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Plant Physiol. (1998) 116: 403-408
Apoplastic Sugars, Fructans, Fructan Exohydrolase, and Invertase
in Winter Oat: Responses to Second-Phase Cold Hardening
David P. Livingston III* and
Cynthia A. Henson
United States Department of Agriculture, Agricultural
Research Service (D.P.L., C.A.H.), and Department of Crop Science,
North Carolina State University, Box 7629, 840 Method Road Unit
III, Raleigh, North Carolina 27695-7629 (D.P.L.); and Department
of Agronomy, University of Wisconsin, 1575 Linden Drive, Madison,
Wisconsin 53706 (C.A.H.)
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ABSTRACT |
Changes in apoplastic carbohydrate
concentrations and activities of carbohydrate-degrading enzymes were
determined in crown tissues of oat (Avena sativa L., cv
Wintok) during cold hardening. During second-phase hardening ( 3°C
for 3 d) levels of fructan, sucrose, glucose, and fructose in the
apoplast increased significantly above that in nonhardened and
first-phase-hardened plants. The extent of the increase in apoplastic
fructan during second-phase hardening varied with the degree of fructan
polymerization (DP) (e.g. DP3 and DP4 increased to a greater extent
than DP7 and DP > 7). Activities of invertase and fructan
exohydrolase in the crown apoplast increased approximately 4-fold over
nonhardened and first-phase-hardened plants. Apoplastic fluid extracted
from nonhardened, first-phase-hardened, and second-phase-hardened crown tissues had low levels, of symplastic contamination, as determined by
malate dehydrogenase activity. The significance of these results in
relation to increases in freezing tolerance from second-phase hardening
is discussed.
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INTRODUCTION |
Cold-hardening winter cereals such as rye (Secale
cereale), wheat (Triticum aestivum), barley
(Hordeum vulgare), and oat (Avena sativa) is
generally accomplished by exposure to temperatures just above freezing.
As early as 1935, Tumenov (cited by Trunova, 1965 ) reported that an
additional phase of hardening by exposure of cold-hardened plants to
nonlethal, below-freezing temperatures resulted in significant
increases in cold hardiness. Tumenov called this 2PH. With a 2PH
treatment, Trunova (1965) and Siminovich (cited by Steponkus, 1978)
induced an increase in freezing tolerance of wheat significantly beyond
that achieved from cold hardening above freezing (1PH). Olien (1984)
reported similar results in rye and barley, and Livingston (1996)
reported a decrease in the LT50 of oats from
13°C with 1PH only to 18°C after a 3-d 2PH period at 3°C.
After 2PH Trunova (1965) found decreased levels of fructan and
increased levels of Glc, Fru, and Suc in whole crown tissue of wheat.
He suggested that the sugar increase during 2PH was providing osmotic
protection to cells in the over-wintering organ (crown) of the plant.
Olien (1984), using a perfusion technique, reported an increase in
apoplastic sugars during 2PH and suggested that these sugars helped
prevent adhesion of ice to critical cellular tissue during
freezing.
In the present study we wanted to determine the percent distribution of
fructan exohydrolase and invertase in the apoplast and symplast of oat
crowns, and what the effect of 1PH and 2PH would be on their activities
in the respective locations. Additionally, we wanted to determine if
fructan was present in the apoplast and the effect of cold hardening on
its presence. Finally, we wanted to re-examine the effect of cold
hardening on simple sugar levels in the apoplast using a different
technique than that used by Olien (1984).
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MATERIALS AND METHODS |
Plant Culture
Seeds of the oat (Avena sativa L.) cv Wintok
were planted in Scotts Metromix 2201
(Scotts-Sierra Horticultural Products Co., Marysville, OH) in plastic
tubes (2.5 cm in diameter × 16 cm in height) with holes in the
bottom to allow drainage. The tubes were suspended in a grid that held
100 tubes. Plants were watered three times weekly with a complete
nutrient solution (Livingston, 1991) and flushed three times weekly
with tap water. Plants were grown for 5 weeks at a day/night
temperature regime of 13/10°C with a 12-h photoperiod in a growth
chamber with 240 µmol m 2
s1 PAR (80% cool fluorescent and 20%
incandescent). These were the NH plants. Five weeks after planting,
plants were transferred to a chamber at 2°C with an 12-h photoperiod
at 310 µmol m2 s1 for
3 weeks. This 3-week period constituted 1PH.
Second-Phase Hardening
After 1PH plants were at the three-leaf stage with two to three
tillers each. They were removed from tubes and washed free of planting
medium in ice water. Roots were trimmed to 2 cm and the plants were
placed in a plastic bag, inoculated with ice, and sealed to help
prevent desiccation. To induce 2PH, plastic bags containing plants were
placed in a freezer at 3°C in the dark for 3 d.
Thermocouples placed near plants were used to monitor temperatures.
Carbohydrate Extraction, Whole Tissue
Six crowns (the tissue remaining after removing leaves and stems
to within 2 cm of the stem base) were bulked and ground for approximately 30 s using a stainless-steel grinder (Livingston, 1990). Measurements were conducted in triplicate. Ten milliliters of
HPLC-grade water at 1°C was used to rinse plant tissue into a beaker.
A 2-mL aliquot was filtered through a 0.45-µm filter and placed on
dry ice. These aliquots were used for determining osmolyte
concentration and the activity of invertase, MDH, and FEH. The
remaining extract was heated to 95°C for 10 min. The lapsed time from
plant harvest to either freezing on dry ice or heating to 95°C was
approximately 3 min. Heat-treated solution was filtered through a
0.45-µm filter in preparation for HPLC analysis. No changes in
carbohydrate composition were observed for 36 h at 2°C in
filtered, heat-treated samples.
Apoplast Extraction
Intact crowns from 15 plants were placed in the bottom 3 cm of a
50-mL syringe barrel. A 4-mL HPLC insert vial was placed on the syringe
tip to collect apoplastic solution. The syringe barrel containing crown
tissue with the 4-mL vial attached was placed in a 50-mL centrifuge
tube and centrifuged at 500g for 10 min at 3°C.
An aliquot of the apoplastic fluid was used for determining osmolyte
concentration and the activities of MDH, FEH, and invertase. The
remaining solution was heated for 10 min at 95°C and analyzed by
HPLC.
The volume of liquid centrifuged averaged around 5 to 7% of the total
liquid in the whole crown tissue (data not shown). By infiltrating the
apoplast with an indicator dye, Tetlow and Farrar (1993) found apoplast
volumes to be about 8% of the total volume in healthy barley
(Hordeum vulgare) leaves.
The viability of crowns after their apoplastic fluid was removed was
examined by observing the regrowth of roots and shoots after a 3-week
growth period under the initial growing conditions described above.
One-hundred percent survival occurred for all three treatments.
Guttation
Guttation was induced in oat plants by placing racks containing
1PH plants in a covered container in the dark with a 4-cm layer of
water in the bottom. After 10 h about 10 droplets of liquid found
on leaf tips of separate plants were pooled for HPLC analysis of
carbohydrates.
Carbohydrate Separation and Quantification
Fructans were separated according to DP using a modified
silver-based analytical HPLC column (7.8 × 300 mm, Aminex
HPX-42A, Bio-Rad). This column was found to hydrolyze fructans and Suc and was therefore permanently modified by passing 0.5 m
NaNO3, at a rate of 2 mL
min1, through the column for approximately
18 h. This treatment eliminated the hydrolysis of smaller fructans
and Suc and improved the resolution of smaller sugars. Resolution of
larger (DP5 and DP6) fructans, however, was slightly reduced in the
modified column. Because samples were not desalted prior to injection,
a cation and anion-exchange guard column immediately preceding the
analytical column was used to prevent co-elution of small ionic
compounds with carbohydrates. The mobile phase was HPLC-grade water at
a flow rate of 0.4 mL min1.
Separated carbohydrates were detected by a 410 refractive index
detector (Waters). Unknown peaks were identified by co-chromatography with external standards. Oligomers were quantified by comparison of
unknown peak areas to peak area-response curves derived from standard
solutions of varying concentrations (Livingston et al., 1993). Peak
area was measured by a Millennium 2000 chromatography workstation
(Waters) with a microcomputer.
Fructan Collection and Hydrolysis
Each size class of fructan is composed of more than one isomer. In
1PH oats DP3 is composed of 1-kestose and neokestose, DP4 is composed
of four isomers, and DP5 is composed of seven isomers (Livingston et
al., 1993). The DP6 and DP7 isomers in oats have not yet been
identified but each size class is composed of numerous isomers.
To confirm that quantified peaks from the apoplastic fluid were Fru
polymers, individual peaks were collected from the 42A column and
hydrolyzed for 1.5 h at 95°C in 0.16 n HCl. These
conditions were found to be ideal for measuring the DP of fructans
(D. P. Livingston, unpublished data). Suc was hydrolyzed with the
samples to confirm that conditions were not too stringent. Hydrolyzed samples were then separated by HPLC using an Aminex 87H column (Bio-Rad). Glc and Fru residues were quantified as above. All peaks
from DP3 to DP7 produced Glc and Fru in ratios consistent with their
suspected DP. The DP > 7 peak is composed of a mixture of sizes,
therefore, its DP could not be determined. No evidence was found from
the hydrolysis data that any of the peaks from whole-tissue extracts or
apoplastic fluid were glucans.
Osmolality, MDH, FEH, and Invertase
Osmolyte concentrations were measured with a vapor pressure
osmometer (model 5100C, Wescor, Logan, UT) calibrated with 100 and 290 mmol/kg NaCl standards. Measurements were conducted in triplicate and
data are expressed as millimoles/gram fresh weight.
NAD-linked MDH (EC 1.1.1. 37) activities were assayed by measuring the
oxaloacetate-dependent oxidation of NADH on a recording spectrophotometer (model 2101-PC, Shimadzu, Tokyo, Japan) at 30°C. Assays were conducted as described by Duke et al. (1975).
FEH activity was assayed using neokestin as the substrate, with
detection of released Fru as described by Henson and Livingston (1996).
Neokestin is a mixture of neokestose-based fructan isomers of DP7 to 14 that is aqueously extracted from oat leaves and precipitated by ethanol
(Livingston, 1989). Invertase activity was measured as described by
Henson (1989). Enzyme activities are expressed as nanomoles or
micromoles of product generated per minute per gram fresh weight of
crown tissue.
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RESULTS AND DISCUSSION |
Carbohydrates in Whole Tissue
The concentrations of Suc and all fructans increased nearly 3-fold
in whole crown tissue during 1PH (Fig.
1), which is consistent with results
reported earlier in oats and other winter cereals (Livingston, 1991;
Pollock and Cairns, 1991).
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| Figure 1.
Carbohydrate concentrations in whole-crown tissue
from the winter oat cv Wintok. Plants were grown under NH (5 weeks at
13°C, black bars), 1PH (3 weeks at 2°C, open bars), or 2PH (1PH
plants were placed in a 3°C freezer for 3 d in the dark,
shaded bars) conditions. The bar above each group of three bars is the
lsd. P = 0.05; n = 3. S, Suc; G, Glc;
F, Fru.
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During 2PH the concentration of all size classes of fructan decreased
in whole crown tissue, whereas the concentration of Glc and Fru
increased. The largest decrease was in DP3 and DP4 fructan, and the
largest increase was in Fru (Fig. 1). This decrease in fructan and
increase in sugars (Suc excepted, Fig. 1) during 2PH agrees with
earlier reports in oats, barley, wheat (Triticum aestivum),
and rye (Secale cereale) (Trunova, 1965; Olien, 1984; Livingston, 1996). Although the increased sugars in whole crown tissue
would lower the freezing point of the cytoplasm by only a fraction of a
degree, if sugars were concentrated in specific regions of the crown,
the freezing point of those regions could be lowered enough to increase
the freezing survival of the whole plant.
Indicators of Cellular Leakage
MDH activity has frequently been used as a specific marker for
cellular integrity, since buffer-soluble MDH activity is inside the
cell when the plasmalemma is intact. MDH activity is readily detectable
in extracellular fluids when the plasmalemma is damaged. The usefulness
of MDH activity as a specific marker of cellular integrity has been
demonstrated in studies of imbibition-induced damage of seeds with
genetically altered testa (Duke and Kakefuda, 1981; Duke et al., 1986)
and in studies of apoplastic fluid composition. In apoplastic
preparations from pea, Brassica napus, and barley (Beers and
Duke, 1988; Tetlow and Farrar, 1993; Husted and Schjoerring, 1995),
levels of MDH contamination of less than or equal to 1% were reported.
In our study the percentages of total MDH activity recovered in
apoplastic preparations from NH, 1PH, and 2PH tissues ranged from 0.7 to 2.8% (Table I), indicating little
cellular rupture. MDH leakage from cells of alfalfa crowns and roots
was 4-fold higher when plants were frozen at 8°C than when they
were frozen at 4°C (Sulc et al., 1991), suggesting an increase in cellular leakage as the temperature was lowered. The small increase in
intracellular leakage from our 2PH treatment could have occurred prior
to or during apoplast extraction, possibly from cellular membranes that
were more susceptible to leakage from centrifugation.
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Table I.
MDH activity and osmolyte concentration of apoplast
and whole crown tissue from plants that were subjected to three
hardening treatments
Shown in parentheses is the amount of marker in the apoplast as a
percentage of the total marker in the crown.
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The second method we used to assess cellular integrity was to compare
the concentration of osmolytes in the apoplast with that of the whole
tissue. Changes in osmolyte concentrations are analogous to changes in
electrical conductivity, which is commonly used as a measure of cell
rupture because the conductivity of extracellular fluids under
nonstressed conditions is low and increases significantly upon loss of
cellular integrity (Dexter et al., 1932; Sulc et al., 1991).
Percentages of total osmolytes present in apoplastic fluid extracted
from NH and 1PH tissues were not significantly different from each
other (Table I). Even though we found no significant difference in
osmolytes in the apoplasts of NH and 1PH oat crowns, others (Marentes
et al., 1993; Antikainen et al., 1996) have shown that quantitative
changes in proteins do occur in the apoplast of rye leaves during 1PH.
We conclude that 2.8% of the cells experienced damage severe enough to
result in the release of macromolecules of at least the size of MDH
because 2.8% of the total MDH was present in the apoplast of 2PH
tissues. This amount of damage was not lethal to the plants, as
evidenced by complete re-growth of roots and stems after apoplast
extraction (data not shown). This rupture of 2.8% of the cells
undoubtedly contributed to the osmolytes present in the apoplast of 2PH
tissues. However, other factors probably contributed to the observed
increase in osmolytes during 2PH. For example, nonlethal perturbations
in the plasmalemma that allow molecules smaller than MDH, such as
carbohydrates (Fig. 2 ), to pass into the apoplast
could have occurred during 2PH. Such leakage would contribute to the
observed increase in osmolytes during 2PH and may have been the result
of specific adaptive responses to the hardening treatment. Some or all
of these adaptive responses may account for the clear increase in
freezing tolerance of oats (Livingston, 1996), as well as other winter
cereals (Trunova, 1965; Olien, 1984).
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| Figure 2.
Carbohydrate concentrations in apoplastic fluid
from crown tissue of the winter oat cv Wintok. Plants were grown under
NH (5 weeks at 13°C, black bars), 1PH (3 weeks at 2°C, open bars), or 2PH (1PH plants were placed in a 3°C freezer for 3 d in the dark, shaded bars) conditions. The error bar above each group of three
bars is the lsd. P = 0.05; n = 3. Numbers above the bars indicate the percentage of the total
carbohydrate (taken from Fig. 1) that was found in the apoplast. S,
Suc; G, Glc; F, Fru.
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Since hydathodes are apparently in direct contact with the apoplast
(Dieffenbach et al., 1980), sampling guttated liquid could be viewed as
an apoplast-sampling method with the least possible perturbation of the
plant. The concentration of Suc in guttated liquid was about 25% of
that in the apoplast. The concentration of Glc in the guttate was
slightly higher than it was in the apoplast, and the concentration of
Fru in guttated liquid was three times that found in the apoplast.
Small quantities of all fructan size classes found in the apoplast were
also found in the solution collected from leaf tips (Fig.
3). Fructan has been found in the guttated liquid
from cold-hardened barley plants, although individual DPs were not
quantified (C.R. Olien, unpublished data). The presence of fructan in
the guttate (Fig. 3) suggests that at least some level of fructan in
the apoplast is not an artifact of extraction.
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| Figure 3.
Carbohydrate concentrations in apoplastic fluid
(shaded bars) in crown tissue in comparison to guttated liquid (open
bars) from the 1PH winter oat cv Wintok. S, Suc; G, Glc; and F, Fru.
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The differences in the composition of fructan, Suc, and Fru in guttated
liquid from that in the apoplast (Fig. 3) could be explained by the
presence of active invertase and FEH in the guttated liquid in contrast
to the apoplastic fluid, which had been heat inactivated. Although we
did not assay for FEH or invertase in guttated liquid, since guttated
liquid passes through hydathodes that are reportedly in contact with
the apoplast (Dieffenbach et al., 1980), it would not be surprising to
find both FEH and invertase in guttated liquid. In addition, the
apoplastic fluid was sampled at the crown and the relative
concentrations of sugars would likely change as they approach
photosynthesizing cells.
Carbohydrates in Apoplast
The amount of Suc, Glc, and Fru in the apoplast of NH and IPH
plants as a percentage of the total in whole crown tissue is similar to
the distribution of osmolytes and MDH. This suggests that their
presence could be attributed to cellular leakage or rupture. A lack of
change during 1PH in the percentage of apoplastic carbohydrates (Fig.
2) suggests that this is not a factor in the universally recognized
increase in freezing tolerance of winter cereals during 1PH; other
mechanisms must be responsible for this adaptation.
In contrast to 1PH, during 2PH the percentage of total sugars in the
apoplast increased to 17.6% for Glc (a 10-fold increase), 14.9% for
Fru (a 20-fold increase), and 6.4% for Suc (a 6-fold increase)
(calculated from Figs. 1 and 2). The increases in these sugars beyond
levels attributable to cellular rupture may be part of a specific
response to the 2PH treatment.
Carbohydrate increases during 2PH do not increase the molarity of the
apoplastic solution enough to lower the freezing point of the apoplast
by more than a fraction of a degree. However, Canny (1995) showed that
the solute concentration in the apoplast is not uniform, but that
solutes tend to accumulate in discrete regions termed sumps. In
addition to sugars being distributed unevenly in the apoplast, the
layer of liquid water into which sugars have apparently been released
would be very small due to the presence of apoplastic ice at 3°C
(Gusta and Fowler, 1977; Single and Marcellos, 1981; Pearce and
Ashworth, 1992). This could lead to regions of very high sugar
concentrations in the apoplast of crown tissue, which could prevent ice
adhesions and possibly provide other forms of protection to cell walls
and membranes.
In both NH and 1PH plants the percentage of fructan in the apoplast was
below that of MDH and osmolytes, suggesting that its presence in the
apoplast could also be attributed to cellular leakage or rupture.
During 2PH all fructans increased 5-fold or more (Fig. 2), but in a
manner that suggested some form of differential leakage into the
apoplast. For example, in NH and 1PH apoplastic fluid the percentage of
fructan did not vary according to size and was always below 1% (Fig.
2). However, after 2PH the percentage of fructan in the apoplast was
inversely related to DP (Fig. 2). In addition to differential leakage,
it is possible that changes in fructan concentrations were influenced
by changes in FEH activity in the apoplast.
Fructan is considered primarily to be a storage carbohydrate, and its
function as a cryoprotectant has always been controversial. Since
freezing in plants begins in the apoplast, it is somewhat easier to
envision a role for fructan in freezing protection, at least where 2PH
is concerned, with the discovery of its presence in the apoplast.
FEH and Invertase in Apoplast and Whole Tissue
Apoplast fluid extracted from crowns at all three stages of
hardening contained readily detectable FEH activity. Although the
location of FEH is generally accepted to be vacuolar (Pollock and
Chatterton, 1988), we considered that it may be present in the apoplast
for several reasons: (a) we found extracellular fructans present in oat
crowns of all three hardening treatments, (b) numerous carbohydrate- hydrolyzing enzymes (acid invertase,  -amylase,  -amylase, -glucosidase, and -glucosidases) are known to be present in the apoplast of many plant species (Hatch et al., 1963; Hawker and Hatch, 1965; Beers and Duke, 1988; Li and McClure, 1989; Beers et al., 1990), and (c) previous localizations of FEH activity relied upon isolation of vacuoles from protoplasts using a
protocol that did not account for extracellular enzyme activities (Wagner and Wiemken, 1986). The levels of FEH activities that we found
in apoplastic fluid from crowns of NH and 1PH oat were not
significantly different from each other and were about 2% of the total
activity in the tissue. This amount of enzyme activity in the apoplast
was probably not significantly different from that resulting from cell
leakage or rupture (Table I). However, FEH in the apoplast of 2PH
plants increased to approximately 11% of the total, which is greater
than that predicted to result simply from cellular rupture. The portion
not attributable to rupture may result from activation of the enzyme
already present in the apoplast, or it may be due to increased
secretion of FEH into the apoplast or a combination of both mechanisms.
FEH activity in whole crown tissue did not significantly (P = 0.05) change as a function of 2PH (Fig. 4), even though
all fructan size classes were lower in whole crown tissue from 2PH plants (Fig. 1). FEH activities reported here cannot be directly correlated with the appearance or disappearance of specific fructans either in whole tissue or in apoplast fluid for several reasons. First,
neokestin, the substrate used in the in vitro assays, is a mixture of
fructan polymers; we measured only the release of free Fru and did not
identify the sizes of individual fructans resulting from the
hydrolysis. Second, the plant extracts assayed were crude and contained
more than one fructan-hydrolyzing enzyme (C. A. Henson,
unpublished data). Third, the in vitro assays were conducted over
several hours, in contrast to the several weeks of hardening treatments
that resulted in the fructan distribution found in whole crown tissue.
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| Figure 4.
Activity of FEH and invertase in apoplast fluid
and whole-crown tissue from the winter oat cv Wintok. Plants were grown
under NH (5 weeks at 13°C, NON), 1PH (3 weeks at 2°C), or 2PH (1PH
plants were placed in a 3°C freezer for 3 d in the dark)
conditions. The bars represent the lsd at P = 0.05 (n = 3). The percentages above the bars in the apoplast
frames indicate the percentage of total activity measured in the
apoplast. G, Glc; fresh wt, Fresh weight; F, Fru. No significant
differences were found in the three treatments of whole crown tissue.
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Apoplastic fluid extracted from crowns at all three stages of hardening
contained invertase activity. Invertase activities in apoplastic fluid
from crowns of NH and 1PH plants were not significantly different from
each other and were about 2 to 2.5% of the total activity in the
tissue. Invertase activity in the apoplast from crowns of 2PH plants
increased to approximately 8% of the total activity in the tissue.
This increase in invertase activity partly explains the increase in Glc
and Fru in the apoplast of 2PH plants (Fig. 2). Invertase activities in
whole crown tissue increased slightly, although not
significantly, as a function of hardening (Fig. 4).
The leakage or secretion of FEH and invertase into the apoplast or the
alteration of their activities during 2PH may be a specific adaptive
response that helps the plant survive freezing temperatures. The
presence of fructan and Suc in the apoplast during 2PH certainly
provides substrates for both enzymes, the products of which are known
cryoprotectants. However, the changes we measured in this study may
also be coincidental to the increase in freezing tolerance during 2PH.
There are undoubtedly other alterations we did not measure that will
help provide answers to the exact nature of the increased freezing
survival during 2PH.
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FOOTNOTES |
*
Corresponding author; e-mail dpl{at}unity.ncsu.edu; fax
1-919-515-2305.
Received August 20, 1997;
accepted October 14, 1997.
1
Mention of a proprietary product does not
constitute an endorsement or recommendation for its use by the U.S.
Department of Agriculture.
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ABBREVIATIONS |
Abbreviations:
DP, degree of polymerization.
FEH, fructan
exohydrolase.
LT50, temperature at which 50% of a
population survives.
MDH, malate dehydrogenase.
NH, nonhardened.
1PH, first phase of cold hardening.
2PH, second phase of cold hardening.
| |
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Abstract
Introduction