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     

The formation and distribution of ice within dormant and deacclimated peach flower buds

Abstract A freeze-fixation technique was used to examine the distribution of ice crystals and the pattern of freezing in peach flower buds. In dormant buds, ice crystals formed at localized sites within the bud axis and scales. Ice crystal formation disrupted tissues and mechanical injury from repetitive freezethaw cycles was apparent. There was evidence of ice formation in the floral organs of dormant buds exposed to ?25°C but none observed in buds exposed to either ?5 or ?10°C. The distribution of ice crystals was different in deacclimated buds. In addition to large ice crystals within the subtending bud axis and scales, evidence of large crystals within the developing floral organs was noted. These crystals were most prominent in the lower portions of the developing flower and peduncle, and caused a separation of the epidermal layer from adjacent cells. The distribution of ice crystals within both dormant and deacclimated peach flower buds corroborated the results of previous thermal analysis experiments.     

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     

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     

Ice crystals formed in tissues during cryosurgery. I. Light microscopy

The size and distribution of ice crystals formed during cryosurgical procedures in intact animals are not clear. In the present experiment oral mucosa was frozen in situ by means of a surface applied cold probe and was excised and freeze substituted while in the frozen state. It was shown that the form of the frozen tissue was preserved during this procedure and the area frozen was divisible into a zone representing the central part of the lesion and a peripheral zone separating this from normal tissue. Ice crystals within the body of the lesion were intracellular in location but varied somewhat in size. Ice crystals in the boundary zone appeared to be intracellular in the epithelium and both intra- and extracellular in the muscle fibres.It is suggested that the intracellular crystals in the body of the frozen area result in cell death while the extracellular ice in the boundary zone results in a less predictable response.     

A Freeze-Fracture Study of the Cell Membranes of Wheat Adapted to Extracellular Freezing and to Growth at Low Temperatures

Seedlings of Triticum aestivum L. cv. Lennox were grown in natural(late autumn) or controlled environments differing in temperature,to give plants differing either only in growth adaptation oralso in hardiness. The frost hardiness of laminae and leaf baseswas measured using an ion leakage method. Pieces of laminaeand leaf bases were freeze-fixed without chemical cryo-protectantor chemical fixative. The number of particles per unit area of the E fracture faceof the plasma membrane was substantially reduced in laminaefrom the environment inducing most hardening, compared to unhardenedcontrols. This difference in frequency of particles on the Eface was not related to adaptation of growth to low temperature(in the leaf bases) and was not an artificial consequence ofchemical treatment prior to freeze-fixation. The density ofparticles on the P face of the plasma membrane was unaffectedby growth environment. More limited data suggested the densityof particles on either fracture face of the thylakoids was alsounaffected. Unlike an earlier report, particle-free areas ofplasma membrane were not found in hardened tissue. Environment did not affect (a) the width of the ‘mouth’at the plasma membrane-plasmodesma junction in the leaf basesor (b) the size of the nuclear pores. Membrane accumulated belowthe plasma membrane only in some cells from the hardiest material. Key words: Hardiness, Membranes, Triticum     

The Formation and Distribution of Ice within Forsythia Flower Buds

Differential thermal analysis detected two freezing events when dormant forsythia (Forsythia viridissima Lindl.) flower buds were cooled. The first occurred just below 0°C, and was coincident with the freezing of adjacent woody tissues. The second exotherm appeared as a spike between −10 and −25°C and was correlated with the lethal low temperature. Although this pattern of freezing was similar to that observed in other woody species, differences were noted. Both direct observations of frozen buds and examination of buds freeze-fixed at −5°C demonstrated that ice formed within the developing flowers at temperatures above the second exotherm and lethal temperature. Ice crystals had formed within the peduncle and in the lower portions of the developing flower. Ice also formed within the scales. In forsythia buds, the developing floral organ did not freeze as a unit as noted in other species. Instead the low temperature exotherm appeared to correspond to the lethal freezing of supercooled water within the anthers and portions of the pistil.     

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.     

Studies on cellular structure and ice location in frozen organs and tissues: the use of freeze-substitution and related techniques

Recent studies have led to the conclusion that extracellular ice per se can damage whole organs and tissues. Thus information on the amount and distribution of ice is an important factor in the design of cooling regimens that avoid intracellular ice formation and attempt to localize the ice formed in areas of the tissue where its disruptive effects can be minimized. Furthermore, ultrastructural studies at subfreezing temperatures can enhance the interpretation of information gained from morphological and function studies conducted before cooling and after rewarming. Although many techniques exist for observing and recording structure in the frozen state, not all are applicable to tissues or organs. Freeze-substitution and isothermal freeze-fixation provide two flexible techniques to explore the frozen state. Isothermal freeze-fixation is most suitable for studies close to the melting point, while freeze-substitution can be used at lower temperatures, extending as far as -120 degrees C. A careful choice of technique can provide an accurate assessment of the amount and distribution of the ice phase and the structure of the tissue matrix.     

Freezing and cryoprotective dehydration in an Antarctic nematode (Panagrolaimus davidi) visualised using a freeze substitution technique

The pattern of ice formation during the freezing of Panagrolaimus davidi, an Antarctic nematode that can survive intracellular ice formation, was visualised using a freeze substitution technique and transmission electron microscopy. Nematodes plunged directly into liquid nitrogen had small ice crystals throughout their tissues, including nuclei and organelles, but did not survive. Those frozen at high subzero temperatures showed three patterns of ice formation: no ice, extracellular ice, and intracellular ice. Nematodes subjected to a slow-freezing regime (at -1 degrees C) had mainly extracellular ice (70.4%), with the bulk of the ice in the pseudocoel. Some (24.8%) had no ice within their bodies, due to cryoprotective dehydration. Nematodes subjected to a fast-freezing regime (at -4 degrees C) had intracellular (54%) and extracellular (42%) ice. Intracellular ice was confined to the cytoplasm of cells, with organelles in the spaces in between ice crystals. The survival of nematodes subjected to the fast-freezing regime (53%) was less than those subjected to the slow-freezing regime (92%).     

Equilibrium freezing of leaf water and extracellular ice formation in Afroalpine ‘giant rosette’ plants

The water potentials of frozen leaves of Afroalpine plants were measured psychrometrically in the field. Comparison of these potentials with the osmotic potentials of an expressed cellular sap and the water potentials of ice indicated almost ideal freezing behaviour and suggested equilibrium freezing. On the basis of the osmotic potentials of expressed cellular sap, the fractions of frozen cellular water which correspond to the measured water potentials of the frozen leaves could be determined (e.g. 74% at -3.0° C). The freezing points of leaves were found to be in the range between 0° C and -0.5° C, rendering evidence for freezing of almost pure water and thus confirming the conclusions drawn from the water-potential measurements. The leaves proved to be frost resistant down to temperatures between -5° C and -15° C, as depending on the species. They tolerated short supercooling periods which were necessary in order to start ice nucleation. Extracellular ice caps and ice crystals in the intercellular space were observed when cross sections of frozen leaves were investigated microscopically at subfreezing temperatures.Symbols T temperature - water potential Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Cryo-scanning electron microscopic study on freezing behaviors of tissue cells in dormant buds of larch (Larix kaempferi)

The freezing behavior of dormant buds in larch, especially at the cellular level, was examined by a Cryo-SEM. The dormant buds exhibited typical extraorgan freezing. Extracellular ice crystals accumulated only in basal areas of scales and beneath crown tissues, areas in which only these living cells had thick walls unlike other tissue cells. By slow cooling (5 °C/day) of dormant buds to −50 °C, all living cells in bud tissues exhibited distinct shrinkage without intracellular ice formation detectable by Cryo-SEM. However, the recrystallization experiment of these slowly cooled tissue cells, which was done by further freezing of slowly cooled buds with LN and then rewarming to −20 °C, confirmed that some of the cells in the leaf primordia, shoot primordia and apical meristem, areas in which cells had thin walls and in which no extracellular ice accumulated, lost freezable water with slow cooling to −30 °C, indicating ability of these cells to adapt by extracellular freezing, whereas other cells in these tissues retained freezable water with slow cooling even to −50 °C, indicating adaptation of these cells by deep supercooling. On the other hand, all cells in crown tissues and in basal areas of scales, areas in which cells had thick walls and in which large masses of ice accumulated, had the ability to adapt by extracellular freezing. It is thought that the presence of two types of cells exhibiting different freezing adaptation abilities within a bud tissue is quite unique and may reflect sophisticated freezing adaptation mechanisms in dormant buds.     

Freezing avoidance by supercooling in Olea europaea cultivars: the role of apoplastic water,solute content and cell wall rigidity

Plants can avoid freezing damage by preventing extracellular ice formation below the equilibrium freezing temperature (supercooling). We used Olea europaea cultivars to assess which traits contribute to avoid ice nucleation at sub‐zero temperatures. Seasonal leaf water relations, non‐structural carbohydrates, nitrogen and tissue damage and ice nucleation temperatures in different plant parts were determined in five cultivars growing in the Patagonian cold desert. Ice seeding in roots occurred at higher temperatures than in stems and leaves. Leaves of cold acclimated cultivars supercooled down to ?13 °C, substantially lower than the minimum air temperatures observed in the study site. During winter, leaf ice nucleation and leaf freezing damage (LT₅₀) occurred at similar temperatures, typical of plant tissues that supercool. Higher leaf density and cell wall rigidity were observed during winter, consistent with a substantial acclimation to sub‐zero temperatures. Larger supercooling capacity and lower LT₅₀ were observed in cold‐acclimated cultivars with higher osmotically active solute content, higher tissue elastic adjustments and lower apoplastic water. Irreversible leaf damage was only observed in laboratory experiments at very low temperatures, but not in the field. A comparative analysis of closely related plants avoids phylogenetic independence bias in a comparative study of adaptations to survive low temperatures.     

Intracellular ice formation and growth in MCF-7 cancer cells

Direct cell injury in cryosurgery is highly related to intracellular ice formation (IIF) during tissue freezing and thawing. Mechanistic understanding of IIF in tumor cells is critical to the development of tumor cryo-ablation protocol. In aid of a high speed CMOS camera system, the events of IIF in MCF-7 cells have been studied using cryomicroscopy. Images of ‘darkening’ type IIF and recrystallization are compared between cells frozen with and without ice seeding. It is found that ice seeding has significant impact on the occurrence and growth of intracellular ice. Without ice seeding, IIF is observed to occur over a very small range of temperature (∼1 °C). The crystal dendrites are indistinguishable, which is independent of the cooling rate. Ice crystal grows much faster and covers the whole intracellular space in comparison to that with ice seeding, which ice stops growing near the cellular nucleus. Recrystallization is observed at the temperature from −13 °C to −9 °C during thawing. On the contrary, IIF occurs from −7 °C to −20 °C with ice seeding at a high subzero temperature (i.e., −2.5 °C). The morphology of intracellular ice frozen is greatly affected by the cooling rate, and no ‘darkening’ type ice formed inside cells during thawing. In addition, the intracellular ice formation is directional, which starts from the plasma membrane and grows toward the cellular nucleus with or without ice seeding. These results can be used to explain some findings of tumor cryosurgery in vivo, especially the causes of insufficient killing of tumor cells in the peripheral area near vessels.     

Ice crystal patterns in artificial gels of extracellular matrix macromolecules after quick-freezing and freeze-substitution

Artificial gels, composed of collagen with or without hyaluronate (HA), a glycosaminoglycan (GAG), and chondroitin sulfate (CS), were prepared and quick-frozen for the purpose of studying the influence of composition and concentration on ice patterns. Dilute gels were spread on coverslips, plunged into a slush of 30% isopentane/70% propane (-185 degrees C), freeze-substituted, and examined by phase-contrast microscopy. Ice patterns were revealed as "ice cavities" in the gel after freeze-substitution. Ice morphology in the gels was gel-type-specific, suggesting that composition in dilute gels can influence ice pattern formation. Crystallization patterns reflecting high, intermediate, and low rates of freezing were observed in all gel types. Intermediate freezing in differentiating gel-type-specific ice patterns. Gels which included hyaluronate (HA) and chondroitin sulfate (CS) altered the ice crystal pattern commonly observed in collagen gels. Ice structure in collagen gels consisted predominantly of long, parallel crystals in the herringbone pattern. Ice crystals separated gel into thin, unbranched fibers with a primary spacing of approximately 2 microns. Ice morphology in HA gels formed a mosaic consisting of packets of ice crystals. Contiguous packets were often oriented at right angles to each other. Periodic crossbridges interconnect primary gel fibers of HA gels and interrupt the lengthwise growth of ice crystals. Smooth beads were visible on primary strands in HA gels frozen at intermediate velocities. The addition of CS to collagen gels resulted in formation of randomly oriented ice crystals in gels frozen at intermediate rates. CS has little influence on ice morphology at low freezing velocities. Primary strands in CS gels were decorated with rough-surfaced, osmiophilic aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Water droplets in intercellular spaces of barley leaves examined by low-temperature scanning electron microscopy

Low-temperature scanning electron microscopy was used to examine fracture faces in leaf blades taken from well-watered or drought-stressed barley (Hordeum vulgare L. cv. Mazurka) seedlings. The leaf blades were freeze-fixed while hydrated and were examined with or without gold-coating. There were droplets (with a smooth surface at the resolution achieved) on the surface of cell walls in leaf blades (0.91 g^-1 water content) from well-watered seedlings grown in an environment of 67% relative humidity. These were mainly on the vascular bundle sheath, the guard and subsidiary cells, and on some mesophyll cells around the substomatal cavity and between the stoma and vascular bundle. The droplets occurred, more abundantly, in the same places in seedlings from 100% relative humidity. They occurred on a few guard cells from wilting leaf blades (0.81 g·g^-1 water content) and were absent from severely drought-stressed leaf blades (0.15 g·g^-1 water content). The droplets sublimed at the same moment as both water which was in leaf cells and water which was allowed to condense (after freeze-fixation) on the wall surface. It is suggested that the droplets are aqueous. Their possible origin and importance is discussed.     

Ultrastructural changes in suspension culture cells of Panicum maximum during cryopreservation

A Panicum maximum cell suspension was used to study ultrastructural changes during cryopreservation. Pregrowing the cells in mannitol caused reduction in the vacuolar volume by redistribution of the large central vacuole into a number of smaller vesicles. Invaginations were formed in the plasma membrane of the cells, to accommodate the reduced cell volume. Swelling of organelles occurred during different stages of cryopreservation. The cisternae of the endoplasmic reticulum dilated and formed vesicles. Although some damage was apparent, organelles were still recognizable in cells frozen slowly and freeze-fixed at –10°C. The cells were able to repair such damage within two days in culture, and regained their normal appearance. Cells frozen slowly without any cryoprotection, and cells frozen rapidly by direct immersion into liquid nitrogen after cryoprotection, were lethally damaged by destruction of membranous structures. Osmiophilic granules were found along the plasma membrane of lethally damaged cells, indicating that their formation is a consequence of freeze damage, rather than a mechanism to prevent injury.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide     

Freezing of tissue-limits for the autoradiographic localization of diffusible substances

Frozen thin sections and sections from freeze-dried and embedded tissue are used for the autoradiographic localization of diffusible substances at the electron microscope level. The presence of ice crystals in such sections may limit the autoradiographic resolution. Ice crystals are formed during freezing and may grow during subsequent processing of tissue. The contribution of ice crystal growth to the final image was estimated by measuring the distribution of the ice crystal sizes in freeze-etch replicas and in sections from freeze-dried and embedded tissues. A surface layer (10-15 mu) without visible ice crystals was present in both preparations. Beneath this surface layer the diameter of ice crystals increased towards the interior with the same relationship between crystal size and distance from the surface in the freeze-etch preparation as in the freeze-dry preparation. Ice crystal growth occurring during a much longer time during freeze-drying compared to freeze-etching does not significantly contribute to the final image in the electron microscope. The formation of ice crystals during freezing determines to a large extent the image (and therefore the autoradiographic resolution) of freeze-dry preparations and this probably holds also for thin cryosections of which examples are given.     

Proteins accumulate in the apoplast of winter rye leaves during cold acclimation

During cold acclimation, winter rye ( Secale cereale L.) plants develop the ability to tolerate freezing temperatures by forming ice in intercellular spaces and xylem vessels. In this study, proteins were extracted from the apoplast of rye leaves to determine their role in controlling extracellular ice formation. Several polypeptides in the 15 to 32 kDa range accumulated in the leaf apoplast during cold acclimation at 5°C and decreased during deacclimation at 20°C. A second group of polypeptides (63, 65 and 68 kDa) appeared only when the leaves were maximally frost tolerant. Ice nucleation activity, as well as the previously reported antifreeze activity, was higher in apoplastic extracts from cold-acclimated than from nonacclimated rye leaves. These results indicate that apoplastic proteins exert a direct influence on the growth of ice. In addition, freezing injury was greater in extracted cold-acclimated leaves than in unextracted cold-acclimated leaves, which suggests that the proteins present in the apoplast are an important component of the mechanism by which winter rye leaves tolerate ice formation     

Heterologous expression, refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae)

Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53 °C ± 0.53 °C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34 °C ± 0.25 °C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability.