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Calcium-dependent protein kinases (CDPKs) serve as calcium ion sensors and play crucial roles in all aspects of plant life cycle. While CDPK gene family has been extensively studied in various plants, there is limited information available for CDPK members in oat, an important cereal crop worldwide. Totally, 60 AsCDPK genes were identified in oat genome and were classified into four subfamilies based on their phylogenetic relationship. The members within each subfamily shared similar gene structure and conserved motifs. Collinearity analysis revealed that AsCDPK gene amplification was attributed to segmental duplication events and underwent strong purifying selection. AsCDPK promoters were predicted to contain cis-acting elements associated with hormones, biotic and abiotic stresses. AsCDPK gene expressions were induced by different salt stresses, exhibiting stress-specific under different salt treatments. Moreover, overexpression of AsCDPK26 gene enhanced salt resistance in C. reinhardtii, a single-cell photoautotrophic model plants. Further analysis revealed a significant correlation between AsCDPK26 and Na+/H+ antiporter 1 (p<0.05), suggesting that AsCDPK26 may interact with ion transporter to modulate salt resistance. These results not only provide valuable insights into AsCDPK genes in response to different salt stresses, but also lay the foundation to mine novel candidates for improving salt tolerance in oat and other crops. [ABSTRACT FROM AUTHOR]

Taproot cracking, a severe and common physiological disorder, markedly reduces radish yield and commercial value. Calcium-dependent protein kinase (CDPK) plays a pivotal role in various plant developmental processes; however, its function in radish taproot cracking remains largely unknown. Here, 37 RsCDPK gene members were identified from the long-read radish genome "QZ-16". Phylogenetic analysis revealed that the CDPK members in radish, tomato, and Arabidopsis were clustered into four groups. Additionally, synteny analysis identified 13 segmental duplication events in the RsCDPK genes. Analysis of paraffin-embedded sections showed that the density and arrangement of fleshy taproot cortex cells are important factors that affect radish cracking. Transcriptome sequencing of the fleshy taproot cortex revealed 5755 differentially expressed genes (DEGs) (3252 upregulated and 2503 downregulated) between non-cracking radish "HongYun" and cracking radish "505". These DEGs were significantly enriched in plant hormone signal transduction, phenylpropanoid biosynthesis, and plant–pathogen interaction KEGG pathways. Furthermore, when comparing the 37 RsCDPK gene family members and RNA-seq DEGs, we identified six RsCDPK genes related to taproot cracking in radish. Soybean hairy root transformation experiments showed that RsCDPK21 significantly and positively regulates root length development. These findings provide valuable insights into the relationship between radish taproot cracking and RsCDPK gene function. [ABSTRACT FROM AUTHOR]

Systemic acquired resistance (SAR) in plants is a defense response that provides resistance against a wide range of pathogens at the whole-plant level following primary infection. Although the molecular mechanisms of SAR have been extensively studied in recent years, the role of phosphorylation that occurs in systemic leaves of SAR-induced plants is poorly understood. We used a data-independent acquisition (DIA) phosphoproteomics platform based on high-resolution mass spectrometry in an Arabidopsis thaliana model to identify phosphoproteins related to SAR establishment. A total of 8011 phosphorylation sites from 3234 proteins were identified in systemic leaves of Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) and mock locally inoculated plants. A total of 859 significantly changed phosphoproteins from 1119 significantly changed phosphopeptides were detected in systemic leaves of Psm ES4326 locally inoculated plants, including numerous transcription factors and kinases. A variety of defense response-related proteins were found to be differentially phosphorylated in systemic leaves of Psm ES4326 locally inoculated leaves, suggesting that these proteins may be functionally involved in SAR through phosphorylation or dephosphorylation. Significantly changed phosphoproteins were enriched mainly in categories related to response to abscisic acid, regulation of stomatal movement, plant–pathogen interaction, MAPK signaling pathway, purine metabolism, photosynthesis-antenna proteins, and flavonoid biosynthesis. A total of 28 proteins were regulated at both protein and phosphorylation levels during SAR. RT-qPCR analysis revealed that changes in phosphorylation levels of proteins during SAR did not result from changes in transcript abundance. This study provides comprehensive details of key phosphoproteins associated with SAR, which will facilitate further research on the molecular mechanisms of SAR.

Systemic acquired resistance (SAR) in plants is a defense response that provides resistance against a wide range of pathogens at the whole-plant level following primary infection. Although the molecular mechanisms of SAR have been extensively studied in recent years, the role of phosphorylation that occurs in systemic leaves of SAR-induced plants is poorly understood. We used a data-independent acquisition (DIA) phosphoproteomics platform based on high-resolution mass spectrometry in an Arabidopsis thaliana model to identify phosphoproteins related to SAR establishment. A total of 8011 phosphorylation sites from 3234 proteins were identified in systemic leaves of Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) and mock locally inoculated plants. A total of 859 significantly changed phosphoproteins from 1119 significantly changed phosphopeptides were detected in systemic leaves of Psm ES4326 locally inoculated plants, including numerous transcription factors and kinases. A variety of defense response-related proteins were found to be differentially phosphorylated in systemic leaves of Psm ES4326 locally inoculated leaves, suggesting that these proteins may be functionally involved in SAR through phosphorylation or dephosphorylation. Significantly changed phosphoproteins were enriched mainly in categories related to response to abscisic acid, regulation of stomatal movement, plant–pathogen interaction, MAPK signaling pathway, purine metabolism, photosynthesis-antenna proteins, and flavonoid biosynthesis. A total of 28 proteins were regulated at both protein and phosphorylation levels during SAR. RT-qPCR analysis revealed that changes in phosphorylation levels of proteins during SAR did not result from changes in transcript abundance. This study provides comprehensive details of key phosphoproteins associated with SAR, which will facilitate further research on the molecular mechanisms of SAR. [ABSTRACT FROM AUTHOR]

Purpose: To identify some natural molecules (inhibitors) against avian coccidia (Eimeria tenella) through molecular docking in order to find the candidate drugs for poultry industry. Methods: The natural compounds, 6’-de-O-acetylcupacinoside, apigenin, artemisinin, cupacinoside, quercetin and rutin, were screened on the basis of previous research data. These biomolecules were selected as potent inhibitors based on extant literature on calcium-dependent protein kinases (CDPKs) in Eimeria tenella. Results: All the compounds showed good interactions with 6’-de-O-acetylcupacinoside. Based on docking studies, quercetin produced the best interaction, with a binding energy of -7.04 kcal/mol, when compared to the other compounds. Conclusions: Based on these in silico results, the screened compounds have great potential for use against E. tenella. In particular, the results suggest that quercetin may be beneficial in the control of avian coccidia through its strong inhibitory potential on CDPKs of E. tenella. [ABSTRACT FROM AUTHOR]

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.

Summary: Receptor for activated C kinase 1 (RACK1) is strictly conserved across eukaryotes and acts as a versatile scaffold protein involved in various signaling pathways. Plant RACK1 is known to exert important functions in innate immunity against fungal and bacterial pathogens. However, the role of the RACK1 in plant–virus interactions remains unknown.Here, we addressed the role of RACK1 of Nicotiana benthamiana during infection by red clover necrotic mosaic virus (RCNMV), a plant positive‐stranded RNA virus. NbRACK1 was shown to be recruited by the p27 viral replication protein into endoplasmic reticulum‐derived aggregated structures (possible replication sites). Downregulation of NbRACK1 by virus‐induced gene silencing inhibited viral cap‐independent translation and p27‐mediated reactive oxygen species (ROS) accumulation, which are prerequisite for RCNMV replication.We also found that NbRACK1 interacted with a host calcium‐dependent protein kinase (NbCDPKiso2) that activated a ROS‐generating enzyme. Interestingly, NbRACK1 was required for the interaction of p27 with NbCDPKiso2, suggesting that NbRACK1 acts as a bridge between the p27 viral replication protein and NbCDPKiso2.Collectively, our findings provide an example of a viral strategy in which a host multifaceted scaffold protein RACK1 is highjacked for promoting viral protein‐triggered ROS production necessary for robust viral replication. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Calcium ion (Ca2+) is a universal second messenger that plays a critical role in plant responses to diverse physiological and environmental stimuli. The stimulus-specific signals are perceived and decoded by a series of Ca2+ binding proteins serving as Ca2+ sensors. The majority of Ca2+ sensors possess the EF-hand motif, a helix-loophelix structure which forms a turn-loop structure. Although EF-hand proteins in model plant such as Arabidopsis have been well described, the identification, classification, and the physiological functions of EF-hand-containing proteins from soybean are not systemically reported. In this study, a total of at least 262 genes possibly encoding proteins containing one to six EF-hand motifs were identified in soybean genome. These genes include 6 calmodulins (CaMs), 144 calmodulin-like proteins (CMLs), 15 calcineurin B-like proteins, 50 calcium-dependent protein kinases (CDPKs), 13 CDPKrelated protein kinases, 2 Ca2+- and CaM-dependent protein kinases, 17 respiratory burst oxidase homologs, and 15 unclassified EF-hand proteins. Most of these genes (87.8%) contain at least one kind of hormonal signaling- and/or stress response-related cis-elements in their -1500 bp promoter regions. Expression analyses by exploring the published microarray and Illumina transcriptome sequencing data revealed that the expression of these EF-hand genes were widely detected in different organs of soybean, and nearly half of the total EF-hand genes were responsive to various environmental or nutritional stresses. Quantitative RT-PCR was used to confirm their responsiveness to several stress treatments. To confirm the Ca2+-binding ability of these EF-hand proteins, four CMLs (CML1, CML13, CML39, and CML95) were randomly selected for SDS- PAGE mobility-shift assay in the presence and absence of Ca2+. Results showed that all of them have the ability to bind Ca2+. This study provided the first comprehensive analyses of genes encoding for EF-hand proteins in soybean. Information on the classification, phylogenetic relationships and expression profiles of soybean EF-hand genes in different tissues and under various environmental and nutritional stresses will be helpful for identifying candidates with potential roles in Ca2+ signal-mediated physiological processes including growth and development, plant-microbe interactions and responses to biotic and abiotic stresses. [ABSTRACT FROM AUTHOR]

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Calcium-dependent protein kinases (CDPKs) and CDPK-related kinases (CRKs) play multiple roles in plant. Nevertheless, genome-wide identification of these two families is limited to several plant species, and role of CRKs in disease resistance remains unclear. In this study, we identified the CDPK and CRK gene families in genome of the economically important crop tomato ( Solanum lycopersicum L.) and analyzed their function in resistance to various pathogens. Twenty-nine CDPK and six CRK genes were identified in tomato genome. Both SlCDPK and SlCRK proteins harbored an STKc_CAMK type protein kinase domain, while only SlCDPKs contained EF-hand type Ca binding domain(s). Phylogenetic analysis revealed that plant CRK family diverged early from CDPKs, and shared a common ancestor gene with subgroup IV CDPKs. Subgroup IV SlCDPK proteins were basic and their genes contained 11 introns, which were distinguished from other subgroups but similar to CRKs. Subgroup I SlCDPKs generally did not carry an N-terminal myristoylation motif while those of the remaining subgroups and SlCRKs universally did. SlCDPK and SlCRK genes were differently responsive to pathogenic stimuli. Furthermore, silencing analyses demonstrated that SlCDPK18 and SlCDPK10 positively regulated nonhost resistance to Xanthomonas oryzae pv. oryzae and host resistance to Pseudomonas syringae pv. tomato ( Pst) DC3000, respectively, while SlCRK6 positively regulated resistance to both Pst DC3000 and Sclerotinia sclerotiorum in tomato. In conclusion, CRKs apparently evolved from CDPK lineage, SlCDPK and SlCRK genes regulate a wide range of resistance and SlCRK6 is the first CRK gene proved to function in plant disease resistance. [ABSTRACT FROM AUTHOR]

Calcium-dependent protein kinases (CDPKs) are multi-functional proteins that combine calcium-binding and signaling capabilities within a single gene product. Current studies have shown that the CDPKs regulate numerous growth and developmental processes and biotic and abiotic stress responses. Nonetheless, knowledge concerning the specific expression patterns and evolutionary history of the CDPK family in cucumber ( Cucumis sativus L.) remains very limited. We, therefore, investigated the phylogenetic relationships and expression profiles of the 19 CDPK genes identified in the cucumber genome sequence, resolving them into four subfamilies based on a phylogenetic tree and gene structures. Tissue-specific expression profiles suggest that cucumber CDPK genes are involved in cucumber tissue development. An expression analysis based on qRT-PCR indicated that cucumber CDPK genes are extensively involved in abscisic acid, salt, cold, drought, heat, and waterlogging responses, possibly by different mechanisms. The fates of two paralogs after divergence were also investigated, suggesting subfunctionalization and neofunctionalization during evolution. These observations lay an important foundation for functional and evolutionary analyses of the CDPK gene family in cucurbitaceae species. [ABSTRACT FROM AUTHOR]

Background In plants, calcium-dependent protein kinases (CDPKs) are involved in tolerance to abiotic stresses and in plant seed development. However, the functions of only a few rice CDPKs have been clarified. At present, it is unclear whether CDPKs also play a role in regulating spikelet fertility. Results We cloned and characterized the rice CDPK gene, OsCPK9. OsCPK9 transcription was induced by abscisic acid (ABA), PEG6000, and NaCl treatments. The results of OsCPK9 overexpression (OsCPK9-OX) and OsCPK9 RNA interference (OsCPK9-RNAi) analyses revealed that OsCPK9 plays a positive role in drought stress tolerance and spikelet fertility. Physiological analyses revealed that OsCPK9 improves drought stress tolerance by enhancing stomatal closure and by improving the osmotic adjustment ability of the plant. It also improves pollen viability, thereby increasing spikelet fertility. In OsCPK9-OX plants, shoot and root elongation showed enhanced sensitivity to ABA, compared with that of wild-type. Overexpression and RNA interference of OsCPK9 affected the transcript levels of ABA- and stress-responsive genes. Conclusions Our results demonstrated that OsCPK9 is a positive regulator of abiotic stress tolerance, spikelet fertility, and ABA sensitivity. [ABSTRACT FROM AUTHOR]

Summary: Plants respond to different environmental cues in a complex way, entailing changes at the cellular and physiological levels. An important step to understand the molecular foundation of stress response in plants is the analysis of stress-responsive proteins. In this work we attempted to investigate and compare changes in the abundance of proteins in the roots of bean (Phaseolus vulgaris L.) germinating under long continuous chilling conditions (10°C, 16 days), exposed to short rapid chilling during germination (10°C, 24h), as well as subjected to recovery from stress (25°C, 24h). The results we obtained indicate that germination under continuous chilling causes alterations in the accumulation of the proteins involved in stress response, energy production, translation, vesicle transport, secondary metabolism and protein degradation. The subsequent recovery influences the accumulation of the proteins implicated in calcium-dependent signal transduction pathways, secondary metabolism and those promoting cell division and expansion. Subjecting the germinating bean seeds to short rapid chilling stress resulted in a transient changes in the relative content of the proteins taking part in energy production, DNA repair, RNA processing and translation. Short stress triggers also the mechanisms of protection against oxidative stress and promotes expression of anti-stress proteins. Subjecting bean seeds to the subsequent recovery influences the abundance of the proteins involved in energy metabolism, protection against stress and production of phytohormones. The exposure to long and short chilling did not result in the alterations of any proteins common to both treatments. The same situation was observed with respect to the recovery after stresses. Bean response to chilling is therefore strongly correlated with the manner and length of exposure to low temperature, which causes divergent proteomic alterations in the roots. [ABSTRACT FROM AUTHOR]

Abstract: We determined the effects of exogenous nitric oxide on photosynthesis and gene expression in transgenic rice plants (PC) over-expressing the maize C4 pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC). Seedlings were subjected to treatments with NO donors, an NO scavenger, phospholipase inhibitors, a Ca2+ chelator, a Ca2+ channel inhibitor, and a hydrogen peroxide (H2O2) inhibitor, individually and in various combinations. The NO donors significantly increased the net photosynthetic rate (PN) of PC and wild-type (WT), especially that of PC. Treatment with an NO scavenger did inhibit the PN of rice plants. The treatments with phospholipase inhibitors and a Ca2+ chelator decreased the PN of WT and PC, and photosynthesis was more strongly inhibited in WT than in PC. Further analyses showed that the NO donors increased endogenous levels of NO and PLD activity, but decreased endogenous levels of Ca2+ both WT and PC. However, there was a greater increase in NO in WT and a greater increase in PLD activity and Ca2+ level in PC. The NO donors also increased both PEPC activity and pepc gene expression in PC. PEPC activity can be increased by SNP alone. But the expression of its encoding gene in PC might be regulated by SNP, together with PA and Ca2+. [Copyright &y& Elsevier]

Abstract: Calcium-dependent protein kinases (CDPKs), which are important sensors of Ca2+ flux in plants, are known to play essential roles in plant development and adaptation to abiotic stresses. In the present work, we studied expression of CDPK genes under osmotic and temperature stress treatments in wild-growing grapevine Vitis amurensis Rupr., which is known to possess high adaptive potential and a high level of resistance against adverse environmental conditions. In this study, using RT-PCR with degenerate primers, DNA sequencing and frequency analysis of RT-PCR products, we identified 13 CDPK genes that are actively expressed in healthy V. amurensis cuttings under high salt, high mannitol, desiccation, and temperature stress conditions. 12 CDPKs, namely VaCPK1, VaCPK2, VaCPK3, VaCPK9, VaCPK13, VaCPK16, VaCPK20, VaCPK21, VaCPK25, VaCPK26, VaCPK29 and VaCPK30, were novel for Vitaceae, and their full cDNAs were obtained and described. Quantitative real-time RT-PCR demonstrated that mRNA levels of 10 VaCPK genes were differentially up-regulated under the osmotic and temperature stress treatments, while the abundance of 3 VaCPK transcript variants, VaCPK3a, VaCPK25, and VaCPK30, was not markedly changed. Expression profiling of the VaCPK genes in leaves, leaf petioles, stems, inflorescences, berries, and seeds of V. amurensis revealed that the genes exhibit different organ-specific expression patterns. The stimulatory effect of abiotic stress on the expression of the VaCPK1, 2, 3, 9, 13, 16, 20, 21, 26, and VaCPK29 genes is suggestive of their implication in the grapevine response to osmotic and temperature stresses, while the variability in their organ-specific expression patterns indicates that the enzymes perform distinct biological functions. [Copyright &y& Elsevier]

Key message: We found that Arabidopsis AtADF1 was phosphorylated by AtCDPK6 at serine 6 predominantly and the phosphoregulation plays a key role in the regulation of ADF1-mediated depolymerization of actin filaments. Abstract: Since actin-depolymerizing factor (ADF) is highly conserved among eukaryotes, it is one of the key modulators for actin organization. In plants, ADF is directly involved in the depolymerization of actin filaments, and therefore important for F-actin-dependent cellular activities. The activity of ADF is tightly controlled through a number of molecular mechanisms, including phosphorylation-mediated inactivation of ADF. To investigate Arabidopsis ADF1 phosphoregulation, we generated AtADF1 phosphorylation site-specific mutants. Using transient expression and stable transgenic approaches, we analyzed the ADF1 phosphorylation mutants in the regulation of actin filament organizations in plant cells. By in vitro phosphorylation assay, we showed that AtADF1 is phosphorylated by AtCDPK6 at serine 6 predominantly. Chemically induced expression of AtCDPK6 can negatively regulate the wild-type AtADF1 in depolymerizing actin filaments, but not those of the mutants AtADF1(S6A) and AtADF1(S6D). These results demonstrate a regulatory function of Arabidopsis CDPK6 in the N-terminal phosphorylation of AtADF1. [ABSTRACT FROM AUTHOR]

Abstract: Genes involved in the calcium signalling pathway have a relationship with cold tolerance in many plants. The primary reaction to many different environmental stresses is an increase in the cytoplasmic Ca2+ concentration. Such variations in the Ca2+ concentration could change the activity of Ca2+-dependent protein functions, further regulating the expression of stress-related genes; therefore, the Ca2+ signalling pathway is involved in the biological stress reaction. The expression of the calcium-modulated protein gene, calmodulin, in Antarctic notothenioid fish (Dissostichus mawsoni) accounts for 0.23% of all transcripts, which is a very high level of expression in this cold-water fish. To elucidate the function of calmodulin (CaM) from Antarctic notothenioid fishes, we introduced the calmodulin (CaM) gene into tobacco plants using a viral vector based on pea early browning virus (PEBV). RT-PCR and Western blot results confirmed that the CaM gene was over-expressed in tobacco. Under low-temperature stress, the CaM transgenic plants exhibited faster growth than wild-type plants. The physiological and biochemical effects of the high-level expression of CaM in tobacco were analysed, and the changes in the electrolyte leakage activity and malondialdehyde content showed that CaM over-expression in tobacco increased the cold tolerance of the plants. These results demonstrate that CaM can possibly be used to enhance the low-temperature tolerance of plants. [Copyright &y& Elsevier]

Plant calcium-dependent protein kinases (CDPKs) play vital roles in calcium signal transduction during various developmental processes and during responses to biotic and abiotic stresses. Here, we isolated and characterized a CDPK gene designated FvCDPK1 from a wild diploid strawberry accession Heilongjiang-3 ( Fragaria vesca L.). The FvCDPK1 gene contains 12 exons and 11 introns, and the sequences of most exons are highly conserved in higher plants. The full-length cDNA of FvCDPK1 contains 1,825 nucleotides with an open reading frame of 1,653 bp encoding a polypeptide of 550 amino acids. The deduced FvCDPK1 protein contains the basic features of typical plant CDPKs: a catalytic kinase domain and a regulatory calmodulin-like domain containing four EF-hand calcium-binding motifs. Phylogenetic analysis confirmed that FvCDPK1 belongs to the plant CDPK family. When transiently expressed in onion epidermal cells, the FvCDPK1-GFP fusion protein was found to be localized in the nucleus. Expression analysis indicated that FvCDPK1 was expressed in fruits at different developmental and ripening stages, as well as in several tissues such as roots, runners, flowers, leaves, and meristems. Moreover, expression levels of FvCDPK1 were higher in meristems than in other vegetative tissues. Under abiotic stress conditions, however, FvCDPK1 was found to be upregulated upon abscisic acid, NaCl, cold-, or high-temperature treatments. Taken together, our data suggest that FvCDPK1 might play a role in various responses to abiotic stresses in strawberry. [ABSTRACT FROM AUTHOR]

CDPKs (calcium-dependent protein kinases), which contain both calmodulin-like calcium binding and serine/threonine protein kinase domains, are only present in plants and some protozoans. Upon activation by a stimulus, they transduce the signal through phosphorylation cascades to induce downstream responses, including transcriptional regulation. To understand the functional specificities of CDPKs, 14 Arabidopsis CPKs (CDPKs in plants) representative of the three main subgroups were characterized at the biochemical level, using HA (haemagglutinin)-tagged CPKs expressed in planta. Most of them were partially or mainly associated with membranes, in agreement with acylation predictions. Importantly, CPKs displayed highly variable calcium-dependences for their kinase activities: seven CPKs from subgroups 1 and 2 were clearly sensitive to calcium with different intensities, whereas six CPKs from subgroup 3 exhibited low or no calcium sensitivity to two generic substrates. Interestingly, this apparent calcium-independence correlated with significant alterations in the predicted EF-hands of these kinases, although they all bound calcium. The noticeable exception, CPK25, was calcium-independent owing to the absence of functional EF-hands. Taken together, the results of the present study suggest that calcium binding differentially affects CDPK isoforms that may be activated by distinct molecularmechanisms. [ABSTRACT FROM AUTHOR]

Juglone (5-hydroxy-1,4-naphthoquinone) is known allelochemical, but its molecular mode of action is not well understood. We found that juglone induced reactive oxygen species production and calcium accumulation. To gain more insight into these cellular responses, we performed large-scale analysis of the rice transcriptome during juglone stress. Exposure to juglone triggered changes in transcript levels of genes related to cell growth, cell wall formation, chemical detoxification, abiotic stress response and epigenesis. The most predominant transcription-factor families were AP2/ERF, HSF, NAC, C2H2, WRKY, MYB and GRAS. Gene expression profiling of juglone-treated rice roots revealed upregulated signaling and biosynthesis of abscisic acid and jasmonic acid and inactivation of gibberellic acid. In addition, juglone upregulated the expression of two calcium-dependent protein kinases (CDPKs), 6 mitogen-activated protein kinase (MAPK) genes and 1 MAPK gene and markedly increased the activities of a CDPK-like kinase and MAPKs. Further characterization of these juglone-responsive genes may be helpful for better understanding the mechanisms of allelochemical tolerance in plants. [ABSTRACT FROM AUTHOR]

Summary: A rice CDPK gene, OsCDPK14 (AY144497), was cloned from developing caryopses of rice (Oryza sativa cv. Zhonghua 15). Its cDNA sequence (1922bp) contains an ORF encoding a 514 amino acids protein (56.7kD, pl 5.18). OsCDPK14 shows the typical structural features of the CDPK family, including a conserved catalytic Ser/Thr kinase domain, an autoinhibitory domain and a CaM-like domain with four putative Ca2+-binding EF hands. Subcellular targeting indicated that OsCDPK14 was located in the cytoplasm, probably due to the absence of myristoylation and palmitoylation motifs. OsCDPK14 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using Syntide-2, a synthetic peptide. Kinase activity was shown to be Ca2+-dependent, and this activation was strongly enhanced by Mn2+ and inhibited by W7 in vitro. These results provide significant insights into the regulation and biochemical properties of OsCDPK14, suggesting OsCDPK14 may be a signal factor of cytoplasm in rice plant. [Copyright &y& Elsevier]

The metabolic plasticity displayed by plants during normal development, and in response to environmental fluctuations and stressors, is essential for their growth and survival. The capacity to regulate metabolic enzymes intricately arises in part from posttranslational modifications that can affect enzymatic activity, intracellular localization, protein–protein interactions, and stability. Protein phosphorylation and thiol/disulfide redox modulation are important modifications in plants, and it is likely that O-glycosylation and S-nitrosylation will also emerge as important mechanisms. Recent advances in the field of proteomics, in particular the development of novel and specific chemistries for the detection of a diverse number of modifications, are rapidly expanding our awareness of possible modifications and our understanding of the enzymes whose functions are likely to be regulated posttranslationally. [Copyright &y& Elsevier]

The serine-170 (S170) calcium-dependent protein kinase phosphorylation site of maize (Zea mays L.) sucrose synthase (SUS) (EC 2.4.1.13) has been implicated in the post-translational regulation of SUS protein stability. To clarify the proteolytic process and the role of phosphorylation, SUS degradation and proteasome activities were studied in the maize leaf elongation zone. Size-exclusion chromatography resolved two peaks of proteasome-like proteolytic activity. The large molecular mass (∼1350 kDa) peak required Mg2+ and ATP for maximal activity and was inhibited by the proteasome inhibitors MG132 and NLVS. Anion-exchange chromatography resolved a similar proteolytic activity that was activated by ATP, characteristics that are consistent with those of a 26S-proteasome. Appropriately, immunoblotting revealed the presence of a 26S-proteasome subunit and highly ubiquitinated proteins within the active fractions eluted from both columns. The smaller molecular mass (∼600 kDa) peak represented only 40% of the total proteasome-like activity and is likely a maize 20S-proteasome as it was activated in vitro by low levels of sodium dodecyl sulfate (SDS). S170 phosphorylated SUS (pS170-SUS) was detected as both high molecular mass (HMM) forms and proteolytic fragments that co-eluted with 26S-proteasome activities on both size-exclusion and anion-exchange columns. Conditions that maintained maximal 26S-proteasome activity reduced the amounts of pS170-SUS recovered. In vitro, the 26S-proteasome degraded SUS and proteasome-specific inhibitors reduced SUS proteolysis. HMM-SUS conjugates were produced in vitro and immunoprecipitations suggested that some SUS might be ubiquitinated in vivo. The results suggest that S170 phosphorylation promotes the formation of HMM, ubiquitin–SUS conjugates that can be targeted for 26S-proteasome-dependent degradation. [Copyright &y& Elsevier]

Insights into the signal transduction of the phytohormone abscisic acid (ABA) have unfolded dramatically in the past few years and reveal an unanticipated complexity. Knockout lines and RNA-interference technology, together with protein interaction analyses, have been used to identify many of the cellular components that regulate or modulate ABA responses. ABA signaling is characterized by a plethora of intracellular messengers. This may reflect the function of ABA in integrating several stress responses and antagonizing pathways via cross-talk, but it hampers the establishment of a unifying concept. Transcriptome analyses have unraveled more than a thousand genes that are differentially regulated by ABA, and these ABA-mediated changes in gene expression translate to major changes in proteome expression. ABA-induced mechanisms that re-adjust cellular protein expression are just surfacing. ABA-response-specific transcription factors have a well-established function in that process and, recently, it has also become clear that phytohormone signaling enforces a sophisticated interference with protein expression at the posttranscriptional level. This interference includes both targeted proteolysis and the regulation of the translation of specific mRNAs by RNA-binding proteins. [Copyright &y& Elsevier]

Several kinases are activated during the defence response following microbial elicitation. While studying the regulation of these kinases in greater detail, it has become clear that the means by which phosphorylation events transmit specific information to the cell is highly complex. To gain a better understanding of the molecular events leading to a response, it will be increasingly important to identify not only the protein targets of phosphorylation but also the specific sites of phosphorylation. New developments in peptide-based phosphoproteome analysis appear to hold the promise of achieving these goals at the whole-cell level. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Pulsed-field gradient (PFG) diffusion NMR spectroscopy studies were conducted with several helix-loop-helix regulatory Ca2+-binding proteins to characterize the conformational changes associated with Ca2+-saturation and/or binding targets. The calmodulin (CaM) system was used as a basis for evaluation, with similar hydrodynamic radii ( R h) obtained for apo- and Ca2+-CaM, consistent with previously reported R h data. In addition, conformational changes associated with CaM binding to target peptides from myosin light chain kinase (MLCK), phosphodiesterase (PDE), and simian immunodeficiency virus (SIV) were accurately determined compared with small-angle X-ray scattering results. Both sets of data demonstrate the well-established collapse of the extended Ca2+-CaM molecule into a globular complex upon peptide binding. The R h of CaM complexes with target peptides from CaM-dependent protein kinase I (CaMKI) and an N-terminal portion of the SIV peptide (SIV-N), as well as the anticancer drug cisplatin were also determined. The CaMKI complex demonstrates a collapse analogous to that observed for MLCK, PDE, and SIV, while the SIV-N shows only a partial collapse. Interestingly, the covalent CaM-cisplatin complex shows a near complete collapse, not expected from previous studies. The method was extended to related calcium binding proteins to show that the R h of calcium and integrin binding protein (CIB), calbrain, and the calcium-binding region from soybean calcium-dependent protein kinase (CDPK) decrease on Ca2+-binding to various extents. Heteronuclear NMR spectroscopy suggests that for CIB and calbrain this is likely because of shifting the equilibrium from unfolded to folded conformations, with calbrain forming a dimer structure. These results demonstrate the utility of PFG-diffusion NMR to rapidly and accurately screen for molecular size changes on protein-ligand and protein-protein interactions for this class of proteins. [ABSTRACT FROM AUTHOR]

Calcium ion is a key messenger in turgor regulation of internodal cells of Lamprothamnium succinctum in response to hypoosmotic treatment. An increase in the concentration of cytosolic free calcium ion ([Ca2+]c) is prerequisite for the turgor regulation [Okazaki and Tazawa (1990) J. Membr. Biol. 114: 189], We examined whether or not a calcium-dependent protein kinase (CDPK) is involved in the Ca2+-mediated turgor regulation of Lamprothamnium cells. A 53-kDa CDPK which phosphorylated preferentially histone H1 but poorly myelin basic protein or casein, was detected in the cell extract of Lamprothamnium by an in-gel protein kinase assay. This protein kinase was detected by Western blotting and was immunoprecipitated using an anti-Dunaliella tertiolecta CDPK antibody which can neutralize the Dunaliella CDPK activity [Yuasa et al. (1995) Plant Cell Physiol. 36: 699]. The 53-kDa CDPK was partially purified from Lamprothamnium and its activity was shown to be inhibited by the antibody and K-252a, a protein kinase inhibitor. Microinjection of the antibody into the cytosbl of Lamprothamnium cells inhibited the decrease in turgor pressure in response to hypoosmotic treatment. However, a transient increase in [Ca2+]c, which was suggested by a transient reduction of the velocity of cytoplasmic streaming, was induced in antibody-injected cells after hypoosmotic treatment. Turgor regulation upon hypoosmotic treatment was inhibited when the cells were treated with K-252a. These results imply that CDPK of Lamprothamnium functions at a down-stream position of Ca2+-mobilization in processing turgor regulation in response to hypoosmotic treatment. [ABSTRACT FROM AUTHOR]

Potato ( Solanum tuberosum) calcium-dependent protein kinase (StCDPK5) has been shown to phosphorylate the N-terminal region of plasma membrane RBOH (respiratory burst oxidase homolog) proteins, and participate in StRBOHB-mediated reactive oxygen species (ROS) burst. The constitutively active form, StCDPK5VK, provides a useful tool for gain-of-function analysis of RBOH in defense responses., StCDPK5- and StCDPK5VK-green fluorescent protein fusion proteins were predominantly targeted to the plasma membrane, and conditional expression of StCDPK5VK activated StRBOHA-D. The interaction was confirmed by bimolecular fluorescence complementation assay. We generated transgenic potato plants containing StCDPK5VK under the control of a pathogen-inducible promoter to investigate the role of ROS burst on defense responses to blight pathogens., Virulent isolates of the late blight pathogen Phytophthora infestans and the early blight pathogen Alternaria solani induced hypersensitive response-like cell death accompanied by ROS production at the infection sites of transgenic plants. Transgenic plants showed resistance to the near-obligate hemibiotrophic pathogen P. infestans and, by contrast, increased susceptibility to the necrotrophic pathogen A. solani., These results indicate that RBOH-dependent ROS contribute to basal defense against near-obligate pathogens, but have a negative role in resistance or have a positive role in expansion of disease lesions caused by necrotrophic pathogens. [ABSTRACT FROM AUTHOR]