a deposition of silica [19], were observed. Such modifications are thought to affect water balance
and the pattern of ice propagation in plant tis- sue rather than the ability of cell walls to un-
dergo deformations.
Our previous experiments performed on cold- grown or on frost pretreated winter oilseed rape
leaves indicated that the activity of cell wall-as- sociated b-galactosidase decreases in cold-accli-
mated winter oilseed rape leaves, whereas it increases rapidly in response to a brief freezing
treatment [20]. The enzyme is responsible for the breakdown of b-galactosyl linkage in pectin and
hemicellulosic polysaccharides [21]. It is involved in the breakage of bonds between cell wall
polysaccharides during cell wall loosening [21,22] and in the degradation of pectic polymers of
galactose during cell growth [23]. The freezing- induced increase in cell capability for the turgor-
dependent
extension growth
was actually
observed in our previous work [5]. Therefore, it may be anticipated that cell wall properties will
differ between cold-acclimated and freeze-af- fected tissues. The major objective of the present
experiments was to verify this supposition by ex- amination of cell wall content and polysaccha-
ride composition in winter rape oilseed rape plants subjected to cold \ 0°C and freezing
B 0°C treatments.
2. Materials and methods
2
.
1
. Plant material Plants of winter oilseed rape Brassica napus
L. var. oleifera L. cv Jantar were grown in a mixture of sand and peat 1:1, vv, under a 16-h
photoperiod, as described earlier [24] at 2015°C daynight temperature for 3 weeks non-accli-
mated NA plants. Light was provided by cool white and daylight fluorescence tubes Pila,
Poland and Tungsram, Hungary, respectively in proportion 1:1, the level of photosynthetic active
radiation at the top of a plant being 200 mmol m
− 2
s
− 1
. After 3 weeks of growth, half of the plants were transferred to an acclimation cham-
ber at 2°C for 21 days, the light conditions be- ing unchanged cold-acclimated CA plants.
Then, CA plants were transferred for 18 h to a freezing chamber Dual Program Illuminated In-
cubator 818, Precision Scientific, USA set at − 5°C. Freezing treatment was followed by plant
thawing and a recovery at 2°C for 6 or 24 h in darkness CAF
1
and CAF
2
plants, respectively. Such a treatment was found previously to in-
crease freezing tolerance of the cold-grown win- ter oilseed rape leaves for 3 – 5 K and to increase
the growth capability of leaf blades [5]. Samples for analyses were cut from the blades of the
fourth and fifth leaves, the expansion of which took place during the experiment and resulted in
eight-fold or two-fold increase of blade surface in NA or CA plants, respectively. All analyses
were performed on leaf discs 2 cm in diameter cut from leaf areas located between the lateral
veins.
2
.
2
. Cell wall isolation The samples of leaf discs in three replicates, 5
g fresh weight each were frozen in liquid nitro- gen. Cell wall isolation and purification was per-
formed according to Waldron and Selvendran [25], using boiling 85 ethanol as the extraction
medium. In preliminary experiments, the cell wall yield from the ethanol-extracted material
was compared with that obtained from benzene- or K-phosphate buffer extracted tissues. The
ethanol-extracted cell wall preparations were sub- jected to 90 vv dimethyl sulfoxide DMSO
treatment for 24 h at 20°C to remove starch [26]. The starch-free as checked with an iodine
test cell wall material was washed free of resid- ual
DMSO by
several washings
with 95
ethanol. Samples were evaporated to dryness and weighed.
2
.
3
. Fractionation of cell wall polysaccharides
2
.
3
.
1
. Pectic substances An air-dried, starch-free pellet approximately
200 mg was extracted with 10 ml mixture of 50 mM trans-1,2-diaminocyclohexane-N,N,N,N-te-
traacetic acid CDTA and 50 mM Na-acetate, pH 6.5, for 6 h and then with 50 mM CDTA
for 2 h at room temperature [27]. The combined CDTA extracts were centrifuged 10 000 × g, 15
min and concentrated by evaporation under re- duced pressure. The concentrate was dialysed for
72 h against deionised water three changes, 4°C, evaporated to dryness under reduced pres-
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
