Dominant Negative Mutants of Opaque2 Suppress Transactivation of a 22-kD Zein Promoter by Opaque2 in Maize Endosperm Cells (original) (raw)
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The maize (Zea mays L.) endosperm specific transcription factor, encoded by the Opaque-2 (02) locus, functions in vivo to activate transcription from its target promoters. 02 regulates the expression of a major storage protein class, the 22 kDa zeins, and of a type I ribosome inactivating protein, b-32, during maturation phase endosperm development. The coding sequence of 02, which indicates it to be a member of the basic region-leucine zipper (bZIP) class of DNA-binding proteins, contains a number of regions rich in either proline or acidic residues which are candidates for activation domains. In functional assays using tobacco mesophyll protoplasts, the level of transactivation conferred by a series of 02-deletion constructs was tested using as a reporter a fusion of the b-32 target promoter to [3-glucuronidase (GUS). The results indicate that 02 has a single acidic activation domain, located near the N-terminus of the protein (amino acids 41-91). The ability of a shorter part of this domain (amino acids 39-82) to confer transactivation was also demonstrated in domain swapping experiments, using fusions of the 02 polypeptide sequence to the DNA-binding domain of the parsley (Petroselinum crispum) transcription factor CPRF1.
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The opaque2 (o2) modifier genes convert the soft endosperm of an o2 mutant to a hard, vitreous phenotype. The primary biochemical change associated with the expression of these genes is a two- to threefold increase in synthesis of the 27-kD gamma-zein storage protein. To investigate the mechanism of modifier gene activity, we examined the level of gamma-zein mRNA and protein synthesis during the early stages of endosperm development in normal, o2, and modified o2 geno-types. Although the o2 mutation was found to reduce expression of the 27-kD gamma-zein genes, the activity of o2 modifier genes dramatically increased the level of both gamma-zein protein and mRNAs as early as 16 days after pollination. At this stage, transcription of gamma-zein genes is reduced by approximately 50% in both o2 and modified o2 genotypes compared to wild type. Thus, it appears that the modifiers regulate gamma-zein synthesis through a post-transcriptional mechanism. Analysis of transcripts from the two nearly identical genes (A and B) encoding the 27-kD gamma-zein protein showed differences in the mRNA ratios in different genotypes. In modified o2 mutants, accumulation of A over B transcript was greatly enhanced during endosperm development. Somatic recombination at this locus was found to reduce the number of B genes in the endosperm, but this could not account for the preferential accumulation of the A transcript. Our results suggest that a product of the o2 modifier genes increases the translation or stability of the A gene mRNA, leading to enhanced synthesis of 27-kD gamma-zein protein.
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