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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 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 1977 American Society of Plant Biologists. All rights reserved. 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). Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 1977 American Society of Plant Biologists. All rights reserved. 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. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 1977 American Society of Plant Biologists. All rights reserved. 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 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 1977 American Society of Plant Biologists. All rights reserved. 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 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 1977 American Society of Plant Biologists. All rights reserved. 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