C4 photosynthesis evolved more than 60 times independently in different plant lineages. Each time, multiple genes were recruited into C4 metabolism. The corresponding promoters acquired new regulatory features such as high expression, light induction, or cell typespecific expression in mesophyll or bundle sheath cells. We have previously shown that histone modifications contribute to the regulation of the model C4 phosphoenolpyruvate carboxylase (C4-Pepc) promoter in maize (Zea mays). We here tested the light-and cell type-specific responses of three selected histone acetylations and two histone methylations on five additional C4 genes (C4-Ca, C4-Ppdk, C4-Me, C4-Pepck, and C4-RbcS2) in maize. Histone acetylation and nucleosome occupancy assays indicated extended promoter regions with regulatory upstream regions more than 1,000 bp from the transcription initiation site for most of these genes. Despite any detectable homology of the promoters on the primary sequence level, histone modification patterns were highly coregulated. Specifically, H3K9ac was regulated by illumination, whereas H3K4me3 was regulated in a cell type-specific manner. We further compared histone modifications on the C4-Pepc and C4-Me genes from maize and the homologous genes from sorghum (Sorghum bicolor) and Setaria italica. Whereas sorghum and maize share a common C4 origin, C4 metabolism evolved independently in S. italica. The distribution of histone modifications over the promoters differed between the species, but differential regulation of light-induced histone acetylation and cell type-specific histone methylation were evident in all three species. We propose that a preexisting histone code was recruited into C4 promoter control during the evolution of C4 metabolism.

SummaryWe have investigated the establishment of histone H3 methylation with respect to environmental and developmental signals for two key genes associated with C 4 photosynthesis in maize. Tri-methylation of histone H3 lysine 4 (H3K4) in roots and leaves was shown to be controlled by autonomous cell-type-specific developmental signals that are independent of illumination and therefore independent of the initiation of transcription. Di-and mono-methylation of H3K4 act antagonistically to this process. The modifications were already established in etiolated seedlings, and remained stable when genes were inactivated by dark treatment or pharmaceutical inhibition of transcription. Constitutive di-methylation of H3K9 was concomitantly detected at specific gene positions. The data support a histone code model whereby cell-type-specific signals induce the formation of a chromatin structure that potentiates gene activation by environmental cues.