C4 photosynthetic pathway fluxes in transgenic rice plants

Most land plants photosynthesize using the C 3 pathway, in which ribulose bisphosphate carboxylase/oxygenase (Rubisco) fixes CO 2 into 3-carbon acids. The C 4 pathway, a biochemical CO 2 -concentrating mechanism that operates in the context of specialized leaf anatomy to concentrate CO 2 around Rubisco, is more efficient. Introduction of the C 4 pathway into the C 3 crop rice could increase yield by 50%. Expression of five C 4 enzymes in transgenic rice previously led to flux through the first step. However, there was no evidence for flux later in the cycle. Here we developed new transgenic rice lines and novel protocols to detect C 4 cycle activity: CO 2 fixation into C 4 acids by carboxylation of a C 3 compound, decarboxylation, refixation of CO 2 by Rubisco, and regeneration of the C 3 donor. We demonstrate that these four core C 4 reactions are operating in rice, establishing the in vivo flux framework needed to progress towards a functional carbon-concentrating mechanism.