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

Abstract

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.

Keywords: 13C labeling; C4 photosynthesis; CO2-concentrating shuttle; carbon flow; maize..

Fig. 1.

Time-course of mass distribution of metabolites involved in the shuttle concentrating CO₂ in the BSCs. The relative abundance of each isotopomer (m_n) for a given metabolite is represented; n is the number of ¹³C atoms incorporated. The graph for malate^a corresponds to the isotopomer distribution after correction for the inactive pool. The x-axis corresponds to the labeling time on a log₁₀ scale. Data are presented in Supplementary Table S6.

Fig. 2.

Overview of ¹³C labeling kinetics by k-means clustering (A) and corresponding ¹³C amounts (B) after correction for inactive pools and for labeling of malate and aspartate in the C4 positon and the C1–C3 positions. Gray lines show the ¹³C enrichment/¹³C amount (in natom ¹³C equivalents g⁻¹ FW) of individual metabolites, and black lines show average ¹³C enrichment of all metabolites in the cluster. The x-axis corresponds to the labeling time on a log₁₀ scale. Carbon position-dependent enrichments and ¹³C amount were separately calculated for the C4 position and C1–C3 positions of malate and aspartate (for further information about calculations, see Supplementary Tables S7 and S8). Data for other ¹³C enrichments and ¹³C amounts are provided in Supplementary Table S5 and S8 (with further information about calculations). The corrected ¹³C enrichment for SBP is shown in brown, and for malate and aspartate in blue and red, respectively. In (B), metabolites are clustered based on enrichment, not the amount of ¹³C in the metabolite. Due to a large amount of ¹³C in sucrose (B, last graph, shown in darker gray), an additional graph for this metabolite is included as an insert.

Fig. 3.

Comparison of ¹³C enrichment (%) of photorespiratory pathway and CBC intermediates in maize at ambient O₂ and Arabidopsis thaliana at ambient and low O₂. The x-axis corresponds to the labeling time on a log₁₀ scale. Values are means ±SD (n=3–13). Data are presented in Supplementary Tables S5 and S10.

Fig. 4.

Distribution of isotopomers between BSCs and MCs. Labeling was performed for 3 min. The number associated with the compounds represents the number of ¹³C incorporated in the molecule. The y-axis shows the percentage of that isotopomer in the BSCs (black) and MCs (gray). The dotted red line indicates 50%. Data are provided in Supplementary Table S12.