Genome-wide identification, classification, and expression analysis of CDPK and its closely related gene families in poplar (Populus trichocarpa)

Calcium-dependent protein kinases (CDPKs) are Ca²⁺-binding proteins known to play crucial roles in Ca²⁺ signal transduction pathways which have been identified throughout plant kingdom and in certain types of protists. Genome-wide analysis of CDPKs have been carried out in Arabidopsis, rice and wheat, and quite a few of CDPKs were proved to play crucial roles in plant stress responsive signature pathways. In this study, a comprehensive analysis of Populus CDPK and its closely related gene families was performed, including phylogeny, chromosome locations, gene structures, and expression profiles. Thirty Populus CDPK genes and twenty closely related kinase genes were identified, which were phylogenetically clustered into eight distinct subfamilies and predominately distributed across fifteen linkage groups (LG). Genomic organization analyses indicated that purifying selection has played a pivotal role in the retention and maintenance of Populus CDPK gene family. Furthermore, microarray analysis showed that a number of Populus CDPK and its closely related genes differentially expressed across disparate tissues and under various stresses. The expression profiles of paralogous pairs were also investigated to reveal their evolution fates. In addition, quantitative real-time RT-PCR was performed on nine selected CDPK genes to confirm their responses to drought stress treatment. These observations may lay the foundation for future functional analysis of Populus CDPK and its closely related gene families to unravel their biological roles.     

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.     

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.     

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     

Cold stress-induced calcium-dependent protein kinase(s) in rice (Oryza sativa L.) seedling stem tissues

Ca²⁺-dependent protein kinases (CDPKs) play an important role in plant signal transduction. Protein kinase(s) activities induced by 5°C cold stress in rice (Oryza sativa L.) seedlings were investigated in both leaf and stem tissues in an early (up to 45 min) and late (up to 12 h) response study. The leaf had 37-, 47- and 55-kDa protein kinase activities, and the stem had 37-, 47- and 55-kDa protein kinase activities. A 16-kDa protein showed constitutive kinase activity in the rice seedling leaf and stem. It was further identified that the 47-kDa protein kinase activity induced by cold in both the cytosolic and membrane fractions of the stem was strictly Ca²⁺-dependent. This CDPK activitiy increased in the presence of the Ca²⁺ ionophore A23187 in stem segments, whereas it was decreased by the Ca²⁺ channel blocker, LaCl₃, and the Ca²⁺ chelator, EGTA. The general protein kinase inhibitor, staurosporine, completely inhibited this CDPK activity in vitro, and both W7, a calmodulin antagonist, and H7, a protein kinase C inhibitor, could only partially decrease this activity. The protein phosphatase inhibitor, okadaic acid, increased CDPK activity. This CDPK activity was also induced by salt, drought stress and the phytohormone abscicic acid. Among the 18 rice varieties tested, this cold-induced 47-kDa CDPK activity was stronger in the cold-tolerant varieties than in the sensitive ones. Received: 13 August 1999 / Accepted: 24 January 2000     

Biotic and abiotic stress responses through calcium-dependent protein kinase (CDPK) signaling in wheat (Triticum aestivum L.)

Calcium-dependent protein kinases (CDPKs) sense the calcium concentration changes in plant cells and play important roles in signaling pathways for disease resistance and various stress responses as indicated by emerging evidences. Among the 20 wheat CDPK genes studied, 10 were found to respond to drought, salinity and ABA treatments. Consistent with previous observations, one CDPK gene was shown to respond to multiple abiotic stresses in wheat suggesting that CDPKs could be converging points for multiple signaling pathways. Among the 12 wheat CDPK genes that were responsive to Blumeria graminis tritici (Bgt) infection or the treatment of hydrogen peroxide (H₂O₂), eight also responded to abiotic stresses, suggesting a cross-talk between biotic and abiotic stress signaling pathways. Phylogenetic analysis indicated that some of these genes were closely related to CDPKs from other species, whose functions have been partially studied, suggesting similar functions wheat CDPK genes. Combining the up-to-date knowledge of CDPK functions and our observations, a model was developed to project the possible roles of wheat CDPK genes in the signaling of biotic and abiotic stress responses.Key words: CDPK, calcium, kinase, stress response, disease resistance, signal transduction, wheatSessile plants have developed sophisticated signaling pathways to deal with dramatic environmental changes that may affect their normal growth, such as pathogen attack, drought, and cold. Calcium is a universal secondary messenger that responds to these stimuli. The fluctuation in cytosolic Ca²⁺ levels can be sensed by calcium-dependent protein kinases (CDPKs), which will modify the phosphorylation status of substrate proteins.^1–3 Accumulating evidence indicate that CDPKs mediate biotic and abiotic stress signaling pathways.^4–7 For example, overexpression of the rice CDPK gene OsCDPK7 provides cold, salt, and drought tolerance for the transgenic rice plants, demonstrating the potential of CDPK engineering to generate stress tolerance enhanced crops.^8,9In wheat, 10 out of 14 CDPK genes appeared to respond to abiotic stresses including drought, NaCl, as well as ABA stimulus (Fig. 1A).¹⁰ Five CDPKs (TaCPK4, 6, 9, 10 and 18) were particularly interesting since they could respond to at least two of the three treatments, among which the expression level of TaCPK9 was enhanced under all three treatments suggesting that TaCPK9 is the point where multiple signaling pathways cross. In wheat, TaCPK4 responded to both ABA treatment and NaCl stress (Fig. 1A). Interestingly, its best Arabidopsis homologs AtCPK4 and AtCPK11, as suggested by a Neighbor-Joining phylogenetic analysis (Fig. 1B), have been postulated as two important positive regulators in CDPK/calcium-mediated ABA signaling pathways.¹¹ Such a correlation strongly supports the idea that TaCPK4 is a good candidate in wheat for ABA signaling. Figure 1A also shows that one wheat CDPK gene could respond to multiple abiotic stresses suggesting that CDPKs are converging points for multiple signaling pathways. On the other hand, multiple CDPKs were involved in single stress response. It is however not clear how these CDPKs are organized in one signaling pathway.Open in a separate windowFigure 1The roles of wheat CDPKs in abiotic and biotic stress responses. (A) One CDPK gene responded to multiple abiotic stresses and multiple CDPKs were required for single stress response. (B) Phylogenetic relationship of wheat CDPKs with functionally studied CDPKs from barley (HvCPKs), Arabidopsis (AtCPKs), and potato (StCDPKs) that are known to be involved in ABA signaling, oxidative burst regulation and defense to powdery mildew pathogenesis. (C) A model depicting CDPK-mediated signaling pathways under biotic and abiotic treatments in wheat (see text for details). Dotted lines with a question mark indicate unknown intermediate steps.Regarding the roles of CDPKs in defense reactions, 12 TaCPKs were found to be responsive to either Blumeria graminis tritici (Bgt) infection or H₂O₂ treatment. The response to H₂O₂ was investigated because cytosolic calcium influx and reactive oxygen species, such as H₂O₂ are known to be implicated in both plant innate immunity and abiotic stresses.^12–17 Among these CDPK genes, five responded to both treatments (Group II) whereas the ones that responded to Bgt infection (Group I) or H₂O₂ treatment (Group III) were four and three respectively. The differential expression patterns suggest different functional modes of these CDPK genes. Involvement of CDPK genes in plant defense response has been shown in multiple species.^5,7 Recently, two barley CDPK paralogs (HvCDPK3 and HvCDPK4) were found to play antagonistic roles during the early phase of powdery mildew pathogenesis.⁵ The close similarity between wheat CDPK genes (TaCPK2 and TaCPK5, Fig. 1B) with these two barley genes may suggest their potential roles in wheat powdery mildew resistance. Surprisingly, we did not detect the responsiveness of TaCPK5 to wheat Bgt infection, indicating the divergence of CDPK functions in these two members of Triticeae family. Recently, one potato (Solanum tuberosum) CDPK gene StCDPK5 has been shown to be directly involved in regulating oxidative burst via phosphorylation of the NADPH oxidase StRBOHB.¹⁸ In light of the close relationship of TaCPK2 with HvCDPK5 and StCDPK5 (Fig. 1B), we speculate that TaCPK2 could be associated with both biotic and abiotic stress response signaling pathways and therefore play multiple roles in wheat.A model was proposed in Figure 1C regarding the positions of wheat CDPK genes in signaling pathways for biotic and abiotic responses. The hypothesis depicted four different roles of wheat CDPK genes: (1) Group I genes that respond only to Bgt infection may, like potato StCDPK5, render defense response through an oxidase like NADPH oxidase that generates increased amount of H₂O₂;¹⁸ (2) At one aspect, Group II genes may participate in defense response in a manner similar to Group I genes; (3) On the other hand, since Group II genes also respond to H₂O₂ treatment directly, an auto-regulation circuit was proposed, which eventually joins the oxidase pathway; (4) Group III CDPK genes and some remaining CDPK genes are considered to be mainly involved in abiotic stress responses. The model positioned CDPKs both upstream and downstream of H₂O₂, presenting a complicated wiring of the signaling pathway network involving wheat CDPKs. Future biochemical, genetic, and transgenic analyses may help elucidate the genuineness of such a rather early model for the functions of wheat CDPK genes.     

CPK12: A Ca2+-dependent protein kinase balancer in abscisic acid signaling

Ca²⁺ is believed to be a critical second messenger in ABA signal transduction. Ca²⁺-dependent protein kinases (CDPKs) are the best characterized Ca²⁺ sensors in plants. Recently, we identified an Arabidopsis CDPK member CPK12 as a negative regulator of ABA signaling in seed germination and post-germination growth, which reveals that different members of the CDPK family may constitute a regulation loop by functioning positively and negatively in ABA signal transduction. We observed that both RNA interference and overexpression of CPK12 gene resulted in ABA-hypersensitive phenotypes in seed germination and post-germination growth, suggesting a high complexity of the CPK12-mediated ABA signaling pathway. CPK12 stimulates a negative ABA-signaling regulator (ABI2) and phosphorylates two positive ABA-signaling regulators (ABF1 and ABF4), which may partly explain the ABA hypersensitivity induced by both downregulation and upregulation of CPK12 expression. Our data indicate that CPK12 appears to function as a balancer in ABA signal transduction in Arabidopsis.     

Crosstalk During Fruit Ripening and Stress Response Among Abscisic Acid,Calcium-Dependent Protein Kinase and Phenylpropanoid

Phenylpropanoids are secondary metabolites produced by plants. They, by differential expression, are involved in responses to biotic and abiotic stresses and confer plant plasticity. In addition, they are synthesized under normal conditions during the fruit-ripening process. Therefore, the understanding of the mechanics involved in the accumulation of these compounds in plants is of extreme importance for the development of plants with greater resistance and tolerance to biotic and abiotic stresses, and plants with greater functional potential. There is evidence that one of the pathways of the induction of phenylpropanoids is dependent on abscisic acid (ABA) and it is generated by a signaling cascade involving calcium (Ca²⁺) and Ca²⁺-dependent protein kinases (CDPKs). Plants have several Ca²⁺ binding proteins that act as cellular sensors and represent the first points of signal transduction. CDPKs are mono-molecular Ca²⁺-sensor/kinase-effector proteins, which perceive Ca²⁺ signals and translate them into protein phosphorylation and thus represent an ideal tool for signal transduction. However, the mechanisms involved in the ABA–CDPK–phenylpropanoids crosstalk under stress conditions and during fruit ripening remains uncertain. Therefore, this review seeks to surface a new line of evidence as an attempt to understand the manner in which the induction of phenylpropanoids occurs in plants.     

Possible involvement of Ca<Superscript>2+</Superscript>-dependent protein kinases in spore germination of the fern<Emphasis Type="Italic">Osmunda Japonica</Emphasis>

Fern gametophyte is a good model system to investigate signal transduction in plant cells. In this work, we examined whether CDPKs are involved in the mechanisms of spore germination of the fernOsmunda japonica. A protein extract from the spores included four CDPK isoforms with relative molecular weights of 56, 53, 49, and 47 kDa, as detected by immunoblot analysis, and they showed CDPK-like activities, as detected by in-gel protein-kinase assay. It was also found that the inhibitors effective on CDPKs, such as a general protein kinase inhibitor, K252a, and a calmodulin antagonist, W-7, largely suppressed the spore germination, and that many proteins of the spores were phosphorylated in vivo in a calcium dependent manner in the period when the spores require external Ca²⁺ for the germination. Furthermore, we showed that Sr²⁺ and Mn²⁺, which could substitute for Ca²⁺ in the spore germination, were also able to activate theOsmunda CDPKs. From these results, we concluded that CDPKs would participate in the spore germination ofO. japonica.     

PbGLR3.3 Regulates Pollen Tube Growth in the Mediation of Ca<Superscript>2+</Superscript> Influx in <Emphasis Type="Italic">Pyrus bretschneideri</Emphasis>

Glutamate receptors (GluRs) are sensors of extracellular signals; they play important roles in the regulation of multiple physiological and developmental processes in eukaryotes. However, their functional roles in fruit trees are largely unknown. Here, based on the pear genome database, which was established in this lab, we identified 34 PbGLRs in pear (Pyrus bretschneideri Rehd), and they were divided into four groups by phylogenetic analysis. In comparisons with other groups, phylogenetic analyses and structural information of the PbGLRs in group 3 suggest that these genes underlie specific characteristics. Among the ten genes in group 3, we observed that the expression of PbGLR3.3 increased gradually during pollen germination and continuous growth, indicating that this gene might play a vital role in the development of pear pollen tubes. Using a combination of antisense oligodeoxy nucleotides and Ca²⁺-sensitive fluorescent probe methods, we verified that PbGLR3.3 participates in DSer-elicited intracellular Ca²⁺ signaling and Ca²⁺ regulation of growth in pear pollen tubes.     

Calcium negatively regulates secretion from dense granules in Toxoplasma gondii

Apicomplexan parasites including Toxoplasma gondii and Plasmodium spp. manufacture a complex arsenal of secreted proteins used to interact with and manipulate their host environment. These proteins are organised into three principle exocytotic compartment types according to their functions: micronemes for extracellular attachment and motility, rhoptries for host cell penetration, and dense granules for subsequent manipulation of the host intracellular environment. The order and timing of these events during the parasite's invasion cycle dictates when exocytosis from each compartment occurs. Tight control of compartment secretion is, therefore, an integral part of apicomplexan biology. Control of microneme exocytosis is best understood, where cytosolic intermediate molecular messengers cGMP and Ca²⁺ act as positive signals. The mechanisms for controlling secretion from rhoptries and dense granules, however, are virtually unknown. Here, we present evidence that dense granule exocytosis is negatively regulated by cytosolic Ca²⁺, and we show that this Ca²⁺‐mediated response is contingent on the function of calcium‐dependent protein kinases TgCDPK1 and TgCDPK3. Reciprocal control of micronemes and dense granules provides an elegant solution to the mutually exclusive functions of these exocytotic compartments in parasite invasion cycles and further demonstrates the central role that Ca²⁺ signalling plays in the invasion biology of apicomplexan parasites.     

TgCDPK3 Regulates Calcium-Dependent Egress of Toxoplasma gondii from Host Cells

The phylum Apicomplexa comprises a group of obligate intracellular parasites of broad medical and agricultural significance, including Toxoplasma gondii and the malaria-causing Plasmodium spp. Key to their parasitic lifestyle is the need to egress from an infected cell, actively move through tissue, and reinvade another cell, thus perpetuating infection. Ca²⁺-mediated signaling events modulate key steps required for host cell egress, invasion and motility, including secretion of microneme organelles and activation of the force-generating actomyosin-based motor. Here we show that a plant-like Calcium-Dependent Protein Kinase (CDPK) in T. gondii, TgCDPK3, which localizes to the inner side of the plasma membrane, is not essential to the parasite but is required for optimal in vitro growth. We demonstrate that TgCDPK3, the orthologue of Plasmodium PfCDPK1, regulates Ca²⁺ ionophore- and DTT-induced host cell egress, but not motility or invasion. Furthermore, we show that targeting to the inner side of the plasma membrane by dual acylation is required for its activity. Interestingly, TgCDPK3 regulates microneme secretion when parasites are intracellular but not extracellular. Indeed, the requirement for TgCDPK3 is most likely determined by the high K⁺ concentration of the host cell. Our results therefore suggest that TgCDPK3''s role differs from that previously hypothesized, and rather support a model where this kinase plays a role in rapidly responding to Ca²⁺ signaling in specific ionic environments to upregulate multiple processes required for gliding motility.     

CDPK-mediated signalling pathways: specificity and cross-talk

Plants are constantly exposed to environmental changes and have to integrate a variety of biotic and abiotic stress stimuli. Calcium-dependent protein kinases (CDPKs) are implicated as important sensors of Ca2+ flux in plants in response to these stresses. CDPKs are encoded by multigene families, and expression levels of these genes are spatially and temporally controlled throughout development. In addition, a subset of CDPK genes responds to external stimuli. Biochemical evidence supports the idea that CDPKs are involved in signal transduction during stress conditions. Furthermore, loss-of-function and gain-of-function studies revealed that signalling pathways leading to cold, salt, drought or pathogen resistance are mediated by specific CDPK isoforms