Mutations of the 22- and 27-kD Zein Promoters Affect Transactivation by the Opaque-2 Protein

Abstract

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.

Analysis of the Methylation Pattern of the Maize Opaque-2 (O2) Promoter and in Vitro Binding Studies Indicate That the O2 B-Zip Protein and Other Endosperm Factors Can Bind to Methylated Target Sequences

“…3 A suggestive hypothesis is that DNA methylation, in addition to the sequence and spacing of the target DNAs, contributes to avoid spurious activation of plant promoters by nonspecific transcription factors. For example, O2 has been shown to bind to sequences with a CATG instead of an ACGT core (24,33) or to an odd base palindromic C-box sequence (23). This possibility is consistent with the loss of meristem determinacy observed in hypomethylated transgenic Arabidopsis plants (6).…”

“…However, in this respect, the O2 protein was shown to tolerate some changes in its recognition sequence. Replacement of ACGT by ACAT in a 22-kDa zein promoter fragment greatly reduced O2 in vitro binding activity (31) and abolished its in vivo transactivation (33). Strong O2 in vitro binding to an odd base C-box, ATGAGTCAT, was reported by de Pater et al (23).…”

A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2

“…The analysis of both 22-kDa and 27-kDa zein gene promoter activity in transient expression assays with endosperm culture cells (6,11) suggested that factors binding the P-box participate in the coordinate activation of zein genes during endosperm development. At least three observations regarding PBF and the P-box support this view: (i) PBF mRNA is specific to the endosperm and accumulates immediately before the activation (25) and probably represent the full-length OHP1 protein (upper band) and a smaller product (lower band) produced by translation initiation from a methionine 37 residues internal to the start codon.…”

“…Much of what is known about this process in maize is based on the molecular, genetic, and biochemical analysis of the opaque2 locus. This work has demonstrated that the Opaque2 (O2) gene encodes a basic leucine zipper (bZIP) transcription factor (3,4) that binds to a promoter element in the 22-kDa class of zein genes to activate their expression (5,6).…”

A 168 bp derivative of Suppressor-mutator/Enhancer is responsible for the maize o2?23 mutation

“…Studies in yeast have shown that the 02 protein can activate transcription of a chimeric promoter containing multimers of the 02 binding site that is normally found in the 22 kDa zein promoter [20]. In endosperm suspension cells, 02 can activate the transcription of a luciferase reporter gene fused to a 22 kDa zein promoter [23 ]. These results strongly suggest that the product of the 02 locus normally functions as a transcriptional activator of a subset of the zein genes.…”