Unraveling the Molecular Basis of Kranz Anatomy in C4 Grasses

C₄ photosynthesis relies on both biochemical and anatomical adaptations; and while the biochemistry is well established, the regulatory networks underlying kranz anatomy are largely unknown. Recently, a number of candidates underlying kranz anatomy development has been identified and a regulatory network involving the transcriptional regulation of SCARECROW/SHORT-ROOT (SCR/SHR) together with IDDs has been hypothesized to determine cell identity in the leaves of C₄ species. We explored numerous transcriptomics data and extensive literature survey to prioritize candidates and identified four members of the IDDs that are co-expressed in the leaf gradient, enriched to the mesophyll (M) cells and to the bundle sheath (BS). These candidates were tested using Y1H and Y2H assays to define an interaction network of SHR/SCR and IDDs. The complexity of the gene networks and time to generate mutations in several genes have hampered functional analyses of these candidates until now. In the Brutnell lab, we successfully applied CRISPR/Cas9 to generate null alleles in S. viridis. In parallel, translational fusion constructs tagged with YPet were developed using the Golden Gate system for cellular localization and ChIPseq. Extensive microscopy in single and higher order mutants are being explored to evaluate any defects in cellular patterning or differentiation of the vascular bundle in the independently evolved Setaria viridis and also in maize. We hypothesize that BS- or M-cells enriched IDDs act together with SHR and SCR define a network that contributes to the differentiation of photosynthetic cells in C₄ grasses.