Spin-Probe Studies during Freezing of Cells Isolated from Cold-Hardened and Nonhardened Winter Rye : MOLECULAR MECHANISM OF MEMBRANE FREEZING INJURY

Mesophyll cells isolated from cold-hardened and nonhardened winter rye (Secale cereale L. cv. Puma) were spin-labeled with the fatty-acid spin probe N-oxyl-4,4-dimethyloxazolidine 5-ketostearic acid. The probe was intercalated within the cellular membranes and changes in probe motion were followed during extracellular freezing of the cells. A correlation was observed between the lethal freezing temperatures (LT₅₀) of the cells and the maximum hyperfine splitting value achieved by the incorporated probe. Rigid limit spectra indicated that a more ordered average packing was attained by membranes of hardened cells which survived freezing to lower temperatures.     

Structural Changes in Thylakoid Proteins during Cold Acclimation and Freezing of Winter Rye (Secale cereale L. cv. Puma)

Thylakoids were isolated from nonhardened and cold-hardened winter rye (Secale cereale L. cv. Puma), and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of sulfhydryl reagents. Electrophoresis of cold-hardened rye thylakoid proteins revealed the presence of a 35 kilodalton polypeptide and the absence of a 51 kilodalton polypeptide found in nonhardened rye thylakoid proteins. The 35 kilodalton band could be induced by adding β-mercaptoethanol to nonhardened rye thylakoid proteins, whereas the 51 kilodalton band could be formed by adding cupric phenanthroline to these same proteins. Sulfhydryl group titration showed that cold-hardened rye thylakoid proteins contained more free sulfhydryls than nonhardened rye proteins. Although amino acid analysis of thylakoid proteins revealed quantitative differences in several amino acid residues, the polarity of thylakoid proteins did not change during cold acclimation. No significant changes in sodium dodecyl sulfate-polyacrylamide gels of thylakoid proteins appeared when either nonhardened or cold-hardened plants were frozen in vivo or in vitro. However, thylakoid proteins did aggregate when frozen in the presence of β-mercaptoethanol. Although thylakoid proteins isolated from cold-hardened rye contained more reduced thiols, a general state of reduction did not act as a cryoprotectant. It is hypothesized that conformational changes of specific proteins may be important for low temperature growth of rye.     

Mitochondrial Activity and Ethanol Accumulation in Ice-encased Winter Cereal Seedlings

Cold-hardened dark-grown seedlings of winter wheat (Triticum aestivum L.) and winter rye (Secale cereale L.) are killed during total encasement in ice at −1 C at a rate related to the initial cold hardiness of the cultivars. Few plants remain alive after 7 days of encasement. Nonhardened seedlings are rapidly killed in ice. The respiratory properties of mitochondria isolated from plants after increasing periods of ice encasement decline slowly, and activity is little impaired when intact plants are about 50% killed. Electron microscopy indicates that mitochondrial structure is not disrupted until 3 weeks of ice encasement. Ethanol accumulates in hardened and nonhardened plants in ice, but at levels which are not toxic to the plants.     

Resistance to low temperature photoinhibition is not associated with isolated thylakoid membranes of winter rye

In vivo measurements of chlorophyll a fluorescence indicate that cold-hardened winter rye (Secale cereale L. cv Musketeer) develops a resistance to low temperature-induced photoinhibition compared with nonhardened rye. After 7.2 hours at 5°C and 1550 micromoles per square meter per second, the ratio of variable fluorescence/maximum fluorescence was depressed by only 23% in cold-hardened rye compared with 46% in nonhardened rye. We have tested the hypothesis that the principal site of this resistance to photoinhibition resides at the level of rye thylakoid membranes. Thylakoids were isolated from cold-hardened and nonhardened rye and exposed to high irradiance (1000-2600 micromoles per square meter per second) at either 5 or 20°C. The photoinhibitory response measured by room temperature fluorescence induction, photosystem II electron transport, photoacoustic spectroscopy, or [¹⁴C]atrazine binding indicates that the differential resistance to low temperature-induced photoinhibition in vivo is not observed in isolated thylakoids. Similar results were obtained whether isolated rye thylakoids were photoinhibited or thylakoids were isolated from rye leaves preexposed to a photoinhibitory treatment. Thus, we conclude that increased resistance to low temperature-induced photoinhibition is not a property of thylakoid membranes but is associated with a higher level of cellular organization.     

Characterization of partially purified glutathione reductase from cold-hardened and nonhardened spinach leaf tissue

Glutathione reductase (GR) (EC 1.6.4.2) was studied in crude and partially purified extracts from nonhardened (25/20 °C D/N) and hardened (5/5 °C D/N) spinach-leaf tissue. Crude extracts of hardened tissue showed a 66% increase in glutathione reductase activity over that of nonhardened tissue. The enzyme was purified by ammonium sulfate precipitation, Sephadex G-150 chromatography, 2′, 5′ ADP-Sepharose affinity chromatography, and DEAE-Sephadex A-50 ion-exchange chromatography. The partially purified enzyme from the two sources showed different kinetic characteristics, heat inactivation, freezing inactivation, and electrophoretic mobilities. Hardened leaves contain different forms of glutathione reductase than do nonhardened leaves. GR from hardened spinach has greater stability against freezing and a higher affinity for substrates at low temperature than does GR from nonhardened spinach.     

Membrane alterations in winter rye and Brassica napus cells during lethal freezing and plasmolysis

Abstract Cells fixed during freezing or plasmolysis were used to study membrane alterations in hardened and non-hardened Brassica napus suspension-cultured cells and rye leaf mesophyll cells. The plasmalemma in non-hardened rye mesophyll cells formed multilamellar vesicles during lethal freezing at high subzero temperatures (–5°C). These vesicles became highly condensed at lower subzero temperatures (–10°C). Conversely, cold-hardened rye mesophyll cells did not undergo membrane alterations at these temperatures. The results from plasmolysis of B. napus and rye mesophyll cells hardened by ABA at 25 °C and low temperature (2°C), respectively, verify the cell response to lethal freezing. Again there was a continuum of responses with 1 kmol m^?3 balanced salt causing multilamellar protrusions. Appression of the plasmalemma against the tonoplast to form multilamellar vesicles and the invagination of these vesicles into the tonoplast were also observed in rye cells undergoing lethal plasmolysis. Increasing the plasmolysing solution to 3 kmol m^?3 occasionally caused the formation of multilamellar vesicles on the cell surface of hardened rye mesophyll cells.     

Spin-label Studies of Membranes in Rye Protoplasts during Extracellular Freezing

Protoplasts isolated from epicotyls of nonhardened winter rye seedlings were spin-labeled with the N-oxyl-4-4-dimethyloxazolidine derivatives of 5-ketostearic (5NS) and 16-ketostearic (16NS) acids. Spectra of the membrane-bound labels showed motional broadening with a rotational correlation time of 1.5 × 10⁻⁸ second for 5NS and 1.5 × 10⁻¹⁰ second for 16NS at 0 C. A procedure was developed to follow membrane changes in these protoplasts during extracellular freezing. With freezing, molecular motion of 5NS, but not of 16NS, spin probes was restricted. The increase in molecular order near the hydrated end of the membrane did not result from lowered temperatures inasmuch as no such change was observed in supercooled samples. These changes are probably due to dehydration of protoplast membranes during extracellular freezing. Similar results were obtained with multilayered egg yolk lecithin and are consistent with previous observations of changes in lecithin multibilayers during dehydration. Such alterations in membrane order might lead to irreversible membrane damage during extracellular freezing of plant cells.     

Adaptive Changes in ATPase Activity in the Cells of Winter Wheat Seedlings during Cold Hardening

A cytochemical study of ATPase activity in the cells of cold hardened and nonhardened winter wheat (Triticum aestivum L. cv. Nongke No. 1) seedlings was carried out by electron microscopic observation of lead phosphate precipitation. ATPase activity associated with various cellular organelles was altered during cold hardening. (a) At 22°C, high plasmalemma ATPase activity was observed in both cold hardened and nonhardened tissues; at 5°C, high activity of plasmalemma ATPase was observed in hardened tissues, but not in unhardened tissues. (b) In nonhardened tissues, tonoplast and vacuoles did not exhibit high ATPase activity at either 22 or 5°C, while in hardened tissues high activity was observed at both temperatures. (c) At 5°C, ATPase activity of nucleoli and chromatin was decreased in hardened tissues, but not in nonhardened tissues. It is suggested that adaptive changes in ATPase activity associated with a particular cellular organelle or membrane may be associated with the development of frost resistance of winter wheat seedlings.     

Freezing damage to isolated tomato fruit mitochondria as modified by cryoprotective agents and storage temperature

Isolated tomato (Lycopersicon esculentum var. Kc 146) fruit mitochondria could be stored successfully in the frozen state without a cryoprotective agent if the mitochondria were frozen quickly by immersion in liquid nitrogen and later thawed quickly at 30 C. Criteria of freezing damage were rate of respiration, adenosine diphosphate to oxygen ratio, and respiratory control ratio. Marked reduction in respiration and loss of respiratory control occurred when mitochondria were transferred from liquid nitrogen to −5, −10, or −18 C for 15 minutes prior to thawing at 30 C. Dimethylsulfoxide (5%) prevented freezing damage when mitochondria were incubated at −5 C but did not prevent freezing damage at −10 or −18 C. Isolated tomato mitochondria show promise as a model system for studying the nature of freezing damage and the mode of action of cryo-protective agents.     

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

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

Isolation and Freezing Tolerances of Mesophyll Cells from Cold-hardened and Nonhardened Winter Rye

A method is described for the release of large quantities of mesophyll cells from leaves of both cold-hardened and nonhardened winter rye seedlings by a combination of enzymic and physical maceration. Such preparations usually contain a certain percentage of nonviable cells and are thus not suitable for quantitative biochemical studies. A method is also described whereby pure preparations of viable cells could be obtained by centrifugation on Percoll, using the observation that upon replacement of the isolation medium by water the viable and dead cells exhibited very different buoyant densities. The buoyant density of cells isolated from cold-hardened seedlings differed significantly from their nonhardened counter-parts. Survival of the isolated cold-hardened and nonhardened cells following extracellular freezing in water and following plasmolysis in balanced salt solutions was found to be in very close agreement with that of the plants and tissues from which they were isolated.     

Freezing behavior of free protoplasts of winter rye

Free protoplasts prepared from the epicotyls of nonhardened rye seedlings were subjected to fast and slow freezing on a microscope-adapted thermoelectric stage. During rapid freezing to ?12 °C, ice formation occurred inside the protoplasts causing lethal disruption of cell and membrane organization. Under slow freezing to ?12 °C, ice formation occurred outside the protoplast with accompanying dehydration and contraction of the protoplast. Complete rehydration and recovery of the protoplasts occurred upon thawing after slow freezing. Free protoplasts therefore afford a new system for the study of mechanisms of plant cell freezing injury and resistance free of the complications presented by a cell wall.     

Swelling and Contraction of Mitochondria from Cold-hardened and Nonhardened Wheat and Rye Seedlings

A comparison of mitochondria isolated from 2 and 24 C grown winter wheat (Triticum aestivum L.) and winter rye (Secale cereale L.) seedlings revealed no correlation between changes in swelling and contraction characteristics and extent of cold hardiness. The swelling response changed markedly due to growth at low temperature, but the change was similar for the four cultivars examined. The swelling response was also observed to change rapidly during aging of isolated mitochondria, either at 2 or 24 C. Spontaneously swollen mitochondria, isolated from 24 C grown seedlings, contracted abruptly upon addition of certain oxidizable substrates, but this response was lost when seedlings were transferred from 24 to 2 C. Studies on the effect of various substrates and respiratory inhibitors on the swelling and contraction responses indicate that inhibitors which reduce or stop electron flow through the electron transport chain also inhibit substrate induced mitochondrial contraction.     

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.     

Leucine transport in cells isolated from cold-hardened and nonhardened winter rye

The properties of the leucine transport systems of cells isolated from dark-grown cold-hardened and nonhardened winter rye (Secale cereale L. cv. Puma) epicotyls were remarkably similar. After 1 hour of incubation, leucine was accumulated in the cells 80- to 100-fold above that of the external medium, but the transported leucine was not metabolized. Approximately one-third of the accumulated leucine was present in the vacuole after 40 minutes of incubation. At 25°C, efflux of leucine from the vacuole was 6 to 10 times slower than it was from the cytoplasm, while at 5°C efflux from the cells was inhibited.     

Fluorescence Properties Indicate that Photosystem II Reaction Centers and Light-Harvesting Complex Are Modified by Low Temperature Growth in Winter Rye

Thylakoids isolated from winter rye (Secale cereale L. cv Puma) grown at 20°C (nonhardened rye, RNH) or 5°C (cold-hardened rye, RH) were characterized using chlorophyll (Chl) fluorescence. Low temperature fluorescence emission spectra of RH thylakoids contained emission bands at 680 and 695 nanometers not present in RNH thylakoids which were interpreted as changes in the association of light-harvesting Chl a/b proteins and photosystem II (PSII) reaction centers. RH thylakoids also exhibited a decrease in the emission ratio of 742/685 nanometers relative to RNH thylakoids.

Room temperature fluorescence induction revealed that a larger proportion of Chl in RH thylakoids was inactive in transferring energy to PSII reaction centers when compared with RNH thylakoids. Fluorescence induction kinetics at 20°C indicated that RNH and RH thylakoids contained the same proportions of fast (α) and slow (β) components of the biphasic induction curve. In RH thylakoids, however, the rate constant for α components increased and the rate constant for β components decreased relative to RNH thylakoids. Thus, energy was transferred more quickly within a PSII reaction center complex in RH thylakoids. In addition, PSII reaction centers in RH thylakoids were less connected, thus reducing energy transfers between reaction center complexes. We concluded that both PSII reaction centers and light-harvesting Chl a/b proteins had been modified during development of rye chloroplasts at 5°C.

     

Role of Abscisic Acid in the Induction of Freezing Tolerance in Brassica napus Suspension-Cultured Cells

Brassica napus suspension-cultured cells could be hardened in 6 days at 25°C by the addition of mefluidide or ABA to the culture medium. Cells treated with mefluidide (10 milligrams per liter) or ABA (50 micromolar) attained an LT₅₀ of −17.5°C or −18°C, respectively, while the LT₅₀ for the comparable nonhardened control (sucrose) was −10°C. The increased freezing tolerance of mefluidide-treated cells was paralleled by a 4- to 23-fold increase in ABA, as measured by gas-liquid chromatography using electron capture detection. Application of 1 milligram per liter of fluridone, an inhibitor of abscisic acid biosynthesis, prevented the mefluidide-induced increase in freezing tolerance and the accumulation of ABA. Both these inhibitory effects of fluridone were overridden by 50 micromolar ABA in the culture medium. On the basis of these results, we concluded that increased ABA levels are important for the induction of freezing tolerance in suspension-cultured cells.     

Freezing injury and root development in winter cereals

Upon exposure to 2°C, the leaves and crowns of rye (Secale cereale L. cv `Puma') and wheat (Triticum aestivum L. cv `Norstar' and `Cappelle') increased in cold hardiness, whereas little change in root cold hardiness was observed. Both root and shoot growth were severely reduced in cold-hardened Norstar wheat plants frozen to −11°C or lower and transplanted to soil. In contrast, shoot growth of plants grown in a nutrient agar medium and subjected to the same hardening and freezing conditions was not affected by freezing temperatures of −20°C while root growth was reduced at −15°C. Thus, it was apparent that lack of root development limited the ability of plants to survive freezing under natural conditions.

Generally, the temperatures at which 50% of the plants were killed as determined by the conductivity method were lower than those obtained by regrowth. A simple explanation for this difference is that the majority of cells in the crown are still alive while a small portion of the cells which are critical for regrowth are injured or killed.

Suspension cultures of Norstar wheat grown in B-5 liquid medium supplemented with 3 milligrams per liter of 2,4-dichlorophenoxyacetic acid could be cold hardened to the same levels as soil growth plants. These cultures produce roots when transferred to the same growth medium supplemented with a low rate of 2,4-dichlorophenoxyacetic acid (<1 milligram per liter). When frozen to −15°C regrowth of cultures was 50% of the control, whereas the percentage of calli with root development was reduced 50% in cultures frozen to −11°C. These results suggest that freezing affects root morphogenesis rather than just killing the cells responsible for root regeneration.

     

Heat Tolerance of Cold Acclimated Puma Winter Rye Seedlings and the Effect of a Heat Shock on Freezing Tolerance

An increase in tolerance to one form of abiotic stress oftenresults in an increase in tolerance to another stress. The heattolerance of Puma rye (Secale cereale) was determined for seedlingseither not cold hardened or hardened under either controlledenvironmental or natural conditions. The heat tolerance wasdetermined either as a function of time at 42°C or the abilityto tolerate a maximum temperature. The seedlings were eithernot heat preconditioned or heat preconditioned before the heatstress. In all cases cold hardened seedlings were more heattolerant than non or partially cold hardened seedlings. Heatpreconditioning had no effect on the heat tolerance of naturallycold hardened seedlings. In contrast, seedlings cold hardenedin a controlled environment chamber, then heat preconditioned,were more heat tolerant than non preconditioned seedlings. Aheat shock of 36°C for 2 h increased the freezing toleranceof non hardened seedlings from –2.5°C to –4.5°C.Analysis of heat shock protein 70 (HSP70) gene expression indicatedthat the HSP70 gene was not induced by cold acclimation andtherefore not directly involved in the increased thermo toleranceobserved. A number of heat stable proteins, simple sugars andlong chain carbohydrate polymers accumulated during the coldacclimation process and may have a role in increasing heat toleranceas well as freezing tolerance. These data suggest cold hardeningincreases heat tolerance, however, a heat shock to non acclimatedseedlings only marginally increased the freezing tolerance ofPuma rye seedlings. ³Present address: Agriculture and Agri-Food Canada, 107 SciencePlace, Saskatoon SK S7N 0X2, Canada.     

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.