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.     

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.     

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.

     

Freezing tolerance of citrus, spinach, and petunia leaf tissue : osmotic adjustment and sensitivity to freeze induced cellular dehydration

Seasonal variations in freezing tolerance, water content, water and osmotic potential, and levels of soluble sugars of leaves of field-grown Valencia orange (Citrus sinensis) trees were studied to determine the ability of citrus trees to cold acclimate under natural conditions. Controlled environmental studies of young potted citrus trees, spinach (Spinacia pleracea), and petunia (Petunia hybrids) were carried out to study the water relations during cold acclimation under less variable conditions. During the coolest weeks of the winter, leaf water content and osmotic potential of field-grown trees decreased about 20 to 25%, while soluble sugars increased by 100%. At the same time, freezing tolerance increased from lethal temperature for 50% (LT₅₀) of −2.8 to −3.8°C. In contrast, citrus leaves cold acclimated at a constant 10°C in growth chambers were freezing tolerant to about −6°C. The calculated freezing induced cellular dehydration at the LT₅₀ remained relatively constant for field-grown leaves throughout the year, but increased for leaves of plants cold acclimated at 10°C in a controlled environment. Spinach leaves cold acclimated at 5°C tolerated increased cellular dehydration compared to nonacclimated leaves. Cold acclimated petunia leaves increased in freezing tolerance by decreasing osmotic potential, but had no capacity to change cellular dehydration sensitivity. The result suggest that two cold acclimation mechanisms are involved in both citrus and spinach leaves and only one in petunia leaves. The common mechanism in all three species tested was a minor increase in tolerance (about −1°C) resulting from low temperature induced osmotic adjustment, and the second in citrus and spinach was a noncolligative mechanism that increased the cellular resistance to freeze hydration.     

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.     

Relationship between Freezing Tolerance of Root-Tip Cells and Cold Stability of Microtubules in Rye (Secale cereale L. cv Puma)

The response of cortical microtubules to low temperature and freezing was assessed for root tips of cold-acclimated and non-acclimated winter rye (Secale cereale L. cv Puma) seedlings using indirect immunofluorescence microscopy with antitubulin antibodies. Roots cooled to 0 or −3°C were fixed for immunofluorescence microscopy at these temperatures or after an additional hour at 4°C. Typical arrays of cortical microtubules were present in root-tip cells of seedlings exposed to the cold-acclimation treatment of 4°C for 2 days. Microtubules in these cold-acclimated cells were more easily depolymerized by a 0°C treatment than microtubules in root-tip cells of nonacclimated, 22°C-grown seedlings. Microtubules were still present in some cells of both nonacclimated and cold-acclimated roots at 0 and −3°C; however, the number of microtubules in these cells was lower than in controls. Microtubules remaining during the −3°C freeze were shorter than microtubules in unfrozen control cells. Repolymerization of microtubules after both the 0 and −3°C treatments occurred within 1 h. Root tips of nonacclimated seedlings had an LT-50 of −9°C. Cold acclimation lowered this value to −14°C. Treatment of 22°C-grown seedlings for 24 h with the microtubule-stabilizing drug taxol caused a decrease in the freezing tolerance of root tips, indicated by a LT-50 of −3°C. Treatment with D-secotaxol, an analog of taxol that was less effective in stabilizing microtubules, did not alter the freezing tolerance. We interpret these data to indicate that a degree of depolymerization of microtubules is necessary for realization of maximum freezing tolerance in root-tip cells of rye.     

Changes in adenine nucleotides and energy charge in isolated winter wheat cells during low temperature stress

Adenylate energy charge (AEC) and adenine nucleotide levels of isolated winter wheat (Triticum aestivum L. cv Kharkov 22 MC) cells exposed to various low temperature stresses were determined. During ice encasement at −1°C, nucleotide levels decreased gradually in approximate relation to a decline in cell viability. AEC values remained high even after 5 weeks of icing when cell viability was severely reduced. When isolated cell suspensions were exposed to various cooling and freezing regimes ranging from −10 to −30°C, cell damage was dependent on the minimum temperature imposed and the duration of exposure to the freezing stress. The levels of all three adenine nucleotides declined with increasing severity of the imposed stress, but AEC values remained high even at −30°C when nearly all of the cells were killed. The addition of 10 millimolar Ca²⁺ to cell suspensions enhanced survival during low temperature stresses, but did not influence nucleotide levels other than through its effect on cell viability. These results indicate that impairment of the ion transport system during the early stages of ice encasement prior to a detectable decline in cell viability cannot be attributed to changes in the adenylate energy charge system of the cell.     

Relaxed tarantula skeletal muscle has two ATP energy-saving mechanisms

Myosin molecules in the relaxed thick filaments of striated muscle have a helical arrangement in which the heads of each molecule interact with each other, forming the interacting-heads motif (IHM). In relaxed mammalian skeletal muscle, this helical ordering occurs only at temperatures >20°C and is disrupted when temperature is decreased. Recent x-ray diffraction studies of live tarantula skeletal muscle have suggested that the two myosin heads of the IHM (blocked heads [BHs] and free heads [FHs]) have very different roles and dynamics during contraction. Here, we explore temperature-induced changes in the BHs and FHs in relaxed tarantula skeletal muscle. We find a change with decreasing temperature that is similar to that in mammals, while increasing temperature induces a different behavior in the heads. At 22.5°C, the BHs and FHs containing ADP.P_i are fully helically organized, but they become progressively disordered as temperature is lowered or raised. Our interpretation suggests that at low temperature, while the BHs remain ordered the FHs become disordered due to transition of the heads to a straight conformation containing Mg.ATP. Above 27.5°C, the nucleotide remains as ADP.P_i, but while BHs remain ordered, half of the FHs become progressively disordered, released semipermanently at a midway distance to the thin filaments while the remaining FHs are docked as swaying heads. We propose a thermosensing mechanism for tarantula skeletal muscle to explain these changes. Our results suggest that tarantula skeletal muscle thick filaments, in addition to having a superrelaxation–based ATP energy-saving mechanism in the range of 8.5–40°C, also exhibit energy saving at lower temperatures (<22.5°C), similar to the proposed refractory state in mammals.     

Involvement of Two Specific Causes of Cell Mortality in Freeze-Thaw Cycles with Freezing to −196°C

The purpose of this study was to examine cell viability after freezing. Two distinct ranges of temperature were identified as corresponding to stages at which yeast cell mortality occurred during freezing to −196°C. The upper temperature range was related to the temperature of crystallization of the medium, which was dependent on the solute concentration; in this range mortality was prevented by high solute concentrations, and the proportion of the medium in the vitreous state was greater than the proportion in the crystallized state. The lower temperature range was related to recrystallization that occurred during thawing. Mortality in this temperature range was increased by a high cooling rate and/or high solute concentration in the freezing medium and a low temperature (less than −70°C). However, a high rate of thawing prevented yeast mortality in this lower temperature range. Overall, it was found that cell viability could be conserved better under freezing conditions by increasing the osmotic pressure of the medium and by using an increased warming rate.     

Studies on Freezing Injury in Plant Cells : II. Protein and Lipid Changes in the Plasma Membranes of Jerusalem Artichoke Tubers during a Lethal Freezing in Vivo

Plasma membranes were isolated from both unfrozen and frozen tissues of Jerusalem artichoke tubers (Helianthus tuberosus L.) in high purity utilizing an aqueous two-polymer phase partition system. Although the recovery of the plasma membranes was decreased significantly by freezing of tissues even at the nonlethal temperature (−5°C), the isolated plasma membrane samples were considered to be representative of the plasma membranes in situ. Freezing of the tissues at sublethal temperatures resulted in marked changes in the chemical composition of the plasma membrane. Those are losses of sterols and phosphatidylethanolamine from the plasma membranes, and a change of specific proteins with relatively high molecular weights into low molecular weight peptides. These specific proteins were designated as frost susceptible proteins. The properties of the plasma membrane ATPase seem to be not affected so much by the in vivo freezing of cells. However, inhibition of the plasma membrane ATPase by N,N′-dicyclohexylcarbodiimide (DCCD) was relatively low before and after freezing in vivo at the nonlethal temperature at −5°C, but was markedly enhanced by freezing in vivo at sublethal temperatures below −10°C. From the results, it is assumed either that the enzyme molecule was partially modified, especially at the presumed DCCD binding sites or that the DCCD had become more accessible to the enzyme as a result of increased permeability of the plasma membranes. These observed changes are discussed in connection with the mechanism of cell injury.     

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.

     

The characterization of myosin–product complexes and of product-release steps during the magnesium ion-dependent adenosine triphosphatase reaction

Evidence is presented that the myosin subfragment-1–ADP complex, generated by the addition of Mg²⁺ and ADP to subfragment 1, is an intermediate within the myosin Mg²⁺-dependent adenosine triphosphatase (ATPase) turnover cycle. The existence of this species as a steady-state intermediate at pH8 and 5°C is demonstrated by fluorescence measurements, but its concentration becomes too low to measure at 21°C. This arises because there is a marked temperature-dependence on the rate of the process controlling ADP dissociation from subfragment 1 (rate=1.4s⁻¹ at 21°C, 0.07s⁻¹ at 5°C). In the ATPase pathway this reaction is in series with a relatively temperature-insensitive process, namely an isomerization of the subfragment-1–product complex (rate=0.055s⁻¹ at 21°C, 0.036s⁻¹ at 5°C). By means of studies on the P_i inhibition of nucleotide-association rates, a myosin subfragment-1–P_i complex was characterized with a dissociation equilibrium constant of 1.5mm. P_i appears to bind more weakly to the myosin subfragment-1–ADP complex. The studies indicate that P_i dissociates from subfragment 1 at a rate greater than 40s⁻¹, and substantiates the existence of a myosin-product isomerization before product release in the elementary processes of the Mg²⁺-dependent ATPase. In this ATPase mechanism Mg²⁺ associates as a complex with ATP and is released as a complex with ADP. In 0.1m-KCl at pH8 1.0mol of H⁺ is released/mol of subfragment 1 concomitant with the myosin-product isomerization or P_i dissociation, and 0.23 mol of H⁺ is released/mol of subfragment when ATP binds to the protein, but 0.23 mol of H⁺ is taken up again from the medium when ADP dissociates. Within experimental sensitivity no H⁺ is released into the medium in the step involving ATP cleavage.     

CO(2) Photoassimilation by the Spinach Chloroplast at Low Temperature

Spinach chloroplasts were used to study the relationship between photosynthetic CO₂ fixation and temperature from 30 to −15°C. In saturating light and high concentrations of CO₂, the temperature coefficients (Q₁₀) above 20°C were less than 2 in the intact chloroplast. Below 15°C, the Q₁₀ values were greater than 2 and gradually increased with decreasing (down to 0°C) temperature to approximately 4.4. Photosynthesis responded similarly to temperature in a reconstituted chloroplast preparation fortified with ribose 5-phosphate. In the intact chloroplast, temperature did not alter the Q₁₀ value in low light and high CO₂. Elevating the temperature to 25°C after photosynthesizing at −15°C (46 minutes) or 0°C (17 minutes) restored the temperature-depressed photosynthetic rate without a lag in the intact chloroplast to the rate of a chloroplast continually at 25°C. At 0°C, the intact chloroplast photosynthetic rate responded slightly to the inorganic phosphate concentration (0.1-1.0 millimolar) and to pH (7.0-8.6). Relative to 25°C, the levels of ribulose 1,5-bisphosphate and glycerate 3-phosphate were increased 1300 and 200%, respectively, whereas glycolate decreased 57% during intact chloroplast photosynthesis at 0°C. Chilling temperature impeded the transport of photosynthetic intermediates from the stromal compartment to the external medium. Ethylene glycol was shown to be an appropriate additive to prevent freezing of the reaction mixture down to −15°C for photosynthetic CO₂ assimilation.     

Redox State of Cytochromes in Frozen Yeast Cells Probed by Resonance Raman Spectroscopy

Cryopreservation is a well-established technique used for the long-term storage of biological materials whose biological activity is effectively stopped under low temperatures (suspended animation). Since most biological methods do not work in a low-temperature frozen environment, the mechanism and details of the depression of cellular activity in the frozen state remain largely uncharacterized. In this work, we propose, to our knowledge, a new approach to study the downregulation of the redox activity of cytochromes b and c in freezing yeast cells in a contactless, label-free manner. Our approach is based on cytochrome photobleaching effects observed in the resonance Raman spectra of live cells. Photoinduced and native redox reactions that contributed to the photobleaching rate were studied over a wide temperature range (from −173 to +25°C). We found that ice formation influences both the rate of cytochrome redox reactions and the balance between the reduced and oxidized cytochromes. We demonstrate that the temperature dependence of native redox reaction rates can be well described by the thermal activation law with an apparent energy of 32.5 kJ/mol, showing that the redox reaction rate is ∼10¹⁵ times slower at liquid nitrogen temperature than at room temperature.     

In Vivo Perturbation of Membrane-Associated Calcium by Freeze-Thaw Stress in Onion Bulb Cells : Simulation of This Perturbation in Extracellular KCl and Alleviation by Calcium

Incipient freeze-thaw stress in onion bulb scale tissue is known to cause enhanced efflux of K⁺, along with small but significant loss of cellular Ca²⁺. During the post-thaw period, irreversibly injured cells undergo a cytological aberration, namely, `protoplasmic swelling.' This cellular symptom is thought to be caused by replacement of Ca²⁺ from membrane by extracellular K⁺ and subsequent perturbation of K⁺ transport properties of plasma membrane. In the present study, onion (Allium cepa L. cv Sweet Sandwich) bulbs were slowly frozen to either −8.5°C or −11.5°C and thawed over ice. Inner epidermal peels from bulb scales were treated with fluorescein diacetate for assessing viability. In these cells, membrane-associated calcium was determined using chlorotetracycline fluorescence microscopy combined with image analysis. Increased freezing stress and tissue infiltration (visual water-soaking) were paralleled by increased ion leakage. Freezing injury (−11.5°C; irreversible) caused a specific and substantial loss of membrane-associated Ca²⁺ compared to control. Loss of membrane-associated Ca²⁺ caused by moderate stress (−8.5°C; reversible) was much less relative to −11.5°C treatment. Ion efflux and Ca²⁺-chlorotetracycline fluorescence showed a negative relationship. Extracellular KCl treatment simulated freeze-thaw stress by causing a similar loss of membrane-associated calcium. This loss was dramatically reduced by presence of extracellular CaCl₂. Our results suggest that the loss of membrane-associated Ca²⁺, in part, plays a role in initiation and progression of freezing injury.     

A Low Temperature Limit for Life on Earth

There is no generally accepted value for the lower temperature limit for life on Earth. We present empirical evidence that free-living microbial cells cooling in the presence of external ice will undergo freeze-induced desiccation and a glass transition (vitrification) at a temperature between −10°C and −26°C. In contrast to intracellular freezing, vitrification does not result in death and cells may survive very low temperatures once vitrified. The high internal viscosity following vitrification means that diffusion of oxygen and metabolites is slowed to such an extent that cellular metabolism ceases. The temperature range for intracellular vitrification makes this a process of fundamental ecological significance for free-living microbes. It is only where extracellular ice is not present that cells can continue to metabolise below these temperatures, and water droplets in clouds provide an important example of such a habitat. In multicellular organisms the cells are isolated from ice in the environment, and the major factor dictating how they respond to low temperature is the physical state of the extracellular fluid. Where this fluid freezes, then the cells will dehydrate and vitrify in a manner analogous to free-living microbes. Where the extracellular fluid undercools then cells can continue to metabolise, albeit slowly, to temperatures below the vitrification temperature of free-living microbes. Evidence suggests that these cells do also eventually vitrify, but at lower temperatures that may be below −50°C. Since cells must return to a fluid state to resume metabolism and complete their life cycle, and ice is almost universally present in environments at sub-zero temperatures, we propose that the vitrification temperature represents a general lower thermal limit to life on Earth, though its precise value differs between unicellular (typically above −20°C) and multicellular organisms (typically below −20°C). Few multicellular organisms can, however, complete their life cycle at temperatures below ∼−2°C.     

Effect of Growth Conditions and Trehalose Content on Cryotolerance of Bakers' Yeast in Frozen Doughs

The cryotolerance in frozen doughs and in water suspensions of bakers' yeast (Saccharomyces cerevisiae) previously grown under various industrial conditions was evaluated on a laboratory scale. Fed-batch cultures were very superior to batch cultures, and strong aeration enhanced cryoresistance in both cases for freezing rates of 1 to 56°C min⁻¹. Loss of cell viability in frozen dough or water was related to the duration of the dissolved-oxygen deficit during fed-batch growth. Strongly aerobic fed-batch cultures grown at a reduced average specific rate (μ = 0.088 h⁻¹ compared with 0.117 h⁻¹) also showed greater trehalose synthesis and improved frozen-dough stability. Insufficient aeration (dissolved-oxygen deficit) and lower growth temperature (20°C instead of 30°C) decreased both fed-batch-grown yeast cryoresistance and trehalose content. Although trehalose had a cryoprotective effect in S. cerevisiae, its effect was neutralized by even a momentary lack of excess dissolved oxygen in the fed-batch growth medium.     

Optimization of Procedures for Counting Viruses by Flow Cytometry

The development of sensitive nucleic acid stains, in combination with flow cytometric techniques, has allowed the identification and enumeration of viruses in aquatic systems. However, the methods used in flow cytometric analyses of viruses have not been consistent to date. A detailed evaluation of a broad range of sample preparations to optimize counts and to promote the consistency of methods used is presented here. The types and concentrations of dyes, fixatives, dilution media, and additives, as well as temperature and length of incubation, dilution factor, and storage conditions were tested. A variety of different viruses, including representatives of phytoplankton viruses, cyanobacteriophages, coliphages, marine bacteriophages, and natural mixed marine virus communities were examined. The conditions that produced optimal counting results were fixation with glutaraldehyde (0.5% final concentration, 15 to 30 min), freezing in liquid nitrogen, and storage at −80°C. Upon thawing, samples should be diluted in Tris-EDTA buffer (pH 8), stained with SYBR Green I (a 5 × 10⁻⁵ dilution of commercial stock), incubated for 10 min in the dark at 80°C, and cooled for 5 min prior to analysis. The results from examinations of storage conditions clearly demonstrated the importance of low storage temperatures (at least −80°C) to prevent strong decreases (occasionally 50 to 80% of the total) in measured total virus abundance with time.     

Effect of stem water content on sap flow from dormant maple and butternut stems: induction of sap flow in butternut

Sap flow from excised maple stems collected over the winter (1986/87) was correlated with stem water content. Stem water content was high in the fall (>0.80) and decreased rapidly during 2 weeks of continuous freezing temperatures in late winter (<0.60). Exudation of sap from stem segments subjected to freeze/thaw cycles was small (<10 mL/kg) in the fall, but substantial exudation (45-50 mL/kg) occurred following the decline in water content. These observations are consistent with Milburn's and O'Malley's models (J.A. Milburn, P.E.R. O'Malley [1984] Can J Bot 62: 2101-2106; P.E.R. O'Malley, J.A. Milburn [1983] Can J Bot 61:3100-3106) of sap absorption into gas-filled fibers during freezing. Exudation volume was increased 200 to 300% in maple stems originally at high water content (>0.80) after perfusion with sucrose and dehydration at −12°C. Sap flow was also induced in butternut stem segments after the same treatment. Thus, sap flow may not be unique to maples. Sap flow could not be increased in stem segments dehydrated at 4°C. Migration of water molecules from small ice crystals in fibers to larger crystals in vessels while stems were frozen may account for increase exudation after dehydration at −12°C. This would result in preferential dehydration of fibers and a distribution of gas and sap favorable for stem-based sap flow.     

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.