Post-transcriptional regulation of expression of the Bronze2 gene of Zea mays L.

The glutathione S-transferase encoded by Bronze2 performs the last genetically defined step in maize anthocyanin biosynthesis, being required for pigment sequestration into vacuoles. The Bz2 primary transcript contains a single intron; in maize leaves both spliced and unspliced Bz2 transcripts are usually present and are predicted to encode 26 and 14 kDa proteins, respectively. To increase understanding of the role and regulation of Bz2 transcript splicing, we examined Bz2 expression during development in transgenic maize plants expressing a 35S:Bz2 (35S:mycBz2i) gene and, by transient expression analysis, in Black Mexican Sweet maize protoplasts. We show here that the gene is expressed in diverse tissues that lack functional copies of one or both transcription factors regulating anthocyanin synthesis, that transcript levels are much higher when the R/B plus C1/Pl transcription factors are present, and that the splicing decision depends on local sequence context. The predicted 14 kDa protein was never detected indicating that unspliced transcripts are likely to be non-coding. The native 26 kDa BZ2 protein is loosely membrane-bound, but it was detectable only in tissues accumulating anthocyanin. Consequently, BZ2 accumulation appears to be limited by stringent post-transcriptional regulation.