Comparative cell-specific transcriptomics reveals differentiation of C4 photosynthesis pathways in switchgrass and other C4 lineages

Almost all C-4 plants require the co-ordination of the adjacent and fully differentiated cell types, mesophyll (M) and bundle sheath (BS). The C-4 photosynthetic pathway operates through two distinct subtypes based on how malate is decarboxylated in BS cells; through NAD-malic enzyme (NAD-ME) or NADP-malic enzyme (NADP-ME). The diverse or unique cell-specific molecular features of M and BS cells from separate C-4 subtypes of independent lineages remain to be determined. We here provide an M/BS cell type-specific transcriptome data set from the monocot NAD-ME subtype switchgrass (Panicum virgatum). A comparative transcriptomics approach was then applied to compare the M/BS mRNA profiles of switchgrass, monocot NADP-ME subtype C-4 plants maize and Setaria viridis, and dicot NAD-ME subtype Cleome gynandra. We evaluated the convergence in the transcript abundance of core components in C-4 photosynthesis and transcription factors to establish Kranz anatomy, as well as gene distribution of biological functions, in these four independent C-4 lineages. We also estimated the divergence between NAD-ME and NADP-ME subtypes of C-4 photosynthesis in the two cell types within C-4 species, including differences in genes encoding decarboxylating enzymes, aminotransferases, and metabolite transporters, and differences in the cell-specific functional enrichment of RNA regulation and protein biogenesis/homeostasis. We suggest that C-4 plants of independent lineages in both monocots and dicots underwent convergent evolution to establish C-4 photosynthesis, while distinct C-4 subtypes also underwent divergent processes for the optimization of M and BS cell co-ordination. The comprehensive data sets in our study provide a basis for further research on evolution of C-4 species.