The role of photorespiration during the evolution of C4 photosynthesis in the genus Flaveria

C-4 photosynthesis represents a most remarkable case of convergent evolution of a complex trait, which includes the reprogramming of the expression patterns of thousands of genes. Anatomical, physiological, and phylogenetic analyses as well as computational modeling indicate that the establishment of a photorespiratory carbon pump (termed C-2 photosynthesis) is a prerequisite for the evolution of C-4. However, a mechanistic model explaining the tight connection between the evolution of C-4 and C-2 photosynthesis is currently lacking. Here we address this question through comparative transcriptomic and biochemical analyses of closely related C-3, C-3-C-4, and C-4 species, combined with Flux Balance Analysis constrained through a mechanistic model of carbon fixation. We show that C-2 photosynthesis creates a misbalance in nitrogen metabolism between bundle sheath and mesophyll cells. Rebalancing nitrogen metabolism requires an anaplerotic carbon cycle that resembles at least parts of a basic C-4 cycle. Our findings thus show how C-2 photosynthesis represents a pre-adaptation for the C-4 system, where the evolution of the C-2 system establishes important C-4 components as a side effect.