Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family

In plants, numerous Ca(2+)-stimulated protein kinase activities occur through calcium-dependent protein kinases (CDPKs). These novel calcium sensors are likely to be crucial mediators of responses to diverse endogenous and environmental cues. However, the precise biological function(s) of most CDPKs remains elusive. The Arabidopsis genome is predicted to encode 34 different CDPKs. In this Update, we analyze the Arabidopsis CDPK gene family and review the expression, regulation, and possible functions of plant CDPKs. By combining emerging cellular and genomic technologies with genetic and biochemical approaches, the characterization of Arabidopsis CDPKs provides a valuable opportunity to understand the plant calcium-signaling network.     

Molecular evolution of calmodulin-like domain protein kinases (CDPKs) in plants and protists

Many genes for calmodulin-like domain protein kinases (CDPKs) have been identified in plants and Alveolate protists. To study the molecular evolution of the CDPK gene family, we performed a phylogenetic analysis of CDPK genomic sequences. Analysis of introns supports the phylogenetic analysis; CDPK genes with similar intron/exon structure are grouped together on the phylogenetic tree. Conserved introns support a monophyletic origin for plant CDPKs, CDPK-related kinases, and phosphoenolpyruvate carboxylase kinases. Plant CDPKs divide into two major branches. Plant CDPK genes on one branch share common intron positions with protist CDPK genes. The introns shared between protist and plant CDPKs presumably originated before the divergence of plants from Alveolates. Additionally, the calmodulin-like domains of protist CDPKs have intron positions in common with animal and fungal calmodulin genes. These results, together with the presence of a highly conserved phase zero intron located precisely at the beginning of the calmodulin-like domain, suggest that the ancestral CDPK gene could have originated from the fusion of protein kinase and calmodulin genes facilitated by recombination of ancient introns. Received: 11 July 2000 / Accepted: 18 April 2001     

Genome-wide identification of the rice calcium-dependent protein kinase and its closely related kinase gene families: comprehensive analysis of the CDPKs gene family in rice

In plants, calcium acts as a universal second messenger in various signal transduction pathways. The plant-specific calcium-dependent protein kinases (CDPKs) play important roles regulating downstream components of calcium signaling. We conducted a genome-wide analysis of rice CDPKs and identified 29 CDPK genes and eight closely related kinase genes, including five CDPK-related kinases (CRKs), one calcium and calmodulin-dependent protein kinase (CCaMK) and two phosphoenolpyruvate (PEP) carboxylase kinase-related kinases (PEPRKs). The mRNA splicing sites of the rice CDPKs, CRKs and PEPRKs (but not OsCCaMK) are highly conserved, suggesting that these kinases are derived from a common ancestor. RNA gel blot analyses revealed that the majority of rice CDPK genes exhibited tissue-specific expression. Expression of OsCPK9 was elevated in seedlings infected by rice blast, indicating that this gene plays an important role in signaling in response to rice blast treatment. Our genomic and bioinformatic analyses will provide an important foundation for further functional dissection of the rice CDPK gene family.     

Tetraploids Isatis indigotica are more responsive and adaptable to stresses than the diploid progenitor based on changes in expression patterns of a cold inducible Ii CPK1

In plants, Ca²⁺-dependent protein kinases (CDPKs) are characterized as important sensors of Ca²⁺ flux in response to varieties of biotic and abiotic stress. A comprehensive survey of global gene expression performed by using an Arabidopsis thaliana whole genome Affymetrix gene chip revealed that CDPK tends to be significantly higher in tetraploid Isatis indigotica than in diploid ones. To investigate different CDPK expression in response to polyploidy, a full-length cDNA clone (IiCPK1) encoding CDPK was isolated from the traditional Chinese medicinal herb I. indigotica cDNA library. IiCPK1 contains some basic features of CDPKs: a catalytic kinase domain including an ATP-binding domain and four EFhand calcium-binding motifs. Real-time PCR analysis indicated the expression of IiCPK1 from two kinds of I. indigotica (tetraploid and diploid). They both were induced in response to cold stress, but tetraploids I. indigotica which has good fertility, exhibited an enhanced resistance and higher yield, and presented to be more responsive and adaptable. Our results suggest that IiCPK1 gene plays a role in adapting to the environmental stress.     

VfCPK1, a gene encoding calcium-dependent protein kinase from Vicia faba, is induced by drought and abscisic acid

Calcium, one of the most ubiquitous second messengers, has been shown to be involved in a wide variety of responses in plants. Calcium-dependent protein kinases (CDPKs) (EC 2.7.1.37) are the predominant Ca(2+)-regulated serine/threonine protein kinase in plants and play an important role in plant calcium signal transduction. CDPKs are encoded by a large multigene family in many plants, which has been showed so far; however, the precise role of each specific CDPK is still largely unknown. A novel CDPK gene designated as VfCPK1 was cloned from epidermal peels of broad bean (Vicia faba L.) leaves using the rapid amplification of cDNA ends (RACE)-PCR technique and its expression was studied in detail. The VfCPK1 cDNA is 1783 bp long and contains an open reading frame of 1482 bp encoding 493 amino acids. VfCPK1 contains all conserved regions found in CDPKs and shows a high level of sequence similarity to many other plant CDPKs. VfCPK1 was highly expressed in leaves, especially in leaf epidermal peels of broad bean in mRNA and protein levels. Expressions of VfCPK1 at both the mRNA and protein levels were increased in leaves treated with abscisic acid or subjected to drought stress. Potential roles of VfCPK1 in epidermal peels are discussed. The nucleotide sequence data reported here were deposited in the GenBank database under accession number AY753552.     

Two mRNA species encoding calcium-dependent protein kinases are differentially expressed in sexual organs of Marchantia polymorpha through alternative splicing

In plants, calcium-dependent calmodulin-independent protein kinases (CDPKs) are the predominant calcium-regulated protein kinases and their genes are encoded by a multigene family. A CDPK gene was cloned from a liverwort, Marchantia polymorpha, which showed a high level of sequence similarities to other higher plant CDPK genes. The liverwort CDPK gene consisted of 9 exons and 8 introns. The 6th and 7th exons (Exon 6A and Exon 6B) were almost identical except for 4-amino acid substitutions, both of which coded for EF-hands in the calcium-binding domain. RT-PCR analysis revealed that two species of mature mRNA containing either Exon 6A or Exon 6B were generated from a single CDPK gene by mutually exclusive alternative splicing. Both histidine-tagged fusion proteins derived from cDNAs containing either Exon 6A or Exon 6B exhibited calcium-dependent protein kinase activity in vitro. Preferential accumulation of the mature mRNA with Exon 6A detected in male sexual organ implies possible sexual control of the ratio between the two CDPK isozymes through alternative splicing. Functions and evolution of CDPKs are discussed based on the structure and expression of the liverwort CDPK gene.     

Plants, symbiosis and parasites: a calcium signalling connection

A unique family of protein kinases has evolved with regulatory domains containing sequences that are related to Ca(2+)-binding EF-hands. In this family, the archetypal Ca(2+)-dependent protein kinases (CDPKs) have been found in plants and some protists, including the malarial parasite, Plasmodium falciparum. Recent genetic evidence has revealed isoform-specific functions for a CDPK that is essential for Plasmodium berghei gametogenesis, and for a related chimeric Ca(2+) and calmodulin-dependent protein kinase (CCaMK) that is essential to the formation of symbiotic nitrogen-fixing nodules in plants. In Arabidopsis thaliana, the analysis of 42 isoforms of CDPK and related kinases is expected to delineate Ca(2+) signalling pathways in all aspects of plant biology.     

A phyloproteomic characterization of in vitro autophosphorylation in calcium-dependent protein kinases

Calcium-dependent protein kinases (CDPKs) are a novel class of signaling molecules that have been broadly implicated in relaying specific calcium-mediated responses to biotic and abiotic stress as well as developmental cues in both plants and protists. Calcium-dependent autophosphorylation has been observed in almost all CDPKs examined, but a physiological role for autophosphorylation has not been demonstrated. To date, only a handful of autophosphorylation sites have been mapped to specific residues within CDPK amino acid sequences. In an attempt to gain further insight into this phenomenon, we have mapped autophosphorylation sites and compared these phosphorylation patterns among multiple CDPK isoforms. From eight CDPKs and two CDPK-related kinases from Arabidopsis thaliana and Plasmodium falciparum, 31 new autophosphorylation sites were characterized, which in addition to the previously described sites, allowed the identification of five conserved loci. Of the 35 total sites analyzed approximately one-half were observed in the N-terminal variable domain. Homology models were generated for the protein kinase and calmodulin-like domains, each containing two of the five conserved sites, to allow intelligent speculation regarding subsequent lines of investigation.     

Calcium-dependent protein kinase gene expression in response to physical and chemical stimuli in mungbean (Vigna radiata)

Protein kinases are important in eukaryotic signal transduction pathways. In this study we designed degenerate oligonucleotides corresponding to two conserved regions of protein kinases and using the polymerase chain reaction (PCR) have amplified a 141 bp fragment of DNA from mungbeans (Vigna radiata Rwilcz cv. Berken). Sequence analysis of the PCR products indicates that they encode several putative protein kinases with respect to their identity with other known plant protein kinases. Using one of the six fragments (CPK3-8), we isolated a 2022 bp cDNA (VrCDPK-1) from a Vigna radiata gt11 library. VrCDPK-1 has a 96 bp 5-untranslated region and a 465 bp 3-untranslated region and an open reading frame of 1461 bp. VrCDPK-1 contains all of the conserved regions commonly found in calcium dependent protein kinases (CDPK). VrCDPK-1 shares 24 to 89% sequence identity with previously reported sequences for plant CDPKs at the protein level. southern analysis revealed the presence of several copies of the CDPK gene. VrCDPK-1 expression was stimulated when mungbean cuttings were treated with CaCl₂, while treatment with MgCl₂ had no effect. We are reporting for the first time a CDPK gene in mungbean which is inducible by mechanical strain. Cuttings treated with indole-3-acetic acid (IAA) or subjected to salt stress showed an increase in VrCDPK-1 expression. There was a dramatic stimulation in VrCDPK-1 expression 6 h after cuttings were treated with cycloheximide.     

Isolation and sequence analysis of a cDNA clone for a carrot calcium-dependent protein kinase: homology to calcium/calmodulin-dependent protein kinases and to calmodulin

Recently, a novel type of calcium-dependent protein kinase (CDPK) that requires neither calmodulin nor phospholipids for activation, has been described in plants. We have isolated a cDNA clone for carrot CDPK by probing a library of somatic embryo cDNAs with oligonucleotides corresponding to highly conserved regions of protein kinases. The product of this gene overexpressed in Escherichia coli reacted strongly with monoclonal antibodies to soybean CDPK. The deduced amino acid sequence of carrot CDPK reveals two major functional domains. An N-terminal catalytic domain with greatest homology to calcium/calmodulin-dependent protein kinase type II from rat brain is coupled to a C-terminal calcium-binding domain resembling calmodulin. These features of the primary sequence explain how CDPK binds calcium and suggest a model for CDPK regulation based on similarities to animal calcium/calmodulin-dependent protein kinases.     

OsCDPK13, a calcium-dependent protein kinase gene from rice,is induced in response to cold and gibberellin

Ca²⁺-dependent protein kinases (CDPKs) (EC 2.7.1.37) are the predominant Ca²⁺-regulated serine/threonine protein kinase in plants and their genes are encoded by a multigene family. CDPKs are important components in signal transduction, but the precise role of each individual CDPK is still largely unknown. A CDPK gene designated as OsCDPK13 was cloned from rice seedlings and it showed a high level of sequence similarities to rice and other plant CDPK genes. OsCDPK13 contains all conserved regions found in CDPKs. It was a single copy gene and was highly expressed in root and leaf sheath tissues of rice seedlings. OsCDPK13 expression was increased in leaf sheath segments treated with gibberellin or subjected to cold stress. The results in this investigation, together with our previous studies, suggest that OsCDPK13 may be an important signaling component in rice seedlings under cold stress condition and in response to gibberellin.     

Expression of a grape calcium-dependent protein kinase ACPK1 in Arabidopsis thaliana promotes plant growth and confers abscisic acid-hypersensitivity in germination, postgermination growth, and stomatal movement

Calcium is an important second messenger involved in abscisic acid (ABA) signal transduction. Calcium-dependent protein kinases (CDPKs) are the best characterized calcium sensor in plants and are believed to be important components in plant hormone signaling. However, in planta genetic evidence has been lacking to link CDPK with ABA-regulated biological functions. We previously identified an ABA-stimulated CDPK from grape berry, which is potentially involved in ABA signaling. Here we report that heterologous overexpression of ACPK1 in Arabidopsis promotes significantly plant growth and enhances ABA-sensitivity in seed germination, early seedling growth and stomatal movement, providing evidence that ACPK1 is involved in ABA signal transduction as a positive regulator, and suggesting that the ACPK1 gene may be potentially used for elevating plant biomass production. The authors Xiang-Chun Yu, Sai-Yong Zhu, and Gui-Feng Gao contributed equally to this work.