Investigating the cis -Regulatory Basis of C 3 and C 4 Photosynthesis in Grasses at Single-Cell Resolution.

While considerable knowledge exists about the enzymes pivotal for C 4 photosynthesis, much less is known about the 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 C 4 enzymes for five different grass species. This study spans four C 4 species, covering three distinct photosynthetic subtypes: Zea mays and Sorghum bicolor (NADP-ME), Panicum miliaceum (NAD-ME), Urochloa fusca (PEPCK), along with the C 3 outgroup Oryza sativa . We studied the cis -regulatory landscape of enzymes essential across all C 4 species and those unique to C 4 subtypes, measuring cell-type-specific biases for C 4 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 4 evolution. Besides promoter proximal ACRs, we found that, on average, C 4 genes have two to three distal cell-type-specific ACRs, highlighting the complexity and divergent nature of C 4 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 cis -regulation at these C 4 loci. This study illuminates the dynamic and complex nature of CRE evolution in C 4 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 C 3 crop performance under changing climatic conditions.