FREEZE TOLERANCE

K.B. Storey and J.M. Storey, Institute of Biochemistry, Carleton University, Ottawa, Canada

2005. In: Extremophiles (Gerday, C. and Glansdorff, N., eds.) Encyclopedia of Life Support Systems (EOLSS), Developed under the Auspices of the UNESCO, Eolss Publishers, Oxford, UK http://www.eolss.net

Summary Many organisms on Earth must endure prolonged exposures to subzero temperatures below the equilibrium freezing point of their body fluids. Uncontrolled ice formation in biological tissues has multiple damaging effects, both physical and metabolic. Hence, organisms have developed strategies to preserve life at temperatures below 0°C. These include: (1) anhydrobiosis - extreme desiccation removes all bulk water that can freeze, (2) vitrification - instead of crystallizing into ice, water solidifies into an amphorous glass, (3) freeze avoidance - multiple mechanisms, including the use of antifreeze agents, are used to greatly suppress the temperature at which biological water freezes, and (4) freeze tolerance - the growth of ice in extracellular fluid spaces is regulated while the liquid state of the cytoplasm is preserved. Freeze tolerance is the winter hardiness strategy of many types of microorganisms, plants and animals and the present article considers the phylogenetic diversity of freeze tolerant organisms and summarizes our current understanding of the genetic, biochemical and physiological adaptations that support freezing survival. These include the use of ice nucleating proteins to initiate and regulate ice formation, the action of antifreeze proteins in molding crystal shape and inhibiting recrystallization, low molecular weight carbohydrate cryoprotectants that provide colligative resistance to cell volume shrinkage during extracellular ice formation while protecting and stabilizing cellular macromolecules, the role of freeze-induced gene expression in producing proteins that aid survival, metabolic adaptations that provide ischemia resistance when freezing cuts off external oxygen supplies to cells, and mechanisms that allow heart beat, breathing, and other vital signs to be interrupted during freezing but restart spontaneously after thawing. Studies of the biochemical mechanisms of natural freezing survival also have multiple applications such as for the development of tissue/organ cryopreservation technology, the improvement of frost resistance in agricultural crops, and commercial snow-making.

1. Introduction The Earth is a cold place. About 90% of the volume of the oceans is colder than 5°C and the surface waters of polar seas drop to about -1.9°C (the freezing point of seawater) for much of the year. Terrestrial environments are even colder. Polar regions cover about 14% of the Earth's surface with winter temperatures that can range from -30 to -70°C; winter in temperate zones can also reach lows of -30°C or below. Yet despite the lethal effect of freezing for many organisms (think of the devastation of a late autumn garden after the first hard frost), life endures in all cold places. Indeed, there are many examples of organisms that can live only in cold environments. For example, psychrophilic bacteria typically cannot grow at temperatures much above 15°C but can grow well at subzero temperatures. Some snow algae cannot grow above 10°C whereas the growth of certain yeasts has been reported at temperatures as low as -18°C. Viable bacteria have been isolated from subsurface permafrost in the Antarctic where they may have spend millenia in a continuous deep freeze at -25 to -30°C and the

1 Antarctic cold deserts are home to an amazing variety of yeasts and cyanobacteria that endure extreme desiccation and wide yearly temperature variation (-55 to +10°C).

Organisms that live at subzero temperatures fall into two major groups - those that sustain all normal life functions at these low temperatures and those that interrupt normal life processes, often entering dormant life phases, in order to endure seasonal or intermittent subzero exposures. The vibrant community of organisms - microbes, plants, animals - living in polar seas are an excellent example of the former. Teleost fish in this environment spend all or most of their lives at -1.9°C even though the osmolality of their body fluids suggests that they should freeze at about -0.5°C. However, some unique adaptations, notably the presence of antifreeze proteins in their body fluids, keep them from freezing. For many terrestrial organisms, however, the often extreme subzero temperatures of winter are met with an interruption of feeding, growth and activity and adaptive strategies including dormancy and cold hardiness are employed to preserve and protect the organism until warmer temperatures return in the spring.

One of the strategies of winter cold hardiness is freeze tolerance, the ability to endure the freezing of extracellular body fluids. Before discussing freeze tolerance as the main topic of this article, a summary of other options is important. As a general rule, organisms deal with any environmental stress at multiple levels including behavioural, physiological, and biochemical responses. Behavioural responses are typically the first line of defense and that is broadly illustrated by organismal responses to subzero temperatures. In advance of winter most organisms seek full or partial shelter from the impending cold. For some, this is accomplished by migration, both long (birds or butterflies may fly thousands of miles) or short (garter snakes in Manitoba, Canada travel several kilometers to mass by the thousands in frost-free underground caves). Many animals go underground to hibernate below the frostline whereas perennial plants concentrate their reserves in underground roots and tubers while sacrificing above-ground foliage. Some animals hibernate underwater (e.g. many frogs and turtles) or winter as aquatic life stages (e.g. dragonfly nymphs) and are safe unless the water in their pond or stream freezes to the bottom. Other organisms gain partial protection from the full force of winter by hibernating at or near the ground surface. With a layer of leaf litter and a deep blanket of snow, temperatures at the soil surface can often remain close to 0°C even when air temperatures above the snowpack drop below -20°C. Furthermore, in this microhabitat, organisms are well buffered from rapid changes in temperature. Many organisms take refuge in this subnivean (under the snow) environment, some in well-developed forms (e.g. insects, spiders, frogs; many perennial plants grow new leaf rosettes before the old foliage dies back) and some as embryonic forms (seeds, spores, cysts). Although temperatures under the snow can still fall to subzero values, the insulation of the snowpack means that organisms have less severe thermal challenges to endure. In northern boreal forests, for example, temperatures under the snowpack may not drop below about -8°C all winter. A final group of organisms experience virtually no thermal buffering and must endure deep and prolonged cold. The most obvious members in this group are trees and shrubs that require protection for their twigs and buds as well as many insects and other arthropods that winter in arboreal sites.

1.1 Low temperature and freezing injury Why is protection from subzero temperatures necessary? Chilling injuries are one factor. Death due to chilling below 0°C without freezing has been well investigated in insects and hypothermia can often be lethal for non-hibernating species of mammals. Indeed, humans cannot withstand the cooling of core organs much below 25°C. Chilling injuries derive from a disruption of the normal integration of metabolic functions. All metabolic reactions are temperature dependent and most increase/decrease in rate by about 2-fold for every 10°C increase/decrease in temperature (Q10 = 2). Some reactions have lower Q10 values and some substantially higher values of 3 or 4. Reactions with high Q10 values are

2 substantially impaired during cooling, often causing disruption of the metabolic pathway or function in which they participate. The temperature sensitivity of metabolic reactions arises because temperature alters the strength of the hydrophobic and hydrophilic weak bonds that are key to the structure and function of macromolecules. For example, weak bonds are major determinants of protein conformation, the association of subunits within a protein or of multiple proteins within a polymer, enzyme-substrate binding, and enzyme-membrane interactions.

Differential effects of low temperatures on opposing metabolic functions are often the most devastating. For example, the electrical potential difference across the plasma membrane, which is key to functions such as cell sensitivity to stimuli, nerve transmission, or muscle contraction, is maintained by the opposing actions of ATP-driven ion pumps that move ions (e.g. Na+, K+, Ca2+, H+) across membranes "uphill" against their chemical gradient versus ion channels that facilitate "downhill" movements along concentration gradients. Collectively, ion pumps use a huge proportion of total cellular energy; the Na+/K + ATPase alone is responsible for 5-40% of total ATP turn-over depending on cell type. For organisms that are not cold adapted, cold exposure can seriously impair the production of ATP, the cellular energy currency, and this, in turn, impairs the activity of ion pumps, with little effect on ion channels, and quickly leads to a dissipation of membrane potential difference. This causes further problems, particularly uncontrolled Ca2+ influx into cells which triggers the action of numerous Ca2+-stimulated proteases that cause major destruction.

These problems caused by chilling can cause major injury or even death for non-adapted organisms if they are cooled below their normal low temperature limits. For example, summer-active ground squirrels are just as sensitive to hypothermia as are humans but during a period of autumn cold acclimation various adjustments are made to their macromolecules that allow them to maintain metabolic integration when their body temperature sinks to near 0°C during winter hibernation. Similar processes of cold acclimation prepare organisms to maintain metabolic integration at subzero temperatures. The biochemistry of cold acclimation is an enormous topic on its own and will receive little attention in this article. Briefly, the process can include changes in the relative activities of various enzymes and metabolic pathways, synthesis of new protein/enzyme variants that function optimally at low temperatures, and adjustments to membrane composition and lipid fuel depots to raise their content of unsaturated lipids so that fluidity is maintained at very low temperatures. Thus, properly cold-acclimated organisms have little to fear from a decrease in temperature to subzero values. The major risk is from freezing.

For most organisms, internal ice formation has multiple damaging or lethal effects (Figure 1). (1) Ice crystals can cause direct physical damage to cells and tissues. Shearing and squeezing stresses can damage or break individual cells that are trapped among growing ice crystals. In multicellular organisms, ice expansion can burst delicate capillaries so that upon thawing vascular integrity is compromised and the organism suffers severe internal bleeding. (2) Ice growth inside of cells damages subcellular architecture and disrupts or destroys the compartmentation of metabolic pathways. The severity of this damage is why cytoplasmic freezing is avoided even by freeze tolerant organisms. Only a couple of exceptions to this have ever been found; the most conclusive demonstration of survival after intracellular freezing coming from studies of the Antarctic nematode, Panagrolaimus davidi. (3) Delivery of oxygen and fuel supplies from extracellular sources is cut off when cells are entrapped in ice or when the blood plasma of multicellular organisms freezes. (4) Freezing in extracellular spaces removes pure water into growing ice crystals and leaves behind a highly concentrated extracellular fluid. This sets up a steep osmotic gradient across cell membranes which causes an outflow of water and net shrinkage of cells. Dehydration, elevated intracellular ionic strength and cell volume reduction all have negative effects on cell morphology and metabolic function, and this is compounded by

3 problems of too rapid swelling of cells during thawing. For organisms in nature, cell volume effects are typically the first and most important consequences of freeze/thaw. Shrinkage below a critical minimum cell volume can cause irreparable damage to cell membranes due to compression stress. Most freeze tolerant organisms in nature do not experience dehydration or ionic strength changes extreme enough to directly affect macromolecules but these factors are of consequence in the field of cryopreservation and have been addressed during the development of protocols for cell freezing (e.g. sperm, blood) in liquid nitrogen (-196°C).

Place Figure 1 about here

2 Strategies for survival at subzero temperatures The potential problems caused by ice in cellular systems have lead to the development of several strategies for subzero survival. Four general categories can be defined although they include some overlap. Each takes a different approach to preserving life at subzero temperatures and addressing the potentially serious injuries that can be done by ice. These are: (1) Anhydrobiosis - literally meaning "life without water", this strategy involves extreme desiccation to eliminate all bulk water from the system and place organisms in a dormant, virtually ametabolic state. With no free water, no ice can form. (2) Vitrification - instead of freezing as crystals of ice, water solidifies into an amorphous glass. (3) Freeze avoidance - potent antifreezes are used to maintain the liquid state of body fluids, often to temperatures approaching -40°C. (4) Freeze tolerance - controlled ice growth in extracellular body fluids is permitted although the liquid state of the cytoplasm is preserved. Freeze tolerance is the focus of the current article but before discussing the mechanisms of freezing survival in detail, a brief summary of the three alternatives is instructive.

2.1 Anhydrobiosis One way to avoid damage due to internal freezing is to minimize or even eliminate all freezable, bulk water from an organism leaving behind only miniscule amounts of water that form bound or vicinal water shells around macromolecules. Hence, there is no free water left to form ice crystals. This is a drastic option that involves extreme dehydration and during which an organism cannot remain active or maintain any normal metabolic functions. It is unheard of among animals of greater than microscopic size and is rarely seen in the vegetative parts of plants except for a small group of "resurrection" plants. However, anhydrobiosis is a common way of ensuring the survival of the dormant stages of many organisms including eggs, cysts, spores and seeds. In a dry state these can remain viable for many years, enduring both heat and cold. Many can be stored without harm in liquid nitrogen.

Anhydrobiosis (also called cryptobiosis) is best known as a means of survival in seasonally arid environments, the classic example being the encysted embryos of brine shrimp (Artemia). These can be dried to the point where water content is less than 0.1 g per g cysts (compared with about 4 g H2O per g dry mass or 80% water for most active organisms). Anhydrobiotic organisms are virtually ametabolic since there is no bulk water remaining to support the diffusion of metabolic intermediates or the activity of enzymes. Desiccation places extreme stress on cellular macromolecules because hydration shells are normally very important for maintaining their functional conformation. To deal with this, high concentrations of polyhydric carbohydrates are synthesized during drying; for example, in brine shrimp cysts the 3-carbon polyol, glycerol, and the disaccharide, trehalose, accumulate to about 4 and 14 %, respectively, of the dry weight. Glycerol stabilizes protein structure whereas trehalose is particularly effective in stabilizing the lipid bilayers of membranes.

2.2 Vitrification

4 Vitrification is a process by which water is solidified, not into a crystal, but into an amorphous glass. The glass incorporates all of the dissolved solutes present in the water and hence vitrified cells are not under osmotic, ionic strength, or volume stresses as frozen ones are. In the laboratory, the requirements for achieving a glass transition are daunting and include the need for extremely high concentrations of solutes (about 40 % solutions), rapid cooling to the glass transition temperature (Tg) that is often well below -30°C, and warming rates in the order of 30-50°C/min to prevent devitrification, the instantaneous crystallization of ice that can occur during warming at any temperature between the Tg and the melting point (MP) of the solution.

In nature, vitrification occurs under less rigorous conditions probably because of other contributing factors such as the substantial dehydration of systems that undergo natural vitrification and the accumulation of high concentrations of sugars that have particularly high glass transition temperatures (e.g. trehalose in animals, sucrose, raffinose or stachyose in plants). Indeed, in both desiccation tolerant resurrection plants and Artemia there is strong evidence that vitrification occurs during drying at temperatures well above 0°C and that the sugars have dual protective roles in both promoting vitrification of any remaining water and directly stabilizing proteins and membranes through hydrogen bonding to polar residues in the dry macromolecular assemblages. Sugar glasses also form in many plant seeds and, of direct relevance to cold-hardiness, are key to subzero survival in the twigs of various subarctic woody plants such as poplar and birch. In poplar, for example, sugar glasses form below -20°C and twigs that are cold-hardened at -20°C to optimize sugar production can subsequently endure exposure to liquid nitrogen.

Vitrification, unlike freezing, is not physically destructive of tissue organization or subcellular architecture and, hence, the technique has been pursued as method for the ultralow storage of medically-important cells, tissues and organs. Although vitrified storage of cell suspensions has been relatively easy to accomplish, the successful preservation of vitrified organs remains elusive. There are several reasons for this including the low tolerance of mammalian organs for substantial dehydration, the requirement for extremely high amounts of cryoprotective agents (such as dimethylsulfoxide, ethylene glycol, glycerol) which may have cytotoxic effects, and the need for very fast and even cooling and warming throughout the entire organ mass to both induce vitrification during cooling and avoid devitrification during warming.

2.3 Freeze avoidance

Most animals and plants of greater than microscopic size (with the possible exception of their eggs or seeds) cannot endure dehydration to the extent needed to utilize either anhydrobiosis or vitrification as a means of subzero survival. A third option for winter survival is freeze avoidance which involves suppression of the temperature at which biological water freezes. One or more mechanisms are employed to lower the temperature at which body fluids freeze to a value that is well below the anticipated environmental minima experienced by the organism. Many insects and other arthropods have optimized this strategy and some show the exceptional ability to remain liquid down to -40°C or even lower. Cold-water marine fish also use this strategy to survive in seawater that can cool to -1.9°C compared with the normal freezing point of the blood of teleost fishes which is about -0.5°C. Deep supercooling also occurs within the primordium tissue of the buds and xylem tissues of many woody plants.

The freeze avoidance strategy is built upon two of the physical properties of water solutions. The first is a colligative property of solutions -- the greater the concentration of dissolved solutes, the lower the freezing point (FP) of the solution. By accumulating high concentrations of solutes, freeze avoiding

5 organisms can substantially reduce the equilibrium FP of their body fluids. The equilibrium FP is the temperature at which an ice crystal added to a solution begins to grow. Typically, compatible solutes are used that are non-ionic, soluble at very high concentrations, readily equilibrate across biological membranes, and do not perturb protein conformation. The solutes used are often the same ones used in anhydrobiosis or natural vitrification and, apart from their colligative effects, they are also highly effective at stabilizing macromolecules against low temperature and low water stresses. In animals, glycerol is by far the most common of the naturally-occurring low molecular weight antifreezes but various other polyhydric alcohols and sugars are used by some species.

The second property of water solutions that is important to freeze avoidance is the phenomenon of supercooling (also called undercooling), the ability of water solutions to chill to temperatures below the equilibrium FP without freezing. All biological fluids supercool to some extent; for example, small volumes of human plasma can be chilled to near -15°C before freezing. Indeed, under controlled laboratory conditions pure water can actually be cooled to -40°C before the random assembly of water molecules into proto-ice nuclei occurs so frequently that homogeneous nucleation (spontaneous crystallization) cannot be avoided. However, very deep supercooling of most biological fluids does not normally occur because crystallization is typically stimulated by the action of heterogeneous nucleators including solutes, particles or surfaces that act to orient water molecules into the crystal lattice shape and trigger freezing. Thus, supercooled solutions are inherently unstable and successful long term freeze avoidance requires the removal or masking of nucleators as well as strategies to block the growth of ice crystals beyond a microscopic size. Strategies that address both of these principles of freeze-avoidance - colligative suppression of the temperature at which body fluids freeze and stabilization of the supercooled state - have been developed by cold-hardy organisms.

2.3.1 Nucleator control: The first requirement for successful freeze avoidance is to limit contact with the most potent nucleator of all, ice. When possible, organisms may winter in sites that, although cold, are relatively ice-free and external waterproofing in the form of a waxy cuticle, a silk cocoon, etc. is frequently used to help prevent the seeding of body fluids by contact with external ice. In tree buds, the thick cell walls, small size and tight packing of cells that surround the supercooled primordium tissue as well as the presence of nonesterified pectins in intercellular spaces all appear to contribute to blocking ice penetration into these tissues even though ice growth is actually promoted in the surrounding bud scales. Secondly, organisms eliminate potential heterogeneous nucleators from their body fluids by such means as voiding the gut (containing bacteria and other foreign particles) or altering the composition of body fluids. For example, the seasonal deletion of one particular lipoprotein from the blood of stag beetle larvae causes a drop in the supercooling point (SCP; the temperature at which a supercooled solution spontaneously freezes) of the larvae from -7°C in summer to below -20°C in winter. A reduction in total body water content also suppresses the FP and SCP of an organism and, not surprisingly, partial dehydration is quite common in overwintering life stages, particularly dormant ones as discussed above. Dehydration reduces the relative amount of freezable, bulk water as compared with the unfreezable water that is bound or ordered by association with macromolecules However, the greatest contributions to supercooling come from the use of specific antifreezes which are of two types, antifreeze proteins and low molecular weight carbohydrate antifreezes.

2.3.2 Antifreeze proteins Antifreeze proteins (AFPs) have arisen independently in multiple phylogenetic groups including plants, coldwater marine fishes, cold-hardy insects and other invertebrates but only one study suggests their possible presence in bacteria. AFPs are diverse in structure, size, type and genetic origin but are unified in function by their ability to adsorb onto the crystal lattice of ice via hydrogen-bonding.

6 Bonding interactions occur between water molecules and the hydrophilic side chains of selected amino acids on the proteins or the carbohydrate residues on glycoproteins. AFPs do not actually prevent ice formation but by adhering to the prism face of crystals, they break up the growth planes and engineer growth into hexagonal bipyramids that cannot grow beyond a microscopic size (Figure 2). Their presence causes a thermal hysteresis, a noncolligative reduction of the FP of body fluids without changing the MP and this characteristic is used for their detection and assay.

Place Figure 2 about here.

AFPs of marine fish show a small thermal hysteresis (only 1-1.5°C) but that is enough to reduce the FP of their body fluids below that of the seawater in which they swim. AFPs are present year-round in polar fish but appear seasonally in temperate species, their production triggered by changes in photoperiod with the shortening days of autumn. AFPs are particularly important for species that routinely come into physical contact with ice such as polar species that swim just under the pack ice in a zone that is filled with floating ice crystals and in-shore temperate species that seasonally encounter surface ice. AFPs are found in the blood of fish as well as in the stomach and intestines (fish ingest ice crystals with their food) and in body tissues that are exposed to the environment (skin, gill). AFPs are synthesized by liver and exported into the plasma for uptake by other tissues although skin produces its own AFPs.

AFPs of marine fish have been extensively studied and five distinct classes are known. One class consists of glycopeptides (AFGPs) of varying lengths which are found in Antarctic nototheniid fish and in temperate cod. AFGPs are composed of a tandemly repeated glycopeptide, alanine-alanine- threonine, linked through the hydroxyl group of threonine to galactosyl-N-acetyl galactosamine. Type 1 AFPs (found in flounders and sculpins) contain alanine-rich single a-helices; type II AFPs (herring, smelt, sea raven) are cystine-rich proteins, type III AFPs (eel pout) are compact beta-proteins, and type IV AFP from the longhorn sculpin has a glutamate/glutamine rich four-helix bundle structure. The structural variety of AFPs reflect their affinity for different planes on the crystallographic surface of ice but they are all unified in the placement of their critical amino acids or carbohydrate residues into alignments that match up precisely with the spacing of water molecules in the ice lattice.

From an evolutionary point of view, fish AFPs are relatively recent inventions. The first evidence of sea level glaciation dates back only about 10-30 million years in the southern hemisphere and only about 2 million years in the north. Thus, the evolutionary pressure to develop AFPs was relatively sudden and recent and, in many cases, occurred after the emergence of present day genera or species. Not surprisingly, then, no common ancestral protein can be found and within in each fish group rapid selective pressure was exerted on some pre-existing protein to bend it to an AFP function. For example, type II AFPs are homologues of the carbohydrate-recognition domain of calcium-dependent lectins whereas the AFGPs of Antarctic nototheniid fish seem to have been derived by duplication and amplification of a small segment of the gene for trypsinogen, a digestive protease that is secreted into the gut. However, northern cod, which have AFGPs that are virtually identical with those of nototheniids, have a completely different AFP gene structure which indicates that their AFGPs were derived from a very different progenitor gene.

AFPs from terrestrial insects are much more powerful than fish antifreezes and may suppress FP by as much as 5-9°C below MP. Many of the insect AFPs are cysteine-rich. For example, the AFP of mealworm beetles has high amounts of both cysteine (18-19%) and threonine (21-26%) residues and is deficient in hydrophobic amino acids. AFP synthesis in insects is induced by shortening photoperiod (typically below 10-11 h of light), facilitated by cool temperatures and mediated internally by the

7 release of juvenile hormone from the insect brain which stimulates AFP synthesis by the fat body (the liver equivalent of insects). AFPs are also found in numerous cold-hardy plants and, surprisingly, many of these are freeze tolerant species. Their role in a freeze tolerant systems emphasizes a second function of AFPs which is the inhibition of ice recrystallization, a phenomenon that will be discussed in greater detail under freeze tolerance.

For some organisms, the removal/masking of nucleators coupled with the addition of AFPs provides sufficient supercooling potential to prevent winter freezing. A reduction in SCP to -10 or -15°C is generally sufficient for organisms that winter at or near the soil surface and that are insulated by thick layers of leaf litter and snow. For other organisms, however, additional protection is needed and this typically comes from carbohydrate antifreezes.

2.3.3 Carbohydrate antifreezes Organisms that winter in more exposed sites or in very cold polar or alpine environments need more powerful antifreeze protection and, for this, colligative protectants are employed. Both FP and SCP of body fluids decrease as the concentration of dissolved solutes increases - this natural process is functionally equivalent to the effect of adding ethylene glycol antifreeze to the radiator of a car. Dissolved solutes offer colligative suppression of FP (by -1.86°C per mole) and, as a bonus, in a nucleator-free system (such as one containing AFPs) the effect of these solutes on SCP is double this value. In combination, the effects of colligative protectants, AFPs, and nucleator clearance can often push the SCP of cold-hardy insects to nearly -40°C (Figure 3) and, in a few instances, SCP values as low as -50 to -55°C have been reported for Arctic species. The most common carbohydrate antifreeze is the three-carbon polyhydric alcohol, glycerol. Its concentration can rise to 2 molar or more in insect body fluids and may constitute as much as 20 % of the fresh weight of the organism over the winter. Other polyols (sorbitol, mannitol, erythritol, threitol, ethylene glycol) and a variety of sugars (glucose, sucrose, trehalose) are also used in some species. The favored position of glycerol may be due to several factors. One is the pre-existence of the pathway for glycerol biosynthesis in all cells because glycerol is a key intermediate in triglyceride synthesis and degradation. Another is the fact that glycerol production makes the most efficient use the carbohydrate reserves (glycogen) from which it is derived; a maximum output of osmotically active particles (2 glycerol produced per glucose unit cleaved off glycogen) is achieved with a very low net loss of carbon from the system. Glycerol also equilibrates rapidly across biological membranes both by diffusion and by facilitated transport which is a key requirement for its distribution into all organs.

Place Figure 3 about here.

In insects, carbohydrate antifreezes are synthesized from huge reserves of the polysaccharide, glycogen, that are laid down in the fat body during late summer feeding. Production is triggered by cold activation of the enzyme glycogen phosphorylase with a trigger temperature that is typically about 5°C. This ensures that the protectants are built up during the cool autumn weeks in advance of the first early winter exposures to subzero temperatures. A metabolic pathway known as the hexose monophosphate shunt has a central role in glycerol synthesis. Units of glucose-1-phosphate are cleaved off glycogen and fed into the shunt; a portion is catabolized (releasing CO2) in the production of reducing power in the form of NADPH whereas the rest exits as the 3-carbon sugar, glyceraldehyde. NADPH-dependent reduction of glyceraldehyde via the enzyme polyol dehydrogenase produces glycerol. Unidirectional synthesis of glycerol continues over several weeks in the autumn and the pool is then maintained throughout the winter until rising temperature or developmental cues initiate its catabolism in the spring. Breakdown is dependent upon the induction of the ATP-dependent enzymes

8 glycerol kinase or glyceraldehyde kinase which convert glycerol back into a sugar phosphate that can re-enter central metabolic pathways.

3. Freeze tolerance Desiccation, vitrification, and freeze avoidance ensure winter survival for many organisms but others have developed a fourth option, freeze tolerance. Freeze tolerance arose as a response to the same biological problems as did freeze avoidance. That is, body fluids naturally supercool to some extent but long term maintenance of a supercooled state is unpredictable due to the potential for random nucleation, and freezing from a deeply supercooled state is highly damaging or lethal because of the speed with which ice courses through a body. Freeze avoiding organisms dealt with these problems by stabilizing the liquid state whereas freeze tolerant organisms accepted freezing but abandoned random nucleation in favor of a controlled freezing that they regulate themselves. For many species, the factors that determined the initial "choice" of strategy are buried in evolutionary time. For example, each strategy is exemplified by insect species that winter on goldenrod - a fly larva is freeze tolerant whereas a moth larva is freeze avoiding. Their winter habitat is ostensibly the same (the galls of both species may even be found on the same plant), yet their strategies are diametrically opposed. Furthermore, there are even reports that certain species of bark beetles can change strategies; animals sampled from the same forest site were freeze tolerant in one winter but freeze avoiding in another. Why this is so is still unknown.

For other species the "choice" of freeze tolerance seems clearer. The necessity to survive freezing probably arose out of a combination of factors including the organism's physiology, choice of winter microhabitat, and the depth and fluctuation of winter temperatures experienced. In addition, all of these factors were probably mitigated by an inability to meet all of the criteria necessary for freeze avoidance. For example, several species of woodland frogs hibernate in the leaf litter of the forest floor. Terrestrial hibernation allows them to breed very early in the spring using meltwater ponds in the forest at a time when aquatic frogs may still be locked under ice-covered ponds. Because frog skin is highly permeable to water, these frogs must winter in damp places to avoid death due to desiccation. However, high permeability also means that when ice penetrates into their hibernacula, they are helpless to prevent its propagation across their skin. Hence, freeze tolerance became a necessity. Salamanders living in the same forests have the same problem but their solution has been to evade freezing exposure by following natural tunnels downwards to underground hibernation sites that are situated well below the frost line.

Intertidal marine invertebrates present another interesting case. When the tides recede, sessile animals such as barnacles and mussels as well as high intertide grazers such as periwinkle snails are exposed to ambient air temperatures that can be well below 0°C on northern shorelines in winter. Although they can close or seal their shells to prevent contact with external ice, they cannot do anything about the seawater trapped within their shells and when that water freezes, their tissues are nucleated. What is interesting, however, is that because ice crystallization is an exothermic event, the body temperature of an organism is actual higher (temperature rises to just under the FP) during the time that ice is growing than if the same organism remained supercooled. Indeed, studies with mussels have shown that the animals can gain significant thermal buffering by letting their body fluids begin to freeze and for intertidal animals that are exposed to subzero air temperatures for only a few hours at a time, freezing can substantially reduce their net thermal stress during each low tide aerial exposure. Notably, the same principle works on a larger scale for organisms stranded in tide pools. Ice forming on the surface of the pool stabilizes the temperature of the remaining seawater at its freezing point (-1.9°C) and greatly reduces the chance that any of the organisms in the tide pool will freeze before they are again

9 submerged by the returning tide. Interestingly, intertidal seaweeds use the freeze avoidance strategy with SCP values as low as -15°C for species in the high intertide that receive the greatest aerial exposure.

Freeze tolerance has arisen multiple times in phylogeny. Many types of microorganisms are freeze tolerant although tolerance in bacteria does not necessarily correlate with psychrophily. Numerous plants from northern and alpine regions are freeze tolerant and interest in imparting or improving freeze tolerance in crop species makes plant cold hardiness a major field of study; key model species include Arabadopsis, winter wheat, barley and rye. Many insects, arthropods and terrestrial microfauna, various types of intertidal invertebrates, and a range of terrestrially-hibernating amphibians and reptiles also tolerate freezing during winter hibernation. The latter include the wood frog Rana sylvatica (Figure 4), four other North American frog species (Hyla versicolor, H. chrysoscelis, Pseudacris crucifer, P. triseriata), two species of turtles (Chrysemys picta, Terrapene carolina), and the European common lizard (Lacerta vivipara) as well as low tolerances by others such as garter snakes (Thamnophis sirtalis). Painted turtles, C. picta, are an interesting case because although adults winter underwater, newly hatched juveniles remain for their first winter within their shallow terrestrial nests on exposed riverbanks and nest temperatures can fall substantially below 0°C. There appears to be considerable geographic variation across North America in juvenile cold hardiness with some authors describing freeze tolerance in certain populations and others contending that survival is based on supercooling, often to lower than -10°C. Wood frogs have become a major model for research on vertebrate freeze tolerance and studies of organ freezing in this species provide lessons for medical researchers interested in the cryopreservation of human organs.

Place Figure 4 about here.

Most animals that are acknowledged as having ecologically-relevant freeze tolerance can endure the conve rsion of at least 50 % and generally as much as 65 % of their total body water into ice. Low amounts of ice (e.g. <20%) are often survivable by organisms but in these cases ice formation seems to be limited to peripheral sites (skin, muscle) and to very brief periods (a few hours at most). What distinguishes freeze tolerant species is the ability to endure long term freezing (weeks or months during the winter) with ice penetration throughout the whole body and with a cessation of all vital functions - no heart beat, no circulation, no breathing, no muscle movement. Recovery after thawing involves the restoration of all these parameters. In frogs, heart beat is the first vital sign that is detectable after thawing followed by breathing movements. The first signs of neuromuscular recovery are twitch responses to the pinching of limb muscles, followed by some voluntary movements and finally by coordinated righting responses and sitting postures.

Although they can endure high amounts of body ice, freeze tolerant organisms are not necessarily tolerant of all freezing conditions. Organisms are highly attuned to the types of freezing exposures that they encounter naturally. Freezing at a rate that is too fast or to a temperature that is too low is lethal. Maximum ice content is reached at very different temperatures in different species, reflecting the normal environmental exposure that the species endures over the winter. For example, wood frogs that winter in the protected subnivean environment reach 65% ice at about -3°C and endure temperatures only a little lower (-6 to -8°C) whereas gall fly larvae that winter above the snowpack reach 65% at temperatures below -20°C and readily endure freezing down to about -30°C (Figure 3). The amount of ice formed at any given subzero temperature is a function of the osmolality of body fluids, the higher the osmolality, the lower the ice content. Hence, a key criterion for freezing survival by most species is the synthesis of high concentrations of low molecular weight sugars or polyols. Marine invertebrates are a notable exception to this. The osmolality of their body fluids is already very high and must

10 remain isotonic with seawater (1000 mOsmol l-1). Intracellular osmotic balance is provided by high levels of amino acids that balance the ionic strength of the hemolymph and seawater and these probably also provide all of the cryoprotection needed to defend cells during freezing.

3.1 Adaptations for freeze tolerance The adaptations needed for freezing survival are numerous because they must address the multiple forms of injury, both physical and metabolic, that can arise when fluids surrounding cells freeze. At present, the known principles of freeze tolerance are these. (A). Freezing is initiated close to the equilibrium FP so that the rate of ice formation is slow and there is plenty of time to initiate protective metabolic adjustments. (B). Ice growth is confined to extracellular and extra-organ spaces (such as the abdominal cavity in frogs or extracellular spaces in leaves); intracellular fluid is concentrated by water loss into external ice but does not freeze. (C). High concentrations of low molecular weight carbohydrates are often accumulated to minimize cell volume reduction during extracellular freezing, prevent intracellular freezing, and protect/stabilize cellular macromolecules, including both proteins and membranes. (D). Because freezing cuts off oxygen delivery to cells, mechanisms of ischemia/anoxia resistance are needed that include pathways of fermentative ATP generation, metabolic rate depression, and antioxidant defenses to provide protection against reperfusion damage during thawing. (E). Physiological adjustments coordinate the cessation and reactivation of vital signs during freeze/thaw and damage repair mechanisms deal with physical injuries caused by ice.

What is interesting, however, is that although freeze tolerance demands many biochemical and physiological adaptations, the majority of these adaptations are "borrowed" from the responses of organisms to other environmental stresses. For example, woodland frogs and marine molluscs are well- endowed with pre-existing abilities to tolerate wide variation in body water content and the osmolality of body fluids and can also endure extended oxygen deprivation. These underlying abilities have been harnessed to support freeze tolerance. The synthesis of glycerol as a cryoprotectant by freeze-tolerant insects is equally seen in freeze-avoiding species and in various desiccation-resistant species living in dry environments. Indeed, studies with wood frogs have shown that one of the key features of amphibian freeze tolerance, the synthesis of glucose as a cryoprotectant, is in fact triggered just as effectively by desiccation as by freezing. Hence, the novel parts of freeze tolerance may actually be fairly few. The development of specific ice nucleating proteins that trigger crystallization is one and another is the addition of novel components to signal transduction cascades to allow a freezing signal to activate a coordinated suite of freeze-protective metabolic responses. In plants, for example, the development of the CBF transcription factor by freeze tolerant species allows them to activate a variety of genes that contain the drought-responsive element and use these gene products in the defense of cells against freeze-induced cellular dehydration. In wood frogs a recently isolated 10 kilodalton peptide, named FR10, may have a similar function. The peptide, that is strongly induced as an early response to freezing in multiple tissues, has characteristics that could allow it to function as a nuclear transcription factor in the activation of genes whose protein products could aid freezing survival.

3.2 Ice nucleators The negative part of the freeze avoidance strategy is its unpredictability. If environmental temperature drops below the SCP, freezing is instantaneous and lethal; the same is true if organisms come in contact with a nucleator at any temperature between the FP and the SCP. Ice growth from a deeply supercooled state is swift, unforgiving and lethal, even for freeze tolerant organisms. Indeed, a number of insect species that were first classified as freeze avoiding because they did not recover after cooling on a dry substrate and flash freezing at their low SCP, have subsequently been shown to endure

11 freezing very well when cooled on a wet substrate so that inoculation by environmental ice occurred at a high subzero temperature. Thus, extensive supercooling is detrimental for freeze tolerant organisms and, therefore, most of these have taken control of the freezing process by employing ice nucleators that initiate freezing close to the FP.

When freezing occurs just under the FP, the initial surge of ice formation and the subsequent rate of ice accumulation are minimized and the time available to implement adaptations to aid freezing survival is maximized. The importance of minimizing the initial ice surge was demonstrated in studies with wall lizards (Podarcis muralis, a species with very poor freezing survival) that were frozen at temperatures between -0.6 and -6.5°C. Lizards only survived when freezing was at temperatures above -3°C when the initial ice surge (% of body water instantly frozen when nucleation is triggered) was 5% or less of total body water. Slow freezing is also critical. When wood frogs are nucleated at -2°C, the exothermic reaction of crystallization causes an immediate jump in body temperature to just under the FP (about -0.5°C). Body temperature holds at this value for several hours while ice slowly forms at a mean rate that is typically <5% of total body water per hour and then body temperature gradually drops back down to ambient. Because the rate of freezing is so slow, the frogs may show nothing but a slight stiffness in some parts of their skin and limbs for the first hour or more and maximal ice content (about 65 % of total body water) may not be reached until 12-24 hours after freezing began.

Because slow freezing is highly beneficial to survival, freeze tolerant animals all appear to use some form of nucleation control. In some cases, a reliance on inoculative freezing due to contact with environmental ice may be sufficient. Frogs may rely heavily on this mechanism. Indeed, wood frogs chilled below their FP on a wet substrate began to freeze within 30 seconds after an ice crystal was dropped onto the substrate showing that they are highly susceptible to ice propagation across their water-permeable skins. However, if direct contact with ice is problematic or if the integument is far less permeable than frog skin, then other types of nucleators are employed. Some organisms "borrow" the ice nucleating abilities of microorganisms. Several types of gram-negative bacteria, particularly strains of Pseudomonas, and at least one fungus (Fusarium sp.) have been identified as nucleators in freeze tolerant insect and plant species. Ice-nucleating Pseudomonas and Enterobacter species are also found in the gut and on the skin of wood frogs and seem to be responsible for the characteristic SCP of about -2°C of the species. Hence, if frogs cool without direct contact with external ice, the ice nucleating bacteria in gut and skin will trigger crystallization before supercooling becomes extensive. Thus, rather than clearing these microorganisms from their gut, as freeze-avoiding species do, freeze tolerant organisms maintain (or possibly even cultivate) ice-nucleating microbes during cold- hardening. Ice nucleation has also been linked with calcium phosphate spherules in the Malpighian tubules of insects as well as with other crystalloid deposits that are common in diapausing and overwintering insects.

An interesting use of bacterial ice nucleators occurs in some tropical alpine plants; their lifestyle can include high daytime temperatures combined with subzero nights. Lobelia telekii grows in the high alpine zone on Mount Kenya. Its leaves are arranged in a rosette and it has a >2 meter tall inflorescence which is hollow and contains about 1 liter of fluid. As temperatures fall each night this fluid cools and starts to freeze at 0°C. The heat of fusion released by the freezing of this large mass of water holds the temperature within the inflorescence at about 0°C all night long whereas ambient air temperature can fall as low as -10°C. Nucleation at 0°C with no supercooling is achieved by two mechanisms: (1) the central fluid has very low osmolality, and (2) potent bacterial ice nucleators in the fluid trigger crystallization. This symbiosis provides the plant with frost resistance and the bacteria with nutrition and shelter. Other Afro-alpine plants gain thermal buffering from the freezing of rain water that is trapped between the rosette leaves and another variation is seen in Espeletia plants from

12 South America that use the freezing of wet dead leaves covering their stems to gain thermal buffering for the underlying frost sensitive tissues.

For other organisms, controlled nucleation is assured by the use of specific ice nucleating proteins (INPs) that are synthesized during autumn cold-hardening. Many insects add INPs to their hemolymph during the winter season in order to raise the SCP of their body fluids above -10°C. Their small body mass and relatively impermeable cuticle means that most insects can supercool quite extensively; even in summer many species can supercool to between -5 and -15°C. However, for optimal freezing survival, SCP must be adjusted upwards so that the rate and sites of ice growth can be managed. INPs have also been found in marine molluscs, in the plasma of freeze tolerant frogs, and in the extracellular fluid of many temperate plants (e.g. fir, winter rye) and lichens. Plant INPs trigger freezing between -5 and -12°C and in winter rye the nucleators appear to be complexes of proteins, carbohydrates and phospholipids. In wood frogs the role of proteinaceous INPs has been questioned because these trigger freezing of plasma at about -8°C, whereas frogs in nature tend to freeze at temperatures between -0.5 and -2°C, as discussed above. Experimental studies remain to be done but it has been proposed that the frog plasma INP does not actually trigger nucleation but is present to guide the propagation of ice crystals through the vascular bed.

Insect hemolymph INPs have received considerable study. INPs function by providing a surface structure that orients water molecules into the crystal lattice shape. As with AFPs there are a variety of types of INPs. For example, a high content of hydrophilic amino acids (20 % glutamate/glutamine) is important for the function of hornet INP whereas a high phosphatidylinositol content is key to the nucleating ability of cranefly and bacterial lipoprotein INPs. The INP of the cranefly has a molecular mass of about 800 kilodaltons and is composed of two proteins (265 and 80 kD) and lipids of about 350 kD. Nucleation temperature is proportional to the molecular mass of the nucleator and calculations suggest that aggregates of INP monomers are necessary to account for nucleation at the temperatures observed in vivo. For example, nucleation at about -4°C would require aggregates of cranefly INP of about 6000 kD. In insect hemolymph the INPs aggregate in long chains with the phosphatidylinositol component involved in the linkage.

Bacterial INPs have been well studied primarily because they are a major cause of frost damage to crop plants. Bacteria such as Pseudomonas species possess powerful INPs in their outer membranes that trigger ice formation in the epithelia of plants. Their function seems to be to induce frost injury to plant cells which results in the release of nutrients that can be used by the bacteria. However, it has also been hypothesized that the INPs may provide resistance to desiccation for the bacteria by sequestering a reserve of water close to the cell surface as well as efficiently dispersing ice crystals away from the cell to limit the chance of intracellular inoculation. Nucleating ability and strength can be quite variable between individual cells in a bacterial population and between different strains of the same species. It is suggested that the differences between cells in a population may be because cells only transiently express nucleating activity at some point in their life cycle. Bacterial INPs can stimulate ice formation anywhere between -2 and -12°C with the percentage of cells able to produce "warm-temperature" nuclei that are active at -5°C or above being quite low in most cases. As in insects, bacterial INP activity is also dependent on molecular mass. The INP monomers of P. syringae have a molecular mass of 120-180 kD yet the relationship between nucleation temperature and molecular mass predicts that a nucleator must be ~20,000 kD to trigger crystallization at -2°C. Therefore, aggregation of INP momoners in the bacterial cell membrane is key to their function and the phosphatidylinositol component seems to anchor the INP monomers into groups on the membrane surface. Bacterial INPs have been put to commercial use in snow-making for ski resorts. Freeze-dried

13 bacterial cells are used rather than the purified protein because of the greater potency of INPs when they are aggregated in polymers.

3.3 Ice management and AFPs Another problem in freeze tolerance is to contain ice where it will do the least harm. Physical damage due to ice expansion within the lumen of capillaries is one of the big problems faced in medical organ cryopreservation; after thawing the punctured capillaries cause massive internal bleeding. One of the ways to limit physical damage by ice is to sequester a high proportion of total ice into spaces where it can do relatively little tissue damage. In freeze tolerant frogs, a huge mass of ice forms within the abdominal cavity and large sheets of ice are sandwiched between skin and skeletal muscle. All organs lose a considerable fraction of their total water (as much as 24 %) into these extra-organ ice masses and this greatly reduces the remaining amount of extracellular water that can freeze within organs or within the lumen of blood vessels. Furthermore, partial dehydration of organs elevates the concentrations of cryoprotectants and other solutes in cells which lowers their FP and reduces the risk of intracellular freezing. The same principle has been demonstrated in plants. In the dormant buds of some cold-hardy woody plants ice forms within the bud scales where it is physically separated from contact with the more delicate shoot apex or flower primordium tissues which do not freeze. The shoot and floral apex lose internal water into these extra-organ ice masses and mortality, if it occurs, is associated with dehydration, not low temperature or ice.

Surprisingly, AFPs have been found in freeze tolerant insects and plants although their presence seems contradictory. Recall that AFPs bind to the surface of growing ice crystals and in freeze avoiding species arrest their growth while crystals are still of microscopic size. AFPs in freeze tolerant animals have the same action but for a different purpose. In freeze tolerant systems AFPs inhibit recrystallization, the phenomenon by which small ice crystals regroup over time into larger and larger crystals. Left unchecked in organisms that may be frozen for weeks or months at a time, ice will recrystallize into very large crystals that could cause serious physical injuries to tissues. Hence, AFPs are employed to provide long term stability of crystal size and shape. Carrot AFP has interesting features. It is a 36 kD glycoprotein that has three N-glycosylation sites although contrary to fish AFGPs, removal of the carbohydrate moiety does not affect recrystallization inhibition by the protein. Carrot AFP is leucine rich and shows high sequence similarity with polygalacturonase inhibitor proteins that are secreted by plants as antifungal agents. AFPs in winter rye also show derivation from pathogenesis-related secretory proteins; in rye the progenitor proteins seem to be ß(1-3)endoglucanase and chitinase. As in fish, therefore, plant AFPs appear to have arisen quite recently on the evolutionary time scale through modification of pre-existing protein types.

Despite the above mechanisms for ice management, some ice damage to tissues may still occur (e.g. bruising is sometimes noted in leg muscles of thawed frogs) and so another ancillary adaptation that aids freeze tolerance appears to be an enhancement of damage repair mechanisms. The first evidence for this came from an analysis of the genes that are up-regulated by freezing in wood frogs. Prominent among these were the genes coding for the three subunits the blood clotting protein, fibrinogen. Fibrinogen is synthesized by liver and secreted into the blood where, as the final step in the coagulation cascade, thrombin cleaves near the N-termini of the Aa and Bb chains to release the A and B fibrinopeptides and expose sites for polymerization into the fibrin mesh of a growing blood clot. The up-regulation of fibrinogen gene activity during freezing in frogs suggests that an improvement to blood clotting capacity, due to enhanced plasma levels of fibriongen (and maybe other clotting factors as well) would allow frogs to deal rapidly with any internal bleeding that is detected upon thawing.

14 3.4 Cryoprotectants The survivable limits of ice accumulation tend to be about 65% of total body water in all species. This is because amounts higher than this remove too much water from the cytoplasm and cause cell shrinkage below a critical minimum cell volume (CMCV). Below the CMCV the compression stress on cell and organelle membranes becomes too great and the biologically-necessary bilayer arrangement of membrane lipids can collapse into an amorpho us gel from which it cannot recover during thawing. Hence, the function of the osmotically-active low molecular weight cryoprotectants is to retard the loss of cytoplasmic water into growing extracellular ice crystals by increasing the osmotic pressure inside cells.

Low molecular weight carbohydrates are used by most species to provide colligative resistance against a reduction in cell volume below the CMCV. Their action in preventing freezing of intracellular fluids is just the same as the whole body protection that they provide for freeze-avoiding organisms. Indeed, the same types of polyols and sugars are used by both freeze tolerant and freeze avoiding organisms. Soil microbes show a similar behaviour. They release soluble organic matter, primarily polyols, into the environment around them. By doing so, they surround themselves with a localized area of highly concentrated unfrozen liquid that helps them to resist ice growth into their microenvironment and limit the freeze-induced dehydration of the cells.

Various sugars are used as cryoprotectants in freeze tolerant plant species including sucrose, raffinose and stachyose as well as selected polyols (sorbitol, mannitol); reports on many species have correlated sugar content, osmotic concentration and frost hardiness. Among animals, glycerol is by far the most common cryoprotectant for the reasons discussed earlier. In freeze tolerant insects, a combination of glycerol and sorbitol (a 6-carbon polyol) occurs frequently (Figure 5). The two polyols are synthesized with distinct time-dependent patterns which suggests that they have different freeze-specific functions (as yet unknown). Glycerol is synthesized first in the early autumn (and cleared last in the spring) and its production is triggered by cool temperatures in the range of 15°C. By contrast, sorbitol production is triggered by 5°C exposure and high levels of this polyol are maintained only during the mid -winter months. The two polyols also have different fates when cryoprotectant pools are catabolized in the spring. Sorbitol is quantitatively converted back to glycogen whereas glycerol is fed into other catabolic or anabolic fates as the insects resume development.

Place Figure 5 about here.

Freeze tolerant vertebrates have different solutions to cryoprotection. In most cases the cryoprotectant is glucose, the normal blood sugar of vertebrates although the gray tree frog (H. versicolor) builds up high levels of glycerol in its tissues. Most freeze tolerant frogs produce high concentrations of glucose with levels rising to 150-300 millimolar (mM) in central organs, compared with control values of only about 5 mM in unfrozen frogs. By contrast, freeze tolerant reptiles typically accumulate very little cryoprotectant, a factor that probably contributes to their relatively low tolerance of freezing. Glucose levels in reptile plasma and organs rarely exceed 30 mM, representing only a 5-10 fold rise during freezing.

Glucose is produced from glycogen, just as glycerol is, but instead of the slow accumulation of glycerol that occurs in insects over several weeks of autumn cold hardening, glucose production by frog liver is directly activated by the initiation of freezing in peripheral tissues. As soon as nucleation occurs (typically on the skin surface), signals are immediately transmitted to the liver. ß-Adrenergic receptors on hepatocyte plasma membranes are activated, stimulating a rise in intracellular cyclic AMP levels, an activation of protein kinase A (PKA), and then a cascade of PKA-mediated events. The

15 most important of these is an activation of glycogen phosphorylase but other enzymes are also targeted to direct the flow of carbon into the desired end product. Glucose levels in liver and blood begin to rise within 2 minutes post-nucleation and over the next several hours, glucose pours out into the blood and is distributed to all organs (Figure 6). Peripheral tissues such as skeletal muscle and skin receive the least glucose (~50 mM) whereas glucose in liver, heart and brain often exceeds 200 mM. This gradient of glucose concentration in the body results because at the same time that liver is pumping glucose out, ice is propagating inward from peripheral tissues and gradually cutting off circulation to organs until liver and heart are the last to freeze. Interestingly, the very high levels of glucose in core organs of frozen frogs create a unique circumstance when the animal thaws. The higher the osmolyte concentration in body fluids, the lower the melting point and so, during thawing, the heart melts first. The consequence of this is that a renewed heart beat is the first vital sign detected as frogs thaw and this allows circulation to be restored as quickly as possible to organs as they melt.

Place Figure 6 about here.

As diabetics know, high glucose causes serious metabolic damage and glucose levels in vertebrates are normally held within strict limits by the opposing actions of two hormones, insulin and glucagon. In healthy humans, glucose typically rises to only about 7.5 mM after a meal and sustained values over 10 mM indicate diabetes. Metabolic problems caused by high glucose include the nonenzymatic attachment of glucose to proteins which impairs their function (e.g. this glycation is the cause of diabetic cataract) and the actions of glucose (or glucosyl moieties on glycated proteins) as pro-oxidants that stimulate the production of oxygen free radicals. Freeze tolerant frogs have somehow overcome these problems. Whether protein glycation is a problem for freeze tolerant frogs is not yet known but wood frogs have very high activities of antioxidant enzymes compared with other frog species and these may keep glucose-mediated oxyradical damage to a minimum during freezing and over the 1-2 weeks that it takes for glucose to return to normal after thawing.

3.5 Membrane protection Maintenance of the integrity of cell membranes during freezing and thawing is critical for cell survival. If membranes break then cell contents leak out. If compression stress on membranes becomes too great, then the lipid bilayer collapses into an amorphous gel and the membrane can no longer carry out its vital activities. The action of colligative cryoprotectants such as glycerol and glucose are key to preventing cell volume from dropping below the CMVC but other protectants are also used that specifically target and stabilize membranes. The two prominent protectants that have this role are trehalose (a disaccharide) and proline (an amino acid). Levels of both compounds rise during cold- hardening in insects and proline accumulates in cold-hardy plants following the cold induction of genes involved in the proline biosynthetic pathway. Both compounds also protect membranes from desiccation stress and their actions have been most thoroughly studied in Artemia. Trehalose inhibits the membrane phase transitions associated with drying or freezing by forming hydrogen bonds between the -OH groups on trehalose and the phosphate moities of the polar head groups of phospholipids or with the hydration sphere centered around them. In doing so, trehalose fits between the phospholipid monolayers and inhibits a transition to the gel phase. Proline interacts with the hydration layer surrounding phospholipids and also intercalates between phospholipid head groups but its exact mechanism must be somewhat different from that of trehalose since proline is an effective stabilizer of phospholipid bilayers during freezing but not during drying.

In plants, numerous studies have identified membrane systems as the primary sites of freezing injury, with damage primarily arising due to severe dehydration during freezing; for example, at -10°C more than 90% of osmotically active water can have moved into extracellular ice. Freezing damage to plant

16 membranes can include expansion-induced-lysis during thawing, lamellar-to-hexagonal-II phase transitions, and fraction jump lesions. The former two types of injury are preventable with cold acclimation in freeze tolerant species. Additional freezing damage to plant membranes has been traced to chemical damage by reactive oxygen species and physical damage by intercellular ice that can form adhesions with cell walls and membranes and cause cell rupture. Freeze protection of plant membranes has been traced to a number of factors including changes in lipid composition, deletion of some membrane material into endocytotic vesicles during cell shrinkage, accumulation of sugar cryoprotectants and proline that have both osmotic effects on cell volume and direct effects as membrane stabilizers, and most recently, the synthesis of proteins that stabilize membranes has been identified. One of these proteins, COR15a, is targeted to chloroplasts. Its constitutive expression in transgenic Arabidopsis significantly lowered the lethal temperature for the plants and reduced the incidence of lamellar-to-hexagonal-II phase transitions that occurred in regions where the plasma membrane was brought into close apposition with the chloroplast envelope as a result of freeze- induced cell volume reduction. COR15a and other related proteins contain amphipathic a-helices that have a strong effect on the intrinsic curvature of phospholipid monolayers and reduce their propensity to form the undesirable hexagonal II phase.

3.6 Gene and protein adaptations Freeze tolerance is a seasonal phenomenon for most animals and plants and during a period of autumn cold acclimation or cold-hardening, a variety of metabolic adjustments are made that support both cold and freezing survival. Recent work with both animal and plant systems has used new technology in molecular biology to identify numerous genes and their protein products that are induced or up- regulated in response to cold and/or freezing stimuli. The literature is rapidly becoming extensive and tends to fall into three categories: (1) genes that are responsive to cold exposure in organisms that are not inherently cold tolerant, (2) genes that are responsive to cold exposure and that aid cold tolerance in cold-hardy organisms, and (3) genes that are responsive to freezing and that aid freeze tolerance. The latter will be the primary focus of this section.

3.6.1 Animals Many of the protein adaptations that support freeze tolerance are put in place during the period of autumn cold-hardening. Because metabolic rates are low in the cold and because energy-expensive processes such as protein biosynthesis are restricted by low ATP supplies in oxygen-limited frozen state, animals must typically be prepared for freezing survival well in advance of the first exposures to freezing temperatures. For example, AFPs and INPs begin to accumulate in the hemolymph of insects in the early autumn, triggered by photoperiod cues. The enzymes involved in polyol synthesis are also elevated at this time, ready to respond to the 5°C trigger temperature that initiates the mass conversion of glycogen to polyols. For some insects these preparations for cold hardiness are linked with a life stage transition to the overwintering form (e.g. larval to pupal transition) and/or with entry into diapause, a state of arrested development that often persists through the mid-winter months. To date, there is relatively little evidence for changes in gene and protein expression that are directly stimulated by freezing in insects. This may be because, with a very small body mass, freezing occurs too fast to be accompanied by significant protein biosynthesis.

In freeze tolerant frogs, however, the situation is different. Some of the protein adaptations needed for freeze tolerance are put in place well before animals experience their first freezing episodes. For example, glycogen phosphorylase activity rises in liver over the autumn and the number of glucose transporters in the plasma membranes of all frog organs also increase to allow rapid uptake of the glucose cryoprotectant. But, as discussed earlier, glucose synthesis is postponed until body fluids actually start to freeze and so is the expression of selected genes. Initial studies using radiotracers to

17 tag the synthesis of new proteins detected both freeze- and thaw-specific proteins made in wood frog organs but did not identify them. Subsequent work using techniques such as cDNA library screening and differential display PCR have now identified several freeze-specific genes. In wood frog liver these included freeze up-regulation of the subunits of fibrinogen (discussed earlier), the ADP/ATP translocase (AAT), subunit 4 of NADH-ubiquinone oxidoreductase (ND4) and several as yet unidentified genes, among them FR10 which may have a role as a freeze-specific transcription factor (Figure 7). AAT catalyzes the transmembrane exchange of cytosolic ADP with mitochondrial ATP generated via oxidative phosphorylation. ND4 was also up-regulated in wood frog brain during freezing as were mitochondrial ATPases 6 and 8; all three of these are encoded on the mitochondrial genome. The time course of induction of these genes during freezing and the fact that transcript levels of all except ND4 also rose when frogs were given anoxia exposure (under a nitrogen gas atmosphere at 5°C) suggests that these proteins may have a role in dealing with freeze-induced ischemia - the interruption of oxygen and fuel deliveries to organs caused by plasma freezing. Other genes that are encoded on the mitochondrial genome are also up-regulated in response to anoxia exposure in adult turtles and are stimulated by both freezing and anoxia in freeze tolerant painted turtles hatchlings (C. picta). These include subunit 5 of ND and subunit 1 of cytochrome oxidase. It appears, then, that regulation of enzymes involved in mitochondrial energy metabolism is of importance to the endurance of both anoxia and freezing stresses in lower vertebrates that are tolerant of these stresses.

Place Figure 7 about here.

3.6.2 Plants Cold-induced genes have been identified in a variety of plant species although not every cold- responsive gene contributes to freeze tolerance. Over 40 cold-induced genes have been identified in Arabidopsis. Several are kinases with roles in the regulation of other proteins or genes. A desaturase is induced as is also common in other cold-stressed plants and animals; desaturases function in cold acclimation to increase membrane fluidity by raising their content of polyunsaturated lipids. Several cold-stimulated genes in barley also have roles in membrane and/or lipid metabolism. Other cold- induced genes in Arabidopsis have obvious functions in freezing survival. These include several enzymes involved in proline biosynthesis as well as dehydrins. Proline is a membrane protectant that stabilizes lipid bilayer structure during freeze-induced cell volume reduction. Dehydrins are hydrophilic proteins that appear to have a structural role in membrane stabilization during dehydration.

Other studies have shown that mutation of a number of genes in Arabidopsis causes a deficiency in freezing tolerance and an increase in electrolyte leakage from cells that indicates damaged membranes. The functions and identities of most of these are not yet known but, for example, mutation of the sfr4 gene in Arabidopsis caused depletion, instead of accumulation, of the low molecular weight cryoprotectant sugars. Other mutations cause an elevation of freezing tolerance. For example, mutation of the esk1 gene in Arabidopsis lowered the lethal freezing temperature of the plants, probably due to the enhanced membrane stabilization afforded by the 30-fold higher levels of proline and 2-fold higher sugar levels in these plants compared with the wild-type. Deletion analysis of the promotors of some cold-induced genes in Arabidopsis resulted in the identification a motif that was also found in dehydration responsive genes. The CRT/DRE (cold repeat/drought-responsive element) is present in a variety of genes (including the COR genes) that are cold-induced and a transcription factor (CBF) that binds to this element has been identified (Figure 8). However, the CBF gene itself is responsive only to freezing whereas another transcription factor (DREB) responds to dehydration signals. Thus, two different stresses, acting via two different transcription factors can activate the same set of freezing/dehydration responsive genes that have roles in regulating problems common to the two stresses (cell volume reduction, cell water loss, membrane compression).

18

Place Figure 8 about here.

Of interest to agriculture is the possibility of using transgenics to enhance freeze tolerance or induce constitutive freeze tolerance in various crop species, thereby reducing economic losses to frost damage. Transgenic Arabidopsis lines have been produced that constitutively express the CBF transcription factor and, therefore, constitutively express a number of genes that are normally cold- inducible. In the absence of cold acclimation, one line that showed constitutive expression of the CBF1 gene showed a level of freeze tolerance comparable to that of the cold-acclimated wild type. However, another study showed that constitutive expression of the CBF3 gene, although producing constitutive freeze tolerance, produced transgenic plants that were dwarfs. Hence, there is a growth penalty for constitutive freeze tolerance that will have to be resolved before this trait could be practically introduced into crop species. Of note is the fact that the resurrection plants whose vegetative parts show constitutive desiccation tolerance are all small and slow-growing. It has been proposed that the energetic cost for the continual maintenance of the many metabolic adaptations needed for desiccation tolerance is very high and limits both yield and productivity, characteristics that are generally positively selected in nature and in agriculture. The same may be true of plants showing constitutive freeze tolerance. An even greater set of problems could arise in trying to use transgenics to induce freeze tolerance in species that are nonhardy. Although the introduction and expression of the CBF transcription factor genes may not be difficult, CBF can work only if the appropriate set of cold-hardy structural genes are present and if these genes contain a CBF response element.

3.6.3 Bacterial Cold Shock Proteins Both heat and cold induce shock responses by organisms. The heat shock response has been extensively studied and its prominent feature is the synthesis of protein chaperones that aid protein refolding and act to protect/rescue proteins whose active conformation is disrupted/denatured by heat exposure. In recent years, a cold shock response has also been identified. In a mesophilic bacterial species such as Escherichia coli, the shift from warm to cold conditions results in an immediate limitation of growth, primarily the result of cold effects on ribosomes that cause a break-up of polysomes into monosomes. However, after a lag period during which cold shock proteins (Csps) are synthesized, protein translation and bacterial growth resumes. Most Csps are small (~7 kDa) proteins that have roles in gene expression, mRNA folding, transcriptional initiation and regulation and/or freeze-protection. The total number of Csps can vary from 12-37 in different bacterial species. Some are known cellular proteins whose levels are up-regulated by cold whereas others are apparently unique to cold shock. For example, CspA, one of the latter group, appears to function as a RNA chaperone to guide efficient translation either by preventing the formation of secondary structure in mRNA that would make it inaccessible for translation at low temperature or by protecting the message from enzymatic degradation. The response of psychrotolerant and psychrophilic bacteria to cold shock is somewhat different. The number of proteins that respond to cold is greater but their level of expression is weaker than the mesophilic response which suggests, not unexpectedly, that these species are better prepared constitutively for dealing with low temperature. A subgroup of these proteins, termed cold-acclimation proteins (Caps), is continuously produced during low temperature growth and may have roles key to life in the cold.

Effects of Csps on freezing survival have received some attention. A single cspB deleted mutant of Bacillus subtilis showed lower freeze survival than wild-type cells. A freeze-sensitive phenotype of Lactococcus lactis was observed upon deletion of the cspA, cspB and cspE genes whereas overproduction of the protein products of these genes increased freezing survival. Since Csps are

19 recognized as nucleic acid binding proteins, they might protect RNA and DNA during the freezing process by binding to these nucleic acids and, hence, increase the survival of the bacterial cells.

3.7 Ischemia resistance, metabolic rate depression, and antioxidant defenses Freezing of extracellular fluids isolates each cell from external supplies of oxygen and fuels, imposing an ischemic state that requires each cell to survive over the long term using only its endogenous fuel reserves and relying on anaerobic pathways of ATP generation. Although subzero temperatures mean that metabolic rates are very low, mechanisms must still be in place to allow all cells and organs (especially highly oxygen-sensitive ones such as brain) to survive for extended times without oxygen. Anoxia tolerance is well developed in many species of insects, intertidal invertebrates and lower vertebrates and the pre-existence of a good anaerobic capacity seems to be a contributing factor to freeze tolerance. Freeze tolerant wood frogs and insects show slow declines in tissue energy reserves (ATP, phosphagen) over time during freezing, but a slow consumption of endogenous glycogen and an accumulation of lactate and alanine as end products is also observed which indicates that ATP production by anaerobic glycolysis is still taking place. The low levels of cellular ATP remaining after extended freezing would be highly injurious to mammals but are not to freeze tolerant species and, upon thawing, an early rebound in ATP levels and other metabolic indices shows that a strong renewal of aerobic metabolism occurs quickly. Freeze tolerant plants use similar mechanisms of fermentative metabolism with ethanol accumulating as the end product during freezing.

Another factor in freezing survival appears to be metabolic rate depression. By strongly suppressing basal metabolic rate (often to values <10% of normal resting rates), organisms gain an extension of the time that a fixed reserve of endogenous fuels can support anaerobic metabolism. Metabolic depression is always a component of facultative anoxia tolerance, and is also a widespread response to other stresses including heat, cold, and desiccation. Winter dormancy or diapause is also common for many organisms. Hence, a pre-existing state of metabolic suppression or an anoxia- or dehydration-induced metabolic depression triggered by freezing is probably employed by most freeze tolerant organisms to extend their potential survival time while frozen.

Studies of ischemia/reperfusion in mammals have identified injuries to cellular macromolecules due to a burst of reactive oxygen species (ROS) generated when oxygen is reintroduced at the end of an ischemic episode. Freeze tolerant animals similarly experience a rapid return of oxygenation during thawing. All animals maintain antioxidant defenses in the form of enzymes such as superoxide dismutase, catalase and glutathione peroxidase and metabolites such as glutathione and vitamin E. These were compared in freeze tolerant (R. sylvatica) and freeze intolerant (R. pipiens) frogs to determine whether tolerant frogs are better prepared to deal with ROS insult. Analysis of lipid peroxidation damage products in wood frog tissues showed no evidence of freeze/thaw related oxidative damage. The metabolic basis for this was traced to high constitutive activities of antioxidant enzymes in wood frog tissues, 2-3 fold higher than in the same organs of R. pipiens. Freeze/thaw also modified the activities of some antioxidant enzymes in wood frog organs. Thus, it appears that mechanisms to deal with potential damage due to ROS formation during thawing are another aspect of natural freeze tolerance.

Bibliography

Franks F. (1985). Biophysics and Biochemistry at Low Temperatures, 208 pp. Cambridge: Cambridge University Press. [Analysis of the physics and chemistry of water and ice, the behaviour of water in

20 biological systems, the freezing of water solutions, vitrification, physical chemistry of freeze avoidance and tolerance in living organisms].

Li P. and Chen T., eds. (1997) Plant Cold Hardiness, 368 pp. New York, Plenum Press. [Numerous articles reviewing multiple aspects of plant cold hardiness and freezing survival].

Marchand P.J. (1991). Life in the Cold: an Introduction to Winter Ecology, 239 pp. Hanover, CT: University of New England Press. [Aimed at science-oriented general readers, the book examines the ecology of winter life for plants and animals including chapters on snow, living conditions under the snowpack or in ice-locked ponds, the challenges of food scarcity and staying warm, and an analysis of humans in cold places]

Margesin R. and Schinner F., eds. (1999). Cold -Adapted Organisms: Ecology, Physiology, Enzymology and Molecular Biology, 416 pp. Berlin: Springer. [Articles contribute current knowledge on cold-hardiness in animals, plants and microorganisms ranging from life in microbial communities in Antarctic lakes to the regulation of freeze-induced gene expression]

Somme L. (1999) The physiology of cold hardiness in terrestrial arthropods. European Journal of Entomology 96, 1-10. [Review of the diversity and phylogeny of cold tolerance among insects and other arthopods]

Storey K.B., ed. (1999). Environmental Stress and Gene Regulation, 181 pp. Oxford: BIOS Scientific Publishers. [Articles provide up-to-date information on fish antifreeze proteins, genes and proteins involved in cold -acclimation in animals and plants, and freeze-induced gene expression]

Storey K.B. and Storey J.M. (1996) Natural freezing survival in animals. Annual Review of Ecology and Systematics. 27, 365-386. [Focus on the principles and recent advances in understanding freezing survival in animals with a discussion of ecologically-relevant freeze tolerance and a review of studies linking freeze tolerance and desiccation tolerance in animals.'

Storey J.M. and Storey K.B. (1998). Life in the freezer - how animals survive winter. Science Spectra 13, 36-42. [Magazine article in a popular format explaining the principles of animal freeze tolerance with color photos of freeze tolerant animals.]

Thomashow, M.F. (2001). So what's new in the field of plant cold acclimation? Lots! Plant Physiology 125, 89-93. [Current analysis of genes involved in plant cold hardiness and freeze tolerance and their regulation with an particular emphasis on membrane cryoprotection in a simple format]

Zachariassen K.E. and Kristiansen E. (2000). Ice nucleation and antinucleation in nature. Cryobiology 41, 257-279. [Review of the principles of nucleation and recrystallization, biological ice nucleation in microbes, plants and animals and the structure and action of ice nucleating and antifreeze proteins].

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Figure 1. Cell responses to extracellular freezing. For an unprotected cell, extracellular ice nucleation results in a rapid growth of ice in large crystals. Solutes are excluded from the crystal and the osmolality of the remaining extracellular fluid rises quickly. Cells respond by loosing water. Cell shrinkage beyond a critical minimum cell volume results in permanent damage to cell membranes so that upon thawing the integrity of the plasma membrane is lost. Freeze tolerant organisms use various protective strategies. Freezing is seeded by ice-nucleating proteins (P) at a temperature just under the equilibrium FP of body fluids so that ice growth is slow and controlled. Antifreeze proteins (brown circles) regulate the shape of crystal growth and inhibit recrystallization so that crystal size stays small. Low molecular weight carbohydrate cryoprotectants (orange circles) such as glycerol or glucose act in a colligative manner to minimize cell volume reduction and others such as trehalose (T) or proline stabilize the membrane bilayer structure. From Storey and Storey (1998) with permission.

Bipyramidal crystal with AFP

Figure 2. Schematic of the interaction of fish antifreeze protein (AFP) with ice. AFP binds to prism faces of ice crystals halting further growth in that plane. Growth continues in the basal plane but AFPs bind to new ice fronts creating a bipyramidal crystal. Modified from Hew and Yang (1992) with permission.

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Figure 3. The final instar larvae of two gall-forming insects exemplify the freeze avoiding (goldenrod gall moth, Epiblema scudderiana) (left) and freeze tolerant (goldenrod gall fly, Eurosta solidaginis) (right) strategies of winter hardiness. Gall moth caterpillars form elliptical galls on the stems of goldenrod which they line with silk after they finish summer feeding. During the autumn, gall moth caterpillars build up glycerol to high levels (~2 M, representing about 20% of their body mass) and can supercool to about -38°C. Gall fly larvae form ball galls. Just before settling into the central cavity for the winter, the larvae eat out a tunnel to the epidermis which provides the exit for the non-feeding adult flies the next spring. Gall fly larvae reezef at about -10°C and use a combination of glycerol and sorbitol as cryoprotectants to prevent intracellular freezing and limit total ice accumulation to about 65% of total body water. Photo: J.M. Storey.

Figure 4. The wood frog, Rana sylvatica, (A) is one of five species of North American frogs that endures long term freezing while wintering under the leaf litter of the forest floor. Frozen frogs (B) adopt a crouched position with limbs tucked close to the body that serves to minimize evaporative water loss. Frozen frogs have no heart beat, no circulation and no breathing; limbs are stiff, the skin is crunchy, the eye looks white due to the frozen lens, and a large mass of ice can be felt in the abdomen. Photos: J.M. Storey.

23 Winter profile of cryoprotectants and glycogen in freeze tolerant gall fly larvae Glycogen Glycerol 400

300

200 Sorbitol µmol/g wet weight 100

0 Aug Sep Oct Nov Dec Jan Feb Mar Apr

20 Mean daily temperature 10 0°C 0 -10

Figure 5. Glycerol (blue line), a 3-carbon polyhydric alcohol, and sorbitol (red line), its 6 -carbon equivalent, are produced as cryoprotectants by freeze tolerant gall fly larvae (Eurosta solidaginis). Each accumulates on a different time course and to a different maximum. Glycerol synthesis is triggered when environmental temperatures drop to a mean of ~15°C whereas sorbitol synthesis comes later when outdoor temperature drops below 5°C. Synthesis of both is derived from the catabolism of the polysaccharide, glycogen (green line), which accumulates in high amounts in the larvae during late summer feeding. When it is time for cryoprotectants to be cleared in the spring, sorbitol is reconverted into glycogen whereas glycerol is mostly catabolized to fuel the renewed development and pupation of the larvae.

Figure 6. The initiation of ice formation on the skin of the wood frog sends immediate signals to the liver that activate glycogenolysis and initiate glucose synthesis; the pathway involves ß-adrenergic receptors on the liver plasma membrane and is mediated intracellularly by cyclic AMP and protein kinase A. Glucose is released into the blood and is taken up in high concentrations by all tissues (levels reach 150-300 mM in core organs) where it helps to limit cell volume reduction due to water loss into extracellular and extra -organ ice masses. In the fully frozen frog, internal organs are visibly shrunken in size and as much as 65% of total body water is converted into ice. From Storey and Storey (1998) with permission.

24 100

80

60

40

20

Relative mRNA band intensity 0 0 1 8 12 24 Thaw Freeze Time (hours)

Figure 7. Freeze-induced gene up-regulation in wood frog liver over a course of freeze/thaw. Changes in the levels of mRNA transcripts for four genes were determined from relative band intensities on northern blots. Symbols show transcripts for: (black circles), fibrinogen a; (red triangles) fibrinogen g; (yellow diamonds), AAT; (green squares), FR10. Control frogs (0 h) were held at 5°C; freezing was at -2.5°C for up to 24 h, and thawed frogs were sampled after 24 h back at 5°C after 24 h frozen. Data fro m Cai and Storey (1997a,b) and Cai et al. (1997) with permission.

Figure 8. Schematic illustrating how cold initiates COR gene expression in plant cells. Chilling stimulates the conversion of an inactive protein "ICE" (meaning "inducer of CBP expression") into its active form which then presumably recognizes a cold-regulatory element, the "ICE-box", present in the promoter regions of CBF (meaning "CRT binding factor") genes. ICE induces the expression of the transcription factor CBF which, in turn, binds to the CRT/DRE element (cold repeat / drought-responsive element: TACCGACAT) that is present in the promoter region of CBF-responsive genes to stimulate the expression of COR and other cold/drought responsive genes. Best studied, to date, is the COR15a gene and its protein product, COR15a, which appears to have a role in stabilizing the chloroplast membrane during freeze-induced cell volume reduction. From Thomashow (1999) with permission.

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