Increased α-ketoglutarate as a missing link from the C3-C4 intermediate state to C4 photosynthesis in the genus Flaveria

As a complex trait, C4 photosynthesis has multiple independent origins in evolution. Phylogenetic evidence and theoretical analysis suggest that C2 photosynthesis, which is driven by glycine decarboxylation in the bundle sheath cell, may function as a bridge from C3 towards C4 photosynthesis. However, the exact molecular mechanism underlying the transition between C2 photosynthesis towards C4 photosynthesis remains elusive. Here, we provide multiple evidence suggesting a role of higher -ketoglutarate (AKG) concentration during this transition. Metabolomic data of 12 Flaveria species, including multiple photosynthetic types, show that AKG concentration initially increases in the C3-C4 intermediate with a further increase in C4 species. Petiole feeding of AKG increased the concentrations of C4 related metabolites in C3-C4 and C4 species but not the activity of C4 related enzymes. Sequence analysis shows that glutamate synthase (Fd-GOGAT), which catalyzes the generation of glutamate using AKG, was under strong positive selection during the evolution of C4 photosynthesis. Simulations with a constraint-based model for C3-C4 intermediate further show that decreasing the activity of Fd-GOGAT facilitates the transition from a C2-dominant to a C4-dominant CO2 concentrating mechanisms. All these provide an insight into the mechanistic switch from C3-C4 intermediate to C4 photosynthesis.