Antifreeze proteins are secreted by winter rye cells in suspension culture

During cold-acclimation, winter rye ( Secale cereale L) leaves secrete antifreeze proteins (AFPs) into the apoplast. The AFPs bind to ice and modify its growth, which is easily observed in vitro . However, it is not yet known whether in planta AFPs interact with ice or whether they exert cryoprotective effects. These experiments are difficult to conduct with intact plants, so the aim of this work was to determine whether AFPs are produced in response to cold temperature in cell culture and to examine their function by using suspension cells. We showed that suspension cells secreted three of the six known winter rye AFPs into the culture medium during acclimation at 4°C. These AFPs were not present in washed suspension cells, thus indicating that they are not firmly bound to the cell walls. In order to examine the function of extracellular AFPs, non-acclimated (NA) winter rye suspension cells and protoplasts isolated from NA winter rye leaves were then frozen and thawed in the presence of AFPs extracted from cold-acclimated winter rye leaves. The AFPs had no effect on the survival of NA protoplasts after freezing; however, they lowered the lethal temperature at which 50% of the cells are killed by freezing (LT₅₀) of NA suspension cells by 2.5°C. We conclude that low above-zero temperatures induce winter rye suspension cells to secrete AFPs free in solution where they can protect intact suspension cells, but not protoplasts, from freezing injury, presumably by interacting with extracellular ice.     

Reorganization of interphase microtubules in root cells of <Emphasis Type="Italic">Medicago sativa</Emphasis> L. during acclimation to osmotic and salt stress

We examined the organization of microtubule system of interphase cells in roots of Medicago sativa L. during acclimation to salt and osmotic stress at different concentrations of NaCl, Na₂SO₄, and mannitol. We identified morphological changes of tubulin cytoskeleton in different root tissues during the acclimation to salt and osmotic stress: (1) decreased density of the cortical microtubule network, (2) random orientation of cortical microtubule bundles, (4) thickening of the bundles, (3) nonuniform density of the bundles, (4) fragmentation of the bundles, and (5) formation of microtubule converging centers. Network thinning and thickening of the bundles were observed both under osmotic and salt stress. Random orientation of cortical microtubules was visualized under osmotic stress but not during salt stress. Fragmentation of microtubule bundles took place under salt stress with a high concentration of mannitol. Formation of microtubule converging centers was common under prolonged action of sodium sulfate, less evident under sodium chloride, and not found after mannitol treatment. Our data show that, in alfalfa root cells, cortical microtubules rearrange not only in response to different ions, but also to osmotic pressure. Thus, the signaling pathways and molecular mechanisms inducing reorganization of the microtubule system may be triggered by sodium cations, as well as by sulfate and chloride anions at concentrations that do not cause irreversible cell damage.     

The effect of low temperature on the microtubules in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L

We have studied the response of the interphase and mitotis microtubule arrays in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L. (Moskovskaya 35 and Moskovskaya 39) during cold stress (1 h at 0 degrees C) and acclimation to cold (3-48 h at 0 degrees C). Our data show that interphase microtubules are more resistant to cold than mitotic arrays in both cultivars. During cold stress the density of endoplasmic microtubules increases in interphase cells of winter plants, yet no changes are detected in cells of spring plants. In mitotic cells of both wheat cultivars the density of microtubules within the kinetochore fibers decreases, yet this effect is more evident in the cells of spring plants. During acclimation to cold of both cultivars, we have observed the disorganization of the interphase cortical arrays and the enhanced growth of endoplasmic microtubule arrays, composed of microtubule converging centers. However, the reaction of mitotic microtubule arrays differs in the cells of winter and spring plants. In winter plants, during prophase diffuse tubulin "halo" accumulates first at perinuclear area, followed by the appearance of the microtubule converging centers. In spring plants, we have observed the formation of the prophase spindle, yet later the prophase spindle is not detected. Metaphase cells of both cultivars show similar aberrations of the mitotic spindle, accumulation of abnormal metaphases and the excessive formation of microtubule converging centers. In telophase cells of both cultivars, acclimation induces similar reaction, resulting in the disorganization of the phragmoplast and the formation of multiple microtubule converging centers. The latter are detected in the perinuclear areas of the daughter cells in winter plants and in the cortical cytoplasm of cells in spring plants. Our data point to the common pathways of microtubule response to cold treatment (0 degrees C). The excessive formation of the microtubule converging centers indicates the activation of microtubule assembly during prolonged cold treatment.     

Microtubules in maize leaf protoplasts in relation to donor tissue and in vitro culture

Summary Maize (Zea mays) leaf protoplasts were isolated from various leaves of two-week (4-leaf) seedlings and from sections of the third leaf blades. Microtubules (MTs) were visualized using immunofluorescence microscopy. Only freshly isolated protoplasts from the third and fourth leaf blades contained MTs, with protoplasts from the fourth leaf containing the most i.e. 13% of fourth-leaf protoplasts contained MTs. In general, protoplasts with fewer and smaller chloroplasts had more MTs. Initially 90–95% of protoplasts from basal portions of leaves had MTs but the percentage decreased slightly during culture particularly after 10 days. The antioxidant n-propyl gallate was beneficial in maintaining MT content. Few protoplasts from older sections intitially contained MTs but in all sections at least some protoplasts regained a significant MT content during culture (e.g., 10% of protoplast from the tip section possessed microtubules after 7 days of culture). Far fewer MTs were observed in individual leaf protoplasts than those isolated from suspension culture.Abbreviations BMS Black Mexican Sweet - MT microtubule - MtSB microtubule stabilizing buffer - PBS phosphate buffered saline     

The Mechanics of Injury to Isolated Protoplasts following Osmotic Contraction and Expansion

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

Microtubules in mesophyll cells of nonacclimated and cold-acclimated spinach : visualization and responses to freezing, low temperature, and dehydration

Responses of cortical microtubules in spinach (Spinacia oleracea L. cv Bloomsdale) mesophyll cells to freezing, thawing, supercooling, and dehydration were assessed. Microtubules were visualized using a modified procedure for indirect immunofluorescence microscopy. Leaf sections of nonacclimated and cold-acclimated spinach were slowly frozen to various temperatures, fixed while frozen, and microtubules immunolabelled. Both nonacclimated and cold-acclimated cells exhibited nearly complete microtubule depolymerization after ice formation. After 1 hour thawing at 23°C, microtubules in both nonacclimated and cold-acclimated cells repolymerized. With time, however, microtubules in nonacclimated cells again depolymerized. Since microtubules in cells of leaf tissue frozen slowly are subjected to dehydration as well as subzero temperatures, these stresses were applied separately and their effects on microtubules noted. Supercooling induced microtubule depolymerization in both nonacclimated and cold-acclimated cells, but to a smaller extent than did freezing. Exposing leaf sections to solutions of sorbitol (a cell wall-penetrating osmoticum) or polyethylene glycol 10,000 (a nonpenetrating osmoticum) at room temperature caused microtubule depolymerization. The effects of low temperature and dehydration are roughly additive in producing the observed microtubule responses during freezing. Only small differences in microtubule stability were resolved between nonacclimated and cold-acclimated cells.     

Behavior of the Plasma Membrane of Isolated Protoplasts during a Freeze-Thaw Cycle

Cryomicroscopy of protoplasts isolated from nonacclimated (NA) rye leaves (Secale cereale L. cv Puma) revealed that the predominant form of injury following cooling to the minimum temperature for 50% survival (LT₅₀) (−5°C) was expansion-induced lysis of the plasma membrane during warming and thawing of the suspending medium when the decreasing osmolality resulted in osmotic expansion of the protoplasts. When cooled to temperatures below the LT₅₀, the predominant form of injury was loss of osmotic responsiveness following cooling so that the protoplasts were osmotically inactive during warming. Only a low incidence (<10%) of expansion-induced lysis was observed in protoplasts isolated from acclimated (ACC) leaves, and the predominant form of injury following cooling to the LT₅₀ (−25°C) was loss of osmotic responsiveness. The tolerable surface area increment (TSAI) which resulted in lysis of 50% of a population (TSAI₅₀) of NA protoplasts osmotically expanded from isotonic solutions was 1122 ± 172 square micrometers. Similar values were obtained when the protoplasts were osmotically expanded from hypertonic solutions. The TSAI determined from cryomicroscopic measurements of individual NA protoplasts was similar to the TSAI₅₀ values obtained from osmotic manipulation. The TSAI₅₀ of ACC protoplasts expanded from isotonic solutions (2145 ± 235 square micrometers) was approximately double that of NA protoplasts and increased following osmotic contraction. Osmotic contractions were readily reversible upon return to isotonic solutions. During freeze-induced dehydration, endocytotic vesicles formed in NA protoplasts whereas exocytotic extrusions formed on the surface of ACC protoplasts. During osmotic expansion following thawing of the suspending medium, the endocytotic vesicles remained in the cytoplasm of NA protoplasts and the protoplasts lysed before their original volume and surface area were regained. In contrast, the exocytotic extrusions were drawn back into the surface of ACC protoplasts as the protoplasts regained their original volume and surface area.     

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

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

Microtubule dynamics in relation to osmotic stress-induced ABA accumulation in Zea mays roots

Microtubules play important roles in many physiological processes such as plant responses to drought stress. Abscisic acid (ABA) accumulates significantly in plants in response to drought conditions, which has been considered as a major response for plants to enhance drought tolerance. In this work, the focus was on the possible roles of microtubules in the induction of ABA biosynthesis in the roots of Zea mays when subjected to osmotic stress. The dynamic changes of microtubules in response to the stress were investigated by immunofluorescence staining, enzyme-linked immunosorbent assay, and a pharmacological approach. Disruption and stabilization of microtubules both significantly stimulated ABA accumulation in maize root cells, although this stimulation was markedly lower than that caused by osmotic stress. Cells in which the microtubule stability had been changed did not respond further to osmotic stress in terms of ABA biosynthesis. However, treatment with both a microtubule de-stabilizer and a stabilizer enhanced the sensitivity of cells to osmotic stress in terms of ABA accumulation. It is suggested that both osmotic stress and changes in microtubule dynamics would trigger maize root cells to biosynthesize ABA, and interactions between osmotic stress and microtubule dynamics would have an effect on ABA accumulation in root cells, although the exact mechanism is not clear at present.     

Protein, leucine aminopeptidase, esterase, acid phosphatase and photosynthetic responses of oleander (Nerium oleander L.) during cold acclimation and freezing treatments

Six-month-old oleander (Nerium oleander L.) pot plants, derived from vegetative propagation by cuttings, were tested for their ability to cold hardening. Damage of the non-acclimated (NA) plants was visible when treated by low freezing temperatures (below -2 degrees C). The responses of total proteins, leucine aminopeptidase (LAP), esterase (EST) and acid phosphatase (ACP) isoforms of NA and cold-acclimated (CA; 4 degrees C for 14 days) plants were compared using polyacrylamide gel electrophoresis. These molecular markers were also compared in NA and CA plants which received for 2h temperatures of 0, -2, -4, -6 and -8 degrees C. A new 38-kDa polypeptide appeared from day 7 to 14 during the acclimation treatment in the bark extracts and on day 14 in the leaf extracts. The above-mentioned polypeptide band (38 kDa) strongly appeared in all freezing treatments (0, -2, -4, -6 and -8 degrees C) in both bark and leaf extracts of the CA plants. Alterations in the number and the intensity of LAP and EST isoforms as well as in the intensity of ACP isoforms were observed in both bark and leaf of the CA oleander plants. A newly expressed EST isoform is proposed as biochemical marker for the cold acclimation treatment. CO2 assimilation rates (A) as well as transpiration rates (E) in NA plants were positive in 0 degrees C and negative in all temperatures below zero in the freezing treatments. In contrast, CO2 assimilation rates (A) and transpiration rates (E) were positive in CA plants in all temperatures of freezing treatment. A significant decrease (P<0.05) in chlorophyll (Chl) a, Chl a+b concentration and Chl a/b ratio were noticed in oleander plants during the acclimation treatment (from day 0 to 14), while Chl b concentration was unchanged at the respective time. On the other hand, no significant (P<0.05) differences were observed in the freezing treatments.     

Cryopreservation of rye protoplasts by vitrification

A procedure has been developed for the vitrification of mesophyll protoplasts isolated from leaves of nonacclimated (NA) and cold-acclimated (ACC) winter rye seedlings (Secale cereale L. cv Puma). The procedure involves (a) equilibration (loading) of the protoplasts with an intermediate concentration (1.5, 1.75, or 2.0 molar) of ethylene glycol (EG) at 20°C; (b) dehydration of the protoplasts in a concentrated vitrification solution made of 7 molar EG + 0.88 molar sorbitol + 6% (w/v) bovine serum albumin (BSA) at 0°C; (c) placing the protoplasts into polypropylene straws and quenching in liquid nitrogen (LN₂); and (d) recovery of the protoplasts from LN₂ and removal (unloading) of the vitrification solution. For NA protoplasts, 47 + 1% survival was obtained following recovery from LN₂ if the protoplasts were first loaded with 1.75 molar EG prior to the dehydration step. However, to achieve this level of survival, NA protoplasts had to be unloaded in a hypertonic (2.0 osmolal [osm]) sorbitol solution. If they were unloaded in an isotonic solution (0.53 osm), survival was 3±2%. In contrast, survival of ACC protoplasts following recovery from LN₂ was 34 ± 10% when the protoplasts were loaded in a 2.0 molar EG solution and unloaded in an isotonic sorbitol solution (1.03 osm). If ACC protoplasts were unloaded in an hypertonic sorbitol solution (1.5 osm), survival was 51 ± 9%. These results indicate that the osmotic excursions incurred during the procedure are a major factor affecting survival.     

Primary and secondary induction requirements for flowering of Festuca rubra

Root and shoot temperatures were varied independently to determine the importance of root temperature during cold acclimation. Spinach ( Spinacia oleracea L. cvs Harbin and Bloomsdale) plants were subjected to 20/20°C. 20/5°C, 5/20°C, and 5/5°C (shoot/root) temperature treatments. Leaf freezing tolerance, water potential, stomatal resistance, osmotic potential, and water content were measured at 0.25. 1.25, 3.25, and 7.25 days of treatment. There was no change in freezing tolerance or the water relations of the 20/20°C treated plants during the course of the experiment. Freezing tolerance was increased by the 5°C shoot temperature treatments, but was not enhanced by water stress induced by the low root temperature. Leaf water potential and water content decreased and stomatal resistance increased within 6 h in the 20/5°C plants. By day 3, osmotic potential began decreasing in the 20/5°C plants. Leaf water content, osmotic potential, and water potential decreased more gradually in plants grown with 5°C shoot temperature, irrespective of root temperature. Decreased water content and osmotic potential were not correlated with increased freezing tolerance as reported for other herbaceous crop plants.     

Stabilization of cortical microtubules by the cell wall in cultured tobacco cells

Cortical microtubules (MTs) in protoplasts prepared from tobacco (Nicotiana tabacum L.) BY-2 cells were found to be sensitive to cold. However, as the protoplasts regenerated cell walls they became resistant to cold, indicating that the cell wall stabilizes cortical MTs against the effects of cold. Since poly-l-lysine was found to stabilize MTs in protoplasts, we examined extensin, an important polycationic component of the cell wall, and found it also to be effective in stabilizing the MTs of protoplasts. Both extensin isolated from culture filtrates of tobacco BY-2 cells and extensin isolated in a similar way from cultures of tobacco XD-6S cells rendered the cortical MTs in protoplasts resistant to cold. Extensin at 0.1 mg·ml⁻¹ was as effective as the cell wall in this respect. It is probable that extensin in the cell wall plays an important role in stabilizing cortical MTs in tobacco BY-2 cells.     

Induction of random microtubule polymerization in cold and drug-treated PtK1 cells following hyperosmotic shock treatment

The effects of hypertonic sucrose on spindle and interphase microtubule (MT) arrays of PtK1 cells were investigated by incubating cells in complete culture medium at 4 degrees or 37 degrees C, with or without hypertonic sucrose, nocodazole or vinblastine (VLB). Results from anti-tubulin immunofluorescence showed that sucrose-induced alterations of spindle morphology seen at 37 degrees C did not occur at cold temperatures, but cold-induced MT loss was diminished. Application of warm hypertonic sucrose following depolymerization of MTs by nocodazole or cold resulted in the formation of a "feltwork" of randomly oriented, short MTs throughout the cytoplasm. These results, and those obtained substituting VLB for nocodazole, suggest that the effects of sucrose depend on the cytoplasmic concentration of soluble tubulin and support the hypothesis that osmotic factors are involved in effects of hypertonic sucrose on MT organization.     

Cell proliferation,cell shape,and microtubule and cellulose microfibril organization of tobacco BY-2 cells are not altered by exposure to near weightlessness in space

The microtubule cytoskeleton and the cell wall both play key roles in plant cell growth and division, determining the plant’s final stature. At near weightlessness, tubulin polymerizes into microtubules in vitro, but these microtubules do not self-organize in the ordered patterns observed at 1g. Likewise, at near weightlessness cortical microtubules in protoplasts have difficulty organizing into parallel arrays, which are required for proper plant cell elongation. However, intact plants do grow in space and therefore should have a normally functioning microtubule cytoskeleton. Since the main difference between protoplasts and plant cells in a tissue is the presence of a cell wall, we studied single, but walled, tobacco BY-2 suspension-cultured cells during an 8-day space-flight experiment on board of the Soyuz capsule and the International Space Station during the 12S mission (March–April 2006). We show that the cortical microtubule density, ordering and orientation in isolated walled plant cells are unaffected by near weightlessness, as are the orientation of the cellulose microfibrils, cell proliferation, and cell shape. Likely, tissue organization is not essential for the organization of these structures in space. When combined with the fact that many recovering protoplasts have an aberrant cortical microtubule cytoskeleton, the results suggest a role for the cell wall, or its production machinery, in structuring the microtubule cytoskeleton.     

The phospholipase A inhibitor, aristolochic acid, disrupts cortical microtubule arrays and root growth in Arabidopsis

The role of phospholipase A(2) in Arabidopsis root growth and microtubule organisation was investigated using a specific inhibitor, aristolochic acid. At 0.5-1.5 microm concentrations, this inhibitor reduced root elongation and caused radial swelling of the root tip. The normally transverse cortical microtubules in root tip cells became progressively more disorganised with increasing concentrations of the inhibitor. Microtubule disorganisation also occurred in leaf epidermal cells of Allium porrum. We propose that phospholipase A(2) is involved in microtubule organisation and anisotropic growth in a manner similar to that reported previously for phospholipase D, thus broadening the significance of phospholipid signalling in microtubule organisation in plants.     

Reorganization of the tubulin and actin cytoskeleton under acclimation and abscisic acid treatment of Triticum aestivum L. plants

Only scanty and contradictory data are available concerning effects of low temperatures and ABA on the structural organization of microtubules (MTs) and microfilaments (MFs), and no information exists on the interaction of these parameters at cold acclimation of plants. Therefore, in cold acclimate and ABA-treated winter wheat plants, a comparative study was made of the state (localization, orientation, structure) and stability of actin and tubulin cytoskeleton in root cells taken from different zones, using indirect immunofluorescent microscope. The plant cold acclimation caused MT aggregation, the rise of MT and MF fluorescence, and the increase of their stability (a decrease of oryzalin effect) mainly in the root differentiation zone, that may testify to the strengthening of contacts between MTs and MFs. Like the cold acclimation, ABA induced the formation of MT bunches only in meristem and elongation zone cells. However in the zone of differentiation, the hormone stimulated the increase of tubulin structure stability, well correlating with a decrease in MT content, aggregation degree, and immunofluorescence, and, in addition with a complete depolymerization of MFs. Low temperatures removed the hormone effect on the structural organization of tubulin and actin cytoskeleton in the zone of differentiation. It is suggested that MT destruction, the decrease of instable MT populations, and the increase of stable MT populations may slow down growth processes in ABA-treated plants, similarly as in seedlings being on the initial stages of cold acclimation. By the end of this process, the induction of plant growth is determined evidently by the increase in the number of instable, highly labile MT populations, and in the status of MF polymerization.     

Root contraction in hyacinth III. Orientation of cortical microtubules visualized by immunofluorescence microscopy

Summary Cortical microtubules (MTs) were visualized in root cortex cells ofHyacinthus orientalis L. using immunofluorescence techniques. Cellular MT orientation was determined adjacent to radial longitudinal and transverse walls of root tip, uncontracted, contracting, and fully contracted regions. As seen in longitudinal views, MTs formed parallel, apparently helical arrays which were oriented transversely, axially or obliquely depending upon the region. Transverse sectional views showed that MTs adjacent to transverse cell walls formed a variety of patterns which varied with developmental stage and cell location. Microtubules were oriented in crisscross or parallel arrays. The parallel arrays were oriented either parallel, perpendicular or oblique to the radius of the root. There was an apparent temporal progression in MT reorientation from outer cortical to inner cortical cell layers. A resultant progression of reoriented cell growth could account for root contraction. These findings corroborate earlier electron microscopic observations of changing MT orientation accompanying root contraction, and provide cytological evidence to test mathematical and biophysical models of the mechanics of cell expansion.Abbreviations MT microtubule - MF microfibril - MTSB microtubule stabilizing buffer - PBS phosphate buffered saline     

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.     

Immunogold localization of glucanase-like antifreeze protein in cold acclimated winter rye

Summary Apoplastic antifreeze proteins (AFPs) accumulate in winter rye (Secale cereale L. cv. Musketeer) leaves during cold acclimation. Two of the rye AFPs with molecular masses of 32 and 35 kDa are similar in their amino acid sequences and epitopes to -1, 3-endoglucanase. Localization of these AFPs, which we refer to as glucanase-like proteins (GLPs), was carried out with antiserum raised against the 32 kDa AFP. Specimens from leaves and roots of non-acclimated (NA) plants and cold acclimated (CA) plants were prepared by freeze-substitution for high resolution immunoelectron microscopy. In CA leaves, high levels of GLPs were observed in cell walls of mesophyll cells adjacent to intercellular spaces and in secondary thickenings of xylem vessels. Taken together with the absence of GLPs in vacuoles, these results confirm the apoplastic accumulation of AFPs in CA winter rye. Within the cells of CA leaves, GLPs were localized in cisternae of the rough endoplasmic reticulum, the Golgi apparatus and the plasma membrane, which indicates that GLPs are secreted via an exocytic bulk-flow pathway. The occurrence of high levels of GLPs in CA leaves, their low presence in NA leaves and the lack of GLPs in roots all suggest that there is a correlation between increased accumulation of GLPs and increased freezing tolerance of these plant materials. Furthermore, the localization of GLPs in the immediate vicinity of pathways for free water within the tissues supports the view that these proteins have an important role in the crystallization and/or recrystallization of water when the leaves of CA winter rye are exposed to freezing temperatures.Abbreviations AFP antifreeze protein - BSA bovine serum albumin - CA cold acclimated - GAR goat antirabbit antiserum conjugated with colloidal gold - GLP glucanase-like protein - NA non-acclimated - PBS phosphate buffered saline - PR pathogenesis related