A Rice Functional Transcriptional Activator, RISBZ1, Responsible for Endosperm-specific Expression of Storage Protein Genes through GCN4 Motif

Abstract

The GCN4 motif, a cis-element that is highly conserved in the promoters of cereal seed storage protein genes, plays a central role in controlling endospermspecific expression. This motif is the recognition site for a basic leucine zipper transcriptional factor that belongs to the group of maize Opaque-2 (O2)-like proteins. Five different basic leucine zipper cDNA clones, designated RISBZ1-5, have been isolated from a rice seed cDNA library. The predicted gene products can be divided into two groups based on their amino acid sequences. Although all the RISBZ proteins are able to interact with the GCN4 motif, only RISBZ1 is capable of activating (more than 100-fold expression) the expression of a reporter gene under a minimal promoter fused to a pentamer of the GCN4 motif. Loss-of-function and gain-of-function experiments using the yeast GAL4 DNA binding domain revealed that the proline-rich N-terminal domain (27 amino acids in length) is responsible for transactivation. The RISBZ1 protein is capable of forming homodimers as well as heterodimers with other RISBZ subunit proteins. RISBZ1 gene expression is restricted to the seed, where it precedes the expression of storage protein genes. When the RISBZ1 promoter was transcriptionally fused to the ␤-glucuronidase reporter gene and the chimeric gene was introduced into rice, the ␤-glucuronidase gene is specifically expressed in aleurone and subaleurone layer of the developing endosperm. These findings suggest that the specific expression of transcriptional activator RISBZ1 gene may determine the endosperm specificity of the storage protein genes.Regulated gene expression is mediated by the combinatorial interactions of multiple cis-elements in the gene's promoter. Specific binding of transcriptional factors to the cognate ciselements constitute a crucial step in transcription initiation and, in turn, on the spatial and temporal expression of genes.Seed storage protein genes provide a model system for the study on the regulatory mechanisms of plant genes (1), since their expression is restricted to a specific tissue and stage during seed development. These specific temporal and spatial expression patterns may be explained as the result of regulatory assemblies of several transcriptional activators that recognize the cis-elements implicated in seed-specific expression. Therefore, to understand such molecular mechanisms, characterization of cis-elements and transcription factors has been performed on many storage protein genes of several crop plants (2,3). Despite numerous studies, the mechanism by which these genes are regulated are poorly understood, since many of the essential cis-elements have not been identified. This is especially true in the case of monocot plants, where many of the promoter analyses of cereal storage protein genes have carried out by transient assays using particle bombardment or heterologous transgenic tobacco system (4 -6). Dissection analyses of promoter using homologous stable transgenic plant have been carried out only on glutelin genes of ...

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