complex developmental expression of a novel stress-responsive barley Ltp gene is determined by a shortened promoter sequence

The search for a cereal promoter capable of driving preferential transgene expression in the pericarp epidermis (epicarp) of developing barley (Hordeum vulgare L.) resulted in the cloning of a novel gene. This encoded a polypeptide of 124 amino acids showing 87% identity with WBP1A, a wheat lipid transfer protein (LTP), but much lower homology to other barley LTPs. In addition to the epicarp, this Ltp-like gene, Ltp6, is highly expressed in coleoptiles and embryos under normal growth conditions. Messenger RNA levels increased in seedling tissues during salt and cold treatments and under applied abscisic acid (ABA) and salicylic acid (SA). Taken together, Ltp6 tissue-specific and response patterns are distinct from other known barley Ltp genes. Inverse PCR was used to derive 2345 bp of upstream Ltp6 sequence. The level of transcription conferred by different promoter deletion constructs was assessed by quantitative real time RT-PCR using gfp as a reporter in transient expression assays. All constructs containing at least 192 bp of upstream sequence and the 5'UTR conferred tissue-specific expression and retained most of the promoter strength. Deletion of 64 bp (-192/-128) from this upstream sequence reduced expression levels by 80%. Moreover, a minimal 247 bp Ltp6 promoter continuously drove gfp expression during spike development, from early ovary differentiation through its final expression in the epicarp and during embryogenesis and germination in transgenic barley, reproducing the expression pattern of the native gene. The potential use of this promoter sequence for targeting transgene-mediated disease resistance in barley and wheat is discussed.