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

Abstract

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.

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

“…The two motifs collectively constitute the so called bifactorial endosperm box found in nearly all cereal prolamin genes such as wheat glutenin, barley hordein, rye secalin, sorghum kafirin, and Coix coixin (4,10,11). In most rice glutelin genes, these two motifs are also associated with the AACA motif (12). 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).…”

Avidin Expressed in Transgenic Rice Confers Resistance to the Stored-Product Insect PestsTribolium confusumandSitotroga cerealella

“…This result is consistent with the evidence that the GluB-1 promoter directs endospermspecific expression. 11) The estimated molecular weight of avidin expressed in the transgenic rice grain was 16 kDa, which was the almost same as for standard avidin.…”

A 45‐bp proximal region containing AACA and GCN4 motif is sufficient to confer endosperm‐specific expression of the rice storage protein glutelin gene,GluA‐3

“…Significant difference between the mean value of each construct and that of the corresponding forward orientation was examined by t-test (p < 0.05) and is designated by an asterisk beside the relative GUS activity. the 5'-flanking region of the GluB subfamily genes [4,33]. We h~tve recently shown by 5'-deletion analysis of the GluB-1 gene that both sets of the AACA and GCN4 motifs are required to c(~nfer endosperm-specific expression [33].…”

“…However, little is known about the function of the AACA motif. More than one AACA motif is repeated in the 5'-flanking region of some glutelin genes, in addition to the AACA motif around -70 conserved in all the glutelin genes [4,33]. Homology search of the AACA motif indicates that the sequence of the AACA motif is very similar to that of a gibberellic acid response element.…”

“…the 5'-flanking region of the GluB subfamily genes [4,33]. We h~tve recently shown by 5'-deletion analysis of the GluB-1 gene that both sets of the AACA and GCN4 motifs are required to c(~nfer endosperm-specific expression [33]. It is possible that the difference in motifs combined with one set of the AACA al~d GCN4 motif makes a difference in the gene expression level between the two glutelin gene families.…”