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_(4) photosynthesis evolved from ancestral C_(3) photosynthesis by recruiting pre-existing genes to fulfill new functions.The enzymes and transporters required for the C_(4) metabolic pathway have been intensively studied and well documented;however,the transcription factors(TFs)that regulate these C_(4) metabolic genes are not yet well understood.In particular,how the TF regulatory network of C_(4) 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_(4) photosynthesis:C_(3)(F.robusta),type Ⅰ C_(3)-C_(4)(F.sonorensis),type Ⅱ C_(3)-C_(4)(F.ramosissima),and C_(4)(F.trinervia).Our results show that more than half of the co-regulatory relationships between TFs and core C_(4) metabolic genes are species specific.The counterparts of the C_(4) genes in C_(3) species were already co-regulated with photosynthesis-related genes,whereas the required TFs for C_(4) photosynthesis were recruited later.The TFs involved in C_(4) photosynthesis were widely recruited in the type Ⅰ C_(3)-C_(4) species;nevertheless,type Ⅱ C_(3)-C_(4) species showed a divergent GRN from C_(4) species.In line with these findings,a ^(13)CO_(2) pulse-labeling experiment showed that the CO_(2) initially fixed into C_(4) acid was not directly released to the Calvin–Benson–Bassham cycle in the type Ⅱ C_(3)-C_(4) species.Therefore,our study uncovered dynamic changes in C_(4) genes and TF co-regulation during the evolutionary process;furthermore,we showed that the metabolic pathway of the type Ⅱ C_(3)-C_(4) species F.ramosissima represents an alternative evolutionary solution to the ammonia imbalance in C_(3)-C_(4) intermediate species.