The GCN4 motif in a rice glutelin gene is essential for endosperm‐specific gene expression and is activated by Opaque‐2 in transgenic rice plants

Abstract

SummaryThe GCN4 motif is conserved in a number of seed storage protein genes, and promoter fragments containing this motif have been shown to be involved in controlling seed-specific expression of the genes studied. All genes encoding the rice seed storage protein glutelin contain the GCN4 motif at similar sites in their 5Ј flanking regions. Using a stable homologous transgenic system, we have analysed the promoter of the rice glutelin gene GluB-1 and demonstrated that the GCN4 motif functions as an essential cis-element for endosperm-specific gene expression. Moreover, a 21 bp GluB-1 promoter fragment spanning the GCN4 motif, as a multimer, directed GUS gene expression in endosperm of transgenic rice plants, when fused directly to the core promoter (-46) of CaMV 35S. In transiently transfected rice protoplasts, over a hundredfold transactivation was observed from the 21 bp sequence by the bZIP type transcriptional activator Opaque-2 (O2) co-expressed under a CaMV 35S promoter. The transactivation was also evident in transgenic plants containing both O2 and the 21 bp sequence/GUS fusion. The O2-mediated activation requires binding of O2 to an intact GCN4 motif. Our results suggest that a bZIP protein functionally similar to O2 may exist in rice and participate in controlling the endosperm-specific expression of GluB-1 through the GCN4 motif.

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

“…In vivo foootprinting of the promoter region of wheat low molecular weight glutenin (16) and maize ␥-zein genes (6) in developing endosperm revealed that the region covering the GCN4 motif and prolamin box are occupied by nuclear proteins from maturing seeds. In vitro DNase I footprinting with nuclear proteins from rice maturing seeds and GST-O2 fusion protein also showed that the sequence covering the GCN4 motif in the promoter of glutelin genes is specifically protected from DNase I digestion (8,17). These findings suggest that O2-like transcription factor may exist and participate in controlling the endosperm-specific expression of many storage protein genes in cereal seed through the GCN4 motif.…”

“…Multimers of the rice glutelin GCN4 motif can direct endosperm-specific expression in stable transgenic rice. In contrast, deletion or base substitution of the GCN4 motif in the rice glutelin promoter reduces promoter activity and alters gene expression pattern (8). The GCN4 motif is often linked together with the prolamin box (TGTAAAG), which are separated by a few nucleotides.…”

“…It has been also reported to be involved in activation of endosperm-specific transcription of b32 ribosome-inactivating protein gene via a distinct cis-sequence (G(a/t)TGAPyPuTGPu) (15), thus indicating that the O2 has broad binding specificity. The GCN4 motif has been reported to be recognized by the O2, resulting in the activation of transcription (8,10,15). In vivo foootprinting of the promoter region of wheat low molecular weight glutenin (16) and maize ␥-zein genes (6) in developing endosperm revealed that the region covering the GCN4 motif and prolamin box are occupied by nuclear proteins from maturing seeds.…”

“…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 the rice (7)(8)(9). Endosperm-specific expression of cereal storage protein genes is regulated by the combinatorial interactions of several cis-elements (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). It has been recently demonstrated in the rice glutelin GluB-1 gene that at least three cis-elements containing the GCN4, ACGT, and AACA motifs within the Ϫ197 bp 1 proximal promoter are required as minimal elements to direct endosperm-specific expression (8,9).…”