CBL1, a calcium sensor that differentially regulates salt, drought, and cold responses in Arabidopsis

Abstract

Although calcium is a critical component in the signal transduction pathways that lead to stress gene expression in higher plants, little is known about the molecular mechanism underlying calcium function. It is believed that cellular calcium changes are perceived by sensor molecules, including calcium binding proteins. The calcineurin B-like (CBL) protein family represents a unique group of calcium sensors in plants. A member of the family, CBL1, is highly inducible by multiple stress signals, implicating CBL1 in stress response pathways. When the CBL1 protein level was increased in transgenic Arabidopsis plants, it altered the stress response pathways in these plants. Although drought-induced gene expression was enhanced, gene induction by cold was inhibited. In addition, CBL1-overexpressing plants showed enhanced tolerance to salt and drought but reduced tolerance to freezing. By contrast, cbl1 null mutant plants showed enhanced cold induction and reduced drought induction of stress genes. The mutant plants displayed less tolerance to salt and drought but enhanced tolerance to freezing. These studies suggest that CBL1 functions as a positive regulator of salt and drought responses and a negative regulator of cold response in plants.

Figure 1.

Stress and ABA Induction of CBL1 Protein and CBL1 Gene Expression in Arabidopsis Tissues. (A) Protein gel blot analysis of CBL1 levels under stress and ABA treatments. Plants were grown on Murashige and Skoog (1962) (MS) agar medium for 3 weeks and were treated with cold (4°C), NaCl (300 mM), ABA (100 μM), mannitol (300 mM), water (as a control), or wounding. Total protein was extracted at the indicated time points. Each lane was loaded with 30 μg of total protein for SDS-PAGE followed by protein gel blot analysis using anti-CBL1 antibody. (B) RT-PCR analysis of CBL1 transcripts in different tissues of Arabidopsis plants. Total RNA was isolated from various tissues (root, leaf, stem, flower, or silique) of 4-week-old wild-type plants grown under long-day conditions or from germinating seeds and young seedlings (3, 7, and 21 days after sowing). RT-PCR was performed with either CBL1-specific primers (top gel) or Actin2-specific primers (bottom gel).

Figure 2.

Constitutive Expression of CBL1 in Arabidopsis Alters Stress-Responsive Gene Expression under Normal Conditions. (A) RNA gel blot analysis in wild-type (WT) and CBL1-expressing (MAS-CBL1) plants. Total RNAs (10 μg) from 3-week-old plants grown on MS medium under normal conditions were used for RNA analysis. rRNA on the membrane was visualized by staining with methylene blue as an equal loading control. The gene markers are described in the text. (B) CBL1 protein levels in wild-type and CBL1-overexpressing lines as revealed by protein gel blot analysis. The top gel indicates CBL1 protein level. The bottom gel shows ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit on the same blot stained with Ponceau red as an equal loading indicator.

Figure 3.

Expression of Stress-Responsive Genes in Wild-Type and CBL1-Overexpressing Arabidopsis Plants Induced by NaCl, Drought, and Cold Treatments. rRNA on the membrane stained with methylene blue served as an equal loading control. All RNA gel blot experiments were repeated at least three times, and results from one representative experiment are shown. WT, wild type. (A) NaCl treatment (300 mM). (B) Drought treatment. (C) Cold treatment.

Figure 4.

Altered Stress Tolerance in CBL1-Overexpressing Plants. (A) Increased salt tolerance. Three-week-old plants were treated with 300 mM NaCl (see Methods) and monitored for bleaching during the subsequent 2 weeks. The photographs were taken on the 10th day. WT, wild type. (B) Increased drought tolerance. Watering was withheld from 3-week-old plants for 23 days before the photographs were taken. (C) Reduced freezing tolerance. After freezing treatment (see Methods), plants were transferred to a growth chamber. The photographs show the difference on the 8th day after transfer to the growth chamber. (D) Survival rate. The survival rate (%) and standard errors were calculated based on results from three independent experiments (see Methods).

Figure 5.

Germination Assays of CBL1-Overexpressing Plants. (A), (C), and (D) Germination time course (days of incubation at 23°C) on MS medium (A), MS medium containing NaCl (C), or MS medium containing mannitol (D) was recorded for wild-type (closed circles) and MAS-CBL1 (open squares [plant 5] and open triangles [plant 17]) seeds. (B) Germination rates of wild-type and MAS-CBL1 seeds after 3 days of incubation at 23°C on MS medium containing different concentrations of ABA. Results are presented as average values ± se from three experiments.

Figure 6.

Isolation and Complementation of the cbl1 T-DNA Insertional Mutant. (A) Scheme of the Arabidopsis CBL1 gene. Exons (closed boxes) and introns (lines) are indicated. The position and orientation of the T-DNA insertion are shown (not to scale). The numbers represent nucleotides, and +1 indicates the transcriptional start site for the CBL1 gene. LB, left border; RB, right border. (B) Complementation genomic DNA fragment. A 4.26-kb DNA region containing the CBL1 gene was amplified by PCR and cloned into the pCAMBIA1300 vector for plant transformation. (C) RT-PCR analysis of CBL1 from wild-type (Wassilewskija [Ws]), cbl1, and cbl1/CBL1 plants. RT-PCR was performed with either CBL1-specific primers (top gel) or Actin2-specific primers (bottom gel). (D) RNA gel blot analysis of CBL1 mRNA in the wild type (Ws), the mutant (cbl1), and a complementation transgenic line (cbl1/CBL1) under cold treatment. Four independent complementation lines were analyzed with similar results, and the result from one line is shown here. Ten micrograms of total RNA from 3-week-old seedlings was probed with CBL1 cDNA. rRNA on the membrane was visualized by methylene blue as an equal loading control.

Figure 7.

Expression of Stress-Responsive Genes in Wild-Type, cbl1, and cbl1/CBL1 Plants Induced by NaCl, Drought, and Cold Treatments. rRNA on the membrane was visualized by staining with methylene blue as an equal loading control. All RNA gel blot experiments were repeated at least three times, and results from one representative experiment are shown. Ws, Wassilewskija. (A) NaCl treatment (300 mM). (B) Drought treatment. (C) Cold treatment.

Figure 8.

Altered Stress Tolerance in cbl1 Mutant Plants. For salt tolerance, 3-week-old plants were exposed to 300 mM NaCl solution every 3 days for three repetitions and monitored subsequently for bleaching during the next 2 weeks. For drought tolerance, watering was withheld for 23 days from 3-week-old soil-grown plants before survival rates were counted. For freezing tolerance, plants treated with freezing stress were returned to the growth chamber. Surviving plants were scored on the 8th day in the growth chamber. Survival rates and standard errors were calculated from results of three independent experiments. Ws, Wassilewskija.

Figure 9.

Germination of cbl1 Mutant Seeds Is Hypersensitive to Osmotic Stress Conditions But Not to ABA. (A), (C), and (D) Germination time course (days after incubation at 23°C) was recorded as described in Figure 5 on MS medium (A), MS medium containing NaCl (C), or MS medium containing mannitol (D) for wild-type (Wassilewskija) and cbl1 mutant seeds. (B) Germination rates of wild-type and cbl1 mutant seeds at 3 days after transfer to 23°C in the presence of different concentrations of ABA. (E) Seed germination rates in the wild type (Wassilewskija [Ws]), cbl1 mutant (cbl1), and four complementation lines (cbl1/CBL1) on MS medium (control) or MS medium containing 125 or 150 mM NaCl. Germination was scored at 3 days after incubation at 23°C. Data from one representative complementation line are shown. Results are presented as average values ± se from three experiments. Closed circles, wild-type; open squares, cbl1 mutant.