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priate light condition. Depending on the plant species, itmay take a few days to several weeks to reach maximumlevels of freezing tolerance and the tolerance producedranges from –10 °C to below –60 °C (Gilmour, Hajela &Thomashow 1988; Webb, Uemura & Steponkus 1994). Coldacclimation is very complex, involving many biochemicaland physiological changes. It has been estimated that theexpression of hundreds of genes can be altered (Guy 1990).These characteristics, and indeed the existence of aninducible cold acclimation process, indicate that the bio-logical costs of constitutive freezing tolerance must be highenough to be a negative factor in evolutionary selection.Despite intensive research for nearly a century, fundamen-tal questions of this field still remain to be answered. Howdo plants sense the decreasing temperature? What are thesignalling cascades that transduce the low temperature cueto the cell nucleus and bring about changes in gene expres-sion? What is the relative importance, to freezing tolerance,of each of the changes in metabolism and gene expressionthat occur during cold acclimation? More complete answerto these questions is of basic scientific interest and has enormous practical applications as well.

In the last few years, exciting progress has been achievedthrough the introduction of molecular and genetic tech-nologies, especially the adoption of Arabidopsis as a modelplant in studying cold acclimation. Here, we summarizedthe recent progress and discuss the major challenges in thisfield. More detailed information on cold acclimation andfreezing tolerance can be found in the following excellentreviews (Steponkus 1984; Guy 1990; Thomashow 1994,1999).

FREEZING INJURY IN PLANTS

Under most circumstances, freezing temperatures inducethe formation of ice in the intercellular spaces and cell wallsof plant tissue. This extracellular ice formation occursbecause the intercellular fluid has a higher freezing pointthan the cytoplasm. In addition, the intercellular liquid nor-mally contains heterogeneous ice nucleating agents, such asdust and ice-nucleating bacterial proteins (Brush, Griffith& Mlynarz 1994). (In the absence of heterogeneous ice-nucleating agents, pure water remains as a supercooledliquid until –39 °C – the homogeneous nucleation tempera-ture at which water freezes in absence of any heteroge-

ABSTRACT

Plant acclimation to freezing temperatures is very complex.Many temperate plants increase in freezing tolerance uponexposure to a period of low but non-freezing temperatures,an adaptive process known as cold acclimation. This ac-climation phenomenon has encouraged investigations ofphysiological, biochemical, and molecular changes that areassociated with the development of freezing tolerance.Although many biochemical and gene-expression changesoccur during cold acclimation, few have been unequivocallydemonstrated to contribute to the development of freezingtolerance. However, in the last few years, exciting newprogress has been made through the use of mutationalanalysis and molecular genetic approaches. We now recog-nize that several interacting signal pathways are activatedto bring about cold acclimation and ensure the winter sur-vival of plants. The challenge for the future is to understandthese pathways at a mechanistic level. Facile map-basedcloning in Arabidopsis and techniques (such as DNAmicro-arrays) for transcript profiling will provide the toolsneeded for this task.

Key-words: cold tolerance; environmental stress; freezingtolerance; gene regulation; signal transduction; winter hardiness.

INTRODUCTION

Freezing temperature represents a major environmentalconstraint limiting growth, development, and distributionof plants. Most tropical and subtropical plant species lackthe ability to adapt to freezing temperature and are typi-cally injured by temperatures below 10 °C. In contrast, tem-perate plants have evolved mechanisms by which they canincrease their ability to withstand the subsequent freezingtemperatures in response to a period of low but non-freezing temperatures. This process is called cold acclima-tion (CA) (Levitt 1980). In nature, cold acclimation is initi-ated by the decreasing temperatures in late autumn or earlywinter. It can be duplicated in the laboratory simply byexposing plants to low temperatures (2–6 °C) under appro-

Correspondence: Zhanguo Xin. Tel: +1 617 5518219; e-mail:ZHANGUO.XIN@cereon.com

INVITED REVIEW

Cold comfort farm: the acclimation of plants to freezing temperatures

Z. XIN1 & J. BROWSE2

1Cereon Genomics, 45 Sidney Street, Cambridge, MA 02139, USA and 2Institute of Biological Chemistry,Washington State University, Pullman, WA 99164, USA

neous nucleating event.) Upon freezing, the ice-state ofwater has a much lower water potential than liquid solutionand this difference increases as temperature decreases(Guy 1990). Thus, when ice forms extracellularly, there is asudden drop in water potential outside the cell. Conse-quently, water from the cytoplasm moves through theplasma membrane by osmosis, leading to cellular dehydra-tion. The net amount of water removed from a cell dependson both the initial solute concentration of the cytoplasmand the freezing temperature which directly determines thewater potential of the extracellular ice. For example, freez-ing at –10 °C results in a water potential of –11·6 MPa. Thiswill remove 90% of the osmotically active water from thecell, assuming the initial osmotic potential of cell is –1 MPa.If the initial osmotic potential of cell is –2 MPa due to accu-mulation of solutes during cold acclimation, the same freez-ing only removes 80% of cellular water. Thus, freezinginjury is largely an injury caused by cellular dehydrationand, for this reason, freezing stress, drought stress and saltstress share many features (and genes) in common.

Dehydration may damage cellular functions in a varietyof ways. However, at least in the case of freezing stress,injury normally involves effects on membrane structure andfunction (Webb et al. 1994; Uemura, Joseph & Steponkus1995). The plasma membrane has been considered as theprimary site of freezing injury since 1912 (Levitt 1980). Theworks from Steponkus and colleagues (Webb et al. 1994;Uemura et al. 1995) provide evidence that freezing-induceddestabilization of plasma membrane involves three differ-ent lesions. In protoplasts from non-acclimated rye leaves,the reduction in cell volume at temperatures down to –5 °Cis accompanied by loss of plasma membrane surface areadue to invagination of the plasma membrane followed bybudding off of endocytotic vesicles. Upon rewarming, themelted water is drawn back into the cells. Because of theirreversible loss of plasma membrane, the protoplast burstsbefore it regains the original volume due to the hydrosta-tic pressure created by the incoming water. This type oflesion is known as ‘expansion-induced lysis’ (Webb et al.1994). In contrast, protoplasts prepared from cold-acclimated leaves do not form endocytotic vesicles. Instead,the plasma membrane is retained as exocytotic extrusionsthat allow re-expansion of the cell upon thawing.

At colder temperatures (and greater dehydration), dif-ferent cellular membranes are brought into close apposi-tion. Membrane lipids in non-acclimated tissues undergolateral phase separations. Certain lipids aggregate to forman inverted structure with hexagonal packing symmetry,called HexII phase. The lipid molecules are arranged incylinders with the head groups oriented toward an aqueouscore, 20 Å in diameter, which disrupts the membranebilayer. The plasma membrane becomes permeable towater and solutes upon rewarming and loses osmoticresponsiveness. Formation of the HexII phase is consideredas an interbilayer event and may involve the participationof two or more bilayers because the HexII phase is onlyobserved in multilamellar or stacked bilayer regions. Mostfrequently, the HexII phase is observed in regions where

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plasma membrane is brought into close apposition with theouter membrane of chloroplasts (Uemura et al. 1995).Thesetwo types of freezing-induced membrane destablization areassociated with protoplasts isolated from non-acclimatedtissues and are largely precluded by cold acclimation(Webb et al. 1994; Uemura et al. 1995).

In cold-acclimated protoplasts, freezing injury is associ-ated with ‘fracture-jump lesion’. This type of membranelesion is exemplified by localized deviation in the freeze-fracture plane of the plasma membrane during cryo-electron microscopy, probably due to the localized fusionof plasma membrane with other cellular membranes,especially the chloroplast envelopes (Webb et al. 1994). Itis believed that both HexII and fracture-jump lesions areformed from a common structural intermediate of mem-branes (Uemura et al. 1995). However, it is unclear whyHexII lesions are observed only in protoplasts isolatedfrom non-acclimated tissues whereas fracture-jump lesionsare observed exclusively in protoplasts from cold-acclimated tissues. Furthermore, the temperatures at whichfracture-jump lesions are observed vary greatly amongplant species (Webb et al. 1994; Uemura et al. 1995). Littleis known about the physiological, biochemical, and molecu-lar basis accounting for this variation, but it is considered ameasure of the relative freezing tolerance that can beachieved in different species. It is worth noting that thesefreezing-induced membrane lesions are primarily observedin isolated protoplasts, and may not reflect what occurs inintact plant cells.

COMPARATIVE BIOCHEMICAL ANDBIOPHYSICAL APPROACHES HAVEIDENTIFIED MANY COMPONENTS OFFREEZING TOLERANCE

A common approach to determine the biochemical basis offreezing tolerance has been to compare the metabolitepools or structural components from cold-acclimated andnon-acclimated plants, or from plant varieties with differ-ent abilities to cold acclimate. Numerous physiological andbiochemical changes are known to occur during cold accli-mation, and these are shown schematically in Fig. 1. Themost notable changes include a reduction or cessation ofgrowth, reduction of tissue water content (Levitt 1980),transient increase in abscisic acid (ABA) levels (Chen,Brenner & Li 1983), changes in membrane lipid composi-tion (Lynch & Steponkus 1987; Uemura & Steponkus1994), the accumulation of compatible osmolytes such asproline, betaine, polyols and soluble sugars, and increasedlevels of antioxidants (Koster & Lynch 1992; Kishitani et al.1994; Murelli et al. 1995; Nomura et al. 1995; Dörffling et al.1997; Tao, Oquist & Wingsle 1998). These complicatedresponses have made it very difficult to separate theprocesses responsible for freezing tolerance from those thatmerely represent responses to low, non-freezing tempera-tures. However, it is clear from these comparativeapproaches that cold acclimation probably requires manychanges in cell biology and metabolism. Here, we will

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briefly review evidence for some of the most importantchanges that have been revealed by such approaches.

Since cell membranes are thought to be the primary sitesof freezing injury, changes in membrane behavior duringcold acclimation must be critical to the development offreezing tolerance. Ultrastructural changes in the plasmamembrane can be observed within 6 h of the start of coldacclimation in Arabidopsis (Ristic & Ashworth 1993). Theunderlying biochemical bases for these changes in mem-brane behavior are not completely known. Alterations inmembrane lipid composition are correlated with mem-brane cryostability as they have been observed during coldacclimation in all species examined (Steponkus 1984;Uemura & Steponkus 1994; Uemura et al. 1995). There isalso direct evidence for the involvement of membranelipids in freezing and dehydration tolerance. For example,when non-acclimated rye protoplasts are placed in hyper-osmotic medium, their plasma membrane buds off endo-cytotic vesicles as they do during freezing. However,Steponkus et al. (1988) demonstrated that when non-acclimated protoplasts were pre-incubated with mono-unsaturated or di-unsaturated species of phosphatidyl-choline so that the phospholipid was incorporated into theplasma membrane, hyperosmotic treatment resulted in theformation of exocytotic extrusions. Disaturated species ofphosphatidylcholine did not induce this change. On theother hand, alteration in lipid composition typically lags farbehind increases in freezing tolerance (Uemura et al. 1995;Wanner & Junttila 1999), suggesting that other changesmust also contribute to membrane stability (Lineberger &Steponkus 1980; Steponkus et al. 1998).

Accumulation of soluble sugars during cold acclimationis well documented in many plants including Arabidopsis,and the time course of sugar accumulation correlates wellwith development of freezing tolerance during cold accli-mation (Ristic & Ashworth 1993; Wanner & Junttila 1999).In addition, genetic evidence is available to support the roleof soluble sugars in freezing tolerance. For example, a

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mutant of Arabidopsis, sfr4 (see below), impaired in itsability to cold acclimate, does not accumulate sugars inresponse to low temperature (McKown, Kuroki & Warren1996). In contrast, esk1, a constitutively freezing-tolerantmutant, accumulates sugars at warm temperatures (Xin &Browse 1998). Several roles for sugars in protecting cellsfrom freezing injury have been proposed, including func-tioning as cryoprotectants for specific enzymes (Carpenteret al. 1986), as molecules promoting membrane stability(Lineberger & Steponkus 1980), and as osmolytes toprevent excessive dehydration during freezing (Steponkus1984). However, soluble sugars alone are insufficient for full freezing tolerance. Several sfr (sensitive to freezing)mutants that accumulate soluble sugars normally duringcold acclimation are, nevertheless, defective in freezing tolerance. Transformation of tobacco with a bacterialpyrophosphatase or invertase gene increased the levels ofsoluble sugars but did not provide any increase in freezingtolerance (Hincha et al. 1996).

Accumulation of betaines has been reported in severalplant species in response to low temperature (Kishitaniet al. 1994; Nomura et al. 1995). Arabidopsis does not accu-mulate betaines. However, overexpression of a single bacterial gene, choline oxidase, in Arabidopsis results inaccumulation of betaine to between 0·1 and 1 mmol g-1

fresh weight (Hayashi et al. 1997). Although low comparedto the levels (20–300 mmol g-1 fresh weight) found in plantsthat naturally accumulate betaines, this increase signifi-cantly improved the tolerance to various stresses includingcold and freezing (Hayashi et al. 1997 and Chen T.H.H., per-sonal communication). Even though betaines improvedfreezing tolerance in Arabidopsis, it is also true that manychilling- and freezing-sensitive species also accumulatebetaines (Yang et al. 1995).

Free proline also increases in plants in response to manystresses (Delauney & Verma 1993). However, its role instress tolerance remains equivocal. Selection of somaticmutants that accumulate proline has provided correlations

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Figure 1. Cold acclimation induces changesin many different cellular processes. Shown inthe figure are some of the changes commonlyobserved when plants are subjected to low,non-freezing temperatures.

with enhanced freezing tolerance in potato (Van Swaaijet al. 1986) and winter wheat (Dörffling et al. 1997). Ara-bidopsis accumulates proline during cold acclimation butthe increase in proline content lags behind the developmentof freezing tolerance (Wanner & Junttila 1999). Thestrongest genetic evidence that proline may contribute tothe increased freezing tolerance comes from the isolationof several constitutively freezing-tolerant mutants in Ara-bidopsis that accumulate proline in the absence of low-temperature treatment (Xin & Browse 1998). Prolinecontent increases by 10-fold during two days of cold accli-mation at 4 °C in wild-type Arabidopsis. In the absence ofacclimation, the esk1–1 mutant contains proline at levels30-fold higher than non-acclimated wild-type plants. Atleast two other freezing-tolerant mutants also contain highlevels of proline (Xin & Browse unpublished) suggestingthat proline does play an important role in freezing toler-ance. However, proline accumulation is not required forfreezing tolerance since some constitutively freezing-tolerant mutants contain the same low levels of proline asnon-acclimated wild-type plants.

Apparently, plants employ multiple mechanisms toensure freezing tolerance. At present, our knowledge of the metabolic changes that contribute to freezing toleranceis incomplete, but information about the biochemicalprocesses contributing to freezing tolerance is essential forsuccessful engineering of freezing tolerance in crop plants.Fortunately, the genetic approaches outlined below offernew ways to evaluate the contribution of individual com-ponents toward overall freezing tolerance.

DIFFERENTIAL SCREENING HASUNCOVERED FAMILIES OF COLD-INDUCED GENES

A major effort in cold acclimation research in the pastdecade has been to identify cold-induced genes and todetermine whether they have roles in freezing tolerance(Guy 1990; Palva 1994;Thomashow 1994, 1999). Many cold-induced genes have been cloned from a variety of plantspecies by differential screening of cDNA libraries andother techniques (Hughes & Pearce 1988; Dunn et al. 1990;Hajela et al. 1990; Kurkela & Franck 1990; Danyluk, Rassart& Sarhan 1991; Lee & Chen 1993; Wolfraim & Dhindsa1993; Zhu, Chen & Li 1993; Castonguay et al. 1995; Ferulloet al. 1997). Some of these genes encode proteins withknown enzymatic functions, such as alcohol dehydrogenase(Jarillo et al. 1993), phenylalanine ammonia lyase, chalconesynthase (Leyva et al. 1995) the fatty acid desaturase, FAD8(Gibson et al. 1994), lipid transfer protein (Hughes &Pearce 1988), a translation initiation factor (Dunn et al.1993), a thiol protease (Shaffer & Fischer 1988), catalases(Prasad et al. 1994) and D-pyrroline-5-carboxylate synthase(the first enzyme committed to proline biosynthesis)(Yoshiba et al. 1995). Some show similarity to a group ofproteins involved in dehydration such as dehydrin- orLEA-like proteins (Gilmour. Artus & Thomashow 1992;Lin & Thomashow 1992), antifreeze proteins (Kurkela &

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Franck 1990), heat shock proteins or molecular chaperones(Anderson et al. 1994a, 1994b; Ukaji et al. 1999). Someencode various signal transduction or regulatory proteins,such as MAP kinases (Jonak et al. 1996; Mizoguchi et al.1996) and calcium-dependent protein kinases (Tahtiharjuet al. 1997). A set of cold-induced genes that have receivedparticular attention are extremely strongly induced by coldtreatment (typically 50- to 100-fold). Because these geneswere very easily identified by differential screening theyhave been characterized by several groups and have beengiven different names.We shall refer to them as COR (cold-regulated) genes (Thomashow 1999), but the same genes orhomologues are referred to as LTI (low-temperature-induced) (Nordin, Vahala & Palva 1993), CAS (cold acclimation-specific) (Monroy et al. 1993), Kin (cold-induced) (Kurkela & Franck 1990) and RD (responsive todessication) genes (Yamaguchi-Shinozaki et al. 1992). Asdiscussed below, the COR genes have been particularlyuseful in investigating the signal pathways associated withcold acclimation.

For the cold-induced genes as a whole, the challenge isto assess the relative contribution of each of them inincreasing freezing tolerance. The first question is whethera particular gene has a significant role in freezing tolerance.The answer, clearly, is that some cold-induced genes can beeliminated without measurably affecting tolerance. Forexample, phenylalanine ammonia lyase and chalcone syn-thase, which are involved in biosynthesis of anthocyanin,are induced to high levels of expression during cold accli-mation. Arabidopsis mutants deficient in either of thesegenes are not measurably affected in their ability todevelop full levels of freezing tolerance (Leyva et al. 1995).Similarly, a null mutant of Arabidopsis, which lacks alcoholdehydrogenase can develop the full level of freezing toler-ance (Jarillo et al. 1993).

The second question is to identify the genes that do con-tribute to freezing tolerance and to determine their relativeimportance. To date, only one gene, COR15a, has beendemonstrated to offer some protection to chloroplasts andprotoplasts derived from non-acclimated transgenic plantsoverexpressing this gene (Artus et al. 1996). Being localizedin the chloroplast stroma, the mature COR15a protein isproposed to function by deferring freezing-induced hexag-onal II phase formation to lower temperature through thealteration of intrinsic curvature of the inner membrane ofthe chloroplast envelope (Steponkus et al. 1998). Theenhancement of freezing tolerance in a COR15a transgenicline is subtle since no obvious improvement is observed atthe whole plant level (Artus et al. 1996; Jaglo-Ottosen et al.1998). Overexpression of other cold-induced genes in trans-genic plants also showed little or no enhancement in freez-ing tolerance (Zhu, Chen & Li 1996; Kaye et al. 1998).

This limited success in demonstrating a role for cold-induced genes could be explained if multiple genes act inconcert to increase freezing tolerance. This hypothesis wasrecently tested by Jaglo-Ottosen et al. (1998) and Liu et al.(1998). CBF1 (CRT-repeat binding factor) is a transcriptionactivator that binds to a low-temperature-inducible DNA

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element, the CRT-repeat, which has a core sequenceAAGAC in promoters of several COR genes (Stockinger,Gilmour & Thomashow 1997). (CBF1 is also calledDREB1B for drought-regulated element binding factor(Liu et al. 1998).) Overexpression of CBF1 in transgenicArabidopsis induces the expression at warm temperaturesof the entire battery of COR genes that have the commonDNA-elements in their promoter region (Jaglo-Ottosenet al. 1998; Liu et al. 1998). When tolerance was assayed byan ion-leakage test, the transgenic plants showed 3·3 °Cimprovement in freezing tolerance over non-transgeniccontrol plants in the absence of cold acclimation. Thisincrease in freezing tolerance is much more dramatic thanthat achieved by overexpression of COR15a alone. Indeed,overexpression of CBF1 increased freezing tolerance at thewhole-plant level (Jaglo-Ottosen et al. 1998), although nodetectable increase in freezing tolerance is observed intransgenic plants overexpressing COR15a alone. Theseexperiments demonstrate that CBF1 and CBF1-mediatedcold-induced genes play significant roles in freezing toler-ance. However, it is by no means clear that the COR genesare the major contributors to this constitutive tolerancesince CBF1 very probably induces other genes that containthe CRT element in their promoters. Indeed, many lines ofevidence now indicate that the CBF1-COR pathwayresponse is only one of several signalling pathways involvedin cold acclimation (see below).

FREEZING TOLERANCE AND PLANT BREEDING

Freezing tolerance is a complex trait with multigeneticinheritance (Thomashow 1990). Plants vary in inherentfreezing tolerance before cold acclimation and in the poten-tial to acquire freezing tolerance during cold acclimation.Genetic analysis of freezing tolerance in crosses of twopotato species with contrasting freezing tolerance demon-strated that these two traits are controlled by different setsof genes (Stone et al. 1993). This finding is important as itimplies that it may be possible to genetically manipulatedifferent aspects of freezing tolerance and combine themto make a significant improvement in freezing tolerance.Although it is not clear how many genes are involved infreezing tolerance, recent advancement in mapping ofquantitative trait loci has permitted the identification ofmajor loci that have a large effect on freezing tolerance(Pan et al. 1994; Galiba et al. 1995). Four of the five majorloci that control vernalization and freezing tolerance inwinter wheat have been mapped on chromosome 5A(Vrn1), 5D (Vrn3), 5B (Vrn4), and 7B (Vrn5) (Galiba et al.1995). It is interesting to note that the location of Vrn1 onchromosome 5A is homologous to the location of barleygene Sh2 on chromosome 7 (5H) and Sp1 of rye on chro-mosome 5R. Although the genes that control vernalizationresponse are closely linked with those that control freezingtolerance, these two traits are controlled by separate genes(Galiba et al. 1995). Further fine-mapping of these majorloci may eventually lead to the identification of genes con-

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tributing to freezing tolerance. Identification of these geneswill permit the study of naturally evolved mechanisms offreezing tolerance. Due to the complexity of the genomeand paucity of genetic and physical markers in these cropplants, the cloning of these quantitative trait loci could bea huge endeavour.

MUTATIONAL ANALYSIS OF FREEZING TOLERANCE

Recently, three mutational screens have been employed inArabidopsis to dissect the mechanisms of freezing toler-ance. One genetic approach, as reported by Ishitani et al.(1997), was to generate transgenic plants harbouring a con-struct of the COR 78 (RD29) gene promoter fused to areporter gene, firefly luciferase. After mutagenesis of thetransgenic plants, mutants with aberrant expression of thereporter gene were isolated. Since the promoter of COR78has cold-, drought-, and ABA-responsive elements, thisapproach allows the simultaneous study of the signal path-ways for cold, drought, and ABA. Hundreds of mutantswith altered luciferase activity have been identified. Thesemutants fall into three major categories: the cos mutantsshow constitutive expression of osmotically responsivegenes; the los mutants show loss of expression of thesegenes; whereas hos mutants show hyper-expression afterinduction by drought, cold or ABA. Analysis of thesemutants suggests that multiple signalling pathways cross-talk and converge to activate the COR78 gene. Freezing tol-erance has been determined for two of the hos mutants,hos1 and hos2 (Ishitani et al. 1998; Lee et al. 1999). Com-pared with wild-type plants, these mutants are either lessfreezing tolerant or are less capable of developing freezingtolerance after cold acclimation even though they hyper-accumulate COR gene transcripts when exposed to cold.The defect in freezing tolerance is unlikely to be due toother mutations that decrease plant vigor in generalbecause similar phenotypes are observed in multiple alleles of mutants isolated from different mutagenized populations.

A second approach involved isolating mutants that aredefective in developing freezing tolerance after cold accli-mation. Warren et al. (1996) have isolated seven Arabidop-sis mutants that fail to develop full freezing tolerance evenafter 2 weeks of cold acclimation.These mutants are namedsfr for sensitive to freezing. This approach offers the poten-tial to identify a wide range of signalling componentsmediating cold acclimation. Some of the sfr mutants aredeficient in accumulation of soluble sugars during coldacclimation (McKown et al. 1996). One of these freezing-sensitive mutants, sfr6, was shown to be deficient in CBF1-mediated induction of COR genes (Knight, Trewas &Knight 1999). Characterization of sfr6 confirms the impor-tance of the CBF1 pathway in cold acclimation. However,except for sfr6, the remaining classes of sfr mutants all showstrong induction of the COR genes even though they arepartly deficient in the ability to cold acclimate. Thus, CORgene induction is only one component of freezing tolerance.

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A third approach involved the isolation of constitutivelyfreezing-tolerant (cft) mutants, i.e. mutants that are morefreezing tolerant than wild-type plants in the absence ofcold acclimation. Twenty-six constitutive freezing-tolerantmutants were isolated from 800 000 EMS-mutagenized M2seedlings of Arabidopsis (Xin & Browse 1998). Due to thecomplexity of cold acclimation, a single gene mutation thatresults in significant increases in freezing tolerance is mostlikely a mutation in a signalling component of cold accli-mation, which mediates to the expression of a suite of ter-minal genes to provide increased freezing tolerance. One ofthe best characterized cft mutants, eskimo1, tolerates freez-ing to –10·6 °C without cold acclimation. This improvementin freezing tolerance (5·1 °C over non-acclimated wild type)represents 70% of the freezing tolerance found in fullyacclimated wild-type plants. The esk1 mutants contain highlevels of proline and soluble sugars but do not express theCOR genes in the absence of cold acclimation (Xin &Browse 1998). This implies that CBF1-regulated genes arenot required for the development of certain aspects offreezing tolerance. Interestingly, cold acclimation of esk1produces plants that are more than 2 °C more freezing tolerant than fully acclimated wild type. Apparently theesk1 mutation is hyperactivating some aspects of freezingtolerance.

Since the cft mutants are freezing tolerant when grownat warm temperatures, the genes that are constitutively acti-vated in these mutants may contribute the freezing toler-ance observed. It is possible that genes which are merelyresponsive to low temperature but play no role in freezingtolerance are not constitutively expressed in the cft mutantsat warm temperature.Thus, these mutants may also provideunique opportunities to separate genes that are essential tofreezing tolerance from those that are merely responsive tolow-temperature exposure. The biochemical and molecularbasis for the constitutive freezing tolerance is unknown inany of the cft mutants. Molecular cloning of the genesdefined by the cft mutations and extensive analysis of thebiochemical and molecular basis of the freezing tolerancein each mutant will likely result in new insight into themechanisms of plant acclimation to freezing temperatures.

SIGNALLING PROCESSES ARE KEY TOUNDERSTANDING COLD ACCLIMATION

The diverse responses of plants to low temperatures andthe complicated biochemical and molecular changes asso-ciated with cold acclimation indicate that a complete under-standing of freezing tolerance will not be possible until thesignalling cascades specific to cold acclimation are defined.In broad terms, it can be assumed that cold acclimationinvolves a temperature sensor to perceive the low tempera-tures and a signal transducer to activate the biochemicaland gene expression events required for increased freezingtolerance. In this respect, major advances have been madeby the identification of DNA sequences, such as CRT, DRE,and ABRE in the 5¢-regulatory regions of several CORgenes (Baker, Wilhelm & Thomashow 1994; Yamaguchi-

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Shinozaki & Shinozaki 1994; Wang & Cutler 1995), and bythe cloning of transcription factors that bind to theseCRT/DRE elements (CBF or DREB) upstream of COR15aand COR78 (Stockinger et al. 1997; Liu et al. 1998). Thesetranscriptional factors belong to small gene families con-sisting of three members, CBF1, CBF2, and CBF3, orga-nized in a direct repeat on chromosome 4 (Gilmour et al.1998; Shinwari et al. 1998). The transcript levels for all threeCBF genes increased within 15 min after plants are exposedto cold temperatures, which is followed by the induction ofCBF1-mediated COR genes at about 3 h. Distinct signalpathway(s) must be involved in the induction of the CBFgenes since these genes do not have CRT/DRE elements in their promoter region.

It is still unclear how plants perceive the decreasing tem-perature and relay this signal to the nucleus to activatethese transcription factors. Several lines of evidence indi-cate that changes in cellular calcium levels may be involvedin temperature sensing. First, a transient increase in cytoso-lic calcium occurs almost immediately when plants areexposed to low temperatures (Monroy & Dhindsa 1995;Knight et al. 1996; Sheen 1996), implying that calcium maybe involved in the transduction of the low temperaturesignal. Second, the temperature that increases calciuminflux coincides with the temperature that induces coldacclimation (Monroy & Dhindsa 1995). Furthermore,calcium channel blockers or chelators inhibit the expres-sion of COR (CAS) genes at low temperature whereascalcium ionophore causes calcium influx and induces theexpression of COR (CAS) genes in warm temperatures(Monroy & Dhindsa 1995; Knight et al. 1996). Other cellu-lar processes may be required to relay the low-temperaturesignal since similar calcium increases are also observed inchilling-sensitive plants which are injured by exposure tolow temperature (Knight et al. 1996; Sheen 1996). Theunsaturation level of membrane lipids or membrane fluid-ity, has been suggested as a temperature-sensing mecha-nism in cyanobacteria (Vigh et al. 1993). Whether thisprocess occurs in higher plants is still subject to debate(Gibson et al. 1994).

The plant hormone ABA has also been shown to mediatethe development of freezing tolerance. Four lines of evidence suggest that ABA may play a central role in thesignal transduction of cold acclimation. First, ABA treat-ment at normal growth temperatures can increase thefreezing tolerance of a wide range of plants including Ara-bidopsis (Lang et al. 1994). Second, endogenous ABA levelsincrease in certain plants in response to low temperatures(Chen et al. 1983). Third, ABA-deficient mutants areimpaired in developing freezing tolerance during cold accli-mation, however, the freezing tolerance can be restored tothe wild-type level by adding ABA into the culture medium(Heino et al. 1990; Gilmour & Thomashow 1991). Fourth,ABA treatment can induce all the COR genes (Heino et al.1990; Gilmour & Thomashow 1991). In contrast to theseresults, all of the ABA-insensitive mutants examined cancold acclimate to the same level as wild type Arabidopsis(Gilmour & Thomashow 1991). One possible explanation

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for these observations is that cold acclimation is mediatedby a different ABA receptor or pathway from that regulat-ing other ABA responses. COR genes can be also inducedby low temperature in ABA-deficient and ABA-insensitivemutants (Gilmour & Thomashow 1991; Nordin, Heino &Tapio Palva 1991), indicating that there are low-temperature signalling pathways independent of ABA.Indeed, the CRT/DRE elements present in the promoterregions of COR genes are not responsive to ABA treatmentthrough promoter analysis (Yamaguchi-Shinozaki & Shinozaki 1994). Rather, a different DNA element, ABRE,is responsible for the ABA inducibility of these COR genes.

Increasing evidence indicates that protein kinases andphosphatases are also involved in transduction of low-temperature signals during cold acclimation in plants.Several protein kinases responsive to low temperature havebeen isolated from higher plants based on sequence homol-ogy or cross-reaction of antibodies (Anderberg & Walker-Simmons 1992; Jonak et al. 1996; Mizoguchi et al. 1996;Tahtiharju et al. 1997). Using specific inhibitors, Monroy,Sangwan & Dhindsa (1998) demonstrated that proteinkinase inhibitor, stauosporine, could prevent the inductionof CAS15 by low temperature whereas a protein phos-phatase inhibitor, okadaic acid, could induce the expressionof CAS15 at 25 °C. They further identified a protein phos-phatase 2A as an early target for cold-inactivation. Theseexperiments demonstrate that plants may use many com-ponents that are similar to those associated with other signalling pathways to control cold acclimation.

The biochemical and genetic studies discussed aboveindicate that the signal cascades controlling cold acclima-tion are likely to be very complex. It is not appropriate toconsider cold acclimation as a simple, linear signallingpathway activating the full set of processes required forincreased freezing tolerance. Instead, we need to considera model for cold acclimation in which parallel or branched

Acclimation of plants to freezing temperatures 899

signalling pathways activate distinct suites of cold acclima-tion responses (Fig. 2). Constitutive activation of one ofthese pathways can result in considerable freezing toler-ance without support from other components. Previousstudies have shown that the expression of COR genes isinduced by both the CBF1 (ABA-independent) pathwayand by the bZIP-mediated ABA-dependent pathway(Gilmour & Thomashow 1991; Nordin et al. 1991; Mantyla,Lang & Palva 1995). As discussed above, it is likely thateach of these pathways induces other genes as well. It islikely that these two pathways induce overlapping sets ofgenes rather than providing parallel induction of a singleset of genes. The constitutively freezing-tolerant mutantesk1 accumulates high levels of proline but does not con-stitutively express the COR genes. Proline accumulation inesk1 is achieved by both constitutive activation of D-pyrroline-5-carboxylate synthase and the prevention ofproline oxidase gene induction by proline, indicating thatESK1 is a signalling or regulatory component. Since esk1mutant plants do not exhibit induction of the COR genes,ESK1 must define a third signal pathway (Fig. 2). Severalof the cft mutants neither accumulate proline nor activateCOR genes (unpublished results). They must define one ormore additional pathways that are distinct from the onesdescribed above. Thus, there are at least four separate sig-nalling pathways involved in cold acclimation. In each ofthe constitutively freezing-tolerant mutants, only one of thesignal pathways is activated, and therefore, only partialfreezing tolerance is achieved. This conclusion is also sup-ported by the analysis of sfr mutants that are not able tofully acclimate (Warren et al. 1996). Most of the sfr mutantsretain over 50% capacity to cold acclimate. The simplestexplanation is that each sfr mutation blocks one signallingpathway. Therefore, each mutant is still able to partiallycold acclimate through signalling pathways that are not dis-rupted in the mutant plant. Cloning of the genes defined by

© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 893–902

Figure 2. Several signalling pathways areinvolved in temperature perception and signaltransduction of cold acclimation. As discussedin the text, at least four pathways (andprobably more) are involved. The figure showsthe relatively rudimentary information that iscurrently known or surmised. There is much to be discovered about the details of eachpathway and the inevitable cross-talk between them.

the sfr and cft mutants will provide unique insights into theregulation of signalling pathways mediating the develop-ment of freezing tolerance.

CONCLUDING REMARKS

At present, it is unclear which genes or biochemicalprocesses are essential to the development of freezing tolerance and which are general responses to low, non-freezing temperatures but are not required for freezing tol-erance. Except for the induction of a few COR genes, thesignal cascades mediating most aspects of cold acclimation,such as increases in ABA, synthesis of compatibleosmolytes, and changes in membrane lipid composition, areunknown. The isolation of several series of mutants withaltered freezing tolerance now opens new routes to studythe processes required for freezing tolerance and to iden-tify components of the signalling pathways that mediatethese processes. For example, using micro-arrays or othergenomics techniques for transcript profiling, it will be pos-sible to compare expression patterns in cft and sfr mutantswith those in wild-type Arabidopsis and thereby identifygenes and proteins required for freezing tolerance. At thesame time, map-based cloning of the cft and sfr loci, usinginformation from the Arabidopsis genome sequence, willallow characterization of additional steps in the signallingcascade. We thank Vicki Racicot for her critical reading ofthe manuscript.

ACKNOWLEDGMENTS

This work was supported by US National Science Founda-tion grant no. IBN-940790 and by the Agricultural ResearchCenter, Washington State University. We would like tothank the two anonymous reviewers for their constructivesuggestions.

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Received 14 December 1999; received in revised form 9 April 2000;accepted for publication 9 April 2000

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