C ₄ photosynthetic pathway fluxes in transgenic rice plants

Most land plants photosynthesize using the ancestral C ₃ pathway, in which ribulose bisphosphate carboxylase/oxygenase (Rubisco) fixes CO ₂ into 3-carbon acids in mesophyll cells. The derived C ₄ pathway, in which a carbon concentrating mechanism operates in the context of specialized leaf anatomy, is more efficient than the C ₃ pathway. Introduction of the C ₄ pathway into the C ₃ crop rice ( Oryza sativa ) could increase yield by 50% and expression of five C ₄ enzymes from maize previously led to flux through the first step in transgenic rice. However, there was no evidence for flux later in the cycle. Here we developed new transgenic lines and novel protocols to detect C ₄ cycle activity against a background of C ₃ photosynthesis and leaf anatomy. Significantly, we demonstrate that the three core C ₄ reactions and CO ₂ refixation are operating in transgenic C ₃ plants, establishing the in vivo flux framework needed to progress towards a functional carbon-concentrating mechanism.