The Transcriptional Activator Opaque2 Recognizes Two Different Target Sequences in the 22-kD-Like a-Prolamin Genes

Abstract

The maize Opaque2 (O2) protein is a "leucine zipper" DNA binding factor that interacts with the sequence TCCACGTAGA in the promoters of the 22-kD alpha-zein genes and activates its transcription. A completely different consensus sequence (GATGAPyPuTGPu) identified in b-32, a gene that encodes an abundant albumin that is also under control of the O2 locus, can also be bound by the O2 protein. We showed that the gene encoding the 22-kD-like alpha-coixin, the alpha-prolamin of the maize-related grass Coix, can also be transactivated by the O2 protein. A binding assay in vitro and footprint analysis demonstrated that the GACATGTC sequence of the alpha-coixin promoter can be recognized and protected by the maize O2 protein. Employing transient expression experiments in immature maize endosperm and tobacco mesophyll protoplasts, we demonstrated that the O2 protein can activate expression of the beta-glucuronidase reporter gene placed under the control of the 22-kD-like alpha-coixin promoter. We also demonstrated that a 22-kD-like alpha-coixin pseudogene promoter is transactivated by the maize O2 protein.

The wheat transcriptional activator SPA: a seed-specific bZIP protein that recognizes the GCN4-like motif in the bifactorial endosperm box of prolamin genes.

“…In view of the conservation of putative cis elements in the promoters of these 22-kD-like o~-prolamin genes, the presence of O2-related proteins in Coix and sorghum is not surprising. These proteins show 70% (Cljop2) and 63% (Sbopa2) amino acid identity to 02, but their pattern of expression has not been reported (Pirovano et al, 1994;Yunes et al, 1994). Nevertheless, their high sequence similarity to 02 and the ability of the sorghum clone to transactivate the maize b-32 promoter to a similar extent as 02 in a transient expression assay strongly suggest that they are likely to be orthologs of 02.…”

The GCN4 motif in a rice glutelin gene is essential for endosperm‐specific gene expression and is activated by Opaque‐2 in transgenic rice plants

“…The O2 protein recognizes only the GCN4 motif in a short promoter of The O2 protein binds diverse DNA sequences and there can be more than one O2 site in the same promoter (Lohmer et al, 1991;Muth et al, 1996;Schmidt et al, 1992;Yunes et al, 1994). To examine whether the O2 protein recognizes any other DNA sequences besides the GCN4 motif in the GluB-1 promoter, we performed methylation interference analysis on a promoter fragment of 245 bp using the GST-O2 55 fusion protein.…”

“…Consistent with these phenotypic effects, the promoters of the 22 kDa zein gene and b-32 contain O2 binding sites and can be transcriptionally activated by O2 co-expressed in transient assays (Lohmer et al, 1991;Schmidt et al, 1992). O2 also binds and activates promoters of seed storage protein genes from other species such as the wheat LMWG-1D1 and Coix α-coixin genes (Holdsworth et al, 1995;Yunes et al, 1994).…”

“…O2 target sites can be roughly classified into the sequences containing the ACGT core and those without the ACGT core (Cord Neto et al, 1995;Holdsworth et al, 1995;Lohmer et al, 1991;Muth et al, 1996;de Pater et al, 1994;Schmidt et al, 1992;Vincentz et al, 1997;Yunes et al, 1994). It is difficult to draw a consensus sequence through the alignment of all O2 target sites identified so far.…”

Barley BLZ1: a bZIP transcriptional activator that interacts with endosperm‐specific gene promoters

“…The endosperm box is a conserved cis-acting element, located around 300 bp upstream of the transcription initiation site of cereal prolamin genes, which contains two motives: the prolamin box (PB) and the GCN4-like motive (GCN). The bZIP transcription factors Opaque-2 (O2) from maize and SPA from wheat, respectively, have been shown to activate prolamin genes through binding to the GCN motive in the bifactorial endosperm box (Albani et al, 1997;Lohmer et al, 1991;Schmidt et al, 1992;Yunes et al, 1994).…”