SummaryCold stress on plants induces changes in the transcription of cold response genes. A cDNA clone encoding C 2 H 2 -type zinc ®nger protein, SCOF-1, was isolated from soybean. The transcription of SCOF-1 is speci®cally induced by low temperature and abscisic acid (ABA) but not by dehydration or high salinity. Constitutive overexpression of SCOF-1 induced cold-regulated (COR) gene expression and enhanced cold tolerance of non-acclimated transgenic Arabidopsis and tobacco plants. SCOF-1 localized to the nucleus but did not bind directly to either C-repeat/dehydration (CRT/DRE) or ABA responsive element (ABRE), cis-acting DNA regulatory elements present in COR gene promoters. However, SCOF-1 greatly enhanced the DNA binding activity of SGBF-1, a soybean G-box binding bZIP transcription factor, to ABRE in vitro. SCOF-1 also interacted with SGBF-1 in a yeast two-hybrid system. The SGBF-1 transactivated the b-glucuronidase reporter gene driven by the ABRE element in Arabidopsis leaf protoplasts. Furthermore, the SCOF-1 enhanced ABRE-dependent gene expression mediated by SGBF-1. These results suggest that SCOF-1 may function as a positive regulator of COR gene expression mediated by ABRE via protein±protein interaction, which in turn enhances cold tolerance of plants.

The Ca 2؉ signal is essential for the activation of plant defense responses, but downstream components of the signaling pathway are still poorly defined. Here we demonstrate that specific calmodulin (CaM) isoforms are activated by infection or pathogen-derived elicitors and participate in These results suggest that specific CaM isoforms are components of a SA-independent signal transduction chain leading to disease resistance.

Calmodulin plays pivotal roles in the transduction of various Ca 2؉-mediated signals and is one of the most highly conserved proteins in eukaryotic cells. In plants, multiple calmodulin isoforms with minor amino acid sequence differences were identified but their functional significances are unknown. To investigate the biological function of calmodulins in the regulation of calmodulin-dependent enzymes, we cloned cDNAs encoding calmodulins in soybean. Among the five cDNAs isolated from soybean, designated as SCaM-1 to -5, SCaM-4 and -5 encoded very divergent calmodulin isoforms which have 32 amino acid substitutions from the highly conserved calmodulin, SCaM-1 encoded by SCaM-1 and SCaM-3. SCaM-4 protein produced in Escherichia coli showed typical characteristics of calmodulin such as Ca 2؉ -dependent electrophoretic mobility shift and the ability to activate phosphodiesterase. However, the extent of mobility shift and antigenicity of SCaM-4 were different from those of SCaM-1. Moreover, SCaM-4 did not activate NAD kinase at all in contrast to SCaM-1. Also there were differences in the expression pattern of SCaM-1 and SCaM-4. Expression levels of SCaM-4 were approximately 5-fold lower than those of SCaM-1 in apical and elongating regions of hypocotyls. In addition, SCaM-4 transcripts were barely detectable in root whereas SCaM-1 transcripts were as abundant as in apical and elongating regions of hypocotyls. In conclusion, the different biochemical properties together with differential expression of SCaM-4 suggest that this novel calmodulin may have different functions in plant cells.Calmodulin, a highly conserved and ubiquitous protein in eukaryotes, mediates Ca 2ϩ signals to various target proteins (1). A variety of regulatory enzymes and proteins such as protein kinases, ion channels, Ca 2ϩ pumps, nitric oxide synthetase, inositol trisphosphate kinase, cyclic nucleotide phosphodiesterase, and NAD kinase are known to be regulated by Ca 2ϩ and calmodulin (2-4). While a great deal of information has been known for biological roles of calmodulin in animal cells, very little is known about the roles of calmodulin in plant cells. This is mainly due to the absence of purified calmodulindependent enzymes and/or their genes in plants. As an effort to investigate the biological role(s) of calmodulin in plants, calmodulin genes in various plant species have been cloned and characterized recently (4). Interestingly, in Arabidopsis, cDNAs encoding multiple calmodulin isoforms have been isolated although the degree of sequence divergence is minor, only 6 amino acid differences between the two most divergent isoforms among Arabidopsis calmodulins (5-7). This is very notable because, in animal cells, only a single form of calmodulin is produced by a calmodulin multigene family (8, 9). However, it has not been determined whether plant calmodulin isoforms have the same biochemical properties such as calcium-binding abilities and activation of calmodulin-dependent enzymes. Also the biological role of multiple calmodulin isoforms...

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 (Delta1-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.

NAD kinase is a CaTo determine which domains were responsible for this differential activation of NAD kinase, a series of chimeric SCaMs were generated by exchanging functional domains between SCaM-4 and SCaM-1. SCaM-4111, a chimeric SCaM-1 that contains the first domain of SCaM-4, was severely impaired (only 40% of maximal) in its ability to activate NAD kinase. SCaM-1444, a chimeric SCaM-4 that contains the first domain of SCaM-1 exhibited nearly full (ϳ70%) activation of NAD kinase. Only chimeras containing domain I of SCaM-1 produced greater than half-maximal activation of NAD kinase. To define the amino acid residue(s) in domain I that were responsible for this differential activation, seven single residue substitution mutants of SCaM-1 were generated and tested for NAD kinase activation. Among these mutants, only K30E and G40D showed greatly reduced NAD kinase activation. Also a double residue substitution mutant, K30E/G40D, containing these two mutations in combination was severely impaired in its NAD kinaseactivating potential, reaching only 20% of maximal activation. Furthermore, a triple mutation, K30E/M36I/ G40D, completely abolished NAD kinase activation. Thus, our data suggest that domain I of CaM plays a key role in the differential activation of NAD kinase exhibited by SCaM-1 and SCaM-4. Further, the residues Lys 30 and Among them, pea NAD kinase, the enzyme capable of converting cellular NAD to NADP, is a strict CaM-requiring enzyme for its activity. Since NAD and NADP are utilized as coenzymes by a variety of enzymes involved in cellular metabolic processes, it is very important for organisms to precisely regulate their cellular NAD(H)/NADP(H) ratio. Therefore, control of NAD kinase activity by CaM is crucial for cellular metabolic homeostasis. For example, trimethylation of lysine 115 in CaM reduces its ability to activate NAD kinase 4 -5-fold (9). Thus, the perturbation of NAD kinase regulation by overexpression of a nonmethylatable K115R mutant CaM resulted in a decreased growth rate, reduced seed production, and reduced pollen viability in transgenic plants (10). Despite the importance of NAD kinase in plant cells, currently it is not well understood how CaM binds and activates the enzyme. To resolve this question, it would be a prerequisite to purify NAD kinase homogeneously and/or to isolate the corresponding gene. However, attempts at this have not been successful, mainly because of the intrinsic instability of the enzyme (11).In an effort to learn about the CaM and target protein interaction mechanism, crystal and solution structures of CaM and CaM-binding target peptide complexes have been resolved (12)(13)(14). These studies provided important information about the mechanism of CaM binding to target peptides and the structural changes that CaM undergoes upon binding. In these structures, the central helix of CaM bends and twists to envelope CaM-binding peptide in a hydrophobic tunnel. However, these structures alone cannot reveal how the surface of CaM interacts with specific functiona...