Previously, it has been shown that a trimer of a 22 bp fragment of the promoter of the seed-specific pea lectin gene confers high expression in seed. Here it is reported that this fragment contains... Show morePreviously, it has been shown that a trimer of a 22 bp fragment of the promoter of the seed-specific pea lectin gene confers high expression in seed. Here it is reported that this fragment contains a binding site for the cloned basic domain/leucine zipper (bZIP) proteins TGA1a and Opaque-2 (O2). Gel shift assays, DNasel footprinting and methylation interference assays using purified TGA1a were performed to determine whether additional binding sites are present in the psl promoter. Within the 469 bp upstream region only one other TGA1a binding site was found, which is much weaker than the one present in the 22 bp element. Both O2 and TGA1a bound to the odd base palindromic C-box sequence, ATGAGTCAT, present within the 22 bp fragment. The 22 bp fragment also contains the sequence CACGTA, which contains the ACGT core usually found in binding sites for bZIP proteins. However, this sequence did not significantly contribute to bZIP protein binding. The binding affinity of TGA1a for the odd base palindromic sequence was low relative to a high-affinity C-box (ATGACGTCAT). By contrast, O2 strongly bound to the odd base C-box; the affinity was comparable with that for high-affinity G-(GACACGTGTC) and C-boxes. It is concluded that the presence of an ACGT core sequence is not a prerequisite for high-affinity binding of O2. Show less
To elucidate the molecular mechanisms responsible for seed-specific gene expression in plants, the promoter of the pea lectin (psl) gene, encoding an abundant seed protein, was used as a model.... Show moreTo elucidate the molecular mechanisms responsible for seed-specific gene expression in plants, the promoter of the pea lectin (psl) gene, encoding an abundant seed protein, was used as a model. Leaf and seed nuclear proteins bound to a region in the psl promoter containing three overlapping TGAC-like motifs, which have been shown to be a binding site for basic/leucine zipper proteins, including TGA1a. A trimer of a 22-bp region of the psl promoter, containing the TGAC-like motifs, coupled to a heterologous minimal promoter conferred low reporter gene expression in root, stem, and leaf and high expression in seed of transgenic tobacco. Expression increased during the midmaturation stage of seed development and was observed in the endosperm as well as in the embryo, where it strongly decreased within a few days after germination. This expression pattern is qualitatively identical to the expression pattern conferred by a 2000-bp fragment of the psl promoter. Nucleotides within the TGAC-like motifs important for in vitro binding are also essential for in vivo transcription activation in vegetative tissue as well as in seed. The electrophoretic mobility of a DNA-protein complex containing seed nuclear protein was different from that formed with leaf nuclear protein. Furthermore, the TGA1a steady state mRNA level in immature seed was relatively low. These results suggest that a seed-specific factor different from TGA1a, but with similar binding specificity, is responsible for gene activation in seed. We conclude that the 22-bp region contains all the information, including an essential TGAGTCATCA sequence, necessary for seed-specific expression and very likely plays an essential role in the seed-specific expression pattern of the psl gene. Show less
