A Tobacco Calcium-Dependent Protein Kinase, CDPK1, Regulates the Transcription Factor REPRESSION OF SHOOT GROWTH in Response to Gibberellins

Identification of RSG Kinase

To identify an RSG kinase that selectively phosphorylates Ser-114 of RSG (350 amino acids, calculated molecular mass of 38,000), we performed an in-gel kinase assay with tobacco leaf cell extracts using an affinity-purified glutathione S-transferase (GST)–RSG fusion protein as a substrate and the S114A mutant version of GST–RSG (GST–S114A) as a negative control. In the in-gel kinase assay, we detected a few bands that represented protein kinase activities with molecular masses in the range of 45 to 60 kD. These kinases phosphorylated GST–RSG much more effectively than GST–S114A, and their kinase activities were obviously enhanced by Ca²⁺ (Figure 1A ). Furthermore, the RSG kinase activities were enriched in the Triton X-100–solubilized fraction but barely detected in the detergent-free soluble fraction. These results suggested that the protein kinases that selectively phosphorylate Ser-114 of RSG are activated by Ca²⁺ and associated with membrane components.

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Figure 1.

RSG Kinase Activities in Tobacco.

(A) Enhancement of RSG kinase activities in the detergent-solubilized fraction by Ca²⁺. Tobacco leaf cell extract was fractioned into a detergent-free soluble fraction (Soluble) and a Triton X-100–solubilized fraction (TX100-solubilized) followed by an in-gel kinase assay with recombinant RSG (GST–RSG) or its S114A mutant version (GST–S114A) as substrates in the presence or absence of Ca²⁺ (+Ca²⁺ or +EGTA). The same proportion of the detergent-free soluble and the Triton X-100–solubilized fractions of the plant proteins were loaded on the gel.

(B) Enhancement of the interaction between RSG kinase and RSG by Ca²⁺. Tobacco leaf cell extract was incubated with glutathione beads immobilized with GST or GST–RSG in the presence (+) or the absence (−) of Ca²⁺ as indicated. The precipitates with beads (Pull down) and the leaf cell extract (Extract) were subjected to an in-gel kinase assay with acrylamide gels containing the GST-tagged phosphorylation domain of RSG [residues 69 to 140, GST–RSG(69-140)] as a substrate with or without Ca²⁺ (+Ca²⁺ or +EGTA). The precipitates were also subjected to SDS-PAGE (Tris/Glycine buffer) and stained by Coomassie blue (CBB; right panel). The experiments were repeated twice with similar results.

Recent studies showed that protein kinases form apparently stable complexes with transcription factors to facilitate signaling beyond the transient interaction between the enzyme and its substrate. Such examples include the binding of Hog1p to mitogen-activated protein kinases in yeasts (Park et al., 2003), PIAS1 to IKKα in mammalian cells (Liu et al., 2007), and ERF7 to PKS3 in Arabidopsis (Song et al., 2005). To examine if the RSG kinase forms a stable complex with RSG, we investigated the RSG-associating protein kinase activities of plant cell extracts. Recombinant GST–RSG was absorbed to glutathione beads. The immobilized RSG fusion protein was incubated with tobacco leaf cell extracts in the presence or absence of Ca²⁺, and precipitated. RSG-associating proteins were subjected to an in-gel kinase assay to detect the RSG-associated kinase activity with the GST–RSG fusion protein as a substrate. Kinases with molecular masses of 50 and 60 kD phosphorylated GST–RSG, and their activities were enhanced by the addition of Ca²⁺ in the reaction (Figure 1B). In addition, Ca²⁺ promoted the interaction between RSG and the major kinase with a molecular mass of 60 kD (Figure 1B). These results suggested that Ca²⁺ enhances both the binding of RSG kinase to RSG and its kinase activity. Minor bands with molecular masses of 45 and 50 kD were also detected by the in-gel kinase assay. Although the exact nature of the proteins is unknown, these bands could represent a degraded form of major RSG kinases and minor RSG kinases.

CDPK1 Phosphorylates Ser-114 of RSG

Ca²⁺ is a common second messenger in eukaryotic signal transduction cascades. In yeasts and mammalian cells, the increase of cytosolic Ca²⁺ concentration activates both protein kinase C and Ca²⁺/Calmodulin-dependent protein kinases that phosphorylate various proteins, including transcription factors (Chawla et al., 1998; McKinsey et al., 2000; Firulli et al., 2003; Kumar et al., 2006). Unlike other eukaryotes, plants do not appear to have homologs of protein kinase C; thus, CDPKs and other calcium-regulated protein kinases are proposed to play key roles in many diverse physiological processes of plants that are regulated by Ca²⁺ signaling. Furthermore, it has been demonstrated that basic-hydrophobic (ϕ)-X-basic-X-X-Ser/Thr-X-X-X-ϕ-basic is one of an optimal target sequence of CDPKs (Sebastià et al., 2004; Harper and Harmon, 2005). The amino acid sequence surrounding Ser-114 of RSG (basic-ϕ-basic-X-X-Ser-X-X-X-X-ϕ) is closely matched to the target motif of CDPKs (Fukazawa et al., 2000). These findings prompted us to consider whether tobacco CDPK might regulate the 14-3-3 binding of RSG through the phosphorylation of Ser-114 of RSG.

When we started our work, known tobacco CDPKs were CDPK1 (Yoon et al., 1999); CDPK2, which is involved in the response to pathogen; and CDPK3, which is a homoeologous gene of CDPK2 (Romeis et al., 2001). The predicted molecular masses of CDPK1 and 2 were 61.6 and 64.7 kD, respectively, which were consistent with the RSG kinase activity observed in the in-gel kinase assay (Figure 1). We tried to examine whether recombinant CDPK1 and 2 phosphorylate Ser-114 of RSG by means of an in vitro kinase assay using antibodies that specifically recognize phosphor-Ser-114 of RSG (Ishida et al., 2004). While CDPK1 phosphorylated Ser-114 of RSG in a Ca²⁺-dependent manner, CDPK2 phosphorylated it only slightly even in the presence of a 200-fold excess of CDPK2 (Figure 2A ). Thus, we focused our attention on CDPK1.

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Figure 2.

Phosphorylation of RSG by CDPK1 in Vitro.

(A) Comparison of the catalytic activities of CDPK1 and CDPK2 on RSG. The GST-tagged phosphorylation domain of RSG [residues 69-140, GST–RSG(69-140)] was phosphorylated by different concentrations of recombinant CDPK1 and CDPK2 (GST–CDPK1–His and GST–CDPK2–His, respectively) for indicated periods of time with or without Ca²⁺. Aliquots of reactions were subjected to SDS-PAGE (Tris/Glycine buffer) followed by immunoblot analysis with anti-pentad histidine (α·His, to detect CDPKs, top), anti-pS114, which specifically recognizes phosphorylated Ser-114 of RSG (α·pS114, middle), and anti-GST [α·GST, to detect GST–RSG(69-140), bottom]. Note that the recombinant CDPK1 amounts were too low for simultaneous detection with recombinant CDPK2. The enzymatic activity of recombinant CDPKs were confirmed using an in vitro phosphorylation assay on the general substrates myelin basic protein or casein.

(B) Comparison of the catalytic activities of CDPK1 and Arabidopsis CKIIA on RSG. Control GST, GST-tagged phosphorylation domains of RSG [GST–RSG(69-140), GST–S114A(69-140), and GST–RSG(88-140)], GST-tagged tobacco mosaic virus–derived polypeptide (GST–TMV-MP-CT), and myelin basic protein were phosphorylated by recombinant Nt CDPK1 or recombinant At CKIIA (GST–CDPK1 and GST–CKIIA, respectively) in the presence of [γ-³²P]ATP. Equal amounts of substrates were subjected to SDS-PAGE (Tris/Glycine buffer), and ³²P-labeled proteins were visualized with a phosphor imager. The experiments were repeated twice with similar results.

We examined the substrate specificity of CDPK1 by means of an in vitro kinase assay using [γ-³²P]ATP. Recombinant CDPK1 effectively phosphorylated GST–RSG fusion proteins as well as a conventional substrate of Ser/Thr kinase, the myelin basic protein, but only slightly phosphorylated the S114A mutant version of GST–RSG and the GST-tagged tobacco mosaic virus movement protein (GST–TMV-MP-CT) (Figure 2B). The protein kinase activity of CDPK1 was not detected in the absence of Ca²⁺ in the reaction (Figure 2A). By contrast, another Ser/Thr kinase, Arabidopsis casein kinase II (At CKIIA) (Mizoguchi et al., 1993), phosphorylated GST–TMV-MP-CT and the myelin basic protein but not GST–RSG fusion proteins. These results indicated that CDPK1 selectively phosphorylates Ser-114 of RSG in vitro.

CDPK1 Binds RSG

It was known that protein kinases and phosphatases may form a complex with their physiological substrates in various cellular signaling cascades (Posas and Saito, 1997; Margolis et al., 2006). To examine whether CDPK1 binds to RSG, we performed an in vitro pull-down assay in the presence or absence of Ca²⁺ using recombinant fusion proteins of maltose binding protein (MBP)–RSG and GST–CDPKs. MBP–RSG bound to GST–CDPK1 in a Ca²⁺-dependent manner but not to the control GST or to GST–CDPK2 (Figure 3A ).

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Figure 3.

Interaction between CDPK1 and RSG in Vitro and in Vivo.

(A) RSG interacts with CDPK1 in a Ca²⁺-dependent manner but not with CDPK2 in vitro. Control GST and GST-tagged recombinant CDPKs (GST–CDPK1 or GST–CDPK2) were immobilized to glutathione beads and incubated with MBP-tagged recombinant RSG [residues 1 to 260, MBP–RSG(1-260)] in the presence (+) or the absence (−) of Ca²⁺ as indicated. After the binding reactions, glutathione bead-bound proteins were subjected to SDS-PAGE (Tris/Glycine buffer) followed by immunoblot analysis with anti-RSG (top) or Coomassie blue (CBB) staining (bottom).

(B) RSG interacts with CDPKs in plant cells. Agrobacterium carrying the expression construct for GST–RSG or control GST driven by the 35S promoter of CaMV was infiltrated into tobacco leaves. Two days after infiltration, leaf extracts were prepared. The expression of GST fusion proteins was determined by immunoblot with the anti-GST antibody (IB by α·GST). The RSG-interacting proteins were immunoprecipitated by anti-GST in the presence (+) or absence (−) of Ca²⁺ as indicated (Ca²⁺ @ IP) and subjected to an in-gel kinase assay with the GST-tagged phosphorylation domain of RSG [residues 69 to 140, GST–RSG(69-140)] as a substrate in the presence or absence of Ca²⁺ (+Ca²⁺ or +EGTA box of In-gel kinase assay, respectively).

(C) Constructs of DEX-inducible CDPK1–His and 35S-driven GST–RSG. pro., promoter.

(D) Coexpression of CDPK1–His and GST–RSG by agroinfiltration. The expression constructs shown in (C) were introduced simultaneously into tobacco leaves by Agrobacterium-mediated infiltration. One day after infiltration, double-infiltrated leaves and control plants (Mock) were sprayed with DEX or water as indicated, and leaf cell extracts were prepared 1 d after the treatment. The leaf cell extract and immunoprecipitates by anti-pentad histidine in the presence of Ca²⁺ (IP by α·His) were subjected to an in-gel kinase assay with GST–RSG(69-140) as a substrate with or without Ca²⁺ (In-gel kinase assay with Ca²⁺ or EGTA, respectively). The minor 50-kD band might be caused by proteolytic degradation and/or posttranslational modifications. The expression of GST and GST–RSG was detected by immunoblot analysis with the anti-GST antibody (IB by α·GST). The arrows indicate an unknown tobacco endogenous protein that interacted with anti-GST.

(E) RSG interacts with CDPK1 in plant cells. The double-infiltrated plants or control plants (Mock) were treated with DEX or water, and leaf cell extracts were prepared as described in (D). RSG-interacting proteins were immunoprecipitated from the leaf cell extracts with anti-GST in the presence or absence of Ca²⁺ (Ca²⁺ @ IP by α·GST) and subjected to an in-gel kinase assay with GST–RSG(69-140) as a substrate with or without Ca²⁺ (In-gel kinase assay with Ca²⁺ or EGTA, respectively). The experiments were repeated twice with similar results.

To confirm the interaction between CDPK and RSG in plant cells, GST–RSG was expressed in Nicotiana benthamiana under the control of the cauliflower mosaic virus 35S promoter (CaMV 35S) by the Agrobacterium tumefaciens–mediated infiltration method (Yang et al., 2000). The GST–RSG fusion protein was immunoprecipitated with anti-GST antibodies in the presence or absence of Ca²⁺, and RSG-bound proteins were subjected to an in-gel kinase assay using GST–RSG as a substrate. A protein kinase with the same molecular mass as CDPK1 was coprecipitated with GST–RSG in a Ca²⁺-dependent manner but not with the control GST (Figure 3B), indicating the interaction between GST–RSG and native CDPK proteins in plant cells. The in-gel kinase assay without Ca²⁺ showed reduced RSG kinase activity in the RSG immunoprecipitates.

We then tried to determine whether RSG interacts with CDPK1 in plant cells by coexpressing GST–RSG and recombinant CDPK1 tagged with a hexa-histidine epitope at its C terminus (CDPK1–His) (Figure 3C). GST–RSG and CDPK1–His were expressed from either the CaMV 35S or a dexamethasone (DEX)–inducible promoter, respectively, following agroinfiltration (Figure 3D). DEX-induced CDPK1–His was immunoprecipitated with anti-pentad histidine antibody followed by detection by means of an in-gel kinase assay. After DEX induction, RSG-associating proteins were immunoprecipitated with anti-GST antibodies in the presence or absence of Ca²⁺, and RSG-bound proteins were subjected to an in-gel kinase assay. The activity of the RSG kinase that was coprecipitated with GST–RSG in a Ca²⁺-dependent manner was remarkably increased with the induction of CDPK1–His by DEX treatment (Figure 3E), indicating that exogenously expressed CDPK1–His and GST–RSG associates in plant cells.

CDPK1 Is a Major Ser-114 Kinase

To test whether CDPK1 participates in RSG phosphorylation on Ser-114 in plant cells, we generated transgenic plants in which CDPK1 expression was specifically repressed by RNA interference (RNAi). We also generated transgenic plants overexpressing CDPK1 under the control of the CaMV 35S promoter. To suppress the expression of CDPK1, the 5′ region of the CDPK1 cDNA covering the N-terminal variable domain was expressed under the control of the CaMV 35S promoter in sense and antisense directions interrupted by the spacer sequence. The expression level of CDPK1 protein was detected with an antibody raised against the N-terminal variable region of CDPK1 (anti-CDPK1), which specifically recognizes CDPK1 (Figure 4A ). In the RNAi transgenic tobacco plants, the protein level of CDPK1 was reduced to 20 to 30% of the control wild-type SR1 tobacco plants (Figure 4B, middle panel). The kinase activities for Ser-114 of RSG in the cell extracts of the transgenic plants were examined by means of an in vitro kinase assay using antibodies that specifically recognize phosphor-Ser-114 of RSG. The data for Ser-114 kinase activity (Figure 4B, top panel) mirror expression levels of CDPK1 in control, RNAi, and overexpresser plants (Figure 4B, middle panel) and support the argument that it is CDPK1 activity in the SR1 extract, and more significantly, not another kinase that phosphorylates Ser-114 of RSG.

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Figure 4.

CDPK1 Is a Major Kinase for Ser-114 of RSG.

(A) Anti-CDPK1 specifically recognizes CDPK1. Recombinant CDPK1 and CDPK2 (GST–CDPK1–His and GST–CDPK2–His, respectively) were blotted onto membrane as indicated and immunodetected with anti-CDPK1 and anti-GST.

(B) The leaf cell extracts from control wild-type SR1, CDPK1 RNAi transgenic plants, or CDPK1–His–overexpressing transgenic plants were incubated with MBP-tagged recombinant RSG (MBP–RSG) in an in vitro phosphorylation buffer. Aliquots of the reactions were subjected to SDS-PAGE (Tris/Glycine buffer) followed by immunoblot analysis with anti-pS114 (top) or anti-CDPK1 (middle). Images of Coomassie blue (CBB) staining were used as a loading control (bottom). The values at the bottoms of the panels indicate the relative level of strengths of signals after standardization. The values of SR1 were set to 1.0. The experiments were repeated twice with similar results. SR1, control wild-type SR1 tobacco plants; CDPK1rnai, transgenic plants in which expression of CDPK1 was decreased by RNAi; CDPK1–His, transgenic plants overexpressing CDPK1–His under the control of the CaMV 35S promoter.

Inhibition of CDPK Affected the Intracellular Localization of RSG

We previously found that the GA levels regulate the intracellular localization of RSG; the regulation of the intracellular localization of RSG by GA requires 14-3-3 binding, which depends on the phosphorylation of Ser-114 in RSG (Ishida et al., 2004). These observations and our results that CDPK phosphorylates Ser-114 of RSG suggested that CDPK regulates the intracellular localization of RSG through the phosphorylation of Ser-114 in response to GAs. To test this possibility, we examined the effects of calmodulin antagonists, Compound 48/80 and Trifluoperazine. By means of an in vitro kinase assay (Figure 5A ), we confirmed that the enzymatic activity of recombinant CDPK1 was effectively inhibited by Compound 48/80 and Trifluoperazine. Using antibodies that specifically recognize phospho-Ser-114 of RSG, we examined the phosphorylation status of Ser-114 in RSG in transgenic plants expressing recombinant RSG tagged with green fluorescent protein at its C terminus (RSG–GFP) and in wild-type SR1, which were treated with a GA biosynthetic inhibitor, Uniconazole P, to reduce endogenous GA, and then were treated with GA₃. An immunoblot analysis clearly showed that GA₃ treatment promoted the phosphorylation of Ser-114 of RSG–GFP (Figure 5B). We confirmed the results with the GFP fusion protein by analyzing the behavior of the endogenous RSG protein in wild-type SR1 plants. In addition, phosphorylation of Ser-114 by GA₃ treatment was further promoted in transgenic plants overexpressing CDPK1, whereas it was repressed in transgenic plants in which CDPK1 expression was decreased by RNAi, showing that CDPK1 plays a central role in Ser-114 phosphorylation in vivo. Subsequently, we investigated the effects of a calmodulin antagonist, Compound 48/80 and Trifluoperazine, on the phosphorylation of Ser-114 in response to GAs. As expected, the GA-induced phosphorylation of Ser-114 was inhibited by the application of GA₃ with Compound 48/80 and Trifluoperazine (Figure 5B). A small amount of phosphorylation of Ser-114 of RSG–GFP was observed before GA treatment, suggesting that low activity of CDPK1 is maintained in Uniconazole P–treated plants.

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Figure 5.

The Calmodulin Antagonists Suppress GA-Induced Phosphorylation of Ser-114 and 14-3-3 Binding to RSG.

(A) The calmodulin antagonist Compound 48/80 and Trifluoperazine inhibit the catalytic activity of CDPK1 in vitro. The recombinant RSG [GST–RSG(69-140)] was phosphorylated by recombinant CDPK1 (GST–CDPK1–His) in vitro in the presence of Compound 48/80 or Trifluoperazine at the concentrations as indicated. Reactions were subjected to immunoblot analysis with anti-pS114 (top) or anti-GST (bottom).

(B) Compound 48/80 and Trifluoperazine inhibit the GA-induced phosphorylation of Ser-114 of RSG. Tobacco plants were treated with the GA biosynthesis inhibitor Uniconazole P for 1 week to reduce endogenous GA and then sprayed with a GA₃ solution or a GA₃ solution containing the calmodulin antagonist Compound 48/80 or Trifluoperazine. At the time points indicated after GA₃ application, leaves were harvested, and total leaf cell extracts were prepared. Aliquots of leaf cell extract were separated by SDS-PAGE (Tris/Glycine buffer) followed by immunoblot analysis with anti-pS114 (top) or anti-RSG (middle). Both anti-RSG antibodies and pS114 antibodies recognized multiple bands of GFP fusion proteins in the immunoblotting. Although the exact nature of the proteins is unknown, multiple bands could be caused by proteolytic degradation and/or posttranslational modifications. Images of Coomassie blue (CBB) staining (ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco]) were demonstrated to indicate RSG is not degraded by GA₃ treatment (bottom). The values at the bottom of the top panels indicate the relative level of strengths of signals after standardization using signals of RSG–GFP (transgenic panels) or RSG (SR1 panel) as a control. The values of signals at 0 h were set to 1.0. The experiments were repeated five times with similar results.

(C) Compound 48/80 and Trifluoperazine suppress GA-induced interaction between RSG and 14-3-3. RSG-interacting proteins were immunoprecipitated with anti-GFP from the leaf cell extract prepared as in (B) and separated by SDS-PAGE (Tris/Glycine buffer) followed by immunoblot analysis with anti-14-3-3 (top), anti-pS114 (middle), or anti-RSG (bottom). The values at the bottoms of the anti-14-3-3 panels indicate the relative level of strengths of signals after standardization using signals of immunoprecipitated RSG–GFP as a loading control. The values of signals at 0 h were set to 1.0. The experiments were repeated five times with similar results. RSG–GFP, GA-treated transgenic plants expressing RSG–GFP under the control of the CaMV 35S promoter; RSG–GFP + C48/80, GA- and Compound 48/80–treated transgenic plants expressing RSG–GFP under the control of the CaMV 35S promoter; RSG–GFP + Tfz, GA- and Trifluoperazine-treated transgenic plants expressing RSG–GFP under the control of the CaMV 35S promoter; S114A–GFP, GA-treated transgenic plants expressing S114A–GFP under the control of the CaMV 35S promoter; RSG–GFP CDPK1rnai, GA-treated RSG–GFP transgenic plants in which expression of CDPK1 was decreased by RNAi; RSG–GFP CDPK1–His, GA-treated transgenic plants expressing RSG–GFP and CDPK1–His under the control of the CaMV 35S promoter; SR1, GA-treated control wild-type SR1 plants.

Since the phosphorylation of Ser-114 promoted 14-3-3 binding to RSG (Ishida et al., 2004), we anticipated that the application of GA₃ facilitates the interaction between RSG and 14-3-3 in vivo. The immunoprecipitation of RSG–GFP with anti-GFP showed that GAs enhanced the 14-3-3 binding to RSG (Figure 5C). The applications of Compound 48/80 and Trifluoperazine with GA₃ inhibited this GA-induced complex formation, suggesting that CDPK activity is involved in the 14-3-3 binding to RSG through the phosphorylation of Ser-114 on RSG in response to GAs.

RSG translocated into the nucleus in response to a reduction in GA levels, and GA treatment could reverse this nuclear accumulation (Ishida et al., 2004). If a CDPK is involved in the regulation of the intracellular localization of RSG, Compound 48/80 and Trifluoperazine should inhibit GA-induced nuclear export of RSG. The applications of Compound 48/80 and Trifluoperazine to transgenic tobacco plants in which RSG–GFP was expressed resulted in the retention of RSG–GFP in the nucleus upon treatment with GA₃ (Figure 6 ). Due to the nature of the calmodulin antagonists, we cannot completely rule out the possibility that they also affected other calcium-regulated kinases. However, RNAi experiments showed that CDPK1 plays a central role in the phosphorylation of Ser-114 of RSG in plants (Figures 4 and 5). Collectively, these results were consistent with the notion that CDPK is involved in the GA-induced nuclear export of RSG through the phosphorylation of Ser-114 on RSG and 14-3-3 binding.

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Figure 6.

The Calmodulin Antagonists Repress GA-Induced Nuclear Export of RSG.

Transgenic tobacco expressing RSG–GFP was treated with the GA biosynthesis inhibitor Uniconazole P for 1 week to reduce endogenous GA and then sprayed with a GA₃ solution or a GA₃ solution containing the calmodulin antagonist Compound 48/80 or Trifluoperazine. Leaves were harvested at the time points indicated, and fluorescence due to GFP was observed with an epifluorescence microscope. GA, GA-treated transgenic plants expressing RSG–GFP under the control of the CaMV 35S promoter; GA + C48/80, GA- and Compound 48/80–treated transgenic plants expressing RSG–GFP under the control of the CaMV 35S promoter; GA + Tfz, GA- and Trifluoperazine-treated transgenic plants expressing RSG–GFP under the control of the CaMV 35S promoter. Bar = 50 μm.

GA Promotes the Phosphorylation of CDPK1

The catalytic activities of CDPKs modulated by not only the Ca²⁺ milieu but also posttranslational regulation, including phosphorylation (Harper et al., 2004). To understand the mechanism of functional activation of CDPK1 by GAs, we first tried to examine if GA affected the expression of CDPK1. The immunoblot analysis showed that the protein level of endogenous CDPK1 was not changed by GA₃ treatment (Figure 7A ). This result suggested that the activation of CDPK by GAs depends on posttranslational mechanisms rather than de novo protein synthesis.

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Figure 7.

CDPK1 Was Modified as a Consequence of the GA Signal.

(A) Protein gel blot analysis of the endogenous CDPK1. SR1 plants were treated with Uniconazole P for 1 week and then sprayed with a GA₃ solution. At the indicated time points after GA₃ application, leaves were harvested, and total leaf cell extracts were prepared. The levels of CDPK1 protein in each total leaf cell extract were visualized by immunoblot with anti-CDPK1 (top). Images of Coomassie blue (CBB) staining (Rubisco) were used as a loading control (bottom). The values at the bottom of the top panel indicate the relative level of strengths of signals after standardization using CBB staining of Rubisco as a loading control. The values of signals at 0 h were set to 1.0. The experiments were repeated twice with similar results.

(B) The intracellular localization pattern of CDPK1 was not apparently affected by GA treatment. Top panel: Transgenic plants expressing CDPK1–His under the control of the CaMV 35S promoter were treated as in (A), and cells were fractionated to the Triton X-100–solubilized fraction (TX100-solubilized) and the detergent-free soluble fraction (Soluble). The same proportion of the detergent-free soluble and the Triton X-100–solubilized fractions of the plant proteins were loaded on the gel. The levels of CDPK1 were visualized by immunoblots with anti-CDPK1. Each value at the bottom of the panel indicates the relative level of strength of signal in a soluble fraction when the value of signal in a Triton X-100–solubilized fraction was set to 1.0. The experiments were repeated three times with similar results. Bottom panels: Transgenic plants expressing CDPK1–GFP under the control of the CaMV 35S promoter were treated as in (A), and fluorescence due to GFP was observed with an epifluorescence microscope at the indicated time points after GA₃ application. Bar = 50 μm.

(C) GA induces the phosphorylation of CDPK1. Aliquots of the Triton X-100–solubilized fraction prepared as in (B) were treated with or without a phosphatase (CIAP and Control, respectively) at 30°C for 1 h. Reactions were separated by SDS-PAGE (Tris/Glycine buffer) and subjected to immunoblot analysis with anti-CDPK1. To facilitate the detection of mobility shift of CDPK1, electrophoresis was performed using a gel of low concentration of acrylamide with extended running time. CIAP, calf intestine alkali phosphatase. The experiments were repeated five times with similar results.

(D) The GA signal promotes the interaction between CDPK1 and RSG. Transgenic plants expressing either CDPK1–His and RSG–GFP under the control of the CaMV 35S promoter or RSG–GFP only under the control of the CaMV 35S promoter were treated as in (A). At the indicated time points after GA₃ application, leaves were harvested, and leaf cell extracts were prepared. RSG-interacting proteins were immunoprecipitated with anti-RSG and analyzed by immunoblots with anti-CDPK1 (top) and anti-RSG (bottom). The values at the bottom of the top panels indicate the relative level of strengths of signals after standardization using signals of immunoprecipitated RSG–GFP as a loading control. The values of signals at 0 h were set to 1.0. The experiments were repeated five times with similar results.

We tested the possibility that GAs affected the intracellular localization of CDPK1. A potential N-myristoylation motif for membrane association is found at the beginning of the N-terminal region of CDPK1. The immunoblot analysis showed that CDPK1 was enriched in the Triton X-100–solubilized fraction, and its fractionation pattern was not apparently affected by GA₃ treatment (Figure 7B, top panel), suggesting that a large portion of CDPK1 was associated with the membrane component irrespective of the GA levels in cells. We confirmed this result by observation of intracellular localization of CDPK1 using transgenic plants expressing a tagged version of CDPK1 including GFP sequence at its C terminus (CDPK1–GFP) (Figure 7B, bottom panel).

Next, we examined the covalent modification state of CDPK1 by immunoblot analysis after GA₃ treatment. When SDS-PAGE was done using a gel of low acrylamide concentration and extended running time, the electrophoretic mobility of CDPK1 was apparently decreased after GA₃ treatment (Figure 7C). Treatment by calf intestine alkaline phosphatase could reverse this mobility shift of CDPK1, showing that GA application promotes the phosphorylation of CDPK1.

We then tried to determine whether GA treatment could affect the interaction between RSG and CDPK1. We generated transgenic tobacco plants expressing RSG–GFP with or without CDPK1–His to investigate the effect of GAs on the interaction between RSG and CDPK1 in vivo. Immunoprecipitation with anti-RSG antibodies showed that the associations of RSG–GFP and CDPK1–His, or endogenous CDPK1, were enhanced by GA₃ treatment (Figure 7D). Furthermore, the results confirmed in vivo interaction of endogenous CDPK1 with RSG–GFP. These results suggested that GAs affect CDPK1 through posttranslational mechanisms, including phosphorylation and promotion of RSG binding.

Overexpression of CDPK1 Affected the Feedback Regulation of GA 20-Oxidase

It has been shown that GA biosynthesis is affected by the activity of the GA response pathway through a feedback mechanism (Hedden and Phillips, 2000). Previously, we found that RSG is involved in the feedback regulation of the GA 20-oxidase gene but not of the GA 3-oxidase gene (Ishida et al., 2004; Matsushita et al., 2007). Because CDPK1 negatively regulates RSG by promoting 14-3-3 binding to RSG through the phosphorylation of Ser-114 of RSG, we expected that the overexpression of CDPK1 would inhibit the feedback regulation of the GA 20-oxidase gene in response to a decrease in the GA levels in cells. To confirm this, we used transgenic tobacco plants in which CDPK1–His was expressed under the control of CaMV 35S.

As shown in Figure 8A , the tobacco GA 20-oxidase gene (GA20ox) was upregulated in a GA-negative feedback manner in the GA-deficient (i.e., Uniconazole P–treated) control wild-type SR1 tobacco plants; however, upregulation by treatment with uniconazole P was suppressed in the transgenic tobacco plants overexpressing CDPK1–His. These results suggest that CDPK1 is involved in the feedback regulation of the GA 20-oxidase gene through the phosphorylation of RSG. Meanwhile, expression of GA 20-oxidase was not apparently enhanced in the transgenic plants in which expression of CDPK1 was reduced by RNAi.

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Figure 8.

Overexpression of CDPK1 Inhibited GA Homeostasis.

(A) Feedback regulation of the GA 20-oxidase gene was suppressed in transgenic plants overexpressing CDPK1–His. Transgenic tobacco overexpressing CDPK1–His under the control of the CaMV 35S promoter and control wild-type SR1 plants were grown with or without the GA biosynthesis inhibitor Uniconazole P for 1 week as indicated. Transgenic tobacco in which expression of CDPK1 was repressed by RNAi and control wild-type SR1 plants were grown without chemical treatment. The mRNA levels of GA 20-oxidase (GA20ox) were examined by quantitative RT-PCR. After PCR, the products were detected by DNA gel blot hybridization. Tobacco arcA was amplified in the same reaction and used as an internal control for RT-PCR. The values at the bottom of the top panel indicate the relative level of strengths of signals after standardization using signals of arcA as a loading control. The values of SR1 were set to 1.0. The experiments were repeated three times with similar results.

(B) Uniconazole P resistance was reduced in transgenic plants overexpressing CDPK1. Seeds of transgenic tobacco overexpressing CDPK1–His and control wild-type SR1 plants were germinated in a medium containing 1 μM Uniconazole P with or without 1 μM GA₃ for 7 d at 28°C. The values at the bottom of panels indicate the germination ratio calculated from three independent plates (±sd, 80 to 100 seeds/plate). The experiments were repeated three times with similar results. SR1, control wild-type SR1 tobacco plants; CDPK1–His, transgenic tobacco plants overexpressing CDPK1–His under the control of the CaMV 35S promoter; CDPK1rnai, transgenic plants in which expression of CDPK1 was decreased by RNAi; +UniP, seedlings germinated on agarose containing Uniconazole P; +Uni+GA, seedlings germinated on agarose containing Uniconazole P and GA₃.

Expression profiling of GA20ox in Uniconazole P–treated CDPK1 overexpressing plants also suggested that the overexpresser might be more sensitive to the GA biosynthetic inhibitor than control plant. To test this possibility, we examined the effect of Uniconazole P on the germination of tobacco. As expected, the CDPK1 overexpresser was more sensitive to Uniconazole P than control wild-type SR1 tobacco plants (Figure 8B). Treatment with GA₃ reversed the inhibition of germination. These results suggested that CDPK1 plays a role in the homeostasis of GAs.

Plant Growth Conditions and Chemical Treatments

The effects of chemicals were examined on plants grown in soil individually in 0.5-liter beakers as described previously (Ishida et al., 2004). Young plants (Nicotiana tabacum cv Petite Havana SR1) received 125 mL of 68 μM Uniconazole P, an inhibitor of GA biosynthesis (Wako), for 5 to 7 d and were then sprayed with a 100 μM GA₃ (Wako) solution with or without 350 to 750 mg/mL Compound 48/80 (Sigma-Aldrich) or Trifluoperazine (MP Biomedicals). Plants were grown under continuous light at 28°C.

Plasmid Construction

Total RNA from tobacco suspension cultured cells (BY-2) was converted into cDNA with SuperScript II (Invitrogen) and oligo(dT)12-18 (Amersham Bioscience). CDPK1 and CDPK2 were cloned by PCR with specific primers (CDPK1, 5′-CCTAGGATCCATGGGTGGTTGTTTTAGCAAGAAG-3′ and 5′-CAGATATCTAGAAAAGCTTCGGCTGTGGTTG-3′; CDPK2, 5′-CCTAGGATCCATGGGGAACACTTGTGTTGGACCA-3′ and 5′-CAGATATCTAGTCTTAGAGCCTCTCTAAATCCGG-3′) from the cDNA and inserted into a pGEX-4T-2 vector (Amersham Bioscience). cDNA encoding a casein kinase II of Arabidopsis thaliana (Mizoguchi et al., 1993) was inserted into a pGEX-4T-1 vector. The expression vectors of the pGEX-4T series, pET32 series (Novagen), and pMal-c (New England Biolabs) were used to prepare GST, His (hexa-histidine), and MBP-tagged versions of the recombinant proteins, respectively. To visualize RSG and CDPK1 protein, a synthetic GFP tag was placed at its C terminus (Chiu et al., 1996). To generate transgenic plants, each expression cassette for recombinant protein was inserted to pBI101.1 (Clontech).

To repress the expression of CDPK1 by RNAi, constructs were prepared as follows: The variable domain of CDPK1, amplified by PCR with primers 5′-GGATCCATGGGTGGTTGTTTTTAGC-3′ and 5′-CTCGAGTTGCTTCCTAATATCTTCAA-3′, was cloned into pENTR-1A (Invitrogen). Cloned fragment was transferred to pPI1 vector, which contains the CaMV 35S promoter, two attR cassettes that oriented to the opposite direction each other, and a fragment of GPA1 intron placed in between the attR cassettes (pPI1 vector was provided by M. Hasebe, National Institute for Basic Biology, Japan). The expression cassette was excised from the plasmid and inserted to pBI101.1.

Generation of the Anti-CDPK1–Specific Antiserum

The N-terminal variable region of CDPK1 (residues 1 to 129) was expressed in Escherichia coli BL21(DE3) cells as a tagged version including a hexa-histidine epitope at its C terminus and purified with nickel-nitrilotriacetic acid agarose (Qiagen) as specified by the manufacturer. The purified polypeptides were further resolved by SDS-PAGE (Tris/Glycine buffer), electroeluted and dialyzed in Tris-buffered saline, and then used as an antigen to immunize rabbits.

Preparation of Protein Extracts

A total cell extract of young tobacco leaves was prepared as follows: treated leaves were harvested at different time points after treatments, disrupted in liquid nitrogen by grinding with a mortar and pestle, and then extracted in 3 volumes of extraction buffer (40 mM MOPS-NaOH, pH 6.5, 20 mM glycerol 2-phosphate disodium, 20 mM NaF, 5 mM Na₃VO₄, 10 mM MgSO₄, 5 mM EGTA, 10% glycerol, 2% Triton X-100, 1 mM DTT, a protease inhibitor cocktail [Pierce], and a phosphatase inhibitor cocktail [Sigma-Aldrich]).

Cells were fractionated as follows: tobacco leaves disrupted as above were extracted in 3 volumes of 50 mM HEPES-NaOH, pH 7.0, 1 mM DTT, 2 mM EDTA, 2 mM EGTA, 10 mM Na₃VO₄, 10 mM NaF, 50 mM glycerol 2-phosphate disodium, the protease inhibitor cocktail, and the phosphatase inhibitor cocktail and then centrifuged at 100,000g for 30 min at 4°C. The resultant supernatant was the detergent-free soluble fraction. The pellet was resuspended in the same volume as that of the supernatant (soluble fraction) with the buffer containing 20 mM HEPES-NaOH, pH 7.0, 1 mM DTT, 1 mM Na₃VO₄, 1 mM NaF, 5 mM glycerol 2-phosphate disodium, 2% Triton X-100, the protease inhibitor cocktail, and the phosphatase inhibitor cocktail and lightly sonicated to promote solubilization, and then centrifuged again as above. The resultant supernatant was the Triton X-100–solubilized fraction.

In-Gel Kinase Assay

An in-gel protein kinase assay was performed as described by Kameshita and Fujisawa (1989) with 10% polyacrylamide gels containing 0.2 mg/mL of recombinant RSG tagged with the GST epitope at its N terminus (GST–RSG) or the S114A mutant version of GST–RSG (GST–S114A) or the phosphorylation domain of RSG (residues 69 to 140) tagged with the GST epitope at its N terminus [GST–RSG(69-140)] or the S114A mutant version of GST–RSG(69-140) [GST–S114A(69-140)]. The detergent-free soluble fraction (30 μg of protein) and the corresponding volume of Triton X-100–solubilized fraction were applied to the gels (the same proportion of the soluble and the Triton X-100–solubilized fractions of the plant proteins were used). After electrophoresis (Tris/Glycine buffer), the gels were washed successively with 50 mM Tris-HCl, pH 8.0, containing 20% isopropanol and then with 50 mM Tris-HCl, pH 8.0, containing 0.05% β-mercaptoethanol (the washing buffer). The denaturation of polypeptides in the gels was completed by shaking the gels in a washing buffer containing 6 M guanidine-HCl. To renature proteins in the gels, the gels were gently shaken in a renaturation buffer containing 50 mM Tris-HCl, pH 8.0, 0.05% Tween 40, and 0.05% β-mercaptoethanol at 4°C for 15 min to 20 h. After equilibration with 40 mM HEPES-KOH, pH 7.6, 10 mM MgCl₂, 1 mM DTT, and 0.5 mM CaCl₂ for the Ca²⁺-plus condition or 0.5 mM EGTA for the Ca²⁺-minus condition at 4°C for 30 min, the gels were incubated with 1 μM [γ-³²P]ATP (37 kBq/mL, 5000 Ci/mmol) at 30°C for 2 h to start the phosphorylation reaction. The reaction was terminated by transferring the gels into 5% (w/v) CCl₃COOH containing 1% Na₄P₂O₇·10H₂O, and gels were successively washed to remove free radioactive ATP. After the gels were dried, the radioactive signals due to ³²Pi-phosphorylated polypeptides in the gels were visualized using a phosphor imager BAS 2500 (Fuji Film).

Immunoblot and Immunoprecipitation Analysis

Aliquots of each protein sample were resolved by SDS-PAGE (Tris/Glycine buffer) and transferred onto a polyvinylidene fluoride membrane (Millipore) and reacted with rat antiserum raised against tobacco RSG (anti-RSG; Igarashi et al., 2001) or tobacco 14-3-3 (anti-14-3-3; Igarashi et al., 2001), or the phosphorylated Ser-114 of RSG (anti-pS114; Ishida et al., 2004) or anti-CDPK1, or anti-pentad histidine (Qiagen) or anti-GST (Amersham Bioscience) followed by the horseradish peroxidase–conjugated second antibody. Chemiluminescence was detected (SuperSignal West Femto Maximum Sensitivity Substrate; Thermo Scientific) and quantified with a CCD camera imaging system (LAS-3000; Fuji Film). Total protein was visualized by Coomassie Brilliant Blue staining for the loading control in some experiments.

Aliquots of each protein sample were immunoprecipitated with antipentad histidine, anti-GST, anti-RSG, or anti-GFP (MBL Nagoya) for 3 to 16 h at 4°C, and then Protein G-conjugated beads (Amersham Bioscience) were added to each reaction. Immunoprecipitates were washed three times at 4°C, and coprecipitated proteins were subjected to immunoblot analysis or an in-gel kinase assay as described above. Reactions without antibodies gave no signal.

Detection of an RSG-Interacting Kinase

The total cell extract was prepared from young tobacco leaves without a chemical treatment as described above. Glutathione beads (Amersham Bioscience) carrying GST (50 μg) or GST-RSG fusion (50 μg) were added to 4 mL of the total cell extract with or without 5.5 mM CaCl₂ (for the Ca²⁺-plus condition or for the Ca²⁺-minus condition, respectively). Each set of reactions was incubated at 4°C for 2 h and washed three times with the extraction buffer with or without CaCl₂. Proteins associated with the beads were subjected to an in-gel kinase assay as described above.

In Vitro Phosphorylation Assay

The catalytic activities of the purified recombinant CDPK1 and CDPK2 tagged with a GST epitope at their N terminus and a hexa-histidine epitope at their C terminus (GST–CDPK1–His and GST–CDPK2–His, respectively) were assayed in a reaction mixture containing 25 mM Tris-HCl, pH 7.6, 10 mM MgCl₂, and 0.5 mM CaCl₂ for the Ca²⁺-plus condition or 2 mM EGTA for the Ca²⁺-minus condition, 0.1% Triton X-100, 10 mM β-mercaptoethanol, and 0.1 μg/μL GST–RSG(69-140) as the substrate with 1 mM ATP (cold assay) or 100 μM ATP supplemented with [γ-³²P]ATP (5000 Ci/mmol, 10 to 20 Ci/reaction, hot assay) at 30°C for 30 to 60 min.

For comparison of the catalytic activities of Nt CDPK1 and Arabidopsis CKIIA, control GST, GST-tagged phosphorylation domains of RSG [GST–RSG(69-140), GST–S114A(69-140), and GST–RSG(88-140)], GST-tagged tobacco mosaic virus–derived polypeptide (GST–TMV-MP-CT), and myelin basic protein (Wako) were phosphorylated by GST-tagged recombinant CDPK1 or recombinant CKIIA (GST–CDPK1 or GST–CKIIA, respectively) in the presence of [γ-³²P]ATP.

Reactions were subjected to SDS-PAGE (Tris/Glycine buffer) followed by immunoblot analysis for the cold assay or visualization of ³²Pi-phosphorylated polypeptides by the phosphor imager for the hot assay as described above.

In Vitro Binding Assay

Purified recombinant CDPK1 and CDPK2 tagged with GST epitope at their N terminus (GST–CDPK1 and GST–CDPK2, respectively, each 5 μg) and the recombinant RSG (residues 1 to 260) tagged with MBP epitope at its N terminus [MBP–RSG(1-260), 7.5 μg] and glutathione beads (Amersham Bioscience) were incubated in Tris-buffered saline containing 0.1% Triton X-100 and 0.05% β-mercaptoethanol with 0.5 mM CaCl₂ for the Ca²⁺-plus condition or 2 mM EGTA for the Ca²⁺-minus condition at 4°C for 60 min and washed with Tris-buffered saline. Proteins binding to the beads (quarter of total amount) were resolved by SDS-PAGE (Tris/Glycine buffer) and visualized by Coomassie Brilliant Blue staining or immunoblot analysis described as above.

Agrobacterium tumefaciens–Mediated Infiltration

Agrobacterium-mediated infiltration was performed according to Van de Hoorn's work (Van de Hoorn et al., 2000; Yang et al., 2000) with a minor modification. The Agrobacterium strain EHA105 was transformed by the freeze-thaw method (Jyothishwaran et al., 2007) with a binary vector of pTA7002 (Aoyama and Chua, 1997) containing CDPK1 tagged with a hexa-histidine epitope at its C terminus (CDPK1–His) and pBE2113-GST (Yuasa et al., 2005) containing RSG. The transformants were grown overnight at 28°C in 10 mL of a YEB medium containing 30 μg/mL kanamycin and 0.25 mM acetosyringone and harvested by centrifugation at 3000g for 15 min. The cells were washed three times with 10 mL of MMS (10 mM MES-KOH, pH 5.5, 10 mM MgSO₄, 2% sucrose, and 0.25 mM acetosyringone) and resuspended in MMS at a final OD₆₀₀ of 0.6. Cultures of Agrobacterium were infiltrated into leaves of Nicotiana benthamiana, and plants were maintained at 25°C under a photoperiod of 16 h light and 8 h dark for 48 h. The inoculated leaves were sprayed with 10 mL of 0.25 mM DEX 24 h after infiltration and then further incubated for 24 h.

In Vitro Phosphatase Treatment

Aliquots of each Triton X-100–solubilized fraction were treated with 45 units of calf intestine alkaline phosphatase (TaKaRa Bio) in a buffer supplied by the manufacturer at 30°C for 30 to 60 min. Reactions were stopped by adding an SDS-PAGE (Tris/Glycine buffer) sample buffer. To observe mobility shift by phosphorylation, SDS-PAGE was performed using the 8.0% acrylamide gel with an extended running time (constant current 20 mA, 1.5 to 2.0 h), and CDPK1 was detected by immunoblot analysis as described above.

Microscopy

Leaves of transgenic tobacco expressing GFP-tagged version of RSG or CDPK1 (RSG–GFP or CDPK1–GFP, respectively) under the control of the CaMV 35S promoter were observed. Fluorescence signals were investigated using an epifluorescence microscope [ECLIPSE 80i (Nikon) and MZ 16 F (Leica Mycrosystems)]. Images were captured with a CCD camera and exported as TIFF format files and further processed with bit map–based image editing software.

RT-PCR

For quantitative RT-RCR studies, total RNA from the stem of transgenic tobacco expressing CDPK1–His under the control of the CaMV 35S promoter or transgenic tobacco in which expression of CDPK1 was repressed by RNAi or control wild-type SR1 were converted into cDNA with SuperScript II (Invitrogen) and oligo(dT)12-18 (Amersham Bioscience). PCR was performed with cDNA derived from 0.5 μg of total RNA (or 10 ng of genomic DNA as a control) with ExTaq (TaKaRa). The primer sequences were 5′-CACTTTGATAGAAAATGTATCTAAG-3′ and 5′-GCCCATTCTGTGCTCACTACTGTGG-3′ for NtGA20ox and 5′-ATTCTAGAACCATGGCGCAAGAATCACTAGTACTC-3′ and 5′-ATGGATCCATAACGGCCAATACCCCA-3′ for tobacco arcA (Ishida et al., 1993), an internal control for RT-PCR. The PCR was run for 18, 21, and 24 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 90 s to ensure that amplifications were within the linear range. The PCR products were size-separated on a 1% (w/v) agarose gel, blotted onto Biodyne B (Pall), and hybridized with ³²P-labeled gene-specific DNA probes. After overnight hybridization in 6× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate) containing 0.1% SDS at 65°C, blots were washed twice in 2× SSC containing 0.1% SDS at 65°C, and then the radioactive signals were visualized with the phosphor imager as described above.

Uniconazole P Tolerance Assay

Seeds of transgenic tobacco expressing CDPK1–His under the control of the CaMV 35S promoter or control wild-type SR1 were germinated on plates with agarose gel containing 1 μM Uniconazole P with or without GA₃ for 7 d at 28°C. Plates were photographed with a CCD camera, and images were exported and further processed as described as above.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: AB040471 (RSG), AB071967 (14-3-3), AF072908 (Nt CDPK1), AJ344154 (Nt CDPK2), AB012856 (Nt GA20ox), D17526 (arcA), and D10246 (At CKIIA, AT5G67380).