Regulatory elements required for light-mediated expression of the Petroselinum crispum chalcone synthase gene

Chalcone synthase (CHS) catalyzes the committed enzymatic step in flavonoid biosynthesis. In parsley (Petroselinum crispum), CHS is encoded by a single gene locus. Transcriptional activation of the gene in response to UV-containing white light has been demonstrated. Analysis of the CHS gene promoter by in vivo footprinting revealed four short sequences, designated Boxes I, II, III, and IV, which contain guanosine residues with altered reactivity to the methylating agent dimethylsulfate in UV-treated versus untreated parsley cells. Studies were performed to characterize the functional components of the CHS gene promoter using a parsley protoplast transient expression system. By deletion and block-mutation analyses it was shown that Boxes I and II act together as a cis-acting unit and are necessary components of the minimal, light-responsive CHS gene promoter. The Box II sequence, which is similar to the conserved G Box sequence defined in promoters of ribulose 1,5-bisphosphate carboxylase small subunit (RBCS) genes, has been subjected to detailed analysis by site-directed mutagenesis. The heptameric sequence 5'-ACGTGGC-3' has been defined as the critical core of Box II required for light induction in the context of the CHS gene minimal promoter. Box II is functionally equivalent to a second, sequence-related element (Box III) that can replace Box II in an orientation-dependent manner. Chimaeric promoter-fusion constructs to the GUS reporter gene demonstrated that Boxes I and II, together constituting a cis-acting unit, are necessary and sufficient for light-mediated activation of the CHS gene promoter.