The behavior of the plasma membrane following osmotic contraction of isolated protoplasts: Implications in freezing injury

Summary Following osmotic contraction of isolated rye protoplast (Secale cereale L. cv. Puma) that results in nearly a 50% reduction in volume, the plasma membrane was smooth, with no folding or pleating. Instead, deletion of plasma membrane occurred and numerous cytoplasmic vesicles were observed. As a result, the area of the plasma membrane was reduced by approximately 40%. Thin sections revealed that the cytoplasmic vesicles were membrane bound and not merely voids in the cytoplasm. High resolution video microscopy revealed the extent of vesiculation showing large clusters of cytoplasmic vesicles following osmotic contraction. Labeling the plasma membrane with fluorescein-Con-A prior to hypertonic contraction suggested that the cytoplasmic vesicles were derived from the plasma membrane. Freeze-fracture particle density on both the protoplasmic (PFp) and exoplasmic face (EFp) of the plasma membrane remained unchanged following contraction, which is consistent with a unit-membrane deletion into cytoplasmic vesicles. Upon partial re-expansion of the protoplasts, thin sections showed that the vesicles remained in the cytoplasm. These results using osmotic manipulation confirm earlier observations of isolated protoplasts at the light microscope level. Upon contraction plasma membrane is deleted into cytoplasmic vesicles, which are not readily reincorporated into the plasma membrane upon expansion. Lysis occurs before the original volume and surface area are regained.Department of Agronomy Series Paper no. 1456.     

The stress-strain relation of the plasma membrane of isolated plant protoplasts

Over periods of up to a few seconds the plasma membrane of isolated rye protoplasts behaves elastically with an area modulus of $\text{230}\text{mN}\text{·}\text{m}{}^{\mathrm{?1}}$. Over longer periods, the area increases with time under large tension and decreases under sufficiently small tension, suggesting that material is incorporated into or depleted from the plane of the membrane.     

Freeze-induced phase transitions in the plasma membrane of isolated protoplasts

Protoplasts were enzymically isolated from 2-week-old non-acclimated rye ( Secale cereale L. cv. Puma) seedlings. They were resuspended in isotonic sorbitol with different concentrations (0–10%) of dimethyl sulfoxide (DMSO). The survival of the protoplasts frozen in isotonic sorbitol solutions declined at temperatures below the freezing point with the LT₅₀ being -8°C. Addition of DMSO to the osmoticum increased survival at freezing temperatures. The optimum concentration of DMSO was 4% and lowered the LT₅₀ to -19°C. Freeze-fracture studies of the plasma membrane revealed aparticulate lipid lamellae at -4°C, but the first appearance of lateral phase separations, striations and inverted cylindrical micelles (hexagonal₁₁-type structures) occurred at -6°C. At lower temperatures, -8 and -10°C, the occurrence of nonbilayer structures became more common. The addition of DMSO decreased the incidence of the ultrastructural changes. With 2 or 4% DMSO, non-bilayer structures were not observed at temperatures above -10°C. Instead, striations and H₁₁-type structures were observed at - 15 and -20°C.     

Destabilization of the plasma membrane of isolated plant protoplasts during a freeze-thaw cycle: the influence of cold acclimation

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.     

Freeze-thaw injury to isolated spinach protoplasts and its simulation at above freezing temperatures

Possibilities to account for the mechanism of freeze-thaw injury to isolated protoplasts of Spinacia oleracea L. cv. Winter Bloomsdale were investigated. A freeze-thaw cycle to −3.9 C resulted in 80% lysis of the protoplasts. At −3.9 C, protoplasts are exposed to the equivalent of a 2.1 osmolal solution. Isolated protoplasts behave as ideal osmometers in the range of concentrations tested (0.35 to 2.75 osmolal), arguing against a minimum critical volume as a mechanism of injury. Average protoplast volume after a freeze-thaw cycle was not greatly different than the volume before freezing, arguing against an irreversible influx of solutes while frozen. A wide variety of sugars and sugar alcohols, none of which was freely permeant, were capable of protecting against injury which occurred when protoplasts were frozen in salt solutions. The extent of injury was also dependent upon the type of monovalent ions present, with Li = Na > K = Rb = Cs and Cl ≥ Br > I, in order of decreasing protoplast survival. Osmotic conditions encountered during a freeze-thaw cycle were established at room temperature by exposing protoplasts to high salt concentrations and then diluting the osmoticum. Injury occurred only after dilution of the osmoticum and was correlated with the expansion of the plasma membrane. Injury observed in frozen-thawed protoplasts was correlated with the increase in surface area the plasma membrane should have undergone during thawing, supporting the contention that contraction of the plasma membrane during freezing and its expansion during thawing are two interacting lesions which cause protoplast lysis during a freezethaw cycle.     

Plant plasma membrane water permeability and slow freezing injury

Abstract Evaluation of existing experimental evidence supports the hypothesis that, under natural or slow rates of freezing, plasma membrane permeability does not significantly limit water efflux from plant cells. Water efflux at slow freezing rates is controlled largely by the rate of heat removal from the plant cells. These conclusions are in direct contrast to the recent views of Levitt (1978).     

Cryomicroscopy of isolated plant protoplasts

It has been nearly 100 years since Müller-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.     

Uptake of isolated plant chromosomes by plant protoplasts

For mass isolation of plant metaphase chromosomes, cultured cells of wheat (Triticum monococcum) and parsley (Petroselinum hortense) were synchronized by hydroxyurea and colchicine treatment. This synchronization procedure resulted in high mitotic synchrony, especially in suspension cultures of parsley in which 80% of the cells were found to be at the metaphase stage. Mitotic protoplasts isolated from these synchronized cell cultures served as a source for isolation of chromosomes. The described isolation and purification method yielded relatively pure chromosome suspension. The uptake of the isolated plant chromosomes into recipient wheat, parsley, and maize protoplasts was induced by polyethylene-glycol treatment. Cytological studies provided evidences for uptake of plant chromosomes into plant protoplasts.Abbreviations PEG polyethylene glycol - HU hydroxyruea - C colchicine - HUC hydroxyurea and colchicine - CIM chromosome isolation medium - TCM Tris chromosome medium     

Polyethylene glycol and electric field treatment of plant protoplasts: characterization of some membrane properties

Petiolar protoplasts of a dihaploid line of winter oilseed rape Brassica napus L. ssp. oleifera were exposed to fusogenic polyethylene glycol (PEG) and electric field treatments. The surface properties and stability of membrane components of the treated protoplasts were investigated by contact angle measurements in aqueous two-phase systems and differential scanning calorimetry, respectively. The leakage of intracellular components was estimated with respect to amino acids, proteins and DNA. Both fusogenic treatments resulted in the same apparent changes in membrane surface hydrophobicity and the same destabilization of membrane components. However, the PEG-treated protoplasts were more leaky than both the control and the electric field-treated protoplasts. The results indicate that the molecular mechanisms of PEG- and electrical field-induced fusion are similar. However, the effects of the latter appear to be less harmful presumably because the parameters for electric field treatment are more easily controlled.     

Dynamics of membrane exchange of the plasma membrane and the lysis of isolated protoplasts during rapid expansions in area

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 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.     

Alterations in membrane transport properties by freezing injury in herbaceous plants:

Leakage of ions from a thawed tissue is a common phenomenon of freezing injury. This leakage is usually assumed to be due to loss of membrane semipermeability or membrane rupture by freezing injury. Freeze injured, yet living, onion (Allium cepa L.) epidermal cells were used to study alterations in cell membranes that result in leakage of ions. In spite of a large efflux of ions, freeze injured cells could be plasmolysed and they remained plasmolysed for several days just like the unfrozen control cells. Injured cells also exhibited protoplasmic streaming. Passive transport of KCl, urea and methyl urea across the cell membranes of injured and control cells was also studied. No difference could be detected for the transport rates of urea and methyl urea between control and injured cells. However, a dramatic increase in the transport rate of KCl was found for the injured cells. Depending upon the extent of initial freezing injury, an increase or a decrease in injury symptoms was found in the post-thaw period. During the progress of freezing injury, 10 days after thawing, a swelling of the protoplasm was seen in the irreversibly injured cells. In spite of this swelling, these cells could be plasmolysed. It appears that the high amount of K⁺ that leaks out into the extracellular water, due to freezing injury, causes protoplasmic swelling by replacing Ca²⁺ in the plasma membrane. We conclude that protoplasmic swelling is a sign of secondary injury. The results presented in this study show that membrane semipermeability is not completely lost and membrane rupture does not occur during the initial stage of freezing injury. In fact, the cells have the ability to repair damage depending upon the degree of injury. Our results show there are specific alterations in membrane semipermeability (e.g., transport of K⁺) which could be repaired completely depending on the degree of injury. These findings suggest that ion leakage due to freezing injury is due to alteration in the membrane proteins and not in the membrane lipids.     

Ultrastructure of nuclei isolated from plant protoplasts

Summary A simple method, involving selective Triton X-100 membrane solubilization, has been developed for the isolation of nuclei from barley and tobacco protoplasts which gives a high yield of essentially pure nuclei. The isolated nuclei resembled those in leaf cells and protoplasts when the isolated nuclei were fixed for short times (2 hours, Medium II), except that their chromatin appeared to be more highly condensed and barley nuclei also lacked the outer nuclear membrane. When longer times of fixation (12 hours, Medium I) were used, the isolated nuclei lacked the characteristic condensed chromatin appearance.     

Transplantation of isolated nuclei into plant protoplasts

The uptake of isolated nuclei from Vicia hajastana Grossh. cells into protoplasts of an auxotrophic cell line of Datura innoxia P. Mill. was induced under the influence of polyethylene glycol and Ca²⁺ at pH 6.8. The frequency of nuclear uptake varied from 0.8 to 2.3% and that of the recovery of prototrophic clones from 10^-5 to 6·10^-4. The prototrophic nuclear fusion products following nuclear uptake could be rescued by initial culture of the protoplasts in non-selective conditions and by the subsequent use of feeder cell layers to support the growth of surviving colonies on a selective medium. The presence of Vicia genomic DNA in some prototrophic clones was confirmed by dot-blot hybridization using Datura and Vicia DNA probes. In certain transformed clones, the recovery of prototrophy was accompanied by the restoration of morphogenetic potential. Welldeveloped shoots typical of wild-type Datura could be regenerated employing an appropriate regeneration medium.Abbreviations MS Murashige and Skoog (1962) - PEG polyethylene glycol     

The plasma membrane of Avena coleoptile protoplasts

Treatment of living protoplasts from the Avena coleoptile with enzymes and chemicals has provided new information about the external surface of the plasma membrane. Treatments with selected detergents and polyene antibiotics indicate that little sterol is present. The lysis of protoplasts in carboxymethyl-RNase which is enzymatically almost inactive provides strong evidence that the lysis previously observed in RNase is not an indication of RNA in the membrane. Divalent cations inhibit the RNase-induced lysis, indicating that such lysis involves the interaction of RNase with negatively charged sites on the plasma membrane surface. Tyrosinase treatment gives no lysis, showing that tyrosine does not play the role in these plasma membranes attributed to it in some animal cells. Peroxidase does not harm coleoptile protoplasts.     

Effect of cold acclimation on the incidence of two forms of freezing injury in protoplasts isolated from rye leaves

The freezing tolerance and incidence of two forms of freezing injury (expansion-induced lysis and loss of osmotic responsiveness) were determined for protoplasts isolated from rye leaves (Secale cereale L. cv Puma) at various times during cold acclimation. During the first 4 weeks of the cold acclimation period, the LT₅₀ (i.e. the minimum temperature at which 50% of the protoplasts survived) decreased from −5°C to −25°C. In protoplasts isolated from nonacclimated leaves (NA protoplasts), expansion-induced lysis (EIL) was the predominant form of injury at the LT₅₀. However, after only 1 week of cold acclimation, the incidence of EIL was reduced to less than 10% at any subzero temperature; and loss of osmotic responsiveness was the predominant form of injury, regardless of the freezing temperature. Fusion of either NA protoplasts or protoplasts isolated from leaves of seedlings cold acclimated for 1 week (1-week ACC protoplasts) with liposomes of dilinoleoylphosphatidylcholine also decreased the incidence of EIL to less than 10%. Fusion of protoplasts with dilinoleoylphosphatidylcholine diminished the incidence of loss of osmotic responsiveness, but only in NA protoplasts or 1-week ACC protoplasts that were frozen to temperatures over the range of -5 to -10°C. These results suggest that the cold acclimation process, which results in a quantitative increase in freezing resistance, involves several different qualitative changes in the cryobehavior of the plasma membrane.     

Roles of the plasma membrane and the cell wall in the responses of plant cells to freezing

In an effort to clarify the responses of a wide range of plant cells to freezing, we examined the responses to freezing of the cells of chilling-sensitive and chilling-resistant tropical and subtropical plants. Among the cells of the plants that we examined, those of African violet ( Saintpaulia grotei Engl.) leaves were most chilling-sensitive, those of hypocotyls in mungbean [ Vigna radiata (L.) R. Wilcz.] seedlings were moderately chilling-sensitive, and those of orchid [ Paphiopedilum insigne (Wallich ex Lindl.) Pfitz.] leaves were chilling-resistant, when all were chilled at -2 degrees C. By contrast, all these plant cells were freezing-sensitive and suffered extensive damage when they were frozen at -2 degrees C. Cryo-scanning electron microscopy (Cryo-SEM) confirmed that, upon chilling at -2 degrees C, both chilling-sensitive and chilling-resistant plant cells were supercooled. Upon freezing at -2 degrees C, by contrast, intracellular freezing occurred in Saintpaulia leaf cells, frost plasmolysis followed by intracellular freezing occurred in mungbean seedling cells, and extracellular freezing (cytorrhysis) occurred in orchid leaf cells. We postulate that chilling-related destabilization of membranes might result in the loss of the ability of the plasma membrane to act as a barrier against the propagation of extracellular ice in chilling-sensitive plant cells. We also examined the role of cell walls in the response to freezing using cells in which the plasma membrane had been disrupted by repeated freezing and thawing. In chilling-sensitive Saintpaulia and mungbean cells, the cells with a disrupted plasma membrane responded to freezing at -2 degrees C by intracellular freezing. By contrast, in chilling-resistant orchid cells, as well as in other cells of chilling-resistant and freezing-resistant plant tissues, including leaves of orchard grass ( Dactylis glomerata L.), leaves of Arabidopsis thaliana (L.) Heynh. and cortical tissues of mulberry ( Morus bombycis Koids.), cells with a disrupted plasma membrane responded to freezing by extracellular freezing. Our results indicate that, in the chilling-sensitive plants cells that we examined, not only the plasma membrane but also the cell wall lacked the ability to serve as a barrier against the propagation of extracellular ice, whereas in the chilling-resistant plant cells that we examined, not only the plasma membrane but also the cell wall acted as a barrier against the propagation of extracellular ice. It appears, therefore, that not only the plasma membrane but also the cell wall greatly influences the freezing behavior of plant cells.     

Acidification of culture media by isolated plant protoplasts

Summary We report the observation of a decrease in media pH caused by isolated protoplasts after alkalinization of the culture medium. Additions of other cations or anions did not produce a similar response. Dinitrophenol immediately terminated the response. The acidification response was larger in suspensions that were cultured in auxins. The responses of protoplasts to changes in external pH may provide a means for assessing viability of nondividing protoplasts.