Family Of CBL-interacting Protein Kinases Research Articles

Molecular and expression analysis indicate the role of CBL interacting protein kinases (CIPKs) in abiotic stress signaling and development in chickpea

Calcineurin B-like proteins (CBL)-interacting protein kinases (CIPKs) regulate the developmental processes, hormone signal transduction and stress responses in plants. Although the genome sequence of chickpea is available, information related to the CIPK gene family is missing in this important crop plant. Here, a total of 22 CIPK genes were identified and characterized in chickpea. We found a high degree of structural and evolutionary conservation in the chickpea CIPK family. Our analysis showed that chickpea CIPKs have evolved with dicots such as Arabidopsis and soybean, and extensive gene duplication events have played an important role in the evolution and expansion of the CIPK gene family in chickpea. The three-dimensional structure of chickpea CIPKs was described by protein homology modelling. Most CIPK proteins are localized in the cytoplasm and nucleus, as predicted by subcellular localization analysis. Promoter analysis revealed various cis-regulatory elements related to plant development, hormone signaling, and abiotic stresses. RNA-seq expression analysis indicated that CIPKs are significantly expressed through a spectrum of developmental stages, tissue/organs that hinted at their important role in plant development. The qRT-PCR analysis revealed that several CaCIPK genes had specific and overlapping expressions in different abiotic stresses like drought, salt, and ABA, suggesting the important role of this gene family in abiotic stress signaling in chickpea. Thus, this study provides an avenue for detailed functional characterization of the CIPK gene family in chickpea and other legume crops.

Evolution, gene expression, and protein‒protein interaction analyses identify candidate CBL-CIPK signalling networks implicated in stress responses to cold and bacterial infection in citrus

BackgroundCold is a major abiotic stress and Huanglongbing and citrus canker disease are two devastating bacterial diseases for citrus. The Ca2+-CBL-CIPK network is known to regulate different types of stress signalling in plants. How do CBL–CIPK signalling networks function in response to cold and infection by CLas or Xcc in citrus?ResultsEight calcineurin B-like proteins (CBLs) and seventeen CBL-interacting protein kinases (CIPKs) were identified from the cold-tolerant satsuma mandarin ‘Guijing2501’ (Citrus. unshiu) and CLas/Xcc-sensitive sweet orange (C. sinensis). Phylogenetic analysis revealed that both CBL and CIPK family members in citrus were classified into an ancient and a recent clade according to their conserved domain characteristics and/or intron/exon structures. Genome duplication analysis suggested that both tandem and segmental duplications contributed to the amplification of the CBL and CIPK gene families in citrus under intense purifying selection, and the duplication events only existed in the recent clades. Expression comparison of the duplicated gene pairs indicated that the duplicated CBL and CIPK genes underwent functional differentiation. Further expression analysis identified that CBL1, 5, 6, and 8 and CIPK2, 8, 12, 15, 16, and 17 were significantly regulated by multiple stresses, including cold, Xcc infection and/or CLas infection, in citrus, whereas CBL2/7 and CIPK1/4/5/11/13/14 were independently highly regulated by cold and CIPK3 was uniquely responsive to Xcc infection. The combination analyses of targeted Y2H assay and expression analysis revealed that CBL6-CIPK8 was the common signalling network in response to cold and Xcc infection, while CBL6/CBL8-CIPK14 was uniquely responsive to cold in citrus. Further stable transformation and cold tolerance assay indicated that overexpression of CuCIPK16 enhanced the cold tolerance of transgenic Arabidopsis with higher POD activity and lower MDA content.ConclusionsIn this study, evolution, gene expression and protein‒protein interaction analyses of citrus CBLs and CIPKs were comprehensively conducted over a genome-wide range. The results will facilitate future functional characterization of individual citrus CBLs and CIPKs under specific stresses and provide clues for the clarification of cold tolerance and disease susceptibility mechanisms in corresponding citrus cultivars.

Halophyte Nitraria billardieri CIPK25 mitigates salinity-induced cell damage by alleviating H2O2 accumulation.

The plant-specific module of calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) play a crucial role in plant adaptation to different biotic and abiotic stresses in various plant species. Despite the importance of the CBL-CIPK module in regulating plant salt tolerance, few halophyte CIPK orthologs have been studied. We identified NbCIPK25 in the halophyte Nitraria billardieri as a salt-responsive gene that may improve salt tolerance in glycophytes. Sequence analyses indicated that NbCIPK25 is a typical CIPK family member with a conserved NAF motif, which contains the amino acids: asparagine, alanine, and phenylalanine. NbCIPK25 overexpression in salt-stressed transgenic Arabidopsis seedlings resulted in enhanced tolerance to salinity, a higher survival rate, longer newly grown roots, more root meristem cells, and less damaged root cells in comparison to wild-type (WT) plants. H2O2 accumulation and malondialdehyde (MDA) content were both deceased in NbCIPK25-transgenic plants under salt treatment. Furthermore, their proline content, an important factor for scavenging reactive oxygen species, accumulated at a significantly higher level. In concordance, the transcription of genes related to proline accumulation was positively regulated in transgenic plants under salt condition. Finally, we observed a stronger auxin response in salt-treated transgenic roots. These results provide evidence for NbCIPK25 improving salt tolerance by mediating scavenging of reactive oxygen species, thereby protecting cells from oxidation and maintaining plant development under salt stress. These findings suggest the potential application of salt-responsive NbCIPK25 for cultivating glycophytes with a higher salt tolerance through genetic engineering.

The CBL-interacting protein kinase CaCIPK13 positively regulates defence mechanisms against cold stress in pepper.

Cold stress is one of the main factors limiting growth and development in pepper. Calcineurin B-like proteins (CBLs) are specific calcium sensors with non-canonical EF-hands to capture calcium signals, and interact with CBL-interacting protein kinases (CIPKs) in the regulation of various stresses. In this study, we isolated a cold-induced CIPK gene from pepper named CaCIPK13, which encodes a protein of 487 amino acids. In silico analyses indicated that CaCIPK13 is a typical CIPK family member with a conserved NAF motif, which consists of the amino acids asparagine, alanine, and phenylalanine. The CaCIPK13 protein was located in the nucleus and plasma membrane. Knock down of CaCIPK13 resulted in enhanced sensitivity to cold stress in pepper, with increased malondialdehyde content, H2O2 accumulation, and electrolyte leakage, while the catalase, peroxidase, superoxide dismutase activities and anthocyanin content were decreased. The transcript level of cold and anthocyanin-related genes was substantially decreased in CaCIPK13-silenced pepper leaves relative to the empty vector control. On the contrary, overexpression of CaCIPK13 in tomato improved cold tolerance via increasing anthocyanin content and activities of reactive oxygen species scavenging enzymes. Furthermore, the interaction of CaCIPK13 with CaCBL1/6/7/8 was Ca2+-dependent. These results indicate that CaCIPK13 plays a positive role in cold tolerance mechanism via CBL-CIPK signalling.

Genome-wide analysis of CBL and CIPK family genes in cotton: conserved structures with divergent interactions and expression.

The online version contains supplementary material available at (doi:10.1007/s12298-021-00943-1).

Foxtail millet (Setaria italica (L.) P. Beauv) CIPKs are responsive to ABA and abiotic stresses.

CBL-interacting protein kinases (CIPKs) have been shown to regulate a variety of environmental stress-related signalling pathways in plants. Foxtail millet (Setaria italica (L.) P. Beauv) is known worldwide as a relatively stress-tolerant C4 crop species. Although the foxtail millet genome sequence has been released, little is known about the functions of CIPKs in foxtail millet. Therefore, a systematic genome-wide analysis of CIPK genes in foxtail millet was performed. In total, 35 CIPK members were identified in foxtail millet and divided into four subgroups (I to IV) on the basis of their phylogenetic relationships. Phylogenetic and gene structure analyses clearly divided all SiCIPKs into intron-poor and intron-rich clades. Cis-element analysis subsequently indicated that these SiCIPKs may be involved in responses to abiotic stimuli, hormones, and light signalling during plant growth and development, and stress-induced expression profile analysis revealed that all the SiCIPKs are involved in various stress signalling pathways. These results suggest that the CIPK genes in foxtail millet exhibit the basic characteristics of CIPK family members and play important roles in response to abiotic stresses. The results of this study will contribute to future functional characterization of abiotic stress responses mediated by CIPKs in foxtail millet.

Salt stress triggers augmented levels of Na+, Ca2+ and ROS and alter stress-responsive gene expression in roots of CBL9 and CIPK23 knockout mutants of Arabidopsis thaliana

Salinity stress adversely affects plant growth and development. Calcineurin B-like proteins (CBLs) are a family of plant calcium sensors that relay signals by interacting with a family of CBL-interacting protein kinases (CIPKs). The current work entails a reverse genetic approach for deciphering the roles of CBL9 and CIPK23 in the roots of Arabidopsis thaliana (Arabidopsis) during salinity stress. The knockout mutants cbl9 and cipk23 showed salt sensitivity and significantly higher levels of sodium ion (Na+), cytosolic calcium [Ca2+]cyt, and reactive oxygen species (ROS) and an increased death of the cells in root tips compared to the wild-type (WT) under salinity stress (100 mM NaCl). Further, we intended to decipher the effect of salinity stress on the relative expression levels of few key genes that play pivotal role in salinity stress responses and ROS-scavenging activities in the roots of the WT and the mutant seedlings. Compared with the WT, the relative expression levels of the genes in cbl9 and/or cipk23 were significantly augmented (CAX1, 2 and 4, ACA1, CNGC14 and 19, 5PTase2 and DHAR2), attenuated (SOS1, APX1, ALX8, CAT2 and 3, DHAR1, SQD1 and 2) and remained unaffected (NHX1–6 and 8 and GST11). These findings were further corroborated with the increased level of stress markers namely Hydrogen peroxide (H2O2) and Malondialdehyde (MDA) and reduction in chlorophyll, biomass and ascorbate peroxidase (APX) activity in mutants under salt stress condition. The results thus highlighted pivotal roles of CBL9 and CIPK23 on salinity stress responses and ROS-scavenging activities in the roots during salinity stress and a schematic model is presented.

Evolution of an intron-poor cluster of the CIPK gene family and expression in response to drought stress in soybean.

Calcium ion is an intracellular messenger that plays a central role in signal transduction pathways. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) signal network have shown different functions in the Ca2+ signaling process. In this work, we identified the entire soybean (Glycine max) CIPK gene family, which comprised 52 genes and divided into four subgroups (I to IV) based on phylogeny. The gene structural analysis separated these 52 genes into an intron-rich clade and an intron-poor clade. Chromosomal location analysis resulted in the identification of 22 duplicated blocks and six tandem duplication events. Phylogenetic classification of 193 CIPK proteins from representative plant species suggested that the intron-poor clade of CIPKs originated in seed plants. Analysis of global gene expression patterns of soybean CIPK family revealed that most intron-poor soybean CIPK genes are drought-inducible; a finding that was further confirmed using qRT-PCR. Our study provides a foundation for further functional analysis to reveal the roles that CIPKs and more specifically the intron-poor clade play in drought tolerance in soybean.

Genome-wide gene phylogeny of CIPK family in cassava and expression analysis of partial drought-induced genes.

Cassava is an important food and potential biofuel crop that is tolerant to multiple abiotic stressors. The mechanisms underlying these tolerances are currently less known. CBL-interacting protein kinases (CIPKs) have been shown to play crucial roles in plant developmental processes, hormone signaling transduction, and in the response to abiotic stress. However, no data is currently available about the CPK family in cassava. In this study, a total of 25 CIPK genes were identified from cassava genome based on our previous genome sequencing data. Phylogenetic analysis suggested that 25 MeCIPKs could be classified into four subfamilies, which was supported by exon-intron organizations and the architectures of conserved protein motifs. Transcriptomic analysis of a wild subspecies and two cultivated varieties showed that most MeCIPKs had different expression patterns between wild subspecies and cultivatars in different tissues or in response to drought stress. Some orthologous genes involved in CIPK interaction networks were identified between Arabidopsis and cassava. The interaction networks and co-expression patterns of these orthologous genes revealed that the crucial pathways controlled by CIPK networks may be involved in the differential response to drought stress in different accessions of cassava. Nine MeCIPK genes were selected to investigate their transcriptional response to various stimuli and the results showed the comprehensive response of the tested MeCIPK genes to osmotic, salt, cold, oxidative stressors, and ABA signaling. The identification and expression analysis of CIPK family suggested that CIPK genes are important components of development and multiple signal transduction pathways in cassava. The findings of this study will help lay a foundation for the functional characterization of the CIPK gene family and provide an improved understanding of abiotic stress responses and signaling transduction in cassava.

Characterization of Alkali Stress‐Responsive Genes of the CIPK Family in Sweet Sorghum [Sorghum bicolor (L.) Moench

ABSTRACTCalcineurin B–like protein (CBL) interacting protein kinases (CIPKs) are critical components in various stress signal transduction pathways. In previous studies, 32 putative CIPK genes (SbCIPKs) were identified in sweet sorghum [Sorghum bicolor (L.) Moench]. In the present study, a conserved sucrose nonfermenting 1 (SNF1)–like kinase domain in the N terminus and a NAF motif (a 24‐amino‐acid domain with the conserved amino acids N, A, and F required for the CBL–CIPK interaction) in C terminus were identified in all SbCIPKs using sequence analysis. Stress‐ and light‐responsive cis‐elements were also identified in the promoter regions of sorghum CIPKs. The real‐time polymerase chain reaction analysis revealed different expression patterns among the 32 SbCIPKs under sodium carbonate (Na2CO3) stress in sweet sorghum seedlings. Based on the expression patterns, six SbCIPKs were upregulated >10‐fold at the transcriptional level. Interestingly, these six CIPKs showed leaf‐specific expression under normal growth conditions, suggesting a relationship between various regulations in leaf function and alkali stress responses in sweet sorghum. Systemic yeast two‐hybrid assay demonstrated the specific interaction between multiple SbCIPKs and SbCBLs. Thus, this study provides the first characterization of the role of the CIPK family proteins in response to alkali stress.

A systematic view of the rice calcineurin B-like protein interacting protein kinase family

Calcium (Ca2+), as a universal second messenger, plays an important role in various signal transduction pathway networks in plants. There are several components of Ca2+ mediating signaling pathways including calcium-dependent protein kinases, calmodulin and calcineurin B-like proteins (CBLs), and CBL-interacting protein kinases (CIPK). Here, we provide a systematic view of 33 rice CIPK (OsCIPK) family members, which are further classified into seven subgroups. The integration of various meta-expression data such as anatomy, diurnal regulation, and phylogenomic tree context of the OsCIPK family reveals that 11 of the family members show preferred expression in leaves during vegetative growth. In addition, 20 OsCIPK genes show peak expression during the day, while none of the genes peaked expression in the dark. Regarding abiotic stress, 16 OsCIPK genes were induced by drought stress in leaf organs; of these, 14 OsCIPK genes showed up-regulation and two OsCIPK genes were down-regulated based on quantitative RT-PCR analyses. We then developed a functional gene network that consists of 15 out of the 16 genes. This network proposes a useful hypothetical model to understand the molecular mechanism of drought response and circadian regulation associated with OsCIPK family genes.

Two poplar calcineurin B-like proteins confer enhanced tolerance to abiotic stresses in transgenic Arabidopsis thaliana

Calcium is a critical component in a number of plant signal transduction pathways and the calcineurin B-like protein (CBL) family is a unique group of calcium sensors regulating a family of CBL-interacting protein kinases (CIPKs). In this study, two poplar CBL genes, PeCBL6 (GenBank acc. No. DQ907710) and PeCBL10 (GenBank acc. No. DQ899956), were characterized in transgenic Arabidopsis thaliana, particularly with regard to its role in abiotic stress resistance. Expression of the two CBL genes in poplar was induced by cold, drought, or high salinity, but not by abscisic acid (ABA) treatment. In Arabidopsis thaliana, PeCBL6 was found in the nucleus and PeCBL10 in the tonoplast. Transgenic Arabidopsis plants overexpressing PeCBL6 or PeCBL10 showed enhanced tolerance to high salinity, drought and low temperature. These results suggested that PeCBL6 and PeCBL10 may function as positive regulators of salt, drought and temperature responses.

Two CBL genes from Populus euphratica confer multiple stress tolerance in transgenic triploid white poplar

In plants, the calcineurin B-like protein (CBL) family is a unique group of calcium sensors that play a key role in decoding calcium transients by specifically interacting with and regulating a family of CBL-interacting protein kinases. In this study, two CBL genes (PeCBL6 and PeCBL10) from Populus euphratica were isolated by reverse transcription-polymerase chain reaction. To examine their functions, the corresponding cDNAs, which expression was driven by the cauliflower mosaic virus 35S promoter, were cloned into a plant expression vector pCAMBIA1301 and introduced into the triploid white poplar (Popolus tomentosa ‘YiXianCiZhu B385’). The physiological parameters, including the percentage of wilted leaves, height growth rate, chlorophyll content, malondialdehyde (MDA) content and relative electrical conductivity (REC) of transgenic lines and wild type (WT) plants, were measured and compared. The results showed that the MDA content and REC of transgenic plants were significantly lower in transgenic plants compared to WT plants when exposed to cold stress and that the presence of the PeCBL gene can induce an increase in height growth rate and a reduction in the number of wilted leaves in comparison to non-transgenic poplars under high salt, drought and cold stress. These results clearly demonstrate that transgenic plants had a greater tolerance to stresses than non-transgenic seedlings, suggesting that PeCBLs might play an important role in stress tolerance.

Molecular cloning and functional characterization of MdSOS2 reveals its involvement in salt tolerance in apple callus and Arabidopsis

Plants respond to various environmental stresses by activating "stress genes". CIPKs (CBL-interacting protein kinases) family genes play an important role in the process of stress response. In this study, a CIPK gene MdSOS2 was isolated from apple (Malus × Domestica). Sequence alignment and phylogenetic analysis showed that it is highly similar with Arabidopsis AtSOS2 and contained the conserved domains and motifs. Expression analysis demonstrated that MdSOS2 expressed in all tested organs at different levels, and positively in response to salt stress. Furthermore, the ectopic expression of MdSOS2 complemented the function of Arabidopsis sos2 mutant, and conferred enhanced salt tolerance to the transgenic Arabidopsis. Yeast two-hybrid assay indicated that the N-terminal of MdSOS2 protein physically interacted with MdSOS3 and AtSOS3, respectively, suggesting that SOS pathway operates in apple tree. Finally, MdSOS2 overexpression enhanced, while its suppression reduced the tolerance to salt in transgenic apple calluses, indicating that MdSOS2 acts as a positive regulator in response to salt stress in apple.

CIPK7 is involved in cold response by interacting with CBL1 in Arabidopsis thaliana

The family of calcineurin B-like (CBL) proteins is a unique group of Ca 2+ sensors in plants. CBLs relay the calcium signal by interacting with and regulating the family of CBL-interacting protein kinases (CIPKs). Extensive studies have demonstrated that the CBL–CIPK complexes mediate plant responses to a variety of external stresses. However, there are few reports on the CBL–CIPK involved in cold stress responses. In this study, we analyzed expression of CIPK7 and CBL1 in Arabidopsis during cold treatments. Expression of CIPK7 was induced by cold, and CIPK7 interacted with CBL1 in vitro. Moreover, affinity chromatography purification of CIPK7 from Arabidopsis plants using CBL1 suggested that CIPK7 may associate with CBL1 in vivo. Expression of CBL1 was cold inducible, and CBL1 had a role in regulating cold response. By comparing expression patterns of CIPK7 between wild-type and cbl1 mutant plants, we found the induction of CIPK7 by cold stress was influenced by CBL1. This is the first report to demonstrate that CIPK7 may play a role in cold response via its interaction with CBL1.

Identification and characterization of putative CIPK genes in maize

Calcium (Ca) plays a crucial role as a second messenger in intracellular signaling elicited by developmental and environmental cues. Calcineurin B-like proteins (CBLs) and their target proteins, CBL-interacting protein kinases (CIPKs) have emerged as a key Ca(2+)-mediated signaling network in response to stresses in plants. Bioinformatic analysis was used to identify 43 putative ZmCIPK (Zea mays CIPK) genes in the genome of maize inbred line B73. Based on gene structures, these ZmCIPKs were divided into intron-rich and intron-poor groups. Phylogenetic analysis indicated that the ZmCIPK family had a high evolutionary relationship with the rice CIPK family of 30 members. Microarray data and RT-PCR assay showed that ZmCIPK genes transcriptionally responded to abiotic stresses, and that 24, 31, 20 and 19 ZmCIPK genes were up-regulated by salt, drought, heat and cold stresses, respectively. There were different expression patterns of ZmCIPKs between cold-tolerant inbred line B73 and cold-sensitive inbred line Mo17 under cold stress. Our findings will aid further molecular dissection of biological functions of the CIPKs in maize, and provide new insight into the CBL-CIPK signaling network in plants.

Constitutive Overexpression of the Calcium Sensor CBL5 Confers Osmotic or Drought Stress Tolerance in Arabidopsis

Calcium serves as a critical messenger in many adaptation and developmental processes. Cellular calcium signals are detected and transmitted by sensor molecules such as calcium-binding proteins. In plants, the calcineurin B-like protein (CBL) family represents a unique group of calcium sensors and plays a key role in decoding calcium transients by specifically interacting with and regulating a family of CBL-interacting protein kinases (CIPKs). In this study, we report the role of Arabidopsis CBL5 gene in high salt or drought tolerance. CBL5 gene is expressed significantly in green tissues, but not in roots. CBL5 was not induced by abiotic stress conditions such as high salt, drought or low temperature. To determine whether the CBL5 gene plays a role in stress response pathways, we ectopically expressed the CBL5 protein in transgenic Arabidopsis plants (35S-CBL5) and examined plant responses to abiotic stresses. CBL5-overexpressing plants displayed enhanced tolerance to high salt or drought stress. CBL5 overexpression also rendered plants more resistant to high salt or hyperosmotic stress during early development (i.e., seed germination) but did not alter their response to abiscisic acid (ABA). Furthermore, overexpression of CBL5 alters the gene expression of stress gene markers, such as RD29A, RD29B and Kin1 etc. These results suggest that CBL5 may function as a positive regulator of salt or drought responses in plants.

The CBL–CIPK Ca2+‐decoding signaling network: function and perspectives

Calcium serves as a versatile messenger in many adaptation and developmental processes in plants. Cellular calcium signals are detected and transmitted by calcium-binding proteins functioning as sensor molecules. The family of calcineurin B-like (CBL) proteins represents a unique group of calcium sensors and contributes to the decoding of calcium transients by interacting with and regulating the family of CBL-interacting protein kinases (CIPKs). In higher plants, CBL proteins and CIPKs form a complex signaling network that allows for flexible but specific signal-response coupling during environmental adaptation reactions. This review presents novel findings concerning the evolution of this signaling network and key insights into the physiological function of CBL-CIPK complexes. These aspects will be presented and discussed in the context of emerging functional principles governing efficient and specific information processing in this signaling system.

Comparative genomic analysis of CIPK gene family in Arabidopsis and Populus

Calcium serves as a second messenger in various signal transduction pathways in plants. CBL-interacting protein kinases (CIPKs), which have a variety of functions, are involved in calcium signal transduction. Previous, the studies on CIPK family members focused on Arabidopsis and rice. Here, we present a comparative genomic analysis of the CIPK gene family in Arabidopsis and poplar, a model tree species. Twenty-seven potential CIPKs were identified from poplar using genome-wide analysis. Like the CIPK gene family from Arabidopsis, CIPK genes from poplar were also divided into intron-free and intron-harboring groups. In the intron-harboring group, the intron distribution of CIPKs is rather conserved during the genome evolutionary process. Many homologous gene pairs were found in the CIPK gene family, indicating duplication events might contribute to the amplification of this gene family. The phylogenetic comparison of CIPKs in combination with intron distribution analysis revealed that CIPK genes from both Arabidopsis and poplar might have an ancient origin, which formed earlier than the separation of these two eudicot species. Our genomic and bioinformatic analysis will provide an important foundation for further functional dissection of the CBL-CIPK signaling network in poplars.

Cloning and characterization of CBL-CIPK signalling components from a legume (Pisum sativum).

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 recombinant PsCIPK protein was autophosphorylated at Thr residue(s). Immunoprecipitation 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 phosphorylated 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.