are presented as a function of seedling age;
B, the growth rate was calculated as follows:
Growth rate (mm-mm1 -d-1)
=
(La
-
Lb)-La-1' .t
where La is the coleoptile length at the beginning of the growth
interval and Lb is the length at time t.
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Copyright © 1990 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 93, 1990
NISHITANI AND NEVINS
398
The purpose of the present study was to identify structural
changes in the wall polymers associated with elongation. The
emphasis was primarily on the analysis of structural features
of ( 1-+3),( l-+4)-/3-D-glucan and feraxan. To accomplish these
objectives, coleoptile cell walls were subjected to enzymatic
fragmentation procedures at different developmental stages.
20
200
0)
E
E
0)
I.0
1Z 100
.10
'aIioo
4~
Compositional Changes in the Wall Matrix
Total neutral sugars liberated by 2 M TFA hydrolysis and
ferulic acid released by saponification with 0.5 M NaOH from
matrix polymers in 1 mg of dry cell wall, are shown in Figure
2. The data reveal a sustained increase in the content of ferulic
acid, a transient increase in the abundance ofglucose, greatest
abundance at about d 6, and smaller increases in xylose and
arabinose during coleoptile growth.
Wall fragments dissociated by the action of feraxanase (Ffraction) consisted mainly of ferulic acid, xylose, arabinose,
galactose, glucose, and uronic acids. The quantities of ferulic
acid, xylose, and arabinose in the F-fraction account for most
(about 70%) of these sugars in the cell wall matrix (cf Figs. 2
and 3). The quantity of ferulic acid released from the total
wall and from the F-fraction are similar through d 5. A
divergence of those values is noted after d 5.
Analysis of the Structural Features of the G-Fraction
As expected, the G-fraction was composed chiefly of glucose. The abundance of this fraction reached a maximum on
d 6. Changes in glucose in the G-fraction account for the
dynamic pattern of 3-D-glucan in the wall matrix (cf. Figs. 2
and 4). Oligosaccharides comprising the G-fraction (Table I)
are predominately trimers, tetramers, and pentamers of glucose. This fragmentation pattern is consistent with their origin
from the f3-D-glucan. The relative compositions of these frag-
C)
0
Days
Figure 3. Changes in sugar and ferulic acid content of the F-fractions
derived from maize cell walls as a function of coleoptile development.
Dry cell walls were treated with feraxanase to obtain the F-fractions.
Sugars and ferulic acid of the F-fractions were analyzed (see "Materials and Methods"). The quantities of sugars and ferulic acid derived
from 1 mg of dry cell wall are shown.
ments in the G-fraction remained nearly constant throughout
coleoptile development (Table I).
Changes in the Structural Features of F-Fractions
Throughout coleoptile development the mol % of 4-xylosyl
residues increased in the F-fraction. In contrast, the mol % of
t-galactosyl and 5-arabinosyl moieties decreased during the
first 5 to 6 d. The mol % of t-arabinosyl and 2,4/3,4-xylosyl
increased initially and then decreased (Fig. 5, A and B).
Fraction F was further subdivided into F-A, F-B, and F-C
by reversed phase chromatography. F-A and F-B did not
contain phenolic acids whereas F-C was feruloylated. The
allocation pattern into these fractions clearly suggest structural
changes as a function of development (Fig. 6). The relative
abundance of the nonferuloylated fraction (F-B) is highest
200
0)
c)
E
CD
0)
E
E
0)
0)
,6 100
a;
f
L-
,
.0o
100
0
'a
.0
C-)
A
v
0
2
4
6
8
10
Days
in
Figure 2. Changes noncellulosic neutral sugars and ferulic acid
content of maize cell walls as a function of coleoptile development.
Dry cell walls were hydrolyzed with 2 N TFA at 1 200C for 1 h and the
neutral sugar content in the hydrolyzate was determined. Separate
cell wall samples were saponified with 0.5 M NaOH at 600C for 90
min to release ferulate residues. The amount of liberated ferulic acid
was determined by HPLC. The content of 1 mg of dry cell walls is
shown.
0
1-
0
2
4
-1
I
6
8
10
Days
Figure 4. Glucose content of the G-fraction as a function of coleoptile
development. Dry cell walls were treated with #-D-glucanase to
liberate the G-fraction. The sugar composition was analyzed (see
"Materials and Methods"). Fractions were composed predominantly
of glucose and the glucose content derived from 1 mg of dry cell
walls is shown.
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Copyright © 1990 American Society of Plant Biologists. All rights reserved.
399
FERAXAN STRUCTURAL CHANGES DURING COLEOPTILE ELONGATION
Table 1. Oligomer Composition of the G-Fraction Derived from
Maize Cell Walls
The G-fraction was liberated from developing coleoptiles by hydrolysis with 3-D-glucanase and the products analyzed by HPLC.
Weight ratios are shown.
Ratio of Oligomer Species
Coleoptile
age
Trimer
Tetramer
0.64
0.61
0.61
0.63
0.62
0.59
0.62
0.27
0.28
0.28
0.29
0.29
0.30
0.30
Pentamer
A
F-C
v .0/1, \?-F
L..
10
E 50
F-a
d
2
3
4
5
6
8
10
A
0.07
0.09
0.08
0.07
0.07
0.10
0.07
2 4/3 4-xyl
40 _
4
t-ara
0
*
Figure 6. Changes in the allocation of arabinoxylan oligomers into
different fractions. Fractions liberated by feraxanase treatment of cell
walls (F-A, F-B, and F-C) were separated by reversed phase chromatography. A, Total carbohydrate content of individual subfractions
derived from 1 mg dry cell wall is shown as a function of coleoptile
age; B, carbohydrate content of individual subfractions derived from
the equivalent of a single coleoptile as a function of age. Values
calculated from data presented in Figures 1 A and 6A.
20
0
so 20
4-xyl
5-ara
0
'V
2
)
4
nuays
._-
I
6
8
6 -B
/
@4
2 lzt-a a \2.4/3,4 - xyl
------
c
m
~~~
--
,,
y t-gal
)2
2
0
6
6
5- ara
-4
10
from d 2 through d 4 but by d 3 a sharp decline is apparent
(Fig. 6A). Meanwhile, from d 2 through d 8 the quantity of
F-C increases. Expressed on the basis of the amount of F-B
per coleoptile an increase is observed from d 2 through d 5
then the level remains constant (Fig. 6B). Correlations with
growth are revealed by this analysis. F-C steadily increases
w
E
.c
4
4
Days
-
8
8
throughout growth and continues until well after elongation
ceases while F-B increases during the phase of maximum
elongation then is sustained at a constant net level when
growth ceases (cf. Figs. 1 and 6). Figure 7 reveals that changes
in F-B and F-C are due to rearrangements in the major sugar
°
components of feraxan, i.e. xylose, arabinose, and uronic
acid. Moreover, the content of ferulic acid increases sharply
within fraction F-C (Fig. 8).
,The molecular size distribution of F-B and F-C fractions
10
were estimated from samples derived from 4-d-old (elongating
10
Days
Figure 5. Major glycosidic linkage arrangements of the F-fractions
as a fuinction of coleoptile development. A, Glycosidic linkages of the
F-fracttions were analyzed by permethylation (see "Materials and
Methods"). Mol percentages of individual glycosidic linkages are
showni. B, The relative proportion of respective glycosidic linkages
were (,as mol %) determined by subtracting the value (mol %) for the
d-2 collIeoptile sample from each subsequent value during coleoptiledevelotpment. The data are shown as a function of age in days.
stage) and 10-d-old (nonelongating stage) coleoptiles. Gel
permeation chromatography was employed (Fig. 9). The F-B
fraction resolved into two major peaks (Fig. 9A). The elution
profile of the F-B derived from coleoptiles at 4 d of age is
. .
similar to that obtained from day 10 samples. There are
quantitative differences (Fig. 6A) and the samples from older
tissues exhibit a less distinct profile. In contrast, the F-C
fraction was resolved into three peaks with characteristic
ferulic acid compositions: the ferulic acid composition is
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Copyright © 1990 American Society of Plant Biologists. All rights reserved.
400
NISHITANI AND NEVINS
Plant Physiol. Vol. 93, 1990
lowest in the first peak, that which was excluded by the BioGel A 0.5 m column, and is highest in the third peak (tubes
77-90). Figure 9B also shows that the relative quantity of the
first peak decreases and that of the third peak increases during
coleoptile development (from d 4 to d 1O).
A
E 40
E
Qf
DISCUSSION
ara
Ferulic acid residues are clearly linked to arabinoxylans in
the maize coleoptile cell wall and the extent of ferulic acid
association with the polysaccharide increases dramatically
during growth and maturation of the tissue. Increases in
ferulic acid residues are due to both augmentation of the
proportion of feruloylated components (Fig. 6) and to an
enhancement in the extent to which ferulic acid is added to
.0
. 20
'a 0
p
c
UA
20
0
2
6
4
8
10
Days
40
1-
0
U
0
E
z
20
CD
U
C3,1
c
-
0
c
c"0
5
0
rv}
0
0
2
4
Days
6
8
0
10
50
30
Figure 7. Changes in the sugar composition of F-B and F-C subfractions. Sugar and ferulic acid comprising the F-B (A) and F-C (B)
subfractions were analyzed and the components derived from 1 mg
of dry cell wall is shown as a function of time.
90
70
Tube
No.
I
E
m
0-
E
0.4 0)
:a,4
c
m
.0I
I
I
I
I
c
C
0)
2
151-
0
30
U
LL 10
5F
0L
M-
0
H
IL
0.2 °
2
6
4
10
8
Days
Days
Figure 8. Ferulic acid content in fraction F-C as a function of age.
Ferulic acDid content (ug.100 gg dry weight-1) of individual F-C fractions was; determined by HPLC analysis after alkaline saponification.
-i
--
----j---
w
50
Tube
No.
70
90
Figure 9. Gel chromatography profiles of F-B and F-C subfractions
derived from coleoptiles from 4- and 10-d-old coleoptiles. A, F-B
subfractions derived from 4- and 1 0-d-old coleoptiles were applied to
a Bio Gel P-10 column (1.5 x 83 cm) and eluted with 50 mm Naacetate buffer at pH 5.4. Fractions (1.5 mL) were collected and
assayed for total sugar by the phenol sulfuric acid method. The total
sugars derived from an initial 1 mg of dry cell wall was calculated. B,
F-C subfractions from 4- and 1 0-d-old coleoptiles were applied to a
Bio Gel A 0.5 M column (1.5 x 8.3 cm) and eluted with 50 mm Naacetate buffer (pH 5.4). Two mL fractions were collected and assayed
for sugar by the phenol sulfuric acid method and the ferulic acid
content was assessed by UV absorption at 324 nm. The total sugars
and ferulic acid derived from 1 mg of dry cell wall were calculated
and shown.
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FERAXAN STRUCTURAL CHANGES DURING COLEOPTILE ELONGATION
feruloylated components (Fig. 9). The results suggest that
certain nonferuloylated domains (F-B fraction) in the feraxan
are successively feruloylated to yield phenolic domains (F-C
fractions), and the feruloylation of these regions continues
throughout the coleoptile aging.
Recent studies (a) suggest that ferulic acid residues in cell
walls may be oxidatively coupled by the action of peroxidase
to form a diferulate cross-links. Diferulate residues are known
to be present in maize (4) and other cereal plants (18, 20).
Thus, it seems plausible that the increase in ferulate residues
during the coleoptile development contributes to an enhanced
opportunity for cross-link formation and facilitates wall matrix coherency. Disruption of these labile bonds would account, at least in part, for the extraction of noncellulosic
polymers by alkaline solutions.
The quantity of nonferuloylated fractions (F-B and to a
lesser extent F-A) increase during the most rapid stages of
elongation. The amount of the feruloylated fraction (F-C)
increases throughout development but becomes most abundant after elongation is complete. We also observe that the
extent of ferulic acid substitution within the F-C fraction
increases in a manner that parallels the increased abundance
of the fraction itself. The observations represent different
aspects of feraxan modification and the data support the
contention that feruloylation of existing domains in F-C and
a net increase in the F-C fraction of the arabinoxylan occurs.
These transitions in arabinoxylan structure may have a role
in regulating cell extension.
The mol % of side chains in feraxan, i.e. 5-arabinosyl, tgalactosyl residues, etc., decreases during the coleoptile growth
(Fig. 5B). The data suggest that adjustments in wall metabolism, including synthesis, degradation and in situ modification, imparts feraxans with fewer side chains during coleoptile
development. This conclusion is in general agreement with
that reported by Carpita (3) based on alkaline extraction
procedures. The degree of polymer branching may influence
rheological behavior. Although we have no direct evidence
for changes in physical properties of feraxan in situ caused by
the deletion of side chains, it is probable that such structural
changes will contribute to the mechanical properties of the
cell wall.
Luttenegger and Nevins ( 14) reported the transitory nature
of (1--3),( l-*4)-(3-D-glucan in maize coleoptile walls during
the coleoptile development. The results of the present study
are consistent with those observations. We find, however, that
there are no significant changes in glycosidic linkage arrangement of the f3-D-glucan during development. Therefore, a
possible role for quantitative changes, rather than structural
changes, is suggested for the ,B-glucan in extension growth of
cereals (13, 19).
The discrepancy in sugar recovery when one compares
yields derived following TFA hydrolysis with the combined
feraxanase and glucanase digestion products (F- and G-fractions) may account, in part, for other polysaccharides, viz.
xyloglucans and arabinogalactans in coleoptile walls. These
polymers should be resistant to hydrolysis by feraxanase and
glucanase and are not solubilized. However, since about 30%
of ferulic acid residues remain undissociated from the wall
after treatment with these enzymes, we may consider the
401
presence of other feruloylated associations. Residual ferulate
may be the consequence of incomplete digestion by the enzyme and/or retained fragments within the wall. The significance of undissociated feruloylated oligomers warrents additional investigation.
The study suggests dynamic adjustments in both feraxan
and f3-D-glucan components in the wall. The correlation of
these events with growth implies a potential role in elongation
of the maize coleoptile. The mechanisms giving rise to specific
changes in matrix polymers and the extent to which these
changes contribute to the physical properties of the cell wall
are being sought.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
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