ACGT and vicilin core sequences in a promoter domain required for seed-specific expression of a 2S storage protein gene are recognized by the opaque-2 regulatory protein

The expression of Brazil nut storage albumin genes is highly regulated during seed development. Several sequences in the promoter of one of these genes show homologies with the target sites of the maize O2 bZIP regulatory protein. We therefore asked whether the O2 protein would recognize these promoter sequences. We show that the O2 protein binds to three different sequences (F1, F2 and F3). F1 and F3 are hybrid C/G and A/G boxes, respectively, that are homologous to the O2-binding site of a maize α-zein gene. F2 is a new O2-binding sequence related to the O2 target sites of the Coix α-coxin, the maize b-32 genes and the AP-1 pseudopalindrome. Molecular modelling showed that an Asn and a Ser in the 02 DNA binding domain make different base-specific contacts with each operator. 5′ Promoter deletions of the be2S1 gene showed that the domain containing the O2 target sites F1 and F2 is required for detectable reporter gene expression in transgenic tobacco seeds. Moreover, the homologous coix O2 protein was shown to in situ transactivate the promoter region encompassing the three O2-binding sites F1, F2 and F3. Thus, these sites may be in vivo regulatory sequences mediating activation by bZIP regulatory proteins.