The C4Ppc promoters of many C4 grass species share a common regulatory mechanism for gene expression in the mesophyll cell

C-4 photosynthetic plants have evolved from C-3 ancestors and are characterized by differential expression of several hundred genes. Strict compartmentalization of key C-4 enzymes either to mesophyll (M) or bundle sheath cells is considered a crucial step towards the evolution of C-4 photosynthesis. In this study, we demonstrate that the 5 '-flanking sequences of the C-4 type phosphoenolpyruvate carboxylase (Ppc) gene from three C-4 grass species could drive M-cell-specific expression of a reporter gene in rice. In addition to that, we identified about 450 bp (upstream of their transcription start site) of the analyzed C(4)Ppc promoters contain all the essential regulatory elements for driving M-cell-specific expression in rice leaves. Importantly, four motifs of conserved nucleotide sequences (CNSs) were also determined, which are essential for the activity of the promoter. A putative interaction between the CNSs and an unknown upstream element(s) is required for driving M-cell-specific expression. This work identifies the evolutionary conservation of C(4)Ppc regulatory mechanisms of multiple closely related C-4 grass species.