The maize EmBP-1 orthologue differentially regulates opaque2-dependent gene expression in yeast and cultured maize endosperm cells.

In addition to the bZIP protein Opaque2 (O2), there are other maize endosperm nuclear proteins that recognize the O2 box in 22 kDa zein gene promoters. In an effort to understand the effect of these factors on 22 kDa zein expression, we have cloned one of these and identified it as the putative maize (Zea mays L.) orthologue of the wheat bZIP protein EmBP-1 (mEmBP-1). The mEmBP-1 protein exhibits 52% sequence identity and 68% similarity with the wheat protein and recognizes a similar spectrum of DNA sequences, albeit with slightly altered specificity. The mEmBP-1 gene exists as duplicate loci in maize on chromosomes 7S (mEmBP-1a) and 2L (mEmBP-1b). The mEmBP-1 genes are expressed in endosperm, embryo, immature ears, tassel, roots, and seedling shoots at low levels. Although mEmBP-1 binds to the O2 box from the 22 kDa zein gene promoter as a homodimer, it is unable to heterodimerize with O2. The mEmBP-1 protein can activate transcription from a truncated promoter containing a pentamer of the O2 site in yeast cells; however, it inhibited regulated transcription of a 22 kDa zein promoter in a transient expression assay using cultured maize endosperm cells.

Mutations of the 22- and 27-kD zein promoters affect transactivation by the Opaque-2 protein.

By utilizing a homologous transient expression system, we have demonstrated that the Opaque-2 (O2) gene product O2 confers positive trans-regulation on a 22-kD zein promoter. This trans-acting function of the O2 protein is mediated by its sequence-specific binding to a cis element (the O2 target site) present in the 22-kD zein promoter. A multimer of a 32-bp promoter fragment containing this O2 target site confers transactivation by O2. A single nucleotide substitution in the O2 target sequence not only abolishes O2 binding in vitro, but also its response to transactivation by O2 in vivo. We have also demonstrated that an amino acid domain including the contiguous basic region and the heptameric leucine repeat is essential for the trans-acting function of the O2 protein. Similar but not identical O2 target sequence motifs can be found in the promoters of zein genes of different molecular weight classes. Conversion of such a motif in the 27-kD zein promoter to an exact O2 target sequence by site-directed mutagenesis was sufficient to increase the binding affinity of the O2 protein in vitro and to confer transactivation by O2 in vivo.

Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes.

opaque-2 (o2) is a regulatory locus in maize that plays an essential role in controlling the expression of genes encoding the 22-kD zein proteins. Through DNase I footprinting and DNA binding analyses, we have identified the binding site for the O2 protein (O2) in the promoter of 22-kD zein genes. The sequence in the 22-kD zein gene promoter that is recognized by O2 is similar to the target site recognized by other "basic/leucine zipper" (bZIP) proteins in that it contains an ACGT core that is necessary for DNA binding. The site is located in the -300 region relative to the translation start and lies about 20 bp downstream of the highly conserved zein gene sequence motif known as the "prolamin box." Employing gel mobility shift assays, we used O2 antibodies and nuclear extracts from an o2 null mutant to demonstrate that the O2 protein in maize endosperm nuclei recognizes the target site in the zein gene promoter. Mobility shift assays using nuclear proteins from an o2 null mutant indicated that other endosperm proteins in addition to O2 can bind the O2 target site and that O2 may be associated with one of these proteins. We also demonstrated that in yeast cells the O2 protein can activate expression of a lacZ gene containing a multimer of the O2 target sequence as part of its promoter, thus confirming its role as a transcriptional activator. A computer-assisted search indicated that the O2 target site is not present in the promoters of zein genes other than those of the 22-kD class. These data suggest a likely explanation at the molecular level for the differential effect of o2 mutations on expression of certain members of the zein gene family.