Characterization of common cis-regulatory elements responsible for the endosperm-specific expression of members of the rice glutelin multigene family

Glutelin is the most abundant storage protein in rice, which is expressed specifically in the endosperm of maturing seed. Glutelin is encoded by about 10 genes per haploid genome, which are clearly divided into two subfamilies (GluA and GluB). Most of them are coordinately expressed during seed maturation in spite of the remarkable divergence in the 5'-flanking regions between members of two subfamilies. In order to identify the common regulatory mechanisms responsible for the endosperm-specific expression, various cis-regulatory elements in the 5'-flanking region of the glutelin GluB-1 gene were characterized by studying the expression of chimeric genes that consisted of the sequentially deleted or mutagenized promoter and a beta-glucuronidase (GUS) reporter gene in transgenic tobacco seeds. The essential cis-regulatory elements governing the spatially and temporally specific expression of the glutelin gene expression were located within the first 245 bp of the promoter region of the GluB-1 gene from the site of initiation of transcription. The AACA motif between positions -73 and -61 common to all the six genes for glutelin sequenced to date and is repeated between positions -212 and -200 is implicated in the seed-specific expression. The GCN4 motif between positions -165 and -158 and between positions -96 and -92 that is conserved at homologous sites in all the members of glutelin gene family is also involved in the seed-specific regulation. However, both are required for the high level of seed-specific expression, because deletion of the region containing one set of both elements or substitution mutation of the AACA or GCN4 motif substantially reduced the activity. As a whole, our results suggest the combinatorial interaction of the elements in regulation of the glutelin gene expression.