A rice calcium-dependent protein kinase OsCPK12 oppositely modulates salt-stress tolerance and blast disease resistance

Calcium-dependent protein kinases (CDPKs) regulate the downstream components in calcium signaling pathways. We investigated the effects of overexpression and disruption of an Oryza sativa (rice) CDPK (OsCPK12) on the plant's response to abiotic and biotic stresses. OsCPK12-overexpressing (OsCPK12-OX) plants exhibited increased tolerance to salt stress. The accumulation of hydrogen peroxide (H(2) O(2) ) in the leaves was less in OsCPK12-OX plants than in wild-type (WT) plants. Genes encoding reactive oxygen species (ROS) scavenging enzymes (OsAPx2 and OsAPx8) were more highly expressed in OsCPK12-OX plants than in WT plants, whereas the expression of the NADPH oxidase gene, OsrbohI, was decreased in OsCPK12-OX plants compared with WT plants. Conversely, a retrotransposon (Tos17) insertion mutant, oscpk12, and plants transformed with an OsCPK12 RNA interference (RNAi) construct were more sensitive to high salinity than were WT plants. The level of H(2) O(2) accumulation was greater in oscpk12 and OsCPK12 RNAi plants than in the WT. These results suggest that OsCPK12 promotes tolerance to salt stress by reducing the accumulation of ROS. We also observed that OsCPK12-OX seedlings had increased sensitivity to abscisic acid (ABA) and increased susceptibility to blast fungus, probably resulting from the repression of ROS production and/or the involvement of OsCPK12 in the ABA signaling pathway. Collectively, our results suggest that OsCPK12 functions in multiple signaling pathways, positively regulating salt tolerance and negatively modulating blast resistance.     

Expression of rice Ca(2+)-dependent protein kinases (CDPKs) genes under different environmental stresses

Ca(2+)-dependent protein kinases (CDPKs) play an essential role in plant Ca(2+)-mediated signal transduction. Twenty-nine CDPK genes have been identified in the rice genome through a complete search of genome and full-length cDNA databases. Eight of them were reported previously to be inducible by different stress stimuli. Sequence comparison revealed that all 29 CDPK genes (OsCPK1-29) contain multiple stress-responsive cis-elements in the promoter region (1kb) upstream of genes. Analysis of the information extracted from the Rice Expression Database indicates that 11 of the CDPK genes are regulated by chilling temperature, dehydration, salt, rice blast infection and chitin treatment. RT-PCR and RNA gel blot hybridization were performed in this study to detect the expression 19 of the CDPK genes. Twelve CDPK genes exhibited cultivar- and tissue-specific expression; four CDPK genes (OsCPK6, OsCPK13, OsCPK17 and OsCPK25) were induced by chilling temperature, dehydration and salt stresses in the rice seedlings. While OsCPK13 (OsCDPK7) was already known to be inducible by chilling temperature and high salt, this is the first report that the other three genes are stress-regulated. OsCPK6 and OsCPK25 are up-regulated by dehydration and heat shock, respectively, while OsCPK17 is down-regulated by chilling temperature, dehydration and high salt stresses. Based on this evidence, rice CDPK genes may be important components in the signal transduction pathways for stress responses. Findings from this research are important for further dissecting mechanisms of stress response and functions of CDPK genes in rice.     

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.     

The rice cold-responsive calcium-dependent protein kinase OsCPK17 is regulated by alternative splicing and post-translational modifications

Plant calcium-dependent protein kinases (CDPKs) are key proteins implicated in calcium-mediated signaling pathways of a wide range of biological events in the organism. The action of each particular CDPK is strictly regulated by many mechanisms in order to ensure an accurate signal translation and the activation of the adequate response processes. In this work, we investigated the regulation of a CDPK involved in rice cold stress response, OsCPK17, to better understand its mode of action. We identified two new alternative splicing (AS) mRNA forms of OsCPK17 encoding truncated versions of the protein, missing the CDPK activation domain. We analyzed the expression patterns of all AS variants in rice tissues and examined their subcellular localization in onion epidermal cells. The results indicate that the AS of OsCPK17 putatively originates truncated forms of the protein with distinct functions, and different subcellular and tissue distributions. Additionally, we addressed the regulation of OsCPK17 by post-translational modifications in several in vitro experiments. Our analysis indicated that OsCPK17 activity depends on its structural rearrangement induced by calcium binding, and that the protein can be autophosphorylated. The identified phosphorylation sites mostly populate the OsCPK17 N-terminal domain. Exceptions are phosphosites T107 and S136 in the kinase domain and S558 in the C-terminal domain. These phosphosites seem conserved in CDPKs and may reflect a common regulatory mechanism for this protein family.     

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.     

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     

Rice calcium‐dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose‐phosphate synthase and is required for a proper cold stress response

Calcium‐dependent protein kinases (CDPKs) are involved in plant tolerance mechanisms to abiotic stresses. Although CDPKs are recognized as key messengers in signal transduction, the specific role of most members of this family remains unknown. Here, we test the hypothesis that OsCPK17 plays a role in rice cold stress response by analysing OsCPK17 knockout, silencing and overexpressing rice lines under low temperature. Altered OsCPK17 gene expression compromises cold tolerance performance, without affecting the expression of key cold stress‐inducible genes. A comparative phosphoproteomic approach led to the identification of six potential in vivo OsCPK17 targets, which are associated with sugar and nitrogen metabolism, and with osmotic regulation. To test direct interaction, in vitro kinase assays were performed, showing that the sucrose‐phosphate synthase OsSPS4 and the aquaporin OsPIP2;1/OsPIP2;6 are phosphorylated by OsCPK17 in a calcium‐dependent manner. Altogether, our data indicates that OsCPK17 is required for a proper cold stress response in rice, likely affecting the activity of membrane channels and sugar metabolism.     

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.     

Molecular evolution of two duplicated CDPK genes CPK7 and CPK12 in grass species: a case study in wheat (Triticum aestivum L.)

Gene duplication contributes to the expansion of gene families and subsequent functional diversification. Calcium-dependent protein kinases (CDPKs) are members of an important calcium sensor family involved in abiotic and biotic stress signaling in plants. We report here the molecular evolution and expression analysis of a pair of duplicated CDPK genes CPK7 and CPK12 that arose in the common ancestor of grass species. With higher nonsynonymous/synonymous ratios (dN/dS, or ω), CPK12 genes appear to diverge more rapidly than CPK7s, suggesting relaxed selection constraints on CPK12s. Sliding window analysis revealed increased dN and ω values at N-terminal regions and the calcium-binding EF hand loops. Likelihood analyses using various models in PAML 4.0 showed purifying selection on both CPK7 and CPK12 lineages. In addition to the divergence in cis-element combinations on their promoters, functional divergence of CPK7 and CPK12 genes was also observed in wheat where TaCPK7 was found to respond to drought (PEG), salt (NaCl), cold, and hydrogen peroxide (H(2)O(2)) while TaCPK12 responded only to the treatment of ABA, a feature that may complement or expand TaCPK7-mediated stress signaling networks of wheat. The contrasting expression patterns of CPK7 and CPK12 genes under stress conditions were also observed in rice, suggesting conservative functional evolution of these genes. Since no positive selection was detected between the two lineages, the divergence of CPK7 and CPK12 genes should be ascribed to subfunctionalization, rather than neofunctionalization. Thus, our work demonstrates another case of evolutionary employment of duplicated genes via subfunctionalization for better adaptation.     

Involvement of Calcium-dependent Protein Kinases in ABA regulation of Stomatal Movement

Patch-clamp techniques were employed to investigate if calcium-dependent protein kinases (CDPKs) be involved in the signal transduction pathways of stomatal movement regulation by the phytohormone abscisic acid (ABA) in Vicia faba. Stomatal opening was completely inhibited by external application of 1 μmol/L ABA, and such ABA inhibition was significantly reversed by the addition of CDPK inhibitor trifluoper- azine (TFP). The inward whole-cell K+-currents were inhibited by 60% in the presence of 1 μmoL/L intracellular ABA, and this inhibition was completely abolished by the addition of CDPK competitive substrate histone Ⅲ-S. The results suggest that CDPKs may be involved in the signal transduction cascades of ABA-regulated stomatal movements.     

Over-expression of calcium-dependent protein kinase 13 and calreticulin interacting protein 1 confers cold tolerance on rice plants

Calcium is a ubiquitous signaling molecule and changes in cytosolic calcium concentration are involved in plant responses to various stimuli. The rice calcium-dependent protein kinase 13 (CDPK13) and calreticulin interacting protein 1 (CRTintP1) have previously been reported to be involved in cold stress response in rice. In this study, rice lines transformed with sense CDPK13 or CRTintP1 constructs were produced and used to investigate the function of these proteins. When the plants were incubated at 5°C for 3 days, leaf blades of both the sense transgenic and vector control rice plants became wilted and curled. When the plants were transferred back to non-stress conditions after cold treatment, the leaf blades died, but the sheaths remained green in the sense transgenic rice plants. Expression of CDPK13 or CRTintP1 was further examined in several rice varieties including cold-tolerant rice varieties. Accumulation of these proteins in the cold-tolerant rice variety was higher than that in rice varieties that are intermediate in their cold tolerance. To examine whether over-expression of CDPK13 and CRTintP1 would have any effect on the proteins or not, sense transgenic rice plants were analyzed using proteomics. The 2D-PAGE profiles of proteins from the vector control were compared with those of the sense transgenic rice plants. Two of the proteins that differed between these lines were calreticulins. The results suggest that CDPK13, calreticulin and CRTintP1 might be important signaling components for response to cold stress in rice.