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

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 CO2-concentrating shuttles in maize.

aminotransferase (AspAT) to aspartate, which moves to the BSC mitochondria where it is converted by AspAT to OAA, reduced by NAD-MDH to malate and decarboxylated by NAD-ME to yield CO2, pyruvate and NADH. The NADH is consumed for OAA reduction. The pyruvate is converted by alanine aminotransferase (AlaAT) to alanine, which moves back to the MC, where it is deaminated by AlaAT to pyruvate that is used by PPDK to regenerate PEP. In the PEPCK subtype (C), some of the OAA is converted to aspartate, which moves to the BSC cytosol, is deaminated to OAA and converted to CO2 and PEP by PEPCK. PEP then returns to the MC.
The remainder of the OAA is reduced to malate by NADP-MDH and moves to the BSC mitochondria where it is decarboxylated by NAD-ME, yielding CO2, NADH and pyruvate.
Pyruvate is converted to alanine which returns to MC and is deaminated to pyruvate, which is used to regenerate PEP. This NAD-ME and PEPCK variants additionally require intercellular movement of amino acids to maintain the NH3 balance. In addition to CO2, some of these shuttles transfer energy to the BSC; in the NADP-ME subtype NADPH is transferred allowing a decrease in photosystem II activity in the BSC chloroplasts, and in PEPCK subtypes the NADH that is released in the BSC mitochondria can be utilized to generate ATP. In some C4 plants, especially NADP-ME subtypes, another intercellular shuttle transfers NADPH and ATP from the MC to the BSC; 3PGA moves from the BSC to the MC and is reduced by phosphoglycerate kinase and NADP-glyceraldehyde-3-phosphate dehydrogenase to triose phosphates, which then move back to the BSC (shown in A). For a given compound, amounts of all isotopomers were summed. Amounts are expressed as nmol g -1 FW except if an asterisk is present (µmol g -1 FW). The x-axis corresponds to the order in which samples were harvested during the day, spanning over a time period of 9 hour and half. For a better comprehension the times of sampling have been numbered and the corresponding average time of sampling (h:min after beginning of the light period) and the 13 C labeling duration are indicated. n.d. stands for not determined. PEP, phosphoenolpyruvate;

Maier
3PGA, 3-phosphoglyceric acid; DHAP, dihydroxyacetone phosphate; FBP, fructose-1,6bisphosphate; F6P, fructose-6-phosphate; SBP, sedoheptulose-1,7-bisphosphate; S7P, sedoheptulose-7-phosphate; R5P, ribose-5-phosphate; Ru5P+Xu5P, ribulose-5-phosphate + xylulose-5-phosphate; RuBP, ribulose-1,5-bisphosphate; G6P, glucose-6-phosphate; G1P, glucose-1-phosphate; ADPG, ADP-glucose; UDPG, UDP-glucose; 2PG, 2-phosphoglycolate; 2OG, 2-oxoglutarate. The amounts of the unlabeled form and each 13 C-isotopomer are provided in Supplementary Table S3, and the summed amounts in Supplementary Table S4.  Supplementary   Table S5. Figure S5. Time-course of mass distribution of metabolites from CBC, starch and sucrose pathways. The relative abundance of each isotopomer (mn) for a given metabolite is represented; n is the number of 13 C atoms incorporated. The graph for SBP 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. Figure S6. Scheme of positional carbon incorporation in compounds from the CO 2 shuttle. (A) Carbon fixation in the CCS. 13 CO2 is incorporated into the C4 positon of OAA and passed on to the C4 positons of malate and aspartate, and 13 C is released from the C4 position by decarboxylation reactions in the BSC. Consequently no label is introduced into the 3-carbon products of the decarboxylation reaction like pyruvate and alanine (B) Carbon transfer between 3PGA and the CCS. Once 13 C is incorporated in the CBC label is randomized due to rapid turnover of the CBC. 3PGA moves to the MC and exchanges via 2PGA with the PEP formed by PPDK, leading to 13 C being incorporated into positions C1-3 of OAA, malate and aspartate, and thence into pyruvate and alanine.

Supplementary Figure S7. Time-course of mass distribution of metabolites in malate and
aspartate during a chase. After 60 min labeling (time 0), 13 CO2 was replaced by 12 CO2 and chase performed for 5 and 20 min. The relative abundance of each isotopomer (mn) for a given metabolite is represented; n is the number of 13 C 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. Figure S8. Regression plots of 13 C amounts calculated with two approaches. 13 C amounts (natom 13 C equivalents g -1 FW) in metabolites were estimated by multiplying the number of 13 C atoms per isotopomer by the corresponding isotopomer amount, and then summing the results. In approach A, isotopomer amounts were calculated by multiplying isotopomer abundance by the average pool size of the intermediate (or the active pool size as determined at 60 min for malate). Approach B used the isotopomer amounts determined in each sample. Amounts for malate, aspartate, malate minus C4 position and aspartate minus C4 position were calculated with both approaches until time point 180 sec