Many plants, including Arabidopsis, show increased resistance to freezing after they have been exposed to low nonfreezing temperatures. This response, termed cold acclimation, is associated with the induction of COR (cold-regulated) genes mediated by the C-repeat/drought-responsive element (CRT/DRE) DNA regulatory element. Increased expression of Arabidopsis CBF1, a transcriptional activator that binds to the CRT/DRE sequence, induced COR gene expression and increased the freezing tolerance of nonacclimated Arabidopsis plants. We conclude that CBF1 is a likely regulator of the cold acclimation response, controlling the level of COR gene expression, which in turn promotes tolerance to freezing.

Summary The CBF cold response pathway has a prominent role in cold acclimation. The pathway includes action of three transcription factors, CBF1, 2 and 3 (also known as DREB1b, c and a, respectively), that are rapidly induced in response to low temperature followed by expression of the CBF-targeted genes (the CBF regulon) that act in concert to increase plant-freezing tolerance. The results of transcriptome profiling and mutagenesis experiments, however, indicate that additional cold response pathways exist and may have important roles in life at low temperature. To further understand the roles that the CBF proteins play in configuring the low temperature transcriptome and to identify additional transcription factors with roles in cold acclimation, we used the Affymetrix GeneChip containing probe sets for approximately 24,000 Arabidopsis genes to define a core set of cold-responsive genes and to determine which genes were targets of CBF2 and 6 other transcription factors that appeared to be coordinately regulated with CBF2. A total of 514 genes were placed in the core set of cold-responsive genes, 302 of which were upregulated and 212 downregulated. Hierarchical clustering and bioinformatic analysis indicated that the 514 cold-responsive transcripts could be assigned to one of seven distinct expression classes and identified multiple potential novel cis-acting cold-regulatory elements. Eighty-five cold-induced genes and eight cold-repressed genes were assigned to the CBF2 regulon. An additional nine cold-induced genes and 15 cold-repressed genes were assigned to a regulon controlled by ZAT12. Of the 25 core cold-induced genes that were most highly upregulated (induced over 15-fold), 19 genes (84%) were induced by CBF2 and another two genes (8%) were regulated by both CBF2 and ZAT12. Thus, the large majority (92%) of the most highly induced genes belong to the CBF and ZAT12 regulons. Constitutive expression of ZAT12 in Arabidopsis caused a small, but reproducible, increase in freezing tolerance, indicating a role for the ZAT12 regulon in cold acclimation. In addition, ZAT12 downregulated the expression of the CBF genes indicating a role for ZAT12 in a negative regulatory circuit that dampens expression of the CBF cold response pathway.

SummaryCold-induced expression of the Arabidopsis COR (coldregulated) genes is mediated by a DNA regulatory element termed the CRT (C-repeat)/DRE (dehydrationresponsive element). Recently, we identified a transcriptional activator, CBF1, that binds to the CRT/DRE and demonstrated that its overexpression in transgenic Arabidopsis plants at non-acclimating temperatures induces COR gene expression and increases plant freezing tolerance. Here we report that CBF1 belongs to a small family of closely related proteins which includes CBF2 and CBF3. DNA sequencing of an 8.7 kb region of the Arabidopsis genome along with genetic mapping experiments indicated that the three CBF genes are organized in direct repeat on chromosome 4 at 72.8 cM, closely linked to molecular markers PG11 and m600. Like CBF1, both CBF2 and CBF3 activated expression of reporter genes in yeast that contained the CRT/DRE as an upstream activator sequence. The transcript levels for all three CBF genes increased within 15 min of transferring plants to low temperature, followed by accumulation of COR gene transcripts at about 2 h. CBF transcripts also accumulated rapidly in response to mechanical agitation. The promoter regions of the CBF genes do not contain the CRT sequence, CCGAC, and overexpression of CBF1 did not have a detectable effect on CBF3 transcript levels, suggesting that the CBF gene family is not subject to autoregulation. We propose that cold-induced expression of CRT/DREcontaining COR genes involves a low temperaturestimulated signalling cascade in which CBF gene induction is an early event.

The Arabidopsis CBF1, 2, and 3 genes (also known as DREB1b, c, and a, respectively) encode transcriptional activators that have a central role in cold tolerance. CBF1-3 are rapidly induced upon exposing plants to low temperature, followed by expression of CBF-targeted genes, the CBF regulon, resulting in an increase in plant freezing tolerance. At present, little is known about the cold-sensing mechanism that controls CBF expression. Results presented here indicate that this mechanism does not require a cold shock to bring about the accumulation of CBF transcripts, but instead, absolute temperature is monitored with a greater degree of input, i.e. lower temperature, resulting in a greater output, i.e. higher levels of CBF transcripts. Temperature-shift experiments also indicate that the cold-sensing mechanism becomes desensitized to a given low temperature, such as 4°C, and that resensitization to that temperature requires between 8 and 24 h at warm temperature. Gene fusion experiments identified a 125-bp section of the CBF2 promoter that is sufficient to impart cold-responsive gene expression. Mutational analysis of this cold-responsive region identified two promoter segments that work in concert to impart robust cold-regulated gene expression. These sequences, designated ICEr1 and ICEr2 (induction of CBF expression region 1 or 2), were also shown to stimulate transcription in response to mechanical agitation and the protein synthesis inhibitor, cycloheximide.Many plants increase in freezing tolerance in response to low nonfreezing temperatures, a phenomenon known as cold acclimation (Guy, 1990;Thomashow, 1999). In Arabidopsis, cold acclimation involves action of the CBF cold-response pathway (Thomashow, 2001). Within 15 min of exposing plants to low temperatures, transcripts accumulate for a family of genes designated CBF1, CBF2, and CBF3 Jaglo-Ottosen et al., 1998; Medina et al., 1999), or DREB1b, DREB1c, and DREB1a (Liu et al., 1998), respectively, which encode transcriptional activators that are members of the AP2/EREBP family of DNA-binding proteins (Riechmann and Meyerowitz, 1998). These transcription factors bind the cold-and dehydration-responsive DNA regulatory element designated the CRT (C-repeat)/ DRE (dehydration response element); (Baker et al., 1994;Yamaguchi-Shinozaki and Shinozaki, 1994;Stockinger et al., 1997) that is present in the promoters of COR and many other cold-responsive genes and stimulate their transcription. Expression of the CBF regulon of target genes then leads to an increase in freezing tolerance Jaglo-Ottosen et al., 1998;Liu et al., 1998;Kasuga et al., 1999). Multiple mechanisms appear to contribute to the enhancement of freezing tolerance, including the synthesis of cryoprotective polypeptides, such as COR15a (Artus et al., 1996;Steponkus et al., 1998), and the accumulation of compatible solutes that have cryoprotective properties, including Suc, raffinose, and Pro (Nanjo et al., 1999;Gilmour et al., 2000;Taji et al., 2002).Currently, little is known about how the CBF gen...

Many cold-regulated genes of Arabidopsis are inducible by abscisic acid (ABA) as well as by cold. This has been thought to occur via two separate signaling pathways, with ABA acting via ABA-responsive promoter elements and low temperature activating the C-repeat element (CRT; dehydration-responsive) promoter element via CBF (DREB1) transcription factors. We show here that ABA is also capable of activating the CRT promoter element. Although the more recently discovered ABAinducible CBF4 transcription factor might have accounted for this, we show here that CBF1-3 transcript levels also increase in response to elevated ABA levels. This increase in CBF1-3 transcript levels appears to be at least in part due to increased activity of the CBF promoters in response to ABA. A total of 125 bp of the CBF2 promoter, which has previously been shown to be sufficient for cold-, mechanical-, and cycloheximide-induced expression, was also sufficient for ABA-induced expression. However, the ABA-responsive promoter element-like motif within this region is not needed for ABA-induced expression. An observed increase in CBF protein levels after ABA treatment, together with previous data showing that increased CBF levels are sufficient for cold-regulated gene induction, suggests that ABA-induced increases in CBF1-3 transcript levels do have the potential to activate the CRT. Our data indicate therefore that activation of the CRT may also occur via a novel ABA-inducible signaling pathway using the normally cold-inducible CBFs.The phytohormone abscisic acid (ABA) is involved in mediating responses to a number of environmental stresses including drought (Leung and Giraudat, 1998). Cellular ABA accumulates in response to such stresses, and increases in its concentration can lead to a number of physiological adaptations, including stomatal closure and growth inhibition, as well as up-regulation of specific genes. Many of the genes that are inducible by ABA are also expressed in response to cold and/or drought conditions; for example, the Arabidopsis cold-regulated (COR) genes RAB18, LTI78, and KIN2 (Kurkela and Franck, 1990; Lång and Palva, 1992;Nordin et al., 1993;Mäntylä et al., 1995). Several signaling pathways leading to COR gene expression have been described, including both ABA-dependent and ABA-independent pathways (Shinozaki and Yamaguchi-Shinozaki, 2000). The gene LTI78 (also known as COR78 or RD29A) has been studied as a paradigm system for a gene whose expression can be controlled by osmotic stress, low temperature, and ABA (Yamaguchi-Shinozaki and Shinozaki, 1994; Ishitani et al., 1997; Liu et al., 1998) via either ABAdependent or ABA-independent routes .Two cis-acting elements have been identified in the promoter of LTI78 and other COR genes that control expression under different stress conditions. The dehydration-responsive (DRE) element (YamaguchiShinozaki and Shinozaki, 1994) has been shown to mediate both cold-and osmotic stress-inducible ABA-independent gene expression (Liu et al., 1998;Shinozaki and Yamaguchi-Shinozak...

The effects of selection on genome variation were investigated and visualized in tomato using a high-density single nucleotide polymorphism (SNP) array. 7,720 SNPs were genotyped on a collection of 426 tomato accessions (410 inbreds and 16 hybrids) and over 97% of the markers were polymorphic in the entire collection. Principal component analysis (PCA) and pairwise estimates of F st supported that the inbred accessions represented seven sub-populations including processing, large-fruited fresh market, large-fruited vintage, cultivated cherry, landrace, wild cherry, and S. pimpinellifolium. Further divisions were found within both the contemporary processing and fresh market sub-populations. These sub-populations showed higher levels of genetic diversity relative to the vintage sub-population. The array provided a large number of polymorphic SNP markers across each sub-population, ranging from 3,159 in the vintage accessions to 6,234 in the cultivated cherry accessions. Visualization of minor allele frequency revealed regions of the genome that distinguished three representative sub-populations of cultivated tomato (processing, fresh market, and vintage), particularly on chromosomes 2, 4, 5, 6, and 11. The PCA loadings and F st outlier analysis between these three sub-populations identified a large number of candidate loci under positive selection on chromosomes 4, 5, and 11. The extent of linkage disequilibrium (LD) was examined within each chromosome for these sub-populations. LD decay varied between chromosomes and sub-populations, with large differences reflective of breeding history. For example, on chromosome 11, decay occurred over 0.8 cM for processing accessions and over 19.7 cM for fresh market accessions. The observed SNP variation and LD decay suggest that different patterns of genetic variation in cultivated tomato are due to introgression from wild species and selection for market specialization.

Many plants increase in freezing tolerance upon exposure to low nonfreezing temperatures, a phenomenon known as cold acclimation. A fundamental goal of cold acclimation research is to identify genes with key roles in this response. Here we review results from our laboratory regarding the discovery of a family of transcriptional activators in Arabidopsis (Arabidopsis thaliana) that regulates the expression of freezing tolerance genes. Specifically, we have identified 3 genes that encode nearly identical transcriptional activators that bind to the CRT (C‐repeat)/DRE (dehydration responsive element) DNA regulatory element present in the promoters of many cold‐ and drought‐inducible genes, including those designated COR (cold‐regulated). These regulatory genes, CBF1, CBF2 and CBF3 (CRT/DRE binding factor), are located in tandem array on chromosome 4. Overexpression of the CBF genes in Arabidopsis induces expression of the entire battery of known COR genes and increases freezing tolerance without a low temperature stimulus. We have, therefore, proposed that the CBF genes are ‘master switches’ that activate a regulon of genes involved in cold acclimation. Significantly, the CBF genes themselves are responsive to low temperature; the levels of CBF transcripts begin increasing within 15 min of transferring plants to low temperature followed by accumulation of COR gene transcripts at 2–4 h. The CBF genes do not appear to be subject to autoregulation as the promoter regions have no evident CRT/DRE elements and overexpression of CBF1 does not induce expression of CBF3. Thus, we have suggested that COR gene induction involves a two‐step cascade of transcriptional activators: the first step, CBF induction, involving an unknown activator present at normal growth temperature and the second step, COR gene induction, involving the action of the CBF activators.

The objective of this study was to construct a single nucleotide polymorphism (SNP)-based genetic map at the cultivated tetraploid level to locate quantitative trait loci (QTL) contributing to economically important traits in potato (Solanum tuberosum L.). The 156 F1 progeny and parents of a cross (MSL603) between “Jacqueline Lee” and “MSG227-2” were genotyped using the Infinium 8303 Potato Array. Furthermore, the progeny and parents were evaluated for foliar late blight reaction to isolates of the US-8 genotype of Phytophthora infestans (Mont.) de Bary and vine maturity. Linkage analyses and QTL mapping were performed using a novel approach that incorporates allele dosage information. The resulting genetic maps contained 1972 SNP markers with an average density of 1.36 marker per cM. QTL mapping identified the major source of late blight resistance in “Jacqueline Lee.” The best SNP marker mapped ∼0.54 Mb from a resistance hotspot on the long arm of chromosome 9. For vine maturity, the major-effect QTL was located on chromosome 5 with allelic effects from both parents. A candidate SNP marker for this trait mapped ∼0.25 Mb from the StCDF1 gene, which is a candidate gene for the maturity trait. The identification of markers for P. infestans resistance will enable the introgression of multiple sources of resistance through marker-assisted selection. Moreover, the discovery of a QTL for late blight resistance not linked to the QTL for vine maturity provides the opportunity to use marker-assisted selection for resistance independent of the selection for vine maturity classifications.