978.full.pdf

Plant Physiol. (1983) 72, 978-988
0032-0889/83/72/0978/1 1/$00.50/0
Effect of Cold Acclimation on Intracellular Ice Formation in
Isolated Protoplastst
MICHAEL F. DOWGERT AND PETER L. STEPONKUS
Department ofAgronomy, Cornell University, Ithaca, New York 14853
ABSTRACT
When cooled at rapid rates to temperatures between -10 and -30°C,
the incidence of intracellular ice formation was less in protoplasts enzymically
isolated from cold acclimated leaves of rye (Secale cereale L. cv
Puma) than that observed in protoplasts isolated from nonacclimated
leaves. The extent of supercooling of the intracellular solution at any given
temperature increased in both nonacclimated and acclimated protoplasts
as the rate of cooling increased. There was no unique relationship between
the extent of supercooling and the incidence of intracellular ice formation
in either nonacclimated or acclimated protoplasts. In both nonacclimated
and acclimated protoplasts, the extent of intracellular supercooling was
snimlar under conditions that resulted in the greatest difference in the
incidence of intracellular ice formation-cooling to -15 or -20°C at rates
of 10 or 16'C/mitute. Further, the hydraulic conductivity determined
during freeze-induced dehydration at -5°C was similar for both nonacclimated
and acclimated protoplasts. A major distinction between nonacclimated
and acclimated protoplasts was the temperature at which nucleation
occurred. In nonacclimated protoplasts, nucleation occurred over a relatively
narrow temperature range with a median nucleation temperature of
-15°C, whereas in acclimated protoplasts, nucleation occurred over a
broader temperature range with a median nucleation temperature of
-42°C. We conclude that the decreased incidence of intracellular ice
formation in acclimated protoplasts is attributable to an increase in the
stability of the plasma membrane which precludes nucleation of the supercooled
intracellular solution and is not attributable to an increase in
hydraulic conductivity of the plasma membrane which purportedly precludes
supercooling of the intracellular solution.
It is commonly observed that the incidence of intracellular ice
formation is a function of the cooling rate. As early as 1886,
Muller-Thurgau (20) observed that rapid cooling resulted in intracellular
ice formation whereas during slow cooling ice crystals
were confined to extracellular spaces. Similar observations were
reported in 1897 by Molisch (19). Since that time, a cooling rate
dependence of intracellular ice formation has been demonstrated
in a diverse range of biological cell types (17).
Levitt and Scarth (10) were among the first to offer a mechanistic
explanation for the cooling rate dependence of intracellular
ice formation. They reasoned that intracellular ice formation
occurs only if the cytoplasm cools faster than its freezing point is
lowered by solute concentration due to water efflux. They concluded
that the rate of water efflux is limiting at rapid cooling
'This material is, in part, based on work supported by the National
Science Foundation under Grant PCM-8012688 and the United States
Department of Energy under Contract DE-AC02-81ER 10917. Department
of Agronomy Series Paper 1450.
Received for publication December 27, 1982 and in revised form April 8, 1983
rates; and the higher the permeability to water, the lower the
probability of intracellular ice formation. Further, because the
deplasmolysis time of cells in cold hardy tissues was less than that
for cells in nonhardy tissues, they inferred that water permeability
increased following cold acclimation and that the incidence of
intracellular ice formation would be less in acclimated tissues. In
1938, Siminovitch and Scarth (23) experimentally demonstrated
that intracellular ice formation occurs less frequently in hardy
tissues than nonhardy tissues when cooled at comparable rates.
They too attributed this difference to the purported increase in
water permeability of the plasma membrane following cold acclimation
as proposed by Levitt and Scarth (10).
Although this concept has been widely accepted and frequently
cited, direct experimental verification of an increase in water
permeability following cold acclimation has been lacking, and the
proposed diminished extent of supercooling in acclimated tissues
has never been experimentally documented during a freeze-thaw
cycle. In fact, there have been few experimental studies of the
relationship between water permeability and cold acclimation.
Sukumaran and Weiser (29) determined water permeability constants
by following the time course of leaf protoplast expansion
during deplasmolysis, which is the same technique used by Levitt
and Scarth (10), and found no difference in the water permeability
of hardy and nonhardy potato cultivars. McKenzie et al. (18)
determined a 'permeability ratio,' ie. the diffusive flux of tritiated
water in live tissue relative to that of dead tissue, for cortex cells
of Cornus stolonifera. The permeability ratio decreased (a higher
permeability) during the initial photoperiodically induced increase
in hardiness from -3 to - 12°C but changed little, if any, during
subsequent acclimation to -65°C. Stout et al. (28), used a nuclear
magnetic resonance technique to determine the rate of ice formation
and diffusional water permeability in Hedera helix. They
concluded that water permeability was not limiting in nonacclimated
tissues.
In spite of these reports, Levitt (9) has steadfastly championed
the notion that the lower incidence of intracellular ice formation
in acclimated tissues is the result of an increase in water permeability
of the plasma membrane. This is disconcerting because the
original report (10) acknowledged that 'on account of the complications
in the methods so far used, the relation between water
permeability and hardiness is not completely worked out. Some
more satisfactory procedure will have to be evolved in order to get
accurate results.' Similarly, neither the method used by McKenzie
et al. (18) nor that of Stout et al. (28) is unequivocal.
More importantly, however, the mechanistic interpretation of
intracellular ice formation proposed by Levitt and Scarth (10) did
not consider the manner by which the supercooled intracellular
solution was nucleated. Our interpretation of their analysis is that
the extent of supercooling of the intracellular solution was considered
the primary determinant of intracellular ice formation and
that any degree of supercooling would result in intracellular ice
formation. A more appropriate explanation for intracellular ice
978

INTRACELLULAR ICE FORMATION IN ISOLATED PROTOPLASTS
Table I. Incidence of Intracellular Ice Formation in Nonacclimated and Acclimated Protoplasts as a Function of
Cooling Rate and Minimum Temperature Imposed
-5
-10
-15
Cooling Rate ( C/min)
3.1 5 10 16
non-acc. acc. non-acc. acc. non-acc. acc. non-acc. acc.
15% 0% 23% 0% 0% 0% 14% 0%
(7/46) (0/24) (6/26) (0/23) (0/20) (0/18) (4/28) (0/20)
18% 19% 24% 8% 41% 31% 86% 11%
(9/50) (4/21) (8/34) (2/25) (7/17) (5/16) (19/22) (2/18)
5% 12% 39% 19% 63% 19% 87% 38%
(1/22) (2/17) (7/18) (3/16) (10/16) (4/21) (26/30) (6/16)
t -20 24% 33% 58% 16% 92% 18% 96% 30%
' -20
E- (5/21) (8/24) (15/26) (3/19) (12/13) (4/22) (22/23) (12/40)
-30
-40
-50
16% 33% 50% 6% 81% 36% 95% 43%
(3/19) (3/9) (12/24) (1/16) (17/21) (5/14) (19/20) (6/14)
20% 21% 52% 45% 95% 79% 97% 86%
(4/20) (4/19) (12/23) (13/29) (21/22) (31/39) (32/33) (19/22)
15% 28% 45% 61% 95% 84% 100% 96%
(3/20) (9/32) (10/22) (14/23) (18/19) (27/32) (19/19) (24/25)
The incidence is given as the percentage of protoplasts in which intracellular ice formation was observed using
the cryomicroscope. Values in parentheses represent the number of protoplasts that froze intracellularly and the
total number of protoplasts observed at each temperature and cooling rate. The total number of protoplasts
observed was in excess of 1200.
formation has been provided by Mazur (14, 17). To wit, at some
finite cooling rate, supercooling of the intracellular solution will
occur, but the probability of intracellular ice formation will increase
only if the cells are supercooled below their nucleation
temperature. Therefore, it is possible that the decreased incidence
of intracellular ice formation in acclimated tissues may be the
result of an alteration in the nucleation temperature rather than
a decrease in the extent of supercooling.
The objective of this study was to determine by cryomicroscopy
the incidence of intracellular ice formation in protoplasts isolated
from nonacclimated and acclimated rye leaves as a function of
cooling rate and temperature. From measurements of volumetric
behavior under rigorously controlled cooling rates, the hydraulic
conductivity, extent of intracellular supercooling, and temperature
at which intracellular ice formation occurred were determined.
MATERIALS AND METHODS
Seeds of Secale cereale L. cv Puma were germinated and grown
for 7 d in vermiculite under a controlled environment (16-h light
period at 20°C and 8-h dark period at 15°C). Nonacclimated
plants, LTso -3 to -5°C, were grown an additional 7 d. Plants to
be acclimated were transferred to 13°C/7°C (11.5-h photoperiod)
for 1 week and then to 2°C (10-h photoperiod) for 4 weeks after
which they were fully acclimated, LT50-25 to -30°C. Protoplasts
were enzymically isolated from leaves in a solution of 1.5% (w/v)
979
Cellulysin (Calbiochem) 0.5% Macerase (Calbiochem), and 0.3%
potassium dextran sulfate as described previously (30). Protoplasts
from nonacclimated tissue were isolated in 0.1 M CaCl2 + 0.15 M
NaCl (0.53 osm2) while those from acclimated tissue were isolated
in 0.9 M sorbitol, then washed 3 times and suspended in 0.19 M
CaCl2 + 0.29 M NaCl (1.033 osm). The different concentrations
were required to maintain the in situ volume of the protoplasts
because of an increase in the internal solute concentration during
cold acclimation. All solution osmolalities were determined using
a freezing point osmometer. Hereafter, protoplasts isolated from
nonacclimated or acclimated leaves will be referred to as nonacclimated
and acclimated protoplasts respectively and should not
be construed to mean that the isolated protoplasts per se were
subjected to the cold acclimation regime in vitro.
Direct observation of protoplast suspensions during cooling and
warming was accomplished using an electronically programmable
cryomicroscope (27). Nonacclimated and acclimated protoplasts
were cooled at rates of 3.1, 5, 10, and 16°C/min to temperatures
of -5, -10, -15, -20, -30, -40, and -50°C. These cooling rates
and temperatures were chosen such that the incidence of intracellular
ice formation ranged from a low to high value.
Individual freeze/thaw runs, along with sample temperature
2Abbreviations: osm, osmolal; PBS, phosphate-buffered saline; DMSO,
dimethyl sulfoxide.

980 DOWGERT AND STEPONKUS
Plant Physiol. Vol. 72, 1983
a NON-ACCLIMATED
i-so TE ERATURE E'C)
f.75
FIG. 1. Incidence of intracellular ice formation in (a) nonacclimated
and (b) acclimated protoplasts as a function of the cooling rate and
minimum temperature imposed (from Table I).
and time after initiation of cooling, were recorded on a video
cassette recorder and projected on a 19-inch color monitor. A x
20 objective and x 5 photo-ocular produced a total magnification
of x 2380 on the monitor. Typically, two or three protoplasts were
viewed during each cooling/warming cycle. Protoplast diameter
and intracellular ice formation as a function of temperature and
time were measured at intervals during replay of the video cassettes
using the 'freeze-frame' on the video cassette recorder.
Intracellular ice formation was identified as the sudden darkening
of the cellular contents ('black flashing') due to gas bubble formation
(24).
Cell volume was calculated from diameter measurements assuming
sphericity. Fractional cell volumes (relative to the isotonic
volume) were calculated in order to compare the volumetric
behavior of protoplasts of different initial volumes. Generally,
protoplasts remained spherical at the higher fractional volumes
(>0.4) with an increasing incidence of irregular shapes at lower
fractional volumes (0.4 were not considered
for volumetric determinations. Diameter measurements were
made at temperature intervals ranging from 1.5°C when cooled at
3.1 °C/min to 3°C intervals when cooled at 16°C/min. The precise
measurement interval for individual protoplasts varied somewhat
depending on the focus of the cells and the observed volumetric
change.
Osmometric behavior of protoplasts was determined by equilibrating
protoplasts in solutions of varied tonicities at 0°C and
calculating volumes from diameter measurements. Approximately
100 protoplasts were measured at 22 different osmolalities over
the range of 0.3 to 3.0 osm for nonacclimated and at 31 different
osmolalities over the range of 0.4 to 6.0 osm for acclimated
protoplasts. The average volume at each osmolality was plotted as
a Boyle-van't Hoff plot and the linear regression and 95% confidence
belts were calculated.
The extent of intracellular supercooling was determined from
the pooled volumetric response at each cooling rate. Fractional
volumes were pooled at 1 °C intervals and the mean and standard
deviation calculated. From the mean fractional volume, the su-
percooling of the intracellular solution was calculated as the extent
in °C to which a given volumetric curve departed from the
equilibrium curve at 1°C intervals. In addition, the range of
supercooling about the mean was calculated for the mean fractional
volume plus or minus 1 SD.
Net water flux (J,) out of the protoplasts during cooling was
calculated using the formula J, = i V/At where A V is the change
in water volume, A is the surface area of the protoplast averaged
over the time of the experiment, and t is the time in seconds.
Hydraulic conductivity (Lp) was determined using the equation
Lp = J,I/A4, where Ai\ is the osmotic potential difference between
the internal and external solutions averaged over the time of the
experiment. The internal osmotic potential at any protoplast volume
was determined from the Boyle-van't Hoff relationship while
the external osmotic potential was estimated by the relationship m
- 273 - T/1.86 where m is the osmolality of the partially frozen
solution and T is the temperature in 'K. Because a comparison
was made of J. and Lp in acclimated and nonacclimated protoplasts
under identical conditions, a more complicated analysis was
considered unnecessary. To determine J. and Lp, protoplasts were
cooled to -5°C at 10°C/min and cellular volumetric changes
were measured during the first 30 s after -5°C was attained. The
cooling rate and temperature were chosen to allow for determinations
of water efflux at high fractional volumes (>0.4) during
the measurement period, thus improving measurement accuracy.
Measurements were made after -5°C was achieved so that the
external osmotic concentration could be assumed constant.
RESULTS
Incidence of Intracellular Ice Formation. When cooled to temperatures
in the range of-5 to -30°C at rapid cooling rates (5°C/
min or faster) and held at the particular temperature for 3 min,
the incidence of intracellular ice formation was consistently greater
in nonacclimated protoplasts than in acclimated protoplasts (Table
I). Over this temperature range, maximal differences occurred at
faster cooling rates and lower temperatures. For example, when
cooled to -20°C at 16°C/min, intracellular ice formation was
observed in 96% of the nonacclimated protoplasts but in only 30%
of the acclimated protoplasts. Only upon cooling to -50°C at
16°C/min was a high incidence of intracellular ice formation
(96%) observed in acclimated protoplasts.
In nonacclimated protoplasts, the incidence of intracellular ice
formation was strongly dependent on both the cooling rate and
minimum temperature imposed (Fig. la). If the protoplasts were
cooled to only -5°C, the incidence was low and independent of
the cooling rate. When cooled to temperatures of -10°C or lower,
the incidence of intracellular ice formation increased greatly as
the cooling rate increased. At a cooling rate of 3.1 °C/min, the
incidence was largely independent of the temperatures imposed
(-5 to -50°C). At cooling rates of 5°C/min and greater, the
incidence of intracellular ice formation increased greatly as the
minimum temperature imposed was decreased over the range of
-3 to -20°C. The incidence of intracellular ice formation, however,
did not increase over the range of -20 to -50°C at any
cooling rate.
In acclimated protoplasts, a cooling rate dependence of the
incidence of intracellular ice formation was observed only when
the protoplasts were cooled to -40 or -50°C (Fig. lb). Over the
range of -5 to -30°C, the incidence was low and independent of
the cooling rate. The temperature dependence of the incidence of
intracellular ice formation was less pronounced in acclimated
protoplasts and occurred over a broader temperature range extending
to -50°C rather than being limited to the range of -3 to
-20°C as observed for nonacclimated protoplasts.
Equilibrium Osmometric Behavior. Protoplasts equilibrated in
solutions of NaCl + CaCl2 of varied tonicities at 0°C behaved as
osmometers over a wide range of osmolalities from 0.3 to 3.0 osm

INTRACELLULAR ICE FORMATION IN ISOLATED PROTOPLASTS
OSMOLALITY
FIG. 2. Boyle-van't Hoff plot of the fractional protoplast volume as a function of osmolality-' for nonacclimated and acclimated protoplasts. For
calculation of the linear regression of volume versus osm-' that is shown, approximately 100 protoplasts were measured at 22 different osmolalities over
the range of 0.3 to 3.0 osm for nonacclimated (A) and at 31 different osmolalities over the range of 0.4 to 6.0 osm for acclimated protoplasts (A). Vb
(nonosmotic volume) was determined by extrapolation of the regression to infinite osmolality. (0) Nonacclimated protoplast volumes ±SD determined
following volumetric equilibration at subzero temperatures of -3, -5, -10, -15, and -20°C. (0) Acclimated protoplast volumes determined following
volumetric equilibration at subzero temperatures of -5, -10, -15, and -20°C.
and 0.4 to 6.0 osm for nonacclimated and acclimated protoplasts,
respectively (Fig. 2). The greater slope observed in the Boyle-van't
Hoff relationship for acclimated protoplasts is the result of the
increase in internal solute concentration that occurs during cold
acclimation. Acclimated protoplasts also have a greater Vb (nonosmotic
volume) which was inferred from extrapolation of the
Boyle-van't Hoff relationship to an infinite osmolality.
Following cooling to subzero temperatures, protoplasts decreased
in volume due to the increase in the osmolality of the
partially frozen suspending medium. The extent of contraction
varied as a function of the minimum temperature imposed. The
average equilibrium volumes attained, plotted as a function of the
osmolality encountered at each subzero temperature (m - T/1.86)
(Fig. 2, circles), were within the 95% confidence belts of the
regressions for the Boyle-van't Hoff relationships calculated from
volumetric measurements at 0°C (Fig. 2). The agreement was very
good considering measurements at subzero temperatures were
limited because protoplasts may depart from sphericity at low
temperatures. The close agreement of the equilibrium volumetric
behavior at subzero temperatures and the osmometric behavior at
0°C in both acclimated and nonacclimated protoplasts strongly
suggests that protoplasts cooled to subzero temperatures attain a
volume consistent with that predicted by Boyle-van't Hoff behavior
at 0°C.
Volumetric Behavior during Cooling to -20°C. Volumetric
changes during cooling were determined for nonacclimated and
acclimated protoplasts cooled to -5, -10, -15, and -20°C at
cooling rates of 3.1, 5, 10, and 16°C/min. For a given cooling rate,
the volumetric behavior over a comparable temperature range was
similar whether the protoplasts were cooled to -5, -10, -15, or
-20°C. As the greatest difference in the incidence of intracellular
ice formation between acclimated and nonacclimated protoplasts
occurred in protoplasts cooled to -20°C, only these results are
presented (Fig. 3). Protoplast volumes were normalized with respect
to the initial volume before cooling. No attempt was made
to present an average or 'typical' response for a particular cooling
981
rate. Instead, the volumetric response of all protoplasts observed
is presented. The range of fractional volumes among protoplasts
at any given temperature and cooling rate was admittedly large.
As the specimen temperature was controlled within ±0.050C, and
the precision of the linear measurements corresponded to ±0.25
P.m, neither contributed greatly to the variability observed. Instead,
the variation is attributed to the inherent variability in water efflux
from individual protoplasts and, at low fractional volumes (

982
I
id
;I
9
I..
I 'K
.4
c
DI~~~~~~~~~~~~~~
NON-ACCLIMATED
Cookin Note lC/uww
.qblIum
09 -I -b -1
1CI
TEMPERATURE
DOWGERT AND STEPONKUS Plant Physiol. Vol. 72, 1983
FIG. 3. Volumetric behavior of nonacclimated and acclimated protoplasts during cooling to -20°C. An asterisk indicates that the protoplast froze
intracellularly at the indicated temperature and fractional volume.

a
Z
u-
a
a
10
'K
a
I IC I!
r
fin
a
-1
M0R-CLIMATED
Cooing Room. 3jlCMM
C - tttI
INTRACELLULAR ICE FORMATION IN ISOLATED PROTOPLASTS
_I
-- -to -5 -20
TEMPERATURE (0
IL
it
r
NON-ACCLIATED
CodWg Room: 5*CMO
,ttttt2l
0~~~~~~~~~4
L ' R4-- in -20
TEMERATURE PC1
ttittttt,
NO-ACCLIMATED
CoOig Rate: 0IC_
~~t
II
-D -iLO -;5 -20
TEMPERATURE PC)
MON-ACCLIMATED
Coding Rot: 16 'CAt
0~~~~~
0
0
0
0
_ _ _ I
_t -.a _ -a0
&
TEMPEiXTURE (-C) -t
9
t
I
I
w'
.
la
, S0
-i
.
8
Z;
L.
Ig
Ito
I
_ACLIMATED
Cooli" Reot. 3.Cni
p
0
ACCLIMATED
Cooing Rote 5C/min
ti
t I
It I
-5 -10 -IS -20
TEMPERATURE rC)
-5 -1 -IS -20
TEMPERATURE (IC)
FIG. 4. Extent of intracellular supercooling in nonacclimated and acclimated protoplasts during cooling to -20°C. Circles represent the supercooling
calculated for the means of the fractional volumes at I °C intervals for each of the cooling rates shown in Figure 3. The vertical bars represent the range
of supercooling calculated for the mean fractional volume + 1 SD (upper limit of the bars) and the mean fractional volume -I SD (lower unit of the bars).
0
1!
-1
n0
0
E5 - 0 nk -I R -M~~~~~~~~~~~~_
(C
TEMPERATURE (I C )
II II
ACCLIMATED 4 -
Coinin ROt 10C/
0
0
0
04 I
0 O
n I I
° -S -10 -15 -20
TEMPERATURE rC)
r
I 1IT ----I
ACCLIMATED
CoDolPAgRot SC/mlOt t
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t
0
0
0
0
0~~~~
0
0
I
;
983
I I~~~~~~~~~~~~~~~~~~~~~~~~~~~

984
oc
6C
4a4
20
I
DOWGERT AND STEPONKUS Plant Physiol. Vol. 72, 1983
Table II. Water Flux and Hydraulic Conductivity of Nonacclimated and Acclimated Protoplasts at -5°C
Protoplasts were suspended in either 0.53 or 1.03 osm solutions of CaC12 + NaCl (0.1 M CaCl2 + 0.15 M NaCl
and 0.19 M CaCl2 + 0.29 M NaCI, respectively) and cooled to -5°C at 1O°C/min. Protoplast volumes were
calculated from measurements of protoplast diameters taken at 5-s intervals during the first 30 s after -50C was
attained. See "Materials and Methods" for calculations of J. and Lp.
Nonacclimated Acclimated
*v
m 0
a,£ 0
y
Z* -v
0'07
0.53 osm 1.03 osm 0.53 osm 1.03 osm
J. (x 10-6 cm s') 5.55 ± 1.77 3.08 ± 1.86 4.66 ± 1.92 3.84 ± 1.06
Lp (x 10-7 cm s-' bar-) 1.22 ± 0.40 0.99 ± 0.56 1.04 ± 0.44 1.20 ± 0.32
.
y v
V V
v~~~~~~
v
v
a
v
F v 0
a v
v
* v
V V0 0
A
vo 0
000
voO
-000
ao0 00
v ° 0° Cooling Rate:
v 0
f
v o A
0 0 non-accimoted
A 0 31CArin A
A ° 10*rfin OC'II
AO
A 0
16TCAnin
ooimoted
uN
-10 -20 -30 -40
NUCLEATION TEMPERATURE
-50
EC)
-60
FIG. 5. Cumulative frequency of intracellular ice formation as a function
of the temperature at which intracellular ice formation was observed
to occur in nonacclimated and acclimated protoplasts cooled to -50°C.
For nonacclimated protoplasts, the total number of protoplasts observed
at each cooling rate was 20, 23, 22, and 33 for the rates of 3.1, 5, 10, and
16°C/min, respectively. For acclimated protoplasts, the total number of
protoplasts observed at each cooling rate was 32, 23, 32, and 25 for the
rates of 3.1, 5, 10, and 16°C/min, respectively.
cooling is equal to the extent in °C to which a given volumetric
curve departed from the equilibrium curve (17). In all instances,
protoplasts that froze intracellularly were supercooled. This was
especially obvious in nonacclimated protoplasts (Fig. 3). Nonetheless,
protoplasts which were supercooled did not always freeze
intracellularly. This was especially obvious in acclimated protoplasts
(Fig. 3).
To provide a general contrast between nonacclimated and
acclimated protoplasts cooled at the various rates, the average
extent of supercooling was calculated from a pooled volumetric
response of nonacclimated or acclimated protoplasts. For each of
the cooling rates, individual fractional volumes were averaged at
1°C intervals to yield the average volumetric response. Supercooling
was then calculated for the average volume and for ±SD
(Fig. 4). Estimates of supercooling at large fractional volumes
(>0.4) were reasonably accurate because of the initial steep decline
in the equilibrium volume at relatively warm subzero temperatures.
As a result, the accuracy of calculation of the extent of
supercooling is largely a function of the accuracy of the protoplast
temperature inferred and is relatively insensitive to errors in the
estimate of volume. Nonetheless, because departure of the protoplasts
from sphericity was minimal at fractional volumes >0.4,
estimates of volume are quite good. In contrast, at small fractional
volumes (

0.2_
0.4
INTRACELLULAR ICE FORMATION IN ISOLATED PROTOPLASTS
0'0 -0 -3 -so
TEMPERATURE (rC)
1.0 ~~~~~~~~ ~CCLII"ATED
0.2 equilbrium -
-
-10 --40 -50
TEMPERATURE I*C)
VouercbhvoIfaciatdpoolssdrn
~ |.
to -50°C. An asterisk indicates that the protoplast froze intracellularly at
the indicated temperature and fractional volume.
985
cooled at 5°C/min was not considered too compelling to warrant
the inference that the protoplasts were supercooled. Although the
greatest difference in the incidence of intracellular ice formation
between nonacclimated and acclimated protoplasts occurred at
cooling rates of 10 and 16°C/min, there was little difference in
the extent of supercooling between nonacclimated and acclimated
protoplasts at these rates (Fig. 4). At these rates, the fractional
volumes were greater than 0.3 and, hence, estimates of supercooling
were most accurate.
Plasma Membrane Hydraulic Conductivity and Water Flux.
The volumetric behavior during cooling suggested that the water
permeability of the plasma membrane did not increase during
cold acclimation. Furthermore, at the slowest cooling rate, water
flux from acclimated protoplasts appeared to be less than that
from nonacclimated protoplasts. This apparent difference could,
in part, be attributable to the differences in the internal solute
concentration between nonacclimated (0.53 osm) and acclimated
(1.03 osm) protoplasts. Therefore, a more direct measure of water
flux and hydraulic conductivity was attempted. Nonacclimated
and acclimated protoplasts were cooled to -5°C at 10°C/min and
cellular volumetric changes were measured during the first 30 s
after -5°C was achieved. Experiments were conducted for both
nonacclimated and acclimated protoplasts cooled in either 0.53 or
1.03 osm solutions.
When cooled in the respective isotonic solutions (nonaccimated,
0.53 osm; acclimated, 1.03 osm), water flux (J.) was
significantly less for acclimated protoplasts (Table II). This difference,
however, was entirely due to the difference in internal solute
concentration because the calculated hydraulic conductivities (4),
which compensated for the difference in solute concentration,
were not significantly different. Further, when considered at equal
osmolalities (0.53 or 1.03), J. and Lp for nonacclimated and
acclimated protoplasts were not significantly different.
Nucleation Temperature. In Table I and Figure 1, a and b, the
incidence of intracellular ice formation is reported as a function
of the cooling rate and minimum temperature imposed and presents
the percentage of protoplasts that froze intracellularly during
cooling and the subsequent isothermal period at the minimum
temperature. These results indicated that the incidence of intracellular
ice formation was strongly temperature dependent. They
did not, however, indicate at what temperature intracellular ice
formation occurred. A more direct approach for determining the
temperature at which intracellular ice was nucleated involved
cooling at the various rates to a single low temperature (-50°C)
and recording only the number of protoplasts that froze intracellularly
during cooling (Fig. 5).
For nonacclimated protoplasts, nucleation occurred over a relatively
narrow temperature range independent of the cooling rate
(from 3.1 to 16°C/min) with the median nucleation temperature
at -15°C. For acclimated protoplasts cooled at 3.1 or 5°C/min,
nucleation occurred over a narrow temperature range with a
median nucleation temperature of -42°C. For those cooled at 10
and 16°C/min, the range of nucleation temperatures was much
broader, but still the median nucleation temperature was -40°C.
Effect of Tonicity of the Suspending Medium on the Nucleation
Temperature. In the preceding experiments, nonacclimated and
acclimated protoplasts were routinely suspended in 0.53 and 1.03
osm solutions of NaCl + CaCl2, respectively, in order to maintain
similar volumes and surface areas. Because the volumetric response
was of interest, it was considered not appropriate to
compare nonacclimated and acclimated protoplasts at equal osmolalities
because of the large difference in volumes which would
result. For example, the fractional volume of acclimated protoplasts
suspended in 0.53 osm was 1.8 compared to 1.0 for nonacclimated
protoplasts (Fig. 2). Conversely, the fractional volume of
nonacclimated protoplasts suspended in 1.03 osm was 0.55 compared
to 1.0 for acclimated protoplasts. Despite these large differ-

986 DOWGERT AND STEPONKUS
Plant Physiol. Vol. 72, 1983
ences in fractional volumes at equal osmolalities, the large difference
in the median nucleation temperature between nonacclimated
and acclimated protoplasts prompted consideration of the
influence of the osmolality of the suspending medium on the
nucleation temperature and the incidence of intracellular ice
formation.
Because the incidence of intracellular ice formation was greatest
at cooling rates of 10 and 16'C/min, the effect of osmolality on
the nucleation temperature was considered only at these cooling
rates (Fig. 6). For both nonacclimated and acclimated protoplasts
at either cooling rate, the median nucleation temperature was
decreased at the higher (1.03) osmolality. For example, the median
nucleation temperature for nonacclimated protoplasts cooled at
10IC/min decreased from -14C when suspended in 0.53 osm to
-280C when suspended in 1.03 osm. When cooled at 16'C/min,
the nucleation temperature decreased from - 16'C in 0.53 osm to
-230C in 1.03 osm solutions. In acclimated protoplasts cooled at
10IC/min, the median nucleation temperature increased from
-420C when suspended in 1.03 osm to -19'C when suspended in
0.53 osm. When cooled at 16'C/min, the increase was from
-420C in 1.03 osm to -280C in 0.53 osm. Nevertheless, when
compared at equal osmolalities, the median nucleation temperature
was consistently lower in acclimated than nonacclimated
protoplasts whether suspended in 0.53 or 1.03 osm solutions.
Volumetric Behavior of Acclimated Protoplasts Cooled to
-50°C. Contrasting the volumetric behavior of nonacclimated
and acclimated protoplasts during cooling to -20°C (Fig. 3)
indicated that the extent of supercooling was not reduced in
acclimated protoplasts as proposed by Levitt and Scarth (10).
Instead, the results with nonacclimated protoplasts were consistent
with the hypothesis of Mazur (17) that intracellular ice formation
occurs if cells are supercooled below their nucleation temperature.
A median nucleation temperature of -42°C in acclimated protoplasts
was not anticipated and thus precluded testing of Mazur's
hypothesis in acclimated protoplasts cooled only to -20°C. Therefore,
the volumetric behavior of acclimated protoplasts was determined
during cooling to -50°C (Fig. 7). For these experiments,
the volumetric behavior was determined at more frequent intervals
during cooling using a digital video image processor as described
by Steponkuset al. (27) rather than video tapes.
The proportion of protoplasts that approached volumetric equilibrium
during cooling decreased as the rate of cooling increased.
At 3.1°C/min, the protoplasts did not approach osmotic equilibrium
or a volumetric plateau until temperatures of -25°C or
lower. At 5°C/min, some protoplasts appeared to approach equilibrium
at -30°C. At either 10 or 16°C/min, few, if any, of the
protoplasts approached osmotic equilibrium at -50°C. Intracellular
ice formation occurred over a relatively narrow temperature
range beginning at approximately -35 to -40°C in acclimated
protoplasts cooled at 3.1 or 5°C/min. In contrast, when acclimated
protoplasts were cooled at rates of 10 or 16°C/min, nucleation
occurred over a broader range which began at approximately
-20°C but with a high frequency in the range of -35°C or lower.
At each cooling rate, the proportion of protoplasts that froze
intracellularly and the temperature range over which nucleation
occurred were consistent with the results reported in Figure 5. For
example, at a cooling rate of 5°C/min, intracellular ice formation
occurred in approximately 50%o of the protoplasts cooled to -50°C,
whereas at a cooling rate of 16°C/min intracellular ice formation
occurred in all of the protoplasts cooled to -50°C. The results of
the separate experiments presented in Figures 5 and 7 also were
consistent with the results of the initial experiment presented in
Table I. A minor inconsistency was the somewhat higher incidence
of intracellular ice formation at the 3.1 and 5°C/min cooling rates
reported in Table I relative to that reported in Figures 5 and 7.
This was attributed to the fact that in Figure 1 the results report
the total incidence of intracellular ice formation during cooling
and the subsequent isothermal period (3 min) at the indicated
temperature. In Figures 5 and 7, only the incidence during cooling
was reported. As intracellular ice formation is a probabilistic
phenomenon, a greater incidence would be expected if supercooled
protoplasts were subjected to the lower temperatures for a
longer period of time.
DISCUSSION
The observation that the incidence of intracellular ice formation
was less in acclimated protoplasts compared to nonacclimated
protoplasts subjected to rapid cooling rates to temperatures be-
tween-10 and -30'C is consistent with the report of Siminovitch
and Scarth (23) that intracellular ice formation is less likely to
occur in acclimated tissues. The lower incidence of intracellular
ice formation, however, was not the result of an increase in water
permeability of the plasma membrane following cold acclimation
as commonly inferred and originally suggested by Levitt and
Scarth (10) and Siminovitch and Scarth (23). The hydraulic
conductivity measured during freeze-induced dehydration at
-5SC was similar for both acclimated and nonacclimated protoplasts
(Table II). Speculation that intracellular supercooling is
precluded during cooling of acclimated cells because of the purported
increase in water permeability (10, 23) was not confirmed
with isolated protoplasts. On the contrary, there was no unique
relationship between the extent of supercooling and the incidence
of intracellular ice formation in either nonacclimated or acclimated
protoplasts. In both nonacclimated and acclimated protoplasts,
the extent of intracellular supercooling was similar under
conditions that resulted in the greatest difference in the incidence
of intracellular ice formation and which provided for the most
accurate estimate of supercooling-cooling to -15 or -20'C at
rates of 10 or 16'C/min.
The major distinction between nonacclimated and acclimated
protoplasts was the temperature at which nucleation occurred, a
point not considered in the analysis of Levitt and Scarth (10). In
nonacclimated protoplasts, nucleation occurred over a relatively
narrow temperature range with a median nucleation temperature
of - 15'C, whereas in acclimated protoplasts, nucleation occurred
over a broader temperature range with a median nucleation
temperature of -420C. We submit that the large difference in the
incidence of intracellular ice formation between nonacclimated
and acclimated protoplasts cooled at rapid rates over the range of
-10 to-30'C is due to a lower nucleation temperature of acclimated
protoplasts.
In contrast to the mechanistic interpretation proposed by Levitt
and Scarth (10), Mazur (14, 17) reasoned that intracellular ice
formation occurs if cells do not approach osmotic equilibrium
before reaching a characteristic nucleation temperature. For cells
that have achieved osmotic equilibrium prior to reaching the
nucleation temperature, the probability of intracellular ice formation
is zero. For cells that are extensively supercooled when
they reach the nucleation temperature, the probability approaches
1.0. Implied is the tacit assumption that at temperatures above the
nucleation temperature the probability of intracellular ice formation
is zero regardless of the extent of supercooling.
Over the range of -10 to -30'C, the incidence of intracellular
ice formation in both nonacclimated and acclimated protoplasts
is entirely consistent with Mazur's proposal. For nonacclimated
protoplasts, the low incidence of intracellular ice formation at a
cooling rate of 3.1 °C/min reflected the proportion of protoplasts
that did not achieve osmotic equilibrium at -15°C. At the faster
cooling rates, the increased incidence of intracellular ice formation
reflected the increasingly higher proportion that did not approach
osmotic equilibrium before the characteristic nucleation temperature
was attained. The constancy of the incidence of intracellular
ice formation at temperatures lower than the nucleation temperature
(Fig. Ia) is consistent with the interpretation that protoplasts

INTRACELLULAR ICE FORMATION IN ISOLATED PROTOPLASTS
which attain osmotic equilibrium before attaining the characteristic
nucleation temperature would not be subject to intracellular
ice formation.
For acclimated protoplasts cooled over the range of -10 to
-30°C, the incidence was relatively low at all cooling rates in
spite of extensive supercooling of the intracellular solution because
the nucleation temperature was not encountered. Only upon cooling
to temperatures of -40 or -50°C did the incidence of intracellular
ice formation in acclimated protoplasts increase in proportion
to the cooling rate.
Other than presenting an average value for the extent of supercooling
at each of the cooling rates (Fig. 4), no further quantitative
analysis has been attempted. A more quantitative analysis of the
incidence of intracellular ice formation as a function of the extent
of supercooling at low fractional osmotic volumes is limited
because of the asymptotic nature of the equilibrium volumetric
response. Under these conditions, small variations in volume may
result in the inference of substantial variations in supercooling of
the protoplast contents (compare Figs. 3 and 4 at slow cooling
rates). These limitations are inconsequential at high fractional cell
volumes where, because of the initial sharp decline in the equilibrium
volumetric response, calculations of the extent of supercooling
are insensitive to errors in the estimate of the fractional
volume. Therefore, the conclusion that little difference exists
between the supercooling of nonacclimated and acclimated protoplasts
cooled at rapid rates is not subject to this limitation. The
limitation of inferring supercooling at low fractional cell volumes
is a consequence of the nature of the equilibrium volumetric
response at low temperatures which is not unique to the protoplast
system but applies to all systems where supercooling is inferred
from volumetric changes during freezing.
Mazur (17) suggests that the lower the nucleation temperature
the higher the critical cooling rate necessary for intracellular ice
formation to occur, assuming all other parameters are constant.
For example, for red blood cells, he has predicted that a 10°C
difference in the assumed nucleation temperature will alter the
critical cooling rate by approximately 1 order of magnitude (17).
Although the median nucleation temperature of acclimated protoplasts
(-42°C) was substantially lower than that of nonaccimated
protoplasts (-15°C), there was no difference in the cooling
rate dependence for intracellular ice formation if the protoplasts
were cooled to -50°C (Table I). This suggests that 'all other
parameters' were not constant. To maintain similar surface areas
and volumes, nonacclimated and acclimated protoplasts were
suspended in 0.53 and 1.03 osm solutions, respectively. The difference
in the internal solute concentration would lessen the
difference in the osmotic gradient during cooling, but this would
have a minimal effect on the extent of supercooling because less
water would have to be removed from the acclimated protoplasts
at any given subzero temperature. In fact, the extent of supercooling
to -20°C was similar for both nonacclimated and acclimated
protoplasts cooled at either 10 or 16°C/min. Although, the
Lp measured at -5°C was similar for both nonacclimated and
acclimated protoplasts, we suspect that impeded water efflux is
occurring at the lower temperatures imposed on the acclimated
protoplasts. This is suggested by theoretical considerations that
reduced water efflux at low temperatures may occur due to the
influence of temperature and increased osmolality on the hydraulic
conductivity of the plasma membrane (8, 22).
Insight into the mechanism of nucleation is required to provide
an explanation for the large difference in the nucleation temperature
of nonacclimated and acclimated protoplasts. As early as
1932, Chambers and Hale (4) deduced that the plasma membrane
acts as a barrier to intracellular ice formation and, so long as the
plasma membrane remains intact, the intracellular solution can
be supercooled below its freezing point. The critical temperature
for a tissue was considered to be the temperature at which the
987
plasma membrane breaks down to permit the external ice to
nucleate the intracellular solution. Subsequently, Asahina (1) also
suggested that intracellular ice formation depends on whether or
not the plasma membrane can prevent nucleation by external ice.
More recently, Mazur (15, 17) has provided a more comprehensive
analysis and also concluded that intracellular ice formation results
from seeding by extracellular ice rather than spontaneous crystallization
within the cell. He cites many reports that nucleation
occurs at lower temperatures in the absence of external ice than in
its presence. Further, in the presence of extracellular ice, nucleation
occurs in the range of -10 to -20° C in a diverse array of cell
types.
The behavior of nonacclimated protoplasts (Fig. 5) is consistent
with the interpretation that intracellular ice formation is the result
of heterogeneous nucleation by external ice in that nucleation
occurred at temperatures well above the expected homogeneous
nucleation temperature of the intracellular solution or the eutectic
of the extracellular solution.
Although the median nucleation temperature of acclimated
protoplasts (-42°C) is close to the homogeneous nucleation temperature
of pure water, we do not believe that this is responsible
for the nucleation of acclimated protoplasts. First, the intracellular
solution is not pure water, and a lower homogeneous nucleation
temperature for such a solution would be expected (5, 21). Second,
if homogeneous nucleation were responsible, a sharper temperature
dependency at -40°C would have been expected and was
not observed, especially at the faster cooling rates. Finally, when
suspended in a 0.53 osm solution, the median nucleation temperature
was substantially higher.
Under some circumstances, nucleation of acclimated protoplasts
(Fig. 5), was associated with a crystallization event. During cooling
of the NaCl + CaCl2 solution to -50°C, a crystallization event
occurred at approximately -35°C during cooling at 3.1°C/min,
at approximately -40°C during cooling at 5°C/min, and only
after (2 to 3 min) -50°C was achieved at cooling rates of 10 or
16°C/min. During warming, melting of the crystals occurred
between -20 and -25°C, regardless of the previous cooling rate.
Because crystallization of NaCl in such a solution is expected to
occur in the range of -20 to -25°C, we conclude that this was
responsible for the crystallization event observed.
In some instances, intracellular ice formation was associated
with the crystallization event. In Figure 5, the sharp initial increases
in the cumulative frequency of intracellular ice formation
in protoplasts cooled at either 3.1 or 5°C/min corresponded to
the temperatures at which the crystallization event occurred, -35
and -40°C, respectively. At these rates, the crystallization event
proceeded directionally across the microscope field due to thermal
characteristics of the cryostage (see Ref. 27). In many cases,
nucleation was observed to occur when the advancing front of
crystallization encountered a protoplast. At the faster cooling rates
(10 and 16°C/min), however, the steep increase in the frequency
was not associated with any visible crystallization event, because
at these rates the crystallization event only occurred after -50°C
was attained. These results indicate that eutectic crystallization
of the suspending medium may influence the nucleation temperature
under certain conditions, but cannot be considered to be the
singular cause of intracellular nucleation. Thus, although nucleation
of yeast cells (13) or sea urchin eggs (Strongyocentrotus nudus)
(2) is not associated with eutectic crystallization under the conditions
imposed, nucleation of eggs of another species of sea urchin
(Hemicentrotus pulcherrimus) is associated with the eutectic crystallization
(3).
Although Mazur (15, 17) suggests that nucleation is largely a
consequence of temperature effects on ice crystals, we have reason
to believe that nucleation is primarily the result of a membrane
alteration per se. Previously, we have presented a preliminary
report of the phenomenology of intracellular ice formation with

988 DOWGERT AND STEPONKUS
Plant Physiol. Vol. 72, 1983
respect to observed perturbations of the plasma membrane (25).
Specifically, with high resolution video cryomicroscopy, the following
phenomena have been observed; immediately before (0.03
to 1 s) intracellular ice formation the following events have been
observed: (a) outward flow of the intracellular solution; (b) fluttering
of the plasma membrane; or (c) eddy-like flow patterns in
the cytoplasm. Upon ice nucleation and attendant gas bubble
formation (see Ref. 24), gas bubbles emerge from the protoplast
in the region of the plasma membrane where the above phenomena
were observed. Upon thawing, gas bubbles and cellular contents
(cytoplasm and chloroplasts) emerge from the same region.
Collectively, these observations suggest that mechanical breakdown
of the plasma membrane precedes intracellular ice formation
and exposes the supercooled intracellular solution to the
external ice matrix. (See Steponkus et al. [26] for a further discussion
of mechanical breakdown of the plasma membrane.)
That nucleation is a consequence of mechanical breakdown of
the plasma membrane is consistent with a literal interpretation of
the comment by Chambers and Hale (4) that the critical temperature
is 'the one at which the barrier (plasma membrane) breaks
down.' It differs considerably, however, from the suggestion of
Mazur (15, 17) that only below -10°C will extracellular ice
crystals of a suitably small size be sufficiently stable to pass
through aqueous channels in the membrane. Mazur's analysis
supposes that an organized crystalline surface is passing through
the membrane. If, however, mechanical breakdown of the membrane
occurs, then ice need not pass through the membrane.
Instead the barrier ceases to exist at the locus of breakdown.
Inferring that the primary cause of nucleation is mechanical
breakdown of the plasma membrane does not exclude the possibility
that the composition of the suspending medium can affect
the nucleation temperature or, in our interpretation, the temperature
at which membrane breakdown occurs. Whereas Mazur (12)
observed no effect of the initial concentration of the suspending
medium on the survival of yeast cells cooled at rapid rates, the
initial concentration of the suspending medium does influence the
nucleation temperature of isolated protoplasts (Fig. 6). An obvious
effect of increasing the external concentration of the suspending
medium is to increase the fraction of unfrozen solution at any
subzero temperature (16). Therefore, one possibility to account for
the lower nucleation temperature in protoplasts suspended in the
higher concentration is that the proximity of ice to the plasma
membrane is decreased. Alternatively, differences in the composition
of the suspending medium may directly affect the stability
of the plasma membrane. Recently, Leibo (6, 7) reported that the
nucleation temperature of mouse ova suspended in PBS is -7°C
whereas those suspended in PBS + 1 M DMSO nucleate below
-40°C. Considering the report that the stability of the plasma
membrane of Friend leukemia cells is increased by the addition of
DMSO to the suspending medium (11), it is possible that DMSO
acts, in part, by increasing the stability of the plasma membrane
at lower temperatures.
A corollary to the hypothesis that nucleation is a result of
destabilization of the plasma membrane is that cold acclimation
increases the stability of the plasma membrane during a freezethaw
cycle. The nucleation temperature of acclimated protoplasts
suspended in a 1.03 osm solution is considerably lower than the
nucleation temperature of nonacclimated protoplasts suspended
in a 0.53 osm solution (Fig. 5). This difference is greater than what
can be accounted for by differences in the tonicity of the suspending
medium. Whether contrasted at 0.53 or 1.03 osm, the median
nucleation temperature is lower in acclimated protoplasts. Considering
that cold acclimation increases the tolerance of the plasma
membrane of isolated protoplasts to mechanical and chemical
stresses (26), it is not unexpected that its efficacy in precluding
intracellular ice nucleation is greater in acclimated protoplasts.
In summary, in previous studies of the cooling rate dependence
of intracellular ice formation and the influence of cold acclimation,
emphasis has been placed on water permeability and cellular
volumetric behavior during cooling. In contrast, relatively little
attention has been directed to the nucleation event, either systematic
determinations of the temperature at which nucleation
occurs or the mechanism of nucleation. An accurate analysis of
factors affecting intracellular ice formation requires the experimental
determination of both cellular volumetric behavior and
the nucleation event. For this, cryomicroscopy offers many advantages.
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