Figure 1.

Mesophyll and bundle sheath clusters show canonical expression of C₄ photosynthesis-related genes. A, Workflow for expression analysis in maize cells. Illustration of protoplasting, 10× chromium gel bead-in-emulsion (GEM) partitioning and cDNA synthesis, RNA sequencing, and data analysis. B, UMAP plot showing a two-dimensional representation of cell relationships from scRNA-seq data set A in multidimensional space. Bundle sheath cells separate into two subclusters at higher resolution (inset). The upper and lower clusters were later determined to correspond to abaxial and adaxial BS cells (Figure 2), and are therefore named ^abBS and ^adBS (C) Violin plots showing distribution of normalized mRNA counts of marker genes for cells in each cluster. Genes listed are known to be differentially expressed in MS and BS cells in C₄ maize (PEP1, MDH6, CA, ME1, RBCS1) or are genes that identify unique clusters (NAAT1, TAAT). D, Violin plots showing normalized mRNA levels both of genes differentially expressed between ^abBS and BS^ad subclusters (SWEET13a, SWEET13b, and SWEET13c, CC3 (cystatin3) and of genes highly expressed in both clusters (ME1, RBCS1). Gene symbols are shown in Figure 1, C and D, and corresponding gene IDs from the most complete current B73 genome annotation available on the Ensembl genome browser (B73 RefGen_v4), as well as full names, are provided in Supplemental Table S2.