Quantitative nature of the Prolamin‐box, ACGT and AACA motifs in a rice glutelin gene promoter: minimal cis‐element requirements for endosperm‐specific gene expression

Abstract

SummaryThe ±197 bp promoter of the rice seed storage protein gene, GluB-1, is capable of conferring endospermspeci®c gene expression. This proximal 5¢¯anking region contains four motifs, GCN4, AACA, ACGT and Prolamin-box, which are conserved in many seed storage protein genes. We previously showed that multiple copies of GCN4 conferred endosperm expression pattern when fused to the ±46 core promoter of CaMV 35S. In this paper we demonstrate, using a similar approach, that tandem repeated copies of any of the other three motifs are unable to direct expression in seeds as well as other tissues of transgenic rice plants. Mutational analysis of individual motifs in the ±197 bp promoter resulted in remarkable reductions in promoter activity. These results indicate that the GCN4 motif acts as an essential element determining endosperm-speci®c expression and that the AACA, ACGT and Prolaminbox are involved in quantitative regulation of the GluB-1 gene. A set of gain-of-function experiments using transgenic rice showed that either the Prolamin-box or AACA, although often coupled with GCN4 in many genes, is insuf®cient to form a functional promoter unit with GCN4, whereas a combination of GCN4, AACA and ACGT motifs was found suf®cient to confer a detectable level of endosperm expression. Taken together, our results provide direct insight into the importance of combinatorial interplay between cis-elements in regulating the expression of seed storage protein genes.

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

“…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).…”

Functional analysis of a cotton glucuronosyltransferase promoter in transgenic tobaccos

“…GUS activity was hardly detected in the corresponding tissues of the negative control plants (data not shown). Consistent with the GUS activity levels, the promoter region contains several important elements, such as an amylase-box, CARE, GARE, GCN4-motif, ACGT motif and AACA motif, which are involved in seed-specific and normal embryo-specific expression [24,[28][29][30][31]39]. Quantitative GUS assays showed that the highest expression was in Functional analysis of a GhGlcAT1 promoter 180 npg the anthers, with relatively high expression in the stigmas and styles.…”

“…The CARE and GARE boxes were required during seed germination in Arabidopsis and rice [28,29]. In addition, a combination of GCN4, ACGT and AACA motifs was sufficient to confer a detectable level of endosperm expression in rice [30,31]. Several motifs including AGAAA, AAATGA and GTGA, and six MYB npg response elements are also present in the promoter region ( Table 1).…”

Molecular characterization of SCARECROW (CsSCR) gene expressed during somatic embryo development and in root of cucumber (Cucumis sativus L.)

“…By screening the 1,736-bp upstream regulatory region of the CsSCR gene with three cis-element recognition programs, we detected a number of potentially interesting sequence motifs: TGCAAAG and AACAAAC, which are necessary for endosperm-specific gene expression (Wu et al 2000); TATCCA and TATCCAC, responsible for the gibberellin response (Lanahan et al 1992;Gubler and Jacobsen 1992); ATTTCAAA, an ethylene-responsive element (Itzhaki et al 1994;Montgomery et al 1993); CCTTTT, which is found in the promoter of the a-amylase gene expressed in embryos and induced by gibberellins (Morita et al 1998;Mena et al 2002); and TGCATG and ACGTG, two ABAresponsive elements (Hattori et al 1992;Hobo et al 1999) (Tab. S2).…”