If the highly efficient C ₄ photosynthesis pathway could be transferred to crops with the C ₃ pathway there could be yield gains of up to 50%. It has been proposed that the multiple metabolic and developmental modifications associated with C ₄ photosynthesis are underpinned by relatively few master regulators that have allowed the evolution of C ₄ photosynthesis more than 60 times in flowering plants. Here we identify a component of one such regulator that consists of a pair of cis -elements located in coding sequence of multiple genes that are preferentially expressed in bundle sheath cells of C ₄ leaves. These motifs represent duons as they play a dual role in coding for amino acids as well as controlling the spatial patterning of gene expression associated with the C ₄ leaf. They act to repress transcription of C ₄ photosynthesis genes in mesophyll cells. These duons are also present in the C ₃ model Arabidopsis thaliana , and, in fact, are conserved in all land plants and even some algae that use C ₃ photosynthesis. C ₄ photosynthesis therefore appears to have coopted an ancient regulatory code to generate the spatial patterning of gene expression that is a hallmark of C ₄ photosynthesis. This intragenic transcriptional regulatory sequence could be exploited in the engineering of efficient photosynthesis of crops.