A novel parsley 4CL1 cis-element is required for developmentally regulated expression and protein-DNA complex formation

The phenylpropanoid enzyme 4-coumarate:coenzyme A ligase (4CL) participates in the biosynthesis of a wide range of secondary products with specialized function and tissue distribution in plants. The parsley 4CL1 promoter directs a complex tissue- and cell-specific pattern of reporter gene expression in transgenic tobacco, consistent with the distribution of phenylpropanoid products and sites of 4CL expression in tobacco vegetative and floral organs. We generated mutants in a 4CL1 promoter element previously implicated as a site for protein-DNA complex formation to analyze its role in vivo. Mutation of this element (FP56) reduced expression in some organs/tissues up to several hundredfold, with little effect on cell-specific expression patterns. Electrophoretic mobility shift assays indicated that the FP56 cis-element is the binding site for tobacco and parsley nuclear proteins, and that mutations in the same element that reduce reporter gene expression in transgenic plants greatly reduce or abolish protein-DNA complex formation. DNAse I protection assays showed that the region of the 4CL1 promoter surrounding the FP56 element is the site for formation of two large protein-DNA complexes, and that an intact FP56 element is required for formation of these complexes. Finally, the detergent deoxycholate was used to investigate the role of protein-protein interactions in FP56 complex formation. Our data suggest that the FP56 cis-element plays a central role in transcriptional activation from the 4CL1 promoter, and that its role may be to nucleate formation of a large protein complex on the promoter.