Two calcium-dependent protein kinases, CPK4 and CPK11, regulate abscisic acid signal transduction in Arabidopsis

Abstract

Many biochemical approaches show functions of calcium-dependent protein kinases (CDPKs) in abscisic acid (ABA) signal transduction, but molecular genetic evidence linking defined CDPK genes with ABA-regulated biological functions at the whole-plant level has been lacking. Here, we report that ABA stimulated two homologous CDPKs in Arabidopsis thaliana, CPK4 and CPK11. Loss-of-function mutations of CPK4 and CPK11 resulted in pleiotropic ABA-insensitive phenotypes in seed germination, seedling growth, and stomatal movement and led to salt insensitivity in seed germination and decreased tolerance of seedlings to salt stress. Double mutants of the two CDPK genes had stronger ABA- and salt-responsive phenotypes than the single mutants. CPK4- or CPK11-overexpressing plants generally showed inverse ABA-related phenotypes relative to those of the loss-of-function mutants. Expression levels of many ABA-responsive genes were altered in the loss-of-function mutants and overexpression lines. The CPK4 and CPK11 kinases both phosphorylated two ABA-responsive transcription factors, ABF1 and ABF4, in vitro, suggesting that the two kinases may regulate ABA signaling through these transcription factors. These data provide in planta genetic evidence for the involvement of CDPK/calcium in ABA signaling at the whole-plant level and show that CPK4 and CPK11 are two important positive regulators in CDPK/calcium-mediated ABA signaling pathways.

Figure 1.

Molecular Analysis of T-DNA Insertion Mutants and CPK4- and CPK11-Transgenic Lines. (A) T-DNA insertion site in cpk4-1 (Col ecotype; SALK_081860 from ABRC). Tandem T-DNA of two copies was inserted into the genome in an inverted fashion at the same locus, which generates an 11-bp deletion from −67 to −57 bp 5′ upstream of the translation start codon (ATG). Boxes and lines represent exons and introns, respectively (figure not drawn to scale). The locations of the primers for identification of the mutants are indicated by arrows. LB and RB, left and right borders of T-DNA insertion, respectively; LBa1, left border primer for T-DNA; LP2 and RP2, left and right genomic primers for the CPK4 gene, respectively; and T-DNA1 and T-DNA2, first and second copies of the inserted T-DNAs, respectively, noting that the two copies were inserted in an inverted manner. nt, nucleotides. (B) T-DNA insertion sites in cpk11-1 (Col ecotype; SALK_023086, ABRC) and cpk11-2 (Col ecotype; SALK_054495, ABRC). Tandem T-DNA of two copies was inserted into the genome for the cpk11-1 mutant in an inverted fashion at the same locus, which generates a 34-bp deletion from −120 to −87 bp 5′ upstream of the translation start codon (ATG). A single copy of T-DNA was inserted for the cpk11-2 mutant, generating a 39-bp deletion from 320 to 358 bp downstream of the translation start codon (ATG). LP1 and LP3, two left genomic primers for the CPK11 gene; RBa1, right border primer for T-DNA; RP1, right genomic primer for CPK11 gene. Other abbreviations are the same as in (A). (C) RT-PCR analysis of CPK4 (indicated by CPK4) and CPK11 (CPK11) expression in wild-type Col and homozygous mutants cpk4-1, cpk11-1, and cpk11-2 and double mutants cpk4-1 cpk11-1 and cpk4-1 cpk11-2. Actin2/8 primers served as control. (D) Immunoblotting analysis with anti-CPK11^C serum, which recognizes both CPK11 and CPK4, in the total proteins (20 μg for each line) extracted from leaves in wild-type Col and the CPK4-overexpressing line 12 (4OE12) and CPK11-overexpressing line 2 (11OE2). Relative band intensities, normalized relative to the intensity of Col, are indicated by numbers in boxes below the bands. Tubulin was used as a control. (E) Real-time PCR and immunoblotting analysis of CPK4 and CPK11 during early stages before and after germination. Immunoblotting was performed with anti-CPK11^C serum in the total proteins extracted from the leaves of the seedlings grown in MS medium from 1 to 10 d after stratification in homozygous mutants cpk4-1 (possessing CPK11) and cpk11-2 (possessing CPK4). Relative band intensities, normalized relative to the intensity with the seedling 3 d after stratification, are indicated by numbers in boxes below the bands. Tubulin was used as a control. For the real-time PCR analysis for each gene, the assays were repeated three times with the independent biological experiments. The value obtained from the seedlings 3 d after stratification was taken as 100%, and all the other values were normalized relative to this value. Each value for real-time PCR is the mean ± se of three independent biological determinations.

Figure 2.

ABA Stimulates both CPK4 and CPK11. ABA treatment enhances both protein amounts (A) and enzymatic activities (B) of CPK4 and CPK11, which depends on ABA dose and displays a time course. In the ABA-dose assays, germinating seeds were transferred, 48 h after stratification, to MS medium containing (±)ABA (0, 0.5, 1, 2, and 5 μM), and 10-d-old seedlings were used for preparation of total proteins. The CPK4 and CPK11 were immunodetected with the anti-CPK4^C serum in the total proteins from Col plants (left panel in [A], indicated by CPK4+CPK11 in Col), the CPK4 with the anti-CPK4^C serum in the total proteins from the cpk11-2 mutant (left panel in [A], indicated by CPK4 in cpk11-2), and the CPK11 with the anti-CPK11^C serum in the total proteins from the cpk4-1 mutant (left panel in [A], indicated by CPK11 in cpk4-1). A 20-μg portion of the total proteins was used in each line for this immunoblotting. The in-gel histone-phosphorylating activity was assayed in the pure CPK4 protein obtained by immunoprecipitation with the anti-CPK4^C serum from the total proteins of the cpk11-2 mutant (left panel in [B], indicated by CPK4 in cpk11-1) and in the pure CPK11 with the anti-CPK11^C serum from the total proteins of the cpk4-1 mutant (left panel in [B], indicated by CPK11 in cpk4-1). A 50-μg portion of the total protein was used in each line for the immunoprecipitation. In the time-course assays, the 3-week-old seedlings of the cpk11-2 and cpk4-1 mutants were sprayed with 50 μM (±)ABA solution, and the leaves were harvested for preparing total proteins at the indicated time after the treatment (0, 30, 60, 120, and 300 min). Immunoblotting was performed as described above for CPK4 in the total protein of the cpk11-2 mutant (right panel in [A], indicated by CPK4 in cpk11-2) and for CPK11 in the total protein of the cpk4-1 mutant (right panel in [A], indicated by CPK11 in cpk4-1). The in-gel histone-phosphorylating activity was assayed as described above in the immunoprecipitated CPK4 protein from the cpk11-2 mutant (right panel in [B], indicated by CPK4 in cpk11-2) and in the immunoprecipitated CPK11 from the cpk4-1 mutant (right panel in [B], indicated by CPK11 in cpk4-1). The assays described in the left panels of (A) and (B) were performed with the same total protein, and those in the right panels with another batch of the same total protein. Tubulin was used as a loading control. In the case of the immunoprecipitation, immunoblotting for tubulin was performed with the total protein sample prior to the immunoprecipitation. Relative band intensities, normalized relative to the corresponding intensity with 0 μM ABA or at 0 min time point, are indicated by numbers in boxes below the bands. The experiments were repeated three times with similar results.

Figure 3.

Loss-of-Function Mutation in the CPK4 or CPK11 Gene Results in ABA-Insensitive Phenotypes, and Overexpression of the Two CDPK Genes Leads to ABA-Hypersensitive Phenotypes in ABA-Induced Inhibition of Seed Germination and Seedling Growth. (A) Seed germination. The germination rates were recorded in MS medium supplemented with 0 μM (top panel), 0.5 μM (middle panel), or 3 μM (bottom panel) (±)ABA during a period from 24 to 72 h after stratification for wild-type Col, cpk4-1, cpk11-1, and cpk11-2 mutants, cpk4-1 cpk11-1 and cpk4-1 cpk11-2 double mutants, mutant complementation lines 35S:CPK4/cpk4-1 and 35S:CPK11/cpk11-2, and two lines overexpressing CPK4 (4OE12) or CPK11 (11OE2). Each value is the mean ± se of three biological determinations. (B) Seedling growth 10 d after transfer from ABA-free MS medium to MS medium supplemented with different concentrations of (±)ABA for the plants as mentioned in (A). Seedlings were transferred from ABA-free medium to ABA-containing medium 48 h after stratification. (C) Primary root growth for the same lines as mentioned in (B) in medium containing 0, 1, 5, 10, 20, or 40 μM ABA. Each value is the mean ± se of at least 50 seedlings. (D) Postgermination growth in MS medium containing 0.8 μM (±)ABA 16 d after stratification for the plants as mentioned in (B). Seeds were planted in the ABA-containing medium, and the postgermination growth was directly investigated 16 d after stratification without transferring the seedlings. (E) Lateral root growth in MS medium containing 1 μM (±)ABA 10 d after transfer from the ABA-free medium for the plants as mentioned in (B). Seedlings were transferred from ABA-free medium to ABA-containing medium 4 d after stratification. Top panel, status of lateral root growth. Bottom panel, statistics of lateral root growth. White columns indicate ABA-free treatment and hatched columns ABA treatment. Each value in the bottom panel is the mean ± se of at least 50 seedlings.

Figure 4.

Loss-of-Function Mutation in CPK4 or CPK11 Results in NaCl-Insensitive Phenotypes in NaCl-Induced Inhibition of Seed Germination and Decreases Tolerance of Seedlings to Salt Stress. (A) Seed germination. Germination rates were recorded at 48, 60, and 72 h in MS medium supplemented with different concentrations of NaCl from 0 to 200 mM for wild-type Col, cpk4-1, and cpk11-2 mutants, the cpk4-1 cpk11-2 double mutant, and two lines overexpressing CPK4 (4OE12) or CPK11 (11OE2). Each value is the mean ± se of three biological determinations. (B) to (D) Tolerance of seedlings to salt stress. The status of seedling growth was recorded 7 d after transfer of the 4-d-old seedlings from medium containing 170 (B) or 200 (C) mM NaCl. A map is presented in (D) for the distribution of wild-type Col, cpk4-1, and cpk11-2 mutants, the cpk4-1 cpk11-2 double mutant, and two lines overexpressing CPK4 (4OE12) or CPK11 (11OE2) in (B) and (C). The entire experiment was replicated three times with similar results.

Figure 5.

Loss-of-Function Mutation in CPK4 or CPK11 Gene Decreases, but Overexpression of the Two CDPK Genes Enhances, Stomata Responsiveness to ABA and the Ability to Preserve Water in Leaves. (A) ABA-induced stomatal closure (top panel) and inhibition of stomatal opening (bottom panel) for wild-type Col, cpk4-1, and cpk11-2 mutants, the cpk4-1 cpk11-2 double mutant, and a line overexpressing CPK11 (11OE2). Values are the means ± se from three independent experiments; n = 60 apertures per experiment. (B) Water loss rates during a 6-h period from the detached leaves of wild-type Col, cpk4-1, cpk11-1, and cpk11-2 mutants, cpk4-1 cpk11-1 and cpk4-1 cpk11-2 double mutants, mutant complementation lines 35S:CPK4/cpk4-1 and 35S:CPK11/cpk11-1, and two lines overexpressing CPK4 (4OE12) or CPK11 (11OE2). Values are the means ± se of five individual plants per genotype. The entire experiment was replicated five times with similar results. (C) Survival rate for wild-type and different mutant lines as mentioned in (B). Drought was imposed on the 3-week-old plants by withholding water until the lethal effect was observed on the knockout mutant plants, then the plants were rewatered and survival rate was recorded 1 week later. Values are the means ± se from three independent experiments; n = 50 plants per line for each experiment. (D) and (E) Whole-plant status in the water loss assays. For assaying water loss from whole plants of the different lines as mentioned in (B), intact plants were well watered (control) or drought stressed by withholding water (drought) for 15 d (D) or for 18 d for assaying water loss of the two lines overexpressing CPK4 (4OE12) or CPK11 (11OE2) in comparison with wild-type Col (E). The entire experiment was replicated three times with similar results.

Figure 6.

Two Protein Kinases, CPK4 and CPK11, Phosphorylate both ABF1 and ABF4. The 3-week-old seedlings of the different genotypes were sprayed with 0 or 50 μM (±)ABA solution and were sampled 1 h after the spraying. The quantity of the total proteins, prepared from leaves and used in each lane of the following assays, was 50 μg. Tubulin was used as a protein loading control. (A) and (B) Mapping of protein kinases phosphorylating ABF1 (A) and ABF4 (B). The recombinant ABF1 or ABF4 (0.5 mg/mL) was embedded in the separating polyacrylamide SDS gel. Total proteins from wide-type Col and the cpk4-1 cpk11-2 double mutant were separated on the gel and assayed to in-gel phosphorylate the two substrates. At the same time after electrophoresis, gels harboring the total proteins from the ABA-free-treated wild-type plants (other gels than those for phosphorylation) were used to detect immunosignals with anti-CPK4^C serum to provide a reference for the position of the CPK4/CPK11 proteins in the lanes of phosphorylation (58 kD CPK4/CPK11). −ABA and +ABA indicate the treatments with 0 or 50 μM (±)ABA, respectively. The assays were repeated three times with the same results. (C) and (D) Phosphorylation of ABF1 (C) or ABF4 (D) by CPK4 and CPK11. The mixed proteins of two kinases (CPK4 + CPK11 in Col) were obtained by immunoprecipitation with anti-CPK4^C serum from the total proteins of wild-type Col, and the pure CPK11 (CPK11 in cpk4-1) and CPK4 (CPK4 in cpk11-2) were immunoprecipitated with the anti-CPK11^C serum from the total proteins of cpk4-1 mutant and with anti-CPK4^C serum from the total proteins of cpk11-2 mutant, respectively. The total proteins from the double mutant cpk4-1 cpk11-2 were also immunoprecipitated with anti-CPK4^C serum for obtaining background in cpk4-1 cpk11-2 as a negative control to show the absence of activity other than CPK4/11 in these assays. The ABF1 and ABF4 were in-gel phosphorylated by the immunoprecipitated proteins as described in (A) and (B). Top panels (columns) represent the relative band intensities of the phosphorylated ABF1 or ABF4 shown in middle panels, normalized relative to the corresponding intensity of the wild-type Col with 0 μM (±)ABA treatment (100%). Values are the means ± se from three biological independent experiments. Immunoblotting for tubulin (bottom panels) was performed with the total proteins prior to the immunoprecipitation. The − and + indicate the treatments with 0 and 50 μM (±)ABA, respectively.

Figure 7.

Expression of ABA-Responsive Genes in the CPK4 and CPK11 Loss-of-Function Mutants and Overexpressing Lines. Expression of ABA-responsive genes was assayed by real-time PCR in the leaves of wild-type Col, cpk4-1, and cpk11-2 mutants, the cpk4-1 cpk11-2 double mutant, and two lines overexpressing CPK4 (4OE12) or CPK11 (11OE2). −ABA, ABA-free treatment; +ABA, 50 μM (±)ABA treatment. The expression levels are presented as relative units with the levels of ABA-treated Col leaves being taken as 100%. Each value is the mean ± se of three independent biological determinations.