Inferring early regulators of maize Kranz anatomy development using transcriptomes of LCM-isolated cells from embryonic leaves

Leaves of C4 plants are characterized by the Kranz anatomy, in which the vascular bundles are surrounded by one layer of organelle-rich bundle sheath (BS) cells and one layer of radially arranged mesophyll (M) cells. Past histological and cell lineage studies maize leaves revealed that Kranz development starts from three contiguous ground meristem (GM) cells, but little is known about the regulators and the molecular mechanism underlying Kranz anatomy development. To identify key regulators, we compared the cell-type transcriptomes of different developmental stages of maize embryonic leaves: 5 stages (primordium 1 (P1) and 3BS+V, 4BS+V, 5BS+V and 6-BS+V cells) of Kranz ground meristem (GM) cells; 4 stages of pre-palisade M (P1 and 3-, 4-, and 5-palisade-like ground meristem (PM) stage) cells and 2 stages of undifferentiated M (1M, 2M) PM cells. All cells were isolated by laser-capture microdissection. We obtained high-quality RNAs and RNA-seq data. Principal components analysis of cell-type transcriptomes showed that pre-Kranz and pre-M cells exhibite distinct mRNA populations. Differential gene expression and regulatory network analyses identified candidate coexpression modules and gene coexpression networks involved in Kranz development. In situ hybridization validated several transcription factor (TF) genes expressed in early Kranz anatomy. Finally, we used DNA affinity purification sequencing (DAP-seq) and cis-element prediction to identify putative cis-regulatory elements in the promoter sequence of each candidate TF gene and obtained a putative network related to Kranz anatomy development. These results provide insights into the transcriptional regulation of Kranz anatomy development.