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.

Histone modifications contribute to gene regulation in eukaryotes. We analyzed genome-wide histone H3 Lysine (Lys) 4 trimethylation and histone H3 Lys 9 acetylation (two modifications typically associated with active genes) in meristematic cells at the base and expanded cells in the blade of the maize (Zea mays) leaf. These data were compared with transcript levels of associated genes. For individual genes, regulations (fold changes) of histone modifications and transcript levels were much better correlated than absolute intensities. When focusing on regulated histone modification sites, we identified highly regulated secondary H3 Lys 9 acetylation peaks on upstream promoters (regulated secondary upstream peaks [R-SUPs]) on 10% of all genes. R-SUPs were more often found on genes that were up-regulated toward the blade than on down-regulated genes and specifically, photosynthetic genes. Among those genes, we identified six genes encoding enzymes of the C4 cycle and a significant enrichment of genes associated with the C4 trait derived from transcriptomic studies. On the DNA level, R-SUPs are frequently associated with ethylene-responsive elements. Based on these data, we suggest coevolution of epigenetic promoter elements during the establishment of C4 photosynthesis.

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