Initial Events during the Evolution of C4 Photosynthesis in C3 Species of Flaveria

The evolution of C-4 photosynthesis in many taxa involves the establishment of a two-celled photorespiratory CO2 pump, termed C-2 photosynthesis. How C-3 species evolved C-2 metabolism is critical to understanding the initial phases of C-4 plant evolution. To evaluate early events in C-4 evolution, we compared leaf anatomy, ultrastructure, and gas-exchange responses of closely related C-3 and C-2 species of Flaveria, a model genus for C-4 evolution. We hypothesized that Flaveria pringlei and Flaveria robusta, two C-3 species that are most closely related to the C-2 Flaveria species, would show rudimentary characteristics of C-2 physiology. Compared with less-related C-3 species, bundle sheath (BS) cells of F. pringlei and F. robusta had more mitochondria and chloroplasts, larger mitochondria, and proportionally more of these organelles located along the inner cell periphery. These patterns were similar, although generally less in magnitude, than those observed in the C-2 species Flaveria angustifolia and Flaveria sonorensis. In F. pringlei and F. robusta, the CO2 compensation point of photosynthesis was slightly lower than in the less-related C-3 species, indicating an increase in photosynthetic efficiency. This could occur because of enhanced refixation of photorespired CO2 by the centripetally positioned organelles in the BS cells. If the phylogenetic positions of F. pringlei and F. robusta reflect ancestral states, these results support a hypothesis that increased numbers of centripetally located organelles initiated a metabolic scavenging of photorespired CO2 within the BS. This could have facilitated the formation of a glycine shuttle between mesophyll and BS cells that characterizes C-2 photosynthesis.