Behavior of the Plasma Membrane of Isolated Protoplasts during a Freeze-Thaw Cycle

Abstract

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...

Current Issues in Plant Cryopreservation

“…Any damage to the cell membrane can alter the delicate balance between intra and extracellular solutes, leading to cell death (Anchordoguy et al, 1987;Dowgert & Steponkus, 1984;Gordon-Kamm & Steponkus, 1984;Lynch & Steponkus, 1987). Lipid peroxidation of fatty acids (FA) in phospholipids can cause extensive damage to the cell membrane if the chain reaction is not controlled, leading to large areas where the semi-permeability of the membrane is altered and thus can no longer function normally (Benson et al, 1992;Halliwell & Gutteridge, 2007).…”

Effect of Cold Acclimation on the Incidence of Two Forms of Freezing Injury in Protoplasts Isolated from Rye Leaves

“…The final osmolality of the solution was adjusted so that it was isotonic to the protoplast sample. The reaction was carried out at 28°C for 3 min after which 5 From our previous studies (10), we know that, in NA protoplasts, injury over the range of 0 to -5°C is a consequence of EIL; whereas at lower temperatures, injury is manifested as LOR and is a consequence of lamellar-to-II hexagonalI, phase transitions in the plasma membrane. Further, in 4-week ACC protoplasts, EIL occurs at only a low frequency and LOR is not associated with lamellar-tohexagonal11 phase transitions.…”

“…When frozen to temperatures over the range of 0 to -5°C, injury is a consequence of osmotic excursions incurred during the freeze/thaw cycle. Freeze-induced osmotic contraction results in endocytotic vesiculation of the plasma membrane and the surface area of the plasma membrane is reduced (5,7). Sufficiently large area reductions are irreversible and the protoplasts lyse during osmotic expansion following thawing of the suspending medium-before regaining their initial size.…”

“…When frozen to lower temperatures (e.g. -10°C), dehydration is more severe and injury is manifested as a complete loss of osmotic responsiveness (LOR) (5). This form of injury is a consequence of dehydration-induced alterations in the ultrastructure of the plasma membrane that include the formation of large aparticulate domains and lamellar-to-hexagonal,1 phase transitions (6).…”

“…Following cold acclimation the behavior of the plasma membrane during a freeze/thaw cycle is altered such that freeze-induced osmotic contraction results in the reversible formation of exocytotic extrusions of the plasma membrane (8) and, as a result, EIL occurs at only a very low frequency in ACC protoplasts (5). Further, although injury in ACC protoplasts at the LT50(-50C) is manifested as a loss ofosmotic responsiveness, dehydration-induced lamellar-to-hexagonal11 phase transitions are not observed at any subzero temperature-even at -35°C (6).…”