Identification of a novel low-temperature-response element in the promoterof the barley (Hordeum vulgare L) gene blt101.1

Two winter barley (Hordeum vulgare L. cv. Igri) genomic clones, lambda gblt 101.1 and lambda gblt101.2, encoding the blt101 gene family, were isolated from a genomic library. Deletion analysis of the blt101.1 promoter, using t ransient fl-glucuronidase (GUS) reporter expression assays, indicated that it contains at least three regulatory regions. A 107-bp region between nucl eotides -168 and -275 with respect to the translation initiation codon, con fers high -level GUS reporter expression at low temperature and contains a sequence (designated CRI) that is highly conserved in equivalent positions within the promoters of both members of the blt101 gene family. A 10-bp mot if contained within CRI binds proteins present in nuclear extracts from bot h control and low temperature-treated barley tissue. Loss-of-function exper iments, using transient-expression analysis, confirmed that this motif acts as a previously unreported low-temperature-responsive element. Nuclease se nsitivity analysis of intact chromatin indicated that the blt101.1 promoter becomes more susceptible to DNase and micrococcal nuclease at low temperat ure, consistent with chromatin reorganisation upon transcriptional inductio n. It is proposed that both the 10-bp motif and chromatin reorganisation ar e involved in the regulation of blt101.1 at low temperature. This is the fi rst detailed analysis of a low- temperature-specific plant promoter and ide ntifies a novel low-temperature-response element.