Cold-loving microbes, plants, and animals—fundamental and applied aspects
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- Margesin, R., Neuner, G. & Storey, K.B. Naturwissenschaften (2007) 94: 77. doi:10.1007/s00114-006-0162-6
Microorganisms, plants, and animals have successfully colonized cold environments, which represent the majority of the biosphere on Earth. They have evolved special mechanisms to overcome the life-endangering influence of low temperature and to survive freezing. Cold adaptation includes a complex range of structural and functional adaptations at the level of all cellular constituents, such as membranes, proteins, metabolic activity, and mechanisms to avoid the destructive effect of intracellular ice formation. These strategies offer multiple biotechnological applications of cold-adapted organisms and/or their products in various fields. In this review, we describe the mechanisms of microorganisms, plants, and animals to cope with the cold and the resulting biotechnological perspectives.
KeywordCryoprotectantsCold adaptationFreeze toleranceSupercoolingProteinsMembranesAntioxidant defensesGene expression
Low temperatures and freezing conditions influence the lives of all organisms in multiple ways, e.g., reduced biochemical reaction rates, increased viscosity of the medium, changes in membrane fluidity and protein conformation, nutrient availability, ability to successfully reproduce, and need for protection against freezing. Because active life requires liquid water, organisms living at the lower temperature scale face a physical limit. The lower temperature limit of life is commonly defined as the freezing point (FP) of cellular water. This limit can drop significantly below 0°C for many organisms, for example, due to the synthesis of antifreeze agents that depress the FP of cellular water. The basis of the lower growth temperature limit is clearly distinct from that of the upper growth temperature limit (heat denaturation of proteins). As water is the basis for all forms of life, effective water management is basic to all cold survival strategies. Water management involves the control of membrane composition and transmembrane osmotic equilibrium, the biosynthesis of compounds that afford protection against injury from freeze desiccation, and the availability of biogenic ice nucleation systems (Franks et al. 1990). Cold adaptation requires a complex range of structural and functional adaptations, and these adaptations render cold-adapted organisms particularly useful for a number of biotechnological applications. This review describes adaptive strategies and the resulting biotechnological perspectives of microorganisms, plants, and animals inhabiting low-temperature environments.
Definition and occurrence
Cold-adapted microorganisms exhibit distinctly different properties than representatives of other thermal classes (mesophiles, thermophiles). Most researchers distinguish between psychrophilic (cold-loving) or psychrotolerant (also named cold-tolerant or psychrotrophic) microorganisms, on the basis of their cardinal temperatures. According to the most widely accepted definition, psychrophiles are unable to grow above 20°C and grow fastest at 15°C or below. They persist in permanently cold habitats, such as in polar regions, at high altitudes, or in the deep sea. Environments with periodic, diurnal, or seasonal temperature fluctuations (e.g., areas in continental climates with high summer and low winter temperatures) are favorable to psychrotolerants, which grow over a wide temperature range and have fastest growth rates above 20°C. In this review, the term “cold-adapted” covers both groups.
The lower growth temperature limit is fixed by the physical properties of aqueous solvent systems inside and outside the cell. The lowest temperature at which microbial growth is possible is assumed to be −12°C. Below −10 to −15°C, the cell water begins to freeze and intracellular salt concentrations increase due to the progressive removal of water into ice crystals. The resulting ionic imbalances, lowering of water activity, and desiccation have a toxic effect on cells (Ingraham and Stokes 1959; Russell 1990).
Phylogenetically diverse microorganisms have remained viable within glacial ice cores for over 120,000 years (Miteva et al. 2004). In the past, special focus has been given to microbial life in frozen natural habitats (snow, glacial and sea ice, permafrost, ice clouds) due to the increasing interest in the question of whether life exists elsewhere in the universe (astrobiology; Price 2004; Mautner 2005). Eutectophiles that live at the critical interface between the solid and liquid phases of water may play a special role (Deming 2002).
There is a wide diversity of representatives of all three domains (Bacteria, Archaea, and Eukarya) in cold ecosystems. Bacteria dominate and are present in greater diversity than Archaea in polar environments, while Archaea are widespread in cold, deep ocean water (Karner et al. 2001; Deming 2002). Many microorganisms have to cope not only with low temperature but also with additional stress factors, such as high pressure (psychro-piezophiles; Margesin and Nogi 2004), high salinity (psychro-halophiles; Romanenko et al. 2002), or high irradiance (Mueller et al. 2005). Cold-adapted microorganisms contribute essentially to the processes of nutrient turnover, biomass production, and litter decomposition in cold ecosystems. There is evidence of a wide range of metabolic activities in cold habitats, e.g., nitrogen fixation, photosynthesis, methanogenesis, and degradation of natural or xenobiotic organic compounds such as proteins, carbohydrates, lignin, and hydrocarbons (Cummings and Black 1999; Margesin et al. 2002a; Trotsenko and Khmelenina 2005). Metabolic fluxes at low temperatures are comparable to those displayed by mesophiles at moderate temperatures. Yeasts may be better adapted to low temperatures than bacteria (Margesin et al. 2003a; Turkiewicz et al. 2003). Metabolic activity does not cease at subzero temperatures, as shown by microbial synthesis of DNA and protein precursors in glacial ice at −15°C (Christner 2002) or in snow at −12 to −17°C (Carpenter et al. 2000), or metabolic activity of permafrost bacteria at temperatures down to −20°C (Rivkina et al. 2000). Bacteria perform basic functions at temperatures far below 0°C. The arctic bacterium Colwellia psychrerythraea is motile at temperatures down to −10°C, and its swimming speeds are comparable at −5 and −10°C (Junge et al. 2003).
To grow successfully in cold habitats, cold-adapted microorganisms have evolved a complex range of adaptations of all their cellular constituents, including membranes, proteins, energy-generating systems, components responsible for nutrient uptake, and the synthesis of compounds conferring cryotolerance (Russell 1998; Margesin and Schinner 1999a; Cavicchioli et al. 2002; Deming 2002; Margesin et al. 2002b; Benson et al. 2004; Shivaji 2004; Häggblom and Margesin 2005). The recent publication of the first genomes of cold-adapted bacteria (Desulfotalea psychrophila, C. psychrerythraea, Methanococcoides burtonii, Pseudoalteromonas haloplanktis, and Shewanella violacea; Goodchild et al. 2004; Nakasone 2004; Rabus et al. 2004; Medigue et al. 2005; Methe et al. 2005) is a big step forward because it allows the comparison of psychrophilic, mesophilic, and thermophilic counterparts, which can facilitate the understanding of the evolution of cold adaptation in bacteria. Comparative genome analyses suggest that the psychrophilic lifestyle is most likely conferred not by a unique set of genes but by a collection of synergistic changes in overall genome content and amino acid composition of proteins (Methe et al. 2005). Colwellia psychrerythraea genome analysis (Methe et al. 2005) revealed several bacterial strategies to cope effectively with the cold: maintenance of membrane fluidity; production and uptake of compounds for cryoprotection (extracellular polysaccharides, compatible solutes); synthesis of enzymes involved in the regulation of key biosynthetic pathways (such as purine and lipid biosynthesis) and degradation of various organic compounds (extra- and intracellular enzymes); production of intracellular carbon and energy reserves (polyhydroxxyalkanoates), as well as nitrogen reserves (polyamides); and adaptation of the molecular structure of proteins to ensure increased flexibility at low temperatures (see below). Another study involving P. haloplanktis genome analysis (Medigue et al. 2005) demonstrated that this organism is particularly well adapted for protection against reactive oxygen species (ROS), which is important for survival at low temperatures where the solubility of gases is increased. The organism entirely lacks pathways (molybdopterin metabolism) that produce ROS. In addition, the bacterium produces dioxygen-consuming lipid desaturases to achieve protection against oxygen and to maintain membrane fluidity at the same time. Colwellia psychrerythraea achieves enhanced antioxidant capacity through the presence of catalase and superoxide dismutases (Methe et al. 2005).
Temperature, growth rate, and metabolic activity
According to the Arrhenius equation, any decrease in temperature causes an exponential decrease of the reaction rate, the magnitude of which depends on the value of the activation energy. Consequently, most biological systems display a reaction rate 2–3 times lower when the temperature is decreased by 10°C (Q10 value). Temperatures outside the linear range of the Arrhenius plot (log of growth rate vs the reciprocal of the absolute temperature) are stress-inducing temperatures. For psychrophiles, Arrhenius plots remain linear down to 0°C, for psychrotolerants and mesophiles they deviate from linearity at 5–10 and at 20°C, respectively (Gounot and Russell 1999).
The term “optimal” growth temperature is often erroneously correlated to the maximal growth rate. The temperature at which the growth rate is maximal reflects only kinetic effects and occurs above the linear part of the Arrhenius curve, which means that the physiological conditions are not ideal (Gounot and Russell 1999; Glansdorff and Xu 2002). Growth rate may not be as relevant as growth yield. The production and/or activity of cold-active enzymes is usually significantly below the “optimal” growth temperature (as determined from growth rate) of the enzyme producer, which reflects the thermal characteristics of the secretion process. Highest cell and enzyme yields are generally obtained at cultivation temperatures that correspond to those of the natural environment of the strains, which should be considered for large-scale production of cold-active enzymes in biotechnology. For example, protease production by Bacillus sp. was almost twice as high at 4°C compared to 25°C, despite the significantly slower growth rate at low temperatures (Feller et al. 1996). Similarly, protease production by Pseudomonas fluorescens was reduced by 50% at 20°C and was completely absent at 30°C (Margesin and Schinner 1992). The growth temperature does not affect thermal characteristics of enzymes secreted at low or moderate temperatures (e.g., at 4 and 20°C).
The membrane is both the interface and the barrier between the internal and external environment of the cell. Cold-adapted bacteria respond and adapt to low temperature by modulating the fluidity of their membrane to maintain the function of membrane proteins involved in respiration and nutrient transport (reviewed by Russell 1998; Russell and Nichols 1999; Chintalapati et al. 2004). This is mainly achieved by altering the fatty acid composition. The most important strategy is to increase the proportion of unsaturated fatty acids, which help to maintain a semifluid state of the membrane at low temperatures (Aguilar et al. 1998; Suzuki et al. 2001). Membranes composed of predominantly saturated fatty acids would become waxy and nonfunctional at low temperatures. Changes in the fatty acid chain length are another commonly observed response to fluctuating temperature conditions. Short-chain fatty acids (especially those with less than 12 carbons) maintain the fluid state of the membrane. Other low-temperature-induced changes include an increase in methyl branching of fatty acids (especially in Gram-positive bacteria) and changes in fatty acid isomerization (more anteiso-branched fatty acids and less iso-branched forms; more cis than trans unsaturated fatty acids). Additional strategies are changes in the lipid head group, in the protein content of the cell membrane [e.g., production of cold-shock proteins (CSPs)], and in the composition of carotenoids. Antarctic bacteria increase the synthesis of polar carotenoids to stabilize the membrane during growth at low temperatures, and at the same time decrease the synthesis of nonpolar carotenoids (Jagannadham et al. 2000).
At the molecular level, a wide range of determinants confer conformational flexibility to proteins. All known structural factors involved in protein stability are either reduced in number or modified to increase flexibility and to reduce rigidity in proteins from cold-adapted microorganisms. These factors can include changes in the frequency of particular molecular bonds (fewer ion pairs, arginine-mediated hydrogen bonds, and aromatic interactions) and amino acid side chains (more polar and less hydrophobic residues, an increased number and clustering of glycine residues, a decrease in proline residues in loops, a reduction in arginine residues resulting in a low arginine/lysine ratio), increased/improved interactions with the solvent (water and associated ions), reduced hydrophobic interactions between subunits, and loose anchoring of N and C termini (Feller and Gerday 1997; Russell 2000; Sheridan et al. 2000; Marx et al. 2004). Obviously, no cold-active enzyme displays all of these features; the strategy can differ from enzyme to enzyme.
CSPs and cold-acclimation proteins
Sudden temperature decreases induce or increase the synthesis of several CSPs in mesophilic bacteria, such as members of the CspA family and RNA- or RNA/DNA-binding proteins (chaperons). Expression of class I CSPs at moderate temperatures occurs at very low levels and is drastically (more than tenfold) induced by cold shock. Class II CSPs are synthesized at moderate temperatures and are less strongly induced after a shift to low temperature. Contrary to heat-shock response, cold-shock response does not require the synthesis of a new sigma factor (prokaryotic initiation enzyme factor) for the control of the expression of genes that are required to cope with cold-induced alteration of protein conformation; this provides a rapid reaction to face the cold stress (Weber and Marahiel 2002). The synthesis of these inducible CSPs slows down when the cell becomes adapted to the low temperature. In contrast, cold-adapted bacteria produce permanently one set of proteins [cold-acclimation proteins (CAPs)] during continuous growth at low temperature and increase the steady-state level of CAPs when the temperature is lowered. CAPs may be fundamental to life in the cold and ensure improved protein synthesis at low temperature. The cold-shock response in cold-adapted bacteria differs from that in mesophilic or thermophilic bacteria in two major aspects: cold shock does not inhibit the synthesis of housekeeping gene products, and the number of CSPs is higher and increases with the severity of the cold shock (reviewed by Gounot and Russell 1999; Margesin et al. 2002b).
Cryoprotection and antifreeze protection
Microorganisms, plants, and animals produce various compounds to protect themselves or the extracellular environment against intracellular freezing. Microbial antifreeze proteins (AFPs) have been detected in basidiomycetes such as snow mold fungi (Coprinus psychromorbidus and Typhula species) and Flammulina velutipes. AFP genes of Typhula ishikariensis do not have any similarity with known proteins and may constitute a new class of AFPs. They may prevent freezing of the extracellular environment to ensure mycelial growth (Hoshino et al. 2006). Bacterial ice-nucleating agents (INAs) serve as templates for ice crystallization and provide resistance to desiccation. They comprise outer membrane proteins, lipids, phospholipids, and carbohydrates (Lundheim 2002). The induction of frost damage in plants by INA-producing bacteria can be an adaptive advantage to get access to nutrients from plants. Osmoprotection of the microbial cell is achieved by the production of intracellular compatible solutes such as polyols and sugars (Gounot and Russell 1999).
Over the last decade, studies on cold-adapted microbes have increased considerably, which can be attributed to several factors, such as the awareness of accelerated environmental changes in polar regions, a strong interest in the habitability of frozen areas elsewhere in the universe (astrobiology), and a realization of the considerable biotechnological potential of these organisms (Russell 1998; Margesin and Schinner 1999b; Cavicchioli et al. 2002; Benson et al. 2004). Scientific publications dedicated to psychrophilic or psychrotolerant microorganisms over the past 10 years have increased by a factor of ten, with almost one-third belonging to the category of biotechnology and applied microbiology.
Applications of cold-active enzymes in biotechnology (Margesin and Schinner 1999b)
Washing at low temperature (energy-saving and applicable to synthetic fibers); contact lens cleaning
Protease (Kannase©, Polarzyme©), lipase (Lipolase©, LipoPrime©), amylase (Stainzyme©), cellulase, oxygenase
Reduced incubation time for lactose hydrolysis in milk and dairy products
β-Galactosidase (Gerday et al. 2001)
Improved juice clarification, increased juice yield
Efficient and gentle removal of fish skin, meat tenderization
Cold pasteurization, food preservation
Catalase, lysozyme, glucose oxidase
Improved taste and aroma of fermentation products (e.g., cheese, dry sausages, alcoholic beverages)
Enzymes involved in fermentation and ripening
Continuous production of wine and beer at 5°C without loss of productivity (Kourkoutas et al. 2003)
Synthesis of volatile and heat-sensitive compounds (e.g., flavors and fragrances)
Lipase, esterase, protease, etc.
Synthesis of acrylamide
Organic phase biocatalysis (increased solvent choice, product yield, and biocatalysis stability)
Enzymes operating under low water conditions
Mild heat inactivation of enzymes without interference with subsequent reactions
Selective enzyme inhibition
Efficient low-temperature ligation
Prevention of carry-over contamination in PCR
Uracil DNA glycosilase
Rapid 5′ end-labeling of nucleic acids
Efficient protoplast formation
Cellulase, xylanase, etc.
Debridement of necrotic tissue, digestion promotion, chemonucleolytic agents
Improved quality after desizing, biopolishing, and stone-washing of fabrics
Amylase, laccase, cellulase
Selective, sensitive, and rapid on-line monitoring of low-temperature processes, quality control
Various enzymes, e.g., dehydrogenase
In situ/on-site bioremediation of organic contaminants
Mono- and dioxygenases, transferases, hydrolases, etc.
Low-energy wastewater treatment
Enzymes involved in mineralization, nitrification, and denitrification
Low-energy anaerobic wastewater treatment
Enzymes involved in anaerobic degradation
Low-temperature biogas (methane) production
Enzymes involved in anaerobic degradation
Enzymes involved in litter degradation
Protein expression systems
The production level of cold-active enzymes by wild-type microbial strains is usually too low for industrial-scale production. Therefore, genes encoding these enzymes have been cloned and expressed in host bacteria, such as Escherichia coli, for which efficient expression systems have been designed to obtain high enzyme yields. However, overexpression at typical growth temperatures of these hosts (30–37°C) often results in inclusion bodies and in the inactivation of heat-sensitive gene products. Tutino et al. (2001) developed promising systems for efficient gene expression and recombinant protein production in cold-adapted bacteria (Tutino et al. 2001). The cold-adapted promoters showed a strong similarity with mesophilic (E. coli) counterparts (Duilio et al. 2004). An inducible expression vector was recently constructed. The expression system was effective in the production of cold-active β-galactosidase and mesophilic alpha-glucosidase in a fully soluble and active form (Papa et al. 2006). Chaperonins play a crucial role in the growth of E. coli at low temperatures. Introduction of the cold-adapted cpn60/10 chaperonin genes of the psychrophilic bacterium Oleispira antarctica, which encode GroEL/ES chaperonin homologues, enabled E. coli to grow well at temperatures down to 0°C. This system may be useful for the expression of cold-adapted proteins. Expression of an O. antarctica esterase was 100-fold higher in E. coli Cpn+ cells grown at 4°C than at 37°C (Ferrer et al. 2003).
Increased legume production in cold regions
Legumes constitute high-quality protein sources for human and animal nutrition, and derive most of their nitrogen requirement from a symbiotic association with rhizobia. Cold periods during the growing season can significantly limit the establishment of this symbiosis. Arctic rhizobia increased the production of legumes by 30% through improved nitrogen fixation and are more efficient than commercial rhizobia (Prevost et al. 2003).
Biocontrol of plant diseases
Cold-adapted fungi that produce antibiotics, cell-wall digestive enzymes, and toxins, or induce host resistance at low temperatures (0–5°C), are commercially available as biocontrol agents (Bio-Green©, Plant-Helper©). They are an alternative to chemical pesticides for the control of diseases and pests in cold climates, of winter crops, and during cold storage (Wong and McBeath 1999). Antibiotics produced by P. fluorescens have been commercialized for the biological control of fire blight in pears and apples (Blightban®; Lindow and Leveau 2002).
Frost protection of plants
A number of bacteria, such as Pseudomonas syringae and Erwinia herbicola, cause frost injury to plants by triggering ice crystal formation through the action of INAs at subzero temperatures (−2 to −10°C) when water might otherwise remain supercooled and liquid. The competitive exclusion of such Ice+ bacteria with naturally occurring or genetically modified “ice-minus” mutants is claimed to be an effective means of frost control. A commercial product (Frostban®) consisting of a mixture of three bacterial strains (P. fluorescens and P. syringae) can be sprayed on crops to protect plants from frost (Skirvin et al. 2000; Lindow and Leveau 2002).
Commercial uses of bacterial INAs for energy- and cost-saving applications include the production of artificial snow (Snomax®; the addition of INA to water in snow-making machines raises the critical temperature for artificial snow making by several degrees), the production of ice as a construction material for installations in the Arctic and Antarctica, the manufacture of ice-cream and other frozen food (Yin et al. 2005), and the substitution for silver iodide in cloud seeding (Lundheim 2002).
Petroleum hydrocarbons are the most widespread contaminants in the environment. Because vast petroleum reserves occur in the Arctic and Antarctica, there is a need to optimize treatment technologies for contaminated sites in these areas. Low-temperature biodegradation of petroleum hydrocarbons in a variety of terrestrial and marine cold ecosystems (reviewed by Margesin and Schinner 2001) is a result of the degradation capacity of indigenous cold-adapted microorganisms. They transform or mineralize organic pollutants into less harmful, nonhazardous substances, which are then integrated into natural biogeochemical cycles. High numbers of hydrocarbon degraders, the prevalence of genotypes with catabolic pathways for the degradation of a wide range of hydrocarbons, and high mineralization potentials in contaminated polar and alpine soils, sediments, and sea provide evidence for the biodegradation activity of indigenous bacteria and fungi (Braddock et al. 1997; Margesin et al. 2003b; Delille et al. 2004). Special challenges to microorganisms in polluted cold regions include reduced enzymatic reaction rates, increased viscosity of liquid hydrocarbons, reduced volatility of toxic compounds, limited bioavailability of nutrients and contaminants. Depending on the local conditions, water activity, oxygen, contents of nutrients (nitrogen and phosphorus), soil moisture, and extremes in pH and salinity are also often limiting factors (Alexander 1999). The oil tanker accident of the Exxon Valdez in Alaska (Wolfe et al. 1994) demonstrated that temperature was not the main limiting factor for petroleum hydrocarbon biodegradation, but instead, the availability of nutrients restricted the effectiveness of biodegradation. Microbial biostimulation via supplementation of nutrients (bioremediation) and oxygen is an efficient method to accelerate the biodegradation process in cold regions. In situ treatment is often the only viable management strategy in remote contaminated sites. Successful on-site treatments of contaminated cold soils include land farming, biopiles, and engineered biopiles (thermal insulations systems) (reviewed by Margesin 2004).
The use of cold-adapted microbial communities for low-energy wastewater treatment leads to a significant decrease in operational costs. In cold climates, industrial wastewater temperature often decreases to 10°C and below. Processes developed for the anaerobic treatment of industrial wastewaters at 8–12°C resulted in chemical oxygen demand (COD) removal efficiencies comparable to those seen during mesophilic or thermophilic anaerobic treatment (Lettinga et al. 1999). Upward-flow anaerobic sludge blanket reactors gave a stable performance (70–90% COD removal) at an operation temperature of 11°C. At 6°C, COD removal was still 30–50% (Singh and Viraraghavan 2004). Bacteria and fungi that degrade high amounts of organic compounds within a short time at temperatures down to 1°C represent a promising source as inocula for accelerated wastewater treatment and also for the construction of biosensors for the rapid monitoring or in situ analysis of pollution (Alkasrawi et al. 1999; Margesin et al. 2004).
Bioleaching is the extraction of specific valuable metals from their ores through the use of bacteria. Several mines worldwide operate at average temperatures of 8–10°C with satisfactory bioleaching performance. Cold-adapted strains of Acidothiobacillus ferrooxidans mediate the bioleaching of metal sulfides at such temperatures (Rossi 1999).
Definition and occurrence
Low temperatures, particularly, freezing temperatures, can dramatically affect plants at cellular to ecosystem scales (Loik et al. 2004). These factors are important filters on recruitment, survival, productivity, and latitudinal and altitudinal distribution of wild plants (see Sakai and Larcher 1987). Low temperatures and frost set agricultural borders for crop species and, in marginal areas, can cause severe yield losses. The intensity and occurrence of freezing temperatures (Fig. 1), their annual timing, and whether they commence episodically, periodically, or regularly (e.g., at night) has led to a variety of low-temperature and frost-survival mechanisms in plants. Tropical rainforest species, such as the horticultural plant Saintpaulia ionantha, (Bodner and Larcher 1987) do not tolerate ice formation in their tissues and are damaged by exposure to low temperatures above 0°C. Plants that may be damaged by temperatures between +12 and 0°C have been termed chilling-sensitive. Plants from outside the tropics and plants from high tropical mountains are usually frost-resistant, i.e., they remain undamaged upon exposure to subzero tissue temperatures. However, the extent of frost damage varies greatly: some species suffer frost damage at just below 0°C, whereas others survive dipping in liquid nitrogen (−196°C; see Sakai and Larcher 1987).
Chilling stress is a direct result of low temperature effects on cellular macromolecules that cause a slowdown of metabolism, solidification of cell membranes, and loss of membrane functions. Freezing stress acts indirectly via extracellular ice crystals that cause freeze dehydration, concentrate the cell sap, and have major mechanical impacts. There is a consensus that the primary cause of freezing injury in plants is most frequently an irreversible dysfunction of the plasma membrane as a consequence of freeze-induced cellular dehydration (Levitt 1980; Webb et al. 1994; Uemura et al. 1995; Xin and Browse 2000).
In contrast to chilling-sensitive plants that show limited potential to acclimate to cold (Sakai and Larcher 1987), frost-resistant plants have evolved mechanisms by which they can increase their resistance to freezing temperatures. This process is called cold acclimation (Levitt 1980). In most plants, natural cold acclimation is induced by exposure to low temperatures. Woody species also respond to shortening day length (Sakai and Larcher 1987).
Mechanisms of frost resistance in plants
Avoidance of ice formation appears to be made possible by FP depression of the cell sap due to the accumulation of solutes. The gain in frost tolerance is, however, usually not more than 2 K. Another mechanism to avoid ice formation is thought to be persistent supercooling (Sakai and Larcher 1987). Supercooling is the ability of tissues to cool distinctly below the FP, sometimes as low as about −38°C, without ice formation. Many woody plants have exploited supercooling, particularly in the xylem ray parenchyma cells or in buds (for review see Wisniewski and Fuller 1999), as a primary strategy to avoid the desiccation of the cytoplasm that would otherwise occur during freezing. However, the protection cannot exceed the homogeneous ice nucleation temperature of water, which is −38.1°C. This supercooling specific threshold temperature limits the geographic distribution of supercooling species to regions where this minimum temperature is not exceeded (Burke et al. 1976). In biological systems, the homogeneous FP of water may be lowered further, down to approximately −41°C, by the osmotic effects of dissolved solutes or by the hydration effects of macromolecules or biological ultra structures, such as membranes.
Survival at temperatures lower than −38°C (−41°C) can only be achieved by tolerance of extracellular ice. Any intracellular ice formation is considered lethal, as it ruptures membranes. In many species, apoplastic ice forms in the extracellular space or extraorgan space in bud scales or lacunes. Formation of apoplastic ice causes freeze dehydration of cells during so-called equilibrium freezing. For this to be tolerated, ice nucleation in the apoplast must proceed in a controlled way, and the movement of water from the protoplast to the extracellular space must be controlled. This requires a barrier between the interior of the cell and the extracellular ice, i.e., the plasma membrane (which must retain its fluidity), and the ability to tolerate freeze-induced cellular dehydration and cell collapse. Freeze-dehydration occurs because the water potential of ice is lower than that of liquid water. Extracellular ice crystals grow by drawing water from cells until the water potential of the ice and the cell sap are equal. The water potential of ice decreases with decreasing temperature (Gusta et al. 1975), down to a limit set by vitrification. The formation of so-called glassed cell solutions is thought to be a natural adaptation of woody plant cells (for a recent review, see Wisniewski et al. 2003). It occurs in extremely hardy plant species that can survive cooling to −196°C in liquid nitrogen. In poplar, glasses can form below −28°C (Hirsh et al. 1985). Although glassed solutions are extremely metastable and exhibit a high degree of supercooling and high hydrostatic tension, they are not subject to ice nucleation, solute crystallization, or water vapour cavitation so long as the solution remains below the melting temperature of the glass (Wisniewski et al. 2003). Thus, when the water in cells forms a glass, diffusion, freezing, and biochemical processes are virtually stopped and the cytoplasm and its contents are extremely stable and relatively unaffected by stresses associated with low temperature and the presence of ice.
Equilibrium freezing does not occur in all cells. In some species, the cell walls partially resist the collapse in cellular volume, creating a divergence from equilibrium (nonideal-equilibrium; Zhu and Beck 1991) and reducing the extent of dehydration (Rajashekar and Burke 1996). However, substantial cellular dehydration still occurs (Zhu and Beck 1991).
Ice nucleation at temperatures just below 0°C (Pearce 2001; Taschler and Neuner 2004) occurs heterogeneously, as it is catalyzed by nucleators. These are INA bacteria, other biological molecules, and organic and inorganic debris (Pearce 2001). Ice nucleation may start via extrinsic nucleators on the plant surface and then grow via stomata into the plant, or may be initiated inside the plant (intrinsic; Wisniewski et al. 1997; Wisniewski and Fuller 1999). Effective intracellular ice nucleators are absent. Once ice has nucleated somewhere, it spreads at high rates of between 4 and 40 mm s−1 throughout the apoplast of the plant (Pearce and Fuller 2001). In some species, internal barriers against the spread of ice have been detected. These barriers can be important in protecting freezing-sensitive tissues such as flowers and fruits (e.g., Carter et al. 2001). Where plants do avoid freezing by supercooling substantially, the mechanisms involved are not fully understood but, at least in the case of buds and the xylem parenchyma of woody species, they include structural features (Wisniewski and Fuller 1999).
Our understanding of the process of ice nucleation and propagation in whole plants under field conditions is incomplete (Wisniewski and Fuller 1999). Whether freezing initially occurs in the xylem vessels themselves or extracellularly (as in peach) where ice is nucleated in the cortex and grows from there to the xylem is still controversial (see Pearce 2001). Little is generally known about how plant structure affects ice nucleation and propagation (Wisniewski and Fuller 1999).
Whether or not freezing injury occurs in plants depends on the cold acclimation state. Freezing injury is caused by cellular freeze dehydration and cell contraction and normally involves damage to plasma membrane structure and function (Levitt 1980; Steponkus and Webb 1992; Webb et al. 1994; Uemura et al. 1995; Xin and Browse 2000). In isolated nonacclimated protoplasts, freezing stress and cell dehydration cause the formation of endocytocic plasma membrane vesicles (recently reviewed by Uemura et al. 2006). Under mild injurious stress during thawing, expansion-induced lysis occurs. Under severe stress, formation of hexagonal II (HII) phase (lamellar-to-nonlamellar phase transition) of the plasma membrane can be observed in regions where the membrane is brought into close apposition with various endomembranes. This is most often the chloroplast envelope and tonoplast. As a result, the protoplasts lose osmotic responsiveness (LOR) during thawing. In cold-acclimated protoplasts, the formation of HII phase is not observed at any injurious temperature, but freeze-induced dehydration results in exocytotic extrusions of the plasma membrane and fracture-jump lesions (FJLs) occur. FJLs are characterized by localized deviations of the fracture plane of the plasma membrane in freeze-fracture electron micrographs and the manifestation of injury by LOR.
Components of increased frost resistance/cold acclimation
Changes in growth and photosynthesis
In evergreen leaves, cold acclimation of photosynthesis can overcome the combined effects of low temperature and high irradiation (for recent reviews, see Adams et al. 2004; Öquist and Huner 2003). In woody plant species, photosynthetic capacity is strongly suppressed during winter (e.g., Ottander et al. 1995; Neuner et al. 1999; Savitch et al. 2002), while herbaceous plants show no sustained downregulation of photosynthetic capacity (cereals: Öquist and Huner 2003; other herbaceous: Hacker and Neuner 2006). Winter cereals continue to grow during cold acclimation and, hence, maintain a strong sink capacity, which is in contrast to woody plants that cease growing and are dormant in winter (Savitch et al. 2002). This balance between photosynthetic energy absorbance and consumption through metabolism and growth has been termed photostasis (Huner et al. 2003).
ROS detoxifying substances
Increased antioxidant content (ROS detoxifying substances) is observed during cold acclimation in plants (for a recent review, see Schulze et al. 2005). ROS are generated in situations with highly energized primary photochemistry but impaired stromal metabolism. ROS can cause membrane damage by the formation of radicals. ROS detoxifying substances may be localized in membranes (e.g., tocopherol and xanthophyll in the thylakoid membrane) or found in the cytosol or the stroma of the chloroplast (mainly ascorbate and glutathione, but also flavonoids).
The functional integrity of biological membranes at low temperatures is necessary for active life processes. For that reason, membrane lipids need to be present in the liquid phase. During cold acclimation of frost-resistant species, changes in membrane lipid composition occur (Senser and Beck 1982; Webb et al. 1994; Uemura et al. 1995; Uemura et al. 2006). The proportion of phospholipids increases. In many plant species, this is primarily the result of an increase in the proportion of unsaturated molecular species of phosphatidylcholine and phosphatidylethanolamine and a decrease of cerebrosides (Uemura et al. 2006).
Also, chilling tolerance increases with increased fatty acid unsaturation (Nishida and Murata 1996). The melting point of membrane lipids is affected by the degree of unsaturation of fatty acids. The greater the number of double bonds and the shorter the fatty acids, the lower the melting point and the lower the temperature at which solidification occurs. By replacing fatty acids, membranes can adapt to the prevailing temperatures. Genetic manipulation of the chloroplast enzyme glycerol-3-phosphate acyltransferase (GPAT), which is involved in phosphatidylglycerol fatty acid unsaturation, changed the chilling tolerance of transgenic tobacco plants to low temperature exposure (Murata et al. 1992).
Changes in membranes may also include proteins (Uemura et al. 2006). During cold acclimation, some plasma membrane polypeptides have been shown to disappear, decrease, or be substituted by others (Uemura and Yoshida 1984). Membrane proteins are surrounded by less mobile lipids and the zone of less mobile lipids around proteins increases during cooling. Reduction of the protein–lipid ratio has been found to be important in cold acclimation of thylakoid membranes by maintaining an adequate membrane fluidity (Schulze et al. 2005).
Several proteins are expressed upon exposure to low temperature and may occur in the cytosol or be secreted to the apoplast. They have various putative functions, including cryoprotection, altered lipid metabolism, protein protection, desiccation tolerance, and sugar metabolism (reviewed by Hiilovaara-Teijo and Palva 1999). Three types of proteins have been shown to accumulate outside the cells in the apoplast during cold acclimation: (1) cell wall-modifying proteins, (2) a group of pathogenesis-related proteins that might be a component of the signal transduction pathway triggered during general stress response, and (3) AFPs that interact with extracellular ice (Atici and Nalbantoglu 2003). AFPs have been found in a considerable number of woody and herbaceous plant species (Atici and Nalbantoglu 2003), including an Antarctic plant (Bravo and Griffith 2005). The effect of AFPs in plants on FP depression appears to be negligible, as it is less than 1°C (Schulze et al. 2005). However, AFPs adsorb onto the surface of ice crystals and modify their shape and growth in a beneficial manner: instead of one large single ice crystal, more but smaller and slower-growing ones develop. During thawing, AFPs may inhibit recrystallization and formation of larger ice crystals. Larger ice crystals increase the possibility of physical damage within frozen plant tissue (Griffith et al. 1997). Together with INA proteins, AFPs are thought to control extracellular ice formation (Marentes et al. 1993).
During cold acclimation, several stress proteins that may function as chaperones and membrane stabilizers during freeze dehydration are expressed in the cytosol (Puhakainen et al. 2004). These proteins have been assigned to the group of late-embryogenesis-abundant proteins (LEA proteins; Wise and Tunnacliffe 2004). LEA proteins are divided into several different structural groups, one such group (LEA II) consists of dehydrins (Allagulova et al. 2003) that are usually expressed in cells in response to dehydration stress. The proposed functions of dehydrins are considerable. Dehydrins seem to operate as membrane stabilizers; to posses cryoprotective function or antifreeze activity; to improve enzyme activity under conditions of low water availability; to act in osmoregulation and as radical scavengers; and, as recently shown, to have Ca2+-binding activity suggesting action as a Ca2+ buffer or Ca2+-dependent chaperone (for a review, see Puhakainen et al. 2004).
Heat shock proteins (HSPs) also accumulate in response to low temperature. Usually, these proteins are synthesized in response to high temperatures or other environmental stresses such as drought, salinity, or flooding. The major putative functions of HSPs include involvement in membrane protection, refolding of denatured proteins, prevention of aggregation of denatured proteins, facilitating correct protein folding during translation, and aiding protein translocation into organelles (for a recent review, see Renaut et al. 2006). In addition, uncoupling proteins may be produced. These are integral proteins of the mitochondrial inner membrane that can potentially cause a transient release of heat by the uncoupling of oxidation from phosphorylation in mitochondria. It has been suggested that they permit plants to retain above-zero tissue temperatures for some time, providing time to prepare to subzero ambient temperatures (Kolesnichenko et al. 2000).
In general, the expression and activity of many enzymes involved in several different metabolic pathways, such as carbon metabolism, photosynthesis, the detoxifying systems, and proline and lignin metabolism, have been shown to change in response to low-temperature exposure (Renaut et al. 2006).
Osmoprotectants or compatible solutes
During cold acclimation, osmotic water potential increases (Ulmer 1937) due to an accumulation of osmoprotectants or compatible solutes such as polyols and soluble sugars (e.g., Ristic and Ashworth 1993); several groups of amino acids, e.g., proline (e.g., Tantau et al. 2004); and quaternary ammonium compounds such as betaine (e.g., Nomura et al. 1995). These substances can accumulate to osmotically significant levels without disrupting plant metabolism. Solute accumulation in cells has a colligative effect that reduces the cell volumetric collapse at any given subzero temperature (Crowe et al. 1992). Direct solute-specific beneficial actions are thought to be stabilization of macromolecules and membranes and protection of membranes against the deleterious effects of increasingly higher concentrations of electrolytes during freeze dehydration.
Some plant species can tolerate extremely low temperatures, even being dipped in liquid nitrogen (Sakai and Larcher 1987), but how easy it will be to transfer such tolerance between species is not yet clear (Pearce 1999). Until now, most research into the molecular basis of frost resistance has focused on the moderately frost-resistant species, Arabidopsis thaliana, and, to some extent, on cereals (barley, wheat, rye, rice). Having the broad spectrum of plant frost-resistance mechanisms in mind, this must be considered a shortcoming, although undoubtedly, this research has yielded already extremely valuable insights into plant frost resistance (Thomashow 1999).
The most prominent biotechnological attempts to alter plant freezing tolerance are briefly addressed in the following section. Plant acclimation to freezing temperatures is very complex. In Arabidopsis, for example, the expression of hundreds of genes is altered in response to low temperature, as demonstrated from a recent large-scale microarray analysis (Van Buskirk and Thomashow 2006). Hence, the transfer of a single cold-responsive gene is unlikely to have a major effect on low temperature resistance. However, overexpression and antisense inhibition studies in transgenic plants are important in providing evidence for the involvement of individual genes in low-temperature resistance. For instance, the overexpression of the GPAT gene from Arabidopsis increased the chilling resistance of tobacco by increasing fatty acid unsaturation in chloroplast membranes. On the other hand, overexpression of GPAT from chilling-sensitive cucumber caused a decrease in fatty acid unsaturation and a concomitant increase in chilling sensitivity of tobacco (Murata et al. 1992; Nishida and Murata 1996). These studies clearly show that changes in fatty acid unsaturation in membranes are a key feature of low-temperature tolerance.
Other promising research has focused on transforming plants with fish (winter flounder) AFPs to improve plant frost resistance (e.g., Hightower et al. 1991). AFPs need to be secreted to the apoplast space in transgenic plants if they are to protect membranes from freeze-induced damage (Wallis et al. 1997) because fish AFPs and antifreeze glycoproteins have been shown to be cryotoxic to thylakoid membranes (Hincha et al. 1993). Hence, the targeting of the encoded gene product must be correct in transgenic plants if it is to improve freezing tolerance.
Temperature sensing in plants is not yet understood; however, a decline in temperature decreases membrane fluidity, which could activate plasma membrane calcium channels (Vigh et al. 1993) and cause the observed increase in cytosolic Ca2+ concentrations. Higher cytosolic Ca2+ concentrations seem not only to affect direct temperature signaling but, additionally, ABA signaling. This suggests that higher cytosolic Ca2+ concentrations are involved in the activation of master genes (Tahtiharju et al. 1997). There is increasing evidence that the cold-induced increase in frost resistance is regulated by a multisignal transduction network leading to the expression of numerous genes (Shinozaki and Yamaguchi-Shinozaki 2000). In the following section, they are referred to as COR genes, although many cold-responsive genes may have been termed differently, such as, for example, LTI (low-temperature induced), KIN (cold inducible), RD (responsive to desiccation), and ERD (early-dehydration inducible). Master genes controlling the expression of many COR genes appear to be the transcription factors or activators of the CRT-repeat binding factor (CBF) group that bind to the promoter-element AAGAC of many COR genes and other transcription factors, such as, e.g., Eskimo 1 (esk1), with an unknown different activation pathway. Transcription of CBFs is induced by cold stress that activates a protein called ICE1, i.e., a potential master regulator of cold acclimation (Chinnusamy et al. 2006). ABA seems to induce COR genes via the transcription activator bZip.
Cloning of the CBF transcription factors advanced the understanding of the molecular genetics of cold acclimation significantly: when the genes encoding these proteins were overexpressed in transgenic Arabidopsis plants, the COR genes were expressed at higher levels and the frost resistance of the plants increased (Jaglo-Ottosen et al. 1998; Kasuga et al. 1999). Another promising approach is to find a promoter (such as WSC120: Ouellet et al. 1998) that is low-temperature-inducible in species belonging to different plant families. Such a promoter would allow the expression of genes only when the plant is cold-stressed, thus alleviating the detrimental effects of constitutive gene expression (Ouellet 2002).
There is one major drawback of the current molecular frost resistance research. Engineering of the freezing resistance trait in plants has, until now, focused primarily on the moderately frost-resistant species, A. thaliana, and some cereals. Future research should reconsider the whole diversity and ultimate potential of low-temperature and frost-resistance mechanisms in plants. Nevertheless, studies on the molecular frost resistance of A. thaliana have yielded very valuable insights into plant frost resistance (Thomashow 1999) and there appear to be some very promising future prospects.
Definition and occurrence
Temperature rules the lives of ectothermic (cold-blooded) animals in multiple ways, not just the effects of temperature change on biochemistry (e.g., metabolic reaction rates, membrane fluidity, protein conformation) but also the effects of seasonal cold temperatures on factors such as changes in food availability, ability to successfully reproduce, and the need for protective insulation or adaptation when ambient temperatures are below 0°C and put animals at risk of freezing. Many ectotherms live in environments where temperature is constantly near 0°C (e.g., deep oceans, polar seas), and show evolutionary adjustments that optimize biochemistry for cold-temperature function (Johnston 2003; Somero 2004). Others must deal with seasonal cold in the winter, and in this section, we focus on animal survival at temperatures below the FP of body fluids (about −0.5°C for most terrestrial and freshwater animals and −1.9°C for marine invertebrates). Freezing is lethal for most organisms, and yet, winter temperatures on land can fall to −30°C in temperate regions and to −70°C in the Arctic or Antarctic. Many species avoid subzero exposure by spending the winter in thermally buffered sites under water or deep underground, but others need an effective mechanism of cold hardiness. Two basic strategies exist: freeze avoidance and freeze tolerance (Block 2003; Sinclair et al. 2003; Storey and Storey 2004a). Both have arisen multiple times in phylogeny.
Freeze-avoiding animals have optimized strategies for supercooling—the ability to maintain body fluids in a liquid state at temperatures below their equilibrium FP. Freeze avoidance occurs widely among terrestrial invertebrates (particularly arthropods) and some reptiles, and allows marine teleost fish (plasma FP about −0.5°C) to keep from freezing when seawater chills to −1.9°C in winter (Duman 2001; Fletcher et al. 2001; Davies et al. 2002; Block 2003; Storey and Storey 2004a). Freeze-tolerant animals endure ice formation in extracellular fluid spaces (often, 65–70% of total body water freezes out) but defend the liquid state of cytoplasm. However, a few cases of survivable intracellular freezing have been reported, the best substantiated being the Antarctic nematode, Panagrolaimus davidi (Wharton 2003), but this is not common. Freeze tolerance is used by many insect species, various intertidal marine invertebrates (e.g., barnacles, snails, bivalves), and selected terrestrially hibernating amphibians and reptiles (Costanzo et al. 1995; Duman 2001; Block 2003; Storey and Storey 1996, 2004a; Storey 2006). Among vertebrates, the North American wood frog, Rana sylvatica, is by far the best-studied (Storey and Storey 2004b).
Freeze-avoiding animals use one or both of two main mechanisms to enhance supercooling: (a) production of AFPs that inhibit the growth of embryonic ice crystals (Duman 2001; Duman et al. 2004; Davies et al. 2002) and (b) accumulation of high levels of low-molecular-weight cryoprotectants (typically polyhydric alcohols or sugars) that provide colligative suppression of FP and supercooling point (SCP) (Storey and Storey 1996). Freeze-tolerant animals take a different strategy and actually use INAs [either nonspecific nucleators or special ice-nucleating proteins (INPs)] to manage the freezing of extracellular body fluids (Duman 2001; Zachariassen and Kristiansen 2000; Storey and Storey 2004a). Other ice-active proteins are employed as recrystallization inhibitors (RIs), providing long-term stability of crystal size and shape in organisms that could be frozen for weeks or months (Wharton et al. 2005). Low-molecular-weight cryoprotectants are also made by freeze-tolerant animals, in this case to protect against intracellular freezing and provide colligative resistance to prevent cell volume from dropping below a critical minimum during water outflow into extracellular ice masses (Fuller and Paynter 2004). A critical minimum cell volume must be retained, because otherwise, the compression stress on membranes becomes too great and the bilayer structure breaks down. Other protectants (e.g., trehalose, proline) are specifically accumulated to stabilize bilayer structure by intercalating within the phospholipids (Anchordoguy et al. 1987). Freezing of extracellular fluids also halts blood flow (causing ischemia and anoxia in tissues) and muscle activity (e.g., heartbeat, breathing, and skeletal muscle movement). Hence, freeze-tolerant animals also need well-developed anoxia tolerance and a strategy for reactivating vital processes after thawing.
AFPs are believed to act by adsorption onto the surface of a growing crystal, disrupting the growth plane, and lowering the temperature at which further growth can occur (Duman 2001; Davies et al. 2002). However, some new ideas on AFP action were put forward recently (Kristiansen and Zachariassen 2005). Animal AFP structure is diverse, with five classes in marine fish and many types in insects (Graether and Sykes 2004; Davies et al. 2002). The variety reflects their affinity for different planes on the ice crystal but all are unified in the placement of critical amino acid or carbohydrate residues into alignments that match up with the spacing of water molecules in the ice lattice. For example, two nonhomologous insect sequences both fold into beta-helices to present an array of threonine residues and bound water molecules to the ice surface (Graether and Sykes 2004). Tissue-specific forms of AFPs occur in both fish and insects (Fletcher et al. 2001; Duman et al. 2002). In fish, the secreted form(s) found in plasma and gut are made by liver, whereas skin and gills that have direct contact with environmental ice make AFPs that stay within their tissues.
Recent studies of AFPs have provided key lessons in protein evolution (Fletcher et al. 2001; Davies et al. 2002). Fish AFPs are recent inventions because sea level glaciation dates back only ∼2 million years ago in the Arctic. There is no common ancestral protein and, instead, AFPs arose in each fish group when rapid selective pressure was placed on a preexisting protein to bend it to antifreeze function. For example, type II AFPs are homologues of the carbohydrate-recognition domain of calcium-dependent lectins, whereas the antifreeze glycopeptides (AFGPs) of Antarctic nototheniid fish were derived by duplication and amplification of a small segment of the trypsinogen gene. However, AFGPs in northern cod, with identical amino acid sequences to nototheniid AFGPs, have a totally different gene structure, indicating that they arose from a very different progenitor gene.
A few insect INPs have been characterized and, like AFPs, have a diverse structure, but all act by providing a surface that orients water molecules into the crystal lattice (Duman 2001). A high content of hydrophilic amino acids (20% glutamate or glutamine) is key to the function of hornet INP, whereas a high phosphatidylinositol content is needed for nucleating action by cranefly INP. The phosphatidylinositol component links INPs (molecular mass ∼800 kD) into the aggregates of at least 6,000 kD that are needed to trigger nucleation.
A variety of polyhydric alcohols and sugars are used as cryoprotectants by cold-hardy animals, but glycerol is the most common. This is due to several favorable factors (Storey 1997), such as the ease of synthesis from glycogen, extremely high solubility, and rapid movement of glycerol across cell membranes including high rates of transport through modified aquaporin channels (Stroud et al. 2003). Midwinter glycerol levels in freeze-avoiding insects can rise over 2 M and constitute ∼20% of total body mass (Storey 1997). The marine smelt has ∼500 mM glycerol in plasma to help lower SCP below −1.9°C, but constant synthesis is needed to replace glycerol lost by diffusion into seawater (Lewis et al. 2004). Freeze-tolerant species have lower cryoprotectant concentrations overall, but levels inside cells soar when water freezes out in extracellular ice masses. Dual cryoprotectant systems, e.g., glycerol and sorbitol, are quite common in freeze-tolerant insects, each polyol accumulated with different seasonal patterns and trigger temperatures (Storey 1997). Sorbitol may have an added role in helping to maintain winter diapause in insects (Yaginuma and Yamashita 1979). The accumulation and regulation of polyols has been extensively studied in insects. Major regulatory control comes from low temperature activation of glycogen phosphorylase and carbon flow through the pentose phosphate cycle is key to the output of both sugars and NADPH for polyol synthesis (Storey 1997; Storey and Storey 2004a). In freeze-tolerant frogs, the cryoprotectant is glucose. Freezing triggers a rapid activation of liver glycogen breakdown so that glucose rises rapidly to 150–300 mM in core organs, overriding homeostatic controls that typically hold glucose at ∼5 mM in vertebrates (Storey and Storey 2004b).
Anoxia tolerance, antioxidant defense, and metabolic rate depression
All freeze-tolerant animals show well-developed anoxia tolerance to maintain viability when plasma freezing interrupts the delivery of oxygen and substrates. Cellular ATP generation while frozen depends on glycolysis with lactate and alanine made as end products (Storey and Storey 2004a). Specific mechanisms of metabolic rate depression are also used to lower energy demand to a level that can be maintained over the whole winter by endogenous fuel reserves. Many insects spend most of the winter in diapause, and freeze-tolerant insects and frogs show phosphorylation-mediated suppression of the activities of key enzymes in ATP-expensive cell functions such as protein synthesis and transmembrane ion pumping (Storey and Storey 2004a).
Good antioxidant defenses also aid freezing survival. Direct evaluation of ROS generation using a fluorescence method found that ROS levels rose in yeast cells during freezing, with highest levels in cells lacking superoxide dismutase (Du and Takagi 2005). Oxidative damage products did not accumulate during freeze/thaw in wood frog organs, but these frogs have much higher activities of antioxidant enzymes (AOEs) than do freeze-intolerant frogs, showing good preparation for dealing with ROS insults (Joanisse and Storey 1996). Damage by ROS is well known in cryopreserved systems, and the addition of antioxidants improves viability during cryostorage (Benson and Bremner 2004). Freeze-responsive gene/enzyme expression can also increase antioxidant defenses when they are needed. Both wood frogs and turtle hatchlings show freeze-stimulated up-regulation of selected AOE genes and enzyme activities (Joanisse and Storey 1996; Storey 2004, 2006). Voituron et al. (2005) also reported cold-induced increases in AOE activities during supercooling in lizards.
Cold- and freeze-induced gene expression
For many years, studies of animal survival below 0°C focused mainly on several highly “visible” components of the phenotype, such as AFPs and cryoprotectants. However, cold hardiness involves many other molecular adaptations, and recent advances in gene screening technology are providing the way to explore these. Two techniques are particularly important: (a) the construction and screening of cDNA libraries and (b) heterologous screening of DNA microarrays (Storey 2004, 2006; Storey and McMullen 2004). Not unexpectedly, these studies are showing that multiple different cell functions are involved in conferring cold hardiness, many of them previously unsuspected.
DNA microarray screening is another excellent method for identifying genes involved in cold hardiness. Multiple benefits of array screening include: (1) simultaneous assessment of hundreds of genes, most of them identified; (2) detection of transcripts that are present in low copy number; (3) relative ease of sample preparation and data quantification; and (4) ability to assess overall responses by groups of related genes (e.g., families, pathways, or cascades) (Eddy and Storey 2002). To date, only heterologous screening can be done for freeze-tolerant species. We have successfully used human arrays to screen for freeze-responsive genes in wood frogs and hatchling turtles (Storey 2004, 2006) and Drosophila melanogaster arrays to find cold-responsive genes in insects (Storey and McMullen 2004). Heterologous screening has potential drawbacks in that cross-hybridization is never 100% and both false-positive and false-negative responses could occur. However, its advantages for gene discovery are enormous; e.g., screening of 19,000 human gene DNA arrays with wood frog cDNA gave 60–80% cross-hybridization to reveal the status of thousands of genes. Screening of wood frog heart revealed multiple genes that were never before linked to freeze tolerance, including genes involved in hypoxia tolerance, adenosine receptor signaling (adenosine is a regulator of metabolic rate depression), natriuretic peptide control of fluid dynamics, protection against advanced glycation end products (AGEs), and antioxidant defense (Storey 2004). In hatchling turtles, freezing triggered the expression of iron-binding proteins, antioxidant defenses, and serine protease inhibitors (Storey 2006). Note that cross-species hybridization needs to be used with additional validation techniques (e.g., PCR or Western blotting to quantify species-specific mRNA transcript or protein levels, respectively) to confirm cold- or freeze-responsive gene up-regulation, but our experience is that the results from array screening are rarely wrong.
DNA array screening and cryopreservation
Cryopreservation has multiple applied uses, and multiple biotechnological applications are being developed from studies of cold-hardy organisms (Benson et al. 2004). Uses of cryopreservation in animal biology include the preservation of gametes for medical and veterinary use, cell/tissue banking for transplantation, and the preservation of stocks of genetically diverse material for endangered species management (Wildt 2000), selective breeding programs (Tervit et al. 2005), and laboratory experimentation (Glenister et al. 1990; Buchholz et al. 2004). Traditionally, advances in cryopreservation have come from empirical studies that systematically alter/optimize a range of parameters (e.g., rates/stages of freeze/thaw, amounts/types of cryoprotectants) and focus mainly on preserving the physical integrity of cells. However, a new approach to cryopreservation is now possible. DNA array screening can be used to document the actual gene-based responses of cells to freezing to identify genes that are cold- or freeze-induced in tolerant species (potentially enhancing hardiness) or in nonhardy species (potentially maladaptive). For example, cryopreservation stress in yeast induced genes encoding HSPs, oxidative stress scavengers, and enzymes of glucose metabolism (Odani et al. 2003), whereas screening of cryopreserved ovarian tissue showed expression of HSPs, DNA-damage-inducible protein 45, and apoptosis genes (Liu et al. 2003). As mentioned above, array screening for freeze-responsive genes in wood frogs found multiple genes whose protein products have never before been associated with freezing survival (Storey 2004). Furthermore, wood frogs produce at least three novel freeze-responsive proteins (FR10, FR47, and Li16) that could have applied uses in cryopreservation (Storey 2004); e.g., when Li16 was overexpressed in an insect cell line, freezing survival was vastly improved (Kotani and Storey, unpublished data). All of these approaches offer new ideas of metabolic targets that need attention for successful cryopreservation.
Improved cold hardiness through transgenics
There is strong interest in the use of transgenics to transfer selected genes from hardy to nonhardy species to improve cold hardiness or freeze tolerance. To date, a main focus of such research with animals has been in marine aquaculture (e.g., salmon and oysters) because the inshore location of aquaculture facilities can expose nonhardy animals to sea ice and −1.9°C water temperatures during winter. Microarray screening of coldwater species might highlight gene targets that could improve cold hardiness when used in a transgenic approach. To date, studies have focused on transgenic expression of AFPs with limited success. For example, a transgenic line of Atlantic salmon was produced that harbored the winter flounder AFP gene. The F3 generation showed gene expression in liver, the presence of AFP precursor in serum, and a hexagonal pattern of plasma ice crystal growth indicative of AFP action (Hew et al. 1999; Zbikowska 2003). Lines of transgenic D. melanogaster also express AFP genes from winter flounder, Atlantic wolf fish, or spruce budworm (Peters et al. 1993; Duncker et al. 1999; Tyshenko and Walker 2004).
Cryopreservation success can also be improved by enhancing cell capacity for rapid transmembrane transport of water or cryoprotectants. Microinjection of aquaporin cRNA into zebrafish embryos did not improve freezing survival (Hagedorn et al. 2004), but similar treatment of immature mouse oocytes resulted in improved water and glycerol transport and higher viability after freezing (Edashige et al. 2003). Studies with Saccharomyces cerevisiae found that both homologous and heterologous overexpression of aquaporin genes enhanced survival during fast freezing of yeast in small batches (Tanghe et al. 2005). However, freezing survival was not improved in large dough batches where cooling rates are much slower (Tanghe et al. 2004); hence, transgenic enhancements that work in a lab may not always be applicable in their intended industrial use. Another approach to maintaining yeast viability in frozen doughs was more effective (Izawa et al. 2004). A deletion mutant of S. cerevisiae lacking the FPS1 gene (encoding a glycerol channel) accumulated but did not export glycerol, and the resulting high intracellular glycerol levels enhanced viability after freezing.
Three classes of ice-active proteins are known: AFPs, INPs, and RIs. Bacterial INPs are already widely used in industry and multiple applied uses can be envisioned for all three types. For example, the wood frog INP (Storey et al. 1992) might be effective for managing ice formation in the microvasculature of organs during medical cryopreservation, and RIs could help to minimize ice damage during long-term storage of cryopreserved materials. Insect INPs might be overexpressed in bacterial systems to produce a protein that could be sprayed on pest species to trigger nucleation and death; this might be effective in food storage facilities such as grain silos. Silencing RNA technology could be used on insect pests to knock out their natural production of AFPs or INPs. AFPs and RIs also have uses in the frozen food industry, including helping to maintain food integrity during long-term freezing and, for foods that are eaten frozen, preserving a smooth texture (Griffith and Ewart 1995).
Several disorders in human diabetes are directly caused by high glucose, including the production of AGEs and the pro-oxidant actions of glucose in generating ROS. Damage occurs with chronic exposure to glucose in the diabetic range (10–45 mM), and yet, wood frogs endure glucose at 200–300 mM during long-term freezing. Frogs must have mechanisms to inhibit or prevent nonenzymatic glycation damage to their proteins. Genes detected as freeze-responsive on DNA arrays included several involved in glucose management, including the receptor for AGEs (Storey 2004). Studies of the antiglycation mechanisms in frogs will provide new insights into the natural mechanisms of AGE control and suggest new strategies for high glucose management in diabetes.
Microorganisms, plants, and animals have evolved a number of common strategies to thrive at low temperatures and to survive or even maintain metabolic activity (bacteria) at subzero temperatures. New progress has shown that multiple adjustments to wide areas of cellular metabolism are required for cell survival and activity in the cold.
Strategies to compensate for the negative effects of low temperatures on biochemical reactions include the production of cold-active enzymes that display a high catalytic efficiency associated with low thermal stability, which originates from an increased flexibility of some or all of the protein structure. This is especially known for enzymes from microorganisms and animals. To preserve membrane function and to maintain membrane fluidity at low temperatures, organisms have adapted their membrane lipid composition. Decreases in temperature result in increased fatty acid unsaturation and/or methyl branching, a shortening of acyl chain length, and may include a reduction of the protein/lipid ratio in certain membranes of plants.
The synthesis of inducible CSPs (providing chaperone actions to stabilize other proteins) and CAPs (addressing selected specific cellular needs) ensures a rapid reaction to sudden temperature decreases. Microorganisms, plants, and animals produce various compounds to protect themselves against freezing. High levels of compatible solutes (osmoprotectants, e.g., sugars, polyols) are accumulated to protect cells against the toxic effects of increased ions and other solutes arising due to freeze dehydration and help to minimize cell volume reduction in animals and microorganisms. AFPs inhibit, slow down, or control the growth of ice crystals. INAs (proteins or nonspecific nucleators) manage ice crystallization in extracellular compartments; in freeze-tolerant plants and animals they help to control ice crystal formation to a tolerable level, in bacteria they can induce frost damage on their hosts (plants) whereby bacteria obtain access to nutrients from the plant.
Any intracellular ice formation is considered lethal. Some plants and animals completely avoid freezing at subzero temperatures by deep supercooling, whereas other species accept ice formation in extracellular spaces but defend the liquid state of the cytoplasm. Both strategies typically utilize high concentrations of colligative protectants to prevent freezing in liquid spaces but use different proteins to manage ice in extracellular spaces—AFPs to prevent ice formation in freeze-avoiding tissues or species and INPs to manage ice formation in freeze-tolerant species.
Antioxidant defense also aids survival at low temperatures. Strategies for the detoxification of ROS that cause membrane damage include the production of high amounts of AOEs (catalase, superoxide dismutases, and dioxygen-consuming lipid desaturases) in microorganisms and animals, special detoxifying substances in plants, or the absence of pathways that produce ROS (bacteria).
The strategies used by various organisms to cope with the cold provide fascinating insights into the adaptability of life on Earth. Continuing research into the cellular and molecular mechanisms of cold adaptation and freezing survival offer valuable insights into the molecular mechanisms of structure–function relationships. Furthermore, these studies provide unique opportunities to identify new key applications for biotechnology advancement, such as improved freeze tolerance in plants and cold hardiness in animals, organ cryopreservation in medicine, and the use of cold-active enzymes for energy-saving processes in the detergent and food industries, for the production of fine chemicals, and in the biological decontamination of polluted sites.