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.