[en] Isocitrate lyase is a key enzyme of the glyoxylate cycle. This cycle plays an essential role in cell growth on acetate, and is important for gluconeogenesis as it bypasses the two oxidative steps of the tricarboxylic acid (TCA) cycle in which CO2 is evolved. In this paper, a null icl mutant of the green microalga Chlamydomonas reinhardtii is described. Our data show that isocitrate lyase is required for growth in darkness on acetate (heterotrophic conditions), as well as for efficient growth in the light when acetate is supplied (mixotrophic conditions). Under these latter conditions, reduced acetate assimilation and concomitant reduced respiration occur, and biomass composition analysis reveals an increase in total fatty acid content, including neutral lipids and free fatty acids. Quantitative proteomic analysis by 14 N/15 N labelling was performed, and more than 1600 proteins were identified. These analyses reveal a strong decrease in the amounts of enzymes of the glyoxylate cycle and gluconeogenesis in parallel with a shift of the TCA cycle towards amino acid synthesis, accompanied by an increase in free amino acids. The decrease of the glyoxylate cycle and gluconeogenesis, as well as the decrease in enzymes involved in beta-oxidation of fatty acids in the icl mutant are probably major factors that contribute to remodelling of lipids in the icl mutant. These modifications are probably responsible for the elevation of the response to oxidative stress, with significantly augmented levels and activities of superoxide dismutase and ascorbate peroxidase, and increased resistance to paraquat.
Bantan-Polak, T., Kassai, M., and, Grant, K.B., (2001) A comparison of fluorescamine and naphthalene-2,3-dicarboxaldehyde fluorogenic reagents for microplate-based detection of amino acids. Anal. Biochem. 297, 128-136. (Pubitemid 32989342)
Barbieri, M.R., Larosa, V., Nouet, C., Subrahmanian, N., Remacle, C., and, Hamel, P.P., (2011) A forward genetic screen identifies mutants deficient for mitochondrial complex I assembly in Chlamydomonas reinhardtii. Genetics, 188, 349-358.
Baud, S., and, Lepiniec, L., (2008) Compared analysis of the regulatory systems controlling lipogenesis in hepatocytes of mice and in maturing oilseeds of Arabidopsis. C. R. Biol. 331, 737-745.
Bedhomme, M., Zaffagnini, M., Marchand, C.H., Gao, X.H., Moslonka-Lefebvre, M., Michelet, L., Decottignies, P., and, Lemaire, S.D., (2009) Regulation by glutathionylation of isocitrate lyase from Chlamydomonas reinhardtii. J. Biol. Chem. 284, 36282-36291.
Behari, R., and, Baker, A., (1993) The carboxyl terminus of isocitrate lyase is not essential for import into glyoxysomes in an in vitro system. J. Biol. Chem. 268, 7315-7322. (Pubitemid 23105623)
Benning, C., and, Somerville, C.R., (1992) Identification of an operon involved in sulfolipid biosynthesis in Rhodobacter sphaeroides. J. Bacteriol. 174, 6479-6487.
Berthold, P., Schmitt, R., and, Mages, W., (2002) An engineered Streptomyces hygroscopicus aph 7" gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii. Protist, 153, 401-412. (Pubitemid 36141164)
Bradford, M.M., (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.
Cipak, A., Hasslacher, M., Tehlivets, O., et al. (2006) Saccharomyces cerevisiae strain expressing a plant fatty acid desaturase produces polyunsaturated fatty acids and is susceptible to oxidative stress induced by lipid peroxidation. Free Radic. Biol. Med. 40, 897-906. (Pubitemid 43327323)
Cipak, A., Jaganjac, M., Tehlivets, O., Kohlwein, S.D., and, Zarkovic, N., (2008) Adaptation to oxidative stress induced by polyunsaturated fatty acids in yeast. Biochim. Biophys. Acta, 1781, 283-287.
Degu, A., Hatew, B., Nunes-Nesi, A., Shlizerman, L., Zur, N., Katz, E., Fernie, A.R., Blumwald, E., and, Sadka, A., (2011) Inhibition of aconitase in citrus fruit callus results in a metabolic shift towards amino acid biosynthesis. Planta, 234, 501-513.
Delrue, B., Fontaine, T., Routier, F., Decq, A., Wieruszeski, J.M., Van Den Koornhuyse, N., Maddelein, M.L., Fournet, B., and, Ball, S., (1992) Waxy Chlamydomonas reinhardtii: monocellular algal mutants defective in amylose biosynthesis and granule-bound starch synthase activity accumulate a structurally modified amylopectin. J. Bacteriol. 174, 3612-3620.
Doebbe, A., Keck, M., La Russa, M., Mussgnug, J.H., Hankamer, B., Tekce, E., Niehaus, K., and, Kruse, O., (2010) The interplay of proton, electron, and metabolite supply for photosynthetic H2 production in Chlamydomonas reinhardtii. J. Biol. Chem. 285, 30247-30260.
Duby, F., and, Matagne, R.F., (2001) Alteration of dark respiration and reduction of phototrophic growth in a mitochondrial DNA deletion mutant of Chlamydomonas lacking cob, nd4, and the 3' end of nd5. Plant Cell, 11, 115-125.
Duby, F., Cardol, P., Matagne, R.F., and, Remacle, C., (2001) Structure of the telomeric ends of mt DNA, transcriptional analysis and complex I assembly in the dum24 mitochondrial mutant of Chlamydomonas reinhardtii. Mol. Genet. Genomics, 266, 109-114. (Pubitemid 32778669)
Eastmond, P.J., and, Graham, I.A., (2001) Re-examining the role of the glyoxylate cycle in oilseeds. Trends Plant Sci. 6, 72-78.
Fan, J., Yan, C., Andre, C., Shanklin, J., Schwender, J., and, Xu, C., (2012) Oil accumulation is controlled by carbon precursor supply for fatty acid synthesis in Chlamydomonas reinhardtii. Plant Cell Physiol. 53, 1380-1390.
Flavell, R.B., and, Fincham, J.R., (1968) Acetate-nonutilizing mutants of Neurospora crassa. II. Biochemical deficiencies and the roles of certain enzymes. J. Bacteriol. 95, 1063-1068.
Harris, E., (1989) The Chlamydomonas Sourcebook. San Diego, CA: Academic Press.
Hayashi, Y., and, Shinozaki, A., (2012) Visualization of microbodies in Chlamydomonas reinhardtii. J. Plant. Res. 125, 579-586.
Hsieh, S.I., Castruita, M., Malasarn, D., et al. (2013) The proteome of copper, iron, zinc, and manganese micronutrient deficiency in Chlamydomonas reinhardtii. Mol. Cell. Proteomics, 12, 65-86.
Hutchings, D., Rawsthorne, S., and, Emes, M.J., (2005) Fatty acid synthesis and the oxidative pentose phosphate pathway in developing embryos of oilseed rape (Brassica napus L.). J. Exp. Bot. 56, 577-585. (Pubitemid 40295144)
Kornberg, H.L., and, Krebs, H.A., (1957) Synthesis of cell constituents from C2-units by a modified tricarboxylic acid cycle. Nature, 179, 988-991.
Kunze, M., Pracharoenwattana, I., Smith, S.M., and, Hartig, A., (2006) A central role for the peroxisomal membrane in glyoxylate cycle function. Biochim. Biophys. Acta, 1763, 1441-1452. (Pubitemid 44883390)
Lecler, R., Godaux, D., Vigeolas, H., Hiligsmann, S., Thonart, P., Franck, F., Cardol, P., and, Remacle, C., (2011) Functional analysis of hydrogen photoproduction of respiratory-deficient mutants of Chlamydomonas reinhardtii. Int. J. Hydrogen Energy, 36, 9562-9570.
Li, Y., Han, D., Hu, G., Dauvillee, D., Sommerfeld, M., Ball, S., and, Hu, Q., (2010) Chlamydomonas starchless mutant defective in ADP-glucose pyrophosphorylase hyper-accumulates triacylglycerol. Metab. Eng. 12, 387-391.
Masset, J., Hiligsmann, S., Hamilton, C., Beckers, L., Franck, F., and, Thonart, P., (2010) Effect of pH on glucose and starch fermentation in batch and sequenced batch mode by new isolated strain of hydrogen producers of Clostridium butyricum. Int. J. Hydrogen Energy, 35, 3371-3378.
Mathy, G., Cardol, P., Dinant, M., et al. (2010) Proteomic and functional characterization of a Chlamydomonas reinhardtii mutant lacking the mitochondrial alternative oxidase 1. J. Proteome Res. 9, 2825-2838.
Matsuoka, M., Ueda, Y., and, Aiba, S., (1980) Role and control of isocitrate lyase in Candida lipolytica. J. Bacteriol. 144, 692-697. (Pubitemid 11164189)
Michinaka, Y., Shimauchi, T., Aki, T., Nakajima, T., Kawamoto, S., Shigeta, S., Suzuki, O., and, Ono, K., (2003) Extracellular secretion of free fatty acids by disruption of a fatty acyl-CoA synthetase gene in Saccharomyces cerevisiae. J. Biosci. Bioeng. 95, 435-440. (Pubitemid 36688164)
Miller, R., Wu, G., Deshpande, R.R., et al. (2010) Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism. Plant Physiol. 154, 1737-1752.
Moellering, E.R., and, Benning, C., (2010) RNA interference silencing of a major lipid droplet protein affects lipid droplet size in Chlamydomonas reinhardtii. Eukaryot. Cell, 9, 97-106.
Murshed, R., Lopez-Lauri, F., and, Sallanon, H., (2008) Microplate quantification of enzymes of the plant ascorbate-glutathione cycle. Anal. Biochem. 383, 320-322.
Naumann, B., Stauber, E.J., Busch, A., Sommer, F., and, Hippler, M., (2005) N-terminal processing of Lhca3 is a key step in remodeling of the photosystem I-light-harvesting complex under iron deficiency in Chlamydomonas reinhardtii. J. Biol. Chem. 280, 20431-20441. (Pubitemid 40776742)
Nguyen, A.V., Toepel, J., Burgess, S., Uhmeyer, A., Blifernez, O., Doebbe, A., Hankamer, B., Nixon, P., Wobbe, L., and, Kruse, O., (2011) Time-course global expression profiles of Chlamydomonas reinhardtii during photo-biological H2 production. PLoS ONE, 6, e29364.
Nogales, J., Guijo, M.I., Quesada, A., and, Merchan, F., (2004) Functional analysis and regulation of the malate synthase from Chlamydomonas reinhardtii. Planta, 219, 325-331. (Pubitemid 39428790)
Nojima, Y., Kibayashi, A., Matsuzaki, H., Hatano, T., and, Fukui, S., (1999) Isolation and characterization of triacylglycerol-secreting mutant strain from yeast, Saccharomyces cerevisiae. J. Gen. Appl. Microbiol. 45, 1-6. (Pubitemid 29213636)
Peskin, A.V., and, Winterbourn, C.C., (2000) A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1). Clin. Chim. Acta, 293, 157-166. (Pubitemid 30103563)
Petridou, S., Foster, K., and, Kindle, K., (1997) Light induces accumulation of isocitrate lyase mRNA in a carotenoid-deficient mutant of Chlamydomonas reinhardtii. Plant Mol. Biol. 33, 381-392. (Pubitemid 27115271)
Porra, R.J., Thompson, W.A., and, Kriedelmann, P.E., (1989) Determination of accurate extractions and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of concentrations of chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta, 975, 384-394.
Pracharoenwattana, I., Cornah, J.E., and, Smith, S.M., (2005) Arabidopsis peroxisomal citrate synthase is required for fatty acid respiration and seed germination. Plant Cell, 17, 2037-2048.
Santiago, B., MacGilvray, M., Faustoferri, R.C., and, Quivey, R.G. Jr, (2012) The branched-chain amino acid aminotransferase encoded by ilvE is involved in acid tolerance in Streptococcus mutans. J. Bacteriol. 194, 2010-2019.
Scharnewski, M., Pongdontri, P., Mora, G., Hoppert, M., and, Fulda, M., (2008) Mutants of Saccharomyces cerevisiae deficient in acyl-CoA synthetases secrete fatty acids due to interrupted fatty acid recycling. FEBS J. 275, 2765-2778. (Pubitemid 351678664)
Schnarrenberger, C., and, Martin, W., (2002) Evolution of the enzymes of the citric acid cycle and the glyoxylate cycle of higher plants. A case study of endosymbiotic gene transfer. Eur. J. Biochem. 269, 868-883. (Pubitemid 34175407)
Silverberg, B.A., (1975) An ultrastructural and cytochemical characterization of microbodies in the green algae. Protoplasma, 83, 269-295.
Slater, T.F., (1984) Overview of methods used for detecting lipid peroxidation. Methods Enzymol. 105, 283-293. (Pubitemid 14165388)
Specht, M., Kuhlgert, S., Fufezan, C., and, Hippler, M., (2011) Proteomics to go: proteomatic enables the user-friendly creation of versatile MS/MS data evaluation workflows. Bioinformatics, 27, 1183-1184.
Tanaka, S., Ikeda, K., Miyasaka, H., Shioi, Y., Suzuki, Y., Tamoi, M., Takeda, T., Shigeoka, S., Harada, K., and, Hirata, K., (2011) Comparison of three Chlamydomonas strains which show distinctive oxidative stress tolerance. J. Biosci. Bioeng. 112, 462-468.
Terashima, M., Specht, M., Naumann, B., and, Hippler, M., (2010) Characterizing the anaerobic response of Chlamydomonas reinhardtii by quantitative proteomics. Mol. Cell. Proteomics, 9, 1514-1532.
Vigeolas, H., Duby, F., Kaymak, E., Niessen, G., Motte, P., Franck, F., and, Remacle, C., (2012) Isolation and partial characterization of mutants with elevated lipid content in Chlorella sorokiniana and Scenedesmus obliquus. J. Biotechnol. 162, 3-12.
Work, V.H., Radakovits, R., Jinkerson, R.E., Meuser, J.E., Elliott, L.G., Vinyard, D.J., Laurens, L.M., Dismukes, G.C., and, Posewitz, M.C., (2010) Increased lipid accumulation in the Chlamydomonas reinhardtii sta7-10 starchless isoamylase mutant and increased carbohydrate synthesis in complemented strains. Eukaryot. Cell, 9, 1251-1261.
Yu, L., Jiang, J., Zhang, C., et al. (2010) Glyoxylate rather than ascorbate is an efficient precursor for oxalate biosynthesis in rice. J. Exp. Bot. 61, 1625-1634.
Zaffagnini, M., Bedhomme, M., Groni, H., Marchand, C.H., Puppo, C., Gontero, B., Cassier-Chauvat, C., Decottignies, P., and, Lemaire, S.D., (2012) Glutathionylation in the photosynthetic model organism Chlamydomonas reinhardtii: a proteomic survey. Mol. Cell. Proteomics, 11, M111.014142.
Zhang, L., Happe, T., and, Melis, A., (2002) Biochemical and morphological characterization of sulfur-deprived and H2-producing Chlamydomonas reinhardtii (green alga). Planta, 214, 552-561. (Pubitemid 40831770)