Ist. Sperimentale Cerealicoltura

Transcriptional properties of the DNA binding protein encoded by the opaque-2 locus

--M. Maddaloni, S. Lohmer¹, N. Di Fonzo, H. Hartings, M. Motto, F. Salamini¹ and R. D. Thompson¹

¹Max-Planck-Inst. Züchtungsf., Köln

To understand the molecular details of the role played by the trans-acting regulatory gene opaque-2 (O2), in promoting the coordinated gene expression of zein and b-32 during endosperm development, the O2 and b-32 loci have been recently isolated and characterized in our laboratory (Hartings et al., EMBO J. 8:2895, 1989; Hartings et al., Plant Mol. Biol. 14:1031, 1990).

The genetic data indicated that the expression of b-32 gene is under the control of the O2 gene product. Therefore, DNA binding studies with a purified O2 fusion protein containing 70% of the wildtype protein sequence were carried out. Band shift assays were performed in the presence of purified glutathione-S-transferase (GST)-O2 fusion protein and a 250 long b-32 promoter fragment. Under the binding condition applied two complexes with different electrophoretic mobilities appeared. That means that the GST-O2 fusion protein was able to bind the b-32 promoter. To demonstrate that the complexes formed were not the results of a potential DNA binding activity of the GST, the labelled b-32 promoter fragment was incubated in the presence of GST alone. Under this condition no complex formation was seen. It was concluded that the retarded complexes are produced through the DNA binding activity of the O2 component of the fusion protein.

To verify experimentally the trans-acting property of the O2 protein on a b-32 promoter an assay for transient gene expression in tobacco protoplasts has been employed. This assay was based on cotransfection of mesophyll protoplasts with an expression and reporter plasmid. The expression plasmid consisted of the full length O2 cDNA under the control of promoter of the 35S gene from Cauliflower mosaic virus. The reporter plasmid was assembled by fusing the b-32 promoter region to the coding region of the bacterial b-glucoronidase (GUS) gene.

The results of the transient expression experiments demonstrated a strong activation of the b-32 promoter in the presence of the O2 gene product. The site of interaction of GST-O2 fusion protein on the b-32 promoter fragment was mapped using DNAse I footprinting. The promoter fragment tested contains five regions which were protected by O2 against DNAse I digestion. These experiments show conclusively that the O2 gene product binds to the promoter region of a b-32 gene, a finding supported by the cotransfection experiments and consistent with the available genetic data.

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