Metabolite pools and carbon flow during C4 photosynthesis in maize: 13CO2 labeling kinetics and cell type fractionation

Worldwide efforts to engineer C₄ photosynthesis into C₃ crops require a deep understanding of how this complex pathway operates. CO₂ is incorporated into four-carbon metabolites in the mesophyll, which move to the bundle sheath where they are decarboxylated to concentrate CO₂ around RuBisCO. We performed dynamic ¹³CO₂ labeling in maize to analyze C flow in C₄ photosynthesis. The overall labeling kinetics reflected the topology of C₄ photosynthesis. Analyses of cell-specific labeling patterns after fractionation to enrich bundle sheath and mesophyll cells revealed concentration gradients to drive intercellular diffusion of malate, but not pyruvate, in the major CO₂-concentrating shuttle. They also revealed intercellular concentration gradients of aspartate, alanine, and phosphenolpyruvate to drive a second phosphoenolpyruvate carboxykinase (PEPCK)-type shuttle, which carries 10-14% of the carbon into the bundle sheath. Gradients also exist to drive intercellular exchange of 3-phosphoglycerate and triose-phosphate. There is rapid carbon exchange between the Calvin-Benson cycle and the CO₂-concentrating shuttle, equivalent to ~10% of carbon gain. In contrast, very little C leaks from the large pools of metabolites in the C concentration shuttle into respiratory metabolism. We postulate that the presence of multiple shuttles, alongside carbon transfer between them and the Calvin-Benson cycle, confers great flexibility in C₄ photosynthesis.