Dominant Negative Mutants of Opaque2 Suppress Transactivation of a 22-kD Zein Promoter by Opaque2 in Maize Endosperm Cells.

Abstract

In maize endosperm, genes encoding the 22-kD zein class of storage proteins are regulated by the OPAQUE2 locus. The Opaque2 (O2) protein shares homology with the basic domain/leucine zipper class of transcriptional activators. Using microprojectile bombardment, we have shown that O2 is capable of transactivating a 22-kD zein promoter in maize endosperm suspension cultures and in longitudinal sections of intact endosperm. Two mutant forms of the O2 gene were constructed by deleting regions that encode either the basic domain or the first 175 N-terminal residues of the O2 protein. When either of these mutant O2 genes was coexpressed with wild-type O2 in a maize endosperm expression system, O2-mediated transactivation of the 22-kD zein promoter was inhibited specifically and in a dose-dependent manner. Electrophoretic mobility shift assays and immunoprecipitation studies indicated that the mutant O2 proteins form heterodimers with wild-type O2 in vitro. The mutant lacking the basic domain forms heterodimers with wild-type O2, which can no longer bind DNA. In contrast, the product of the N-terminal truncation allele forms homodimers and heterodimers with wild-type O2, both of which can still bind DNA. Because the N-terminal region contains an activation domain, it is likely that these latter complexes are deficient in transactivation. Dominant negative inhibitors of gene expression, such as those constructed here, provide an alternative to antisense RNA approaches for inactivation of gene function in plants.