ABSTRACICryomicroscopy of protoplasts isolated from nonacclimated (NA) rye leaves (Secak cereal L. cv Puma) revealed that the predominant form of injury following cooling to the minimum temperature for 50% survival (LT5s) (-5C) was expansion-induced lysis of the plasma membrane during warming and thawing of the suspending medium when the decreasing osmolality resulted in osmotic expansion of the protoplasts. When cooled to temperatures below the LT50, the predominant form of injury was loss of osmotic responsiveness following cooling so that the protoplasts were osmotically inactive during warming. Only a low incidence (<10%) of expansion-induced lysis was observed in protoplasts isolated from acclimated (ACC) leaves, and the predominant form of injury following cooling to the LT50 (-25°C) was loss of osmotic responsiveness. The tolerable surface area increment (TSAI) which resulted in lysis of 50% of a population (TSAI5.) of NA protoplasts osmotically expanded from isotonic solutions was 1122 ± 172 square micrometers. Similar values were obtained when the protoplasts were osmotically expanded from hypertonic solutions. The TSAI determined from cryomicroscopic measurements of individual NA protoplasts was similar to the TSAI5, values obtained from osmotic manipulation. The TSAIso of ACC protoplasts expanded from isotonic solutions (2145 ± 235 square micrometers) was approximately double that of NA protoplasts and increased following osmotic contraction. Osmotic contractions were readily reversible upon return to isotonic solutions. During freeze-induced dehydration, endocytotic vesicles formed in NA protoplasts whereas exocytotic extrusions formed on the surface of ACC protoplasts. During osmotic expansion following thawing of the suspending medium, the endocytotic vesicles remained in the cytoplasm of NA protoplasts and the protoplasts lysed before their original volume and surface area were regained. In contrast, the exocytotic extrusions were drawn back into the surface of ACC protoplasts as the protoplasts regained their original volume and surface area.One form offreeze-thaw injury to isolated protoplasts is termed expansion-induced lysis (19,20,22). Following osmotically induced contraction, the plasma membrane lyses upon reexpansion before the original surface area is regained. Levitt and Scarth (7,9) were among the first to note plasmolysis and deplasmolysis may result in lysis of the plasma membrane, and they referred to this form of injury as 'deplasmolysis injury'. They observed that protoplasts of intact cells lysed during expansion following osmotically induced contraction. The (20). For a population of protoplasts, the absolute magnitude of the increase in surface area that results in lysis of 50% of the population is denoted as the TSAI1o. On the basis of the TSAI5o and the characteristic Boyle-van't Hoff behavior ofa population ofspinach protoplasts, it has been inferred that expansion-induced lysis quantitatively accounts for freezethaw injury to these protoplasts cooled to the LT5o (22). Pre...

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.Key Words membrane mechanics 9 plant protoplasts 9 osmotic expansion 9 cold acclimation

Summary.The plasma membrane of protoplasts isolated from rye leaves (Secale cereale L. cv. Puma) can withstand a maximum elastic stretching of about 2%. Larger area expansions involve the incorporation of new material into the membrane. The dynamics of this process during expansion from isotonic solutions and the probable frequency of lysis have been measured as a function of membrane tension in populations of protoplasts isolated from both co/d-acclimated and nonacclimated plants. To a first approximation, both increase exponentially with tension. An analytical solution is reported for the membrane tension as a function of time during an arbitrary expansion in area.

When cooled at rapid rates to temperatures between -10 and -30°C, the incidence of intracellular ice formation was less in protoplasts enzymically isolated from cold acclimated leaves of rye (Secale cereale L. cv Puma) than that observed in protoplasts isolated from nonacclimated leaves. The extent of supercooling of the intracellular solution at any given temperature increased in both nonacclimated and acclimated protoplasts as the rate of cooling increased. There was no unique relationship between the extent of supercooling and the incidence of intracellular ice formation in either nonacclimated or acclimated protoplasts. In both nonacclimated and acclimated protoplasts, the extent of intracellular supercooling was snimlar under conditions that resulted in the greatest difference in the incidence of intracellular ice formation-cooling to -15 or -20°C at rates of 10 or 16'C/mitute. Further, the hydraulic conductivity determined during freeze-induced dehydration at -5°C was similar for both nonacclimated and acclimated protoplasts. A major distinction between nonacclimated and acclimated protoplasts was the temperature at which nucleation occurred. In nonacclimated protoplasts, nucleation occurred over a relatively narrow temperature range with a median nucleation temperature of -15°C, whereas in acclimated protoplasts, nucleation occurred over a broader temperature range with a median nucleation temperature of -42°C. We conclude that the decreased incidence of intracellular ice formation in acclimated protoplasts is attributable to an increase in the stability of the plasma membrane which precludes nucleation of the supercooled intracellular solution and is not attributable to an increase in hydraulic conductivity of the plasma membrane which purportedly precludes supercooling of the intracellular solution.It is commonly observed that the incidence of intracellular ice formation is a function of the cooling rate. As early as 1886, observed that rapid cooling resulted in intracellular ice formation whereas during slow cooling ice crystals were confined to extracellular spaces. Similar rates; and the higher the permeability to water, the lower the probability of intracellular ice formation. Further, because the deplasmolysis time of cells in cold hardy tissues was less than that for cells in nonhardy tissues, they inferred that water permeability increased following cold acclimation and that the incidence of intracellular ice formation would be less in acclimated tissues. In 1938, Siminovitch and Scarth (23) experimentally demonstrated that intracellular ice formation occurs less frequently in hardy tissues than nonhardy tissues when cooled at comparable rates. They too attributed this difference to the purported increase in water permeability of the plasma membrane following cold acclimation as proposed by Levitt and Scarth (10). Although this concept has been widely accepted and frequently cited, direct experimental verification of an increase in water permeability following cold acclimation has been...

No abstract