[Show abstract][Hide abstract] ABSTRACT: The transcription of soybean (Glycine max) calmodulin isoform-4 (GmCaM-4) is dramatically induced within 0.5 h of exposure to pathogen or NaCl. Core cis-acting elements that regulate the expression of the GmCaM-4 gene in response to pathogen and salt stress were previously identified, between -1,207 and -1,128 bp, and between -858 and -728 bp, in the GmCaM-4 promoter. Here, we characterized the properties of the DNA-binding complexes that form at the two core cis-acting elements of the GmCaM-4 promoter in pathogen-treated nuclear extracts. We generated GUS reporter constructs harboring various deletions of approximately 1.3-kb GmCaM-4 promoter, and analyzed GUS expression in tobacco plants transformed with these constructs. The GUS expression analysis suggested that the two previously identified core regions are involved in inducing GmCaM-4 expression in the heterologous system. Finally, a transient expression assay of Arabidopsis protoplasts showed that the GmCaM-4 promoter produced greater levels of GUS activity than did the CaMV35S promoter after pathogen or NaCl treatments, suggesting that the GmCaM-4 promoter may be useful in the production of conditional gene expression systems.
[Show abstract][Hide abstract] ABSTRACT: Calmodulin (CaM), a key Ca(2+) sensor in eukaryotes, regulates diverse cellular processes by interacting with many proteins. To identify Ca(2+)/CaM-mediated signaling components, we screened an Arabidopsis expression library with horseradish peroxidase-conjugated Arabidopsis calmodulin2 (AtCaM2) and isolated a homolog of the UBP6 deubiquitinating enzyme family (AtUBP6) containing a Ca(2+)-dependent CaM-binding domain (CaMBD). The CaM-binding activity of the AtUBP6 CaMBD was confirmed by CaM mobility shift assay, phosphodiesterase competition assay and site-directed mutagenesis. Furthermore, expression of AtUBP6 restored canavanine resistance to the Deltaubp6 yeast mutant. This is the first demonstration that Ca(2+) signaling via CaM is involved in ubiquitin-mediated protein degradation and/or stabilization in plants.
[Show abstract][Hide abstract] ABSTRACT: Calmodulin (CaM) is a ubiquitous Ca(2+)-binding protein known to regulate diverse cellular functions by modulating the activity of various target proteins. We isolated a cDNA encoding AtWRKY7, a novel CaM-binding transcription factor, from an Arabidopsis expression library with horseradish peroxidase-conjugated CaM. CaM binds specifically to the Ca(2+)-dependent CaM-binding domain (CaMBD) of AtWRKY7, as shown by site-directed mutagenesis, a gel mobility shift assay, a split-ubiquitin assay, and a competition assay using a Ca2+/CaM-dependent enzyme. Furthermore, we show that the CaMBD of AtWRKY7 is a conserved structural motif (C-motif) found in group IId of the WRKY protein family.
[Show abstract][Hide abstract] ABSTRACT: Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of
a variety of enzymes and proteins. Plants express numerous CaM isoforms that exhibit differential activation and/or inhibition
of CaM-dependent enzymes in vitro. However, the specific biological functions of plant CaM are not well known. In this study, we isolated a cDNA encoding a
CaM binding transcription factor, MYB2, that regulates the expression of salt- and dehydration-responsive genes in Arabidopsis. This was achieved using a salt-inducible CaM isoform (GmCaM4) as a probe from a salt-treated Arabidopsis expression library. Using domain mapping, we identified a Ca2+-dependent CaM binding domain in MYB2. The specific binding of CaM to CaM binding domain was confirmed by site-directed mutagenesis,
a gel mobility shift assay, split ubiquitin assay, and a competition assay using a Ca2+/CaM-dependent enzyme. Interestingly, the specific CaM isoform GmCaM4 enhances the DNA binding activity of AtMYB2, whereas
this was inhibited by a closely related CaM isoform (GmCaM1). Overexpression of Gm-CaM4 in Arabidopsis up-regulates the transcription rate of AtMYB2-regulated genes, including the proline-synthesizing enzyme P5CS1 (Δ1-pyrroline-5-carboxylate synthetase-1), which confers salt tolerance by facilitating proline accumulation. Therefore, we suggest
that a specific CaM isoform mediates salt-induced Ca2+ signaling through the activation of an MYB transcriptional activator, thereby resulting in salt tolerance in plants.
[Show abstract][Hide abstract] ABSTRACT: The Ca(2+)-binding protein calmodulin mediates cellular Ca(2+) signals in response to a wide array of stimuli in higher eukaryotes. Plants express numerous CaM isoforms. Transcription of one soybean (Glycine max) CaM isoform, SCaM-4, is dramatically induced within 30 min of pathogen or NaCl stresses. To characterize the cis-acting element(s) of this gene, we isolated an approximately 2-kb promoter sequence of the gene. Deletion analysis of the promoter revealed that a 130-bp region located between nucleotide positions -858 and -728 is required for the stressors to induce expression of SCaM-4. A hexameric DNA sequence within this region, GAAAAA (GT-1 cis-element), was identified as a core cis-acting element for the induction of the SCaM-4 gene. The GT-1 cis-element interacts with an Arabidopsis GT-1-like transcription factor, AtGT-3b, in vitro and in a yeast selection system. Transcription of AtGT-3b is also rapidly induced within 30 min after pathogen and NaCl treatment. These results suggest that an interaction between a GT-1 cis-element and a GT-1-like transcription factor plays a role in pathogen- and salt-induced SCaM-4 gene expression in both soybean and Arabidopsis.
[Show abstract][Hide abstract] ABSTRACT: Reversible phosphorylation is a key mechanism for the control of intercellular events in eukaryotic cells. In animal cells, Ca2+/CaM-dependent protein phosphorylation and dephosphorylation are implicated in the regulation of a number of cellular processes. However, little is known on the functions of Ca2+/CaM-dependent protein kinases and phosphatases in Ca2+ signaling in plants. From an Arabidopsis expression library, we isolated cDNA encoding a dual specificity protein phosphatase 1, which is capable of hydrolyzing both phosphoserine/threonine and phosphotyrosine residues of the substrates. Using a gel overlay assay, we identified two Ca2+-dependent CaM binding domains (CaMBDI in the N terminus and CaMBDII in the C terminus). Specific binding of CaM to two CaMBD was confirmed by site-directed mutagenesis, a gel mobility shift assay, and a competition assay using a Ca2+/CaM-dependent enzyme. At increasing concentrations of CaM, the biochemical activity of dual specificity protein phosphatase 1 on the p-nitrophenyl phosphate (pNPP) substrate was increased, whereas activity on the phosphotyrosine of myelin basic protein (MBP) was inhibited. Our results collectively indicate that calmodulin differentially regulates the activity of protein phosphatase, dependent on the substrate. Based on these findings, we propose that the Ca2+ signaling pathway is mediated by CaM cross-talks with a protein phosphorylation signal pathway in plants via protein dephosphorylation.
[Show abstract][Hide abstract] ABSTRACT: We isolated a stamen-specific cDNA, BSD1 (Brassica stamen specific plant defensin 1) that encodes a novel plant defensin peptide in Chinese cabbage (Brassica campestris L. ssp. pekinensis). Plant defensins are antimicrobial peptides containing eight highly conserved cysteine residues linked by disulfide bridges. In BSD1, the eight cysteine residues and a glutamate residue at position 29 are conserved whereas other amino acid residues of the plant defensins consensus sequence are substituted. BSD1 transcripts accumulate specifically in the stamen of developing flowers and its level drops as the flowers mature. The recombinant BSD1 produced in Escherichia coli showed antifungal activity against several phytopathogenic fungi. Furthermore, constitutive over-expression of the BSD1 gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter conferred enhanced tolerance against the Phytophthora parasitica in the transgenic tobacco plants.
[Show abstract][Hide abstract] ABSTRACT: Transient influx of Ca2+ constitutes an early event in the signaling cascades that trigger plant defense responses. However, the downstream components
of defense-associated Ca2+ signaling are largely unknown. Because Ca2+ signals are mediated by Ca2+-binding proteins, including calmodulin (CaM), identification and characterization of CaM-binding proteins elicited by pathogens
should provide insights into the mechanism by which Ca2+ 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 inEscherichia coli binds to soybean CaM isoform-1 (SCaM-1) in a Ca2+-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 Ca2+-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 Ca2+/CaM-dependent enzyme. Expression ofOsMlo was strongly induced by a fungal pathogen and by plant defense signaling molecules. We propose that binding of Ca2+-loaded CaM to the C-terminal tail may be a common feature of Mlo proteins.