Metabolite pools and carbon flow during C ₄ photosynthesis in maize: ¹³CO ₂ labeling kinetics and cell type fractionation

Abstract

Analysis of labeling kinetics, pool sizes, and concentration gradients of metabolites reveals the operation of multiple decarboxylation pathways and rapid movement of carbon between the Calvin–Benson cycle and the CO ₂-concentrating shuttles in maize.

Abstract

Worldwide efforts to engineer C ₄ photosynthesis into C ₃ crops require a deep understanding of how this complex pathway operates. CO ₂ is incorporated into four-carbon metabolites in the mesophyll, which move to the bundle sheath where they are decarboxylated to concentrate CO ₂ around RuBisCO. We performed dynamic ¹³CO ₂ labeling in maize to analyze C flow in C ₄ photosynthesis. The overall labeling kinetics reflected the topology of C ₄ 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 CO ₂-concentrating shuttle. They also revealed intercellular concentration gradients of aspartate, alanine, and phosph enolpyruvate to drive a second phospho enolpyruvate 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 CO ₂-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 C ₄ photosynthesis.