Investigating the <i>cis-</i> regulatory basis of C ₃ and C ₄ photosynthesis in grasses at single-cell resolution

While considerable knowledge exists about the enzymes pivotal for C₄ photosynthesis, much less is known about the <i>cis-</i>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: <i>Zea mays</i> and <i>Sorghum bicolor</i> (NADP-dependent malic enzyme), <i>Panicum miliaceum</i> (NAD-dependent malic enzyme), <i>Urochloa fusca</i> (phosphoenolpyruvate carboxykinase), along with the C₃ outgroup <i>Oryza sativa</i>. We studied the <i>cis-</i>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 phylogenetics revealed diverse co-option of gene family members between species, showcasing the various paths of C₄ evolution. Besides promoter proximal ACRs, we found that, on average, C₄ genes have two to three distal cell-type-specific ACRs, highlighting the complexity and divergent nature of C₄ 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 <i>cis</i>-regulation at these C₄ loci. This study illuminates the dynamic and complex nature of <i>cis</i>-regulatory elements evolution in C₄ photosynthesis, particularly highlighting the intricate <i>cis-</i>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.