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A synthetic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), was studied by differential scanning calorimetry at different hydration levels. A glass transition was observed in the lamellar gel (Lβ) phase but was not detected in a more ordered lamellar crystalline phase. The glassy state of the Lβ phase of POPE has properties that are typical of other glass-forming materials, i.e., nonexponential relaxation behavior below the glass transition temperature, a broad distribution of relaxation times, and plasticization by water. The significance of the glass transition in biological membranes is discussed.

Sugar content and freezing tolerance of protoplasts of Arabidopsis thaliana leaves were manipulated by incubating seedlings in a sucrose solution before protoplast isolation. Incubation in a 400 mM sucrose solution at 2 °C in the dark increased their freezing tolerance equivalent to that achieved after a conventional cold acclimation at 2 °C. The increased freezing tolerance was due to a decrease in the incidence of freeze-induced lesions: expansion-induced lysis (EIL) between −2 and −4 °C and loss of osmotic responsiveness (LOR) between −5 and −12 °C. The concentration of sucrose in the incubation medium required to minimize the incidence of the lesions was substantially different: 10–35 mM for EIL and 30–400 mM for LOR. Incubation in the sucrose solution at 23 °C decreased LOR only at −5 and −6 °C but less than that incubated at 2 °C, and there was no effect on EIL. Incubation in sorbitol solutions at 2 °C also decreased LOR at −5 and −6 °C but much less than in the sucrose solution. These results suggest that low concentrations of sucrose act as a metabolic substrate for the low-temperature-induced alterations required for the amelioration of EIL and, at higher concentrations, sucrose has a direct cryoprotective effect to minimize LOR.

Protoplasts were tested to determine whether the freezing sensitivity of the sfr4 (sensitive to freezing) mutant of Arabidopsis was due to the mutant's deficiency in soluble sugars after cold acclimation. When grown under nonacclimated conditions,sfr4 protoplasts possessed freezing tolerance similar to that of wild type, with the temperature at which 50% of protoplasts are injured (LT50) of −4.5°C. In both wild-type andsfr4 protoplasts, expansion-induced lysis was the predominant lesion between −2°C and −4°C, but its incidence was low (approximately 10%); below −5°C, loss of osmotic responsiveness (LOR) was the predominant lesion. After cold acclimation, the LT50 was decreased to only −5.6°C forsfr4 protoplasts, compared with −9.1°C for wild-type protoplasts. Although expansion-induced lysis was precluded in both types of protoplasts, the sfr4 protoplasts remained susceptible to LOR. After incubation of seedlings in Suc solution in the dark at 2°C, freezing tolerance and the incidence of freeze-induced lesions in sfr4 protoplasts were examined. The freezing tolerance of isolated protoplasts (LT50 of −9°C) and the incidence of LOR were now similar for wild type and sfr4. These results indicate that the freezing sensitivity of cold-acclimated sfr4 is due to its continued susceptibility to LOR (associated with lyotropic formation of the hexagonal II phase) and associated with the low sugar content of its cells.

The effect of sucrose on the phase behavior of 1,2-dioleoylphosphatidylethanolamine (DOPE) as a function of hydration was studied using differential scanning calorimetry and X-ray diffraction. DOPE/sucrose/water dispersions were dehydrated at osmotic pressures (Pi) ranging from 2 to 300 MPa at 30 degrees C and 0 degrees C. The hexagonal II-to-lamellar gel (H(II)--L(beta)) thermotropic phase transition was observed during cooling in mixtures dehydrated at Pior=35 MPa. After dehydration at Pior=57 MPa, the H(II)--L(beta) thermotropic phase transition was precluded when sucrose entered the rigid glassy state while the lipid was in the H(II) phase. Sucrose also hindered the H(II)-to-lamellar crystalline (L(c)), and H(II)-to-inverted ribbon (P(delta)) lyotropic phase transitions, which occurred in pure DOPE. Although the L(c) phase was observed in dehydrated 2:1 (mole ratio) DOPE/sucrose mixtures, it did not form in mixtures with higher sucrose contents (1:1 and 1:2 mixtures). The impact of sucrose on formation of the ordered phases (i.e., the L(c), L(beta), and P(delta) phases) of DOPE was explained as a trapping of DOPE in a metastable H(II) phase due to increased viscosity of the sucrose matrix. In addition, a glass transition of DOPE in the H(II) phase was observed, which we believe is the first report of a glass transition in phospholipids.

The glass transition of sucrose in 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE)/sucrose mixtures was studied by differential scanning calorimetry. Mole ratios of 1:2, 1:1, and 2:1 DOPE/sucrose mixtures were dehydrated at osmotic pressures ranging from 41 to 311 MPa through vapor-phase equilibration. Two glass transitions were observed in 1:2 DOPE/sucrose mixtures that were dehydrated at osmotic pressures >81 MPa. The first glass transition temperature (Tg) was lower than that of pure sucrose solutions that were dehydrated under identical conditions. The second Tg of the 1:2 DOPE/sucrose mixture was similar to the Tg of sucrose solutions that were dehydrated in the absence of DOPE. In the 2:1 and 1:1 DOPE/sucrose mixtures, only one Tg was observed at a temperature lower than that of pure sucrose. We propose that depression of the Tg of sucrose in the DOPE/sucrose mixtures is the result of a confinement effect that occurs in the sucrose solution that is sequestered in the aqueous cores of the h...

The phase behavior of partially hydrated 1, 2-dioleoylphosphatidylethanolamine (DOPE) has been studied using differential scanning calorimetry and X-ray diffraction methods together with water sorption isotherms. DOPE liposomes were dehydrated in the H(II) phase at 29 degrees C and in the L(alpha) phase at 0 degrees C by vapor phase equilibration over saturated salt solutions. Other samples were prepared by hydration of dried DOPE by vapor phase equilibration at 29 degrees C and 0 degrees C. Five lipid phases (lamellar liquid crystalline, L(alpha); lamellar gel, L(beta); inverted hexagonal, H(II); inverted ribbon, P(delta); and lamellar crystalline, L(c)) and the ice phase were observed depending on the water content and temperature. The ice phase did not form in DOPE suspensions containing9 wt% water. The L(c) phase was observed in samples with a water content of 2-6 wt% that were annealed at 0 degrees C for 2 or more days. The L(c) phase melted at 5-20 degrees C producing the H(II) phase. The P(delta) phase was observed at water contents of0.5 wt%. The phase diagram, which includes five lipid phases and two water phases (ice and liquid water), has been constructed. The freeze-induced dehydration of DOPE has been described with the aid of the phase diagram.

The lipid composition of the inner and outer membranes of the chloroplast envelope isolated from winter rye (Secale cereale L. cv Puma) leaves was characterized before and after cold acclimation. In nonacclimated leaves the inner membrane contained high proportions of monogalactosyldiacylglycerols (MGDG, 47.9 mol% of the total lipids) and digalactosyldiacylglycerols (DGDG, 31.1 mol%) and a low proportion of phosphatidylcholine (PC, 8.1 mol%). The outer membrane contained a similar proportion of DGDG (30.0 mol%); however, the proportion of MGDG was much lower (20.1 mol%) and the proportion of PC was much higher (31.5 mol%). After 4 weeks of cold acclimation, the proportions of these lipid classes were significantly altered in both of the inner and outer membranes. In the inner membrane the proportion of MGDG decreased (from 47.9 to 38.4 mol%) and the proportion of DGDG increased (from 31.1 to 39.3 mol%), with only a slight change in the proportion of PC (from 8.1 to 8.8 mol%). In the outer membrane MGDG decreased from 20.1 to 14.8 mol%, DGDG increased from 30.0 to 39.9 mol%, and PC decreased from 31.5 to 25.4 mol%. Thus, both before and after cold acclimation, the proportion of MGDG was much higher in the inner membrane than in the outer membrane. In contrast, the proportion of PC was higher in the outer membrane than in the inner membrane. The relationship between the lipid composition of the inner and outer membranes of the chloroplast envelope and freeze-induced membrane lesions is discussed.

Although cryopreservation is routinely used for the storage of a range of biological organisms, few studies have been conducted to determine whether cryopreservation increases the frequency of mutation. A procedure for the cryopreservation of Drosophila melanogaster embryos has recently been developed. Cryopreservation of D. melanogaster is of special interest to geneticists and evolutionary biologists because it would make it possible to assay control and experimental populations simultaneously during long-term studies. Before cryopreserved embryos can be used for such studies, it is first necessary to show that cryopreservation is not mutagenic. We tested for mutagenic effects of cryopreservation in D. melanogaster embryos with an X-linked, recessive lethal assay. The mutation rates of cryopreserved and control flies were not significantly different. We can be 95% certain that cryopreservation does not increase mutation by a factor greater than 2·39. This is the first quantitative estimate of the mutagenic effect of cryopreservation on the germ line of a metazoan. The results are reassuring when considering the genetic impact of cryopreservation on mammalian gametes and embryos.

Freezing injury in rye and oat is a consequence of the formation of the inverted hexagonal (H(II)) phase in regions where the plasma membrane is brought into close proximity with cytoplasmic membranes during freeze-induced dehydration. Susceptibility to plasma membrane destabilization and H(II) phase formation during freezing is associated with alterations in plasma membrane lipid composition. This paper examines the influence of lipid composition and hydration on the propensity of lipid mixtures of DOPE:DOPC and DOPE:DOPC:sterols with added cerebrosides (CER) to form the H(II) phase during dehydration. The addition of CER to DOPE:DOPC:beta-sitosterol mixtures decreased the water content of the dispersions in a manner suggesting that most or all of the water in the dehydrated mixtures was associated with the phospholipids. The addition of CER significantly decreased the osmotic pressure at which the L(alpha) --H(II) phase transition occurred from an osmotic pressure of 76.1 MPa for DOPE:DOPC (50:50) to 20 MPa in DOPE:DOPC:beta-sitosterol:CER (22.5:22.5:50:5) and 8 MPa in DOPE:DOPC:beta-sitosterol:CER (15:15:50:20). Experiments examining the effects of CER on the thermally-induced formation of the H(II) phase in fully hydrated mixtures and examining the influence of CER on the formation of the H(II) phase in DOPE:DOPC mixtures lacking beta-sitosterol suggested that CER facilitated the L(alpha) --H(II) phase transition by effecting a decrease in bilayer hydration and by increased lateral packing pressures within the acyl domain of the bilayer. Taken in sum, these data indicate that the differential propensity of the rye and oat plasma membranes to undergo freeze-induced formation of the L(alpha) --H(II) phase cannot be attributed to one lipid species. Rather, the propensity towards freeze-induced membrane destabilization is a consequence of the summation of physical characteristics of the membrane lipid components that included bilayer hydration, packing pressures within the hydrophobic domain of the membrane, the propensity of the lipid components to demix, and the relative proportions of the various lipid components.

Several cold-regulated (COR) polypeptides, which have little or no amino acid sequence identity with known proteins, are synthesized during cold acclimation of Arabidopsis thaliana. However, the function of the polypeptides has yet to be elucidated. The objective of this study was to determine if COR6.6 and COR15am influence the incidence of either freeze-induced fusion or freeze-induced leakage of small unilamellar vesicles (SUVs) composed of either a single species of phosphatidylcholine (either 1-palmitoyl-2-oleoyl-, dioleoyl-, or dilinoleoylphosphatidylcholine), a mixture of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, and free sterols (1:1:1, mol:mol), or the total lipid extract of the plasma membrane of either nonacclimated or cold-acclimated rye leaves. When the SUVs were suspended in a dilute tris(hydroxymethyl)-aminomethane/2-(N-morpholino)ethanesulfonic acid buffer, both COR6.6 and COR15am invariably decreased the incidence of freeze-induced fusion regardless of the lipid composition. However, if the SUVs were suspended in a dilute solution of either sucrose or NaCl, the COR polypeptides had little or no effect on the incidence of freeze-induced fusion. Moreover, the COR polypeptides did not decrease the incidence of freeze-induced leakage[mdash]regardless of whether the SUVs were suspended in either the dilute buffer alone or with added sucrose or NaCl. In fact, with SUVs composed of a single species of phosphatidylcholine suspended in the dilute buffer, the COR polypeptides resulted in an anomalous increase in freeze-induced leakage. When considered collectively, these results suggest that neither COR6.6 nor COR15am has a direct cryoprotective effect on SUVs frozen in vitro.

Freeze-induced injury of protoplasts of non-acclimated rye and oat is associated with the formation of the inverted hexagonal (HII) phase in regions where the plasma membrane and various endomembranes are brought into close apposition as a result of freeze-induced dehydration. The influence of lipid composition and hydration on the propensity of mixtures of DOPE:DOPC containing either sterols or acylated steryl glucosides to form the HII phase was determined by DSC, freeze-fracture electron microscopy and X-ray diffraction. The addition of plant sterols to a mixture of DOPE/DOPC (either 1:1:1 or 1:1:2 mole ratio of DOPE/DOPC/sterols) reduced the total hydration of the mixture (expressed as wt% water) after desorption over a range of osmotic pressures of 2.8 to 286 MPa. However, most or all of the water remaining in the dehydrated lipid mixtures was associated predominantly with the phospholipids. Both sterols and acylated steryl glucosides significantly promoted both the dehydration-induced and thermally induced L alpha--HII phase transitions in DOPE/DOPC mixtures however, acylated steryl glucosides were much more effective. In mixtures containing plant sterols, the HII phase occurred after dehydration at 20 MPa (20 degrees C), which resulted in a water content of 11.7 wt%. In contrast, mixtures containing acylated steryl glucosides were in the HII phase in excess water, i.e., they did not require dehydration to effect the L alpha--HII phase transition. The results indicate that genotypic differences in the lipid composition of the plasma membrane of rye and oat leaves have a significant influence on the propensity for formation of the HII phase during freeze-induced dehydration.

Ice formation in concentrated solutions of ethylene glycol (EG; 48 to 53 wt%) was studied after various thermal histories. To characterize the rate of ice nucleation as a function of temperature (to - 150°C), the solutions were cooled at a constant rate to the nadir temperature and then warmed to a storage temperature (Ts) at which ice formation was studied under isothermal conditions using differential scanning calorimetry (DSC) and cryomicroscopy. The DSC data were analyzed using the Johnson-Mehl-Avrami (JMA) model of isothermal transformation. The model contains two parameters: a kinetic constant K and a reaction-order parameter n. The kinetic constant was used to quantify the relative stability of the amorphous vs frozen state and rates of ice nucleation and growth in highly supercooled solutions of EG. Our theoretical analysis suggests that the kinetic constant is proportional to the diffusion coefficient D of water molecules and to the 23 power of the concentration of ice nuclei that form in a solution prior to storage at Ts. Experimentally, the kinetic constant is shown to be the product of two values, the first of which is dependent on the nadir temperature and the second on storage temperature. Increases in the kinetic constant corresponding to two nucleation events occurred with decreasing nadir temperature. During cooling the second ice nucleation event began at a temperature approximately 20°C lower than that at which the first nucleation event was completed. For example, in solutions containing 50 to 51.5 wt% EG, the first nucleation event occurred at temperatures between -80 and -100°C and the second nucleation event occurred at temperatures between 120 and - 135°C, which is in the vicinity of the glass formation temperature (Tg). Cryomicroscopic measurements of the spherulite concentration confirmed the appearance of the first and second nucleation events in EG solutions. DSC studies showed that the total amount of nucleation increased approximately 20-fold if the concentration of EG was decreased from 51 to 48 wt%. Only about 10% of this increase was associated with the first nucleation event and the remainder was the result of the second nucleation event. Furthermore, the proportion of nucleation attributable to the first and second nucleation events was dependent on the EG concentration. At concentrations ⩾51 wt% EG, the majority of ice nuclei formed during the first nucleation event. However, in a 48 wt% EG solution the kinetic constant measured after cooling to Tn ⩾ -100°C, when only the first nucleation event occurred, was about 16 times smaller than that measured after cooling to Tn = - 150°C, when both the first and second nucleation events occurred. For this reason, the second nucleation event is the principal cause of the decrease in stability of the amorphous state in EG solutions in which the concentration is decreased from 51 to 48 wt%. Therefore, the minimum concentration of cryoprotectant needed for vitrification at a given cooling and warming rate can be substantially reduced if the rate of ice nucleation can be decreased. Addition of bovine serum albumin (BSA) decreased the value of the kinetic constant determined for concentrated solutions of EG; however, the effect of BSA was small in comparison to that which could be effected by a small increase in the concentration of EG. For example, the decrease in the nucleation parameter, which was affected by addition of 6 g of BSA to 100 g of a 48 wt% EG solution, could also be achieved by increasing the EG concentration by 0.4 wt%.

Freezing injury in protoplasts isolated from leaves of nonaccli-mated rye (Secale cereale cv Puma) is associated with the formation of the inverted hexagonal (HII) phase. However, in protoplasts from cold-acclimated rye, injury is associated with the occurrence of localized deviations in the fracture plane, a lesion referred to as the "fracture-jump lesion." To establish that these ultrastructural consequences of freezing are not unique to protoplasts, we have examined the manifestations of freezing injury in leaves of non-acclimated and cold-acclimated rye by freeze-fracture electron microscopy. At -10[deg]C, injury in nonacclimated leaves was manifested by the appearance of aparticulate domains in the plasma membrane, aparticulate lamellae subtending the plasma membrane, and by the frequent occurrence of the HII phase. The HII phase was not observed in leaves of cold-acclimated rye frozen to -35[deg]C. Rather, injury was associated with the occurrence of the fracture-jump lesion between the plasma membrane and closely appressed cytoplasmic membranes. Studies of the time dependence of HII phase formation in nonacclimated leaves indicated that freeze-induced dehydration requires longer times in leaves than in isolated protoplasts. These results demonstrate that the freeze-induced formation of the HII phase in nonacclimated rye and the fracture-jump lesion in cold-acclimated rye are not unique to protoplasts but also occur in the leaves from which the protoplasts are isolated.

Several phosphatidylcholine (PC) species were studied by differential scanning calorimetry at different levels of hydration. A glass transition was observed in both lamellar gel and nonlamellar phases, with the glass transition temperature, T(g), decreasing as water content was increased. The structure of the lipid mesophase has a major impact on T(g), with the lamellar gel phase having a higher T(g) than that of nonlamellar phases of the same lipid. While the headgroup has a noticeable influence on the T(g), changing the chain length, on the other hand, has less of an impact. The values of the calorimteric T(g) were compared with other measures of molecular mobility in the PC species at comparable water contents reported in the literature. Observation of a T(g) in different phosphatidylethanolamines (PE), as previously reported, and PC species in this study suggests that a glass transition can be expected to be a common feature of biological membranes and phospholipid bilayer preparations, such as liposomes.

The weight of a DSC sample is routinely determined as the difference between the weight of the pan containing the sample and the empty pan. If the sample is sensitive to atmospheric moisture, weighing is usually performed after sealing the pan. However, we have observed that sealing induced changes in the measured weight of the pan when aluminum DSC pans were hermetically sealed with a Perkin-Elmer Volatile Sealer Assembly. The difference between the weight of sealed and non-sealed DSC pans was about 10 μg and independent of the presence of a sample in the DSC pan. Correction for this difference is important in applications that require precise weights of DSC samples.

Cryomicroscopic observations were made of the volumetric behavior and kinetics of intracellular ice formation (IIF) in Drosophila melanogaster embryos in a modified cell culture medium (BD.20) or BD.20 + 2 M ethylene glycol. After rapid cooling to a given temperature, transient volumetric contraction of the embryos during the isothermal period was quantified by computerized video image analysis. Fitting these data to the numerical solution of the volume flux equation yielded estimates of the hydraulic permeability coefficient (Lp) for individual embryos at various subfreezing temperatures. Lp approximately followed an Arrhenius relation between -2 and -9 degrees C, with a value of 0.168 microns/(min-atm) extrapolated to 0 degrees C and an apparent activation energy delta E of 38.9 kcal/mol. IIF during an isothermal period occurred at random times whose characteristic temperature range and kinetics were affected by the presence of ethylene glycol. A stochastic process model developed to fit these data indicated the influence of both time-dependent and instantaneous components of IIF, presumed to be the result of seeding and heterogeneous nucleation, respectively. The presence of 2 M ethylene glycol depressed the characteristic temperature of instantaneous IIF by about 12 degrees C and reduced the rate constant for time-dependent IIF. Comparison with observed incidences of IIF yielded an estimate of the supercooling tolerance of 3 to 5 degrees C.

A procedure has been developed for the vitrification of mesophyll protoplasts isolated from leaves of nonacclimated (NA) and cold-acclimated (ACC) winter rye seedlings (Secale cereale L. cv Puma). The procedure involves (a) equilibration (loading) of the protoplasts with an intermediate concentration (1.5, 1.75, or 2.0 molar) of ethylene glycol (EG) at 20 degrees C; (b) dehydration of the protoplasts in a concentrated vitrification solution made of 7 molar EG + 0.88 molar sorbitol + 6% (w/v) bovine serum albumin (BSA) at 0 degrees C; (c) placing the protoplasts into polypropylene straws and quenching in liquid nitrogen (LN(2)); and (d) recovery of the protoplasts from LN(2) and removal (unloading) of the vitrification solution. For NA protoplasts, 47 + 1% survival was obtained following recovery from LN(2) if the protoplasts were first loaded with 1.75 molar EG prior to the dehydration step. However, to achieve this level of survival, NA protoplasts had to be unloaded in a hypertonic (2.0 osmolal [osm]) sorbitol solution. If they were unloaded in an isotonic solution (0.53 osm), survival was 3+/-2%. In contrast, survival of ACC protoplasts following recovery from LN(2) was 34 +/- 10% when the protoplasts were loaded in a 2.0 molar EG solution and unloaded in an isotonic sorbitol solution (1.03 osm). If ACC protoplasts were unloaded in an hypertonic sorbitol solution (1.5 osm), survival was 51 +/- 9%. These results indicate that the osmotic excursions incurred during the procedure are a major factor affecting survival.

Destabilization of the plasma membrane, which is a primary cause of freezing injury, is a consequence of freeze-induced osmotic stresses and cell dehydration. However, the mechanism of injury depends on the magnitude of the osmotic stress and the extent of cell dehydration. Over the range of 0 to -5 $^\circ$ C, destabilization of the plasma membrane in protoplasts isolated from non-acclimated rye leaves is a result of osmotic excursions, because freeze-induced osmotic contraction results in endocytotic vesiculation of the plasma membrane and sufficiently large area reductions are irreversible. At lower temperatures, the protoplasts are subjected to extremely large osmotic pressures (-12 MPa at -10 $^\circ$ C), and there are several changes in the ultrastructure of the plasma membrane, including the formation of aparticulate domains and lamellar- to hexagonal $\_{II}$ -phase transitions. These changes, which are manifestations of demixing of the membrane components, are predicted by a theory of bilayer interactions at low levels of hydration. During cold acclimation, the cryobehaviour of the plasma membrane is altered; osmotic contraction results in the reversible formation of exocytotic extrusions and the propensity for dehydration-induced demixing and lamellar- to hexagonal $\_{II}$ -phase transitions is decreased. In both cases, the differential behaviour is also observed in liposomes prepared from plasma membrane lipids isolated from non-acclimated and cold-acclimated leaves. However, as no lipid species are unique to the plasma membrane of either non-acclimated or cold-acclimated leaves, the differential behaviour is caused by altered lipid-lipid interactions because of different proportions of the lipid species. Hence the behaviour of the plasma membrane can be altered by using a protoplast-liposome fusion procedure to selectively modify the lipid composition of the plasma membrane. These studies provide direct evidence that the increased cryostability of the plasma membrane is a consequence of alterations in its lipid composition.

There is an urgent need to preserve the ever-increasing number (greater than 30,000) of different genetic strains of D. melanogaster that are maintained in national and international stock centres and in the laboratories of individual investigators. In all cases, the stocks are maintained as adult populations and require transfer to fresh medium every two to four weeks. This is not only costly in terms of materials, labour and space, but unique strains are vulnerable to accidental loss, contamination, and changes in genotype that can occur during continuous culture through mutation, genetic drift or selection. Although cryopreservation of Drosophila germ-plasm would be an enormous advantage, many attempts using conventional procedures have been unsuccessful. D. melanogaster embryos are refractory to conventional cryopreservation procedures because of the contravening conditions required to minimize mortality resulting from both intracellular ice formation and chilling injury at subzero temperatures. To overcome these obstacles, we have developed a vitrification procedure that precludes intracellular ice formation so that the embryos can be cooled and warmed at ultra-rapid rates to minimize chilling injury, and have recovered viable embryos following storage in liquid nitrogen. In a series of 53 experiments, a total of 3,711 larvae emerged from 17,280 eggs that were cooled in liquid nitrogen (18.4 +/- 8.8%). Further, using a subset from this population, approximately 3% of the surviving larvae (24/800) developed into adults. These adults were fertile and produced an F1 generation.

Constitutive expression of the co ld- r egulated COR15a gene of Arabidopsis thaliana results in a significant increase in the survival of isolated protoplasts frozen over the range of −4.5 to −7°C. The increased freezing tolerance is the result of a decreased incidence of freeze-induced lamellar-to-hexagonal II phase transitions that occur in regions where the plasma membrane is brought into close apposition with the chloroplast envelope as a result of freeze-induced dehydration. Moreover, the mature polypeptide encoded by this gene, COR15am, increases the lamellar-to-hexagonal II phase transition temperature of dioleoylphosphatidylethanolamine and promotes formation of the lamellar phase in a lipid mixture composed of the major lipid species that comprise the chloroplast envelope. We propose that COR15am, which is located in the chloroplast stroma, defers freeze-induced formation of the hexagonal II phase to lower temperatures (lower hydrations) by altering the intrinsic curvature of the inner membrane of the chloroplast envelope.

Freezing injury is primarily a consequence of membrane destabilization resulting from freeze-induced dehydration (Steponkus, 1984). Although all cellular membranes are vulnerable to freeze-induced destabilization, the plasma membrane is of primary importance because of the critical role it plays during a freeze/thaw cycle. The plasma membrane is the principal interface between the extracellular medium and the cytoplasm and acts as a semipermeable barrier allowing for the efflux/influx of water during a freeze/ thaw cycle. In addition, the plasma membrane prevents seeding of the intracellular solution by extracellular ice. Thus, whether the cell survives during a freeze/thaw cycle is ultimately a consequence of the stability of the plasma membrane.

Cold acclimation in plants is associated with the expression of COR (cold-regulated) genes that encode polypeptides of unknown function. It has been widely speculated that products of these genes might have roles in freezing tolerance. Here we provide direct evidence in support of this hypothesis. We show that constitutive expression of COR15a , a cold-regulated gene of Arabidopsis thaliana that encodes a chloroplast-targeted polypeptide, enhances the in vivo freezing tolerance of chloroplasts in nonacclimated plants by almost 2°C, nearly one-third of the increase that occurs upon cold acclimation of wild-type plants. Significantly, constitutive expression of COR15a also affects the in vitro freezing tolerance of protoplasts. At temperatures between −5 and −8°C, the survival of protoplasts isolated from leaves of nonacclimated transgenic plants expressing COR15a was greater than that of protoplasts isolated from leaves of nonacclimated wild-type plants. At temperatures between −2 and −4°C, constitutive expression of COR15a had a slight negative effect on survival. The implications of these data regarding possible modes of COR15a action are discussed.

Cold acclimation of Arabidopsis thaliana includes the expression of cold-regulated (COR) genes and the accumulation of COR polypeptides. The hydration characteristics of two COR polypeptides, COR6.6 and COR15am, have been determined and their effects on the dehydration-induced liquid crystalline-to-gel and lamellar-to-hexagonal II phase transitions in phospholipid mixtures have been examined. After dehydration at osmotic pressures between 8 and 150 MPa, the water content of the COR polypeptides was less than that of bovine serum albumin, with COR15am the least hydrated: bovine serum albumin > COR6.6 > COR15am. Neither COR6.6 nor COR15am altered the dehydration-induced gel lamellar -> fluid lamellar phase transition temperature of either dipalmitoylphosphatidylcholine or dioleoylphosphatidylcholine (DOPC). In multilamellar vesicles of dioleoylphosphatidylethanolamine:DOPC (1:1, mol:mol) prepared by either freeze-thaw or reverse-phase evaporation methods, neither COR6.6, COR15am, nor bovine serum albumin altered the incidence of the dehydration-induced formation of the inverted hexagonal phase as a function of osmotic pressure. However, a specific ultrastructural alteration[mdash] the formation of a striated surface morphology in the lamellar domains[mdash]was observed in mixtures of dioleoylphosphatidylethanolamine:DOPC that were dehydrated in the presence of COR15am. Nevertheless, neither COR6.6 nor COR15am appears to participate in a specific protein-phospholipid interaction that alters the dehydration-induced phase behavior of phospholipid vesicles.

Arabidospis thaliana alters gene expression and increases in freezing tolerance in response to low non-freezing temperatures. One of our long range goals has been to determine whether certain COR (cold-regulated) genes that encode hydrophilic, “boiling-soluble” polypeptides have a role in freezing tolerance; another has been to determine the mechanism(s) by which A. thaliana senses low temperature and alters gene expression. Most of our work on COR gene function has focused on COR15. We have found that this gene encodes a 15 kD polypeptide that is targeted to the stromal compartment of chloroplasts. During import, the polypeptide is processed to a hydrophilic 9 kD polypeptide, designated COR15am, that shares a low degree of amino acid sequence similarity with group III LEA (late-embryogenesis-abundant) proteins Transgenic A. thaliana plants that constitutively produce COR15am have been created and tested for freezing tolerance. The data indicate that COR15am enhances the freezing tolerance of chloroplasts in non-acclimated plants by abundant 2°C, nearly one-third of the increase that occurs upon cold acclimation of both transgenic and wild type plants. Constitutive expression of COR15a was also found to affect freezing tolerance at the cellular level: at freezing temperatures between -6 and -8 °C, the survival of protoplasts isolated from leaves of non-acclimated plants expressing CORl5a was greater than that of protoplasts isolated from leaves of plants not expressing CORl5a. The implications of these data regarding possible COR15a functions are discussed. In regarding to gene regulation, the promoters of COR15a and COR78 were found to be up-regulated in response to low temperature, drought and ABA. An examination of COR gene expression in the A. thaliana abi (ABA-insensitive) mutants indicated that low temperature and ABA regulation involve independent pathways. Gene fusion experiments established that COR15a has a functional DRE (drought-regulatory-element) that imparts cold- and drought-regulated gene expression in tobacco. Gel retardation studies indicated that nuclear extracts prepared from both cold-acclimated and non-acclimated Arabidopsis plants contain a protein(s) that binds to the COR15a DRE and that the avidity of the protein(s) for the element is not significantly altered with cold acclimation.

Maximum freezing tolerance of Arabidopsis thaliana L. Heyn (Columbia) was attained after 1 week of cold acclimation at 2[deg]C. During this time, there were significant changes in both the lipid composition of the plasma membrane and the freeze-induced lesions that were associated with injury. The proportion of phospholipids increased from 46.8 to 57.1 mol% of the total lipids with little change in the proportions of the phospholipid classes. Although the proportion of di-unsaturated species of phosphatidylcholine and phosphatidylethanolamine increased, mono-unsaturated species were still the preponderant species. The proportion of cerebrosides decreased from 7.3 to 4.3 mol% with only small changes in the proportions of the various molecular species. The proportion of free sterols decreased from 37.7 to 31.2 mol%, but there were only small changes in the proportions of sterylglucosides and acylated sterylglucosides. Freezing tolerance of protoplasts isolated from either nonacclimated or cold-acclimated leaves was similar to that of leaves from which the protoplasts were isolated (-3.5[deg]C for nonacclimated leaves; -10[deg]C for cold-acclimated leaves). In protoplasts isolated from nonacclimated leaves, the incidence of expansion-induced lysis was [less than or equal to]10% at any subzero temperature. Instead, freezing injury was associated with formation of the hexagonal II phase in the plasma membrane and subtending lamellae. In protoplasts isolated from cold-acclimated leaves, neither expansion-induced lysis nor freeze-induced formation of the hexagonal II phase occurred. Instead, injury was associated with the "fracture-jump lesion," which is manifested as localized deviations of the plasma membrane fracture plane to subtending lamellae. The relationship between the freeze-induced lesions and alterations in the lipid composition of the plasma membrane during cold acclimation is discussed.

A detailed analysis of cold acclimation of a winter rye (Secale cereale L. cv Puma), a winter oat (Avena sativa L. cv Kanota), and a spring oat cultivar (Ogle) revealed that freezing injury of leaves of nonacclimated seedlings occurred at -2[deg]C in both the winter and spring cultivars of oat but did not occur in winter rye leaves until after freezing at -4[deg]C. The maximum freezing tolerance was attained in all cultivars after 4 weeks of cold acclimation, and the temperature at which 50% electrolyte leakage occurred decreased to -8[deg]C for spring oat, -10[deg]C for winter oat, and -21[deg]C for winter rye. In protoplasts isolated from leaves of nonacclimated spring oat, expansion-induced lysis was the predominant form of injury over the range of -2 to -4[deg]C. At temperatures lower than -4[deg]C, loss of osmotic responsiveness, which was associated with the formation of the hexagonal II phase in the plasma membrane and subtending lamellae, was the predominant form of injury. In protoplasts isolated from leaves of cold-acclimated oat, loss of osmotic responsiveness was the predominant form of injury at all injurious temperatures; however, the hexagonal II phase was not observed. Rather, injury was associated with the occurrence of localized deviations of the plasma membrane fracture plane to closely appressed lamellae, which we refer to as the "fracture-jump lesion." Although the freeze-induced lesions in the plasma membrane of protoplasts of spring oat were identical with those reported previously for protoplasts of winter rye, they occurred at significantly higher temperatures that correspond to the lethal freezing temperature.

The lipid composition of the plasma membrane isolated from leaves of spring oat (Avena sativa L. cv Ogle) was vastly different from that of winter rye (Secale cereale L. cv Puma). The plasma membrane of spring oat contained large proportions of phospholipids (28.8 mol% of the total lipids), cerebrosides (27.2 mol%), and acylated sterylglucosides (27.3 mol%) with lesser proportions of free sterols (8.4 mol%) and sterylglucosides (5.6 mol%). In contrast, the plasma membrane of winter rye contained a greater proportion of phospholipids (36.6 mol%), and there was a lower proportion of cerebrosides (16.4 mol%); free sterols (38.1 mol%) were the predominant sterols, with lesser proportions of sterylglucosides (5.6 mol%) and acylated sterylglucosides (2.9 mol%). Although the relative proportions of individual phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine, and the molecular species of these two phospholipids were similar in oat and rye, the relative proportions of di-unsaturated species of these two phospholipids were substantially lower in oat than in rye. The relative proportions of sterol species in oat were different from those in rye; the molecular species of cerebrosides were similar in oat and rye, with only slight differences in the proportions of the individual species. After 4 weeks of cold acclimation, the proportion of phospholipids increased significantly in both oat (from 28.8 to 36.8 mol%) and rye (from 36.6 to 43.3 mol%) as a result of increases in the proportions of phosphatidylcholine and phosphatidylethanolamine. For both oat and rye, the relative proportions of di-unsaturated species increased after cold acclimation, but the increase was greater in rye than in oat. In both oat and rye, this increase occurred largely during the first week of cold acclimation. During the 4 weeks of cold acclimation, there was a progressive decrease in the proportion of cerebrosides in the plasma membrane of rye (from 16.4 to 10.5 mol%), but there was only a small decrease in oat (from 27.2 to 24.2 mol%). In both oat and rye, there were only small changes in the proportions of free sterols and sterol derivatives during cold acclimation. Consequently, the proportions of both acylated sterylglucosides and cerebrosides remained substantially higher in oat than in rye after cold acclimation. The relationship between these differences in the plasma membrane lipid composition of oat and rye and their freezing tolerance is presented.

Bovine oocytes that were immature (IMM), matured in vitro (IVM) or in vivo (MAT), or matured and fertilized in vitro (IVF) were studied using a microscope diffusion chamber to estimate osmotic parameters and a cryomicroscope to characterize intracellular ice formation (IIF). Linear Boyle van't Hoff relationships were observed with all four types of oocytes between 0.265 and 0.799 osm NaCl. At 20 degrees C, estimates of hydraulic conductivity (Lp) were significantly higher for IVM oocytes than IMM and MAT oocytes (0.84 micron/(min.atm) vs 0.45 and 0.47, respectively). IVM oocytes also tended to have higher Lp values than IVF oocytes (0.55 micron/(min.atm)). At 5 degrees C, the Lp of IVM oocytes decreased to 0.36 micron/min.atm) corresponding to an Arrhenius activation energy of 7.84 kcal/mol. The incidence of IIF in MAT oocytes suspended in salt solution and subjected to linear cooling to -60 degrees C was 45% at 4 degrees C/min, 75% at 8 degrees C/min, and 93% at 16 degrees C/min; with IVF oocytes, the incidence of IIF was 40% at 4 degrees C/min, 92% at 8 degrees C/min, and 100% at 16 degrees C/min. Comparisons involving median IIF temperatures (TIIF50s) and the distributions of the observed IIF temperatures for IMM (Myers et al., Cryo-Lett. 8, 260), IVM (Chandrasekaran et al., Cryobiology 27, 676), MAT and IVF oocytes indicated that the IIF incidence in IMM oocytes cooled at 4 degrees C/min was greater than that of oocytes at the other developmental stages cooled at the same rate. The TIIF50s of IVM and IVF oocytes were lowered by equilibration in 1.5 M ethylene glycol (EG), glycerol, or propylene glycol (PG) prior to cooling, with EG tending to lower the TIIF50s more than glycerol or PG. For all three cryoprotectants, the TIIF50s and IFF temperature distributions were cooling-rate dependent. The Weibull probability distribution was fitted to the distributions of the IIF temperatures of oocytes suspended in salt solutions with and without cryoprotectants yielding R2 values ranging from 0.70 to 0.98.

Plasma membranes of protoplasts isolated from non-acclimated rye plants undergo a transition from the bilayer to the inverted hexagonal (HII) phase during freeze-induced dehydration at -10 degrees C. It has been suggested (Bryant, G. and Wolfe, J. (1989) Eur. Biophys. J. 16, 369-372) that the differential hydration of various membrane components may induce fluid-fluid demixing of highly hydrated (e.g., PC) from poorly hydrated (PE) components during dehydration. This could yield a PE-enriched domain more prone to form the HII phase. We have examined the lyotropic phase behavior of mixtures of DOPE and DOPC at 20 degrees C by freeze-fracture electron microscopy, differential scanning calorimetry, and X-ray diffraction. HII phase formation was favored by higher proportions of DOPE and lower water contents. Mixtures of 1:1 and 1:3 DOPE/DOPC had a hydration-dependent appearance of two L alpha phases at water contents just above those at which the HII phase occurred. The hydration-dependence of the lamellar repeat spacings suggested that the DOPE-enriched domains preferentially underwent the L alpha-to-HII phase transition. Mixtures of 3:1 DOPE/DOPC did not separate into two L alpha phases during dehydration. These data suggest that the differential hydration characteristics of various membrane components may induce their lateral fluid-fluid demixing during dehydration.

The process of cold acclimation is a complex developmental phenomenon that invokes the orchestration of many different processes, including hormonal responses to environmental cues, altered gene activity, the synthesis of new gene products, and alterations that influence virtually every facet of cellular metabolism. Although seemingly disparate, many of these metabolic changes ultimately contribute to the increased cryostability of cellular membranes, destabilisation of which is the primary cause of freezing injury. Therefore, to provide a rational integration of the many different facets of the cold acclimation process, it is necessary to first identify the freeze/thaw-induced lesions in cellular membranes and to elucidate the biophysical mechanisms and biochemical alterations responsible for their occurrence. Although all cellular membranes are vulnerable to freeze-induced destabilisation, maintenance of the structural integrity of the plasma membrane is a prerequisite for survival because of the central role that it plays during a freeze-thaw cycle. Cryomicroscopy studies of isolated protoplasts complemented with electron microscopy studies of freeze- induced ultrastructural changes are yielding a comprehensive analysis of the phenomenology of freezing injury and the identification of specific ‘lesions’ in the plasma membrane, which vary depending on the stage of acclimation and the nadir temperature to which the protoplasts are cooled. Of the lesions identified to date (expansion-induced lysis, lamellar-to-hexagonal II phase transitions, and the fracture-jump lesion), all are a consequence of freeze- induced dehydration; however, whereas expansion-induced lysis is the result of cellular dehydration and the large osmotic excursions incurred during a freeze-thaw cycle, lamellar-to-hexagonal II phase transitions and the fracture-jump lesion are consequences of the removal of water that is closely associated with cellular membranes. These studies, together with a detailed analysis of the plasma membrane lipid composition at the molecular species level and procedures to alter selectively the lipid composition of the plasma membrane have provided a foundation for mechanistic studies to establish directly the relationships between alterations in the plasma membrane lipid composition and its increased cryostability after cold acclimation. In order to determine if genotypic diversity in freezing tolerance is associated with genotypic differences in membrane lipid composition, we are extending these studies to other cereals (e.g., oat and barley), which are less freezing tolerant than winter rye. These studies reveal that the freeze-induced membrane lesions in a spring oat cultivar (Ogle) are qualitatively similar to those observed in rye, but occur at substantially higher temperatures. Moreover, the lipid composition of the plasma membrane isolated from leaves of the spring oat is vastly different from that of winter rye leaves. These studies suggest that strategies for improving the freezing tolerance of cereals should include techniques to modify membrane lipid composition.

The common fruit fly Drosophila melanogaster is the subject of investigation in many diverse areas of biology. It has been studied intensively by geneticists, developmental and molecular biologists, neurobiologists, population and evolutionary biologists, entomologists, and chronobiologists. Currently, interest in D. melanogaster is most intense among molecular biologists, but studies of D. melanogaster have a long and distinguished history, dating back to Thomas Hunt Morgan in the first decade of this century. As a result of both past and present activity, there is an enormous number of D. melanogaster genetic stocks. In 1985 it was estimated that the number of different stocks was in excess of 30,000 and was rapidly increasing because of the increased number of investigators studying Drosophila, the increased number of large scale mutant screens, and the generation of new stocks by DNA transformation. Since then, the number of mutant stocks is even greater, especially since so many germ line transformants have been obtained; for example, in Drosophila Information Service (June 1988), some 1350 entries were recorded in the “clone list.” Many of these clones have been reinserted in several different places in the germ line via P-element mediated transformation. We estimate that over 50,000 different genetic lines of D. melanogaster are now maintained in national and international stock centers and in the laboratories of individual investigators.

The three high-molecular-weight subunits of chloroplast coupling factor (CF 1 ) are the primary proteins released from pyrophosphate-washed thylakoids exposed to freezing. Identical subunit profiles are found in the supernatant proteins of thylakoids exposed to different intensities of freezing stress by the inclusion of sugars with varying degrees of cryoprotective efficiency. Isolated CF 1 is inactivated by freezing in the presence of NaCl, glucose, and sucrose but raffinose can protect against loss of enzymatic activity during freezing. The low specific activity of the supernatant proteins released from the thylakoid and the inability to recover the Ca 2+ -dependent ATPase activity lost from the membrane suggest that inactivation accompanies release of CF 1 during freezing.

Highly enriched plasma membrane fractions were isolated from leaves of nonacclimated (NA) and acclimated (ACC) rye (Secale cereale L. cv Puma) seedlings. Collectively, free sterols, steryl glucosides, and acylated steryl glucosides constituted >50 mole% of the total lipid in both NA and ACC plasma membrane fractions. Glucocerebrosides containing hydroxy fatty acids constituted the major glycolipid class of the plasma membrane, accounting for 16 mole% of the total lipid. Phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine with lesser amounts of phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and phosphatidylinositol, comprised only 32 mole% of the total lipid in NA samples. Following cold acclimation, free sterols increased from 33 to 44 mole%, while steryl glucosides and acylated steryl glucosides decreased from 15 to 6 mole% and 4 to 1 mole%, respectively. Sterol analyses of these lipid classes demonstrated that free beta-sitosterol increased from 21 to 32 mole% (accounting for the increase in free sterols as a class) at the expense of sterol derivatives containing beta-sitosterol. Glucocerebrosides decreased from 16 to 7 mole% of the total lipid following cold acclimation. In addition, the relative proportions of associated hydroxy fatty acids, including 22:0 (h), 24:0 (h), 22:1 (h), and 24:1 (h), were altered. The phospholipid content of the plasma membrane fraction increased to 42 mole% of the total lipid following cold acclimation. Although the relative proportions of the individual phospholipids did not change appreciably after cold acclimation, there were substantial differences in the molecular species. Di-unsaturated molecular species (18:2/18:2, 18:2/18:3, 18:3/18:3) of phosphatidylcholine and phosphatidylethanolamine increased following acclimation. These results demonstrate that cold acclimation results in substantial changes in the lipid composition of the plasma membrane.

Identification of chloroplast coupling factor particles, by the freeze-etching and negative-staining techniques, was made utilizing chloroplast thylakoids isolated from spinach leaves. Complete removal of particles, comparable in diameter to purified coupling factor particles, from the outer surface of freeze-etched thylakoids was achieved by treatment with 0.8% silicotungstate. Reappearance of particles, comparable in diameter to purified coupling factor particles, on the outer surface of freeze-etched thylakoids was demonstrated by combining silicotungstate-treated thylakoids with purified chloroplast coupling factor. Negative-staining results were in agreement with the freeze-etch data. The results demonstrate that the chloroplast coupling factor particles are exposed on the outer surface.

Possibilities to account for the mechanism of freeze-thaw injury to isolated protoplasts of Spinacia oleracea L. cv. Winter Bloomsdale were investigated. A freeze-thaw cycle to −3.9 C resulted in 80% lysis of the protoplasts. At −3.9 C, protoplasts are exposed to the equivalent of a 2.1 osmolal solution. Isolated protoplasts behave as ideal osmometers in the range of concentrations tested (0.35 to 2.75 osmolal), arguing against a minimum critical volume as a mechanism of injury. Average protoplast volume after a freeze-thaw cycle was not greatly different than the volume before freezing, arguing against an irreversible influx of solutes while frozen. A wide variety of sugars and sugar alcohols, none of which was freely permeant, were capable of protecting against injury which occurred when protoplasts were frozen in salt solutions. The extent of injury was also dependent upon the type of monovalent ions present, with Li = Na > K = Rb = Cs and Cl ≥ Br > I, in order of decreasing protoplast survival. Osmotic conditions encountered during a freeze-thaw cycle were established at room temperature by exposing protoplasts to high salt concentrations and then diluting the osmoticum. Injury occurred only after dilution of the osmoticum and was correlated with the expansion of the plasma membrane. Injury observed in frozen-thawed protoplasts was correlated with the increase in surface area the plasma membrane should have undergone during thawing, supporting the contention that contraction of the plasma membrane during freezing and its expansion during thawing are two interacting lesions which cause protoplast lysis during a freezethaw cycle.

Four cultivars of kurume azalea (Rhododendron sp.), ‘Hersey Red’, ‘Snow’, ‘Coral Bells’ and ‘Hino Crimson’ reported to have different susceptibilities to winter injury in the nursery were compared for their sensitivities to direct freezing injury (both to roots and leaves) and desiccation injury under controlled conditions. Sensitivity to root freezing injury was inversely correlated with winter injury. A positive association between leaf hardiness to freezing injury and resistance to winter injury was found only in ‘Hino Crimson’. Susceptibility to winter injury was most closely associated with desiccation, as indicated by the minimum water potentials under conditions of frozen soil and high evaporative demand.

The transverse relaxation rate of water protons of nonacclimated Hedera helix L. cv. Thorndale (ivy) bark decreased in the presence of added extracellular Ca2+, Mg2+, or CO2. Evidence that the decreased transverse relaxation rate is due to decreased plasmalemma water permeability is presented. The decrease in plasmalemma water permeability induced by Ca2+ and CO2 was less in cold-acclimated ivy bark than in nonacclimated ivy bark, indicating that the process of cold acclimation altered a functional property of the plasmalemma. It is not known if the functional change in the plasmalemma is related to the degree of plant cold hardiness or is simply a low temperature response.

The stress and strain (surface tension and fractional change in area) in the plasma membrane of protoplasts isolated from rye leaves (Secale cereale L. cv Puma) were measured during osmotic expansions from isotonic into a range of more dilute solutions. The membrane surface tension increases rapidly to a maximum and then decreases slowly with some protoplasts lysing in all phases of the expansion. The maximum surface tension is greater for rapid expansions, and protoplasts lyse earlier during rapid expansion. Over the range of expansion rates investigated, the area at which lysis occurs is not strongly dependent on expansion rate. The value of the maximum tension is determined by the expansion rate and the rate at which new material is incorporated into the membrane. During osmotic expansion, protoplasts isolated from cold-acclimated plants incorporate material faster than do those from nonacclimated plants and thus incur lower membrane tensions.

The freezing tolerance of protoplasts isolated from nonacclimated rye leaves ( Secale cereale L. cv Puma) was significantly altered by using a pH-induced protoplastliposome fusion technique to modify the lipid composition of the plasma membrane. The increase in freezing tolerance was elicited by fusion with liposomes composed of either the total phospholipid fraction isolated from the plasma membrane of cold-acclimated leaves or single mono- or diunsaturated species of phosphatidylcholine (PtdCho). Of the PtdCho species tested, dilinoleoylphosphatidylcholine ([Lin 2 ]PtdCho) and dilinolenoylphosphatidylcholine ([Lnn 2 ]PtdCho) liposomes were the most effective; 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-linoleoylphosphatidylcholine, or dioleoylphosphatidylcholine liposomes were somewhat less effective; dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine liposomes had no effect. The increased freezing tolerance was the result of a transformation in the cryobehavior of the plasma membrane during freeze-induced osmotic contraction. In control nonacclimated protoplasts, osmotic contraction resulted in endocytotic vesiculation of the plasma membrane which was irreversible and resulted in lysis during osmotic expansion after melting of the suspending medium. In nonacclimated protoplasts fused with mono- or diunsaturated species of PtdCho, osmotic contraction resulted in the reversible formation of exocytotic extrusions of the plasma membrane—as normally occurs in protoplasts isolated from cold-acclimated leaves (acclimated protoplasts). In scanning electron micrographs, the morphology of the extrusions of nonacclimated protoplasts fused with [Lin 2 ]PtdCho was virtually indistinguishable from that of the extrusions formed in acclimated protoplasts. These studies provide direct evidence that changes in the lipid composition of the plasma membrane are causally related to one facet of the cold-acclimation process.

In conclusion, isolated protoplasts are an excellent arena in which destabilization of the plasma membrane can be directly observed during a freeze-thaw cycle by cryomicroscopy. Destabilization is manifested in various ways—Intracellular ice formation, loss of osmotic responsiveness, or expansion-induced lysis. The incidence of any particular form of injury will depend on the freeze-thaw protocol and hardiness of the tissue from which the protoplasts were isolated. In all cases, however, cold acclimation directly increases the stability of the plasma membrane to the multiple stresses that arise during a freeze-thaw cycle. Such observations provide for functional differences in the plasma membrane that may now be used to consider the significance of any compositional changes in the membrane that might be determined.

The plasma membrane of isolated protoplasts from non-acclimated but not from acclimated rye leaves undergoes lamellar-to-hexagonal H II phase transitions after freeze-induced dehydration (Gordon-Kamm, W.J. and P.L. Steponkus (1984) Proc. Natl. Acad. Sci. USA 81, 6373–6377). An effort was made to determine whether plasma membrane lipid components show similar behavior when dehydrated. Liposomes were prepared from the total lipid mixture extracted from plasma membrane fractions isolated from non-acclimated (NA) or cold acclimated (ACC) rye leaves ( Secale cereale L. cv Puma). Freeze-fracture electron microscopy was used to identify phase changes in liposomes after osmotic dehydration. When NA liposomes prepared in 1.3 wt% sorbitol were incubated at 4°C in 73 wt% sorbitol for 15 h (overnight), hexagonal H II phase was observed in aggregates of apparently fused liposomes with H II observed in approximately 75% of the fracture faces. In contrast, neither aggregates of apparently fused liposomes nor H II phase was observed in ACC liposomes after dehydration under the same conditions Dehydration of NA or ACC liposomes in 40 or 60 wt% sorbitol resulted in the formation of intermediate morphologies such as loosely ordered cylinders and ripple structures. Lipidic particles and pits of various sizes and morphologies were observed after dehydraton of both NA and ACC liposomes in 40, 60, or 73 wt% sorbitol. These results indicate that lipid composition determines the propensity of the plasma membrane to undergo dehydration-induced phase transitions and that alterations in the lipid composition of the plasma membrane play a causal role in the process of cold acclimation.

Differential cryoprotection is afforded to chloroplast thylakoids against freeze-induced uncoupling of cyclic photophosphorylation by equimolar concentrations of glucose, sucrose, and raffinose. This differential protective effect appears to be due to nonideal activity-concentration profiles exhibited by the sugars during freezing. When cryoprotection is analyzed as a function of the mole fraction of NaCl to which the membranes are exposed during freezing, the pattern of protection to cyclic photophosphorylation and its component reactions is not dependent upon the chemical identity of the protective solute. Cryoprotective efficiency of glucose, sucrose, and raffinose can be accounted for by proposing an activity dependent alteration in the freezing environment rather than specific solute-membrane interactions.