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Fumarate and cytosolic pH as modulators of the synthesis or consumption of C4 organic acids through NADP-malic enzyme in Arabidopsis thaliana

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Abstract

Arabidopsis thaliana is a plant species that accumulates high levels of organic acids and uses them as carbon, energy and reducing power sources. Among the enzymes that metabolize these compounds, one of the most important ones is malic enzyme (ME). A. thaliana contains four malic enzymes (NADP-ME 1–4) to catalyze the reversible oxidative decarboxylation of malate in the presence of NADP. NADP-ME2 is the only one located in the cell cytosol of all Arabidopsis organs providing most of the total NADP-ME activity. In the present work, the regulation of this key enzyme by fumarate was investigated by kinetic assays, structural analysis and a site-directed mutagenesis approach. The final effect of this metabolite on NADP-ME2 forward activity not only depends on fumarate and substrate concentrations but also on the pH of the reaction medium. Fumarate produced an increase in NADP-ME2 activity by binding to an allosteric site. However at higher concentrations, fumarate caused a competitive inhibition, excluding the substrate malate from binding to the active site. The characterization of ME2-R115A mutant, which is not activated by fumarate, confirms this hypothesis. In addition, the reverse reaction (reductive carboxylation of pyruvate) is also modulated by fumarate, but in a different way. The results indicate pH-dependence of the fumarate modulation with opposite behavior on the two activities analyzed. Thereby, the coordinated action of fumarate over the direct and reverse reactions would allow a precise and specific modulation of the metabolic flux through this enzyme, leading to the synthesis or degradation of C4 compounds under certain conditions. Thus, the physiological context might be exerting an accurate control of ME activity in planta, through changes in metabolite and substrate concentrations and cytosolic pH.

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References

  • Araújo WL, Nunes-Nesi A, Fernie AR (2011) Fumarate: multiple functions of a simple metabolite. Phytochemistry 72:838–843

    Article  Google Scholar 

  • Brown NJ, Palmer BG, Stanley S, Hajaji H, Janacek SH, Astley HM, Parsley K, Kajala K, Quick WP, Trenkamp S, Fernie AR, Maurino VG, Hibberd JM (2010) C4 acid decarboxylases required for C4 photosynthesis are active in the mid-vein of the C3 species Arabidopsis thaliana, and are important in sugar and amino acid metabolism. Plant J 61:122–133

    Article  PubMed  CAS  Google Scholar 

  • Chang GG, Tong L (2003) Structure and function of malic enzymes, a new class of oxidative decarboxylases. Biochemistry 42:12721–12733

    Article  PubMed  CAS  Google Scholar 

  • Chia DW, Yoder TJ, Reiter WD, Gibson SI (2000) Fumaric acid: an overlooked form of fixed carbon in Arabidopsis and other plant species. Planta 211:743–751

    Article  PubMed  CAS  Google Scholar 

  • Dawson RM, Elliott DC, Elliott WH, Jones KM (1986) Data for biochemical research. Oxford Science Publication, Oxford

    Google Scholar 

  • Detarsio E, Gerrard Wheeler MC, Campos Bermúdez VA, Andreo CS, Drincovich MF (2003) Maize C4 NADP-malic enzyme. Expression in Escherichia coli and characterization of site-direct mutants at the putative nucleotide-binding sites. J Biol Chem 278:13757–13764

    Article  PubMed  CAS  Google Scholar 

  • Drincovich MF, Lara MV, Maurino VG, Andreo CS (2010) C4 decarboxylases. Different solutions for the same biochemical problem, the provision of CO2 in the bundle sheath cells. C4 photosynthesis and related CO2 concentrating mechanisms. Springer, Heidelberg

    Google Scholar 

  • Fahnenstich H, Saigo M, Niessen M, Drincovich MF, Zanor MI, Fernie A, Andreo CS, Flügge UI, Maurino VG (2007) Low levels of malate and fumarate cause accelerated senescence during extended darkness in Arabidopsis thaliana overexpressing maize C4 NADP-malic enzyme. Plant Physiol 145:640–655

    Article  PubMed  CAS  Google Scholar 

  • Famiani F, Walker RP, Tecsi L, Chen ZH, Proietti P, Leegood RC (2000) An immunohistochemical study of the compartmentation of metabolism during the development of grape (Vitis vinifera L.) berries. J Exp Bot 51:675–683

    Article  PubMed  CAS  Google Scholar 

  • Felle HH (2001) pH: signal and messenger in plant cells. Plant Biol 3:577–591

    Article  CAS  Google Scholar 

  • Fernie AR, Martinoia E (2009) Malate. Jack of all trades or master of a few? Phytochemistry 70:828–832

    Article  PubMed  CAS  Google Scholar 

  • Gerrard Wheeler MC, Tronconi MA, Drincovich MF, Andreo CS, Flügge UI, Maurino VG (2005) A comprehensive analysis of the NADP-malic enzyme gene family of Arabidopsis thaliana. Plant Physiol 139:39–51

    Article  Google Scholar 

  • Gerrard Wheeler MC, Arias CL, Tronconi MA, Maurino VG, Andreo CS, Drincovich MF (2008) Arabidopsis thaliana NADP-malic enzyme isoforms: high degree of identity but clearly distinct properties. Plant Mol Biol 67:231–242

    Article  CAS  Google Scholar 

  • Gerrard Wheeler MC, Arias CL, Maurino VG, Andreo CS, Drincovich MF (2009) Identification of domains involved in the allosteric regulation of cytosolic Arabidopsis thaliana NADP-malic enzymes. FEBS J 276:5665–5677

    Article  PubMed  Google Scholar 

  • Gout E, Blignyp R, Pascal N, Doucel R (1993) 13C Nuclear magnetic resonance studies of malate and citrate synthesis and compartmentation in higher plant cells. J Biol Chem 268:3986–3992

    PubMed  CAS  Google Scholar 

  • Hsieh JY, Chiang YH, Chang KY, Hung HC (2009) Functional role of fumarate site Glu59 involved in allosteric regulation and subunit–subunit interaction of human mitochondrial NAD(P)+-dependent malic enzyme. FEBS J 276:983–994

    Article  PubMed  CAS  Google Scholar 

  • Hung HC, Kuo MW, Chang GG, Liu GY (2005) Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme). Biochem J 392:39–45

    Article  PubMed  CAS  Google Scholar 

  • Hurth MA, Suh SJ, Kretzschmar T, Geis T, Bregante M, Gambale F, Martinoia E, Neuhaus HE (2005) Impaired pH homeostasis in Arabidopsis lacking the vacuolar dicarboxylate transporter and analysis of carboxylic acid transport across the tonoplast. Plant Physiol 137:901–910

    Article  PubMed  CAS  Google Scholar 

  • Jump AS, Marchant R, Peñuelas J (2009) Environmental change and the option value of genetic diversity. Trends Plant Sci 14:51–58

    Article  PubMed  CAS  Google Scholar 

  • Karsten WE, Pais JE, Rao GSJ, Harris BG, Cook PF (2003) Ascaris summ NAD-malic enzyme is activated by malate and fumarate binding to separate allosteric sites. Biochemistry 42:9712–9721

    Article  PubMed  CAS  Google Scholar 

  • Kilian J, Whitehead D, Horak J, Wanke D, Weinl S, Batistic O, D’Angelo C, Bornberg-Bauer E, Kudla J, Harter K (2007) The AtGenExpress global stress expression data set: protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses. Plant J 50:347–363

    Article  PubMed  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Lai LB, Tausta SL, Nelson TM (2002) Differential regulation of transcripts encoding cytosolic NADP-malic enzyme in C3 and C4 Flaveria species. Plant Physiol 128:140–149

    Article  PubMed  CAS  Google Scholar 

  • Laporte MM, Shen B, Tarczynnsky MC (2002) Engineering for drought avoidance: expression of maize NADP-malic enzyme in tobacco results in altered stomatal function. J Exp Bot 59:699–705

    Article  Google Scholar 

  • Laskowski RA, Gerick F, Thornton JM (2009) The structural basis of allosteric regulation in proteins. FEBS Lett 583:1692–1698

    Article  PubMed  CAS  Google Scholar 

  • Liu S, Cheng Y, Zhang X, Guan Q, Nishiuchi S, Hase K, Takano T (2007) Expression of an NADP-malic enzyme gene in rice (Oryza sativa L.) is induced by environmental stresses; over-expression of the gene in Arabidopsis confer salt and osmotic stress tolerance. Plant Mol Biol 64:49–58

    Article  PubMed  CAS  Google Scholar 

  • Maurino VG, Gerrard Wheeler MC, Andreo CS, Drincovich MF (2009) Redundancy is sometimes seen only by the uncritical: does Arabidopsis need six malic enzyme isoforms? Plant Sci 176:715–721

    Article  CAS  Google Scholar 

  • Okazaki Y, Tazawa M, Iwasaki N (1994) Light-induced changes in cytosolic pH in leaf cells of Egeria densa: measurements with pH-sensitive microelectrodes. Plant Cell Physiol 35:943–950

    CAS  Google Scholar 

  • Parker D, Beckmann M, Zubair H, Enot DP, Caracuel-Rios Z, Overy DP, Snowdon S, Talbot NJ, Draper J (2009) Metabolomic analysis reveals a common pattern of metabolic re-programming during invasion of three host plant species by Magnaporthe grisea. Plant J 59:723–737

    Article  PubMed  CAS  Google Scholar 

  • Pracharoenwattana I, Zhou WX, Keech O, Francisco PB, Udomchalothorn T, Tschoep H, Stitt M, Gibon Y, Smith SM (2010) Arabidopsis has a cytosolic fumarase required for the massive allocation of photosynthate into fumaric acid and for rapid plant growth on high nitrogen. Plant J 62:785–795

    Article  PubMed  CAS  Google Scholar 

  • Rienmüller F, Dreyer I, Schönknecht G, Schulz A, Schumacher K, Nagy R, Martinoia E, Marten I, Hedrich R (2012) Luminal and cytosolic pH feedback on proton pump activity and ATP affinity of the V-type ATPase from Arabidopsis. J Biol Chem 287:8989–8993

    Article  Google Scholar 

  • Roverts DV (1975) Enzyme kinetics. Cambridge Chemistry Texts, Cambridge

    Google Scholar 

  • Shearer HL, Turpin DH, Dennos DT (2004) Characterization of NADP-dependent malic enzyme from developing castor oil seed endosperm. Arch Biochem Biophys 429:134–144

    Article  PubMed  CAS  Google Scholar 

  • Tronconi MA, Fahnenstich H, Gerrard Wheeler MC, Andreo CS, Flügge UI, Drincovich MF, Maurino VG (2008) Arabidopsis NAD-malic enzyme functions as a homodimer and heterodimer and has a major impact during nocturnal metabolism. Plant Physiol 146:1540–1552

    Article  PubMed  CAS  Google Scholar 

  • Tronconi MA, Maurino VG, Andreo CS, Drincovich MF (2010) Three different and tissue-specific NAD-malic enzyme generated by alternative subunit association in Arabidopsis thaliana. J Biol Chem 285:11870–11879

    Article  PubMed  CAS  Google Scholar 

  • Voll LM, Zell MB, Engelsdorf T, Saur A, Gerrard Wheeler MC, Drincovich MF, Weber APM, Maurino VG (2012) Loss of cytosolic NADP-malic enzyme 2 in Arabidopsis is associated with enhanced susceptibility towards Colletotrichum higginsianum. New Phytol 195:189–202

    Article  PubMed  CAS  Google Scholar 

  • Yang Z, Lanks CW, Tong L (2002) Molecular mechanism for the regulation of human mitochondrial NAD(P)+-dependent malic enzyme by ATP and fumarate. Structure 10:951–960

    Article  PubMed  CAS  Google Scholar 

  • Zell MB, Fahnenstich H, Maier A, Saigo M, Voznesenskaya EV, Edwards GE, Andreo CS, Schleifenbaum F, Zell C, Drincovich MF, Maurino VG (2010) Analysis of A. thaliana with highly reduced levels of malate and fumarate sheds light on the role of these organic acids as storage carbon molecules. Plant Physiol 152:1251–1262

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

CSA, MFD and MGW are members of the Researcher Career of National Council of Scientific and Technical Research (CONICET) and CLA is a fellow of the same institution. This work has been supported by National Agency for Promotion of Science and Technology (ANPCyT).

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Authors and Affiliations

  1. Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Universidad Nacional de Rosario, Suipacha 531, Rosario, Argentina

    Cintia Lucía Arias, Carlos Santiago Andreo, María Fabiana Drincovich & Mariel Claudia Gerrard Wheeler

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  1. Cintia Lucía Arias
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  2. Carlos Santiago Andreo
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  3. María Fabiana Drincovich
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  4. Mariel Claudia Gerrard Wheeler
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Correspondence to Carlos Santiago Andreo.

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Arias, C.L., Andreo, C.S., Drincovich, M.F. et al. Fumarate and cytosolic pH as modulators of the synthesis or consumption of C4 organic acids through NADP-malic enzyme in Arabidopsis thaliana . Plant Mol Biol 81, 297–307 (2013). https://doi.org/10.1007/s11103-012-9999-6

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