Induction of C ₄ genes evolved through changes in cis allowing integration into ancestral C ₃ gene regulatory networks

C ₄ photosynthesis has evolved independently in over sixty lineages and in so doing repurposed existing enzymes to drive a carbon pump that limits the RuBisCO oxygenation reaction. In all cases, gene expression is modified such that C ₄ proteins accumulate to levels matching those of the photosynthetic apparatus. To better understand this rewiring of gene expression we undertook RNA- and DNaseI-SEQ on de-etiolating seedlings of C ₄ Gynandropsis gynandra , which is sister to C ₃ Arabidopsis. Changes in chloroplast ultrastructure and C ₄ gene expression were coordinated and rapid. C ₃ photosynthesis and C ₄ genes showed similar induction patterns, but C ₄ genes from G. gynandra were more strongly induced than orthologs from Arabidopsis. A gene regulatory network predicted transcription factors operating at the top of the de-etiolation network, including those responding to light, act upstream of C ₄ genes. Light responsive elements, especially G-, E- and GT-boxes were over-represented in accessible chromatin around C ₄ genes. Moreover, in vivo binding of many G-, E- and GT-boxes was detected. Overall, the data support a model in which rapid and robust C ₄ gene expression following light exposure is generated through modifications in cis to allow integration into high-level transcriptional networks including those underpinned by conserved light responsive elements.