Exaptation of ancestral cell-identity networks enables C4 photosynthesis

C₄ photosynthesis is used by the most productive plants on the planet, and compared with the ancestral C₃ pathway, it confers a 50% increase in efficiency¹. In more than 60 C₄ lineages, CO₂ fixation is compartmentalized between tissues, and bundle-sheath cells become photosynthetically activated². 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 C₄ leaves are associated with the gain of a pre-existing cis-code found in the C₃ 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 C₃ rice and C₄ sorghum. Photosynthesis genes that are rewired to be strongly expressed in the bundle-sheath cells of C₄ sorghum acquire cis-elements that are recognized by DOFs. Our findings are consistent with a simple model in which C₄ 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 C₃ and C₄ leaves to activate photosynthesis in the bundle sheath. Our findings provide molecular insights into the evolution of the complex C₄ pathway, and might also guide the rational engineering of C₄ photosynthesis in C₃ crops to improve crop productivity and resilience^3,4.