Investigating thecis-Regulatory Basis of C3and C4Photosynthesis in Grasses at Single-Cell Resolution

Abstract

AbstractWhile considerable knowledge exists about the enzymes pivotal for C4photosynthesis, much less is known about thecis-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 C4enzymes for five different grass species. This study spans four C4species, covering three distinct photosynthetic subtypes:Zea maysandSorghum bicolor(NADP-ME),Panicum miliaceum(NAD-ME),Urochloa fusca(PEPCK), along with the C3outgroupOryza sativa. We studied thecis-regulatory landscape of enzymes essential across all C4species and those unique to C4subtypes, measuring cell-type-specific biases for C4enzymes using chromatin accessibility data. Integrating these data with phylogenetics revealed diverse co-option of gene family members between species, showcasing the various paths of C4evolution. Besides promoter proximal ACRs, we found that, on average, C4genes have two to three distal cell-type-specific ACRs, highlighting the complexity and divergent nature of C4evolution. 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 ofcis-regulation at these C4loci. This study illuminates the dynamic and complex nature of CRE evolution in C4photosynthesis, particularly highlighting the intricatecis-regulatory evolution of key loci. Our findings offer a valuable resource for future investigations, potentially aiding in the optimization of C3crop performance under changing climatic conditions.