C₄ gene induction during de-etiolation evolved through changes in cis to allow integration with ancestral C₃ gene regulatory networks

C₄ photosynthesis has evolved by repurposing enzymes found in C₃ plants. Compared with the ancestral C₃ state, accumulation of C₄ cycle proteins is enhanced. We used de-etiolation of C₄ Gynandropsis gynandra and C₃ Arabidopsis thaliana to understand this process. C₄ gene expression and chloroplast biogenesis in G. gynandra were tightly coordinated. Although C₃ and C₄ photosynthesis genes showed similar induction patterns, in G. gynandra, C₄ genes were more strongly induced than orthologs from A. thaliana. In vivo binding of TGA and homeodomain as well as light-responsive elements such as G- and I-box motifs were associated with the rapid increase in transcripts of C₄ genes. Deletion analysis confirmed that regions containing G- and I-boxes were necessary for high expression. The data support a model in which accumulation of transcripts derived from C₄ photosynthesis genes in C₄ leaves is enhanced because modifications in cis allowed integration into ancestral transcriptional networks.