In vitro and functional analysis of the Arabidopsis Adh 5' flanking sequence

The Adh (alcohol dehydrogenase) gene of Arabidopsis is expressed constitutively in immature seedlings and cells in suspension, and may be induced in roots of mature plants. In vivo DMS footprinting of the 5$\sp\prime$ flanking region of this gene has identified sites of protein-DNA interaction which include the G-box element also found in several light regulated genes (RbcS, Cab, and Chs). The Adh G-box binds specifically to a protein component of Arabidopsis extracts from cell culture and mature leaves, and mutations of footprinted G-box bases partially or completely disrupt binding. Corresponding G-box mutations in the full length Adh promoter resulted in $>$60% reduction in reporter gene activity when assayed in Arabidopsis protoplasts. Adh promoter mediated expression was observed in both 4-day-old and 15-day-old Arabidopsis seedlings transformed by microprojectile bombardment. Deletions of the Adh promoter were assayed in Arabidopsis seedlings by particle bombardment and in Arabidopsis protoplasts. Sequence domains which are necessary for wild type gene activity were confined to first 390 bp 5$\sp\prime$ to the transcription start site and included all sites of in vivo protein-DNA interaction previously identified by Ferl and Laughner (1989). Deletion of 5$\sp\prime$ sequences from $-$390 to $-$289 decreased activity by 50%, and further deletion to $-$177 resulted in a decrease to less than 10% of full gene activity.