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.     

Freezing tolerance and avoidance in high-elevation Hawaiian plants

Freezing resistance mechanisms were studied in five endemic Hawaiian species growing at high elevations on Haleakala volcano, Hawaii, where nocturnal subzero (°C) air temperatures frequently occur. Extracellular freezing occurred at around -5°C in leaves of Argyroxiphium sandwicense and Sophora chrysophylla, but these leaves can tolerate extracellular ice accumulation to -15°C and -12°C, respectively. Mucilage, which apparently acted as an ice nucleator, comprised 9 to 11% of the dry weight of leaf tissue in these two species. Leaves of Vaccinium reticulatum and Styphelia tameiameiae were also found to tolerate substantial extracellular freezing. Dubautia menziesii, on the other hand, exhibited the characteristics of permanent supercooling; a very rapid decline in liquid water content associated with simultaneous intracellular and extracellular freezing. However, in those species that tolerate extracellular freezing, the decline in liquid water content during freezing is relatively slow. Osmotic potential was lower at pre-dawn than at midday in four of the species studied. Nocturnal production of osmotically active solutes may have helped to prevent intracellular freeze dehydration as well as to provide non-colligative protection of cell membranes. Styphelia tameiameiae supercooled to -9·3°C and tolerated tissue freezing to below -15°C, a unique combination of physiological characteristics related to freezing. Tolerance of extracellular ice formation after considerable supercooling may have resulted from low tissue water content and a high degree of intracellular water binding in this species, as determined by nuclear magnetic resonance studies. The climate at high elevations in Hawaii is relatively unpredictable in terms of the duration of subzero temperatures and the lowest subzero temperature reached during the night. It appears that plants growing in this tropical alpine habitat have been under selective pressures for the evolution of freezing tolerance mechanisms.     

Ultrastructure before freezing,while frozen,and after thawing in assessing cryoinjury of mouse epididymal spermatozoa

Tails of mouse epididymides were treated as follows: control, unfrozen with and without cryoprotective agents (CPA); frozen (to below ?80 °C), slowly (8 °C/min), and rapidly (18 °C/sec), with and without CPA. Intracellular and/or extracellular location of CPA, at least glycerol, was influenced, respectively, by high (22 °C) or low (0 °C) exposure temperature. Standard procedures in electron microscopy were employed and the frozen state preserved by freeze-substitution. Motility before freezing and after thawing was the criterion of cryosurvival.Results showed no evidence of deleterious ultrastructural effects of freezing at rates compared, or of benefits of CPA, regardless of their cellular location. Differences were noted, however, in the appearance of spermatozoa in the frozen state, as a function of the rate of freezing but not as a function of the presence, absence, or location of either glycerol of DMSO. Rapidly frozen cells showed intracellular ice formation in the acrosome, neck, midpiece, and tail regions; there was no intranuclear ice, and extracellular ice artifacts were small. Slowly frozen cells showed large extracellular ice artifacts with evidence of shrinkage distortion due to the dehydration induced by extracellular ice. No spermatozoa survived any of the freezing treatments, showing the lethal effect of both extracellular ice during slow freezing and of intracellular and/or extracellular ice during rapid freezing.     

Subcellular and immunocytochemical localization of the enzymes involved in ammonia assimilation in mesophyll and bundle-sheath cells of maize leaves

The cellular localization of the enzymes involved in primary nitrogen assimilation was investigated following separation of mesophyll protoplasts and bundle-sheath cells of maize (Zea mays L.) leaves. Determination of the enzymatic activities in the two types of cell revealed that nitrate and nitrite reductase are principally located in the mesophyll cells whereas glutamine synthetase (GS) and ferredoxin-dependent glutamate synthase (Fd-GOGAT) are present in both tissues with a preferential location in the bundle-sheath strands. In order to confirm the results obtained by this conventional biochemical method we have used an in-situ immunofluorescence technique to unambiguously localize GS and Fd-GOGAT at the cellular level. Thin-sectioned maize leaves treated with specific GS and Fd-GOGAT antisera followed by conjugation with fluorescein-isothiocyanate-labelled sheep anti-rabbit immunoglobulins clearly show that GS is equally distributed within the leaf whereas Fd-GOGAT is mostly present in the chloroplasts of the bundle-sheath cells. The cellular localization of nitrate reductase, nitrite reductase, GS-2 and Fd-GOGAT in maize leaf cell types strongly indicates that primary nitrogen assimilation functions in the mesophyll cells while photorespiratory nitrogen recycling is restricted to the bundle-sheath cells.     

Deep supercooling enabled by surface impregnation with lipophilic substances explains the survival of overwintering buds at extreme freezing

The frost survival mechanism of vegetative buds of angiosperms was suggested to be extracellular freezing causing dehydration, elevated osmotic potential to prevent freezing. However, extreme dehydration would be needed to avoid freezing at the temperatures down to ?45°C encountered by many trees. Buds of Alnus alnobetula, in common with other frost hardy angiosperms, excrete a lipophilic substance, whose functional role remains unclear. Freezing of buds was studied by infrared thermography, psychrometry, and cryomicroscopy. Buds of A. alnobetula did not survive by extracellular ice tolerance but by deep supercooling, down to ?45°C. An internal ice barrier prevented ice penetration from the frozen stem into the bud. Cryomicroscopy revealed a new freezing mechanism. Until now, supercooled buds lost water towards ice masses that form in the subtending stem and/or bud scales. In A. alnobetula, ice forms harmlessly inside the bud between the supercooled leaves. This would immediately trigger intracellular freezing and kill the supercooled bud in other species. In A. alnobetula, lipophilic substances (triterpenoids and flavonoid aglycones) impregnate the surface of bud leaves. These prevent extrinsic ice nucleation so allowing supercooling. This suggests a means to protect forestry and agricultural crops from extrinsic ice nucleation allowing transient supercooling during night frosts.     

Relation of ice formation to ultrastructural cryoinjury and cryoprotection of rough endoplasmic reticulum

Ultrastructural observations on the frozen state of pancreatic acinar cells were correlated with results of parallel studies before freezing and after thawing, as to cryoinjury and cryoprotection.Data support an hypothesis of freezing injury based upon intracellular ice and solution effects during rapid and slow freezing, respectively. The basis for superiority of extracellular over intracellular glycerol in cryoprotection was demonstrated in terms of these factors.Evidence is offered to explain the ultrastructural cryoinjury and cryoprotection of rough endoplasmic reticulum (RER) seen after thawing, relative to the combined effects of freezing rate and glycerol. Slow freezing, in combination with the presence of extracellular glycerol, provided sufficient dehydration to almost completely suppress intracellular ice formation, yielding minimal ultrastructural alteration of RER. Greatest cryoinjury, expressed as extensive conversion of RER into sphere-like vesicles, was induced by the extensive intracellular ice formation which accompanied rapid freezing. A mechanism is suggested to explain physical damage of RER by intracellular ice.     

A microscale model for prediction of breast cancer cell damage during cryosurgery

The morphology of cancerous breast tissue is characterized by tightly packed groups of small malignant cells, as found in most duct cell carcinoma. This special structure affects the osmotic responses of the cells to freezing and hence their probability of damage from cellular dehydration or intracellular ice formation. A mathematical model has been developed to study the microscale damage to these breast cancer cells during cryosurgery by accounting for their special structure. The model is based on a spherical unit comprised of an extracellular region that surrounds several layers of cancer cells, as experimentally observed of breast duct cell carcinoma by other researchers. Temperature transients in the breast cancer undergoing cryosurgery are calculated numerically using the Pennes equation. When subjected to various thermal histories, both cellular dehydration and intracellular ice formation in the unit structure are examined by considering the cell-to-cell contact and water transport at the microscale level. It is found that the cells in the inner layers hardly dehydrated while those in the outermost layer do greatly. The results help interpret the previously observed experimental phenomena that breast cancer tissues exhibit intracellular ice formation even at a slow cooling rate of -3 degrees C/min. In the attempt to better define an optimal procedure for breast cancer cryosurgery, various freezing protocols are simulated. The constant heat flux protocol induces greater cellular dehydration and higher intracellular ice formation probability simultaneously compared to the other protocols studied.     

Avoidance of intracellular freezing by the freezing-tolerant New Zealand Alpine weta Hemideina maori (Orthoptera: Stenopelmatidae)

Calorimetric analysis indicates that 82% of the body water of Hemideina maori is converted into ice at 10 degrees C. This is a high proportion and led us to investigate whether intracellular freezing occurs in H. maori tissue. Malpighian tubules and fat bodies were frozen in haemolymph on a microscope cold stage. No fat body cells, and 2% of Malpighian tubule cells froze during cooling to -8 degrees C. Unfrozen cells appeared shrunken after ice formed in the extracellular medium. There was no difference between the survival of control tissues and those frozen to -8 degrees C. At temperatures below -15 degrees C (lethal temperatures for weta), there was a decline in survival, which was strongly correlated with temperature, but no change in the appearance of tissue. It is concluded that intracellular freezing is avoided by Hemideina maori through osmotic dehydration and freeze concentration effects, but the reasons for low temperature mortality remain unclear. The freezing process in H. maori appears to rely on extracellular ice nucleation, possibly with the aid of an ice nucleating protein, to osmotically dehydrate the cells and avoid intracellular freezing. The lower lethal temperature of H. maori (-10 degrees C) is high compared to organisms that survive intracellular freezing. This suggests that the category of 'freezing tolerance' is an oversimplification, and that it may encompass at least two strategies: intracellular freezing tolerance and avoidance.     

Freezing Characteristics of Rigid Plant Tissues (Development of Cell Tension during Extracellular Freezing)

The freezing characteristics and development of cell tension during extracellular freezing were examined in supercooling stem tissues of riverbank grapes (Vitis riparia) and cold-hardened leaves of live oak (Quercus virginiana) and mountain cranberry (Vaccinium vitis-idaea). Dormant stem xylem and pith tissues of river-bank grapes were resistant to freeze-induced dehydration above the homogeneous nucleation temperature, and they developed cell tension reaching a maximum of 27 MPa. Similarly, extracellular freezing induced cell tension in the leaves of live oak and mountain cranberry. Maximum cell tension in the leaves of live oak was 16.8 MPa and 8.3 MPa in the leaves of mountain cranberry. Following peak tensions in the leaves, a decline in the pressure was observed with progressive freezing. The results suggest that resistance to cell deformation during extracellular freezing due to cell-wall rigidity can lead to reduced cell dehydration and increased cell tension. A relationship to predict freezing behavior in plant tissues based on cell rigidity is presented. Based on cell-water relations and ice nucleation rates, cell-wall rigidity has been shown to effect the freezing characteristics of plant tissues, including freeze-induced dehydration, supercooling, and homogeneous nucleation temperatures.     

Changes in Osmotic Pressure and Mucilage during Low-Temperature Acclimation of Opuntia ficus-indica

Opuntia ficus-indica, a Crassulacean acid metabolism plant cultivated for its fruits and cladodes, was used to examine chemical and physiological events accompanying low-temperature acclimation. Changes in osmotic pressure, water content, low molecular weight solutes, and extracellular mucilage were monitored in the photosynthetic chlorenchyma and the water-storage parenchyma when plants maintained at day/night air temperatures of 30/20°C were shifted to 10/0°C. An increase in osmotic pressure of 0.13 megapascal occurred after 13 days at 10/0°C. Synthesis of glucose, fructose, and glycerol accounted for most of the observed increase in osmotic pressure during the low-temperature acclimation. Extracellular mucilage and the relative apoplastic water content increased by 24 and 10%, respectively, during exposure to low temperatures. These increases apparently favor the extracellular nucleation of ice closer to the equilibrium freezing temperature for plants at 10/0°C, which could make the cellular dehydration more gradual and less damaging. Nuclear magnetic resonance studies helped elucidate the cellular processes during ice formation, such as those revealed by changes in the relaxation times of two water fractions in the chlorenchyma. The latter results suggested a restricted mobility of intracellular water and an increased mobility of extracellular water for plants at 10/0°C compared with those at 30/20°C. Increased mobility of extracellular water could facilitate extracellular ice growth and thus delay the potentially lethal intracellular freezing during low-temperature acclimation.     

Freezing stress response in woody tissues observed using low-temperature scanning electron microscopy and freeze substitution techniques

The objective of the current research was to examine the response of woody plant tissues to freezing stress by using scanning electron microscopy (SEM). Nonsupercooling species red osier dogwood (Cornus stolonifera Michx.), weeping willow (Salix babylonica L.), and corkscrew willow (Salix matsudana Koidz. f. tortuosa Rehd.) survived freezing stress as low as −60°C. Cell collapse of ray parenchyma cells of these species was expected but did not occur. It was concluded that ray parenchyma cells of these species do not fit into either the supercooling or extracellular freezing classifications. Tissues from flowering dogwood (Cornus florida L.), apple (Malus domestica Borkh. cv “Starking III”), red oak (Quercus rubra L.), scarlet oak (Quercus coccinea Muench.), and red ash (Fraxinus pennsylvanica Marsh) were confirmed as supercooling species, and did not survive exposures below −40°C. Ray parenchyma cells of these species did not collapse in response to freezing stress, as was expected. Cell collapse along the margins of voids were observed in bark of all seven species. Voids were the result of extracellular ice crystals formed in the bark during exposure to freezing stress. Tissues prepared by freeze substitution techniques were found to be adequately preserved when compared to those prepared by conventional fixation and low temperature SEM techniques. A freezing protocol for imposing freezing stress at temperatures lower than experienced naturally in the area where the study was conducted was developed that produced responses comparable to those observed in specimens collected in the field during natural freezing events.     

Phylogenetic analyses in cornus substantiate ancestry of xylem supercooling freezing behavior and reveal lineage of desiccation related proteins

The response of woody plant tissues to freezing temperature has evolved into two distinct behaviors: an avoidance strategy, in which intracellular water supercools, and a freeze-tolerance strategy, where cells tolerate the loss of water to extracellular ice. Although both strategies involve extracellular ice formation, supercooling cells are thought to resist freeze-induced dehydration. Dehydrin proteins, which accumulate during cold acclimation in numerous herbaceous and woody plants, have been speculated to provide, among other things, protection from desiccative extracellular ice formation. Here we use Cornus as a model system to provide the first phylogenetic characterization of xylem freezing behavior and dehydrin-like proteins. Our data suggest that both freezing behavior and the accumulation of dehydrin-like proteins in Cornus are lineage related; supercooling and nonaccumulation of dehydrin-like proteins are ancestral within the genus. The nonsupercooling strategy evolved within the blue- or white-fruited subgroup where representative species exhibit high levels of freeze tolerance. Within the blue- or white-fruited lineage, a single origin of dehydrin-like proteins was documented and displayed a trend for size increase in molecular mass. Phylogenetic analyses revealed that an early divergent group of red-fruited supercooling dogwoods lack a similar protein. Dehydrin-like proteins were limited to neither nonsupercooling species nor to those that possess extreme freeze tolerance.     

Cell shape and localisation of ice in leaves of overwintering wheat during frost stress in the field

Wheat leaf pieces were excised and freeze-fixed in the field, preparatory to low-temperature scanning electron microscopy to study distribution of ice within leaf blades, and associated cell shapes, during natural frosts. Pieces of leaf blades from wheat plants (Triticum aestivum L. 7942H1-20-8) overwintering in Indiana, USA (January, 1991), were excised and immediately freeze-fixed by manually plunging in melting freon. Cells in controls were turgid and extracellular ice was absent. The leaves of the frost-stressed plants froze at about — 2.4° C, and at that temperature extracellular ice was mainly located sub-epidermally, including in the substomatal cavity, and occupied about 14% of the fracture faces. The frequency of ice particles per unit leaf area in two specimens was 14 and 210 · mm^–2 (about 140 and 2100 · g^–1 leaf fresh-weight basis). At -9.0° C, ice filled the extracellular spaces, occupying 61% of the fracture faces. Cells were somewhat collapsed at -2.4° C and were much more collapsed at -9.0° C. The epidermal cells were more collapsed than the mesophyll cells. Tissue structure (connections with adjacent cells), wall flexibility, and ice growth may all have influenced the shapes of the collapsing cells. The experiments demonstrate the feasibility of freeze-fixation in the field. The sub-epidermal location of most ice indicates that in the field either (i) ice is nucleated sub-epidermally (implying both the presence of nucleators and the presence of liquid water in the sub-epidermal spaces) or (ii) ice is nucleated on the leaf surface, then propagates into the leaf probably through stomata.Abbreviations LTSEM low-temperature scanning electron microscopy     

Inter- and intracellular distribution of amino acids and other metabolites in maize (Zea mays L.) leaves

In illuminated maize (Zea mays L.) leaves, the distribution of triose phosphates, 3-phosphoglycerate, malate and various amino acids between the chloroplastic and the extrachloroplastic compartments of mesophyll and bundle-sheath cells, and the total vacuolar fraction of the leaves, was determined by a combination of previously published methods, for separating mesophyll from bundle-sheath material, and for nonaqueous subcellular fractionation. The results show that the triose phosphate/3-phosphoglycerate ratio in the extrachloroplastic fraction of the mesophyll cells is about 20-fold higher than in the bundle-sheath cells, which is in accordance with a triose phosphate/phosphoglycerate shuttle postulated previously. Whereas the vacuolar compartment was shown to contain most of the cellular malate, amino acids were found to be almost absent from this compartment. The amino-acid pattern in the extrachloroplastic fraction of the bundle-sheath cells largely resembled the pattern in whole leaves. These results show that for future studies the analysis of amino-acid contents in whole maize leaves can be used as a measure for the amino-acid levels in the cytosol of bundle-sheath cells.Abbreviations BS bundle sheath - Chl chlorophyll - Man -mannosidase - ME malic enzyme - MDH malate dehydrogenase - MS mesophyll - PEPCase phosphoenolpyruvate carboxylase - PGA 3-phosphoglycerate - trioseP triose phosphate This work was supported by the Bundesminister für Forschung und Technologie.     

Salt treatment induces frost hardiness in leaves and isolated thylakoids from spinach

Frost hardiness of spinach (Spinacia oleracea L.) leaves was increased by high concentrations of NaCl in the hydroponic culture medium. Freezing damage was determined by measurement of slow chlorophyll fluorescence quenching after freezing of leaves. Both the osmolality of the leaf sap and forst hardiness of the leaves were linearly correlated with the salt concentration in the hydroponic culture medium. Freezing damage occurred, irrespective of the extent of frost hardening, when dehydration of cells during extracellular ice formation decreased cellular volume to approximately 14% of the volume of unfrozen cells. The resistance of isolated, washed thylakoids against mechanical and chemical damage by freezing was investigated. Chemical damage by freezing caused by salt accumulation was measured as release of chloroplast coupling factor (CF1; EC 3.6.1.3), and mechanical damage was measured as release of the lumenal protein plastocyanin from the membranes during an in-vitro freeze-thaw cycle. Isolated thylakoids from salt-treated frost-hardy spinach and those from plants hardened under natural conditions did not exhibit improved tolerance against chemical freezing stress exerted by high salt concentrations. They were, however, more hardy than thylakoids from unhardened control leaves against mechanical damage by freezing.Abbreviation CF1 peripheral part of chloroplast coupling factor ATPase     

Cyst(e)ine Is the Transport Metabolite of Assimilated Sulfur from Bundle-Sheath to Mesophyll Cells in Maize Leaves

The intercellular distribution of the enzymes and metabolites of assimilatory sulfate reduction and glutathione synthesis was analyzed in maize (Zea mays L. cv LG 9) leaves. Mesophyll cells and strands of bundle-sheath cells from second leaves of 11-d-old maize seedlings were obtained by two different mechanical-isolation methods. Cross-contamination of cell preparations was determined using ribulose bisphosphate carboxylase (EC 4.1.1.39) and nitrate reductase (EC 1.6.6.1) as marker enzymes for bundle-sheath and mesophyll cells, respectively. ATP sulfurylase (EC 2.7.7.4) and adenosine 5′-phosphosulfate sulfotransferase activities were detected almost exclusively in the bundle-sheath cells, whereas GSH synthetase (EC 6.3.2.3) and cyst(e)ine, γ-glutamylcysteine, and glutathione were located predominantly in the mesophyll cells. Feeding experiments using [³⁵S]sulfate with intact leaves indicated that cyst(e)ine was the transport metabolite of reduced sulfur from bundle-sheath to mesophyll cells. This result was corroborated by tracer experiments, which showed that isolated bundle-sheath strands fed with [³⁵S]sulfate secreted radioactive cyst(e)ine as the sole thiol into the resuspending medium. The results presented in this paper show that assimilatory sulfate reduction is restricted to the bundle-sheath cells, whereas the formation of glutathione takes place predominantly in the mesophyll cells, with cyst(e)ine functioning as a transport metabolite between the two cell types.     

Ice crystals formed in tissue during cryosurgery. II. Electron microscopy

Tissues frozen by means of a cryosurgical probe have been examined by electron microscopy following techniques designed to preserve the ice crystal spaces.Ice crystals appeared similar whether tissues were quenched or not following cryosurgery and the various techniques of dehydration resulted in similar ice crystal architecture.Ice crystal spaces in the area deep to the freezing probe were intracellular both in epithelium and muscle although in the muscle zone some fibers contained large and others small crystal spaces. It is suggested that this might be due to variations in the local blood supply.At the periphery of the frozen area ice crystals were usually extracellular producing gross distortion of the cells which, however, retained intracellular structural integrity. These results are consistent with the belief of many workers that intracellular ice is lethal while extracellular ice is not, but no evidence of penetration of cell membrane by ice crystals was seen.     

Extracellular ice and cell shape in frost-stressed cereal leaves: A low-temperature scanning-electron-microscopy study

Low-temperature scanning electron microscopy was used to examine transverse fracture faces through cereal leaf pieces subjected to frost. Specimens were studied before and after sublimation of the ice. The position of extracellular ice in the leaf was inferred from the difference between the specimen before and after sublimation and from ridges and points which occurred in the extracellular ice during sublimation. Steps in the fracture surface indicated that the fracture plane passed through the extracellular ice crystals as well as through cells and also helped identify extracellular ice. The cells in controls were turgid and extracellular ice was absent. Leaf pieces from hardened rye were excised and frost-stressed to-3.3°,-21° and-72°C, cooling at 2–12°·h^-1. Cell collapse and extracellular ice were evident at-3.3°C and increased considerably by-21° C. At-21° and-72°C the leaf pieces were mainly filled with extracellular ice and there were few remaining gas spaces. The epidermal and mesophyll cells were laterally flattened, perpendicular to their attachment to adjacent cells, and phloem and vascular sheath cells were more irregularly deformed. Leaf pieces from tender barley were cooled at 2°C·min^-1 to-20° C; they were then mainly filled with extracellular ice, and the cells were highly collapsed as in the rye. In rye leaves frozen to-3.6° C before excision, ice crystals occurred in peri-vascular, sub-epidermal and intervening mesophyll spaces. In rye leaf pieces frozen to-3.3° C after excision or to-3.6° C before excision, mesophyll cells were partly collapsed even when not covered by ice, indicating that collapse of the cell wall, as well as the enclosed protoplast, was driven by dehydration. No gas or ice-filled spaces were found between wall and the enclosed protoplast. It is suggested that this can be explained without invoking chemical bonding between cell wall and plasma membrane: when the wall pores are filled by water, the pore size would reduce vapour pressure so making penetration of the wall by ice or gas less likely.Abbreviations SEM scanning electron microscopy     

Effect of microtubule stabilization on the freezing tolerance of mesophyll cells of spinach

Freezing, dehydration, and supercooling cause microtubules in mesophyll cells of spinach (Spinacia oleracea L. cv Bloomsdale) to depolymerize (ME Bartolo, JV Carter, Plant Physiol [1991] 97: 175-181). The objective of this study was to determine whether the LT₅₀ (lethal temperature: the freezing temperature at which 50% of the tissue is killed) of spinach leaf tissue can be changed by diminishing the extent of microtubule depolymerization in response to freezing. Also examined was how tolerance to the components of extracellular freezing, low temperature and dehydration, is affected by microtubule stabilization. Leaf sections of nonacclimated and cold-acclimated spinach were treated with 20 micromolar taxol, a microtubule-stabilizing compound, prior to freezing, supercooling, or dehydration. Taxol stabilized microtubules against depolymerization in cells subjected to these stresses. When pretreated with taxol both nonacclimated and cold-acclimated cells exhibited increased injury during freezing and dehydration. In contrast, supercooling did not injure cells with taxol-stabilized microtubules. Electrolyte leakage, visual appearance of the cells, or a microtubule repolymerization assay were used to assess injury. As leaves were cold-acclimated beyond the normal period of 2 weeks taxol had less of an effect on cell survival during freezing. In leaves acclimated for up to 2 weeks, stabilizing microtubules with taxol resulted in death at a higher freezing temperature. At certain stages of cold acclimation, it appears that if microtubule depolymerization does not occur during a freeze-thaw cycle the plant cell will be killed at a higher temperature than if microtubule depolymerization proceeds normally. An alternative explanation of these results is that taxol may generate abnormal microtubules, and connections between microtubules and the plasma membrane, such that normal cellular responses to freeze-induced dehydration and subsequent rehydration are blocked, with resultant enhanced freezing injury.