IDENTIFICATION OF PROMOTER ELEMENTS IN A LOW-TEMPERATURE-RESPONSIVE GENE (BLT4.9) FROM BARLEY (HORDEUM-VULGARE L.)

The blt4 barley gene family encodes non-specific lipid transfer protei ns and has been shown, by in situ localisation, to be expressed in the epidermal cells of leaves. The transcriptionally controlled, low-temp erature-responsive member of this gene family, blt4.9, is predominantl y 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. Deleti on analysis showed that a 42 bp sequence proximal to, but not includin g, the CAAT and TATA boxes, confers enhanced low-temperature response to a reporter gene in a barley shoot explant transient expression syst em. Other promoter regions were shown to contain negative and positive regulatory regions. Electrophoretic mobility shift analysis (EMSA) wa s used with nuclear proteins from either low-temperature or control-te mperature-treated plants to further investigate the blt4.9 promoter. S ynthetic oligonucleotides were used to identify a hexanucleotide, CCGA AA, within the 42 bp, low-temperature-responsive promoter region, as t he binding site of a low-mobility nuclear protein complex. This comple x was present in nuclear extracts from both low-temperature-treated an d control plants and was the only complex formed within this region. M utation of the CCGAAA motif within the low-temperature-responsive 42 b p promoter sequence reduced low-temperature responsiveness to basal le vels. 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 blt 4.9 in barley.