Induction of C₄genes evolved through changes incisallowing integration into ancestral C₃gene regulatory networks

C₄photosynthesis 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 C₄proteins 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 C₄Gynandropsis gynandra, which is sister to C₃Arabidopsis. Changes in chloroplast ultrastructure and C₄gene expression were coordinated and rapid. C₃photosynthesis and C₄genes showed similar induction patterns, but C₄genes fromG. gynandrawere 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 C₄genes. Light responsive elements, especially G-, E- and GT-boxes were over-represented in accessible chromatin around C₄genes. Moreover,in vivobinding of many G-, E- and GT-boxes was detected. Overall, the data support a model in which rapid and robust C₄gene expression following light exposure is generated through modifications incisto allow integration into high-level transcriptional networks including those underpinned by conserved light responsive elements.