Interacting cis elements in the plastocyanin promoter from spinach ensure regulated high-level expression

The spinach plastocyanin promoter contains most, if not all, cis elements crucial for its activity downstream of -259 bp relative to the transcription start site. The -259/-79 bp promoter fragment is capable of conferring glucuronidase (GUS) gene expression on the minimal -90/+3 bp 35S RNA promoter of CaMV and -51/+60 bp plastocyanin promoter, regardless of its orientation. Using 5' promoter deletion analysis and site directed mutagenesis we identified three regions, designated PC-1 (-195/-188), PC-2 (-179/-164) and PC-3 (-90/-77) for promoter function. An interaction between PC-3 and the upstream elements is required for high levels of expression. All these sequences contain binding sites for protein factors, as shown by gel shift assays. PC-3 includes a binding site with some resemblance to GT-1 box II, but additional nucleotide sequences immediately downstream of this motif, which are conserved among all published plastocyanin promoters, are required as well. The sequence interval -168/-79 bp is sufficient to confer light-responsive, organ-specific and chloroplast-dependent GUS gene expression on minimal promoters.