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.     

Protein kinase activity in Cucumis sativus cotyledons: effect of calcium and light

Light signals received by phytochromes in plants may be transduced through protein phosphorylation. Ca(2+) as second messenger was involved in phytochrome-mediated cellular events. Our experiments with Cucumis sativus cotyledons, treated with red (R) and far-red (FR) light, showed a stimulatory effect on in vitro protein phosphorylation of histone, added as exogenous substrate to the cotyledon extracts, and also modified the phosphorylation of endogenous polypeptides. The effect of light treatments was mimicked by the addition of Ca(2+) to the phosphorylation buffer, indicating phytochrome- and Ca(2+)-dependence on activity of some protein kinases (PKs). In-gel kinase assays were performed to characterize the PKs involved at the cotyledon stage of cucumber plants. Three proteins of about 75, 57 and 47kDa with PK activity were detected between M(r) markers of 94 and 45kDa. All three were able to phosphorylate histone and undergo autophosphorylation. However, only the 75 and 57kDa proteins autophosphorylated and phosphorylated the substrate in a Ca(2+)-dependent manner, and were inhibited when calmodulin (CaM) antagonists were added to the incubation buffer. Western-blot analysis with polyclonal antibodies directed against calcium-dependent protein kinase of rice (OsCDPK11) or Arabidopsis (AtCPK2) recognised 57 and 75kDa polypeptides, respectively. These results indicate the presence in cucumber cotyledons of at least two proteins (ca. 75 and 57kDa) with activity of PKs that could be calcium-dependent protein kinases (CDPKs). Both CDPKs could be modulated by phytochromes throughout FR-HIR and VLFR responses.     

Plants, symbiosis and parasites: a calcium signalling connection

A unique family of protein kinases has evolved with regulatory domains containing sequences that are related to Ca(2+)-binding EF-hands. In this family, the archetypal Ca(2+)-dependent protein kinases (CDPKs) have been found in plants and some protists, including the malarial parasite, Plasmodium falciparum. Recent genetic evidence has revealed isoform-specific functions for a CDPK that is essential for Plasmodium berghei gametogenesis, and for a related chimeric Ca(2+) and calmodulin-dependent protein kinase (CCaMK) that is essential to the formation of symbiotic nitrogen-fixing nodules in plants. In Arabidopsis thaliana, the analysis of 42 isoforms of CDPK and related kinases is expected to delineate Ca(2+) signalling pathways in all aspects of plant biology.     

Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family

In plants, numerous Ca(2+)-stimulated protein kinase activities occur through calcium-dependent protein kinases (CDPKs). These novel calcium sensors are likely to be crucial mediators of responses to diverse endogenous and environmental cues. However, the precise biological function(s) of most CDPKs remains elusive. The Arabidopsis genome is predicted to encode 34 different CDPKs. In this Update, we analyze the Arabidopsis CDPK gene family and review the expression, regulation, and possible functions of plant CDPKs. By combining emerging cellular and genomic technologies with genetic and biochemical approaches, the characterization of Arabidopsis CDPKs provides a valuable opportunity to understand the plant calcium-signaling network.     

Identification of a novel phosphorylation motif for CDPKs: phosphorylation of synthetic peptides lacking basic residues at P-3/P-4

The Ca(2+)-dependent protein kinases (CDPKs) are members of a large subfamily of protein kinases in plants that have been implicated in the control of numerous aspects of plant growth and development. One known substrate of the CDPKs is the ER-located ACA2 calcium pump, which is regulated by phosphorylation of Ser(45). In the present study, a synthetic peptide based on the known regulatory phosphorylation site (RRFRFTANLS(45)KRYEA) was efficiently phosphorylated in vitro by CDPKs but not a plant SNF1-related protein kinase. Phosphorylation of the Ser(45)-ACA2 peptide was surprising because the sequence lacks basic residues at P-3/P-4 (relative to the phosphorylated Ser at position P) that are considered to be essential recognition elements for CDPKs. We demonstrate that phosphorylation of the Ser(45)-ACA2 peptide is dependent on the cluster of basic residues found N-terminal (P-6 to P-9) as well as C-terminal (P + 1/P + 2) to the phosphorylated Ser. The results establish a new general phosphorylation motif for CDPKs: [Basic-Basic-X-Basic]-phi-X(4)-S/T-X-Basic (where phi is a hydrophobic residue). The motif predicts a number of new phosphorylation sites in plant proteins. Evidence is presented that the novel motif may explain the phosphorylation by CDPKs of Ser271 in the aquaporin PM28A.     

巴西橡胶树HbCMK基因的克隆及表达

根据植物CMK(4-Diphosphocytidyl-2C-methyl-D-erythritol Kinase,CMK)的同源序列设计引物,通过RT-PCR结合RACE的方法在橡胶树中获得了与其相应的CMK基因,命名为HbCMK.序列分析表明HbCMK长1 415 bp,编码388个氨基酸,该氨基酸序列与长春花(Catharanthus roseus)、拟南芥(Arabidopsis thaliana)、甜菊(Stevia rebaudiana)、丹参(Salvia miltiorrhiza)、烟草(Nicotiana benthamiana)和水稻(Oryza sativa)的CMK相似性分别达到72.6%、72.5%、71.9%、70.9%、69.0%和66.9%.半定量RT-PCR结果显示,HbCMK的表达具有组织差异性,在愈伤组织中大量表达,在叶片和胶乳中微量表达,乙烯诱导胶乳HbCMK的表达,伤害对HbCMK的表达几乎没有影响.本实验结果为进一步了解MEP途径在橡胶树胶乳中的作用和天然橡胶生物合成的调控奠定了基础.     

巴西橡胶树一个编码含有C2结构域蛋白cDNA的克隆及表达分析

本研究根据从巴西橡树胶乳cDNA文库中获得的一个EST片段的序列信息设计引物,通过RACE的方法获得了橡胶树编码含有C2结构域蛋白的cDNA(命名为HbC2)。序列分析表明,HbC2长为1185bp,含有813bp的阅读框,140bp的5'-UTR和232bp的3'-UTR,编码270个氨基酸,分子量为30.9KD,等电点为6.29,含有保守的C2结构域。半定量RT-PCR分析表明HbC2在花、芽、叶、胶乳和树皮中都有表达,其中在胶乳中表达量最高。茉莉酸可抑制HbC2的表达,乙烯对HbC2的表达没有影响。此研究为进一步研究C2蛋白基因在橡胶树中的生物学功能奠定基础。     

Unexpected structure of the Ca2+-regulatory region from soybean calcium-dependent protein kinase-alpha

Calcium-dependent protein kinases (CDPKs) are an extensive class of multidomain Ca(2+)-regulated enzymes from plants and protozoa. In vivo the so-called calmodulin-like domain (CLD) of CDPK binds intramolecularly to the junction domain (JD), which exhibits both kinase-inhibitory and CLD binding properties. Here we report the high resolution solution structure of the calcium-regulatory region from soybean CDPK-alpha determined in the presence of a peptide encompassing the JD. The structure of both lobes of CLD resembles that of related helix-loop-helix Ca(2+)-binding proteins. NMR chemical shift mapping studies demonstrate that the JD induces significant structural changes in isolated Ca(2+)-CLD, particularly the C-terminal domain, although a stable complex is not formed. A CLD solution structure calculated on the basis of NMR data and long range fluorescence resonance energy transfer distances reveals an activated state with both lobes positioned side by side, similar to calcineurin B rather than calmodulin, highlighting the possible pitfall of assigning function purely from sequence information.     

Comparative modeling studies of the calmodulin-like domain of calcium-dependent protein kinase from soybean

Calmodulin-like domain protein kinases (CDPKs) represent a new class of calcium-dependent protein-phosphorylating enzymes that are not activated by calmodulin or phospholipid compounds. They have been found exclusively in plant and protozoal tissues. CDPKs are typified by four distinct domains: an N-terminal leader sequence, a protein kinase (PK) domain, a calmodulin-like domain (CLD), and a junction domain (JD) between the PK domain and CLD. Structural characterization of the CLD of CDPKalpha from soybean was undertaken based on the amino acid sequence homology of CLD to the structurally well-characterized calmodulin (CaM) family of structures. Tertiary models of apo-CLD, Ca(2+)-CLD complex, and intermolecularly bound Ca(2+)-CLD-JD complexes were obtained via automated and non-automated homology building methods. The resulting structures were compared and validated based on energy differences, phi-psi angle distribution, solvent accessibility, and hydrophobic potential. Circular dichroism, one-dimensional, and two-dimensional nuclear magnetic resonance spectroscopy studies of the CLD and peptides encompassing the JD provide experimental support to the models. The results suggest that there is a possible interaction between the CLD and JD domain similar to that of the CaM/calmodulin-dependent protein kinase II system. At low Ca(2+) levels, the JD may act as an autoinhibitory domain for kinase activity, and during calcium activation an intramolecular CLD-JD complex may form, relieving inhibition of the PK domain. Interactions between the JD and the C terminus of the CLD appear to be particularly important. The outcome of this study supports an intramolecular binding model for calcium activation of CDPK, although not exclusively.     

Activation and inhibition of rubber transferases by metal cofactors and pyrophosphate substrates

Metal cofactors are necessary for the activity of alkylation by prenyl transfer in enzyme-catalyzed reactions. Rubber transferase (RuT, a cis-prenyl transferase) associated with purified rubber particles from Hevea brasiliensis, Parthenium argentatum and Ficus elastica can use magnesium and manganese interchangably to achieve maximum velocity. We define the concentration of activator required for maximum velocity as [A](max). The [A](max)(Mg2+) in F. elastica (100 mM) is 10 times the [A](max)(Mg2+) for either H. brasiliensis (10 mM) or P. argentatum (8 mM). The [A](max)(Mn2+) in F. elastica (11 mM), H. brasiliensis (3.8 mM) and P. argentatum (6.8 mM) and the [A](max)(Mg2+) in H. brasiliensis (10 mM) and P. argentatum (8 mM) are similar. The differences in [A](max)(Mg2+) correlate with the actual endogenous Mg(2+) concentrations in the latex of living plants. Extremely low Mn(2+) levels in vivo indicate that Mg(2+) is the RuT cofactor in living H. brasiliensis and F. elastica trees. Kinetic analyses demonstrate that FPP-Mg(2+) and FPP-Mn(2+) are active substrates for rubber molecule initiation, although free FPP and metal cations, Mg(2+) and Mn(2+), can interact independently at the active site with the following relative dissociation constants K(d)(FPP) 相似文献   

Calcium and signal transduction in plants

Environmental and hormonal signals control diverse physiological processes in plants. The mechanisms by which plant cells perceive and transduce these signals are poorly understood. Understanding biochemical and molecular events involved in signal transduction pathways has become one of the most active areas of plant research. Research during the last 15 years has established that Ca2+ acts as a messenger in transducing external signals. The evidence in support of Ca2+ as a messenger is unequivocal and fulfills all the requirements of a messenger. The role of Ca2+ becomes even more important because it is the only messenger known so far in plants. Since our last review on the Ca2+ messenger system in 1987, there has been tremendous progress in elucidating various aspects of Ca(2+) -signaling pathways in plants. These include demonstration of signal-induced changes in cytosolic Ca2+, calmodulin and calmodulin-like proteins, identification of different Ca2+ channels, characterization of Ca(2+) -dependent protein kinases (CDPKs) both at the biochemical and molecular levels, evidence for the presence of calmodulin-dependent protein kinases, and increased evidence in support of the role of inositol phospholipids in the Ca(2+) -signaling system. Despite the progress in Ca2+ research in plants, it is still in its infancy and much more needs to be done to understand the precise mechanisms by which Ca2+ regulates a wide variety of physiological processes. The purpose of this review is to summarize some of these recent developments in Ca2+ research as it relates to signal transduction in plants.     

Variations in the level of enzyme activity and immunolocalization of calcium-dependent protein kinases in the phloem of different cucumber organs

Calcium-dependent protein kinases (CDPKs) constitute a unique family of enzymes in plants that are characterized by a C-terminal calmodulin (CaM)-like domain. Through protein kinase assays, we have examined the levels of cucumber calcium-dependent kinase (CsCDPK) activity in various organs of cucumber seedlings and plants. The activity of CsCDPK was highest in cucumber plant leaves followed by seedling roots and hypocotyls; however, cucumber plant flowers, seedling cotyledons, and hooks had levels that were barely detectable. The CsCDPKs were immunolocalized using polyclonal antibodies that are highly specific against a part of the kinase domain of a calcium-dependent protein kinase (CsCDPKS) in the phloem sieve elements (SEs) in various organs of cucumber. In addition, this study indicates the presence of CsCDPKs in organelle-like bodies associated with the plasma membrane of sieve elements in mature stems and roots as well as in the storage bodies of immature seeds. These findings are discussed in terms of the likely roles played by CDPKs in the signal transduction pathways for Ca2+-regulated phloem transport of assimilates from leaves to various organs during growth and development of cucumber seedlings and plants.     

Structure of the regulatory apparatus of a calcium-dependent protein kinase (CDPK): a novel mode of calmodulin-target recognition

Calcium-dependent protein kinases (CDPKs) are a class of calcium-binding sensory proteins that are found in plants and certain protozoa, including the causative agent of malaria, Plasmodium falciparum. CDPKs have diverse regulatory functions, including involvement in the triggering of the lytic cycle of malarial infection. CDPKs contain an autoinhibitory junction (J) region whose calcium-dependent interaction with the tethered regulatory calmodulin-like domain (CaM-LD) activates the catalytic kinase domain. We report here the X-ray crystal structure of the J-CaM-LD region of CDPK from Arabidopsis thaliana (AtCPK1), determined to 2.0 A resolution using multiple-wavelength anomalous dispersion (MAD). The structure reveals a symmetric dimer of calcium-bound J-CaM-LD with domain-swap interactions, in which the J region of one protomer interacts extensively with the carboxy-terminal EF-hand domain (C-lobe) of the partner protomer. However, as the J-CaM-LD is monomeric in solution, the activated monomer was modelled to account for the intra-molecular recognition of the two domains. While the J-CaM-LD segment mimics certain aspects of target motif recognition by CaM other features are specific to CDPKs, in particular the combination of the strong interaction between the N and C-lobes of the CaM-LD and the exclusive use of only the C-lobe in the recognition of the covalently tethered target region. Combined with our previous observations showing that there is likely to be strong interactions between this tethered J region and the CaM-LD even at basal Ca(2+) concentrations, the new structural data indicate that the response to calcium of CDPKs is clearly unique among the CaM family.     

A thapsigargin-sensitive Ca(2+) pump is present in the pea Golgi apparatus membrane

The Golgi apparatus behaves as a bona fide Ca(2+) store in animal cells and yeast (Saccharomyces cerevisiae); however, it is not known whether this organelle plays a similar role in plant cells. In this work, we investigated the presence of an active Ca(2+) accumulation mechanism in the plant cell Golgi apparatus. Toward this end, we measured Ca(2+) uptake in subcellular fractions isolated from the elongating zone of etiolated pea (Pisum sativum) epicotyls. Separation of organelles using sucrose gradients showed a strong correlation between the distribution of an ATP-dependent Ca(2+) uptake activity and the Golgi apparatus marker enzyme, xyloglucan-fucosyltransferase. The kinetic parameters obtained for this activity were: the rate of maximum Ca(2+) uptake of 2.5 nmol mg min(-1) and an apparent K(m) for Ca(2+) of 209 nM. The ATP-dependent Ca(2+) uptake was strongly inhibited by vanadate (inhibitor concentration causing 50% inhibition [I(50)] = 126 microM) and cyclopiazonic acid (I(50) = 0.36 nmol mg protein(-1)) and was not stimulated by calmodulin (1 microM). Addition of Cd(2+) and Cu(2+) at nanomolar concentration inhibited the Ca(2+) uptake, whereas Mn(2+), Fe(2+), and Co(2+) had no significant effect. Interestingly, the active calcium uptake was inhibited by thapsigargin (apparent I(50) = 88 nM), a well-known inhibitor of the endoplasmic reticulum and Golgi sarco-endoplasmic reticulum Ca(2+) ATPase from mammalian cells. A thapsigargin-sensitive Ca(2+) uptake activity was also detected in a cauliflower (Brassica oleracea) Golgi-enriched fraction, suggesting that other plants may also possess thapsigargin-sensitive Golgi Ca(2+) pumps. To our knowledge, this is the first report of a plant Ca(2+) pump activity that shows sensitivity to low concentrations of thapsigargin.     

The formation of 5-phosphomevalonate by mevalonate kinase in Hevea brasiliensis latex

1. Evidence has been produced for the formation of 5-phosphomevalonate from potassium dl-mevalonate by the latex of Hevea brasiliensis and by reconstituted freeze-dried serum obtained from this latex. 2. The enzyme, mevalonate kinase, catalysing the formation of 5-phosphomevalonate from potassium dl-mevalonate and ATP has been partially purified. 3. 5-Phosphomevalonate formed by the purified mevalonate kinase from potassium [2-(14)C]mevalonate has been shown to be incorporated by latex into rubber to about 2.4 times the extent of dl-mevalonate. 4. The enzyme can utilize inosine triphosphate as effectively as adenosine triphosphate as a phosphate donor and is also slightly active with uridine triphosphate. 5. The enzyme was fairly stable to a range of pH values and temperatures, the activity being optimum at pH7.5 and 60-70 degrees . The energy of activation was 10.7kcal./mole. The K(m) values were 0.13mm for potassium dl-mevalonate and 2.0mm for ATP at 30 degrees . 6. The enzyme required the presence of Mn(2+) (1mm) for maximum activity; this could be replaced by Mg(2+) (4mm), which was less effective, and by Ca(2+), which was far less effective. 6. Although the enzyme did not require cysteine or reduced glutathione for activation in aerobic conditions, it was inhibited by reagents known to react with thiol groups.     

巴西橡胶树磷脂酰肌醇转移蛋白cDNA的克隆及其序列分析

本文从巴西橡胶树(Hevea brasiliensis)差减cDNA文库中筛选到一个与磷脂酰肌醇转移蛋白(phos-phatidylinositol transfer protein)同源性较高的基因片段,并根据该基因片段序列信息,设计特异性引物,采用cDNA末端快速扩增技术RACE(rapid amplification of cDNA ends)进行差异片段的5'和3'端的扩增,并获得长度为1081bp的全长cDNA克隆R291(GenBank登陆号:AY589690)。序列分析表明,该基因包含702bp的开放阅读框,编码234个氨基酸,推测其蛋白质的分子量为26.8kD,等电点为6.51,有一个的跨膜螺旋区(氨基酸位点为83～103)。R291基因含有一个脂质结合保守区(Sec14p-like lipid-binding domain),具有CRAL-TRIO脂质结合结构域,推测该基因是一个磷脂酰肌醇转移蛋白基因。该基因的克隆将为橡胶树磷脂酰肌醇代谢的研究奠定了基础,将有助于进一步了解磷脂酰肌醇代谢与胶乳再生之间的关系。     

巴西橡胶树一个AP2／EREBP转录因子的克隆及表达分析

本文采用RACE技术从巴西橡胶树中鉴定出一个AP2／EREBP转录因子。该转录因子全长cDNANl225bp,最长开放阅读框699bp,预测编码蛋白包含232个氨基酸;序列比对分析发现,该转录因子具有一段保守的AP2／EREBP结构域,与拟南芥Soloist（At4g13040）具有较高的相似性,将该基因命名HbSoloist。半定量胙PcR分析结果表明,HbSoloist在巴西橡胶树的胶乳、叶片、树皮和花中均有表达。与健康橡胶树相比,死皮树胶乳HbSoloist表达量明显下降。研究发HbSoloist表达受乙烯利、茉莉酸和低温处理调控,表明HbSoloist基因可能在巴西橡胶树乙烯、茉莉酸和低温反应中发挥作用。     

A Ca2+/calmodulin-binding peroxidase from Euphorbia latex: novel aspects of calcium-hydrogen peroxide cross-talk in the regulation of plant defenses

Calmodulin (CaM) is a ubiquitous Ca(2+) sensor found in all eukaryotes, where it participates in the regulation of diverse calcium-dependent physiological processes. In response to fluctuations of the intracellular concentration of Ca(2+), CaM binds to a set of unrelated target proteins and modulates their activity. In plants, a growing number of CaM-binding proteins have been identified that apparently do not have a counterpart in animals. Some of these plant-specific Ca(2+)/CaM-activated proteins are known to tune the interaction between calcium and H(2)O(2) in orchestrating plant defenses against biotic and abiotic stresses. We previously characterized a calcium-dependent peroxidase isolated from the latex of the Mediterranean shrub Euphorbia characias (ELP) [Medda et al. (2003) Biochemistry 42, 8909-8918]. Here we report the cDNA nucleotide sequence of Euphorbia latex peroxidase, showing that the derived protein has two distinct amino acid sequences recognized as CaM-binding sites. The cDNA encoding for an E. characias CaM was also found and sequenced, and its protein product was detected in the latex. Results obtained from different CaM-binding assays and the determination of steady-state parameters showed unequivocally that ELP is a CaM-binding protein activated by the Ca(2+)/CaM system. To the best of our knowledge, this is the first example of a peroxidase regulated by this classic signal transduction mechanism. These findings suggest that peroxidase might be another node in the Ca(2+)/H(2)O(2)-mediated plant defense system, having both positive and negative effects in regulating H(2)O(2) homeostasis.