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Cryobiology of isolated plant protoplasts: IV. Cellular injury
ABSTRACTS,
16th
ANNUAL
related to the elevated salt (CaCl, + NaCl) concentration which the protoplasts encountered during the freeze-thaw cycle. Sorbitol was apparently able to colligatively reduce the concentration of salt and, thus, reduce the amount of injury. Bovine serum albumin acted as a cryoprotectant, reducing the sensitivity of the protoplasts to both expansion-induced lysis and senescence, while having no effect on the sensitivity of the protoplasts to high salt concentrations. Thus it is likely that the nature of the strain(s) which cause expansion-induced lysis is quite different from those which cause injury by protracted exposure to high salt concentrations. 35.
Cryobiology of Isolated Plant Thermodynamic Considerations.
Protoplasts:
I.
R. L. LEVIN, J. R. FERGUSON, M. F. DOWGERT, AND P. L. STEP~NKUS (Sibley School of Mechanical and Aerospace Engineering and Department of Agronomy, Cornell University, Ithaca, New York 14853).
During a freeze-thaw cycle of isolated plant protoplast suspensions, ice will initially nucleate extracellularly. This results in a disequilibrium in the chemical potential of water in the intracellular solution vs the partially frozen extracellular medium. Equilibrium may be achieved by cellular dehydration or intracellular ice formation. The manner of equilibration is influenced by the cell water permeability, cellular volume to surface area ratio, cooling rate, and the minimum temperature imposed. Of these factors, cellular water permeability is the major unknown. Cellular volumetric responses during a freeze-thaw cycle were directly determined and a thermodynamic model of cell water transport was used to statistically determine the subzero temperature dependence of cell water permeability. Use of these experimentally obtained values for the cell water permeability at subzero temperatures enables a more accurate estimation of the extent of disequilibrium which cells experience during various freeze-thaw regimes to be made. 36. Cryobiology tracellular
of Isolated Plant Ice Formation.
Protoplasts: M. F. LEVIN,
II. In-
DOWGERT, AND J.
R. L. R. (Department of Agronomy and Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, New York 14853). P. L.
STEPONKUS,
FERCUSON
Direct observations of isolated cereal protoplasts exposed to varied freeze-thaw regimes using a cryomicroscope document that the incidence of intracellular ice formation is dependent on the cooling rate and the minimum temperature imposed. If cells are cooled to temperatures between -2 and -5°C the incidence of intracellular ice formation, as manifested by “black flashing” of the cell, is very low-regardless
593
MEETING
of the cooling rate (in the range of 2 to 120”Cmin). When cooled to temperatures between -5 and -2O”C, intracellular ice formation is strongly influenced by the cooling rate (in the range of 2 to 8O”Cimin). The incidence is close to zero at rates less than 3”Cimin and increases as cooling rate increases. At cooling rates greater than lS”C/min, the incidence of intracellular ice formation is greater than 95%. If the cells are cooled to temperatures lower than -2o”C, the incidence of intracellular ice formation is greater than 95% regardless of the cooling rate imposed (in the range of 4 to 80°C min). Analysis of cellular volume, extent of supercooling of the intracellular solution, and temperature at the time of intracellular ice formation indicate that intracellular ice formation is primarily a function of a temperature-dependent nucleation event whose probability is low at temperatures higher than -5°C and high at temperatures lower than -20°C. Within this temperature range, the extent of supercooling increases the probability of intracellular ice formation. 37. Cryobiolog-y of Isolated Plant Altered Cellular Gas Content.
Protoplasts: J.
R.
III.
FERGUSON,
P. L. STEPONKUS, M. F. DOWGERT, AND R. L. LEVIN (Sibley School of Mechanical and Aerospace Engineering and Department of Agronomy, Cornell University, Ithaca, New York 14853).
The optical “blackening” or “flashing” of cells during intracellular ice formation has been purported to be due to the diffraction of light by very small ice crystals formed within the cells. Our observations, however, suggest that the primary cause of “black flashing” is the formation of many small gas bubbles with radii near the wavelength of the impinging light. These numerous gas bubbles then scatter the impinging light producing a black image. Thermodynamic models of gas solubility and cellular dehydration are combined and used to determine the temperature transition (-8 to - 12°C) over which gas bubble formation will occur. Although gas solubility increases with decreasing temperature, the concentration of the dissolved intracellular gases increases and exceeds the solubility limit of the system. Rapid phase changes of intracellular water preclude the gradual dissipation of the evolving gases and visible bubbles result. If intracellular ice formation occurs at temperatures above -5”C, gas bubble formation is not observed. At these temperatures, the amount of ice formed is not suffi cient to cause gas to exceed its solubility limit. Intracellular ice formation without gas bubble formation has been termed “white flashing.” 38. Cryobiology Cellular
of
Isolated
Plant
Protoplasts:
IV.
P. L. STEPONKUS, M. F. DOWGERT, R. L. LEVIN, AND J. F. FERGUSON (Department of Agronomy and Sibley School of Injury.
594
ABSTRACTS,
16th ANNUAL
Mechanical and Aerospace Engineering, Cornell University, Ithaca, New York 14853). During a freeze-thaw cycle, a cell encounters a spectrum of cellular stresses, and injury may be manifested as different cellular or membrane lesions as a result of the different stresses. The spectrum of lesions may be directly observed as they are manifested in altered cellular volumetric responses during a freeze-thaw cycle. Intracellular ice formation at high fractional cell volumes will result in the immediate physical disruption of the plasma membrane. Cellular dehydration may result in several lesions depending on the freeze-thaw protocol. If cells are cooled to -5”C, and subsequently warmed to +5”C, they will exhibit characteristic Boyle van’t Hoff behavior during both cooling and warming. During warming, however, they will lyse before regaining their initial size. If cells are cooled to temperatures lower than -5°C two additional forms of injury are observed. Following cooling, cells may exhibit a total loss of osmotic responsiveness and remain in the contracted state during warming. Such a manifestation is suggestive of a complete loss of semipermeability of the plasma membrane. Ahernatively, cells may exhibit altered osmometric behavior during warming. Such cells expand during warming but to a lower extent at any given osmolality than normal cells. While the cells remain physically intact, this manifestation is suggestive of a partial loss of semipermeability or leakiness of the plasma membrane. Each type of injury is probabilistic in nature such that its frequency of occurrence depends upon the conditions of the freeze-thaw regime. In view of the various lesions which result in cellular injury, proposals regarding the mechanism of freezing injury should consider the nature of cellular lesions in their interpretation of freezing injury. 39. Cryobiology of Isolated Plant Protoplasts: V. Contraction -Expansion-Induced Alterations in the Plasma Membrane. P. L. STEPONKUS,
M. F. DOWGERT, AND S. C. WIEST (Department of Agronomy, Cornell University, Ithaca, New York 14853). One form of freeze-thaw injury manifested by isolated protoplasts in an expansion-induced lysis during warming. Although osmometric behavior is best described as a function of volume, the lytic lesion resulting from volumetric changes is best described as a function of surface area of the plasma membrane. Protoplasts do not possess a fixed maximum surface area at which lysis occurs. Instead, the surface area at which lysis occurs is decreased if the protoplasts have been previously contracted. Freeze-thaw injury is the result of two major strains: a freeze- or contractioninduced membrane alteration which decreases the maximum critical surface area of the plasma membrane and a thaw- or expansion-induced dissolution of
MEETING
the plasma membrane which occurs when the maximum critical surface area is exceeded. In a population of cells, the absolute magnitude of change in the mean surface area of the population which results in lysis of 50% of the cells appears to be constant and independent of the extent of contraction. This value varies among species examined to date: 900 pm2 for spinach, 400 pm* for wheat, and 600 pm” for rye. Direct measurements of individual cells during a freeze-thaw cycle document the fact that cells contracted during freezing lyse before achieving their original surface area. Larger cells, however, undergo larger surface area increments than do smaller cells. Thus, the tolerable surface area increment is determined by the initial cell size but remains constant regardless of the extent of contraction experienced by cells of a given size. 40. Cryobiology fluence
of Isolated Plant Acclimation.
Protoplasts:
VI. In-
of Cold
P. L. STEPONKUS, M. F. DOWGERT, AND S. R. ROBERTS (Department of Agronomy, Cornell University, Ithaca, New York 14853).
Sensitivity of isolated protoplasts to freezing injury parallels that of the parent tissue from which they were derived and can be ameliorated by cold acclimation. When rye seedlings are cold acclimated, their hardiness increases from -3 to -26°C as determined by freezing of isolated crowns. Protoplasts isolated from these plants exhibit comparable levels of hardiness: 50% survival of those isolated from nonacclimated tissue occurs at -2.5”C while 50% survival of protoplasts isolated from plants acclimated for 4 weeks occurs at -28°C. Thus, the hardiness level of the parent tissue is manifested by the same degree of hardiness in the derived protoplasts frozen in vitro. Isolated protoplasts afford the possibility to determine which of several alterations associated with the cold acclimation contribute to the increased tolerance to a freeze-thaw cycle. These alterations include: (1) changes in the water permeability of the plasma membrane; (2) changes in cellular characteristics which influence osmometric behavior, specifically, solute content and the osmotically inactive volume: and (3) tolerance of the plasma membrane to contraction and expansion. The influence of cold acclimation on each of these factors is discussed. SESSION
4. CRYOSURGERY
41. Determination of the Influence of Experimental Local Freezing on Total Femoral Greater Trochanteric Apophyseal Grobtath Arrest. JACOB
F. KATZ AND ROBERT S. SIFFERT (Orthopaedic Research Laboratory, Department of Orthopaedics, Mt. Sinai School of Medicine of the City University of New York, 100th Street and Fifth Avenue, New York, New York 10029).
16th
ANNUAL
related to the elevated salt (CaCl, + NaCl) concentration which the protoplasts encountered during the freeze-thaw cycle. Sorbitol was apparently able to colligatively reduce the concentration of salt and, thus, reduce the amount of injury. Bovine serum albumin acted as a cryoprotectant, reducing the sensitivity of the protoplasts to both expansion-induced lysis and senescence, while having no effect on the sensitivity of the protoplasts to high salt concentrations. Thus it is likely that the nature of the strain(s) which cause expansion-induced lysis is quite different from those which cause injury by protracted exposure to high salt concentrations. 35.
Cryobiology of Isolated Plant Thermodynamic Considerations.
Protoplasts:
I.
R. L. LEVIN, J. R. FERGUSON, M. F. DOWGERT, AND P. L. STEP~NKUS (Sibley School of Mechanical and Aerospace Engineering and Department of Agronomy, Cornell University, Ithaca, New York 14853).
During a freeze-thaw cycle of isolated plant protoplast suspensions, ice will initially nucleate extracellularly. This results in a disequilibrium in the chemical potential of water in the intracellular solution vs the partially frozen extracellular medium. Equilibrium may be achieved by cellular dehydration or intracellular ice formation. The manner of equilibration is influenced by the cell water permeability, cellular volume to surface area ratio, cooling rate, and the minimum temperature imposed. Of these factors, cellular water permeability is the major unknown. Cellular volumetric responses during a freeze-thaw cycle were directly determined and a thermodynamic model of cell water transport was used to statistically determine the subzero temperature dependence of cell water permeability. Use of these experimentally obtained values for the cell water permeability at subzero temperatures enables a more accurate estimation of the extent of disequilibrium which cells experience during various freeze-thaw regimes to be made. 36. Cryobiology tracellular
of Isolated Plant Ice Formation.
Protoplasts: M. F. LEVIN,
II. In-
DOWGERT, AND J.
R. L. R. (Department of Agronomy and Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, New York 14853). P. L.
STEPONKUS,
FERCUSON
Direct observations of isolated cereal protoplasts exposed to varied freeze-thaw regimes using a cryomicroscope document that the incidence of intracellular ice formation is dependent on the cooling rate and the minimum temperature imposed. If cells are cooled to temperatures between -2 and -5°C the incidence of intracellular ice formation, as manifested by “black flashing” of the cell, is very low-regardless
593
MEETING
of the cooling rate (in the range of 2 to 120”Cmin). When cooled to temperatures between -5 and -2O”C, intracellular ice formation is strongly influenced by the cooling rate (in the range of 2 to 8O”Cimin). The incidence is close to zero at rates less than 3”Cimin and increases as cooling rate increases. At cooling rates greater than lS”C/min, the incidence of intracellular ice formation is greater than 95%. If the cells are cooled to temperatures lower than -2o”C, the incidence of intracellular ice formation is greater than 95% regardless of the cooling rate imposed (in the range of 4 to 80°C min). Analysis of cellular volume, extent of supercooling of the intracellular solution, and temperature at the time of intracellular ice formation indicate that intracellular ice formation is primarily a function of a temperature-dependent nucleation event whose probability is low at temperatures higher than -5°C and high at temperatures lower than -20°C. Within this temperature range, the extent of supercooling increases the probability of intracellular ice formation. 37. Cryobiolog-y of Isolated Plant Altered Cellular Gas Content.
Protoplasts: J.
R.
III.
FERGUSON,
P. L. STEPONKUS, M. F. DOWGERT, AND R. L. LEVIN (Sibley School of Mechanical and Aerospace Engineering and Department of Agronomy, Cornell University, Ithaca, New York 14853).
The optical “blackening” or “flashing” of cells during intracellular ice formation has been purported to be due to the diffraction of light by very small ice crystals formed within the cells. Our observations, however, suggest that the primary cause of “black flashing” is the formation of many small gas bubbles with radii near the wavelength of the impinging light. These numerous gas bubbles then scatter the impinging light producing a black image. Thermodynamic models of gas solubility and cellular dehydration are combined and used to determine the temperature transition (-8 to - 12°C) over which gas bubble formation will occur. Although gas solubility increases with decreasing temperature, the concentration of the dissolved intracellular gases increases and exceeds the solubility limit of the system. Rapid phase changes of intracellular water preclude the gradual dissipation of the evolving gases and visible bubbles result. If intracellular ice formation occurs at temperatures above -5”C, gas bubble formation is not observed. At these temperatures, the amount of ice formed is not suffi cient to cause gas to exceed its solubility limit. Intracellular ice formation without gas bubble formation has been termed “white flashing.” 38. Cryobiology Cellular
of
Isolated
Plant
Protoplasts:
IV.
P. L. STEPONKUS, M. F. DOWGERT, R. L. LEVIN, AND J. F. FERGUSON (Department of Agronomy and Sibley School of Injury.
594
ABSTRACTS,
16th ANNUAL
Mechanical and Aerospace Engineering, Cornell University, Ithaca, New York 14853). During a freeze-thaw cycle, a cell encounters a spectrum of cellular stresses, and injury may be manifested as different cellular or membrane lesions as a result of the different stresses. The spectrum of lesions may be directly observed as they are manifested in altered cellular volumetric responses during a freeze-thaw cycle. Intracellular ice formation at high fractional cell volumes will result in the immediate physical disruption of the plasma membrane. Cellular dehydration may result in several lesions depending on the freeze-thaw protocol. If cells are cooled to -5”C, and subsequently warmed to +5”C, they will exhibit characteristic Boyle van’t Hoff behavior during both cooling and warming. During warming, however, they will lyse before regaining their initial size. If cells are cooled to temperatures lower than -5°C two additional forms of injury are observed. Following cooling, cells may exhibit a total loss of osmotic responsiveness and remain in the contracted state during warming. Such a manifestation is suggestive of a complete loss of semipermeability of the plasma membrane. Ahernatively, cells may exhibit altered osmometric behavior during warming. Such cells expand during warming but to a lower extent at any given osmolality than normal cells. While the cells remain physically intact, this manifestation is suggestive of a partial loss of semipermeability or leakiness of the plasma membrane. Each type of injury is probabilistic in nature such that its frequency of occurrence depends upon the conditions of the freeze-thaw regime. In view of the various lesions which result in cellular injury, proposals regarding the mechanism of freezing injury should consider the nature of cellular lesions in their interpretation of freezing injury. 39. Cryobiology of Isolated Plant Protoplasts: V. Contraction -Expansion-Induced Alterations in the Plasma Membrane. P. L. STEPONKUS,
M. F. DOWGERT, AND S. C. WIEST (Department of Agronomy, Cornell University, Ithaca, New York 14853). One form of freeze-thaw injury manifested by isolated protoplasts in an expansion-induced lysis during warming. Although osmometric behavior is best described as a function of volume, the lytic lesion resulting from volumetric changes is best described as a function of surface area of the plasma membrane. Protoplasts do not possess a fixed maximum surface area at which lysis occurs. Instead, the surface area at which lysis occurs is decreased if the protoplasts have been previously contracted. Freeze-thaw injury is the result of two major strains: a freeze- or contractioninduced membrane alteration which decreases the maximum critical surface area of the plasma membrane and a thaw- or expansion-induced dissolution of
MEETING
the plasma membrane which occurs when the maximum critical surface area is exceeded. In a population of cells, the absolute magnitude of change in the mean surface area of the population which results in lysis of 50% of the cells appears to be constant and independent of the extent of contraction. This value varies among species examined to date: 900 pm2 for spinach, 400 pm* for wheat, and 600 pm” for rye. Direct measurements of individual cells during a freeze-thaw cycle document the fact that cells contracted during freezing lyse before achieving their original surface area. Larger cells, however, undergo larger surface area increments than do smaller cells. Thus, the tolerable surface area increment is determined by the initial cell size but remains constant regardless of the extent of contraction experienced by cells of a given size. 40. Cryobiology fluence
of Isolated Plant Acclimation.
Protoplasts:
VI. In-
of Cold
P. L. STEPONKUS, M. F. DOWGERT, AND S. R. ROBERTS (Department of Agronomy, Cornell University, Ithaca, New York 14853).
Sensitivity of isolated protoplasts to freezing injury parallels that of the parent tissue from which they were derived and can be ameliorated by cold acclimation. When rye seedlings are cold acclimated, their hardiness increases from -3 to -26°C as determined by freezing of isolated crowns. Protoplasts isolated from these plants exhibit comparable levels of hardiness: 50% survival of those isolated from nonacclimated tissue occurs at -2.5”C while 50% survival of protoplasts isolated from plants acclimated for 4 weeks occurs at -28°C. Thus, the hardiness level of the parent tissue is manifested by the same degree of hardiness in the derived protoplasts frozen in vitro. Isolated protoplasts afford the possibility to determine which of several alterations associated with the cold acclimation contribute to the increased tolerance to a freeze-thaw cycle. These alterations include: (1) changes in the water permeability of the plasma membrane; (2) changes in cellular characteristics which influence osmometric behavior, specifically, solute content and the osmotically inactive volume: and (3) tolerance of the plasma membrane to contraction and expansion. The influence of cold acclimation on each of these factors is discussed. SESSION
4. CRYOSURGERY
41. Determination of the Influence of Experimental Local Freezing on Total Femoral Greater Trochanteric Apophyseal Grobtath Arrest. JACOB
F. KATZ AND ROBERT S. SIFFERT (Orthopaedic Research Laboratory, Department of Orthopaedics, Mt. Sinai School of Medicine of the City University of New York, 100th Street and Fifth Avenue, New York, New York 10029).