Comparative spatiotemporal single cell transcriptomes reveal rewiring of pre-existing regulations during emergence of Kranz anatomy in C ₄ grasses

Many of the world’s most productive food and bioenergy crops use C ₄ photosynthesis, which have high photosynthetic efficiency due to a Kranz anatomy-based CO ₂ concentrating mechanism. Here, we took a comparative transcriptomics approach using single cell spatial transcriptomes of leaf primordia for maize ( Zea mays ) and single cell RNA-seq (scRNA-seq) atlases of corresponding leaf tissues for three C ₄ species ( Zea mays , Sorghum bicolor , Setaria viridis ) and one C ₃ rice ( Oryza sativa ) to study regulatory networks involved in the development and evolution of Kranz anatomy. We show that the formation of Kranz anatomy involves extensive recruitment and modification of pre-existing regulatory modules, especially the SHR-SCR module and the auxin signaling pathway. Members of the INDETERMINATE DOMAIN ( IDD ) family transcription factors, including IDD7 and IDDP1 , contribute to the modification of the involved modules. These extensive recruitment and modification of existing genetic regulatory programs underlie the repeated emergence of C ₄ photosynthesis during evolution.