Effect of cold acclimation on bulk tissue electrical impedance: I. Measurements with birdsfoot trefoil at subfreezing temperatures

The resistive and reactive components of electrical impedance were measured for birdsfoot trefoil (Lotus corniculatus L.) stems at freezing temperatures to −8°C. As temperature decreased the specific resistance at frequencies between 49 hertz and 1.11 megahertz of stems from cold acclimated plants increased more rapidly than from nonacclimated plants. This temperature dependence of specific resistance could be characterized by an Arrhenius activation energy; cold acclimated stems had a larger Arrhenius activation energy than nonacclimated stems. The low frequency resistance is believed to characterize the extracellular region of the stems and the high frequency resistance is believed to characterize the intracellular region of the stems. Cold acclimation increased the intracellular but not the extracellular resistance at nonfreezing temperatures. Cold acclimated stems were not injured by freezing to −8°C and thawing, but nonacclimated stems were injured by freezing to temperatures between −2.2 and −5.6°C and thawing. Injury to nonacclimated stems at freezing temperatures below −2.2°C was indicated by a decrease in the ratio of resistance at 49 Hz to that at 1.11 megahertz.     

Frost injury and heterogeneous ice nucleation in leaves of tuber-bearing solanum species : ice nucleation activity of external source of nucleants

The heterogeneous ice nucleation characteristics and frost injury in supercooled leaves upon ice formation were studied in nonhardened and cold-hardened species and crosses of tuber-bearing Solanum. The ice nucleation activity of the leaves was low at temperatures just below 0°C and further decreased as a result of cold acclimation. In the absence of supercooling, the nonhardened and cold-hardened leaves tolerated extracellular freezing between −3.5° and −8.5°C. However, if ice initiation in the supercooled leaves occurred at any temperature below −2.6°C, the leaves were lethally injured.

To prevent supercooling in these leaves, various nucleants were tested for their ice nucleating ability. One% aqueous suspensions of fluorophlogopite and acetoacetanilide were found to be effective in ice nucleation of the Solanum leaves above −1°C. They had threshold temperatures of −0.7° and −0.8°C, respectively, for freezing in distilled H₂O. Although freezing could be initiated in the Solanum leaves above −1°C with both the nucleants, 1% aqueous fluorophlogopite suspension showed overall higher ice nucleation activity than acetoacetanilide and was nontoxic to the leaves. The cold-hardened leaves survived between −2.5° and −6.5° using 1% aqueous fluorophlogopite suspension as a nucleant. The killing temperatures in the cold-hardened leaves were similar to those determined using ice as a nucleant. However, in the nonhardened leaves, use of fluorophlogopite as a nucleant resulted in lethal injury at higher temperatures than those estimated using ice as a nucleant.

     

Interactions among Flooding, Freezing, and Ice Encasement in Winter Wheat

Exposure of winter wheat (Triticum aestivum L.) to various combinations of flooding and freezing stresses induces much greater damage than the individual stresses. Cold-hardened plants flooded for 1 week or exposed to −6°C for 1 week show 100% survival, while survival of plants exposed to both stresses simultaneously is reduced by 20 to 30%, and cold hardiness decreases by several degrees. The level of nonstructural carbohydrates increases in crown tissue during cold acclimation, but decreases when the plants are exposed to flooding or to −6°C for 1 week. The respiratory capacity of crown tissue segments declines when the plants are stressed. Uptake of ⁸⁶Rb by the roots of intact seedlings declines after exposure to either freezing or flooding, whereas passive efflux of amino acids is observed after freezing but not following flooding. This study has shown that detectable stress-induced metabolic changes occur in winter wheat before the applied stress is severe enough to reduce survival.     

Freezing injury and root development in winter cereals

Upon exposure to 2°C, the leaves and crowns of rye (Secale cereale L. cv `Puma') and wheat (Triticum aestivum L. cv `Norstar' and `Cappelle') increased in cold hardiness, whereas little change in root cold hardiness was observed. Both root and shoot growth were severely reduced in cold-hardened Norstar wheat plants frozen to −11°C or lower and transplanted to soil. In contrast, shoot growth of plants grown in a nutrient agar medium and subjected to the same hardening and freezing conditions was not affected by freezing temperatures of −20°C while root growth was reduced at −15°C. Thus, it was apparent that lack of root development limited the ability of plants to survive freezing under natural conditions.

Generally, the temperatures at which 50% of the plants were killed as determined by the conductivity method were lower than those obtained by regrowth. A simple explanation for this difference is that the majority of cells in the crown are still alive while a small portion of the cells which are critical for regrowth are injured or killed.

Suspension cultures of Norstar wheat grown in B-5 liquid medium supplemented with 3 milligrams per liter of 2,4-dichlorophenoxyacetic acid could be cold hardened to the same levels as soil growth plants. These cultures produce roots when transferred to the same growth medium supplemented with a low rate of 2,4-dichlorophenoxyacetic acid (<1 milligram per liter). When frozen to −15°C regrowth of cultures was 50% of the control, whereas the percentage of calli with root development was reduced 50% in cultures frozen to −11°C. These results suggest that freezing affects root morphogenesis rather than just killing the cells responsible for root regeneration.

     

Relationship between Freezing Tolerance of Root-Tip Cells and Cold Stability of Microtubules in Rye (Secale cereale L. cv Puma)

The response of cortical microtubules to low temperature and freezing was assessed for root tips of cold-acclimated and non-acclimated winter rye (Secale cereale L. cv Puma) seedlings using indirect immunofluorescence microscopy with antitubulin antibodies. Roots cooled to 0 or −3°C were fixed for immunofluorescence microscopy at these temperatures or after an additional hour at 4°C. Typical arrays of cortical microtubules were present in root-tip cells of seedlings exposed to the cold-acclimation treatment of 4°C for 2 days. Microtubules in these cold-acclimated cells were more easily depolymerized by a 0°C treatment than microtubules in root-tip cells of nonacclimated, 22°C-grown seedlings. Microtubules were still present in some cells of both nonacclimated and cold-acclimated roots at 0 and −3°C; however, the number of microtubules in these cells was lower than in controls. Microtubules remaining during the −3°C freeze were shorter than microtubules in unfrozen control cells. Repolymerization of microtubules after both the 0 and −3°C treatments occurred within 1 h. Root tips of nonacclimated seedlings had an LT-50 of −9°C. Cold acclimation lowered this value to −14°C. Treatment of 22°C-grown seedlings for 24 h with the microtubule-stabilizing drug taxol caused a decrease in the freezing tolerance of root tips, indicated by a LT-50 of −3°C. Treatment with D-secotaxol, an analog of taxol that was less effective in stabilizing microtubules, did not alter the freezing tolerance. We interpret these data to indicate that a degree of depolymerization of microtubules is necessary for realization of maximum freezing tolerance in root-tip cells of rye.     

Temperature Dependence of Photosynthesis in Agropyron smithii Rydb. : II. CONTRIBUTION FROM ELECTRON TRANSPORT AND PHOTOPHOSPHORYLATION

As part of an analysis of the factors regulating photosynthesis in Agropyron smithii Rydb., a C₃ grass, the response of electron transport and photophosphorylation to temperature in isolated chloroplast thylakoids has been examined. The response of the light reactions to temperature was found to depend strongly on the preincubation time especially at temperatures above 35°C. Using methyl viologen as a noncyclic electron acceptor, coupled electron transport was found to be stable to 38°C; however, uncoupled electron transport was inhibited above 38°C. Photophosphorylation became unstable at lower temperatures, becoming progressively inhibited from 35 to 42°C. The coupling ratio, ATP/2e⁻, decreased continuously with temperature above 35°C. Likewise, photosystem I electron transport was stable up to 48°C, while cyclic photophosphorylation became inhibited above 35°C. Net proton uptake was found to decrease with temperatures above 35°C supporting the hypothesis that high temperature produces thermal uncoupling in these chloroplast thylakoids. Previously determined limitations of net photosynthesis in whole leaves in the temperature region from 35 to 40°C may be due to thermal uncoupling that limits ATP and/or changes the stromal environment required for photosynthetic carbon reduction. Previously determined limitations to photosynthesis in whole leaves above 40°C correlate with inhibition of photosynthetic electron transport at photosystem II along with the cessation of photophosphorylation.     

Survival of Ice Nucleation-Active and Genetically Engineered Non-Ice-Nucleating Pseudomonas syringae Strains after Freezing

The survival after freezing of ice nucleation-active (INA) and genetically engineered non-INA strains of Pseudomonas syringae was compared. Each strain was applied to oat seedlings and allowed to colonize for 3 days, and the plants were subjected to various freezing temperatures. Plant leaves were harvested before and after freezing on two consecutive days, and bacterial populations were determined. Populations of the INA wild-type strain increased 15-fold in the 18 h after the oat plants incurred frost damage at −5 and −12°C. Plants colonized by the non-INA strain were undamaged at −5°C and exhibited no changes in population size after two freeze trials. As freezing temperatures were lowered (−7, −9, and −12°C), oat plants colonized by the non-INA strain suffered increased frost damage concomitant with bacterial population increases following 18 h. At −12°C, both strains behaved identically. The data show a relationship between frost damage to plants and increased bacterial population size during the following 18 h, indicating a potential competitive advantage of INA strains of P. syringae over non-INA strains in mild freezing environments.     

Freezing tolerance of citrus, spinach, and petunia leaf tissue : osmotic adjustment and sensitivity to freeze induced cellular dehydration

Seasonal variations in freezing tolerance, water content, water and osmotic potential, and levels of soluble sugars of leaves of field-grown Valencia orange (Citrus sinensis) trees were studied to determine the ability of citrus trees to cold acclimate under natural conditions. Controlled environmental studies of young potted citrus trees, spinach (Spinacia pleracea), and petunia (Petunia hybrids) were carried out to study the water relations during cold acclimation under less variable conditions. During the coolest weeks of the winter, leaf water content and osmotic potential of field-grown trees decreased about 20 to 25%, while soluble sugars increased by 100%. At the same time, freezing tolerance increased from lethal temperature for 50% (LT₅₀) of −2.8 to −3.8°C. In contrast, citrus leaves cold acclimated at a constant 10°C in growth chambers were freezing tolerant to about −6°C. The calculated freezing induced cellular dehydration at the LT₅₀ remained relatively constant for field-grown leaves throughout the year, but increased for leaves of plants cold acclimated at 10°C in a controlled environment. Spinach leaves cold acclimated at 5°C tolerated increased cellular dehydration compared to nonacclimated leaves. Cold acclimated petunia leaves increased in freezing tolerance by decreasing osmotic potential, but had no capacity to change cellular dehydration sensitivity. The result suggest that two cold acclimation mechanisms are involved in both citrus and spinach leaves and only one in petunia leaves. The common mechanism in all three species tested was a minor increase in tolerance (about −1°C) resulting from low temperature induced osmotic adjustment, and the second in citrus and spinach was a noncolligative mechanism that increased the cellular resistance to freeze hydration.     

High Survivability of Cheese Whey-Grown Rhizobium meliloti Cells upon Exposure to Physical Stress

Whey, a by-product of the dairy industry, has been found to protect the rhizobia cells during freezing and thawing. Cells of rhizobia grown on whey sustained freezing better at −18°C than did cells grown on mannitol or sucrose. Suspensions of cells grown on whey or mannitol that were suspended in whey performed equally well at −18 and −80°C, with 94 and 100% survival, respectively. Whey-grown rhizobia in pellets withstood desiccation better than did their mannitol-grown equivalents. Rhizobia that were grown on whey and then inoculated onto commercial peat showed a survival rate of 100% after 23 weeks at −4°C. Whey-grown cells in peat performed better at various temperatures during storage, even when they were exposed to desiccation, than did mannitol-grown cells in peat. Whey, therefore, offers interesting possibilities as a Rhizobium protectant for the inoculum industry.     

Stabilization of Frozen Lactobacillus delbrueckii subsp. bulgaricus in Glycerol Suspensions: Freezing Kinetics and Storage Temperature Effects

The interactions between freezing kinetics and subsequent storage temperatures and their effects on the biological activity of lactic acid bacteria have not been examined in studies to date. This paper investigates the effects of three freezing protocols and two storage temperatures on the viability and acidification activity of Lactobacillus delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol. Samples were examined at −196°C and −20°C by freeze fracture and freeze substitution electron microscopy. Differential scanning calorimetry was used to measure proportions of ice and glass transition temperatures for each freezing condition tested. Following storage at low temperatures (−196°C and −80°C), the viability and acidification activity of L. delbrueckii subsp. bulgaricus decreased after freezing and were strongly dependent on freezing kinetics. High cooling rates obtained by direct immersion in liquid nitrogen resulted in the minimum loss of acidification activity and viability. The amount of ice formed in the freeze-concentrated matrix was determined by the freezing protocol, but no intracellular ice was observed in cells suspended in glycerol at any cooling rate. For samples stored at −20°C, the maximum loss of viability and acidification activity was observed with rapidly cooled cells. By scanning electron microscopy, these cells were not observed to contain intracellular ice, and they were observed to be plasmolyzed. It is suggested that the cell damage which occurs in rapidly cooled cells during storage at high subzero temperatures is caused by an osmotic imbalance during warming, not the formation of intracellular ice.     

Relationship between Thermal Transitions and Freezing Injury in Pea and Soybean Seeds

In an attempt to correlate freezable water with freezing injury, the thermal behavior of pea (Pisum sativum L.) and soybean (Glycine max L. Merr) seed parts at different moisture contents were compared with survival of the seeds when exposed to low temperatures. Thermal transitions between −150 and 10°C were studied using differential scanning calorimetry. In pea, reduction of germinability, after exposure of seeds to temperatures between − 18 and − 180°C, occurred at a constant moisture content (about 0.33 gram H₂O/gram dry weight) regardless of the temperature; this moisture level was above that at which freezable water was first detectable by differential scanning calorimetry (0.26 gram H₂O/gram dry weight). In contrast, damage to soybean seeds was observed at progressively lower moisture contents (from 0.33 to 0.20 gram H₂O/gram dry weight) when the temperature was decreased from −18°C to −50°C. At −18 and −30°C, moisture contents at which damage to soybean seeds was evident were above that at which freezable water was first detectable (0.23 gram H₂O/gram dry weight). However, at −50, −80, and −180°C, damage was evident even when freezable water was not detectable. The data suggest that, while the quantity of water is important in the expression of freezing injury, the presence of freezable water does not account for the damage.     

A Low Temperature Limit for Life on Earth

There is no generally accepted value for the lower temperature limit for life on Earth. We present empirical evidence that free-living microbial cells cooling in the presence of external ice will undergo freeze-induced desiccation and a glass transition (vitrification) at a temperature between −10°C and −26°C. In contrast to intracellular freezing, vitrification does not result in death and cells may survive very low temperatures once vitrified. The high internal viscosity following vitrification means that diffusion of oxygen and metabolites is slowed to such an extent that cellular metabolism ceases. The temperature range for intracellular vitrification makes this a process of fundamental ecological significance for free-living microbes. It is only where extracellular ice is not present that cells can continue to metabolise below these temperatures, and water droplets in clouds provide an important example of such a habitat. In multicellular organisms the cells are isolated from ice in the environment, and the major factor dictating how they respond to low temperature is the physical state of the extracellular fluid. Where this fluid freezes, then the cells will dehydrate and vitrify in a manner analogous to free-living microbes. Where the extracellular fluid undercools then cells can continue to metabolise, albeit slowly, to temperatures below the vitrification temperature of free-living microbes. Evidence suggests that these cells do also eventually vitrify, but at lower temperatures that may be below −50°C. Since cells must return to a fluid state to resume metabolism and complete their life cycle, and ice is almost universally present in environments at sub-zero temperatures, we propose that the vitrification temperature represents a general lower thermal limit to life on Earth, though its precise value differs between unicellular (typically above −20°C) and multicellular organisms (typically below −20°C). Few multicellular organisms can, however, complete their life cycle at temperatures below ∼−2°C.     

A Leech Capable of Surviving Exposure to Extremely Low Temperatures

It is widely considered that most organisms cannot survive prolonged exposure to temperatures below 0°C, primarily because of the damage caused by the water in cells as it freezes. However, some organisms are capable of surviving extreme variations in environmental conditions. In the case of temperature, the ability to survive subzero temperatures is referred to as cryobiosis. We show that the ozobranchid leech, Ozobranchus jantseanus, a parasite of freshwater turtles, has a surprisingly high tolerance to freezing and thawing. This finding is particularly interesting because the leach can survive these temperatures without any acclimation period or pretreatment. Specifically, the leech survived exposure to super-low temperatures by storage in liquid nitrogen (−196°C) for 24 hours, as well as long-term storage at temperatures as low as −90°C for up to 32 months. The leech was also capable of enduring repeated freeze-thaw cycles in the temperature range 20°C to −100°C and then back to 20°C. The results demonstrated that the novel cryotolerance mechanisms employed by O. jantseanus enable the leech to withstand a wider range of temperatures than those reported previously for cryobiotic organisms. We anticipate that the mechanism for the observed tolerance to freezing and thawing in O. jantseanus will prove useful for future studies of cryopreservation.     

Topical Application of Ice-Nucleating-Active Bacteria Decreases Insect Cold Tolerance

The majority of overwintering insects avoid lethal freezing by lowering the temperature at which ice spontaneously nucleates within their body fluids. We examined the effect of ice-nucleating-active bacteria on the cold-hardiness of the lady beetle, Hippodamia convergens, a freeze-intolerant species that overwinters by supercooling to ca. −16°C. Topical application of the ice-nucleating-active bacteria Pseudomonas syringae increased the supercooling point to temperatures as high as −3°C. This decrease in cold tolerance was maintained for at least 3 days after treatment. Various treatment doses (10⁸, 10⁶, and 10⁴ bacteria per ml) and modes of action (bacterial ingestion and topical application) were also compared. At the highest concentration of topically applied P. syringae, 50% of the beetles froze between −2 and −4°C. After topical application at the lowest concentration, 50% of the individuals froze by −11°C. In contrast, beetles fed bacteria at this concentration did not begin to freeze until −10°C, and 50% were frozen only at temperatures of −13°C or less. In addition to reducing the supercooling capacity in H. convergens, ice-nucleating-active bacteria also significantly reduced the cold-hardiness of four additional insects. These data demonstrate that ice-nucleating-active bacteria can be used to elevate the supercooling point and thereby decrease insect cold tolerance. The results of this study support the proposition that ice-nucleating-active bacteria may be used as a biological insecticide for the control of insect pests during the winter.     

The role of antioxidant defense in freezing tolerance of resurrection plant Haberlea rhodopensis

Haberlea rhodopensis Friv. is unique with its ability to survive two extreme environmental stresses—desiccation to air-dry state and subzero temperatures. In contrast to desiccation tolerance, the mechanisms of freezing tolerance of resurrection plants are scarcely investigated. In the present study, the role of antioxidant defense in the acquisition of cold acclimation and freezing tolerance in this resurrection plant was investigated comparing the results of two sets of experiments—short term freezing stress after cold acclimation in controlled conditions and long term freezing stress as a part of seasonal temperature fluctuations in an outdoor ex situ experiment. Significant enhancement in flavonoids and anthocyanin content was observed only as a result of freezing-induced desiccation. The total amount of polyphenols increased upon cold acclimation and it was similar to the control in post freezing stress and freezing-induced desiccation. The main role of phenylethanoid glucoside, myconoside and hispidulin 8-C-(2-O-syringoyl-b-glucopyranoside) in cold acclimation and freezing tolerance was elucidated. The treatments under controlled conditions in a growth chamber showed enhancement in antioxidant enzymes activity upon cold acclimation but it declined after subsequent exposure to −10 °C. Although it varied under ex situ conditions, the activity of antioxidant enzymes was high, indicating their important role in overcoming oxidative stress under all treatments. In addition, the activity of specific isoenzymes was upregulated as compared to the control plants, which could be more useful for stress counteraction compared to changes in the total enzyme activity, due to the action of these isoforms in the specific cellular compartments.Supplementary informationThe online version contains supplementary material available at 10.1007/s12298-021-00998-0.     

Changes in adenine nucleotides and energy charge in isolated winter wheat cells during low temperature stress

Adenylate energy charge (AEC) and adenine nucleotide levels of isolated winter wheat (Triticum aestivum L. cv Kharkov 22 MC) cells exposed to various low temperature stresses were determined. During ice encasement at −1°C, nucleotide levels decreased gradually in approximate relation to a decline in cell viability. AEC values remained high even after 5 weeks of icing when cell viability was severely reduced. When isolated cell suspensions were exposed to various cooling and freezing regimes ranging from −10 to −30°C, cell damage was dependent on the minimum temperature imposed and the duration of exposure to the freezing stress. The levels of all three adenine nucleotides declined with increasing severity of the imposed stress, but AEC values remained high even at −30°C when nearly all of the cells were killed. The addition of 10 millimolar Ca²⁺ to cell suspensions enhanced survival during low temperature stresses, but did not influence nucleotide levels other than through its effect on cell viability. These results indicate that impairment of the ion transport system during the early stages of ice encasement prior to a detectable decline in cell viability cannot be attributed to changes in the adenylate energy charge system of the cell.     

Effect of a Magnetic Field on Drosophila under Supercooled Conditions

Under subzero degree conditions, free water contained in biological cells tends to freeze and then most living things die due to low temperatures. We examined the effect of a variable magnetic field on Drosophila under supercooled conditions (a state in which freezing is not caused even below the freezing point). Under such supercooled conditions with the magnetic field at 0°C for 72 hours, −4°C for 24 hours and −8°C for 1 hour, the Drosophila all survived, while all conversely died under the supercooled conditions without the magnetic field. This result indicates a possibility that the magnetic field can reduce cell damage caused due to low temperatures in living things.     

Freezing Characteristics of Cultured Catharanthus roseus (L). G. Don Cells Treated with Dimethylsulfoxide and Sorbitol in Relation to Cryopreservation

The freezing behavior of dimethylsulfoxide (DMSO) and sorbitol solutions and periwinkle (Catharanthus roseus) cells treated with DMSO and sorbitol alone and in combination was examined by nuclear magnetic resonance and differential thermal analysis. Incorporation of DMSO or sorbitol into the liquid growth medium had a significant effect in the temperature range for initiation to completion of ice crystallization. Compared to the control, less water crystallized at temperatures below −30°C in DMSO-treated cells. Similar results were obtained with sorbitol-treated cells, except sorbitol had less effect on the amount of water crystallized at temperatures below −25°C. There was a close association between the per cent unfrozen water at −40°C and per cent cell survival after freezing for 1 hour in liquid nitrogen. It appears that, in periwinkle suspension cultures, the amount of liquid water at −40°C is critical for a successful cryopreservation. The combination of DMSO and sorbitol was the most effective in preventing water from freezing. The results obtained may explain the cryoprotective properties of DMSO and sorbitol and why DMSO and sorbitol in combination are more effective as cryoprotectants than when used alone.     

Survival of Clinical and Poultry-Derived Isolates of Campylobacter jejuni at a Low Temperature (4°C)

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in humans, and contamination of poultry has been implicated in illness. The bacteria are fastidious in terms of their temperature requirements, being unable to grow below ca. 31°C, but have been found to be physiologically active at lower temperatures and to tolerate exposure to low temperatures in a strain-dependent manner. In this study, 19 field isolates of C. jejuni (10 of clinical and 9 of poultry origin) were studied for their ability to tolerate prolonged exposure to low temperature (4°C). Although substantial variability was found among different strains, clinical isolates tended to be significantly more likely to remain viable following cold exposure than poultry-derived strains. In contrast, the relative degree of tolerance of the bacteria to freezing at −20°C and freeze-thawing was strain specific but independent of strain source (poultry versus clinical) and degree of cold (4°C) tolerance.     

Effect of cold acclimation on the incidence of two forms of freezing injury in protoplasts isolated from rye leaves

The freezing tolerance and incidence of two forms of freezing injury (expansion-induced lysis and loss of osmotic responsiveness) were determined for protoplasts isolated from rye leaves (Secale cereale L. cv Puma) at various times during cold acclimation. During the first 4 weeks of the cold acclimation period, the LT₅₀ (i.e. the minimum temperature at which 50% of the protoplasts survived) decreased from −5°C to −25°C. In protoplasts isolated from nonacclimated leaves (NA protoplasts), expansion-induced lysis (EIL) was the predominant form of injury at the LT₅₀. However, after only 1 week of cold acclimation, the incidence of EIL was reduced to less than 10% at any subzero temperature; and loss of osmotic responsiveness was the predominant form of injury, regardless of the freezing temperature. Fusion of either NA protoplasts or protoplasts isolated from leaves of seedlings cold acclimated for 1 week (1-week ACC protoplasts) with liposomes of dilinoleoylphosphatidylcholine also decreased the incidence of EIL to less than 10%. Fusion of protoplasts with dilinoleoylphosphatidylcholine diminished the incidence of loss of osmotic responsiveness, but only in NA protoplasts or 1-week ACC protoplasts that were frozen to temperatures over the range of -5 to -10°C. These results suggest that the cold acclimation process, which results in a quantitative increase in freezing resistance, involves several different qualitative changes in the cryobehavior of the plasma membrane.