Binding of cell type-specific nuclear proteins to the 5 '-flanking region of maize C-4 phosphoenolpyruvate carboxylase gene confers its differential transcription in mesophyll cells

C-4-type phosphenolpyruvate carboxylase (C4PEPC) acts as a primary carbon a ssimilatory enzyme in the C-4 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 chime ric maize C4Ppc1 5'-flanking region/beta -glucuronidase (GUS) gene into mai ze plants by Agrobacterium-mediated transformation. Activity assay and hist ochemical staining showed that GUS is almost exclusively localized in leaf MC of transgenic maize plants. This observation suggests that the introduce d 5' region of maize C4Ppc1 contains the necessary cis element(s) for its s pecific expression in MC. Next, we investigated whether the 5' region of th e maize gene interacts with nuclear proteins in a cell type-specific manner . By gel shift assays with nuclear extracts prepared from MC or bundle shea th cells (BSC), cell type-specific DNA-protein interactions were detected: nuclear factors PEPIb and PEPIc are specific to MC whereas PEPIa and PEPIIa are specific to BSC. Light alters the binding activity of these factors. T hese interactions were not detected in the assay with nuclear extract prepa red from root, or competed out by oligonucleotides corresponding to the bin ding sites for the maize nuclear protein, PEP-I, which is known to bind spe cifically 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.