Investigating the cis-Regulatory Basis of C3 and C4 Photosynthesis in Grasses at Single-Cell Resolution. C3 C4 grasses

While considerable knowledge exists about the enzymes pivotal for C4 photosynthesis, much less is known about cis-regulation important for specifying their expression in distinct cell types. Here, we use single-cell-indexed ATAC-seq to identify cell-type-specific accessible chromatin regions (ACRs) associated with C4 enzymes for five different grass species. This study spans four C4 species, covering three distinct photosynthetic subtypes: Zea mays and Sorghum bicolor (NADP-ME), Panicum miliaceum (NAD-ME), Urochloa fusca (PEPCK), along with the C3 outgroup Oryza sativa. We studied the cis-regulatory landscape of enzymes essential across all C4 species and those unique to C4 subtypes, measuring cell-type-specific biases for C4 enzymes using chromatin accessibility data. Integrating these data with phylogenetic trees revealed diverse co-option of gene family members between species, showcasing the various paths of C4 evolution. We mapped cell-type-specific ACRs surrounding each C4 gene, discovering that, on average, these genes have two to three cell-type-specific ACRs located beyond the core promoter. These results provide a detailed genomic map of potential regulatory sequences operating on key C4 genes. Examining the evolutionary history of these cell-type-specific ACRs revealed a spectrum of conserved and novel ACRs, even among closely related species, indicating ongoing evolution of cis-regulation at these C4 loci. This work illuminates the dynamic and complex nature of CRE evolution in C4 photosynthesis, particularly highlighting the intricate cis-regulatory evolution of key loci. Our findings offer a valuable resource for future investigations, potentially aiding in the optimization of C3 crop performance under changing climatic conditions.