Calcium signaling-mediated and differential induction of calmodulin gene expression by stress in Oryza sativa L

Ca(2+)/calmodulin transduction pathways have been implicated in mediating stress response and tolerance in plants. Here, three genes encoding calmodulin (Cam) members of the EF-hand family of Ca(2+)-binding proteins were identified from Oryza sativa L. databases. Complementary DNA for each of the calmodulin genes, OsCam1, OsCam2, and OsCam3 were sequenced. OsCam1 and OsCam2 encode a conventional 148-amino acid calmodulin protein that contains four characteristic Ca(2+)-binding motifs. OsCam3 encode a similar protein with a 38-amino-acid extension containing a putative prenylation site (CVIL) at the carboxyl terminus. RT-PCR showed that each of the genes is expressed in leaves and roots of 2-week old rice seedlings. By RNA gel blot analysis, OsCam1 mRNA levels strongly increased in response to NaCl, mannitol and wounding treatments. In contrast, OsCam2 mRNA levels were relatively unchanged under all conditions investigated. NaCl treatment and wounding also increased the OsCam3 mRNA level, but in a more transient manner. Our results indicate that although the expression of genes encoding different calmodulin isoforms is ubiquitous, they are differentially regulated by various stress signals. In addition, we have demonstrated that the calcium-channel blocker lanthanum chloride inhibited the induction of OsCam1 gene expression by both NaCl and mannitol treatments. These results suggest that osmotic stressinduced expression of OsCam1 gene requires the [Ca(2+)]cyt elevation that is known to occur in response to these stimuli.     

Distinct calcium signaling pathways regulate calmodulin gene expression in tobacco

Cold shock and wind stimuli initiate Ca(2+) transients in transgenic tobacco (Nicotiana plumbaginifolia) seedlings (named MAQ 2.4) containing cytoplasmic aequorin. To investigate whether these stimuli initiate Ca(2+) pathways that are spatially distinct, stress-induced nuclear and cytoplasmic Ca(2+) transients and the expression of a stress-induced calmodulin gene were compared. Tobacco seedlings were transformed with a construct that encodes a fusion protein between nucleoplasmin (a major oocyte nuclear protein) and aequorin. Immunocytochemical evidence indicated targeting of the fusion protein to the nucleus in these plants, which were named MAQ 7.11. Comparison between MAQ 7.11 and MAQ 2.4 seedlings confirmed that wind stimuli and cold shock invoke separate Ca(2+) signaling pathways. Partial cDNAs encoding two tobacco calmodulin genes, NpCaM-1 and NpCaM-2, were identified and shown to have distinct nucleotide sequences that encode identical polypeptides. Expression of NpCaM-1, but not NpCaM-2, responded to wind and cold shock stimulation. Comparison of the Ca(2+) dynamics with NpCaM-1 expression after stimulation suggested that wind-induced NpCaM-1 expression is regulated by a Ca(2+) signaling pathway operational predominantly in the nucleus. In contrast, expression of NpCaM-1 in response to cold shock is regulated by a pathway operational predominantly in the cytoplasm.     

Nucleomorphin. A novel, acidic, nuclear calmodulin-binding protein from dictyostelium that regulates nuclear number

Probing of Dictyostelium discoideum cell extracts after SDS-PAGE using (35)S-recombinant calmodulin (CaM) as a probe has revealed approximately three-dozen Ca(2+)-dependent calmodulin binding proteins. Here, we report the molecular cloning, expression, and subcellular localization of a gene encoding a novel calmodulin-binding protein (CaMBP); we have called nucleomorphin, from D. discoideum. A lambdaZAP cDNA expression library of cells from multicellular development was screened using a recombinant calmodulin probe ((35)S-VU1-CaM). The open reading frame of 1119 nucleotides encodes a polypeptide of 340 amino acids with a calculated molecular mass of 38.7 kDa and is constitutively expressed throughout the Dictyostelium life cycle. Nucleomorphin contains a highly acidic glutamic/aspartic acid inverted repeat (DEED) with significant similarity to the conserved nucleoplasmin domain and a putative transmembrane domain in the carboxyl-terminal region. Southern blotting reveals that nucleomorphin exists as a single copy gene. Using gel overlay assays and CaM-agarose we show that bacterially expressed nucleomorphin binds to bovine CaM in a Ca(2+)-dependent manner. Amino-terminal fusion to the green fluorescence protein (GFP) showed that GFP-NumA localized to the nucleus as distinct arc-like patterns similar to heterochromatin regions. GFP-NumA lacking the acidic DEED repeat still showed arc-like accumulations at the nuclear periphery, but the number of nuclei in these cells was increased markedly compared with control cells. Cells expressing GFP-NumA lacking the transmembrane domain localized to the nuclear periphery but did not affect nuclear number or gross morphology. Nucleomorphin is the first nuclear CaMBP to be identified in Dictyostelium. Furthermore, these data present the first identification of a member of the nucleoplasmin family as a calmodulin-binding protein and suggest nucleomorphin has a role in nuclear structure in Dictyostelium.     

Ca2+-dependent GTPase, extra-large G protein 2 (XLG2), promotes activation of DNA-binding protein related to vernalization 1 (RTV1), leading to activation of floral integrator genes and early flowering in Arabidopsis

Heterotrimeric G proteins, consisting of Gα, Gβ, and Gγ subunits, play important roles in plant development and cell signaling. In Arabidopsis, in addition to one prototypical G protein α subunit, GPA1, there are three extra-large G proteins, XLG1, XLG2, and XLG3, of largely unknown function. Each extra-large G (XLG) protein has a C-terminal Gα-like region and a ～400 amino acid N-terminal extension. Here we show that the three XLG proteins specifically bind and hydrolyze GTP, despite the fact that these plant-specific proteins lack key conserved amino acid residues important for GTP binding and hydrolysis of GTP in mammalian Gα proteins. Moreover, unlike other known Gα proteins, these activities require Ca(2+) instead of Mg(2+) as a cofactor. Yeast two-hybrid library screening and in vitro protein pull-down assays revealed that XLG2 interacts with the nuclear protein RTV1 (related to vernalization 1). Electrophoretic mobility shift assays show that RTV1 binds to DNA in vitro in a non-sequence-specific manner and that GTP-bound XLG2 promotes the DNA binding activity of RTV1. Overexpression of RTV1 results in early flowering. Combined overexpression of XLG2 and RTV1 enhances this early flowering phenotype and elevates expression of the floral pathway integrator genes, FT and SOC1, but does not repress expression of the floral repressor, FLC. Chromatin immunoprecipitation assays show that XLG2 increases RTV1 binding to FT and SOC1 promoters. Thus, a Ca(2+)-dependent G protein, XLG2, promotes RTV1 DNA binding activity for a subset of floral integrator genes and contributes to floral transition.     

Arabidopsis Calmodulin-binding Protein IQ67-Domain 1 Localizes to Microtubules and Interacts with Kinesin Light Chain-related Protein-1

Calcium (Ca²⁺) is a key second messenger in eukaryotes and regulates diverse cellular processes, most notably via calmodulin (CaM). In Arabidopsis thaliana, IQD1 (IQ67 domain 1) is the founding member of the IQD family of putative CaM targets. The 33 predicted IQD proteins share a conserved domain of 67 amino acids that is characterized by a unique arrangement of multiple CaM recruitment motifs, including so-called IQ motifs. Whereas IQD1 has been implicated in the regulation of defense metabolism, the biochemical functions of IQD proteins remain to be elucidated. In this study we show that IQD1 binds to multiple Arabidopsis CaM and CaM-like (CML) proteins in vitro and in yeast two-hybrid interaction assays. CaM overlay assays revealed moderate affinity of IQD1 to CaM2 (K_d ∼ 0.6 μm). Deletion mapping of IQD1 demonstrated the importance of the IQ67 domain for CaM2 binding in vitro, which is corroborated by interaction of the shortest IQD member, IQD20, with Arabidopsis CaM/CMLs in yeast. A genetic screen of a cDNA library identified Arabidopsis kinesin light chain-related protein-1 (KLCR1) as an IQD1 interactor. The subcellular localization of GFP-tagged IQD1 proteins to microtubules and the cell nucleus in transiently and stably transformed plant tissues (tobacco leaves and Arabidopsis seedlings) suggests direct interaction of IQD1 and KLCR1 in planta that is supported by GFP∼IQD1-dependent recruitment of RFP∼KLCR1 and RFP∼CaM2 to microtubules. Collectively, the prospect arises that IQD1 and related proteins provide Ca²⁺/CaM-regulated scaffolds for facilitating cellular transport of specific cargo along microtubular tracks via kinesin motor proteins.     

The IQD Gene Family in Soybean: Structure,Phylogeny, Evolution and Expression

Members of the plant-specific IQ67-domain (IQD) protein family are involved in plant development and the basal defense response. Although systematic characterization of this family has been carried out in Arabidopsis, tomato (Solanum lycopersicum), Brachypodium distachyon and rice (Oryza sativa), systematic analysis and expression profiling of this gene family in soybean (Glycine max) have not previously been reported. In this study, we identified and structurally characterized IQD genes in the soybean genome. A complete set of 67 soybean IQD genes (GmIQD1–67) was identified using Blast search tools, and the genes were clustered into four subfamilies (IQD I–IV) based on phylogeny. These soybean IQD genes are distributed unevenly across all 20 chromosomes, with 30 segmental duplication events, suggesting that segmental duplication has played a major role in the expansion of the soybean IQD gene family. Analysis of the Ka/Ks ratios showed that the duplicated genes of the GmIQD family primarily underwent purifying selection. Microsynteny was detected in most pairs: genes in clade 1–3 might be present in genome regions that were inverted, expanded or contracted after the divergence; most gene pairs in clade 4 showed high conservation with little rearrangement among these gene-residing regions. Of the soybean IQD genes examined, six were most highly expressed in young leaves, six in flowers, one in roots and two in nodules. Our qRT-PCR analysis of 24 soybean IQD III genes confirmed that these genes are regulated by MeJA stress. Our findings present a comprehensive overview of the soybean IQD gene family and provide insights into the evolution of this family. In addition, this work lays a solid foundation for further experiments aimed at determining the biological functions of soybean IQD genes in growth and development.     

Isolation and characterization of a novel calcium/calmodulin-dependent protein kinase, AtCK, from arabidopsis

Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin (Ca(2+)/CaM)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about Ca(2+)/CaM-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative Ca(2+)-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a Ca(2+)-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM Mn(2+). The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other Ca(2+)/CaM-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and Ca(2+)/CaM-dependent protein kinase), increasing the concentration of calmodulin to more than 3 microgram suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis Ca(2+)/CaM-dependent protein kinase which is presumably involved in CaM-mediated signaling.     

Mlo, a modulator of plant defense and cell death, is a novel calmodulin-binding protein. Isolation and characterization of a rice Mlo homologue

Transient influx of Ca(2+) constitutes an early event in the signaling cascades that trigger plant defense responses. However, the downstream components of defense-associated Ca(2+) signaling are largely unknown. Because Ca(2+) signals are mediated by Ca(2+)-binding proteins, including calmodulin (CaM), identification and characterization of CaM-binding proteins elicited by pathogens should provide insights into the mechanism by which Ca(2+) regulates defense responses. In this study, we isolated a gene encoding rice Mlo (Oryza sativa Mlo; OsMlo) using a protein-protein interaction-based screening of a cDNA expression library constructed from pathogen-elicited rice suspension cells. OsMlo has a molecular mass of 62 kDa and shares 65% sequence identity and scaffold topology with barley Mlo, a heptahelical transmembrane protein known to function as a negative regulator of broad spectrum disease resistance and leaf cell death. By using gel overlay assays, we showed that OsMlo produced in Escherichia coli binds to soybean CaM isoform-1 (SCaM-1) in a Ca(2+)-dependent manner. We located a 20-amino acid CaM-binding domain (CaMBD) in the OsMlo C-terminal cytoplasmic tail that is necessary and sufficient for Ca(2+)-dependent CaM complex formation. Specific binding of the conserved CaMBD to CaM was corroborated by site-directed mutagenesis, a gel mobility shift assay, and a competition assay with a Ca(2+)/CaM-dependent enzyme. Expression of OsMlo was strongly induced by a fungal pathogen and by plant defense signaling molecules. We propose that binding of Ca(2+)-loaded CaM to the C-terminal tail may be a common feature of Mlo proteins.     

Regulation of nuclear Ca2+ signaling by translocation of the Ca2+ messenger synthesizing enzyme ADP-ribosyl cyclase during neuronal depolarization

In neurons, voltage-gated Ca(2+) channels and nuclear Ca(2+) signaling play important roles, such as in the regulation of gene expression. However, the link between electrical activity and biochemical cascade activation involved in the generation of the nuclear Ca(2+) signaling is poorly understood. Here we show that depolarization of Aplysia neurons induces the translocation of ADP-ribosyl cyclase, a Ca(2+) messenger synthesizing enzyme, from the cytosol into the nucleus. The translocation is dependent on Ca(2+) influx mainly through the voltage-dependent L-type Ca(2+) channels. We report also that specific nucleoplasmic Ca(2+) signals can be induced by three different calcium messengers, cyclic ADP-ribose, nicotinic acid adenine dinucleotide phosphate (NAADP), both produced by the ADP-ribosyl cyclase, and inositol 1,4,5-trisphosphate (IP(3)). Moreover, our pharmacological data show that NAADP acts on its own receptor, which cooperates with the IP(3) and the ryanodine receptors to generate nucleoplasmic Ca(2+) oscillations. We propose a new model where voltage-dependent L-type Ca(2+) channel-induced nuclear translocation of the cytosolic cyclase is a crucial step in the fine tuning of nuclear Ca(2+) signals in neurons.     

Isolation and characterization of a novel calmodulin-binding protein from potato.

Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.     

Large store-operated calcium selective currents due to co-expression of Orai1 or Orai2 with the intracellular calcium sensor, Stim1

The molecular nature of store-operated Ca(2+)-selective channels has remained an enigma, due largely to the continued inability to convincingly demonstrate Ca(2+)-selective store-operated currents resulting from exogenous expression of known genes. Recent findings have implicated two proteins, Stim1 and Orai1, as having essential roles in store-operated Ca(2+) entry across the plasma membrane. However, transient overexpression of these proteins on their own results in little or no increase in store-operated entry. Here we demonstrate dramatic synergism between these two mediators; co-transfection of HEK293 cells with Stim1 and Orai1 results in an approximate 20-fold increase in store-operated Ca(2+) entry and Ca(2+)-selective current. This demonstrates that these two proteins are limiting for both the signaling and permeation mechanisms for Ca(2+)-selective store-operated Ca(2+) entry. There are three mammalian homologs of Orai1, and in expression experiments they all produced or augmented store-operated Ca(2+) entry with efficacies in the order Orai1 > Orai2 > Orai3. Stim1 apparently initiates the signaling process by acting as a Ca(2+) sensor in the endoplasmic reticulum. This results in rearrangement of Stim1 within the cell and migration toward the plasma membrane to regulate in some manner Orai1 located in the plasma membrane. However, we demonstrate that Stim1 does not incorporate in the surface membrane, and thus likely regulates or interacts with Orai1 at sites of close apposition between the plasma membrane and an intracellular Stim1-containing organelle.     

The domain of unknown function 4005 (DUF4005) in an Arabidopsis IQD protein functions in microtubule binding

The dynamic responses of microtubules (MTs) to internal and external signals are modulated by a plethora of microtubule-associated proteins (MAPs). In higher plants, many plant-specific MAPs have emerged during evolution as advantageous to their sessile lifestyle. Some members of the IQ67 domain (IQD) protein family have been shown to be plant-specific MAPs. However, the mechanisms of interaction between IQD proteins and MTs remain elusive. Here we demonstrate that the domain of unknown function 4005 (DUF4005) of the Arabidopsis IQD family protein ABS6/AtIQD16 is a novel MT-binding domain. Cosedimentation assays showed that the DUF4005 domain binds directly to MTs in vitro. GFP-labeled DUF4005 also decorates all types of MT arrays tested in vivo. Furthermore, we showed that a conserved stretch of 15 amino acid residues within the DUF4005 domain, which shares sequence similarity with the C-terminal MT-binding domain of human MAP Kif18A, is required for the binding to MTs. Transgenic lines overexpressing the DUF4005 domain displayed a spectrum of developmental defects, including spiral growth and stunted growth at the organismal level. At the cellular level, DUF4005 overexpression caused defects in epidermal pavement cell and trichome morphogenesis, as well as abnormal anisotropic cell elongation in the hypocotyls of dark-grown seedlings. These data establish that the DUF4005 domain of ABS6/AtIQD16 is a new MT-binding domain, overexpression of which perturbs MT homeostasis in plants. Our findings provide new insights into the MT-binding mechanisms of plant IQD proteins.     

Calcium, nucleotide, and actin affect the interaction of mammalian Myo1c with its light chain calmodulin

To investigate the interaction of mammalian class I myosin, Myo1c, with its light chain calmodulin, we expressed (with calmodulin) truncation mutants consisting of the Myo1c motor domain followed by 0-4 presumed calmodulin-binding (IQ) domains (Myo1c (0IQ)-Myo1c (4IQ)). The amount of calmodulin associating with the Myo1c heavy chain increased with increasing number of IQ domains from Myo1c (0IQ) to Myo1c (3IQ). No calmodulin beyond that associated with Myo1c (3IQ) was found with Myo1c (4IQ) despite its availability, showing that Myo1c binds three molecules of calmodulin with no evidence of a fourth IQ domain. Unlike Myo1c (0IQ), the basal ATPase activity of Myo1c (1IQ) was >10-fold higher in Ca (2+) vs EGTA +/- exogenous calmodulin, showing that regulation is by Ca (2+) binding to calmodulin on the first IQ domain. The K m and V max of the actin-activated Mg (2+)-ATPase activity were largely independent of the number of IQ domains present and moderately affected by Ca (2+). In binding assays, some calmodulin pelleted with Myo1c heavy chain when actin was present, but a considerable fraction remained in the supernatant, suggesting that calmodulin is displaced most likely from the second IQ domain. The Myo1c heavy chain associated with actin in a nucleotide-dependent fashion. In ATP a smaller proportion of calmodulin pelleted with the heavy chain, suggesting that Myo1c undergoes nucleotide-dependent conformational changes that affect the affinity of calmodulin for the heavy chain. The studies support a model in which Myo1c in the inner ear is regulated by both Ca (2+) and nucleotide, which exert their effects on motor activity through the light-chain-binding region.     

Genome-wide comparative analysis of the <Emphasis Type="Italic">IQD</Emphasis> gene families in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

Background  

Calcium signaling plays a prominent role in plants for coordinating a wide range of developmental processes and responses to environmental cues. Stimulus-specific generation of intracellular calcium transients, decoding of calcium signatures, and transformation of the signal into cellular responses are integral modules of the transduction process. Several hundred proteins with functions in calcium signaling circuits have been identified, and the number of downstream targets of calcium sensors is expected to increase. We previously identified a novel, calmodulin-binding nuclear protein, IQD1, which stimulates glucosinolate accumulation and plant defense in Arabidopsis thaliana. Here, we present a comparative genome-wide analysis of a new class of putative calmodulin target proteins in Arabidopsis and rice.