Download Ice Adhesions in Relation to Freeze Stress1

Plant Physiol. (1977) 60, 499-503
Ice Adhesions in Relation to Freeze Stress1
Received for publication February 8, 1977 and in revised form May 4, 1977
C. R. OLIEN AND MYRTLE N. SMITH
Agricultural Research Service, United States Department of Agriculture, and Crop and Soil Sciences
Department, Michigan State University, East Lansing, Michigan 48824
ABSTRACT
In freezing, competitive interaction between ice and hydrophilic
plant substances causes an energy of adhesion to develop through the
intersitial liquid. The thermodynamic basis for the adhesion energy is
disussed, with estimates of the energies involved. In this research,
effects of adhesion energy were observed microscopicaUly in conjunction
with energies of crystaflization and frost desication. The complex
character of ice in intact crown tissue of winter barley (Hordeum
vulgare L.) and the problems of sectioning frozen tissue without
producing artifacts led to an alternative study of single barley ceUs in a
mesh of ice and cell wall polymers. Adhesions between ice, cell wall
polymers, and the plasmalemma form a complexly interacting system in
which the pattern of crystallization is a major factor in determination of
stress and injury.
Freezing of water within crown tissues of cereals causes
several different forms of stress energy to develop. These
stresses have been distinguished by differences in the dissipation
of crystallization energy, the temperature range in which the
stress becomes injurious, and the histological pattern of injury
in the plant (10). The cause of injury in the temperature range
between -8 and -16 C has been the most elusive. Direct
injury from growth of ice crystals requires a large free energy of
crystallization. This can only develop from supercooling or from
rapid transfer of latent heat in very wet tissue (6), and therefore
is most often effective in winter cereals, above -8 C. Frost
desiccation, where ice acts independently through the vapor
phase as a water accumulator, does not injure leaf tissues of
hardened "Hudson" barley (Hordeum vulgare L.) until the ice
temperature is below -16 C (7).
A thermodynamic study of equilibrium freezing in complex
interfaces between ice and hydrophilic substances led to the
conclusion that competition for the interfacial liquid water
caused an energy of adhesion to develop (8-10). The adhesion
energy developed through competitive structuring of the interfacial liquid and was predicted to cause significant stress in the
temperature range near -10 C.
Effects of adhesion energy were observed microscopically in
conjunction with effects of crystallization and frost desiccation.
The complex character of ice in intact crown tissue and the
problems of sectioning frozen tissue without producing artifacts
led to an alternative study of single winter barley cells in a mesh
of ice and cell wall polymers.
MATERIALS AND METHODS
Free cell cultures of Hudson barley were produced by initiating calluses from barley embryos on Petri plates of Gamborg
1 Cooperative investigations of the Agricultural Research Service,
U.S.D.A. and Michigan Agricultural Experiment Station, East Lansing,
Mich. 48824; Journal Article No. 7811.
B5 medium containing 2 ,uglml 2,4-dichlorophenoxyacetic acid
(4). The embryos were obtained from washed seeds surfacesterilized 18 to 24 hr in a covered jar of 0.1% sodium hypochlorite. The callus-producing embryos were transferred from the
initial 2,4-D Petri plates to 125-ml flasks of the same medium.
Here they were increased to a diameter of 1 to 1.5 mm and
then transferred to liquid B5 medium in 125-ml flasks grown at
25 C in shake culture (125 rpm). In shake culture, newly
formed cells were continually displaced from the callus, producing a suspension of predominantly single cells. Not all cells
persisted as singles; two to four celled clumps were not uncommon. To maintain culture uniformity subculturing was eliminated, and cultures were kept on shake only until a cell count
of approximately 25 cells/mm3 was attained.
Cultures of such count were ready for cold hardening. The
culture flasks were transferred to a 2 C chamber. Here the
flasks were immersed in an ice bath on a rotary shaker. At 2 C,
the ice melted slowly providing a uniform 0 C hardening
temperature.
Responses of cells to several forms of stress were used to
compare tender with hardened cells in suspension and to compare them with leaf tissue (11). Mean cell response was evaluated from a conductivity technique used as described previously
(7). Uniformity of response was determined by neutral red vital
stain. Initially the cell suspension was distributed on ashless
filter paper, and the moisture content of the paper was adjusted
to give a phase transition pattern similar to the pattern that
occurs in the intercellular liquid when Hudson leaf tissue freezes
(Fig. 1). The heat sink and thermal contact used for testing
cells were the same as those used for testing leaves in order to
generate equivalent crystallization energies in the intercellular
liquid for a specified degree of supercooling in the nonequilibrium freeze test (Fig. 1) (6). The primary distinction between
tender and hardened cells involves the ease with which ice can
grow from the intercellular space into the protoplast (Fig. 2).
The free energy for crystal growth into the protoplast is determined by the degree of supercooling below the freezing point of
liquid in the protoplast. This freezing point was determined
with a cell mass obtained by centrifugation. Ten ,u of the cell
mass was placed in a wire loop that had a thermocouple in its
center, supercooled in a still-air chamber at -10 C, inoculated
with ice, and the temperature plateau recorded. Freezing point
was determined by comparison with a reference series of sucrose
solutions. The freezing point of cells centrifuged from tissue
culture was nearly the same as that of mesophyll cells from leaf
tissue (-1.1 C). Crystallization energy across the plasmalemma
of cells dispersed on filter paper was controlled by regulating
the displacement of temperature from the equilibrium freezing
point. In other tests, vapor pressure and temperature were
regulated independently and held constant until equilibrium
was attained to vectorize equilibrium freezing and desiccation
stresses (7, 9).
To observe interactions with ice, suspensions of hardened
cells were washed with water and resuspended in either water
or a 2% araboxylan solution. This polymer was extracted from
499
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500
OLIEN AND SMITH
H 1.0
3 .9
0
Z .8
0
.7
I
o .6
H
z .5
H .4
oz .3
J
H
.2
.1
uO-j
Im
0 -I -2 -3 -4 -5 -6 -7 -8 -9 -10
TEMPERATURE (C)
Equilibrium Transition
---Nonequilibrium Transition
-
FIG. 1. Relative content of liquid intercellular water at equilibrium
), and the nonequilibrium
with ice as a function of temperature (
freezing pattern for intercellular liquid that results from ice inoculation
after 5 C supercooling (- - -) for leaf tissue of Hudson barley (11).
These patterns were approximated for the intercellular liquid of a cell
suspension on ashless filter paper by adjusting the liquid content to
50% (9).
0
l0
20-
30
~~~~~~~L
w
'x 40z 50
Z 60-
TL
TC
cr 70\
\
80
90I
1000 -I -2 -3 -4 -5 -6 -7 -8 -9
TEMPERATURE (C)
FIG. 2. Injury as evaluated by conductivity of leach water versus
initial temperature of supercooling before deliberate ice nucleation in
the nonequilibrium freeze test. Per cent injured equaled the ratio of
increase in conductivity after the test to the increase in conductivity
after a rapid freeze to -60 C, times 100. TL: tender leaf; TC: tender
cells; HC: hardened cells; HL: hardened leaf.
seeds of 'Rosen' rye (Secale cereale L.) (6). Drops of the cell
suspensions were frozen on glass slides. Ice was permitted to
sublime from uncovered slides to observe the grosser effects of
freezing. Observations were made at 160 and 400x with normal
substage illumination and with epidarkfield. Slides with cell
suspensions frozen under cover glasses were studied at 1,000x
with oil immersion.
young cells generated in abundance from fresh callus. In culture,
under neither light nor dark conditions, was there any development of Chl.
Hardy cells were distinguished from tender cells bv their
survival of -5 C supercooling. Maximum hardiness was attained
after 3 weeks at 0 C. From seed to hardened cells was a
sequence taking 9 to 10 weeks, about the same time required to
produce a hardened plant from seed.
Adhesion Energy. Three factors are involved in the interaction between ice, cell wall polymers, and the plasmalemma.
These are: (a) free energy of crystal growth which causes
growth of ice crystals with extended interfaces through the
intercellular liquid of barley plant tissues; (b) freeze desiccation,
which draws hydrophilic substances into interactions where
competition occurs for structuring of the remaining liquid water;
and (c) energy of adhesion. The first two factors have been
discussed frequently. The third factor, adhesion, requires some
explanation.
In liquid water, the potential energy range of the H-bond
overlaps the kinetic energy distribution of the water molecules
(2, 5, 12, 13). Rapid redistribution of kinetic and potential
energies results in instability with a half-life of 10-11 sec for
structure in liquid water. Even in ice at the liquid interface, the
half-life is only 10-5 sec (3). This instability gives water fluid
properties so that it is relatively ineffective as an adhesive even
between hydrophilic substances to which it is bonded with high
energy. Fluidity permits this energy to be dissipated over a long
distance and so requires only a small force to provide the
energy for displacement or separation.
At 0 C, ice bonds loosely with hydrophilic substances through
the interstitial liquid. Quartz glass is wettable and a plate with a
1-cm2 area required very little force to move it along the liquid
on an ice surface at 0 C or to separate it from the ice.
Decreasing the temperature to -0.5 C stabilized the adhesion
to the extent that in repeated tests an average of 10 kg was
required to break a 1-cm2 junction between ice and quartz glass.
The effective energy of adhesion between ice and substances
that compete for liquid water was evaluated from the free
energies of transition and the means by which these free energies
were dissipated as transitions progressed (8, 9). The energy of
freezing is the potential energy of vectorized bonding that draws
water molecules into an ice lattice structure. This is opposed by
temperature, a measure of the exchangeable kinetic energy.
Freezing and melting involve resonance between potential and
kinetic energy, kinetic energy being expended by a water
molecule in escape from the lattice while acquiring potential
energy and, conversely, for a molecule from the liquid approaching a lattice site (8).
100
90
80
-
70
-25 C
R EOUILIBRIUM FREEZING
I NONEOUILIBRIUM FREEZING
to60
-
RESULTS AND DISCUSSION
Cell Culture and Freezing Response. The tissue culture cells,
ranging in size from 60 to 100 ,um in diameter, had a thin
primary wall with no noticeable secondary thickening. As a
result, the wall was fragile and easily folded or damaged
mechanically. The plasmalemma was strongly appressed against
the restraining cell wall so as to be undifferentiable under oil
immersion. Internally, when the cell was actively growing, the
nucleus was a large, central structure supported by prominent
protoplasmic strands. As the cells matured and vacuolated, the
nucleus was pressed against the wall. Other easily stained
granular and globular bodies were likewise most prominent in
Plant Physiol. Vol. 60, 1977
50
,, 40
30
20
10
+
I1
.
R I 1000 R I 2000
_
3000
4000
5000
C(co /mole)
FIG. 3. Frequency distribution of exchangeable kinetic energy of
water molecules over shifting activation limits for freezing (e1) and
melting (Eh). Frequency distribution is normal distribution in threedimensional space as expressed by polar coordinants (8-10).
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Plant
Physiol.
501
ICE ADHESIONS IN RELATION TO STRESS
Vol. 60, 1977
The free energy of freezing becomes greater than the free
energy of melting as the temperature decreases. The shift in the
frequency distribution of exchangeable kinetic energy for water
molecules is shown in Figure 3 by a comparison of the distributions at 0 and -25 C. The free energies of freezing and melting
return to balance, as equilibrium freezing progresses, principally
by a decrease in the activation energy of melting (10). Competitive equilibrium freezing is quite different from a system where
the ice is separated from the polymer by a gas phase (frost
desiccation) (1). In frost desiccation, the free energies are
balanced by a shift in vapor pressure, a density function, rather
than a shift in activation energies. The shift in activation energy
4pr the ice-liquid-polymer interface results in development of an
adhesion energy between ice and the hydrophilic substance as
they compete for the intervening liquid. Competitive structuring
of the interstitial liquid causes it to bind the hydrophilic substance to the ice. The energies of activation and adhesion can
be calculated (9, 10). The energy of adhesion is determined
mainly by the shift in the activation energy of melting and the
density of water bonding to the polymer. The energy of adhesion
per mole of liquid water below -8 C approximately equals the
reduction in latent heat of equilibrium freezing (9, 10). Thus,
although not a linear function, within the temperature range of
-8 to -16 C adhesion energy approximately equals 20 cal per
mole times the temperature in degrees C. The energy of
adhesion per area of interface depends on the bonding pattern
of the polymer. The force required to break an adhesion equals
sJ~~~~ 2Y~~~K7
~~'t
A
Vi
I-
~~L~~~'
Sri
_
AC-FAr
W
*~~~~~~~~~~~.- -. ,<If
-0.
-M
le
FIG. 5. Enlargment (x 400) of a 'Hudson' barley cell from extreme
left of mesh seen in Figure 5. Photographed with normal substage
lighting showing details of the pattern of ice crystal formations around
the cell.
/~~~~~--
FIG. 4. Mes prdue by frezn a 2%
prpaaio
of araboxylan
from 'Rosen' rye in which a few 'Hudson' barley cells were suspended.
This polymer is an excellent inhibitor of freezing kinetics causing
f smalil ice-A crys,tacs Ice wasn
I-oA
mes
develoment f the--pt tere
sublimed from mesh to improve observation. Seven 'Hudson' cells
(dark areas) form part of the mesh. x 100.
the adhesion energy per bond times the number of bonds
involved divided by the distance over which these bonds are
broken, expressed as a differential equation to determine the
critical force requirement.
Ice-Polymer-Plasmalemma Interactions. A water suspension
of cells on a glass slide at -0.5 C froze very slowly and the rate
of crystal growth could be controlled by slight temperature
adjustments. At low magnifications, single cells could be seen
to be engulfed by the advancing front of a growing crystal
without distortion or injury. Single cells were observed at
1,000 x magnification with various forms of illumination as
freezing progressed. Lowering the temperature of ice-encased
cells resulted in growth of ice inside the wall. The physical
structure of the wall did not appear to be a major barrier to
crystal growth. More subtle effects of ice as it interacted with
the plasmalemma at a lower temperature where the cell died
could not be clearly observed.
Increasing the initial crystallization energy by supercooling a
water suspension of cells more than 5 C resulted in a rapid
growth of skeletal crystals after ice inoculation as the temperature rose toward the freezing point. Many separate crystals
formed disorientations of the skeletal ice. Cells between ice
crystals were severely deformed during secondary crystal
growth, as the temperature again decreased. The cell wall often
was torn and effects of ice on the plasmalemma could be
observed microscopically.
Effects of adhesion with ice depend on the pattern of crystal
growth because adhesions occur at ice interfaces. Freezing must
occur as a nonequilibrium process to provide the energy for the
extension of the ice interface through the intercellular liquid.
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Plant Physiol. Vol. 60, 1977
OLIEN AND SMITH
502
FIG. 6. Same cell and magnification (x 400)
as
graphed with epidarkfield polarized light. This lighti
clearly the amount of peripheral distortion of the cell.
extension of the ice interface without increasing the crystallization energy. The freezing inhibitor caused 10 or more crystals
to develop around each cell with an initial supercooling of -3
C. Although ice did grow into and kill a portion of the tender
cells at this crystallization energy, it did not injure a significant
number of the hardened cells. Affected cells were killed early,
in the first phase of freezing, when the crystallization energy
was highest (greatest displacement from the freezing point).
The conductivity of the cell suspension rose abruptly as electroin the freezing
lyte was released from the tender cells early
of intercelprocess. Adhesion effects developed as the amount
lular liquid diminished and became injurious as the temperature
decreased during the secondary growth of ice crystals.
The mesh-like structure of a preparation of araboxylan extracted from 'Rosen' rye, frozen after suspercooling to -3 C, is
shown in Figure 4. The ice was sublimed from the slide before
photographing the mesh that developed between the ice crystals.
This polymer is an excellent inhibitor of freezing kinetics (6). It
forms an adhesive as well as cohesive film on ice as the crystal
grows, progressively inhibiting crystallization. As a result, water
that had been in the preparation froze as small crystals with a
was contained.
high degree of interface in which the polymerinvestigations
to act
The rye polymer was found in previous
as a cryoprotectant by controlling growth of ice crystals (6). In
ice crystals to grow more
protected plants the polymer causesin critical
regions such as the
form
to
smaller
and
crystals
slowly
meristems and their supporting crown tissues. Large ice crystals
develop only in intercellular spaces or between tissues where
deformation is not injurious. Stress in critical regions also is
relieved by redistribution of water from regions where freezing
is inhibited to regions of free crystal growth. Cells suspended in
the polymer solution were affected as if they were small regions
of denser polymer concentration. The cell walls were torn and
the plasmalemma deformed into a continuation of the polymer
mesh. Figure 5 shows a cell in the polymer mesh with normal
the same cell under
epidarkfield polarized light. The protoplasmic contents can be
distinguished from the rye polymer under the epi lighting
Figure 6v photo- substage
lighting, and Figure 6 shows
but more detail of the mesh can be seen
system,
illumination.
with substage
Adhesions between ice and the plasmalemma prevent the cell
from maintaining a minimum surface configuration as the skeletal crystals grow. The crystals deform the protoplast as they
withdraw the water and concentrate the dehydrated cell substance on the crystal surface as an adhering layer. Distortion of
the cell periphery between adjacent crystals is especially discernible and occurs for cells trapped between ice crystals as they
form in pure water as well as in the solution of polymer used to
increase ice crystals interface.
e'N?
FIG. 7. Hardened cell of 'Hudson' barley killed
Photograph shows the wrinkled nature of the surface ti
contraction.
x
by desiccation.
400.
Freezing cells in their growth medium, that contained sugars
and salts, plus the araboxylan freezing inhibitor produced a
mesh less regularly structured than that from the "pure" polymer preparation. Solutes accumulated in a liquid coating around
each cell as the external water froze. Ice adhesions to the
and this liquid coating
plasmalemma occurred less frequently normal
shape as freezing
also permitted the cell to retain a more
of cells
suspension
in
water
the
One
molar
glycerol
progressed.
resulted in formation of a fluid layer around each cell that
from forming between ice and the cell.
prevented any adhesion
After sublimation of ice from this system at -10 C, the cells
other cells that had been
had a normal shape resembling
water without
freezing.
by evaporation of
dehydrated
A cell killed by desiccation is shown in Figure 7. Desiccated
cells simply contracted. The periphery was not stretched out in
sufficient to an extended surface as happened to cells in Figures 5 and 6.
be
cause growth of ice from the intercellular
egion into the Nor were they grossly contorted as occurred in freezing of
protoplasts. For specified degree of supercooli ing, addition of supercooled water suspensions where the cells were deformed
Effects of ice crystals on cells and in
araboxylan freezing inhibitors to the water in which the cells between fewer
However, the crystallization
energy
must not
t
ri
a
were
suspended slowed the
rate
of crystal
growth iand promoted
crystals.
simple colloid systems were similar. Freezing a polymer solution
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Plant Physiol. Vol. 60, 1977
ICE ADHESIONS IN RELATION TO STRESS
produced a mesh-like structure with an extended interface. By
contrast, evaporating the water from an unfrozen polymer
solution resulted in contraction of the polymer to a simple
precipitate.
As a result of this study and in coordination with other
research on winter hardiness, cryoprotectants are being evaluated with respect to several specific activities:
A. Control of ice crystal growth and redistribution of intercellular water.
1. Control of free energy of intercellular freezing by
modification of transition patterns (reduction of free
energy decreases crystal growth through densely structured tissues).
2. Inhibition of freezing kinetics (reduces rate of crystal
growth and crystal size).
B. Prevent injurious adhesions from forming between ice
and cell substances, especially the plasmalemma.
C. Prevent growth of ice from intercellular water into the
protoplasm by reduction of free energy of freezing across
the plasmalemma or by stabilization of the plasmalemma.
Single cells of any genotype can be grown and hardened in
media that contain a variety of nutrients, toxins, and hormones
to evaluate the range of phenotypic expression of cryoprotectants. The cells can be structured into artificial tissues with
addition of intercellular cryoprotectants obtained from other
genotypes. The artificial tissues can be tested by stress vectorization for response to various combinations of stress energies.
The response of various permutations provides an experimental
basis for characterization of heritable traits and
regulators.
503
exogenous
Acknowledgment-The authors wish to thank P. Carlson for his assistance in culturing
barley and wheat as single cell suspensions.
LITERATURE CITED
1. BOYER JS 1969 Measurement of the water status of plants. Annu Rev Plant Physiol 20:
351-364
2. EYRING H, T REE, N HIRAI 1958 Significant structures in the liquid state. 1. Proc Nat
Acad Sci USA 44: 683-688
3. FLETCHER NH 1970 The Chemical Physics of Ice. Cambridge Univ Press
4. GAMBORG OL, LR WETTER 1975 Plant Tissue Culture Methods. National Research
Council of Canada, Prairie Regional Laboratory, Saskatoon Saskatchewan
5. NEMETHY G, HA SCHERAGA 1962 Structure of water and hydrophobic bonding in proteins.
1. A model for the thermodynamic properties of liquid water. J Chem Physics 36: 33823400
6. OLIEN CR 1965 Interference of cereal polymers and related compounds with freezing.
Cryobiology 2: 47-54
7. OLIEN CR 1971 A comparison of desiccation and freezing as stress vectors. Cryobiology 8:
244-248
8. OLIEN CR 1973 Thermodynamic components of freezing stress. J Theroret Biol 39: 201210
9. OLIEN CR 1974 Energies of freezing and frost desiccation. Plant Physiol 53: 764-767
10. OLIEN CR 1974 Stress analysis. In Winter Hardiness in Barley. Research Report 247.
Michigan State Univ Agricultural Experimental Station, East Lansing Mich
11. OLIEN CR 1977 Barley: Patterns of Response to Freezing Stress. US Department of
Agriculture Technical Bulletin 1558
12. RAHMAN A, FH SnLLINGER 1971 Molecular dynamics study of liquid water. J Chem
Physics 55: 3336-3359
13. VAND V, WA SENIOR 1965 Structure and partition function of liquid water. 111. Development of the partition function for a band model of water. J Chem Physics 43: 1878-1884
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CORRECTIONS
Vol. 60: 150-156. 1977
Okabe, Keiichiro, Georg H. Schmid, and Joseph Straub.
Genetic Characterization and High Efficiency Photosynthesis of an Aurea Mutant of Tobacco.
Page 155, column 2, paragraph 2, lines 13-15 should be corrected to read: Moreover, the production of haploid (su aur)
plants from anther cultures of Su/su var. Aurea or from the
green type su/su Aur/aur of crossing No. 7 should provide
new insights.
doleacetic Acid Levels in Phaseolus, Zea, and Pinus during
Seed Germination.
Vol. 60: 499-503. 1977
Olien, C. R., and Myrtle N. Smith. Ice Adhesions in Relation to
Freeze Stress.
Page 501, column 1, legend of Figure 4, magnification should
be corrected to read: x 50.
Pages 501 and 502, legends of Figures 5 and 6, magnifications
should be corrected to read: x 200. Legends of Figures 5 and
6 should refer to comparison with the preceding figures.
Vol. 60: 317-319. 1977
Tillberg, Elisabeth. The title should be corrected to read: In-
935