In Escherichia coli, a family of cold shock proteins (CSPs) function as transcription antiterminators or translational enhancers at low temperature by destabilizing RNA secondary structure. A wheat nucleic acid-binding protein (WCSP1) was found to contain a cold shock domain (CSD) bearing high similarity to E. coli cold shock proteins. In the present study, a series of mutations were introduced into WCSP1, and its functionality was investigated by using in vivo and in vitro assays in the context of functional conservation with E. coli CSPs. Constitutive expression of WT WCSP1 in an E. coli cspA, cspB, cspE, cspG quadruple deletion mutant complemented its cold-sensitive phenotype, suggesting that WCSP1 shares a function with E. coli CSPs for cold adaptation. In addition, transcription antitermination activity was demonstrated for WCSP1 by using an E. coli strain that has a hairpin loop upstream of a chloramphenicol resistance gene. In vitro dsDNA melting assays clearly demonstrated that WCSP1 melts dsDNA, an activity that was positively correlated to the ability to bind ssDNA. When mutations were introduced at critical residues within the consensus RNA binding motifs (RNP1 and RNP2) of WCSP1, it failed to melt dsDNA. Studies with WCSP1-GFP fusion proteins documented patterns that are consistent with ER and nuclear localization. In vivo and in vitro functional analyses, coupled with subcellular localization data, suggest that WCSP1 may function as a RNA chaperone to destabilize secondary structure and is involved in the regulation of translation under low temperature.cold acclimation ͉ cold shock protein ͉ RNA binding ͉ RNA chaperone ͉ wheat

The molecular mechanisms of cold acclimation are still largely unknown; however, it has been established that overwintering plants such as winter wheat increases freeze tolerance during cold treatments. In prokaryotes, cold shock proteins are induced by temperature downshifts and have been proposed to function as RNA chaperones. A wheat cDNA encoding a putative nucleic acid-binding protein, WCSP1, was isolated and found to be homologous to the predominant CspA of Escherichia coli. The putative WCSP1 protein contains a three-domain structure consisting of an N-terminal cold shock domain with two internal conserved consensus RNA binding domains and an internal glycine-rich region, which is interspersed with three C-terminal CX 2 CX 4 HX 4 C (CCHC) zinc fingers. Each domain has been described independently within several nucleotide-binding proteins. Northern and Western blot analyses showed that WCSP1 mRNA and protein levels steadily increased during cold acclimation, respectively. WCSP1 induction was cold-specific because neither abscisic acid treatment, drought, salinity, nor heat stress induced WCSP1 expression. Nucleotide binding assays determined that WCSP1 binds ssDNA, dsDNA, and RNA homopolymers. The capacity to bind dsDNA was nearly eliminated in a mutant protein lacking C-terminal zinc fingers. Structural and expression similarities to E. coli CspA suggest that WCSP1 may be involved in gene regulation during cold acclimation.

No abstract

The benzenoid ester, methylbenzoate is one of the most abundant scent compounds detected in the majority of snapdragon (Antirrhinum majus) varieties. It is produced in upper and lower lobes of petals by enzymatic methylation of benzoic acid in the reaction catalyzed byS-adenosyl-l-methionine:benzoic acid carboxyl methyltransferase (BAMT). To identify the location of methylbenzoate biosynthesis, we conducted an extensive immunolocalization study by light and electron microscopy at cellular and subcellular levels using antibodies against BAMT protein. BAMT was immunolocalized predominantly in the conical cells of the inner epidermal layer and, to a much lesser extent, in the cells of the outer epidermis of snapdragon flower petal lobes. It was also located in the inner epidermis of the corolla tube with little BAMT protein detected in the outer epidermis and in the yellow hairs within the tube on the bee's way to the nectar. These results strongly suggest that scent biosynthetic genes are expressed almost exclusively in the epidermal cells of floral organs. Immunogold labeling studies reveal that BAMT is a cytosolic enzyme, suggesting cytosolic location of methylbenzoate biosynthesis. The concentration of scent production on flower surfaces that face the pollinators during landing may increase pollination efficiency and also help to minimize the biosynthetic cost of advertising for pollinators.

In plants, Late Embryogenesis Abundant (LEA) proteins typically accumulate in response to low water availability conditions imposed during development or by the environment. Analogous proteins in other organisms are induced when exposed to stress conditions. Most of this diverse set of proteins can be grouped according to properties such as high hydrophilicity and high content of glycine or other small amino acids in what we have termed hydrophilins. Previously, we showed that hydrophilins protect enzyme activities in vitro from low water availability effects. Here, we demonstrate that hydrophilins can also protect enzyme activities from the adverse effects induced by freeze-thaw cycles in vitro. We monitored conformational changes induced by freeze-thaw on the enzyme lactate dehydrogenase (LDH) using the fluorophore 1-anilinonaphthalene-8-sulfonate (ANS). Hydrophilin addition prevents enzyme inactivation and this effect is reflected in changes in the ANS-fluorescence levels determined for LDH. We further show that for selected plant hydrophilins, removal of certain conserved domains affects their protecting capabilities. Thus, we propose that hydrophilins, and in particular specific protein domains, have a role in protecting cell components from the adverse effects caused by low water availability such as those present during freezing conditions by preventing deleterious changes in protein secondary and tertiary structure.