Abstract
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.