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Calorimetric determination of the state of water in the cells of frozen dogwood (Cornus florida L.)
684
ABSTRACTS, PONKUS
14853).
2lst ANNUAL
(Cornell University, Ithaca, New York
MEETING
show. using DSC and EM, that vacuolar contents can form glasses but they are less stable than cytoplasmic glasses. We also demonstrate by direct visualization. that the first appearance of ice in cell cytoplasm coincides with the appearance of devitrification in the DSC record and high mortality in the tissue. Preliminary study of raw extract of superhardy wood by DSC indicates that much of the glass-forming capability may come from very high concentrations of intracellular sucrose. (Supported in part by NIH Grants BSRG RR05737 and GM 17959.)
Sections of differentiating cotyledon cultures of Douglas-fir (Pseudotsrrga menziesii) were cooled to subzero temperatures at a rate of I”C/h in various suspending media and tissue survival was determined by development of viable plantlets in culture after freezing. Freezing the tissue in either proline, trehalose, or DMSO, alone, did not increase survival over the water controls. However, when 10% (v/v) DMSO was added to equiosmolal concentrations (I. I osm) of proline, trehalose, or sucrose, increased sur8. Calorimetric Determination of the State of Water vival was obtained. At temperatures of 0, -5, - IO. in the Cells of Frozen Do~lzwod (Cornwj7oridrr and - 15”C, survival in proline + DMSO was 80, 53, L.). ROBERT J. WILLIAMS (American Red Cross 30, and 35%, respectively. In trehalose + DMSO at Blood Services Laboratories, Bethesda, Marythese same temperatures, survival was 88, 64, 39, and land). 77~. In sucrose + DMSO. survival was 100, 100, IO, and 0%. In water alone, survival was 100, 27, 0, and When first-year stems from hardened dogwood are 0%. To determine at what cooling rate intracellular ice frozen, parenchymal cells resist further plasmolysis formation (IIF) would be precluded in bulk frozen and supercool at temperatures below about -4°C. tissue, cotyledon sections were suspended in cryoproHowever, no second exotherm or cell flashing has tectants. frozen on a cryomicroscope and the median been reported at or below the killing temperature, seeding temperature at which IIF occurred (TIFF,,) - 25°C. and no cell “flashing” has been observed and the cumulative frequency of IIF were determined. under the cryomicroscope. Recent experiments using For tissue sections frozen in either 0.53 osm proline a differential scanning calorimeter have shown thermal or trehalose, a high cumulative frequency of IIF (>.8) events associated with freezing injury. Tissues frozen was observed at cooling rates of 2.5”Cimin or greater and equilibrated at a high subfreezing temperature and with a TIFF,,, of ~ 12°C for proline and - 15°C for cooled at O.Z”C/min show a diffuse second exotherm trehalose. However, when 10% DMSO was added to centered on the killing temperature. During warming, 0.53 osm proline, a high cumulative frequency of IIF two endothermic melting peaks are seen, the first as(.86) was only observed at cooling rates of 40”Cimin sociated with water removed from plasmolyzed cells or greater. At this cooling rate, the TIFF,, was - 20°C. and typical of the melting of an aqueous solution, the Tissue sections frozen in 0.53 osm trehalose + IO7c second produced by the melting of extracellular ice DMSO had a cumulative frequency of IIF of 0.70 at and typical of the melting of pure water. The second cooling rates of 20”Cimin or greater. At this cooling peak is small in tissues uninjured by freezing, but berate, the TIFF,,was - 18°C.These results suggest that comes significant after injury. Thus it appears that. as in tissue sections suspended in cryoprotectants and the killing temperature is approached, the structures cooled at a rate of l”C/h, intracellular ice formation supporting the cell membranes against osmotic forces was precluded and was not responsible for injury. are plastically deformed, but the cell membranes do not lose semipermeability and cells do not freeze intracellularly. The compression is irreversible so that 7. Continued Studies qflntrucellular Gloss Formation part of the cell water lost during freezing is not reas a Method of Natural Cryoprotec,tion in Sirturned upon thawing. (Supported in part by NIH perhardy Populus Balsamifern I’. Virginiunu. Grants BSRG RR05737 and GM 17959.) A. HIRSH, R. J. WILLIAMS, E. EKBE,* R. STEEKE,* AND H. T. MERYMAN (American Red Cross Blood Services Laboratories, Bethesda, 9. An Oil Drop Method for Detecting Inte~fuciul EnMaryland; *Plant Virology Laboratory, U.S. ergy Changes in Osmotic& Stressed Sea C/rchin Eggs. ROBERT J. WILLIAMS AND TSUNEO Department of Agriculture, Beltsville, MaryTAKAHASHI (American Red Cross Blood Serland). vices Laboratories, Bethesda, Maryland). Using differential scanning calorimetry (DSC) and When droplets of two liquids are dispersed in a third freeze-fracture freeze-etch electron microscopy (EM), immiscible phase, the droplets with the lower interwe have previously presented evidence that the intrafacial energy will engulf the droplets with the higher cellular fluids of superhardy Poprclus hnlsam(fera v. interfacial energy [S. Torza and S. G. Mason, Sciencr Virginiana go through several glass transitions as they 163, 813-814 (1969)]. We have applied this phenomare cooled slowly below -20°C. Here is presented further data on these intracellular aqueous glasses. We enon to measurement of the interfacial tension in living
ABSTRACTS, PONKUS
14853).
2lst ANNUAL
(Cornell University, Ithaca, New York
MEETING
show. using DSC and EM, that vacuolar contents can form glasses but they are less stable than cytoplasmic glasses. We also demonstrate by direct visualization. that the first appearance of ice in cell cytoplasm coincides with the appearance of devitrification in the DSC record and high mortality in the tissue. Preliminary study of raw extract of superhardy wood by DSC indicates that much of the glass-forming capability may come from very high concentrations of intracellular sucrose. (Supported in part by NIH Grants BSRG RR05737 and GM 17959.)
Sections of differentiating cotyledon cultures of Douglas-fir (Pseudotsrrga menziesii) were cooled to subzero temperatures at a rate of I”C/h in various suspending media and tissue survival was determined by development of viable plantlets in culture after freezing. Freezing the tissue in either proline, trehalose, or DMSO, alone, did not increase survival over the water controls. However, when 10% (v/v) DMSO was added to equiosmolal concentrations (I. I osm) of proline, trehalose, or sucrose, increased sur8. Calorimetric Determination of the State of Water vival was obtained. At temperatures of 0, -5, - IO. in the Cells of Frozen Do~lzwod (Cornwj7oridrr and - 15”C, survival in proline + DMSO was 80, 53, L.). ROBERT J. WILLIAMS (American Red Cross 30, and 35%, respectively. In trehalose + DMSO at Blood Services Laboratories, Bethesda, Marythese same temperatures, survival was 88, 64, 39, and land). 77~. In sucrose + DMSO. survival was 100, 100, IO, and 0%. In water alone, survival was 100, 27, 0, and When first-year stems from hardened dogwood are 0%. To determine at what cooling rate intracellular ice frozen, parenchymal cells resist further plasmolysis formation (IIF) would be precluded in bulk frozen and supercool at temperatures below about -4°C. tissue, cotyledon sections were suspended in cryoproHowever, no second exotherm or cell flashing has tectants. frozen on a cryomicroscope and the median been reported at or below the killing temperature, seeding temperature at which IIF occurred (TIFF,,) - 25°C. and no cell “flashing” has been observed and the cumulative frequency of IIF were determined. under the cryomicroscope. Recent experiments using For tissue sections frozen in either 0.53 osm proline a differential scanning calorimeter have shown thermal or trehalose, a high cumulative frequency of IIF (>.8) events associated with freezing injury. Tissues frozen was observed at cooling rates of 2.5”Cimin or greater and equilibrated at a high subfreezing temperature and with a TIFF,,, of ~ 12°C for proline and - 15°C for cooled at O.Z”C/min show a diffuse second exotherm trehalose. However, when 10% DMSO was added to centered on the killing temperature. During warming, 0.53 osm proline, a high cumulative frequency of IIF two endothermic melting peaks are seen, the first as(.86) was only observed at cooling rates of 40”Cimin sociated with water removed from plasmolyzed cells or greater. At this cooling rate, the TIFF,, was - 20°C. and typical of the melting of an aqueous solution, the Tissue sections frozen in 0.53 osm trehalose + IO7c second produced by the melting of extracellular ice DMSO had a cumulative frequency of IIF of 0.70 at and typical of the melting of pure water. The second cooling rates of 20”Cimin or greater. At this cooling peak is small in tissues uninjured by freezing, but berate, the TIFF,,was - 18°C.These results suggest that comes significant after injury. Thus it appears that. as in tissue sections suspended in cryoprotectants and the killing temperature is approached, the structures cooled at a rate of l”C/h, intracellular ice formation supporting the cell membranes against osmotic forces was precluded and was not responsible for injury. are plastically deformed, but the cell membranes do not lose semipermeability and cells do not freeze intracellularly. The compression is irreversible so that 7. Continued Studies qflntrucellular Gloss Formation part of the cell water lost during freezing is not reas a Method of Natural Cryoprotec,tion in Sirturned upon thawing. (Supported in part by NIH perhardy Populus Balsamifern I’. Virginiunu. Grants BSRG RR05737 and GM 17959.) A. HIRSH, R. J. WILLIAMS, E. EKBE,* R. STEEKE,* AND H. T. MERYMAN (American Red Cross Blood Services Laboratories, Bethesda, 9. An Oil Drop Method for Detecting Inte~fuciul EnMaryland; *Plant Virology Laboratory, U.S. ergy Changes in Osmotic& Stressed Sea C/rchin Eggs. ROBERT J. WILLIAMS AND TSUNEO Department of Agriculture, Beltsville, MaryTAKAHASHI (American Red Cross Blood Serland). vices Laboratories, Bethesda, Maryland). Using differential scanning calorimetry (DSC) and When droplets of two liquids are dispersed in a third freeze-fracture freeze-etch electron microscopy (EM), immiscible phase, the droplets with the lower interwe have previously presented evidence that the intrafacial energy will engulf the droplets with the higher cellular fluids of superhardy Poprclus hnlsam(fera v. interfacial energy [S. Torza and S. G. Mason, Sciencr Virginiana go through several glass transitions as they 163, 813-814 (1969)]. We have applied this phenomare cooled slowly below -20°C. Here is presented further data on these intracellular aqueous glasses. We enon to measurement of the interfacial tension in living