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.