Binding of cell type-specific nuclear proteins to the 5'-flanking region of maize C4 phosphoenolpyruvate carboxylase gene confers its differential transcription in mesophyll cells.

C4-type phosphenolpyruvate carboxylase (C4PEPC) acts as a primary carbon assimilatory enzyme in the C4 photosynthetic pathway. The maize C4PEPC gene (C4Ppc1) is specifically expressed in mesophyll cells (MC) of light-grown leaves, but the molecular mechanism responsible for its cell type-specific expression has not been characterized. In this study, we introduced a chimeric maize C4Ppc1 5'-flanking region/beta-glucuronidase (GUS) gene into maize plants by Agrobacterium-mediated transformation. Activity assay and histochemical staining showed that GUS is almost exclusively localized in leaf MC of transgenic maize plants. This observation suggests that the introduced 5' region of maize C4Ppc1 contains the necessary cis element(s) for its specific expression in MC. Next, we investigated whether the 5' region of the maize gene interacts with nuclear proteins in a cell type-specific manner. By gel shift assays with nuclear extracts prepared from MC or bundle sheath cells (BSC), cell type-specific DNA-protein interactions were detected: nuclear factors PEP(Ib) and PEP(Ic) are specific to MC whereas PEP(Ia) and PEP(IIa) are specific to BSC. Light alters the binding activity of these factors. These interactions were not detected in the assay with nuclear extract prepared from root, or competed out by oligonucleotides corresponding to the binding sites for the maize nuclear protein, PEP-I, which is known to bind specifically to the promoter region of C4Ppc1. The results suggest that novel cell type-specific positive and negative nuclear factors bind to the maize C4Ppc1 5'-flanking region and regulate its differential transcription in MC in a light-dependent manner.