The Ca 21-binding protein calmodulin mediates cellular Ca 21 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 2858 and 2728 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.Plant cells, like animal cells, elevate their cytosolic free-calcium levels ([Ca 21 ] cyt ) with varying amplitude, frequency, and duration in response to a variety of external stimuli (Thomas et al., 1996; Berridge, 1997;McAinsh and Hetherington, 1998 -bound CaM transduces the signals into many cellular processes through modulation of a variety of CaM-binding proteins, including enzymes such as kinases, phosphatases, and nitric-oxide synthase, as well as receptors, ion channels, G-proteins, and transcription factors (Liao et al., 1996;Snedden and Fromm, 1998;Lee et al., 1999a;Zuhlke et al., 1999).In plant cells, in contrast to mammalian cells, multiple CaM genes code for a number of CaM isoforms. This has been shown in wheat (Triticum aestivum; Yang et al., 1996), potato (Solanum tuberosum; Takezawa et al., 1995;Poovaiah et al., 1996), and soybean (Glycine max; Lee et al., 1995a), among others. Over 30 genes encoding CaM isoforms are found in the Arabidopsis genome (The Arabidopsis Genome Initiative, 2000). We have recently cloned five CaM isoforms from soybean (SCaM-1-5). Although SCaM-1-3 are more than 90% identical to mammalian CaM, SCaM-4 and SCaM-5 exhibit only a 78% homology with SCaM-1 and are therefore the most divergent isoforms reported thus far in the plant and animal kingdoms. SCaM-4 is considered to be a bona fide CaM isoform based on the following characteristics. In its primary protein structure, SCaM-4 has four conserved putative EF-hands and a central linker region, hallmark structural features of CaM (Lee et al., 1995a). In addition, most of the nonconsensus amino acids occur outside the EF-hands, and the total number of Article, publication date, and citation information can be found at www.plantphysiol.org/cgi

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.

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.

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