Evolution of gene regulatory network of C 4 photosynthesis in the genus Flaveria reveals the evolutionary status of C 3 -C 4 intermediate species

C photosynthesis evolved from ancestral C photosynthesis by recruiting pre-existing genes to fulfill new functions. The enzymes and transporters required for the C metabolic pathway have been intensively studied and well documented; however, the transcription factors (TFs) that regulate these C metabolic genes are not yet well understood. In particular, how the TF regulatory network of C metabolic genes was rewired during the evolutionary process is unclear. Here, we constructed gene regulatory networks (GRNs) for four closely evolutionarily related species from the genus Flaveria, which represent four different evolutionary stages of C photosynthesis: C (F. robusta), type I C -C (F. sonorensis), type II C -C (F. ramosissima), and C (F. trinervia). Our results show that more than half of the co-regulatory relationships between TFs and core C metabolic genes are species specific. The counterparts of the C genes in C species were already co-regulated with photosynthesis-related genes, whereas the required TFs for C photosynthesis were recruited later. The TFs involved in C photosynthesis were widely recruited in the type I C -C species; nevertheless, type II C -C species showed a divergent GRN from C species. In line with these findings, a CO pulse-labeling experiment showed that the CO initially fixed into C acid was not directly released to the Calvin-Benson-Bassham cycle in the type II C -C species. Therefore, our study uncovered dynamic changes in C genes and TF co-regulation during the evolutionary process; furthermore, we showed that the metabolic pathway of the type II C -C species F. ramosissima represents an alternative evolutionary solution to the ammonia imbalance in C -C intermediate species.