Interactions of the glutelin Gt3 5' flanking regulatory regions with rice nuclear proteins

Three DNA binding activities, BP-1, BP-2 and BP-3, which interact with the 5' flanking region of the rice glutelin Gt3 gene, were identified by gel retardation assays of rice seed nuclear extracts. The DNA binding activities were seed-specific as identical DNA-protein complexes were not observed when nuclear extracts from leaf tissue or suspension culture cells were analyzed, suggesting that these DNA binding activities are involved in seed-specific expression of the Gt3 gene. The DNA sequences recognized by these DNA binding activities were identified by DNaseI foot-printing and Bal 31 nuclease mapping analyses. BP-1 recognizes DNA sequences located at -272 bp to -259 bp relative to the transcriptional start site. This DNA segment contains a sequence motif that is conserved among several seed protein genes, implicating that the motif may be a common cis-regulatory element determining seed-specific expression of these genes. BP-2 interacts with sequences located between -861 bp to -838 bp while BP-3 interacts with sequences upstream of -788 bp. The temporal levels of BP-2 binding activity parallel the steady state levels of the Gt3 mRNAs during seed development. Overall, these results and those obtained from in vivo promoter analysis in transgenic plants [Zhao et al. (1994) Plant Mol. Biol. 25: 429] indicate that multiple regulatory elements located at two spatially separated regions of the Gt3 promoter are involved in endosperm-specific and temporal regulation.