Genome-wide transcription factor binding in leaves from C₃ and C₄ grasses

The majority of plants use C₃ photosynthesis, but over 60 independent lineages of angiosperms have evolved the C₄ pathway. In most C₄ species, photosynthesis gene expression is compartmented between mesophyll and bundle-sheath cells. We performed DNaseI sequencing to identify genome-wide profiles of transcription factor binding in leaves of the C₄ grasses Zea mays, Sorghum bicolor, and Setaria italica as well as C₃ Brachypodium distachyon. In C₄ species, while bundle-sheath strands and whole leaves shared similarity in the broad regions of DNA accessible to transcription factors, the short sequences bound varied. Transcription factor binding was prevalent in gene bodies as well as promoters, and many of these sites could represent duons that influence gene regulation in addition to amino acid sequence. Although globally there was little correlation between any individual DNaseI footprint and cell-specific gene expression, within individual species transcription factor binding to the same motifs in multiple genes provided evidence for shared mechanisms governing C₄ photosynthesis gene expression. Furthermore, interspecific comparisons identified a small number of highly conserved transcription factor binding sites associated with leaves from species that diverged around 60 million years ago. These data therefore provide insight into the architecture associated with C₄ photosynthesis gene expression in particular and characteristics of transcription factor binding in cereal crops in general.