Jae Hyuk Yoo

Pathogen- and NaCl-Induced Expression of the SCaM-4 Promoter Is Mediated in Part by a GT-1 Box That Interacts with a GT-1-Like Transcription Factor

The Ca2+-binding protein calmodulin mediates cellular Ca2+ 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.

WRKY group IId transcription factors interact with calmodulin

Calmodulin (CaM) is a ubiquitous Ca2+‐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 Ca2+‐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.

Identification of a Calmodulin-binding NAC Protein as a Transcriptional Repressor in Arabidopsis

Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of a variety of proteins. However, little is known about how CaM directly regulates transcription. Screening of an Arabidopsis cDNA expression library using horseradish peroxidase-conjugated calmodulin as a probe identified a calmodulin-binding NAC protein (CBNAC). Using gel overlay assays, a Ca2+-dependent CaM-binding domain was identified in the C terminus of this protein. Specific binding of CaM to CaM-binding domain was corroborated by site-directed mutagenesis and a split-ubiquitin assay. Using a PCR-mediated random binding site selection method, we identified a DNA-binding sequence (CBNACBS) for CBNAC, which consisted of a GCTT core sequence flanked on both sides by other frequently repeating sequences (TTGCTTANNNNNNAAG). CBNAC was able to bind to CBNACBS, which resulted in the repression of transcription in Arabidopsis protoplasts. Interestingly, the transcriptional repression mediated by CBNAC was enhanced by CaM. These results suggest that CBNAC may be a CaM-regulated transcriptional repressor in Arabidopsis.

Characterization of a stamen-specific cDNA encoding a novel plant defensin in Chinese cabbage

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.

Isolation of a Calmodulin-binding Transcription Factor from Rice (Oryza sativa L.)

Calmodulin (CaM) regulates diverse cellular functions by modulating the activities of a variety of enzymes and proteins. However, direct modulation of transcription factors by CaM has been poorly understood. In this study, we isolated a putative transcription factor by screening a rice cDNA expression library by using CaM:horse-radish peroxidase as a probe. This factor, which we have designated OsCBT (Oryza sativa CaM-binding transcription factor), has structural features similar to Arabidopsis AtSRs/AtCAMTAs and encodes a 103-kDa protein because it contains a CG-1 homology DNA-binding domain, three ankyrin repeats, a putative transcriptional activation domain, and five putative CaM-binding motifs. By using a gel overlay assay, gel mobility shift assays, and site-directed mutagenesis, we showed that OsCBT has two different types of functional CaM-binding domains, an IQ motif, and a Ca2+-dependent motif. To determine the DNA binding specificity of OsCBT, we employed a random binding site selection method. This analysis showed that OsCBT preferentially binds to the sequence 5′-TWCG(C/T)GTKKKKTKCG-3′ (W and K represent A or C and T or G, respectively). OsCBT was able to bind this sequence and activate β-glucuronidase reporter gene expression driven by a minimal promoter containing tandem repeats of these sequences in Arabidopsis leaf protoplasts. Green fluorescent protein fusions of two putative nuclear localization signals of OsCBT, a bipartite and a SV40 type, were predominantly localized in the nucleus. Most interestingly, the transcriptional activation mediated by OsCBT was inhibited by co-transfection with a CaM gene. Taken together, our results suggest that OsCBT is a transcription activator modulated by CaM.

Regulation of MAPK Phosphatase 1 (AtMKP1) by Calmodulin in Arabidopsis

The mitogen-activated protein kinases (MAPKs) are key signal transduction molecules, which respond to various external stimuli. The MAPK phosphatases (MKPs) are known to be negative regulators of MAPKs in eukaryotes. We screened an Arabidopsis cDNA library using horseradish peroxidase-conjugated calmodulin (CaM), and isolated AtMKP1 as a CaM-binding protein. Recently, tobacco NtMKP1 and rice OsMKP1, two orthologs of Arabidopsis AtMKP1, were reported to bind CaM via a single putative CaM binding domain (CaMBD). However, little is known about the regulation of phosphatase activity of plant MKP1s by CaM binding. In this study, we identified two Ca2+-dependent CaMBDs within AtMKP1. Specific binding of CaM to two different CaMBDs was verified using a gel mobility shift assay, a competition assay with a Ca2+/CaM-dependent enzyme, and a split-ubiquitin assay. The peptides for two CaMBDs, CaMBDI and CaMBDII, bound CaM in a Ca2+-dependent manner, and the binding affinity of CaMBDII was found to be higher than that of CaMBDI. CaM overlay assays using mutated CaMBDs showed that four amino acids, Trp453 and Leu456 in CaMBDI and Trp678 and Ile684 in CaMBDII, play a pivotal role in CaM binding. Moreover, the phosphatase activity of AtMKP1 was increased by CaM in a Ca2+-dependent manner. Our results suggest that two important signaling pathways, Ca2+ signaling and the MAPK signaling cascade, are connected in plants via the regulation of AtMKP1 activity. To our knowledge, this is the first report to show that the biochemical activity of MKP1 in plants is regulated by CaM.

An S-locus receptor-like kinase in plasma membrane interacts with calmodulin in Arabidopsis

MINT‐6800978:AtCaM2 (uniprotkb:P25069) physically interacts (MI:0218) with CBRLK1 (uniprotkb:Q9ZT06) by cytoplasmic complementation assay (MI:0228)

Arabidopsis ubiquitin-specific protease 6 (AtUBP6) interacts with calmodulin

Calmodulin (CaM), a key Ca2+ sensor in eukaryotes, regulates diverse cellular processes by interacting with many proteins. To identify Ca2+/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 Ca2+‐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 Δubp6 yeast mutant. This is the first demonstration that Ca2+ signaling via CaM is involved in ubiquitin‐mediated protein degradation and/or stabilization in plants.