title: Identification of promoter elements in a low-temperature-responsive gene (blt4.9) from barley (Hordeum vulgare L.).
abstract: The blt4 barley gene family encodes non-specific lipid transfer proteins and has been shown, by in situ localisation, to be expressed in the epidermal cells of leaves. The transcriptionally controlled, low-temperature-responsive member of this gene family, blt4.9, is predominantly expressed in shoot meristems. The promoter region (1938 bp) of blt4.9 contains sequence motifs which have been implicated in responses to low temperature, abscisic acid and other environmental factors. Deletion analysis showed that a 42 bp sequence proximal to, but not including, the CAAT and TATA boxes, confers enhanced low-temperature response to a reporter gene in a barley shoot explant transient expression system. Other promoter regions were shown to contain negative and positive regulatory regions. Electrophoretic mobility shift analysis (EMSA) was used with nuclear proteins from either low-temperature- or control-temperature-treated plants to further investigate the blt4.9 promoter. Synthetic oligonucleotides were used to identify a hexanucleotide, CCGAAA, within the 42 bp, low-temperature-responsive promoter region, as the binding site of a low-mobility nuclear protein complex. This complex was present in nuclear extracts from both low-temperature-treated and control plants and was the only complex formed within this region. Mutation of the CCGAAA motif within the low-temperature-responsive 42 bp promoter sequence reduced low-temperature responsiveness to basal levels. A related upstream element, CCGAC, known to be a low-temperature-responsive element in other plants, did not bind to nuclear proteins in this study. It is proposed that the hexanucleotide CCGAAA, at -195 from the first ATG, is involved in the low-temperature response of blt4.9 in barley.
authors.affiliation: Department of Biochemistry and Genetics, University of Newcastle upon Tyne, UK.
authors.forename: M A
authors.initials: MA
authors.lastname: Dunn
authors.forename: A J
authors.initials: AJ
authors.lastname: White
authors.forename: S
authors.initials: S
authors.lastname: Vural
authors.forename: M A
authors.initials: MA
authors.lastname: Hughes
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003080:Cold Temperature; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D017343:Genes, Plant; D017930:Genes, Reporter; D005966:Glucuronidase / Q000235:genetics; D001467:Hordeum / Q000235:genetics*; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic
chemical_list: D017931:DNA Primers; D018744:DNA, Plant; D009687:Nuclear Proteins; D010940:Plant Proteins; C071568:BLT4 protein, Hordeum vulgare; D005966:Glucuronidase