"Cold Acclimation and Freezing Tolerance in Plants". In

Cold Acclimation andFreezing Tolerance inPlantsFrancois Ouellet, Universite du Quebec a Montreal, Montreal, Quebec, Canada

Cold acclimation is the process leading to the development of freezing tolerance in

plants. It is a complex multigenic process that requires a programmed and integrated

genetic capacity to activate the appropriate mechanisms needed to withstand harsh

winter conditions.

Introduction

Temperature and water availability are among the mostimportant environmental factors affecting plant growthand development. Because most crops of economical im-portance are sensitive to temperatures below 108C, signifi-cant losses can result from sudden frosts in the fall and fromunusual freezing temperatures in the winter. Tolerant an-nual and biannual plants possess the genetic make-up re-quired to acquire freezing tolerance (FT) through a processcalled cold acclimation. This review summarizes the generalconcepts of FT and how the plants adjust their metabolismwhen exposed to a low temperature (LT) stimulus.

Cold Acclimation and FreezingTolerance

The cold acclimation process allows hardy plants to triggerthe efficient mechanisms needed for the acquisition of FT,which determines their capacity to survive winter. Duringthe period of exposure to LT, numerous biochemical,physiological andmetabolic functions are altered in plants.These changes are regulated by LT mostly at the gene ex-pression level. In natural conditions, cold acclimation oc-curs in the fall, when temperatures are still above thefreezing point. The maximal FT is maintained during win-ter, when temperatures are below the freezing point andlost in the spring when temperatures go back above thefreezing point. FT is a complex multigenic trait that re-quires an integrated, genetically programmed capacity tooverwinter. This means that some species, and even somevarieties within each species, possess a greater acclimationcapacity than others. Herbaceous species such as wintercereals achieve an LT50 (lethal temperature 50; tempera-ture that kills 50% of the individuals) of about 2208C to2258C when fully acclimated. Trees use a variety of mech-anisms to withstand much lower freezing temperatures. Incontrast, some tropical species will suffer irreversible dam-ages at temperatures just above the freezing point.

As temperatures fall, ice forms in the apoplastic spaceand cytoplasmic water is drawn from the cells to the

growing mass of extracellular ice. To avoid the loss ofwater, cells of frost-tolerant organisms accumulate lowmolecular weight solutes such as proline, sugars andglycinebetaine. This occurs because LT induces theexpression of genes encoding enzymes required for thebiosynthesis of these compounds, such as d-1-pyrroline-5-carboxylate synthetase, phosphoethanolamine methyl-transferase, betainealdehyde dehydrogenase, sucrosesynthase, galactinol synthase and others. Interestingly,some of the corresponding genes are part of the C-boxbinding factor (CBF) regulon (see the section on Tran-scription factors). Together, the compatible solutescontribute to the increase in osmotic potential and con-comitant decrease of the cytoplasmic freezing point. Thelatter property prevents the formation of deleterious icecrystals that would cause irreversible structural damageand lead to cellular death. The solutes also stabilize thestructure of cell membranes and proteins, thus preservingtheir functional capacities following a freeze–thaw cycle.The improvement of cold tolerance by traditional breed-

ing depends on the availability of species/varieties showinghigh levels of cold tolerance.Although traditional breedingmethods have generated a remarkable improvement in thecold tolerance of certain plants, extensive breeding hascaused the depletion of FT-associated genes available inthe genetic pools of each species. Genetic manipulationoffers the possibility of crossing sexual barriers and trans-ferring tolerance genes identified in cold-tolerant species tocold-sensitive species of any family. To establish a goodimprovement strategy, it is essential to understand themechanisms involved in the cold acclimation process anddevelopment of FT. This involves studying the differentaspects associated with the adaptative mechanisms of tol-erant species. In addition, because freezing stress sharessome common characteristics with other stresses such asdrought and salinity, it is important to distinguish theevents that are specific toFT from those related to a generalstress response. The physiological and biochemical mod-ifications resulting from LT exposure have been describedextensively (Ensminger et al., 2006; Thomashow, 1999).Less understood is how the LT stimulus is perceived by the

Article Contents

Advanced article

. Introduction

. Cold Acclimation and Freezing Tolerance

. Metabolic and Structural Adjustments

. Modifications in Gene Expression

. Influence of Trans Regulators on LT-responsive Gene

Expression

. Cold Perception and Signal Transduction

. Industrial and Medical Applications of Freezing-

associated Proteins

doi: 10.1002/9780470015902.a0020093

ENCYCLOPEDIA OF LIFE SCIENCES & 2007, John Wiley & Sons, Ltd. www.els.net 1

cells, how the signal is transduced and what specific ele-ments are required for proper expression of cold-induciblegenes. Over the last 15 years, a considerable and increasinginterest in the field has led to major breakthroughs in ourunderstanding of LT perception and induction of LT-responsive gene expression. See also: Plant Stress Physiol-ogy; Plant Temperature Stress

Metabolic and Structural Adjustments

Plasma membrane

The plasma membrane is considered to be the primary siteof freezing injury. The lipid content has been extensivelystudied. Both the relative abundance and saturation levelof membrane lipids change upon exposure to LT. The gen-eral desaturation of these lipids ensures that the mem-branes will remain as fluid as possible when temperaturesdrop. Plants that have a better ability to cold acclimate willalso respond better at the molecular level to express andactivate the enzymes required for desaturation or hydrol-ysis of fatty acids. If this adjustment does not take place astemperature is decreased, significant structural damageoccurs when cell membranes undergo phase transitionfrom a liquid crystalline to a gel state. This is importantbecause the physical structure (gel or fluid phase) of lipiddomains in membranes influences cellular signalling andother biological functions. If the temperature at which themembrane lipid phase transition occurs could be lowered,or if the membrane phase transition could be eliminatedcompletely, membranes would remain fluid at low temper-atures andmembrane damagemay be reduced. In additionto lipids, proteins are likely to also play a role in the stab-ilization of membranes. Technologies such as proteomicsare the newest tools used to understand the changes thatoccur in the plasma membrane during acclimation andfreezing (Uemura et al., 2006). Themain finding from thesestudies is that FT-associated proteins such as dehydrinsand lipocalins accumulate at the plasma membrane uponLT exposure. Although their precise mode of action is un-known, evidence suggests that these proteins protect mol-ecules and structures against freeze-induced damage.

Photosynthesis and sugar metabolism

In higher plants subjected to changing environmental con-ditions such as LT, photosynthetic tissues have to adjusttheir photosynthetic capacity (Ensminger et al., 2006). Thisinvolves adjusting the chloroplastic antennas, which arecomposed of pigments (mostly carotenoids) and proteins.Since there is a delicate equilibriumbetween the energy thatis harvested and used, it has been suggested that the redoxstatus of photosynthesis could be an important signallingmechanism especially during cold stress. Exposure ofplants to LT may induce high excitation pressure and cre-ate an energy imbalance. However, photosynthetic

organisms have evolved a number of mechanisms to im-prove such conditions. Chronic exposure to high excitationpressure may lead to photoinhibition, which is defined asthe light-dependent decrease in photosynthetic rate thatoccurs when the photon flux is in excess. This excess lightcan be eliminated by thermal dissipation through a processknown as nonphotochemical quenching, and plants thatcan cold acclimate have more efficient mechanisms to doso. If the absorbed energy exceeds both the photochemicaland nonphotochemical quenching capacities, the result isirreversible photoinhibition or photodamage. Damages tothe chloroplast structure disrupt its photosynthetic capac-ity. It has been suggested that photosynthesis could likelyfunction as a sensor of this imbalance through the redoxstate of photosynthetic electron-transport components, re-sulting in the regulation of photochemical and metabolicprocesses in the chloroplast. Since light is essential for thedevelopment of maximal FT, it appears likely that photo-synthesis, and perhaps other processes taking place in thechloroplast, are crucial to cold acclimation and FT. Anefficient communication channel must therefore exist be-tween the nucleus and chloroplast to ensure the propermetabolic adjustment needed for the development of FT.This however remains to be demonstrated experimentally.In addition to the adjustment of the photosynthetic ap-

paratus, it was suggested that sugar-signalling pathwayscould regulate plant acclimation to LT but this remains tobe determined. What is known though is that overwinter-ing plants accumulate higher levels of soluble carbohy-drates such as sucrose, glucose, fructose, stachyose,raffinose, sorbitol and mannitol. Some of these sugars in-crease the intracellular osmolytes concentration and thuslower the freezing point of the cytoplasm. In the chlorop-last, starch is hydrolysed by b-amylase to produce maltose(Kaplan et al., 2006). The transcript levels and activity ofthe different amylase isoforms are regulated by environ-mental stimuli such as cold, heat and drought stress. Theactivity of the chloroplastic isoform in Arabidopsis isneeded for the protection of photosystem II efficiency fol-lowing freezing stress, presumably because it produces thecryoprotective compound maltose, a precursor of solublesugar metabolism.

Crosstolerance with other stresses

Exposure to LT leads to a loss of water and the productionof reactive oxygen species (ROS). In fact, abiotic stressesthat cause cellular dehydration or ROS accumulation suchas LT, drought and salinity induce common subsets ofgenes, indicating that common responses are required todevelop tolerance to these stresses. It is believed that ROSwould have a role in plant sensing and signalling of thetemperature stimulus (Suzuki and Mittler, 2006). Main-taining or controlling the levels ofROS is critical to preventdetrimental consequences. The superoxide produced byNADPH oxidases upon LT exposure activates stress-response pathways and induces defence mechanisms.

Cold Acclimation and Freezing Tolerance in Plants

ENCYCLOPEDIA OF LIFE SCIENCES & 2007, John Wiley & Sons, Ltd. www.els.net2

This occurs via the activation of an oxidative stress regulonin plants, and it is thought that this regulon overlaps withthe different networks controlling temperature stress accli-mation and tolerance. Antioxidants such as glutathione,ascorbate and Vitamin E, and enzymes involved in detoxi-fication ofROS such as catalase and superoxide dismutase,have been suggested to protect plants against freeze–thawdamage.

Modifications in Gene Expression

Low temperature leads to important modifications in geneexpression (Nakashima and Yamaguchi-Shinozaki, 2006).It is estimated that the expression of nearly 10%of all genesis affected, one way or another, by exposure to cold tem-peratures. Thismodification of the transcriptome results inimportant genetic, metabolic and structural adjustments atthe cellular level. A plethora of genes are expressed in re-sponse to LT and the molecular responses associated withthe induction of their expression are complex. A large-scaleEST (expressed sequence tags) analysis revealed the stressresponse is partly conserved between wheat and Arabido-psis, two phylogenetically distant plant species (Houdeet al., 2006). More than 44% of the 2637 putative wheatstress-regulated genes have a homologue that is regulatedby stress in Arabidopsis. A comprehensive discussion of allthe proteins associated with cold response goes beyond thescope of this review, therefore I will only review some of themost studied LT-associated proteins.

Structural proteins

Dehydrins are the most conspicuous subset of soluble pro-teins induced by dehydrative stresses such as droughtstress, LT and salinity. They have a broad size range, showno significant similarity in amino acid sequence to proteinsof known function and they accumulate to levels in excessof 1% of total soluble proteins. Dehydrins can be classifiedby their content in different amino acid segments namedK,S and Y, which are usually repeated several times.Dehydrins are also peculiar by their high hydrophilicityand boiling solubility, which facilitates their purification.Their widespread cellular distribution and propensity toadopt an a-helical structure in the presence of SDS is con-sistentwith a role in the protection of cellular structures andmolecules against freezing-induced damages. It was shownthat dehydrins canprotect enzymes from freezingdamage invitro. For example, the wheatWCS120 protein at 0.2mM isas efficient as 250mM sucrose in the cryoprotection of lac-tate dehydrogenase (over a million-fold more efficient on amolar basis). First, it was proposed that dehydrins, actingsynergistically with compatible solutes, may replace waterfor the ‘solvation’ of membranes and proteins. This prop-erty would alleviate the dehydration-induced membranedestabilization and protein coagulation. Second, these pro-teins may reduce the incidence of events leading to the

formation of nonbilayer structures between membranes.Finally, dehydrins may counteract the irreversible damagesof increasing concentrations of various ions in the cyto-plasm during dehydration. High ionic concentrations de-crease interbilayer repulsion due to charge screening andlead to an interaction between bilayers and the induction ofnonbilayer structures. Dehydrins have been found in mostplant species exposed to cold temperatures studied so far,whether they show a good FT or not. Overexpression ofdehydrins in Arabidopsis and strawberry only resulted in amodest increase in FT. This suggests that even though theseproteins always accumulate upon LT exposure, they maynot be the major determinants of tolerance.Another LT-responsive gene family encodes antifreeze

proteins (AFPs) (Griffith and Yaish, 2004). These proteinshave a high affinity for ice and possess two typical prop-erties: ice recrystallization inhibition and thermal hyster-esis (the difference between the freezing point and themelting point). They accumulate at high concentrations inthe apoplast, the extracellular space between the plasmamembrane and cell wall, where they bind ice crystals. Thecoating of the crystals restricts their growth, thereby en-abling plants to survive freezing conditions. Interestingly,some AFPs show homology to a group of plant pathogen-esis-related proteins that includes b-1,3-glucanases, end-ochitinases and osmotin-like and thaumatin-like proteins.Other AFPs like the wheat TaIRI ice recrystallization in-hibition proteins have a peculiar bipartite structure. Theyshowa leucine-rich repeat receptor domain of receptor-likeprotein kinases at theirN-terminus and ice recrystallizationinhibition domains at theirC-terminus. The available dataon the characterization of the different AFPs suggest thatthey would have a role in the tolerance of plants to freezingstress by preventing the formation of large ice crystals thatcould damage cellular structures. The homology of someAFPs with disease resistance proteins raises the possibilityof a dual role in abiotic and biotic stress tolerance.

Transcription factors

TheLT-inducible accumulation of transcripts and proteinsindicated the existence of regulatory elements controllinggene expression at the transcriptional level. A major focusof research aimed at the understanding of LT-inducedmodifications in gene expression has thus been directedtowards the identification of cis-acting promoter elementsand transcription factors binding them (van Buskirk andThomashow, 2006). Despite the fact that many groupshave studied promoters of LT-responsive genes in variousplant species, only one cis-regulatory element (and closevariants) has been identified as a genuine LT-responsiveelement (LTRE) with the consensus sequence RCCGAC.The transcription factors binding this element, namedCBF, were first identified in Arabidopsis. They belong to asubset of the AP2 class of transcription factors and havebeen found in most plant species examined so far. Over-expression of CBF genes inArabidopsis increases FT with-out any requirement for LT exposure, indicating that this



transcription factor is amajor player in the development ofFT. Following a rapid induction of CBF transcript accu-mulation in response to LT, the corresponding proteinsaccumulate and induce the subsequent accumulation oftheir target genes, which invariably contain LTREs in theirpromoters. In some cases, the binding of aCBF to its targetLTRE requires the presence of ADA adaptor proteins andthe GCN5 histone acetyltransferase. Adaptor proteins arecofactors that are required for the function of some tran-scription factors whereas, histone acetyltransferase arerequired to release the DNA from the nucleosome coreto allow the binding of transcription factors to promoterelements. Large-scale microarray and genomic studiesidentified the CBF regulon, the set of genes up- or down-regulated by CBFs. Because the accumulation of CBFtranscripts occurs as soon as plants are exposed to LT, itwas speculated that constitutively present transcriptionfactors regulating their expression must exist. Elegantstudies performed using CBF promoter-driven luciferaseexpression allowed the identification of ICE1 (inducer ofCBF expression 1) (Chinnusamy et al., 2006). This basichelix–loop–helix transcription factor regulates the tran-scription of CBFs and other cold-induced regulons, andconsequently regulates the development of FT. ICE1 isconstitutively expressed in Arabidopsis and the level ofcorresponding protein is regulated by the variant RINGfinger protein HOS1, a negative regulator of cold re-sponses. LT exposure induces the ubiquitination and sub-sequent proteolysis of ICE1, preventing a highaccumulation of CBF.

Other transcription factors are also involved in cold re-sponse. Data generated by transcriptomics analyses per-formed inArabidopsis indicated that some cold-responsivegenes are part of the CBF regulon. In addition, other cold-responsive genes are not regulated by the CBF transcrip-tion factors, including 15 genes that encode known or pu-tative transcription factors. These results support theexistence of both CBF-dependent and CBF-independentregulatory pathways associated with LT response inArabi-dopsis. One of the latter pathways involves ZAT12, a zinc-finger protein predicted to encode a transcriptional repres-sor. Overexpression of ZAT12 represses genes that arenegatively associated with LT, and induces the expressionof genes that are positively associated with LT. The overalleffect should have been a fairly good increase in FT of non-acclimatedplants.However, the observed improvementwasmodest, indicating that this pathway is not as important asthe CBF pathway for the development of FT. Interestingly,there is anoverlap in the regulons affected in theZAT12 andCBF cold-response pathways, indicating that they cross-talk. Seven genes for which the expression is affected by LTexposure are part of both the CBF and ZAT12 regulons.Other studies revealed that the transcription factors HOS9(a homeodomain type) andHOS10 (aR2R3myeloblastosistype) are part of other CBF-independent pathways of generegulation in response to LT.

Although global, genomic-scale technologies helped todelimit cold stress-related regulons, the signalling events

from sensors (perception) to transcription factors to cellu-lar responses requires further investigation. Future studiesneed to be focused on determining the genes that are themajor factors regulating FT. However, it could prove in-formative to look at genes that are downregulated by ex-posure to LT.

Influence of Trans Regulators on LT-responsive Gene Expression

Vernalization is the promotion of flowering by a coldtreatment, a physiological process that has been best stud-ied in monocotyledonous winter growth habit Gramineaespecies. Because a plant that has achieved its vernalizationrequirement loses its FT, there appears to be a relationshipbetween the responses related to vernalization and thoserelated to cold tolerance. Spring habit cereal cultivars donot have a vernalization response and are unable to main-tain LT-induced gene expression in an upregulated condi-tionwhen exposed to LT. Consequently, they are unable toachieve levels of FT similar to those ofwinter cultivars. Theclose association between the point of vernalization satu-ration and the start of a decline in messenger ribonucleicacid (mRNA) and protein levels of FT-associated proteins,such as the wheat WCS120 dehydrins, indicates that ver-nalization genes play a key role in determining the durationof expression of the FT-associated genes (Kane et al.,2006). This suggests an explanation for the apparent pleio-tropic effect that the VRN-A1 (vernalization) locus has onboth the vernalization response and FT in wheat. It ap-pears that any factor that delays the transition from veg-etative to reproductive stages, such as a vernalization orphotoperiod requirement for flowering, would be expectedto increase the level of expression of FT genes in cerealsexposed to acclimating temperatures. The VRN-A1 locusmaps to the long arm of chromosome 5A, the same chro-mosome that was shown to bear factors having a positiveinfluence on the regulation of LT-inducible genes. Thecorresponding gene,TaVRN1, encodes aMADS-box tran-scription factor that positively regulates the transition fromthe vegetative to reproductive phase. In winter wheat, atleast two other transcription factors are important for thenegative regulationof flowering.TheMADS-boxTaVRT2and the zinc finger TaVRN2 proteins act by delaying theaccumulation of TaVRN1. The two repressors accumulateduring the vegetative phase in winter cereal varieties.TaVRT2 directly binds its target element, called a CArGbox, in the TaVRN1 promoter. The binding represses theactivity of the promoter and hence the accumulation of theTaVRN1 mRNA and protein. This repression effect is en-hanced or stabilized by TaVRN2. Upon vernalization,TaVRT2 and TaVRN2 transcript levels decline by a mech-anism that is still unknown, and this allows the gradualaccumulation of TaVRN1 and subsequent transition fromvegetative to reproductive phase. Spring growth habit ce-real species do not express the TaVRT2 and TaVRN2



repressors, therefore they show a high level of TaVRN1protein from their early development until the flowers aremature.

Cold Perception and SignalTransduction

Cold acclimation involves proper perception, precise sig-nalling and regulation of the transcriptome. The mecha-nisms by which plant cells perceive the LT stimulus arepoorly understood. The plasmamembrane is recognized asthe primary site of injury and is hypothesized to also be asite of perception of the LT stimulus. Available evidencepoints towards major roles played by the actin cytoskel-eton, the chloroplast and histidine kinases as possible coldsensors. The LT stimulus is transduced via both abscisicacid (ABA)-dependent and ABA-independent pathways,with calciumbeing an important secondarymessenger. Thecalcium signal is likely transduced by a series of phosphor-ylation events that would involve calcium-dependent pro-tein kinases. Although several calcium-dependent andcalcium-independent protein kinases have been cloned todate, there is little information regarding the specific down-stream signalling components leading to the activation ofspecific subsets of genes. A major difficulty lies in the factthat many LT-responsive genes are also induced by otherstimuli such as drought, ABA and salinity, and it is stillunclear how these different stimuli converge to induce theexpression of the same genes. One thing that is well ac-cepted is that the CBF pathway is one of the most impor-tant pathways leading to the expression of LT-responsivegenes.

Evidence is accumulating for the involvement of aphosphoinositides transduction pathway in abiotic stresssignalling (Chinnusamy et al., 2006). An inositol poly-phosphate 1-phosphatase, FIERY1, regulates cytosolic in-ositol-1,4,5-triphosphate levels, a molecule that can triggercalcium release from intracellular stores. Other studieshave provided evidence for the involvement of a pathwayinvolving mitogen-activated protein kinases (AtMEKK1-AtMKK2-AtMPK4/6) cascades in LT and other abioticstress signal transduction. The phosphoinositides andMAPK pathways have first been described in animal sys-tems; therefore, evidence has now clearly established thatthe plant and animal kingdoms share many similar char-acteristics in their signal transduction pathways.

Industrial and Medical Applications ofFreezing-associated Proteins

The ability of FT-associated proteins to protectplants against freezing damage was exploited forseveral industrial and medical applications (Bretonet al., 2000).

Food additives

Freezing is a widely used technique for food preservationduring storage and transportation. It reduces spoilage byinhibitingmicrobial growth andhelps in extending shelf lifeof a wide variety of foods. However, there are many ex-amples of foods that cannot be frozen without unaccept-able changes in quality, particularly the high water–containing tissues such as fruits, vegetables, fish andmeats.Extracellular and intracellular ice formation results in un-desirable changes in taste and texture due to cellular de-hydration, concentration of solutes andmechanical factorsthat can collectively lead to destruction of the plasmamembrane. Ice formation in frozen foods is influenced bythe rate of freezing and by the storage temperature.Although foods are usually flash frozen to produce smallice crystals, these ice crystals may grow larger over time.To find an alternative to the currently available cryo-

protectants, scientists have long considered using sub-stances made by organisms that survive freezingconditions. The first substance possessing properties toprotect an organism from freezing was discovered in fish ofthe southern polar ocean. The characterization of thesesubstances led to the identification of AFPs. Recently,proteins with similar functions were found in cold tolerantplants and insects. AFPs are used at very low concentra-tions to inhibit the growth of ice. Meat treated with AFP-containing solutions, frozen then thawed show smaller icecrystals, less drip and no detrimental effect on flavour, tex-ture, tenderness, juiciness and overall quality. In frozendesserts that are eatenwhile frozen such as popsicles and icecream, AFPs inhibit ice recrystallization and preserve thesmooth, creamy texture of high-quality products.The most studied AFPs from plants are those originally

found in rye. Since then, they were identified and charac-terized from other cereal species such as ryegrass andwheat. These AFPs accumulate only during cold acclimat-ion of cereals andwere found to be similar to pathogenesis-related proteins. The dual activities of these proteins couldserve two functions as additives in frozen foods by inhib-iting the recrystallization of ice and by reducing microbialactivity within the food products (Griffith and Yaish,2004). This will undoubtedly have a tremendous impact onthe quality and shelf life of both frozen foods and freshproduce that need to be stored at low nonfreezingtemperatures.The other group of plant proteins that may become the

target of new biotechnological applications in the food andcosmetic industries is the dehydrins group (Breton et al.,2000). As plants are able to tolerate extreme freezing anddehydration stresses (plant seeds lose 80% of their watercontent duringmaturation and remain viable), andbecausethese processes are highly correlated with dehydrin accu-mulation, these specific plant proteins may find very inno-vative applications in the future. For example, dehydrinsmaybe used in ointment against frostbite because currentlyavailable ointments give at best a negligible protectionagainst frostbite on the face and ears. This application is



promising since frostbites involve the same phenomena asplant cell freezing: ice formation, increased osmolality ofthe extracellular fluid and intracellular dehydration.

Cryopreservation of mammalian cells

The ability to cryopreserve human cells, tissues and organswould have an important impact in medicine, particularlyin organ transplantation and reproduction. Chemical sub-stances such as dimethyl sulfoxide (DMSO), glycerol andsucrose are currently used as cryoprotective agents. Thesesubstances are used at high concentrations, which maycause toxicity and side effects.Anattractive alternative is touse plant extracts containing cryoprotective proteins.It was recently shown that a wheat protein extract has theability to cryoprotect rat hepatocytes and other cell types(Hamel et al., 2006). This novel cryopreservation technol-ogy is efficient and permits long-term storage and recoveryof large quantities of healthy cells that maintain their in-tegrity and metabolic activities. Studies are underway todetermine if the sameprotection canbe conferred tohumanhepatocytes. The nature of the specific proteins conferringthe cryopreservation activity is still unknown. Anotherpotential application of AFPs is the cryopreservation ofhuman blood platelets, which are blood-clotting agents.Platelets stored at room temperature can be kept only forfive days before microbial contamination and progressiveageing-induced activation. Cooling below 58C increasesion leakage and platelet activation by calcium entry.A preliminary study has shown that AFPs can prevention leakage and thereby improved the survival of plateletsat temperatures as low as 48C. The development of an effi-cient technique to prolong the storage of human plateletswould have enormous health benefits. Although the pros-pects of the technology using plant cryoprotective proteinsappear encouraging for the future of cryobiology, furtherresearch must be pursued. An approach that would com-bineAFPs and other FT-associated proteins like dehydrinsmight yield the optimal cryopreservation conditions.

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Further Reading

Huner NPA, Oquist G and Sarhan F (1998) Energy balance and

acclimation to light and cold. Trends in Plant Science 3:

224–230.

Kaplan F, Kopka J, Haskell DW et al. (2004) Exploring the tem-

perature-stress metabolome of Arabidopsis. Plant Physiology

136: 4159–4168.

Lee BH,HendersonDAandZhu JK (2005) TheArabidopsis cold-

responsive transcriptome and its regulation by ICE1. The Plant

Cell 17: 3155–3175.

Oono Y, Seki M, Satou M et al. (2006) Monitoring expression

profiles of Arabidopsis genes during cold acclimation and de-

acclimation usingDNAmicroarrays.Functional and Integrative

Genomics 6: 212–234.

Ouellet F (2002) Out of the cold: unveiling the elements required

for low temperature induction of gene expression in plants.

In vitroCellular andDevelopmental Biology –Plant 38: 396–403.

Sarhan F andDanyluk J (1998) Engineering cold-tolerant crops–

Throwing themaster switch.Trends inPlant Science 3: 289–290.

Sarhan F, Ouellet F and Vazquez-Tello A (1997) The wheat

WCS120 gene family. A useful model to understand the mole-

cular genetics of freezing tolerance in cereals. Physiologia

Plantarum 101: 439–445.



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