Plant Physiol. (1990) 93, 1246-12520032-0889/90/93/1 246/07/$01 .00/0

Received for publication January 26, 1990Accepted March 22, 1990

Molecular Cloning and Expression of cor (Cold-Regulated)Genes in Arabidopsis thalianal

Ravindra K. Hajela, David P. Horvath, Sarah J. Gilmour, and Michael F. Thomashow*Department of Crop and Soil Sciences (R.K.H, S.J.G, M.F. T), Department of Microbiology and Public Health

(M.F. T), and Program in Genetics (D.P.H, M.F. T) Michigan State University, East Lansing, Michigan 48824-1325

ABSTRACT

We have previously shown that changes in gene expressionoccur in Arabidopsis thaliana. L. (Heyn) during cold acclimation(SJ Gilmour, RK Hajela, MF Thomashow [1988] Plant Physiol 87:745-750). Here we report the isolation of cDNA clones of fourcold-regulated (cor) genes from Arabidopsis and examine theirexpression in response to low temperature, abscisic acid (ABA),water stress, and heat shock. The results of Northem analysisindicated that the transcript levels for the four cor genes, repre-sented by clones pHH7.2, pHH28, pHH29, and pHH67, increasedmarkedly between I and 4 hours of cold treatment, reached amaximum at about 8 to 12 hours, and remained at elevated levelsfor as long as the plants were kept in the cold (up to 2 weeks).Retuming cold acclimated plants to control temperature resultedin the levels of the cor transcripts falling rapidly to those foundin nonacclimated plants; this occurred within 4 hours for thetranscrpts represented by pHH7.2 and pHH28, and 8 hours forthose represented by pHH29 and pHH67. Nuclear run-on tran-scription assays indicated that the temperature-regulated expres-sion of the cor genes represented by pHH7.2, pHH28, and pHH29was controlled primarily at the posttranscriptional level while thecor gene represented by pHH67 was regulated largely at thetranscriptional level. Northern analysis also indicated that thelevels of cor gene transcripts increased in response to both ABAapplication and water stress, but not to heat shock. The possiblesignificance of cor genes being regulated by both low tempera-ture and water stress is discussed.

Plants vary widely in their responses to cold temperatures.Many species of tropical and subtropical origin are 'chillingsensitive'; they are injured or killed by exposure to low,nonfreezing temperatures (21). In contrast, plants from tem-perate regions generally acclimate to cold temperatures andsurvive this environmental 'stress' (21). One of the mostdramatic manifestations of cold acclimation, or cold harden-ing, is increased frost tolerance. Other changes that occur

during cold acclimation include alterations in lipid composi-tion, increased sugar and soluble protein content, and theappearance of new isozymes (21, 30, 32). Some of thesechanges, in particular the alterations in lipid composition,appear to contribute directly to the increased frost toleranceof acclimated plants (32). Others potentially increase the

' Supported by grants from the U.S. Department of Agriculture(88-37264-3880), the Michigan Agricultural Experiment Station,and the Michigan Research Excellence Fund.

overall fitness of the plant for low temperature survival. Inmost cases, however, the exact role that a given change has inthe cold acclimation process is uncertain.

In 1970, Weiser (35) suggested that cold acclimation in-volves changes in gene expression. Indeed, there is now evi-dence that altered gene expression occurs during cold accli-mation in a wide variety of plant species (11, 13, 23, 24, 28).Significantly, the number ofchanges that occur are, in general,relatively modest. That is, while differences are evident, theoverall patterns of gene expression in acclimated and nonac-climated plants appear quite similar. This situation is in sharpcontrast to the heat shock and anaerobic stress responseswhich involve extensive changes in gene expression (29).

Establishing the functions of the cold-regulated genes andthe mechanism(s) responsible for their thermoregulation arenow major goals in the study of cold acclimation. To addressthese issues, we have chosen to work with Arabidopsis thal-iana L.(Heyn). We previously established that Arabidopsisbecomes more freezing tolerant in response to low tempera-tures and that a limited number of changes in gene expressionoccur during the cold acclimation process (11, 33); similarresults have been obtained by others (18). Here we report theisolation and initial characterization of four cold-regulated(cor) cDNA clones from Arabidopsis. We show that thetranscript levels for these genes increase dramatically duringcold acclimation, and present evidence that both transcrip-tional and posttranscriptional control mechanisms are in-volved in this regulation. In addition, we show that theexpression of all four cor genes is responsive to drought stressand ABA, a phytohormone that has been implicated as havinga role in the cold acclimation process (2, 3).

MATERIALS AND METHODS

Plant Material

Arabidopsis thaliana L. (Heyn) plants (Landsberg erectaand Columbia) were grown in controlled environment growthchambers as described previously (11). Plants were grownunder continuous illumination of approximately 120 IAE m-2s-' at 22°C for 18 to 20 d, and harvested prior to bolting.

Temperature and Drought Stress Treatments

Cold treatment was at 5°C for varying lengths of time andheat shock was at 37°C for 3 h. In each case, plants weregrown at normal growth temperature (22°C) and placed ingrowth chambers preset to the stress temperature. Light con-

1246

COLD-REGULATED GENES IN Arabidopsis

ditions were the same as those used for normal growth ofplants. Drought stress was induced in plants growing at con-trol temperature (22°C) by withholding water until they be-came visibly wilted (approximately 8 d).

ABA Treatment

Plants grown at 22°C were sprayed to runoff with 100 ,uMABA (mixed isomers) (Sigma) in 0.02% (v/v) Tween-20.2 Thepots were covered with Saran Wrap to slow evaporation, andplaced at 22°C under fluorescent lights in the laboratory or inenvironmental chambers under normal growth conditions.

RNA and DNA Extraction

Total and poly(A+) RNA was extracted as previously de-scribed (11). Total Arabidopsis DNA was prepared from thesupernatant left after LiCl precipitation of RNA from totalnucleic acids (1 1) as follows. The supernatant was diluted to0.5 M LiCl with distilled H20 and the DNA precipitated withethanol. The DNA was then resuspended in buffer containing10 mm Tris-HCl (pH 8.0), 1 mM EDTA, extracted twice withphenol:chloroform:isoamyl alcohol (25:24:1), precipitatedwith ethanol, and resuspended in 10 mM Tris-HCl (pH 8.0),1 mm EDTA. Plasmids were isolated by alkaline lysis andbanding on isopycnic CsCl-ethidium bromide gradients (22).

Preparation and Screening of cDNA Library

Double-stranded cDNA was synthesized from poly(A+)RNA extracted from 3 d cold-treated Arabidopsis (Columbia)plants using the method of Gubler and Hoffman (12). EcoRIlinkers were added to the cDNAs and the DNA fragmentswere inserted into the EcoRI site of X-ZAP (Stratagene).Recombinant phage were packaged in vitro using Pakagene(Promega) and plated on Escherichia coli BB4 (Stratagene).Approximately 105 primary recombinants were obtained. Thelibrary was then amplified once and frozen in SM buffer (SMbuffer is 100 mM NaCl, 8 mM MgSO4, 50 mm Tris-HCl [pH7.5], 0.01% [w/v] gelatin) containing 7% DMSO (v/v) (22).

Differential screening of the cDNA library was done bypreparing plaque lifts on Nytran membranes (Schleicher andSchuell) and hybridizing the lifts with 32P-labeled ss cDNAprobes prepared against poly(A+) RNA isolated from coldacclimated and nonacclimated Arabidopsis (1, 22). Plaqueswhich showed greater hybridization with the probes repre-senting cold acclimated plants were purified and the recom-binant cDNA excised from the phage in pBluescript SK(-)using the biological rescue recommended by the supplier(Stratagene).

Northern and Southern Analysis

Total or poly(A+) RNA was fractionated on denaturingformaldehyde agarose gels (22) and Northern blots preparedon Nytran membranes (Schleicher and Schuell) using 20XSSPE (SSPE is 18 mM NaCl, 10 mM NaPi [pH 7.7], 1 mM

2Abbreviations: Tween 20, polyoxyethylenesorbitan monolaurate20; kbp, kilobase pair; kb, kilobase; ss, single-stranded.

EDTA) as the transfer buffer. The blotted RNA was visualizedby staining with methylene blue (15) as a check for RNAintegrity, efficiency of transfer, and equivalency of loading.The filters were air dried and baked at 80°C under reducedpressure for approximately 2 h.

Total Arabidopsis or plasmid DNA was digested with var-ious restriction enzymes and fractionated by agarose gel elec-trophoresis (22). Southern blots were then prepared on Nytranmembranes using standard methods (22) and baked as de-scribed above.Northern and Southern blots were prewashed in 0.1% (w/

v) SDS, 0.1 X SSPE for 30 min at 65°C before prehybridizationand hybridization. Northern blots were hybridized andwashed using standard methods (22). Southern blots wereprehybridized and hybridized using the nonfat dry milkmethod of Johnson et al. (17) and washed using standardconditions (22). 32P-Labeled probes were made from gel pu-rified cDNA inserts using random oligonucleotide primers(10). Autoradiography was done using AR5 x-ray film (Ko-dak) and Cronex lightning plus intensifying screens (Dupont)at -80°C. In certain Northern analyses, the radioactivity wasquantified using a Betagen 603 Blot Analyzer (Betagen Corp.).

Nuclear Run-On Transcription Experiments

Nuclei were isolated from whole plants that had been coldacclimated for 3 d and from nonacclimated whole plants asdescribed by Feinbaum and Ausubel (9) with the exceptionthat nuclei from nonacclimated plants were harvested at roomtemperature and were not chilled until they were ground inthe nuclei extraction buffer. Run-on transcription assays wereperformed as described by Ausubel et al. (1) using [32P]UTPas the radiolabel. The 32P-RNA was purified and hybridizedas described (1) to either 5 or 10 ,g linearized plasmid DNAthat had been denatured and 'slot blotted' onto Optibindpolyester-backed nitrocellulose (Schleicher and Schuell). Ra-dioactivity hybridizing to the blots was quantified using aBetagen 603 Blot Analyzer.

RESULTS

Isolation of cor cDNA Clones

Previous in vitro RNA translation studies indicated that thelevels of certain mRNAs increased dramatically in cold-accli-mated Arabidopsis (1 1, 18, 33). To isolate clones for such cortranscripts, we constructed a cDNA library in X-ZAP usingpoly(A+) RNA isolated from cold-acclimated plants andscreened the library by differential hybridization using cDNAprobes made against poly(A+) RNA prepared from eitheracclimated or nonacclimated plants. Twenty-five clones thatdisplayed greater hybridization with probes for the cold-accli-mated plants were identified and the inserts 'subcloned' intopBluescript SK(-) using the biological rescue procedure inher-ent in the X-ZAP system. Southern and Northern analysesindicated that the cDNA inserts represented four different cortranscripts. Plasmids pHH28, pHH29, and pHH67 were cho-sen to represent three of the transcripts; each had a singlecDNA insert (Table I). Plasmid pHH7 had two inserts, oneof which, a 1.1 kbp fragment, represented the fourth cor

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Plant Physiol. Vol. 93, 1990

Table I. General Characteristics of cor cDNA ClonesClone Insert Transcript Representation

Designation Size Size in Librarya

kbp kb %

pHH7.2 1.1 1.4 0.02pHH28 1.3 2.5 0.03pHH29 0.6 0.6 0.1pHH67 0.7 0.7 0.1

a Inserts from the various cor cDNA clones were 32P-labeled (10),hybridized with plaque lifts of the cDNA library, and the percentageof the plaques displaying hybridization estimated.

transcript. This fragment was subcloned into pBluescriptSK(-) and the clone designated pHH7.2. A fifth clone, pHH25,like the majority of the clones, did not display differentialhybridization and was picked to represent genes that were notsubject to cold regulation.Northern analysis of total RNA isolated from cold-accli-

mated and nonacclimated plants indicated that the levels ofthe transcripts represented by pHH7.2, pHH28, pHH29, andpHH67 increased dramatically in acclimated plants (Fig. 1).Similar results were obtained when poly(A+) RNA sampleswere analyzed (not shown). When the cold-acclimated plantswere 'deacclimated' by returning them to normal growthtemperature for 1 d, the cor transcripts returned to low levels(Fig. 1). In contrast, the transcript levels for the 'control'clone, pHH25, were approximately equal in acclimated, non-

acclimated, and deacclimated plants (Fig. 1).Additional Northern analyses were performed to determine

the kinetics of accumulation of cor transcripts at low temper-ature (Fig. 2). The data indicate that marked increases in thelevels of the transcripts occurred between 1 and 4 h oftransferring the plants to low temperature (5°C) and that theycontinued to increase in concentration up to about 12 h. Thetranscripts remained at the elevated levels for as long as theplants were kept in the cold (up to 14 d). When the plantswere returned to normal growth temperatures, the levels ofthe transcripts decreased rapidly, returning to those found innonacclimated plants by either 4 h, for pHH7.2 and pHH28,or 8 h, for pHH29 and pHH67.

Southern Analysis of cor Genes

Southern analysis of total Arabidopsis DNA was performedto determine whether the isolated cor genes were present athigh or low copy number. The data show that the hybridiza-tion pattern obtained with each cor cDNA insert was simple;only a single EcoRI or BamHI fragment hybridized with eachprobe (Fig. 3). In addition, the hybridization intensity ob-tained with these probes was only about one-tenth to one-hundredth of that obtained with pAH484 (16), a plasmidcontaining the psbA chloroplast gene (not shown). From thesedata, and the fact that the cor transcripts were polyadenylated,it is probable that the cor genes are low or single copy nucleargenes.

Regulation of cor Gene Expression

Nuclear run-on transcription assays were conducted todetermine whether the temperature regulated expression of

the isolated cor genes involved transcriptional and/or post-transcriptional control mechanisms. The results indicate thatboth forms ofregulation were involved (Table II). Specifically,the transcription rates for the cor genes represented bypHH7.2, pHH28, and pHH29, like the 'constitutive' generepresented by pHH25, increased very little, if at all, in cold-acclimated plants. Thus, the 8.6-, 22-, and 1 -fold increasesdetected in the steady state transcript levels for pHH7.2,pHH28, and pHH29, respectively, in cold-acclimated plantswould appear to be due primarily to posttranscriptional con-trol mechanisms. In contrast, the apparent transcription rateof the cor gene represented by pHH67 increased about10-fold in cold-acclimated plants while the steady state tran-script levels for this gene increased about 26-fold. Thus, itwould appear that the cor gene represented by pHH67 isregulated at both the transcriptional and posttranscriptionallevel, with transcriptional control having a major role.

Expression of cor Genes in Response to ABA, WaterStress, and Heat Shock

It has been shown for a variety of plants (2, 3, 25), includingArabidopsis (20), that exogenous application of ABA at nor-mal growth temperatures can result in increased frost toler-ance. Thus, it was of interest to determine whether the expres-sion of any of the cloned cor genes was responsive to ABA.This was tested by spraying the leaves of Arabidopsis with asolution containing ABA (100 ,tM), isolating total RNA atvarious times, and determining the levels ofthe cor transcriptsby Northern analysis. Remarkably, the results indicate thatthe transcript levels of all four cor genes increased uponapplication of the ABA (Fig. 4). Such increases were notobserved when the solution lacking ABA was sprayed on theplants (not shown). Further, increased transcript levels werenot observed for rbcS or the gene represented by pHH25 (Fig.4). It should be noted that while ABA treatment consistentlyresulted in increased transcript levels for all four cloned corgenes, the absolute level of increase varied substantially fromexperiment to experiment. The cause of this variation isunknown. In addition, the level ofthe increases observed withABA treatment was generally much less than the increasesthat occurred upon cold treatment. Whether this is indicativeof a low 'sensitivity' of the cor genes to ABA or is reflectiveof the method ofABA application is not known.

Because ABA levels are known to increase in water-stressedplants, it was of interest to determine whether cor gene expres-sion was also affected by drought. Thus, plants growing atnormal temperature were subjected to drought stress by with-holding water. At various times, total RNA was isolated andthe levels of the cor transcripts were determined by Northernanalysis. The data indicate that when the relative water con-tent of the plants had fallen to about 80%, a point at whichthe plants still appeared turgid, the cor transcripts were aboutthe same as in the watered plants (Fig. 4). However, when theapproximate relative water content ofthe plants had decreasedto either 70 or 40%, points at which the plants were visiblywilted, the levels of the cor transcripts increased dramatically.In contrast, the level of the transcript represented by pHH25did not increase in the wilted plants.

1 248 HAJELA ET AL.

COLD-REGULATED GENES IN Arabidopsis

25

14 4

0170-7 006 0

N A D

Figure 1. Northern analysis of cor gene tran-scripts. Total RNA (5 ,tg) isolated from Arabidop-sis leaves and stems was fractionated on form-aldehyde agarose gels, transferred to Nytran,and hybridized with 32P-labeled cDNA inserts asindicated. N, Nonacclimated; A, cold acclimatedfor 3 d at 50C; D, deacclimated (3 d at 50Cfollowed by 1 d at 220C). Transcript sizes aregiven in kb.

pH H25

We also examined the expression of the cor genes in re-sponse to another temperature extreme, heat shock. Plantswere transferred from normal growth temperature to 37TC for3 h, conditions that are known to induce the synthesis of heatshock proteins in Arabidopsis (36), total RNA was isolated,and cor transcript levels were determined by Northern analy-sis. As a control, the transcript levels for hsp2O were monitored(using an Arabidopsis hsp2O cDNA clone [14]) and found tobe markedly elevated in the heat-treated plants (Fig. 4). Thelevels of the cor transcripts in these plants, however, were thesame as, or less than, in control plants (Fig. 4). Thus, theaccumulation of cor gene transcripts does not appear to bepart of a general response to environmental stress.

DISCUSSION

Numerous studies with a variety of plant species haveindicated that cold acclimation is associated with changes inprotein synthesis (1 1, 13, 18, 23, 24). Further, in vitro trans-lation experiments using poly(A+) RNA isolated from coldacclimated and nonacclimated plants has suggested that atleast some of these changes in gene expression result fromalterations in the populations of specific transcripts (1 1, 13,18, 23, 24). Here we show directly, by cDNA cloning and

Acclimation De,

Northern analysis, that the transcript levels of four cold-regulated genes from Arabidopsis, referred to as cor genes, doindeed increase (some 10-fold or greater) in cold-acclimatedplants. The levels of the transcripts for these genes increasequickly in response to low temperature, remain at elevatedlevels for as long as the plants are kept in the cold, and quicklydecrease in concentration upon returning the plants to normalgrowth temperatures. Similarly, Mohapatra et al. (25, 26)have isolated cDNA clones of four cold-regulated genes fromalfalfa and have shown that the transcript levels for thesegenes also increase dramatically in cold-acclimated plants.A fundamental goal now is to determine the mechanisms)

responsible for the temperature-regulated accumulation of corgene transcripts. Here, we have begun to address this issue byconducting nuclear run-on transcription experiments. Theresults indicate that both transcriptional and posttranscrip-tional control mechanisms are involved. Specifically, the dataindicate a major transcriptional component for the cold-regulated expression of the cor gene represented by pHH67and a major posttranscriptional component for regulation ofthe cor genes represented by pHH7.2, pHH28, and pHH29.The data also indicate that temperature-regulated expressionof the pHH67 cor gene probably involves posttranscriptional

acclimation

CI< ii¢ <|> s^9 jsb9S( ,c tz(N °°

r'

*"t~~~~~Nwnz xFigure 2. Levels of cor gene transcripts duringcold acclimation and deacclimation. Total RNA(5 qg) isolated from Arabidopsis leaves andstems was fractionated on formaldehyde aga-rose gels, transferred to Nytran, and hybridizedwith 32P-labeled cDNA inserts as indicated. Ac-climation was at 50C and deacclimation was at220C. The plants used in the deacclimation ex-periment had been cold acclimated for 3 d.

N A D N A D N A D N A D

pH H 7-2 pH H28 pH H29 pHH67

pHH-12

pHH28

pHH29

pHH67-W-9wW.law,

I.... 'W"

1 249

Plant Physiol. Vol. 93, 1990

B

..i am

_

Figure 3. Southern analysis of Arabidopsis ge-

nomic DNA. Total DNA was extracted from Ar-abidopsis leaves and stems, digested with eitherBamHl (B) or EcoRI (E), the fragments fraction-ated on agarose gels, and transferred to Nytran.The membranes were then hybridized with 32p_labeled inserts from the cDNA clones as indi-cated. Sizes of the restriction fragments aregiven in kbp.

pH H 2 r/ ) H f

control as well. Efforts will now be directed at characterizingthe cold-inducible promoter of the pHH67 cor gene anddetermining whether the posttranscriptional control mecha-nism(s) involves regulation at the level ofRNA stability, RNAprocessing, or RNA transport.As mentioned previously, it has been shown for a variety

of plant species (2, 3, 25), including Arabidopsis (20), thatexogenous application of ABA can increase the freezing tol-erance of plant cells. It has, therefore, been suggested thatABA may have a fundamental role in the cold acclimationprocess. The hypothesis of Chen et al. (2) is that low temper-ature triggers an elevation of endogenous ABA levels whichin turn induces the synthesis of proteins responsible for in-creased frost tolerance. If this were true, then one wouldexpect that certain genes would be regulated by both lowtemperature and ABA. Indeed, in vivo protein labeling and invitro translation experiments with a number of plant specieshave indicated that the levels of certain transcripts increase in

response to both low temperature and exogenously appliedABA at normal growth temperature (20, 25, 28). In addition,our results directly show that the transcript levels of fourdifferent Arabidopsis cor genes are responsive to both lowtemperature and ABA (Fig. 4). Similarly, Mohapatra et al.(25) have shown that the transcript levels for one of the cold-regulated genes that they isolated from alfalfa increases inresponse to ABA.

It can be concluded that certain genes are regulated by bothABA and low temperature. It is less clear, however, whetherABA normally mediates the changes in gene expression thatoccur in response to low temperature. Endogenous ABA levelsappear to increase in response to low temperature in some

plants (2, 6, 19). However, in the case of Solanum commer-

sonii, the increase is only transient (2). Further, in alfalfa, a

plant that increases in frost tolerance and undergoes changesin gene expression in response to low temperature (25), theABA levels do not appear to increase in response to low

Table II. Relative Transcription Rates and Transcript Levels of cor Genes in Cold Acclimated VersusNonacclimated Plants

cDNA CloneParameter

pHH7.2 pHH28 pHH29 pHH67 pHH25

-fold increase in cold-acclimated plantsTranscription rate 1.2 ± 0.2 1.8 ± 0.3 2.4 ± 1.1 9.6 ± 2.3 1.7 ± 0.3Transcript levelb 8.6 ± 1.9 22 ± 5.0 11 ± 4.3 26 ± 8.4 0.8 ± 0.03

a Three independent sets of nuclei were isolated from control and 3 d cold-acclimated plants and therelative transcription rates were determined by nuclear run-on transcription assays as described in"Materials and Methods." Values are the mean (± SE) fold increase in transcription rate in cold-acclimatedplants. b Total RNA was isolated from control and cold-acclimated plants (either leaves and stemsor whole plants) and the steady state transcript levels of the indicated genes were determined byNorthem analysis as described in "Materials and Methods." Values are the mean (± SE) fold increase intranscript level in cold-acclimated plants. n = 4 for the cor genes; n = 2 for pHH25. Two of thedeterminations for the cor genes and one for pHH25 were from the plants used in the transcription rateassays. The values for the cor genes are minimum estimates since they do not include determinationswhere transcripts were not detected in control plants (i.e. cases in which the increase was 'infinite').

HAJELA ET AL.1 250

COLD-REGULATED GENES IN Arabidopsis

RWC (%)93 80 70 43

pHH7-2 pHH772

pHH28

pHH29 O

pHH28

pHH29

pHH67 pHH67

pHH25 i PHH25s.,w:

.

rbcS

pHH72

pHH28

pHH29

pHH67

pHH25

hsp20

Figure 4. Effect of ABA treatment (A), drought(B), and heat shock (C) on cor transcript levelsABA was applied as a 100 Mm foliar spray asdescribed in "Materials and Methods." Droughttreatment was performed by withholding waterto the indicated relative water content. Heatshock was at 370C for 3 h. Total RNA wasextracted from the plants, 5 Mg fractionated onformaldehyde agarose gels, transferred to Ny-tran, and hybridized with 32P-labeled cDNA in-serts as indicated. Abbreviations: RWC, relativewater content; HS, heat shock; C, control (non-heat shocked).

temperature (34). Clearly, further experimentation will berequired to establish the link between low temperature andABA regulated gene expression.A final point that deserves discussion is the regulation of

cor genes by water stress. Presumably, the increase in cor

transcript levels that occurred in the drought-stressed plants(Fig. 4) was due to increased ABA levels (ABA increases inplants in response to water stress). The intriguing questionthen is whether the expression of cor genes under conditionsof water stress is fortuitous or, alternatively, whether thesegenes might have functions in both cold acclimation anddrought tolerance. There is reason to think that the latter maybe true. When plant tissues are frozen under equilibriumconditions, the water in the extracellular spaces freezes result-ing in a lowered water potential. Consequently, intracellularliquid water moves across the plasma membrane into theextracellular spaces causing cell dehydration. Tolerance tofreezing must, therefore, include tolerance to water stress (thestress can be quite severe as a freeze to -10IC results in a waterpotential of about -12 MPa). Thus, it is reasonable to thinkthat freezing and drought tolerance might have certain mech-anisms in common, and that such mechanisms might includefunctions encoded by common genes.

In this context, it is interesting to note that drought stresshas been observed to increase the frost tolerance of wheat(31), rye (31), and cabbage (5). Moreover, we have found thatArabidopsis cor genes encode four polypeptides, having ap-parent masses of 160, 47, 24, and 15 kD, that share an unusualbiochemical property: they are not precipitated by boiling inaqueous solution (C Lin, WW Guo, E Everson, MFThomashow, unpublished data). The significance here is thata family of proteins that are believed to have roles in desic-cation tolerance, the LEA proteins (late embryogenesis abun-dant), also remain soluble upon boiling (4). Dure et al. (8)

originally described these proteins in cotton where they werefound to be synthesized during late embryogenesis, a timejust prior to when the embryos undergo desiccation. Morerecently, it has been recognized that lea-related genes arepresent in a wide range of plants (7), and that in at least somecases, the levels of lea transcripts increase in response to waterstress or ABA application (4, 27). It will, therefore, be ofinterest to determine the relationship between the cor and leagenes and to determine whether any of the polypeptidesencoded by these genes have cryoprotective or desiccationprotective properties. The Arabidopsis cor cDNA clonespHH7.2 and pHH67 described here should aid greatly in suchefforts since hybrid-select and hybrid-arrest translation exper-iments indicate that they code for the 47 and 15 kD 'boiling-stable' COR polypeptides, respectively (C Lin, WW Guo, EEverson, MF Thomashow, unpublished data).

ACKNOWLEDGMENTS

We wish to thank Harry Klee, Lee McIntosh, and ElizabethVierling for providing clones of rbcS, psbA, and hsp2O, respectively,and Lee McIntosh, Rebecca Grumet, and Suzanne Hugly for criticalreadings of the manuscript.

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