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Metabolic evidence for distinct pyruvate pools inside plant mitochondria

Nature Plants volume 8, pages 694–705 (2022)Cite this article

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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).

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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.

References

  1. Millar, A. H., Small, I. D., Day, D. A. & Whelan, J. Mitochondrial biogenesis and function in Arabidopsis. Arabidopsis Book 6, e0111 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Le, X. H., Lee, C.-P. & Millar, A. H. The mitochondrial pyruvate carrier (MPC) complex mediates one of three pyruvate-supplying pathways that sustain Arabidopsis respiratory metabolism. Plant Cell 33, 2776–2793 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Dieuaide-Noubhani, M., Raffard, G., Canioni, P., Pradet, A. & Raymond, P. Quantification of compartmented metabolic fluxes in maize root tips using isotope distribution from 13C- or 14C-labeled glucose. J. Biol. Chem. 270, 13147–13159 (1995).

    Article  CAS  PubMed  Google Scholar 

  4. Edwards, S., Nguyen, B.-T., Do, B. & Roberts, J. K. M. Contribution of malic enzyme, pyruvate kinase, phosphoenolpyruvate carboxylase, and the Krebs cycle to respiration and biosynthesis and to intracellular pH regulation during hypoxia in maize root tips observed by nuclear magnetic resonance imaging and gas chromatography–mass spectrometry. Plant Physiol. 116, 1073–1081 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Tcherkez, G. et al. In folio respiratory fluxomics revealed by 13C isotopic labeling and H/D isotope effects highlight the noncyclic nature of the tricarboxylic acid “cycle” in illuminated leaves. Plant Physiol. 151, 620–630 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Day, D. A. & Hanson, J. B. Pyruvate and malate transport and oxidation in corn mitochondria. Plant Physiol. 59, 630–635 (1977).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Tronconi, M. A., Maurino, V. G., Andreo, C. S. & Drincovich, M. F. Three different and tissue-specific NAD-malic enzymes generated by alternative subunit association in Arabidopsis thaliana. J. Biol. Chem. 285, 11870–11879 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Tronconi, M. A. et al. Arabidopsis NAD-malic enzyme functions as a homodimer and heterodimer and has a major impact on nocturnal metabolism. Plant Physiol. 146, 1540–1552 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Winkel, B. S. Metabolic channeling in plants. Annu. Rev. Plant Biol. 55, 85–107 (2004).

    Article  CAS  PubMed  Google Scholar 

  10. Srere, P. A. & Mosbach, K. Metabolic compartmentation: symbiotic, organellar, multienzymic, and microenvironmental. Annu. Rev. Microbiol. 28, 61–83 (1974).

    Article  CAS  PubMed  Google Scholar 

  11. Sweetlove, L. J. & Fernie, A. R. The role of dynamic enzyme assemblies and substrate channelling in metabolic regulation. Nat. Commun. 9, 2136 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Srere, P. A. The metabolon. Trends Biochem. Sci. 10, 109–110 (1985).

    Article  Google Scholar 

  13. Morgunov, I. & Srere, P. A. Interaction between citrate synthase and malate dehydrogenase: substrate channeling of oxaloacetate. J. Biol. Chem. 273, 29540–29544 (1998).

    Article  CAS  PubMed  Google Scholar 

  14. Vélot, C., Mixon, M. B., Teige, M. & Srere, P. A. Model of a quinary structure between Krebs TCA cycle enzymes: a model for the metabolon. Biochemistry 36, 14271–14276 (1997).

    Article  PubMed  Google Scholar 

  15. Wu, F. & Minteer, S. Krebs cycle metabolon: structural evidence of substrate channeling revealed by cross‐linking and mass spectrometry. Angew. Chem. Int. Ed. Engl. 54, 1851–1854 (2015).

    Article  CAS  PubMed  Google Scholar 

  16. Robinson, J. B. Jr., Inman, L., Sumegi, B. & Srere, P. A. Further characterization of the Krebs tricarboxylic acid cycle metabolon. J. Biol. Chem. 262, 1786–1790 (1987).

    Article  CAS  PubMed  Google Scholar 

  17. Zhao, H. et al. Quantitative analysis of purine nucleotides indicates that purinosomes increase de novo purine biosynthesis. J. Biol. Chem. 290, 6705–6713 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Castellana, M. et al. Enzyme clustering accelerates processing of intermediates through metabolic channeling. Nat. Biotechnol. 32, 1011–1018 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sonnewald, U. et al. NMR spectroscopic studies of 13C acetate and 13C glucose metabolism in neocortical astrocytes: evidence for mitochondrial heterogeneity. Dev. Neurosci. 15, 351–358 (1993).

    Article  CAS  PubMed  Google Scholar 

  20. Cruz, F. et al. Intracellular compartmentation of pyruvate in primary cultures of cortical neurons as detected by 13C NMR spectroscopy with multiple 13C labels. J. Neurosci. Res. 66, 771–781 (2001).

    Article  CAS  PubMed  Google Scholar 

  21. Waagepetersen, H. S., Sonnewald, U., Larsson, O. M. & Schousboe, A. Multiple compartments with different metabolic characteristics are involved in biosynthesis of intracellular and released glutamine and citrate in astrocytes. Glia 35, 246–252 (2001).

    Article  CAS  PubMed  Google Scholar 

  22. Lee, C. P. et al. The versatility of plant organic acid metabolism in leaves is underpinned by mitochondrial malate–citrate exchange. Plant Cell 33, 3700–3720 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Oliver, D. J. & Walker, G. H. Characterization of the transport of oxaloacetate by pea leaf mitochondria. Plant Physiol. 76, 409–413 (1984).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zhang, Y. et al. Protein–protein interactions and metabolite channelling in the plant tricarboxylic acid cycle. Nat. Commun. 8, 15212 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Arabidopsis Interactome Mapping Consortium Evidence for network evolution in an Arabidopsis interactome map. Science 333, 601–607 (2011).

    Article  PubMed Central  CAS  Google Scholar 

  26. Jin, K. et al. Yeast mitochondrial protein–protein interactions reveal diverse complexes and disease-relevant functional relationships. J. Proteome Res. 14, 1220–1237 (2015).

    Article  CAS  PubMed  Google Scholar 

  27. Linden, A. et al. A cross-linking mass spectrometry approach defines protein interactions in yeast mitochondria. Mol. Cell. Proteom. 19, 1161–1178 (2020).

    Article  CAS  Google Scholar 

  28. Go, C. D. et al. A proximity-dependent biotinylation map of a human cell. Nature 595, 120–124 (2021).

    Article  CAS  PubMed  Google Scholar 

  29. D’Souza, S. F. & Srere, P. A. Cross-linking of mitochondrial matrix proteins in situ. Biochim. Biophys. Acta 724, 40–51 (1983).

    Article  PubMed  Google Scholar 

  30. Liu, F., Lössl, P., Rabbitts, B. M., Balaban, R. S. & Heck, A. J. R. The interactome of intact mitochondria by cross-linking mass spectrometry provides evidence for coexisting respiratory supercomplexes. Mol. Cell. Proteom. 17, 216–232 (2018).

    Article  CAS  Google Scholar 

  31. Schweppe, D. K. et al. Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry. Proc. Natl. Acad. Sci. USA 114, 1732–1737 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. González-Fuente, M. et al. EffectorK, a comprehensive resource to mine for Ralstonia, Xanthomonas, and other published effector interactors in the Arabidopsis proteome. Mol. Plant Pathol. 21, 1257–1270 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Romero-Barrios, N. et al. Advanced cataloging of Lysine-63 polyubiquitin networks by genomic, interactome, and sensor-based proteomic analyses. Plant Cell 32, 123–138 (2020).

    Article  CAS  PubMed  Google Scholar 

  34. Nagampalli, R. et al. Human mitochondrial pyruvate carrier 2 as an autonomous membrane transporter. Sci. Rep. 8, 3510 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Halestrap, A. P. The mechanism of the inhibition of the mitochondrial pyruvate transportater by alpha-cyanocinnamate derivatives. Biochem. J. 156, 181–183 (1976).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Paradies, G. Interaction of α-cyano[14C]cinnamate with the mitochondrial pyruvate translocator. Biochim. Biophys. Acta 766, 446–450 (1984).

    Article  CAS  PubMed  Google Scholar 

  37. Hildyard, J. C., Ammala, C., Dukes, I. D., Thomson, S. A. & Halestrap, A. P. Identification and characterisation of a new class of highly specific and potent inhibitors of the mitochondrial pyruvate carrier. Biochim. Biophys. Acta 1707, 221–230 (2005).

    Article  CAS  PubMed  Google Scholar 

  38. Wheeldon, I. et al. Substrate channelling as an approach to cascade reactions. Nat. Chem. 8, 299–309 (2016).

    Article  CAS  PubMed  Google Scholar 

  39. Lüderitz, R. & Klemme, J. H. [Isolation and characterization of a membrane-bound pyruvate dehydrogenase complex from the phototrophic bacterium Rhodospirillum rubrum (author’s translation)]. Z. Naturforsch. C 32, 351–361 (1977).

    Article  PubMed  Google Scholar 

  40. Phelps, A. & Lindsay, J. G. Mammalian pyruvate dehydrogenase complex binds tightly to the mitochondrial inner membrane. Biochem. Soc. Trans. 14, 893 (1986).

    Article  CAS  Google Scholar 

  41. Millar, A. H., Hill, S. A., & Leaver, C. J. Plant mitochondrial 2-oxoglutarate dehydrogenase complex: purification and characterization in potato. Biochem. J. 343, 327–334 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Li, L., Carrie, C., Nelson, C., Whelan, J. & Millar, A. Accumulation of newly synthesized F1 in vivo in Arabidopsis mitochondria provides evidence for modular assembly of the plant F1Fo ATP synthase. J. Biol. Chem. 287, 25749–25757 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Huang, S., Braun, H.-P., Gawryluk, R. M. R. & Millar, A. H. Mitochondrial complex II of plants: subunit composition, assembly, and function in respiration and signaling. Plant J. 98, 405–417 (2019).

    Article  CAS  PubMed  Google Scholar 

  44. Mansilla, N., Racca, S., Gras, D. E., Gonzalez, D. H. & Welchen, E. The complexity of mitochondrial complex IV: an update of cytochrome c oxidase biogenesis in plants. Int. J. Mol. Sci. 19, 662 (2018).

    Article  PubMed Central  CAS  Google Scholar 

  45. Douce, R., Bourguignon, J., Neuburger, M. & Rébeillé, F. The glycine decarboxylase system: a fascinating complex. Trends Plant Sci. 6, 167–176 (2001).

    Article  CAS  PubMed  Google Scholar 

  46. Morgan, M. J. et al. Decrease in manganese superoxide dismutase leads to reduced root growth and affects tricarboxylic acid cycle flux and mitochondrial redox homeostasis. Plant Physiol. 147, 101–114 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Romero, L. C. et al. Cysteine and cysteine-related signaling pathways in Arabidopsis thaliana. Mol. Plant 7, 264–276 (2014).

    Article  CAS  PubMed  Google Scholar 

  48. Vögtle, F. N. et al. Landscape of submitochondrial protein distribution. Nat. Commun. 8, 290 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Deng, Y., Zou, W., Li, G. & Zhao, J. Translocase of the inner membrane E9 and 10 are essential for maintaining mitochondrial function during early embryo cell and endosperm free nucleus divisions in Arabidopsis. Plant Physiol. 166, 853–868 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. Haggie, P. M. & Verkman, A. S. Diffusion of tricarboxylic acid cycle enzymes in the mitochondrial matrix in vivo: evidence for restricted mobility of a multienzyme complex. J. Biol. Chem. 277, 40782–40788 (2002).

    Article  CAS  PubMed  Google Scholar 

  51. Puchulu-Campanella, E. et al. Identification of the components of a glycolytic enzyme metabolon on the human red blood cell membrane. J. Biol. Chem. 288, 848–858 (2013).

    Article  CAS  PubMed  Google Scholar 

  52. Araiza‐Olivera, D. et al. A glycolytic metabolon in Saccharomyces cerevisiae is stabilized by F‐actin. FEBS J. 280, 3887–3905 (2013).

    Article  PubMed  CAS  Google Scholar 

  53. Graham, J. W. A. et al. Glycolytic enzymes associate dynamically with mitochondria in response to respiratory demand and support substrate channeling. Plant Cell 19, 3723–3738 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Panicot, M. et al. A polyamine metabolon involving aminopropyl transferase complexes in Arabidopsis. Plant Cell 14, 2539–2551 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Stavrinides, A. et al. Unlocking the diversity of alkaloids in Catharanthus roseus: nuclear localization suggests metabolic channeling in secondary metabolism. Chem. Biol. 22, 336–341 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Mucha, S. et al. The formation of a camalexin biosynthetic metabolon. Plant Cell 31, 2697–2710 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Gou, M., Ran, X., Martin, D. W. & Liu, C.-J. The scaffold proteins of lignin biosynthetic cytochrome P450 enzymes. Nat. Plants 4, 299–310 (2018).

    Article  CAS  PubMed  Google Scholar 

  58. Fujino, N. et al. Physical interactions among flavonoid enzymes in snapdragon and torenia reveal the diversity in the flavonoid metabolon organization of different plant species. Plant J. 94, 372–392 (2018).

    Article  CAS  PubMed  Google Scholar 

  59. Fuchs, P. et al. Single organelle function and organization as estimated from Arabidopsis mitochondrial proteomics. Plant J. 101, 420–441 (2020).

    Article  CAS  PubMed  Google Scholar 

  60. Neupert, W. SnapShot: mitochondrial architecture. Cell 149, 722–722 e721 (2012).

    Article  CAS  PubMed  Google Scholar 

  61. Schwarzländer, M. & Fuchs, P. Plant mitochondrial membranes: adding structure and new functions to respiratory physiology. Curr. Opin. Plant Biol. 40, 147–157 (2017).

    Article  PubMed  CAS  Google Scholar 

  62. Choi, Y. H. et al. Are natural deep eutectic solvents the missing link in understanding cellular metabolism and physiology? Plant Physiol. 156, 1701–1705 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Sweetlove, L. J. & Fernie, A. R. The spatial organization of metabolism within the plant cell. Annu. Rev. Plant Biol. 64, 723–746 (2013).

    Article  CAS  PubMed  Google Scholar 

  64. Margineantu, D. H., Brown, R. M., Brown, G. K., Marcus, A. H. & Capaldi, R. A. Heterogeneous distribution of pyruvate dehydrogenase in the matrix of mitochondria. Mitochondrion 1, 327–338 (2002).

    Article  CAS  PubMed  Google Scholar 

  65. Loeber, G., Infante, A. A., Maurer-Fogy, I., Krystek, E. & Dworkin, M. B. Human NAD(+)-dependent mitochondrial malic enzyme: cDNA cloning, primary structure, and expression in Escherichia coli. J. Biol. Chem. 266, 3016–3021 (1991).

    Article  CAS  PubMed  Google Scholar 

  66. Pongratz, R. L., Kibbey, R. G. & Cline, G. W. Investigating the roles of mitochondrial and cytosolic malic enzyme in insulin secretion. Methods Enzymol. 457, 425–450 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Nagel, W. O. & Sauer, L. A. Mitochondrial malic enzymes: purification and properties of the NAD(P)-dependent malic enzyme from canine small intestinal mucosa. J. Biol. Chem. 257, 12405–12411 (1982).

    Article  CAS  PubMed  Google Scholar 

  68. Stefan, P. In vivo evidence for a functional glycolytic compartment in synchronous yeast cells. Z. Naturforsch. C 36, 615–618 (1981).

    Article  Google Scholar 

  69. Raugi, G. J., Liang, T. & Blum, J. J. Structural organization of three pools of acetyl coenzyme A in Tetrahymena. J. Biol. Chem. 248, 8064–8072 (1973).

    Article  CAS  PubMed  Google Scholar 

  70. Srere, P. A. Complexes of sequential metabolic enzymes. Annu. Rev. Biochem. 56, 89–124 (1987).

    Article  CAS  PubMed  Google Scholar 

  71. Giegé, P. et al. Enzymes of glycolysis are functionally associated with the mitochondrion in Arabidopsis cells. Plant Cell 15, 2140–2151 (2003).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  72. Zhang, Y. et al. A moonlighting role for enzymes of glycolysis in the co-localization of mitochondria and chloroplasts. Nat. Commun. 11, 4509 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  73. Zhang, Y. & Fernie, A. On the detection and functional significance of the protein–protein interactions of mitochondrial transport proteins. Biomolecules 10, 1107 (2020).

    Article  CAS  PubMed Central  Google Scholar 

  74. Zwingmann, C., Richter-Landsberg, C. & Leibfritz, D. 13C isotopomer analysis of glucose and alanine metabolism reveals cytosolic pyruvate compartmentation as part of energy metabolism in astrocytes. Glia 34, 200–212 (2001).

    Article  CAS  PubMed  Google Scholar 

  75. Umbach, A. L., Ng, V. S. & Siedow, J. N. Regulation of plant alternative oxidase activity: a tale of two cysteines. Biochim. Biophys. Acta 1757, 135–142 (2006).

    Article  CAS  PubMed  Google Scholar 

  76. Selinski, J., Hartmann, A., Höfler, S., Deckers-Hebestreit, G. & Scheibe, R. Refined method to study the posttranslational regulation of alternative oxidases from Arabidopsis thaliana in vitro. Physiol. Plant. 157, 264–279 (2016).

    Article  CAS  PubMed  Google Scholar 

  77. Millar, A. H., Wiskich, J. T., Whelan, J. & Day, D. A. Organic acid activation of the alternative oxidase of plant mitochondria. FEBS Lett. 329, 259–262 (1993).

    Article  CAS  PubMed  Google Scholar 

  78. Selinski, J. et al. Alternative oxidase isoforms are differentially activated by tricarboxylic acid cycle intermediates. Plant Physiol. 176, 1423–1432 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  79. Tronconi, M. A., Hüdig, M., Schranz, M. E. & Maurino, V. G. Independent recruitment of duplicated β-subunit-coding NAD-ME genes aided the evolution of C4 photosynthesis in Cleomaceae. Front. Plant Sci. 11, 572080 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  80. Aubry, S., Brown, N. J. & Hibberd, J. M. The role of proteins in C3 plants prior to their recruitment into the C4 pathway. J. Exp. Bot. 62, 3049–3059 (2011).

    Article  CAS  PubMed  Google Scholar 

  81. Netting, A. G. pH, abscisic acid and the integration of metabolism in plants under stressed and non‐stressed conditions. II. Modifications in modes of metabolism induced by variation in the tension on the water column and by stress. J. Exp. Bot. 53, 151–173 (2002).

    CAS  PubMed  Google Scholar 

  82. Santelia, D. & Lawson, T. Rethinking guard cell metabolism. Plant Physiol. 172, 1371–1392 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Post-Beittenmiller, D., Roughan, G. & Ohlrogge, J. B. Regulation of plant fatty acid biosynthesis: analysis of acyl-coenzyme a and acyl-acyl carrier protein substrate pools in spinach and pea chloroplasts. Plant Physiol. 100, 923–930 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Camp, P. J. & Randall, D. D. Purification and characterization of the pea chloroplast pyruvate dehydrogenase complex: a source of acetyl-CoA and NADH for fatty acid biosynthesis. Plant Physiol. 77, 571–577 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Ke, J. et al. The role of pyruvate dehydrogenase and acetyl-coenzyme A synthetase in fatty acid synthesis in developing Arabidopsis seeds. Plant Physiol. 123, 497–508 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Diebold, R., Schuster, J., Däschner, K. & Binder, S. The branched-chain amino acid transaminase gene family in Arabidopsis encodes plastid and mitochondrial proteins. Plant Physiol. 129, 540–550 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Hüdig, M. et al. Respiratory and C4-photosynthetic NAD-malic enzyme coexist in bundle sheath cell mitochondria and evolved via association of differentially adapted subunits. Plant Cell 34, 597–615 (2021).

    Article  Google Scholar 

  88. Rao, X. & Dixon, R. A. The differences between NAD-ME and NADP-ME subtypes of C4 photosynthesis: more than decarboxylating enzymes. Front. Plant Sci. 7, 1525 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  89. Achnine, L., Blancaflor, E. B., Rasmussen, S. & Dixon, R. A. Colocalization of l-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase for metabolic channeling in phenylpropanoid biosynthesis. Plant Cell 16, 3098–3109 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Brailsford, M. A., Thompson, A. G., Kaderbhai, N. & Beechey, R. B. Pyruvate metabolism in castor-bean mitochondria. Biochem. J. 239, 355–361 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Grigorenko, E. V., Small, W. C., Persson, L. O. & Srere, P. A. Citrate synthase 1 interacts with the citrate transporter of yeast mitochondria. J. Mol. Recognit. 3, 215–219 (1990).

    Article  CAS  PubMed  Google Scholar 

  92. Wiskich, J. T., Bryce, J. H., Day, D. A. & Dry, I. B. Evidence for metabolic domains within the matrix compartment of pea leaf mitochondria: implications for photorespiratory metabolism. Plant Physiol. 93, 611–616 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Millar, A. H., Liddell, A. & Leaver, C. J. Isolation and subfractionation of mitochondria from plants. Methods Cell. Biol. 80, 65–90 (2007).

    Article  CAS  PubMed  Google Scholar 

  94. Le, X., Millar, A. H. & Lee, C. P. Assessing the kinetics of metabolite uptake and utilization by isolated mitochondria using selective reaction monitoring mass spectrometry (SRM-MS) in plant mitochondria: methods and protocols. Methods Mol. Biol. 2363, 85–100 (2022).

    Article  PubMed  Google Scholar 

  95. Petereit, J. et al. Mitochondrial CLPP2 assists coordination and homeostasis of respiratory complexes. Plant Physiol. 184, 148–164 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Monachello, D., Guillaumot, D. & Lurin, C. A pipeline for systematic yeast 2-hybrid matricial screening in liquid culture. Protoc. Exch. https://doi.org/10.21203/rs.2.9948/v1 (2019).

  97. Dreze, M. et al. High-quality binary interactome mapping. Methods Enzymol. 470, 281–315 (2010).

    Article  CAS  PubMed  Google Scholar 

  98. Couturier, J. et al. Monothiol glutaredoxin–BolA interactions: redox control of Arabidopsis thaliana BolA2 and SufE1. Mol. Plant 7, 187–205 (2014).

    Article  CAS  PubMed  Google Scholar 

  99. Ndamukong, I. et al. SA-inducible Arabidopsis glutaredoxin interacts with TGA factors and suppresses JA-responsive PDF1.2 transcription. Plant J. 50, 128–139 (2007).

    Article  CAS  PubMed  Google Scholar 

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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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Authors and Affiliations

  1. School of Molecular Sciences, The University of Western Australia, Perth, WA, Australia

    Xuyen H. Le, Chun Pong Lee & A. Harvey Millar

  2. The ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, Perth, WA, Australia

    Xuyen H. Le, Chun Pong Lee & A. Harvey Millar

  3. Université Paris-Saclay, CNRS, INRAE, Univ Evry, Institute of Plant Sciences Paris-Saclay (IPS2), Orsay, France

    Dario Monachello

  4. Université de Paris, CNRS, INRAE, Institute of Plant Sciences Paris-Saclay (IPS2), Orsay, France

    Dario Monachello

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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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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).

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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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