Characterization of a novel class of plant homeodomain proteins that bind to the C-4 phosphoenolpyruvate carboxylase gene of Flaveria trinervia

We are interested in the regulatory mechanisms responsible for the mesophyl l-specific expression of C-4 phosphoenolpyruvate carboxylase (PEPCase). A o ne-hybrid screen resulted in the cloning of four different members of a nov el class of plant homeodomain proteins, which are most likely involved in t he mesophyll-specific expression of the C-4 PEPCase gene in C-4 species of the genus Flaveria. inspection of the homeodomains of the four proteins rev eals that they share many common features with homeodomains described so fa r, but there are also significant differences. Interestingly, this class of homeodomain proteins occurs also in Arabidopsis thaliana and other C-3 pla nts. One-hybrid experiments as well as in vitro, DNA binding studies confir med that these novel homeodomain proteins specifically interact with the pr oximal region of the C-4 PEPCase gene. The N-terminal domains of the homeod omain proteins contain highly conserved sequence motifs. Two-hybrid experim ents show that these motifs are sufficient to confer homo- or heterodimer f ormation between the proteins. Mutagenesis of conserved cysteine residues w ithin the dimerization domain indicates that these residues are essential f or dimer formation. Therefore, we designate this novel class of homeobox pr oteins ZF-HD, for zinc finger homeodomain protein. Our data suggest that th e ZF-HD class of homeodomain proteins may be involved in the establishment of the characteristic expression pattern of the C-4 PEPCase gene.