Exaptation of Ancestral Cell-identity Networks Enables C Photosynthesis

C photosynthesis is used by the most productive plants on the planet, and compared with the ancestral C pathway, it confers a 50% increase in efficiency. In more than 60 C lineages, CO fixation is compartmentalized between tissues, and bundle-sheath cells become photosynthetically activated. How the bundle sheath acquires this alternate identity that allows efficient photosynthesis is unclear. Here we show that changes to bundle-sheath gene expression in C leaves are associated with the gain of a pre-existing cis-code found in the C leaf. From single-nucleus gene-expression and chromatin-accessibility atlases, we uncover DNA binding with one finger (DOF) motifs that define bundle-sheath identity in the major crops C rice and C sorghum. Photosynthesis genes that are rewired to be strongly expressed in the bundle-sheath cells of C sorghum acquire cis-elements that are recognized by DOFs. Our findings are consistent with a simple model in which C photosynthesis is based on the recruitment of an ancestral cis-code associated with bundle-sheath identity. Gain of such elements harnessed a stable patterning of transcription factors between cell types that are found in both C and C leaves to activate photosynthesis in the bundle sheath. Our findings provide molecular insights into the evolution of the complex C pathway, and might also guide the rational engineering of C photosynthesis in C crops to improve crop productivity and resilience.