The developing leaf of the wild grass Brachypodium distachyon at single-cell resolution.

Leaves are the plant's main photosynthetic organs and drive Earth's primary production. Grasses form longitudinal leaves with parallel venation and graminoid stomata. Yet, how distinct leaf tissues coordinately develop to build functional grass leaf anatomy is not well understood. Here, we decoded the developing grass leaf from vegetative meristems to mature tissues using single-cell RNA-sequencing in the wild grass Brachypodium distachyon. In-depth analysis of epidermal clusters and multiplexed whole-mount RNA-fluorescence in situ hybridization resolved most epidermal lineages and confirmed them in planta. Gene regulatory network analysis distinguished the targetomes of the 2 co-expressed, yet functionally divergent stomatal transcription factors BdMUTE and BdFAMA. Finally, we used our dataset to identify and functionally describe the role of the transcription factor gene BdGRAS32 in inhibiting cell division and a stomata-specific role for the putative cell wall-modifying enzyme BdPME53-like. Our single-cell grass leaf atlas enables the dissection of developmental processes that make the grass leaf, which sustains global food production.