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. 2004 Mar 16;101(11):3985-90.
doi: 10.1073/pnas.0303029101. Epub 2004 Mar 2.

CBF2/DREB1C is a negative regulator of CBF1/DREB1B and CBF3/DREB1A expression and plays a central role in stress tolerance in Arabidopsis

Fernando Novillo  1 , José M Alonso, Joseph R Ecker, Julio Salinas
Affiliations

CBF2/DREB1C is a negative regulator of CBF1/DREB1B and CBF3/DREB1A expression and plays a central role in stress tolerance in Arabidopsis

Fernando Novillo et al. Proc Natl Acad Sci U S A. .

Abstract

CBF/DREB1 (C-repeat-binding factor/dehydration responsive element-binding factor 1) genes encode a small family of transcriptional activators that have been described as playing an important role in freezing tolerance and cold acclimation in Arabidopsis. To specify this role, we used a reverse genetic approach and identified a mutant, cbf2, in which the CBF2/DREB1C gene was disrupted. Here, we show that cbf2 plants have higher capacity to tolerate freezing than WT ones before and after cold acclimation and are more tolerant to dehydration and salt stress. All these phenotypes correlate with a stronger and more sustained expression of CBF/DREB1-regulated genes, which results from an increased expression of CBF1/DREB1B and CBF3/DREB1A in the mutant. In addition, we show that the expression of CBF1/DREB1B and CBF3/DREB1A in response to low temperature precedes that of CBF2/DREB1C. These results indicate that CBF2/DREB1C negatively regulates CBF1/DREB1B and CBF3/DREB1A, ensuring that their expression is transient and tightly controlled, which, in turn, guarantees the proper induction of downstream genes and the accurate development of Arabidopsis tolerance to freezing and related stresses.

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Figures

Fig. 1.
Fig. 1.
Structure of CBF/DREB1 cluster and T-DNA insertion in the CBF2/DREB1C gene. (A) Schematic representation of the CBF/DREB1 cluster with the T-DNA insertion. The site of insertion in the cbf2 mutant, 179 bp upstream of the CBF2/DREB1C start codon, is represented. Arrows indicate the direction of transcription in the CBF/DREB1 genes. The CBF locus is not drawn to scale. (B) RNA-blot hybridization by using a specific probe for CBF2/DREB1C and total RNA (20 μg) prepared from 3-week-old rosette leaves of WT and cbf2 mutant plants grown under control conditions (C) or exposed to 4°C for 1 h and 3 h. Equal amounts of RNA were present in each sample as confirmed by ethidium bromide staining of rRNAs.
Fig. 2.
Fig. 2.
Freezing tolerance of cbf2 mutant plants. Three-week-old WT and cbf2 plants grown under long-day photoperiods at 20°C were exposed to different freezing temperatures for 6 h. Freezing tolerance was estimated as the percentage of plants surviving each specific temperature after 7 days of recovery under unstressed conditions. (A) Tolerance of nonacclimated plants. (B) Representative nonacclimated WT and cbf2 plants 7 days after being exposed to -6°C for 6 h. (C) Tolerance of cold-acclimated (7 days at 4°C) plants. (D) Representative cold-acclimated WT and cbf2 plants 7 days after being exposed to -10°C for 6 h. In A and C, data are expressed as means of three independent experiments with 50 plants each. Bars indicate SE.
Fig. 3.
Fig. 3.
Tolerance to dehydration and salt stress of cbf2 mutant plants. (A) Dehydration tolerance of 3-week-old WT and cbf2 plants. Tolerance was estimated as the percentage of initial FW that remains after transferring plants to a dry filter paper and allowing them to develop for 2 days without watering. (B) Representative WT and cbf2 plants after dehydration treatment. (C) Salt tolerance of 3-week-old WT and cbf2 plants. Tolerance was estimated by determining the root elongation and FW of plants transferred to a medium containing 100 mM NaCl for 7 days. These values are represented as a percentage of root elongation and FW of WT unstressed plants. (D) Representative WT and cbf2 plants after salt treatment. In A and C, data are expressed as means of three independent experiments with 20 plants each. Bars indicate SE. Values obtained from WT and cbf2 were in all cases significantly different (P < 0.05) as determined by Student's t test.
Fig. 4.
Fig. 4.
Transcript levels of cold-induced genes in the cbf2 mutant and in the complemented cbf2 mutant. RNA-blot hybridizations were performed with total RNA (20 μg) isolated from 3-week-old rosette leaves of WT, cbf2, and complemented cbf2 (cbf2+CBF2) plants grown under control conditions (C) or exposed to 4°C for the indicated times. In all cases, gene-specific probes were used for the hybridizations. (A) Transcript levels of LTI78, KIN1, COR15A, COR47, RCI1A, RCI2A, and DREB2A cold-inducible genes in WT and cbf2. LTI78, KIN1, COR15A, and COR47 contain the CRT/DRE element in their promoters, whereas RCI1A, RCI2A, and DREB2A do not. (B) Transcript levels of CBF/DREB1 genes in WT and cbf2. (C) Transcript levels of CBF/DREB1, KIN1, and COR15A genes in WT, cbf2, and cbf2+CBF2. Equal amounts of RNA were present in each sample as confirmed by ethidium bromide staining of rRNAs.
Fig. 5.
Fig. 5.
Transcript levels of CBF/DREB1 genes in response to low temperature. (A) RNA-blot hybridizations were performed with total RNA (20 μg) isolated from 3-week-old rosette leaves of Columbia plants grown under control conditions (C) or exposed to 4°C for the indicated times. Specific probes were used for the hybridizations. Hybridization with a probe representing L18, a ribosomal gene, was used to normalize signals from the CBF/DREB1 genes. (B) Quantitative representation of the relative expression of CBF/DREB1 genes (CBF/L18) in response to low temperature.
Fig. 6.
Fig. 6.
Proposed model for the function of CBF2/DREB1C in cold acclimation and the regulation of CBF/DREB1 gene expression in response to low temperature. Arrowheads and end lines indicate positive and negative regulation, respectively.
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