Evolution of gene regulatory network of C4 photosynthesis in the genus Flaveria reveals the evolutionary status of C3-C4 intermediate species

C4 photosynthesis evolved from ancestral C3 photosynthesis by recruiting pre-existing genes to fulfill new functions. The enzymes and transporters required for the C4 metabolic pathway have been intensively studied and well documented; however, the transcription factors (TFs) that regulate these C4 metabolic genes are not yet well understood. In particular, how the TF regulatory network of C4 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 C4 photosynthesis: C3 (F. robusta), type I C3-C4 (F. sonorensis), type II C3-C4 (F. ramosissima), and C4 (F. trinervia). Our results show that more than half of the co-regulatory relationships between TFs and core C4 metabolic genes are species specific. The counterparts of the C4 genes in C3 species were already co-regulated with photosynthesis-related genes, whereas the required TFs for C4 photosynthesis were recruited later. The TFs involved in C4 photosynthesis were widely recruited in the type I C3-C4 species; nevertheless, type II C3-C4 species showed a divergent GRN from C4 species. In line with these findings, a 13CO2 pulse-labeling experiment showed that the CO2 initially fixed into C4 acid was not directly released to the Calvin-Benson-Bassham cycle in the type II C3-C4 species. Therefore, our study uncovered dynamic changes in C4 genes and TF co-regulation during the evolutionary process; furthermore, we showed that the metabolic pathway of the type II C3-C4 species F. ramosissima represents an alternative evolutionary solution to the ammonia imbalance in C3-C4 intermediate species.