Fine structure and function of the osmotin gene promoter

The gene encoding osmotin, a tobacco pathogenesis-related protein, has been shown to be regulated by an array of hormonal and environmental signals. The osmotin promoter fragment -248 to -108 upstream of the transcription start site (fragment A), was sufficient to direct reporter gene expression when fused to a minimal CaMV 35S promoter in transient assays using microprojectile bombardment. This was consistent with previous 5'-deletion analyses of the osmotin promoter which showed that the promoter sequence from -248 to -108 is absolutely required for reporter gene activity. Nuclear protein factors from salt-adapted tobacco cells, ABA-treated unadapted cells, and young cultured tobacco leaves were shown to interact with fragment A by gel mobility-shift assays. DNase I footprinting revealed that three conserved promoter elements in fragment A interact specifically with nuclear factors. These elements are: (1) a cluster of G-box-like sequences (G sequence); (2) an AT-1 box-like sequence, 5'-AATTATTTTATG-3' (AT sequence); (3) a sequence highly conserved in ethylene-induced PR gene promoters, 5'-TAAGA/CGCCGCC-3' (PR sequence). Transient expression assays performed with fragment A deletions fused to GUS indicated that osmotin promoter activity correlated with the presence of these elements. UV cross-linking analysis showed that the protein complex bound to fragment A consisted of at least four individual proteins with approximate molecular masses of 28, 29, 40 and 42 kDa. One component of this protein complex, which was associated with the G sequence, was a 14-3-3 like protein.