Upregulation and cell specificity of C4 genes are derived from ancestral C3 gene regulatory networks

C4 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 C4 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 C4 Gynandropsis gynandra, which is sister to C3 Arabidopsis. Changes in chloroplast ultrastructure and C4 gene expression were coordinated and rapid. C3 photosynthesis and C4 genes showed similar induction patterns, but C4 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 C4 genes. Light responsive elements, especially G-, E- and GT-boxes were over-represented in accessible chromatin around C4 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 C4 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.