Molecular Plant Advance Access originally published online on February 8, 2008
Molecular Plant 2008 1(2):238-248; doi:10.1093/mp/ssn003
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Calcineurin-B-Like Protein CBL9 Interacts with Target Kinase CIPK3 in the Regulation of ABA Response in Seed Germination
a Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA
b Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India
c Present address: Department of Bio-Environmental Science, Sunchon National University, Suncheon, Jeonnam 540–742, Korea
d Present address: Department of Molecular Physiology and Biochemistry, National Institute of Agricultural Biotechnology, Suwon 441–707, Korea
1 To whom correspondence should be addressed. E-mail sluan{at}nature.berkeley.edu, fax (510) 642-4995, tel. (510) 642-6306
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Abstract |
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Calcium plays a vital role as a second messenger in many signaling pathways in plants. The calcineurin B-like proteins (CBLs) represent a family of plant calcium-binding proteins that function in calcium signaling by interacting with their interacting protein kinases (CIPKs). In our previous study, we have reported a role for one of the CBLs (CBL9) and one of the CIPKs (CIPK3) in ABA signaling. Here, we have shown that CBL9 and CIPK3 physically and functionally interact with each other in regulating the ABA responses. The CBL9 and CIPK3 proteins interacted with each other in the yeast two-hybrid system and when expressed in plant cells. The double mutant cbl9cipk3 showed the similar hypersensitive response to ABA as observed in single mutants (cbl9 or cipk3). The constitutively active form of CIPK3 genetically complemented the cbl9 mutant, indicating that CIPK3 function downstream of CBL9. Based on these findings, we conclude that CBL9 and CIPK3 act together in the same pathway for regulating ABA responses.
Key Words: ABA • calcium • CBL • CIPK • osmotic stress • signal transduction
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INTRODUCTION |
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It has been established that phytohormone abscisic acid (ABA) plays a vital role in plant growth and developmental processes, including embryo development, seed maturation, dormancy, seed germination, root and shoot growth, and stress responses (Koornneef et al., 1998; Leung and Giraudat, 1998; McCourt, 1999; Finkelstein et al., 2002). In many of the stress responses, the ABA levels increase and application of ABA to plants mimics the stress condition. Furthermore, there is much overlap in the abiotic stress such as salinity, osmotic, cold and drought stress and ABA-mediated responses, especially in the gene expression pattern, indicating significant cross-talk of ABA and stress signaling pathways (Leung and Giraudat, 1998; Thomashow, 1999; Rock, 2000; Shinozaki and Yamaguchi-Shinozaki, 2000; Finkelstein et al., 2002). Based on recent studies, it has been clear that osmotic stress imposed by high salt and drought is transduced by ABA-dependent and ABA-independent pathways. The mechanism by which plants perceive and transduce ABA signal to initiate the changes in gene expression and physiological responses is still poorly understood. Seed germination inhibition has been used as a critical bioassay for screening mutants for ABA responses (Giraudat, 1995; Koornneef et al., 1998; Leung and Giraudat, 1998; McCourt, 1999). A major progress in ABA signaling research involves identification of ABI (ABA Insensitive) mutants (Giraudat, 1995; Finkelstein et al., 2002). The protein phosphatases ABI1 and ABI2 may function as more general regulators that regulate both seed germination and stomatal movement (Pei et al., 1997; Allen et al., 1999). In contrast, the transcription factors ABI3–5 are highly expressed during seed maturation and seed germination and play a major role in seed dormancy (Finkelstein et al., 2002). More recent studies by both forward and reverse genetic approaches have revealed numerous other components involved in ABA response (Giraudat, 1995; Finkelstein et al., 2002). These components range from early signaling intermediates such as G-proteins and protein kinases/phosphatases to late-stage transcription factors and RNA metabolic proteins, implicating a complex molecular network in the modulation of ABA responses in plants. The early ABA signal regulators include ROP10 (the Rho-like small G protein), ERA1 (farnesyltranferase β-subunit ENHANCED RESPONSE TO ABA) (reviewed in Himmelbach et al., 2003), CBL9 (a calcineurin B-like protein (Pandey et al., 2004)), and CIPK3 (calcineurin B-like-interacting protein kinase 3 (Kim et al., 2003)). The transcriptional factors such as ERF7 and ABR1 are AP2-domain-containing transcription factors (Song et al., 2005; Pandey et al., 2005). RNA-processing proteins include HYL1 (HYPONASTIC LEAVES) (a double-stranded RNA-binding protein), ABH1 (ABA-HYPERSENSITIVE1) (an mRNA cap binding protein), and SAD1 (a SM-like snRNP protein, reviewed in Himmelbach et al., 2003).
Calcium is involved in the early steps of ABA-mediated responses in guard cells that control stomatal movements (reviewed by Leung and Giraudat, 1998; Schroeder et al., 2001). In the study of seed germination, calcium has been shown to be a critical factor for GA-induced germination (Bethke and Jones, 1998). In addition, ABA and GA are counteracting with each other in germination control. However, little is known about how calcium plays a role in the ABA–GA cross-talk. The finding that calcium sensor CBL9 and CBL-interacting protein kinase, CIPK3, are negative regulators of ABA response in germination clearly indicates that calcium may play a role as a positive regulator of GA signaling but a negative factor for ABA response in regulating seed germination, consistent with the opposing effect of GA and ABA on the germination process. What are the downstream components that mediate both ‘positive’ and ‘negative’ roles of calcium signal in GA and ABA responses? Studies have shown that calmodulin and calcium-dependent protein kinases (CDPKs) may be important for the GA signaling (Bethke and Jones, 1994; Abo-el-Saad and Wu, 1995; Bethke et al., 1997). Our previous reports on the CBL–CIPK network indicated that CBL-type calcium sensors and their kinases often serve as negative regulators of ABA signaling (Kim et al., 2003; Pandey et al., 2004). For example, CBL9 and CIPK3 both serve as negative regulators for ABA response in seed germination in Arabidopsis (Kim et al., 2003; Pandey et al., 2004). Because Arabidopsis contains at least 10 members in CBL and 25 members in CIPK gene family, it provides a high level of diversity and complexity in the function of the CBL–CIPK network. For instance, studies have shown that one CBL member often interacts with more than one CIPK and each CIPK interacts with more than one CBL. Some CBL members share common target CIPKs and some CIPKs share common CBL regulators (Kim et al., 2000; Luan et al., 2002; Batistic and Kudla, 2004; Kolukisaoglu et al., 2004). Such an interaction network will produce highly flexible control of gene function among the gene members. They can have redundant, specific, and antagonizing effects on each other, depending on the detailed relationship of the gene products in the CBL–CIPK network. Therefore, CBL–CIPK may be involved in more than one signaling pathway in seed germination. Indeed, one study showed that a CBL protein plays a role in GA signaling in rice seeds (Hwang et al., 2005), expanding the functional repertoire of CBL family reported in ABA response in Arabidopsis. In this study, we focus on the functional relationship of CBL9 and CIPK3, both involved in ABA response during seed germination. We show that CBL9 and CIPK3 physically and functionally interact with each other in the regulation of ABA response.
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CBL9 Interacted Physically with CIPK3
Our previous studies showed that cbl9 and cipk3 single mutants each displayed altered phenotype in ABA sensitivity during seed germination (Kim et al., 2003; Pandey et al., 2004). As CBL calcium sensors function by interacting with CIPKs, we speculated that CBL9 and CIPK3 may function in the same pathway by forming a CBL–CIPK pair in the signaling process. We tested this hypothesis by examining whether CBL9 physically interacts with CIPK3 using yeast two-hybrid analysis. As shown in Figure 1A, CBL9 interacted with the full-length CIPK3 but not with truncated CIPK3-kinase domain that lack the regulatory domain responsible for interaction with CBL (Kim et al., 2000; Albrecht et al., 2001; Luan et al., 2002). Similarly, CBL9 did not interact with some other CIPK members such as CIPK13 (Figure 1A). It is important to note that CBL1—a close homologue of CBL9 (with >90% amino acid sequence identity)—did not interact with CIPK3 in the yeast two-hybrid system (Kolukisaoglu et al., 2004; Y. H. Cheong, G. K. Pandey and S. Luan, unpublished). At the same time, CBL9 interacted with 10 other CIPKs, whereas CIPK3 interacted with three other CBLs (Kolukisaoglu et al., 2004; Y. H. Cheong, G. K. Pandey and S. Luan, unpublished). To further confirm the interaction of CBL9 with CIPK3, we have performed immuno-precipitation analysis with c-myc-tagged CBL9 and HA-tagged CIPK3 proteins expressed in transgenic Arabidopsis seedlings. Immuno-precipitation was done either with anti-c-myc or anti-HA antibody followed by Western blotting with the other antibody. We have found that the CBL9–c-myc and CIPK3–HA-tagged protein could be immuno-precipitated by anti-HA and anti-c-myc antibody, respectively (Figure 1B). In the control experiment, CBL9–c-myc, when mixed with CIPK13–HA or CIPK3-kinase domain–HA, could not be immuno-precipitated (Figure 1B), indicating that CBL9 and CIPK3 specifically interact with each other and that the interaction requires the C-terminal regulatory domain of CIPK3.
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Double Mutant cbl9cipk3 Shows Similar ABA Sensitivity to Single Mutants
In the single mutant analyses reported earlier, CBL9 (Pandey et al., 2004) and CIPK3 (Kim et al., 2003) both regulate ABA and osmotic stress signaling in Arabidopsis. In addition, the expression pattern of CBL9 and CIPK3 was highly related. Both are strongly expressed in germinating seeds and highly regulated by ABA and abiotic stress conditions in leaves. We have ascertained above that CBL9 and CIPK3 interact physically (Figure 1). These findings are consistent with the possibility that CBL9 and CIPK3 function in the same pathway as a CBL–CIPK pair. We created the double mutant, cbl9cipk3, by crossing the homozygous single mutants (Figure 2A). Because cbl9 and cipk3 single mutants were in different backgrounds (Col-0 and Ws), we crossed the wild-type plants of Col-0 and Ws to get WsxCol-0 lines to be used as controls in our experiments. RT–PCR analysis of cbl9cipk3 double mutant reveals elimination of transcript of CBL9 and CIPK3 (Figure 2B). We noted that expression of CIPK3 transcript in cbl9 mutant and CBL9 transcript in cipk3 mutant were not affected. As a loading control, the expression of Actin-2 gene was not affected in single and double mutants (Figure 2B).
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To evaluate the consequence of CBL9 and CIPK3 disruption, we examined the double mutant plants under normal growth conditions in MS agar media and in soil and found them to be indistinguishable in development and growth from the control (WsxCol-0) (data not shown). Since cbl9 and cipk3 single mutants displayed hypersensitive behavior under ABA and osmotic stress, we examined three independently segregated lines of cbl9cipk3 (cbl9cipk3-1, cbl9cipk3-2, cbl9cipk3-3) double mutant under similar conditions. In the vertical growth assays on the MS agar for ABA response, the single and double mutant seeds exhibited similar degree of hypersensitivity to ABA when compared to the control seeds (Figure 3A). In addition, double mutant seeds and seedlings were also hypersensitive to osmotic stress (mannitol) and high-salt-like single mutant (Figure 3A). As shown in Figure 3, the germination and subsequent growth of single and double mutant seedlings were similar to the control seedlings (WsxCol-0) on the normal medium (MS) but were significantly more inhibited by ABA and stress media. Results of more detailed analyses of germination rates in the presence of ABA and stress agents are depicted in Figure 3B and 3C. With 0.7 µM ABA, more than 68% control (WsxCol-0) seeds germinated whereas only 22% cbl9cipk3-1 mutant seeds germinated in 3 d after transferring to 23°C. In the presence of 150 mM NaCl, 78% of control (WsxCol-0) seeds germinated within 3 d but the germination frequency for the mutant seeds was only 35%. Similarly, germination frequency of control (WsxCol-0) and double mutant seeds were 60 and 35%, respectively, on the medium containing 350 mM mannitol. It is important to note that the cipk3 is more sensitive to salt than cbl9 and the double mutant cbl9cipk3 follows the pattern of cipk3 mutant (Figure 3B). In all phenotypic analysis of cbl9cipk3 double mutant, we compared the germination and seedling growth on ABA and osmotic stress media along with single mutants, cbl9 and cipk3, and their respective wild-type backgrounds (Figure 3B). In addition, we have included the independently segregated double mutant lines such as cbl9cipk3-1, cbl9cipk3-2, and cbl9cipk3-3 and heterozygous CBL9xCIPK3 lines (CBL9xCIPK3-1 and CBL9xCIPK3-2), which served as another set of controls (Figure 3C). All analyses consistently showed that cbl9cipk3 double mutant lines were similarly sensitive to ABA and osmotic stress as single mutants (Figure 3).
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Constitutively Active CIPK3 Rescued the cbl9 Mutant Phenotype
Physical interaction and phenotypic analysis in cbl9cipk3 double mutant and single mutants indicated that CBL9 and CIPK3 might be working in the same pathway in ABA response during seed germination. To confirm that CBL9-CIPK3 complex is responsible for modulating the ABA signaling pathway, we tested whether CBL9 functions through activation of CIPK3. We generated the constitutively active form of CIPK3 (T/D CIPK3) by mutating the threonine (T173) residue in the activation loop of CIPK3 to aspartate (D173). Both wild-type (Col-0) and cbl9 mutant plants were transformed with either wild-type CIPK3 or T/D-CIPK3 under the control of Cauliflower mosaic virus (CaMV) 35S promoter. By RT–PCR using CIPK3 specific primers, we have verified 15 over-expressing transgenic lines containing T/D-CIPK3 or wild-type CIPK3 in the cbl9 mutant and Col-0 background (data not shown). Four T3 homozygous lines showing higher transcript levels (Figure 4A) were selected for further phenotypic analysis.
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All four cbl9::T/D-CIPK3 transgenic lines did not display any growth-related and developmental phenotype when grown in MS agar or in soil. When grown in MS agar media containing 0.7 µM ABA, 150 mM NaCl, or 375 mM mannitol, these transgenic lines germinated and grew like the wild-type whereas the cbl9 mutant was more inhibited (Figure 4B). A detailed analysis of seed germination also showed that cbl9 mutant phenotype was rescued by expressing constitutively active CIPK3 (Figure 4C).
To determine whether the levels of CIPK3 in cbl9 mutant are limiting in vivo and whether increasing the wild-type CIPK3 level could also rescue the hypersensitive phenotype, we have also created cbl9::CIPK3 (WT) transgenic lines and four independent homozygous lines, which were confirmed by RT–PCR (Figure 5A), were analyzed for their ABA and osmotic stress sensitivity on MS agar plate. We performed the germination assay on MS and MS medium containing ABA or osmotic stress agents but did not find any significant difference between cbl9 mutant and the different transgenic lines of cbl9::CIPK3 (WT) (data not shown). Moreover, germination and vertical growth analysis showed that over-expression of wild-type CIPK3 in cbl9 mutant did not rescue the ABA and osmotic hypersensitivity of the mutant (Figure 5B and 5C). Thus, over-expression of constitutively active CIPK3 kinase could bypass the requirement of calcium sensor CBL9 in the CBL–CIPK signaling cascade, suggesting that CIPK3 might be downstream of CBL9 in the same pathway.
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Over-Expression of T/D CIPK3 in the Wild-Type Plants Did Not Alter ABA or Osmotic Stress Sensitivity
It is conceivable that the abundance of active CIPK3 molecule plays a key role in modulating ABA response. To determine whether levels of active CIPK3 in wild-type Arabidopsis might be limiting and whether increasing their levels by over-expression can lead to enhanced resistance to ABA in germination and growth, we generated T/D CIPK3 over-expressing transgenic lines in the Col-0 background. The transgenic lines over-expressing T/D CIPK3 were confirmed by RT–PCR and four T3 homozygous lines were selected which showed similar over-expression pattern in RT–PCR analysis (Figure 6A). These four lines were further analyzed in MS agar with or without ABA (0.7 µM) and osmotic stress (150 mM NaCl or 350 mM mannitol) for their germination and growth (Figure 6B and 6C). The T/D CIPK3 over-expressers did not show any detectable levels of tolerance or insensitiveness to ABA and osmotic stress. At the same time, we also tested the transgenic lines containing higher levels of CIPK3 (wild-type) in Col-0 background and found no change in ABA and osmotic stress sensitivity in germination and growth assay (data not shown). These results suggest that levels of active CIPK3 are not a limiting factor in ABA response regulation in Arabidopsis.
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODS |
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In this study, we have shown that a calcium sensor, CBL9, and its interacting protein kinase, CIPK3, function in the same pathway that negatively regulates ABA response during seed germination in Arabidopsis. By yeast two-hybrid and immuno-precipitation analysis, we have determined the physical interaction between CBL9 and CIPK3. The phenotypic analysis of cbl9cipk3 double mutant revealed that the function of CBL9 and CIPK3 is not cumulative. Together with transgenic evaluation of cbl9 mutant transformed with constitutively active kinase, our results suggest that CIPK3 functions downstream of CBL9 that regulates the activity of CIPK3 by forming a signaling complex.
Recent studies have revealed the complexity of the CBL–CIPK network. Initial genetics analysis of single mutants of two CBLs (CBL1 and CBL9) with more than 90% amino acid sequence identity revealed specific function of each CBL members. While CBL9 plays a role in ABA response in seed germination, CBL1 does not (Cheong et al., 2003; Pandey et al., 2004). One of the CBL-interacting protein kinase, CIPK3, also functions in ABA response, as shown in the single mutant analysis (Kim et al., 2003). Our study here shows that CBL9, but not CBL1, interacts with CIPK3 and functions in the same pathway. Because CBL1 and CBL9 are both expressed in a number of tissues and regulated by stress conditions, we speculate that the functional specificity of CBL1 and CBL9 in ABA response is determined, at least in part, by the specific partner kinases with which they interact. Our studies on the double mutant analysis of CBL1 and CBL9 revealed new phenotypes that are not displayed by single mutants of CBL1 or CBL9, suggesting that CBL1 and CBL9 clearly have functional redundancy (Cheong et al., unpublished results). In addition, our studies also indicated that over-expression of CBL1 would inhibit the function of CBL9–CIPK3-regulated processes such as cold responses (Cheong et al., 2003). Therefore, CBL1 and CBL9 each has its specific function; they can also antagonize each other by competing for common interacting kinases (Cheong et al., 2003) and they have functional redundancy as well, by interacting with same kinases in the same pathways.
The expression of constitutively active CIPK3 in cbl9 mutant rescued the mutant phenotype in ABA response, demonstrating that CBL9 might be one of the component required for activation of CIPK3. Although we have not determined whether CBL9 directly regulated or influenced the kinase activity of CIPK3 but, based on the genetic evidence in which the hyperactive (T/D) CIPK3 could rescue the cbl9 mutant phenotype in ABA-mediated responses, it is imperative that CBL9 and CIPK3 exist in ABA-mediated signaling pathways. Because of the higher degree of interaction complexity of CBLs and CIPKs, i.e. CBL9 interact with 10 of the CIPKs and CIPK3 is one of them, it is very hard to conclude that CIPK3 is the downstream component in the CBL9-mediated ABA-signaling pathway. Moreover, the expression patterns of some of the marker genes under ABA and other osmotic stress conditions in cbl9 (Pandey et al., 2004) and cipk3 (Kim et al., 2003) do not exactly overlap, which also suggests that some other factors/components might be required in the activation of CIPK3 besides CBL9. By examining the over-expression of wild-type CIPK3 in cbll9 mutant, we realized that the mere over-expression of CIPK3 does not rescue the hypersensitive phenotype of cbl9 mutant, indicating that activation of CIPK3 kinase activity is required for the CBL9–CIPK3 signaling and the level of CIPK3 is not a limiting factor in this pathway. In this study, the expression of T/D CIPK3 (constitutively active kinase) in the cbl9 mutant background can rescue the phenotype in ABA and osmotic stress (NaCl and mannitol) mediated inhibition of seed germination and growth (Figure 4). This finding is reminiscent to the observation that the activated form of SOS2/CIPK24, but not the expression of the wild-type SOS2/CIPK24, could rescue the salt-hypersensitive phenotype of sos3/cbl4 (Guo et al., 2004). As regulatory genes are often considered as the targets for engineering agronomically desired traits, we tested whether hyper-activation of CIPK3 (a negative regulator of seed dormancy) would lead to ABA resistance and premature germination. We found that the over-expression of constitutively active (T/D CIPK3) or wild-type CIPK3 in Col-0 does not affect the sensitivity of these transgenic lines against ABA and osmotic stress. This observation suggests that it is not a simple ‘gain of function’ of the active form of CIPK3 in the plant cell, but a fine control involving other components in the signaling network might be required in order to block ABA sensitivity. Contrary to our observation, previous studies on constitutively active kinases such as PKS11/CIPK8 (Gong et al., 2002a) and PKS18/CIPK20 (Gong et al., 2002b) suggested that over-expression of constitutively active kinase in the wild-type plants leads to response modulation in glucose and ABA-signaling pathways.
Previous studies have already provided several components involved in ABA response in seed dormancy. These include the positive regulators ABI3, 4, and 5 that are transcription factors required for ABA-induced seed dormancy (Finkelstein et al., 2002). The ABI1 and ABI2, like CBL9–CIPK3, are negative regulators of ABA-induced dormancy (Merlot et al., 2001). Because studies have shown that some CIPK members may be able to interact with ABI1 or ABI2 (Ohta et al., 2003), it is conceivable that ABI1/2 phosphatases are somehow associated with the CBL–CIPK network in the modulation of ABA-mediated seed dormancy. Downstream of CBL9–CIPK3 might exist transcription factors such as ABR1, whose expression is regulated by CIPK3 (Pandey et al., 2005). Disruption of ABR1 gene also inhibits ABA response, mimicking the effect of mutations in CBL9 and CIPK3 (Pandey et al., 2005). A simplified model in Figure 7 illustrates the pathways for both positive and negative regulation of ABA-induced seed dormancy and will assist further dissection of the signaling network for ABA response in plants.
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Plant Materials, Stress Treatments, and RNA Analysis
Arabidopsis thaliana plants (ecotype Columbia) were grown in the greenhouse under long-day conditions (16-h light/8-h dark cycle) at 500 µmol m–2 s–1and 21–23°C, with 75% humidity for generation of seeds. For RNA analysis, 2-week-old seedlings grown on MS medium (Murashige and Skoog, 1962) were treated under different stress conditions. Seeds were treated with isopropanol for 5 min and with 50% bleach for 15 min, washed five times with sterile water, and plated on MS medium solidified with 0.9% agar. Total RNA was isolated with Tripure isolation reagent (Roche Diagnostics, Indianapolis, IN). All experiments were repeated at least three times, and results from one representative experiment are shown.
Site-Directed Mutagenesis and Plasmid Construction
Threonine173 (T) in the activation loop of kinase domain was mutated to Aspartate (D) by using a site-directed mutagenesis kit (Stratagene, La Jolla, CA) as per manufacturer's instruction. The T-to-D mutation was introduced in the CIPK3 primers, forward (5'- AGGGATGATGGACTCTTGCATGACTCGTGTGGAACACCAAACTAC-3’) and reverse (5'-GTAGTTTGGTGTTCCACACGAGTCATGCAAGAGTCCATCATCCCT-3’) and 18 cycles of PCR were done by using CIPK3–pGAD.GH plasmid (Kim et al., 2000) as template. T-to-D mutation was confirmed by sequencing and the T/D CIPK3 insert was subsequently cloned into pCAMBIA1300 (CAMBIA, Canberra, Australia) vector under the control of CaMV 35S promoter. Similarly, CIPK3 full-length cDNA without any mutation (wild-type) was also cloned into pCAMBIA vector containing CaMV 35S promoter.
The kinase domain of CIPK3 was amplified by PCR using CIPK3–pGAD.GH template and the forward (5'-TGGTGGATCCAATGAATCGGAGACAGCAA-3’) and reverse (5'-CATTGTCGACCGCTGGCTGTTCTTCTCTCTTCT) primers. The PCR product was digested with BamH1 and Sal1 and cloned into pGAD.GH. For constructing CIPK13–pGAD.GH and CBL9–pGBT9.BS, PCR was performed by using CIPK13-specific forward (5'- AAAGGATCCAATGGCTCAAGTACTATCTACACCCTTGGC-3’) and reverse (5'-AAAGTCGACTCACTGTTCAATTTCAGGTGGCAAACAC-3’) primers, and CBL9-specific forward (5'-AAA GGATCCCATGGGTTGTTTCCATTCCACGGC-3’) and reverse (5'-AAAGTCGACTCACGTCGCAATCTCGTCCACTCCG-3’) primers using the total cDNA of Arabidopsis seedling as template. The BamH1 and Sal1 fragments were cloned into pGAD.GH and pGBT9.BS, respectively.
The epitope tag c-myc or HA was introduced at the 3’ end of the respective open reading frame. The CBL9–c-myc, CIPK3–HA, CIPK3-kinase domain–HA, CIPK13–HA, were cloned into pCAMBIA1300 vector containing CaMV 35S promoter. Pfu (Stratagene, La Jolla, CA) was used for all PCR amplifications and the constructs of the cloned fragments were confirmed by restriction digestion and DNA sequencing.
Yeast Two-Hybrid Interaction Analysis
Saccharomyces cerevisiae strain PJ-69 expressing the CBL9–pGBT9.BS bait was transformed with various preys, as shown in the figure legends. The transformed yeast cells were selected on synthetic complete medium lacking tryptophan and leucine (SC–LT). Interaction was determined on synthetic complete medium lacking tryptophan, leucine, and histidine, and supplemented with 1.5 mM 3-amino-1,2,4-triazole (Sigma, Saint Louis, MO). Auto-activation activity was not found with CBL9–pGBT9.BS bait.
Generation of Transgenic Plants
35S–CIPK3–pCAMBIA 1300, 35S–T/D CIPK3–pCAMBIA1300 (CAMBIA, Canberra, Australia), and empty vector (pCAMBIA 1300) constructs were transferred into Agrobacterium tumefaciens GV3101 and used for Arabidopsis transformation. For cbl9 mutant or Col-0 background plant transformation, plants were grown in a greenhouse under long-day conditions (16-h light/8-h dark cycle) for 4 weeks before a floral-dip procedure (Clough and Bent, 1998). Briefly, Agrobacterium cells were grown in Luria-Bertani broth for 24 h at 30°C. The cells were collected by centrifugation and re-suspended in infiltration medium (half-strength MS medium, 5% sucrose, 1X Gamborg's vitamins, 0.044 µM benzylamino purine, and 0.04% Silwet L77) to an OD 600 of 1.5–2.0. Plants were dipped into this suspension for 30 s and transferred to a greenhouse. Seeds were harvested from these plants and screened on selection medium (half-strength MS medium, 1X Gamborg's vitamins, and 20 µg/mL hygromycin) for transformants. The putative transformants (T1) were rescued from plates and grown in a greenhouse under long-day conditions. T2 plants were raised to produce seeds that were germinated to produce T3 plants. Those T2 plants that produced 100% hygromycin-resistant plants in the T3 generation were considered homozygous transformants and were used for further experiments.
Co-Immunoprecipitation Analysis
The constructs containing CBL9–c-myc, CIPK3–HA, CIPK3 kinase domain–HA, CIPK13–HA, respectively, were transformed into Arabidopsis via Agrobacterium. T1 transgenic lines were grown on MS agar plate for 3 weeks and the total protein was extracted in 50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% NP-40, 1 mM DTT, 1 mM PMSF, plus protease inhibitor cocktail mixture (Sigma, Saint Louis, MO).
The total protein extract of CBL9–c-myc was mixed with CIPK3–HA, CIPK3 kinase domain–HA, or CIPK13–HA, and then immuno-precipitation was conducted at 4°C for 3 h by using either anti-HA or anti-c-myc antibodies (Roche Diagnostics, Indianapolis, IN) followed by incubation with protein A-sepharose beads (Bio-Rad, Hercules, CA). After washing three times with protein extraction buffer containing 75 mM NaCl, immuo-precipitated proteins were eluted from the sepharose beads by 100 mM Glycine, pH 2.5. The immuno-precipitated samples were subjected to SDS-PAGE and Western blotting with either anti-c-myc or anti-HA antibody.
RT–PCR Analysis of Gene Expression
To examine the expression by reverse transcriptase–mediated PCR (RT–PCR), DNase I-treated, total RNA (2.5 µg) was denatured and subjected to reverse transcription reaction using Superscript II (200 units per reaction; Invitrogen, San Diego, CA) at 42°C for 50 min followed by heat inactivation of the reverse transcriptase at 70°C for 15 min. PCR amplification was performed with initial denaturation at 94°C for 3 min followed by 35 cycles of incubations at 94°C for 30 s, 55°C for 45 s, and 72°C for 2 min and a final extension at 72°C for 10 min. For CBL9 gene, specific forward (5'-GATGATGGGGAGTGAGTAATATCAGAA-3’) and reverse (5'-GTCCACCTCCGAGTTAAATACGAAACT-3’) primers were used to amplify a PCR product covering the full ORF of 642 bp. Similarly, CIPK3-specific forward (5'-ATGAATCGGAGACAGCAAGTGAAACGTAGAG-3’) and reverse (5'-TCACTTTGCTGTTTCTTTCTTAACTTCGTTATTC-3’) primers were used to amplify a PCR product of 1.326 kb. Expression levels of Actin-2 were monitored with forward (5'-GGAAAGGATCTGTACGGTAAC-3’) and reverse (5'-TGTGAACGATTCCTGGAC-3’) primers to serve as a quantifying control. Aliquots of individual PCR products were resolved by agarose gel electrophoresis and visualized with ethidium bromide by Gel Doc 1000 (Bio-Rad, Hercules, CA).
Isolation of cbl9cipk3 Double Mutant
Homozygous cbl9 (Pandey et al., 2004) and cipk3 (Kim et al., 2003) mutants were crossed. F1 seeds of double mutant hybrid should contain double antibiotics resistance markers (BAR or Bialaphos resistance gene in the cbl9 mutant and NPTII in cipk3 mutant) and were selected on MS agar containing kanamycin sulphate (50 mg lit–1; Invitrogen, San Diego, CA) and Basta (Glufosinate ammonium 25 mg lit–1; Crescent, NY). The resistant seedlings were transferred to soil for F2 seed production after selfing under greenhouse growth condition. Resistant F2 seedlings were screened by genomic DNA–PCR and, subsequently, cbl9cipk3 double mutant plants were confirmed by RT–PCR. The primers used for PCR screening were CBL9 genomic DNA-specific forward (5'-CTCTTGTCATCCGTAAATGGGTTGTTTC-3’) and reverse (5'-GCTTGTCTTGTCTCTTTCACGTCGCAATCTCG-3’); CIPK3-genomic DNA-specific forward (5'-GGAGAACCTGTTGCTCTCAAGATTCTT-3’) and reverse (5'-TTGAGGTTTCCATAGGAGTCCAATAG-3’). A large number of cbl9cipk3 individual double mutant plants were selected and segregated to subsequent generation for consistency in seed germination and growth assays. As cbl9 and cipk3 mutants were in different ecotype backgrounds, Col-0 and Ws, respectively, the wild-type plants from these two ecotypes were also crossed in parallel and used as control in all the experiments.
Germination Assay
Approximately 100 seeds each from the control and various mutant and transgenic lines, as indicated in figure legends, were planted in triplicate on MS medium or medium containing different concentrations of ABA, NaCl, or mannitol, and incubated at 4°C for 6 d before being placed at 23°C under long-day conditions. Germination (emergence of radicles) was scored daily for 9 d. The vertical germination and growth assays shown in Figures 3–6
were performed in a similar manner except that the plates were placed vertically on a rack. Plant growth was monitored and photographed after 12 d.
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Acknowledgements |
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We are grateful to the ABRC (Ohio State University, Columbus, OH) for Arabidopsis seeds and Syngenta Research and Technology, Torrey Mesa Research Institute (San Diego, CA) for providing the T-DNA insertional lines. This work was supported by a grant from the National Science Foundation (to SL). No conflict of interest declared.
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