The Arabidopsis CBF gene family is composed of three genes encoding AP2 domain-containing proteins whose expression Is regulated by low temperature but not by abscisic acid or dehydration

Abstract

We have identified two genes from Arabidopsis that show high similarity with CBF1, a gene encoding an AP2 domain-containing transcriptional activator that binds to the low-temperature-responsive element CCGAC and induces the expression of some cold-regulated genes, increasing plant freezing tolerance. These two genes, which we have named CBF2 and CBF3, also encode proteins containing AP2 DNA-binding motifs. Furthermore, like CBF1, CBF2 and CBF3 proteins also include putative nuclear-localization signals and potential acidic activation domains. The CBF2 and CBF3 genes are linked to CBF1, constituting a cluster on the bottom arm of chromosome IV. The high level of similarity among the three CBF genes, their tandem organization, and the fact that they have the same transcriptional orientation all suggest a common origin. CBF1, CBF2, and CBF3 show identical expression patterns, being induced very rapidly by low-temperature treatment. However, in contrast to most of the cold-induced plant genes characterized, they are not responsive to abscisic acid or dehydration. Taken together, all of these data suggest that CBF2 and CBF3 may function as transcriptional activators, controlling the level of low-temperature gene expression and promoting freezing tolerance through an abscisic acid-independent pathway.

Figure 1

Genomic organization of the CBF1, CBF2, and CBF3 genes. A, Physical map of CBF genes on chromosome IV of Arabidopsis. The ORFs are shown with open bars. The restriction sites are HindIII (H), XbaI (X), and ScaI (S). The direction of transcriptions is indicated by arrows. B, DNA-blot hybridizations of Arabidopsis genomic DNA (4 μg) digested with ScaI (S), NdeI (N), XbaI (X), BglII (B), and HindIII (H). The probes used were the 692-bp RsaI fragment from CBF2 and the 1124-bp HindIII fragment from CBF3, as described in Methods. The positions of molecular size markers are in the center.

Figure 2

Alignment of the 5′ upstream sequences of CBF1, CBF2, and CBF3. Six-hundred-eighty bases of the 5′-untranslated region of CBF1 were aligned with 720 and 702 bases of the corresponding regions of CBF2 and CBF3, respectively. Asterisks indicate nucleotides identical to the CBF1 sequence. Hyphens indicate gaps inserted in the sequences for better alignment. The initiation ATGs and putative TATA boxes (TATAAA) are double underlined. CANNTG motifs and related sequences CACGTC and TACGTG are shown in gray boxes. Black boxes highlight the CAGCC pentamers. The CCGTC sequence is single underlined.

Figure 3

Amino acid sequences of CBF1, CBF2, and CBF3 proteins. A, Sequence alignment. Amino acid residues identical to the CBF1 sequence at a given position are indicated by asterisks. Points represent gaps inserted in the sequences for better alignment. Black box corresponds to the potential nuclear localization signals. Gray boxes highlight the AP2 domains. Underlined amino acids indicate potential recognition sites for protein kinase C (•) and casein kinase II (⧫). B, Comparison of the AP2 domains from CBF1, CBF2, and CBF3 proteins and the Arabidopsis ethylene-responsive element-binding protein AtEBP (Bütner and Singh, 1997). Identical amino acids are highlighted in black boxes. Points represent gaps inserted in the sequences for better alignment. Residues belonging to the conserved AP2 domain elements YRG and RAYD (Okamuro et al., 1997) are indicated. Amino acids in the RAYD conserved element predicted to form an amphipatic α-helix that might promote DNA binding (Okamuro et al., 1997) are underlined.

Figure 4

CBF1, CBF2, and CBF3 transcripts accumulate in response to low temperature but not in response to ABA or water stress. RNA-blot hybridizations were performed with total RNA (10 μg per lane) isolated from leaves exposed to 4°C for the indicated time, sprayed with 100 μm ABA (A), sprayed with the ABA solvent (C), or dehydrated until losing 50% of their fresh weight (D). The probes used were the 3′ fragments of CBF1, CBF2, and CBF3 and the fragments of the KIN1 and RBP4 genes described in Methods.

Figure 5

Accumulation of CBF1, CBF2, and CBF3 transcripts in different organs of Arabidopsis in response to low temperature. RNA-blot hybridizations were carried out with total RNA (15 μg per lane) isolated from leaves, stems, and roots of plants grown at 22°C (C) or exposed to 4°C for 1 h (4°). The specific probes for the CBF genes and the probe used for RBP4 are described in Methods.