Search

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.

In conclusion, isolated protoplasts are an excellent arena in which destabilization of the plasma membrane can be directly observed during a freeze-thaw cycle by cryomicroscopy. Destabilization is manifested in various ways—Intracellular ice formation, loss of osmotic responsiveness, or expansion-induced lysis. The incidence of any particular form of injury will depend on the freeze-thaw protocol and hardiness of the tissue from which the protoplasts were isolated. In all cases, however, cold acclimation directly increases the stability of the plasma membrane to the multiple stresses that arise during a freeze-thaw cycle. Such observations provide for functional differences in the plasma membrane that may now be used to consider the significance of any compositional changes in the membrane that might be determined.

Micro-osmotic manipulation was used to determine the influence of osmotic contraction on the expansion potential of individual protoplasts isolated from rye ( Secale cereale L. cv Puma) leaves. For protoplasts isolated from leaves of nonacclimated plants (NA protoplasts), osmotic contraction in sufficiently hypertonic solutions (>1.53 osmolal) predisposed the protoplasts to lysis during osmotic expansion when they were returned to isotonic conditions (0.53 osmolal). In contrast, for protoplasts isolated from leaves of cold acclimated plants (ACC protoplasts), osmotic contraction in either 2.6 or 4.0 osmolal solutions was readily reversible. Following osmotic contraction, the resting tension (γ r ) of NA protoplasts was similar to that determined for protoplasts in isotonic solutions ( i.e. 110 ± 22 micronewtons per meter). In contrast, γ r of ACC protoplasts decreased from 164 ± 27 micronewtons per meter in isotonic solutions to values close to zero in hypertonic solutions. Following expansion in hypotonic solutions, γ r 9s of both NA and ACC protoplasts were similar for area expansions over the range of 1.3 to 1.6. Following osmotic contraction and reexpansion of NA protoplasts, hysteresis was observed in the relationship between γ r and surface area—with higher values of γ r at a given surface area. In contrast, no hysteresis was observed in this relationship for ACC protoplasts. Direct measurements of plasma membrane tension (γ) during osmotic expansion of NA protoplasts from hypertonic solutions (1.53 osmolal) revealed that γ increased rapidly after small increments in surface area, and lysis occurred over a range of 1.2 to 8 millinewtons per meter. During osmotic expansion of ACC protoplasts from hypertonic solutions (2.6 osmolal), there was little increase in γ until after the isotonic surface area was exceeded. These results are discussed in relation to the differences in the behavior of the plasma membrane of NA and ACC protoplasts during osmotic contraction ( i.e. endocytotic vesiculation versus exocytotic extrusion) and provide a mechanistic interpretation to account for the differential sensitivity of NA and ACC protoplasts to osmotic expansion from hypertonic solutions.

Cryomicroscopy of protoplasts isolated from nonacclimated (NA) rye leaves ( Secale cereale L. cv Puma) revealed that the predominant form of injury following cooling to the minimum temperature for 50% survival (LT 50 ) (−5°C) 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 LT 50 , 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 ( 50 (−25°C) was loss of osmotic responsiveness. The tolerable surface area increment (TSAI) which resulted in lysis of 50% of a population (TSAI 50 ) 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 TSAI 50 values obtained from osmotic manipulation. The TSAI 50 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.

When cooled at rapid rates to temperatures between -10 and -30 degrees 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 similar under conditions that resulted in the greatest difference in the incidence of intracellular ice formation-cooling to -15 or -20 degrees C at rates of 10 or 16 degrees C/minute. Further, the hydraulic conductivity determined during freeze-induced dehydration at -5 degrees 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 degrees C, whereas in acclimated protoplasts, nucleation occurred over a broader temperature range with a median nucleation temperature of -42 degrees 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 has been nearly 100 years since Muller-Thurgau (26) employed cryomicroscopy to identify the cooling rate dependency of intracellular ice formation. Since that time cryomicroscopy has advanced from the “ice age” when Molisch (23) packed his microscope in ice to the “space age” of today when computer hardware developed for space satellite imagery is used for cryomicroscopic image analysis. Although interest in cryomicroscopy has been sporadic in the intervening period, current interest is at a high level—largely as a result of the refinement in the cryomicroscope design by Diller and Cravalho (9). The increased sophistication in cryostage design and precision of temperature control allow for quantitative studies of cell behavior during a freeze-thaw cycle. Not only does quantitative video image analysis facilitate this task, but it provides for increased resolution of cellular and subcellular responses during the freeze-thaw cycle. Most importantly, cryomicroscopy presents a researcher with a panorama of cellular behavior within which existing facts can be placed in perspective and from which future experiments can be more accurately focused.

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 cold-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.