Nuclear factors binding to the extensin promoter exhibit differential activity in carrot protoplasts and cells.

The expression of the cell wall protein extensin, a hydroxyproline-rich glycoprotein, is induced by several different stimuli, including wounding. The process of protoplast preparation mimics the wounding effect and results in the induction of extensin. Using transient expression in protoplasts we analyzed several deletions of the extensin promoter. We identified an important transcriptional regulatory element located between the two TATA boxes that characterize the extensin promoter. Other regulatory elements, located further upstream between -719 to -658, are necessary for maximum level of expression. Employing electrophoretic mobility shift assays and methylation interference experiments, we demonstrate the interaction of nuclear factors with these upstream regulatory elements. In addition to the previously identified factors EGBF-1 and EGBF-2, which are mainly present in unwounded cells, we identified an additional novel DNA-binding activity that is present in extracts prepared from protoplasts but not in extracts from unwounded cells. This factor, designated EBF (extensin-binding protein), binds to a DNA fragment which when deleted results in a 48% reduction in expression.