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Metabolic evidence for distinct pyruvate pools inside plant mitochondria

Abstract

The majority of the pyruvate inside plant mitochondria is either transported into the matrix from the cytosol via the mitochondria pyruvate carrier (MPC) or synthesized in the matrix by alanine aminotransferase (AlaAT) or NAD-malic enzyme (NAD-ME). Pyruvate from these origins could mix into a single pool in the matrix and contribute indistinguishably to respiration via the pyruvate dehydrogenase complex (PDC), or these molecules could maintain a degree of independence in metabolic regulation. Here we demonstrate that feeding isolated mitochondria with uniformly labelled 13C-pyruvate and unlabelled malate enables the assessment of pyruvate contribution from different sources to intermediate production in the tricarboxylic acid cycle. Imported pyruvate was the preferred source for citrate production even when the synthesis of NAD-ME-derived pyruvate was optimized. Genetic or pharmacological elimination of MPC activity removed this preference and allowed an equivalent amount of citrate to be generated from the pyruvate produced by NAD-ME. Increasing the mitochondrial pyruvate pool size by exogenous addition affected only metabolites from pyruvate transported by MPC, whereas depleting the pyruvate pool size by transamination to alanine affected only metabolic products derived from NAD-ME. PDC was more membrane-associated than AlaAT and NAD-ME, suggesting that the physical organization of metabolic machinery may influence metabolic rates. Together, these data reveal that the respiratory substrate supply in plants involves distinct pyruvate pools inside the matrix that can be flexibly mixed on the basis of the rate of pyruvate transport from the cytosol. These pools are independently regulated and contribute differentially to organic acid export from plant mitochondria.

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Fig. 1: The use of imported pyruvate for the TCA cycle is preferred to the use of ME-derived pyruvate by Col-0.
Fig. 2: The loss of MPC1 changed the pyruvate usage pattern for generating TCA cycle intermediates.
Fig. 3: The rate of accumulation of total extra-mitochondrial ME-derived metabolites in mitochondrial feeding experiments.
Fig. 4: The impact of the removal of pyruvate by AlaAT on citrate production in Col-0, mpc1 and mpc1/gMPC1 mitochondria.
Fig. 5: The degree of mitochondrial membrane association of TCA cycle enzymes, NAD-ME, AlaAT and PDC in Arabidopsis.
Fig. 6: Schematic presentation of the source and sink of mitochondria pyruvate pools via MPC (blue) and NAD-ME (grey).

Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information files. Source data are provided with this paper.

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Acknowledgements

This work is supported by the Australian Research Council Centre of Excellence in Plant Energy Biology (grant nos CE140100008 and FL200100057), and X.H.L. is a Forrest Scholar supported by the Forrest Research Foundation and a receiver of Research Training Program scholarships from the Department of Education, Skills and Employment in the Australian government. Peptide quantitation in this work was performed as a service by E. Ströher from the WA Proteomics Facility as a node of Proteomics Australia, supported by infrastructure funding from the Western Australian state government in partnership with Bioplatforms Australia under the Commonwealth Government National Collaborative Research Infrastructure Strategy.

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X.H.L., C.-P.L. and A.H.M. designed the research. X.H.L. performed most of the experiments and data analysis. C.-P.L. assisted with some of the MS and data analysis. D.M. performed the interactome analyses. X.H.L., C.-P.L. and A.H.M. wrote the paper.

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Correspondence to A. Harvey Millar.

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The authors declare no competing interests.

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Nature Plants thanks Veronica Maurino, Alisdair Fernie, and Markus Schwarzlander for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 13C3-Pyruvate and malate feeding to isolated mitochondria of Col-0, me1.me2 and me1.me2.mpc1.

(a) Time courses of metabolite concentrations in the extra-mitochondrial space of isolated mitochondria from Col-0 incubated with various concentrations of 13C3-pyruvate and 500 µM malate (n = 3). (b) Time courses of metabolite concentrations in the extra-mitochondrial space of isolated mitochondria from Col-0, me1.me2, me1.me2.mpc1 incubated with 500 µM 13C3-pyruvate and 500 µM malate (n = 4). All experiments were conducted in the presence of ADP at pH 6.4 to initiate substrate uptake and consumption by both pathways - the MPC pathway (labelled metabolites) and the NAD-ME pathway (unlabeled metabolites). Metabolic reaction was stopped by centrifugation through a single silicon oil layer by which the mitochondrial pellet was separated from the extra-mitochondrial medium. Unused substrate and exported products in the extra-mitochondrial medium were quantified using LC-SRM-MS. Each data point represents averaged value from three or more biological replicates with error bars indicating standard error. Significant differences between mutants and wildtype are denoted by coloured asterisks based on two-sided Student’s t-tests (*, p < 0.05, See Source data Extended data Fig. 1 for exact p-values).

Source data

Extended Data Fig. 2 13C3-Pyruvate and malate feeding to isolated mitochondria of Col-0, mpc1 and mpc1/gMPC1.

Time courses of metabolite concentrations in the extra-mitochondrial space of isolated mitochondria incubated with 500 µM 13C3-pyruvate and 500 µM malate via MPC pathway (a) and via NAD-ME pathway (b). All experiments were conducted in the presence of ADP at pH 6.4 to initiate substrate uptake and consumption by both pathways. Metabolic reaction was stopped by centrifugation through a single silicon oil layer in which the mitochondrial pellet was separated from the extra-mitochondrial medium. Unused substrate and exported products in the extra-mitochondrial medium were quantified using LC-SRM-MS. Each data point represents averaged value from three biological replicates with error bars indicating standard error (n = 3). Significant differences between mpc1, Col-0 and mpc1/gMPC1 are denoted by asterisks based on two-sided Student’s t-tests (*, p < 0.05, See Source data Extended data Fig. 2 for exact p-values).

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Extended Data Fig. 3 Pyruvate and 13C4-malate feeding to isolated mitochondria of Col-0, mpc1 and mpc1/gMPC1.

Time courses of metabolite concentrations in the extra-mitochondrial space of isolated mitochondria incubated with 500 µM pyruvate and 500 µM 13C4- via MPC pathway (a) and via NAD-ME pathway (b). All experiments were conducted in the presence of ADP at pH 6.4 to initiate substrate uptake and consumption by both pathways. Metabolic reaction was stopped by centrifugation through a single silicon oil layer in which the mitochondrial pellet was separated from the extra-mitochondrial medium. Unused substrates and exported products in the extra-mitochondrial medium were quantified using LC-SRM-MS. Each data point represents averaged value from three biological replicates with error bars indicating standard error (n = 3). Significant differences between mpc1, Col-0 and mpc1/gMPC1 are denoted by asterisks based on Student’s t-tests (*, p < 0.05) (c) Bar graphs show the rates calculated from time course values of metabolite concentration recorded in the extra-mitochondrial space after varying incubation periods. Each bar represents averaged value from three or more replicates represented by data points. Significant differences between controls and treatments are denoted by asterisks based on two-sided Student’s t-tests (*, p < 0.05, See Source data Extended data Fig. 3 for exact p-values).

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Extended Data Fig. 4 The total amount and the rate of metabolites exported from mitochondria that were made via the NAD-ME pathway.

The total amount of NAD-ME derived metabolites was calculated from time course experiments of either pyruvate and 13C4-malate feeding (a, including unlabeled citrate, 2-oxoglutarate, succinate, pyruvate) or pyruvate and 13C4-malate feeding (b, including 13C6-citrate, 13C5-2-oxoglutarate, 13C4-succinate, 13C3-pyruvate) to isolated mitochondria of Col-0, mpc1 and mpc1/gMPC1. All experiments were performed in the presence of ADP at pH 6.4 to initiate substrate uptake and consumption by both pathways. Metabolic reaction was stopped by centrifugation through a single silicon oil layer in which the mitochondrial pellet was separated from the extra-mitochondrial medium. Unused substrates and exported products in the extra-mitochondrial medium were quantified using LC-SRM-MS. Each data point represents averaged value from three biological replicates with error bars indicating standard error (n = 3). (c) Bar graphs show the calculated export rate of all metabolites combined which were made from ME-derived pyruvate after 5 minutes feeding the mitochondria with pyruvate and 13C4-malate. Each stacked bar represents averaged value of the indicated metabolite from three or more replicates. Data points represented the total amount of ME-derived metabolites exported in independent replicates.

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Extended Data Fig. 5 13C3-Pyruvate and malate feeding to isolated mitochondria of Col-0, mpc1 and mpc1/gMPC1 with or without the addition of glutamate.

Time courses of metabolite concentrations in the extra-mitochondrial space of isolated mitochondria incubated with 500 µM 13C3-pyruvate and 500 µM malate with or without the addition of glutamate. All experiments were conducted in the presence of ADP at pH 6.4 to initiate substrate uptake and consumption via both MPC and NAD-ME pathways. Metabolic reaction was stopped by centrifugation through a single silicon oil layer in which the mitochondrial pellet was separated from the extra-mitochondrial medium. Unused substrate and exported products in the extra-mitochondrial medium were quantified using LC-SRM-MS. Line graphs show the amount of 13C2-citrate (a), citrate (b) and pyruvate (c) during 5-minute incubation. Each data point represents averaged value from three biological replicates with error bars indicating standard error (n = 3). Significant differences between controls (straight lines) and treatments (dotted lines) are denoted by coloured asterisks based on two-sided Student’s t-tests (*, p < 0.05, See Source data Extended data Fig. 5 for exact p-values).

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Le, X.H., Lee, C.P., Monachello, D. et al. Metabolic evidence for distinct pyruvate pools inside plant mitochondria. Nat. Plants 8, 694–705 (2022). https://doi.org/10.1038/s41477-022-01165-3

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