sure and weighed. The CDTA-insoluble residues were used for extraction of hemicelluloses.
In an independent set of experiments, pectic substances of the cell walls were fractionated into
three subfractions, according to Iraki et al. [11]. Cell wall preparations 100 mg were extracted
sequentially as follows: once with 10 ml ice-cold 5 mM ethylenediaminetetraacetic acid EDTA for
12 h with constant stirring, twice with 10 ml of 0.5 ammonium oxalate pH 6.5 at 100°C for 1 h
each, and once with 3.5 ml of 0.1 M KOH for 4 h. The EDTA and ammonium oxalate solutions each
extracted pectic substances by chelation of Ca
2 +
crosslinking the galacturonic acid units, and the 0.1 M KOH removed additional pectic substances
by hydrolysis of ester linkages or other weak alkali labile bonds [28].
2
.
3
.
2
. Hemicelluloses The cell wall residues, left after CDTA extrac-
tion, were treated with sodium chlorite 1 to remove lignins which interfere with the extraction
of hemicellulosic substances. Then, they were ex- tracted three times with 4 M KOH 100 ml g
− 1
cell wall preparation, supplemented with NaBH
4
3 mg l
− 1
, at 25°C [27]. The combined KOH extracts, acidified to pH 5.0 with glacial acetic
acid, were
dialysed against
deionised water
overnight and evaporated under reduced pressure. The mass of air-dry samples were determined by
weighing. No uronic acid was left in the hemicellu- lose fractions as checked with the uronic acid
assay see below.
2
.
3
.
3
. Cellulose content The KOH-insoluble residues were centrifuged at
2000 × g for 15 min. The pellets were washed with deionised water, weighed and hydrolysed in 14 M
sulphuric acid for 1 h at room temperature and then in 1 M sulphuric acid for 2 h at 100°C [27].
The glucose content in the hydrolysate was taken as a measure of cellulose content in the sample. It
was determined with the anthrone method [29] using b-
D
-glucose as a standard.
2
.
4
. Determination of sugar content and composition in the pectic and hemicellulose
polysaccharides Pectin and hemicellulose samples were hy-
drolysed in 2 M trifluoroacetic acid TFA at 125°C for 1 h. The liberated monosaccharides
were reduced to their respective alditol acetates and analysed by gas chromatography GC with
erythritol 0.5 mg 0.1 ml
− 1
added as an internal standard [30]. GC analyses was performed using a
Hewlett Packard
5890 gas
chromatograph equipped with the flame ionisation detector, a
splitless injection port, an HP 7673 autosampler and
fused silica
Wide Bore
Open Column
WBOTC, 30 m × 0.53 mm i.d. J W Scientific, USA, coated with 1 mm DB-Wax. The column
temperature program was: 5 min at 195°C, fol- lowed by a rise to 220°C, 5°C per min. Helium
was used as a carrier gas at a flow rate of 20 ml min
− 1
.
2
.
5
. Uronic acid content estimation The studied fractions were hydrolysed in 2 ml of
12 M sulphuric acid at 35°C for 1 h and then in 22 ml of boiling water for 2 h. In cool hydrolysates,
uronic acid contents were determined colorimetri- cally [31], with glucuronic acid as a standard.
Calculation of the uronic acid concentration was based upon the difference in absorbances between
450 and 400 nm.
2
.
6
. Statistics All determinations were performed on three
replicates, in three independently run experiments. The effects of the temperature treatment were
tested by one-way or two-way analysis of variance ANOVA. Means were compared between the
treatments by the least significant difference L.S.D. at the 0.05 probability level using Tukey’s
test.
3. Results
Preliminary experiments showed that extraction of winter oilseed rape leaves with ethanol resulted
in somewhat higher cell wall yield in comparison with that obtained from the benzene- or K-phos-
phate buffer-extracted tissues the respective val- ues were: 270, 259 or 250 mg cell wall g
− 1
leaf dry weight. Therefore, the ethanol extraction was
used for further analyses.
Fig. 1. Effects of low temperature on cell wall content in winter oilseed rape leaves. Significant differences at P B 0.05
are marked with different letters, shown in diagram.
polysaccharide fractions in the studied leaves, whereas hemicellulose content was relatively low
Table 1. The share of these polysaccharide frac- tions in the total cell wall polysaccharide pool in
NA leaves taken as 100 was 46.7, 37.1 and 16, respectively.
Cold acclimation of leaves resulted in the pro- nounced, statistically significant increase of the
pectin content in cell walls, as compared with the NA samples Table 1. The content of this fraction
decreased by 30 in response to the freezethaw treatment in comparison with that in CA tissues
see data for CAF
1
and CAF
2
discs. The differ- ence was statistically significant. The contents of
hemicellulose and cellulose fractions were not af- fected by low temperature treatments Table 1.
Sequential fractionation of pectic substances into three subfractions of different solubility in
calciummagnesium-chelating or diluted alkaline solutions yielded a total amount of pectins ex-
tracted from cell walls similar to that obtained by the CDTA extraction compare the respective data
in Tables 1 and 2. The most abundant fraction consisted of the ammonium-extractable pectins
67, whereas pectins extracted with diluted KOH showed the lowest share 7 in the total
pectin pool. Acclimation of leaves in the cold resulted in statistically significant increases in
pectins extractable by ammonium oxalate or di- luted KOH. The freezing treatment brought about
a pronounced decrease of these pectin subfractions to levels similar to those observed in NA leaves.
The content of the EDTA-soluble subfraction in the frost-thawed cells increased about twice.
Determination of uronic acid UA and neutral monosaccharide contents in pectins indicated that
uronic acid was the most abundant component of these polysaccharides Fig. 2. Its content in
pectins from NA leaves was 65 i.e. 65 mg of UA in 100 mg of pectins. Although the total
content of UA in CA walls increased about twice The pilot experiments showed that there were
no differences in the cell wall content and polysac- charide composition between the non-acclimated
leaf blades sampled from the plants just before the start of cold treatment and after the next 3 weeks
of plant growth at warm temperature 2015°C. Therefore, only the blades sampled just before the
cold treatment were analysed in the further studies and taken as a reference to the cold-acclimated
ones.
3
.
1
. Changes in cell wall content The content of cell wall in the cold-acclimated
CA leaf discs was about two-fold higher than that in the non-acclimated NA ones Fig. 1. The
exposure of CA plants to the freeze-thaw treat- ment resulted in a statistically significant decrease
of cell wall content observed after tissue recovery at 2°C, which resulted in a lower by 23 cell
wall content in CAF
2
compared with the CA discs.
3
.
2
. Changes in cell wall polysaccharide contents and composition
Pectins and cellulose were the most abundant
Table 1 Low temperature-induced changes in polysaccharide contents in leaf cell walls mg [100 mg cell wall]
− 1
a
Pectins CDTA extraction Treatment
Hemicelluloses Cellulose
Total polysaccharide pool 24.9
a
8.6
a
NA 19.8
a
53.3 20.9
a
9.2
a
39.1
b
CA 69.2
32.3
c
8.9
a
CAF
1
20.1
a
61.3 30.1
c
9.0
a
CAF
2
21.3
a
60.4
a
Means marked with same letter within the column do not differ significantly at the 0.5 probability level.
Table 2 Low temperature-induced modifications in pectin fractions mg [100 mg cell wall]
− 1
a
Fraction Treatment
Total polysaccharide pool Ammonium oxalate
KOH 0.1 M EDTA
NA 6.2
a
15.9
a
1.6
a
23.7 6.3
a
CA 29.8
b
4.8
b
40.9 16.2
a
1.4
a
32.2 14.6
b
CAF
− 1
a
Means marked with the same letter within the column do not differ significantly at the 0.05 probability level.
in comparison with the NA walls Fig. 2, compare the surface areas occupied by UA in NA and CA
specimens, its share in the CA pectin pool de- creased to 48. This was due to the increased
concentrations of neutral sugars such as arabinose and galactose or glucose in CA pectins, as com-
pared with NA pectins Fig. 2. The high contents of arabinose and glucose were also noted in
pectins in CAF
1
tissues, whereas galactose content markedly decreased. In consequence, the ara-
binose to galactose ratio in CAF
1
samples in- creased markedly from 1:1 in NA and CA pectins
to 2.5:1 in CAF
1
pectins. In pectins from CAF
2
discs, galactose and glucose contents increased whereas arabinose share decreased. Rhamnose, xy-
lose, fucose and mannose contents were low equal to or below 5 mg 100 mg
− 1
pectins in all the studied leaves and their contents were not
modified by the temperature treatments. The sugar composition of hemicelluloses Fig. 3
was less affected by cold acclimation and freezing treatment than that of pectins. Xylose, galactose,
arabinose and glucose in order of decreasing con- centration in NA samples were the main compo-
nents of hemicelluloses in winter oilseed rape leaves. They made up 80 or 85 of the total
hemicellulose sugars in NA or CA leaves, respec- tively. Low temperature cold and freezing treat-
ments changed the relative proportions of these sugars. The contents of galactose and arabinose
increased in the cold- and frost-treated leaves, changes in the arabinose content becoming statisti-
cally significant in the frost-pretreated discs. The contents of xylose and glucose decreased in re-
sponse to low temperature treatments, the effects of transient freezing resulting in statistically sig-
nificant differences between NA and CAF
1
and CAF
2
discs. The content of rhamnose, relatively high in hemicelluloses from NA discs, decreased in
the CA leaves; the freezing treatment was practi- cally of no further effect.
4. Discussion
