Biochemical mechanisms driving rapid fluxes in C4 photosynthesis

Sufficient flux through pathways is critical to their function within the metabolic network. A high flux cycle called C4 photosynthesis evolved in some plants to combat the slow speed and low specificity of the plants carbon fixation enzyme Rubisco. In these plants, flux through the C4 photosynthetic cycle eclipses flux through Rubisco making it the highest flux in planta. Engineering efforts are underway to transfer this high flux pathway to crops using the ancestral C3 photosynthetic type, such as rice, to improve yield and increase water and nitrogen use efficiencies. Most of the enzymatic and transport steps of the C4 cycle have been characterized. However, well-established features, including altered chloroplast structure, diversity in transfer acids, and the underlying mechanisms driving high flux through this biochemical CO2 pump remain unexplained. We identify three drivers of high fluxes through alternative decarboxylation subsystems in C4 plants: For NAD- and NADP-based malic enzymes, kinetic modeling demonstrates that the oxidation state of mitochondria and chloroplasts, respectively, drive rapid decarboxylation. For phosphoenolpyruvate carboxykinase, RNA-seq and enzyme activity measurements suggest the presence of a new C4 cycle enzyme driving fast decarboxylation. These observations not only suggest a hitherto unknown C4 cycle but also provide mechanistic explanations for decades-old anatomical and biochemical observations.