bZIP proteins bind to a palindromic sequence without an ACGT core located in a seed-specific element of the pea...

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 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.