Plant Physiol. (1986) 81, 361-366
0032-0889/86/81/0361 /06/$0 1.00/0
Changes in Adenine Nucleotides and Energy Charge in Isolated
Winter Wheat Cells During Low Temperature Stress'
Received for publication September 11, 1985 and in revised form January 15, 1986
M. KEITH POMEROY* AND CHRIS J. ANDREWS
Chemistry and Biology Research Institute, Research Branch, Agriculture Canada, Ottawa,
Ontario K1A 0C6 Canada
but smaller changes were observed in the levels of ADP and
AMP (9). AEC did not change appreciably in either winter or
Adenylate energy charge (AEC) and adenine nucleotide levels of spring wheat during cold acclimation, but values were generally
isolated winter wheat (Triticum aestivum L. cv Kharkov 22 MC) cells high and significantly greater in the winter than in the spring
exposed to various low temperature stresses were determined. During ice wheat. ATP levels and AEC values increased in leaves of winter
encasement at -1°C, nucleotide levels decreased gradually in approxi- rape plants during cold acclimation in light, while in roots, not
mate relation to a decline in cell viability. AEC values remained high capable of hardening, a pronounced decrease in ATP was obeven after 5 weeks of icing when cell viability was severely reduced. served during exposure to cold (18). AEC of actively growing,
When isolated cell suspensions were exposed to various cooling and unstressed plants is usually between 0.85 and 0.95, but imposifreezing regimes ranging from -10 to -30°C, cell damage was dependent tion of stress, such as anoxia, frequently results in a large decrease
on the minimum temperature imposed and the duration of exposure to in AEC (16).
the freezing stress. The levels of all three adenine nucleotides declined
The present study was undertaken to determine if low temperwith increasing severity of the imposed stress, but AEC values remained ature-induced injury to isolated winter wheat cells is related to
high even at -30°C when nearly all of the cells were killed. The addition changes in AEC and levels of adenine nucleotides. The role of
of 10 millimolar Ca2 to cell suspensions enhanced survival during low Ca2" in providing protection to the cells from various types,
temperature stresses, but did not influence nucleotide levels other than intensities, and durations of low temperature stresses also was
through its effect on cell viability. These results indicate that impairment examined.
ABSTRACT
of the ion transport system during the early stages of ice encasement
prior to a detectable decline in cell viability cannot be attributed to
changes in the adenylate energy charge system of the cell.
Exposure of enzymically isolated winter wheat cells to low
temperature stresses reduces cell viability and impairs certain
membrane functions (10, 13). Ice encasement stress, which induces partial anaerobiosis in the cells, markedly reduces ion
uptake prior to a significant decline in cell viability or increased
ion efflux, whereas brief exposure to lower subfreezing temperatures, ranging from -10 to -30C, results in more general
membrane damage. The inclusion of 10 mM Ca2" in the suspending medium during stress treatments significantly increases
cell viability and reduces membrane damage (10). The mechanism of damage to the plasma membrane during exposure to
low temperature stresses has not been elucidated, but we have
previously suggested that inhibition of ion uptake during the
early stage of icing may result from a reduction in cellular energy
charge, or from damage to membrane ATPase(s) involved in ion
transport (10).
AEC2 and ATP levels have been determined for many plant
species grown under a wide variety of environmental conditions
( 16), but we are not aware ofany reports relating these parameters
to low temperature-induced injury in winter cereals. ATP levels
increased during the early stages of cold acclimation in winter
wheat, and to a much lesser extent in spring wheat, while similar,
'Contribution No. 1574, Chemistry and Biology Research Institute,
Agriculture Canada.
2 Abbreviation: AEC, adenylate energy charge.
361
MATERIALS AND METHODS
Plant Material. Seed of Kharkov 22 MC winter wheat (Triticum aestivum L.) was surface sterilized and sown on moist filter
paper in glass trays. The trays were placed in 24°C in dark for 24
h and then cold-acclimated in dark with diurnal temperatures of
2°C/16 h and 0°C/8 h for 6 to 8 weeks.
Cells were isolated from the primary leaves of cold-hardened
seedlings by a modification of the enzymic digestion method of
Singh (17), previously described (13). Following digestion in
pectolyase and purification on a Percoll gradient, the purified
cells were suspended either in 3 mm Tris-Mes buffer (pH 6.5) or
in the same buffer plus 10 mM (a2+ (CaCl2.2H20).
Stress Treatments. All stress treatments were carried out in
1.5 ml polypropylene micro tubes. To facilitate freezing of cell
suspensions without supercooling, 0.5 ml of suspension medium
or suspension medium plus 10 mM Ca2' was added to each micro
tube and the tubes were placed in a freezer. When the medium
was frozen, 0.5 ml of cells (equivalent to 25-40 ,ul packed
volume) suspended in media ± Ca2+ was added to appropriate
tubes. The tubes were then immediately transferred to programmable freezers under various temperature regimes in the dark,
as detailed below.
For ice encasement treatments, groups of tubes containing
frozen cell suspensions and other tubes containing cells added to
unfrozen media were placed in a freezer at -1 ± 0.2°C. At this
temperature, no ice formation was observed in the cell suspensions added to the unfrozen media. At various intervals, groups
of tubes were removed from the freezer. Samples to be used for
viability measurements were thawed, centrifuged for 5 min at
200g in a microfuge, plasmolyzed with 1 N NaCl, and viable cells
and total cells counted on a haemacytometer slide. For AEC
determinations, 0.5 ml of 9% TCA was added to the frozen cell
suspensions and the suspensions were then allowed to thaw at
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Copyright © 1986 American Society of Plant Biologists. All rights reserved.
40C.
ence
Plant
POMEROY AND ANDREWS
362
Physiol.
Vol. 81, 1986
Preliminary experiments indicated that thawing in the presof TCA increased nucleotide yield and that the final 3%
concentration of TCA
was
1..0
sufficient to extract all of the nucleo-
significantly inhibit enzyme
activity in subsequent assays.
For the various freezing stress treatments, groups of tubes
containing frozen cell suspensions were transferred to freezers
either at --2 or -30'C. The temperature for all slow cooling
treatments was decreased 20C/h a-nd groups of tubes were removed when the temperature reached
10, -20, and -300C
(slow cool/short duration). An additional set of samples was left
at -10 and -2O'C until the total amount of time (15 h) that
these samples were exposed to freezing stress was equivalent to
that for the -30'C slow cooling treatment (slow cool/long duration). For all rapid cooling treatments, the frozen cell suspensions were transferred directly to -3O'C and samples were removed when the temperatures within the cell suspensions
reached
10, -20, and -3O'C (rapid cool/short duration). A
second set of samples was cooled similarly, but then left at -10,
-20, and -3O'C for 15 h (rapid cool/long duration). The temperature of the cell suspension during rapid cooling was monitored by a thermocouple inserted into a test sample and attached
to a Kaye Instruments, Digistrip II datalogger. The times required
for the suspensions to reach the treatment temperatures were as
follows:
100C, 6 min; -20'C, 8.5 min; and -30'C, 20 min.
Freezing treatments were allowed to warm slowly at O'C for 2 h,
overlaid with 0.5 ml of 9% TCA and thawed at 40C. Cell viability
of samples without TCA was estimated as indicated above.
AEC Measurements. Adenine nucleotides were determined by
tides from the cells and did not
modification of the luciferase bioluminescence assay described
by Pradet (14), which allows determination of all three adenine
a
0.).8
m- -
m
0
1001
0).6
i-
80
-*
60
40
* NON
ICED/-Ca2+
NON ICED/ +Ca2t
20
A
ICED/ -Ca24
A
ICED/
+Ca2+
0
8
co
Lu
0
E
u 4t
()0
?
by using pyruvate kinase and adenylate kinase for
stepwise conversion of ADP and AMP to ATP. After thawing
nucleotides
the
2-
min at 4'C, 00 usl
ml with 0.1Im Tris-acetate (pH
of each sample was diluted to
7.75) containing 2 mm EDTA. Then 100,Ml of this suspension
was added to each of 3 micro tubes containing
00,Ml of Reagent
A (20 mmi Tris-acetate, 7.5 mm Mg-acetate, and 80 mM Kacetate [pH 7.75]), 100 gl of Reagent B (100,gl Reagent A, 5
and extraction of the cells with TCA for 30
and 4 units pyruvate kinase EC
phosphoenolpyruvate,
mm Tris-acetate
7.4]), or 100 M1 of
suspended in
Reagent C (100 Ml Reagent B and 0.62 units myokinase EC
mm EDTA [pH 6.0]).
2.7.4.3 suspended in 3.2 m (NH4)2S04,
nmol
[pH
2.7.1.40
Each tube
for 30
adding
was
min.
mixed
on a vortex
mixer and incubated at 24C
Bioluminescence assay systems were prepared by
Ml of sample from Reagent mixtures A, B, and C
00
(above), and 300 Mil of 0.1Im Tris-acetate, 2 mms EDTA (pH 7.75)
similarly labeled luminometer cuvettes. The cuvettes were
0
I
I
0
a
LKIB
Wallac 1251 Luminometer and bioluminesc-
of ATP
recorded following automatic dispensing of 100
monitoring reagent (obtained from LKB Wallac) containing the
firefly luciferase and D-luciferin. The ATP content of each cuvette was determined by measuring the increase in light emitted
Ml aliquots
following automatic dispensing of three separate
mM
ATP into the sample cuvettes. Energy charge was
of
ence
calculated by the method of Atkinson (1).
Cell suspensions were extracted and precipitated with 5 % TCA,
solubilized with
N
NaOH and total
protein determined by the
method of Lowry et aL. (7).
RESULTS
The viability of noniced isolated winter wheat cells declined
10C, either in the
by less than 40% during 5 weeks exposure to
presence or absence of 10 mm Ca"~(Fig. 1). In contrast, viability
of ice encased cells decreased markedly after 3 weeks in the
absence of Ca21, but declined only slightly in the presence of
Ca"~. After 5 weeks icing, nearly all cells were killed in the
I
I
I
3
4
5
WEEKS AT
FIG.
1.
Cell
viability, AEC, and total
winter wheat cells
following
presence and absence of 10
-ic
'adenine
exposure to
mm~Ca"~.
l'C
nucleotide levels of
(iced and noniced) in
Each value represents
an
the
average
of duplicate determinations from three to five separate experiments.
Bars,
SE.
absence of
Ca"~,
but about 60% survival
suspended in medium containing
was
observed in cells
Ca"~.
prolonged
1C, with the greatest decreases
which sustained the most damage (Fig.
The levels of all adenine nucleotides declined after
to
loaded into
I
2
exposure of cell
occurring in
1; Table I).
suspensions
treatments
to
The presence of
Ca`~in
the
suspending medium
maintained higher levels of adenylates, consistent with greater
viability observed in these preparations. The level of ATP in all
including those which induced severe damage, was
higher than the levels of ADP or AMP, and usually
represented about 75% of total adenylates. This was reflected in
relatively high AEC values under all treatment conditions (Fig.
1). The decline in levels of total adenylates occurred concomitant
with the decline in cell viability, but no significant decrease in
AEC was observed even in treatments where nearly all the cells
were killed, and adenylate levels were extremely low.
The rate of cooling, and the intensity and duration of freezing
stress all affected viability and the levels of adenine nucleotides
in suspensions of isolated winter wheat cells. When cells were
subjected to slow cooling (2'C/h) and removed from the freezing
stress when the treatment temperatures were reached (Fig. 2, and
the -30'C treatment in Fig. 3), viability and nucleotide levels
declined progressively with decreasing temperature. The relative
treatments,
much
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ADENINE NUCLEOTIDE CHANGES DURING LOW TEMPERATURE STRESSES
363
Table I. Changes in Adenine Nucleotide Levels during Ice Encasement at -J°C in Presence and Absence of
Calcium
Each value represents an average ± SE of duplicate determinations from three to five separate experiments.
Treatment
Ca2+
ATP
ADP
AMP
mM
nmol/50 ,g protein
Control
0
5.2 ± 0.6
1.8 ± 0.3
0.6 ± 0.3
10
6.0± 1.1
2.5±0.5
0.8±0.2
Noniced/3 d
Iced/3 d
Noniced/l wk
Iced/l wk
Noniced/3 wk
Iced/3 wk
Noniced/5 wk
Iced/5 wk
I
ATP
2
0
10
0
10
5.4 ± 0.3
5.0±0.7
4.9 ± 0.9
4.7±1.0
1.9 ± 0.8
1.4±0.3
0.7 ± 0.4
1.1±0.2
0.3 ± 0.1
0.4±0.2
0.5 ± 0.2
0.3±0.2
0
10
0
10
4.6 ± 0.3
5.3±0.6
4.2 ± 0.4
4.8 ± 0.4
2.2 ± 0.4
1.9±0.4
1.8 ± 0.4
2.0 ± 0.5
0.5 ± 0.1
0.5±0.1
0.7 ± 0.2
0.6 ± 0.2
0
10
0
10
3.2 ± 0.4
3.3 ± 0.5
2.2 ± 0.1
3.8 ± 0.3
1.0 ± 0.3
1.6 ± 0.4
0.9 ± 0.2
1.4 ± 0.3
1.6 ±
0.6 ±
0.6 ±
0.5 ±
0
10
0
10
1.6 ± 0.1
2.0±0.6
1.0 ± 0.2
2.7±0.2
0.4 ± 0.2
0.5±0.1
0.3 ± 0.1
0.4±0.3
0.2 ± 0
0.3±0.1
0.1 ± 0.1
0.2±0.1
D
EW
E
ADP
Viability
AEC
12r
101
loor
8
801-
0.8
F
0.6
CO)
a 0.5
_
L.
.:.:.T
U 0.
D U'
Z
Z
_
z E
_
6
-
-J
4
F
0.4
20 -
0.2
40
-J
UJ
2
..
0
60
+Ca-Ca
Control
+Ca-Ca
-10 C
i
+Ca-Ca
-200C
C.
o0
O
+Ca -Ca
Control
+Ca-Ca
-1 0°C
FIG. 2. Adenine nucleotide levels, cell
viability, and AEC of winter wheat cells
suspended in buffer or in buffer plus 10
mM Ca2 , cooled slowly to -10 and
and immediately removed from
the freezing stress. Controls were maintained at 0'C. Treatments at -30'C not
presented as these are identical to slow
cool, long duration presented in Figure 3.
For adenine nucleotides, the total height
of histograms represent total nucleotides,
and the histogram is subdivided to show
the proportions of ATP, ADP, and AMP.
For viability and AEC, histograms extend
from zero. Data are averages of duplicate
determinations from three (-10 and
-20°C treatments) or six (controls) experiments. Bars, SE.
-20°C,
7 1.01.0
F1Zm
02
0.4
0.2
0.1
0.2
w
<
+Ca-Ca
-200C
SLOW COOL / SHORT DURATION
magnitude of reduction in nucleotide levels was generally similar
for ATP, ADP, and AMP, and as a consequence the AEC did
not vary significantly among the treatments. The level of ATP
was much greater than that of ADP or AMP in all treatments,
usually constituting from 60 to 80% of the adenylate pool,
resulting in AEC values of 0.8 to 0.9. The presence of 10 mM
Ca2" in the suspending medium did not significantly alter cell
viability, nucleotide levels, or AEC.
When cell suspensions were cooled slowly to the treatment
temperatures and then maintained at these temperatures until
the total duration of the stress treatment reached 15 h (Fig. 3),
cell viability and nucleotide levels in the -10 and -20°C treatments decreased only slightly more than was observed in the
slow cool/short duration treatments. AEC values were maintained at about 0.80 to 0.85, even in cell preparations exposed
to -30°C where both viability and nucleotide levels were severely
reduced. No significant differences were observed in viability,
nucleotide levels, or AEC when 10 mm Ca2" was included in the
suspension medium.
Cells which were cooled rapidly to treatment temperatures and
immediately removed from the freezing stress (Fig. 4) sustained
approximately the same degree of injury at -10°C as the slow
cool/short duration treatments. However, injury in these rapid
cool/short duration preparations was much less pronounced than
in the -20°C slow cool treatments with short or long duration
exposure. At -30°C, viability was reduced to less than 10%, as
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364
POMEROY AND ANDREWS
Plant Physiol. Vol. 81, 1986
Viability
2 ATP
2 ADP
B
AMP
88 K
AEC
100 _
1.0
80
0.8
.-I
-
FIG. 3. Adenine nucleotide levels, cell
viability, and AEC of winter wheat cells
suspended in buffer or buffer plus 10 mm
Ca2+, cooled slowly to -10, -20, and
-30°C, and maintained at these temperatures until the total duration of freezing
stress was 15 h. Controls as in Figure 2.
Data are averages of duplicate determinations from four experiments. Bars, SE.
6
_
a
LU.c
o-
F 60
6
-
0.6 u
m
uLU: t
n
5
40
-
z e
-J
Z E 2
<' 20
-
4-
-J
4
0L
+Ca-Ca
-1 O°C
+Ca-Ca
-200C
0O
+Ca-Ca
-300C
LE
LU
+Ca -Ca
-10lc
+Ca-Ca
-200C
0.4
0.2
+Ca-Ca
-30°C
JO
SLOW COOL / LONG DURATION
E
c
0.
ATP
Viability
m
ADP
14k
AEC
AMP
12 F
-W
Cn
0
CD
FIG. 4. Adenine nucleotide levels, cell
100 r
10k
1.0
viability, and AEC of winter wheat cells
suspended in buffer or buffer plus 10 mm
cooled rapidly to -10, -20, and
Ca2l,
-30°C, and immediately removed from
the freezing stress. Controls as in Figure
c:3
8k
80
0
0
60
6k
0.8
F
0.6
2. Data
-
LU
0
CD
4
z
0
4i
zcz 2
0.4
20
0.2
of duplicate deter-
e
NE.
-J
0i
LUl
,..
O
40
are averages
minations from four experiments. Bars,
L
.....
I_ w3
O L
+Ca -Ca
-10OC
+Ca -Ca
-200C
+Ca -Ca
-30°C
_~
+Ca -Ca
+Ca i-Ca
-10°C
-2CDOC
+Ca -Ca
-30°C
0
RAPID COOL / SHORT DURATION
also was observed in the cells cooled slowly. Adenine nucleotide
levels were generally higher in the rapid cool/short duration
treatment than in the cells cooled slowly. However, AEC values
were similar to those observed in the slow-cool treatments. The
presence of Ca2" in the suspending medium increased viability
only in cells exposed to -30°C, while a small but consistent
increase in nucleotide levels was observed in cell suspensions
containing 10 mm Ca".
Cell viability and adenine nucleotide levels decreased markedly
when the duration of exposure to freezing stresses of rapidly
cooled cell suspensions was extended from a few minutes (Fig.
4) to 15 h (Fig. 5). However, the decline in viability after 15 h
exposure to the stress was significantly less pronounced in cell
suspensions containing Cae' than in those suspended in buffer
alone. This resulted in considerably higher levels of nucleotides
in the presence of Ca2" at both -10 and -20°C, although AEC
values were nearly identical for all treatments, including -30°C
where both viability and nucleotides were severely reduced. The
rapid cool/long duration treatment was the most severe of the
four treatments examined, resulting in the greatest declines in
cell viability and adenine nucleotide levels.
DISCUSSION
Earlier investigations (10, 13) into the effects of low temperature stresses on membrane properties of enzymically isolated
winter wheat cells indicated that the relatively mild stress of ice
encasement at -1C specifically damaged the ion transport system of the cell, while more severe freezing stresses resulted in
general membrane dysfunction, including loss of semipermeability. From these results, it was suggested (10) that inhibition of
ion uptake in the early stages of icing, prior to a detectable
decline in cell viability, might result either from a reduction of
energy charge below that required for active ion uptake, or from
damage to plasma membrane ATPase(s) involved in this process.
The results presented in this paper indicate that the previously
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ADENINE NUCLEOTIDE CHANGES DURING LOW TEMPERATURE STRESSES
O
ATP
U
Viability
S
ADP
E
AEC
E
100
AMP
8r
~
Oo
_ 1.0
80-
A
-
at
-J
6
60 F
J t
:
a
-J
o
- 4
o"06
z~
40 -
-
-JJ
uJ
2
201+Ca-Ca
-1 0°C
+Ca-Ca
-200C
OL
+Ca-Ca
-300C
365
+Ca--Ca
-10°C
O0
+Ca-Ca
-20°c
FIG. 5. Adenine nucleotide levels, cell
viability, and AEC of winter wheat cells
suspended in buffer or buffer plus 10 mm
Ca2+, cooled rapidly to -10, -20, and
0.6 <) -30°C, and maintained at these temperauW tures until the total duration of freezing
0.4
stress was 15 h. Controls as in Figure 2.
Data are averages of duplicate determina0.2
tions from four experiments. Bars, SE.
0.8
-300C
RAPID COOL / LONG DURATION
reported (10) reduction in ion uptake after only 1 d icing is not
due to decreased levels of ATP or to a decline in AEC. ATP
levels declined throughout the ice encasement period, at rates
generally related to levels of cell viability in the various treatments, whereas AEC values remained high even when cell viability was severely reduced. These observations suggest that adenine nucleotide levels do not change appreciably in cells that
remain viable during ice encasement, and that the eventual
decline in nucleotides results from the degradation of these
compounds in cells which had died.
Studies on a wide variety of plant species (16 and references
therein), including wheat, have led to the conclusion that AEC
values of healthy, actively growing plant cells usually vary between 0.80 and 0.95. Furthermore, AEC values are generally
higher for nongreen than for green plant cells and usually remain
high even during growth at low temperature due to a balanced
reduction in ATP-utilizing and ATP-regenerating pathways (16).
The relatively high AECs observed in cells from winter wheat
grown in the dark at low temperature are consistent with these
observations. Exposure to anoxia, on the other hand, has been
reported to rapidly induce significant decreases in AEC values in
plant tissues (15, 16). This was not observed when wheat cells
were subjected to the anaerobic stress of ice encasement even for
several weeks. Total adenine nucleotide levels were severely
depressed, but AEC values remained high. Preliminary experiments in which cell suspensions were subjected to a nitrogen
atmosphere at 0°C revealed only a gradual decline in both
nucleotide levels and AEC values. At a treatment temperatue of
24°C, the AEC decreased to less than 0.5 in only 1 h. These
observations indicate that isolated winter wheat cells do respond
to conditions of anoxia in a manner similar to that reported for
plant tissues (15, 16) and that the response is temperature dependent. The absence of a similar response during prolonged ice
encasement at -1C suggests that during this stress there is little
demand for ATP by synthetic processes, or that the stress does
not induce complete anoxia in the cells to elicit a rapid decline
in AEC levels.
The results obtained in the experiments on the rate of cooling
and intensity and duration of freezing indicate that injury to
isolated winter wheat cells is closely related to the minimum
temperature imposed and to the duration of exposure to the
freezing stress. This is consistent with results obtained with intact
winter wheat plants where it was shown that increased duration
of exposure to sublethal freezing stress reduces survival and cold
hardiness (11, 12). Previous studies by Steponkus et al. (19) on
the freezing characteristics of isolated rye mesophyll cells also
indicated that intracellular ice formation and subsequent injury
was dependent on the minimum temperature of exposure.
It is generally accepted that rapid cooling of whole plants is
more damaging than slow cooling to the same temperature (6).
Furthermore, it has been demonstrated that intracellular ice
formation in isolated winter rye cells is dependent on cooling
rate when the cells are suspended in 0.25 osmolal salt solution,
but not when they are suspended in water (19). In our experiments where the cells were suspended either in 3 mm buffer or 3
mm buffer plus 10 mm CaCl2 (approximately 0.03 osmolal), a
close correlation between cooling rate and viability was not
observed. However, it should be noted that the results obtained
at different cooling rates are confounded by the fact that duration
of exposure to stresses in the slow and rapid cool treatments were
necessarily different due to the nature of the experiments. The
greater level of injury induced by the slow cool/short duration
treatment than by rapid cool/short duration (cf -20°C treatments) is probably due to longer exposure to the freezing stress
in the slow cool treatment. Cells exposed to slow cool/long
duration regime incurred less damage than those subjected to
rapid cool/long duration. However, in the rapid cool treatment,
the temperatures of the cell suspensions were quickly reduced to
the minimum exposure temperatures and retained at these temperatures for 15 h, whereas the temperatures in the slow cool
treatments were gradually reduced and hence the duration of
exposure to the minimum treatment temperature was considerably less than for rapidly cooled cells. In these experiments, the
two factors cooling rate and freezing duration are clearly interrelated, but the observations indicate that the two cooling rates
do not cause an appreciable difference in injury level, whereas
the duration of exposure to the minimum test temperature has
a greater effect on cell survival.
The role of Ca2l in the cellular metabolism of plants is not
well understood, but there are numerous reports that it protects
the plasma membrane from damage due to various stresses, and
that it plays an important role in many membrane associated
processes (2-5). On the other hand, a recent review (8) postulates
that chilling-induced increases in Ca2' in the cytoplasm indicate
a breakdown in calcium homeostasis and may serve as a primary
transducer of chilling injury. In the present experiments, calcium
has a significant protective effect only against the ice encasement
stress, and the rapid cool/long duration freezing treatment. This
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POMEROY AND ANDREWS
366
is consistent with cell viability data obtained previously (10)
where it was concluded that the protective effect of Ca2" was
more pronounced against the ice encasement than against freezing stresses. Whereas in the previous study (10) Ca2" allowed a
significant increase in 86Rb uptake after a few days of ice encasement, followed by consistently higher survival, the pi'esent results
indicate that Ca2" has only a limited effect on increases in total
adenine nucleotide levels, other than those directly related to
changes in cell viability. The relationship between Ca2" and the
promotion of cell survival and adenylate levels specifically in the
rapid cool/long duration freezing treatment is not understood.
The results of this study have demonstrated that AEC values
do not decline significantly following low temperature stress, and
that ATP levels decrease as cell viability is reduced. Consequently, the previously reported rapid decline in ion transport
observed prior to a significant loss in cell viability following icing
stress cannot be attributed to changes in the adenylate energy
charge of the cell or lack of ATP supply. Experiments are
currently in progress to determine if the observed changes in ion
transport properties result from impairment of plasma membrane ATPase activity.
Acknowledgment-The authors wish to acknowledge the excellent technical
assistance of K. P. Stanley.
LITERATURE CITED
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