A partial C₄photosynthetic biochemical pathway in rice

AbstractIntroduction of a C4photosynthetic pathway into C3rice (Oryza sativa) requires installation of a biochemical pump that concentrates CO2at 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 C4photosynthetic enzymes from the NADP-malic enzyme subtype, phosphoenolpyruvate carboxylase (ZmPEPC), NADP-malate dehydrogenase (ZmNADP-MDH), NADP-malic enzyme (ZmNADP-ME) and pyruvate phosphate dikinase (ZmPPDK), 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 of13CO2through photosynthetic metabolism revealed a significant increase in the incorporation of13C into malate, consistent with increased fixation of13CO2via PEP carboxylase in lines expressing the maize PEPC enzyme. We also showed13C labelling of aspartate indicating additional13CO2fixation 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 phosphoenolpyruvate (PEP) indicating limited carbon flux through C4enzymes into the Calvin-Benson cycle. Crossing the quadruple line with a line with reduced glycine decarboxylase H-protein (OsGDCH) abundance led to a photosynthetic phenotype characteristic of the reducedOsGDCH 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 C4cycle, these results demonstrate for the first-time flux through the carboxylation phase of C4metabolism in transgenic rice containing the key metabolic steps in the C4pathway.