Plant Molecular Biology

, Volume 71, Issue 6, pp 569–583 | Cite as

Function and regulation of the glutathione peroxidase homologous gene GPXH/GPX5 in Chlamydomonas reinhardtii

  • Beat B. Fischer
  • Régine Dayer
  • Yvonne Schwarzenbach
  • Stéphane D. Lemaire
  • Renata Behra
  • Anja Liedtke
  • Rik I. L. Eggen


When exposed to strong sunlight, photosynthetic organisms encounter photooxidative stress by the increased production of reactive oxygen species causing harmful damages to proteins and membranes. Consequently, a fast and specific induction of defense mechanisms is required to protect the organism from cell death. In Chlamydomonas reinhardtii, the glutathione peroxidase homologous gene GPXH/GPX5 was shown to be specifically upregulated by singlet oxygen formed during high light conditions presumably to prevent the accumulation of lipid hydroperoxides and membrane damage. We now showed that the GPXH protein is a thioredoxin-dependent peroxidase catalyzing the reduction of hydrogen peroxide and organic hydroperoxides. Furthermore, the GPXH gene seems to encode a dual-targeted protein, predicted to be localized both in the chloroplast and the cytoplasm, which is active with either plastidic TRXy or cytosolic TRXh1. Putative dual-targeting is achieved by alternative transcription and translation start sites expressed independently from either a TATA-box or an Initiator core promoter. Expression of both transcripts was upregulated by photooxidative stress even though with different strengths. The induction required the presence of the core promoter sequences and multiple upstream regulatory elements including a Sp1-like element and an earlier identified CRE/AP-1 homologous sequence. This element was further characterized by mutation analysis but could not be confirmed to be a consensus CRE or AP1 element. Instead, it rather seems to be another member of the large group of TGAC-transcription factor binding sites found to be involved in the response of different genes to oxidative stress.


Glutathione peroxidase Thioredoxin Singlet oxygen Dual-targeting Transcriptional regulation Chlamydomonas reinhardtii 



We thank Hans-Peter Kohler for his help in calculating enzyme kinetics and Myroslawa Miginiac-Maslow for critical reading of the manuscript. This study was financially supported by the Swiss national science foundation (grant 31003A-111919).

Supplementary material

11103_2009_9540_MOESM1_ESM.doc (374 kb)
(DOC 374 kb)


  1. Angel P, Imagawa M, Chiu R, Stein B, Imbra RJ, Rahmsdorf HJ, Jonat C, Herrlich P, Karin M (1987) Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell 49:729–739CrossRefPubMedGoogle Scholar
  2. Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399CrossRefPubMedGoogle Scholar
  3. Ausubel FM (1994) Current protocols in molecular biology. Wiley, New YorkGoogle Scholar
  4. Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472CrossRefPubMedGoogle Scholar
  5. Carrie C, Kuhn K, Murcha MW, Duncan O, Small ID, O’Toole N, Whelan J (2009) Approaches to defining dual-targeted proteins in Arabidopsis. Plant J 57:1128–1139CrossRefPubMedGoogle Scholar
  6. Chen W, Chao G, Singh KB (1996) The promoter of a H2O2-inducible, Arabidopsis glutathione S-transferase gene contains closely linked OBF- and OBP1-binding sites. Plant J 10:955–966CrossRefPubMedGoogle Scholar
  7. Chu S, Ferro TJ (2005) Sp1: regulation of gene expression by phosphorylation. Gene 348:1–11CrossRefPubMedGoogle Scholar
  8. Collin V, Issakidis-Bourguet E, Marchand C, Hirasawa M, Lancelin JM, Knaff DB, Miginiac-Maslow M (2003) The Arabidopsis plastidial thioredoxins: new functions and new insights into specificity. J Biol Chem 278:23747–23752CrossRefPubMedGoogle Scholar
  9. Daniel PB, Walker WH, Habener JF (1998) Cyclic AMP signaling and gene regulation. Annu Rev Nutr 18:353–383CrossRefPubMedGoogle Scholar
  10. Dayer R, Fischer BB, Eggen RI, Lemaire SD (2008) The peroxiredoxin and glutathione peroxidase families in Chlamydomonas reinhardtii. Genetics 179:41–57CrossRefPubMedGoogle Scholar
  11. De Cesare D, Sassone-Corsi P (2000) Transcriptional regulation by cyclic AMP-responsive factors. Prog Nucleic Acid Res Mol Biol 64:343–369CrossRefPubMedGoogle Scholar
  12. Debuchy R, Purton S, Rochaix JD (1989) The argininosuccinate lyase gene of Chlamydomonas reinhardtii: an important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARG7 locus. EMBO J 8:2803–2809PubMedGoogle Scholar
  13. Delaunay A, Pflieger D, Barrault MB, Vinh J, Toledano MB (2002) A thiol peroxidase is an H2O2 receptor and redox-transducer in gene activation. Cell 111:471–481CrossRefPubMedGoogle Scholar
  14. Deutsch PJ, Hoeffler JP, Jameson JL, Lin JC, Habener JF (1988) Structural determinants for transcriptional activation by cAMP- responsive DNA elements. J Biol Chem 263:18466–18472PubMedGoogle Scholar
  15. Droog F, Spek A, van der Kooy A, de Ruyter A, Hoge H, Libbenga K, Hooykaas P, van der Zaal B (1995) Promoter analysis of the auxin-regulated tobacco glutathione S-transferase genes Nt103–1 and Nt103–35. Plant Mol Biol 29:413–429CrossRefPubMedGoogle Scholar
  16. Ellis JG, Tokuhisa JG, Llewellyn DJ, Bouchez D, Singh K, Dennis ES, Peacock WJ (1993) Does the ocs-element occur as a functional component of the promoters of plant genes? Plant J 4:433–443CrossRefPubMedGoogle Scholar
  17. Emami KH, Navarre WW, Smale ST (1995) Core promoter specificities of the Sp1 and VP16 transcriptional activation domains. Mol Cell Biol 15:5906–5916PubMedGoogle Scholar
  18. Emami KH, Jain A, Smale ST (1997) Mechanism of synergy between TATA and initiator: synergistic binding of TFIID following a putative TFIIA-induced isomerization. Genes Dev 11:3007–3019CrossRefPubMedGoogle Scholar
  19. Fischer BB, Krieger-Liszkay A, Eggen RIL (2004) Photosensitizers neutral red (type I) and rose bengal (type II) cause light-dependent toxicity in Chlamydomonas reinhardtii and induce the Gpxh gene via increased singlet oxygen formation. Environ Sci Technol 38:6307–6313CrossRefPubMedGoogle Scholar
  20. Fischer BB, Krieger-Liszkay A, Eggen RIL (2005) Oxitative stress induced by the photosensitizers neutral red (type I) or rose bengal (type II) in the light causes different genetic responses in Chlamydomonas reinhardtii. Plant Sci 168:747–759CrossRefGoogle Scholar
  21. Fischer BB, Eggen RIL, Trebst A, Krieger-Liszkay A (2006) The glutathione peroxidase homologous gene Gpxh in Chlamydomonas reinhardtii is upregulated by singlet oxygen produced in photosystem II. Planta 223:583–590CrossRefPubMedGoogle Scholar
  22. Fischer BB, Dayer R, Wiesendanger M, Eggen RIL (2007a) Independent regulation of the GPXH gene expression by primary and secondary effects of high light stress in Chlamydomonas reinhardtii. Physiol Plant 130:195–206CrossRefGoogle Scholar
  23. Fischer BB, Krieger-Liszkay A, Hideg E, Snyrychova I, Wiesendanger M, Eggen RI (2007b) Role of singlet oxygen in chloroplast to nucleus retrograde signaling in Chlamydomonas reinhardtii. FEBS Lett 581:5555–5560CrossRefPubMedGoogle Scholar
  24. Gaber A, Tamoi M, Takeda T, Nakano Y, Shigeoka S (2001) NADPH-dependent glutathione peroxidase-like proteins (Gpx-1, Gpx-2) reduce unsaturated fatty acid hydroperoxides in Synechocystis PCC 6803. FEBS Lett 499:32–36CrossRefPubMedGoogle Scholar
  25. Garreton V, Carpinelli J, Jordana X, Holuigue L (2002) The as-1 promoter element is an oxidative stress-responsive element and salicylic acid activates it via oxidative species. Plant Physiol 130:1516–1526CrossRefPubMedGoogle Scholar
  26. Ghosh S, Wu YM, Li R, Hu YF (2005) Jun proteins modulate the ovary-specific promoter of aromatase gene in ovarian granulosa cells via a cAMP-responsive element. Oncogene 24:2236–2246CrossRefPubMedGoogle Scholar
  27. Giono LE, Varone CL, Canepa ET (2001) 5-Aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB). Biochem J 353:307–316CrossRefPubMedGoogle Scholar
  28. Gorner W, Durchschlag E, Wolf J, Brown EL, Ammerer G, Ruis H, Schuller C (2002) Acute glucose starvation activates the nuclear localization signal of a stress-specific yeast transcription factor. EMBO J 21:135–144CrossRefPubMedGoogle Scholar
  29. Guberman AS, Scassa ME, Giono LE, Varone CL, Canepa ET (2003) Inhibitory effect of AP-1 complex on 5-aminolevulinate synthase gene expression through sequestration of cAMP-response element protein (CRE)-binding protein (CBP) coactivator. J Biol Chem 278:2317–2326CrossRefPubMedGoogle Scholar
  30. Hai T, Hartman MG (2001) The molecular biology and nomenclature of the activating transcription factor/cAMP responsive element binding family of transcription factors: activating transcription factor proteins and homeostasis. Gene 273:1–11CrossRefPubMedGoogle Scholar
  31. Harris EH (1989) The Chlamydomonas sourcebook: a comprehensive guide to biology and laboratory use. Academic Press, San DiegoGoogle Scholar
  32. Herbette S, Lenne C, Leblanc N, Julien JL, Drevet JR, Roeckel-Drevet P (2002) Two GPX-like proteins from Lycopersicon esculentum and Helianthus annuus are antioxidant enzymes with phospholipid hydroperoxide glutathione peroxidase and thioredoxin peroxidase activities. Eur J Biochem 269:2414–2420CrossRefPubMedGoogle Scholar
  33. Herbette S, Roeckel-Drevet P, Drevet JR (2007) Seleno-independent glutathione peroxidases—more than simple antioxidant scavengers. FEBS J 274:2163–2180CrossRefPubMedGoogle Scholar
  34. Iqbal A, Yabuta Y, Takeda T, Nakano Y, Shigeoka S (2006) Hydroperoxide reduction by thioredoxin-specific glutathione peroxidase isoenzymes of Arabidopsis thaliana. FEBS J 273:5589–5597CrossRefPubMedGoogle Scholar
  35. Jacquot JP, Rivera-Madrid R, Marinho P, Kollarova M, Le Marechal P, Miginiac-Maslow M, Meyer Y (1994) Arabidopsis thaliana NADPH thioredoxin reductase. cDNA characterization and expression of the recombinant protein in Escherichia coli. J Mol Biol 235:1357–1363CrossRefPubMedGoogle Scholar
  36. Jaeger J, Sorensen K, Wolff SP (1994) Peroxide accumulation in detergents. J Biochem Biophys Methods 29:77–81CrossRefPubMedGoogle Scholar
  37. Jung BG, Lee KO, Lee SS, Chi YH, Jang HH, Kang SS, Lee K, Lim D, Yoon SC, Yun DJ, Inoue Y, Cho MJ, Lee SY (2002) A Chinese cabbage cDNA with high sequence identity to phospholipid hydroperoxide glutathione peroxidases encodes a novel isoform of thioredoxin-dependent peroxidase. J Biol Chem 277:12572–12578CrossRefPubMedGoogle Scholar
  38. Juven-Gershon T, Hsu JY, Theisen JW, Kadonaga JT (2008) The RNA polymerase II core promoter—the gateway to transcription. Curr Opin Cell Biol 20:253–259CrossRefPubMedGoogle Scholar
  39. Karin M, Liu Z, Zandi E (1997) AP-1 function and regulation. Curr Opin Cell Biol 9:240–246CrossRefPubMedGoogle Scholar
  40. Karin M, Takahashi T, Kapahi P, Delhase M, Chen Y, Makris C, Rothwarf D, Baud V, Natoli G, Guido F, Li N (2001) Oxidative stress and gene expression: the AP-1 and NF-kappaB connections. Biofactors 15:87–89CrossRefPubMedGoogle Scholar
  41. Kindle KL (1990) High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 87:1228–1232CrossRefPubMedGoogle Scholar
  42. Kirchsteiger K, Pulido P, Gonzalez M, Cejudo FJ (2009) NADPH thioredoxin reductase C controls the redox status of chloroplast 2-Cys peroxiredoxins in Arabidopsis thaliana. Mol Plant 2:298–307CrossRefPubMedGoogle Scholar
  43. Kropat J, von Gromoff ED, Muller FW, Beck CF (1995) Heat shock and light activation of a Chlamydomonas HSP70 gene are mediated by independent regulatory pathways. Mol Gen Genet 248:727–734CrossRefPubMedGoogle Scholar
  44. Lam E, Benfey PN, Gilmartin PM, Fang RX, Chua NH (1989) Site-specific mutations alter in vitro factor binding and change promoter expression pattern in transgenic plants. Proc Natl Acad Sci USA 86:7890–7894CrossRefPubMedGoogle Scholar
  45. Ledford HK, Chin BL, Niyogi KK (2007) Acclimation to singlet oxygen stress in Chlamydomonas reinhardtii. Eukaryot Cell 6:919–930CrossRefPubMedGoogle Scholar
  46. Leisinger U, Rüfenacht K, Zehnder AJB, Eggen RIL (1999) Structure of a glutathione peroxidase homologous gene involved in the oxidative stress response in Chlamydomonas reinhardtii. Plant Sci 149:139–149CrossRefGoogle Scholar
  47. Leisinger U, Rüfenacht K, Fischer B, Pesaro M, Spengler A, Zehnder AJB, Eggen RIL (2001) The glutathione peroxidase homologous gene from Chlamydomonas reinhardtii is transcriptionally up-regulated by singlet oxygen. Plant Mol Biol 46:395–408CrossRefPubMedGoogle Scholar
  48. Lemaire SD, Collin V, Keryer E, Quesada A, Miginiac-Maslow M (2003) Characterization of thioredoxin y, a new type of thioredoxin identified in the genome of Chlamydomonas reinhardtii. FEBS Lett 543:87–92CrossRefPubMedGoogle Scholar
  49. Lemaire SD, Guillon B, Le Marechal P, Keryer E, Miginiac-Maslow M, Decottignies P (2004) New thioredoxin targets in the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 101:7475–7480CrossRefPubMedGoogle Scholar
  50. Lemaire SD, Michelet L, Zaffagnini M, Massot V, Issakidis-Bourguet E (2007) Thioredoxins in chloroplasts. Curr Genet 51:343–365CrossRefPubMedGoogle Scholar
  51. Li Z, Wakao S, Fischer BB, Niyogi KK (2009) Sensing and responding to excess light. Annu Rev Plant Biol 60:239–260CrossRefPubMedGoogle Scholar
  52. Lodha M, Schulz-Raffelt M, Schroda M (2008) A new assay for promoter analysis in Chlamydomonas reveals roles for heat shock elements and the TATA box in HSP70A promoter-mediated activation of transgene expression. Eukaryot Cell 7:172–176CrossRefPubMedGoogle Scholar
  53. Maiorino M, Gregolin C, Ursini F (1990) Phospholipid hydroperoxide glutathione peroxidase. Methods Enzymol 186:448–457CrossRefPubMedGoogle Scholar
  54. Maiorino M, Thomas JP, Girotti AW, Ursini F (1991) Reactivity of phospholipid hydroperoxide glutathione-peroxidase with membrane and lipoprotein lipid hydroperoxides. Free Radic Res Commun 12–3:131–135CrossRefGoogle Scholar
  55. Mayr B, Montminy M (2001) Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol 2:599–609CrossRefPubMedGoogle Scholar
  56. Merchant SS, Prochnik SE, Vallon O et al (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318:245–250CrossRefPubMedGoogle Scholar
  57. Mittag M (1996) Conserved circadian elements in phylogenetically diverse algae. Proc Natl Acad Sci USA 93:14401–14404CrossRefPubMedGoogle Scholar
  58. Nakamura M, Tsunoda T, Obokata J (2002) Photosynthesis nuclear genes generally lack TATA-boxes: a tobacco photosystem I gene responds to light through an initiator. Plant J 29:1–10CrossRefPubMedGoogle Scholar
  59. Narusaka Y, Narusaka M, Seki M, Fujita M, Ishida J, Nakashima M, Enju A, Sakurai T, Satou M, Kamiya A, Park P, Kobayashi M, Shinozaki K (2003) Expression profiles of Arabidopsis phospholipase A IIA gene in response to biotic and abiotic stresses. Plant Cell Physiol 44:1246–1252CrossRefPubMedGoogle Scholar
  60. Navrot N, Collin V, Gualberto J, Gelhaye E, Hirasawa M, Rey P, Knaff DB, Issakidis E, Jacquot JP, Routhier N (2006) Plant glutathione peroxidases are functional peroxiredoxins distributed in several subcellular compartments and regulated during biotic and abiotic stresses. Plant Physiol 142:1364–1379CrossRefPubMedGoogle Scholar
  61. Ndamukong I, Abdallat AA, Thurow C, Fode B, Zander M, Weigel R, Gatz C (2007) SA-inducible Arabidopsis glutaredoxin interacts with TGA factors and suppresses JA-responsive PDF1.2 transcription. Plant J 50:128–139CrossRefPubMedGoogle Scholar
  62. Pérez-Ruiz JM, Spinola MC, Kirchsteiger K, Moreno J, Sahrawy M, Cejudo FJ (2006) Rice NTRC is a high-efficiency redox system for chloroplast protection against oxidative damage. Plant Cell 18:2356–2368CrossRefPubMedGoogle Scholar
  63. Rodriguez Milla MA, Maurer A, Rodriguez Huete A, Gustafson JP (2003) Glutathione peroxidase genes in Arabidopsis are ubiquitous and regulated by abiotic stresses through diverse signaling pathways. Plant J 36:602–615CrossRefPubMedGoogle Scholar
  64. Rouhier N, Jacquot JP (2005) The plant multigenic family of thiol peroxidases. Free Radic Biol Med 38:1413–1421CrossRefPubMedGoogle Scholar
  65. Sambrook J, Maniatis T, Fritsch EF (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  66. Schenk PM, Baumann S, Mattes R, Steinbiss HH (1995) Improved high-level expression system for eukaryotic genes in Escherichia coli using T7 RNA polymerase and rare ArgtRNAs. Biotechniques 19:196–200PubMedGoogle Scholar
  67. Serrato AJ, Pérez-Ruiz JM, Spinola MC, Cejudo FJ (2004) A novel NADPH thioredoxin reductase, localized in the chloroplast, which deficiency causes hypersensitivity to abiotic stress in Arabidopsis thaliana. J Biol Chem 279:43821–43827CrossRefPubMedGoogle Scholar
  68. Shahmuradov IA, Gammerman AJ, Hancock JM, Bramley PM, Solovyev VV (2003) PlantProm: a database of plant promoter sequences. Nucleic Acids Res 31:114–117CrossRefPubMedGoogle Scholar
  69. Smale ST (1997) Transcription initiation from TATA-less promoters within eukaryotic protein-coding genes. Biochim Biophys Acta 1351:73–88PubMedGoogle Scholar
  70. Smale ST, Kadonaga JT (2003) The RNA polymerase II core promoter. Annu Rev Biochem 72:449–479CrossRefPubMedGoogle Scholar
  71. Stein M, Jacquot JP, Jeannette E, Decottignies P, Hodges M, Lancelin JM, Mittard V, Schmitter JM, Miginiac-Maslow M (1995) Chlamydomonas reinhardtii thioredoxins: structure of the genes coding for the chloroplastic m and cytosolic h isoforms; expression in Escherichia coli of the recombinant proteins, purification and biochemical properties. Plant Mol Biol 28:487–503CrossRefPubMedGoogle Scholar
  72. Tanaka T, Izawa S, Inoue Y (2005) GPX2, encoding a phospholipid hydroperoxide glutathione peroxidase homologue, codes for an atypical 2-Cys peroxiredoxin in Saccharomyces cerevisiae. J Biol Chem 280:42078–42087CrossRefPubMedGoogle Scholar
  73. Thatcher LF, Carrie C, Andersson CR, Sivasithamparam K, Whelan J, Singh KB (2007) Differential gene expression and subcellular targeting of Arabidopsis glutathione S-transferase F8 is achieved through alternative transcription start sites. J Biol Chem 282:28915–28928CrossRefPubMedGoogle Scholar
  74. Thomas JP, Maiorino M, Ursini F, Girotti AW (1990) Protective action of phospholipid hydroperoxide glutathione peroxidase against membrane-damaging lipid peroxidation. In situ reduction of phospholipid and cholesterol hydroperoxides. J Biol Chem 265:454–461PubMedGoogle Scholar
  75. Ulmasov T, Hagen G, Guilfoyle T (1994) The ocs element in the soybean GH2/4 promoter is activated by both active and inactive auxin and salicylic acid analogues. Plant Mol Biol 26:1055–1064CrossRefPubMedGoogle Scholar
  76. Ursini F, Maiorino M, Brigelius-Flohe R, Aumann KD, Roveri A, Schomburg D, Flohe L (1995) Diversity of glutathione peroxidases. Methods Enzymol 252:38–53CrossRefPubMedGoogle Scholar
  77. van der Zaal BJ, Droog FN, Pieterse FJ, Hooykaas PJ (1996) Auxin-sensitive elements from promoters of tobacco GST genes and a consensus as-1-like element differ only in relative strength. Plant Physiol 110:79–88CrossRefPubMedGoogle Scholar
  78. Wasserman WW, Fahl WE (1997) Functional antioxidant responsive elements. Proc Natl Acad Sci USA 94:5361–5366CrossRefPubMedGoogle Scholar
  79. Yamamoto YY, Ichida H, Abe T, Suzuki Y, Sugano S, Obokata J (2007) Differentiation of core promoter architecture between plants and mammals revealed by LDSS analysis. Nucleic Acids Res 35:6219–6226CrossRefPubMedGoogle Scholar
  80. Yoshimura K, Miyao K, Gaber A, Takeda T, Kanaboshi H, Miyasaka H, Shigeoka S (2004) Enhancement of stress tolerance in transgenic tobacco plants overexpressing Chlamydomonas glutathione peroxidase in chloroplasts or cytosol. Plant J 37:21–33CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Beat B. Fischer
    • 1
  • Régine Dayer
    • 1
  • Yvonne Schwarzenbach
    • 1
  • Stéphane D. Lemaire
    • 2
  • Renata Behra
    • 1
  • Anja Liedtke
    • 1
  • Rik I. L. Eggen
    • 1
  1. 1.Department of Environmental ToxicologyEawag, Swiss Federal Institute of Aquatic Science and TechnologyDuebendorfSwitzerland
  2. 2.Institut de Biotechnologie des Plantes, Unité Mixte de Recherche 8618Centre National de la Recherche ScientifiqueOrsay CedexFrance

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