Expression and localization of calcium-dependent protein kinase isoforms in chickpea

ARTICLE IN PRESS Journal of Plant Physiology 163 (2006) 1135—1149

www.elsevier.de/jplph

Expression and localization of calcium-dependent protein kinase isoforms in chickpea S.R. Syam Prakash, Chelliah Jayabaskaran Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India Received 13 February 2006; accepted 5 April 2006

KEYWORDS Calcium-dependent protein kinase isoforms; Chloroplast membrane; Immunolocalization; Real-time quantitative PCR; Stresses

Summary Calcium-dependent protein kinases (CPKs) play important roles in multiple signal transduction pathways but the precise role of individual CPK is largely unknown. We isolated two cDNAs encoding two CPK isoforms (Cicer arietinum CPKs—CaCPK1 and CaCPK2) of chickpea. Their expression in various organs and in response to various phytohormones, and dehydration, high salt stress and fungal spore in excised leaves as well as localization in leaf and stem tissues were analyzed in this study. CaCPK1 protein and its activity were ubiquitous in all tissues examined. In contrast, CaCPK2 transcript, CaCPK2 protein and its activity were almost undetectable in flowers and fruits. Both CaCPK1 and CaCPK2 transcripts and proteins were abundant in roots but in minor quantities in leaves and stems. Of the three phytohormones tested, viz. indole-3-acetic acid (IAA), gibberellin (GA3) and benzyladenine (BA), only BA increased both CaCPK1 and CaCPK2 transcripts, proteins and their activities. GA3 induced accumulation of CaCPK2 transcript and protein but CaCPK1 remained unaffected. The expression of CaCPK1 and CaCPK2 in leaves was enhanced in response to high salt stress. Treatments with Aspergillus sp. spores increased expression of CaCPK1 in chickpea leaf tissue but had no effect on CaCPK2. Excised leaves subjected to dehydration showed increase in CaCPK2 expression but not in CaCPK1. Both isoforms were located in the plasma membrane (PM) and chloroplast membrane of leaf mesophyll cells as well as in the PM of stem xylem parenchyma cells. These results suggest specific roles for CaCPK isoforms in phytohormone/ defense/stress signaling pathways. & 2006 Elsevier GmbH. All rights reserved.

Abbreviations: ABA, abscisic acid; AtCPK, Arabidopsis thaliana CPK; BA, benzyladenine; CaCPK, Cicer arietinum CPK; CPK, calciumdependent protein kinase; FITC, fluorescein isothiocyanate; GA, gibberellin; IAA, indole-3-acetic acid; NtCPK, Nicotiana tabacum CPK; OsCPK, Oryza sativa CPK; PBS, phosphate-buffered saline; PM, plasma membrane; VrCPK, Vigna radiata CPK Corresponding author. Tel.: +91 80 22932482; fax: +91 80 3341814/3341683. E-mail address: [email protected] (C. Jayabaskaran). 0176-1617/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2006.04.002

ARTICLE IN PRESS 1136

Introduction In plants, calcium-dependent protein kinases (CPKs) are key intermediates in calcium-mediated signaling that couple changes in Ca2+ levels to a specific response. These enzymes are activated through calcium binding at the calmodulin-like domain and require only micromolar concentrations of free calcium for their activity (Harmon et al., 1986; Harper et al., 1991; Roberts and Harmon, 1992). Activated CPKs alter protein phosphorylation or relative gene expression through transduction of calcium signals (Cheng et al., 2002). The biological properties and locations of different CPKs are proposed to determine their precise cellular activities including defense/stress responses. Moreover, it is not known how specific calcium activation of particular CPK is regulated upon stress and other signals and whether it is regulated in a tissues-specific manner. Plant CPKs are encoded by a large gene family, with 12 subfamilies comprising 34 CPK isoforms in Arabidopsis and 27 in rice (Cheng et al., 2002; Harper et al., 2004). It has been shown that CPKs are found in multiple subcellular locations (Putnam-Evans et al., 1990; Schaller et al., 1992; Pical et al., 1993; Anil et al., 2003; Dammann et al., 2003; Kumar and Jayabaskaran, 2004). The multiple subcellular locations and large number of genes indicate that individual CPK isoforms have different functions and participate in distinct signaling pathways. Recent studies have begun to clarify the specific roles of individual CPK isoforms in signal transduction pathways involving phytohormones and in response to environmental stresses and pathogen attacks. It has been shown in different plant species that treatments with abiotic and biotic stress compounds and phytohormones induce kinase activity and/or gene transcription of various CPKs. For example, in Arabidopsis cold, salinity and drought stresses cause an increase in the transcripts encoding Arabidopsis thaliana CPKs AtCPK10 and AtCPK11 (Urao et al., 1994). In rice, low temperatures have shown to activate a membrane-bound CPK, which suggest a role for this enzyme in adaptation to low temperatures (Martin and Busconi, 2001). Saijo et al. (2000, 2001) demonstrated that overexpression of (Oryza sativa CPK) OsCPK7 protein in transgenic rice plants increases the tolerance of cold, salinity and drought. There is little information concerning the expression of CPK genes, accumulation of their proteins and corresponding enzymatic activities in response to stress and phytohormone signals and most of the data are related to changes in mRNA

S.R. Syam Prakash, C. Jayabaskaran levels. It is not known whether or not the increases in mRNA levels are accompanied by increase in protein levels and/or kinase activities. There are a few reports describing activation of kinase activities of CPKs during stress (Romeis et al., 2001; Chico et al., 2002) or treatment with phytohormones (Abbasi et al., 2004). Furthermore, the expression patterns or activity changes of some CPKs have suggested functional roles in stress- and phytohormone-induced responses. Recently, we isolated and characterized two cDNA clones from chickpea, coding for two isoforms of CPKs (Cicer arietinum CPKs) CaCPK1 and CaCPK2, respectively (Prakash and Jayabaskaran, 2006). In order to get insights on how these two CPK isoforms are regulated and which pathways trigger the expression of these CPKs, we analyzed their mRNA and protein levels as well as their kinase activities by using quantitative real-time RT-PCR analysis, isoform-specific antibodies and measurements of kinase activities, respectively, in response to salinity, dehydration stress, fungal spore and phytohormone treatments. We present the evidence that the expression of CaCPK1 mRNA, protein and kinase activity are rapidly induced by salt (NaCl) stress or by fungal spore treatment but not by dehydration whereas CaCPK2 by dehydration and not by salt (NaCl) or by fungal spore. These findings are discussed in terms of possible involvement of CaCPK1 in the salt- and fungal sporeinduced signal transduction pathways and CaCPK2 in the dehydration-induced signal transduction pathway. Moreover, immunohistochemical experiments using isoform-specific antibodies were performed to analyze cellular and subcellular localizations of both isoforms in chickpea leaves and stems. Our results show that both isoforms are present in the plasma membrane (PM) and chloroplast membrane of leaf mesophyll cells, and PM of xylem parenchyma cells. This study is the first report of localization of CPKs in chloroplast.

Materials and methods Plant growth conditions, and phytohormone and stress treatments Chickpea (Cicer arietinum L. cv. Kabuli) plants were grown in soil in a green house in 70% relative humidity in 16 h of light (25 1C) and 8 h of dark (20 1C) cycle. Leaves, stems, roots, flowers and fruits were collected from 75-d-old mature plants and used for immunolocalization and organ-specific expression studies. For various hormonal and stress

ARTICLE IN PRESS Subcellular localization and differential expression of CaCPK1 and CaCPK2 treatments, leaf tissues from 10-d-old seedlings were used. For hormone experiments, excised leaves were preincubated in sterile Petri dishes containing sterile distilled water for a period of 24 h to account for the stress responses caused by excision and also to deplete the endogenous phytohormones. Subsequent to the preincubation, separate sets of leaves were treated exogenously with 100 mM each of benzyladenine (BA), indole-3acetic acid (IAA) or gibberellin (GA3) in a room for 24 h with 12 h light/12 h dark cycle at 26 1C. For salt stress treatment, leaves were treated with 100 mM NaCl. Placing the excised leaves on Whatman 3 MM filter paper imposed water stress. For fungal spore treatment, excised leaves were preincubated in Murashige and Skoog (MS) liquid medium (Murashige and Skoog, 1962) for 24 h and transferred to MS medium containing Aspergillus conidial suspension (6
104 spores/mL). Leaves were collected at different intervals after various phytohormone and stress treatments (as indicated in figure legends), frozen in liquid nitrogen and stored at 70 1C until use.

Real-time PCR Total RNA from different chickpea tissues (stems, leaves, roots, flowers and fruits) or from leaves subjected to various treatments was isolated with TRIZOL reagent (Life Technologies, USA) following the manufacture’s instructions. Complementary DNA was synthesized using 1 mg total RNA incubated with an oligo (dT) primer followed by a 20-mL reverse transcription reaction with 200 U M-MuLV reverse transcriptase (Fermentas). Conditions used were 37 1C for 5 min, 42 1C for 60 min and 70 1C for 10 min. Real-time PCR reactions were run on a DNA Engine OpticonTM using QuantiTectTM SYBR Green 1 dye and the primer pairs 50 -AGAACAAACCTACGCCTGAT-30 (forward primer) and 50 -AGAGTGGTACTGGTGATTATTAGTG-30 (reverse primer) for CaCPK1, 50 GAAAATCCCTTCGCAATCGA-30 (forward primer) and 50 -CCTTCAAAACGGTGAGTTTTGA-30 (reverse primer) for CaCPK2 and 50 -ATTGTCTTGAGTGGTGGTTCT-30 (forward primer) and 50 -TTCCTCTCTGGTGGTGCTAC-30 (reverse primer) for actin. These primer pairs amplify fragments of 100, 70 and 120 bp for CaCPK1, CaCPK2 and actin, respectively. The expression of CaCPK1 and CaCPK2 mRNA was normalized to the expression of actin mRNA. The PCR conditions were 95 1C for 15 min, followed by 40 cycles of 95 1C for 15 s, 60 1C for 30 s, and 72 1C for 30 s. For each real-time PCR, 25 mL of a mixture containing 2 mL of RT product, 12.5 mL of 2
QuantiTect SYBRs PCR master mix and 2 mL each

1137

forward and reverse primers at a concentration of 10 mM was used. A negative control lacking RT enzyme was also included in each assay. All reactions were performed in triplicate. Following the PCR, a melting curve analysis was performed. Ct or threshold cycle was used for relative quantification of the input target number. Relative fold difference (N) is the number of treated target gene copies relative to the untreated control gene copies determined as N ¼ 2DDCt ¼ 2(DCt treated–DCt control) , where DDCt ¼ DCt of the treated sample minus DCt of the untreated control sample and DCt is the difference in threshold cycles for the CaCPK1 and CaCPK2 targets and the actin internal reference.

Expression and production of polyclonal antibodies against the variable domains of CaCPK1 and CaCPK2 To prepare isoform-specific antibodies, the variable regions of CaCPK1 (GenBank accession ]AY312268) (1–222 bp) and CaCPK2 (GenBank accession ]AY312269) (1–198 bp) corresponding to amino acid residues of 1–74 and 1–66 were RTPCR amplified using total RNA isolated from 10-dold seedling leaf tissues by specific oligonucleotide primers. Primers for CaCPK1 were 50 -ATGGGTTGTCAAGGTAGCAAGG-30 (forward primer) and 50 GTGAACTGTTTTGACATTCTGG-30 (reverse primer) and the primers for CaCPK2 were 50 -ATGGGTAATTGTTGCGCTACCCCTC-30 (forward primer) and 50 -ACGACCTAGCTCGTATCG-30 (reverse primer). The RT-PCR products were cloned into the pQE30 UA expression vector (Qiagen) fused in frame with His6 under the control of phage T5 promoter to give recombinant pQECaCPK1-var and pQECaCPK2-var plasmids. Both the PCR products and the insert DNAs of the expression constructs were sequenced for verification of sequences and orientation of the inserts. The fusion proteins were expressed in Escherichia coli M15 cells and purified by Ni-NTA resin columns. The concentration and purity of the fusion proteins were determined using the method of Bradford (1976) and SDS-PAGE (Laemmli, 1970). The purified His-tagged fusion proteins were used to immunize rabbits using a standard protocol as described (Kumar and Jayabaskaran, 2004) to obtain CaCPK isoform-specific antibodies.

Total protein extraction Total protein was extracted from chickpea tissue samples according to the method of Kumar and Jayabaskaran (2004). Individual frozen chickpea

ARTICLE IN PRESS 1138 tissues were separately ground into a fine powder in liquid nitrogen using a mortar and pestle. The powder was extracted with a buffer containing 10 mM Tris-HCl (pH 7.2), 5 mM NaCl, 1 mM EDTA and 0.1% (v/v) Triton X-100 (3 mL/g fresh weight) and centrifuged at 15,000g for 20 min at 4 1C to remove the insoluble material. The total protein in the supernatant was estimated by the method of Bradford (1976) using BSA as a standard.

Protein gel blot analysis Approximately 10 mg of purified fusion CaCPK1var and CaCPK2-var proteins or 30 mg of total proteins extracted from chickpea various tissue samples were separated on 10% (w/v) SDS-PAGE and analyzed by protein gel blot analysis (Towbin et al., 1979) using (1:5000) either isoform-specific antiCaCPK1-var serum or anti-CaCPK2-var serum.

Immunoprecipitation and immunocomplex kinase assay For immunoprecipitation, 150 mg of the chickpea total protein extract in 450 mL of immunoprecipitation buffer [10 mM Tris-HCl (pH 7.2), 5 mM NaCl, 1 mM EDTA, 0.1% (v/v) Triton X-100 and 1 mM PMSF] was incubated with 20 mL of protein A-sepharose for 2 h at 4 1C. The supernatant was collected by brief centrifugation and incubated with 0.45 mL of isoform-specific CaCPK antibodies at dilution of 1:1000 for 4 h at 4 1C. Then, 50 mL of protein Asepharose was added to the sample and the whole mixture was incubated further for 2 h at 4 1C. The precipitated immunocomplexes were collected by centrifugation and washed thrice with 500 mL of wash buffer [10 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1 mM EDTA and 0.1% (v/v) Triton X-100] and thrice with kinase buffer [50 mM Tris-HCl (pH 7.2), 10 mM MgCl2 and 20% (v/v) glycerol] and suspended in 50 mL of kinase buffer. The kinase assay was carried out in 10 mM MgCl2, 1 mg/mL histone-III S, 2.5 mCi [g-32P] ATP, with or without 0.25 mM CaCl2 with 25 mL of immunoprecipitate. After incubation for 5 min at 37 1C, 20 ml of the supernatants were spotted on P81 phosphocellulose paper and washed with 1% (v/v) phosphoric acid. The filters were dried and counted in a liquid scintillation counter.

Preparation of cytosolic and microsomal fraction proteins Cytosolic and membrane fraction proteins were prepared as described (Lu and Hrabak, 2002) with minor modifications. Ten-d-old chickpea seedling

S.R. Syam Prakash, C. Jayabaskaran leaves were ground in a mortar and pestle at 4 1C and mixed with 1 mL of homogenization buffer [50 mM Tris-HCl (pH 8.2), 20% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride and 1 mM dithiothreitol] per gram wet tissue. The slurry was clarified by passing through Mira cloth followed by centrifugation. Microsomal membranes were pelleted by ultracentrifugation at 125,000g for 30 min and suspended in buffer [50 mM Tris-HCl (pH 8.2), 20% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride and 1 mM dithiothreitol]. The resuspended microsomes and cytosolic fractions were kept at 0–4 1C for immediate use or frozen with liquid N2 and stored in aliquots at 80 1C until use.

Membrane-binding assays Membrane-binding assays were carried out as described previously (Lu and Hrabak, 2002) with modifications. Microsomal membranes were resuspended at 0.3 mg/mL in resuspension buffer [50 mM Tris-HCl (pH 8.2), 20% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride and 1 mM dithiothreitol] alone or in resuspension buffer containing one of the following: 10 mM EDTA, 1 M NaCl, 1% (v/v) Triton X-100 or 0.1% (w/v) SDS. After incubation at 4 1C for 30 min, samples were centrifuged at 125,000g at 4 1C for 30 min to pellet membrane vesicles. The supernatants were saved and the pellets were resuspended in resuspension buffer. The supernatant and the pellet fractions were subjected to SDS-PAGE and immunoblot analysis.

Immunofluorescence microscopy For immunofluorescence microscopy, freshly cut chickpea leaf and stem tissues were fixed with 5% (v/v) formaldehyde for 30 min at room temperature, rinsed with water and washed in 10 mM phosphate buffer (pH 7.4) and 150 mM NaCl (phosphate-buffered saline—PBS) thrice for 10 min. Free-hand sections of chickpea tissues (20 mm thick) were made, mounted on glass slides and treated with blocking solution [3% (w/v) BSA in PBS (pH 7.4)] overnight at 4 1C. Later, the sections were incubated with the anti-CaCPK1 or antiCaCPK2 serum (1:500 dilutions) in blocking solution for 1 h at room temperature. After three 10-min washes in PBS-T [10 mM phosphate buffer (pH 7.4), 150 mM NaCl and 0.2% (v/v) Triton X-100], the sections were incubated with goat anti-rabbit IgGfluorescein isothiocyanate (FITC) conjugate at a dilution of 1:500 in the blocking solution for 1 h at room temperature. The unbound conjugates were washed thrice for 10 min with PBS-T. Sections were

ARTICLE IN PRESS Subcellular localization and differential expression of CaCPK1 and CaCPK2 mounted on glass slides with water or glycerin and were observed on a Leica Fluorescence Microscope (Leica Microsystems AG, Germany). Images were processed using the Adobe Photodeluxe image analysis software.

Immunotransmission electron microscopy For immunotransmission electron microscopy, freshly cut chickpea leaf and stem tissues were fixed with a solution of 1% (w/v) paraformaldhyde and 0.05% (v/v) glutaraldehyde in phosphate buffer (pH 7.2) overnight. Immunostaining was carried out as per the protocol supplied by PELCO International using LR White Resin. Briefly, the fixed material was dehydrated using ethanol and then embedded in LR White Resin at 65 1C for 24 h. Ultrathin sections (100–120 nm) were cut using Leica semiautomatic microtome (Leica Microsystems AG, Germany). The sections were mounted on nickel grids and subjected to immunostaining. BSA, 3% (w/v; sigma), in phosphate buffer (pH 7.2) was used as blocking agent. All incubations with antibodies were carried out in blocking buffer. Primary antibody was used without dilution (neat) and incubated for 1 h at room temperature followed by washing thrice 10 min each in blocking buffer. Goat anti-rabbit IgG conjugated to 20 nm gold particles was used as secondary antibody at 1:100 dilutions and incubated for 1 h at room temperature. The grids containing the sections were washed thrice 10 min each with blocking buffer, and subjected to counter staining with uranyl acetate in 70% (v/v) ethanol for 20 min followed by staining with lead citrate for 30 s. The sections were then viewed using a JEOL 100 CXII transmission electron microscope.

Sequence data Sequence data from this article have been deposited at the EMBL/GenBank under accession numbers AY312268 for CaCPK1 and AY312269 for CaCPK2.

1139

plasmids pQECaCPK1-var and pQECaCPK2-var were transformed into E. coli strain M15 and expression of the recombinant proteins was induced by addition of 0.5 mM IPTG. Expressions of both the variable regions of CaCPK1 and CaCPK2 cDNAs in E. coli were detected in the IPTG-induced crude E. coli extracts (Fig. 1A and D). We have detected His6-tag-CaCPK1-var fusion protein as 9.9 kDa and the His6-tag-CaCPK2-var fusion protein as 9.65 kDa in the IPTG-induced crude E. coli protein extracts which were in agreement with their molecular weights expected from the corresponding DNA fragments cloned in the expression clones. These protein bands were not found in uninduced E. coli extracts containing pQECaCPK1-var or pQECaCPK2var confirming the bands as genuine His6-tagCaCPK1-var and His6-tag-CaCPK2-var fusion proteins. The recombinant fusion proteins were purified to near homogeneity using Ni-NTA affinity tag (Qiagen) and polyclonal antibodies were raised against the fusion proteins. The specificity of polyclonal antisera was checked by protein gel blot analysis. The anti-CaCPK1-var antibodies recognize the His6-CaCPK1-var antigen (Fig. 1B). The antiCaCPK2-var antibodies showed similar specificity for His6-CaCPK2-var (Fig. 1E). The polyclonal antibodies raised against variable domains of CaCPK1 and CaCPK2 were checked for crossreactivity against the opposite recombinant isoform (data not shown). The antiserum containing antibodies against the variable domains of CaCPK1 specifically recognized the CaCPK1 isoform and showed no cross-reactivity with CaCPK2 isoform. Similarly, antiserum-containing antibodies against variable domains of CaCPK2 isoform specifically recognized the CaCPK2 isoform without crossreacting with the CaCPK1 isoform. The CaCPK isoform-specific antibodies were also tested for ability to recognize their respective CaCPK proteins from chickpea seedling leaves. Anti-CaCPK1 antibody detected a 62 kDa (Fig. 1C) band in the total soluble protein extracted from seedling leaves while anti-CaCPK2 antibody detected a 61 kDa (Fig. 1E) band in the protein gel blotting.

Results Production of antisera specific for CaCPK1 and CaCPK2 isoforms

Effect of phytohormones and environmental stresses on expression of CaCPK1 and CaCPK2

The N-terminal variable regions of CaCPK1 and CaCPK2 cDNAs corresponding to the first 1–74 and 1–66 amino acids were RT-PCR amplified using total RNA isolated from seedling leaves and inserted into the expression vector pQE30 UA. The resulting

The accumulation of CaCPK1 and CaCPK2 transcripts, proteins and their kinase activities was monitored in chickpea various organs and in excised leaves treated with phytohormones and environmental stresses.

ARTICLE IN PRESS 1140

S.R. Syam Prakash, C. Jayabaskaran

Figure 1. Expression of recombinant CaCPK1-var and CaCPK2-var fusion protein in E. coli and protein gel blot analyses using the recombinant proteins. Ten micrograms of protein extracts were separated by SDS-PAGE. (A) Expression of the CaCPK1-var. Coomassie-Brilliant Blue R-250 staining of protein extracts from lanes 1 and 2, IPTG-induced E. coli cells containing pQECaCPK1-var; lane 3, IPTG-uninduced E. coli cells containing pQECaCPK1-var; and lane 4, protein molecular weight markers. The position of the His6-tag-CaCPK1 protein is denoted by arrowhead on the left. (B and C) protein gel blot analysis. Separated proteins from IPTG-induced E. coli cells containing pQECaCPK1-var and a protein extract from chickpea seedlings were transferred onto nitrocellulose membrane and protein gel blotted with antiCaCPK1 antibody. Arrows show the molecular masses of two detected proteins bands. (D) Expression of the CaCPK2-var. Coomassie-Brilliant Blue R-250 staining of protein extracts from lane 1, IPTG-uninduced E. coli cells containing pQECaCPK2-var, lane 2 and 3, IPTG-induced E. coli cells containing pQECaCPK2-var; and lane 4, protein molecular weight markers. The position of the His6-tag-CaCPK2 protein is denoted by arrowhead on the left. (E and F) Protein gel blot analysis. Separated proteins from IPTG-induced E. coli cells containing pQECaCPK2-var and a protein extract from chickpea seedlings were transferred onto nitrocellulose membrane and protein gel blotted with anti-CaCPK2 antibody. Arrows show the molecular masses of two detected protein bands.

Organ specificity Figure 2 shows that CaCPK1 and CaCPK2 transcripts, proteins and enzyme activities are present at different levels in all tested organs including roots, leaves, stems, flowers and fruits. No transcripts, proteins and enzyme activity could be detected for CaCPK2 in flowers and fruits. Both CaCPK1 and CaCPK2 genes are expressed in chickpea roots, stems and leaves at approximately similar levels. The highest levels of transcripts, proteins and enzyme activities were found in roots

for both the isoforms and their levels were low in stems and leaves.

Response to phytohormones The level of CaCPK1 and CaCPK2 mRNAs increased approximately 3-fold following BA treatment (Fig. 3A). In GA3-treated leaves, the level of CaCPK2 transcript was about 3-fold higher than water-treated control leaves; however, CaCPK1 transcript level was unchanged. IAA was totally

ARTICLE IN PRESS Subcellular localization and differential expression of CaCPK1 and CaCPK2

Figure 2. In vivo expression analyses of CaCPK1 and CaCPK2 in various chickpea organs (S, stems; L, leaves; R, roots; Fl, flowers and Fr, fruits). (A) Real-time RT-PCR analysis of CaCPK1 and CaCPK2 transcript accumulation patterns from various organs. Total RNAs (1 mg) from various organs were reverse transcribed and subjected to real-time RT-PCR analysis (see Materials and methods). The relative expression value of target was normalized to an endogenous control actin and relative to a calibrator. The lowest value was set to 1.0 and all other values were expressed relative to it. The data shown represent mean values obtained from three independent experiments and error bars indicate the SD of the mean. (B) Protein gel blot analysis of accumulation patterns of CaCPK1 and CaCPK2 proteins using total soluble proteins extracted from various organs. Total soluble proteins were electrophoretically separated by SDS-PAGE on a 10% (w/v) gel, transferred onto nitrocellulose membrane and probed with isoform-specific antibodies raised against the variable domains of CaCPK1 and CaCPK2. Equal amounts of proteins (30 mg) were loaded in each lane. (C) Calciumdependent kinase activity of CaCPK1 and CaCPK2 in total soluble protein extracts of various chickpea organs. CaCPK1 or CaCPK2 was immunoprecipitated from total soluble protein extracts and subjected to immunocomplex kinase assay with 1 mg/mL histone III-S as substrate in the presence of 2.5 mCi [g-32P] ATP as described in Materials and methods.

1141

Figure 3. Phytohormone-induced changes in the abundance of CaCPK1 and CaCPK2 transcripts, proteins and their activities in chickpea seedling leaves. Excised leaves of 10-d-old chickpea seedlings were treated with water, IAA, GA3 or BA for 24 h. All tested phytohormones were used at 100-mM concentrations. (A) Real-time RTPCR analysis of CaCPK1 and CaCPK2 transcript accumulation pattern using total RNAs from each sample. Other explanations are as described in the legend to Fig. 2A. (B) Protein gel blot analysis of accumulation pattern of CaCPK1 and CaCPK2 proteins using total soluble protein extracts from each sample. Inset is the protein gel blots showing immunoreactive CaCPK1 and CaCPK2 bands. Relative amounts of CaCPK1 and CaCPK2 proteins were done by densitometric scan of protein gel blots. (C) Calcium-dependent kinase activity of CaCPK1 and CaCPK2 in total soluble protein extracts of each sample. Other explanations are described in the legend to Fig. 2C.

ARTICLE IN PRESS 1142 ineffective and did not affect the levels of CaCPK1 and CaCPK2 transcripts. Figure 3B (inset) shows anti-CaCPK1 and anti-CaCPK2 immunoblots of crude protein extracts from leaves treated with water, IAA, GA3 and BA. An intensely immunostained CaCPK1 protein band was seen in BA-treated leaves. This band was faint in the protein extract from water-treated (control) as well as IAA- and GA3-treated leaves. The level of CaCPK1 protein is 2.5-fold higher in BA-treated leaves than in watertreated (control) leaves. An intensely immunostained CaCPK2 protein band was also seen in leaves treated with BA and GA3. This band was faint in the protein extract from water-treated (control) leaves as well as IAA-treated leaves. In GA3-treated and BA-treated leaves, the relative level of CaCPK2

S.R. Syam Prakash, C. Jayabaskaran protein was 3- and 2–3-fold higher, respectively, compared with that in water-treated (control) leaves. As shown in Fig. 3C, the activity of CaCPK1 increased 3-fold in response to treatment with BA; however, IAA and GA3 did not show any significant increase or decrease in activity. Furthermore, the activity of CaCPK2 was increased 3-fold in response to treatments with both GA3 and BA, but did not change by IAA treatment.

Response to environmental stresses Figure 4A shows the patterns of the CaCPK1 and CaCPK2 mRNA accumulations in the chickpea seedling leaves in response to dehydration, high salt and fungal spore treatments. The accumulation

Figure 4. Stress-induced changes in the expression of CaCPK1 and CaCPK2 transcripts, proteins and their activities in chickpea seedling leaves. Excised leaves of 10-d-old chickpea seedlings were treated with 100 mM NaCl for salt stress (SS), subjected to dehydration for water stress (WS) or treated with fungal spores (FS) for 1, 2, 4, 8 or 12 h. (SS0, SS1, SS2, SS4, SS8, WS0, WS1, WS4, WS8, WS12, FS0, FS1, FS4, FS8 and FS12). (A) Real-time RT-PCR analysis of CaCPK1 and CaCPK2 transcript accumulation patterns using total RNAs from each sample. Other explanations are as described in the legend to Fig. 2A. (B) Protein gel blot analysis of accumulation patterns of CaCPK1 and CaCPK2 proteins using total soluble protein extracts from each sample. Inset is the protein gel blots showing the immunoreactive CaCPK1 and CaCPK2 bands. Relative amounts of CaCPK1 and CaCPK2 proteins were done by densitometric scan of protein gel blots. (C) Calcium-dependent kinase activity of CaCPK1 and CaCPK2 in total soluble protein extracts of each sample. Other explanations are described in the legend to Fig. 2C.

ARTICLE IN PRESS Subcellular localization and differential expression of CaCPK1 and CaCPK2 of CaCPK1 and CaCPK2 mRNAs enhanced in a timedependent manner upon salinity stress. The level of CaCPK1 transcript reached a maximum of 8-fold higher after 2 h of salt stress-treatment and decreased by 4 and 8 h while the level of CaCPK2 transcript reached a maximum of 4-fold higher at 2 h, and remained constant by 4 and 8 h. The level of CaCPK2 transcript reached a maximum of 10-fold higher at 1 h of dehydration treatment and decreased to 4-fold at 12 h. Furthermore, analysis of CaCPK1 gene expression analysis showed that a basal level of CaCPK1 transcript was present in the control sample, which was not inducible by dehydration treatments. When the seedling leaves were subjected to fungal spore treatment, the accumulation of CaCPK1 mRNA enhanced rapidly and reached a maximum of 10-fold at 1 h of fungal spore treatment. This was followed by a decline to a basal level of accumulation at 12 h and that was maintained over the time of treatment. The mRNA level of CaCPK2 was not affected by fungal spore treatment. Figure 4B shows the patterns of CaCPK1 and CaCPK2 protein accumulation over time in seedling leaves treated to salt stress, dehydration and fungal spore treatment conditions. The accumulation of CaCPK1 protein in leaves increased 3.5-fold at 2 h after salt stress treatment and declined to basal level at 8 h. The accumulation of CaCPK2 protein in leaves increased to 3-fold at 1 h after drought stress treatment and decreased to basal level at 12 h. The accumulation of CaCPK1 protein in leaves increased to 3-fold at 1 h after fungal spore treatment and decreased to basal level at 12 h. Figure 4C shows the pattern of protein kinase activity of CaCPK1 and CaCPK2 over time in the excised chickpea seedling leaves exposed to different stress conditions. The activity of CaCPK1 was increased a maximum of 3-fold after 2 h in response to salt stress treatment. Treatment for 4 and 8 h did not lead to any further increase in CaCPK1 activity. In contrast, there was no significant decrease or increase in activity observed for CaCPK2 upon salt stress treatment. When the leaves were treated to dehydration, there was no significant decrease or increase in the activity observed for CaCPK1. The activity of CaCPK2 increased 3-fold at 1 h in response to dehydration treatment. Treatment for 4, 8 and 12 h did not lead to any further increase in CaCPK2 activity. When the leaves were treated with fungal spores the activity of CaCPK1 increased 3-fold at 1 h. Treatment for 4, 8 and 12 h did not lead to any further increase in CaCPK1 activity. There was no significant decrease or increase in activity

1143

observed for CaCPK2 in response to fungal spore treatment. These results suggest that changes in CaCPK1 and CaCPK2 activities determined reflect the changes of their mRNAs and proteins determined by real-time RT-PCR and protein gel blot analysis, respectively. Taken together, these results strongly suggest that the dehydration-, salt- and/or fungal spore treatment-induced accumulation of CaCPK1 and CaCPK2 transcripts, proteins and protein kinase activities was timedependent.

Membrane localization of CaCPK1 and CaCPK2 in chickpea seedling leaves To gain an understanding of the subcellular localization of the CaCPK1 and CaCPK2 isoforms, we analyzed microsomal and cytosolic fractions of chickpea seedling leaves for the presence of CaCPK1 and CaCPK2 isoforms. The microsomal and soluble cytosolic proteins were subjected to protein gel blot analysis using isoform-specific antibodies raised against the variable domains of CaCPK recombinant proteins. The results depicted in Fig. 5A show that both CaCPK1 and CaCPK2 proteins were present in the membrane fraction, but not in the soluble cytosolic fraction, showing that both the isoforms are membrane-associated CPKs. So, our study clearly demonstrates that CaCPK1 and CaCPK2 are membrane associated in chickpea seedling leaves. To study the interaction of CaCPK1 and CaCPK2 with the microsomal membranes, isolated membranes from the seedling leaves were incubated in the presence of buffer alone or buffer containing a chelating agent (EDTA), high ionic strength (NaCl), a denaturant (urea), a non-ionic detergent (Triton X-100) or an ionic detergent (SDS). After 30 min, the samples were ultracentrifuged and the extracted protein in the pellet and supernatant fractions were subjected to protein gel blot analysis using the isoform-specific CaCPK1 and CaCPK2 antibodies. Treatment with the chelating agent EDTA and NaCl had no effect on the solubilization of CaCPK1 and CaCPK2 proteins from the membranes (Fig. 5B, lanes b and c) whereas treatments with urea, Triton X-100 and SDS had almost completely solubilized the CaCPK1 and CaCPK2 proteins from the microsomal membranes (Fig. 5B, lanes d, e and f). It is clear that treatments that disrupt ionic or electrostatic interactions were not effective at dissociating CaCPK1 and CaCPK2 from the membranes, whereas treatments that disrupted most types of hydrophobic interactions

ARTICLE IN PRESS 1144

S.R. Syam Prakash, C. Jayabaskaran

Cellular localization of CaCPK1 and CaCPK2 in chickpea leaves and stems by immunofluorescence microscopy

Figure 5. Membrane association of CaCPK1 and CaCPK2. (A) Protein gel blot analysis showing the microsomal membrane association of CaCPK1 and CaCPK2. Chickpea seedling leaf extracts were fractionated into soluble (S) and microsomal membrane (M) fractions as described in Materials and methods. Microsomal membrane and soluble proteins were electrophoreticaly separated by SDS-PAGE on a 12.5% (w/v) gel, transferred onto nitrocellulose membrane and probed with isoform-specific antibodies raised against the variable domains of CaCPK1 and CaCPK2 as well as H+-ATPase-specific antibodies as a marker protein of microsomal membrane. Equal amounts of proteins (30 mg) were loaded in each lane. (B) Protein gel blot analysis of CaCPK1 and CaCPK2 proteins in the pellet (P) and supernatant (S) fractions after chemical treatments. Microsomal membranes were isolated from the microsomal fractions and the membrane pellets were homogenized in resuspension buffer alone (a) or resuspension buffer containing 10 mM EDTA (b), 1 M NaCl (c), 8 M urea (d), 3% (v/v) Triton X-100 (e) or 0.1% (w/v) SDS (f) and incubated at 4 1C for 30 min before repelleting. The resulting pellet and supernatant proteins were electrophoreticaly separated by SDS-PAGE on a 12.5% (w/v) gel, transferred onto a nitrocellulose membrane and probed with isoform-specific antibodies raised against the variable domains of CaCPK1 and CaCPK2. Equal amounts of proteins (30 mg) were loaded in each lane.

efficiently solubilized CaCPK1 and CaCPK2. Because Triton X-100 was able to release CaCPK1 and CaCPK2 from membranes, CaCPK1 and CaCPK2 are unlikely to be associated with detergentresistant membranes or lipid rafts (Moffett et al., 2000).

Immunocytochemistry was used to determine the tissue and the cellular localization of CaCPK1 and CaCPK2 in the leaves and stems. A cross-section of a leaf midrib showed the presence of mesophyll cells (Fig. 6A). Details of the longitudinal stem section containing xylem fibers and parenchyma cells are shown in Fig. 6B. To investigate the cellular localization of CaCPK1 and CaCPK2, immunolocalization was carried out using isoformspecific antibodies raised against the variable domains of individual CaCPKs with fixed sections. The immunodetection of CaCPK1 and CaCPK2 were identified by the green fluorescence of FITCconjugated secondary antibody. The sections of chickpea plant leaf midrib and stem showed strong immunofluorescent localization of CaCPK1 and CaCPK2. In the leaf midrib, the fluorescence was localized to the periphery of mesophyll cells for both CaCPK1 (Fig. 6C) and CaCPK2 (Fig. 6E). In stems CaCPK1 was located in the xylem parenchyma cells (Fig. 6D). Similar results were observed for CaCPK2 (Fig. 6F). No green fluorescence was found in all control sections probed with preimmune serum (data not shown).

Subcellular localizations of CaCPK1 and CaCPK2 in chickpea leaves and stems by immunotransmission electron microscopy To investigate subcellular locations for CaCPK1 and CaCPK2, immunogold localization was carried out using the same CaCPK1 and CaCPK2 antibodies and gold goat anti-rabbit secondary labeling with fixed ultrathin cross-sections. The immunodetection of CaCPK1 and CaCPK2 were identified by 20 nm electron dense gold particles under JEOL CXII 100 transmission electron microscope. The leaf midrib and stem showed intense labeling. In the leaf midrib, the labeling corresponding to CaCPK1 was restricted to the plasma and chloroplast membrane of mesophyll cells (Fig. 7A). Identical results were obtained using leaf midrib probed with antiserum against CaCPK2 (Fig. 7D). In the stem cross-sections, we have observed anti-CaCPK1 (Fig. 7B) and anti-CaCPK2 (Fig. 7E) immunolabeling in the PM of xylem parenchyma cells. Figure 7C and F show high-magnification view of a specific region of a stem labeled with CaCPK1 and CaCPK2 antibodies, respectively. No label was found over the cytoplasm and over the cell wall. If immune serum was replaced by preimmune serum, no gold

ARTICLE IN PRESS Subcellular localization and differential expression of CaCPK1 and CaCPK2

1145

Figure 6. Morphological and structural observations of chickpea leaf and stem sections and immunolocalization of CaCPK1 and CaCPK2 by confocal laser scanning microscopy. (A and B) Bright-field images of sections of chickpea organs. (A) Cross-section of a leaf, showing the structure of mesophyll cells. (B) Longitudinal section of a stem, showing the structure of xylem with fibers and parenchyma cells. (C–F) Immunofluorescence images of sections of chickpea organs. Sections of leaf and stem were probed with antibody raised against the variable domain of either CaCPK1 or CaCPK2, followed by an FITC-conjugated goat anti-rabbit secondary antibody. (C and D) Detection of CaCPK1 with a FITCconjugated goat anti-rabbit secondary antibody in cross-section of leaf (C) and longitudinal section of stem (D). (E and F) Detection of CaCPK2 with a FITC-conjugated goat anti-rabbit secondary antibody in cross-section of leaf (E) and longitudinal section of stem (F). The abbreviations used are: M, mesophyll cells; XF, xylem fiber and XP, xylem parenchyma. Bar ¼ 25 mm.

particles were observed over CaCPK1 and CaCPK2 proteins (data not shown). Together, these results indicate that the labeling pattern observed on sections of fixed chickpea leaves and stems incubated with anti-CaCPK1 or antiCaCPK2 antibodies is due to the specific interaction of the antibodies with their respective antigens.

Discussion CPK genes have been shown to be differentially expressed in different organs of certain plant species. It has been shown that among three rice CPK genes, SPK is specifically expressed in developing seeds, and the other two, OsCPK2 and OsCPK11, are abundant in both roots and

ARTICLE IN PRESS 1146

S.R. Syam Prakash, C. Jayabaskaran

Figure 7. Transmission electron microscopic immunogold localization of CaCPK1 (A–C) and CaCPK2 (D–F) proteins in chickpea leaves and stems. Ultrathin sections were probed with antibodies raised against the variable domains of CaCPK1 and CaCPK2, followed by secondary antibodies conjugated to 20-nm gold particles. Gold particles can be seen on chloroplast membrane and plasma membrane (PM) of mesophyll cells in the leaf ((A) for CaCPK1 and (D) for CaCPK2) and PM of parenchyma cells in the stem ((B) and (C) for CaCPK1 and (E) and (F) for CaCPK2). (C) and (F) represent enlargement of selectable regions of (B) and (E), respectively. Arrowheads point to the locations of gold particles. The abbreviations used are: PM, PM; Cp, chloroplast; CM, chloroplast membrane and SG, starch granule. Magnification: (A and D) 27,000
; (B and E) 19,000
; (C and F) 33, 000
.

coleoptiles (Frattini et al., 1999). Distribution of Arabidopsis mRNAs encoding isoforms CPK6, CPK9 and CPK19 have been shown to differ in leaves, roots and flowers (Hong et al., 1996). Similarly, it was recently reported that transcripts for cucumber CPKs and the activity of CPKs in different organs differed and the highest kinase activity and their transcript accumulation were seen in cucumber plant leaves followed by cucumber seedling roots and hypocotyls (Kumar et al., 2004). To answer the question whether or not CaCPK1 and

CaCPK2 transcripts, proteins and activity are present in various organs, we examined the accumulation of their transcripts and proteins as well as activity in various organs of chickpea plants. CaCPK1 transcripts and proteins accumulated in all the organ samples examined. However, maximum expression of transcripts, accumulation of CaCPK1 protein and the activity were detected in roots. The expression of CaCPK2, protein accumulation and activity were found significant quantities in roots, although trace amounts could also be

ARTICLE IN PRESS Subcellular localization and differential expression of CaCPK1 and CaCPK2 detected in leaves and stems. Neither CaCPK2 transcript nor their proteins were detected in flowers and fruits. These results suggest the differential expression of the two CaCPK isoforms in various organs of chickpea plants. Exogenously applied phytohormones, such as cytokinins and auxins are known to induce protein kinase transcripts in certain plants. Botella et al. (1996) reported increased mRNA level of mungbean CPK, Vigna radiata CPK1 (VrCPK1) in the leaves of light-grown 10-d-old seedlings after treatment with 0.5 mM IAA. It has been shown that cytokinins induce WPK4 transcripts in 6-d-old wheat seedlings (Sano and Youssefian, 1994). Ullanat and Jayabaskaran (2002) have shown that cytokinin treatment of excised etiolated cotyledons caused an upregulatory effect on CsCPK3 transcript levels; however, a down-regulatory effect was shown in roots and in etiolated hypocotyls. The authors have further observed that auxin treatment had no significant effect on the CsCPK3 transcript level. In the present work, the two chickpea CPK genes respond differentially to exogenously supplied phytohormones. CaCPK1 was up-regulated in response to BA treatment and CaCPK2 was upregulated in response to both GA3 and BA treatments. In this study, we found that the amounts of CaCPK1 and CaCPK2 proteins and their phosphorylation activity were generally correlates with the amounts of transcripts. These results suggest that CaCPK1 may catalyze an important phosphorylation event in signaling in chickpea leaves in response to BA and CaCPK2 in response to BA and GA3. Recently, it has been demonstrated that the CPK cascades play important roles in stress responses in plants. Expression of OsCPK7, a rice CPK was induced by cold and salinity in both shoots and roots of 10-d-old rice seedlings (Saijo et al., 2001). When the OsCPK7 was overexpressed in rice, plants showed increased tolerance to cold and salt (Saijo et al., 2000). The response of CPK gene expression to various stresses varied among individual members. For example, AtCPK10 and AtCPK11 are involved in mediating drought and salt stress signaling while AtCPK30 (AtCPK1a) is involved in cold, salt and abscisic acid (ABA)-induced pathways (Sheen, 1996). Nicotiana tabacum CPKs—NtCPK2 and NtCPK3—two CPK genes from tobacco, are involved in mediating defense and osmotic stress signaling pathways whereas NtCPK1 mediates an array of signals including GA3, ABA, cytokinin, wounding, fungal elicitors and salt stress (Romeis et al., 2001; Ludwig et al., 2004). In the present work, we examined the expression of the two chickpea CPK isoforms in response to osmotic stress (dehydration), ionic stress (NaCl) as well as fungal

1147

spore treatment. Our data showed that these enzymes are differentially regulated in response to dehydration and salt stresses as well as fungal spore treatment suggesting that they play different physiological functions. Neither of the two tested CaCPK1 and CaCPK2 showed identical patterns of expression upon exposure to dehydration and salt stresses as well as fungal spore treatment. The levels of CaCPK1 transcripts, proteins and activity were increased by NaCl treatment and fungal spore treatment while the levels of CaCPK2 transcripts, proteins and activity were increased by dehydration stress. These observations suggest that the in vivo functions of the two genes are not identical. These results provided evidence that CaCPK1 may catalyze an important phosphorylation event in signaling in chickpea under salt stress conditions and in response to fungal spore and CaCPK2 in dehydration stress. CPKs were shown to be localized in various cellular compartments, PM being in most cases (Schaller et al., 1992; Verhey et al., 1993; BaizabalAguirre and de la Vara, 1997; Iwata et al., 1998; Dammann et al., 2003; Kumar and Jayabaskaran, 2004), and in some plants in the cytosol (Battey, 1990; Klimczak and Hind, 1990; DasGupta, 1994; MacIntosh et al., 1996; Frylinck and Dubery, 1998), endoplasmic reticulum membrane (Lu and Hrabak, 2002), peroxisomes (Dammann et al., 2003) or even in nuclei (Li et al., 1991). Because the localization of CPKs is an important step for the elucidation of biological functions and because CPK isoforms confer different specificities and functions in multiple signaling pathways, we determine the precise localization of CaCPK1 and CaCPK2 in leaf and stem tissues of chickpea. Immunofluorescence and immunogold labeling were used to determine cellular and subcellular localization of the isoforms. Immunofluorescence localization of CaCPK1 and CaCPK2 in leaf midrib and stem tissues showed that both the isoforms are present in leaf mesophyll cells and in stem xylem parenchyma cells. Analysis of immunogold-stained leaf midrib ultrathin sections by electron microscopy revealed that both isoforms are localized at the PM and chloroplast membrane of mesophyll cells. In stems these two isoforms are localized only in the PM of the xylem parenchyma cells. The multiple subcellular localizations suggest that these CPK isoforms are involved in multiple signal transduction pathways. The occurrence of two CPK isoforms in the chloroplast membrane of chickpea leaves suggested that the possible physiological roles of these CPK isoforms in the organelles. There is no previous information about presence of any CPK in the chloroplast membrane. Our results provide the first

ARTICLE IN PRESS 1148 evidence for chloroplast-membrane-associated CPKs. Chloroplasts are major contributors to the biosynthetic activity in plant cells and house many essential biosynthetic pathways, such as biosynthesis of fatty acids, reduction of nitrate and sulfate, and synthesis of prenyl lipids and isoprenoids, to name just a few. Chloroplasts are also tightly interconnected with metabolic pathways localized in different cell compartments, e.g. metabolism of carbohydrates, amino acids and lipids. It was known from earlier studies (Flu ¨gge, 1998) that numerous substrate-specific transporters are present in the inner membrane, reflecting this metabolic interdependence. There is a considerable amount of evidence showing that the chloroplast outer membrane contains different solute-selective ion channels (Pohlmeyer et al., 1998a, b; Bo ¨lter et al., 1999; Bo ¨lter and Soll, 2001). Taken together, the present results and those of previous studies provide a potential mechanism for calcium regulation of various metabolic pathways and/or signal transduction pathways in chloroplasts. In conclusion, we have shown differential expression of CaCPK1 and CaCPK2 genes in various organs and in response to phytohormones, and biotic and abiotic stress treatments. This study shows that in chickpea CaCPK1 and CaCPK2 would be signaling components under dehydration, salt or biotic stresses and in response to hormones, such as BA and GA3. The results of the study on the immunolocalization of these isoforms in leaf and stem tissues showed that they are present in the PM and chloroplast membrane of mesophyll cells in the leaf and PM of xylem parenchyma cells in the stem, suggesting that they are involved in multiple signal transduction and/or metabolic pathways. All these observations draw us to the conclusion that they may play distinct roles in mediating responses to Ca2+ in chickpea. Further work is required to clarify the details about how these CaCPK isoforms recognize their substrates to trigger specific signaling events.

Acknowledgments We are grateful to Prof. Tom W. Okita, Washington State University, Pullman, USA for providing cDNA library of chickpea, Dr. Diego Breviario, Instituto Biosintesi Vegetali, Italy for OsCPK2 cDNA clone and Prof. Ramesh Maheshwari for critical reading of the manuscript. Dr. Chandrasekhar Sagar, NIMHANS, Bangalore helped in immunoelectron microscopy. This work was supported by a grant from Department of Science and Technology,

S.R. Syam Prakash, C. Jayabaskaran Government of India. S.R.S.P is a recipient of a Research Fellowship from CSIR.

References Abbasi F, Onodera H, Toki S, Tanaka H, Komatsu S. OsCDPK13, a calcium-dependent protein kinase gene from rice, is induced by cold and gibberellin in rice leaf sheath. Plant Mol Biol 2004;55:541–52. Anil VS, Harmon AC, Rao KS. Temporal association of Ca2+-dependent protein kinase with oil bodies during seed development in Santalum album L: its biochemical characterization and significance. Plant Cell Physiol 2003;44:367–76. Baizabal-Aguirre VM, de la Vara LEG. Purification and characterization of a calcium-regulated protein kinase from beet root (Beta vulgaris) plasma membranes. Physiol Plant 1997;99:135–43. Battey NH. Calcium-activated protein kinase from soluble and membrane fractions of maize coleoptiles. Biochem Biophys Res Commun 1990;170:17–22. Bo ¨lter B, Soll J. Ion channels in outer membranes of chloroplasts and mitochondria: open doors or regulated gates? EMBO J 2001;20:935–40. Bo ¨lter B, Soll J, Hill K, Hemmler R, Wagner R. A rectifying ATP-regulated solute channel in the chloroplastic outer envelope from pea. EMBO J 1999;18:5505–16. Botella JR, Arteca JM, Somodevilla M, Arteca RN. Calcium-dependent protein kinase gene expression in response to physical and chemical stimuli in mungbean (Vigna radiata). Plant Mol Biol 1996;30:1129–37. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54. Cheng SH, Willmann MR, Chen HC, Sheen J. Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family. Plant Physiol 2002;129:469–85. Chico JM, Raices M, Tellez-Inon MT, Ulloa RM. A calciumdependent protein kinase is systemically induced upon wounding in tomato plants. Plant Physiol 2002;128: 256–70. Dammann C, Ichida A, Hong B, Romanowsky SM, Hrabak EM, Harmon AC, et al. Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis. Plant Physiol 2003;132:1840–8. DasGupta M. Characterization of a calcium-dependent protein kinase from Arachis hypogea (groundnut) seeds. Plant Physiol 1994;140:961–9. Flu ¨gge UI. Metabolite transporters in plastids. Curr Opin Plant Biol 1998;1:201–6. Frattini M, Morello L, Breviario D. Rice calcium-dependent protein kinase isoforms OsCDPK2 and OsCDPK11 show different responses to light and different expression patterns during seed development. Plant Mol Biol 1999;412:753–64. Frylinck L, Dubery IA. Protein kinase activities in ripening mango, Mangifera indica L. fruit tissue III. Purification

ARTICLE IN PRESS Subcellular localization and differential expression of CaCPK1 and CaCPK2 and characterization of a calcium-regulated protein kinase. Biochem Biophys Acta 1998;1387:342–54. Harmon AC, Putnam-Evans C, Cormier MJ. Calciumdependent but calmodulin-independent protein kinase from soybean. Plant Physiol 1986;83:830–7. Harper JF, Sussman MR, Schaller GE, Putnam-Evans C, Charbonneau H, Harmon AC. A calcium-dependent protein kinase with a regulatory domain similar to calmodulin. Science 1991;252:951–4. Harper JF, Breton G, Harmon AC. Decoding Ca2+ signals through plant protein kinase. Annu Rev Plant Biol 2004;55:263–88. Hong Y, Takano M, Liu C-M, Gasch A, Chye ML, Chua N-H. Expression of three members of the calcium-dependent protein kinase gene family in Arabidopsis thaliana. Plant Mol Biol 1996;30:1259–75. Iwata Y, Kuriyama M, Nakakita M, Kojima H, Ohto M, Nakamura K. Characterization of a calcium-dependent protein kinase of tobacco leaves that is associated with the plasma membrane and is inducible by sucrose. Plant Cell Physiol 1998;39:1176–83. Klimczak LJ, Hind G. Biochemical similarities between soluble and membrane-bound calcium-dependent protein kinases of barley. Plant Physiol 1990;92:919–23. Kumar KGS, Jayabaskaran C. Variations in the level of enzyme activity and immunolocalization of calciumdependent protein kinases in the phloem of different cucumber organs. J Plant Physiol 2004;16:889–901. Kumar KGS, Ullanat R, Jayabaskaran C. Molecular cloning, characterization, tissue-specific and phytohormone-induced expression of calcium-dependent protein kinase gene in cucumber (Cucumis sativus L.). J Plant Physiol 2004;161:1061–71. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. Li H, Dauwalder M, Roux SJ. Partial purification and characterization of a Ca2+-dependent protein kinase from pea nuclei. Plant Physiol 1991;96:720–7. Lu SX, Hrabak EM. An Arabidopsis calcium-dependent protein kinase is associated with the endoplasmic reticulum. Plant Physiol 2002;128:1008–21. Ludwig AA, Romeis T, Jones JDG. CDPK-mediated signaling pathways: specificity and cross-talk. J Exp Bot 2004;55:181–8. MacIntosh GC, Ulloa RM, Raices M, Tellez-Inon MT. Changes in calcium-dependent protein kinase activity during in vitro tuberization in potato. Plant Physiol 1996;112:1541–50. Martin ML, Busconi L. A rice membrane-bound calciumdependent protein kinase is activated in response to low temperature. Plant Physiol 2001;125:1442–9. Moffett S, Brown DA, Linder ME. Lipid-dependent targeting of G proteins into rafts. J Biol Chem 2000;275:2191–8. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 1962;15:473–97. Pical C, Fredlund KM, Petit PX, Sommarin M, Moller IM. The outer membrane of plant mitochondria contains a

1149

calcium-dependent protein kinase and multiple phosphoproteins. FEBS Lett 1993;336:347–51. Pohlmeyer K, Soll J, Grimm R, Hill K, Wagner R. A highconductance solute channel in the chloroplastic outer envelope from pea. Plant Cell 1998a;10:1207–16. Pohlmeyer K, Soll J, Steinkamp T, Hinnah SC, Wagner R. Isolation and characterization of an amino-selective channel protein present in the chloroplastic outer envelope membrane. Proc Natl Acad Sci USA 1998b;94:9504–9. Prakash SRS, Jayabaskaran C. Heterologous expression and biochemical characterization of two calciumdependent protein kinase isoforms CaCPK1 and CaCPK2 from chickpea. J Plant Physiol 2006; (in press). Putnam-Evans CL, Harmon AC, Cormier MJ. Purification and characterization of a novel calcium-dependent protein kinase from soybean. Biochemistry 1990;29: 2488–95. Roberts DM, Harmon AC. Calcium-modulated proteins: targets of intracellular calcium signals in higher plants. Annu Rev Plant Physiol Plant Mol Biol 1992;43:375–414. Romeis T, Ludwig AA, Martin R, Jones JDG. Calciumdependent protein kinases play an essential role in a plant defense response. EMBO J 2001;20:5556–67. Saijo Y, Hata S, Kyozuka J, Shimamoto K, Izui K. Overexpression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants. Plant J 2000;23:319–27. Saijo Y, Kinoshita N, Ishiyama K, Hata S, Kyozuka J, Hayakawa T, et al. A Ca2+-dependent protein kinase that endows rice plants with cold- and salt-stress tolerance functions in vascular bundles. Plant Cell Physiol 2001;42:1228–33. Sano H, Youssefian S. Light and nutritional regulation of transcripts encoding a wheat protein kinase homolog is mediated by cytokinins. Proc Natl Acad Sci USA 1994;91:2582–6. Schaller GE, Harmon AC, Sussman MR. Characterization of a calcium- and lipid-dependent protein kinase associated with plasma membrane of oat. Biochemistry 1992;31:1721–7. Sheen J. Ca2+-dependent protein kinases and stress signal transduction in plants. Science 1996;274:1900–2. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979;76:4350–4. Ullanat R, Jayabaskaran C. Distinct light-, cytokinin- and tissue-specific regulation of calcium-dependent protein kinase gene expression in cucumber (Cucumis sativus). Plant Sci 2002;162:153–63. Urao T, Katagiri T, Mizoguchi T, Yamaguchi-Shinozaki K, Hayashida N, Shinozaki K. Two genes that encode Ca2+dependent protein kinases are induced by drought and high-salt stresses in Arabidopsis thaliana. Mol Gen Genet 1994;244:331–40. Verhey SD, Gaiser JC, Lomax TL. Protein kinases in zucchini: characterization of calcium-requiring plasma membrane kinases. Plant Physiol 1993;103:413–9.