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

Sarahmi Ishida, Takashi Yuasa, Masaru Nakata, Yohsuke Takahashi
Sarahmi Ishida
aDepartment of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
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Takashi Yuasa
bDepartment of Plant Resources, Faculty of Agriculture, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
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Masaru Nakata
cDepartment of Biological Science, Graduate School of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima 739-8526, Japan
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Yohsuke Takahashi
cDepartment of Biological Science, Graduate School of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima 739-8526, Japan
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Published December 2008. DOI: https://doi.org/10.1105/tpc.107.057489

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Abstract

The homeostasis of gibberellins (GAs) is maintained by negative feedback in plants. REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcriptional activator that has been suggested to play a role in GA feedback by the regulation of GA biosynthetic enzymes. The 14-3-3 signaling proteins negatively regulate RSG by sequestering it in the cytoplasm in response to GAs. The phosphorylation on Ser-114 of RSG is essential for 14-3-3 binding of RSG. Here, we identified tobacco Ca2+-dependent protein kinase (CDPK1) as an RSG kinase that promotes 14-3-3 binding to RSG by phosphorylation of Ser-114 of RSG. CDPK1 interacts with RSG in a Ca2+-dependent manner in vivo and in vitro and specifically phosphorylates Ser-114 of RSG. Inhibition of CDPK repressed the GA-induced phosphorylation of Ser-114 of RSG and the GA-induced nuclear export of RSG. Overexpression of CDPK1 inhibited the feedback regulation of a GA 20-oxidase gene and resulted in sensitization to the GA biosynthetic inhibitor. Our results suggest that CDPK1 decodes the Ca2+ signal produced by GAs and regulates the intracellular localization of RSG.

INTRODUCTION

As sessile organisms, plants have acquired developmental plasticity during their evolution to integrate their endogenous program for morphogenesis with the ever-fluctuating environment throughout their life cycle. Plant hormones crucially contribute not only to orchestrate innate transcriptional processes in a spatiotemporal specialized manner but also to transduce exterior environmental stimuli to nuclei. Gibberellins (GAs), which are tetracyclic diterpenoid growth factors, are essential regulators of many aspects of plant development, including seed germination, stem elongation, flower induction, and anther development (Davies, 2004). The endogenous levels of GAs are delicately refined by feedback control at several steps in the metabolic pathway, including GA 20-oxidase, GA 3-oxidase, and GA 2-oxidase. Although GA feedback regulation has been shown to depend on GA signaling components, including DELLA regulators, an F-box adaptor subunit of SCF E3 ligase, SPINDLY, PHOTOPERIOD-RESPONSIVE1 (reviewed in Fleet and Sun, 2005), and a GA receptor (Ueguchi-Tanaka et al., 2005; Griffiths et al., 2006), its molecular mechanisms are still largely unknown.

REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcriptional activator with a basic leucine zipper domain and is involved in the regulation of endogenous amounts of GAs (Fukazawa et al., 2000). A dominant-negative form of RSG repressed the expression of the ent-kaurene oxidase gene of the GA biosynthetic pathway in transformed tobacco plants. This downregulation reduced endogenous amounts of GAs and severely inhibited the process of cell elongation of stems, resulting in a dwarf phenotype. The function of RSG is negatively regulated by 14-3-3 signaling proteins (Igarashi et al., 2001), which form a highly conserved family of homo- and heterodimeric proteins in eukaryotes (reviewed in van Hemert et al., 2001; Tzivion and Avruch, 2002). The 14-3-3 proteins bind to phosphorylated motifs containing phosphoserine residues of RSXpSXP and RXY/FXpSXP (pS indicates a critical phosphoserine) in their target proteins (Yaffe et al., 1997). Through these binding reactions, the 14-3-3 proteins appear to act as molecular scaffolds or chaperones. The biological roles of 14-3-3 complexes have been demonstrated in signal transduction, subcellular targeting, and cell cycle control. The 14-3-3 proteins can also act as allosteric cofactors modulating the catalytic activity of their binding partners. The 14-3-3 proteins bind to RSG depending on the RSG phosphorylation of Ser-114 and thereby sequester RSG in the cytoplasm so that it is unable to regulate its target genes in the nucleus (Igarashi et al., 2001; Ishida et al., 2004). We found that GA levels regulate the intracellular localization of RSG. RSG is translocated into the nucleus in response to a reduction in GA levels (Ishida et al., 2004). GA treatment could reverse this nuclear accumulation. Recently, a similar function of 14-3-3 proteins in regulating intracellular localization has been reported in brassinosteroid signaling. 14-3-3s mediate the brassinosteroid regulation of the nuclear localization of a transcriptional repressor BRASSINAZOLE-RESISTANT1 in a manner similar to the regulation of RSG (Gampala et al., 2007). The GA-dependent nuclear export of RSG requires 14-3-3 binding and Ser/Thr kinase activity (Ishida et al., 2004). However, the understanding of the molecular mechanisms whereby GA regulates the intracellular localization of RSG is still limited. Of particular importance is the identification and characterization of the protein kinase that promotes the association of RSG with 14-3-3 proteins through phosphorylation on Ser-114 of RSG in response to GAs.

One of the fastest known responses to GA is an increase in the concentration of cytosolic Ca2+ (Bush, 1996). Ca2+ is a ubiquitous second messenger that is involved in the signal transduction of many environmental and developmental stimuli in eukaryotes (Sanders et al., 2002; Schuster et al., 2002). In response to diverse internal and external stimuli, cells generate transient increases in the concentration of intracellular free Ca2+ that vary in amplitude, frequency, duration, intracellular location, and timing (Berridge et al., 2000; Allen et al., 2001; Evans et al., 2001; Rudd and Franklin-Tong, 2001). Important information regarding the nature of the stimulus may be encoded in the different spatiotemporal profiles of increases in the concentration of Ca2+ (McAinsh and Hetherington, 1998; Sanders et al., 2002; Schuster et al., 2002). Different Ca2+ sensors recognize specific Ca2+ signatures and bring about changes in metabolism and gene expression (Sanders et al., 1999; Rudd and Franklin-Tong, 2001). Among Ca2+ binding sensory proteins in plants, Ca2+-dependent protein kinases (CDPKs) are thought to play central roles in Ca2+ signaling because protein kinase C and conventional calmodulin-dependent protein kinase (CaMK), which represent the two major types of Ca2+-regulated kinases in animal systems, are missing from Arabidopsis thaliana (Hrabak et al., 2003). CDPKs are Ser/Thr protein kinases that are only found in plants and some protozoans. There are 34 genes encoding CDPKs in Arabidopsis (Arabidopsis Genome Initiative, 2000) and 29 genes in rice (Oryza sativa; Asano et al., 2005). CDPK proteins are composed of a variable N-terminal domain, a catalytic domain, an autoinhibitory region, and a calmodulin-like domain (Harper et al., 1991; Suen and Choi, 1991; Cheng et al., 2002; Hrabak et al., 2003). They are activated upon binding Ca2+ to their calmodulin-like domain, which makes them effective switches for the transduction of Ca2+ signals in plant cells. CDPKs have been reported to be involved in diverse physiological processes, including the accumulation of storage starch and protein in immature seeds of rice (Asano et al., 2002), tolerance to cold, salt, and drought stress in rice (Saijo et al., 2000), a defense response in tobacco (Romeis et al., 2000, 2001), root development and regulation of nodule number in Medicago truncatula (Ivashuta et al., 2005; Gargantini et al., 2006), abscisic acid response in Arabidopsis (Choi et al., 2005), and pollen tube growth in petunia (Petunia hybrida; Yoon et al., 2006). There is evidence that CDPKs are targeted to multiple cellular locations, including the cytosol, nucleus, plasma membrane, endoplasmic reticulum, peroxisomes, mitochondrial outer membrane, and oil bodies (Harper et al., 2004), which suggests that CDPKs participate in the phosphorylation of various proteins. While none of the CDPKs appears to be an integral membrane protein, 24 of the 34 Arabidopsis CDPKs have potential N-myristoylation motifs for membrane association in the beginning of their highly variable N-terminal domain. To understand how CDPKs affect plant physiology, their specific target proteins must be clarified. However, very little is known about the physiological target proteins of CDPKs.

In this study, we identified a CDPK as an RSG kinase that promotes 14-3-3 binding of RSG by phosphorylation of Ser-114 of RSG. Inhibition of CDPK repressed the GA-induced phosphorylation of Ser-114 of RSG and GA-induced nuclear export of RSG. Overexpression of CDPK inhibited the feedback regulation of a GA 20-oxidase gene and resulted in sensitization to a GA biosynthetic inhibitor. Our results suggest that a CDPK decodes the Ca2+ signal produced by GAs and regulates the intracellular localization of RSG.

RESULTS

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.

Figure 1.
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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.

Figure 2.
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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 ).

Figure 3.
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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.

Figure 4.
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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.

Figure 5.
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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.

Figure 6.
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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.

Figure 7.
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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.

Figure 8.
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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.

DISCUSSION

Signaling pathways are complex networks of biochemical reactions that result in the alteration in a gene expression pattern mediated by transcriptional machinery. Sequence-specific transcription factors collectively function as the key interface between genetic information encoded in the DNA sequence and the signal transduction systems in response to internal and external stimuli. Intensive studies have revealed the posttranslational regulation of transcription factors, including covalent modifications, and the interaction with coactivators and general transcription factors (reviewed in Kadonaga, 2004). Phosphorylation is one of the most frequent and important posttranslational modifications of transcription factors. However, the knowledge of protein kinases that directly phosphorylate transcription factors is still limited in plants. In this study, we identified the tobacco Ca2+-dependent protein kinase CDPK1 as an RSG kinase that regulates the intracellular localization of RSG by promoting 14-3-3 binding of RSG in response to GAs.

Phylogenetic analyses have proposed that the CDPK gene family arose through the fusion of a CaMK and a calmodulin (Harper et al., 1991; Cheng et al., 2002). The kinase domain of CDPK1 is 46% identical and 75% similar to that of mouse CaMKII at the amino acid sequence level. The 14-3-3 binding motifs (RSXpSXP and RXY/FXpSXP) overlap with consensus phosphorylation sites for CaMK and CDPK (Pinna and Ruzzene, 1996; Cheng et al., 2002). Histone deacetylase (HDAC) is a corepressor of the transcriptional regulation of eukaryotes (reviewed in Struhl, 1998). Mammalian CaMKII phosphorylates HDAC4 and 5 and promotes nuclear exclusion of HDACs through 14-3-3 binding in skeletal muscle differentiation and cardiac growth, respectively (McKinsey et al., 2000; Little et al., 2007). These molecular mechanisms are quite similar to that of the regulation of RSG in plants. Thus, the nuclear-cytoplasmic partitioning of transcriptional regulatory factors by 14-3-3 proteins depending on Ca2+ signaling appears to be an evolutionarily ancient mechanism or to have evolved in parallel in two different kingdoms.

The Arabidopsis genome encodes 1085 typical protein kinases, ∼4% of the predicted protein-coding genes (Arabidopsis Genome Initiative, 2000). Not only is the proportion of Arabidopsis kinase genes about twice that found in Saccharomyces cerevisiae (Hunter and Plowman, 1997) or Caenorhabditis elegans (Plowman et al., 1999), but there are also major differences in the types of kinases found in plants. For example, receptor kinases in plants phosphorylate Ser and Thr residues, whereas, in animals, the predominant type of receptor kinase phosphorylates Tyr residues. In addition, protein kinase C, a major kinase that decodes Ca2+ signals in animals, appears to be missing in plants. Another family of Ca2+-related kinases, Ca2+ and calmodulin-dependent protein kinases (CCaMK), is found in several plant species, including lily (Lilium longiflorum; Patil et al., 1995), maize (Zea mays; Wang et al., 2001), and rice (Zhang et al., 2002); however, Arabidopsis lacks CCaMK (Harper et al., 2004). Thus, CDPK seems to be a major molecular decoder of Ca2+ signals in plants.

In Arabidopsis, CDPKs comprise a gene family with 34 members. The family members break into four major clades in the phylogenic analysis (Cheng et al., 2002). We performed a systematic analysis on the At CDPKs to examine the RSG kinase activity using recombinant proteins. Our recent results suggested that among all the Arabidopsis CDPKs related to Nt CDPK1, only the Arabidopsis CDPK that is most closely related to Nt CDPK1 bound to RSG and phosphorylated it (M. Nakata and Y. Takahashi, unpublished results). Besides, in this work, we found that specific repression of CDPK1 by RNAi caused a marked reduction in the Ser-114 kinase activity in vitro and in vivo (Figures 4 and 5). The results of our study collectively suggest that tobacco CDPK1 and only its most closely related CDPK in tobacco play a central role in the phosphorylation of Ser-114 on RSG in response to GAs.

We found that the overexpression of CDPK1 inhibits the upregulation of the GA 20-oxidase gene in response to a decrease in GA levels and confers higher sensitivity to a GA biosynthetic inhibitor in transgenic plants (Figure 8). Although CDPK1 knockdown caused a reduction in the RSG kinase activity in the kinase assay (Figure 4) and in vivo (Figure 5), the GA 20-oxidase gene was not obviously upregulated in the transgenic plants (Figure 8A). This observation is likely attributable to residual CDPK1 activity in the knockdown plants or to the functional redundancy among CDPKs. Another possibility is that the upregulation of the GA 20-oxidase gene requires cell signaling events in addition to the nuclear accumulation of RSG in response to a decrease in GA levels in cells.

Transcriptional regulation relies on the collective action of sequence-specific factors along with the core RNA polymerase II transcriptional machinery, an assortment of coregulators that bridge the DNA binding factors to the transcriptional machinery, a number of chromatin-remodeling factors that mobilize nucleosomes, and a variety of enzymes that catalyze the covalent modification of histones and other proteins (reviewed in Kadonaga, 2004). In addition to these transcription-related proteins, recent studies revealed that various proteins, including metabolic enzymes (Zheng et al., 2003; Hall et al., 2004), protein kinases (Pokholok et al., 2006; Proft et al., 2006), and proteins of the nuclear pore complex (Tanaka et al., 2007), physically associate with the regulatory region of the genes. Transcriptional regulation might be more complex and require more proteins than previously supposed. In this study, we found an apparent stable interaction between CDPK1 and transcription factor RSG.

Because the primary structures of CDPK isoforms are very similar, especially within their kinase domains, it was considered unlikely that they would have distinguishable substrate specificities. However, our knockdown study using RNAi uncovered a distinct physiological function of CDPK1 in tobacco plants. A particularly interesting question arises, namely, how RSG is specifically recognized by CDPK1. CDPK proteins possess a variable N-terminal domain, a catalytic domain, an autoinhibitory region, and a calmodulin-like domain (Hrabak et al., 2003). Considering the highly conserved amino acid sequences of a catalytic domain, an autoinhibitory region, and a calmodulin-like domain among CDPK isoforms, a variable N-terminal domain could play an important role in the recognition of the specific substrate. Identification of the RSG-interacting region of CDPK1 would provide an insight into the molecular mechanisms whereby an individual CDPK distinguishes its specific substrates.

We found that CDPK1 is associated with a membrane fraction (Figure 7B). This intracellular localization of CDPK1 raises the question of where transcription factor RSG is phosphorylated by CDPK1. A possible explanation is that CDPK1 phosphorylates RSG in the cytoplasm and promotes the cytoplasmic localization of RSG by facilitating 14-3-3 binding because RSG shuttles continuously between the nucleus and the cytoplasm (Igarashi et al., 2001). Another possibility is that CDPK1 translocates into the nucleus and phosphorylates RSG in response to GAs. Forkhead transcription factors FOXOs of animals are exported from the nucleus to the cytoplasm through phosphorylation by protein kinase Akt and 14-3-3 binding in response to growth factors (Brunet et al., 1999; Manning and Cantley, 2007). In this similar signaling mechanism, Akt is activated at the cell membrane by PI3K (phosphatidylinositol 3-kinase) (reviewed in Engelman et al., 2006) and is believed to enter the nucleus to phosphorylate FOXOs. Although our biochemical studies showed that a change in GA levels did not apparently affect the intracellular localization of CDPK1 (Figure 7B), we cannot rule out the possibility that only a small portion of CDPK1 binds to RSG in the nucleus in response to GAs. Lee et al. (2003) reported a possible nuclear localization of CDPK1. We expect that future studies of the dynamic interaction between CDPK1 and RSG in plant cells using fluorescence resonance energy transfer or bimolecular fluorescence complementation will further our understanding of the detailed molecular mechanisms whereby CDPK1 regulates RSG.

Cell signaling is triggered by the perception of ligands or environmental stimuli and results in specific patterns of nuclear gene expression. Upon GA perception, a soluble GA receptor, GA-INSENSITIVE DWARF1 (GID1), causes the degradation of nuclear DELLA proteins that function as repressors of GA-dependent processes (reviewed in Fleet and Sun, 2005). We found that GAs induced the phosphorylation of Ser-114 of RSG by CDPK1 (Figure 5B). If CDPK1 acts downstream of DELLA proteins in GA signaling, the manner in which CDPK1 associated with the cell membrane is activated by posttranslational mechanisms through the degradation of DELLA regulators in the nucleus would be an interesting issue. In such a case, the disappearance of DELLA proteins would induce increases in the concentration of intracellular free Ca2+ in the cytoplasm. This implies a novel type of retrograde signaling pathway (i.e., from the nucleus to the cytoplasm and not through transcription). An additional possibility is that a trace of CDPK1 and/or its close relative in the nucleus could be activated by the disappearance of DELLA proteins through an increase of Ca2+ in the nucleus or by unknown posttranslational modification. In this context, it would be worth noting that nuclear-localized CaMKII phosphorylates HDAC4 and promotes its cytoplasmic localization through 14-3-3 binding in mammalian cardiac cells (Little et al., 2007). Another possibility is that CDPK1 would be activated by GAs through a DELLA-independent pathway. Although it is unambiguous that the soluble GA receptor GID1-mediated degradation of DELLA proteins is a central regulatory switch in GA signaling, several physiological studies suggested an alternative signaling pathway, that is, the existence of a membrane-localized GA receptor (Hooley et al., 1991; Gilroy and Jones, 1994). Furthermore, genetic study showed the involvement of heterotrimeric G protein in GA signaling (Ueguchi-Tanaka et al., 2000), also suggesting a plasma membrane GA receptor. If CDPK is regulated by a DELLA-independent pathway, the increase of Ca2+ levels in the cytoplasm by GAs could be downstream of a plasma membrane GA receptor. Potato (Solanum tuberosum), PHOTOPERIOD-RESPONSIVE1 (PHOR1) is a component of GA signaling that translocates from the cytoplasm to the nucleus by the application of GAs (Amador et al., 2001). This raises the equally interesting question of whether the GA-dependent nucleocytoplasmic redistribution of PHOR1 is downstream of DELLA proteins. To elucidate the molecular pathway activating CDPK1 in response to GAs, we are examining the relationship between DELLA proteins and CDPK1 using a mutant DELLA protein that is resistant to GA-mediated degradation. Our identification of CDPK1 as an RSG kinase provides an important clue for understanding a new aspect of the GA signaling network.

METHODS

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).

Acknowledgments

We thank Naoto Yabe and Yuri Abe for technical assistance with microscopy observation, Takeshi Ito for technical support, and Tsuyoshi Furumoto for helpful discussions. This study was supported in part by grants from Japan Society for the Promotion of Science to S.I. (17570031) and Y.T. (18657017) and by a grant from Ministry of Education, Culture, Sports, Science and Technology to Y.T. (17054029). S.I. was also supported by the Hayashi Memorial Foundation for Female Natural Scientists and the Asahi Glass Foundation.

Footnotes

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Yohsuke Takahashi (ytakahas{at}hiroshima-u.ac.jp).

  • www.plantcell.org/cgi/doi/10.1105/tpc.107.057489

  • ↵1 These authors contributed equally to this work.

  • Received December 27, 2007.
  • Revised November 8, 2008.
  • Accepted December 4, 2008.
  • Published December 23, 2008.

References

  1. ↵
    Allen, G.J., Chu, S.P., Harrington, C.L., Schumacher, K., Hoffmann, T., Tang, Y.Y., Grill, E., and Schroeder, J.I. (2001). A defined range of guard cell calcium oscillation parameters encodes stomatal movements. Nature 411: 1053–1057.
    OpenUrlCrossRefPubMed
  2. ↵
    Amador, V., Monte, E., García-Martínez, J.-L., and Prat, S. (2001). Gibberellins signal nuclear import of PHOR1, a photoperiod-responsive protein with homology to Drosophila armadillo. Cell 106: 343–354.
    OpenUrlCrossRefPubMed
  3. ↵
    Aoyama, T., and Chua, N.-H. (1997). A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J. 11: 605–612.
    OpenUrlCrossRefPubMed
  4. ↵
    Arabidopsis Genome Initiative (2000). Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408: 796–815.
    OpenUrlCrossRefPubMed
  5. ↵
    Asano, T., et al. (2002). Rice SPK, a calmodulin-like domain protein kinase, is required for storage product accumulation during seed development: phosphorylation of sucrose synthase is a possible factor. Plant Cell 14: 619–628.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Asano, T., Tanaka, N., Yang, G., Hayashi, N., and Komatsu, S. (2005). Genome-wide identification of the rice calcium-dependent protein kinase and its closely related kinase gene families: Comprehensive analysis of the CDPKs gene family in rice. Plant Cell Physiol. 46: 356–366.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Berridge, M.J., Lipp, P., and Bootman, M.D. (2000). The versatility and universality of calcium signaling. Nat. Rev. Mol. Cell Biol. 1: 11–21.
    OpenUrlCrossRefPubMed
  8. ↵
    Brunet, A., Bonni, A., Zigmond, M.J., Lin, M.Z., Juo, P., Hu, L.S., Anderson, M.J., Arden, K.C., Blenis, J., and Greenberg, M.E. (1999). Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96: 857–868.
    OpenUrlCrossRefPubMed
  9. ↵
    Bush, D.S. (1996). Effects of gibberellic acid and environmental factors on cytosolic calcium in wheat aleurone cells. Planta 199: 89–99.
    OpenUrl
  10. ↵
    Chawla, S., Hardingham, G.E., Quinn, D.R., and Bading, H. (1998). CBP: A signal-regulated transcriptional coactivator controlled by nuclear calcium and CaM kinase IV. Science 281: 1505–1509.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Cheng, S.-H., Willmann, M.R., Chen, H.-C., and Sheen, J. (2002). Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family. Plant Physiol. 129: 469–485.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Choi, H., Park, H.J., Park, J.H., Kim, S., Im, M.Y., Seo, H.H., Kim, Y.W., Hwang, I., and Kim, S.Y. (2005). Arabidopsis calcium-dependent protein kinase AtCPK32 interacts with ABF4, a transcriptional regulator of abscisic acid-responsive gene expression, and modulates its activity. Plant Physiol. 139: 1750–1761.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Chiu, W.-I., Niwa, Y., Zeng, W., Hirano, T., Kobayashi, H., and Sheen, J. (1996). Engineered GFP as a vital reporter in plants. Curr. Biol. 6: 325–330.
    OpenUrlCrossRefPubMed
  14. ↵
    Davies, P.J., ed. (2004). Plant Hormones: Biosynthesis, Signal Transduction, Action! (Dordrecht, The Netherlands: Kluwer Academic Publishers).
  15. ↵
    Engelman, J.A., Luo, J., and Cantley, L.C. (2006). The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat. Rev. Genet. 7: 606–619.
    OpenUrlCrossRefPubMed
  16. ↵
    Evans, N.H., McAinsh, M.R., and Hetherington, A.M. (2001). Calcium oscillations in higher plants. Curr. Opin. Plant Biol. 4: 415–420.
    OpenUrlCrossRefPubMed
  17. ↵
    Firulli, B.A., Howard, M.J., McDaid, J.R., McIlreavey, L., Dionne, K.M., Centonze, V.E., Cserjesi, P., Virshup, D.M., and Firulli, A.B. (2003). PKA, PKC, and the protein phosphatase 2A influence HAND factor function: A mechanism for tissue-specific transcriptional regulation. Mol. Cell 12: 1225–1237.
    OpenUrlCrossRefPubMed
  18. ↵
    Fleet, C.M., and Sun, T.-p. (2005). A DELLAcate balance: The role of gibberellin in plant morphogenesis. Curr. Opin. Plant Biol. 8: 77–85.
    OpenUrlCrossRefPubMed
  19. ↵
    Fukazawa, J., Sakai, T., Ishida, S., Yamaguchi, I., Kamiya, Y., and Takahashi, Y. (2000). REPRESSION OF SHOOT GROWTH, a bZIP transcriptional activator, regulates cell elongation by controlling the level of gibberellins. Plant Cell 12: 901–915.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Gampala, et al. (2007). An essential role for 14-3-3 proteins in Brassinosteroid signal transduction in Arabidopsis. Dev. Cell 13: 177–189.
    OpenUrlCrossRefPubMed
  21. ↵
    Gargantini, P.R., Gonzalez-Rizzo, S., Chinchilla, D., Raices, M., Giammaria, V., Ulloa, R.M., Frugier, F., and Crespi, M.D. (2006). A CDPK isoform participates in the regulation of nodule number in Medicago truncatula. Plant J. 48: 843–856.
    OpenUrlCrossRefPubMed
  22. ↵
    Gilroy, S., and Jones, R.L. (1994). Perception of gibberellin and abscisic acid at the external face of the plasma membrane of barley (Hordeum vulgare L.) aleurone protoplasts. Plant Physiol. 104: 1185–1192.
    OpenUrlAbstract
  23. ↵
    Griffiths, J., Murase, K., Rieu, I., Zentella, R., Zhang, Z.-L., Powers, S.J., Gong, F., Phillips, A.L., Hedden, P., Sun, T.-p., and Thomas, S.G. (2006). Genetic characterization and functional analysis of the GID1 gibberellin receptors in Arabidopsis. Plant Cell 18: 3399–3414.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Hall, D.A., Zhu, H., Zhu, X., Royce, T., Gerstein, M., and Snyder, M. (2004). Regulation of gene expression by a metabolic enzyme. Science 306: 482–484.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Harper, J.F., Breton, G., and Harmon, A. (2004). Decoding Ca2+ signals through plant protein kinases. Annu. Rev. Plant Biol. 55: 263–288.
    OpenUrlCrossRefPubMed
  26. ↵
    Harper, J.F., and Harmon, A. (2005). Plants, symbiosis and parasites: A calcium signaling connection. Nat. Rev. Mol. Cell Biol. 6: 555–566.
    OpenUrlCrossRefPubMed
  27. ↵
    Harper, J.F., Sussman, M.R., Schaller, G.E., Putnam-Evans, C., Charbonneau, H., and Harmon, A.C. (1991). A calcium-dependent protein kinase with a regulatory domain similar to calmodulin. Science 252: 951–954.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Hedden, P., and Phillips, A.L. (2000). Gibberellin metabolism: New insights revealed by the genes. Trends Plant Sci. 5: 523–530.
    OpenUrlCrossRefPubMed
  29. ↵
    Hooley, R., Beale, M.H., and Smith, S.J. (1991). Gibberellin perception at the plasma membrane of Avena fatua aleurone protoplasts. Planta 183: 274–280.
    OpenUrlPubMed
  30. ↵
    Hrabak, E.M., et al. (2003). The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol. 132: 666–680.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Hunter, T., and Plowman, G.D. (1997). The protein kinases of budding yeast: Six score and more. Trends Biochem. Sci. 22: 18–22.
    OpenUrlCrossRefPubMed
  32. ↵
    Igarashi, D., Ishida, S., Fukazawa, J., and Takahashi, Y. (2001). 14-3-3 proteins regulate intracellular localization of the bZIP transcriptional activator RSG. Plant Cell 13: 2483–2497.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Ishida, S., Fukazawa, J., Yuasa, T., and Takahashi, Y. (2004). Involvement of 14-3-3 signaling protein binding in the functional regulation of the transcriptional activator REPRESSION OF SHOOT GROWTH by gibberellins. Plant Cell 16: 2641–2651.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Ishida, S., Takahashi, Y., and Nagata, T. (1993). Isolation of cDNA of an auxin-regulated gene encoding a G protein β subunit-like protein from tobacco BY-2 cells. Proc. Natl. Acad. Sci. USA 90: 11152–11156.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Ivashuta, S., Liu, J., Lohar, D.P., Haridas, S., Bucciarelli, B., Vandenbosch, K.A., Vance, C.P., Harrison, M.J., and Gantt, J.S. (2005). RNA interference identifies a calcium-dependent protein kinase involved in Medicago truncatula root development. Plant Cell 17: 2911–2921.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Jyothishwaran, G., Kotresha, D., Selvaraj, T., Srideshikan, S.M., Rajvanshi, P.K., and Jayabaskaran, C. (2007). A modified freeze–thaw method for efficient transformation of Agrobacterium tumefaciens. Curr. Sci. 93: 770–772.
    OpenUrl
  37. ↵
    Kadonaga, J.T. (2004). Regulation of RNA polymerase II transcription by sequence-specific DNA binding factors. Cell 116: 247–257.
    OpenUrlCrossRefPubMed
  38. ↵
    Kameshita, I., and Fujisawa, H. (1989). A sensitive method for detection of calmodulin-dependent protein kinase II activity in sodium dodecyl sulfate-polyacrylamide gel. Anal. Biochem. 183: 139–143.
    OpenUrlCrossRefPubMed
  39. ↵
    Kumar, P.P., Purbey, P.K., Sinha, C.K., Notani, D., Limaye, A., Jayani, R.S., and Galande, S. (2006). Phosphorylation of SATB1, a global gene regulator, acts as a molecular switch regulating its transcriptional activity in vivo. Mol. Cell 22: 231–243.
    OpenUrlCrossRefPubMed
  40. ↵
    Lee, S.S., Cho, H.S., Yoon, G.M., Ahn, J.W., Kim, H.H., and Pai, H.S. (2003). Interaction of NtCDPK1 calcium-dependent protein kinase with NtRpn3 regulatory subunit of the 26S proteasome in Nicotiana tabacum. Plant J. 33: 825–840.
    OpenUrlCrossRefPubMed
  41. ↵
    Little, G.H., Bai, Y., Williams, T., and Poizat, C. (2007). Nuclear calcium/calmodulin-dependent protein kinase II preferentially transmits signals to histone deacetylase 4 in cardiac cells. J. Biol. Chem. 282: 7219–7231.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Liu, B., Yang, Y., Chernishof, V., Loo, R., Jang, H., Tahk, S., Yang, R., Mink, S., Shultz, D., and Bellone, C. (2007). Proinflammatory stimuli induce IKKα-mediated phosphorylation of PIAS1 to restrict inflammation and immunity. Cell 129: 903–914.
    OpenUrlCrossRefPubMed
  43. ↵
    Manning, B.D., and Cantley, L.C. (2007). AKT/PKB signaling: Navigating downstream. Cell 129: 1261–1274.
    OpenUrlCrossRefPubMed
  44. ↵
    Margolis, S.S., et al. (2006). Role for the PP2A/B56δ phosphatase in regulating 14-3-3 release from Cdc25 to control mitosis. Cell 127: 759–773.
    OpenUrlCrossRefPubMed
  45. ↵
    Matsushita, A., Furumoto, T., Ishida, S., and Takahashi, Y. (2007). AGF1, an AT-hook protein, is necessary for the negative feedback of AtGA3ox1 encoding GA 3-oxidase. Plant Physiol. 143: 1152–1162.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    McAinsh, M.R., and Hetherington, A.M. (1998). Encoding specificity in Ca2+ signaling systems. Trends Plant Sci. 3: 32–36.
    OpenUrlCrossRef
  47. ↵
    McKinsey, T.A., Zhang, C.L., and Olson, E.N. (2000). Activation of the myocyte enhancer factor-2 transcription factor by calcium/calmodulin-dependent protein kinase-stimulated binding of 14-3-3 to histone deacetylase 5. Proc. Natl. Acad. Sci. USA 97: 14400–14405.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    Mizoguchi, T., Yamaguchi-Shinozaki, K., Hayashida, N., Kamada, H., and Shinozaki, K. (1993). Cloning and characterization of two cDNAs encoding casein kinase II catalytic subunits in Arabidopsis thaliana. Plant Mol. Biol. 21: 279–289.
    OpenUrlCrossRefPubMed
  49. ↵
    Park, S.-H., Zarrinpar, A., and Lim, W.A. (2003). Rewiring MAP kinase pathways using alternative scaffold assembly mechanisms. Science 299: 1061–1064.
    OpenUrlAbstract/FREE Full Text
  50. ↵
    Patil, S., Takezawa, D., and Poovaiah, B.W. (1995). Chimeric plant calcium/calmodulin-dependent protein-kinase gene with a neural visinin-like calcium-binding domain. Proc. Natl. Acad. Sci. USA 92: 4897–4901.
    OpenUrlAbstract/FREE Full Text
  51. ↵
    Pinna, L.A., and Ruzzene, M. (1996). How do protein kinases recognize their substrates? Biochim. Biophys. Acta 1314: 191–225.
    OpenUrlCrossRefPubMed
  52. ↵
    Plowman, G.D., Sudarsanam, S., Bingham, J., Whyte, D., and Hunter, T. (1999). The protein kinases of Caenorhabditis elegans: A model for signal transduction in multicellular organisms. Proc. Natl. Acad. Sci. USA 96: 13603–13610.
    OpenUrlAbstract/FREE Full Text
  53. ↵
    Pokholok, D.K., Zeitlinger, J., Hannett, N.M., Reynolds, D.B., and Young, R.A. (2006). Activated signal transduction kinases frequently occupy target genes. Science 313: 533–536.
    OpenUrlAbstract/FREE Full Text
  54. ↵
    Posas, F., and Saito, H. (1997). Osmotic activation of the HOG MAPK pathway via ste11p MAPKKK: Scaffold role of Pbs2p MAPKK. Science 276: 1702–1705.
    OpenUrlAbstract/FREE Full Text
  55. ↵
    Proft, M., Mas, G., de Nadal, E., Vendrell, A., Noriega, N., Struhl, K., and Posas, F. (2006). The stress-activated Hog1 kinase is a selective transcriptional elongation factor for genes. Responding to osmotic stress. Mol. Cell 23: 241–250.
    OpenUrlCrossRefPubMed
  56. ↵
    Romeis, T., Ludwig, A.A., Martin, R., and Jones, J.D.G. (2001). Calcium-dependent protein kinases play an essential role in a plant defense response. EMBO J. 20: 5556–5567.
    OpenUrlAbstract/FREE Full Text
  57. ↵
    Romeis, T., Piedras, P., and Jones, J.D.G. (2000). Resistance gene-dependent activation of a calcium-dependent protein kinase in the plant defense response. Plant Cell 12: 803–815.
    OpenUrlAbstract/FREE Full Text
  58. ↵
    Rudd, J.J., and Franklin-Tong, V.E. (2001). Unraveling response-specificity in Ca2+ signaling pathways in plant cells. New Phytol. 151: 7–33.
    OpenUrlCrossRef
  59. ↵
    Saijo, Y., Hata, S., Kyozuka, J., Shimamoto, K., and Izui, K. (2000). Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants. Plant J. 23: 319–327.
    OpenUrlCrossRefPubMed
  60. ↵
    Sanders, D., Brownlee, C., and Harper, J.F. (1999). Communicating with calcium. Plant Cell 11: 691–706.
    OpenUrlFREE Full Text
  61. ↵
    Sanders, D., Pelloux, J., Brownlee, C., and Harper, J.F. (2002). Calcium at the crossroads of signaling. Plant Cell 14: S401–S417.
    OpenUrlFREE Full Text
  62. ↵
    Schuster, S., Marhl, M., and Hofer, T. (2002). Modeling of simple and complex calcium oscillations. From single-cell responses to intercellular signaling. Eur. J. Biochem. 269: 1333–1355.
    OpenUrlPubMed
  63. ↵
    Sebastià, C.H., Harden, S.C., Clouse, S.D., Kieber, J.J., and Huber, S.C. (2004). Identification of a new motif for CDPK phosphorylation in vitro that suggests ACC synthase may be a CDPK substrate. Arch. Biochem. Biophys. 428: 81–91.
    OpenUrlCrossRefPubMed
  64. ↵
    Song, C.-P., Agarwal, M., Ohta, M., Guo, Y., Halfter, U., Wang, P., and Zhu, J.-K. (2005). Role of an Arabidopsis AP2/EREBP-type transcriptional repressor in abscisic acid and drought stress responses. Plant Cell 17: 2384–2396.
    OpenUrlAbstract/FREE Full Text
  65. ↵
    Struhl, K. (1998). Histone acetylation and transcriptional regulatory mechanisms. Genes Dev. 12: 599–606.
    OpenUrlFREE Full Text
  66. ↵
    Suen, K.L., and Choi, J.H. (1991). Isolation and sequence analysis of a cDNA clone for a carrot calcium-dependent protein kinase: Homology to calcium/calmodulin-dependent protein kinases and to calmodulin. Plant Mol. Biol. 17: 581–590.
    OpenUrlCrossRefPubMed
  67. ↵
    Tanaka, T., Ohkubo, S., Tatsuno, I., and Prives, C. (2007). hCAS/CSE1L associates with chromatin and regulates expression of select p53 target genes. Cell 130: 638–650.
    OpenUrlCrossRefPubMed
  68. ↵
    Tzivion, G., and Avruch, J. (2002). 14-3-3 proteins: Active cofactors in cellular regulation by serine/threonine phosphorylation. J. Biol. Chem. 277: 3061–3064.
    OpenUrlFREE Full Text
  69. ↵
    Ueguchi-Tanaka, M., Ashikari, M., Nakajima, M., Itoh, H., Katoh, E., Kobayashi, M., Chow, T.Y., Hsing, Y.I., Kitano, H., Yamaguchi, I., and Matsuoka, M. (2005). GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin. Nature 437: 693–698.
    OpenUrlCrossRefPubMed
  70. ↵
    Ueguchi-Tanaka, M., Fujisawa, Y., Kobayashi, M., Ashikari, M., Iwasaki, Y., Kitano, H., and Matsuoka, M. (2000). Rice dwarf mutant d1, which is defective in the subunit of the heterotrimeric G protein, affects gibberellin signal transduction. Proc. Natl. Acad. Sci. USA 97: 11638–11643.
    OpenUrlAbstract/FREE Full Text
  71. ↵
    Van de Hoorn, R.A.L., Laurent, F., Roth, R., and De Wit, P.J.G.M. (2000). Agroinfiltration is a versatile tool that facilitates comparative analyses of Avr 9/Cf-9-induced and Avr 4/Cf-4-induced necrosis. Mol. Plant Microbe Interact. 13: 439–446.
    OpenUrlCrossRefPubMed
  72. ↵
    van Hemert, M.J., Steensma, H.Y., and van Heusden, G.P. (2001). 14-3-3 proteins: Key regulators of cell division, signaling and apoptosis. Bioessays 23: 936–946.
    OpenUrlCrossRefPubMed
  73. ↵
    Wang, L., Liang, S., and Lu, Y.-T. (2001). Characterization, physical location and expression of the genes encoding calcium/calmodulin-dependent protein kinases in maize (Zea mays L.). Planta 213: 556–564.
    OpenUrlCrossRefPubMed
  74. ↵
    Yaffe, M.B., Rittinger, K., Volinia, S., Caron, P.R., Aitken, A., Leffers, H., Gamblin, S.J., Smerdon, S.J., and Cantley, L.C. (1997). The structural basis for 14-3-3: Phosphopeptide binding specificity. Cell 91: 961–971.
    OpenUrlCrossRefPubMed
  75. ↵
    Yang, Y., Li, R., and Qi, M. (2000). In vivo analysis of plant promoters and transcription factors by agroinfiltration. Plant J. 22: 543–551.
    OpenUrlCrossRefPubMed
  76. ↵
    Yoon, G.M., Cho, H.S., Ha, H.J., Liu, J.R., and Lee, H.S. (1999). Characterization of NtCDPK1, a calcium-dependent protein kinase gene in Nicotiana tabacum, and the activity of its encoded protein. Plant Mol. Biol. 39: 991–1001.
    OpenUrlCrossRefPubMed
  77. ↵
    Yoon, G.M., Dowd, P.E., Gilroy, S., and McCubbin, A.G. (2006). Calcium-dependent protein kinase isoforms in petunia have distinct functions in pollen tube growth, including regulating polarity. Plant Cell 18: 867–878.
    OpenUrlAbstract/FREE Full Text
  78. ↵
    Yuasa, T., Sugiki, M., and Watanabe, Y. (2005). Activation of SIPK in response to UV-C irradiation, utility of a glutathione-S transferase-tagged plant MAP kinase by transient expression with agroinfiltration. Plant Biotechnol. 22: 7–12.
    OpenUrlCrossRef
  79. ↵
    Zhang, L., Liu, B.F., Liang, S., Jones, R.L., and Lu, Y.T. (2002). Molecular and biochemical characterization of a calcium/calmodulin-binding protein kinase from rice. Biochem. J. 368: 145–157.
    OpenUrlCrossRefPubMed
  80. ↵
    Zheng, L., Roeder, R.G., and Luo, Y. (2003). S phase activation of the histone H2B promoter by OCA-S, a coactivator complex that contains GAPDH as a key component. Cell 114: 255–266.
    OpenUrlCrossRefPubMed
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A Tobacco Calcium-Dependent Protein Kinase, CDPK1, Regulates the Transcription Factor REPRESSION OF SHOOT GROWTH in Response to Gibberellins
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A Tobacco Calcium-Dependent Protein Kinase, CDPK1, Regulates the Transcription Factor REPRESSION OF SHOOT GROWTH in Response to Gibberellins
Sarahmi Ishida, Takashi Yuasa, Masaru Nakata, Yohsuke Takahashi
The Plant Cell Dec 2008, 20 (12) 3273-3288; DOI: 10.1105/tpc.107.057489

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A Tobacco Calcium-Dependent Protein Kinase, CDPK1, Regulates the Transcription Factor REPRESSION OF SHOOT GROWTH in Response to Gibberellins
Sarahmi Ishida, Takashi Yuasa, Masaru Nakata, Yohsuke Takahashi
The Plant Cell Dec 2008, 20 (12) 3273-3288; DOI: 10.1105/tpc.107.057489
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The Plant Cell Online: 20 (12)
The Plant Cell
Vol. 20, Issue 12
December 2008
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