Mutations in the Ca2+/H+ transporter CAX1 increase CBF/DREB1 expression and the cold-acclimation response in Arabidopsis

Abstract

Transient increases in cytosolic free calcium concentration ([Ca2+]cyt) are essential for plant responses to a variety of environmental stimuli, including low temperature. Subsequent reestablishment of [Ca2+]cyt to resting levels by Ca2+ pumps and antiporters is required for the correct transduction of the signal [corrected]. C-repeat binding factor/dehydration responsive element binding factor 1 (Ca2+/H+) antiporters is required for the correct transduction of the signal. We have isolated a cDNA from Arabidopsis that corresponds to a new cold-inducible gene, rare cold inducible4 (RCI4), which was identical to calcium exchanger 1 (CAX1), a gene that encodes a vacuolar Ca2+/H+ antiporter involved in the regulation of intracellular Ca2+ levels. The expression of CAX1 was induced in response to low temperature through an abscisic acid-independent pathway. To determine the function of CAX1 in Arabidopsis stress tolerance, we identified two T-DNA insertion mutants, cax1-3 and cax1-4, that display reduced tonoplast Ca2+/H+ antiport activity. The mutants showed no significant differences with respect to the wild type when analyzed for dehydration, high-salt, chilling, or constitutive freezing tolerance. However, they exhibited increased freezing tolerance after cold acclimation, demonstrating that CAX1 plays an important role in this adaptive response. This phenotype correlates with the enhanced expression of CBF/DREB1 genes and their corresponding targets in response to low temperature. Our results indicate that CAX1 ensures the accurate development of the cold-acclimation response in Arabidopsis by controlling the induction of CBF/DREB1 and downstream genes.

Figure 1.

Accumulation of CAX1 Transcripts in Response to Low Temperature. (A) RNA gel blot hybridization with total RNA (10 μg) from 4-day-old etiolated seedlings exposed to 4°C for the indicated times. (B) RNA gel blot hybridization with total RNA (10 μg) from leaves of 3-week-old plants exposed to 4°C for the indicated times. (C) RNA gel blot hybridization with total RNA (10 μg) from roots, rosette leaves, stems, flowers, and siliques of 8-week-old plants exposed to 4°C for 1 day. Cold treatment efficacy and equal RNA loading were controlled with probes for KIN1 and 18S rRNA, respectively.

Figure 2.

Accumulation of CAX1 Transcripts in Response to Different Treatments and in ABA-Deficient and ABA-Insensitive Mutants. (A) RNA gel blot hybridization with total RNA (10 μg) obtained from 3-week-old leaves grown at control temperature (C), exposed for 1 day at 4°C (4°C), dehydrated until they lost 50% of their fresh weight (D), or treated with 250 mM NaCl (NaCl), with 100 μM ABA (ABA), or with the ABA solvent (C_ABA). (B) RNA gel blot hybridization with total RNA (10 μg) obtained from 3-week-old leaves of Landsberg erecta (Ler), aba1, and abi1 plants grown at control temperature (C), exposed for 1 day at 4°C (4°C), or dehydrated until they lost 50% of their fresh weight (D). Cold treatment efficacy and equal RNA loading were controlled with probes for KIN1 and 18S rRNA, respectively.

Figure 3.

Localization of T-DNAs and Accumulation of CAX1 Transcripts in cax1-3 and cax1-4. (A) Scheme of the CAX1 gene. Large and small boxes represent exons and introns, respectively. ATG indicates the start codon. T-DNA insertions corresponding to cax1-3 and cax1-4 are shown. The scheme is not drawn to scale. (B) RNA gel blot hybridization with total RNA (10 μg) obtained from 3-week-old leaves of Col wild-type (WT), cax1-3, and cax1-4 plants grown at 20°C (C) or exposed for 1 day at 4°C (4°C). Equal RNA loading was controlled with a probe for 18S rRNA.

Figure 4.

Ca²⁺ Tolerance in cax1-3 and cax1-4. (A) Tolerance to Ca²⁺ was estimated as the percentage of initial fresh weight (FW) that remained after transferring 3-week-old Col wild-type (WT), cax1-3, and cax1-4 plants to a medium containing 80 mM CaCl₂ for 7 days. Data are expressed as means of three independent experiments with 20 plants each. Bars indicate standard errors. In all cases, values obtained from wild-type and mutant plants were significantly different (P < 0.05) as determined by Student's t test. (B) Representative wild-type and mutant plants after CaCl₂ treatment.

Figure 5.

Ca²⁺ Concentration and Ca²⁺/H⁺ Antiport Activity in cax1-3 and cax1-4. (A) Ca²⁺ content of leaves from 3-week-old Col wild-type (WT), cax1-3, and cax1-4 plants was determined by atomic absorption spectrophotometry. Data are expressed as means of three independent experiments. Bars indicate standard errors. In all cases, values obtained from wild-type and mutant plants were significantly different (P < 0.05) as determined by Student's t test. DW, dry weight. (B) Ca²⁺/H⁺ antiport activity was measured in vacuole-enriched membrane vesicles from roots and leaves of Col wild-type, cax1-3, and cax1-4 plants pretreated with 100 mM CaCl₂ by monitoring the quenching of 9-amino-6-chloro-2-methoxyacridine fluorescence. The percentage of fluorescence recovered during a 12-min time course is represented. At 11 min, (NH₄)₂SO₄ (25 mM) was added for full recovery of fluorescence. Representative results from five replicate experiments are shown. Each replicate experiment was performed using independent membrane vesicle preparations.

Figure 6.

Freezing Tolerance of cax1-3 and cax1-4. Three-week-old Col wild-type (WT), cax1-3, and cax1-4 plants were exposed to different freezing temperatures for 1 h. Freezing tolerance was estimated as the percentage of plants surviving each specific temperature after 2 weeks of recovery under unstressed conditions. In (A) and (B), data are expressed as means of three independent experiments with 50 plants each. Bars indicate standard errors. (A) Freezing tolerance of nonacclimated wild-type and cax1 plants. (B) Freezing tolerance of cold-acclimated (4°C, 7 days) wild-type and cax1 plants. (C) Representative cold-acclimated wild-type and cax1 plants after being exposed at −8°C for 1 h and recovering for 2 weeks at 20°C.

Figure 7.

Transcript Levels of Cold-Inducible Genes in cax1-3 and cax1-4. (A) RNA gel blot hybridization with total RNA (10 μg) obtained from 3-week-old Col wild-type (WT), cax1-3, and cax1-4 plants grown under control conditions (C) or exposed for 1 day at 4°C (4°C). (B) RNA gel blot hybridization with total RNA (20 μg) obtained from 3-week-old Col wild-type, cax1-3, and cax1-4 plants grown under control conditions (C) or exposed for 1 h at 4°C (4°C). Equal RNA loading was controlled with a probe for 18S rRNA.

Figure 8.

Hypothetical Model for CAX1 Function in Low-Temperature Signaling.

Figure 9.

Ca²⁺/H⁺ Antiporter Activity in Wild-Type, cax1-3, and cax1-4 Plants in Response to Low Temperature. Ca²⁺/H⁺ antiport activity was measured in vacuole-enriched membrane vesicles from roots and leaves of Ca²⁺-treated Col wild-type (WT), cax1-3, and cax1-4 plants grown at 20°C (C) or exposed for 1 day at 4°C (4°C) by monitoring the quenching of 9-amino-6-chloro-2-methoxyacridine fluorescence. The percentage of fluorescence recovered during a 12-min time course is represented. At 11 min, (NH₄)₂SO₄ (25 mM) was added for full recovery of fluorescence. Representative results from three replicate experiments are shown. Each replicate experiment was performed using independent membrane vesicle preparations. (A) Antiport activity in vesicles from roots and leaves of plants pretreated with 100 mM CaCl₂. (B) Antiport activity in vesicles from roots of plants pretreated with 50 mM CaCl₂.