Abstract
The tricarboxylic acid (TCA) cycle is an important metabolic pathway to underpin stomatal movements, given that respiration is thought to be the main energy source for guard cell (GC) metabolism. However, it is still unclear how the metabolic fluxes throughout the TCA cycle and associated pathways are regulated in GCs. Here we used a 13C-positional isotopomer approach and performed a multi-species/cell-types analysis based on previous 13C-labelling studies carried out using Arabidopsis rosettes, maize leaves, Arabidopsis source and sink leaves, and isolated GCs from Arabidopsis and tobacco. We aimed to compare flux modes through the TCA cycle and associated pathways in GCs and leaves, which are mostly composed by mesophyll cells (MCs). Mesophyll cells showed high 13C-enrichment into alanine and aspartate following provision of 13CO2, whilst GCs and sink MCs showed high 13C-incorporation into glutamate/glutamine following provision of 13C-sucrose. Only GCs showed high 13C-enrichment in the carbon 1 atom of glutamine, which is derived from phosphoenolpyruvate carboxylase (PEPc)-mediated CO2 assimilation. The PEPc-mediated 13C-incorporation into malate was similar between GCs and MCs, but GCs had higher 13C-enrichment and accumulation of fumarate than MCs. The metabolic fluxes throughout the TCA cycle of illuminated GCs resemble those of sink MCs, but with different contribution from PEPc, glycolysis and the TCA cycle to glutamate/glutamine synthesis. We further demonstrate that transamination reactions catalysed by alanine and aspartate amino transferases may support non-cyclic TCA flux modes in illuminated MCs.
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References
Abadie C, Tcherkez G (2019) In vivo phosphoenolpyruvate carboxylase activity is controlled by CO2 and O2 mole fractions and represents a major flux at high photorespiration rates. New Phytol 221:1843–1852. https://doi.org/10.1111/nph.15500
Abadie C, Bathellier C, Tcherkez G (2018) Carbon allocation to major metabolites in illuminated leaves is not just proportional to photosynthesis when gaseous conditions (CO2 and O2) vary. New Phytol 218:94–106. https://doi.org/10.1111/nph.14984
Abadie C, Lothier J, Boex-Fontvieille E et al (2017) Direct assessment of the metabolic origin of carbon atoms in glutamate from illuminated leaves using 13C-NMR. New Phytol 216:1079–1089. https://doi.org/10.1111/nph.14719
Antoniewicz MR (2013) 13C metabolic flux analysis: optimal design of isotopic labeling experiments. Curr Opin Biotechnol 24:1116–1121. https://doi.org/10.1016/j.copbio.2013.02.003
Araújo WL, Nunes-Nesi A, Fernie AR (2011a) Fumarate: Multiple functions of a simple metabolite. Phytochemistry 72:838–843. https://doi.org/10.1016/j.phytochem.2011.02.028
Araújo WL, Nunes-Nesi A, Osorio S et al (2011b) Antisense inhibition of the iron-sulphur subunit of succinate dehydrogenase enhances photosynthesis and growth in tomato via an organic acid-mediated effect on stomatal aperture. Plant Cell 23:600–627. https://doi.org/10.1105/tpc.110.081224
Arrivault S, Obata T, Szecówka M et al (2017) Metabolite pools and carbon flow during C4 photosynthesis in maize: 13CO2 labeling kinetics and cell type fractionation. J Exp Bot 68:283–298. https://doi.org/10.1093/jxb/erw414
Balparda M, Bouzid M, del Martinez M, P, et al (2023) Regulation of plant carbon assimilation metabolism by post-translational modifications. Plant J 114:1059–1079. https://doi.org/10.1111/tpj.16240
Cardoso LL, Freire FBS, Daloso DM (2023) Plant metabolic networks under stress: a multi-species/stress condition meta-analysis. J Soil Sci Plant Nutr 23:4–21. https://doi.org/10.1007/s42729-022-01032-2
Dahmani I, Qin K, Zhang Y, Fernie AR (2023) The formation and function of plant metabolons. Plant J 114:1080–1092. https://doi.org/10.1111/tpj.16179
de Daloso DM, Morais EG, Oliveira e Silva KF, Williams TCR (2023) Cell-type-specific metabolism in plants. Plant J. https://doi.org/10.1111/tpj.16214
Daloso DM, Antunes WC, Pinheiro DP et al (2015a) Tobacco guard cells fix CO2 by both Rubisco and PEPcase while sucrose acts as a substrate during light-induced stomatal opening. Plant Cell Environ 38:2353–2371. https://doi.org/10.1111/pce.12555
Daloso DM, dos Anjos L, Fernie AR (2016) Roles of sucrose in guard cell regulation. New Phytol 211:809–818. https://doi.org/10.1111/nph.13950
Daloso DM, Medeiros DB, dos Anjos L et al (2017) Metabolism within the specialized guard cells of plants. New Phytol 216:1018–1033. https://doi.org/10.1111/nph.14823
Daloso DM, Müller K, Obata T et al (2015b) Thioredoxin, a master regulator of the tricarboxylic acid cycle in plant mitochondria. Proc Natl Acad Sci U S A 112:E1392–E1400. https://doi.org/10.1073/pnas.1424840112
Dethloff F, Orf I, Kopka J (2017) Rapid in situ 13C tracing of sucrose utilization in Arabidopsis sink and source leaves. Plant Methods 13:1–19. https://doi.org/10.1186/s13007-017-0239-6
Dunnett CW (1955) A multiple comparison procedure for comparing several treatments with a control. J Am Stat Assoc 50:1096–1121. https://doi.org/10.1080/01621459.1955.10501294
Eprintsev AT, Fedorin DN, Cherkasskikh MV, Igamberdiev AU (2018) Expression of succinate dehydrogenase and fumarase genes in maize leaves is mediated by cryptochrome. J Plant Physiol 221:81–84. https://doi.org/10.1016/j.jplph.2017.12.004
Eprintsev AT, Fedorin DN, Karabutova LA, Igamberdiev AU (2016a) Expression of genes encoding subunits A and B of succinate dehydrogenase in germinating maize seeds is regulated by methylation of their promoters. J Plant Physiol 205:33–40. https://doi.org/10.1016/j.jplph.2016.08.008
Eprintsev AT, Fedorin DN, Sazonova OV, Igamberdiev AU (2016b) Light inhibition of fumarase in Arabidopsis leaves is phytochrome A-dependent and mediated by calcium. Plant Physiol Biochem 102:161–166. https://doi.org/10.1016/j.plaphy.2016.02.028
Flexas J, Carriquí M (2020) Photosynthesis and photosynthetic efficiencies along the terrestrial plant’s phylogeny: lessons for improving crop photosynthesis. Plant J 101:964–978. https://doi.org/10.1111/tpj.14651
Flütsch S, Horrer D, Santelia D (2022) Starch biosynthesis in guard cells has features of both autotrophic and heterotrophic tissues. Plant Physiol. https://doi.org/10.1093/plphys/kiac087
Flütsch S, Santelia D (2021) Mesophyll-derived sugars are positive regulators of light-driven stomatal opening. New Phytol 230:1754–1760. https://doi.org/10.1111/nph.17322
Flütsch S, Wang Y, Takemiya A et al (2020) Guard cell starch degradation yields glucose for rapid stomatal opening in Arabidopsis. Plant Cell 32:2325–2344. https://doi.org/10.1105/tpc.18.00802
Fonseca-Pereira P, Souza PVL, Fernie AR et al (2021) Thioredoxin-mediated regulation of (photo)respiration and central metabolism. J Exp Bot 72:5987–6002. https://doi.org/10.1093/jxb/erab098
Gauthier PPG, Bligny R, Gout E et al (2010) In folio isotopic tracing demonstrates that nitrogen assimilation into glutamate is mostly independent from current CO2 assimilation in illuminated leaves of Brassica napus. New Phytol 185:988–999. https://doi.org/10.1111/j.1469-8137.2009.03130.x
Gong XY, Tcherkez G, Wenig J et al (2018) Determination of leaf respiration in the light: comparison between an isotopic disequilibrium method and the Laisk method. New Phytol 218:1371–1382. https://doi.org/10.1111/nph.15126
Gotow K, Taylor S, Zeiger E (1988) Photosynthetic carbon fixation in guard cell protoplasts of Vicia faba L. Plant Physiol 86:700–705
Hartman MD, Rojas BE, Iglesias AA, Figueroa CM (2023) The involvement of allosteric effectors and post-translational modifications in the control of plant central carbon metabolism. Plant J 114:1037–1058. https://doi.org/10.1111/tpj.16215
Hatch MD (1971) The C4 -pathway of photosynthesis. Evidence for an intermediate pool of carbon dioxide and the identity of the donor C4-dicarboxylic acid. Biochem J 125:425–432. https://doi.org/10.1042/bj1250425
Hedrich R, Raschke K, Stitt M (1985) A role for fructose 2,6-bisphosphate in regulating carbohydrate metabolism in guard cells. Plant Physiol 79:977–982. https://doi.org/10.1104/pp.79.4.977
Heise R, Arrivault S, Szecowka M et al (2014) Flux profiling of photosynthetic carbon metabolism in intact plants. Nat Protoc 9:1803–1824. https://doi.org/10.1038/nprot.2014.115
Hildebrandt TM, Nunes Nesi A, Araújo WL, Braun HP (2015) Amino acid catabolism in plants. Mol Plant 8:1563–1579. https://doi.org/10.1016/j.molp.2015.09.005
Kopka J, Schauer N, Krueger S et al (2005) GMD@CSB.DB: the Golm metabolome database. Bioinformatics 21:1635–1638. https://doi.org/10.1093/bioinformatics/bti236
Lawson T, Matthews J (2020) Guard cell metabolism and stomatal function. Annu Rev Plant Biol 71:273–302. https://doi.org/10.1146/annurev-arplant-050718-100251
Lawson T, Oxborough K, Morison JIL, Baker NR (2003) The responses of guard and mesophyll cell photosynthesis to CO2, O2, light, and water stress in a range of species are similar. J Exp Bot 54:1743–1752. https://doi.org/10.1093/jxb/erg186
Le XH, Lee C-P, Millar AH (2021) The mitochondrial pyruvate carrier (MPC) complex mediates one of three pyruvate-supplying pathways that sustain Arabidopsis respiratory metabolism. Plant Cell 33:2776–2793. https://doi.org/10.1093/plcell/koab148
Le XH, Lee CP, Monachello D, Millar AH (2022) Metabolic evidence for distinct pyruvate pools inside plant mitochondria. Nat Plants 8:694–705. https://doi.org/10.1038/s41477-022-01165-3
Lee CP, Elsässer M, Fuchs P et al (2021) The versatility of plant organic acid metabolism in leaves is underpinned by mitochondrial malate–citrate exchange. Plant Cell. https://doi.org/10.1093/plcell/koab223
Lee CP, Eubel H, O’Toole N, Millar AH (2008) Heterogeneity of the mitochondrial proteome for photosynthetic and non-photosynthetic Arabidopsis metabolism. Mol Cell Proteomics 7:1297–1316. https://doi.org/10.1074/mcp.M700535-MCP200
Lim SL, Flütsch S, Liu J et al (2022) Arabidopsis guard cell chloroplasts import cytosolic ATP for starch turnover and stomatal opening. Nat Commun 13:1–13. https://doi.org/10.1038/s41467-022-28263-2
Lima VF, de Souza LP, Williams TCR et al (2018) Gas chromatography-mass spectrometry-based 13C-labeling studies in plant metabolomics. Plant metabolomics. Springer, New York, pp 47–58
Lima VF, Erban A, Daubermann AG et al (2021) Establishment of a GC-MS-based 13C-positional isotopomer approach suitable for investigating metabolic fluxes in plant primary metabolism. Plant J 108:1213–1233. https://doi.org/10.1111/tpj.15484
Lima VF, Freire FBS, Cândido-Sobrinho SA et al (2023) Unveiling the dark side of guard cell metabolism. Plant Physiol Biochem 201:107862. https://doi.org/10.1016/j.plaphy.2023.107862
Lima VF, Medeiros DB, Dos Anjos L et al (2018) Toward multifaceted roles of sucrose in the regulation of stomatal movement. Plant Signal Behav. https://doi.org/10.1080/15592324.2018.1494468
Lisec J, Schauer N, Kopka J et al (2006) Gas chromatography mass spectrometry—based metabolite profiling in plants. Nat Protoc 1:387–396. https://doi.org/10.1038/nprot.2006.59
Luedemann A, Strassburg K, Erban A, Kopka J (2008) TagFinder for the quantitative analysis of gas chromatography - Mass spectrometry (GC-MS)-based metabolite profiling experiments. Bioinformatics 24:732–737. https://doi.org/10.1093/bioinformatics/btn023
Martí MC, Jiménez A, Sevilla F (2020) Thioredoxin network in plant mitochondria: cysteine S-posttranslational modifications and stress conditions. Front Plant Sci 11:1–20
Maurice Cheung CY, Poolman MG, Fell DA et al (2014) A diel flux balance model captures interactions between light and dark metabolism during day-night cycles in C3 and crassulacean acid metabolism leaves. Plant Physiol 165:917–929. https://doi.org/10.1104/pp.113.234468
Medeiros DB, Barros KA, Barros JAS et al (2017) Impaired malate and fumarate accumulation due to the mutation of the tonoplast dicarboxylate transporter has little effects on stomatal behavior. Plant Physiol 175:1068–1081. https://doi.org/10.1104/pp.17.00971
Medeiros DB, Perez Souza L, Antunes WC et al (2018) Sucrose breakdown within guard cells provides substrates for glycolysis and glutamine biosynthesis during light-induced stomatal opening. Plant J 94:583–594. https://doi.org/10.1111/tpj.13889
Møller IM, Igamberdiev AU, Bykova NV et al (2020) Matrix redox physiology governs the regulation of plant mitochondrial metabolism through posttranslational protein modifications. Plant Cell 32:573–594. https://doi.org/10.1105/tpc.19.00535
Nunes-Nesi A, Carrari F, Gibon Y et al (2007) Deficiency of mitochondrial fumarase activity in tomato plants impairs photosynthesis via an effect on stomatal function. Plant J 50:1093–1106. https://doi.org/10.1111/j.1365-313X.2007.03115.x
Okahashi N, Kawana S, Iida J et al (2019) Fragmentation of dicarboxylic and tricarboxylic acids in the krebs cycle using GC-EI-MS and GC-EI-MS/MS. Mass Spectrom 8:A0073–A0073. https://doi.org/10.5702/massspectrometry.a0073
Piro L, Flütsch S, Santelia D (2023) Arabidopsis sucrose synthase 3 (SUS3) regulates starch accumulation in guard cells at the end of day. Plant Signal Behav 18:4–8. https://doi.org/10.1080/15592324.2023.2171614
Popov VN, Eprintsev AT, Fedorin DN, Igamberdiev AU (2010) Succinate dehydrogenase in Arabidopsis thaliana is regulated by light via phytochrome A. FEBS Lett 584:199–202. https://doi.org/10.1016/j.febslet.2009.11.057
Rao X, Dixon RA (2016) The differences between NAD-ME and NADP-ME subtypes of C4 photosynthesis: More than decarboxylating enzymes. Front Plant Sci 7:1–9. https://doi.org/10.3389/fpls.2016.01525
Ratcliffe RG, Shachar-Hill Y (2006) Measuring multiple fluxes through plant metabolic networks. Plant J 45:490–511. https://doi.org/10.1111/j.1365-313X.2005.02649.x
Rocha M, Licausi F, Araujo WL et al (2010) Glycolysis and the tricarboxylic acid cycle are linked by alanine aminotransferase during hypoxia induced by waterlogging of lotus japonicus. Plant Physiol 152:1501–1513. https://doi.org/10.1104/pp.109.150045
Schlüter U, Bräutigam A, Droz J-M et al (2019) The role of alanine and aspartate aminotransferases in C4 photosynthesis. Plant Biol 21:64–76. https://doi.org/10.1111/plb.12904
Shameer S, Ratcliffe RG, Sweetlove LJ (2019) Leaf energy balance requires mitochondrial respiration and export of chloroplast NADPH in the light. Plant Physiol 180:1947–1961. https://doi.org/10.1104/pp.19.00624
Sweetlove LJ, Beard KFM, Nunes-Nesi A et al (2010) Not just a circle: flux modes in the plant TCA cycle. Trends Plant Sci 15:462–470. https://doi.org/10.1016/j.tplants.2010.05.006
Sweetlove LJ, Fernie AR (2018) The role of dynamic enzyme assemblies and substrate channelling in metabolic regulation. Nat Commun. https://doi.org/10.1038/s41467-018-04543-8
Szecowka M, Heise R, Tohge T et al (2013) Metabolic fluxes in an illuminated Arabidopsis rosette. Plant Cell 25:694–714. https://doi.org/10.1105/tpc.112.106989
Tan XLJ, Cheung CYM (2020) A multiphase flux balance model reveals flexibility of central carbon metabolism in guard cells of C3 plants. Plant J 104:1648–1656. https://doi.org/10.1111/tpj.15027
Tcherkez G, Boex-Fontvieille E, Mahé A, Hodges M (2012) Respiratory carbon fluxes in leaves. Curr Opin Plant Biol 15:308–314. https://doi.org/10.1016/j.pbi.2011.12.003
Tcherkez G, Cornic G, Bligny R et al (2005) In Vivo Respiratory metabolism of illuminated leaves. Plant Physiol 138:1596–1606. https://doi.org/10.1104/pp.105.062141
Tcherkez G, Mahé A, Gauthier P et al (2009) 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. https://doi.org/10.1104/pp.109.142976
Tovar-Méndez A, Miernyk JA, Randall DD (2003) Regulation of pyruvate dehydrogenase complex activity in plant cells. Eur J Biochem 270:1043–1049. https://doi.org/10.1046/j.1432-1033.2003.03469.x
Wieloch T, Ehlers I, Yu J et al (2018) Intramolecular 13C analysis of tree rings provides multiple plant ecophysiology signals covering decades. Sci Rep 8:1–10. https://doi.org/10.1038/s41598-018-23422-2
Wieloch T, Werner RA, Schleucher J (2021) Carbon flux around leaf-cytosolic glyceraldehyde-3-phosphate dehydrogenase introduces a 13C signal in plant glucose. J Exp Bot 72:7136–7144. https://doi.org/10.1093/jxb/erab316
Willmer C, Fricker M (1996) Stomata. Chapman & Hall, London
Zandalinas SI, Balfagón D, Gómez-Cadenas A, Mittler R (2022) Responses of plants to climate change: Metabolic changes during abiotic stress combination in plants. J Exp Bot. https://doi.org/10.1093/jxb/erac073
Zhang Y, Beard KFM, Swart C et al (2017) Protein-protein interactions and metabolite channelling in the plant tricarboxylic acid cycle. Nat Commun. https://doi.org/10.1038/ncomms15212
Zhang Y, Giese J, Mae-Lin Kerbler S et al (2021) Two mitochondrial phosphatases, PP2c63 and Sal2, are required for posttranslational regulation of the TCA cycle in Arabidopsis. Mol Plant. https://doi.org/10.1016/j.molp.2021.03.023
Zhu M, Geng S, Chakravorty D et al (2020) Metabolomics of red-light-induced stomatal opening in Arabidopsis thaliana: coupling with abscisic acid and jasmonic acid metabolism. Plant J 101:1331–1348. https://doi.org/10.1111/tpj.14594
Zubimendi JP, Martinatto A, Valacco MP et al (2018) The complex allosteric and redox regulation of the fumarate hydratase and malate dehydratase reactions of Arabidopsis thaliana Fumarase 1 and 2 gives clues for understanding the massive accumulation of fumarate. FEBS J 285:2205–2224. https://doi.org/10.1111/febs.14483
Acknowledgements
This work was partially supported by the National Institute of Science and Technology in Plant Physiology under Stress Conditions (INCT Plant Stress Physiology – Grant: 406455/2022-8) and the National Council for Scientific and Technological Development (CNPq, Grant No. 404817/2021-1). J.K. and A.E. acknowledge funding of the German science foundation (DFG) grant (KO 2329/7-1) that is part of the SCyCode research consortium (FOR2816). We also acknowledge the research fellowship granted by CNPq to D.M.D and the scholarships granted by the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES-Brazil) to A.G.D. and V.F.L. We thank the authors who kindly provided their mass spectral data from published work.
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Daubermann, A.G., Lima, V.F., Erban, A. et al. Novel guard cell sink characteristics revealed by a multi-species/cell-types meta-analysis of 13C-labelling experiments. Theor. Exp. Plant Physiol. 36, 1–20 (2024). https://doi.org/10.1007/s40626-023-00299-9
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DOI: https://doi.org/10.1007/s40626-023-00299-9