overexpression of the arabidopsis cbf3 …...overexpression of the arabidopsis cbf3 transcriptional...

Overexpression of the Arabidopsis CBF3 TranscriptionalActivator Mimics Multiple Biochemical ChangesAssociated with Cold Acclimation1

Sarah J. Gilmour, Audrey M. Sebolt, Maite P. Salazar, John D. Everard, and Michael F. Thomashow*

Department of Crop and Soil Sciences, Michigan State University, East Lansing, Michigan 48824–1325(S.J.G., A.M.S., M.P.S., M.F.T.); and Dupont Agricultural Products Biotechnology Experiment Station402/5238, Wilmington, Delaware 19880–0402 (J.D.E.)

We further investigated the role of the Arabidopsis CBF regulatory genes in cold acclimation, the process whereby certainplants increase in freezing tolerance upon exposure to low temperature. The CBF genes, which are rapidly induced inresponse to low temperature, encode transcriptional activators that control the expression of genes containing the C-repeat/dehydration responsive element DNA regulatory element in their promoters. Constitutive expression of either CBF1 or CBF3(also known as DREB1b and DREB1a, respectively) in transgenic Arabidopsis plants has been shown to induce the expressionof target COR (cold-regulated) genes and to enhance freezing tolerance in nonacclimated plants. Here we demonstrate thatoverexpression of CBF3 in Arabidopsis also increases the freezing tolerance of cold-acclimated plants. Moreover, we showthat it results in multiple biochemical changes associated with cold acclimation: CBF3-expressing plants had elevated levelsof proline (Pro) and total soluble sugars, including sucrose, raffinose, glucose, and fructose. Plants overexpressing CBF3 alsohad elevated P5CS transcript levels suggesting that the increase in Pro levels resulted, at least in part, from increasedexpression of the key Pro biosynthetic enzyme D1-pyrroline-5-carboxylate synthase. These results lead us to propose thatCBF3 integrates the activation of multiple components of the cold acclimation response.

Understanding the mechanisms that plants haveevolved to survive freezing is of basic scientific in-terest and has the potential to offer new strategies toimprove the freezing tolerance of agronomic plants.Toward this end, many investigators have focused onstudying the phenomenon of cold acclimation, theprocess whereby certain plants increase in freezingtolerance in response to low temperatures (Hughesand Dunn, 1996; Thomashow, 1999). One line of in-vestigation has been to identify genes that are in-duced at low temperature and to determine whetherthey have roles in freezing tolerance. This has led tothe identification of scores of cold-induced genes,many of which encode either LEA (late embryogen-esis abundant) or novel polypeptides (Thomashow,1998). Recent results obtained with Arabidopsis in-dicate that at least one COR (cold-regulated) gene hasa role in freezing tolerance. Overexpression ofCOR15a, which encodes a novel polypeptide that istargeted to the chloroplasts, has been shown to in-crease the freezing tolerance of chloroplasts in vivoand protoplasts in vitro (Artus et al., 1996). This in-crease in freezing tolerance results from the COR15a-

encoded protein stabilizing membranes against freez-ing injury (Artus et al., 1996; Steponkus et al., 1998).There is evidence that the mature COR15a polypep-tide, COR15am, acts directly as a cryoprotective pro-tein that decreases the propensity of lipid bilayers toform deleterious hexagonal II phase lipids (Steponkuset al., 1998), a major type of freeze-induced membranelesion that occurs in nonacclimated plants (Steponkuset al., 1993).

Whereas overexpression of COR15a has a detect-able effect on freezing tolerance of chloroplasts andprotoplasts, the effect is small (1°C–2°C; Artus et al.,1996; Steponkus et al., 1998). Moreover, it has little ifany effect on whole-plant freezing survival (Jaglo-Ottosen et al., 1998). Expression of the entire batteryof COR genes, which includes the COR6.6, COR47,and COR78 gene pairs (in addition to the COR15 genepair), however, does (Jaglo-Ottosen et al., 1998). Theability to express all of the COR genes in concert atwarm temperature was made possible by the dis-covery of the CBF family of transcriptional activa-tors (Stockinger et al., 1997; Gilmour et al., 1998),also known as DREB1 proteins (Liu et al., 1998; Shin-wari et al., 1998). COR6.6, COR15a, COR47, andCOR78, and presumably other yet to be discoveredCRT (C-repeat)/DRE (dehydration responsiveelement)-regulated COR genes, contain in their pro-moters a cold- and dehydration-responsive DNAregulatory element known as the CRT/DRE (Bakeret al., 1994; Yamaguchi-Shinozaki and Shinozaki,1994). The first CRT/DRE binding factor to be dis-

1 This work was supported in part by a subcontract (no. 593–0219 – 06) under the U.S. Department of Agriculture/CooperativeState Research, Education and Extension Service CooperativeAgreement (no. 96 –34340 –2711) North Central Biotechnology Ini-tiative and by funds from Mendel Biotechnology, Inc., and theMichigan Agricultural Experiment Station.

* Corresponding author; e-mail [email protected]; fax517–353–5174.

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covered, CBF1 (CRT/DRE binding factor 1), wasinitially shown to activate expression of reportergenes in yeast that carried the CRT/DRE element(Stockinger et al., 1997). This indicated that the pro-tein, which has an AP2/EREBP DNA binding mo-tif (Riechmann and Meyerowitz, 1998), was a tran-scriptional activator. Overexpression of CBF1 inArabidopsis was subsequently shown to activateexpression of the entire battery of known CRT/DRE-regulated COR genes and to enhance whole plantfreezing survival without a low temperature stimu-lus (Jaglo-Ottosen et al., 1998).

Additional studies have shown that CBF1 is amember of a small gene family encoding nearly iden-tical proteins (Gilmour et al., 1998; Shinwari et al.,1998). The genes, CBF1, CBF2, and CBF3 (also knownas DREB1b, DREB1c, and DREB1a, respectively), arelocated in tandem on chromosome 4 (Gilmour et al.,1998; Shinwari et al., 1998). Overexpression of CBF3in Arabidopsis, like overexpression of CBF1, acti-vates COR gene expression and enhances freezingtolerance at warm nonacclimating temperatures (Liuet al., 1998; Kasuga et al., 1999). All three CBF genesare cold-induced; CBF transcript levels increasewithin 15 min of transferring plants to low tempera-ture followed at approximately 2 h by accumulationof transcripts for the target CRT/DRE-regulated CORgenes (Gilmour et al., 1998; Shinwari et al., 1998). Themechanism whereby the CBF genes are activated bylow temperature is not known but does not appear toinvolve autoregulation (Gilmour et al., 1998).

Many biochemical changes occur in plants duringcold acclimation in addition to COR gene expressionand are likely to have roles in freezing tolerance. It iswell documented that lipid composition changesduring cold acclimation in a wide range of plants andthere are compelling data to indicate that this con-tributes to greater freezing tolerance (Steponkus etal., 1993). In a similar manner, the levels of Pro andSuc increase in Arabidopsis (McKown et al., 1996;Wanner and Junttila, 1999) and other plants (Guy etal., 1992; Koster and Lynch, 1992) during cold accli-mation and are likely to have roles in freezing toler-ance. There is evidence that Pro can protect bothmembranes and proteins against freeze-induceddamage in vitro (Rudolph and Crowe, 1985; Carpen-ter and Crowe, 1988) and direct evidence that in-creased levels of Pro enhances whole plant freezingtolerance (Nanjo et al., 1999). Suc and other simplesugars have also been shown to be effective cryopro-tectants in vitro (Strauss and Hauser, 1986; Carpenterand Crowe, 1988) and there is correlative evidenceindicating a role in freezing tolerance in cold-acclimated plants (Guy et al., 1992; Koster and Lynch,1992; Wanner and Junttila, 1999). A question thusraised is whether the CBF transcription factors arelimited to activating the expression of COR genesencoding cryoprotective polypeptides, or alterna-tively, have a role in activating multiple components

of the cold acclimation response. The results pre-sented here support the latter model.

RESULTS

Identification of Transgenic Arabidopsis Plants ThatOverexpress CBF3

Transgenic Arabidopsis plants that overexpressCBF3 at normal growth temperature were created byplacing the CBF3 coding sequence under control ofthe cauliflower mosaic virus (CaMV) 35S promoterand transforming the construct into Arabidopsisplants using the floral dip transformation procedure.Twenty-two independent lines were identified inwhich the T2 plants segregated 3:1 for kanamycinresistance (the selectable marker carried on the trans-formation vector). These lines presumably carried asingle active T-DNA locus. The kanamycin resistantT2 plants were then screened by western analysis forconstitutive expression of COR15a, a target gene ofthe CBF transcriptional activators. Eight lines wereidentified that constitutively produced the COR15amprotein at levels that were equal to or greater thanthat which occurred in cold-acclimated wild-typeplants. However, further analysis indicated that infive of these lines, COR15a expression was not uni-form among the plants; i.e. although all of the plantswere kanamycin resistant, not all produced theCOR15am polypeptide. This variation may have re-sulted from gene silencing events. Regardless, theselines were not used further for this study.

Three independent transgenic lines (A28, A30, andA40) were identified that produced the COR15ampolypeptide at high levels uniformly among plantsgrown at normal temperatures. Northern analysis(Fig. 1) indicated that the transcript levels for CBF3were about equal in nonacclimated and cold-treatedA28, A30, and A40 plants and were much greaterthan those observed in either nonacclimated or cold-treated control plants (i.e. non-transformed plants ortransgenic plants carrying the transformation vectorwithout an insert). The transcript levels for two targetCOR genes, COR15a and COR6.6, were also nearlyequal in nonacclimated and cold-treated A28, A30,and A40 plants and approximated the levels ob-served in cold-acclimated control plants (Fig. 1).Western analysis (Fig. 2) indicated that the proteinsencoded by COR15a and COR6.6 were present inboth nonacclimated and cold-acclimated A28, A30,and A40 plants at 3- to 5-fold higher levels than thosefound in cold-acclimated control plants.

Effects of CBF3 Overexpression on Vegetative Growth,Time to Flowering, and Freezing Tolerance

Liu et al. (1998) have reported that transgenic Ara-bidopsis plants overexpressing CBF3 (DREB1a) havea “dwarf” phenotype. This was also true of the A28,A30, and A40 transgenic plants. After the same num-

Role of Arabidopsis CBF3 in Cold Acclimation

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ber of days of vegetative growth at normal temper-ature, the size of the leaves and overall dimensions ofthe CBF3-expressing plants were considerably lessthan those of the control plants (Fig. 3, A and B).Additional effects on growth that were not previ-ously noted were also evident. One was that CBF3-expressing plants had a pronounced prostrategrowth habit; whereas the leaves of the controlplants generally had an upright stature, those of thetransgenic plants laid flat to the soil (Fig. 3C). TheCBF3-expressing plants also had much shorter pet-ioles when compared with those of the controlplants (Fig. 3D) and the leaves had a slight bluish-green tint (Fig. 3, A and B). Also, there was a sub-stantial difference in time to flowering between thecontrol and CBF3-expressing plants; i.e. controlplants bolted and formed flowers well before theCBF3-expressing plants did (Fig. 3E). In one exper-iment, for instance, the control plants began to boltat 17 d, whereas the A40, A30, and A28 plants took21, 26, and 28 d, respectively, to initiate bolting

(Table I). The CBF3-expressing plants went on toform flowers and set seed, although as noted by Liuet al. (1998), the final plant mass and seed yield wereconsiderably less than that obtained with controlplants (Fig. 3F). The lower yield of seed was due atleast in part to the CBF3-expressing plants produc-ing fewer axillary shoots. The delay in floweringobserved in the CBF3-expressing plants, signifi-cantly, did not “simply” involve a slower overallgrowth rate, but appeared to involve a developmen-tal delay in flowering. In one experiment, for in-stance, the control plants produced an average of 4.5and 4.6 leaves per rosette, whereas the A40, A30,and A28 transgenic plants produced 6.0, 9.7, and12.5 leaves per rosette, respectively (Table I).

Overexpression of CBF3 (DREB1a) (Liu et al., 1998;Kasuga et al., 1999), like overexpression of CBF1(Jaglo-Ottosen et al., 1998), has been reported to in-crease the freezing tolerance of nonacclimated plants.Similarly, we found that nonacclimated control plantswere killed by freezing at 26°C for 24 h whereasnonacclimated CBF3-expressing plants were not; re-sults for Arabidopsis (L.) Heynh. ecotype Was-silewskija (Ws)-2 and A30 plants are shown in Figure4A. To quantify the increase in freezing tolerance wecarried out electrolyte leakage tests. The results indi-cated that the freezing tolerance of nonacclimatedCBF3-expressing plants was greater than that of thenonacclimated control plants; nonacclimated controlplants had an EL50 (temperature that caused a 50%leakage of electrolytes) of approximately 24.5°C,whereas the three CBF3-expressing lines had EL50values of approximately 28°C (Fig. 4B). The freezingtolerance of cold-acclimated CBF3-expressing plantswas significantly greater than that of both nonaccli-mated CBF3-expressing plants and cold-acclimatedcontrol plants. CBF3-expressing plants that had beencold acclimated for 7 d had EL50 values of 211°C andlower (Fig. 4, C and D). In these particular experi-ments, the cold-acclimated control plants had EL50values of approximately 26°C (Fig. 4, C and D). Inother experiments, we have obtained EL50 values aslow as 28°C, but have never observed cold-acclimated control Ws-2 plants with EL50 values aslow as those obtained with the cold-acclimated CBF3-expressing plants.

Figure 1. Transcript levels of CBF3 and target COR genes in trans-genic plants overexpressing CBF3. Northern analysis of total RNA (20mg for CBF3; 5 mg for the other genes) prepared from control Arabi-dopsis Ws-2 and B6 plants and from CBF3-expressing A40, A28, andA30 plants. Plants were either grown at 20°C (W) or at 20°C and thencold treated at 5°C for 7 d (C). eIF4a (eukaryotic initiation factor 4a)is a constitutively expressed gene used as a loading control (Metz etal., 1992). Upon long exposure, CBF3 transcripts can be detected inthe total RNA samples prepared from cold-treated Ws-2 and B6plants (not shown).

Figure 2. Protein levels of COR15am and COR6.6in transgenic plants overexpressing CBF3. West-ern analysis of total soluble protein (50 mg) pre-pared from control Arabidopsis Ws-2 and B6plants and from CBF3-expressing A40, A30, andA28 plants. Plants were either grown at 20°C(W), or grown at 20°C and then cold treated at5°C for 7 d (C). Protein transfers were treatedwith antiserum made to recombinant COR15amand COR6.6 polypeptides (Gilmour et al.,1996).

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Overexpression of CBF3 Affects Pro Metabolism

Pro levels increase during cold acclimation in Ara-bidopsis and other plants (Koster and Lynch, 1992;Alberdi et al., 1993; McKown et al., 1996; Wanner andJunttila, 1999). We reasoned that if the increase in Pro

that occurs in Arabidopsis with cold acclimation wasbrought about by genes that were regulated by theCBF activators, then overexpression of CBF3 mightresult in elevated levels of Pro in nonacclimatedplants. This was the case. Under nonacclimating

Figure 3. Growth characteristics of CBF3-expressing transgenic plants. A, Non-transformed Ws-2 plants and transgenicCBF3-expressing A30 plants after 2 weeks growth at 20°C. B, Non-transformed Ws-2 and transgenic B6 “control” plants andtransgenic CBF3-expressing A40, A30, and A28 plants after 15 d growth at 20°C. C and D, Non-transformed Ws-2 plantsand transgenic CBF3-expressing A28 plants after 16 d growth at 20°C. E, Non-transformed Ws-2 plants and CBF3-expressingA30 and A28 plants after 5 weeks growth at 20°C. F, As in E except after 9 weeks of growth at 20°C.




growth conditions, the free Pro levels in the CBF3-expressing plants were approximately 5-fold higherthan they were in the control plants, levels whichwere about the same as those found in cold-acclimated control plants (Fig. 5). The Pro levels inthe CBF3-expressing plants increased further (ap-proximately 2-fold) upon cold acclimation and were2- to 3-fold higher than those found in the cold-acclimated control plants (Fig. 5).

The Pro biosynthetic enzyme D1-pyrroline-5-car-boxylate synthase (P5CS) has a key role in determin-ing Pro levels in plants (Yoshiba et al., 1997). Be-cause of this, and that P5CS transcript levels havebeen shown to increase in Arabidopsis in response tolow temperature (Xin and Browse, 1998), it was ofinterest to determine whether P5CS transcript levelswere elevated in the CBF3-expressing plants. North-ern analysis indicated that they were; P5CS transcriptlevels were approximately 4-fold higher in nonaccli-mated CBF3-expressing plants than they were in non-acclimated control plants and were about equal tothose found in the control plants that had been cold-treated for 1d (Fig. 6). The P5CS transcript levels in 7-dcold-acclimated CBF3-expressing plants were 2- to3-fold higher than in cold-acclimated control plants(Fig. 6), a finding that was consistent with the 2- to3-fold higher levels of Pro found in the cold-acclimated CBF3-expressing plants (Fig. 5).

Overexpression of CBF3 Affects Sugar Metabolism

The accumulation of simple sugars is another com-monly observed biochemical change that occurs withcold acclimation in Arabidopsis and other plants(Guy et al., 1992; Koster and Lynch, 1992; McKown etal., 1996; Wanner and Junttila, 1999). Thus, it was ofinterest to determine whether CBF3 expression af-fected the sugar levels in plants. This was tested bydetermining the amount of total soluble sugars incontrol and CBF3-expressing plants at both nonaccli-mating and cold-acclimating temperatures. The re-sults indicated (Fig. 7) that the levels of total sugarsin nonacclimated CBF3-expressing plants were ap-proximately 3-fold higher than those in nonaccli-mated control plants. Upon cold acclimation, sugarlevels went up in both the control and CBF3-expressing plants approximately 2-fold; thus, sugarlevels in the CBF3-expressing plants remained ap-

Figure 4. Effect of CBF3 overexpression on plant freezing tolerance.A, Seedlings of control Arabidopsis Ws-2 plants and CBF3-expressingA30 plants were grown at 20°C on solid medium and then frozen at22°C for 24 h followed by 24 h at 26°C. B, Control ArabidopsisWs-2 plants and CBF3-expressing A40, A30, and A28 plants weregrown at 20°C, and the freezing tolerance of leaves was measuredusing the electrolyte leakage test. C and D, Same as B except thatplants were grown at 20°C followed by 7-d-cold acclimation at 5°C.

Table I. Effects of CBF3 expression on time to flowering and ro-sette leaf number

Plants Time to Flowering (d)a Rosette Leaves Per Plant (n)

Ws-2 17 4.5 (8)B6 17 4.6 (8)A40 21 6.0 (7)A30 26 9.7 (15)A28 28 12.5 (8)

a Period of time between planting seeds in soil and appearance offirst flower buds in a population of “n” plants.

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proximately 3-fold higher in the control plants. Totalsoluble sugars notably accounted for as much as 20%of the total dry weight of plant material in the cold-acclimated CBF3-expressing plants.

Analysis of the total soluble sugars by gas chroma-tography was performed on samples prepared fromWs-2 and B6 control plants and A28, A30, and A40CBF3-expressing plants. The results indicated thatCBF3 expression brought about increased levels ofseveral sugars, namely Glc, Fru, Suc, and raffinose. Arepresentative experiment comparing nonacclimated

and cold-acclimated Ws-2 and A28 plants is pre-sented in Figure 8. The results indicate that the levelsof Glc, Fru, Suc, and raffinose increased in the Ws-2control plants upon cold acclimation by approxi-mately 2- to 5-fold depending on the particular sugar.In nonacclimated A28 plants, the levels of the foursugars were equal to or higher than they were in thecold-acclimated Ws-2 plants and increased furtherupon cold acclimation. In the cold-acclimated A28plants, Suc plus raffinose accounted for approxi-mately 12% of the total dry weight and Glc plus Fruaccounted for approximately 8%.

Two enzymes that have key roles in determiningthe levels of Suc in plant cells are Suc-phosphate-synthase (SPS) and Suc synthase (SuSy; Winter andHuber, 2000). Transcript levels for genes encodingboth SPS (Strand et al., 1997) and SuSy (Dejardin etal., 1999) have been shown to increase in Arabidopsisin response to low temperature. Thus, it was of in-terest to determine whether CBF3 had an effect onthe expression of these genes. As previously re-ported, we found that the transcript levels for boththe SPS and SuSy genes increased in response to lowtemperature (Fig. 6). However, these increases didnot appear to involve the CBF transcriptional activa-tors as there was little if any difference in the tran-script levels for the SPS and SuSy encoding genes inthe CBF3-expressing and control plants (Fig. 6). Thus,the effects of CBF3 on sugar levels do not appear tobe mediated by altering transcription of the SPS orSuSy encoding genes.

Figure 5. Effect of CBF3 expression on Pro levels. Free Pro levelswere determined in leaf tissue from control Arabidopsis Ws-2 and B6plants and CBF3-expressing A40, A30, and A28 plants grown at 20°C(warm), or plants grown at 20°C and cold-treated at 5°C for 7 d (7-dcold).

Figure 6. Effect of CBF3 expression on transcript levels of genes involved in Pro and sugar metabolism. Northern analysisof total RNA (20 mg for CBF3; 5 mg for other genes) isolated from control Arabidopsis Ws-2 and B6 plants and fromCBF3-expressing A40, A30, and A28 plants. Plants were grown at 20°C then cold-treated at 5°C for the times indicated. Theblots were hybridized with probes for CBF3, COR78, P5CS2, Suc synthase (SuSy), Suc-phosphate synthase (SPS), and eIF4a,a constitutively expressed gene used as a loading control (Metz et al., 1992).




DISCUSSION

The increase in freezing tolerance that occurs withcold acclimation is thought to involve the activation ofmultiple freezing tolerance mechanisms. Here weshow that overexpression of the CBF3 transcriptionalactivator results in multiple biochemical changes thatare commonly observed to occur in plants during coldacclimation. We show specifically that CBF3 overex-pression in Arabidopsis results in the accumulation ofCOR polypeptides, the accumulation of Pro, and theaccumulation of soluble sugars including Suc, raffin-ose, Glc, and Fru. There is evidence to indicate thateach of these “classes” of biochemical alterations—COR polypeptides (Thomashow, 1998), Pro (Rudolphand Crowe, 1985; Carpenter and Crowe, 1988; Nanjoet al., 1999), and sugars (Strauss and Hauser, 1986;Carpenter and Crowe, 1988; Koster and Lynch, 1992;Wanner and Junttila, 1999)—contribute to an enhance-ment of freezing tolerance. It is interesting that we alsofound that Arabidopsis plants overexpressing CBF3displayed a prostrate growth habit, a phenotype thathas been associated with cold acclimation, and in-creased freezing tolerance in other plants (Omran etal., 1968; Roberts, 1990). Taken together, these findingslead us to propose that Arabidopsis CBF3 is a keyregulatory gene that acts to integrate the activation ofmultiple mechanisms that work in concert to enhancefreezing tolerance. Presumably CBF1 and CBF2 haveoverlapping, if not identical, roles to those of CBF3.

How does CBF3 bring about the biochemicalchanges that we observed? In the case of COR geneinduction, the answer is known; CBF3 binds to CRT/DRE regulatory elements located in the promoters ofthese genes and activates their expression (Gilmouret al., 1998). Whether CBF3 also binds to the promot-ers of genes directly involved in sugar and Pro syn-thesis remains to be determined. However, our re-sults indicate that CBF3 overexpression causes anincrease in the transcript levels for at least one of thetwo known P5CS Pro biosynthetic genes (Strizhov et

al., 1997). A CBF3-induced increase in P5CS geneexpression thus seems likely to contribute to the in-crease in Pro levels observed in the CBF3-expressingplants. An examination of the promoter region of theP5CS2 gene (Strizhov et al., 1997; GenBank accessionno. X86788) reveals that the core conserved sequenceof the CRT/DRE regulatory element, CCGAC (Bakeret al., 1994; Yamaguchi-Shinozaki and Shinozaki,1994), is present twice within 350 nucleotides up-stream of the ATG start codon. Whether CBF3 bindsto these sequences and activates expression of theP5CS2 gene, however, remains to be determined. Itshould be noted that Kasuga et al. (1999) did notobserve any effect of CBF3 (DREB1A) expression onthe levels of P5CS transcripts, nor did they observean increase in P5CS transcript levels in response tolow temperature, as we observed here (Fig. 6) and asXin and Browse (1998) previously reported. The rea-son for this difference is not immediately obvious,but may be due, in part, to the P5CS probes used; weused a cDNA for the P5CS2 gene whereas Kasuga etal. (1999) used a probe for P5CS1.

Xin and Browse (1998) have identified an Arabi-dopsis gene, ESKIMO1 (ESK1), that affects the levelsof Pro and sugars and has a major effect on freezingtolerance. Whereas wild-type plants grown at normaltemperatures had a median lethal temperature (LT)50of 22.8°C in a whole plant freeze test, plants carryingan esk1 mutation had a median lethal temperature of210.6°C. The concentrations of free Pro and totalsugars were elevated in the esk1 mutant plants about30-fold and 2-fold, respectively. In addition, P5CStranscript levels in nonacclimated esk1 plants were

Figure 7. Effect of CBF3 expression on levels of total soluble sugars.Total soluble sugars were determined for leaf tissue from controlWs-2 and B6 plants and CBF3-expressing A40, A30, and A28 plantsgrown at 20°C (warm) or plants grown at 20°C and cold treated at5°C for 7 d (7-d cold).

Figure 8. Effect of CBF3 expression on the levels of specific solublesugars. Total sugars were prepared from leaf tissue of control Ws-2plants and CBF3-expressing A28 plants grown at 20°C (white bars) orat 20°C followed by 7 d at 5°C (black bars) and the levels ofindividual sugars determined by gas chromatography.

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approximately 8-fold higher than in nonacclimatedwild-type plants. The esk1 mutation, significantly,did not affect expression of the COR genes; the tran-script levels for COR6.6, COR15a, COR47, and COR78remained low under normal growth conditions in theesk1 plants and were highly induced in response tolow temperature. Therefore, Xin and Browse (1998)reasonably argued that it is not appropriate to con-sider cold acclimation as a simple, linear signalingpathway activating the full set of processes respon-sible for increasing freezing tolerance. Instead, theyproposed a model in which parallel or branchedsignaling pathways activate “distinct suites” of coldacclimation responses. As envisioned, activation ofone pathway would be able to result in considerablefreezing tolerance without support from other com-ponents. Because esk1 plants did not overexpress theCOR genes, Xin and Browse (1998) proposed that esk1defines a signaling pathway of cold acclimation thatis distinct from that which mediates expression of theCOR genes.

Our findings with the CBF3 overexpressing plantsdo not contradict the general cold acclimation modelof Xin and Browse (1998), but suggest a more inte-grated circuitry for cold acclimation signaling. Inparticular, our results provide evidence that the syn-thesis of COR proteins, Pro and sugars are coordi-nately regulated and that CBF3 has a key role in thisregulation. The fact that the esk1 mutation affects thelevels of P5CS expression, but not COR gene expres-sion, can be explained in ways that do not necessitatethe existence of distinct signaling pathways for Proand COR protein synthesis. The two available esk1alleles are recessive and, as mentioned above, causean 8-fold increase in P5CS transcript levels. Thus,ESK1 appears to be a negative regulator of P5CStranscription at warm temperatures. One simple pos-sibility is that ESK1 is a transcriptional repressor thatbinds to the promoter of one or both of the Arabido-spis P5CS genes (Strizhov et al., 1997) at warm tem-perature and keeps transcription at a relatively lowlevel. At low temperature, CBF3 could either directlybind to the P5CS promoter(s) and overcome repres-sion by ESK1, or induce the expression of some otherprotein that inactivates ESK1. In these scenarios, theesk1 mutation would not affect expression of the CORgenes because the COR genes would not have theESK1 binding sequence. Of course, more complexmodels could also explain the current findings. Thefundamental point is that the new information pro-vided by this study argues for an integrated controlof cold acclimation with CBF3, and presumably theother CBF proteins as well, having a key role ininducing multiple biochemical and physiologicalchanges that act to increase plant freezing tolerance.

The finding that CBF3 overexpression results inaccumulation of COR polypeptides, Pro, and sugarssuggests that a considerable portion of the cold ac-climation response falls under control of the CBF

genes. However, our results also indicate that thefreezing tolerance of the CBF3 overexpressing plantsincreased further in response to low temperature(Fig. 4). Thus, overexpression of CBF3 at warm tem-perature did not result in maximum freezing toler-ance even though expression of its target genes, asjudged by the levels of COR proteins (Fig. 2), wassomewhat greater than that which occurred in cold-acclimated control plants. These findings could signifythat CBF3 controls only a subset of low temperature-induced freezing tolerance genes and that additionalnon-CBF3-controlled genes might be induced in re-sponse to low temperature and activate additionalfreezing tolerance mechanisms. This would be akin tothe parallel pathway model proposed by Xin andBrowse (1998). However, it remains possible that CBF3actually regulates all of the genes that are important incold acclimation, but that the activities of the proteinsencoded by these genes might be greater at low tem-perature; the proteins might be activated in response tophosphorylation catalyzed by a temperature-regulatedprotein kinase, for instance, or their action affectedby changes in metabolite pool levels brought aboutby slower plant growth at low temperature. There isalso the more trivial explanation that expression ofCBF3 under control of the CaMV 35S promoter mightnot completely mimic the activation of CBF3 by lowtemperature.

The effect that CBF3 overexpression has on flower-ing is curious. Time to flowering is controlled bymultiple physiological and environmental factors(Koornneef et al., 1998; Levy and Dean, 1998). Insome plants, including Arabidopsis, the transition toflowering is responsive to vernalization, a long pe-riod (weeks) of low temperature treatment. In “fac-ultative” plants such as Arabidopsis, the effect ofvernalization is to shorten the time to flowering (themagnitude of the effect varies greatly among Arabi-dopsis ecotypes). If CBF3 expression at warm tem-perature was fully mimicking exposure to low tem-perature, then one might think that if it had any effecton flowering, that it would decrease the transitiontime and cause a corresponding decrease in the num-ber of rosette leaves per plant. Our results, however,indicate that CBF3 expression had the opposite effect;it increased the time to flowering and increased thenumber of rosette leaves per plant. A mechanism toexplain this effect is not immediately obvious butwould seem to be consistent with the notion thatCBF3 expression does not control all low temperatureresponses in Arabidopsis.

We previously reported that CBF1 overexpressioninduced COR gene expression and increased plantfreezing tolerance without a low temperature stimu-lus (Jaglo-Ottosen et al., 1998). A significant differ-ence in that study and the results presented here isthat we did not note any obvious effect of CBF1overexpression on plant growth. A likely explanationfor this difference is suggested by the results of Liu et




al. (1998). These investigators found a positive corre-lation between the level of CBF3 (DREB1a) expres-sion, the level of expression of the target gene COR78(RD29a), and the degree to which the plants werestunted in growth. In our previous experiments(Jaglo-Ottosen et al., 1998), the level of COR15amprotein in nonacclimated CBF1-expressing plantswas approximately equal to the levels of COR15am incontrol plants that had been cold-acclimated for 7 d.Here, the COR15am levels in the nonacclimatedCBF3-expressing plants was greater than in controlplants that had been cold-acclimated for 7 d (Fig. 2).Thus, it seems likely that the lack of effect on growthand development observed with the CBF1-expressingplants was due to lower levels of CBF-induced targetgene expression. It should be kept in mind, however,that it has not yet been established that all three CBFgenes control the same sets of genes. A detailedcomparison between the levels of CBF1, CBF2, andCBF3 expression in transgenic Arabidopsis plants,the levels of target gene expression assessed on agenomic scale through microarray analysis, and adetailed analysis of the growth and development ofthe plants should help resolve this issue.

MATERIALS AND METHODS

Plant Growth

Arabidopsis (L.) Heynh. ecotype Wassilewskija (Ws)-2and transgenic plants in the Ws-2 background were grownin controlled environment chambers at 20°C under con-stant illumination from cool-white fluorescent lights (100–150 mmol m22 s21) in Baccto planting mix (Michigan Peat,Houston). Pots were subirrigated with deionized water asnecessary. All seeds were cold-treated (5°C) for 4 d imme-diately after planting to ensure uniform germination.Plants were cold acclimated by placing pots at 5°C undercontinuous light (20–60 mmol m22 s21) as described (Gil-mour et al., 1988).

Constructs and Plant Transformation

A 910-bp BamHI/HindIII fragment from a cDNA clonecontaining the whole coding region of CBF3 (Gilmour et al.,1998) was inserted into the BglII and HindIII sites of thebinary transformation vector pGA643 (An et al., 1988). Theresulting plasmid, pMPS13, which contains the CBF3 cod-ing sequence under control of the CaMV 35S promoter, wastransformed into Agrobacterium tumefaciens strain GV3101by electroporation. Arabidopsis plants were transformedwith plasmid pMPS13 or the transformation vector pGA643using the floral dip method (Clough and Bent, 1998). Trans-formed plants were selected on the basis of kanamycinresistance. Homozygous T3 or T4 plants were used in allexperiments.

Western Analysis

Total soluble protein was obtained essentially as de-scribed (Gilmour et al., 1996) by grinding leaf material

(approximately 100 mg) in 0.4 mL of extraction buffer (50mm PIPES (1,4-piperazinediethanesulfonic acid), pH 7.0, 25mm EDTA) containing 2.5% (w/v) polyvinyl-polypyrro-lidone and removing insoluble material by centrifugation(16,000g 3 20 min). Protein concentration in the superna-tant was measured using the dye-binding method of Brad-ford (1976) with bovine serum albumin as the standard.Protein samples (50 mg of total protein) were fractionatedby Tricine (N-[tris(hydroxymethyl)methyl]glycine) SDS/PAGE (Schagger and von Jagow, 1987) and transferred to0.2 micron nitrocellulose membranes by electroblotting(Towbin et al., 1979). COR15am and COR6.6 were detectedusing the enhanced chemiluminescence kit (Amersham,Buckinghamshire, UK) with antiserum raised to recombi-nant COR15am and COR6.6 (Gilmour et al., 1996).

RNA Hybridization and cDNA Probes

Total RNA was extracted from Arabidopsis plants asdescribed previously (Gilmour et al., 1988). Northern trans-fers were prepared and hybridized as described (Hajela etal., 1990) using high stringency wash conditions (Stock-inger et al., 1997). 32P-labeled probes were prepared byrandom priming (Feinberg and Vogelstein, 1983). A gene-specific probe to CBF3 was made to the 39 end of the cDNAclone by PCR as described previously (Gilmour et al.,1998). Arabidopsis cDNA clones encoding Arabidopsis Sucsynthase (182C20T7), corresponding to the SUS1 gene(Martin et al., 1993), and D1-pyrroline-5-carboxylate syn-thase (125M17T7), corresponding to the P5CS2 gene(Strizhov et al., 1997), were obtained from the ArabidopsisBiological Resource Center at Ohio State University (Co-lumbus). Probes made against the P5CS2 coding sequencewould be expected to cross-hybridize with transcripts fromthe highly similar P5CS1 gene (Strizhov et al., 1997) andthus serve as a measure of total P5CS transcripts. A cDNAencoding a putative Suc-phosphate synthase gene fromArabidopsis was obtained from James Zhang (Mendel Bio-technology, Hayward, CA).

Pro Analysis

Lyophilized leaf material (30 mg) was extracted with 3mL of deionized water at 80°C for 15 min. The sampleswere shaken for approximately 1 h at room temperatureand then allowed to stand overnight at 4°C. The extractswere filtered through glass wool and analyzed for Procontent using the acid ninhydrin method (Troll and Lind-sley, 1955). Briefly, 500 mL of the aqueous extract wasmixed with 500 mL of glacial acetic acid and 500 mL of acidninhydrin reagent (12.5 mg of ninhydrin, 0.3 mL of glacialacetic acid, 0.2 mL of 18 n orthophosphoric acid) andheated at 100°C for 45 min. After cooling, 500 mL of thereaction mix was partitioned against toluene (2 mL) andthe absorbance of the organic phase was determined at 515nm. The resulting values were compared with a standardcurve constructed using known amounts of Pro (Sigma, St.

Gilmour et al.



Louis). Pro levels in certain samples were confirmed byamino acid analysis using an amino acid analyzer at theMacromolecular Structure Facility in the Biochemistry De-partment at Michigan State University.

Sugar Analysis

Total soluble sugars were extracted from lyophilized leafmaterial (20 mg) in 80% (v/v) ethanol (2 mL) at 80°C for 15min. The samples were shaken for approximately 1 h atroom temperature and allowed to stand overnight at 4°C.Extracts were filtered through glass wool and chlorophyllremoved by shaking samples (0.4 mL) with water (0.4 mL)and chloroform (0.4 mL). The aqueous extract was assayedfor sugar content using the phenol-sulfuric acid assay(Dubois et al., 1956). Certain samples were analyzed usinga miniaturized version of the gas chromatography methoddescribed in Everard et al. (1994) with the following mod-ifications. Vacuum dried carbohydrates were solubilizedovernight in 0.5 mL of dry pyridine containing 30 mg mL21

hydroxylamine-HCl and 0.1 mg mL21 phenyl b-d-glucoside (derivatization efficiency internal standard). Af-ter heating at 75°C for 1 h, samples were cooled to roomtemperature and derivatized by the addition of 0.5 mL ofhexamethyldisilazane and 0.05 mL of trifluoroacetic acid.Precipitates were allowed to settle for 1 h at room temper-ature prior to transferring the liquid phase to automaticsampler vials (0.3 mL). Samples were analyzed on aHewlett-Packard 6890 gas chromatograph fitted with a0.25-mm film, 30 m 3 320 mm DB17 capillary column (J&WScientific, Folsom, CA), and a flame ionization detector.The chromatography details were as follows: 1-mL injec-tions with a 1:50 split ratio; injector and detector tempera-tures 305°C; initial column temperature 150°C with a rampof 3°C min21 to 200°C, followed immediately by a 20°Cmin21 ramp to 290°C with a 10-min hold at this finaltemperature. The carrier gas was hydrogen at a linearvelocity of 46 cm s21.

Whole Plant Freeze Test

Ws-2 and A30 seedlings were grown (13 and 20 d, re-spectively) on Gamborg’s B-5 medium containing 0.2%(w/v) Suc under sterile conditions in Petri dishes. Theplants were tested for freezing tolerance by first placing theplates at 22°C in the dark for 3 h followed by ice nucleationwith sterile ice chips. The plates were incubated an addi-tional 21 h at 22°C, then the temperature of the freezer wasturned down to 26°C, and the plates were left at thistemperature for an additional 24 h. The plates were takenfrom the freezer and placed at 4°C in the dark for 18 h,followed by 2 d at 22°C under cool-white fluorescent lights(40–50 mmol m22 s21) with an 18-h photoperiod. The plateswere then scored for freezing damage.

Electrolyte Leakage Freeze Test

Electrolyte leakage freeze tests were performed essen-tially as described (Uemura et al., 1995) with minor mod-

ifications. Tubes (16 3 12 mm) containing 3 to 4 leaveswere placed in a low temperature bath set at 22°C in arandomized design. The randomization was performedwith the aid of the SAS system (SAS Institute, Cary, NC).Ice chips were added to each tube after 1 h of incubation.Each tube was capped with foam plugs and incubated afurther 1 h at 22°C. The bath temperature was then low-ered 1°C every 20 min. Tubes were removed at each tem-perature and incubated an additional hour at that sametemperature in a separate bath. Tubes were placed on iceafter removal from the bath until all tubes had been re-moved. The samples were then thawed overnight at 2.5°C,and electrolyte leakage was measured as described (Gil-mour et al., 1988).

ACKNOWLEDGMENTS

We gratefully acknowledge the assistance of DanielCook for the whole plant freeze test and the Macromolec-ular Structure Facility in the Biochemistry Department atMichigan State University for performing amino acid anal-ysis. We also thank James Zhang for providing a clone ofSuc-phosphate synthase and the Arabidopsis Biological Re-source Center at Ohio State University for providing cDNAclones for P5CS2 and Suc synthase.

Received July 11, 2000; modified September 7, 2000; ac-cepted October 2, 2000.

LITERATURE CITED

Alberdi M, Corcuers LJ, Maldonado C, Barrientos M,Fernandez J, Henriquez O (1993) Cold acclimation incultivars of Avena sativa. Phytochemistry 33: 57–60

An G, Ebert PR, Mitra A, Ha SB (1988) Binary vectors. InSB Gelvin, RA Schilperoort, eds, Plant Molecular BiologyManual. Kluwer Academic Publishers, Dordrecht, TheNetherlands, pp A3, 1–19

Artus NN, Uemura M, Steponkus PL, Gilmour SJ, Lin CT,Thomashow MF (1996) Constitutive expression of thecold-regulated Arabidopsis thaliana COR15a gene affectsboth chloroplast and protoplast freezing tolerance. ProcNatl Acad Sci USA 93: 13404–13409

Baker SS, Wilhelm KS, Thomashow MF (1994) The 59-region of Arabidopsis thaliana cor15a has cis-acting ele-ments that confer cold-, drought- and ABA-regulatedgene expression. Plant Mol Biol 24: 701–713

Bradford MM (1976) A rapid and sensitive method for thequantitation of microgram quantities of protein utilizingthe principle of protein-dye dining. Anal Biochem 72:248–254

Carpenter JF, Crowe JH (1988) The mechanism of cryopro-tection of proteins by solutes. Cryobiology 25: 244–255

Clough SJ, Bent AF (1998) Floral dip: a simplified methodfor Agrobacterium-mediated transformation of Arabidopsisthaliana. Plant J 16: 735–743




Dejardin A, Sokolov LN, Kleczkowski LA (1999) Sugar-osmoticum levels modulate differential abscisic acid-independent expression of two stress-responsive sucrosesynthase genes in Arabidopsis. Biochem J 344: 503–509

Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F(1956) Colorimetric method for determination of sugarsand related substances. Anal Chem 28: 350–356

Everard JD, Gucci R, Kann SC, Flore JA, Loescher WH(1994) Gas exchange and carbon partitioning in theleaves of celery (Apium graveolens L.) at various levels ofroot zone salinity. Plant Physiol 106: 281–292

Feinberg AP, Vogelstein B (1983) A technique for radiola-belling restriction endonuclease fragments to high spe-cific activity. Anal Biochem 132: 6–13

Gilmour SJ, Hajela RK, Thomashow MF (1988) Cold accli-mation in Arabidopsis thaliana. Plant Physiol 87: 745–750

Gilmour SJ, Lin C, Thomashow MF (1996) Purificationand properties of Arabidopsis thaliana COR (cold-regulated) gene polypeptides COR15am and COR6.6 ex-pressed in Escherichia coli. Plant Physiol 111: 293–299

Gilmour SJ, Zarka DG, Stockinger EJ, Salazar MP,Houghton JM, Thomashow MF (1998) Low temperatureregulation of the Arabidopsis CBF family of AP2 tran-scriptional activators as an early step in cold-inducedCOR gene expression. Plant J 16: 433–442

Guy CL, Huber JLA, Huber SC (1992) Sucrose phosphatesynthase and sucrose accumulation at low temperature.Plant Physiol 100: 502–508

Hajela RK, Horvath DP, Gilmour SJ, Thomashow MF(1990) Molecular cloning and expression of cor (cold-regulated) genes in Arabidopsis thaliana. Plant Physiol 93:1246–1252

Hughes MA, Dunn MA (1996) The molecular biology ofplant acclimation to low temperature. J Exp Bot 47: 291–305

Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, SchabenbergerO, Thomashow MF (1998) Arabidopsis CBF1 overexpres-sion induces COR genes and enhances freezing tolerance.Science 280: 104–106

Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shi-nozaki K (1999) Improving plant drought, salt, andfreezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotech 17: 287–291

Koornneef M, Alonso-Blanco C, Peeters AJM, Soppe W(1998) Genetic control of flowering time in Arabidopsis.Annu Rev Plant Physiol Plant Mol Biol 49: 345–370

Koster KK, Lynch DV (1992) Solute accumulation andcompartmentation during the cold acclimation of pumarye. Plant Physiol 98: 108–113

Levy YY, Dean C (1998) The transition to flowering. PlantCell 10: 1973–1989

Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S,Yamaguchi-Shinozaki K, Shinozaki K (1998) Two tran-scription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signaltransduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis.Plant Cell 10: 1391–1406

Martin T, Frommer WB, Salanoubat M, Willmitzer L(1993) Expression of an Arabidopsis sucrose synthasegene indicates a role in metabolism of sucrose both

during phloem loading and in sink organs. Plant J 4:367–377

McKown R, Kuroki G, Warren G (1996) Cold responses ofArabidopsis mutants impaired in freezing tolerance. J ExpBot 47: 1919–1925

Metz AM, Timmer RT, Browning KS (1992) Sequences fortwo cDNAs encoding Arabidopsis thaliana eukaryotic pro-tein synthesis initiation factor 4A. Gene 120: 313–314

Nanjo T, Kobayashi M, Yoshiba Y, Kakubari Y, Yamaguchi-Shinozaki K, Shinozaki K (1999) Antisense suppressionof proline degradation improves tolerance to freezing andsalinity in Arabidopsis thaliana. FEBS Lett 461: 205–210

Omran AO, Atkins IM, Gilmour ECJ (1968) Heritability ofcold hardiness in flax. Crop Sci 8: 716–719

Riechmann JL, Meyerowitz EM (1998) The AP2/EREBPfamily of plant transcription factors. Biol Chem 379:633–646

Roberts DWA (1990) Identification of loci on chromosome5A of wheat involved in control of cold hardiness, ver-nalization, leaf length, rosette growth habit, and heightof hardened plants. Genome 33: 247–259

Rudolph AS, Crowe JH (1985) Membrane stabilizationduring freezing: the role of two natural cryoprotectants,trehalose and proline. Cryobiology 22: 367–377

Schagger H, von Jagow G (1987) Tricine-sodium dodecylsulfate-polyacrylamide gel electrophoresis for the sepa-ration of proteins in the range from 1 to 100 kDa. AnalBiochem 166: 368–379

Shinwari ZK, Nakashima K, Miura S, Kasuga M, Seki M,Yamaguchi-Shinozaki K, Shinozaki K (1998) An Arabi-dopsis gene family encoding DRE/CRT binding proteinsinvolved in low-temperature-responsive gene expres-sion. Biochem Biophys Res Commun 250: 161–170

Steponkus PL, Uemura M, Joseph RA, Gilmour SJ, Tho-mashow MF (1998) Mode of action of the COR15a geneon the freezing tolerance of Arabidopsis thaliana. Proc NatlAcad Sci USA 95: 14570–14575

Steponkus PL, Uemura M, Webb MS (1993) A contrast ofthe cryostability of the plasma membrane of winter ryeand spring oat-two species that widely differ in theirfreezing tolerance and plasma membrane lipid composi-tion. In PL Steponkus, ed, Advances in Low-TemperatureBiology, Vol 2. JAI Press, London, pp 211–312

Stockinger EJ, Gilmour SJ, Thomashow MF (1997) Ara-bidopsis thaliana CBF1 encodes an AP2 domain-con-taining transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element thatstimulates transcription in response to low temper-ature and water deficit. Proc Natl Acad Sci USA 94:1035–1040

Strand A, Hurry V, Gustafsson P, Gardstrom P (1997)Development of Arabidopsis thaliana leaves at low tem-peratures releases the suppression of photosynthesis andphotosynthetic gene expression despite the accumula-tion of soluble carbohydrates. Plant J 12: 605–614

Strauss G, Hauser H (1986) Stabilization of lipid bilayervesicles by sucrose during freezing. Proc Natl Acad SciUSA 83: 2422–2426

Gilmour et al.



Strizhov N, Abraham E, Okresz L, Blicking S, ZilbersteinA, Schell J, Koncz C, Szabados L (1997) Differential ex-pression of two P5CS genes controlling proline accumu-lation during salt-stress required ABA and is regulated byABA1, ABI1 and AXR2 in Arabidopsis. Plant J 12: 557–569

Thomashow MF (1998) Role of cold-responsive genes inplant freezing tolerance. Plant Physiol 118: 1–7

Thomashow MF (1999) Plant cold acclimation: freezingtolerance genes and regulatory mechanisms. Annu RevPlant Physiol Plant Mol Biol 50: 571–599

Towbin H, Staehelin T, Gordon J (1979) Electrophoretictransfer of proteins from polyacrylamide gels to nitrocel-lulose sheets: procedure and some applications. ProcNatl Acad Sci USA 76: 4350–4354

Troll W, Lindsley J (1955) A photometric method for thedetermination of proline. J Biol Chem 215: 655–660

Uemura M, Joseph RA, Steponkus PL (1995) Cold accli-mation of Arabidopsis thaliana: effect on plasma mem-

brane lipid composition and freeze-induced lesions.Plant Physiol 109: 15–30

Wanner LA, Junttila O (1999) Cold-induced freezing tol-erance in Arabidopsis. Plant Physiol 120: 391–400

Winter H, Huber SC (2000) Regulation of sucrose metab-olism higher plants: localization and regulation of activ-ity of key enzymes. Crit Rev Plant Sci 19: 31–67

Xin Z, Browse J (1998) eskimo1 mutants of Arabidopsis areconstitutively freezing-tolerant. Proc Natl Acad Sci USA95: 7799–7804

Yamaguchi-Shinozaki K, Shinozaki K (1994) A novel cis-acting element in an Arabidopsis gene is involved inresponsiveness to drought, low-temperature, or high-saltstress. Plant Cell 6: 251–264

Yoshiba Y, Kiyosue T, Nakashima K, Yamaguchi-Shinozaki K, Shinozaki K (1997) Regulation of levels ofproline as an osmolyte in plants under water stress. PlantCell Physiol 38: 1095–1102




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