Single-cell RNA-sequencing provides new insights into the cell-specific expression patterns and transcriptional regulation of photosynthetic genes in bermudagrass leaf blades

As an important warm-season turfgrass species, bermudagrass (Cynodon dactylon L.) flourishes in warm areas around the world due to the existence of the C4 photosynthetic pathway. However, how C4 photosynthesis operates in bermudagrass leaves is still poorly understood. In this study, we performed single-cell RNA-sequencing on 5296 cells from bermudagrass leaf blades. Eight cell clusters corresponding to mesophyll, bundle sheath, epidermis and vascular bundle cells were successfully identified using known cell marker genes. Expression profiling indicated that genes encoding NADP-dependent malic enzymes (NADP-MEs) were highly expressed in bundle sheath cells, whereas NAD-ME genes were weakly expressed in all cell types, suggesting C4 photosynthesis of bermudagrass leaf blades might be NADP-ME type rather than NAD-ME type. The results also indicated that starch synthesis-related genes showed preferential expression in bundle sheath cells, whereas starch degradation-related genes were highly expressed in mesophyll cells, which agrees with the observed accumulation of starch-filled chloroplasts in bundle sheath cells. Gene co-expression analysis further revealed that different families of transcription factors were co-expressed with multiple C4 photosynthesis-related genes, suggesting a complex transcription regulatory network of C4 photosynthesis might exist in bermudagrass leaf blades. These findings collectively provided new insights into the cell-specific expression patterns and transcriptional regulation of photosynthetic genes in bermudagrass.