ABSTRACT As a complex trait, C 4 photosynthesis has multiple independent origins in evolution. Phylogenetic evidence and theoretical analysis suggest that C 2 photosynthesis, which is driven by glycine decarboxylation in the bundle sheath cell, may function as a bridge from C 3 towards C 4 photosynthesis. However, the exact molecular mechanism underlying the transition between C 2 photosynthesis towards C 4 photosynthesis remains elusive. Here, we provide multiple evidence suggesting a role of higher α-ketoglutarate (AKG) concentration during this transition. Metabolomic data of 12 Flaveria species, including multiple photosynthetic types, show that AKG concentration initially increases in the C 3 -C 4 intermediate with a further increase in C 4 species. Petiole feeding of AKG increased the concentrations of C 4 related metabolites in C 3 -C 4 and C 4 species but not the activity of C 4 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 C 4 photosynthesis. Simulations with a constraint-based model for C 3 -C 4 intermediate further show that decreasing the activity of Fd-GOGAT facilitates the transition from a C 2 -dominant to a C 4 -dominant CO 2 concentrating mechanisms. All these provide an insight into the mechanistic switch from C 3 -C 4 intermediate to C 4 photosynthesis.

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