Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant Arabidopsis

Authors

  • Xin Zhang,

    1. MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA
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    • These authors contributed equally to this work.

    • Present address: The National Key Facility on Crop Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, China.

  • Sarah G. Fowler,

    1. MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA
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    • These authors contributed equally to this work.

  • Hongmei Cheng,

    1. Department of Horticulture and Crop Science, 1680 Madison Avenue, The Ohio State University/OARDC, Wooster, OH 44691, USA
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  • Yigong Lou,

    1. Plant Biology Department, Carnegie Institution of Washington, 260 Panama Street, Stanford, CA 94305, USA
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  • Seung Y. Rhee,

    1. Plant Biology Department, Carnegie Institution of Washington, 260 Panama Street, Stanford, CA 94305, USA
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  • Eric J. Stockinger,

    1. Department of Horticulture and Crop Science, 1680 Madison Avenue, The Ohio State University/OARDC, Wooster, OH 44691, USA
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  • Michael F. Thomashow

    Corresponding author
    1. MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA
    2. Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824, USA
      For correspondence (fax +1 517 353 9168; e-mail thomash6@msu.edu).
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For correspondence (fax +1 517 353 9168; e-mail thomash6@msu.edu).

Summary

Many plants increase in freezing tolerance in response to low temperature, a process known as cold acclimation. In Arabidopsis, cold acclimation involves action of the CBF cold response pathway. Key components of the pathway include rapid cold-induced expression of three homologous genes encoding transcriptional activators, CBF1, 2 and 3 (also known as DREB1b, c and a, respectively), followed by expression of CBF-targeted genes, the CBF regulon, that increase freezing tolerance. Unlike Arabidopsis, tomato cannot cold acclimate raising the question of whether it has a functional CBF cold response pathway. Here we show that tomato, like Arabidopsis, encodes three CBF homologs, LeCBF1–3 (Lycopersicon esculentum CBF1–3), that are present in tandem array in the genome. Only the tomato LeCBF1 gene, however, was found to be cold-inducible. As is the case for Arabidopsis CBF1–3, transcripts for LeCBF1–3 did accumulate in response to mechanical agitation, but not in response to drought, ABA or high salinity. Constitutive overexpression of LeCBF1 in transgenic Arabidopsis plants induced expression of CBF-targeted genes and increased freezing tolerance indicating that LeCBF1 encodes a functional homolog of the Arabidopsis CBF1–3 proteins. However, constitutive overexpression of either LeCBF1 or AtCBF3 in transgenic tomato plants did not increase freezing tolerance. Gene expression studies, including the use of a cDNA microarray representing approximately 8000 tomato genes, identified only four genes that were induced 2.5-fold or more in the LeCBF1 or AtCBF3 overexpressing plants, three of which were putative members of the tomato CBF regulon as they were also upregulated in response to low temperature. Additional experiments indicated that of eight tomato genes that were likely orthologs of Arabidopsis CBF regulon genes, none were responsive to CBF overexpression in tomato. From these results, we conclude that tomato has a complete CBF cold response pathway, but that the tomato CBF regulon differs from that of Arabidopsis and appears to be considerably smaller and less diverse in function.

Introduction

Plants vary greatly in their ability to tolerate cold temperatures (Sakai and Larcher, 1987). Many species from tropical regions, such as tomato, maize and rice, are unable to tolerate freezing and suffer chilling injury when exposed to temperatures in the range of 0 to 12°C. In contrast, plants from temperate regions, such as wheat, canola and Arabidopsis, are able tolerate both chilling and freezing temperatures. The ability of these plants to withstand freezing, however, is not a constant property, but increases dramatically upon exposure to low, non-freezing temperatures, a phenomenon known as cold acclimation (Guy, 1990; Thomashow, 1998). Cold acclimation involves the activation of multiple mechanisms that contribute to an increase in freezing tolerance including the accumulation of low-molecular weight cryoprotective molecules, the synthesis of cryoprotective proteins and alterations in membrane lipid composition (Artus et al., 1996; Gilmour et al., 2000; Nanjo et al., 1999; Steponkus et al., 1993, 1998; Taji et al., 2002).

It has recently been established that an important component of cold acclimation in Arabidopsis is the CBF cold response pathway (Thomashow, 2001). Within 15 min of exposing plants to low temperature, genes are induced that encode a small family of transcriptional activators known as either CBF1, CBF2, and CBF3 (Gilmour et al., 1998; Medina et al., 1999; Stockinger et al., 1997) or DREB1b, DREB1c, and DREB1a, respectively (Kasuga et al., 1999; Liu et al., 1998). This is quickly followed by activation of the ‘CBF regulon,’ the set of genes that are induced in response to the CBF/DREB1 transcriptional activators. The immediate target genes of the CBF/DREB1 proteins have CRT (C-repeat)/DRE (dehydration responsive element) elements in their promoters, the DNA regulatory sequence to which the CBF/DREB1 transcriptional activators bind (Baker et al., 1994; Stockinger et al., 1997; Yamaguchi-Shinozaki and Shinozaki, 1994). Expression of the CBF regulon results in an increase in freezing tolerance due, in part, to the induction of genes encoding cryoprotective proteins and enzymes involved in the synthesis of low-molecular weight cryoprotectants (Gilmour et al., 1998; Jaglo-Ottosen et al., 1998; Kasuga et al., 1999; Liu et al., 1998). Expression of the CBF regulon also results in an increase in tolerance to drought and high salinity (Kasuga et al., 1999) and appears to have a role in acclimation to chilling temperatures (Gong et al., 2002).

The CBF/DREB1 proteins belong to the AP2/ERF family of DNA-binding proteins (Stockinger et al., 1997). This protein family, which comprises more than 140 members in Arabidopsis, has in common the AP2/ERF DNA-binding motif (Okamuro et al., 1997; Riechmann and Meyerowitz, 1998). There is, however, a feature of CBF1, 2 and 3, and three other CBF/DREB1 proteins (CBF4/DREB1d, DREB1e and DREB1f) that distinguish them from the rest of the AP2/ERF proteins; a conserved set of amino acid sequences located immediately upstream and downstream of the AP2/ERF domain. These sequences, PKK/RPAGRxKFxETRHP and DSAWR, have been designated the CBF ‘signature sequences’ (Jaglo et al., 2001). The function of these sequences remains to be determined.

CBF-like proteins containing the signature sequences are present in a wide range of plants including those that cold acclimate, such as Brassica napus (Jaglo et al., 2001) and barley (Choi et al., 2002), as well as in plants that do not cold acclimate, such as tomato (Jaglo et al., 2001) and rice (Choi et al., 2002; Dubouzet et al., 2003). Moreover, genes encoding apparent CBF orthologs are induced rapidly in response to low temperature in B. napus (Jaglo et al., 2001), barley (Choi et al., 2002), tomato (Jaglo et al., 2001) and rice (Dubouzet et al., 2003). Thus, components of the CBF cold response pathway are highly conserved in plants. Indeed, overexpression of Arabidopsis CBF genes in transgenic B. napus plants induces expression of orthologs of the Arabidopsis COR genes that have CRT/DRE elements in their promoters and brings about an increase in freezing tolerance (Jaglo et al., 2001). Thus, B. napus, a close relative of Arabidopsis that cold acclimates, appears to have a complete functional CBF cold response pathway, i.e. CBF genes that are induced in response to low temperature; target genes that are induced by the CBF transcriptional activators; and a CBF regulon of genes that act in concert to increase plant freezing tolerance.

The finding that chilling-sensitive plants like tomato and rice have genes that encode CBF-like proteins and are induced in response to low temperature raises fundamental questions about the CBF cold response pathways in these organisms. Do tomato, rice and other freezing-sensitive plants have complete CBF cold response pathways? Alternatively, do they have CBF pathways that are deficient in one or more important feature that contributes to the low-temperature sensitivity of the plants? Here we begin to address this question by examining the CBF cold response pathway in tomato.

Results

Tomato has three CBF genes arranged in tandem array

We previously identified a tomato EST (expressed sequence tag) encoding an apparent CBF homolog, LeCBF1 (Lycopersicon esculentum CBF1) (Jaglo et al., 2001; Figure 1a). A subsequent BLAST search of the tomato EST database using the LeCBF1 sequence as the query resulted in the identification of a second tomato EST (AI487887) encoding an apparent CBF homolog, LeCBF2 (Figure 1a). Southern analysis indicated that the two tomato CBF genes were physically linked and suggested the existence of a third CBF gene (not shown). The isolation and sequencing a 19 kb genomic clone containing the tomato CBF locus confirmed this (Genbank accession no. AY497899). As is the case in Arabidopsis (Gilmour et al., 1998), the tomato LeCBF1, 2 and 3 genes were found to be organized in tandem array with none of the protein-coding sequences being interrupted with introns (Figure 1b). No other closely related CBF genes were detected by Southern analysis using LeCBF2 as the probe and low-stringency hybridization and wash conditions (not shown). The nucleotide sequences upstream and downstream of the LeCBF gene cluster represented in the genomic clone showed no significant sequence similarity with the corresponding regions upstream and downstream of the ArabidopsisCBF1–3 gene cluster, CBF4, DREB1e or DREBf indicating the absence of ‘microsynteny’ between tomato and Arabidopsis beyond the CBF gene clusters. Mapping experiments indicated that the tomato LeCBF genes were located on tomato chromosome 3 between molecular markers TG520 and CT141 (Figure 1c).

Figure 1.

Amino acid sequence comparison, locus structure, mapping and phylogenetic analysis of CBF genes in tomato and Arabidopsis.
(a) Alignment of Arabidopsis CBF1, 2 and 3 protein sequences, AtCBF1, AtCBF2 and AtCBF3, with CBF-like proteins from tomato, LeCBF1, LeCBF2 and LeCBF3. The AP2/EREBP domain is indicated by an over-line and the CBF signature sequences PKK/RPAGRxKFxETRHP and DSAWR are indicated by black dots.
(b) Structure of the CBF locus in tomato. The black bar represents the tomato genomic DNA encompassed by the bacteriophage λ genomic clone Le-3. The arrows indicate the location and extent of the three tomato CBF genes and other potential coding regions identified within λLe-3. Downstream of LeCBF2 are four apparent exons encoding a protein with high identity to human CAB2 and yeast YCR44c, vesicle and vacuolar-targeted proteins, respectively, implicated in cellular Mn2+ homeostasis (Nezu et al., 2002; Paidhungat and Garrett, 1998). Within one of the introns is a retroelement reverse transcriptase-like sequence. Upstream of LeCBF3 is a short stretch of nucleotides with similarity to several tomato ESTs (AW441210; score 996; 1.8e−40) predicted to encode an mRNA glycosyltransferase (a β-fructosidase). The glycosyltransferase and retroelement reverse transcriptase-like sequences contain multiple nonsense codons indicating that they are unlikely to encode functional proteins.
(c) Chromosomal location of the tomato CBF locus relative to other chromosome 3 molecular markers (Van der Knaap and Tanksley, 2001).
(d) Similarity tree showing the relationships between the tomato and Arabidopsis CBF1, 2 and 3 proteins. The tree was generated by using the neighbor-joining algorithm in mega 2.0 software, version 2.1. The bar indicates the scale for branch length.

The amino acid sequences of the tomato LeCBF proteins were 70–84% identical to each other and 51–59% identical to those of the Arabidopsis CBF1, 2 and 3 proteins. Each LeCBF protein has the CBF signature sequences that surround the AP2/ERF-binding domain (Figure 1a). Protein alignments revealed the existence of additional regions outside of the AP2/ERF domain and signature sequences that were highly conserved between the Arabidopsis and tomato CBF proteins (Figure 1a). A comparison of amino acid sequences indicated that the LeCBF1, 2 and 3 proteins were more closely related to each other than to the AtCBF1, 2 and 3 proteins (Figure 1d).

LeCBF1, but not LeCBF2 and LeCBF3, is induced in response to low temperature

We previously presented results indicating that transcripts for the tomato LeCBF1 gene accumulate rapidly in response to low temperature (Jaglo et al., 2001). The results presented here confirm and extend these findings (Figure 2). Using gene-specific probes, it was found that LeCBF1 transcripts accumulate quickly in response to low temperature in plants grown under constant light or under a 16-h photoperiod (Figure 2a). Interestingly, however, differences in expression patterns were observed in these plants. Under constant light, LeCBF1 transcripts accumulated rapidly upon exposure to low temperature reaching a peak level at about 2 h of cold treatment, followed by a return to levels found in warm-grown plants after about 24 h of cold treatment. In contrast, when plants were grown under the 16-h photoperiod, LeCBF1 transcripts accumulated rapidly to a high level (though not as high as in the plants grown under constant light), remained elevated at a constant level for 16 h, and then decreased at 24 h, but remained elevated over the level found in the warm grown plants. Like the Arabidopsis CBF1, 2 and 3 genes, the tomato LeCBF1 gene was responsive to mechanical agitation (Figure 2a) but only weakly (if at all) responsive to dehydration, high salinity and treatment with ABA (Figure 2b).

Figure 2.

Accumulation of LeCBF transcripts in response to abiotic stresses. Tomato seedlings were grown at 26°C and subjected to a variety of abiotic stresses. RNA gel blots were prepared from total RNA and hybridized with gene specific probes for LeCBF1, 2 and 3; eIF4a as a control for RNA loading; and the Le25 (Kahn et al., 1993) tomato gene as a positive control for drought, high salinity and ABA treatments.
(a) RNA was isolated from plants grown in either continuous light (LL) or a 16-h photoperiod (16:8) then treated at 4°C for the times indicated. Samples were also harvested after 5 min mechanical agitation (MA) followed by 15 min rest. Control samples were grown in a 16-h photoperiod at 26°C then harvested in parallel with the environmentally stressed samples.
(b) Plants were grown in a 16-h photoperiod. For the drought, NaCl and ABA treatments, the plants were placed on dry filter paper or transferred to filter paper saturated with MS solution supplemented with 250 mm NaCl or 100 μm ABA, respectively, for varying times as indicated. For the control samples, the plants were transferred to filter paper saturated with MS solution and incubated in parallel with the experimental samples.

In contrast to LeCBF1, neither LeCBF2 nor LeCBF3 was induced in response to low temperature (Figure 2). They were, however, responsive to mechanical agitation indicating that the promoters of these two genes were not completely inactive. Like LeCBF1, the LeCBF2 and LeCBF3 genes were only weakly responsive, if at all, to dehydration, high salinity or ABA treatment.

Two regions within the promoter of the Arabidopsis CBF2 gene, ICEr1 and ICEr2 (induction of CBF expression regions 1 and 2), have been shown to act together to impart cold-responsive gene expression (Zarka et al., 2003). These sequences are within two of six ‘boxes’ that have been identified as being conserved in the promoters of the AtCBF1, 2 and 3 genes (Shinwari et al., 1998). A search of the LeCBF1–3 promoter regions indicated that all three had the ICEr2 sequence ACTCCG at similar locations with respect to the putative start of transcription (−119 to −165). However, no sequences similar to ICEr1 or the other conserved boxes were found in the promoters of the LeCBF1–3 genes.

LeCBF1 functions in Arabidopsis

Constitutive expression of the Arabidopsis CBF1–4 genes in transgenic Arabidopsis plants activates expression of the target CRT/DRE-containing COR (cold-regulated) genes and enhances freezing tolerance in the absence of a low-temperature stimulus (Gilmour et al., 2000; Haake et al., 2002; Jaglo-Ottosen et al., 1998; Kasuga et al., 1999; Liu et al., 1998; S.J. Gilmour and M.F. Thomashow, Michigan State University, East Lansing, MI, USA, unpublished data). If the tomato LeCBF1 protein was a functional homolog of the Arabidopsis CBF protein, then expressing it in transgenic Arabidopsis plants might also activate expression of the COR genes and result in an increase in freezing tolerance. To test this, transgenic Arabidopsis plants were created that overexpressed LeCBF1 under control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Several transgenic T2 lines were identified and three lines, L1, L3 and L50, were selected for further study. Transcript levels for LeCBF1 were present in all three of these lines at similar levels in non-acclimated and cold-acclimated plants (Figure 3a). Plants of each line were very similar in appearance to control plants at the seedling stage, but later in development displayed aberrant growth phenotypes similar to those described for overexpression of Arabidopsis CBF genes (Figure 4). In particular, the LeCBF1-expressing plants produced fewer axillary shoots, less seed, and were delayed in flowering. The delay in flowering did not appear to be simply caused by slower growth of the transgenic plants, but instead, developmental in nature as more rosette leaves were produced by the L1 and L50 plants than the wild-type plants (Table 1).

Figure 3.

Accumulation of COR transcripts and proteins in transgenic Arabidopsis overexpressing LeCBF. Total RNA and protein were prepared from Arabidopsis Ws-2 (wild type), B6 (vector control) and LeCBF1-expressing plants (lines L1, L3 and L50) before (W) and after (C) treatment at 4°C for 7 days in continuous light.
(a) RNA gel blots were hybridized with probes for LeCBF1, the COR genes COR15a and COR6.6, and eIF4a, a control for RNA loading.
(b) Protein transfers were treated with antibodies raised against recombinant COR6.6 and COR15a proteins.

Figure 4.

Growth characteristics of transgenic Arabidopsis plants overexpressing LeCBF1. Arabidopsis Ws-2 (wild type), B6 (vector control) and LeCBF1-expressing (L1, L3 and L50) plants were grown in controlled environment chambers at 20°C with continuous light.
(a) Seedlings after 14 days growth.
(b) Mature plants after 5 weeks growth.
(c) Senescent plants after 9 weeks growth.

Table 1.  Effect of LeCBF1 expression on flowering time of transgenic plants
Transgenic linesDays to boltingRosette leaves at boltinga
MeanSD
  1. aNumber of rosette leaves formed per plant (n = 8) at flowering stage.

Ws-2206.30.7
B6206.30.5
L1238.30.7
L3236.40.5
L502910.50.9

Overexpression of the Arabidopsis CBF/DREB1 genes in transgenic plants induces expression of COR15a, COR6.6 and other CRT/DRE-containing genes (members of the CBF regulon) and increases freezing tolerance without a low-temperature stimulus (Gilmour et al., 2000; Jaglo-Ottosen et al., 1998; Kasuga et al., 1999; Liu et al., 1998; S.J. Gilmour and M.F. Thomashow, unpublished data). This was also the case in the transgenic lines overexpressing the tomato LeCBF1 gene. Transcript and polypeptide levels for the CRT/DRE-containing COR15a and COR6.6 genes were elevated in non-acclimated L1, L3 and L50 transgenic plants (Figure 3) and the freezing tolerance of the plants was greater than that of the control plants (Figure 5). Electrolyte leakage assays indicated that the non-acclimated control plants had an EL50 (the temperature at which leakage of 50% of total electrolytes occurs) of approximately −4°C, whereas the EL50 of non-acclimated LeCBF1-expressing plants was approximately −6.5°C. Taken together, these results indicated that the LeCBF1 protein was a functional homolog of the Arabidopsis CBF proteins.

Figure 5.

Freezing tolerance of transgenic Arabidopsis plants overexpressing LeCBF1. Arabidopsis Ws-2 (wild type), B6 (vector control) and LeCBF1-expressing (L1, L3 and L50) plants were grown for 3 weeks at 20°C in continuous light. Leaves were frozen at various temperatures below zero as indicated and damage assessed by measuring electrolyte leakage.

Tomato and Arabidopsis CBF regulons differ in composition

The data presented above established that tomato had upstream components of the CBF cold response pathway; namely, a functional CBF homolog, LeCBF1, that was induced by a regulatory system responsive to low temperature. We next explored downstream components of the CBF cold response pathway; in particular, we compared the CBF regulons of tomato and Arabidopsis.

Fowler and Thomashow (2002) previously used Affymetrix GeneChips representing approximately 8000 genes to identify genes that were part of the CBF regulon in Arabidopsis. The results indicated that 30 cold-regulated genes were upregulated threefold or more in transgenic Arabidopsis plants that overexpressed either AtCBF1, 2 or 3 (Table 2). Of these, 25 had one or more CRT/DRE elements in their promoters and thus were presumably direct targets of the CBF proteins. The remaining five genes that were both CBF- and cold-responsive were potentially targets of transcription factors that were induced in response to CBF expression (such as the AP2 domain proteins, RAP2.6 and RAP2.1) and as such, were also considered to be members of the CBF regulon. Shinozaki and colleagues (Seki et al., 2001) conducted similar experiments and reported only one additional gene, ERD4 (which encodes a protein of unknown function) that was upregulated threefold or more in CBF3/DREB1a-overexpressing plants.

Table 2. Arabidopsis CBF regulon genes and identification of putative tomato homologs and their expression in response to CBF overexpression
ArabidopsisTomato
AGI identifierDescriptionCategoryNo. of CRTsaTomato TCbCBF-regulatedc
  1. aShows the number of CRT/DRE core elements (CCGAC) found in the 1000 bp upstream sequence for each gene (http://www.arabidopsis.org/tools/bulk/sequences/index.jsp).

  2. b‘x’ indicates that we did not find any matches between the Arabidopsis CBF regulon gene and a tomato sequence that met the criteria of an expect value of <1e−30 associated with a region of similarity comprising greater than 60% of the Arabidopsis protein.

  3. cCBF regulation was tested by microarray (M) or Northern (N) analysis. n.d. indicates that CBF regulation of this genes was not tested.

At1g09350Putative galactinol synthaseMetabolism4TC122376No (N)
At1g60470Putative galactinol synthaseMetabolism0TC122376No (N)
At2g24560Putative GDSL lipase/hydrolaseMetabolism2TC123815n.d.
At3g55610Pyrroline-5-carboxlyate synthetase (P5CS2)Metabolism1TC117423No (M)
At1g62570Putative glutamine synthaseMetabolism0xx
At1g43160AP2 domain protein (RAP2.6)Transcription3xx
At1g46768AP2 domain protein (RAP2.1)Transcription2TC123459No (M)
At1g47710Putative serpinProtein fate2TC127023n.d.
At1g08890Putative sugar transporterTransport facilitation3xx
At4g17550Glycerol-3-phosphate permease-like proteinTransport facilitation2xx
At2g28900Putative water channel proteinCellular biogenesis2xx
At4g12470pEARLI 1-like proteinCellular biogenesis0xx
At1g05170Putative Avr9 elicitor response proteinCell rescue/defense0TC118062No (M)
At2g17840Putative senescence-associated protein 12Cell rescue/defense1TC126203No (N)
At1g20440Dehydrin (COR47)Cell rescue/defense3xx
At2g42530COR15bCell rescue/defense2xx
At1g20450Dehydrin (ERD10)Cell rescue/defense4xx
At5g52310COR78Cell rescue/defense4xx
At4g15910Di21Cell rescue/defense1xx
At5g52300RD29BCell rescue/defense3xx
At1g01470LEA proteinCell rescue/defense3TC116653No (M)
At5g15970COR6.6Cell rescue/defense1xx
At2g43620Putative endochitinaseCell rescue/defense1TC124809No (M)
At3g50970Dehydrin (Xero 2)Cell rescue/defense3xx
At1g64890Hypothetical proteinUnknown protein2xx
At2g23120Unknown protein (COR8.5)Unknown protein2xx
At4g21570Putative proteinUnknown protein2TC125199No (N)
At1g10090Hypothetical proteinUnknown protein0xx
At1g04570Putative proteinUnknown protein2xx
At1g27200Unknown proteinUnknown protein1xx

To identify genes that were members of the tomato CBF regulon, tomato plants were transformed with either LeCBF1 or AtCBF3 under control of the CaMV 35S promoter and the effects on gene expression were assessed. Two independent transgenic tomato lines transformed with either LeCBF1 or AtCBF3 were tested. Northern analysis indicated the presence of CBF transcripts in each of the transgenic lines (Figure 6a). These plants, like Arabidopsis overexpressing CBF genes at high levels, were stunted in growth (Figure 7) and delayed in flowering (data not shown). In addition, they produced fewer fruit per plant than control tomato plants (data not shown). They did not, however, display an increase in freezing tolerance. The EL50 for both control and CBF-expression transgenic tomato plants was approximately −2.0°C (data not shown).

Figure 6.

Transcript levels of putative CBF-inducible tomato genes in transgenic tomato plants overexpressing AtCBF3 or LeCBF1 and in cold-treated wild-type tomato plants.
Wild type and transgenic tomato plants were grown for 3 weeks in a 16-h photoperiod. Total RNA was isolated from these plants and RNA gel blots were prepared and hybridized with probes corresponding to the CBF transgenes AtCBF3 and LeCBF1 and the four genes identified as being CBF-inducible in the microarray analysis (see text for details). A probe for 25S rRNA served as the loading control.
(a) RNA prepared from wild-type tomato plants (DH) and transgenic tomato plants overexpressing either AtCBF3 (lines 14 and 15) or LeCBF1 (lines 12 and 27).
(b) RNA prepared from wild-type tomato plants treated at 4°C for the indicated times.

Figure 7.

Growth characteristics of transgenic tomato plants overexpressing AtCBF3 or LeCBF1. Shown are wild type (DH) tomato plants and AtCBF3-overexpressing (3–14 and 3–15) and LeCBF1-overexpressing (1–12 and 1–27) transgenic tomato plants after 4 weeks growth in a 16-h photoperiod with a daytime temperature of 26°C and a nighttime temperature of 20°C.

A tomato cDNA microarray spotted with more than 12 000 EST clones representing approximately 8700 genes was used to detect CBF-responsive genes (http://bti.cornell.edu/CGEP/CGEP.html). The results presented in Table 3 indicate that transcripts for only four of the 8700 genes increased in levels 2.5-fold (equal to a mean log ratio of 1.3) or more in the transgenic tomato plants overexpressing either LeCBF1 or AtCBF3 (this number only increased to eight genes when the cut-off criterion was lowered to twofold induction). Transcripts for two genes, annotated as encoding a probable proteinase inhibitor (cLER1P6/TC115955) and a dehydrin-like protein (cLER17C11/TC116174), increased more than 2.5-fold in response to expression of both transcription factors. Transcripts for two other genes, annotated as encoding a glycine-rich protein (cLET13N19/TC117384) and a homolog of potato dehydrin Ci7 (cLER1E13/TC116013) also showed an increase of more than 2.5-fold in the AtCBF3 transgenic lines, but their levels of induction in the LeCBF1 overexpressing lines were unclear. In the case of the glycine-rich protein (TC117384), the average induction was more than sevenfold, but one of the replicates had a log ratio of 0.9 equivalent to a fold change of 1.8. As for the Ci7 dehydrin (TC116013), there were technical problems with one of the four slides and thus, a firm conclusion could not be drawn. The entire dataset for the CBF-overexpression experiment can be accessed at http://aztec.stanford.edu/cold/index.html.

Table 3.  Proteins encoded by tomato genes induced in response to overexpression of LeCBF1 (LeCBF1-ox) and AtCBF3 (AtCBF3-ox)
DescriptionClone IDTIGR TGI TCaMean log ratio (mean fold-change)b
AtCBF3-oxLeCBF1-ox
  1. aAvailable from http://www.tigr.org/tdb/tgi/lgi/

  2. bBold text indicates results pass criteria for 2.5-fold upregulation (equivalent to a log ratio of 1.3).

  3. cThe mean log ratio was calculated from less than four slides.

Dehydrin Ci7 homologcLER1E13TC1160132.4 (5.4)0.7c (1.6)
Dehydrin-like proteincLER17C11TC1161742.5 (5.6)2.0 (4.0)
Glycine-rich proteincLET13N19TC1173843.0 (8.2)2.9 (7.3)
Probable proteinase inhibitorcLER1P6TC1159553.5 (11.0)2.6 (6.2)

Northern blot analysis was conducted to confirm the results of the microarray analysis (Figure 6a). The results indicate that transcripts for the dehydrin-like protein (TC116174), the glycine-rich protein (TC117384) and the probable proteinase inhibitor (TC115955) were highly induced in both the LeCBF1 and AtCBF3 transgenic lines. Transcripts for the Ci7 dehydrin (TC116013) were also elevated, although only about two- to threefold.

CBF-responsive genes that are members of a functional CBF cold response pathway should be induced in response to low temperature. This was true of both dehydrins and the proteinase inhibitor (Figure 6b), although the level of induction of the two dehydrins was substantially greater than the level of cold induction of the proteinase inhibitor gene. In addition, the induction profile for the dehydrin genes differed from that of the proteinase inhibitor gene; transcripts for the dehydrins were markedly elevated at 24 and 168 h, whereas those for the proteinase inhibitor were elevated at 2 and 24 h. To further explore the possibility of the two dehydrin genes being direct targets of the tomato LeCBF1 transcriptional activator, we asked whether the promoter regions of these genes included potential CRT/DRE elements. To determine this, the promoter regions of the two dehydrin genes were isolated by inverse PCR and their nucleic acid sequences were determined. The results indicated that within 464 bp of the putative start of transcription of the Ci7-like gene, that there were two putative CRT/DRE elements (i.e. the core sequence of the CRT/DRE element, CCGAC, was present two times) and within 139 bp of the putative start of the dehydrin-like protein there was one putative CRT/DRE element.

The cold- and CBF-inducible dehydrin-like protein and the Ci7 homolog resemble, in sequence, the Arabidopsis dehydrin ERD10 (Kiyosue et al., 1994), a member of the Arabidopsis CBF regulon (Table 2). To compare further the tomato and Arabidopsis CBF regulons, we identified tomato genes that were candidate orthologs of Arabidopsis CBF regulon genes and examined their expression in the transgenic tomato plants overexpressing the CBF proteins. To identify candidate tomato orthologs, we performed BLAST searches against the TIGR Tomato Gene Index (http://www.tigr.org/tdb/tgi/lgi/). Using selection criteria of expect values of <1e−30 associated with regions of similarity comprising greater than 60% of the Arabidopsis protein, we identified 10 tomato tentative consensus or singleton sequences that were the best match for 11 of the Arabidopsis CBF regulon genes (summarized in Table 2). Of these, five genes encoding pyrroline-5-carboxylate synthase, RAP2.1, a putative Avr9 elicitor response protein, a LEA protein and a putative endochitinase, were represented on the tomato cDNA microarray used in the experiments described above. These genes were not found to be affected by CBF expression (summarized in Table 2). To test expression of tomato genes that were potential orthologs of Arabidopsis CBF regulon genes that were not on the cDNA microarray, we identified ESTs (http://www.tigr.org/tdb/tgi/lgi/) for putative tomato orthologs for ArabidopsisCBF regulon genes and conducted Northern analyses. ESTs for three such genes, encoding a putative galactinol synthase, a putative senescence-associated protein 12 and a putative unknown protein, were obtained, but none were responsive to CBF expression (summarized in Table 2). Thus, of eight tomato genes that were putative orthologs of Arabidopsis genes that are members of the CBF regulon, none were responsive to CBF expression in tomato.

Discussion

Arabidopsis can cold acclimate and survive freezing whereas tomato cannot. What accounts for this difference? The answer to this question, of course, lies in the natural history of these plants and the selection pressures that have shaped their genomes. In particular, the difference in freezing tolerance between Arabidopsis and tomato is certain to reflect differences in selection for the genetic systems that impart cold tolerance. At present, the best-described genetic system involved in freezing tolerance is the CBF cold response pathway. Thus, a fundamental question raised is whether the tomato genome encodes this response pathway. The results presented here indicate that it does. Tomato has a CBF locus comprising three CBF genes, one of which, LeCBF1, is induced in response to low temperature. The protein encoded by LeCBF1 is functional since its overexpression in transgenic Arabidopsis results in the induction of CRT/DRE-regulated genes and brings about an increase in freezing tolerance. In addition, overexpression of LeCBF1 (or AtCBF3) in transgenic tomato results in the activation of at least two cold-regulated genes that have putative CRT/DRE elements in their promoters. These results are consistent with tomato having the basic components of a CBF cold response pathway: ‘upstream’ components that include a functional CBF gene and a cold sensing and regulatory system that induces expression of this gene; and ‘downstream’ components which include genes that are activated in response to CBF expression with at least two having CRT/DRE motifs in their promoters.

While tomato appears to have a functional CBF cold response pathway, the tomato pathway clearly differs from that of Arabidopsis. In Arabidopsis, all three linked CBF genes are cold-inducible, whereas in tomato, only LeCBF1 is induced in response to low temperature. What is likely to be more significant, however, is that the composition of the tomato CBF regulon appears to differ considerably from that of Arabidopsis. Fowler and Thomashow (2002) surveyed the expression of about 8000 Arabidopsis genes and identified 30 that were cold-regulated and induced threefold or more by CBF-overexpression. These genes, which were designated members of the Arabidopsis CBF regulon, cover a diverse group of functions: there are genes that encode proteins thought to have roles in cryoprotection, including COR, LEA and dehydrin polypeptides; enzymes involved in the synthesis of proline and raffinose, compatible solutes that have cryoprotective activities; regulatory proteins including the RAP2.1 and RAP2.6 transcription factors; proteins involved in transport and protein fate; and a number of putative or hypothetical proteins with unknown functions.

In sharp contrast, our survey of about 8700 tomato genes resulted in the identification of only three cold-regulated genes that were induced 2.5-fold or more by CBF-overexpression: two encoded dehydrins, which presumably have roles in cryoprotection, and the third encodes a probable proteinase inhibitor. These results suggest that the CBF regulon of tomato may be smaller in size and potentially less diverse in function than that of Arabidopsis. If, as estimated (Van der Hoeven et al., 2002), tomato has about 35 000 genes, then the tomato CBF regulon may be composed of only about 10 genes. Of course, this conclusion assumes that the 8000 or so tomato and Arabidopsis genes that have been surveyed are representative of their respective genomes. This may not be the case. It is possible that probes for genes that comprise the CBF regulon in tomato are grossly underrepresented on the available tomato microarray. If true, further investigation might reveal that the tomato and Arabidopsis CBF regulons are more similar in size and diversity of function than what would appear to be the case from the current survey. It should be pointed out, however, that the transcript levels for more than 8% (1085) of the probes on the microarray have been found to change 2.5-fold or more in tomato plants exposed to low temperature (4°C) (S.G. Fowler, Michigan State University, East Lansing, MI, USA, unpublished results). In comparison, Fowler and Thomashow (2002), using slightly stricter criteria and similar experimental conditions, found that approximately 4% of the Arabidopsis genes on the AtGenome1 GeneChip were cold responsive. Thus, the tomato mircoarray would not appear to be underrepresented generally in cold-responsive genes. Whatever the case, the results presented provide direct evidence that the tomato and Arabidopsis regulons differ in makeup as multiple genes that comprise the Arabidopsis regulon, including those encoding galactinol synthase, pyrroline-5-carboxylate and the RAP2.1 transcription factor, were not found to be members of the tomato CBF regulon.

Hsieh et al. (2002a,b) reported that overexpression of AtCBF1 in transgenic tomato results in upregulated expression of the CATALASE1 (CAT1) gene. Two cDNAs for this gene were on the microarray used in our study (TUS14M14 and TUS25N2). Our results indicated that there might be an increase in CAT1 transcript levels in the AtCBF3 and LeCBF1 overexpressing tomato plants, but if so, it was slight, being well below our 2.5-fold cut off limit. Moreover, analysis of these transcripts using the microarray indicated that transcripts for CAT1 did not increase in response to low temperature (S.G. Fowler, unpublished results). Thus, at this point, it would not appear that the CAT1 gene is a member of the tomato CBF regulon, but additional analysis is needed to draw a firm conclusion.

What accounts for the apparent differences in size and complexity of the tomato and Arabidopsis CBF regulons? One possibility is differences in the proteins required for CBF activity. For instance, in Arabidopsis, the SFR6 gene is required for the CBF transcription factors to stimulate expression of the COR genes (Boyce et al., 2003; Knight et al., 1999). In particular, the sfr6-1 mutation does not affect cold-induced expression of the CBF genes, but results in dramatically lower expression of CBF-targeted genes such as COR15a and COR6.6. Perhaps tomato encodes a ‘weak’ allele of SFR6 (or other gene required for high level CBF function), which contributes to most CBF-targeted genes being expressed at low levels. Alternatively, the apparent differences in tomato and Arabidopsis CBF regulons might reflect differences in the distribution of functional CRT/DRE elements within the genomes of these two plants. Additional study will be required to critically distinguish between these two possibilities.

The fact that both Arabidopsis and tomato have a CBF pathway suggests that this genetic system was present in a common ancestor of these plants. As both pathways include cold-regulated CBF genes, it seems likely that the common ancestor lived in a cold environment and had a CBF pathway that contributed to cold tolerance. As descendents of the common ancestor invaded different environments, changes in selective pressures could have fixed variants of the CBF pathway. In the case of tomato, this may have included loss of CRT/DRE elements from target genes that impart freezing tolerance as well as loss of cold-regulatory elements from the promoters of LeCBF2 and 3. Mutations resulting in weak alleles of SFR6 or other genes required for CBF activity might also have occurred. Such variation could have involved positive selection. Overexpression of CBF genes in Arabidopsis and tomato results in plants that produce less seed than wild-type plants when plants are grown under non-stressed conditions. Thus, mutations resulting in loss of CRT/DRE elements from target genes or a decrease in CBF expression or activity may have been selected for over time in plants that were not under pressure to survive freezing temperatures.

A related point regarding evolution of the CBF cold response pathways in tomato and Arabidopsis regards the fact that both plants have three CBF genes in tandem array. The finding that the three tomato CBF proteins more closely resemble each other than they do the Arabidopsis CBF proteins could be because of the putative common ancestor mentioned above having a single CBF gene and that independently, as both Arabidopsis and tomato evolved, gene duplication events occurred and were fixed in their respective populations. In this case, the existence of three CBF genes arranged in tandem in both Arabidopsis and tomato would be a coincidence resulting from unrelated events. An alternative possibility is that the putative common ancestor had a locus with three CBF genes in tandem and that the current loci in Arabidopsis and tomato have arisen through concerted evolution.

The evolutionary scenarios presented above are highly speculative and represent only a few of many possibilities. Indeed, developing scenarios regarding the evolution of the CBF cold response pathway is complicated by the fact that the CBF regulon of genes can have multiple functions. In Arabidopsis, the CBF regulon not only imparts freezing tolerance, but also imparts tolerance to chilling temperatures, dehydration stress and high salinity. Thus, selective pressures for functional CRT/DRE elements and the number and regulation of CBF genes would not be expected to be limited to freezing temperature, but involve other stresses as well. A survey of the CBF cold response pathways in a range of wild and cultivated tomato species as well as close relatives of tomato that differ in chilling and freezing tolerance such as Solanum tuberosum (chilling tolerant, freezing sensitive) and S. commersonii (chilling and freezing tolerant) should lead to a better understanding of how the CBF cold response pathway evolved in solanaceous plants and whether differences in the pathway contribute to the differences in cold tolerance observed in these plant species.

Finally, the discovery of the CBF cold response pathway and demonstration that overexpression of CBF genes increases freezing, chilling and drought tolerance has opened the possibility of improving the stress tolerance of agriculturally important plants through optimizing expression of the CBF genes. However, this approach has an inherent limitation: the composition of genes that comprise the CBF regulon in a given plant species. Overexpression of AtCBF1 in tomato has been shown to increase the chilling and drought tolerance of transgenic tomato plants (Hsieh et al., 2002a,b). However, as demonstrated here and elsewhere (Hsieh et al., 2002a), CBF overexpression in tomato does not result in a detectible increase in freezing tolerance. If this is because of a lack of functional CRT/DRE elements in the promoters of genes that are required to impart freezing tolerance, then manipulation of CBF expression would have limited potential for improving the freezing tolerance trait of tomato. If, on the other hand, tomato has an SFR6-like deficiency, then rectification of this ‘shortcoming’ might have an effect on freezing tolerance. Understanding more about the conservation and evolution of the CBF cold response pathway in tomato and other solanaceous plants should help address this issue.

Experimental procedures

Plant material, growth conditions and stress treatments

Seeds of tomato (L. esculentum var. D. Huang) were grown under sterile conditions on filter paper in Magenta boxes containing Murashige and Skoog (MS) medium (Life Technologies Inc., Gaithersburg, MD, USA) supplemented with 3% sucrose and solidified with 0.7% phytagar (Life Technologies Inc.). Seedlings were grown for 3 weeks with a 16 h light period under 100 μmol m−2 sec−1 cool-white fluorescent illumination at 26°C and 8 h dark period at 20°C.

Abiotic stress treatments were applied 8 h after the switch to the light phase. Low-temperature treatments were performed at 4°C with a photoperiod of 16 h and a light intensity of 20 μmol m−2 sec−1. Low-temperature treatments were also performed on tomato seedlings that had been grown under continuous illumination of 100 μmol m−2 sec−1, at 26°C, in pots containing Baccto Planting Mix (Michigan Peat, Houston, TX, USA). These plants were cold-treated as above except that illumination (20 μmol m−2 sec−1) at 4°C was continuous. For drought stress, 3-week-old seedlings were transferred to and incubated in Magenta boxes containing two layers of dry filter paper for varying times. For NaCl and ABA treatments, plants were transferred to Magenta boxes containing two layers of filter paper soaked with MS medium supplemented with 250 mm NaCl or 100 μm ABA, respectively. For drought, NaCl and ABA control treatments, seedlings were placed on filter paper saturated with MS medium and incubated for varying times. Mechanical agitation was performed by tapping Magenta boxes containing the plants against a bench for 5 min then allowing the box to remain undisturbed for 15 min (control plants were treated the same, except that the Magenta boxes were not tapped against the bench). Plants were harvested at varying times after stress treatments and immediately frozen in liquid nitrogen. Plant material was stored at −80°C prior to RNA extraction.

Arabidopsis thaliana (L.) Heynh. (ecotype Wassilewskija, Ws-2) and transgenic Arabidopsis plants in the Ws-2 background were grown in pots containing Baccto Planting Mix (Michigan Peat). The pots were placed in controlled environment chambers at 20°C under continuous, cool-white fluorescent illumination of 100–150 μmol m−2 sec−1 for 2–3 weeks. For cold treatments, pots containing the plants were transferred to 4°C under continuous light (20 μmol m−2 sec−1) as described (Gilmour et al., 1998).

For transcript profiling and subsequent Northern analysis, wild type and transgenic tomato plants in the D. Huang background were grown in pots containing Baccto Planting Mix (Michigan Peat). The seedlings were grown for 3 weeks with a photoperiod consisting of a 16 h light period (100–150 μmol m−2 sec−1) at 26°C and 8 h dark period at 20°C. For low-temperature treatments, plants were transferred to 4°C (16-h photoperiod, light intensity: 20 μmol m−2 sec−1) 8 h after the switch to the light phase.

Constructs and plant transformation

The entire cDNA insert for tomato CBF1 (accession no. AY034473) containing the whole coding region of LeCBF1 (Jaglo et al., 2001) was placed under control of the strong constitutive CaMV 35S promoter and transformed into Arabidopsis. Specifically, a 776-bp BamHI/KpnI fragment from a cDNA EST clone (accession no. AI484513) that encodes the tomato CBF1 gene was ligated into the BamHI and KpnI sites of PIC-20H vector. The resulting construct was digested with BamHI and Hind III, and the fragment cloned into the BglII and HindIII sites of the binary transformation vector PGA 643 (An et al., 1988). The resulting plasmid, pXIN, which contains the LeCBF1 coding sequence under control of CaMV 35S promoter, was transformed into Agrobacterium tumefaciens strain GV3101 by electroporation. Arabidopsis plants were transformed with plasmid pXIN1 using the floral dip method (Clough and Bent, 1998). Seeds of the transgenic Arabidopsis line (B6), which carries the T-DNA from the vector pGA643, were kindly provided by Sarah Gilmour. Transformed plants were selected on the basis of kanamycin resistance. Homozygous T3 plants were used for further study.

The plasmids, pXIN (see above) and pMPS13 (Gilmour et al., 2000), which contain the LeCBF1 and AtCBF3 coding sequences, respectively, were introduced into tomato (var. D. Huang) by Agrobacterium-mediated transformation using a leaf-disc transformation procedure (Costa et al., 2000; McCormick et al., 1986). Transformants were selected on plates of MS medium with kanamycin (50 mg−1 ml). The kanamycin-resistant plantlets were grown in magenta boxes, transferred to soil and grown in a greenhouse with a 16-h photoperiod. Two individual lines over-expressing each gene were selected for further study by Northern blot analysis.

Isolation of tomato cDNAs and a genomic clone encoding CBF-like proteins

A tomato EST clone EST246209 (accession no. AI487887) was identified through a BLAST search of the tomato EST database (provided by the National Center for Biotechnology Information) using the LeCBF1 sequence (Jaglo et al., 2001) as a query. This gene was named LeCBF2. The EST clone was obtained from the Clemson University Genomics Institute (Clemson, SC, USA).

To determine the precise structural organization of the tomato CBF locus, we constructed a tomato bacteriophage λ genomic library using DNA isolated from tomato accession TA491 (Van der Knaap and Tanksley, 2001) and screened this library with a LeCBF2 probe. This resulted in the isolation of λ clone Le-3, a 19 kb insert encompassing LeCBF1–3. The complete DNA sequence of λ clone Le-3 (GenBank accession no. AY497899) was determined by primer walking through overlapping NotI and XbaI subclones in plasmid vector pGEM11Z using the facilities of the OSU/OARDC Molecular and Cellular Imaging Center. Additional coding regions outside of the CBF locus were identified using the gene structure prediction software genescan with the Arabidopsis matrix function (Burge and Karlin, 1997), and by conducting BLAST searches against the tomato and Arabidopsis DNA sequence databases (Altschul et al., 1990, 1997; Quackenbush et al., 2001). Comparisons between the 19 kb tomato λ clone Le-3 and the Arabidopsis chromosome segments were conducted using PipMaker (http://bio.cse.psu.edu/pipmaker/) (Schwartz et al., 2000). The chromosomal location of the CBF locus was determined utilizing 100 F2 individuals derived from an L. esculentum × L. pimpinellifolium cross (Van der Knaap and Tanksley, 2001), the polymorphic enzymes EcoRV and ScaI, and the software package mapmaker version 2.0 (Lander et al., 1987) with the Kosambi mapping function (Kosambi, 1944). Searches of the LeCBF1–3 promoter regions were performed using the web-based interface for MotifSampler (Thijs et al., 2002, http://www.esat.kuleuven.ac.be/thijs/Work/MotifSampler.html).

DNA sequencing and analysis

Double-stranded sequencing of plasmid DNA was performed on an Applied Biosystems Automated Fluorescent Sequencer at the Michigan State University Genome Technology Support Facility. Sequence analysis was performed using dnastar software (DNASTAR, Madison, WI, USA).

DNA and RNA hybridization

Total RNA was isolated from tomato and Arabidopsis plants (Howe et al., 1996). The RNA (5–20 μg) was electrophoresed in a 1% agarose formaldehyde gel and transferred to a nylon filter (Hybond-N, Amersham, Buckinghamshire, UK) as described (Stockinger et al., 1997). Tomato genomic DNA was isolated from mature leaves using the Wizard Genomic DNA Purification kit (Promega, Madison, WI, USA). Southern blotting was performed using 10 μg of genomic DNA digested with restriction enzymes, which was transferred to a nylon membrane as previous described (Stockinger et al., 1997). P32-labeled probes were prepared by random priming. The probe for LeCBF1 was prepared from a full-length cDNA of LeCBF1 (Jaglo et al., 2001) and the probe for LeCBF2 was the entire cDNA insert from EST AI487887. The probe for LeCBF3 transcript was obtained by amplifying approximately 900 bp from a subclone of the Le-3 λ clone by PCR using the primers 5′ CCGTGTTCTTTGGTCTCAAC-3′ and 5′-GTCTTATATACGCCAATGATTCTC-3′. Gene-specific probes for LeCBF1 and LeCBF2 were obtained by amplifying approximately 150 bp of the 3′ ends of cDNA inserts by PCR using the primers: LeCBF1: 5′-GTGTGGAAACTGATGCCTAC-3′ and 5′-ATGTTCATGTATCCCGGCCA-3′, LeCBF2: 5′-ACGTTGAATTAGCTGATGTG-3′ and 5′-TTCAAATATTGTTACTACGC-3′. A gene-specific probe for LeCBF3 was obtained by amplifying approximately 150 bp 3′ UTR of the LeCBF3 using a subclone of the Le-3 λ clone as the template. The primers used were 5′-GTGGAAGCTGATGATATGCC-3′ and 5′ CACTAATTCGTACCATATCC-3′. Probes for the tomato genes, Ci7/TC116013, dehydrin-like/TC116174, proteinase inhibitor/TC115955 and glycine-rich protein/TC117384, were the entire cDNA inserts from the EST clones cLEN12G19, cLER17C11, cLER4K22 and cLET13N19 respectively. To estimate relative loading and transfer, filters were hybridized a second time with probes for the constitutively expressed eukaryotic Initiation Factor 4A (eIF-4A) gene (Metz et al., 1992) or 25S rDNA (Delseny et al., 1983) which were isolated from cDNA clones encoding these genes by restriction enzyme digestion. Filters were hybridized, and washed as described (Gilmour et al., 2000; Stockinger et al., 1997).

Protein extraction and Western analysis

Total soluble protein was obtained essentially as described (Gilmour et al., 1996) by grinding leaf material (approximately 100 mg) in 0.4 ml of extraction buffer (50 mm 1,4-piperazinediethanesulfonic acid, pH 7.0, 25 mm EDTA) containing 2.5% (w/v) polyvinyl-polypyrrolidone and removing insoluble material by centrifugation (16 000 g, 20 min). Protein concentration in the supernatant was measured using the dye-binding method of Bradford (1976) with bovine serum albumin as the standard. For Western analysis, 50 μg of total protein was separated by 10% Tricine (N [tris(hydroxymethyl)methyl]glycine)/SDS/PAGE (Schägger and von Jagow, 1987) and electroblotted onto 0.2 μ nitrocellulose membranes (Towbin et al., 1979). COR15am and COR6.6 were detected using the enhanced chemiluminescence kit (Amersham) with antiserum raised to recombinant COR15am and COR6.6 polypeptides (Gilmour et al., 1996).

Electrolyte leakage freeze test

The freezing tolerance of transgenic plants overexpressing LeCBF1 was determined using the electrolyte leakage test as described (Jaglo-Ottosen et al., 1998).

Transcript profiling of tomato

Total RNA was extracted, as described above, from a pool of tissue derived from the aerial parts of eight individual tomato seedlings cold treated as above. Labeled probe cDNA was prepared from 50 μg of total RNA using the 3DNA Submicro EX Expression Array Cy3/Cy5 kit (Genisphere, Montvale, NJ, USA). Briefly, a primer consisting of poly(dT), fused to a ‘capture sequence’ was used to prime first strand cDNA synthesis. The capture sequence is complementary to a nucleotide sequence carried by the 3DNA dye reagent and so allows hybridization of the dye reagent to probe molecules hybridized to the arrays.

An experimental and a control sample, each labeled with either the Cy3 or Cy5 capture sequence were mixed and hybridized overnight at 55°C to a tomato microarray obtained from the Center for Gene Expression Profiling (http://bti.cornell.edu/CGEP/CGEP.html) in a total volume of 67 μl of hybridization buffer as described by the manufacturer of the kit. The arrays were then washed in 2× SSC, 0.2% SDS at 55°C for 10 min, in 2× SSC at room temperature for 10 min and in 0.2× SSC at room temperature for 10 min after which they were rinsed in 95% ethanol and centrifuged dry. Arrays were hybridized a second time for 2 h at 55°C with a 67 μl volume containing Cy3 and Cy5 capture reagents in hybridization buffer as described by the manufacturer of the kit. After the second hybridization, arrays were washed and dried as described above except that the arrays were protected from light during these procedures. Hybridized arrays were scanned in an Affymetrix 428 Array Scanner (Affymetrix, Santa Clara, CA, USA). Each competitive hybridization was repeated four times; two biological replicate samples were prepared with new tissue grown and treated under identical conditions, then each biological replicate pair of samples was hybridized to two chips with the dye labeling reversed.

The intensities of spots were measured using Genepix 3.0 (Axon Instruments Inc., Union City, CA, USA). Log ratios of the Cy3 and Cy3 intensities were normalized with the print-tip group lowess normalization of Terry Speed using the SMA module (Dudoit et al., 2003) for the R statistical package (http://www.r-project.org/). To remove unreliable spots, those caused by dust or background on the array and those with un-normalized intensities of less than 500 in both channels, were flagged and ignored. Spots where <55% of the pixels making up the spot had an intensity greater than two standard deviations above background were removed. The array data were compared and queried using microsoft access software (Microsoft, Redmond, WA, USA).

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Acknowledgements

We thank Paul Debbie from the Center for Gene Expression Profiling, which is funded by the National Science Foundation (DBI 0116076), for advice on microarray hybridization and gene annotation; Jeff Landgraff (MSU Genome Technology Support Facility) for technical advice on microarray hybridization and analysis; Esther van der Knaap, who made her survey and mapping filters available to us, and provided assistance with the genetics; Marianne Huebner, Vincent Melfi, Peter Bergholz and Rebecca Grumet for helpful discussions and advice on data analysis; and Elizabeth Todd, a College of Wooster Independent Studies student whose preliminary data indicated the presence of three linked and intron-less tomato CBF genes. This research was supported in part by a grant from the NSF Plant Genome Program (DBI 0110124). Research in the Thomashow laboratory was also supported in part by grants from the DOE (DEFG0291ER20021) and the Michigan Agricultural Experiment Station.

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