A freeze-etch study of the effects of extracellular freezing on cellular membranes of wheat

Seedlings of Triticum aestivum L. cv. Lennox were grown in different environments to obtain different hardiness. Pieces of laminae and leaf bases were slowly cooled to sub-zero temperatures and the damage caused was assessed by an ion-leakage method. Comparable pieces of tissue were slowly cooled to temperatures between 2° and-14°C and were then freeze-fixed and freeze-etched. Membranes generally retained their lamellar structures indicated by the abundance of typical membrane fracture faces in all treatments, and some membrane fracture faces had patches which lacked the usual scattering of intramembranous particles (IMP). These IMP-free areas were present in the plasma membrane of tissues given a damaging freezing treatment, but were absent from the plasma membrane of room-temperature controls, of supercooled tissues, and of tissues given a non-damaging freezing treatment. The frequency of IMP-free areas and the proportion of the plasma membrane affected increased with increasing damage. In the most damaged tissue (79% damage; leaf bases exposed to-8°C), 20% of the plasma membrane was IMP-free. The frequencies of IMP at a distance from the IMP-free areas were unaffected by freezing treatments. There was a patchy distribution of IMP in other membranes (nuclear envelope, tonoplast, thylakoids, chloroplast envelope), but only in the nuclear envelope did it appear possible that their occurrence coincided with damage. The IMP-free areas of several membranes were sometimes associated together in stacks. Such membranes lay both to the outside and inside of the plasma membrane, indicating that at least some of the adjacent membrane fragments arose as a result of membrane reorganization induced by the damaging treatment. Occasional views of folded IMP-free plasma membrane tended to confirm this conclusion. The following hypothesis is advanced to explain the damage induced by extracellular freezing. Areas of plasma membrane become free of IMP, probably as a result of the freezing-induced cellular dehydration. The lipids in these IMP-free patches may be in the fluid rather than the gel phase. The formation of these IMP-free patches, especially in the plasma membrane, initiates or involves proliferation and possibly fusion of membranes, and during or following this process, the cells become leaky.Abbreviations EF exoplasmatic fracture face - IMP intramembranous particles - PF protoplasmatic fracture face     

The membranes of slowly drought-stressed wheat seedlings: a freeze-fracture study

Seedlings of Triticum aestivum L. cv. Neepawa were slowly drought-stressed by witholding water after sowing in pots. Leaf extension stopped during development of the third leaf. Damage was assessed by rewatering the pots and measuring regrowth; 1–5 d after growth stopped, rewatering induced significant regrowth within several hours; 6–13 d after growth stopped, regrowth was delayed; from 14 d after growth stopped, no regrowth occurred after rewatering. Leaf bases were excised from the drought-stressed seedlings during this period of increasing damage, and were freeze-etched.Intramembranous particles (IMP) were evenly scattered in the plasma membrane in those plants which regrew immediately after rewatering. In the plants which regrew after a delay or which did not regrow on rewatering, there were patches without IMP in plasma membrane, nuclear envelope, and other membranes. Plasma membrane, nuclear envelope and possibly other membranes were sometimes partly replaced by vesicles, possibly formed from the original membrane. Such vesiculation occurred in a few cells in plants which survived the stress with a delayed regrowth, and was commoner in the plants which did not recover. The results support the idea that slow drought induces IMP-free patches in membranes including the plasma membrane, this induces membrane reorganisation including vesiculation of membranes and coagulation of protoplasm, and that these are expressed as delayed or failed regrowth. Some IMP-free patches in the plasma membrane had a faint ordered sub-structure, possibly a hexagonal lipid phase. Such patches were infrequent and IMP sometimes occurred in areas of plasma membrane having an apparently similar sub-structure. Thus the IMP-free patches could not be explained by a lamellar-hexagonal phase transition. As the stress became damaging, vesicles and endoplasmic reticulum accumulated immediately next to the plasma membrane. Mainly during the early period of damaging stress (6–10 d after growth stopped), depressions, invaginations, and rarer lesions occurred in the plasma membrane, sometimes associated with some of the IMP-free patches. In the same period, many nuclear envelopes had exceptionally large nuclear pores.Abbreviations E exoplasmic - IMP intramembranous particles - P protoplasmic     

Reduction of intramembranous particles in the periacrosomal plasma membrane of boar spermatozoa during in vitro capacitation: a statistical study

Membrane remodeling in the periacrosomal plasma membrane (PAPM) of boar spermatozoa during incubation in capacitation medium was examined by the freeze-fracture technique. In the preservation medium (PM) group, the major small (about 8 nm) intramembranous particles (IMP) and the minor large (> 10 nm) IMP were distributed evenly in the PAPM. The IMP-free area increased during capacitation. To correct the IMP-free area, arithmetically redistributed (ARD)-IMP density was used for statistical analysis. In the PM group, the mean density +/- SD of large IMP was 379 +/- 64 and 266 +/- 58/microm2, and that of small IMP was 1450 +/- 155 and 672 +/- 252/microm2 in protoplasmic (P) and external (E) faces, respectively. During capacitation, the significant (P < 0.01) reduction of large IMP density was encountered only in the E face of a few incubation groups, while that of the small IMP density occurred in the P face by 2 h. Consequently, reduction of the total IMP density of both faces was not significant in the large IMP, but it was significant (P < 0.01) in the small IMP. One-fifth of the total small IMP density reduced by 2 h. Filipin-sterol complexes (FSC) were numerous in the PAPM, and FSC-free areas also increased during capacitation. The mechanism of IMP-free area formation and the behavior of the small IMP in the PAPM during capacitation were discussed in relation to membrane stability.     

Wheat tissues freeze-etched during exposure to extracellular freezing: distribution of ice

Pieces excised from leaf bases and laminae of seedlings of Triticum aestivum L. cv. Lennox were slowly frozen, using a specially designed apparatus, to temperatures between 2° and 14° C. These treatments ranged from non-damaging to damaging, based on ion-leakage tests to be found in the accompanying report (Pearce and Willison 1985, Planta 163, 304–316). The frozen tissue pieces were then freeze-fixed by rapidly cooling them, via melting Freon, to liquid-nitrogen temperature. The tissue was subsequently prepared for electron microscopy by freeze-etching. Ice crystals formed during slow freezing would tend to be much larger than those formed during subsequent freeze-fixation. Ice crystals surrounding the excised tissues were much larger in the frozen than in the control tissues (the latter rapidly freeze-fixed from room temperature). Large ice crystals were present between cells of frozen laminae and absent from controls. Intercellular spaces were infrequent in control leaf bases and no ice-filled intercellular spaces were found in frozen leaf bases. Intracellular ice crystals were smaller in frozen tissues than in controls. It is concluded that all ice formation before freeze-fixation was extracellular. This extracellular ice was either only extra-tissue (leaf bases), or extra-tissue and intercellular (laminae). Periplasmic ice was sometimes present, in control as well as slowly frozen tissues, and the crystals were always small; thus they were probably formed during freeze-fixation rather than during slow freezing. The plasma membrane sometimes showed imprints of cell-wall microfibrils. These were less abundant in leaf bases at 8° C than in controls, and were present on only a minority of plasma membranes from laminae. Therefore, extracellular ice probably did not compress the cells substantially, and changes in cell size and shape were possibly primarily a result of freezing-induced dehydration. Fine-scale distortions (wrinkles) in the plasma membrane, while absent from controls, were present, although only rarely, in both damaged and non-damaged tissues; they were therefore ice-induced but not directly related to the process of damage.     

Expression levels of the Na + /K + transporter OsHKT2;1 and vacuolar Na + /H + exchanger OsNHX1 , Na enrichment,maintaining the photosynthetic abilities and growth performances of indica rice seedlings under salt stress

Salt affected soil inhibits plant growth, development and productivity, especially in case of rice crop. Ion homeostasis is a candidate defense mechanism in the salt tolerant plants or halophyte species, where the salt toxic ions are stored in the vacuoles. The aim of this investigation was to determine the OsNHX1 (a vacuolar Na+/H+ exchanger) and OsHKT2;1 (Na+/K+ transporter) regulation by salt stress (200 mM NaCl) in two rice cultivars, i.e. Pokkali (salt tolerant) and IR29 (salt susceptible), the accumulation of Na+ in the root and leaf tissues using CoroNa Green® staining dye and the associated physiological changes in test plants. Na+ content was largely increased in the root tissues of rice seedlings cv. Pokkali (15 min after salt stress) due to the higher expression of OsHKT2;1 gene (by 2.5 folds) in the root tissues. The expression of OsNHX1 gene in the leaf tissues was evidently increased in salt stressed seedlings of Pokkali, whereas it was unchanged in salt stressed seedlings of IR29. Na+ in the root tissues of both Pokkali and IR29 was enriched, when subjected to 200 mM NaCl for 12 h and easily detected in the leaf tissues of salt stressed plants exposed for 24 h, especially in cv. Pokkali. Moreover, the overexpression of OsNHX1 gene regulated the translocation of Na+ from root to leaf tissues, and compartmentation of Na+ into vacuoles, thereby maintaining the photosynthetic abilities in cv. Pokkali. Overall growth performance, maximum quantum yield (Fv/Fm), photon yield of PSII (ΦPSII) and net photosynthetic rate (Pn) was improved in salt stressed leaves of Pokkali than those in salt stressed IR29.     

Freeze-fracture study of the site of attachment of Cryptosporidium muris in gastric glands.

The mode and organization of the attachment site of Cryptosporidium muris to gastric glands of stomach were investigated by the freeze-fracture method. Cryptosporidium muris was enveloped by a double membrane, of host plasma membrane origin, which formed the parasitophorous vacuole. The outer membrane of the double membrane was continuous with host plasma membrane, while the inner membrane was connected with the anterior part of the parasite plasma membrane at the annular ring. The density of intramembranous particles (IMP) was severely altered at the above two junctures. The parasitophorous outer membrane showed low IMP-density when compared to the host plasma membrane, although both membranes were continuous at the dense band. The inner membrane had few IMP, whereas the parasite plasma membrane showed numerous IMP, although both membranes were continuous at the annular ring. The size of dense band and annular ring was similar in diameter. The feeder organelle was clearly visible as membrane folds in freeze-fracture and some of them were connected with small vesicles of cytoplasm, indicating that the feeder organelle may play an important role for incorporation of nutrients from the host cell.     

Intramembrane changes occurring during maturation of herpes simplex virus type 1: freeze-fracture study.

During the maturation of two strains of herpes simplex virus type 1 (VR3 and Patton), intramembrane changes were detected with the freeze-fracture technique in the viral envelope and the infected cell plasma membrane, and these changes were compared with data obtained from thin sections. Regardless of the strain, the inner leaflet of the viral envelope of extracellular virions was characterized by a density of intramembrane particles (IMP) three times larger than the host nuclear and plasma membrane. Addition of IMP, which probably represent virus-coded proteins, was detected in the viral envelope only after budding from the nuclear membrane, whereas it occurred during envelopment of capsids at cytoplasmic vacuoles. Fused membranes also showed one of their fracture faces covered with a high density of IMP similar to that of the mature virion envelope. The internal side of the membrane leaflet bearing these numerous particles was always characterized by the presence of an electron-dense material in thin sections. In addition, the plasma membrane of fibroblasts and Vero cells showed strain-specific changes: patches of closely packed IMP were observed with the VR3 strain, whereas ridges almost devoid of IMP characterized the plasmalemma of cells infected with the Patton strain. These intramembrane changes, however, were not observed as early as herpes membrane antigens. Thus, application of the freeze-fracture technique to herpes simplex virus type 1-infected cells revealed striking structural differences between viral and uninfected cell membranes. These differences are probably related to insertion and clustering of virus-coded proteins in the hydrophobic part of the membrane bilayer.     

Effect of water stress on cotton leaves : I. An electron microscopic stereological study of the palisade cells

Palisade cells from fully expanded leaves from irrigated and nonirrigated, field grown cotton (Gossypium hirsutum L. cv. Paymaster 266) were subjected to a microscopic examination to evaluate the effect of water stress on subcellular structures. The water potential difference between the two treatments was 13 bars at the time of sampling. The dimensions of the palisade cells and their density per unit leaf area were determined by light microscopy. Palisade cells from stressed plants had the same diameter, but were taller than their counterparts in irrigated plants. The density of the palisade cells was the same in both treatments as was the fractional volume of the intercellular space. It was concluded that the reduced leaf area observed in the stressed plants resulted primarily from a mitotic sensitivity to water stress. Further, expansion of palisade cells was not inhibited by the stress imposed in this study.

Morphometric analysis of electron micrographs was used to evaluate the subcellular structure of palisade cells from nonstressed and stressed plants. The fractional volumes of cell walls, total cytoplasm, chloroplasts, starch granules, intrachloroplast bodies, mitochondria, peroxisomes, and central vacuoles were determined. The surface densities of grana and stroma lamellae, outer chloroplast membranes, mitochondrial cristae, endoplasmic reticulum and Golgi cisternae were also measured. The number of chloroplasts, mitochondria, and peroxisomes were determined. These data were expressed as actual volumes, areas, and numbers per palisade cell for each treatment. Palisade cells from stressed plants had thinner cell walls, larger central vacuoles and approximately the same amount of cytoplasm compared to cells from nonstressed plants. Within the cytoplasm, stressed plants had more but smaller chloroplasts with increased grana and stroma lamellae surfaces, larger mithchondria with reduced cristae surfaces, smaller peroxisomes and reduced membrane surfaces of endoplasmic reticulum and Golgi cisternae.

     

Phagocytosis of bacteria by polymorphonuclear leukocytes: a freeze-fracture, scanning electron microscope, and thin-section investigation of membrane structure

The changes in membrane structure of rabbit polymorphonuclear (PMN) leukocytes during bacterial phagocytosis was investigated with scanning electron microscope (SEM), thin-section, and freeze-fracture techniques. SEM observations of bacterial attachment sites showed the involvement of limited areas of PMN membrane surface (0.01-0.25μm(2)). Frequently, these areas of attachment were located on membrane extensions. The membrane extensions were present before, during, and after the engulfment of bacteria, but were diminished in size after bacterial engulfment. In general, the results obtained with SEM and thin-section techniques aided in the interpretation of the three-dimensional freeze-fracture replicas. Freeze-fracture results revealed the PMN leukocytes had two fracture faces as determined by the relative density of intramembranous particles (IMP). Membranous extensions of the plasma membrane, lysosomes, and phagocytic vacuoles contained IMP's with a distribution and density similar to those of the plasma membrane. During phagocytosis, IMPs within the plasma membrane did not undergo a massive aggregation. In fact, structural changes within the membranes were infrequent and localized to regions such as the attachment sites of bacteria, the fusion sites on the plasma membrane, and small scale changes in the phagocytic vacuole membrane during membrane fusion. During the formation of the phagocytic vacuole, the IMPs of the plasma membrane appeared to move in with the lipid bilayer while maintaining a distribution and density of IMPs similar to those of the plasma membranes. Occasionally, IMPs were aligned to linear arrays within phagocytic vacuole membranes. This alignment might be due to an interaction with linearly arranged motile structures on the side of the phagocytic vacuole membranes. IMP-free regions were observed after fusion of lysosomes with the phagocytic vacuoles or plasma membrane. These IMP-free areas probably represent sites where membrane fusion occurred between lysosomal membrane and phagocytic vacuole membrane or plasma membrane. Highly symmetrical patterns of IMPs were not observed during lysosomal membrane fusion.     

Freeze-etching study on membrane ultrastructural changes caused by freezing: cooling rate-dependent intramembrane particle aggregation in erythrocyte stripped ghosts

The changes of membrane ultrastructures by freezing stresses were examined on stripped ghosts which were made by removing almost all peripheral membrane proteins from human erythrocyte membranes. By freezing these stripped ghost membranes showed cooling rate-dependent intramembrane particle (IMP) aggregation. With the cooling rates at and faster than 30,000 degrees C/min, their IMPs were evenly distributed on the fracture faces. However, cooling rates at and slower than 8000 degrees C/min resulted in IMP aggregation. The degree of IMP aggregation increased in parallel with decreasing cooling rates. Without freezing, the IMP aggregation in stripped ghosts could be induced by exposing these ghosts to hypertonic salt solutions, but lowering the temperature did not affect IMP aggregation. The cooling rate-dependent IMP aggregation during freezing was suppressed by adding cryoprotective agents which were known to reduce the salt concentration of the medium during freezing. It is suggested that the IMP aggregation in stripped ghosts by freezing occurs by exposure to concentrated salt solutions during freezing. This result indicates the possibility that IMP aggregation may arise during slow freezing of some biomembranes as a result of an increase in salt concentration rather than as a result of reduction in temperature.     

Parallel effects of freezing and osmotic stress on the ATPase activity and protein composition of the plasma membrane of winter rye seedlings

The objective of this study was to determine the influence of freezing versus hypertonic stress on the ATPase activity and polypeptide profile of the plasma membrane of nonacclimated winter rye leaves (Secale cereale L. cv Puma). Exposure of leaves to hypertonic sorbitol solutions resulted in a similar extent of injury as did freezing to subzero temperatures that resulted in equivalent osmotic stresses. When isolated with a two-phase partition system of aqueous polymers, the plasma membrane fractions of control, frozen, or hypertonically stressed leaves were of similar purity as judged by the distribution of marker enzyme activities. When assayed in the presence of Triton X-100 (0.05% w/w), ATPase activity was decreased only slightly in plasma membrane fractions isolated from either frozen or hypertonically stressed leaves. In contrast, the specific ATPase activity of the plasma membrane fractions assayed in the absence of Triton X-100 increased following freezing or hypertonic stress. As a result, the Triton X-100 stimulation of the ATPase activity decreased significantly from sixfold in control leaves to threefold in lethally stressed leaves and reflects an increase in the permeability of the plasma membrane vesicles. The increased permeability was also manifested as a decrease in H⁺-transport following exposure to freezing or hypertonic stress. Both freezing and hypertonic exposure at subzero temperatures altered the polypeptide profile of the plasma membrane, but with the exception of one polypeptide, there was no difference between the two treatments.     

The plasma-membrance IMP pattern as related to animal/vegetal polarity in the amphibian egg

Freeze-fracture electron microscopy of the plasma membrane of the fertilized, uncleaved Xenopus egg shows that intramembranous particles (IMPs) range in size from ca. 50 to 200 Å and that more IMPs are attached to the E-face than to the P-face. The overall IMP densities of the animal and the vegetal hemisphere do not differ significantly. IMP-free regions (?, ca. 0.1 μm) on the tips of surface protrusions were irregularly distributed in the animal and the vegetal half (E-face) occupying ca. 8.5 and 2%, respectively of the free area. The relative densities for 16 different IMP sizes have been compared, on the basis of seven animal and seven vegetal halves, counting (E-faces only) ca. 10,000 IMPs in each hemisphere. For IMP sizes of ≤81 Å, a significant difference (P < 0.0005) was found, more small IMPs being present in the animal half. Some evidence for IMP-associated thin elements was found. These findings are discussed in relation to plasma membrane anisotropy and the morphogenetic role of the egg cortex.     

Proline accumulation in canola leaf discs subjected to osmotic stress is related to the loss of chlorophylls and to the decrease of mitochondrial activity

Many stress studies use simplified experimental models like leaf discs, but the validity of such approaches remains a matter of debate, especially when attempts are made to obtain suitable biotests. We report here the analysis of the resistance of canola leaf discs to osmotic stress, as related to the accumulation of proline. For that purpose, the explanted tissues were incubated for 24 h under continuous light and at 28°C on polyethylene glycol solutions of external potentials (Π_ext) ranging from −0.1 to −8.0 MPa. Proline content, water deficit and chlorophyll content were quantified. The cell membrane stability, which is a structural component of viability, was assessed according to electrolyte leakage. Biomembrane oxidative damage was measured through the production of malondialdehyde and the mitochondrial activity was quantified by assessing the ability of the tissues to reduce 2,3,5-triphenyltetrazolium chloride. Although the water deficit of the tissues reached high values (i.e. up to 70%), the cell membrane stability remained high. Furthermore, the oxidative damage to biomembranes was quite low, even in highly dehydrated tissues. In contrast, osmotic stress induced a significant decrease in the chlorophyll content and a strong accumulation of proline. These two processes each reached a maximum at a Π_ext of −3.0 MPa, although the tissues appeared to be viable at even lower Π_ext, suggesting they are not responsible for primary resistance. The mitochondrial activity was strongly decreased in the stressed leaf discs, although this activity was at least partially restored in tissues incubated for 24 h on a non-stressing medium. It appears that the cell components of the osmotic stress resistance assessed observed in this study do not reflect the modes of resistance of an intact plant.     

Calcium-Dependent Freezing Tolerance in Arabidopsis Involves Membrane Resealing via Synaptotagmin SYT1

Plant freezing tolerance involves the prevention of lethal freeze-induced damage to the plasma membrane. We hypothesized that plant freezing tolerance involves membrane resealing, which, in animal cells, is accomplished by calcium-dependent exocytosis following mechanical disruption of the plasma membrane. In Arabidopsis thaliana protoplasts, extracellular calcium enhanced not only freezing tolerance but also tolerance to electroporation, which typically punctures the plasma membrane. However, calcium did not enhance survival when protoplasts were exposed to osmotic stress that mimicked freeze-induced dehydration. Calcium-dependent freezing tolerance was also detected with leaf sections in which ice crystals intruded into tissues. Interestingly, calcium-dependent freezing tolerance was inhibited by extracellular addition of an antibody against the cytosolic region of SYT1, a homolog of synaptotagmin known to be a calcium sensor that initiates exocytosis. This inhibition indicates that the puncture allowing the antibody to flow into the cytoplasm occurs during freeze/thawing. Thus, we propose that calcium-dependent freezing tolerance results from resealing of the punctured site. Protoplasts or leaf sections isolated from Arabidopsis SYT1-RNA interference (RNAi) plants lost calcium-dependent freezing tolerance, and intact SYT1-RNAi plants had lower freezing tolerance than control plants. Taken together, these findings suggest that calcium-dependent freezing tolerance results from membrane resealing and that this mechanism involves SYT1 function.     

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.     

Long- and short-term freezing induce different types of injury in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> leaf cells

In nature, intact plant cells are subjected to freezing and can remain frozen for prolonged periods. We assayed the survival of Arabidopsis thaliana leaf cells following freezing and found that short- and long-term exposures produced different types of cellular injury. To identify the cause of these injuries, we examined the ultrastructure of the cell plasma membranes. Our results demonstrate that ultrastructural changes in the plasma membrane due to short-term freezing are associated with interbilayer events, including close apposition of the membranes. In both acclimated and non-acclimated leaf cells, these interbilayer events resulted in “fracture-jump lesions” in the plasma membrane. On the other hand, long-term freezing was associated with the development of extensive protein-free areas caused by the aggregation of intramembrane proteins with consequent vesiculation of the affected membrane regions; this effect was clearly different from the ultrastructural changes induced by interbilayer events. We also found that prolonged exposure of non-acclimated leaf cells to a concentrated electrolyte solution produced effects that were similar to those caused by long-term freezing, suggesting that the ultrastructural changes observed in the plasma membrane following long-term freezing are produced by exposure of the leaf cells to a concentrated electrolyte solution. This study illustrates multiple causes of freezing-induced injury in plant cells and may provide useful information regarding the functional role of the diverse changes that occur during cold acclimation.     

Factors Affecting Polyamine Accumulation in Barley (Hordeum vulgare L.) Leaf Sections during Osmotic Stress

Turner, L. B. and Stewart, G. R. 1988. Factors affecting polyamineaccumulation in barley (Hordeum vulgare L.) leaf sections duringosmotic stress.-J. exp. Bot. 39: 311–316. Polyamine concentrations in peeled leaf sections of Hordeumvulgare were unaffected by decreases in leaf water potentialif osmotic adjustment took place and leaf turgor was maintained.Putrescine accumulation occurred concomitantly with a decreasein leaf turgor. Increases in the order of 3-to 4-fold were observed.An apparently greater putrescine accumulation (7-fold) occurredwhen leaf sections were osmotically stressed in the presenceof exogenous phosphate ions. This was the result of water lossfrom the tissue and the large decline which occurred in theputrescine levels of control tissue sections incubated withphosphate ions. Putrescine accumulation was at its maximum after4 h osmotic stress. In contrast, proline accumulation took placebetween 4 h and 24 h after the imposition of osmotic stress. Key words: Hordeum vulgare, osmotic stress, polyamine     

Freeze-fracture studies of the developing cell surface. II. Particle-free membrane blisters on glutaraldehyde-fixed corneal fibroblasts are artefacts.

We describe, in sections and by freeze-fracture, four classes of intramembrane particle (IMP)-free membrane blebs or "blisters" associated with glutaraldehyde-fixed embryonic corneal fibroblasts: (a) Single blisters attached to the cell membrane; (b) free (detached) vesicles; (c) myelin figures; (d) multivesicular protrusions which resemble the "mounds" described by others on nerve growth cones. The IMP-free, membrane-bounded blisters contain no ground cytoplasm or organelles, in contrast to blebs on trypsin-isolated fibroblasts, which we show here do contain cytoplasm and IMP-rich membranes. That the IMP-free membrane blisters in embryonic corneas are artefacts of fixation is demonstrated by (a) their absence in replicas of fibroblasts frozen and fractured without prior aldehyde fixation and (b) their absence in sections of fibroblasts fixed in a combination of glutaraldehyde and osmium tetroxide. We suggest that the addition of osmium prevents postfixation movement of membrane lipids, especially the negatively charged "fluid" lipids which others have shown are capable of considerable mobility after aldehyde fixation alone. Recent literature has implicated membrane blistering in secretory processes and in growth of nerves, but before the functional significance of such IMP-free blisters is assessed, membrane mobility of the type shown here should be taken into consideration.     

Increased ethylene synthesis enhances chilling tolerance in tomato

Freezing of nonacclimated protoplasts close to lethal temperatures induces alterations in the macromolecular organization of the plasma membrane but the significance of these structural changes in freezing injury is still uncertain. We therefore cooled non-acclimated protoplasts isolated from cultivars of winter rye ( Secale cereale L.) to two sub-zero temperatures using two different cooling rates and analyzed freeze-induced plasma membrane changes by freeze-fracture electron microscopy. When a high cooling rate was used a lipid phase transition was observed in 34% of the total membrane fracture faces of the protoplasts, while with a slow cooling rate it occurred only to a very small extent. Smooth, aparticulate lamellae were approximately three times more frequent at low than at high cooling rate. Lipid phase transition from lamellar to hexagonal_II (H_II) phase occurred at high cooling rate more frequently at −10°C than at −30°C in three cultivars. The results suggest that the greatly increased proportion of phase transition from bilayer to non-bilayer phase is an artifact caused by too fast a cooling rate of protoplasts. Furthermore, lateral phase separation of the plasma membrane with segregation of intramembrane particles and the appearance of membrane associated stacks of lipid lamellae, may cause cellular death by retarding the flow of intracellular water towards extracellular ice crystals formed during freezing.     

Implication of ABA and proline on cell membrane injury of water deficit stressed barley seedlings

The aim of this work was to examine the ability of ABA and proline to counteract the deleterious effect of water deficit stress on cell membrane injuries. Six-day-old seedlings of two barley genotypes (cv. Aramir, line R567) were treated with ABA (2·10⁻⁴ M) or proline (0.1 M) for 24 h, and then subjected to osmotic stress for 24h, by immersing their roots in polyethylene glycol (PEG 6000) solution of osmotic potential of −1.0 MPa and −1.5 MPa or by submerging the leaf pieces in PEG solution of osmotic potential of −1.6 MPa. Pretreatment of plants with ABA and proline caused an increase of free proline level in the leaves. Plants treated with ABA exhibited a lower membrane injury index under water stress conditions than those untreated even when no effect of this hormone on RWC in the leaves of stressed plants was observed. Pretreatment of plants with proline prevented to some extent membrane damage in leaves of the stressed seedlings, but only in the case when stress was imposed to roots. Improvement in water status of leaves was also observed in seedlings pretreatment with proline. The protective effect of both ABA and proline was more pronounced in line R567 that exhibited higher membrane injury under water deficit stress conditions.