Frost injury and heterogeneous ice nucleation in leaves of tuber-bearing solanum species : ice nucleation activity of external source of nucleants

The heterogeneous ice nucleation characteristics and frost injury in supercooled leaves upon ice formation were studied in nonhardened and cold-hardened species and crosses of tuber-bearing Solanum. The ice nucleation activity of the leaves was low at temperatures just below 0°C and further decreased as a result of cold acclimation. In the absence of supercooling, the nonhardened and cold-hardened leaves tolerated extracellular freezing between −3.5° and −8.5°C. However, if ice initiation in the supercooled leaves occurred at any temperature below −2.6°C, the leaves were lethally injured.

To prevent supercooling in these leaves, various nucleants were tested for their ice nucleating ability. One% aqueous suspensions of fluorophlogopite and acetoacetanilide were found to be effective in ice nucleation of the Solanum leaves above −1°C. They had threshold temperatures of −0.7° and −0.8°C, respectively, for freezing in distilled H₂O. Although freezing could be initiated in the Solanum leaves above −1°C with both the nucleants, 1% aqueous fluorophlogopite suspension showed overall higher ice nucleation activity than acetoacetanilide and was nontoxic to the leaves. The cold-hardened leaves survived between −2.5° and −6.5° using 1% aqueous fluorophlogopite suspension as a nucleant. The killing temperatures in the cold-hardened leaves were similar to those determined using ice as a nucleant. However, in the nonhardened leaves, use of fluorophlogopite as a nucleant resulted in lethal injury at higher temperatures than those estimated using ice as a nucleant.

     

Survival of plant tissue at super-low temperatures v. An electron microscope study of ice in cortical cells cooled rapidly

Experiments were carried out with cortical cells in twig bark of mulberry trees in winter in order to clarify the mechanism of survival at super-low temperatures with rapid cooling and rewarming. Attention was given to the relation between the existence of intracellular ice crystals and survival.

Cortical cells were cooled rapidly by direct immersion into liquid nitrogen or isopentane cooled at various temperatures. After immersion, they were freeze-substituted with absolute ethanol at −78°. They were then embedded, sectioned and examined under the electron microscope for the presence and distribution of cavities left after ice removal.

Cells were found to remain alive and contain no ice cavities when immersed rapidly into isopentane baths kept below −60°. Those cells at intermediate temperatures from −20° to −45°, were almost all destroyed. It was also observed that many ice cavities were contained in the cells immersed rapidly into isopentane baths at −30°. The data seem to indicate that no ice crystals were formed when cooled rapidly by direct immersion into isopentane baths below −60° or into liquid nitrogen.

The tissue sections immersed in liquid nitrogen were rapidly transferred to isopentane baths at temperatures ranging from −70° to −10° before rapid rewarming. There was little damage when samples were held at temperatures below −50° for 10 minutes or below −60° for 16 hours. No cavities were found in these cells. Above −45°, and especially at −30°, however, all cells were completely destroyed even when exposed only for 1 minute. Many ice cavities were observed throughout these cells. The results obtained may be explained in terms of the growth rate of intracellular ice crystals.

     

Freezing Characteristics of Cultured Catharanthus roseus (L). G. Don Cells Treated with Dimethylsulfoxide and Sorbitol in Relation to Cryopreservation

The freezing behavior of dimethylsulfoxide (DMSO) and sorbitol solutions and periwinkle (Catharanthus roseus) cells treated with DMSO and sorbitol alone and in combination was examined by nuclear magnetic resonance and differential thermal analysis. Incorporation of DMSO or sorbitol into the liquid growth medium had a significant effect in the temperature range for initiation to completion of ice crystallization. Compared to the control, less water crystallized at temperatures below −30°C in DMSO-treated cells. Similar results were obtained with sorbitol-treated cells, except sorbitol had less effect on the amount of water crystallized at temperatures below −25°C. There was a close association between the per cent unfrozen water at −40°C and per cent cell survival after freezing for 1 hour in liquid nitrogen. It appears that, in periwinkle suspension cultures, the amount of liquid water at −40°C is critical for a successful cryopreservation. The combination of DMSO and sorbitol was the most effective in preventing water from freezing. The results obtained may explain the cryoprotective properties of DMSO and sorbitol and why DMSO and sorbitol in combination are more effective as cryoprotectants than when used alone.     

Bacterial Activity at −2 to −20°C in Arctic Wintertime Sea Ice

Arctic wintertime sea-ice cores, characterized by a temperature gradient of −2 to −20°C, were investigated to better understand constraints on bacterial abundance, activity, and diversity at subzero temperatures. With the fluorescent stains 4′,6′-diamidino-2-phenylindole 2HCl (DAPI) (for DNA) and 5-cyano-2,3-ditoyl tetrazolium chloride (CTC) (for O₂-based respiration), the abundances of total, particle-associated (>3-μm), free-living, and actively respiring bacteria were determined for ice-core samples melted at their in situ temperatures (−2 to −20°C) and at the corresponding salinities of their brine inclusions (38 to 209 ppt). Fluorescence in situ hybridization was applied to determine the proportions of Bacteria, Cytophaga-Flavobacteria-Bacteroides (CFB), and Archaea. Microtome-prepared ice sections also were examined microscopically under in situ conditions to evaluate bacterial abundance (by DAPI staining) and particle associations within the brine-inclusion network of the ice. For both melted and intact ice sections, more than 50% of cells were found to be associated with particles or surfaces (sediment grains, detritus, and ice-crystal boundaries). CTC-active bacteria (0.5 to 4% of the total) and cells detectable by rRNA probes (18 to 86% of the total) were found in all ice samples, including the coldest (−20°C), where virtually all active cells were particle associated. The percentage of active bacteria associated with particles increased with decreasing temperature, as did the percentages of CFB (16 to 82% of Bacteria) and Archaea (0.0 to 3.4% of total cells). These results, combined with correlation analyses between bacterial variables and measures of particulate matter in the ice as well as the increase in CFB at lower temperatures, confirm the importance of particle or surface association to bacterial activity at subzero temperatures. Measuring activity down to −20°C adds to the concept that liquid inclusions in frozen environments provide an adequate habitat for active microbial populations on Earth and possibly elsewhere.     

Ice Nucleation Activity in Fusarium acuminatum and Fusarium avenaceum

Twenty fungal genera, including 14 Fusarium species, were examined for ice nucleation activity at −5.0°C, and this activity was found only in Fusarium acuminatum and Fusarium avenaceum. This characteristic is unique to these two species. Ice nucleation activity of F. avenaceum was compared with ice nucleation activity of a Pseudomonas sp. strain. Cumulative nucleus spectra are similar for both microorganisms, while the maximum temperatures of ice nucleation were −2.5°C for F. avenaceum and −1.0°C for the bacteria. Ice nucleation activity of F. avenaceum was stable at pH levels from 1 to 13 and tolerated temperature treatments up to 60°C, suggesting that these ice nuclei are more similar to lichen ice nuclei than to bacterial ones. Ice nuclei of F. avenaceum, unlike bacterial ice nuclei, pass through a 0.22-μm-pore-size filter. Fusarial nuclei share some characteristics with the so-called leaf-derived nuclei with which they might be identified: they are cell free and stable up to 60°C, and they are found in the same kinds of environment. Highly stable ice nuclei produced by fast-growing microorganisms have potential applications in biotechnology. This is the first report of ice nucleation activity in free-living fungi.     

Microbial Life beneath a High Arctic Glacier

The debris-rich basal ice layers of a high Arctic glacier were shown to contain metabolically diverse microbes that could be cultured oligotrophically at low temperatures (0.3 to 4°C). These organisms included aerobic chemoheterotrophs and anaerobic nitrate reducers, sulfate reducers, and methanogens. Colonies purified from subglacial samples at 4°C appeared to be predominantly psychrophilic. Aerobic chemoheterotrophs were metabolically active in unfrozen basal sediments when they were cultured at 0.3°C in the dark (to simulate nearly in situ conditions), producing ¹⁴CO₂ from radiolabeled sodium acetate with minimal organic amendment (≥38 μM C). In contrast, no activity was observed when samples were cultured at subfreezing temperatures (≤−1.8°C) for 66 days. Electron microscopy of thawed basal ice samples revealed various cell morphologies, including dividing cells. This suggests that the subglacial environment beneath a polythermal glacier provides a viable habitat for life and that microbes may be widespread where the basal ice is temperate and water is present at the base of the glacier and where organic carbon from glacially overridden soils is present. Our observations raise the possibility that in situ microbial production of CO₂ and CH₄ beneath ice masses (e.g., the Northern Hemisphere ice sheets) is an important factor in carbon cycling during glacial periods. Moreover, this terrestrial environment may provide a model for viable habitats for life on Mars, since similar conditions may exist or may have existed in the basal sediments beneath the Martian north polar ice cap.     

Ice nucleation temperature of individual leaves in relation to population sizes of ice nucleation active bacteria and frost injury

Ice nucleation temperatures of individual leaves were determined by a tube nucleation test. With this assay, a direct quantitative relationship was obtained between the temperatures at which ice nucleation occurred on individual oat (Avena sativa L.) leaves and the population sizes of ice nucleation active (INA) bacteria present on those leaves. In the absence of INA bacteria, nucleation of supercooled growth-chamber grown oat leaves did not occur until temperatures were below approximately −5°C. Both nucleation temperature and population size of INA bacteria were determined on the same individual, field-grown oat leaves. Leaves with higher ice nucleation temperatures harbored larger populations of INA bacteria than did leaves with lower nucleation temperatures. Log₁₀ mean populations of INA bacteria per leaf were 5.14 and 3.51 for leaves with nucleation temperatures of −2.5°C and −3.0°C, respectively. Nucleation frequencies (the ratio of ice nuclei to viable cells) of INA bacteria on leaves were lognormally distributed. Strains from two very different collections of Pseudomonas syringae and one of Erwinia herbicola were cultured on nutrient glycerol agar and tested for nucleation frequency at −5°C. Nucleation frequencies of these bacterial strains were also lognormally distributed within each of the three sets. The tube nucleation test was used to determine the frequency with which individual leaves in an oat canopy harbored large populations of INA bacteria throughout the growing season. This test also predicted relative frost hazard to tomato (Lycopersicon esculentum Mill) plants.     

Bacterial ice nucleation: a factor in frost injury to plants

Heterogeneous ice nuclei are necessary, and the common epiphytic ice nucleation active (INA) bacteria Pseudomonas syringae van Hall and Erwinia herbicola (Löhnis) Dye are sufficient to incite frost injury to sensitive plants at −5°C. The ice nucleation activity of the bacteria occurs at the same temperatures at which frost injury to sensitive plants occurs in nature. Bacterial ice nucleation on leaves can be detected at about −2°C, whereas the leaves themselves, i.e. without INA bacteria, contain nuclei active only at much lower temperatures. The temperature at which injury to plants occurs is predictable on the basis of the ice nucleation activity of leaf discs, which in turn depends on the number and ice nucleation activity of their resident bacteria. Bacterial isolates which are able to incite injury to corn at −5°C are always active as ice nuclei at −5°C. INA bacteria incited frost injury to all of the species of sensitive plants tested.     

Stabilization of Frozen Lactobacillus delbrueckii subsp. bulgaricus in Glycerol Suspensions: Freezing Kinetics and Storage Temperature Effects

The interactions between freezing kinetics and subsequent storage temperatures and their effects on the biological activity of lactic acid bacteria have not been examined in studies to date. This paper investigates the effects of three freezing protocols and two storage temperatures on the viability and acidification activity of Lactobacillus delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol. Samples were examined at −196°C and −20°C by freeze fracture and freeze substitution electron microscopy. Differential scanning calorimetry was used to measure proportions of ice and glass transition temperatures for each freezing condition tested. Following storage at low temperatures (−196°C and −80°C), the viability and acidification activity of L. delbrueckii subsp. bulgaricus decreased after freezing and were strongly dependent on freezing kinetics. High cooling rates obtained by direct immersion in liquid nitrogen resulted in the minimum loss of acidification activity and viability. The amount of ice formed in the freeze-concentrated matrix was determined by the freezing protocol, but no intracellular ice was observed in cells suspended in glycerol at any cooling rate. For samples stored at −20°C, the maximum loss of viability and acidification activity was observed with rapidly cooled cells. By scanning electron microscopy, these cells were not observed to contain intracellular ice, and they were observed to be plasmolyzed. It is suggested that the cell damage which occurs in rapidly cooled cells during storage at high subzero temperatures is caused by an osmotic imbalance during warming, not the formation of intracellular ice.     

Effect of cold acclimation on intracellular ice formation in isolated protoplasts

When cooled at rapid rates to temperatures between −10 and −30°C, the incidence of intracellular ice formation was less in protoplasts enzymically isolated from cold acclimated leaves of rye (Secale cereale L. cv Puma) than that observed in protoplasts isolated from nonacclimated leaves. The extent of supercooling of the intracellular solution at any given temperature increased in both nonacclimated and acclimated protoplasts as the rate of cooling increased. There was no unique relationship between the extent of supercooling and the incidence of intracellular ice formation in either nonacclimated or acclimated protoplasts. In both nonacclimated and acclimated protoplasts, the extent of intracellular supercooling was similar under conditions that resulted in the greatest difference in the incidence of intracellular ice formation—cooling to −15 or −20°C at rates of 10 or 16°C/minute. Further, the hydraulic conductivity determined during freeze-induced dehydration at −5°C was similar for both nonacclimated and acclimated protoplasts. A major distinction between nonacclimated and acclimated protoplasts was the temperature at which nucleation occurred. In nonacclimated protoplasts, nucleation occurred over a relatively narrow temperature range with a median nucleation temperature of −15°C, whereas in acclimated protoplasts, nucleation occurred over a broader temperature range with a median nucleation temperature of −42°C. We conclude that the decreased incidence of intracellular ice formation in acclimated protoplasts is attributable to an increase in the stability of the plasma membrane which precludes nucleation of the supercooled intracellular solution and is not attributable to an increase in hydraulic conductivity of the plasma membrane which purportedly precludes supercooling of the intracellular solution.     

Miscanthus Establishment and Overwintering in the Midwest USA: A Regional Modeling Study of Crop Residue Management on Critical Minimum Soil Temperatures

Miscanthus is an intriguing cellulosic bioenergy feedstock because its aboveground productivity is high for low amounts of agrochemical inputs, but soil temperatures below −3.5°C could threaten successful cultivation in temperate regions. We used a combination of observed soil temperatures and the Agro-IBIS model to investigate how strategic residue management could reduce the risk of rhizome threatening soil temperatures. This objective was addressed using a historical (1978–2007) reconstruction of extreme minimum 10 cm soil temperatures experienced across the Midwest US and model sensitivity studies that quantified the impact of crop residue on soil temperatures. At observation sites and for simulations that had bare soil, two critical soil temperature thresholds (50% rhizome winterkill at −3.5°C and −6.0°C for different Miscanthus genotypes) were reached at rhizome planting depth (10 cm) over large geographic areas. The coldest average annual extreme 10 cm soil temperatures were between −8°C to −11°C across North Dakota, South Dakota, and Minnesota. Large portions of the region experienced 10 cm soil temperatures below −3.5°C in 75% or greater for all years, and portions of North and South Dakota, Minnesota, and Wisconsin experienced soil temperatures below −6.0°C in 50–60% of all years. For simulated management options that established varied thicknesses (1–5 cm) of miscanthus straw following harvest, extreme minimum soil temperatures increased by 2.5°C to 6°C compared to bare soil, with the greatest warming associated with thicker residue layers. While the likelihood of 10 cm soil temperatures reaching −3.5°C was greatly reduced with 2–5 cm of surface residue, portions of the Dakotas, Nebraska, Minnesota, and Wisconsin still experienced temperatures colder than −3.5°C in 50–80% of all years. Nonetheless, strategic residue management could help increase the likelihood of overwintering of miscanthus rhizomes in the first few years after establishment, although low productivity and biomass availability during these early stages could hamper such efforts.     

Intracellular Freezing in the Infective Juveniles of Steinernema feltiae: An Entomopathogenic Nematode

Taking advantage of their optical transparency, we clearly observed the third stage infective juveniles (IJs) of Steinernema feltiae freezing under a cryo-stage microscope. The IJs froze when the water surrounding them froze at −2°C and below. However, they avoid inoculative freezing at −1°C, suggesting cryoprotective dehydration. Freezing was evident as a sudden darkening and cessation of IJs'' movement. Freeze substitution and transmission electron microscopy confirmed that the IJs of S. feltiae freeze intracellularly. Ice crystals were found in every compartment of the body. IJs frozen at high sub-zero temperatures (−1 and −3°C) survived and had small ice crystals. Those frozen at −10°C had large ice crystals and did not survive. However, the pattern of ice formation was not well-controlled and individual nematodes frozen at −3°C had both small and large ice crystals. IJs frozen by plunging directly into liquid nitrogen had small ice crystals, but did not survive. This study thus presents the evidence that S. feltiae is only the second freeze tolerant animal, after the Antarctic nematode Panagrolaimus davidi, shown to withstand extensive intracellular freezing.     

A Leech Capable of Surviving Exposure to Extremely Low Temperatures

It is widely considered that most organisms cannot survive prolonged exposure to temperatures below 0°C, primarily because of the damage caused by the water in cells as it freezes. However, some organisms are capable of surviving extreme variations in environmental conditions. In the case of temperature, the ability to survive subzero temperatures is referred to as cryobiosis. We show that the ozobranchid leech, Ozobranchus jantseanus, a parasite of freshwater turtles, has a surprisingly high tolerance to freezing and thawing. This finding is particularly interesting because the leach can survive these temperatures without any acclimation period or pretreatment. Specifically, the leech survived exposure to super-low temperatures by storage in liquid nitrogen (−196°C) for 24 hours, as well as long-term storage at temperatures as low as −90°C for up to 32 months. The leech was also capable of enduring repeated freeze-thaw cycles in the temperature range 20°C to −100°C and then back to 20°C. The results demonstrated that the novel cryotolerance mechanisms employed by O. jantseanus enable the leech to withstand a wider range of temperatures than those reported previously for cryobiotic organisms. We anticipate that the mechanism for the observed tolerance to freezing and thawing in O. jantseanus will prove useful for future studies of cryopreservation.     

Cold related mortalities and protection against cold in Yakutsk,eastern Siberia: observation and interview study

Objective To assess how effectively measures adopted in extreme cold in Yakutsk control winter mortality.Design Interviews to assess outdoor clothing and measure indoor temperatures; regressions of these and of delayed cause-specific mortalities on temperature.Setting Yakutsk, east Siberia, Russia.Subjects: All people aged 50-59 and 65-74 years living within 400 km of Yakutsk during 1989-95 and sample of 1002 men and women who agreed to be interviewed.Main outcome measures Daily mortality from all causes and from ischaemic heart, cerebrovascular, and respiratory disease.Results Mean temperature for October-March 1989-95 was −26.6°C. At 10.2°C people wore 3.30 (95% confidence interval 3.08 to 3.53) layers of clothing outdoors, increasing to 4.39 (4.13 to 4.66; P<0.0001) layers at −20°C. Thick coats, often of fur, replaced anoraks as temperature fell to −48.2°C. 82% of people went out each day when temperatures were 10.2°C to −20°C, but below −20°C the proportion fell steadily to 44% (35% to 53%) at −48.2°C (P<0.001), and overall shivering outdoors did not increase. Living room temperature was 17.9 (17.2 to 18.5)°C at 10.2°C outdoors, 19.6 (18.8 to 20.4)°C at −20°C, and 19.1 (18.6 to 19.6)°C at −48.2°C. Mortality from all causes and from ischaemic heart and respiratory disease was unaffected by the fall in temperature. Mortality from respiratory disease (daily deaths per million) rose from 4.7 (4.3 to 5.1) to 5.1 (4.4 to 5.7) (P=0.03), but this was offset by a fall in deaths from injury.Conclusions People in Yakutsk wore very warm clothing, and in extremely cold weather stayed indoors in warm housing, preventing the increases in mortality seen in winter in milder regions of the world. Only respiratory mortality rose, perhaps because of breathing cold air.

Key messages

• Death rates from ischaemic heart, cerebrovascular, and respiratory disease and all causes have been shown to increase as air temperature falls
• In Yakutsk, Russia, mortality from cerebrovascular and ischaemic heart disease and all causes among people aged 50-59 and 65-74 was unchanged as temperature fell to −48.2°C
• Mortality from respiratory disease increased as temperatures fell below −20°C but this was more than offset by a decrease in deaths from accidents
• Exceptionally warm clothing, with reduction of outdoor excursions at temperatures below −20°C, prevented overall outdoor cold stress

     

Effect of low temperature and calcium on survival and membrane properties of isolated winter wheat cells

Isolated cells obtained by enzymic digestion of young primary leaves of cold-hardened, dark-grown Kharkov winter wheat (Triticum aestivum L.) were exposed to various low temperature stresses. The initial uptake of ⁸⁶Rb was generally decreased by increasing concentrations of Ca²⁺, but after longer periods of incubation, the inhibiting effect of high Ca²⁺ levels diminished. Viability of isolated cells suspended in water declined rapidly when ice encased at −1°C, while in the presence of 10 millimolar Ca²⁺ viability declined only gradually over a 5-week period. Ice encasement markedly reduced ⁸⁶Rb uptake prior to a significant decline in cell viability or increased ion efflux. Cell damage increased progressively when the icing temperature was reduced from −1 to −2 and −3°C, but the presence of Ca²⁺ in the suspending medium reduced injury. Cell viability and ion uptake were reduced to a greater extent following slow cooling than after rapid cooling to subfreezing temperatures ranging from −10 to −30°C. The results from this study support the view that an early change in cellular properties due to prolonged ice encasement at −1°C involves the ion transport system, whereas cooling to lower subfreezing temperatures for only a few hours results in more general membrane damage, including loss of semipermeability of the plasma membrane.     

The Formation and Distribution of Ice within Forsythia Flower Buds

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

Ice Nucleation Activity in Lichens

A newly discovered form of biological ice nucleus associated with lichens is described. Ice nucleation spectra of a variety of lichens from the southwestern United States were measured by the drop-freezing method. Several epilithic lichen samples of the genera Rhizoplaca, Xanthoparmelia, and Xanthoria had nuclei active at temperatures as warm as −2.3°C and had densities of 2.3 × 10⁶ to more than 1 × 10⁸ nuclei g⁻¹ at −5°C (2 to 4 orders of magnitude higher than any plants infected with ice nucleation-active bacteria). Most lichens tested had nucleation activity above −8°C. Lichen substrates (rocks, plants, and soil) showed negligible activity above −8°C. Ice nucleation-active bacteria were not isolated from the lichens, and activity was not destroyed by heat (70°C) or sonication, indicating that lichen-associated ice nuclei are nonbacterial in origin and differ chemically from previously described biological ice nuclei. An axenic culture of the lichen fungus Rhizoplaca chrysoleuca showed detectable ice nucleation activity at −1.9°C and an ice nucleation density of 4.5 × 10⁶ nuclei g⁻¹ at −5°C. It is hypothesized that these lichens, which are both frost tolerant and dependent on atmospheric moisture, derive benefit in the form of increased moisture deposition as a result of ice nucleation.     

Growth Temperature-Induced Alterations in the Thermotropic Properties of Nerium oleander Membrane Lipids

The temperature boundary for phase separation of membrane lipids extracted from Nerium oleander leaves was determined by analysis of spin label motion using electron spin resonance spectroscopy and by analysis of polarization of fluorescence from the probe, trans-parinaric acid. A discontinuity of the temperature coefficient for spin label motion, and for trans-parinaric acid fluorescence was detected at 7°C and −3°C with membrane lipids from plants grown at 45°C/32°C (day/night) and 20°C/15°C, respectively. This change was associated with a sharp increase in the polarization of fluorescence from trans-parinaric acid indicating that significant domains of solid lipid form below 7°C or −3°C in these preparations but not above these temperatures. In addition, spin label motion indicated that the lipids of plants grown at low temperatures are more fluid than those of plants grown at higher temperatures.

A change in the molecular ordering of lipids was also detected by analysis of the separation of the hyperfine extrema of electron spin resonance spectra. This occurred at 2°C and 33°C with lipids from the high and low temperature grown plants, respectively. According to previous interpretation of spin label data the change at 29°C (or 33°C) would have indicated the temperature for the initiation of the phase separation process, and the change at 7°C (or −3°C) its completion. Because of the present results, however, this interpretation needs to be modified.

Differences in the physical properties of membrane lipids of plants grown at the hot or cool temperatures correlate with differences in the physiological characteristics of plants and with changes in the fatty acid composition of the corresponding membrane lipids. Environmentally induced modification of membrane lipids could thus account, in part, for the apparently beneficial adjustments of physiological properties of this plant when grown in these regimes.

     

Effect of cold acclimation on bulk tissue electrical impedance: I. Measurements with birdsfoot trefoil at subfreezing temperatures

The resistive and reactive components of electrical impedance were measured for birdsfoot trefoil (Lotus corniculatus L.) stems at freezing temperatures to −8°C. As temperature decreased the specific resistance at frequencies between 49 hertz and 1.11 megahertz of stems from cold acclimated plants increased more rapidly than from nonacclimated plants. This temperature dependence of specific resistance could be characterized by an Arrhenius activation energy; cold acclimated stems had a larger Arrhenius activation energy than nonacclimated stems. The low frequency resistance is believed to characterize the extracellular region of the stems and the high frequency resistance is believed to characterize the intracellular region of the stems. Cold acclimation increased the intracellular but not the extracellular resistance at nonfreezing temperatures. Cold acclimated stems were not injured by freezing to −8°C and thawing, but nonacclimated stems were injured by freezing to temperatures between −2.2 and −5.6°C and thawing. Injury to nonacclimated stems at freezing temperatures below −2.2°C was indicated by a decrease in the ratio of resistance at 49 Hz to that at 1.11 megahertz.     

A novel method of natural cryoprotection : intracellular glass formation in deeply frozen populus

Correlating measurements from differential scanning calorimetry, freeze-fracture freeze-etch electron microscopy, and survival of twigs after two-step cooling experiments, we provide strong evidence that winter-hardened Populus balsamifera v. virginiana (Sarg.) resists the stresses of freezing below −28°C by amorphous solidification (glass formation) of most of its intracellular contents during slow cooling (≤5°C per hour). It is shown that other components of the intracellular medium go through glass transitions during slow cooling at about −45°C and below −70°C. This `three glass' model was then used to predict the results of differential scanning calorimetry, freeze-fracture freeze-etch electron microscopy, and biological experiments. This model is the first definitive explanation for the resistance of a woody plant to liquid N₂ temperatures even if quench cooling (1200°C per minute) begins at temperatures as high as −20°C and warming is very slow (≤5°C per hour). It is also the first time high temperature natural intracellular glass formation has been demonstrated.