A Calcium-Dependent Protein Kinase Is Systemically Induced upon Wounding in Tomato Plants

A full-length cDNA clone (LeCDPK1) from tomato (Lycopersicon esculentum) encoding a calcium-dependent protein kinase (CDPK) was isolated by screening a cDNA library from tomato cell cultures exposed to Cladosporium fulvum elicitor preparations. The predicted amino acid sequence of the cDNA reveals a high degree of similarity with other members of the CDPK family. LeCDPK1 has a putative N-terminal myristoylation sequence and presents a possible palmitoylation site. The in vitro translated protein conserves the biochemical properties of a member of the CDPK family. In addition, CDPK activity was detected in soluble and particulate extracts of tomato leaves. Basal levels of LeCDPK1 mRNA were detected by northern-blot analysis in roots, stems, leaves, and flowers of tomato plants. The expression of LeCDPK1 was rapidly and transiently enhanced in detached tomato leaves treated with pathogen elicitors and H2O2. Moreover, when tomato greenhouse plants were subjected to mechanical wounding, a transient increase of LeCDPK1 steady-state mRNA levels was detected locally at the site of the injury and systemically in distant non-wounded leaves. The increase observed in LeCDPK1 mRNA upon wounding correlates with an increase in the amount and in the activity of a soluble CDPK detected in extracts of tomato leaves, suggesting that this kinase is part of physiological plant defense mechanisms against biotic or abiotic attacks.     

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

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

<Emphasis Type="Italic">St</Emphasis>CDPK2 expression and activity reveal a highly responsive potato calcium-dependent protein kinase involved in light signalling

Calcium-dependent protein kinases (CDPKs) are essential calcium sensors. In this work, we have studied StCDPK2 isoform from potato both at gene and protein level. StCdpk2 genomic sequence contains eight exons and seven introns, as was observed for StCdpk1. There is one copy of the gene per genome located in chromosome 7. StCDPK2 encodes an active CDPK of 515 aminoacids, with an apparent MW of 57 kDa, which presents myristoylation and palmitoylation consensus in its N-terminus. StCDPK2 is highly expressed in leaves and green sprouts; enhanced expression was detected under light treatment, which corresponds well with light responsive cis-acting elements found in its promoter sequence. Antibodies against the recombinant StCDPK2::6xHis protein detected this isoform in soluble and particulate fractions from leaves. StCDPK2 autophosphorylation and kinase activity are both calcium dependent reaching half maximal activation at 0.6 μM calcium. The active kinase is autophosphorylated on serine and tyrosine residues and its activity is negatively modulated by phosphatidic acid (PA). Our results reveal StCDPK2 as a signalling element involved in plant growth and development and show that its activity is tightly regulated.     

Membrane-associated protein kinase activities in developing apple fruit

Fruit development is a process involving various signals and gene expression. Protein phosphorylation catalysed by protein kinases is known to play a key role in eukaryotic cell signalling and so may be involved in the regulation of fruit development. Using the method of exogenous substrate phosphorylation, the activity of calcium-dependent and calmodulin-independent protein kinase (CDPK) that was stimulated by phosphatidylserine, and the myelin basic protein (MBP)-phosphorylating activity that could be due to a calcium-independent mitogen-activated protein kinase-like (MAPK-like) activity in the developing apple fruits were identified. The CDPK activity was shown to be predominantly localized in the plasma membrane, whereas in the presence of phosphatidylserine, the high activity of CDPK was detected in both plasma membrane and endomembranes. The MAPK-like activity was predominantly associated with endomembranes. The assays of bivalent cation requirement showed that Mn²⁺ could replace Mg²⁺ in the incubation system for the protein kinase activities and stimulate CDPK activity more than Mg²⁺. Heat treatment abolished CDPK but stimulated MAPK-like activity. The activities of the phosphatidylserine-stimulated CDPK and of the MAPK-like were fruit developmental stage-specific with higher activities of both enzymes in the early and middle developmental stages in comparison with the late developmental stage. These data suggest that the detected protein kinases may play an important role in the fruit development.     

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.     

A rice membrane calcium-dependent protein kinase is induced by gibberellin.

A rice (Oryza sativa) seed plasma-membrane calcium-dependent serine/threonine protein kinase (CDPK) has been partially purified. Comparing results in seeds that were treated with and without the plant hormone gibberellin (GA) for 10 min showed that rice CDPK was highly induced by GA. After separating solubilized membrane proteins by sodium dodecyl sulfate-gel electrophoresis, followed by renaturation, a radiolabeled phosphoprotein band of approximately 58 kD was detected, and it was apparently produced by autophosphorylation. There are five aspects of the rice CDPK that show similarity to mammalian protein kinase C (PKC) and to other plant CDPKs: (a) Histone IIIS and PKC peptide-ser25 (19-31) are phosphorylated by rice CDPK. (b) The phosphorylation reaction is strictly dependent on calcium. (c) The activity of the rice CDPK is inhibited by either staurosporine or the PKC inhibitory peptide (19-36). (d) Addition of calmodulin has no effect on the activity of the enzyme; however, the CDPK is inhibited by the calmodulin antagonists trifluoperazine and W-7. (e) The rice CDPK reacts with a mammalian anti-PKC antibody in immunoblotting analysis. However, there is one major difference between the rice CDPK and other CDPKs: the rice CDPK is induced by GA, whereas no mammalian PKC or other plant CDPKs are known to be induced by any hormone.     

Calcium-dependent protein kinase from maize seedlings activated by phospholipids.

A calcium- and phospholipid-dependent protein kinase of apparent molecular mass 54 kDa (designated ZmCPKp54) was partially purified from etiolated maize seedlings. Activity of ZmCPKp54 is stimulated by phosphatidylserine and phosphatidylinositol, but is not essentially affected by diolein and phorbol esters. The enzyme cross-reacts with polyclonal antibodies against the calmodulin like-domain of the calcium-dependent protein kinase, but not with antibodies against catalytic or regulatory domains of protein kinase C. ZmCPKp54 is not able to phosphorylate the specific substrates of protein kinase C (MARCKS peptide and protein kinase C substrate peptide derived from pseudosubstrate sequence) and its activity is not inhibited by specific PKC inhibitors (bisindolylmaleimide, protein kinase C pseudosubstrate inhibitory peptide). The substrate specificity and sensitivity to the inhibitors of the maize enzyme resembles calcium-dependent protein kinase. The biochemical and immunological properties indicate that ZmCPKp54 belongs to the calcium-dependent protein kinase family.     

Inhibition of plant calcium-dependent protein kinases by basic polypeptides

Wheat embryo Ca2+-dependent protein kinase (CDPK) is inhibited by a variety of polypeptides including actin, gramicidin S, melittin, protamine, various histone preparations, histone H4 and by basic amino-acid homopolymers. Melittin (Ki 9 microM) is a non-competitive inhibitor of wheat germ CDPK and also inhibits wheat leaf CDPK and silver beet leaf CDPKs. Protamine inhibits wheat germ CDPK in an apparently competitive fashion (Ki 0.2 microM) and is also a potent, albeit less effective, inhibitor of the leaf CDPKs. Various basic amino-acid homopolymers are also potent, apparently competitive inhibitors of wheat embryo CDPK, namely poly(L-lysine) (IC50 2 nM), poly(L-ornithine) (IC50 3 nM) and poly(L-arginine) (IC50 17 nM) and also inhibit the leaf CDPKs, albeit at higher concentrations. Histone H4 and various calf thymus histone preparations inhibit wheat embryo CDPK in a fashion that is not competitive and calmodulin can substantially reverse such inhibition.     

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

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

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.