The transcriptional repressor activity of ASYMMETRIC LEAVES1 is inhibited by direct interaction with calmodulin in Arabidopsis.

Calmodulin (CaM), a key Ca2+ sensor, regulates diverse cellular processes by modulating the activity of a variety of enzymes and proteins. However, little is known about the biological function of CaM in plant development. In this study, an ASYMMETRIC LEAVES1 (AS1) transcription factor was isolated as a CaM-binding protein. AS1 contains two putative CaM-binding domains (CaMBDs) at the N-terminus. Using domain mapping analysis, both predicted domains were identified as authentic Ca2+ -dependent CaMBDs. We identified three hydrophobic amino acid residues for CaM binding, Trp49 in CaMBDI, and Trp81 and Phe103 in CaMBDII. The interactions of AS1 with CaM were verified in yeast and plant cells. Based on electrophoretic mobility shift assays, CaM inhibited the DNA-binding activity of the AS1/AS2 complex to two cis-regulatory motifs in the KNAT1 promoter. Furthermore, CaM relieved the suppression of KNAT1 transcription by AS1 not only in transient expression assays of protoplasts but also by the overexpression of a CaM-binding negative form of AS1 in as1 mutant plant. Our study suggests that CaM, a calcium sensor, can be involved in the transcriptional control of meristem cell-specific genes by the inhibition of AS1 under the condition of higher levels of Ca2+ in plants.

An S-locus receptor-like kinase plays a role as a negative regulator in plant defense responses.

Plant cells often use cell surface receptors to sense environmental changes and then transduce external signals via activated signaling pathways to trigger adaptive responses. In Arabidopsis, the receptor-like protein kinase (RLK) gene family contains more than 600 members, and some of these are induced by pathogen infection, suggesting a possible role in plant defense responses. We previously characterized an S-locus RLK (CBRLK1) at the biochemical level. In this study, we examined the physiological function of CBRLK1 in defense responses. CBRLK1 mutant and CBRLK1-overexpressing transgenic plants showed enhanced and reduced resistance against a virulent bacterial pathogen, respectively. The altered pathogen resistances of the mutant and overexpressing transgenic plants were associated with increased and reduced induction of the pathogenesis-related gene PR1, respectively. These results suggest that CBRLK1 plays a negative role in the disease resistance signaling pathway in Arabidopsis.

OsBWMK1 mediates SA-dependent defense responses by activating the transcription factor OsWRKY33.

Mitogen-activated protein kinases (MAPKs) play important roles in responses to various environmental stresses. In a previous study, we demonstrated that OsBWMK1, which localizes in the nucleus, mediates PR gene expression by activating the OsEREBP1 transcription factor, and that the constitutive expression of OsBWMK1 also enhances resistance against pathogen infections [Y.H. Cheong, B.C. Moon, J.K. Kim, C.Y. Kim, M.C. Kim, I.H. Kim, C.Y. Park, J.C. Kim, B.O. Park, S.C. Koo, H.W. Yoon, W.S. Chung, C.O. Lim, S.Y. Lee, M.J. Cho, BWMK1, rice mitogen-activated protein kinase, locates in the nucleus and mediates pathogenesis-related gene expression by activation of a transcription factor, Plant Physiol. 132 (2003) 1961--1972]. Here, we report that OsBWMK1 phosphorylates OsWRKY33, which binds to the W-box element (TTGACCA) in several PR gene promoters, thereby enhancing DNA-binding activity of the factor to its in vitro cognate binding site. Transient coexpression of OsBWMK1 and OsWRKY33 in Arabidopsis protoplasts elevates SA-dependent expression of the GUS-reporter gene driven by the W-box element and the PR1 promoter. Furthermore, the levels of SA and H(2)O(2) are elevated in 35S-OsBWMK1 transgenic plants that show HR-like cell death. Altogether, OsBWMK1 may mediate SA-dependent defense responses by activating the WRKY transcription factor in plants.

Pathogen-induced binding of the soybean zinc finger homeodomain proteins GmZF-HD1 and GmZF-HD2 to two repeats of ATTA homeodomain binding site in the calmodulin isoform 4 (GmCaM4) promoter.

Calmodulin (CaM) is involved in defense responses in plants. In soybean (Glycine max), transcription of calmodulin isoform 4 (GmCaM4) is rapidly induced within 30 min after pathogen stimulation, but regulation of the GmCaM4 gene in response to pathogen is poorly understood. Here, we used the yeast one-hybrid system to isolate two cDNA clones encoding proteins that bind to a 30-nt A/T-rich sequence in the GmCaM4 promoter, a region that contains two repeats of a conserved homeodomain binding site, ATTA. The two proteins, GmZF-HD1 and GmZF-HD2, belong to the zinc finger homeodomain (ZF-HD) transcription factor family. Domain deletion analysis showed that a homeodomain motif can bind to the 30-nt GmCaM4 promoter sequence, whereas the two zinc finger domains cannot. Critically, the formation of super-shifted complexes by an anti-GmZF-HD1 antibody incubated with nuclear extracts from pathogen-treated cells suggests that the interaction between GmZF-HD1 and two homeodomain binding site repeats is regulated by pathogen stimulation. Finally, a transient expression assay with Arabidopsis protoplasts confirmed that GmZF-HD1 can activate the expression of GmCaM4 by specifically interacting with the two repeats. These results suggest that the GmZF-HD1 and -2 proteins function as ZF-HD transcription factors to activate GmCaM4 gene expression in response to pathogen.

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.

Phosphorylation and concomitant structural changes in human 2-Cys peroxiredoxin isotype I differentially regulate its peroxidase and molecular chaperone functions.

The H2O2-catabolizing peroxidase activity of human peroxiredoxin I (hPrxI) was previously shown to be regulated by phosphorylation of Thr90. Here, we show that hPrxI forms multiple oligomers with distinct secondary structures. HPrxI is a dual function protein, since it can behave either as a peroxidase or as a molecular chaperone. The effects of phosphorylation of hPrxI on its protein structure and dual functions were determined using site-directed mutagenesis, in which the phosphorylation site was substituted with aspartate to mimic the phosphorylated status of the protein (T90D-hPrxI). Phosphorylation of the protein induces significant changes in its protein structure from low molecular weight (MW) protein species to high MW protein complexes as well as its dual functions. In contrast to the wild type (WT)- and T90A-hPrxI, the T90D-hPrxI exhibited a markedly reduced peroxidase activity, but showed about sixfold higher chaperone activity than WT-hPrxI.

WRKY group IId transcription factors interact with calmodulin.

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.

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

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.

Partial purification and properties of a phosphatidylinositol 4,5-bisphosphate hydrolyzing phospholipase C from the soluble fraction of soybean sprouts.

Three soluble enzyme fractions (F-I, F-II, and F-III) that hydrolyze phophoinositides were separated from soybean sprouts by using Matrex green gel column chromatography. Among the three phosphatidylinositol (PI)-specific phopholipsase C (PLC) enzymes, only the third fraction (F-III) was able to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) as well as phosphatidylinositol (PI) and phosphatidylinositol phosphate (PIP) as substrates. The F-I and F-II fractions only showed enzymatic activities for PI and PIP. The PIP2-hydrolyzing PLC protein, F-III, was partially purified using the chromatographic steps of the Matrex green gel, phenyl Toyopearl, Matrex orange gel, Mono S cation exchange, and superose 6 gel filtration columns. The molecular weight of the F-III protein was estimated to be about 64 kDa on SDS-PAGE. The protein showed immunocross-reactivity with a polyclonal antibody that was prepared against the X and Y motifs of animal PLC enzymes, the conserved catalytic domains. Ca2+ ion critically affected the PIP2-hydrolyzing PLC activity of the F-III protein, representing maximal activity at 10 microM Ca2+ concentration. The PIP2-hydrolyzing PLC activity of the protein was also significantly increased by sodium deoxycholate (SDC) from 0.05 to 0.08%. However, the activity was greatly reduced above the concentration, and no activity was detected at 0.3% SDC. In addition, the protein exhibited maximal PIP2-hydrolyzing PLC activity at pH, in the range of 6.5-7.5.

Pathogenesis-related gene expression by specific calmodulin isoforms is dependent on NIM1, a key regulator of systemic acquired resistance.

Plants produce numerous calmodulin isoforms that exhibit differential gene expression patterns and sense different Ca2+ signals. This diversity results in different physiological responses to particular stimuli. Gm-CaM-4 and -5 are two divergent calmodulin isoforms from the soybean (Glycine max) that have been reported to be involved in plant disease resistance. However, little is known about the pathway by which these specific isoforms transduce the defense signal and up-regulate pathogenesis-related (PR) genes. Here we report that overexpression of GmCaM-4/-5 induces constitutive PR gene expression and enhances disease resistance in wild-type Arabidopsis, but not in the nim1 mutant of Arabidopsis. GmCaM-4/-5 also appear to activate trans-acting elements that bind to cis-acting elements in the Arabidopsis PR-1 promoter. Thus up-regulation of PR genes by these GmCaM isoforms is dependent on NIM1 (Non immunity 1) and unknown transcription factors.

Pn-AMPs, the hevein-like proteins from Pharbitis nil confers disease resistance against phytopathogenic fungi in tomato, Lycopersicum esculentum.

The antifungal activity of hevein-like proteins has been associated with their chitin-binding activities. Pn-AMP1 and Pn-AMP2, two hevein homologues from Pharbitis nil, show in vitro antifungal activities against both chitin and non-chitin containing fungi. Purified Pn-AMPs retained antifungal activities only under non-reducing conditions. When Pn-AMP2 cDNA was constitutively expressed in tomato (Lycopersicon esculentum) plants under the control of CaMV35S promoter, the transgenic plants showed enhanced resistance against both the non-chitinous fungus Phytophthora capsici, and the chitin-containing fungus Fusarium oxysporum. Thus, the chitin component in the fungal cell wall is not an absolute requirement for Pn-AMP's antifungal activities. These results when considered together suggest that Pn-AMPs have the potential for developing transgenic plants resistant to a wide range of phytopathogenic fungi.

Constitutive expression of mammalian nitric oxide synthase in tobacco plants triggers disease resistance to pathogens.

Nitric oxide (NO) is known for its role in the activation of plant defense responses. To examine the involvement and mode of action of NO in plant defense responses, we introduced calmodulin-dependent mammalian neuronal nitric oxide synthase (nNOS), which controls the CaMV35S promoter, into wild-type and NahG tobacco plants. Constitutive expression of nNOS led to NO production and triggered spontaneous induction of leaf lesions. Transgenic plants accumulated high amounts of H(2)O(2), with catalase activity lower than that in the wild type. nNOS transgenic plants contained high levels of salicylic acid (SA), and they induced an array of SA-, jasmonic acid (JA)-, and/or ethylene (ET)-related genes. Consequently, NahG co-expression blocked the induction of systemic acquired resistance (SAR)-associated genes in transgenic plants, implying SA is involved in NO-mediated induction of SAR genes. The transgenic plants exhibited enhanced resistance to a spectrum of pathogens, including bacteria, fungi, and viruses. Our results suggest a highly ranked regulatory role for NO in SA-, JA-, and/or ET-dependent pathways that lead to disease resistance.

Specificity of DNA sequences recognized by the zinc-finger homeodomain protein, GmZF-HD1 in soybean.

Zinc finger-homeodomain proteins (ZF-HDs) have been identified in many plant species. In soybean (Glycine max), GmZF-HD1 functions as a transcription factor that activates the soybean calmodulin isoform-4 (GmCaM-4) gene in response to pathogens. Recently, we reported specific binding of GmZF-HD1 to a 30-nt A/T-rich cis-element which constitutes two repeats of a conserved homeodomain binding site, ATTA, within -1207 to -1128bp of the GmCaM-4 promoter. Herein, homeodomain sequences of the GmZF-HD1 protein were compared to those of other homeodomain proteins and characterized the specificity of DNA sequences in the interaction of the GmCaM-4 promoter with GmZF-HD1 protein. Considering the conservation of homeodomains in plants, the AG sequence within a 30-nt A/T-rich cis-element is required for binding of the GmZF-HD1 protein. Approximately 25-bp of A/T-rich DNA sequences containing an AG sequence is necessary for effective binding to the GmZF-HD1 protein. Taken together, the results support the notion that the GmZF-HD1 protein specifically functions in plant stress signalling by interacting with the promoter of GmCaM-4.

Calcium and calmodulin-mediated regulation of gene expression in plants.

Sessile plants have developed a very delicate system to sense diverse kinds of endogenous developmental cues and exogenous environmental stimuli by using a simple Ca2+ ion. Calmodulin (CaM) is the predominant Ca2+ sensor and plays a crucial role in decoding the Ca2+ signatures into proper cellular responses in various cellular compartments in eukaryotes. A growing body of evidence points to the importance of Ca2+ and CaM in the regulation of the transcriptional process during plant responses to endogenous and exogenous stimuli. Here, we review recent progress in the identification of transcriptional regulators modulated by Ca2+ and CaM and in the assessment of their functional significance during plant signal transduction in response to biotic and abiotic stresses and developmental cues.

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

Calmodulin-regulated protein phosphorylation plays a pivotal role in amplifying and diversifying the action of calcium ion. In this study, we identified a calmodulin-binding receptor-like protein kinase (CBRLK1) that was classified into an S-locus RLK family. The plasma membrane localization was determined by the localization of CBRLK1 tagged with a green fluorescence protein. Calmodulin bound specifically to a Ca(2+)-dependent calmodulin binding domain in the C-terminus of CBRLK1. The bacterially expressed CBRLK1 kinase domain could autophosphorylate and phosphorylates general kinase substrates, such as myelin basic proteins. The autophosphorylation sites of CBRLK1 were identified by mass spectrometric analysis of phosphopeptides.

Functional analysis of the stress-inducible soybean calmodulin isoform-4 (GmCaM-4) promoter in transgenic tobacco plants.

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.

Identification and characterization of alternative promoters of the rice MAP kinase gene OsBWMK1.

Our previous study suggested that OsBWMK1, a gene which encodes a member of the rice MAP kinase family, generates transcript variants which show distinct expression patterns in response to environmental stresses. The transcript variants are generated by alternative splicing and by use of alternative promoters. To test whether the two alternative promoters, pOsBWMK1L (promoter for the OsBWMK1L splice variant) and pOsBWMK1S (promoter for the OsBWMK1S splice variant), are biologically functional, we analyzed transgenic plants expressing GUS fusion constructs for each promoter. Both pOsBWMK1L and pOsBWMK1S are biologically active, although the activity of pOsBWMK1S is lower than that of pOsBWMK1L. Histochemical analysis revealed that pOsBWMK1L is constitutively active in most tissues at various developmental stages in rice and Arabidopsis, whereas pOsBWMK1S activity is spatially and temporally restricted. Furthermore, the expression of pOsBWMK1S::GUS was upregulated in response to hydrogen peroxide, a plant defense signaling molecule, in both plant species. These results suggest that the differential expression of OsBWMK1 splice variants is the result of alternative promoter usage and, moreover, that the mechanisms controlling OsBWMK1 gene expression are conserved in both monocot and dicot plants.

The calmodulin-binding transcription factor OsCBT suppresses defense responses to pathogens in rice.

We previously isolated the OsCBT gene, which encodes a calmodulin (CaM)-binding protein, from a rice expression library constructed from fungal elicitor-treated rice suspension cells. In order to understand the function of OsCBT in rice, we isolated and characterized a T-DNA insertion mutant allele named oscbt-1. The oscbt-1 mutant exhibits reduced levels of OsCBT transcripts and no significant morphological changes compared to wild-type plant although the growth of the mutant is stunted. However, oscbt-1 mutants showed significant resistance to two major rice pathogens. The growth of the rice blast fungus Magnaporthe grisea, as well as the bacterial pathogen Xanthomonas oryzae pv. oryzae was significantly suppressed in oscbt-1 plants. Histochemical analysis indicated that the hypersensitive-response was induced in the oscbt-1 mutant in response to compatible strains of fungal pathogens. OsCBT expression was induced upon challenge with fungal elicitor. We also observed significant increase in the level of pathogenesis-related genes in the oscbt-1 mutant even under pathogen-free condition. Taken together, the results support an idea that OsCBT might act as a negative regulator on plant defense.

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 Ca(2+)-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 Ca(2+)/CaM-dependent enzyme, and a split-ubiquitin assay. The peptides for two CaMBDs, CaMBDI and CaMBDII, bound CaM in a Ca(2+)-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, Trp(453) and Leu(456) in CaMBDI and Trp(678) and Ile(684) in CaMBDII, play a pivotal role in CaM binding. Moreover, the phosphatase activity of AtMKP1 was increased by CaM in a Ca(2+)-dependent manner. Our results suggest that two important signaling pathways, Ca(2+) 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.

Alternative splicing of the OsBWMK1 gene generates three transcript variants showing differential subcellular localizations.

In eukaryotes, mitogen-activated protein kinases (MAPKs) play important roles in various developmental processes and in environmental stress responses. Here, we show that alternative splicing of the OsBWMK1, a member of the rice MAPK family, generates three transcript variants, OsBWMK1L, OsBWMK1M, and OsBWMK1S. The OsBWMK1L transcript variant was highly and constitutively expressed in all rice tissues tested and its expression was not altered by various stress conditions, whereas OsBWMK1M and OsBWMK1S were normally expressed at low levels but were induced by various stresses. A transient expression assay demonstrated that OsBWMK1L::GFP and OsBWMK1M::GFP were predominantly localized in the cytoplasm, whereas most OsBWMK1S::GFP was localized in the nucleus. Moreover, treatment with defense signaling related molecules, such as H(2)O(2) and SA, induced translocation of OsBWMK1 isoforms from the cytoplasm to the nucleus. Thus, our results suggest that alternative splicing of OsBWMK1 generates three different transcript variants that produce proteins with different subcellular localizations.