ZmPBF and ZmGAMYB transcription factors independently transactivate the promoter of the maize (Zea mays) β-carotene hydroxylase 2 gene.

The maize (Zea mays) enzyme beta-carotene hydroxylase 2 (ZmBCH2) controls key steps in the conversion of beta-carotene to zeaxanthin in the endosperm. The ZmBCH2 gene has an endosperm-preferred and developmentally regulated expression profile, but the detailed regulatory mechanism is unknown. To gain insight into the regulation of ZmBCH2, we isolated 2036 bp of the 5'-flanking region containing the 263 bp 5'-untranslated region (5'-UTR) including the first intron. We linked this to the beta-glucuronidase reporter gene gusA. We found that high-level expression of gusA in rice seeds requires the 5'-UTR for enhanced activation. Truncated variants of the ZmBCH2 promoter retained their seed-preferred expression profile as long as a prolamin box and AACA motif were present. We identified candidate genes encoding the corresponding transcription factors (ZmPBF and ZmGAMYB) and confirmed that their spatiotemporal expression profiles are similar to ZmBCH2. Both ZmPBF and ZmGAMYB can transactivate ZmBCH2 expression in maize endosperm. To eliminate potential confounding effects in maize, we characterized the regulation of the minimal promoter region of ZmBCH2 in transgenic rice. This revealed that ZmPBF and ZmGAMYB independently transactivate the ZmBCH2 promoter. The mechanism that underpins our data provides an exciting new strategy for the control of target gene expression in engineered plants.