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•Ca2+-dependent protein kinase (CDPK) is believed to be involved in abscisic acid (ABA) signaling, and several members of the Arabidopsis CDPK superfamily have been identified as positive ABA signaling regulators, but it remains unknown if CDPK negatively regulates ABA signaling.
•Here, we investigated the function of an Arabidopsis (Arabidopsis thaliana) CDPK, CPK12, in ABA signaling pathway.
•We generated Arabidopsis CPK12-RNAi lines, and observed that downregulation of CPK12 resulted in ABA hypersensitivity in seed germination and post-germination growth, and altered expression of a set of ABA-responsive genes. Expression assay showed that CPK12 was ubiquitously expressed and localized to both cytosol and nucleus. Biochemical assays showed that CPK12 interacted with, phosphorylated and stimulated a type 2C protein phosphatase ABI2, and phosphorylated two ABA-responsive transcription factors (ABF1 and ABF4) in vitro.
•Our findings show that the Arabidopsis CPK12 is a negative ABA-signaling regulator in seed germination and post-germination growth, suggesting that different members of the CDPK family may constitute a regulation loop by functioning positively and negatively in ABA signal transduction.
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The CDPKs are encoded by large multigene family with possible redundancy and/or diversity in their functions (Harmon et al., 2001; Cheng et al., 2002; Hrabak et al., 2003), and they are believed to be important components in plant hormone signaling (Cheng et al., 2002; Ludwig et al., 2004). Two Arabidopsis (Arabidopsis thaliana) CDPKs (CPK), CPK10, and CPK30, were demonstrated to activate a stress- and ABA-inducible promoter, indicating connection of the CDPKs to the ABA signaling pathway (Sheen, 1996). In addition, expression of some members of CDPK family was shown to be stimulated by exogenous ABA in rice (Oryza sativa; Li & Komatsu, 2000) and in tobacco (Nicotiana tabacum; Yoon et al., 1999). Genetic evidence has been provided for involvement of several members of the Arabidopsis CDPK family in ABA signal transduction. The Arabidopsis CPK32 is positively involved in ABA-regulated seed germination (Choi et al., 2005). Arabidopsis CPK3 and CPK6 are positive regulators in ABA-mediated stomatal closure (Mori et al., 2006); CPK6 is also positively involved in methyl jasmonate signaling in guard cells (Munemasa et al., 2011). Arabidopsis CPK4 and CPK11, two homologous CDPKs, positively regulate three major ABA-mediated physiological responses, including seed germination, post-germination growth, stomatal movement and plant stress tolerance (Zhu et al., 2007). Recently, genetic and physiological evidence was provided to reveal that the Arabidopsis CPK10 functions in ABA- and Ca2+-mediated stomatal regulation in response to drought stress (Zou et al., 2010), and that the guard cell anion channel SLAC1 is regulated by CPK21 and CPK23, which may constitute key Ca2+-dependent-steps in ABA-responsive guard-cell signaling pathway (Geiger et al., 2010).
In recent years, several candidate downstream substrates of CDPKs involved in ABA signaling have been identified. The ABA-responsive transcription factor ABF4 was suggested as a candidate substrate of the Arabidopsis ABA signaling regulators CPK4, CPK11 (Zhu et al., 2007) and CPK32 (Choi et al., 2005). An ABF4 homologue, ABF1, was also a potential downstream target of CPK4 and CPK11 (Zhu et al., 2007). In addition, CPK11 interacts physically with a nuclear zinc finger protein AtDi19-1 (Rodriguez et al., 2006) and a heat-shock protein HSP1 (Uno et al., 2009), suggesting that this protein kinase may have multiple substrates. Recently, HSP1 was identified as a functionally interacting partner of CPK10 (Zou et al., 2010), and the guard cell anion channel SLAC1 was shown to interact with CPK21 and CPK23, suggesting that SLAC1 may be potential target of the two CDPKs (Geiger et al., 2010). However, our knowledge about the functionally downstream targets of CDPKs regulating ABA signaling is still limited.
Whereas a subset of CDPKs and their candidate targets were identified to be positively involved in ABA signaling, it is unclear whether CDPK negatively regulates ABA signaling. Here, we report that the Arabidopsis CPK12, which interacts with, phosphorylates and stimulates a type 2C protein phosphatase, ABI2, and phosphorylates two ABA-responsive transcription factors, ABF1 and ABF4 in vitro, is involved negatively in ABA signaling in seed germination and post-germination growth. These findings suggest that different members of the CDPK family may constitute a regulation loop by functioning positively and negatively in ABA signal transduction.
Materials and Methods
Plant materials, constructs, and Arabidopsis transformation
Arabidopsis thaliana (L.) Heynh. Columbia ecotype gl1 (Col-5) was used in the generation of transgenic plants. The gl1 is A. thaliana ecotype Col-0 carrying the homozygous recessive glabrous mutation. The gl1 plants have substantially the same ABA sensitivity in seed germination and postgermination growth as the Col-0 plants (see the Supporting Information, Fig. S1). To generate RNA interference (RNAi) lines downregulating CPK12 gene (Arabidopsis genomic locus At5g23580) expression, a gene-specific 242-bp fragment corresponding to the region of nt 6 to 247 of the CPK12 cDNA was amplified using PCR with forward primer 5′-ACGCGTCGACGAACAAACCAAGAACCAGATGGGTT-3′ and reverse primer 5′-CCGCTCGAGCGTTGGGGTATTCAGACAAGTGATG-3′. This fragment was inserted in sense orientation into the XhoI/SalI sites of pSK-int vector. The same fragment, amplified with forward primer 5′-AACTGCAGGAACAAACCAAGAACCAGATGGGTT-3′ and reverse primer 5′-GGACTAGTCGTTGGGGTATTCAGACAAGTGATG-3′, was subsequently placed in antisense orientation into the PstI/SpeI sites of pSK-int already carrying the sense fragment. The entire RNAi cassette comprising the sense and antisense fragments interspersed by the actin 11 intron was excised from pSK-int using the flanking SacI/ApaI sites and inserted into the SacI/ApaI site of pSUPER1300(+) vector yielding the pSUPER1300(+)-CPK12 RNAi construct. The pSUPER1300(+) Super Promoter is a hybrid promoter combining a triple repeat of the Agrobacterium tumefaciens octopine synthase (ocs) activator sequences along with the mannopine synthase (mas) activator elements fused to the mas promoter, termed (Aocs)3AmasPmas (Ni et al., 1995). The construct was introduced into A. tumefaciens GV3101 and transformed into gl1 by floral dip method (Clough & Bent, 1998). Transgenic plants were grown on Murashige–Skoog (MS) agar plates containing hygromycin (50 μg ml−1) in order to screen the positive seedlings. Fourteen homozygote lines containing single insert were obtained. The homozygous T3 seeds of the transgenic plants were used for further analysis Plants were grown in a growth chamber at 20–21°C on MS medium at c. 80 μmol photons m−2 s−1, or in compost soil at c. 120 μmol photons m−2 s−1 over a 16-h photoperiod.
Real-time PCR analysis
To assay the gene expression in the transgenic plants, quantitative real-time PCR analysis was performed with the RNA samples isolated from 10-d-old seedlings. Amplification by PCR was performed with primers specific for three Arabidopsis CPKs: forward and reverse primers for CPK4 and CPK11 were the same as we used previously (Zhu et al., 2007); for CPK12, the forward primer was 5′-CGAAACCCTCAAAGAAATAA-3′ and the reverse primer was 5′-TGGTGTCCTCGTACGCACTCTC-3′. The primers specific for ABA-responsive genes were: forward 5′-AGAGTGTGCCTTTGTATGGTTTTA-3′ and reverse 5′-CATCCTCTCTCTACAATAGTTCGCT-3′ for ABI1 (At4g26080); forward 5′-GATGGAAGATTCTGTCTCAACGATT-3′ and reverse 5′-GTTTCTCCTTCACTATCTCCTCCG-3′ for ABI2 (At5g57050); forward 5′-CATCTTAGACAGCAGTCAAGGTTT-3′ and reverse 5′-GTCGTGTCAAAGAACTCGTTGCTATC-3′ for ABI3 (At3g24650); forward 5′-GGGCAGGAACAAGGAGGAAGTG-3′ and reverse 5′-ACGGCGGTGGATGAGTTATTGAT-3′ for ABI4 (At2g40220); forward 5′-CAATAAGAGAGGGATAGCGAACGAG-3′ and reverse 5′-CGTCCATTGCTGTCTCCTCCA-3′ for ABI5 (At2g36270); forward 5′-TCAACAACTTAGGCGGCGATAC-3′ and reverse 5′-GC-AACCGAAGATGTAGTAGTCA-3′ for ABF1 (At1g49720); forward 5′-TTGGGGAATGAGCCACCAGGAG-3′ and reverse 5′-GACCCAAAATCTTTCCCTACAC-3′ for ABF2 (At1g45249); forward 5′-CTTTGTTGATGGTGTGAGTGAG-3′ and reverse 5′-GTGTTTCCACTATTACCATTGC-3′ for ABF3 (At4g34000); forward 5′-AACAACTTAGGAGGTGGTGGTC-3′ and reverse 5′-CTTCAGGAGTTCATCCATGTTC-3′ for ABF4 (At3g19290); forward 5′-GATCAGCCTGTCTCAATTTC-3′ and reverse 5′-CTTCTGCCATATTAGCCAAC-3′ for DREB1A (At4g25480); forward 5′-TCTCTGAACCAGAGTCGTTT-3′ and reverse 5′-CTTCTTCTCACCGTCTTCAC-3′ for ERD10 (At1g20450); forward 5′-ACCAACAAGAATGCCTTCCA-3′ and reverse 5′-CCGCATCCGATACACTCTTT-3′ for KIN1 (At5g15960); forward 5′-ACCAACAAGAATGCCTTCCA-3′ and reverse 5′-ACTGCCGCATCCGATATACT-3′ for KIN2 (At5g15970); forward 5′-TCATACGACGGTTGCCAGAA-3′ and reverse 5′-AGCAACGTTTACAAGCTTTGATTG-3′ for MYC2 (At1g32640); and forward 5′-ATCACTTGGCTCCACTGTTGTTC-3′ and reverse 5′-ACAAAACACACATAAACATCCAAAGT-3′ for RD29A (At5g52310). Amplification of ACTIN2/8 (forward primer 5′-GGTAACATTGTGCTCAGTGGTGG-3′, reverse primer 5′-AACGACCTTAATCTTCATGCTGC-3′) genes was used as an internal control (Charrier et al., 2002). The suitability of the primers sequences in term of efficiency of annealing was evaluated in advance using the primer premier 5.0 program (PREMIER Biosoft International, California, USA). The experiments were repeated thrice independently, and the data were averaged. The cDNA was amplified by using SYBR Premix Ex TaqTM (Takara, Dalian Division, Dalian, China) with a C1000 Thermal Cycler (Bio-Rad).
Transient expression in Arabidopsis protoplasts
To observe subcellular localization of CPK12, the corresponding cDNA of an open reading frame (ORF) was amplified by PCR with forward primer 5′-GACTAGTATGGCGAACAAACCAAGAACCAG-3′ and reverse primer 5′-AGGCGCGCCAGACATTCATAGACTCATCAG-3′. The cDNA of the ORF was driven by the Cauliflower mosaic virus (CaMV) 35S promoter and downstream-tagged by green fluorescent protein (GFP). The 35S promoter-driven and GFP-cDNA was fused to the pMD 19-T vector (Takara) at the PstI (5′-end) and EcoRI (3′-end) sites.
We used two marker proteins for observations of precise localization of CPK12: a nuclear-localized FBI1/HFR1 basic helix–loop–helix (bHLH) transcription factor (Arabidopsis genomic locus At1g02340; Fairchild et al., 2000; Jang et al., 2005), and a cytosol-nuclear double-localized PYR1 protein (At4g17870; Park et al., 2009). Both proteins were tagged by mCherry (a red fluorescent protein, RFP). For the FBI1–mCherry construct, the ORF of FBI1 was amplified by PCR with forward primer 5′-CCTTAATTAAATGTCGAATAATCAAGCTTT-3′ and reverse primer 5′-AGGCGCGCCATAGTCTTCTCATCGCATGGG-3′ and cloned to the PacI (5′-end) and AscI (3′-end) sites of the pMD 19-T vector harboring mCherry. For the PYR1–mCherry construct, the ORF of PRY1 was amplified by PCR with forward primer 5′-CCTTAATTAAATGCCTTCGGAGTTAACACC-3′ and reverse primer 5′-AGGCGCGCCACGTCACCTGAGAACCACTTC-3′, and cloned to the PacI (5′-end) and AscI (3′-end) sites of the pMD 19-T vector harboring mCherry.
Protoplasts were isolated from the leaves of 3- to 4-wk-old Arabidopsis plants (ecotype Columbia-0) and transiently transformed by using polyethylene glycol (PEG) essentially according to Sheen’s protocol (http://genetics.mgh.harvard.edu/sheenweb/). The protoplasts were cotransformed by either the CPK12–GFP and FBI1–mCherry construct pair or CPK12–GFP and PYR1–mCherry construct pair to observe whether CPK12 colocalized with these two proteins. Fluorescence of GFP was observed by a confocal laser scanning microscope (Nikon EZ-C1, Nikon Corporation, Tokyo, Japan) after incubation at 23°C for 16 h. The GFP fluorescence and mCherry fluorescence were excited, respectively, with a 488 nm and 543 nm argon-ion laser, filtered with 545 nm spectroscope. The GFP fluorescence was detected with a 505–530 nm filter set and the mCherry fluorescence was detected with a 585–615 nm filter set.
Analysis of gene expression by promoter-GUS (β-glucuronidase) transformation
A 982 bp promoter fragment (upstream of ATG) of Arabidopsis gene At5g23580 (CPK12) was isolated by PCR using forward primer 5′-GGAATTCCCAAACGAGAGGAGGCAAAGA-3′ and reverse primer 5′-ACGCGTCGACTTTGTTCGCCATTCTTGAGAGC-3′ and cloned into the EcoRI (5′-end)–SalI (3′-end) sites of pCAMBIA1391 vector. This construct was introduced into the GV3101 strain A. tumefaciens and transformed into Arabidopsis (Col) plants by floral infiltration. Homologous plants of the T3 generation were used for the analysis of GUS activity. The GUS staining was performed essentially according to Jefferson et al. (1987). Whole plants or tissues were immersed in 1 mM 5-bromo-4-chloro-3-indolyl-b-GlcUA (X-gluc, Sigma-Aldrich, Beijing, China) solution in 100 mM sodium phosphate (pH 7.0), 2 mM EDTA, 0.05 mM ferricyanide, 0.05 mM ferrocyanide, and 0.1% (v : v) Triton X-100 for 12 h at 37°C. Chlorophyll was cleared from the tissues with a mixture of 30% acetic acid and 70% ethanol.
Creation of deletion mutations of ABI2
Wild type ABI2 (ABI2-WT) was amplified by using primers I2-F1 (5′-GCTCTAGAATGGACGAAGTTTCTCCTGC-AGTCGCTG-3′) and I2-R1 (5′-GGGGTACCATTCAA-GGATTTGCTCTTGAATTTCC-3′) with total cDNA as template, then fused to the pMD-19-T vector (Takara) and confirmed by sequencing. The three putative CDPK phosphorylation sites (CPS) deletion mutation of the ABI2 were generated by Overlap PCR using KOD-PLUS- (Toyobo, Osaka, Japan) with ABI2-WT plastid as template.
For the first putative CPS R-X-X-S/T (RPFT) deletion mutation (amino acid residues 13–16) of ABI2, PCR was done with primers I2F2 (5′-TGCAGTCGCTGTTCCA-TTCGACCCTCACGCCGGACTTAG-3′) and I2R1 with ABI2-WT plastid as template to generate product P1, then another PCR was done with primers I2F1 and I2R1 by using P1 as template to generate product ABI2 with the first CPS deletion (dF).
For the deletion of both the first and second putative CPS R-X-X-S/T deletion mutation of ABI2 (RPFT, amino acid residues 13–16; RTES, amino acid residues 98–101), PCR was done with primer pairs I2F1 plus I2R3 (5′-CTCAAACAGACTTCTACTAAGTACTTTCTTC-3′) and I2F3 (5′-GAAGAAAGTACTTAGTAGAAGTCTGTTTGAG-3′) plus I2R1 primer by using dF as template and generated the products P2 and P3, respectively. Another PCR was performed with primers I2F1 and I2R1 by using P2 and P3 as templates, which allowed us to obtain a product, ABI2, with the first and second CPS deletion (dFS).
For the deletion of all the three CPS sites (RPFT, amino acid residues 13–16; RTES, amino acid residues 98–101; RGKT, amino acid residues 258–261), primer pairs I2F1 plus I2R4 (5′-GACAACGCGAGTGGACACAAAACCG-CCC-3′), and I2F4 (5′-GGGCGGTTTTGTGTCCACTCGCGTTGTC-3′) plus I2R1 were used for the first PCR by using dFS as template and generated the product P4 and P5, respectively. Another PCR was performed with primers I2F1 and I2R1 by using P4 and P5 as templates, which produced ABI2−3CPS (with all the three CPS deletion).
Preparation of recombinant CPK12, ABI2, mutated ABI2, ABF1 and ABF4 proteins
The procedures for production of the recombinant ABF1 and ABF4 proteins were described previously (Zhu et al., 2007). To prepare recombinant CPK12, the cDNA of its coding regions were amplified by using forward primer 5′-GGAATTCTATGGCGAACAAACCAAGAACCAG-3′ and reverse primer 5′-ACGCGTCGACTCAGACATTCATAGACTCATCAG-3′. The PCR product was digested with EcoRI and SalI and cloned into pET-48 b(+) vector (Novagen, San Diego, California, USA). To prepare recombinant ABI2 and the deletion mutation of ABI2−3CPS (deletion of three putative CDPK phosphorylation sites, abbreviated as –3CPS), the cDNA of their coding regions were amplified with forward primer 5′-GGAATTCATGGACGAAGTTTCTCCTGC-3′ and reverse primer 5′-CCGCTCGAGTCAATTCAAGGATTTGCTC-3′. The PCR template for the wild-type ABI2 cDNA cloning was the ABI2-WT, but the template for ABI2−3CPS cDNA was the ABI2−3CPS product, both of which were described earlier (see the section Creation of deletion mutations of ABI2′). The PCR product was digested with EcoRI and XhoI and cloned into pET-28 a-c(+) vector (Novagen). The constructs were expressed in E. coli strain BL21(DE3). The individual proteins were purified according to manufacture’s instructions (Novagen). It is noteworthy that, for the expression of ABI2s to assay PP2C activity, 5 mM MgCl2·6H2O were added to the E. coli culture medium, and the cells were collected and lysed in a buffer containing 500 mM NaCl, 5 mM MgCl2·6H2O and 25 mM Tris, pH 8.0.
In-gel kinase assays
In-gel kinase activity assay of proteins was performed essentially as described in Yu et al. (2006). Briefly, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a gel that was polymerized in the presence of 0.5 mg ml−1 substrate (ABF1, ABF4 or ABI2 protein), the gels were washed twice with 50 mM Tris-HCl, pH 8.0, containing 20% (v : v) 2-propanol for 1 h per wash and then with buffer A composed of 50 mM Tris-HCl, pH 8.0, 5 mM 2-mercaptoethanol and 0.1 mM EDTA for 1 h at room temperature. Proteins in the gels were denatured by incubating the gels in buffer A containing 6 M guanidine hydrochloride for two incubations of 1 h each at room temperature. Proteins were then renatured using buffer A containing 0.05% (v : v) Tween 20 for six incubations of 3 h each at 4°C. After preincubation at room temperature for 30 min with buffer B composed of 40 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-NaOH, pH 7.5, 10 mM MgCl2, 0.45 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N‘,N‘-tetraacetic acid (EGTA) (1 mM in the Ca++-free medium), and 2 mM dithiothreitol (DTT) in the absence or presence of 0.55 mM CaCl2, the gels were incubated with buffer B containing 50 μM ATP and 10 μCi ml−1 [r32-P]-ATP (3000 Ci mmol–1) for 1 h at room temperature. The gels were then washed extensively with 5% trichloroacetic acid and 1% sodium pyrophosphate until radioactivity in the used wash solution was barely detectable and then stained with Coomassie Brilliant Blue R-250 (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, England). After destaining, the gels were air dried between two sheets of cellophane, and the substrate in gel phosphorylated by CDPK was detected by autoradiography after exposition of the dried gels to Kodak X-Omat BT film (Eastman Kodak Company, Rochester, New York, USA).
Analysis of protein interaction by yeast two-hybrid system
Interaction between proteins was assayed by a yeast Gal4-based two-hybrid system by using procedures as described by the manufacturer (Clontech, Mountain View, California, USA). The ORF of CPK12 was amplified by PCR with forward primer 5′-GGAATTCATGGCGAACAAACCAAGAACCAG-3′ and reverse primer 5′-ACGCGTCGACTCAGACATTCATAGACTCATCAG-3′ and cloned to EcoRI (5′-end) and SalI (3′-end) sites of the pGBKT7 vector. The ORF of ABI2 and ABI2−3CPS was amplified by PCR with forward primer 5′-GGAATTCATGGACGAAGTTTCTCCTGC-3′ and reverse primer 5′-CCGCTCGAGTCAATTCAAGGATTTGCTC-3′ and cloned to EcoRI (5′-end) and XhoI (3′-end) sites of the pGADT7 vector. The PCR template for the wild-type ABI2 cDNA cloning was the ABI2-WT, but template for ABI2−3CPS cDNA was the ABI2−3CPS product. The ABI2-WT and ABI2−3CPS templates for PCR were described earlier (see the section Creation of deletion mutations of ABI2).
PP2C phosphatase assay
The phosphatase activity was measured by the serine-threonine phosphatase assay system (Promega, Madison, Wisconsin, USA). Each reaction was performed in a 50 μl reaction volume containing 10 μg ABI2 or ABI2−3CPS, 20 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM MgCl2·6H2O and different concentrations of CPK12. After incubation with peptide substrate (supplied with the Promega kit) at 30°C for 30 min, the reaction was stopped by addition of 50 μl molybdate dye. Absorbance at 630 nm was measured 30 min after the addition of molybdate dye.
The seeds were surface-sterilized in 5% (v : v) sodium hypochlorite, and then rinsed five times with sterile water. Approximately 100 seeds each from wild type (gl1) and different transgenic lines were planted on MS medium (product# M5524Sigma) with different concentrations of ABA, and incubated at 4°C for 3 d before being placed at 22°C under light conditions (80 μmol photons m−2 s−1), and germination and post-germination growth were investigated at the times indicated.
Downregulation of CPK12 results in ABA hypersensitivity in seed germination and post-germination growth
We previously identified an ABA-stimulated CDPK, ACPK1, from grape (Yu et al., 2006), and further showed that the homologues of ACPK1 in Arabidopsis, CPK4 and CPK11, are positive regulators in ABA signaling (Zhu et al., 2007). The Arabidopsis CPK12 is the closed homologue of ACPK1 (Yu et al., 2006) and CPK4 and CPK11 (Hrabak et al., 2003; see also Fig. S2). We wondered whether CPK12 is also involved in ABA signaling. However, we failed to isolate T-DNA insertion mutants of CPK12 gene from the mutant pool of Arabidopsis Biological Resource Center and other public biological resource services. The stock numbers of the putative T-DNA insertion mutants we obtained and tested by PCR genotyping were SALK_010212, SALK_090011, CS852493, CS852493, CS852429 and CS850937, and all the putative mutants were finally shown to be wild types, among which two lines, SALK_010212 and SALK_ 090011, were shown to harbor one copy of T-DNA insertion in the promoter region (SALK_010212) or 3′-end untranslated region (SALK_090011) of CPK12 gene, but expression of CPK12 was not affected in either of these mutants. Therefore, to study the function of CPK12 in ABA signaling, we generated CPK12-RNAi lines. We screened 14 homozygote RNAi lines, and selected six (lines R7, R8, R9, R10, R11 and R12) as examples for this study. Expression of CPK12 gene was downregulated in these RNAi lines, and the amounts of CPK12 mRNA showed a gradual decrease from the line R12 to line R7, which creates a gradient of the CPK12 expression levels (Fig. 1a). However, expression of CPK4 and CPK11 genes was not affected in these CPK12-RNAi lines (Fig. 1a), showing the specificity of the RNAi construct to CPK12.
The seeds of the CPK12-RNAi lines germinated normally as the wild-type seeds did in the ABA-free media, but in the media supplemented with different concentrations of (±)-ABA (0.1, 0.2, 0.3, 0.4 and 0.5 μM), their germination rate was significantly less than that of the wild-type seeds (Fig. 1b). It is particularly noteworthy that the intensity of this ABA hypersensitivity in seed germination was positively correlated to the CPK12 mRNA levels (Fig. 1b).
It is noteworthy that the CPK12-RNAi lines have a strong hypersensitivity to ABA in seed germination and post-germination growth. We therefore used a range of low ABA concentrations to assay ABA sensitivity in seed germination. At 0.1 μM and 0.2 μM ABA, the ABA hypersensitivity of the CPK12-RNAi lines in seed germination was more obvious 24 h after stratification (Fig. 1b), but this hypersensitivity became less apparent 36 h or 48 h after stratification (data not shown), likely because the applied ABA concentrations were too low to inhibit seed germination after an early period of ABA-sensitive window (Lopez-Molina et al., 2001). However, ABA concentrations over 0.3 μM (0.3–0.5 μM) showed more durable-inhibiting effects on seed germination of the CPK12-RNAi lines 36 or 48h after stratification, while these low concentrations of ABA had much less inhibiting-effect on germination of wild-type seeds (Fig. 1b).
The CPK12-RNAi lines showed early seedling growth comparable to that of the wild-type plants in the ABA-free medium, but this early growth of the CPK12-RNAi lines was reduced much more by ABA treatments (0.3, 0.4 or 0.5 μM) than that of the wild-type seedlings (Fig. 2a–d). As we observed in seed germination (Fig. 1), the intensity of this ABA hypersensitivity in post-germination growth was also positively correlated to the CPK12 mRNA levels in the media containing 0.3 μM ABA and 0.4 μM ABA (Fig. 2b,c), while in the medium containing 0.5 μM ABA-, the post-germination growth of all the CPK12-RNAi lines was almost completely inhibited (Fig. 2d), suggesting that CPK12 plays an important role in ABA signaling process controlling early seedling growth.
CPK12 is ubiquitously expressed and localizes to both cytosol and nucleus
Using GUS as a reporter, driven by the CPK12 promoter, CPK12 was shown to be expressed ubiquitously in all the organs/tissues (Fig. 3c–h) except for seeds, including dry seeds (Fig. 3a), seeds during stratification (Fig. 3b, at low temperature) and mature seeds residing in silique (Fig. 3h). These data are essentially consistent with the microarray data published online at the public website Genevestigator (https://www.genevestigator.com) (Fig. 3i). It is notable that the two closet CPK12-homologues, CPK4 and CPK11, are also ubiquitously expressed (see also Zhu et al., 2007) like CPK12, but with different profiles relative to expression levels in different organs (Fig. 3i).
A transient expression in Arabidopsis protoplasts showed that the CPK12 protein colocalizes with a nuclear-localized FBI1/HFR1 bHLH transcription factor (Arabidopsis genomic locus At1g02340; Fairchild et al., 2000; Jang et al., 2005) and with a cytosol-nuclear double-localized PYR1 protein (At4g17870; Park et al., 2009) (Fig. 4a). The control images showed that the fluorescence of GFP or RFP was distributed evenly in the cytosolic space, which is different from that of GFP-tagged CPK12 or RFP-tagged FBI1 or PYR1 (Fig. 4b), showing the specificity and reliability of the subcellular localization assays. These data showed clearly that CPK12 localizes to both cytosol and nucleus. PYR1 is a member of the START-domain family proteins, which have been shown to be ABA receptors (called PYR/PYL/RCAR) interacting with the type 2C protein phosphatases such as ABI1 and ABI2 to transduce ABA signal to downstream gene expression (Ma et al., 2009; Park et al., 2009).
It should be noted that we used the mesophyll protoplast to transiently express CPK12 to observe its subcellular localization. Given that CPK12 is ubiquitously expressed, we do not exclude possibility that it could localize differently in other tissues/organs. Further studies will be required to elucidate this.
CPK12 phosphorylates two transcription factors ABF1 and ABF4 and a type 2C protein phosphatase ABI2 in vitro
We screened the potential substrates of CPK12, and observed that the ABA-responsive transcription factor ABF1 and ABF4 (Choi et al., 2000; Uno et al., 2000) and a negative ABA signaling regulator, type 2C protein phosphatase ABI2 (Leung et al., 1997), could be phosphorylated in vitro by CPK12 in the presence of Ca2+, but this phosphorylation activity was completely abolished in the absence of Ca2+ (Fig. 5a). A mutated form of ABI2, ABI2−3CPS, with deletion of all the three putative CDPK phosphorylation sites (Fig. 5b), could not be phosphorylated by CPK12 (Fig. 5a), showing the specificity and reliability of the phosphorylation assays. These results suggest that CPK12 could function in ABA signaling by phosphorylating downstream ABA signaling regulators.
CPK12 interacts with ABI2 and stimulates ABI2 phosphatase activity in vitro
We focused the further assays on ABI2, and tested whether CPK12 can interact with ABI2 and influence ABI2 phosphatase activity in vitro, given that ABI2 is an important player in the ABA signaling pathway directly downstream of the PYR/PYL/RCAR ABA receptors (Ma et al., 2009; Park et al., 2009). Consistent with the observation that CPK12 phosphorylates ABI2 in vitro, we showed that CPK12 interacts with ABI2 in yeast two-hybrid system (Fig. 6a). Interestingly, deletion of all the three putative CDPK phosphorylation sites (ABI2−3CPS) did not affect the interaction between CPK12 and ABI2 (Fig. 6a), suggesting that the bi-molecular interaction between CPK12 and ABI2 is a distinct process from the phosphorylation event of ABI2 by CPK12 (see Fig. 5).
Furthermore, we observed that CPK12 stimulates the phosphatase activity of ABI2 in a CPK12-dose-dependent manner (Fig. 6b,c). The deletion mutation of the three putative CDPK phosphorylation sites (ABI2−3CPS) significantly reduced the PP2C activity of ABI2, but did not significantly change the stimulation pattern of the PP2C activity by CPK12 (Fig. 6c), which suggests that the physical interaction is important for functional interaction between CPK12 and ABI2.
Downregulation of CPK12 alters expression of a set of ABA-responsive genes
CPK12 negatively regulates ABA signaling in seed germination and post-germination growth
The three members of Arabidopsis CDPK superfamily, CPK4, CPK11 and CPK12, belong to the same subgroup (Hrabak et al., 2003). We previously showed that CPK4 and CPK11 positively regulate ABA signaling in all the three major ABA responses, including seed germination, post-germination growth and stomatal movement (Zhu et al., 2007). In the present study, we have shown that their closet homologue, CPK12, functions in ABA signaling as a negative regulator in seed germination and post-germination growth (Figs 1, 2), which apparently antagonizes CPK4 and CPK11 in ABA signaling pathway. However, we did not observe any ABA-related phenotype in stomatal movement when downregulating CPK12 expression by RNAi (data not shown), whereas CPK4 and CPK11 regulate stomatal signaling in response to ABA (Zhu et al., 2007). These findings indicate that the function of CPK12 is distinct from that of its homologues CPK4 and CPK11. CPK12 has c. 30% amino acid residues different from CPK4/CPK11 (Fig. S2), which may be responsible for these differences in function of ABA signaling pathway.
CPK12 may function in a complex signaling network through regulating ABA signaling components ABI2, ABF1 and ABF4
We showed that CPK12 phosphorylates two ABA-responsive transcription factors ABF1 and ABF4 (Choi et al., 2000; Uno et al., 2000). Most interestingly, CPK12 interacts with, phosphorylates a negative ABA signaling regulator PP2C ABI2 (Leung et al., 1997), and stimulates the catalytic activities of ABI2 phosphatase (Figs 5, 6). Arabidopsis clade A PP2Cs, including ABI1 and ABI2 (Leung et al., 1994, 1997) were recently reported to relay the ABA signal directly downstream of ABA receptors PYR/PYL/RCAR (Ma et al., 2009; Park et al., 2009). Stimulation of the ABI2 activity by CPK12 is consistent with the negative role of CPK12 in the ABA signaling pathway, and suggests that the CPK12 could be negatively involved in the early events of ABA signaling pathways. CPK12 localizes to the same subcellular compartments (cytosol and nucleus, Fig. 4) as a member of the PYR/PYL/RCAR ABA receptors PYR1 and ABI2 PP2C (Ma et al., 2009; Park et al., 2009), providing CPK12 with opportunity to meet, and interact with, ABI2 in cells. CPK12 may improve ABI2 activity by directly interacting with ABI2 (Fig. 6a). A mutated ABI2 with deletion of all the three putative Ca2+-dependent protein kinase sites, ABI2−3CPS, significantly reduced PP2C catalytic activity, while its activity was still stimulated by CPK12 (Fig. 6b,c), which supports the idea that CPK12 stimulates ABI2 activity at least partly through direct, physical, interaction with ABI2. However, based on the present data (Fig. 6), it is difficult to suggest that CPK12 functions through phosphorylating ABI2, because, owing to technical difficulties, we failed to test the effects of CPK12 on ABI2 activity in the presence of ATP, which allows CPK12 to phosphorylate its substrate such as ABI2. Further studies will be needed to assess whether CPK12 functions through phosphorylating ABI2.
It is notable that the two CPK12 homologues, CPK4 and CPK11, also phosphorylate ABF1 and ABF4, but positively regulate ABA signaling (Zhu et al., 2007). CPK12 is expressed ubiquitously (Fig. 3), which is similar to CPK4 and CPK11 (Zhu et al., 2007), although the profiles of expression levels of the three CPKs in different organs are different (Fig. 3), and all the three CPKs localizes to the same subcellular compartments (cytoplasm and nucleus) (Fig. 4; see also Zhu et al., 2007). How may we understand the functional distinction between CPK12 and CPK4/CPK11 with these similarities between them in structure, substrates, expression profile and subcellular localization? According to the present data, we suggest that CPK12 may function, in concert with the PYR/PYL/RCAR ABA receptors and its closet homologues CPK4 and CPK11, to balance ABA signaling through regulation of both positive regulator ABF1/4 and negative regulator ABI2, which may be different from the function of CPK4 and CPK11 (Fig. 8).
Consistent with a negative ABA signaling regulator, downregulation of CPK12 expression enhanced expression of a set of ABA-positive regulator genes, including ABI3, ABI5, ABF1, ABF2, ERD10, KIN1, KIN2 and RD29A (Fig. 7), but in contrast to this gene expression profile, the expression levels of all these genes were reduced in the cpk4 and cpk11 loss-of-function mutants (knockout mutants of CPK4 or CPK11 gene) except for ABI3, whose expression was not affected in these cpk4/cpk11 mutants (Zhu et al., 2007), which is essentially in accord with the role of positive ABA signaling regulators of CPK4 and CPK11 (Zhu et al., 2007). It is noteworthy, however, that two negative regulators, ABI1 and ABI2, were upregulated in the CPK12-RNAi lines (Fig. 7), suggesting that CPK12-mediated ABA signaling may be balanced by a series of positive and negative signaling events in this complex ABA signaling network.
In addition to the positive ABA signaling regulators CPK4, CPK11 (Zhu et al., 2007) and CPK32 (Choi et al., 2005) in seed germination and post-germination growth, the identification of CPK12 as a negative ABA signaling regulator indicates that different members of CDPK family may constitute a regulation loop by functioning positively and negatively in ABA signal transduction (Fig. 8). Further studies will be needed to explore the interconnections among these CDPKs in ABA signal transduction pathways.
This research was supported by National Natural Science Foundation of China (grant nos. 30671444 and 90817104 to D-P.Z. and 30700053 to X-F.W.) and by a grant from Agricultural Ministry of China (grant no. 2008ZX08009-003 to D.P.Z.).