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Abstract

This thesis is intended to further our understanding of the mechanisms by which expression of phenylpropanoid genes, specifically parsley 4-coumarate : CoA ligase (4CL1), are developmental^ regulated. The 4CL enzyme is the third and final step in the core phenylpropanoid metabolic pathway. 4CL1-GUS fusions are specifically expressed in the xylem and floral organs in transgenic tobacco. In-vivo footprinting and sitedirected mutagenesis revealed a region spanning -78 to -120, known as footprint 56, of the 4CL1 promoter which is critical for 4CL expression. DNasel footprinting and gel shift assays using tobacco nuclear extracts indicated that a nuclear factor or complex of factors bind to this cis-element on the 4CL1 promoter in vitro. A putative transcription factor which binds to this footprint 56, termed 56BF, was isolated by using a footprint 56 oligonucleotide as a probe to screen a tobacco cDNA expression library. To test whether 56BF is involved in regulating expression of the 4CL1 gene, transgenic tobacco plants that over-expressed or were suppressed for its' accumulation were generated. 56BF recombinant protein was expressed in E. Coli to test the DNA-binding specificity of the protein. Results discussed in this thesis strongly suggest that 56BF does not regulate the 4CL1 gene in vivo.