Calcium-dependent Protein Kinases Family Research Articles

Genome-wide analysis of calcium-dependent protein kinase (CDPK) family and functional characterization of TaCDPK25-U in response to drought stress in wheat

Calcium (Ca2+)-dependent protein kinase (CDPK) is one kind of biomacromolecule that can not only act as the unique Ca2+ sensors for the increase and decrease of calcium concentrations, but also as an effector for calcium signaling. It plays a vital role in regulating plant growth and development as well as stress responses. Although it has been extensively characterized in many plants, the importance of CDPK family in wheat is not well studied to date. In this study, a total of 85, 57, 27, and 27 CDPKs were identified in Triticum aestivum (T. aestivum), Triticum dicoccoides (T. dicoccoides), Triticum urartu (T. urartu) and Aegilops tauschii (A. tauschii), respectively, and grouped them into four phylogenetic subclades. Then, the gene structure, protein domains, conserved motifs and cis-acting elements of them were comprehensively investigated. Synteny analysis demonstrated that segmental duplication events and polyploidization contributed to the expansion of TaCDPK family. Furthermore, the tissue-specific as well as stress-responsive candidates were detected based on RNA-seq analysis. Results showed that under drought stress, TaCDPK25-U was induced to transcribe two alternative spliced isoforms (TaCDPK25-U-AS1 and TaCDPK25-U-AS2), which displayed differential expression levels and different subcellular localization. Finally, over-expression of TaCDPK25-U-AS1 or TaCDPK25-U-AS2 enhanced the drought tolerance in Arabidopsis thaliana (A. thaliana). Additionally, the expression of TaCDPK25-U-AS2 was demonstrated to be positively regulated by TaDREB3 to improve drought tolerance in vivo and in vitro. This study laid the foundation to better understand the evolution and function of CDPK family in wheat.

Foxtail millet SiCDPK7 gene enhances tolerance to extreme temperature stress in transgenic plants

Global climate change has resulted in increasingly harsh environments that have become the restriction factors for plants growth and development. Calcium-dependent protein kinases (CDPKs) in plants play critical roles in resisting various abiotic stresses, such as heat and cold stresses. Using the expression pattern analysis of foxtail millet CDPK family members under heat- and cold-treatments, respectively, and the SiCDPK7 was found to respond to extreme temperature stress and was selected for further study. The results suggested that overexpression of SiCDPK7 in Arabidopsis conferred tolerance to heat stress by increasing seedling survival rates and hypocotyl elongation compared with wild type (WT) plants, and also can enhance heat stress tolerance in foxtail millet. Analysis of physiological and biochemical indexes showed that SiCDPK7 transgenic plant lines had the markedly higher catalase (CAT) activity and the significantly lower malonaldehyde (MDA) content than WT plants. In addition, qRT-PCR analysis showed that the transcription levels of heat and cold stress-responsive genes were significantly increased in SiCDPK7 transgenic Arabidopsis and foxtail millet under stress conditions. This supporting evidence suggests that SiCDPK7 facilitated the extreme temperature tolerance capabilities in plants

The role of CDPKs in plant development, nutrient and stress signaling.

The second messenger calcium (Ca2+) is a ubiquitous intracellular signaling molecule found in eukaryotic cells. In plants, the multigene family of calcium-dependent protein kinases (CDPKs) plays an important role in regulating plant growth, development, and stress tolerance. CDPKs sense changes in intracellular Ca2+ concentration and translate them into phosphorylation events that initiate downstream signaling processes. Several functional and expression studies on different CDPKs and their encoding genes have confirmed their multifunctional role in stress. Here, we provide an overview of the signal transduction mechanisms and functional roles of CDPKs. This review includes details on the regulation of secondary metabolites, nutrient uptake, regulation of flower development, hormonal regulation, and biotic and abiotic stress responses.

The Calcium-Dependent Protein Kinase TaCDPK27 Positively Regulates Salt Tolerance in Wheat.

As essential calcium ion (Ca2+) sensors in plants, calcium-dependent protein kinases (CDPKs) function in regulating the environmental adaptation of plants. However, the response mechanism of CDPKs to salt stress is not well understood. In the current study, the wheat salt-responsive gene TaCDPK27 was identified. The open reading frame (ORF) of TaCDPK27 was 1875 bp, coding 624 amino acids. The predicted molecular weight and isoelectric point were 68.905 kDa and 5.6, respectively. TaCDPK27 has the closest relationship with subgroup III members of the CDPK family of rice. Increased expression of TaCDPK27 in wheat seedling roots and leaves was triggered by 150 mM NaCl treatment. TaCDPK27 was mainly located in the cytoplasm. After NaCl treatment, some of this protein was transferred to the membrane. The inhibitory effect of TaCDPK27 silencing on the growth of wheat seedlings was slight. After exposure to 150 mM NaCl for 6 days, the NaCl stress tolerance of TaCDPK27-silenced wheat seedlings was reduced, with shorter lengths of both roots and leaves compared with those of the control seedlings. Moreover, silencing of TaCDPK27 further promoted the generation of reactive oxygen species (ROS); reduced the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT); aggravated the injury to photosystem II (PS II); and increased programmed cell death (PCD) in wheat leaves under NaCl treatment, confirming that the TaCDPK27-silenced seedlings exhibited more NaCl injury than control seedlings. Taken together, the decrease in NaCl tolerance in TaCDPK27-silenced seedlings was due to excessive ROS accumulation and subsequent aggravation of the NaCl-induced PCD. TaCDPK27 may be essential for positively regulating salt tolerance in wheat seedlings.

Molecular Characterization Reveals the Involvement of Calcium Dependent Protein Kinases in Abiotic Stress Signaling and Development in Chickpea (Cicer arietinum).

Calcium-dependent protein kinases (CDPKs) are a major group of calcium (Ca2+) sensors in plants. CDPKs play a dual function of “Ca2+ sensor and responder.” These sensors decode the “Ca2+ signatures” generated in response to adverse growth conditions such as drought, salinity, and cold and developmental processes. However, knowledge of the CDPK family in the legume crop chickpea is missing. Here, we have identified a total of 22 CDPK genes in the chickpea genome. The phylogenetic analysis of the chickpea CDPK family with other plants revealed their evolutionary conservation. Protein homology modeling described the three-dimensional structure of chickpea CDPKs. Defined arrangements of α-helix, β-strands, and transmembrane-helix represent important structures like kinase domain, inhibitory junction domain, N and C-lobes of EF-hand motifs. Subcellular localization analysis revealed that CaCDPK proteins are localized mainly at the cytoplasm and in the nucleus. Most of the CaCDPK promoters had abiotic stress and development-related cis-regulatory elements, suggesting the functional role of CaCDPKs in abiotic stress and development-related signaling. RNA sequencing (RNA-seq) expression analysis indicated the role of the CaCDPK family in various developmental stages, including vegetative, reproductive development, senescence stages, and during seed stages of early embryogenesis, late embryogenesis, mid and late seed maturity. The real-time quantitative PCR (qRT-PCR) analysis revealed that several CaCDPK genes are specifically as well as commonly induced by drought, salt, and Abscisic acid (ABA). Overall, these findings indicate that the CDPK family is probably involved in abiotic stress responses and development in chickpeas. This study provides crucial information on the CDPK family that will be utilized in generating abiotic stress-tolerant and high-yielding chickpea varieties.

Identification of CDPK Gene Family in Solanum habrochaites and Its Function Analysis under Stress.

Tomato is an important vegetable crop. In the process of tomato production, it will encounter abiotic stress, such as low temperature, drought, and high salt, and biotic stress, such as pathogen infection, which will seriously affect the yield of tomato. Calcium-dependent protein kinase (CDPK) is a class of major calcium signal receptor which has an important regulatory effect on the perception and decoding of calcium signals. CDPK plays a key role in many aspects of plant growth, such as the elongation of pollen tubes, plant growth, and response to biotic and abiotic stress. While some studies have concentrated on Arabidopsis and pepper, Solanum habrochaites is a wild species relative of cultivated tomato and there is no report on CDPK in Solanum habrochaites to date. Using tomato genomic data, this study identified 33 members of the CDPK gene family. Evolutionary analysis divides family members into four Asian groups, of which the CDPK family members have 11 gene replication pairs. Subcellular location analysis showed that most proteins were predicted to be located in the cytoplasm, and less protein existed on the cell membrane. Not all CDPK family members have a transmembrane domain. Cis regulatory elements relating to light, hormones, and drought stress are overrepresented in the promoter region of the CDPK genes in Solanum habrochaites. The expression levels of each gene under biotic stress and abiotic stress were quantified by qRT-PCR. The results showed that members of the CDPK family in Solanum habrochaites respond to different biotic and abiotic stresses. Among them, the expression of ShCDPK6 and ShCDPK26 genes change significantly. ShCDPK6 and ShCDPK26 genes were silenced using VIGS (virus-induced gene silencing), and the silenced plants illustrated reduced stress resistance to Botrytis cinerea, cold, and drought stress. The results of this study will provide a basis for the in-depth study of the CDPK gene family in Solanum habrochaites, laying the foundation for further analysis of the function of the gene family.

Genome-Wide Identification and Characterization of CDPK Family Reveal Their Involvements in Growth and Development and Abiotic Stress in Sweet Potato and Its Two Diploid Relatives.

Calcium-dependent protein kinase (CDPKs) is one of the calcium-sensing proteins in plants. They are likely to play important roles in growth and development and abiotic stress responses. However, these functions have not been explored in sweet potato. In this study, we identified 39 CDPKs in cultivated hexaploid sweet potato (Ipomoea batatas, 2n = 6x = 90), 35 CDPKs in diploid relative Ipomoea trifida (2n = 2x = 30), and 35 CDPKs in Ipomoea triloba (2n = 2x = 30) via genome structure analysis and phylogenetic characterization, respectively. The protein physiological property, chromosome localization, phylogenetic relationship, gene structure, promoter cis-acting regulatory elements, and protein interaction network were systematically investigated to explore the possible roles of homologous CDPKs in the growth and development and abiotic stress responses of sweet potato. The expression profiles of the identified CDPKs in different tissues and treatments revealed tissue specificity and various expression patterns in sweet potato and its two diploid relatives, supporting the difference in the evolutionary trajectories of hexaploid sweet potato. These results are a critical first step in understanding the functions of sweet potato CDPK genes and provide more candidate genes for improving yield and abiotic stress tolerance in cultivated sweet potato.

Identification and expression assay of calcium-dependent protein kinase family genes in Hevea brasiliensis and determination of HbCDPK5 functions in disease resistance

Calcium (Ca2+) signaling is one of the earliest factors to coordinate plant adaptive responses. As direct sensors and activators of Ca2+ signals, calcium-dependent protein kinases (CDPKs) were reported to be widely involved in regulating different biotic and abiotic stress stimuli. In this study, 32 Hevea brasiliensis CDPK (HbCDPK) genes were predicted and classified into four subgroups. Among them, the full-length coding sequences of 28 HbCDPK genes were confirmed by RT-PCR and verified by sequencing. Putative cis-elements assay in the promoters of HbCDPKs showed that most of the HbCDPK genes contained gibberellic acid-responsive element (GARE), abscisic acid-responsive element (ABRE), salicylic acid-responsive element (SARE), defense and stress responsive element (TC-rich repeats) and low-temperature response element (LTR), which could be activated by different biotic and abiotic stresses. Real-time PCR analysis indicated that 28 HbCDPK genes respond to infection of pathogenic fungi and a variety of phytohormones. Subcellular localization was observed with most HbCDPKs located in cell membrane, cytoplasm or organelles. Some HbCDPKs were confirmed to cause reactive oxygen species (ROS) production and accumulation in rubber tree mesophyll protoplast directly. HbCDPK5 was strongly induced by the inoculation with Colletotrichum gloeosporioides and was chosen for further analysis. HbCDPK5 localized to the cell membrane and cytoplasm, and obviously regulated the accumulation of ROS in rubber tree mesophyll protoplast. Overexpression of HbCDPK5 in Arabidopsis enhanced the resistance to Botrytis cinerea. These results indicate that rubber tree CDPK genes play important roles in plant disease resistance.

Calcium-dependent Protein Kinase 5 (CPK5) positively modulates drought tolerance through phosphorylating ABA-Responsive Element Binding Factors in oilseed rape (Brassica napus L.)

Drought is an environmental stress that causes severe crop loss. Drought stress can induce abscisic acid (ABA) accumulation and cytoplasmic calcium oscillation. Calcium-dependent protein kinases (CPKs) constitute a group of Ser/Thr protein kinases decoding calcium signals. However, the function and molecular mechanisms of most CPKs in oilseed rape (Brassica napus) remain unknown. Here, we report the functional characterization of BnaCPK5 in drought stress tolerance. BnaCPK5 belongs to Group I of the CPK family and was localized at the plasma membrane and nuclei. Overexpression of BnaCPK5 enhanced drought stress tolerance compared with the control. A screening of interacting proteins identified that BnaCPK5 interacted strongly with two ABA-Responsive Element Binding Factors (ABF/AREBs), BnaABF3 and BnaABF4. BnaCPK5 was shown to phosphorylate both BnaABF3 and BnaABF4 in a kinase assay. Further, it was found that the phosphorylation of BnaABF3 and BnaABF4 by BnaCPK5 increased their transcriptional activities against the famous drought stress marker gene, Responsive to Dehydration (RD) 29B and protein stability. Taken together, these data demonstrate that BnaCPK5 acts as a positive regulator of drought tolerance by, at least in part, phosphorylating two core ABA-signaling components to modulate Late-Embryogenesis Abundant (LEA)-like RD29B expression.

Isotopically Dimethyl Labeling-Based Quantitative Proteomic Analysis of Phosphoproteomes of Soybean Cultivars.

Isotopically dimethyl labeling was applied in a quantitative post-translational modification (PTM) proteomic study of phosphoproteomic changes in the drought responses of two contrasting soybean cultivars. A total of 9457 phosphopeptides were identified subsequently, corresponding to 4571 phosphoprotein groups and 3889 leading phosphoproteins, which contained nine kinase families consisting of 279 kinases. These phosphoproteins contained a total of 8087 phosphosites, 6106 of which were newly identified and constituted 54% of the current soybean phosphosite repository. These phosphosites were converted into the highly conserved kinase docking sites by bioinformatics analysis, which predicted six kinase families that matched with those newly found nine kinase families. The overly post-translationally modified proteins (OPP) occupies 2.1% of these leading phosphoproteins. Most of these OPPs are photoreceptors, mRNA-, histone-, and phospholipid-binding proteins, as well as protein kinase/phosphatases. The subgroup population distribution of phosphoproteins over the number of phosphosites of phosphoproteins follows the exponential decay law, Y = 4.13e−0.098X − 0.04. Out of 218 significantly regulated unique phosphopeptide groups, 188 phosphoproteins were regulated by the drought-tolerant cultivar under the water loss condition. These significantly regulated phosphoproteins (SRP) are mainly enriched in the biological functions of water transport and deprivation, methionine metabolic processes, photosynthesis/light reaction, and response to cadmium ion, osmotic stress, and ABA response. Seventeen and 15 SRPs are protein kinases/phosphatases and transcription factors, respectively. Bioinformatics analysis again revealed that three members of the calcium dependent protein kinase family (CAMK family), GmSRK2I, GmCIPK25, and GmAKINβ1 kinases, constitute a phosphor-relay-mediated signal transduction network, regulating ion channel activities and many nuclear events in this drought-tolerant cultivar, which presumably contributes to the development of the soybean drought tolerance under water deprivation process.

Evolution and expression analysis of CDPK genes under drought stress in two varieties of potato

Calcium-dependent protein kinases (CDPKs) function directly in plant development and stress responses. We used whole genome sequences and mRNA expression data to analyze the phylogenetic relationships, gene structure, collinearity, and differential expression of CDPKs in two differentially drought-tolerant potato varieties. In total, we identified 25 CDPK proteins belonging to four subfamilies. There was a significant collinear relationship among 13 CDPK genes belonging to four segmentally duplicated pairs. Subcellular prediction implied that all StCDPKs were localized at the plasma membrane. Analysis of promoter regions revealed that StCDPKs were photosensitive and responsive to biotic stress, abiotic stress, and hormone stimuli. RNA-seq analysis showed differential expression of StCDPKs among various potato tissues, and qPCR analysis revealed that 20 StCDPKs exhibited differential expression patterns under drought stress between drought-tolerant (QS9) and drought sensitive (Atl) potato varieties. Among these, the most strongly drought-induced genes were respectively StCDPK3 and StCDPK23, highlighting these as attractive candidate genes for further functional analyses of drought-stress responses in potato. Our results demonstrating the tissue specific and drought stress-responsive StCDPK genes of potato both provide a reference for further research about the functions of CDPK family proteins and should support ongoing efforts for the further genetic improvement of potato.

A Structural Bioinformatics Study of Calcium Dependent Protein Kinase 6 (CDPK6) of Cryptosporidium Parvum for Identification of Novel Therapeutics

Cryptosporidium parvum is an obligate intracellular protozoan parasite that infects the intestinal tract of mammals and causes cryptosporidiosis. C. parvum oocysts are ingested by a host that reproduces sexually and asexually in the intestinal lining cells, resulting in diarrhea. Calcium Dependent Protein Kinases (CDPKs) are found in the apicomplexan, algae, and plants; however, they are reported in vertebrates and are regarded as excellent drug targets for pharmaceutical interventions. CDPKs of Cryptosporidium are probably involved in the regulation of invasion and egress process during the infection of the host cells. Calcium-dependent protein kinase6 (CDPK6) (cgd4_3330) is a hypothetical protein of the CDPKs family which was originated from the plants and also a few species of protozoa. These proteins regulate several biological processes of apicomplexan for example Cryptosporidium, Plasmodium, and Toxoplasma. In this study, an attempt is made to address the sequences similarity and phylogenetic study of CDPK6 among apicomplexans CDPKs family. Furthermore, the three-dimensional structure determination of the target protein was performed through a molecular modeling approach (MODELLER v9.21 software) followed by virtual screening (GOLD software suite & Glide software) of small molecule inhibitors from five different datasets. The best inhibitor Tres Cantos Antimalarial Set (TCAMS) _11730 reported a binding affinity of -8.2 kcal/mol towards the CDPK6 of C. parvum through molecular docking studies (AutoDock v4.2.6 software). Furthermore, the reliability and validation of the binding mode of the inhibitor were performed through a complex molecular dynamics simulation in an aqueous environment with GROMACS 2018.3 software for a time interval of 100 ns. The findings of the study advocate that the best inhibitor may be considered for industrial pharmaceutical research in future.

Genome-wide survey and expression analysis of calcium-dependent protein kinase (CDPK) in grass Brachypodium distachyon

BackgroundCa2+ played as a ubiquitous secondary messenger involved in plant growth, development, and responses to various environmental stimuli. Calcium-dependent protein kinases (CDPK) were important Ca2+ sensors, which could directly translate Ca2+ signals into downstream phosphorylation signals. Considering the importance of CDPKs as Ca2+ effectors for regulation of plant stress tolerance and few studies on Brachypodium distachyon were available, it was of interest for us to isolate CDPKs from B. distachyon.ResultsA systemic analysis of 30 CDPK family genes in B. distachyon was performed. Results showed that all BdCDPK family members contained conserved catalytic Ser/Thr protein kinase domain, autoinhibitory domain, and EF-hand domain, and a variable N-terminal domain, could be divided into four subgroup (I-IV), based upon sequence homology. Most BdCDPKs had four EF-hands, in which EF2 and EF4 revealed high variability and strong divergence from EF-hand in AtCDPKs. Synteny results indicated that large number of syntenic relationship events existed between rice and B. distachyon, implying their high conservation. Expression profiles indicated that most of BdCDPK genes were involved in phytohormones signal transduction pathways and regulated physiological process in responding to multiple environmental stresses. Moreover, the co-expression network implied that BdCDPKs might be both the activator and the repressor involved in WRKY transcription factors or MAPK cascade genes mediated stress response processes, base on their complex regulatory network.ConclusionsBdCDPKs might play multiple function in WRKY or MAPK mediated abiotic stresses response and phytohormone signaling transduction in B. distachyon. Our genomics analysis of BdCDPKs could provide fundamental information for further investigation the functions of CDPKs in integrating Ca2+ signalling pathways in response to environments stresses in B. distachyon.

Expression, Subcellular Localization, and Interactions of CPK Family Genes in Maize.

Calcium-dependent protein kinase (CPKs) is a key player in the calcium signaling pathway to decode calcium signals into various physiological responses. cDNA sequences of 9 ZmCPK genes were successfully cloned from all four phylogenetic groups in maize. qRT-PCR analysis showed the expression variation of these selected genes under abscisic acid (ABA) and calcium chloride (CaCl2) treatment. Due to the presence of N-myristoylation/palmitoylation sites, the selected ZmCPK members were localized in a plasma membrane. To clarify whether ZmCPK, a key player in calcium signaling, interacts with key players of ABA, protein phosphatase 2Cs (PP2Cs) and the SNF1-related protein kinase 2s (SnRK2s) and mitogen-activated protein kinase (MAPK) signaling pathways in maize, we examined the interaction between 9 CPKs, 8 PP2Cs, 5 SnRKs, and 20 members of the MPK family in maize by using yeast two-hybrid assay. Our results showed that three ZmCPKs interact with three different members of ZmSnRKs while four ZmCPK members had a positive interaction with 13 members of ZmMPKs in different combinations. These four ZmCPK proteins are from three different groups in maize. These findings of physical interactions between ZmCPKs, ZmSnRKs, and ZmMPKs suggested that these signaling pathways do not only have indirect influence but also have direct crosstalk that may involve the defense mechanism in maize. The present study may improve the understanding of signal transduction in plants.

PtrCDPK10 of Poncirus trifoliata functions in dehydration and drought tolerance by reducing ROS accumulation via phosphorylating PtrAPX

Calcium-dependent protein kinases (CDPKs) are important calcium signaling components that have been shown to play crucial roles in modulating plant abiotic stress responses. However, the physiological and regulatory roles of most CDPKs are still poorly understood. Here, we report the functional characterization of PtrCDPK10 from trifoliate orange (Poncirus trifoliata (L.) Raf.) in dehydration and drought stress tolerance. PtrCDPK10, categorized in the Type III subgroup of the CDPK family, was localized to the nucleus and plasma membrane. Transcript levels of PtrCDPK10 were up-regulated by dehydration, salt and ABA treatments. Transgenic trifoliate orange plants overexpressing PtrCDPK10 showed enhanced dehydration tolerance compared with the wild type (WT), whereas VIGS (virus-induced gene silencing)-mediated knockdown of PtrCDPK10 resulted in elevated susceptibility to dehydration and drought stresses. Yeast two-hybrid screening identified several proteins that interacted with PtrCDPK10, including an ascorbate peroxidase (PtrAPX). PtrCDPK10 was shown to phosphorylate PtrAPX based on an in vitro kinase assay. PtrCDPK10-overexpressing transgenic lines exhibited higher PtrAPX mRNA abundance and APX activity and accumulated dramatically less ROS in comparison with the WT, while PtrCDPK10-silenced VIGS lines showed decreased PtrAPX expression and increased ROS level. Taken together, these results demonstrate that PtrCDPK10 promotes dehydration and drought tolerance by, at least in part, phosphorylating APX to modulate ROS homeostasis.

Genome-Wide Identification of Calcium-Dependent Protein Kinases in Chlamydomonas reinhardtii and Functional Analyses in Nitrogen Deficiency-Induced Oil Accumulation.

Calcium-dependent protein kinases (CDPKs) are recognized as important calcium (Ca2+) sensors in signal transduction and play multiple roles in plant growth and developmental processes, as well as in response to various environmental stresses. However, little information is available about the CDPK family in the green microalga Chlamydomonas reinhardtii. In this study, 15 CrCDPK genes were identified in C. reinhardtii genome, and their functions in nitrogen (N) deficiency-induced oil accumulation were analyzed. Our results showed that all CrCDPK proteins harbored the typical elongation factor (EF)-hand Ca2+-binding and protein kinase domains. Phylogenetic analysis revealed that these CrCDPKs were clustered into one group together with a subclade of several CPKs from Arabidopsis and rice, clearly separating from the remaining AtCPKs and OsCPKs. These genes were located in 10 chromosomes and one scaffold of C. reinhardtii and contained 6–17 exons. RNA sequencing and quantitative reverse transcription (qRT)-PCR assays indicated that most of these CrCDPKs were significantly induced by N deficiency and salt stress. Lanthanum chloride (LaCl3), a plasma membrane Ca2+ channel blocker, limited oil accumulation in C. reinhardtii under N-deficient conditions, suggesting that Ca2+ was involved in N deficiency-induced oil accumulation. Furthermore, RNA interference (RNAi) silencing analyses demonstrated that six CrCDPKs played positive roles and three CrCDPKs played negative roles in N deficiency-induced oil accumulation in C. reinhardtii.

Evaluation of protective immunity induced by recombinant calcium-dependent protein kinase 1 (TgCDPK1) protein against acute toxoplasmosis in mice

Toxoplasma gondii is an intracellular zoonotic parasite that causes toxoplasmosis, which can cause economic losses and serious public health problems worldwide. A member of the T. gondii calcium-dependent protein kinases family, TgCDPK1 was recently identified as an essential regulator of exocytosis in T. gondii, and participated in direct parasite motility, host-cell invasion and egress. In the present study, the protective immunity of recombinant TgCDPK1 protein (rTgCDPK1) was evaluated against acute toxoplasmosis in mice. rTgCDPK1 were expressed and purified, BABL/c mice were intraperitoneally immunized with rTgCDPK1 and challenged with the highly virulent RH strain of T. gondii. The specific immune responses were analyzed by measuring the cytokine and serum antibody, and lymphocyte proliferation assays, flow cytometry of lymphocytes and the survival curve were employed to evaluate the protective efficacy. From the results we found that special humoral and cellular responses could be elicited in vaccine mice, and higher level of IgG antibody, and the significant increased levels of Th1-type cytokines IFN-γ, IL-12 (p70), IL10 and CD3+CD4+CD8− and CD3+CD8+CD4− T cells could also be detected comparing to control mice (P < 0.05). All vaccinated mice prolonged survival time (14.90 ± 2.89 days) challenge with 1000 tachyzoites of RH, while the control mice died within 8 days. These results indicated that TgCDPK1 protein was a potential vaccine candidate against acute toxoplasmosis.

Calcium-Dependent Protein Kinase Genes in Glycyrrhiza Uralensis Appear to be Involved in Promoting the Biosynthesis of Glycyrrhizic Acid and Flavonoids under Salt Stress.

As calcium signal sensors, calcium-dependent protein kinases (CPKs) play vital roles in stimulating the production of secondary metabolites to participate in plant development and response to environmental stress. However, investigations of the Glycyrrhiza uralensis CPK family genes and their multiple functions are rarely reported. In this study, a total of 23 GuCPK genes in G. uralensis were identified, and their phylogenetic relationships, evolutionary characteristics, gene structure, motif distribution, and promoter cis-acting elements were analyzed. Ten GuCPKs showed root-specific preferential expressions, and GuCPKs indicated different expression patterns under treatments of CaCl2 and NaCl. In addition, under 2.5 mM of CaCl2 and 30 mM of NaCl treatments, the diverse, induced expression of GuCPKs and significant accumulations of glycyrrhizic acid and flavonoids suggested the possible important function of GuCPKs in regulating the production of glycyrrhizic acid and flavonoids. Our results provide a genome-wide characterization of CPK family genes in G. uralensis, and serve as a foundation for understanding the potential function and regulatory mechanism of GuCPKs in promoting the biosynthesis of glycyrrhizic acid and flavonoids under salt stress.

Genome-Wide Analysis of CDPK Family in Foxtail Millet and Determination of SiCDPK24 Functions in Drought Stress.

Plant calcium-dependent protein kinases (CDPKs) were reported to play important roles in plant resistance to abiotic stress. Foxtail millet cultivation “H138” was used for RNA-seq analysis. The data from drought-induced de novo transcriptomic sequences of foxtail millet showed that CDPKs were up- or down-regulated by drought to different degrees. In this study, 29 foxtail millet CDPKs were classified into four subgroups. These genes were unevenly distributed on nine foxtail millet chromosomes, and chromosomes 2, 3, and 9 contained the most SiCDPK members. Analysis of putative cis-acting elements showed that most foxtail millet CDPK genes contained the ABRE, LTR, HSE, MYB, MYC, DRE, CGTCA-motif, and TGACG-motif cis-acting elements, which could be activated by abiotic stresses. Real-time PCR analysis indicated that 29 SiCDPK genes experienced different degrees of induction under drought and ABA stresses. SiCDPK24 had the highest expression levels at 6 and 12 h of drought treatment and was chosen for further analysis. SiCDPK24 localized to the cell membrane and the nucleus of Arabidopsis mesophyll protoplasts. Western blot analysis showed that SiCDPK24 protein had autophosphorylation activity. Overexpression of SiCDPK24 in Arabidopsis enhanced drought resistance and improved the survival rate under drought stress. It also activated the expressions of nine stress-related genes, namely RD29A, RD29B, RD22, KIN1, COR15, COR47, LEA14, CBF3/DREB1A, and DREB2A. These genes are involved in resistance to abiotic stresses in Arabidopsis. These results indicate that foxtail millet CDPK genes play important roles in resisting drought stress.

Calcium-Dependent Protein Kinase Family Genes Involved in Ethylene-Induced Natural Rubber Production in Different Hevea brasiliensis Cultivars.

Natural rubber latex production can be improved by ethylene stimulation in the rubber tree (Hevea brasiliensis). However, the expression levels of most functional proteins for natural rubber biosynthesis are not induced after ethylene application, indicating that post-translational modifications, especially protein phosphorylation, may play important roles in ethylene signaling in Hevea. Here, we performed a comprehensive investigation on evolution, ethylene-induced expression and protein–protein interaction of calcium-dependent protein kinases (CPKs), an important serine/threonine protein kinase family, in Hevea. Nine duplication events were determined in the 30 identified HbCPK genes. Expression profiling of HbCPKs in three rubber tree cultivars with low, medium and high ethylene sensitivity showed that HbCPK6, 17, 20, 22, 24, 28 and 30 are induced by ethylene in at least one cultivar. Evolution rate analysis suggested accelerated evolution rates in two paralogue pairs, HbCPK9/18 and HbCPK19/20. Analysis of proteomic data for rubber latex after ethylene treatment showed that seven HbCPK proteins could be detected, including six ethylene-induced ones. Protein–protein interaction analysis of the 493 different abundant proteins revealed that protein kinases, especially calcium-dependent protein kinases, possess most key nodes of the interaction network, indicating that protein kinase and protein phosphorylation play important roles in ethylene signaling in latex of Hevea. In summary, our data revealed the expression patterns of HbCPK family members and functional divergence of two HbCPK paralogue pairs, as well as the potential important roles of HbCPKs in ethylene-induced rubber production improvement in Hevea.