Advertisement

Plant Molecular Biology

, Volume 63, Issue 2, pp 273–287 | Cite as

Aconitase plays a role in regulating resistance to oxidative stress and cell death in Arabidopsis and Nicotiana benthamiana

  • Wolfgang Moeder
  • Olga del Pozo
  • Duroy A. Navarre
  • Gregory B. Martin
  • Daniel F. Klessig
Article

Abstract

In animals, aconitase is a bifunctional protein. When an iron-sulfur cluster is present in its catalytic center, aconitase displays enzymatic activity; when this cluster is lost, it switches to an RNA-binding protein that regulates the translatability or stability of certain transcripts. To investigate the role of aconitase in plants, we assessed its ability to bind mRNA. Recombinant aconitase failed to bind an iron responsive element (IRE) from the human ferritin gene. However, it bound the 5′ UTR of the Arabidopsis chloroplastic CuZn superoxide dismutase 2 (CSD2) mRNA, and this binding was specific. Arabidopsis aconitase knockout (KO) plants were found to have significantly less chlorosis after treatment with the superoxide-generating compound, paraquat. This phenotype correlated with delayed induction of the antioxidant gene GST1, suggesting that these KO lines are more tolerant to oxidative stress. Increased levels of CSD2 mRNAs were observed in the KO lines, although the level of CSD2 protein was not affected. Virus-induced gene silencing (VIGS) of aconitase in Nicotiana benthamiana caused a 90% reduction in aconitase activity, stunting, spontaneous necrotic lesions, and increased resistance to paraquat. The silenced plants also had less cell death after transient co-expression of the AvrPto and Pto proteins or the pro-apoptotic protein Bax. Following inoculation with Pseudomonas syringae pv. tabaci carrying avrPto, aconitase-silenced N. benthamiana plants expressing the Pto transgene displayed a delayed hypersensitive response (HR) and supported higher levels of bacterial growth. Disease-associated cell death in N. benthamiana inoculated with P. s. pv. tabaci was also reduced. Taken together, these results suggest that aconitase plays a role in mediating oxidative stress and regulating cell death.

Keywords

Aconitase Arabidopsis Cell death Nicotiana benthamiana Oxidative stress Superoxide dismutase RNA-binding VIGS 

Abbreviations

CSD2

CuZn superoxide dismutase 2

GMSA

Gel mobility shift assay

IRE

Iron responsive element

IRP-1

Iron regulatory protein 1

mt

mitochondrial

UTR

Untranslated region

VIGS

Virus-induced gene silencing

Notes

Acknowledgments

We thank Dr. P. Perez for the Arabidopsis aconitase clone, Dr. Kühn for the IRP-1 clone and the pSPT-fer plasmid, Dr. D. Kliebenstein for the CSD2 antibodies, and D’Maris Dempsey for critical reading of the manuscript. This work was supported by grant MCB-0110404 (D.F.K.) and grant DBI-116076 (G.B.M.) from the National Science Foundation and by a fellowship from the Deutsche Forschungsgemeinschaft (MO 955/1-1) to W.M.

References

  1. Abarca D, Roldán M, Martín M, Sabater B (2001) Arabidopsis thaliana ecotype Cvi shows increased tolerance to photo-oxidative stress and contains a new chloroplastic copper/zinc superoxide dismutase isoenzyme. J Exp Bot 52:1417–1425PubMedCrossRefGoogle Scholar
  2. Ahlfors R, Lang S, Overmyer K, Jaspers P, Brosche M, Tauriainen A, Kollist H, Tuominen H, Belles-Boix E, Piippo M, Inze D, Palva ET, Kangasjarvi J (2004) Arabidopsis radical-induced cell death1 belongs to the WWE protein–protein interaction domain protein family and modulates abscisic acid, ethylene, and methyl jasmonate responses. Plant Cell 16:1925–1937PubMedCrossRefGoogle Scholar
  3. Alonso JM, Stepanova AN, Leisse TJ et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301:653–657PubMedCrossRefGoogle Scholar
  4. Alvarez ME, Pennell RI, Meijer P-J, Ishikawa A, Dixon RA, Lamb C (1998) Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity. Cell 92:773–784PubMedCrossRefGoogle Scholar
  5. Arnon DI (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol 24:1–15PubMedGoogle Scholar
  6. Basilion JP, Rouault TA, Massinople CM, Klausner RD, Burgess WH (1994) The iron-responsive element-binding protein: localization of the RNA-binding site to the aconitase active-site cleft. Proc Natl Acad Sci USA 91:574–578PubMedCrossRefGoogle Scholar
  7. Bowling SA, Guo A, Cao H, Gordon AS, Klessig DF, Dong X (1994) A mutation in Arabidopsis that leads to constitutive expression of systemic acquired resistance. Plant Cell 6:1845–1857PubMedCrossRefGoogle Scholar
  8. Brouquisse R, Nishimura M, Gaillard J, Douce R (1987) Characterization of a cytosolic aconitase in higher plant cells. Plant Physiol 84:1402–1407PubMedGoogle Scholar
  9. Cabiscol E, Piulats E, Echave P, Herrero E, Ros J (2000) Oxidative stress promotes specific damage in Saccharomyces cerevisiae. J Biol Chem 275:27393–27398PubMedGoogle Scholar
  10. Carrari F, Nunes-Nesi A, Gibon Y, Lytovchenko A, Loureiro EM, Fernie AR (2003) Reduced expression of aconitase results in an enhanced rate of photosynthesis and marked shifts in carbon partitioning in illuminated leaves of wild species tomato. Plant Physiol 133:1322–1335PubMedCrossRefGoogle Scholar
  11. Castro L, Rodriguez M, Radi R (1994) Aconitase is readily inactivated by peroxynitrite, but not by its precursor, nitric oxide. J Biol Chem 269:29409–29415PubMedGoogle Scholar
  12. Chen XJ, Wang X, Kaufman BA, Butow RA (2005) Aconitase couples metabolic regulation to mitochondrial DNA maintenance. Science 307:714–717PubMedCrossRefGoogle Scholar
  13. Courtois-Verniquet F, Douce R (1993) Lack of aconitase in glyoxisomes and peroxisomes. Biochem J 294:103–107PubMedGoogle Scholar
  14. De Bellis L, Tsugeki R, Alpi A, Nishimura M (1993) Purification and characterization of aconitase isoforms from etiolated pumpkin cotyledons. Physiol Plant 88:485–492CrossRefGoogle Scholar
  15. del Pozo O, Pedley K, Martin GB (2004) MAPKKKα is a positive regulator of cell death associated with both plant immunity and disease. EMBO J 23:3072–3082PubMedCrossRefGoogle Scholar
  16. Dempsey D, Shah J, Klessig DF (1999) Salicylic acid and disease resistance in plants. Crit Rev Plant Sci 18:547–575CrossRefGoogle Scholar
  17. Eanes RZ, Kun E (1971) Separation and characterization of aconitate hydratase isoenzymes from pig tissues. Biochim Biophys Acta 227:204–210PubMedGoogle Scholar
  18. Fujibe T, Saji H, Arakawa K, Yabe N, Takeuchi Y, Yamamoto KT (2004) A methyl viologen-resistant mutant of Arabidopsis, which is allelic to ozone-sensitive rcd1, is tolerant to supplemental ultraviolet-B irradiation. Plant Physiol 134:275–285PubMedCrossRefGoogle Scholar
  19. Gangloff SP, Marguet D, Lauquin GJ (1990) Molecular cloning of the yeast mitochondrial aconitase gene (ACO1) and evidence of a synergistic regulation of expression by glucose plus glutamate. Mol Cell Biol 10:3552–3561Google Scholar
  20. Grant JJ, Yun B-W, Loake GJ (2000) Oxidative burst and cognate redox signalling reported by luciferase imaging: identification of a signal network that functions independently of ethylene, SA and Me-JA but is dependent on MAPKK activity. Plant J 24:569–582PubMedCrossRefGoogle Scholar
  21. Hayashi M, De Bellis L, Alpi A, Nishimura M (1995) Cytosolic aconitase participates in the glyoxylate cycle in etiolated pumpkin cotyledons. Plant Cell Physiol 36:669–680PubMedGoogle Scholar
  22. He X, Anderson JC, del Pozo O, Gu Y-Q, Tang X, Martin GB (2004) Silencing of subfamily I of protein phosphatase 2A catalytic subunits results in inactivation of plant defense responses and localized cell death. Plant J 38:563–577PubMedCrossRefGoogle Scholar
  23. Heazlewood JL, Tonti-Filippini JS, Gout AM, Day DA, Whelan J, Millar AH (2004) Experimental analysis of the Arabidopsis mitochondrial proteome highlights signaling and regulatory components, provides assessment of targeting prediction programs, and indicates plant-specific mitochondrial proteins. Plant Cell 16:241–256PubMedCrossRefGoogle Scholar
  24. Hentze MW, Kühn LC (1996) Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc Natl Acad Sci USA 93:8175–8182PubMedCrossRefGoogle Scholar
  25. Hirling H, Henderson BR, Kühn LC (1994) Mutational analysis of the (4Fe-4S)-cluster converting iron regulatory factor from its RNA-binding form to cytoplasmic aconitase. EMBO J 13:453–461PubMedGoogle Scholar
  26. Kaldy P, Menotti E, Moret R, Kühn LC (1999) Identification of RNA-binding surfaces in IRP-1. EMBO J 18:6073–6083PubMedCrossRefGoogle Scholar
  27. Kennedy MC, Mende-Mueller L, Blondin GA, Beinert H (1992) Purification and characterization of cytosolic aconitase from beef liver and its relationship to the iron-responsive element binding protein. Proc Natl Acad Sci USA 89:11730–11734PubMedCrossRefGoogle Scholar
  28. Klausner RD, Rouault TA, Harford JB (1993) Regulating the fate of mRNA: control of cellular iron metabolism. Cell 72:19–26PubMedCrossRefGoogle Scholar
  29. Kliebenstein DJ, Monde RA, Last RL (1998) Superoxide dismutase in Arabidopsis: an eclectic enzyme family with disparate regulation and protein localization. Plant Physiol 118:637–650PubMedCrossRefGoogle Scholar
  30. Kruft V, Eubel H, Jansch L, Werhahn W, Braun HP (2001) Proteomic approach to identify novel mitochondrial proteins in Arabidopsis. Plant Physiol 127:1694–1710PubMedCrossRefGoogle Scholar
  31. Lacomme C, Santa Cruz S (1999) Bax-induced cell death in tobacco is similar to the hypersensitive response. Proc Natl Acad Sci USA 96:7956–7961PubMedCrossRefGoogle Scholar
  32. Lamb CJ, Dixon RA (1997) The oxidative burst in plant disease resistance. Annu Rev Plant Physiol Plant Mol Biol 48:251–275PubMedCrossRefGoogle Scholar
  33. Lescure AM, Proudhon D, Pesey H, Ragland M, Theil EC, Briat JF (1991) Ferritin gene transcription is regulated by iron in soybean cell cultures. Proc Natl Acad Sci USA 88:8222–8226PubMedCrossRefGoogle Scholar
  34. Lobréaux S, Massenet O, Briat JF (1992) Iron induces ferritin synthesis in maize plantlets. Plant Mol Biol 19:563–575PubMedCrossRefGoogle Scholar
  35. Mathews DH, Sabina J, Zuker M, Turner DH (1999) Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol 288:911–940PubMedCrossRefGoogle Scholar
  36. Millar AH, Sweetlove LJ, Giege P, Leaver CJ (2001) Analysis of the Arabidopsis mitochondrial proteome. Plant Physiol 127:1711–1727PubMedCrossRefGoogle Scholar
  37. Müllner EW, Neupert B, Kühn LC (1989) A specific mRNA binding factor regulates the iron-dependent stability of cytoplasmic transferring receptor mRNA. Cell 58:373–382PubMedCrossRefGoogle Scholar
  38. Navarre DA, Wendehenne D, Durner J, Noad R, Klessig DF (2000) Nitric oxide modulates the activity of tobacco aconitase. Plant Physiol 122:573–582PubMedCrossRefGoogle Scholar
  39. Overmyer K, Tuominen H, Kettunen R, Betz C, Langebartels C, Sandermann H, Kangasjarvi J (2000) Ozone-sensitive Arabidopsis rcd1 mutant reveals opposite roles for ethylene and jasmonate signaling pathways in regulating superoxide-dependent cell death. Plant Cell 12:1849–1862PubMedCrossRefGoogle Scholar
  40. Pedley KF, Martin GB (2003) Molecular basis of Pto-mediated resistance to bacterial speck disease in tomato. Annu Rev Phytopathol 41:215–243PubMedCrossRefGoogle Scholar
  41. Pesole G, Liuni S, D’Souza M (2000) PatSearch: a pattern matcher software that finds functional elements in nucleotide and protein sequences and assesses their statistical significance. Bioinformatics 16:439–450PubMedCrossRefGoogle Scholar
  42. Peyret P, Perez P, Alric M (1995) Structure, genomic organization and expression of the Arabidopsis thaliana aconitase gene. J Biol Chem 270:8131–8137PubMedCrossRefGoogle Scholar
  43. Philpott CC, Klausner RD, Rouault TA (1994) The bifunctional iron-responsive element binding protein/cytosolic aconitase: the role of active site residues in ligand binding and regulation. Proc Natl Acad Sci USA 91:7321–7325PubMedCrossRefGoogle Scholar
  44. Povey S, Slaughter CA, Wilson DE, Gormley IP, Buckton KE, Perry P, Bobrow M (1976) Evidence for the assignment of the loci AK1, AK3 and ACONs to chromosome 9 in man. Ann Hum Genet 39:413–422PubMedGoogle Scholar
  45. Regev-Rudzki N, Karniely S, Ben-Haim NN, Pinnes O (2005) Yeast aconitase in two locations and two metabolic pathways: seeing small amounts is believing. Mol Biol Cell 16:4163–4171PubMedCrossRefGoogle Scholar
  46. Rizhsky L, Liang H, Mittler R (2003) The water-water cycle is essential for chloroplast protection in the absence of stress. J Biol Chem 278:38921–38925PubMedCrossRefGoogle Scholar
  47. Rommens CMT, Salmeron JM, Oldroyd GED, Staskawicz BJ (1995) Intergenic transfer and functional expression of the tomato disease resistance gene Pto. Plant Cell 7:1537–1544PubMedCrossRefGoogle Scholar
  48. Rosso MG, Li Y, Strizhov N, Reiss B, Dekker K, Weisshaar B (2003) An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics. Plant Mol Biol 53:247–259PubMedCrossRefGoogle Scholar
  49. Rothenberger S, Müllner EW, Kühn LC (1990) The mRNA binding protein which controls ferritin and transferrin receptor expression is conserved during evolution. Nucleic Acid Res 18:1175–1179PubMedGoogle Scholar
  50. Schnarrenberger C, Martin W (2002) Evolution of the enzymes of the citric acid cycle and the glyoxylate cycle of higher plants. Eur J Biochem 269:868–883PubMedCrossRefGoogle Scholar
  51. Sen Gupta A, Heinen JL, Holaday AS, Burke JJ, Allen RD (1993) Increased resistance to oxidative stress in transgenic plants that overexpress chloroplastic Cu/Zn superoxide dismutase. Proc Natl Acad Sci USA 90:1629–1633CrossRefGoogle Scholar
  52. Shah J, Kachroo P, Klessig DF (1999) The Arabidopsis ssi1 mutation restores pathogenesis-related gene expression in npr1 plants and renders defensin gene expression SA dependent. Plant Cell 11:191–206PubMedCrossRefGoogle Scholar
  53. Slaughter CA, Povey S, Carritt B, Solomon E, Bobrow M (1978) Assignment of the locus ACONM to chromosome 22. Cytogenet Cell Genet 22:223–225PubMedGoogle Scholar
  54. Slaymaker DH, Navarre DA, Clark D, del Pozo O, Martin GB, Klessig DF (2002) The tobacco salicylic acid-binding protein (SABP) 3 is the chloroplast carbonic anhydrase, which exhibits antioxidant activity and plays a role in the hypersensitive defense response. Proc Natl Acad Sci USA 99:11640–11645PubMedCrossRefGoogle Scholar
  55. Sweetlove LJ, Heazlewood JL, Herald V, Holtzapffel R, Day DA, Leaver CJ, Millar AH (2002) The impact of oxidative stress on Arabidopsis mitochondria. Plant J 32:891–904PubMedCrossRefGoogle Scholar
  56. Tang Y, Guest JR (1999) Direct evidence for mRNA binding and post-transcriptional regulation by Escherichia coli aconitases. Microbiology 145:3069–3079PubMedGoogle Scholar
  57. Tang Y, Quail MA, Artymiuk PJ, Guest JR, Green J (2002) Escherichia coli aconitases and oxidative stress: post-transcriptional regulation of sodA expression. Microbiology 148:1027–1037PubMedGoogle Scholar
  58. Verniquet F, Gaillard J, Neuburger M, Douce R (1991) Rapid inactivation of plant aconitase by hydrogen peroxide. Biochem J 276:643–648PubMedGoogle Scholar
  59. Wei J, Theil EC (2000) Identification and characterization of the iron regulatory elementin the ferritin gene of a plant (soybean). J Biol Chem 275:17488–17493PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Wolfgang Moeder
    • 1
    • 2
  • Olga del Pozo
    • 1
    • 3
  • Duroy A. Navarre
    • 4
    • 5
  • Gregory B. Martin
    • 1
  • Daniel F. Klessig
    • 1
  1. 1.Boyce Thompson Institute for Plant ResearchIthacaUSA
  2. 2.Department of Cell and Systems BiologyUniversity of TorontoTorontoCanada
  3. 3.Instituto de Bioquímica Vegetal y FotosíntesisSevillaSpain
  4. 4.Agricultural Research ServiceUSDAProsserUSA
  5. 5.Department Plant PathologyWashington State UniversityProsserUSA

Personalised recommendations