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.     

Seasonal variation in freezing tolerance of the New Zealand alpine cockroach Celatoblatta quinquemaculata

1. The cold hardiness of the alpine cockroach Celatoblatta quinquemaculata was investigated. This species is found at 1360 m a.s.l. beneath schist slabs on the Rock and Pillar Range (Central Otago, New Zealand). Cockroaches were collected monthly from January to December 1996, and their LT₅₀ and supercooling points determined.
2. Celatoblatta quinquemaculata was freezing tolerant throughout the year, with a lower lethal temperature in winter of – 8.9 °C. Celatoblatta quinquemaculata was also found frozen under rocks in the field when the under-rock temperature was below – 3 °C, and could survive being frozen at – 5 °C for 4 days in the laboratory.
3. There was a marked decrease in LT₅₀ temperature from – 5.5 °C in April to – 7.5 °C in May. This coincides with decreasing temperatures from summer through autumn to winter, during which temperatures beneath snow-covered rocks may reach – 7.3 °C.
4. Supercooling points fluctuated during the year, with an increase from – 4.2 °C in autumn to – 3.4 °C in winter. Supercooling point was highest in spring, and changes in supercooling point do not appear to be related to changes in LT₅₀.
5. Recordings of environmental temperatures from the Rock and Pillar Range suggest that cockroaches may undergo up to twenty-three freeze–thaw cycles in the coldest month of the year, and that they may remain frozen for periods of up to 21 h. Maximum cooling rates recorded in the field (0.01 °C min^–1) were 100-fold slower than laboratory cooling rates, so survival estimates from laboratory experiments may be underestimates.     

Differential cold hardiness in adults and nymphs of the peach-potato aphid Myzus persicae

The LT₅₀ (lethal temperature) of first instar and adult stages of the peach-potato aphid Myzus persicae was lowered following long term acclimation at low temperatures.
First instars consistently showed greater cold hardiness than adult stages at each acclimation temperature, with the differential increasing as the temperature was lowered. When maintained at 5°C (the lowest acclimation regime) nymphs and adults had dLT₅₀8.3°C and 4.7°C respectively lower than those for non-acclimated individuals.
When 10°C acclimated adults were returned to 20°C, the acclimation effect was retained in full for 6 days but complete deacclimation occurred by day 10. In contrast the LT₅₀ of their progeny increased gradually from the first day of adult deacclimation towards the level of the unacclimated control over a period of 10 days.
A change in cold hardiness was observed in first instars according to their position in the birth sequence. The LT₅₀ of first-born nymphs (day 1 of reproduction) from 20°C parents was - 15.9°C rising to - 8.3°C by day 4 and remaining at this level until the end of the reproductive period.
The differential mortality between nymphs and adults observed in the laboratory was supported by the results of a field experiment. Adult aphids kept in clip-cages on a crop of oilseed rape showed greater mortality compared with those introduced as nymphs when the minimum temperature fell below -4°C for the first time in winter. At - 10°C mortality of aphids introduced as adults approached 100% whereas more than 50% of those introduced as nymphs were still alive at this temperature.     

Role of cell wall in freezing tolerance of cultured potato cells and their protoplasts

Cultured potato ( Solanum tuberosum L., cv. Red Pontiac) cells suspended in PEG 1000 solutions of 0.6 and O.S osmol exhibited significantly different freezing tolerance from the same cells when suspended in PEG 6000 solutions of the same osmolalities. Cells suspended in PEG 6000 showed cytorhysis instead of plasmolysis. Cells in 0.2 and 0.4 osmol PEG 1000 had LT₅₀(1 of −2.5°C, but the LT₅₀ decreased to −7.50C as the osmolality increased to 0.8 osmol. In PEG 6000 the LT₅₀ remained at −2.50C for all osmolalities used, up to and including 0.8 osmol.
Released protoplasts suspended in 0.5 M sucrose had LT₅₀ of −21.5°C, compared to −12°C for whole cells suspended in the same medium. These results lend credence to an involvement of the cell wall in freezing injury of cultured potato cells, and are interpreted in terms of the generation of a mechanical stress between cell wall and plasma membrane during the freeze-thaw cycle.     

Cold-acclimation induced changes in freezing tolerance and translatable RNA content in Citrus grandis and Poncirus trifoliata

Cold-acclimation-induced changes in freezing tolerance and translatable RNA content were compared in seedlings of a relatively cold sensitive citrus species, Citrus grandis L. Osb. cv. Thong Dee (pummelo), and the cold-hardy citrus relative, Poncirus trifoliata L. Raf. cv. Pomeroy (trifoliate orange). Cold acclimation of pummelo (10 days at 15°C followed by 4 weeks at 10°/5°C, day/night) resulted in a decrease in LT₅₀ from −6 to −8°C, while in trifoliate orange (acclimated for 7 weeks at 5°C), the LT₅₀ decreased from −9 to −18°C. Qualitative changes in the in vitro translation profile, revealed by two-dimensional gel electrophoresis, were observed following cold acclimation in both species. An mRNA for a large polypeptide (ca 160 kDa) was detected following cold acclimation of trifoliate orange. A similar change was not observed in pummelo following cold acclimation.     

Intergenerational acclimation in aphid overwintering

Abstract.  1. When first instar nymphs and adults of the grain aphid Sitobion avenae (Fabricius) (Hemiptera: Aphidiae) were maintained in long-term cultures (>6 months) at 20 °C and 10 °C, the LT₅₀ decreased from −8 and −8.8 °C to −16.0 and −13.5 °C, respectively.
2. When aphids from the 20 °C culture were transferred to 10 °C, there was a progressive increase in cold tolerance through three successive generations. Transfer of newly moulted pre-reproductive adults reared at 10 °C for three generations back to 20 °C resulted in a rapid loss of cold hardiness in their nymphal offspring.
3. In all generations reared at 10 °C, first born nymphs were more cold hardy than those born later in the birth sequence. The LT₅₀ of nymphs produced on the first day of reproduction in the first, second and third generations maintained at 10 °C were −14.8, −17.0 and −16.6 °C, respectively. Thereafter, nymphal cold hardiness decreased over the subsequent 14 days of reproduction in each generation at 10 °C with mean LT₅₀ values of −10.3, −12.6 and −14.8 °C, respectively. By contrast, the cold tolerance of first born nymphs of aphids reared continuously at 20 °C did not differ in comparison with later born siblings. The LT₅₀ of adult aphids was also unaffected by ageing.
4. The ecological relevance of these findings is discussed in relation to the overwintering survival of aphids such as S. avenae .     

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.     

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-induced changes in the polysome pattern and protein synthesis in winter rye (Secale cereale) leaves

Cold-induced changes in the polysome pattern and protein synthesis were analyzed in winter rye, Secale cereale L. cv. Voima, during one week's cold stress treatment, which was performed by transferring the 7-day-old plants from the greenhouse (25°C, long-day conditions) to 3°C and a photoperiod of 10. 5 h. Freezing resistance determined by electrolyte leakage increased significantly upon cold stress starting from LT₅₀ value –5°C. and reaching –9°C on the day 7 of cold exposure. After 4 weeks at low temperature, plants reached an LT₅₀ of –12°C. The polysome content increased markedly during cold stress compared to the control plants. After 2 weeks of cold treatment the polysome content decreased to the same level as that in control plants. The size-class distribution of polysomes showed a high proportion of large protein synthesizing polysomes in cold-stressed plants. After 2 weeks the values were comparable to those in control plants. Cold-induced proteins were detected using ³⁵S-labelled methionine for in vitro translations. At least 2 new polypeptides, M_r 30000 and 18000, were induced on the first day of cold stress and continued to be expressed at low temperatures 4 weeks later.     

The role of ABA in freezing tolerance and cold acclimation in barley

The role of ABA in freezing resistance in nonacclimated and cold‐acclimated barley ( Hordeum vulgare L.) was studied. Eleven nonacclimated cultivars differed in their LT₅₀, ranging from −10.8 to −4.8°C. Sugars, free proline, soluble proteins and ABA were analyzed in nonacclimated cultivars and during cold acclimation of one cultivar. There was an inverse correlation between LT₅₀ and both ABA and sucrose contents. Exogenous ABA caused a decrease in the freezing point of leaf tissue in the cultivar with the lowest level of endogenous ABA, but not in the cultivar with the highest level, suggesting that ABA in the latter may be near the optimum endogenous level to induce freezing tolerance. Plants of cv. Aramir treated with ABA or allowed to acclimate to cold temperature increased their soluble sugar content to a similar level. The LT₅₀ of leaves of cold‐acclimated cv. Aramir decreased from −5.8 to −11.4°C, with biphasic kinetics, accumulating proline and soluble sugars with similar kinetics. The biphasic profile observed during cold acclimation could be a direct consequence of cryoprotectant accumulation kinetics. ABA and soluble protein accumulation showed a single step profile, associated mainly with the second phase of the LT₅₀ decrease. Thus, a significant increase in endogenous ABA is part of the response of barley to low temperature and may be required as a signal for the second phase of cold acclimation. Endogenous ABA contents in the nonacclimated state may determine constitutive freezing tolerance.     

Inositol 1,4,5-trisphosphate formation in leaves of winter oilseed rape plants in response to freezing, tissue water potential aed abscisic acid

Time courses of formation of inositol 1,4,5-trisphosphate (IP₃) were followed in the leaves of non-acclimated and cold (2°C)-acclimated winter oilseed rape ( Brassica napus L. var. oleifera ) plants, subjected to different freezing temperatures or to polyethylene glycol 8000 (PEG) and abscisic acid (ABA) treatments. Changes in water potential (Ψ_w) and in ABA level in the frost- and PEG-treated tissues were also determined. Results obtained indicate that temperatures sligthly higher than LT₅₀ induced a transient and substantial increase in IP₃ level, both in non-acclimated and cold-acclimated tissues. At comparable freezing temperature (–5°C) the response of cold-acclimated leaves was lower than that of non-acclimated ones. The PEG-depedent decrease in Ψ_w to –0.9 MPa or ABA (0.1 m M ) treatment gave rise to a transient increase in IP₃ content in non-acclimated tissues only. Collectively, the data indicate that cold acclimation of plants may lead to lower cell responsiveness to the factors studied in terms of induction of IP₃ formation. Changes in the IP₃ content, observed in the present experiments, support our previous suggestion that non-killing freezing temperatures may induce the phosphoinositide pathway, both in non-acclimated and cold-acclimated tissues. Lowering of tissue water potential to some threshold value or a high exogenous ABA supply may mimic the freezing-dependent reaction in the non-acclimated leaves.     

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.     

Overwintering problems of newly established Miscanthus plantations can be overcome by identifying genotypes with improved rhizome cold tolerance

Miscanthus , a perennial rhizomatous C₄ grass, is a potential biomass crop in Europe, mainly because of its high yield potential and low demand for inputs. However, until recently only a single clone, M. × giganteus , was available for the extensive field trials performed across Europe and this showed poor overwintering in the first year after planting at some locations in Northern Europe. Therefore, field trials with five Miscanthus genotypes, including two acquisitions of Miscanthus × giganteus , one of M. sacchariflorus and two hybrids of M. sinensis were planted in early summer 1997 at four sites, in Sweden, Denmark, England and Germany. The field trials showed that better overwintering of newly established plants at a site was not apparently connected with size or early senescence. An artificial freezing test with rhizomes removed from the field in January 1998 showed that the lethal temperature at which 50% were killed (LT₅₀) for M. × giganteus and M. sacchariflorus genotypes was −3.4 °C. However, LT₅₀ in one of the M. sinensis hybrid genotypes tested was −6.5 °C and this genotype had the highest survival rates in the field in Sweden and Denmark. Although the carbohydrate content of rhizomes, osmotic potential of cell sap and mineral composition were not found to explain differences in frost tolerance adequately, moisture contents correlated with frost hardiness (LT₅₀) in most cases. The results obtained form a basis for identifying suitable Miscanthus genotypes for biomass production in the differing climatic regions of Europe.     

The effects of medium and rate of freezing on the survival of chlamydias after lyophilization

The effects of suspension media and rate of freezing on the survival of Chlamydia trachomatis LGV₂ and Chlamydia pneumoniae after lyophilization were assessed. The highest loss in infectious elementary bodies (EBs) occurred during lyophilization. The survival was higher after freezing at a rate of 1°C min^-1 and lyophilization than that after rapid freezing at - 70°C or - 196°C. The recovery (± 5%) was higher when fetal calf serum (FCS) containing glucose, saccharose or lactose were used as lyophilization media than that (0.5–3%) when yolk-sac, skimmed milk or phosphate buffer containing sucrose, glutamine and 10% FCS (SPG) were used. After lyophilization, the survival was not affected in the tested range from 10⁴ to 5 times 10⁶ inclusion-forming units (ifu) ml^-1 prior to freezing. After storage for 4 months at 4°C, the numbers of ifu of both Chlamydia serovars that were recovered were identical to the numbers of ifu immediately after lyophilization. It was concluded that chlamydias can be stored and transported in lyophilized form. However, a loss of 95% in infectious EBs should be taken into account.     

Drought stress-induced changes in the composition and physical state of phospholipids in wheat

Seedlings of winter wheat ( Triticum aestivum L. cv. Jubilejnaja 50) were grown under normal and dry conditions. Frost resistance (LT₅₀) of 10-day-old control seedlings was −6°C. LT₅₀ of the subsequently drought-stressed leaves shifted to −16°C. In plants of the same physiological age (28 days) but grown without stress, LT₅₀ was −12°C. Phosphatidylcholine accumulated and phosphatidylethanolamine decreased in drought-stressed leaves. Fatty acid unsaturation of these phospholipids increased with leaf age, independently of water supply. Both ageing and drought stress produced a decrease in the apparent phase separation temperature of isolated total phospholipids as determined by electron spin resonance. The possible role of dehydration-induced structural changes in the bilayer matrix in triggering adaptive alterations in membrane composition, similar to those observed during cold hardening, is discussed.     

Hypometabolism in torpid goldsinny wrasse subjected to rapid reductions in seawater temperature

Goldsinny Ctenolabrus rupestris were subjected to rapid, environmentally realistic, reductions in temperature at 2° C increments from 10 to 4° C over a 3-day period in full-strength sea water. In separate experiments, oxygen uptake measurements and ultrasound recordings of heart rate and opercular motion were carried out at regular intervals over the same temperature regime. Mean oxygen uptake rates fell from 0.042 to 0.028 ml O₂ g⁻¹ h⁻¹ between 10 and 6° C respectively (Q₁₀=2.71). Between 6 and 4° C mean rates decreased from 0.028 to 0.008 ml O₂ g⁻¹ h⁻¹ (Q₁₀=542). Mean opercular motion and heart beat rates decreased from 49.5 and 60.3 beats min⁻¹ respectively at 10° C to 18.7 and 18.0 beats min⁻¹ respectively at 4° C. Most goldsinny subjected to 4° C were observed in a torpid state and would not react to external stimulation. Opercular motion was erratic at 4° C and would at times cease altogether for periods up to 1.3 min duration. Heart movement was diffcult to detect at 4° C and may also have ceased for prolonged periods. Q₁₀ values for opercular motion and heart beat rates recorded between 6 and 4° C were 6.39 and 24.52 respectively compared with values of 2.42 and 2.93 respectively recorded between 10 and 8° C. Such large depressions in metabolism appear not to have been reported previously for a marine fish species. No goldsinny mortalities were recorded at any temperature. The possibility that hypometabolic torpor is an adaptive strategy for goldsinny survival at low environmental temperatures is discussed.     

The consequences of freezing temperatures followed by high irradiance on in vivo chlorophyll fluorescence and growth in Picea abies

Picea abies (L.) Karst. plants, propagated by cuttings, were subjected to one night of freezing temperatures (-5°C), high irradiance (1 200 or 1 800 μmol m⁻² s⁻¹), or freezing temperatures followed by high irradiance. The treatments were applied at bud burst, at time of shoot elongation, and when the shoots had ceased to elongate. The maximum quantum yield of photosynthesis, F_v/F_m, dry weight of branches and needles, and length and survival of shoots were measured. F_v/F_m and growth decreased after a night of freezing temperatures followed by high irradiance, at the time of bud burst and shoot elongation. High irradiance alone influenced F_v/F_m, but not growth. Freezing temperatures affected F_v/F_m, and growth at the time of shoot elongation. F₀ increased after a night of freezing temperatures and decreased with age of the current-year needles. It was concluded that the use of short-term measurements of chlorophyll fluorescence induction to predict changes in growth after a night of frost and subsequent high light was not a reliable method.     

Sensitivity of Bacillus coagulans spores to combinations of high hydrostatic pressure, heat, acidity and nisin

We investigated the combined effects of pressure, temperature, pH, initial spore concentration and the presence of nisin on the survival of spores of Bacillus coagulans. Spores were more sensitive to pressure both at lower pH and at higher treatment temperatures. An additional 1.5-log₁₀ reduction in cfu ml^-1 was observed when pH was lowered from 7.0 to 4.0 during pressurization at 400 Mpa and 45°C. A 4-log₁₀ cfu ml^-1 reduction was observed when the temperature was increased from 25°C to 70°C during pressurization at 400 Mpa. The spores were sensitive to nisin at concentrations as low as 0.2 IU ml^-1. At least a 6-log₁₀ reduction was generally achieved with pressurization at 400 Mpa in pH 4.0 buffer at 70°C for 30 min when plated in nutrient agar containing 0.8 IU ml^-1 nisin.     

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.     

Induction of freezing tolerance in potato (Solanum commersonü) suspension cultured cells

The induction of freezing tolerance by abscisic acid (ABA) or cold treatment in suspension cultured cells of Solanum commersonii was studied. Both ABA (50–100 μ M ) at 23°C and low temperature (4°C) increased freezing tolerance in cultured Solanum commersonii cells from a LT₅₀ (freezing temperature at which 50% cells were killed) of —5°C (control) to —11.5°C in 2 days. Cold-induced freezing tolerance reached its maximum at 2 days and remained constant throughout the cold acclimation period of 11 days. The freezing tolerance induced by ABA, however, showed a rapid decline 2 to 5 days after initiation of ABA treatments. Addition of ABA (100 μ M ) to the culture medium at the inception of low temperature treatment did not enhance freezing tolerance of the cells beyond the level attainable by either treatment singly. Poly(A⁺)-RNA was isolated from the respective treatments, translated in a rabbit reticulocyte lysate cell free system, and the translation products were resolved by two dimensional polyacrylamide gel electrophoresis (ID-PAGE). Analysis of the in vitro translated products revealed changes in the abundance of approximately 26 products (encoding for polypeptides with M, of 14 to 69 kDa and pl of 4.90 to 6.60) in ABA-treated cells 12 h after treatment, and 20 (encoding for polypeptides with M_r of 12 to 69 kDa, with pl of 4.80 to 6.42) in cells exposed to 4°C for 12 h. There were only 5 novel translation products observed when the ABA-treated cells reached the highest level of freezing tolerance (2 days after the initiation of ABA treatment). Changes in translatable RNA populations during the induction of freezing tolerance in cells treated with either ABA or low temperature are discussed.