C 4 gene induction during de-etiolation evolved through changes in cis to allow integration with ancestral C 3 gene regulatory networks.

C 4 photosynthesis has evolved by repurposing enzymes found in C 3 plants. Compared with the ancestral C 3 state, accumulation of C 4 cycle proteins is enhanced. We used de-etiolation of C 4 Gynandropsis gynandra and C 3 Arabidopsis thaliana to understand this process. C 4 gene expression and chloroplast biogenesis in G. gynandra were tightly coordinated. Although C 3 and C 4 photosynthesis genes showed similar induction patterns, in G. gynandra , C 4 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 C 4 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 C 4 photosynthesis genes in C 4 leaves is enhanced because modifications in cis allowed integration into ancestral transcriptional networks.