Transient embryo-specific expression of anthocyanin in wheat

Abstract

Three barley tissue-specific promoters (Dhn12, Itr1, and Ltp1) were transcriptionally fused to the C1 and/or the B-peru genes involved in anthocyanin biosynthesis. Transient expression of these constructs was examined in different wheat tissues. Constructs with tissue-specific promoters were only active in embryos. A green fluorescent protein (GFP) driven by the actin promoter was used as an internal standard to monitor the effectiveness of each bombardment. Further, normalization of the transient expression assay using the GFP reference significantly reduced the variability between separate bombardments, and the intensity of anthocyanin pigmentation quantified by image analysis allowed for a rapid and accurate evaluation of different promoters. Compared to CaMV35S promoter, tissue-specific promoters were more effective in directing anthocyanin production, specifically in wheat embryos, and the C1 gene was more effective than B-peru. Among the tissue-specific promoters, the Ltp1 was superior to ltr1 and Dhn12 in combination with C1 and/or B-peru gene(s). Analysis of nucleotide sequences of all three tissue-specific promoters revealed the presence of G-box, E-box, and RY elements, which might trigger embryo-specific expression in wheat.