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

PEG-mediated transformation was used for gene delivery and evaluation of various parameters affecting the transient expression of a gene for ß-glucuronidase (gus) in mesophyll protoplasts of Capsicum annuum. Transient expression was found to be dependent on PEG concentration and exposure time of plasmid DNA to protoplasts as well as the amount of plasmid DNA. Maximum GUS activity was obtained when protoplasts were applied to 40% concentration and molecular weight was 6,000 of PEG solution with 30 min of exposure time. Protoplasts of pepper were transformed with a vector, pCAMBIA::Ac, which contained a pCAMBIA1302 T-DNA vector carrying a maize transposable element, Ac (activator), a selection marker HPT (hygromycin phosphotransferase), and a GFP-coding region driven by the 35S promoter in the presence of PEG. Approximately 30% of the protoplasts expressed GFP. Visibly transformed colonies were obtained from protoplasts after 2 months of culture and GFP was expressed. Southern hybridization confirmed the presence of Ac in the pepper genome.

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The synthesis of the suitably protected form (1) of the tetrasaccharide repeat unit, →2)-α-D-Manp-(1→2)-β-D-Manp-(1→3)-α-D-GlcpNAc-(1→6)-α-D-Manp-(1→ (A), of the O-antigen polysaccharide of the lipopolysaccharide from Escherichia coli O77 has been accomplished by latent–active glycosylation employing the 2′-carboxybenzyl (CB) gly coside method. In addition to previously used latent glycosyl donors, 2′-(benzyloxycarbonyl)benzyl (BCB) glycosides, new latent glycosyl donors, 2'-(allyloxycarbonyl)benzyl (ACB) glycosides, have been introduced as a direct precursor for the active CB glycosides. We also demonstrate that 4,6-O-benzylidene-2-azido-2-deoxy-α-D-mannopyranoside (7) has been readily prepared from D-glucosamine in good yield.Key words: Escherichia coli O77, glycosylation, 2′-carboxybenzyl (CB) glycosides, 2′-(allyloxycarbonyl)benzyl (ACB) glycosides, glycosyl donor.

A maize transposable family, Ac/Ds, has been successfully utilized as a mutagenizing agent not only in monocot but also in dicot. In order to develop insertional mutagenesis system in pepper, the mobility of Ac/Ds has been examined. In this study, the excision of the elements was monitored via transient assay system with protoplasts. Two different systems were developed and compared; one-and two-elements systems. In a one-element system, Ac alone was introduced into cells. As a two-element system, Ac and Ds were cloned into a single vector and were expressed in protoplasts. Our data showed that both Ac and Ds elements were highly mobile in pepper cells. This is the first report suggesting that Ac/Ds mediated gene tagging system could be successfully operated in pepper.