Naturwissenschaften (2007) 94:77–99
DOI 10.1007/s00114-006-0162-6
REVIEW
Cold-loving microbes, plants, and animals—fundamental
and applied aspects
R. Margesin & G. Neuner & K. B. Storey
Received: 30 November 2005 / Revised: 11 August 2006 / Accepted: 22 August 2006 / Published online: 13 October 2006
# Springer-Verlag 2006
Abstract 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.
Keyword Cryoprotectants . Cold adaptation . Freeze
tolerance . Supercooling . Proteins . Membranes .
Antioxidant defenses . Gene expression
R. Margesin (*)
Institute of Microbiology, Leopold Franzens University,
Technikerstrasse 25,
6020 Innsbruck, Austria
e-mail: [email protected]
G. Neuner
Institute of Botany, Leopold Franzens University,
Sternwartestrasse 15,
6020 Innsbruck, Austria
K. B. Storey
Department of Biology, Carleton University,
1125 Colonel By Drive,
Ottawa, Ontario K1S 5B6, Canada
Introduction
The majority (>80%) of the Earth’s biosphere is cold (Fig. 1)
and exposed to temperatures below 5°C throughout the year.
Vast areas of the soil ecosystem are permanently frozen or
are unfrozen for only a few weeks in summer, and 90% of
the ocean volume is below 5°C. Cold-adapted organisms
have successfully colonized these cold environments because they have evolved special mechanisms to overcome
the life-endangering influence of low temperature. Unlike
thermophiles, cold-adapted organisms include not only
prokaryotes, but also eukaryotes, plants and ectothermic
animals. However, the current knowledge is rarely assembled and compared (Margesin and Schinner 1999a; Bowles
et al. 2002).
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
78
Naturwissenschaften (2007) 94:77–99
Fig. 1 Occurrence of low temperatures and frost on Earth. A annual
minimum temperatures above +5°C, B annual minimum temperature
above 0°C, C episodic frosts with temperatures down to −10°C, D
regions with cold winters and mean annual minimum temperatures
between −10 and −40°C (white lines −30°C minimum isotherm), E
mean annual minimum temperatures below −40°C, F polar ice. The
above zones correspond to the areas of plant species with different
types of frost resistance. Zone A chilling-sensitive plants of the
equatorial tropics, zone B extremely freezing-sensitive plants, zone C
plants protected by effective supercooling and depression of the FP,
zone D plants with limited freezing tolerance and trees with wood
capable of deep supercooling, zone E completely freezing-tolerant
plants (from Larcher 2003)
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.
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
Cold-adapted microorganisms
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
Naturwissenschaften (2007) 94:77–99
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).
Adaptive strategies
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)
79
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 dioxygenconsuming 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 stressinducing 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
80
100
Relativeactivity (%)
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).
Naturwissenschaften (2007) 94:77–99
80
60
40
20
0
0
10
20
30
40
50
60
70
80
Membrane lipids
Enzymes
Cold-adapted organisms produce cold-active enzymes with
high catalytic efficiency (Kcat/Km) at low and moderate
temperatures (0–30°C) at which homologous enzymes
produced by microorganisms from other thermal classes
are poorly active or not active at all. In addition, these
enzymes are generally thermolabile; their apparent maximal
Residualactivity (%)
100
The membrane is both the interface and the barrier between
the internal and external environment of the cell. Coldadapted 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).
80
60
40
20
0
0
20
40
60
Temperature (˚C)
Fig. 2 Effect of temperature on activity (top) and stability (bottom;
residual activity after 15 min of incubation, determined at 25°C and
pH 9) of cold-active pectate lyase produced by the alpine Mrakia
frigida strain A15 (circles) and its mesophilic counterpart produced by
Bacillus subtilis (squares). Modified from Margesin et al. (2005)
activity is shifted towards low temperatures and denaturation occurs at higher temperatures (Fig. 2). Comparisons
between crystallographic structures or molecular models of
enzymes with different temperature optima indicate a higher
flexibility of cold-active enzymes, whereas thermostable
proteins have a rigid structure. According to the currently
accepted hypothesis, cold-active enzymes must increase the
flexibility of some or all parts of the protein to compensate for
the lower thermal energy provided by the low temperature
habitat (Hochachka and Somero 1984; Somero 2004). Genome
analysis confirmed this theory (Methe et al. 2005). Flexibility
induces a decrease in the activation energy and thus provides
high catalytic efficiency at low temperature, but at the expense
of activity loss at higher temperatures. In return, this increased
flexibility is responsible for the generally low stability of the
protein structure of cold-active enzymes, due to an inverse
relationship between stability and activity (D’Amico et al.
2002; Marx et al. 2004). Enzymes from psychro-piezophiles
are a good example of a molecular compromise between two
conflicting regimes. These enzymes are active and stable at
high pressure (10–30 MPa), but the catalytic efficiency at low
Naturwissenschaften (2007) 94:77–99
temperatures is suboptimal. On one hand, efficient catalysis at
low temperatures requires enzyme flexibility, whereas, on the
other hand, enhanced rigidity is necessary at high pressure
(Glansdorff and Xu 2002).
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, argininemediated 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.
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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 INAproducing 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).
Biotechnological perspectives
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 coldshock 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).
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.
Enzyme market
The characteristic features of cold-active enzymes (high
catalytic efficiency at low and moderate temperatures;
thermolability) offer a number of advantages for biotechnology processes, such as the shortening of process times,
saving of energy costs, prevention of the loss of volatile
compounds, performance of reactions that involve thermosensitive compounds, and reduced risk of contamination.
However, the weak thermostability can also be a drawback
when enzyme stability is required for storability reasons.
Protein engineering is a successful method to improve
thermostability of cold-active enzymes without impairing
catalytic activity (Kristjansdottir and Gudmundsdottir
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Table 1 Applications of cold-active enzymes in biotechnology (Margesin and Schinner 1999b)
Application field
Advantage
Involved enzymes
Detergents
Washing at low temperature (energy-saving and
applicable to synthetic fibers); contact lens cleaning
Reduced incubation time for lactose hydrolysis in milk
and dairy products
Improved juice clarification, increased juice yield
Efficient and gentle removal of fish skin, meat
tenderization
Cold pasteurization, food preservation
Improved taste and aroma of fermentation products
(e.g., cheese, dry sausages, alcoholic beverages)
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)
Synthesis of acrylamide
Organic phase biocatalysis (increased solvent choice,
product yield, and biocatalysis stability)
Mild heat inactivation of enzymes without interference
with subsequent reactions
Selective enzyme inhibition
Efficient low-temperature ligation
Prevention of carry-over contamination in PCR
Rapid 5′ end-labeling of nucleic acids
Efficient protoplast formation
Debridement of necrotic tissue, digestion promotion,
chemonucleolytic agents
Improved quality after desizing, biopolishing, and
stone-washing of fabrics
Selective, sensitive, and rapid on-line monitoring
of low-temperature processes, quality control
In situ/on-site bioremediation of organic contaminants
Low-energy wastewater treatment
Protease (Kannase©, Polarzyme©), lipase (Lipolase©,
LipoPrime©), amylase (Stainzyme©), cellulase, oxygenase
β-Galactosidase (Gerday et al. 2001)
Food industry
Organic synthesis
Molecular biology
Pharmaceuticals
Textiles
Biosensors
Environment
Low-energy anaerobic wastewater treatment
Low-temperature biogas (methane) production
Low-temperature composting
2000; Russell 2000; Yokoigawa et al. 2003; Siddiqui
et al. 2004). There are a wide range of applications for
cold-active enzymes (Table 1), but only a few have been
commercialized. The major current application field is in
the detergent industry. Other application areas are the
pharmaceutical and food industries (reviewed by Ohgiya
et al. 1999; Cavicchioli et al. 2002).
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
Pectinase, cellulase
Protease, carbohydrase
Catalase, lysozyme, glucose oxidase
Enzymes involved in fermentation and ripening
Lipase, esterase, protease, etc.
Nitrile hydratase
Enzymes operating under low water conditions
Various enzymes
Protease
DNA ligase
Uracil DNA glycosilase
Alkaline phosphatase
Cellulase, xylanase, etc.
Multienzyme systems
Amylase, laccase, cellulase
Various enzymes, e.g., dehydrogenase
Mono- and dioxygenases, transferases, hydrolases, etc.
Enzymes involved in mineralization, nitrification, and
denitrification
Enzymes involved in anaerobic degradation
Enzymes involved in anaerobic degradation
Enzymes involved in litter degradation
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 coldadapted 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
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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).
Agriculture
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 costsaving 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).
83
Environmental biotechnology
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 lowenergy 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
84
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).
Cold-acclimated plants
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 frostsurvival 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),
Naturwissenschaften (2007) 94:77–99
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).
Adaptive strategies
Mechanisms of frost resistance in plants
Frost resistance can be achieved by two main mechanisms:
(1) avoidance of ice formation in tissues or (2) tolerance of
apoplastic, extracellular ice. It is important to note that an
individual plant may employ both mechanisms of frost
resistance but in different tissues (Sakai and Larcher 1987).
This can even occur within one single leaf: freezing injury
in maize leaves apparently resulted from a combination of
freezing-induced cellular dehydration of some tissues and
intracellular ice formation in epidermal and bundle-sheath
cells (Ashworth and Pearce 2002).
(1) 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.
(2) 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
Naturwissenschaften (2007) 94:77–99
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 (nonidealequilibrium; 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).
85
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).
Freezing injury
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
Factors influencing tolerance of freeze dehydration and
membrane stability and factors controlling growth of ice in
freezing plants are important, as is whether extracellular
freezing or supercooling occurs. Prominent physiological
and biochemical changes during cold acclimation are
outlined in Fig. 3. In the cold acclimated state, reduction
or cessation of growth and photosynthesis is often
observed, tissue water content is reduced, solutes accumu-
86
Naturwissenschaften (2007) 94:77–99
Fig. 3 Most prominent physiological and biochemical changes
often accompanying increased
frost resistance in plants (after
Xin and Browse 2000)
late (Ulmer 1937; Levitt 1980), cell wall modifications take
place (e.g., cereals: Hiilovaara-Teijo and Palva 1999), and
abscisic acid levels may transiently increase (Chen and
Gusta 1983). Other changes include the modification of
lipids and lipid/protein ratios in membranes, the expression
of cold-related (COR) proteins, an increase of osmolytes
and ROS detoxifying substances. Some of the changes
induced by cold acclimation will be addressed in more
detail below.
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).
Changes in growth and photosynthesis
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).
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
Membrane modifications
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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).
Protein modifications
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 wallmodifying 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
87
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.
Biotechnological perspectives
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).
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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 lowtemperature 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 follow-
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ing 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.
Cold-hardy animals
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
Naturwissenschaften (2007) 94:77–99
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 coldtemperature 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 beststudied (Storey and Storey 2004b).
Adaptive strategies
General principles
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
89
[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.
Ice-active proteins
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
90
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.
Cryoprotectants
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
Naturwissenschaften (2007) 94:77–99
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 coldinduced 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
Naturwissenschaften (2007) 94:77–99
multiple different cell functions are involved in conferring
cold hardiness, many of them previously unsuspected.
For example, screening of a cDNA library made from
cold-exposed insect larvae highlighted the up-regulation
of EsMlp, encoding a muscle LIM protein that may
function in cold temperature restructuring of muscle
(Bilgen et al. 2001). Screening of cDNA libraries made
from the liver or brain of wood frogs revealed freezeresponsive genes including fibrinogen, mitochondrial
membrane transporters (ADP–ATP translocase, inorganic
phosphate carrier), NADH-ubiquinone oxidoreductase
subunit 4 and ribosomal elongation factor 1 gamma subunit
(reviewed in Storey 2004). Each suggests a different
metabolic function that needs to be addressed for freezing
survival. For example, fibrinogen up-regulation suggests
that enhanced blood clotting capacity is important to deal
with any physical damage to the microvasculature caused
by ice expansion; such damage is a primary injury
encountered in medical organ cryopreservation. Screening
of cDNA libraries also has the unique ability to reveal
novel genes that may be key to the freeze-tolerance
phenotype. To date, three novel genes are known in wood
frog liver that have no counterparts in gene sequence data
banks (Storey 2004). Figure 4 shows the methodological
approaches that can be applied to find and characterize
novel genes, illustrating the process for li16 from wood
frog liver (Storey and Storey 2004a). The functions of these
novel genes are not yet known, but each shows different
organ and time course patterns of expression, responds to
different signal transduction pathways, and is differently
affected by the anoxia or dehydration stresses that mimic
elements of freezing.
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 freezeresponsive 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
91
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 freezeresponsive gene up-regulation, but our experience is that
the results from array screening are rarely wrong.
Biotechnological perspectives
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 freezeinduced 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
92
Naturwissenschaften (2007) 94:77–99
Fig. 4 Methods for discovery and analysis of freeze-induced genes
highlighting the novel gene, li16, from the liver of the freeze-tolerant
wood frog. A cDNA library made from the liver of frozen frogs was
screened with cDNA probes made from control vs frozen frogs,
revealing a freeze-responsive clone, liver 16. The full nucleotide
sequence was retrieved using 5′ rapid amplification of cDNA ends.
Northern and Western blotting revealed the pattern of freezeresponsive li16 mRNA and Li16 protein expression and the nuclear
run-off technique confirmed that elevated levels of li16 mRNA arose
due to enhanced gene transcription. From Storey and Storey (2004a)
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
Naturwissenschaften (2007) 94:77–99
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.
Ice-active proteins
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
93
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).
Diabetes research
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 freezeresponsive 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.
Conclusion
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 (osmo-
94
protectants, 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 dioxygenconsuming 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.
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