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

Yasuyuki Onodera,Akihiro Suzuki,Chuan-Yin Wu,Haruhiko Washida,Fumio Takaiwa

doi:10.1074/jbc.m007405200

Yasuyuki Onodera, Akihiro Suzuki + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m007405200

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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 endosperm-specific 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 beta-glucuronidase reporter gene and the chimeric gene was introduced into rice, the beta-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.

Highlights

Regulated gene expression is mediated by the combinatorial interactions of multiple cis-elements in the gene’s promoter
Prolamin box (TGTAAAG), GCN4 motif (TGA(G/C)TCA), AACA motif (AACAAAA), and ACGT motif, which are conserved in many promoters of cereal seed storage protein genes, have been characterized as cis-elements involved in endosperm-specific expression by loss-of-function and gain-of-function experiments [3]
Gain-of-function experiments showed that combinations of two motifs (GCN4 motif and prolamin box, GCN4 motif and AACA motif) are not sufficient to confer endosperm-specific expression, suggesting that participation of additional element is required to form a functional complex of trans-acting factors [6, 13]

Summary

EXPERIMENTAL PROCEDURES

To construct fusion plasmids with the GAL4 DNA-binding domain (positions 1–147), various N-terminal regions of the RISBZ1 and RISBZ2 proteins were amplified by PCR with Pfu Taq polymerase (Stratagene) using a forward primer and the reverse primer containing a BamHI site and a termination codon ϩ SacI site at the 5Ј ends, respectively. For construction of monomer and tetramer of 12 bp of normal GCN4 (GCTGAGTCATGA) and mutant GCN4 (GCTtccTCATGA), double-stranded oligonucleotides were generated by annealing the 12- and 48-base complementary oligonucleotide pairs containing the sticky ACGT sequence at the 5Ј end of the sense strand, respectively These double-stranded oligonucleotides were inserted into the SalI and StuI sites of Ϫ46 CaMV/GUS reporter gene. Histochemical analysis of GUS gene expression was carried out as described previously [8]

RESULTS

DISCUSSION

Effector