Freezing stress and membrane injury of Norway spruce (Picea abies) tissues

Effects of sub-zero temperatures (−5 to −35°C) on the tissues of needles, buds and shoots of Norway spruce [ Picea abies (L.) Karst.] were studied. The freezing caused increased efflux of cellular electrolytes. Freezing injury of the primordial shoots and 1-year-old shoots was the result of the spontaneous freezing of a deep supercooled cellular water. The crystallization injures the cellular membranes leading to the loss of semipermeability and to the drastic efflux of K⁺. In the needles there was no deep supercooling of water and two patterns of changes in the membranes, depending upon the range of the applied temperatures, could be distinguished. At 0 to – 25°C, which do not kill the cells, we observed a disturbance in the membrane semipermeability as monitored by electrolytes efflux within a few hours after thawing of the needles. At lower temperatures (−35°C) we observed irreversible loss of the membrane semipermeability, and death of the tissue. Those changes occurred 10 h after thawing and were probably caused by the released lytic enzymes and some toxic compounds, which acted on the cellular membranes.     

Ultrastructural changes arising from freezing of leaf blade cells of rye (Secale cereale): an investigation using freeze-substitution

The survival at sub-zero temperatures of leaf blade cells of rye ( Secale cereale L. cv. Voima), which had not been cold acclimated, was determined by measuring the efflux of ninhydrin-positive substances: 50% of the cells were dead at −4°C (LT₅₀) and none survived at −12°C or below. Examination of ultrastructural changes during cold hardening and freezing injury requires frozen tissues prepared for transmission electron microscopy without thawing. Specimens were prepared from leaf blade segments at room temperature, −4°C or −12°C by plunge freezing at 3 m s⁻¹ into a cooling medium at −170°C followed by freeze-substitution in acetone with OsO₄ fixation. Comparisons of room temperature specimens were made with those prepared by chemical fixation using glutaraldehyde/paraformaldehyde/tannic acid. On freezing to −12°C, the cells were severely dehydrated and distorted, the vacuoles severely shrunken and the cytoplasm and mitochondria disorganized whereas the chloroplasts were little affected. On freezing to −4°C, some cells were as disorganized as those at −12°C, others were relatively intact, and some showed evidence of intracellular ice crystal formation.     

Freezing and desiccation tolerance of imbibed canola seed

Depending on the environmental conditions, imbibed seeds survive subzero temperatures either by supercooling or by tolerating freezing-induced desiccation. We investigated what the predominant survival mechanism is in freezing canola ( Brassica napus cv. Quest) and concluded that it depends on the cooling rate. Seeds cooled at 3°C h⁻¹ or faster supercooled, whereas seeds cooled over a 4-day period to −12°C and then cooled at 3°C h⁻¹ to−40°C did not display low temperature exotherms. Both differential thermal analysis and nuclear magnetic resonance (NMR) spectroscopy confirmed that imbibed canola seeds undergo freezing-induced desiccation at slow cooling rates. The freezing tolerance of imbibed canola seed (LT₅₀) was determined by slowly cooling to −12°C for 48 h, followed with cooling at 3°C h⁻¹ to −40°C, or by holding at a constant −6°C (LD₅₀). For both tests, the loss in freezing tolerance of imbibed seeds was a function of time and temperature of imbibition. Freezing tolerance was rapidly lost after radicle emergence. Seeds imbibed in 100 μ M abscisic acid (ABA), particularly at 2°C, lost freezing tolerance at a slower rate compared with water-imbibed seeds. Seeds imbibed in water either at 23°C for 16 h, or 8°C for 6 days, or 2°C for 6 days were not germinable after storage at −6°C for 10 days. Seeds imbibed in ABA at 23°C for 24 h, or 8°C for 8 days, or 2°C for 15 days were highly germinable after 40 days at a constant −6°C. Desiccation injury induced at a high temperature (60°C), as with injury induced by freezing, was found to be a function of imbibition temperature and time.     

THE COMBINED EFFECTS OF LOW TEMPERATURE AND SO2+NO2 POLLUTION ON THE NEW SEASON'S GROWTH AND WATER RELATIONS OF PICEA SITCHENSIS

Picea sitchensis (Bong.) Carr. seedlings were exposed to SO₂, NO₂ and SO₂+ NO₂ during dormancy in controlled environments, and were taken to night temperatures of 4, 0, −5, −10 and −15 °C in a freezer. Conditions in the freezer were carefully monitored during the low–temperature treatments. In two experiments, different photoenvironments and temperature regimes were imposed prior to the cold treatments, and different effects were observed. In the first, only limited frost hardiness was achieved and night temperatures of −15 °C were lethal. Temperatures of −5 and − 10 °C led to poor survival of lateral buds, particularly in plants exposed to 45 ppb SO₂. The poor bud break in plants exposed to SO₂ and to − 5 °C resulted in a loss of the effectiveness of this temperature as a chill requirement. Pressure-volume analysis showed that the shoots of plants exposed to NO₂ had greater elasticity (lower elastic moduli, e), so that loss of turgor occurred at lower relative water contents. In contrast, a hardening period (2 weeks in night/day temperatures of 3/10 °C and 8 h days at 50 μmol m⁻² s⁻¹ PAR) gave decreased elasticity and lower solute potentials of spruce shoots. In the second experiment, exposure to 30 ppb SO₂ and SO₂+ NO₂ led to slight, but consistent, increases in frost injury to the needles of plants frozen to − 5 and − 10 °C. The results suggest that the main interaction of low temperatures and winter pollutants may be on bud survival rather than on needle damage, but that effects are subtle, only occurring with certain combinations of pollutant dose and cold treatment.     

Identification of tissues showing the lowest tolerance to freezing in larvae of the rice stem borer, Chilo suppressalis

Abstract.  Even though overwintering larvae of the rice stem borer, Chilo suppressalis , are freeze-tolerant, they cannot survive below −30 °C. Furthermore, nondiapausing larvae cannot survive freezing. However, the cause of death due to freezing is unclear. To identify the cause of death by freezing in larvae, those tissues most injured by low temperatures are identified using the vital stain trypan blue. In overwintering larvae, the midgut of dead larvae stains blue, and remarkable colour density differences between dead and surviving larvae are observed in the midgut. In nondiapausing larvae incubated at −10 °C for several hours, the fat body of dead larvae is strongly stained. Furthermore, increases in mortality with treatment time correspond with increases in the area of the fat body stained. Sterile nondiapausing larvae with lower supercooling points, below −20 °C, do not freeze at −10 °C and survive the treatment. However, all the larvae die when subjected to inoculative freezing at −10 °C, and the fat body stains blue. These results suggest that the midgut in overwintering larvae and the fat body in nondiapausing larvae have the lowest tolerance to freezing.     

Structural changes and impairment of function associated with freezing and thawing in muscle, nerve, and leucocytes

In some tissues, especially of skeletal muscle, leucocytes, and peripheral nerves, the effects of freezing to low temperatures, e.g., −78 or −150 °C, and thawing have been a total loss of function and a severe cellular structural disturbance. In the case of muscle, the fibers are converted during thawing into a highly condensed form, called thaw-rigor, in which the individual sarcomeres are shortened beyond a supercontracted state. This effect must be avoided if muscle is to be frozen and stored successfully. Leucocytes and especially the neutrophils are seriously affected by exposure to low temperature. The result is a general swelling of the cell and of its nucleus which is suggestive of damage to the membranes of the cell. Rat cutaneous nerves frozen and exposed to temperature below −15 °C showed after thawing a general disturbance of the myelin involving rupture and separaration of the lamellae. In the axoplasm the neurofilaments and neurotuabules are converted into small aggregates and the mitochondria appear to be swollen. Nerves frozen at −5 and some at −10 °C showed little change in structure, the myelin and axoplasm exhibiting a normal appearance. A check on viability by excitation from an electric stimulus indicated survival after exposure to −5 and in a few cases to −10 °C.     

Rapid cold-hardening in a Karoo beetle, Afrinus sp.

Abstract.  In the insect rapid cold-hardening response, survival at subzero temperatures is greatly improved by a brief pre-exposure at a milder temperature. It is predicted that insects with minimal cold tolerance capabilities living in variable environments should use rapid cold-hardening to survive sudden cold snaps. This is tested in Afrinus sp., a beetle that lives in an exposed habitat on rock outcrops in the Karoo Desert, South Africa, where microclimate temperatures drop infrequently to below freezing. Afrinus sp. shows a significant rapid cold-hardening response: survival of a 2-h exposure to −6.5 °C is much improved after pre-exposure to −2 °C, to 0 °C with a 2-h return to the rearing temperature, and to 40 °C, but not after pre-exposure to 0 °C. Little is known about the mechanism of the rapid cold-hardening response, although the data suggest that rapid cold-hardening may be mediated via several different mechanisms.     

Ultrastructure-function correlative studies for cardiac cryopreservation. II. Hearts frozen to various temperatures without a cryoprotectant

Isolated rat hearts were perfused with balanced salt solution (BSS) for 20 min, sealed in a metal cannister, and cooled in a −20 °C acetone bath at a rate of 1 °C/min to one of four subzero core temperatures (−10, −12, −17, or −20 °C). Upon attainment of the desired temperature the hearts were rapidly thawed (40–50 ° C/min) and reperfused with BSS for an additional 20 min. Approximately half of the hearts cooled to −10 or −12 °C resumed spontaneous contractile activity after thawing. One of 16 hearts survived cooling to −17 °C, while no heart survived cooling to −20 °C. Nonfrozen controls gave a positive inotropic response to a standard test dose of ouabain; none of the thawed survivors did.     

Effect of temperature on sulphate reduction, growth rate and growth yield in five psychrophilic sulphate-reducing bacteria from Arctic sediments

Five psychrophilic sulphate-reducing bacteria (strains ASv26, LSv21, PSv29, LSv54 and LSv514) isolated from Arctic sediments were examined for their adaptation to permanently low temperatures. All strains grew at −1.8°C, the freezing point of sea water, but their optimum temperature for growth ( T _opt) were 7°C (PSv29), 10°C (ASv26, LSv54) and 18°C (LSv21, LSv514). Although T _opt was considerably above the in situ temperatures of their habitats (−1.7°C and 2.6°C), relative growth rates were still high at 0°C, accounting for 25–41% of those at T _opt. Short-term incubations of exponentially growing cultures showed that the highest sulphate reduction rates occurred 2–9°C above T _opt. In contrast to growth and sulphate reduction rates, growth yields of strains ASv26, LSv54 and PSv29 were almost constant between −1.8°C and T _opt. For strains LSv21 and LSv514, however, growth yields were highest at the lowest temperatures, around 0°C. The results indicate that psychrophilic sulphate-reducing bacteria are specially adapted to permanently low temperatures by high relative growth rates and high growth yields at in situ conditions.     

SEASONALITY AND THERMAL ACCLIMATION OF REACTIVE OXYGEN METABOLISM IN FUCUS VESICULOSUS (PHAEOPHYCEAE)

The intertidal brown macroalga Fucus vesiculosus L. acclimates its defense against reactive oxygen in response to both (1) growth at different temperatures in laboratory culture and (2) seasonal changes in environmental conditions. Fucus vesiculosus was grown in seawater at 0° C, 20° C, and at 0° C with a 3-h daily emersion at −10° C. Algae grown at low temperature, both with and without freezing, produced less reactive oxygen after severe freezing stress than those grown at 20° C. These differences were correlated with growth temperature-induced changes in activities of superoxide dismutase (SOD), glutathione reductase, and ascorbate peroxidase. The contents of tocopherols increased with increased cultivation temperature, whereas the activity of catalase and the content of glutathione and ascorbate did not change. Growth at 0° C increased the resistance of photosynthesis to freezing and reduced photoinhibition in high light at 5° C; the latter effect was further increased in algae subject to daily freezing. These data suggest that elevated activity of reactive oxygen scavenging enzymes, especially SOD, increases the resistance to photoinhibition, at least at low temperature, as well as being important for freezing tolerance. Seasonal changes in reactive oxygen metabolism showed a similar pattern to those elicited by temperature in laboratory culture. Summer samples had lower activities of most reactive oxygen scavenging enzymes than algae collected in autumn and winter when water temperatures were lower. In contrast to the laboratory experiments, ascorbate content did change and was lower during the winter than summer, whereas the content of glutathione was not influenced by season. Overall, the data not only indicate that temperature plays an important role in the regulation of stress tolerance and reactive oxygen metabolism but also suggest that other factors are also involved.     

Cold hardening of raspberry plants in vitro is enhanced by increasing sucrose in the culture medium

The influence of exogenously applied sucrose on cold hardening of raspberry ( Rubus idaeus L.) in vitro was examined. Raspberry plants (cv. Preussen) were cultured on Murashige-Skoog (MS) media with different levels (1, 3, 5 and 7%) of sucrose and subjected to low-temperature acclimation (3/−3°C day/night temperature, 8-h photoperiod) for 14 days. Cold hardiness (LT₅₀ in controlled freezing), shoot moisture content, osmolality and the amounts of sucrose, glucose and fructose were determined. Exogenously applied sucrose was taken up by plants, but the uptake corresponded to less than 10% of total sugar reserves in the culture. Cold hardiness was primarily affected by acclimation treatment, but sucrose increased cold hardiness of nonacclimated plants and significantly enhanced the effect of acclimation treatment, 5% sucrose in the culture medium being optimal for cold hardening. LT₅₀ values ranged between −4.1 and −7.1°C for nonacclimated, and between −14.2 and −20.7°C for cold-acclimated shoots. Shoot moisture content was inversely related to medium sucrose level and declined only slightly during cold acclimation. After cold acclimation, plant osmolality predicted hardiness better than shoot moisture content. Plant osmolality and sugar content were increased by increasing the medium sucrose level and, to a greater extent, by cold acclimation. Sucrose, glucose and fructose accumulated during hardening. Sucrose was the predominant sugar, and the rate of sucrose accumulation during cold acclimation was independent of the medium sucrose level or the initial plant sucrose content. A close correlation between cold hardiness and total sugars, sucrose, glucose and fructose was established. These results suggest that sugars have more than a purely osmotic effect in protecting acclimated raspberry plants from cold.     

Vegetative growth and frost hardiness of cloudberry (Rubus chamaemorus) as affected by temperature and photoperiod

The effect of photoperiod and temperature on growth and induction and development of frost hardiness in cloudberry ( Rubus chamaemorus L.) was examined in two experiments. The photoperiods were 8, 12 or 24 h and the temperatures were 18, 15, 12, 9, 4, 3, –3 or –4°C depending on the experiment. The level of hardiness was expressed as LT₆₆ or LT₅₀ (the lethal temperature for 66 or 50% of the plant material) for percentage of bud break and for the degree of coloring by triphenyltetrazolium chloride for rhizomes. The vegetative growth was clearly affected by daylength; petiole elongation, leaf growth, shoot dry weight and number of shoots per plant were all reduced under short days compared with long days. However, the photoperiod had no significant effect on hardening of buds or rhizomes. Hardening increased with successively decreasing temperatures. To get the maximum hardiness, plants had to be exposed to freezing temperatures.     

Tannin impacts on microbial diversity and the functioning of alpine soils: a multidisciplinary approach

In alpine ecosystems, tannin-rich-litter decomposition occurs mainly under snow. With global change, variations in snowfall might affect soil temperature and microbial diversity with biogeochemical consequences on ecosystem processes. However, the relationships linking soil temperature and tannin degradation with soil microorganisms and nutrients fluxes remain poorly understood. Here, we combined biogeochemical and molecular profiling approaches to monitor tannin degradation, nutrient cycling and microbial communities (Bacteria, Crenarcheotes, Fungi) in undisturbed wintertime soil cores exposed to low temperature (0°C/−6°C), amended or not with tannins, extracted from Dryas octopetala . No toxic effect of tannins on microbial populations was found, indicating that they withstand phenolics from alpine vegetation litter. Additionally at −6°C, higher carbon mineralization, higher protocatechuic acid concentration (intermediary metabolite of tannin catabolism), and changes in fungal phylogenetic composition showed that freezing temperatures may select fungi able to degrade D. octopetala 's tannins. In contrast, negative net nitrogen mineralization rates were observed at −6°C possibly due to a more efficient N immobilization by tannins than N production by microbial activities, and suggesting a decoupling between C and N mineralization. Our results confirmed tannins and soil temperatures as relevant controls of microbial catabolism which are crucial for alpine ecosystems functioning and carbon storage.     

Field ecology of freeze tolerance: interannual variation in cooling rates, freeze-thaw and thermal stress in the microhabitat of the alpine cockroach Celatoblatta quinquemaculata

Microclimate recordings were made over four years on the Rock and Pillar Range, New Zealand. These are used in conjunction with mortality and freezing data derived in the laboratory to make inferences about the winter thermal ecology of the freeze-tolerant alpine cockroach Celatoblatta quinquemaculata (Dictyoptera: Blattidae). Threshold temperatures are identified through laboratory experiments at ecologically relevant cooling rates: 0°C; −3.1°C (FP₅: the temperature at which 5% of cockroaches are expected to be frozen); and −4.5°C (FP₅₀). The maximum cooling rate in the microhabitat across any of the thresholds was 0.06°C min⁻¹, considerably slower than the 1°C min⁻¹ normally used in laboratory studies. Freeze-thaw events occurred regularly in the field, and temperature minima occasionally fell to temperatures lethal to C. quinquemaculata . Variability in snow cover contributed to interannual variation in microclimate temperatures. Decreased snow cover is predicted with climate change scenarios, and this will probably result in more freeze-thaw cycles and more extreme minimum temperatures in this environment. It is concluded that the limited environmental tolerances of the animals living in this habitat make the few degrees of interannual variation ecologically relevant.     

Survival of the first stage larva of the metastrongyloid nematode Elaphostrongylus rangiferi under various conditions of temperature and humidity

First-stage larvae of E. rangiferi kept in water at 50°C died within 80 minutes, while at 6° the last larvae died between day 180 and 210. The time it took to reach 1_x= 0.5 (half of the larvae dead) at various temperatures between 6° and 50° was well described by the exponential function y = 614.6e^−0.15x, giving a value of 615 days to reach 1_x= 0.5 at 0°C. There was no clear decrease in the survival of larvae frozen at −20° in faeces and in water, and at −80° in faeces after 360 days. When subjected to repealed freezing and thawing, all larvae died within 77 days. When kept in air at RH = 20% and 22°C, all larvae died within 11 days, while when frozen (−20°C) in air at RH approx. 0%, 1_x stayed at approx. 0.5 from day 5 to day 16.     

Altitudinal and seasonal variation of frost resistance of Fagus crenata and Betula ermanii along the Pacific slope of Mt. Fuji, Japan

1 Frost resistance of Fagus crenata (Siebold's beech) and Betula ermanii (Japanese mountain birch) was investigated with respect to the species' altitudinal distribution on the Pacific slope of Mt. Fuji from 1996 to 1997. Flint's Index of Injury, which is based on electrolyte leakage from freeze-injured tissue, was used to assess frost hardiness of shoots produced in the previous growing season.
2 Fagus crenata is found on the lower slopes (700–1600 m a.s.l.). Mid- to late November hardening of shoots was enhanced, midwinter damage below −30 °C reduced and dehardening delayed nearer the upper limit. To here temperatures began to rise at least 3 weeks before dehardening began. Shade crown shoots were more susceptible to deep-freeze damage than light crown shoots. If the ultimate upper distribution limit was determined by frost hardiness, F. crenata would be expected to occur up to 1800 m altitude.
3 Betula ermanii is found between 1600 m and 2800 m, and intensive hardening occurred at all altitudes during the second half of October. Frost hardiness increased considerably with altitude up to the forest limit, where frost acclimation preceded the temperature decline by 2 weeks. Once maximum frost resistance had been attained freezing to −47 °C failed to cause tissue injury. Dehardening began slightly later at the tree line, but the time–course was the same at all altitudes. Main and lateral shoots did not differ in frost hardiness.
4 Comparison of monthly air temperature minima over the past 66 years with the course of frost resistance showed that F. crenata and B. ermanii found on the Pacific slope of Mt. Fuji were unlikely to suffer damage by frost.
5 The observed uppermost distribution limit for B. ermanii at 2800 m altitude on Mt. Fuji is considered both with our observations and with previous hypotheses.     

CO2 enrichment and development of freezing tolerance in Norway spruce

Plant growth and adaptation to cold and freezing temperatures in a CO₂-enriched atmosphere have received little attention despite their predicted effects on plant distribution and productivity. In this study we looked at the interaction between elevated CO₂ and development of freezing tolerance in Norway spruce ( Picea abies (L.) Karst.). First-year seedlings were grown under controlled conditions in an atmosphere enriched in CO₂ (70 Pa) for one simulated growth season. We measured shoot growth, registered the timing of growth cessation and bud set, measured needle net photosynthetic rate, and determined needle carbohydrate concentration (fructose+pinitol, glucose, sucrose, inositol, raffinose and starch). Freezing tolerance (LT₅₀) was determined after exposing whole seedlings to temperatures ranging from −6.5 to −36.0°C and scoring for visual needle browning. Elevated CO₂ did not affect height growth or the timing of growth cessation and bud set. The only statistically significant effects of CO₂ treatment were on seedling dry weight, percent dry matter and starch content. During the three weeks after growth cessation and bud set, freezing tolerance increased from −10 to −35°C, and there was a marked increase in all soluble sugars except inositol. However, neither freezing tolerance nor the concentration of soluble sugars was significantly influenced by elevated CO₂.     

Cold acclimation of Ilex aquifolium under natural conditions with special regard to the photosynthetic apparatus

Frost resistance of leaves of holly ( Ilex aquifolium L.) increased from about −9°C in late summer to −24°C in mid-winter. The gradual rise in cold hardiness occurred when the minimum air temperature dropped to 0°C or below and was closely related to increase in the cellular sap concentration. Predominantly, the decrease in the osmotic potential of the cellular sap was caused by sugar accumulation, mainly of sucrose. The capacity of net photosynthesis of the leaves, as well as the total lipid and protein content and the proportion of individual lipids of the thylakoid membranes, did not significantly change during cold acclimation. The gradual shift towards desaturation in the fatty acids of the thylakoid lipids during the hardening period was neither correlated with alterations in the frost resistance nor did it affect the potential efficiency for various light-induced chloroplast membrane reactions such as linear photosynthetic electron transport, photophosphorylation and the proton gradient (ΔpH). It is suggested that in holly leaves reduction in cell volume changes during freeze-thawing and cryoprotection by sugars could play a dominant role for the increase in frost resistance. Seasonal changes in the degree of unsaturation of polar lipids of the thylakoids could contribute to maintain optimal functional efficiency of the membranes at low temperatures rather than to avoid freezing damage.     

Cryopreservation of the sperm of the Japanese bitterling

Sperm of the Japanese bitterling Tanakia limbata that had been cryopreserved with 5 or 10% methanol plus 95 or 90% foetal bovine serum (FBS) showed higher percentage and longer duration of motility than those that had been cryopreserved with 90% FBS and 10% DMSO, glycerol, N,N-dimethylacetamide or N, N-dimethylformamide. Foetal bovine serum, used as extender, had some cryoprotective effects when spermatozoa were cooled either with 10% methanol or without methanol. Spermatozoa, cooled to −40° C and then immersed in liquid nitrogen, had greater post-thaw motility than those cooled to −20, −60, or −80° C. The post-thaw percentage of motile spermatozoa increased significantly ( P < 0·001) with decreases in the freezing rate from 60 to 5°C min⁻¹. These results indicate that 10% methanol plus 90% foetal bovine serum is a suitable diluent for cryopreservation of bitterling spermatozoa and that samples should be cooled to -40°C at a low freezing rate for effective storage.     

Macromolecular synthesis by yeasts under frozen conditions

Although viable fungi have been recovered from a wide variety of icy environments, their metabolic capabilities under frozen conditions are still largely unknown. We investigated basidiomycetous yeasts isolated from an Antarctic ice core and showed that after freezing at a relatively slow rate (0.8°C min⁻¹), the cells are excluded into veins of liquid at the triple junctions of ice crystals. These strains were capable of reproductive growth at −5°C under liquid conditions. Under frozen conditions, metabolic activity was assessed by measuring rates of [³H]leucine incorporation into the acid-insoluble macromolecular fraction, which decreased exponentially at temperatures between 15°C and −15°C and was inhibited by the protein synthesis inhibitor cycloheximide. Experiments at −5°C under frozen and liquid conditions revealed 2–3 orders of magnitude lower rates of endogenous metabolism in ice, likely due to the high salinity in the liquid fraction of the ice (equivalent of ≈ 1.4 mol l⁻¹ of NaCl at −5°C). The mesophile Saccharomyces cerevisae also incorporated [³H]leucine at −5°C and −15°C, indicating that this activity is not exclusive to cold-adapted microorganisms. The ability of yeast cells to incorporate amino acid substrates into macromolecules and remain metabolically active under these conditions has implications for understanding the survival of Eukarya in icy environments.