Received for publication September 9, 1987
and in revised form August 26, 1988
Plant Physiol. (1989) 89, 792-797
0032-0889/89/89/0792/06/$01 .00/0
Structure of a Pectic Polysaccharide Fraction from Zea
Shoots1
Yoji Kato and Donald J. Nevins*
Laboratory of Food Science, Faculty of Education, Hirosaki University, Hirosaki 036 Japan (Y.K.), and
Department of Vegetable Crops, University of California, Davis, California 95616 (D.J.N.)
ABSTRACT
with a-amylase (Sigma, type lA from porcine pancreas), the
hot water extract (water-soluble polysaccharide fraction III,
WSP-III) was resolved into seven fractions by (NH4)2SO4
precipitation. These procedures were described in a previous
paper (9). One fraction, 20P-S (362 mL of aqueous solution),
precipitated in 20% (NH4)2SO4 but soluble in water (see Table
I in Ref. 9), was used for structural characterization.
Erwinia carotovora PL, purified to the electrophoretically
pure state, was a generous gift of Mr. Naganuma of Tohoku
University (1 1).
A pectic fraction, accounting for about 0.3% of the total cell
wall polysaccharide, was derived from the hot water extract of
an insoluble fraction of the buffer-homogenate of Zea shoots. The
pectic polysaccharide fraction was characterized by fragmentation analysis after hydrolysis with acid and Erwinia carotovora
pectate lyase. The results suggest that the fraction consists of
mostly a linear homopolygalacturonan with neutral sugar components or a homogalacturonan and a rhamnogalacturonan with
neutral sugar components.
General Methods
Concentration of carbohydrate solutions was performed
under reduced pressure at 35 to 40°C. Paper chromatography
was performed on Toyo No. 50 filter paper by the multiple
ascending method using butanol:pyridine:water (6:4:3, v/v)
(method A), or by the descending method using ethyl acetate:water:acetic acid:formic acid (18:4:3:1, v/v) (method B).
Sugars on the chromatogram were detected with alkaline silver
nitrate (13). Total carbohydrate and uronic acid were determined by the phenol-H2SO4 method (4) and the carbazoleH2SO4 method (1), respectively. GLC was conducted with a
Yanagimoto model G-80 gas chromatograph equipped with
a flame ionization detector at a nitrogen gas flow rate of 15
mL/min.
To learn the nature of structural features of cell walls in
elongating monocot tissues, we initiated a detailed study on
Zea shoot cell-wall polysaccharides. In previous reports we
reported on the structural characterization of the arabino-3,6galactan obtained from a soluble fraction of the buffer-homogenate of Zea shoots (6), and of the (1-s3),(l-)-,3-D
glucan and (l-3)-f-D-glucan, which were obtained by treatment of an insoluble fraction of the buffer-homogenate of
Zea shoots with 3 M LiCl and hot water (8, 9). In the course
of structural studies of polysaccharides obtained by treatment
of an insoluble fraction of the buffer-homogenate of Zea
shoots with hot water, we prepared a pectic polysaccharide
fraction.
The pectic polysaccharides of the primary cell walls of
monocots have not been studied extensively, although it is
clear that monocot cell walls possess relatively small amounts
of GalUA2 (3, 12).
This report is the fourth in a series of dealing with the
water-soluble polysaccharides of Zea shoots and describes the
structure of a pectic polysaccharide fraction from Zea shoots.
Analysis of Neutral Sugars in Poly- or Oligosaccharides
Polysaccharide (10-50 ,ug) was hydrolyzed with 2 M TFA
for 5 to 6 h at 100°C. Oligosaccharide (5-10 ug) was hydrolyzed with 1 M TFA for 4 to 5 h at 100°C. Each hydrolysate
was evaporated to dryness. Sugars were converted to alditol
trifluoroacetates and analyzed by GLC on a column (0.4 x
200 cm) packed with 1.5% QF- 1 on Chromosorb W at
140°C (5).
MATERIALS AND METHODS
Materials
Zea mays L. (B73 x Mo 17) shoots (fresh weight, 3800 g),
excised at the coleoptile node 96 h after imbibition, were
homogenized with 10 mm Na-phosphate buffer (pH 6.5) and
centrifuged. The precipitate (wet weight, 310 g) was treated
successively with 3 M LiCl and hot water. After hydrolysis
Resolution of Fraction 20P-S by DEAE-Sephadex A-25
Chromatography
Fraction 20P-S (475.6 mg as GalUA equivalent/350 mL of
water) was mixed with 40 mL of 0.25 M Na-phosphate buffer
(pH 6.0) containing 1% NaN3 and the mixture was applied
to a column (2 x 29.5 cm) of DEAE-Sephadex A-25 preequilibrated with 25 mm Na-phosphate buffer (pH 6.2) containing
1% NaN3. The column was washed with the same buffer (390
mL), then stepwise elution was carried out with buffers containing 0.1 M (523 mL), 0.2 M (400 mL), 0.5 M (362 mL), and
' Supported in part by National Science Foundation research grants
PCM 7818588 and DMB850901.
2
Abbreviations: GalUA, galacturonic acid; Rha, rhamnose; PL,
pectate lyase.
792
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Copyright © 1989 American Society of Plant Biologists. All rights reserved.
ZEA PECTIC POLYSACCHARIDE
793
II were 376.2 mg and 23.7 mg GalUA equivalent respectively.
20P-S-I was designated as Zea pectin.
Chromatography on a Sepharose CL-6B column of
fractions 20P-S-1 and 20p-S-II.
Fraction 20P-S-I (1061 ,ug
as
GaIUA equivalent/L of 20
mM Na-acetate buffer, pH 5.0) and fraction 20P-S-II
(610 jig as GalUA equivalent/l mL of 20 mm Na-acetate
buffer, pH 5.0) were individually applied to a column (1.3 x
40 cm) of Sepharose CL-6B preequilibrated with 20 mm Naacetate buffer (pH 5.0), followed by filtration through the
E
-I
0
o
0
column with the same buffer. Fractions of 0.5 mL each were
collected and assayed for uronic acid. Leuconostoc dextran
(10) Dextran 110, Dextran T-40, and glucose were used for
column calibration.
4I
Acid Hydrolysis of Zea Pectin
0
20
40
60
80
Tube No. (0.5ml/tube)
100
Figure 1. Resolution of the components of a low mol wt neutral
fraction prepared from partial acid hydrolysate of Zea pectin. The
fraction (1 10 ,g as Xyl equivalent/i mL of water) was derived from a
partial acid hydrolysate of of Zea pectin (see "Materials and Methods")
and applied to a column (1 x 60) of Bio-Gel P-2 operated at 450C.
One-half mL fractions were eluted with water and assayed for carbohydrate. Tubes 31 to 39, 40 to 44, 45 to 50, 51 to 55, 56 to 58,
59 to 64, 65 to 72, 73 to 82 were separately combined and concentrated to give fractions PAH-N-a to -h.
1.0 M NaCl (428 mL), 0.5 M NaOH (513 mL), and 0.5 M HC1
(238 mL); 9.5 mL fractions were collected. Fractions were
assayed for carbohydrate by the phenol-H2SO4 method. Unadsorbed fractions (20P-S-I) and fractions eluted with 0.5 M
NaCl (20P-S-II) were separately combined and concentrated
to about 100 mL. Methanol (400 mL) was added to the
respective concentrates and mixed, the mixture was maintained at 4°C overnight and centrifuged. The precipitate was
successively washed with 80% methanol, methanol and ether,
and finally dried. The yields of fractions 20P-S-I and 20P-S-
Zea pectin (about 60 mg as GalUA equivalent) was hydrolyzed with 20 mL of 1 M TFA for 5 h at IOO°C and evaporated
to dryness in the presence of methanol. The dried material
was dissolved in 5 mL of water and applied to a column (1.8
x 8.5 cm) of Dowex- (acetate form). The column was washed
with water (45 mL), then by stepwise elution with 0.1 M (145
mL), 0.2 M (220 mL), 0.5 M (160 mL), and 4 M (215 mL)
acetic acid and 2 M NaOH (210 mL). Fractions of 5 mL were
collected and assayed for carbohydrate. Tubes 4 to 10, 20 to
52, 53 to 115, 121 to 160, and 163 to 204 were separately
combined and concentrated to give AH-1 through AH-5.
Partial Acid Hydrolysis of Zea Pectin
Zea pectin (about 32 mg as GalUA equivalent) was hydrolyzed sequentially with 0.1 M (6 mL), 0.5 M (4 mL), and 2 M
(1 mL) TFA for 2 h at 100°C. After each hydrolysis, the
residue was separated from the supernatant by centrifugation.
The supernatant obtained in each step was evaporated to
dryness, and then dissolved in 1 mL of water. Four mL of
methanol was added to the solution, it was mixed and the
mixture was maintained for 24 h at -20C. The resulting
Table I. Yields and Neutral Sugar Composition of Fractions Obtained from a Low Mol Wt Neutral
Fraction Prepared from the Partial Acid Hydrolysate of Zea Pectin
Neutral Sugar Composition
Tube Nos.
Fraction
.
Yield"a Rha
In Figure 3
Ara
Xyl
Man
Gal
Fuc
Glc
%
mo%/0
9.1
8.1
23.0
3.9
7.6
50.6
4.9
31-39
27.7
3.2
10.3
19.3
7.8
6.6
10.6
40-44
-c
3.1
3.8
13.2
6.6
22.5
25.4
45-50
5.6
-d
14.1
2.7
3.1
29.8
1.9
10.8
10.2
51-55
8.4
15.5
23.0
38.6
-e
56-58
0.3
3.4
20.6
-f
6.6
30.2
16.3
59-64
4.4
11.4
12.0
-g
65-72
12.5
15.0
21.0
15.9
44.7
27.2
10.1
7.9
7.5
-h
13.0
73-82
14.6
aThe amount of carbohydrate material in each peak, expressed as percent of the total
carbohydrate, was determined by the phenol-H2SO4 method.
PAH-N-a
-b
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Copyright © 1989 American Society of Plant Biologists. All rights reserved.
15.8
22.3
25.3
30.1
14.5
26.3
20.3
19.7
recovered
Plant Physiol. Vol. 89, 1989
KATO AND NEVINS
794
the incubation mixture was monitored for 4,5-unsaturated
galacturonic acid by measuring absorbance at 235 nm. After
1 h incubation, a portion (0.4 mL) of the incubation mixture
was subjected to gel-filtration on Sepharose CL-6B, and another portion (1.5 mL) was subjected to DEAE-Sephadex A25 chromatography.
.
1.5
0~~~~~~
RESULTS
Isolation and Purification of a Pectic Polysaccharide
Fraction
I01
\-
E
c1.0
r)C\M
10
0
0
20 30
Time
(min)
f
60
|Z
0
0~~
5
It
LOQ.5
C.)
OC
0
0
-o
o
cn
0,
O.
50
50
70
90
110
130
150
Tube No.(05mI/tube)
Figure 2. Resolution of the components of Zea pectin after hydrolysis
with pectate lyase. Zea pectin (5.3 mg as GalUA equivalent/2 mL of
50 mm Tris-HCI buffer, pH 8.2) was incubated with E. carotovora
pectate lyase. At intervals, the incubation mixture was monitored for
the presence of 4,5-unsaturated GalUA (inset). After 1 h incubation,
a portion (0.4 mL) of the hydrolysate was applied to a column (1.3 x
40 cm) of Sepharose CL-6B preequilibrated with 20 mm Na-acetate
buffer (pH 5.0), followed by elution of fractions with the same buffer.
Fractions of 0.5 mL were collected and assayed for uronic acid (A&v,
*-*), total carbohydrate (A4w, X-X), and 4,5 unsaturated
galacturonic acid (A2m, O---O). The arrows on the figure index the
elution positions of Leuconostoc dextran (VO), Dextran 110 (110),
Dextran T-40 (40), and glucose (G) used for column calibration.
mixture was centrifuged to remove the methanol-precipitated
materials. The supernatant (low mol wt fraction) was evaporated to dryness. The dried material was dissolved in 1 mL of
water and passed through a column (0.4 x 6 cm) of Dowex1 (acetate form), yielding the neutral fraction. Acidic sugars
were eluted from the column with 8.5 M acetic acid.
The neutral fraction obtained from the low mol wt fraction
of 0.1 M TFA hydrolysate was designated as a low mol wt
neutral fraction (yield: 110 ,ug as Xyl equivalent). The acidic
fractions obtained from the low mol wt fractions in the 0.1,
0.5, and 2 M TFA hydrolysates were combined to give a low
mol wt acidic fraction (yield: 788 Mg as GalUA equivalent).
Enzymic Hydrolysis of Zeo Pectin
Zea pectin (5.3 mg as GalUA equivalent) was dissolved in
2 mL of 50 mm Tris-HCl buffer (pH 8.2) and incubated with
20 ML of Erwinia carotovora pectate lyase (PL, 1 mg/50 mm
Tris-HCl buffer, pH 8.2) at room temperature. At intervals,
Carbohydrate (as Glc equivalent), uronic acid (as GalUA
equivalent), and protein content in WSP-III in 2.6 L of Naphosphate buffer obtained from Zea shoots (fresh weight,
3800 g) were 2444, 906, and 20.8 mg, respectively. Sugar
composition analysis showed that WSP-III consisted of Rha,
Rib, Ara, Xyl, Man, Gal, Glc, and uronic acid in the molar
proportions of 0.3: 2.2: 3.8: 2.2: trace: 4.6: 39.9: 47.0. WSPIII was fractionated into seven fractions by the graded
(NH4)2S04 precipitation method as described previously (9).
Studies on certain fractions reveal that most of the glucose
residues are derived from (1- 3), (1-+4)-fl-D-glucan and (13)#-D-glucan (8, 9).
Uronic acid content in fraction 20P-S (362 mL of aqueous
solution), obtained after resolution of WSP-III by the graded
(NH4)2SO4 precipitation method, was 492.3 mg. About 55%
of the total uronic acid in WSP-III was recovered as fraction
20P-S. Sugar composition analysis of fraction 20P-S showed
that it also consisted of Rha, Ara, Xyl, Gal, Glc, and uronic
acid in the molar proportions of 0.4: 0.5: 0.4: 2.0: 1.1: 95.6.
When fraction 20P-S (350 mL of the sample) was chromatographed on DEAE-Sephadex A-25 (phosphate form), it was
separated into two fractions: one that was not retained by the
column (fraction 20P-S-I) and another that eluted from
DEAE-Sephadex at 0.5 M NaCl (fraction 20P-S-II) when
subjected to elution by stepwise increments of salt solutions.
Polysaccharides from both fractions were recovered by centrifugation after precipitation with four volumes of methanol.
Yields of fractions 20P-S-I and -II were 376.2 and 23.7 mg as
GalUA equivalent. The total yield accounted for approximately 10% ofthe total WSP-III. Previous studies have shown
that WSP-III accounts for approximately 2.5% of the total
Zea cell wall polysaccharides (6, 7). Therefore, one may
esLimate that the pectic polysaccharide recovered in these
fractions accounts for about 0.3% of the total Zea cell wall
polysaccharides.
To attempt purification, fraction 20P-S-I was chromatographed on DEAE-Sephadex A-25 (acetate form). However,
the fraction was strongly absorbed on the gel and no carbohydrate eluted during stepwise elution with 0 to 1, 1, and 2 M
NaCl in the same buffer or with 0.1, 0.5, 2 M NaOH. The
results suggest that fraction 20P-S-I does not contain neutral
polysaccharide(s). Fraction 20P-S-I was insoluble in cold
water but became soluble after heating in a boiling water bath
for 10 min. The aqueous solution exhibited a high viscosity
and formed a gel upon addition of acid. When fractions 20PS-I and -II were separately subjected to gel-filtration chromatography on Sepharose CL-6B, they both eluted near the void
volume of the column. Sugar composition analysis showed
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Copyright © 1989 American Society of Plant Biologists. All rights reserved.
ZEA PECTIC POLYSACCHARIDE
795
Figure 3. Fractionation of the pectate
lyase hydrolysate of the Zea pectin. The
pectate lyase hydrolysate of Zea pectin (1.5
mL, see "Materials and Methods") was apE plied to a column (1.8 x 7.0 cm) of DEAEC
Sephadex A-25 preequilibrated with 20 mM
Ul)
Na-acetate buffer (pH 5.0). After washing
ro the column with the same buffer, the ad.0
sorbed materials were eluted with a linear
a
w
C salt gradient imposed with the same buffer
0
u
and assayed for carbohydrate (A4so) and
b.00 4,5-unsaturated
galacturonic acid (A2W).
(0
.Ja Tubes4to6,11 to20,21 to27,28to36,
'4
38 to 46, 47 to 50, 51 to 57, and 58 to 75
were separately combined and concentrated to give fractions EH-a to -h.
40
60
50
Tube No. (3 ml/tube)
Table 11. Yields and Neutral Sugar Composition of Fractions Obtained from the Enzymic Hydrolysate
of Zea Pectin after Chromatography on DEAE-Sephadex A-25
Tube Nos.
in Figure
Fraction3
Rha
Fuc
Neutral Sugar Composition
Xyl
Man
Ara
Glc
Gal
mo%/O
5.1 Trace 11.1 28.8 4.7 33.0 17.3
0.9 55/45
1.1 Trace 12.0 46.6 4.4 23.5 12.4
0.4 45/55
7.6
0.4 69/31
7.5 Trace 20.0 45.5 7.8 11.6
7.7
2.4 Trace 18.0 44.8 2.1 25.0
1.0 53/47
81.1
0/100
7.4 Trace 17.0 36.7 4.4 24.5 10.1
1.0 40/60
Trace Trace 13.7 39.3 8.4 26.3 12.4
2.0 40/60
51-57
EH-g
6.1
EH-h
1.3 Trace 20.4 47.2 6.1 19.0
58-75
13.3 49/51
a The amount of carbohydrate material in each peak, expressed as percent of the total recovered
b Ratio of neutral sugar (NS) and acidic
carbohydrate, was determined by the phenol-H2SO4 method.
sugar (AS). NS content was determined by GLC of the alditol trifluoroacetates of the acid hydrolysate.
AS content was determined by the carbazole-H2S04 method.
%
EH-a
EH-b
EH-c
EH-d
EH-e
EH-f
4-6
11-20
21-27
28-36
38-46
47-50
that fractions 20P-S-I and -II consisted of Rha, Fuc, Ara, Xyl,
Man, Glc, Gal, and GalUA in the molar proportions of 0.7:
0.2: 1.1: 1.23: 0.2: 1.3: 1.5: 93.9 and of 0.6:0.: 0.:0.: 0.9:
1.2: 4.2: 92.7, respectively. Both fractions contained a small
amount of unidentified sugars (about 0.5%). Fraction 20P-SI is hereafter designated as Zea pectin.
Acid Hydrolysis of Zea Pectin and Analysis of the
Hydrolysate
Zea pectin (about 60 mg as GalUA equivalent) was hydrolyzed with 1 M TFA. The hydrolysate was chromatographed
on a Dowex- 1 column, and five carbohydrate fractions AH- 1
(unadsorbed fractions), AH-2 (eluted with 0.1 M CH3COOH),
AH-3 (eluted with 0.2 and 0.5 M CH3COOH), AH-4 (eluted
with 4.0 M CH3COOH) and AH-5 (eluted with 2.0 M NaOH)
were obtained at yields of 8.3, 33.5, 2.8, 3.1, and 2.0 mg as
GalUA equivalents, respectively.
The major fraction, AH-2, was indistinguishable from authentic galacturonic acid upon paper chromatographic analysis (method B).
AH-3 resolved into two components (RGaJUA 0.14 and 0.04.
method B), and the major compound, AH-3-i (RGalUA 0.14,
about 70% of AH-3) was isolated by preparative paper chromatography. Neutral sugar analysis of the acid hydrolysate by
GLC and paper chromatography (method A) indicated that
rhamnose is the sole neutral sugar in AH-3-i, and paper
chromatographic analysis (method B) showed that galacturonic acid is a sole acidic sugar. The ratio of galacturonic acid
and rhamnose of AH-3-i was approximately 5:1.
The unadsorbed fraction, AH-1, was chromatographed on
a column (1.0 x 60 cm) of Bio-Gel P-2, and fractions corre-
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Copyright © 1989 American Society of Plant Biologists. All rights reserved.
796
KATO AND NEVINS
Plant Physiol. Vol. 89, 1989
was chromatographed on Sepharose CL-6B. Fig2 reveals that the enzymic hydrolysate of Zea pectin
exhibits a much lower degree of polymerization compared
with that of the original Zea pectin which eluted near the V0
with a peak at tube No. 45.
Another portion of the enzymic hydrolysate of Zea pectin
was chromatographed on DEAE-Sephadex A-25 (Fig. 3), and
eight fractions, EH-a to EH-h were obtained. The elution
pattern of a substance with absorption at 235 nm, corresponding to the 4,5-unsaturated GalUA, is associated with that of
carbohydrate (A480) (Fig. 3). Yields, ratios, of neutral and
acidic sugars and neutral sugar compositions of the fractions
are summarized in Table II. All except for fraction EH-e
consist of neutral and acidic sugars and their neutral sugar
composition is nearly the same.
The major fraction, EH-e, was chromatographed on BioGel P-2 (Fig. 4). Three fractions, EH-e-l, -2, and -3 obtained
were rechromatographed on the same column. When each of
the fractions was subjected to paper chromatography (method
hydrolysate
Vo
GalUA
1.0
I
no Lr,
E
I
,
ure
.,
I#
C
11
rf)CM
I
0.6
0
_
Co
co°
,
0.4
It
~~~~~~~~~~~~~~~~~~~~~~~~I
.0.
0
C0 0.2
0
,J
0 20
E B0
40
60
Tube No. (0.5 ml /tube)
100
1l20
120
Figure 4. Fractionation of fraction EH-e. A concentr rate of fraction
EH-e obtained in Figure 3 was applied to a column ( [1 x 55 cm) of
Bio-Gel P-2 preequilibrated with 1 M ammonium acetat4 e and operated
at
The column was eluted with 1 M ammc mnium acetate.
Fractions of 0.5 mL were collected and assayed for carbohydrate
(A4so) and 4,5-unsaturated galacturonic acid (A235). The arrows on the
figure index the elution positions of apple pectin (VO) arnd GalUA used
for column calibration. Tubes 39 to 46 (fraction EH- e-1), 47 to 53
(EH-e-2), and 54 to 59 (EH-e-3) were separately coml ibined, concen-
45°C.
trated, and refined by rechromatography
on
the
same
single
observed;
ROaIUA
values
were
0.06 for
EH-e-l, 0.21 for EH-e-2, and 0.79 for EH-e-3. Paper chromatographic analysis (method B) of the acid hydrolysates of
EH-e-1 to -3 showed that these three fractions contained
GalUA. Sugiura (15) reported that E. carotovora PL hydrolyzed pectic acid into mono-, di-, tri-, tetra-, pentasaccharide
and higher oligomers. EH-e-l, -2, and -3 probably represent
tetra-, tri-, and disaccharides of GalUA, the nonreducing
terminals of which
are
4,5-unsaturated GalUA.
DISCUSSION
column.
sponding to monosaccharide were collected and I subjected to
neutral sugar analysis. Rha, Fuc, Ara, Xyl, Man, Glc and Gal
were detected in the molar proportions of 21 0 5.2: 19.2:
18.1: 2.6: 12.0: 22.8.
Partial Acid Hydrolysis of Zea Pectin and Ana lysis of the
Hydrolysate
Zea pectin (about 32 mg as GalUA equivalen It) was hydrolyzed sequentially with 0.1, 0.5, and 2 M TFA. i k low mol wt
acidic fraction, and a low mol wt neutral fracti ion were prepared from the hydrolysates as described in "`' daterials and
Methods."
The low mol wt acidic fraction was analyz Eed by paper
chromatography (method B). Three component s (RGaiUA 1.0,
0.28, and 0.1 11) were detected; the RGacUA valuesvwere identical
with those of authentic GalUA, GalUA-(l -4) -GalUA, and
GalUA-(1-4)-GalUA-( I >4)-GalUA.
The low mol wt neutral fraction was chromal tographed on
a column (1.0 x 60 cm) of Bio-Gel P-2 (Fig. 1). INeutral sugar
composition of fractions PAH-N-a to -h (Fig. 1) vvere analyzed
by GLC, and the results are summarized in Tabl le I. All of the
fractions consisted of Rha, Fuc, Ara, Xyl, Man, 4 Glc, and Gal.
Enzymic Hydrolysis of Zea Pectin and Analys
Hydrolysate
B),
sis of the
Ray and Rottenberg (12) isolated a GalUA disaccharide
from oat coleoptile cell walls, and Wada and Ray (16) extracted polygalacturonic acid from oat coleptile cell walls by
ammonium oxalate. Methylation analyses of maize coleoptile
cell walls have demonstrated the presence of 4-linked galacturonosyl residues (2). The results of these studies suggest that
monocot cell walls contain small amounts of 4-linked galacturonans. More recently, Shibuya and Nakane (14) isolated
and characterized pectic polysaccharide of rice endosperm
cell walls.
The isolation of rhamnose-containing oligogalacturonic
acid from the acid-hydrolysate of Zea pectin clearly indicates
that the Zea pectin contains rhamnogalacturonan. However,
it is most likely that the Zea pectin preparation obtained here
is a mixture of rhamnogalacturonan and polygalacturonic
acid.
Zea pectin was hydrolyzed with pectate lyase. Fractions
EH-a, -b, -c, -d, -f, -g, and -h obtained from the hydrolysate
contained GalUA, Rha, Fuc, Ara, Xyl, Man, Glc, and Gal in
similar concentrations (Table II). Undoubtedly, these fractions were derived from the Zea pectin preparation. The
fractions exhibit absorption at 235 nm because of the presence
of 4,5-unsaturated GalUA (Fig. 3). Moreover, fractions PAHN-a to -h obtained by Bio-Gel P-2 chromatography after
partial acid hydrolysis of Zea pectin revealed oligosaccharides
comprised of Rha, Fuc, Ara, Xyl, Man, Glc, and Gal (Table
I). Although detailed structural studies of fractions EH-a to
h and PAH-N-a to -h have not been completed, the results
-
Zea Pectin (about 5.3 mg as GalUA equivalen it) was hydrolyzed with Erwinia carotovora PL. A portion of fthe enzymic
suggest
glycosidic bonds between other cell wall polysaccha-
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Copyright © 1989 American Society of Plant Biologists. All rights reserved.
ZEA PECTIC POLYSACCHARIDE
rides (including pectic neutral oligo- and polysaccharides) and
rhamnogalacturonan in Zea shoot cell walls.
1.
2.
3.
4.
5.
6.
7.
LITERATURE CITED
Bitter T, Muir HM (1962) A modified uronic acid carbazole
reaction. Anal Biochem 4: 330-334
Darvill AG, Smith CJ, Hall MA (1978) Cell wall structure and
elongation growth in Zea mays coleoptile tissue. New Phytol
80: 503-516
Darvill AG, McNeil M, Albersheim P, Delmer DP (1980) The
primary cell walls of flowering plants. In NE Tolbert, ed, The
Biochemistry of Plants, Vol 1. Academic Press, New York, pp
91-162
Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956)
Colorimetric method for determination of sugars and related
substances. Anal Chem 28: 350-356
Kato Y, Ito S, Iki K, Matsuda K (1982) Xyloglucan and l-Dglucan in cell walls of rice seedlings. Plant Cell Physiol 23:
351-364
Kato Y, Nevins DJ (1984) Structure of the arabinogalactan from
Zea shoots. Plant Physiol 74: 562-568
Kato Y, Nevins DJ (1984) Enzymic dissociation of Zea shoot
cell wall polysaccharides. I. Preliminary characterization of the
8.
9.
10.
11.
12.
13.
14.
15.
16.
797
water-insoluble fraction of Zea shoot cell walls. Plant Physiol
75: 740-744
Kato Y, Nevins DJ (1985) A (1--3)-j#-D-glucan isolated from Zea
shoot cell wall preparations. Plant Physiol 78: 20-24
Kato Y, Nevins DJ (1986) Fine structure of (13),(14)-D
glucan from Zea shoot cell-walls. Carbohydr Res 147: 69-85
Kabayashi M, Shishido K, Kicuchi T, Matsuda K (1973) Fractionation of the Leuconostoc mesenterides NRRL B- 1299 dextran and preliminary characterization of the fractions. Agric
Biol Chem 37: 357-365
Naganuma T (1984) Pectic substances-degrading enzymes produced by Erwinia carotovora and Pseudomonas marginallis.
Masters thesis, Tohoku University, Sendai, Japan
Ray PM, Rottenberg DA (1964) Uronic acid constituents of oat
coleoptile cell walls. Biochem J 90: 646-655
Robyt J, French D (1963) Action pattern and specificity of an
amylase from Bacillus subtilis. Arch Biochem Biophys 100:
451-467
Shibuya N, Nakane R (1984) Pectic polysaccharides of rice
endosperm cell walls. Phytochemistry 23: 1425-1429
Sugiura J (1982) Pectin and pectate lyase produced by Erwinia
carotovora. Doctoral thesis, Tohoku University, Sendai, Japan
Wada S, Ray PM (1978) Matrix polysaccharides of oat coleoptile
cell walls. Phytochemistry 17: 923-931
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