Abstract Introduction of a C 4 photosynthetic pathway into C 3 rice ( Oryza sativa ) requires installation of a biochemical pump that concentrates CO 2 at the site of carboxylation in modified bundle sheath cells. To investigate the feasibility of this, we generated a quadruple line that simultaneously expresses four of the core C 4 photosynthetic enzymes from the NADP-malic enzyme subtype, phospho enol pyruvate carboxylase ( Zm PEPC), NADP-malate dehydrogenase ( Zm NADP-MDH), NADP-malic enzyme ( Zm NADP-ME) and pyruvate phosphate dikinase ( Zm PPDK), in a cell-specific manner. This led to enhanced enzyme activity but was largely neutral in its effects on photosynthetic rate and growth. Measurements of the flux of 13 CO 2 through photosynthetic metabolism revealed a significant increase in the incorporation of 13 C into malate, consistent with increased fixation of 13 CO 2 via PEP carboxylase in lines expressing the maize PEPC enzyme. We also showed 13 C labelling of aspartate indicating additional 13 CO 2 fixation into oxaloacetate by PEPC and conversion to aspartate by the endogenous aspartate aminotransferase activity. However, there were no significant differences in labelling of 3-phosphoglycerate (3PGA) or phospho enol pyruvate (PEP) indicating limited carbon flux through C 4 enzymes into the Calvin-Benson cycle. Crossing the quadruple line with a line with reduced glycine decarboxylase H-protein ( Os GDCH) abundance led to a photosynthetic phenotype characteristic of the reduced Os GDCH line and higher labelling of malate, aspartate and citrate. While Kranz anatomy or other anatomical modifications have not yet been installed in these plants to enable a fully functional C 4 cycle, these results demonstrate for the first-time flux through the carboxylation phase of C 4 metabolism in transgenic rice containing the key metabolic steps in the C 4 pathway.

A partial C4photosynthetic biochemical pathway in rice