Structures of the three homoeologous loci of wheat benzoxazinone biosynthetic genes TaBx3 and TaBx4 and characterization of their promoter sequences

Common wheat (2n = 6x = 42, genome formula AABBDD) accumulates benzoxazinones (Bxs) as defensive compounds. There are five Bx biosynthetic genes (TaBx1-TaBx5), and their homoeologous alleles are located on all three homoeologous chromosomes of the A, B and D genomes. Here the molecular structures of the TaBx3 and TaBx4 loci, both of which are located on chromosomes 5A, 5B and 5D, were revealed by sequencing transformation-competent artificial chromosome (TAC) clones. In all homoeologous chromosomes, TaBx3 existed downstream of TaBx4 in a tail-to-head manner, and the two genes were separated from each other by 9.0 kb in 5A, 7.3 kb in 5B and 11.3 kb in 5D. Among the three homoeologs of TaBx3 and TaBx4, the promoter sequences were less conserved than the coding sequences. The promoter sequences of TaBx3 and TaBx4 were highly similar to those of their respective orthologs in the diploid progenitors of common wheat, but were not similar to those of the maize orthologs. Sequence similarity was found between the TaBx3 and TaBx4 coding sequences, but not between their promoter sequences despite their similar transcription pattern at the seedling stage. Some putative cis-elements were found to be shared by all TaBx3 and TaBx4 promoter regions. These results imply that stage-specific transcription of TaBx3 and TaBx4 is not controlled by global sequence similarity of their promoters but by some essential cis-elements. The promoter activity measured by transient assays in wheat protoplasts was similar among the three homoeologs of TaBx3 and TaBx4 in spite of their differential transcript levels in wheat seedlings.