Genetic and molecular analyses of natural variation indicate CBF2 as a candidate gene for underlying a freezing tolerance quantitative trait locus in Arabidopsis

Abstract

Natural variation for freezing tolerance is a major component of adaptation and geographic distribution of plant species. However, little is known about the genes and molecular mechanisms that determine its naturally occurring diversity. We have analyzed the intraspecific freezing tolerance variation existent between two geographically distant accessions of Arabidopsis (Arabidopsis thaliana), Cape Verde Islands (Cvi) and Landsberg erecta (Ler). They differed in their freezing tolerance before and after cold acclimation, as well as in the cold acclimation response in relation to photoperiod conditions. Using a quantitative genetic approach, we found that freezing tolerance differences after cold acclimation were determined by seven quantitative trait loci (QTL), named FREEZING TOLERANCE QTL 1 (FTQ1) to FTQ7. FTQ4 was the QTL with the largest effect detected in two photoperiod conditions, while five other FTQ loci behaved as photoperiod dependent. FTQ4 colocated with the tandem repeated genes C-REPEAT BINDING FACTOR 1 (CBF1), CBF2, and CBF3, which encode transcriptional activators involved in the cold acclimation response. The low freezing tolerance of FTQ4-Cvi alleles was associated with a deletion of the promoter region of Cvi CBF2, and with low RNA expression of CBF2 and of several CBF target genes. Genetic complementation of FTQ4-Cvi plants with a CBF2-Ler transgene suggests that such CBF2 allelic variation is the cause of CBF2 misexpression and the molecular basis of FTQ4.

Figure 1.

Freezing tolerance of Ler and Cvi accessions. A, Percentage of surviving plants to different freezing temperatures with or without previous cold acclimation. Bars correspond to the mean ± se of three replicates. Sigmoidal curves were fitted by a distance-weighted least-squares procedure, from which the LT₅₀ values shown in the legend were calculated. A, Cold acclimated; NA, non-cold acclimated. B, Cold-acclimated plants of Ler and Cvi (surrounded by circles) and various RIL and transgenic lines 2 weeks after freezing at −8°C.

Figure 2.

QTL likelihood maps for freezing tolerance in the Ler-Cvi RIL population grown under two photoperiod conditions. Genetic maps of the Arabidopsis linkage groups are shown in the abscissa and LOD scores in the ordinate. Chromosome numbers are indicated in the top right corner of each image. The LOD threshold used for QTL detection is shown as a hatched horizontal gray line and the 2-LOD support intervals of the detected QTL are depicted as thick lines on the genetic maps. Continuous or hatched LOD profiles and 2-LOD intervals correspond to QTL mapping under LD or SD photoperiods, respectively. For each QTL, its name, the percentage of phenotypic variance explained, and its allele additive effect are shown. Allele effects are given in survival percentage units, positive values indicating that Ler genotypes show higher freezing tolerance than Cvi.

Figure 3.

Expression of cold-induced genes in Ler, Cvi, and Col. A, RNA-blot hybridizations of CBF1, CBF2, and CBF3 in plants exposed to 4°C for 0, 1, 2, or 4 h. Ethidium bromide staining of the ribosomal RNA (rRNA) is shown as a loading control. B, RNA-blot hybridizations of CBF target genes in Ler and Cvi control plants (0 h) and plants exposed to 4°C for 24 h. Hybridization of a RBP4 probe is shown as a loading control.

Figure 4.

DNA sequence comparison of the genomic regions containing CBF1, CBF2, and CBF3 genes from Ler, Cvi, and Col. Genomic organization of the CBF cluster is shown in the top part. Single-nucleotide substitutions are depicted as vertical bars, different colors denoting allele compositions (indicated in the bottom left box). Indels are depicted as vertical arrows with similar color codes. Thirteen indels affect single nucleotides, one involves two nucleotides, and another five nucleotides. A large Ler-Cvi deletion (Del) is represented as a yellow color box. Asterisks (*) indicate amino acid changes. A summary of the number of polymorphisms is presented in the bottom left box. Ler-Col polymorphisms in the 1.6-kb genomic deletion of Cvi are not considered and are depicted in black. Numbers in the top part correspond to the Ler-Cvi polymorphisms in the 0.6-kb coding regions of the CBF genes and in the first kilobase 5′ upstream from the start codons. GenBank accession numbers of Cvi, Ler, and Col sequences are AY667247, AF076155, and AL022197, respectively.

Figure 5.

Expression of a CBF2 transgene and freezing tolerance of transgenic lines. A, Expression of CBF2 in Cvi, RIL44, and Ler untransformed plants and in transgenic lines containing the Ler CBF2 transgene. Expression was analyzed in control plants not exposed to cold (0) and in plants of the same line exposed to 4°C during 2 h (2). The ribosomal RNA (rRNA) stained with ethidium bromide is shown as a loading control. B, Freezing tolerance after cold acclimation under a LD photoperiod of transgenic lines overexpressing a Ler CBF2 gene. Dark gray color bars depict transgenic lines, and light gray bars represent untransformed controls. Bars correspond to mean ± se of each transgenic line derived from four replicates. Numbers in the top part of the images show the mean ± se of the various transgenic lines analyzed for each background. T2 Cvi, T2 RIL42, T2 RIL44, and T2 Ler denote different independent T2 transgenic homozygous lines in the corresponding genetic backgrounds.