Investigating the regulatory basis of C and C photosynthesis in grasses at single-cell resolution.

While considerable knowledge exists about the enzymes pivotal for C photosynthesis, much less is known about the 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 C enzymes for five different grass species. This study spans four C species, covering three distinct photosynthetic subtypes: and (NADP-dependent malic enzyme), (NAD-dependent malic enzyme), (phosphoenolpyruvate carboxykinase), along with the C outgroup . We studied the regulatory landscape of enzymes essential across all C species and those unique to C subtypes, measuring cell-type-specific biases for C enzymes using chromatin accessibility data. Integrating these data with phylogenetics revealed diverse co-option of gene family members between species, showcasing the various paths of C evolution. Besides promoter proximal ACRs, we found that, on average, C genes have two to three distal cell-type-specific ACRs, highlighting the complexity and divergent nature of C evolution. 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 -regulation at these C loci. This study illuminates the dynamic and complex nature of -regulatory elements evolution in C photosynthesis, particularly highlighting the intricate regulatory evolution of key loci. Our findings offer a valuable resource for future investigations, potentially aiding in the optimization of C crop performance under changing climatic conditions.