Upregulation and Cell Specificity of C4 Genes Are Derived from Ancestral C3 Gene Regulatory Networks

The efficient C4 pathway is based on strong up-regulation of genes found in C3 plants, but also compartmentation of their expression into distinct cell-types such as the mesophyll and bundle sheath. Transcription factors associated with these phenomena have not been identified. To address this, we undertook genome-wide analysis of transcript accumulation, chromatin accessibility and transcription factor binding in C4 Gynandropsis gynandra. From these data, two models relating to the molecular evolution of C4 photosynthesis are proposed. First, increased expression of C4 genes is associated with increased binding by MYB-related transcription factors. Second, mesophyll specific expression is associated with binding of homeodomain transcription factors. Overall, we conclude that during evolution of the complex C4 trait, C4 cycle genes gain cis-elements that operate in the C3 leaf such that they become integrated into existing gene regulatory networks associated with cell specificity and photosynthesis.