Cloning and characterization of CBL-CIPK signalling components from a legume (Pisum sativum) Shilpi Mahajan, Sudhir K. Sopory and Narendra Tuteja Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, New Delhi, India In plants, calcium plays an important role in regula- ting gene expression and many other processes inclu- ding abiotic stress signalling. However, the molecular mechanisms underlying the role of calcium in cellular functions are not well established. Many external stimuli including light and various stress factors can bring out changes in cellular Ca 2+ level, which can affect plant growth and development [1,2]. The Ca 2+ serves as second messenger and its concentration is delicately balanced by the presence of ‘Ca 2+ stores’ such as vacuoles, endoplasmic reticulum, mitochon- dria and cell wall. Ca 2+ signals exhibit a high degree of specificity and are decoded by Ca 2+ sensing proteins known as Ca 2+ sensors, which are small proteins interacting with their target proteins to relay the signal. In plant cells many Ca 2+ sensors have been identified which include calmodulin (CaM) and calmodulin-related proteins [3,4], Ca 2+ -dependent protein kinases (CDPKs) [5,6], and calcineurin B-like proteins (CBLs) [4]. The first plant CBL to be iden- tified was from Arabidopsis thaliana, and is known as both AtCBL and ScaBP (SOS3-like calcium- binding protein) [7,8]. CBL proteins contain four Ca 2+ -binding EF hand motifs [9] and functions by interacting and regulating a group of Ser ⁄ Thr pro- tein kinases called CBL-interacting protein kinases Keywords abscisic acid; abiotic stress; biotic stress; calcium sensor CBL; CIPK Correspondence N. Tuteja, Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India Fax: +91 11 26162316 E-mail: narendra@icgeb.res.in Note The sequences reported in this paper have been deposited in the General Bank database (accession nos. AY134619 (pea CBL cDNA); AY883569 (pea CBL genomic clone); AY191840 (pea CIPK cDNA). (Received 7 October 2005, revised 11 December 2005, accepted 19 December 2005) doi:10.1111/j.1742-4658.2006.05111.x The studies on calcium sensor calcineurin B-like protein (CBL) and CBL interacting protein kinases (CIPK) are limited to Arabidopsis and rice and their functional role is only beginning to emerge. Here, we present cloning and characterization of a protein kinase (PsCIPK) from a legume, pea, with novel properties. The PsCIPK gene is intronless and encodes a protein that showed partial homology to the members of CIPK family. The recom- binant PsCIPK protein was autophosphorylated at Thr residue(s). Immu- noprecipitation and yeast two-hybrid analysis showed direct interaction of PsCIPK with PsCBL, whose cDNA and genomic DNA were also cloned in this study. PsCBL showed homology to AtCBL3 and contained calcium- binding activity. We demonstrate for the first time that PsCBL is phos- phorylated at its Thr residue(s) by PsCIPK. Immunofluorescence ⁄ confocal microscopy showed that PsCBL is exclusively localized in the cytosol, whereas PsCIPK is localized in the cytosol and the outer membrane. The exposure of plants to NaCl, cold and wounding co-ordinately upregulated the expression of PsCBL and PsCIPK genes. The transcript levels of both genes were also coordinately stimulated in response to calcium and salicylic acid. However, drought and abscisic acid had no effect on the expression of these genes. These studies show the ubiquitous presence of CBL ⁄ CIPK in higher plants and enhance our understanding of their role in abiotic and biotic stress signalling. Abbreviations 3-AT, 3-aminotrizole; ABA, abscisic acid; CaM, calmodulin; CBL, calcineurin B-like protein; CDPK, Ca 2+ -dependent protein kinase; CIPK, CBL interacting protein kinases; DAPI, diamidino-2phenylindole hydrochloride; DTT, dithiothreitol; IPTG, isopropyl thio-b- D-galactoside; SA, salicylic acid; SD, synthetic dextrose; UAS, upstream activating sequences; UTR, 5¢ untranslated region; YPD, yeast extract–peptone–dextrose. FEBS Journal 273 (2006) 907–925 ª 2006 The Authors Journal compilation ª 2006 FEBS 907 (CIPKs) [4,10,11]. CIPKs most likely represent tar- gets of Ca 2+ signals sensed and transduced by CBL proteins. CIPK consist of a catalytic domain (at the N ter- minus) and a regulatory domain (at the C terminus) that interact with each other to keep the enzyme inactive (autoinhibition), presumably by preventing substrate access to the catalytic site [12]. CBL binds to the FISL motif (or NAF domain) ) an autoinhib- itory domain present in the regulatory domain of the CIPK – and thereby makes the enzyme active by disrupting the intramolecular domain interaction of CIPK [12–14]. A database search revealed 10 AtCBLs and 25 CIPKs in the Arabidopsis genome and 10 CBLs and 30 CIPKs in the rice genome [4,12,15]. An analysis of genome evolution suggested that a large number of gene family members resulted from segmental duplications [15]. Furthermore, dif- ferential affinities among different AtCBL–CIPK members have been reported [11,12]. For example, AtCBL1 is known to interact only with a subset of six CIPKs (AtCIPK 1, 7, 8, 17, 18 and 24) [15]. The multiple combinations of CBL–CIPK complexes might provide a novel mechanism to integrate and specifically decode signals in plants [12,13]. Recent studies in Arabidopsis indicated that several such genes function in stress [12,14–21]. Except in Arabid- opsis and rice the CBL–CIPK pathways have not been well studied in higher plants. In this report, we describe the cloning and charac- terization of a novel CIPK and its interacting partner CBL from Pisum sativum. PsCIPK showed auto- phosphorylation and could phosphorylate pea CBL and other substrates such as casein. The mRNA lev- els of PsCIPK were coordinately upregulated along with CBL, in response to various abiotic and biotic stresses, and to calcium and salicylic acid, but not to abscisic acid (ABA) or dehydration. PsCIPK showed dual localization (in the cytosol and the plasma mem- brane) while CBL was localized exclusively in the cytosol. Results Isolation and sequence analysis of PsCIPK and CBL cDNAs and genomic clones For cDNA cloning, first partial fragments of 550 bp for PsCIPK and 335 bp for PsCBL were amplified by PCR using double-stranded cDNAs (prepared from mRNA isolated from NaCl-stressed pea seedlings) as template and the degenerate primers, designed from the conserved areas of AtCIPK and AtCBL of Arabidopsis, respect- ively (data not shown). The cDNA clones of CIPK (pBS-PsCIPK) and CBL (pBS-PsCBL) were obtained by screening the pea cDNA library with respective par- tial DNA fragments as probes. Sequence analysis of pBS-CIPK cDNA (Accession no. AY191840) shows that it encodes a full length cDNA, 1842 bp in size with an ORF of 1553 bp, a 5¢ untranslated region (UTR) of 47 bp and a 3¢ UTR of 242 bp including 39 bp of poly(A) tail. The PsCIPK ORF encodes a protein of 516 amino acid residues with a predicted molecular mass of  57.9 kDa and pI 8.23. Sequence analysis of pBS-CBL cDNA (Accession no. AY134619) shows that it encodes a full length cDNA, 972 bp in size with an ORF of 678 bp, a 5¢ UTR of 131 bp and a 3¢ UTR of 163 bp including 20 bp of poly(A) tail. The PsCBL ORF encodes a protein of 225 amino acid residues with a calculated molecular mass of 25.9 kDa and pI 4.67. The amino acid sequence alignment of PsCIPK with AtCIPK12, AtCBL19, Gossypium hirsutum (Gh) kin- ase, and AtCIPK18 is shown in Fig. 1A. The N-ter- minal domain of PsCIPK contains an activation domain starting from the conserved DFG and ending at APE; the C-terminal domain contains the NAF (FISL) motif (Fig. 1A). Phylogenetic analysis indicated 67% sequence identity with AtCIPK12 (Accession no. NP_193605), 66% with AtCIPK24 ⁄ SOS2-like (AAK26847), and 66% with GhCIPK (AAT64036) (data not shown). The identity of PsCIPK with other AtCIPKs is: 64% with AtCIPK19 (NP199393), and 62% with AtCIPK18 (NP174217) (data not shown). Fig. 1. Multiple amino acid sequence alignment. (A) Comparison of predicted amino acid sequences of PsCIPK with AtCIPK12 (Accession no.NP_193605), AtCIPK19 (NP199393), GhCIPK (AAT64036) and AtCIPK18 (NP174217). The activation and NAF domains are shown in the boxes. (B) The deduced amino acid sequence of PsCBL is aligned with rice CBL (OsCBL, Accession no. AAR01663) and AtCBL3 (AAM91280). The calcium binding domains (EF1–4) and calcineurin A binding domain are shown in the box. The dot in the EF1 box repre- sents the modified amino acids alanine (A) as compared to the oxygen containing-calcium binding residue aspartate (D). The conserved dis- tances between EF hands are marked. Multiple alignments were performed using CLUSTAL W. The program recognizes a consensus residue and based on that residue other amino acids that fall in that consensus position are marked. The most identical amino acids at each protein are dark shaded and similar ones are light shaded whereas nonsimilar ones are left unshaded. The amino acids marked by red, blue, green and pink lines indicates the putative casein kinase II, protein kinase C, the cAMP- and cGMP-dependent protein kinase and putative tyrosine kinase phosphorylation sites, respectively. Stress-induced CIPK from pea phosphorylate CBL S. Mahajan et al. 908 FEBS Journal 273 (2006) 907–925 ª 2006 The Authors Journal compilation ª 2006 FEBS A B S. Mahajan et al. Stress-induced CIPK from pea phosphorylate CBL FEBS Journal 273 (2006) 907–925 ª 2006 The Authors Journal compilation ª 2006 FEBS 909 The amino acid sequence alignment of PsCBL with rice CBL (OsCBL) and Arabibopsis CBL (AtCBL3) is shown in Fig. 1B. It lacks the myristoylation site in the N-terminal sequence. PsCBL contains four EF hand Ca 2+ -binding domains (Fig. 1B). The EF1 shows variation from the canonical EF hand. The amino acid D at position 1, of EF1 is replaced by amino acid A (Fig. 1B). The EF1 and EF2 are 22 amino acids apart, whereas EF2 and EF3, and EF3 and EF4 are 25 and 32 amino acids apart, respectively (Fig. 1B). The cal- cineurin A binding domain is also present between positions 155 and 172 (Fig. 1B). Phylogenetic analysis indicated the identity of PsCBL with OsCBL (Acces- sion no. AAR01663), AtCBL3 (AAM91280), AtCBL2 (AAM65177), AtCBL6 (AAG28400), AtCBL4 ⁄ SOS3- like (BAD43952), and AtCBL1 (BAC43389) as 92, 90, 89, 71, 68, and 66%, respectively (data not shown). Genomic organization of PsCIPK and PsCBL For PsCIPK, a genomic fragment (1.84 Kb) was amplified by PCR from the pea genomic DNA as a template with the 5¢ UTR and 3¢ UTR specific primers of PsCIPK gene. As a control the primers were used to amplify a cDNA fragment of expected size 1.84 Kb using cDNA as a template. To confirm the specificity of the PCR products a nested PCR (2nd PCR) was performed using PsCIPK gene-specific internal prim- ers. These fragments were then cloned and sequenced (data not shown). The same size and sequence of the genomic fragment of PsCIPK and the cDNA show that PsCIPK is an intron-less gene. For the PsCBL gene, a genomic fragment (3.22 Kb) was amplified by PCR using the pea genomic DNA as a template with the 5¢ UTR and 3¢ UTR specific prim- ers of the PsCBL gene. As a control, the same set of primers was used to amplify a cDNA fragment of expected size 0.97 Kb using cDNA as a template. To confirm the specificity of the PCR products a nested PCR (2nd PCR) was performed using gene-specific internal primers. As a result 2.547 Kb genomic and 0.67 Kb (expected size) cDNA fragments were obtained, which were cloned and sequenced (data not shown). The higher size of the genomic fragment of PsCBL as compared to the cDNA indicates that this gene contains introns. Sequence analysis of the genomic clone reveals that the PsCBL genomic clone spans 2.547 Kb (Accession no. AY883569) (from ATG to TAA). Alignment of the genomic sequence with the cDNA sequence identified eight exons (121, 82, 59, 108, 52, 80, 112, and 58 bp in size) and seven introns (331, 223, 682, 346, 80, 109, and 92 bp in size) (Fig. 2A). Two introns of 401 and 81 bp were found localized in the 5¢ UTR region (Fig. 2A). Most of the 3¢ and 5¢ splice junctions follow the typical canonical consensus dinucleotide sequence GU-AG found in other plant in- trons. Figure 2B shows the genomic organization of AtCBL3 (Accession no. AT4G265702) containing seven exons and six introns. The sizes of all the exons except exon 5 were found to be mostly conserved between PsCBL and AtCBL3 (Fig. 2A and B). The PsCBL gene has an additional splice site at the fifth exon. Accord- ingly; there was one intron fewer in AtCBL3 as com- pared to PsCBL (Fig. 2A and B). The sizes of introns are not conserved between the two species (Fig. 2A and B). Tissue distribution of PsCBL and CIPK and their copy number in pea genome The transcript levels of PsCIPK and PsCBL in different tissues of pea were studied by northern A B Fig. 2. Genomic organization of PsCBL. The schematic representation of the exon–intron organization of genomic PsCBL clone (A) and the Arabidopsis homologue (AtCBL3) clone (B). Closed boxes represent exons, and lines between closed boxes represent introns. The dark boxes represent the UTRs. The position of ATG and TAA are marked. The numbers below the lines and the above boxes indicate the sizes (bp) of introns and exons, respectively. Stress-induced CIPK from pea phosphorylate CBL S. Mahajan et al. 910 FEBS Journal 273 (2006) 907–925 ª 2006 The Authors Journal compilation ª 2006 FEBS hybridization. PsCIPK (1.8 Kb) and PsCBL (1.0 Kb) were ubiquitously present in all the tissues examined including root, shoot, tendril and flower, but at relat- ively higher levels in leaves and roots as compared to the other tissues (data not shown). The pattern of Southern genomic hybridization bands under low (data not shown) and high strin- gency washing conditions suggests that both PsCIPK and PsCBL exist as single-copy genes in the pea genome (Fig. 3A and B, respectively). Restriction enzymes which either had a specific site in the gene or which had no restriction site were used. Some of the enzymes such as SpeI and XbaI, which had no recognition site in PsCIPK cDNA and genomic DNA sequence gave a single band after hybridiza- tion (Fig. 3A, lanes 1 and 2), whereas enzymes such as BglII and NdeI, which had a single specific site in the gene gave two bands after hybridization (lanes 3 and 4). However, with HindIII, which has a single site in the gene towards the 3¢ end (that would result in 3127 and 94 bp fragments) gave single band around the 5 Kb region (Fig. 3B, lane 6). It is poss- ible that the second fragment containing a very small part of the gene did not hybridize under the condi- tions used. Enzymes such as EcoRI, BglII and NdeI, which had no recognition site in the PsCBL cDNA and genomic DNA sequence gave a single band after hybridization (Fig. 3B, lanes 3, 4 and 7), whereas enzymes such as SpeI and SacI, which had a single specific restriction site in the gene gave two bands after hybridization (lanes 2 and 5). Expression and purification of PsCIPK and PsCBL The pea cDNA encoding CIPK and CBL were cloned into the expression vector pET28a and the recombin- ant proteins were expressed in Escherichia coli. SDS ⁄ PAGE analysis showed a highly expressed a 58 kDa additional polypeptide for PsCIPK (Fig. 4A, lane 2) and a 26 kDa additional polypeptide for PsCBL (Fig. 4G, lane 2) in isopropyl thio-b-d-gal- actoside (IPTG) induced fractions, respectively, as compared to uninduced (lane 1). The recombinant PsCIPK and PsCBL were present in the soluble frac- tions and therefore purified in the soluble form through a single Ni 2+ –NTA–agarose column chroma- tography step. PsCIPK and PsCBL proteins, purified to near homogeneity, showed a 58-kDa (Fig. 4A, lane 3) and a 26-kDa band (Fig. 4G, lane 3), respectively. In western blotting, the anti-PsCIPK and PsCBL antibodies detected PsCIPK as a single band of 58 kDa (Fig. 4B, lane 2 and 3, respectively) and a single band 26 kDa of PsCBL (Fig. 4H, lane 2 and 3) in the IPTG-induced fraction and in the purified fraction. There was no signal in the uninduced frac- tion of PsCIPK (Fig. 4B, lane 1) or PsCBL (Fig. 4H, lane 1). The purified PsCIPK and PsCBL proteins were also recognized by anti-His antibody (data not shown). PsCBL encodes a functional Ca 2+ -binding protein The presence of conserved EF-hand motifs in the predicted protein sequence of PsCBL suggests that it may function as Ca 2+ -binding protein. To check the Ca 2+ -binding activity of PsCBL, the purified protein in two different concentrations (3 and 4 lg) along with positive and negative controls were fractionated by SDS ⁄ PAGE (Fig. 4I), electro-blotted onto mem- brane and incubated with radioactive 45 CaCl 2 (Fig. 4J). The same sets of proteins were also spotted on a membrane (dot blot) and treated as above (Fig. 4K). The results show that PsCBL binds to 45 Ca 2+ (Fig. 4J, lanes 2 and 3). The positive control Entamoeba histolytica calcium binding protein (EhCaBP) [22], showed binding to 45 Ca 2+ (Fig. 4J, lane 1), while negative controls (glutathione S-trans- ferase and BSA) lack the binding (Fig. 4J, lanes 4 and 5). Similar results were obtained with dot blot analysis (Fig. 4K). In Fig. 4K, spots 1 and 2 are PsCBL protein (3 and 4 lg), lanes 3 and 4 are the same negative controls and lane 5 is the positive NU 1caS 1RocE 11lgB 1ed N 111dn i H A CIPK 1abX 12.0 0.5 1.0 1.6 2.0 3.0 4.0 5.0 7.0 1epS B CBL kbkb 12.0 0.5 1.0 1.6 2.0 3.0 4.0 5.0 7.0 NU 11lgB 1edN 1epS Fig. 3. Southern blot analysis of PsCIPK and PsCBL to determine copy number in the pea genome. (A, B) Genomic DNA gel blots analysis. Pea genomic DNA (30 lg) was completely digested with the enzyme indicated, separated by electrophoresis, blotted and hybridized with the [a- 32 P]dCTP-labelled PsCIPK (1.2-Kb fragment from the 3¢ end containing the 3¢ UTR) (A) and [a- 32 P]dCTP-labelled PsCBL cDNA (0.97 Kb, full-length) (B), cDNAs as probes. Un, Uncut DNA. The DNA size (Kb) is indicated at the left. S. Mahajan et al. Stress-induced CIPK from pea phosphorylate CBL FEBS Journal 273 (2006) 907–925 ª 2006 The Authors Journal compilation ª 2006 FEBS 911 control. The CD spectrum of purified recombinant PsCBL (1.2 mgÆmL )1 ) in the presence and absence of Ca 2+ was markedly different (Fig. 4L). The spectrum of PsCBL changed significantly when Ca 2+ was either added or depleted by the addition of EGTA (Fig. 4L). No significant change in the spectra of the AB CD E F H IJ L K G Fig. 4. Purification of PsCIPK and PsCBL proteins and their activities. (A) Induction and purification of overexpressed PsCIPK in E. coli is shown on SDS ⁄ PAGE. Lane M, Molecular weight marker; lane 1, uninduced; lane 2, IPTG induced; lane 3, PsCIPK protein after Ni 2+ –NTA– agarose column chromatography. The protein size markers are indicated at the left side of the gel. (B) Western blot analysis of the same protein fractions of lanes 1–3 as shown in panel (A) using polyclonal anti-PsCIPK antiserum. (C, D) Autophosphorylation of PsCIPK and phos- phorylation of PsCBL by PsCIPK. PsCIPK protein in the presence of Mn 2+ (lane 1), Mg 2+ (lane 2), PsCBL plus Mn 2+ (lane 3), PsCBL plus Mg 2+ (lane 4) and casein plus Mg 2+ (lane 5) incubated with [c- 32 P]ATP in kinase buffer, electrophoresed on SDS ⁄ PAGE and stained with Coomassie blue (C) followed by autoradiography (D). (E) Phosphoamino acid analysis of PsCIPK autophosphorylation (lane 2) and PsCBL phosphorylation by PsCIPK (lane 1). Positions of phosphoserine (P-Ser), phosphothreonine (P-Thr), and phosphotyrosine (P-Tyr) are marked at right side of autoradiogram. (F) Immunodepletion of kinase activity of PsCIPK. PsCIPK protein was immunodepleted using anti-PsCIPK anti- bodies. Lane 1, Phosphorylation of PsCBL by PsCIPK (control without any IgG); lane 2, PsCIPK pretreated with preimmune IgG; lane 3, PsCIPK pretreated with anti-PsCIPK IgG. (G) Induction and purification of overexpressed PsCBL in E. coli is shown on SDS ⁄ PAGE. Lane M, Molecular weight marker; lane 1, uninduced; lane 2, IPTG induced; lane 3, PsCBL protein after Ni 2+ –NTA–agarose column chromatography. The protein size markers are indicated at the left side of the gel. (H) Western blot analysis of the same protein fractions of lanes 1–3 as shown in panel (G) using polyclonal anti-PsCBL antiserum. (I, J, K) 45 Ca 2+ overlay assay showing that PsCBL is a functional Ca 2+ binding pro- tein. (I) PsCBL along with the controls were run on 12% SDS ⁄ PAGE and stained with Coomassie blue. Lane 1, EhCaBP protein (positive control); lanes 2 and 3, PsCBL (3 and 4 lg); lanes 4 and 5, GST and BSA (negative controls). Lane M, Pre-stained marker. (J) The same sam- ples (as in panel I) transferred to nitrocellulose membrane and assayed by 45 Ca 2+ binding. Only PsCBL (lane 2 and 3) and the positive control (lane 1) showed Ca 2+ -binding capability. (K) Dot blot analysis of the same protein samples (as in panel I) followed by 45 Ca 2+ overlay assay to confirm the 45 Ca 2+ binding data. Spots 1 and 2, PsCBL proteins (3 and 4 lg); lanes 3 and 4, negative controls; lane 5, positive control. (L) CD spectra of PsCBL, calcium-bound PsCBL and the calcium-bound PsCBL treated with 1.25 m M EGTA. Stress-induced CIPK from pea phosphorylate CBL S. Mahajan et al. 912 FEBS Journal 273 (2006) 907–925 ª 2006 The Authors Journal compilation ª 2006 FEBS protein was observed by the addition or depletion of Mg 2+ (data not shown). These results suggest that PsCBL changes its conformation in a Ca 2+ -depend- ent manner. PsCIPK phosphorylates PsCBL at Thr residue(s) To determine whether PsCIPK is a functional protein kinase, the autophosphorylation and substrate phos- phorylation activities of the enzyme were checked by incubating the enzyme with [c- 32 P]ATP in the absence or presence of the substrates. After incubation, the phosphorylation of the proteins was examined by SDS ⁄ PAGE (Fig. 4C) followed by autoradiography (Fig. 4D). The result shows that PsCIPK autophos- phorylated (58 kDa) in the presence of Mn 2+ (Fig. 4D, lane 1) as well as Mg 2+ (Fig. 4D, lane 2). The sequence analysis of PsCBL revealed that it has putative phosphorylation sites (Fig. 1B). We therefore tested whether PsCBL is a substrate for the PsCIPK enzyme. The result shows that PsCBL is phosphorylat- ed strongly by PsCIPK in the presence of the divalent cations Mn 2+ and Mg 2+ (Fig. 4D lanes 3 and 4, respectively). We have shown that PsCIPK also phos- phorylates casein in the presence of Mg 2+ (Fig. 4D, lane 5). CBL has no effect on the autophosphorylation of CIPK (Fig. 4D, lane 3 and 4). PsCIPK phosphoryl- ated PsCBL even in the absence of exogenous Ca 2+ in the reaction buffer (data not shown). This data is sim- ilar to that reported earlier for AtCIPK1, where no effect was noted on substrate phosphorylation (MBP and casein) in the presence or absence of any exogen- ously supplied Ca 2+ in the reaction buffer [10]. For phosphoamino acid analysis, the radioactive autophosphorylated 58-kDa band of PsCIPK and the 26-kDa band of PsCBL from the above gel were excised, acid hydrolysed and subjected to paper chro- matography. The results show that PsCIPK phos- phorylates PsCBL at Thr residue(s) (Fig. 4E, lane 1) and also becomes autophosphorylated at its Thr resi- due(s) (Fig. 4E, lane 2). PsCBL did not show any autophosphorylation, as without any kinase there was no phosphorylation of CBL (data not shown). To confirm the phosphorylation activity of PsCIPK, an immunodepletion experiment was per- formed as follows. Purified PsCIPK was reacted sepa- rately with IgG purified from the sera of preimmune rabbit and a rabbit immunized with PsCIPK. The antigen–antibody complex was removed by protein A- Sepharose. The supernatant was analysed for PsCIPK activity to phosphorylate PsCBL. Results revealed that immunodepletion of PsCIPK in the extract decreased the phosphorylation of PsCBL significantly (Fig. 4F, lane 3),whereas there was no reduction of PsCIPK activity to phosphorylate PsCBL in the sam- ple treated with preimmune IgG (Fig. 4F, lane 2). Lane 1 is the control reaction without the addition of IgG. Regulation of transcript levels of PsCIPK and PsCBL in response to stress To analyse PsCIPK and PsCBL expression under various abiotic and biotic stresses, 7-day-old pea seedlings were stressed for different times. The control plants were grown without any stress treatment. Total RNAs were extracted from control and treated shoot tissues and hybridized with PsCIPK (1.2-Kb fragment from the 3¢ end containing the 3¢ UTR) and PsCBL (0.97 Kb, full-length) cDNA probes. As shown in Fig. 5, the transcript levels of both PsCIPK (panels A, C, E, G, I, K, and N) and PsCBL (panels B, D, F, H, J, L, and O) are coordinately regulated following a similar trend. Following low temperature treatment the transcripts of both genes started increasing from 9 h, reaching a maximum at 12–24 h (Fig. 5A and B). After NaCl treatment the levels increased after 12 h and were maintained high at least until 24 h (Fig. 5C and D). In response to wounding stress, both the genes showed an early induction at 3 h; however, the levels decreased by 6 h (Fig. 5E and F). The transcript levels in salicylic acid (SA) stress were increased after 8 h of treatment and then decreased at 12 h (Fig. 5I and J). Interestingly, the transcript levels of both the genes did not alter in response to dehydration stress (Fig. 5G and H) and after the exogenous application of ABA hormone (Fig. 5K and L). As a positive con- trol, an ABA responsive gene PDH45 (see Fig. 5 leg- ends). was used. The transcript level of PDH45 strongly increased from 12 to 24 h under similar experimental conditions (Fig. 5M). Calcium upregulates PsCIPK and PsCBL in a dose-dependent manner As PsCIPK and PsCBL are strongly upregulated in response to various abiotic and biotic stresses and as the signalling pathway for these stresses are often mediated by Ca 2+ , the effect of exogenous Ca 2+ was analysed on the transcript levels of both the genes. As shown in Fig. 5N the transcript level of PsCIPK was upregulated in response to Ca 2+ , reaching a maximum at 10 mm and declined at higher Ca 2+ concentrations (Fig. 5N). The transcript level of PsCBL was strongly upregulated by Ca 2+ . The level started increasing at 5 mm of exo- genously supplied Ca 2+ and the maximum level was S. Mahajan et al. Stress-induced CIPK from pea phosphorylate CBL FEBS Journal 273 (2006) 907–925 ª 2006 The Authors Journal compilation ª 2006 FEBS 913 observed at 50 mm and remained constant thereafter (Fig. 5O). To exclude the possibility of this upregulation being mediated via any divalent cation, the effect of Mg 2+ was also tested. Plants treated with 50 mm Mg 2+ for 24 h did not show any upregulation of transcripts of either of the genes (Fig. 5N and O, second lane). PsCBL B D F H J L O 18 S 1.0 kb 18 S 1.0 kb 18 S 1.0 kb 18 S 1.0 kb 18 S 1.0 kb 18 S 1.0 kb 18 S 1.0 kb PsCIPK A C E G I K N 18 S 1.8 kb 18 S 1.8 kb 18 S 1.8 kb 18 S 1.8 kb 18 S 1.8 kb 18 S 1.8 kb 18 S 1.8 kb CaCl 2 SALICYLIC ACID (150 µ M) COLD (4 0 C) SALINITY (150 m M NaCl ) WOUNDING DEHYDRATION ABSICIC ACID (100 µ M) M M M Fig. 5. Expression pattern of PsCIPK and PsCBL genes in response to various abiotic and biotic stresses. The total RNAs were extracted from leaf tissue after the stress treatment. The various abiotic stresses used for treatment of pea seedlings were cold (A and B), salinity (C and D), wounding (E and F), drought (G and H), SA (I and J), ABA (K and L) and calcium (N and O). Panel M is the control for ABA responsive gene, PDH45 [35]. The RNAs (50 lg) samples were separated by electrophoresis, blotted and hybridized with the [a- 32 P]dCTP-labelled PsCIPK (1.2-Kb fragment from 3¢ end containing the 3¢ UTR) (panels A, C, E, G, I, K and N), and [a- 32 P]dCTP-labelled PsCBL cDNA (0.97 Kb, full-length) probes (panels B, D, F, H, J, L, and O). For each stress examined the upper panel shows the autoradiograph of transcript (1.8 Kb for PsCIPK and 1 Kb for PsCBL), while the lower panel shows the hybridization of same blot with 18S rRNA gene (loading control). In each panel, lane 1 is the control (C) without any treatment while other lanes are the RNAs samples collected after stress treatments at the indicated time points. Stress-induced CIPK from pea phosphorylate CBL S. Mahajan et al. 914 FEBS Journal 273 (2006) 907–925 ª 2006 The Authors Journal compilation ª 2006 FEBS In vitro interaction of PsCBL with PsCIPK protein by far-western blotting As the two genes (PsCBL and PsCIPK) showed a sim- ilar and synchronized transcript profile, we speculated that these may interact with each other. We studied the interaction of PsCBL with PsCIPK by the far- western method (see Experimental procedures). Briefly, the two proteins and controls were separated by SDS ⁄ PAGE, transferred to nylon membrane and then renatured on the membrane. Next they were incubated with the second protein PsCBL in the presence or absence of CaCl 2 (1 mm), followed by western blotting with anti-CBL IgG. The results of far-western blotting showed that PsCBL binds to PsCIPK, which was recognized by anti-CBL IgG (Fig. 6B, lanes 1 and 2). This binding is calcium dependent as no signal was observed when the experiment was performed in the absence of calcium (data not shown). As a negative control, 47-kDa pea helicase (lane 3) and 80-kDa pea MCM7 protein (lane 4) were used; these produced no signal. Figure 6A, shows a Ponceau-S stained mem- brane in which lane 1 contains prephosphorylated CIPK which suggests that CBL can interact with both phosphorylated and nonphosphorylated forms of CIPK. To further confirm binding, the same experi- (-Leu,- Trp). (-Leu,-Trp,-His + 15 mM 3AT.) YPD MEDIA β galactosidase assa y . Pea CBL in pGBKT7 1.6 Pea CIPK in pGADT7 A C B D EFG HI JK 58 47 26 80 58 47 26 80 kDa kDa Ponseu-S Immuno blot Ponseu-S Immuno blot 0.5 1.0 1 2 3 4 5 6 1.6 1.0 2.0 3.0 2.0 kb kb Fig. 6. Direct interaction of PsCBL and PsCIPK proteins, in vitro,as well as via a yeast two-hybrid system. (A, B) PsCBL interacts with PsCIPK in vitro. PsCIPK prephosphorylated (2 lg, lane 1), PsCIPK (2 lg, lane 2), pea helicase (PDH47) [36] (6 lg, lane 3, negative control), pea MCM7 (3 lg, lane 4, negative control) and PsCBL (5 lg, lane 5) were run on SDS ⁄ PAGE, transferred to PVDF mem- branes, stained with Ponceau-S (A). The proteins on the same blot were denatured ⁄ renatured, blocked with BSA, incubated with 1 lgÆmL )1 CBL protein followed by standard western using anti- PsCBL antibodies (B). (C, D) PsCIPK interacts with PsCBL in vitro. The same set of proteins as (A) were stained with Ponceau-S (C) treated as above until the BSA blocking step, and then incubated with 1 lgÆmL )1 of PsCIPK protein and detected with anti-PsCIPK antibodies (D). (E–K) PsCIPK interacts with PsCBL in a yeast two- hybrid system. (E) The ORF of PsCBL was cloned in pGBKT7 and checked by restriction (NcoI and EcoRI) to show the insert size of 0.67 Kb (lane 2), lane 1 is the DNA marker. (F) The ORF of PsCIPK was cloned in pGADT7 vector and checked by restriction (EcoRI and XhoI) to give the insert size of 1.55 Kb (lane 2), lane 1 is DNA marker. (G) Template for panels H–K. (H) Phenotype on YPD plate showing uninhibited growth of all the above. (I) Phenotype on syn- thetic dextrose lacking Leucine and Trytophan (SD –Leu–Trp) plate; this is selection medium for double transformants. (J) Phenotype on synthetic dextrose lacking Leucine, Trytophan, and Histidine containing 15 m M 3-AT (SD–Leu–Trp–His+3AT) plate; here growth represent the interaction of PsCBL with PsCIPK. (K) b-galactosidase filter assay further confirms the interaction. The blue colour repre- sents interaction of both the proteins (PsCBL-CIPK) resulting in the expression of b-galactosidase reporter gene. S. Mahajan et al. Stress-induced CIPK from pea phosphorylate CBL FEBS Journal 273 (2006) 907–925 ª 2006 The Authors Journal compilation ª 2006 FEBS 915 ment was performed by incubating the same proteins on the membrane with the PsCIPK followed by West- ern blotting with anti-CIPK antibodies (Fig. 6C and D). The results show that PsCIPK can also bind to PsCBL (Fig. 6D, lane 5). PsCIPK did not bind to the negative controls (Fig. 6D). Figure 6C is a Ponceau-S stained membrane. Interaction of PsCBL with PsCIPK via yeast two-hybrid system The complete ORF of PsCBL (678 bp) was cloned into the NcoI and EcoRI sites of yeast two-hybrid binding domain vector (pGBKT7). The resulting construct (pGBKT7-PsCBL or BD-CBL) was verified by sequen- cing and digestion with NcoI and EcoRI to give a band of 678 bp (Fig. 6E, lane 2). On the other hand the com- plete ORF of PsCIPK (1.5 Kb) was cloned into the EcoRI and XhoI sites of yeast two-hybrid activating domain vector (pGADT7). The resulting construct (pGADT7-PsCIPK or AD-CIPK) was verified by sequencing and digestion with EcoRI and XhoI to give a band of 1.5 Kb on gel electrophoresis (Fig. 6F, lane 2). The Saccharomyces cervisiae AH109 cells were co- transformed with both the constructs (BD-PsCBL plus AD-PsCIPK) as well as with several combinations of plasmids which served as controls for this experiment. Interactions between PsCBL and PsCIPK were deter- mined by growth of the cotransformants on the selec- tion media of synthetic dextrose (SD) lacking Leu, Trp, and His (SD-Leu – Trp – His – and containing 15 mm 3-aminotrizole, 3-AT). 3-AT is a competitive inhibitor of histidine and checks any leaky expression of histi- dine. Yeast cells could survive due to the activation of the nutritional marker gene HIS3. Activation of the sec- ond reporter gene (lacZ), was monitored by measuring b-galactosidase activity. The results are shown in Fig. 6G–K. Figure 6G is a template for panels H to K showing the clones streaked: clone 1, Yeast (AH109) cells containing co- transformants of BD-PsCBL plus AD-PsCIPK; clone 2, cotransformants of BD-PsCBL and AD vector alone; clone 3, AD-PsCIPK and BD vector alone; clone 4, cotransformants of empty AD and BD vec- tors; clone 5, cotransformants of Pea Gb and Gc served as a positive control (unpublished data); clone 6, yeast AH109 cells alone. All these transformants and AH109 cells grew on the yeast extract–peptone– dextrose (YPD) plate (nonselective medium) (Fig. 6H). Except AH109 cells, all of the cotransformants con- taining AD and BD vectors showed growth on SD- Leu – Trp – medium (Fig. 6I). In a selection medium lacking Leu, Trp and His (SD-Leu – Trp – His – +15mm 3-AT) only selected clones of cotransformants (BD- PsCBL plus AD-PsCIPK) and the positive control, in which the HIS3 gene was transactivated grew (Fig. 6J). This confirmed the interaction of PsCBL and PsCIPK proteins. The results from b-galactosidase filter assay of colonies of cotransformants (BD-PsCBL plus AD- PsCIPK) further confirmed the interaction between PsCBL and PsCIPK (Fig. 6K, blue colonies). Domain swapping was also performed in which PsCBL was cloned in pGADT7 and PsCIPK was cloned in pGBKT7 and similar interaction results were obtained (data not shown). The results show that PsCIPK inter- acts with PsCBL in a yeast two-hybrid system. A PsCIPK mutant with a deletion in the autoinhibitory (NAF) motif failed to interact with PsCBL thus con- firming the authenticity of these proteins and emphasi- zing the importance NAF in the interaction between them (data not shown). Localization by immunofluorescence labelling and confocal microscopy Localization of PsCIPK and PsCBL was analysed by immunofluorescence labelling of tobacco BY2 cells fol- lowed by confocal microscopy. Cell cultures were found to better for these studies than a whole-plant system. Immunofluorescence labelling of tobacco BY2 cells with anti-PsCIPK (Fig. 7B) and anti-PsCBL (Fig. 7J) antibodies showed the localization of both proteins in the cytosol of all cells. In addition, PsCIPK protein was also localized in the outer membrane (Fig. 7B). A single enlarged cell showing PsCIPK localization is shown in Fig. 7E, whereas Fig. 7M is the single enlarged cell showing PsCBL localization. Figure 7A, D, I and L are diamidino-2phenylindole hydrochloride (DAPI) stained cells showing the nuc- leus, and Fig. 7C, F, K and N are the merged images of A and B, D and E, I and J, and L and M, respect- ively. Discussion Nature has developed many pathways for combating and tolerating the the various stress signals that cross- talk with each other. The CBL ⁄ CIPK pathway is one of these; it emerged as a novel pathway for deciphering calcium signatures and initiating a series of phosphory- lation cascades. This ultimately results in the expres- sion and regulation of stress genes mediating plant adaptation in response to array of stresses. The exist- ence of a large family of CIPK and CBL genes has been reported in Arabidopsis and rice [15]. However, the details of the role of these proteins and the inter- Stress-induced CIPK from pea phosphorylate CBL S. Mahajan et al. 916 FEBS Journal 273 (2006) 907–925 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... C )A( A ⁄ G) (A ⁄ G ⁄ T )A( C ⁄ T) (A ⁄ C ⁄ G)ACACCACAAGACC)3¢ 5¢-CTTAT(C ⁄ G)AACAAGGAA (A ⁄ C)AATTTC-3¢ 5¢-GTATCAGCTTC(C ⁄ T)TCAAATGTC-3¢ 5¢-CCATCACAAGAAACTAGAGAAAC-3 5¢-TTAAGTACTATAAAT-ACACAGCCTA-3¢ 5¢-CGAGCTCACTGCCTCTCAAC-3¢ 5¢-ACTCGTAGC-ACAGAGACAGAG-3¢ 5¢-ATGGCAGTAGTAGCAG-CTCC-3¢ 5¢-TCAGGTGTCT-AAGTTCAGAGATTC-3¢ 5¢-ATGTTGCAGTGCTTAGAGGGA-3¢ 5¢-TTAAGTATCATCTACTTGTGAATG-3¢ 5¢-CCTCCGGAATTCATGGCAGTAGTAGCAGCTCC-3¢... Chandok MR, Prasad J, Bhattacharya S, Sopory SK & Bhattacharya A (1997) Characterization FEBS Journal 273 (2006) 907–925 ª 2006 The Authors Journal compilation ª 2006 FEBS S Mahajan et al of EhCaBP, a calcium binding protein of Entamoeba histolytica and its binding proteins Mol Biochem Parasitol 84, 69–82 33 Pham XH, Reddy MK, Ehtesham NZ, Matta B & Tuteja N (2000) A DNA helicase from Pisum sativum is homologous... its kinase activity and C-terminal regulatory domain for salt tolerance in Arabidopsis thaliana Plant Cell 16, 435–449 Pandey GK, Cheonga YH, Kim K-N, Granta JJ, Li L, Hunga W, D’Angeloc C, Weinl S, Kudla J & Luana S (2004) The calcium sensor calcineurin B-like 9 modulates abscisic acid sensitivity and biosynthesis in Arabidopsis Plant Cell 16, 1912–1924 Nagae M, Nozawas A, Koizumi N, Sano H, Hashimoto... autoradiography PsCIPK was immunodepleted by addition of PsCIPK antibodies (IgG) in a standard phosphorylation reaction containing 1 mm CaCl2 and 5 mm MgCl2 as described [31] PsCBL phosphorylation by PsCIPK was also carried out in the kinase buffer containing 5 mm MgCl2 without addition of any exogenous Ca2+ 922 Phospho-amino acid analysis The phosphorylated 26-kDa band of PsCBL and the 58-kDa band of. .. SOS1, a plasma membrane Na+ ⁄ H+ exchanger in Arabidopsis thaliana, by SOS2 and SOS3 Proc Natl Acad Sci USA 99, 8436–8441 Shah J & Klessig DF (1999) In Biochemistry & Molecular Biology of Plant Hormones (Hooykaas PPJ, Hall MA & Libbenga KR, eds), pp 513–541 Elsevier, Amstradam Dat JF, Lopez-Delgado H, Foyer CH & Scott IM (1998) Parallel changes in H2O2 and catalase during thermotolerance induced by salicylic... This kinase showed dual localization in the cytosol and in the plasma membrane Characterization of PsCIPK and its interacting PsCBL Sequence analysis of PsCIPK showed that it has low similarity (67%) to AtCIPK12, which also has not been well characterized Regarding PsCBL, the amino acid sequence alignments revealed that PsCBL is more similar to rice CBL (92%) and AtCBL3 (90%) All 10 AtCBL genes are reported...S Mahajan et al Stress-induced CIPK from pea phosphorylate CBL A B C D Fig 7 Subcellular localization of PsCIPK and PsCBL in tobacco BY2 cells by immunofluorescence labelling and confocal microscopy The BY2 cells were fixed, permeabilized and treated with primary antibodies against PsCIPK (A F) and PsCBL (I–N) followed by Alexafluor488-labelled secondary antibodies and then counterstained with DAPI Multiple... salicylic acid or heat acclimation in mustard seedlings Plant Physiol 116, 1351– 1357 Borsani O, Valpuesta V & Botella MA (2001) Evidence for a role of salicylic acid in the oxidative damage generated by NaCl and osmotic stress in Arabidopsis seedlings Plant Physiol 126, 1024–1030 Pandey S, Tiwari SB, Tyagi W, Reddy MK, Upadhyaya C & Sopory SK (2002) A Ca2+ ⁄ CaM-dependent kinase from pea is stress regulated... controlling the major genes in the signalling pathway, much more effort should be invested in analysing the role of these genes in higher plants Here, we report the isolation and characterization of a novel CIPK from a legume, Pisum sativum (pea) and show that it interacts and phosphorylates a CBL having homology to AtCBL3 Both PsCIPK and PsCBL genes showed delayed transcriptional upregulation in response... cells are shown in panel A C for PsCIPK and I–K for PsCBL Confocal image of single enlarged cell is also shown in panel D–F for PsCIPK and panel L–N for PsCBL (A, D, I and L) Images of cells stained with DAPI (blue) (B, E, J and M) Alexafluor488-labelled immunostained cells showing florescence (green) Anti-PsCIPK labelling is seen in the cytosol as well as plasma membrane (B and E), while Anti-PsCBL labelling . (degenerate reverse) 55¢-CCATCACAAGAAACTAGAGAAAC-3 PsCIPK (5¢UTR forward) 65¢-TTAAGTACTATAAAT-ACACAGCCTA-3¢ PsCIPK (3¢UTR reverse) 75¢-CGAGCTCACTGCCTCTCAAC-3¢. Cloning and characterization of CBL-CIPK signalling components from a legume (Pisum sativum) Shilpi Mahajan, Sudhir K. Sopory and Narendra Tuteja Plant