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Temporal aspects of alterations in the cryobehavior and lipid composition of the plasma membrane during cold acclimation of winter rye
ABSTRACTS,
26th ANNUAL
at 2O”C, and there is less than 20% survival after 30 min. When equilibrated in isotonic (0.6 Osm) sorbitol solutions containing increasing concentrations of permeating cryoprotectants (CPAs), such as ethylene glyco1 (EG), dimethyl sulfoxide (DMSO), or glycerol, the large decrease in survival occurs at somewhat higher concentrations. For example, survival of protoplasts equilibrated in a sorbitol solution containing 1.5 M EG (2.09 Osm) or DMSO (2.19 Osm) is approx 70% after 15 min with an additional 10% decrease after 60 min. At higher concentrations (e.g., 2.0 M CPA), the initial decrease in survival is much greater, with less than 40% survival of the protoplasts after just 15 min in EG or DMSO. In solutions containing glycerol, the decrease in survival is even greater. For example, in the isotonic sorbitol solution containing 1.5 M glycerol (2.16 Osm), survival decreases to less than 20% in 15 min. Somewhat paradoxically, the apparent toxicity of the CPAs is greater (i.e., there are larger decreases in survival) if the protoplasts are equilibrated in the hypertonic solutions at O’C, especially in the case of DMSO. The extreme sensitivity to hypertonic stresses is further exacerbated because of the relatively low solute permeabilities in tobacco protoplasts in comparison to rye protoplasts. For example, volumetric equilibration in 1 M EG solutions requires approx 45 min in tobacco; whereas only 12 min is required in rye protoplasts. Similarly, equilibration in DMSO requires approx 30 min in tobacco, but approx 10 min in rye. Several factors suggest that the apparent toxicity is associated with the extent and duration of protoplast dehydration. For example, the decrease in survival occurs within the first 15 min of exposure and only declines slightly after longer durations-when the extent of volumetric contraction is decreasing because of the influx of solute. Second, the order of apparent toxicity (DMSO < EG < glycerol) follows the order of the permeation rates of the CPAs. Also, the paradoxical situation that the apparent toxicity is greater if the protoplasts are equilibrated at 0 rather than 20°C is also consistent with the interpretation that apparent toxicity is greater if the influx of solutes is slowed. Finally, a two-step loading procedure is less injurious than a one-step procedure, i.e., survival is approximately twofold greater in protoplasts loaded with 1 M/2 EG or DMSO than in those loaded directly in solutions containing 2 M CPA. These observations indicate that the apparent toxicity is associated with protoplast dehydration that occurs during the initial stage of the loading procedure. (Supported by a gift from RJR-Nabisco, Inc.) 144. Alteration of the Freezing Tolerance of Rye Protoplasts by Selective Lipid Enrichment of the Plasma Membrane. MATSUO UEMURA AND PETER L. STEPONKUS (Department of Agronomy, Cornell University, Ithaca, New York 14853).
MEETING
585
The freezing tolerance of protoplasts isolated from nonacclimated rye leaves (Secale cereale L. cv Puma) can be significantly altered by using a protoplastliposome fusion technique to selectively modify the lipid composition of the plasma membrane. Enrichment of the plasma membrane of NA protoplasts (protoplasts isolated from leaves of nonacclimated seedlings) with mono- or diunsaturated species of phosphatidylcholine (PC) increases the freezing tolerance because of the resultant transformation in the cryobehavior of the plasma membrane during freeze-induced osmotic contraction. With NA protoplasts, osmotic contraction results in endocytotic vesiculation of the plasma membrane; whereas with NA protoplasts fused with liposomes of dilinoleoylphosphatidylcholine (DL,PC), osmotic contraction results in the formation of exocytotic extrusions, as occurs in protoplasts isolated from cold-acclimated leaves. The increase in freezing tolerance is dependent on the concentration of DL,PC used during fusion and plateaus at a concentration of 1 umol of lipid/lo6 protoplasts. Fusion with disaturated PC species (e.g., dimyristoyl-PC or dipahnitoyl-PC) has no effect on the freezing tolerance. Similarly, when fused with hposomes composed of various species of phosphatidylethanolamine (e.g., dimyristoyl-PE, I-palmitoyl-2-myristoyl-PE, or DL,PE) in mixtures with either DbPC or DMPC to facilitate liposome formation and fusion, there is no effect on the freezing tolerance. When fused with liposomes composed of DL,PC and sterols (B-sitosterol and campesterol), there is a negative effect on the freezing tolerance that cannot be entirely accounted for by a decrease in the amount of PC that is incorporated into the plasma membrane. These studies demonstrate that the transformation in the cryobehavior that occurs during natural cold acclimation is a consequence of the increases in mono- and diunsaturated PC species. However, the increase in freezing tolerance can also be elicited with diphytanoyl PC, which is not a natural component of the plasma membrane of rye. This lipid contains long-chain, saturated acyl chains (3,7,11, IS-tetramethylhexadecanoyl), which are branched because of the four methyl groups, and remains in a liquid crystalline phase at temperatures above - 55°C. These results suggest that the transformation in the behavior of the plasma membrane is related to the molecular packing characteristics of the lipid components or the disorder parameter of the bilayer and not necessarily specific for mono- or diunsaturated species of PC. (Supported by the United States Department of Agriculture Competitive Grants 85-CRCR-l-1651 and 88-37264-3988.) 145. Temporal Aspects of Alterations in the Cryobehavior and Lipid Composition of the Plasma Membrane during Cold Acclimation of Winter Rye. M. UEMURA, E. B. CAHOON, D. V.
586
ABSTRACTS,
26th ANNUAL
LYNCH, AND P. L. STEP~NKUS (Department of Agronomy, Cornell University, Ithaca, New York 14853). Typically, cold acclimation of rye seedlings is induced by exposure to temperatures of 5°C for periods of 4 to 6 weeks. During this time, the freezing tolerance of the leaves increases progressively from - 5°C to -25”C, and protoplasts isolated from the leaves reflect the same increase in freezing tolerance. When protoplasts isolated from nonacclimated leaves (NA protoplasts) are frozen to temperatures over the range of 0 to -5°C injury is a consequence of osmotic excursions incurred during the freeze/thaw cycle because freeze-induced osmotic contraction results in endocytotic vesiculation of the plasma membrane. Sufficiently large area reductions are irreversible, and the protoplasts lyse during osmotic expansion following thawing of the suspending medium. This form of injury is referred to as expansion-induced lysis (EIL). In protoplasts isolated from fully acclimated leaves (ACC protoplasts), EIL is not observed because the plasma membrane forms exocytotic extrusions during osmotic contraction, and the surface area of the plasma membrane is conserved such that ACC protoplasts can withstand freeze/thaw-induced osmotic excursions. This change in the cryobehavior of the plasma membrane (formation of exocytotic extrusions rather than endocytotic vesicles) occurs within the first 7 to 10 days of the cold acclimation period. After only 1 week of cold acclimation, the incidence of EIL is reduced to
MEETING
146. Cryomicroscopy of Plant Cell Suspensions Resistant and Recalcitrant to Cryopreservation. F. VAN IREN, E. W. M. SCHRJJNEMAKERS, AND M. R. MCLELLAN* (Department of Plant Molecular Biology, Leiden University, Nonnensteeg 3,231l VI Leiden, The Netherlands, and *Culture Collection of Algae and Protozoa (NERC), Freshwater Biology Association Ferry House, Ambleside, Cumbria, LA22 OLP, United Kingdom (presently Cell Systems, Cambridge, United Kingdom)). Plant cell suspension cultures are studied with various aims in many laboratories. Stable storage of such lines is important, e.g., with respect to the tendency of such lines to (genetic) instability, because maintenance is costly, and for patent applications. Cryopreservation of plant cell suspensions is routine now for about half of the lines tried, the other half appearing recalcitrant to (semihoutine protocols. The latter generally include pretreatment with a sugar alcohol for several days, cryoprotection with a two or three component mixture, two-step freezing, and rapid thawing. In this study, two plant cell suspension lines (Catharanthus roseus and Tabaernaemontana divaricata) were successfully cryopreserved. Two other lines (Cinchona robusta and another Tabaernaemontana divaricata) were recalcitrant to these procedures. Cryomicroscopy indicated that the recalcitrant lines suffered a range of lesions, including rapid deplasmolysis during thawing. The same phenomena were observed when Catharanthus was cryoprotected with a mixture from which it does not recover after cryopreservation. A significant proportion of the cells of resistant lines maintained plasmolysis during freezing and thawing, a small proportion of the population (- 10%) froze intracellularly, and the remainder showed the same phenomena as the recalcitrant lines. The results are tentatively explained. Excessive uptake of cryoprotectants is presumed to be one of the major phenomena leading to cell death. 147. Inhibition of Insect Ice Nucleator Proteins by Znsect Antifreeze Proteins. JOHN G. DUMAN (Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556). A number of overwintering insect species have thermal hysteresis producing antifreeze proteins in the hemolymph and gut fluids. Antifreeze proteins were purifted from the hemolymph of the larvae of the beetle Dendroides canadensis. From the same hemolymph a high-molecular-weight protein fraction with ice nucleator activity was isolated. The ice nucleator concentration of a buffer solution containing the protein ice nucleator was determined according to the method of Vali (1972). Additions of antifreeze protein to these
26th ANNUAL
at 2O”C, and there is less than 20% survival after 30 min. When equilibrated in isotonic (0.6 Osm) sorbitol solutions containing increasing concentrations of permeating cryoprotectants (CPAs), such as ethylene glyco1 (EG), dimethyl sulfoxide (DMSO), or glycerol, the large decrease in survival occurs at somewhat higher concentrations. For example, survival of protoplasts equilibrated in a sorbitol solution containing 1.5 M EG (2.09 Osm) or DMSO (2.19 Osm) is approx 70% after 15 min with an additional 10% decrease after 60 min. At higher concentrations (e.g., 2.0 M CPA), the initial decrease in survival is much greater, with less than 40% survival of the protoplasts after just 15 min in EG or DMSO. In solutions containing glycerol, the decrease in survival is even greater. For example, in the isotonic sorbitol solution containing 1.5 M glycerol (2.16 Osm), survival decreases to less than 20% in 15 min. Somewhat paradoxically, the apparent toxicity of the CPAs is greater (i.e., there are larger decreases in survival) if the protoplasts are equilibrated in the hypertonic solutions at O’C, especially in the case of DMSO. The extreme sensitivity to hypertonic stresses is further exacerbated because of the relatively low solute permeabilities in tobacco protoplasts in comparison to rye protoplasts. For example, volumetric equilibration in 1 M EG solutions requires approx 45 min in tobacco; whereas only 12 min is required in rye protoplasts. Similarly, equilibration in DMSO requires approx 30 min in tobacco, but approx 10 min in rye. Several factors suggest that the apparent toxicity is associated with the extent and duration of protoplast dehydration. For example, the decrease in survival occurs within the first 15 min of exposure and only declines slightly after longer durations-when the extent of volumetric contraction is decreasing because of the influx of solute. Second, the order of apparent toxicity (DMSO < EG < glycerol) follows the order of the permeation rates of the CPAs. Also, the paradoxical situation that the apparent toxicity is greater if the protoplasts are equilibrated at 0 rather than 20°C is also consistent with the interpretation that apparent toxicity is greater if the influx of solutes is slowed. Finally, a two-step loading procedure is less injurious than a one-step procedure, i.e., survival is approximately twofold greater in protoplasts loaded with 1 M/2 EG or DMSO than in those loaded directly in solutions containing 2 M CPA. These observations indicate that the apparent toxicity is associated with protoplast dehydration that occurs during the initial stage of the loading procedure. (Supported by a gift from RJR-Nabisco, Inc.) 144. Alteration of the Freezing Tolerance of Rye Protoplasts by Selective Lipid Enrichment of the Plasma Membrane. MATSUO UEMURA AND PETER L. STEPONKUS (Department of Agronomy, Cornell University, Ithaca, New York 14853).
MEETING
585
The freezing tolerance of protoplasts isolated from nonacclimated rye leaves (Secale cereale L. cv Puma) can be significantly altered by using a protoplastliposome fusion technique to selectively modify the lipid composition of the plasma membrane. Enrichment of the plasma membrane of NA protoplasts (protoplasts isolated from leaves of nonacclimated seedlings) with mono- or diunsaturated species of phosphatidylcholine (PC) increases the freezing tolerance because of the resultant transformation in the cryobehavior of the plasma membrane during freeze-induced osmotic contraction. With NA protoplasts, osmotic contraction results in endocytotic vesiculation of the plasma membrane; whereas with NA protoplasts fused with liposomes of dilinoleoylphosphatidylcholine (DL,PC), osmotic contraction results in the formation of exocytotic extrusions, as occurs in protoplasts isolated from cold-acclimated leaves. The increase in freezing tolerance is dependent on the concentration of DL,PC used during fusion and plateaus at a concentration of 1 umol of lipid/lo6 protoplasts. Fusion with disaturated PC species (e.g., dimyristoyl-PC or dipahnitoyl-PC) has no effect on the freezing tolerance. Similarly, when fused with hposomes composed of various species of phosphatidylethanolamine (e.g., dimyristoyl-PE, I-palmitoyl-2-myristoyl-PE, or DL,PE) in mixtures with either DbPC or DMPC to facilitate liposome formation and fusion, there is no effect on the freezing tolerance. When fused with liposomes composed of DL,PC and sterols (B-sitosterol and campesterol), there is a negative effect on the freezing tolerance that cannot be entirely accounted for by a decrease in the amount of PC that is incorporated into the plasma membrane. These studies demonstrate that the transformation in the cryobehavior that occurs during natural cold acclimation is a consequence of the increases in mono- and diunsaturated PC species. However, the increase in freezing tolerance can also be elicited with diphytanoyl PC, which is not a natural component of the plasma membrane of rye. This lipid contains long-chain, saturated acyl chains (3,7,11, IS-tetramethylhexadecanoyl), which are branched because of the four methyl groups, and remains in a liquid crystalline phase at temperatures above - 55°C. These results suggest that the transformation in the behavior of the plasma membrane is related to the molecular packing characteristics of the lipid components or the disorder parameter of the bilayer and not necessarily specific for mono- or diunsaturated species of PC. (Supported by the United States Department of Agriculture Competitive Grants 85-CRCR-l-1651 and 88-37264-3988.) 145. Temporal Aspects of Alterations in the Cryobehavior and Lipid Composition of the Plasma Membrane during Cold Acclimation of Winter Rye. M. UEMURA, E. B. CAHOON, D. V.
586
ABSTRACTS,
26th ANNUAL
LYNCH, AND P. L. STEP~NKUS (Department of Agronomy, Cornell University, Ithaca, New York 14853). Typically, cold acclimation of rye seedlings is induced by exposure to temperatures of 5°C for periods of 4 to 6 weeks. During this time, the freezing tolerance of the leaves increases progressively from - 5°C to -25”C, and protoplasts isolated from the leaves reflect the same increase in freezing tolerance. When protoplasts isolated from nonacclimated leaves (NA protoplasts) are frozen to temperatures over the range of 0 to -5°C injury is a consequence of osmotic excursions incurred during the freeze/thaw cycle because freeze-induced osmotic contraction results in endocytotic vesiculation of the plasma membrane. Sufficiently large area reductions are irreversible, and the protoplasts lyse during osmotic expansion following thawing of the suspending medium. This form of injury is referred to as expansion-induced lysis (EIL). In protoplasts isolated from fully acclimated leaves (ACC protoplasts), EIL is not observed because the plasma membrane forms exocytotic extrusions during osmotic contraction, and the surface area of the plasma membrane is conserved such that ACC protoplasts can withstand freeze/thaw-induced osmotic excursions. This change in the cryobehavior of the plasma membrane (formation of exocytotic extrusions rather than endocytotic vesicles) occurs within the first 7 to 10 days of the cold acclimation period. After only 1 week of cold acclimation, the incidence of EIL is reduced to
MEETING
146. Cryomicroscopy of Plant Cell Suspensions Resistant and Recalcitrant to Cryopreservation. F. VAN IREN, E. W. M. SCHRJJNEMAKERS, AND M. R. MCLELLAN* (Department of Plant Molecular Biology, Leiden University, Nonnensteeg 3,231l VI Leiden, The Netherlands, and *Culture Collection of Algae and Protozoa (NERC), Freshwater Biology Association Ferry House, Ambleside, Cumbria, LA22 OLP, United Kingdom (presently Cell Systems, Cambridge, United Kingdom)). Plant cell suspension cultures are studied with various aims in many laboratories. Stable storage of such lines is important, e.g., with respect to the tendency of such lines to (genetic) instability, because maintenance is costly, and for patent applications. Cryopreservation of plant cell suspensions is routine now for about half of the lines tried, the other half appearing recalcitrant to (semihoutine protocols. The latter generally include pretreatment with a sugar alcohol for several days, cryoprotection with a two or three component mixture, two-step freezing, and rapid thawing. In this study, two plant cell suspension lines (Catharanthus roseus and Tabaernaemontana divaricata) were successfully cryopreserved. Two other lines (Cinchona robusta and another Tabaernaemontana divaricata) were recalcitrant to these procedures. Cryomicroscopy indicated that the recalcitrant lines suffered a range of lesions, including rapid deplasmolysis during thawing. The same phenomena were observed when Catharanthus was cryoprotected with a mixture from which it does not recover after cryopreservation. A significant proportion of the cells of resistant lines maintained plasmolysis during freezing and thawing, a small proportion of the population (- 10%) froze intracellularly, and the remainder showed the same phenomena as the recalcitrant lines. The results are tentatively explained. Excessive uptake of cryoprotectants is presumed to be one of the major phenomena leading to cell death. 147. Inhibition of Insect Ice Nucleator Proteins by Znsect Antifreeze Proteins. JOHN G. DUMAN (Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556). A number of overwintering insect species have thermal hysteresis producing antifreeze proteins in the hemolymph and gut fluids. Antifreeze proteins were purifted from the hemolymph of the larvae of the beetle Dendroides canadensis. From the same hemolymph a high-molecular-weight protein fraction with ice nucleator activity was isolated. The ice nucleator concentration of a buffer solution containing the protein ice nucleator was determined according to the method of Vali (1972). Additions of antifreeze protein to these