GhCaM7-like, a calcium sensor gene, influences cotton fiber elongation and biomass production.

Calcium signaling regulates many developmental processes in plants. Calmodulin (CaM) is one of the most conserved calcium sensors and has a flexible conformation in eukaryotes. The molecular functions of CaM are unknown in cotton, which is a major source of natural fiber. In this study, a Gossypium hirsutum L.CaM7-like gene was isolated from upland cotton. Bioinformatics analysis indicated that the GhCaM7-like gene was highly conserved as compared with Arabidopsis AtCaM7. The GhCaM7-like gene showed a high expression level in elongating fibers. Expression of ß-glucuronidase was observed in trichomes on the stem, leaf and root in transgenic Arabidopsis plants of a PROGhCaM7-like:GUS fusion. Silencing of the GhCaM7-like gene resulted in decreased fiber length, but also caused reduction in stem height, leaf dimensions, seed length and 100-seed weight, in comparison with those of the control. Reduced expression of the GhCaM7-like gene caused decreased Ca2+ influx in cells of the leaf hypodermis and stem apex, and down-regulation of GhIQD1 (IQ67-domain containing protein), GhAnn2 (Annexins) and GhEXP2 (Expansin). These results indicate that the GhCaM7-like gene plays a vital role in calcium signaling pathways, and may regulate cotton fiber elongation and biomass production by affecting Ca2+ signatures and downstream signaling pathways of CaM.

Loss of calmodulin binding to Bax inhibitor-1 affects Pseudomonas-mediated hypersensitive response-associated cell death in Arabidopsis thaliana.

Bax inhibitor-1 (BI-1) is a cell death suppressor protein conserved across a variety of organisms. The Arabidopsis atbi1-1 plant is a mutant in which the C-terminal 6 amino acids of the expressed BI-1 protein have been replaced by T-DNA insertion. This mutant BI-1 protein (AtBI-CM) produced in Escherichia coli can no longer bind to calmodulin. A promoter-reporter assay demonstrated compartmentalized expression of BI-1 during hypersensitive response, introduced by the inoculation of Pseudomonas syringae possessing the avrRTP2 gene, Pst(avrRPT2). In addition, both BI-1 knockdown plants and atbi1-1 showed increased sensitivity to Pst(avrRPT2)-induced cell death. The results indicated that the loss of calmodulin binding reduces the cell death suppressor activity of BI-1 in planta.

Crystal-structure and biochemical characterization of recombinant human calcyphosine delineates a novel EF-hand-containing protein family.

Calcyphosine is an EF-hand protein involved in both Ca(2+)-phosphatidylinositol and cyclic AMP signal cascades, as well as in other cellular functions. The crystal structure of Ca(2+)-loaded calcyphosine was determined up to 2.65 A resolution and reveals a protein containing two pairs of Ca(2+)-binding EF-hand motifs. Calcyphosine shares a highly similar overall topology with calmodulin. However, there are striking differences between EF-hand 4, both N-terminal and C-terminal regions, and interdomain linkers. The C-terminal domain of calcyphosine possesses a large hydrophobic pocket in the presence of calcium ions that might be implicated in ligand binding, while its N-terminal hydrophobic pocket is almost shielded by an additional terminal helix. Calcyphosine is largely monomeric, regardless of the presence of Ca(2+). Differences in structure, oligomeric state in the presence and in the absence of Ca(2+), a highly conserved sequence with low similarity to other proteins, and phylogeny define a new EF-hand-containing family of calcyphosine proteins that extends from arthropods to humans.

Analysis of Acropora muricata calmodulin (CaM) indicates that scleractinian corals possess the ancestral exon/intron organization of the eumetazoan CaM gene.

Calmodulin (CaM), belonging to the tropinin C (TnC) superfamily, is one of the calcium-binding proteins that are highly conserved in their protein and gene structure. Based on the structure comparison among published vertebrate and invertebrate CaM, it is proposed that the ancestral form of eumetazoan CaM genes should have five exons and four introns (four-intron hypothesis). In this study, we determined the gene structure of CaM in the coral Acropora muricata, an anthozoan cnidarian representing the basal position in animal evolution. A CaM clone was isolated from a cDNA library constructed from the spawned eggs of A. muricata. This clone was composed of 908 nucleotides, including 162 base pairs (bp) of 5'-untranslated region (UTR), 296 bp of 3'-UTR, and an open reading frame 450 bp in length. The deduced amino acid indicated that the Acropora CaM protein is identical to that of the actiniarian, Metridinium senile, and has four putative calcium-binding domains highly similar to those of other vertebrate or invertebrate CaMs. Southern blot analysis revealed that Acropora CaM is a putative single-copy gene in the nuclear genome. Genomic sequencing showed that Acropora CaM was composed of five exons and four introns, with intron II not corresponding to any region in the actiniarian CaM gene, which possesses only four exons and three introns. Our results highlight that the coral CaM gene isolated from A. muricata has four introns at the predicted positions of the early metazoan CaM gene organization, providing the first evidence from the basal eumetazoan phylum to support the four-intron hypothesis.

Characterization of the spasmin 1 gene in Zoothamnium arbuscula strain Kawagoe (protozoa, ciliophora) and its relation to other spasmins and centrins.

Zoothamnium arbuscula strain Kawagoe is a giant sessile peritrich ciliated protozoa that possesses a contractile organelle called a spasmoneme. We report here on the molecular characterization and provide an opportunity to discuss the evolutionary relationships of the Z. arbuscula spasmin; spasmins belong to the calmodulin superfamily and are the major components of spasmoneme filaments. We analysed and obtained the whole sequence of the spasmin 1 gene and a partial sequence of the spasmin 2 gene. It is surprising that the sequence of spasmin 1 does not contain introns and encodes an open reading frame of 531 bp. It predicts a product of 177 amino acids with a calculated molecular mass of 19659 Da and a pI of 4.68. The amino acid sequence has two putative calcium-binding domains. One of them is a functional domain, as defined by the EF-hand consensus. The varieties of spasmins were revealed by comparison with amino acid components and molecular relationships of spasmin 1 protein and other spasmins. A comparison of the amino acid sequence between the Z. arbuscula spasmin and known centrins indicates that spasmins have a one residue deletion in the EF-hand domain-2 and four residue insertions in domain-4, as does the Vorticella spasmin. However, there are large variations in the amino acid sequence at domain-4 within spasmin 1, spasmin 2 and the Vorticella spasmin.

Calmodulin target database.

The intracellular calcium sensor protein calmodulin (CaM) interacts with a large number of proteins to regulate their biological functions in response to calcium stimulus. This molecular recognition process is diverse in its mechanism, but can be grouped into several classes based on structural and sequence information. We have developed a web-based database (http://calcium.uhnres.utoronto.ca/ctdb) for this family of proteins containing CaM binding sites or, as we propose to call it herein, CaM recruitment signaling (CRS) motifs. At present the CRS motif found in approximately 180 protein sequences in the databases can be divided into four subclasses, each subclass representing a distinct structural mode of molecular recognition involving CaM. The database can predict a putative CRS location within a given protein sequence, identify the subclass to which it may belong, and structural and biophysical parameters such as hydrophobicity, hydrophobic moment, and propensity for alpha-helix formation.

Comparative study of the pattern of expression of calmodulin messenger RNAs in the mouse brain.

Calmodulin is a major calcium-binding protein in the mammalian brain, playing an important role in neuronal cell function. Its amino acid sequence is highly conserved and the protein is encoded by multiple genes. In the mouse brain, as well as in the rat and the human brain, three different genes have been detected for calmodulin, CaM I, CaM II and CaM III, all of which encode an identical protein. We studied the pattern of expression of the three calmodulin genes and the pattern of calmodulin distribution in the mouse brain by in situ hybridization histochemistry and immunohistochemistry. We found that calmodulin messenger RNAs from the three calmodulin genes were widely expressed in the mouse brain. Nevertheless, there were differences in their patterns of distribution. In general, all calmodulin messenger RNAs were preferentially distributed in hippocampus, cerebral cortex and cerebellar cortex, and CaM II messenger RNA also in caudate-putamen. However, all messenger RNAs showed clearly differentiated patterns of distribution in the hippocampus and the cerebellar cortex. Calmodulin immunoreactivity was present in all cells so far examined. Immunostaining was observed both in the cell nucleus, where it was especially strong, and in the cytoplasm. Our results suggest that the three calmodulin genes are differentially regulated in the mouse brain and also that, although all calmodulin genes have a basal expression, precise regulation of calmodulin levels might be attained through the different contribution of the three calmodulin genes.

Comparative structural analysis of calmodulins from Trypanosoma brucei, T. congolense, T. vivax, Tetrahymena thermophila and bovine brain.

Calmodulin is an intracellular calcium receptor protein utilized extensively by eukaryotic cells to mediate responsiveness to calcium signals. The present study evaluates the effects on protein structure of amino acid substitutions in trypanosome calmodulin. Calmodulin conformation, hydrophobicity and antigenic determinants are compared among Trypanosoma brucei, Trypanosoma congolense, Trypanosoma vivax, Tetrahymena thermophila and bovine brain. Trypanosome calmodulin differs from brain and Tetrahymena calmodulins based upon isoelectric point, retention time on a C-2/C-18 reverse phase column and interaction with polyclonal antibodies against trypanosome calmodulin by radioimmunoassay or Western procedures. These same analyses do not distinguish trypanosome calmodulins from each other. Polyclonal antibodies against Tetrahymena calmodulin are equally specific and do not recognize the trypanosome or brain calmodulins. Calcium-induced exposure of hydrophobic binding sites are quantitated using the fluorescent probe, N-phenyl-1-naphthylamine. All calmodulins, regardless of source, enhance the fluorescence of N-phenyl-1-naphthylamine 3-4 fold in the presence of calcium. These data demonstrate the extent to which functional calmodulins vary in their structures. We conclude that African trypanosomes share a common calmodulin that is structurally distinct from calmodulin of vertebrates or Tetrahymena.