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 (02) protein shares homology with the basic domainlleucine zipper class of transcriptional activators. Using microprojectile bombardment, we have shown that 0 2 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 02 gene were constructed by deleting regions that encode either the basic domain or the first 175 N-terminal residues.of the 0 2 protein.When either of these mutant 02 genes was coexpressed with wild-type 02 in a maize endosperm expression system, 02-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 0 2 proteins form heterodimers with wild-type 0 2 in vitro. The mutant lacking the basic domain forms heterodimers with wild-type 02, which can no longer bind DNA. In contrast, the product of the N-terminal truncation allele forms homodimers and heterodimers with wild-type 02, 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.

Specific Combinations of Zein Genes and Genetic Backgrounds Influence the Transcription of the Heavy-Chain Zein Genes in Maizeopaque-2 Endosperms

“…This subset, identified by the M1 family of sequences, actually codes for mature zein polypeptides of 246 to 241 amino acids and explains the size variability within the heavy zein subclass that is subdivided into zH1 and zH2, respectively (Viotti et al, 1985). In vitro experiments showed that Opaque-2 binds as homodimer to the motif 5Ј-TCCACGTAGA-3Ј (O2 box) that occurs once in the promoter of the heavy chain zein genes about Ϫ300 bp from the ATG start codon (Schmidt et al, 1990) and data from in vivo experiments support this binding specificity (Unger et al, 1993). However, evidences for promiscuity in DNAmotif binding for this type of bZIP factor (Izawa et al, 1993) have been reported (Yunes et al, 1994(Yunes et al, , 1998.…”

The Transcription Factors HvABI5 and HvVP1 Are Required for the Abscisic Acid Induction of Gene Expression in Barley Aleurone Cells

“…In the case of barley, for which mutants of HvABI5 are not available, one way to study the effect of a transcription factor is through the expression of a dominant-negative form of the protein to repress the function of the wild-type gene. This has been particularly suitable for bZIP factors (Unger et al, 1993;Fukazawa et al, 2000;Sprenger-Haussels and Weisshaar, 2000). We constructed a dominant-negative form of HvABI5, and when introduced into aleurones, it efficiently repressed the ABA induction of the ABRC3-GUS reporter construct (Figure 4).…”

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

“…The moderate increase in GUS activity observed with the sense effector was that expected in a wild type endosperm expressing its physiological level of transcription factors (BLZ1 among them), where optimal (or close to optimal) concentrations of BLZ1 will be present. The five to 10 times enhancement reported with other bZIP, such as maize O2 and wheat SPA (Albani et al, 1997;Unger et al, 1993), are probably due to the use of heterologous systems such as tobacco protoplasts or cultured cell lines, where background levels of these effectors are probably different from the native tissue. The effects observed for the Blz1 antisense constructs in co-bombarded barley endosperms are of particular relevance since avoiding the synthesis of a crucial transcription factor would be expected to have dramatic effects on the expression of the gene(s) that it regulates.…”