Planta

, 224:300 | Cite as

Rice ascorbate peroxidase gene family encodes functionally diverse isoforms localized in different subcellular compartments

  • Felipe Karam Teixeira
  • Larissa Menezes-Benavente
  • Vinícius Costa Galvão
  • Rogério Margis
  • Márcia Margis-Pinheiro
Original Article

Abstract

Aerobic organisms evolved a complex antioxidant system, which protect the cells against oxidative damage caused by partially reduced oxygen intermediates, also known as reactive oxygen species. In plants, ascorbate peroxidases (EC, 1.11.1.11) catalyze the conversion of H2O2 to H2O, using ascorbate as the specific electron donor in this enzymatic reaction. Previously, eight APx genes were identified in the rice (Oryza sativa L.) genome through in silico analysis: two cytosolic isoforms, two putative peroxisomal isoforms, and four putative chloroplastic ones. Using gene-specific probes, we confirmed the presence of the eight APx genes in the rice genome by Southern blot hybridization. Transcript accumulation analysis showed specific expression patterns for each member of the APx family according to developmental stage and in response to salt stress, revealing the complexity of the antioxidant system in plants. Finally, the subcellular localization of rice APx isoforms was determined using GFP-fusion proteins in BY-2 tobacco cells. In agreement with the initial prediction, OSAPX3 was localized in the peroxisomes. On the other hand, the OSAPX6-GFP fusion protein was found in mitochondria of the BY-2 cells, in contrast to the chloroplastic location predicted by sequence analysis. Our findings reveal the functional diversity of the rice APx genes and suggest complementation and coordination of the antioxidant defenses in different cellular compartments during development and abiotic stress.

Keywords

Antioxidant metabolism Ascorbate peroxidase BY-2 tobacco cells Green fluorescent protein Oryza 

Abbreviations

APx

Ascorbate peroxidase

CaMV

Cauliflower mosaic virus

CAT

Catalase

DHAR

Dehydroascorbate reductase

DIC

Differential interference contrast

EST

Expressed sequence tag

GFP

Green fluorescent protein

GPx

Glutathione peroxidase

GR

Glutathione reductase

KO

Knock-out mutants

MDHAR

Monodehydroascorbate reductase

PCD

Programmed cell death

pER

Peroxisomal endoplasmatic reticulum

Prx

Peroxidase

ROS

Reactive oxygen species

SOD

Superoxide dismutase

SRE

Splicing regulatory cis-element

TMRE

Tetramethylrhodamine ethyl ester

3´-UTR

3´untranslated region

References

  1. Agrawal GK, Jwa N, Iwahashi H, Rakwal R (2003) Importance of ascorbate peroxidases OsAPx1 and OsAPx2 in the rice pathogen response pathway and growth and reproduction revealed by their transcriptional profiling. Gene 322:93–103CrossRefPubMedGoogle Scholar
  2. Alscher RG, Donahue JL, Cramer CL (1997) Reactive oxygen species and antioxidants: relationships in green cells. Physiol Plant 100:224–233CrossRefGoogle Scholar
  3. Asada K (1999) The water-water cycle in chloroplasts: scavenging of active oxygen and dissipation of excess photons. Annu Rev Plant Physiol Plant Mol Biol 50:601–639CrossRefPubMedGoogle Scholar
  4. Causse MA et al (1994) Saturated molecular map of the rice genome based on an interspecific backcross population. Genetics 138:1251–1274PubMedGoogle Scholar
  5. Chang CC, Ball L, Fryer MJ, Baker N, Karpinski S, Mullineaux PM (2004) Induction of ASCORBATE PEROXIDASE 2 expression in wounded Arabidopsis leaves does not involve known wound-signalling pathways but is associated with changes in photosynthesis. Plant J 38:499–511CrossRefPubMedGoogle Scholar
  6. Chew O, Whelan J (2004) Just read the message: a model for sorting of proteins between mitochondria and chloroplasts. Trends Plant Sci 9:318–319CrossRefPubMedGoogle Scholar
  7. Chew O, Rudhe C, Glaser E, Whelan J (2003a) Characterization of the targeting signal of dual-targeted pea glutathione reductase. Plant Mol Biol 53:341–356CrossRefGoogle Scholar
  8. Chew O, Whelan J, Millar H (2003b) Molecular definition of the ascorbate-glutathione cycle in Arabidopsis mitochondria reveals dual targeting of antioxidant defenses in plants. J Biol Chem 278:46869–46877CrossRefGoogle Scholar
  9. Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci 81:1991–1995PubMedCrossRefGoogle Scholar
  10. Danna CH, Bartoli CG, Sacco F, Ingala LR, Santa-María GE, Guiamet JJ, Ugalde RA (2003) Thylakoid-bound ascorbate peroxidase mutant exhibits impaired electron transport and photosynthetic activity. Plant Physiol 132:2116–2125CrossRefPubMedGoogle Scholar
  11. Dat J, Vandenabeele S, Vranová E, Van Montagu M, Inzé D, Van Breusegem F (2000) Dual action of the active oxygen species during plant stress response. Cell Mol Life Sci 57:779–795CrossRefPubMedGoogle Scholar
  12. Davletova S, Rizhsky L, Liang H, Shengqiang Z, Oliver DJ, Coutu J, Shulaev V, Schlauch K, Mittler R (2005) Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of arabidopsis. Plant Cell 17:268–281CrossRefPubMedGoogle Scholar
  13. De Leonardis S, Dipierro N, Dipierro S (2000) Purification and characterization of an ascorbate peroxidase from potato tuber mitochondria. Plant Physiol Biochem 38:773–779CrossRefGoogle Scholar
  14. Feinberg AP, Vogelstein B (1983). A technique for radiolabelling DNA restriction endonuclease fragments to high specific affinity. Anal Biochem 132:6–13CrossRefPubMedGoogle Scholar
  15. van der Fits L, Deakin E, Hoge H, Memelink J (2000) The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation. Plant Mol Biol 43:495–502CrossRefPubMedGoogle Scholar
  16. Fourcroy P, Vansuyt G, Kushnir S, Inzé D, Briat J (2004) Iron-regulated expression of a cytosolic ascorbate peroxidase encoded by the APX1 gene in arabidopsis seedlings. Plant Physiol 134:605–613CrossRefPubMedGoogle Scholar
  17. Fridovich I (1998) Oxygen toxicity: a radical explanation. J Exp Bot 201:1203–1209Google Scholar
  18. Fryer MJ, Ball L, Oxborough K, Karpinski S, Mullineaux PM, Baker NR (2003) Control of Ascorbate Peroxidase 2 expression by hydrogen peroxide and leaf water status during excess light stress reveals a functional organization of Arabidopsis leaves. Plant J 33:691–705CrossRefPubMedGoogle Scholar
  19. Furlani AMC, Furlani PR (1998) Composição e pH de soluções nutritivas para estudos fisiológicos e de seleção de plantas em condições nutricionais adversas. Technical bulletin 121, Instituto Agronômico de Campinas pp 1–34Google Scholar
  20. Gadea J, Conejero V, Vera P (1999) Developmental regulation of a cytosolic ascorbate peroxidase gene from tomato plants. Mol Gen Genet 262:212–219CrossRefPubMedGoogle Scholar
  21. Halliwell B, Gutteridge JM (1999) Free radicals in biology and medicine. Oxford University Press, New York p 936Google Scholar
  22. Höfgen R, Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res 16:9877PubMedCrossRefGoogle Scholar
  23. Ishikawa T, Sakai K, Takeda T, Shigeoka S (1995) Cloning and expression of cDNA encoding a new type of ascorbate peroxidase in spinach. FEBS Lett 367:28–32CrossRefPubMedGoogle Scholar
  24. Ishikawa T, Sakai K, Yoshimura K, Takeda T, Shigeoka S (1996) cDNAs encoding spinach stromal and thylakoid-bound ascorbate peroxidase, differing in the presence or absence of their 3′-coding regions. FEBS Lett 384:289–293CrossRefPubMedGoogle Scholar
  25. Ishikawa T, Yoshimura K, Sakai K, Tamoi M, Takeda T, Shigeoka S (1998) Molecular characterization and physiological role of a glyoxysome-bound ascorbate peroxidase from spinach. Plant Cell Physiol 39:23–34PubMedGoogle Scholar
  26. Jiménez A, Hernández JA, del Río LA, Sevilla F (1997) Evidence for the presence of the ascorbate-glutathione cycle in mitochondria and peroxisomes of pea leaves. Plant Physiol 114:275–284PubMedGoogle Scholar
  27. Jiménez A, Hernández JA, Pastori G, del Río LA, Sevilla F (1998) Role of the ascorbate-glutathione cycle of mitochondria and peroxisomes in the senescence of pea leaves. Plant Physiol 118:1327–1335CrossRefPubMedGoogle Scholar
  28. Kamada T, Nito K, Hayashi H, Mano S, Hayashi M, Nishimura M (2003) Functional differentiation of peroxisomes revealed by expression profiles of peroxisomal genes in Arabidopsis thaliana. Plant Cell Physiol 44:1275–1289CrossRefPubMedGoogle Scholar
  29. Karimi M, Inzé D, Depicker A (2002) GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195CrossRefPubMedGoogle Scholar
  30. Karpinski S, Escobar C, Karpinska B, Creissen G, Mullineaux PM (1997) Photosynthetic electron transport regulates the expression of cytosolic ascorbate peroxidase genes in arabidopsis during excess light stress. Plant Cell 9:627–640CrossRefPubMedGoogle Scholar
  31. Kato K, Matsumoto T, Koiwai S, Mizusaki S, Nishida K, Nogushi M, Tamaki E (1972) Liquid suspension culture of tobacco cells. In: Terui G (ed) Ferment technology today. Society of Fermentation Technology, Osaka, pp 689–695Google Scholar
  32. Kunce CM, Trelease RN, Turley RB (1988) Purification and biosynthesis of cottonseed (Gossypium hirsutum L). Biochem J 251:147–155PubMedGoogle Scholar
  33. Lee HD, Kim YS, Lee CB (2001) The inductive responses of the antioxidant enzymes by salt stress in the rice (Oryza sativa L.). J Plant Physiol 158:737–745CrossRefGoogle Scholar
  34. Lisenbee CS, Heinze M, Trelease RN (2003a) Peroxisomal ascorbate peroxidase resides within a subdomain of rough endoplasmic reticulum in wild-type arabidopsis cells. Plant Physiol 132:870–882CrossRefGoogle Scholar
  35. Lisenbee CS, Karnik SK, Trelease RN (2003b) Overexpression and mislocalization of a tail-anchored GFP redefines the identity of peroxisomal ER. Traffic 4:491–501CrossRefGoogle Scholar
  36. Livak KJ, Schmittgen TD (2001) Analysis of relative expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  37. Madhusudhan R, Ishikawa T, Sawa Y, Shigeoka S, Shibata H (2003) Characterization of an ascorbate peroxidase in plastids of tobacco BY-2 cells. Physiol Plant 117:550–557CrossRefPubMedGoogle Scholar
  38. Mano S, Yamaguchi K, Hayashi M, Nishimura M (1997) Stromal and thylakoid-bound ascorbate peroxidases are produced by alternative splicing in pumpkin. FEBS Lett 413:21–26CrossRefPubMedGoogle Scholar
  39. Margis-Pinheiro M, Zhou XR, Zhu QH, Dennis ES, Upadhyaya NM (2005) Isolation and characterization of a Ds-tagged rice (Oryza sativa L.) GA-responsive dwarf mutant defective in an early step of the gibberellin biosynthesis pathway. Plant Cell Rep 23:819–33CrossRefPubMedGoogle Scholar
  40. Menezes-Benavente L, Kernodle SP, Margis-Pinheiro M, Scandalios JG (2004a) Salt-induced antioxidant metabolism defenses in maize (Zea mays L.) seedlings. Redox Rep 9:29–36CrossRefGoogle Scholar
  41. Menezes-Benavente L, Teixeira FK, Kamei CL, Margis-Pinheiro M (2004b) Salt stress induces expression of genes encoding antioxidant enzymes in seedlings of a Brazilian indica rice (Oryza sativa L.). Plant Sci 166:323–331CrossRefGoogle Scholar
  42. Mittler R, Feng X, Cohen M (1998) Post-transcriptional suppression of cytosolic ascorbate peroxidase expression during pathogen-induced programmed cell death in tobacco. Plant Cell 10:461–473CrossRefPubMedGoogle Scholar
  43. Mittler R, Vanderauwera S, Gallery M, Van Breusegem F (2004) Reactive oxygen gene network of plants. Trends Plant Sci 9:490–498CrossRefPubMedGoogle Scholar
  44. Mittova V, Guy M, Tal M, Volokita M (2004a) Salinity up-regulates the antioxidative system in root mitochondria and peroxisomes of the wild salt-tolerant tomato species Lycopersicon pennellii. J Exp Bot 55:1105–1113CrossRefGoogle Scholar
  45. Mittova V, Theodoulou FL, Kiddle G, Volokita M, Tal M, Foyer CH, Guy M (2004b) Comparison of mitochondrial ascorbate peroxidase in the cultivated tomato, Lycopersicon esculentum, and its wild, salt-tolerant relative, L. pennellii—a role for matrix isoforms in protection against oxidative damage. Plant Cell Environ 27:237–250CrossRefGoogle Scholar
  46. Miyake C, Cao WH, Asada K (1993) Purification and molecular properties of thylakoid-bound acorbate peroxidase in spinach chloroplasts. Plant Cell Physiol 34:881–889Google Scholar
  47. Mullen RT, Trelease RN (2000) The sorting signals for peroxisomal membrane-bound ascorbate peroxidase are within its C-terminal tail. J Biol Chem 275:16337–16344CrossRefPubMedGoogle Scholar
  48. Mullen RT, Lisenbee CS, Miernyk JA, Trelease RN (1999) Peroxisomal membrane ascorbate peroxidase is sorted to a membranous network that resembles a subdomain of the endoplasmic reticulum. Plant Cell 11:2167–2185CrossRefPubMedGoogle Scholar
  49. Mullineaux P, Karpinski S (2002) Signal transduction in response to excess light: getting out of the chloroplast. Curr Opin Plant Biol 5:43–48CrossRefPubMedGoogle Scholar
  50. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol 15:473–497CrossRefGoogle Scholar
  51. Nishimura M, Hayashi M, Kato A, Yamaguchi H, Mano S (1996) Functional transformation of microbodies in higher plant cells. Cell Struct Funct 21:387–393PubMedCrossRefGoogle Scholar
  52. Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol 49:249–79CrossRefPubMedGoogle Scholar
  53. Obara K, Sumi K, Fukuda H (2002) The use of multiple transcription starts causes the dual targeting of Arabidopsis putative mondehydroascorbate reductase to both mitochondria and chloroplasts. Plant Cell Physiol 43:697–705CrossRefPubMedGoogle Scholar
  54. Orozco-Cárdenas ML, Narváez-Vasquéz J, Ryan CA (2001) Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl-jasmonate. Plant Cell 13:179–191CrossRefPubMedGoogle Scholar
  55. Pnueli L, Liang H, Rozenberg M, Mittler R (2003) Growth suppression, altered stomatal responses, and augmented induction of heat shock protein in cytosolic ascobate peroxidase (Apx1)-deficient Arabidopsis plants. Plant J 34:187–203CrossRefPubMedGoogle Scholar
  56. Ragueh F, Fescure N, Roby D, Marco Y (1989) Gene expression in Nicotiana tabacum in response to compatible and incompatible isolates of Pseudomonas solonaciarum. Physiol Mol Plant Pathol 35:23–33CrossRefGoogle Scholar
  57. Rizhsky L, Davletova S, Liang H, Mittler R (2004) The zing finger protein Zat12 is required for cytosolic ascorbate peroxidase 1 expression during oxidative stress in Arabidopsis. J Biol Chem 279:11736–11743CrossRefPubMedGoogle Scholar
  58. Sato Y, Murakami T, Funatsuki H, Matsuba S, Saruyama H, Tanida M (2001) Heat shock-mediated APX gene expression and protection against chilling injury in rice seedlings. J Exp Bot 52:145–151CrossRefPubMedGoogle Scholar
  59. Savouré A, Thorin D, Xeu-Jun H, Van Montagu M, Inzé D, Verbruggen N (1999) NaCl and CuSO4 treatments trigger distinct oxidative defense mechanisms in Nicotiana plumbaginifolia L. Plant Cell Environ 22:387–396CrossRefGoogle Scholar
  60. Scandalios JG (2002) The rise of ROS. Trends Biochem Sci 27:483–486CrossRefPubMedGoogle Scholar
  61. Shigeoka S, Nakano Y, Kitaoka S (1980) Metabolism of hydrogen peroxide in Euglena gracilis Z by L-ascorbic acid peroxidase. Biochem J 186:377–380PubMedGoogle Scholar
  62. Shigeoka S, Ishikawa T, Tamoi M, Miyagawa Y, Takeda T, Yabuta Y, Yoshimura K (2002) Regulation and function of ascorbate peroxidase isoenzymes. J Exp Bot 53:1305–1319CrossRefPubMedGoogle Scholar
  63. Silva-Filho MC (2003) One ticket for multiple destinations: dual targeting of proteins to distinct subcellular locations. Curr Opin Plant Biol 6:589–595CrossRefPubMedGoogle Scholar
  64. Teixeira FK, Menezes-Benavente L, Margis R, Margis-Pinheiro M (2004) Analysis of the molecular evolutionary history of the ascorbate peroxidase gene family: inferences from the rice genome. J Mol Evol 59:761–770CrossRefPubMedGoogle Scholar
  65. Vandenabeele S, Van Der Kelen K, Dat J, Gadjev I, Boonefaes T, Morsa S, Rottiers P, Slooten L, Van Montagu M, Zabeau M, Inzé D, Van Breusegem F (2003) A comprehensive analysis of hydrogen peroxide-induced gene expression in tobacco. Proc Natl Acad Sci USA 100:16113–16118CrossRefPubMedGoogle Scholar
  66. Yabuta Y, Motoki T, Yoshimura K, Takeda T, Ishikawa, Shigeoka S (2002) Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress. Plant J 32:915–925CrossRefPubMedGoogle Scholar
  67. Yoshimura K, Yabuta Y, Ishikawa T, Shigeoka S (2000) Expression of spinach ascorbate peroxidase isoenzymes in response to oxidative stresses. Plant Physiol 123:223–233CrossRefPubMedGoogle Scholar
  68. Yoshimura K, Yabuta Y, Ishikawa T, Shigeoka S (2002) Identification of a cis element for tissue-specific alternative splicing of chloroplast ascorbate peroxidase pre-mRNA in higher plants. J Biol Chem 277:40623–40632CrossRefPubMedGoogle Scholar
  69. Zhang H, Wang J, Nickel U, Allen RD, Goodman HM (1997) Cloning and expression of an Arabidopsis gene encoding a putative peroxisomal ascorbate peroxidase. Plant Mol Biol 34:967–971CrossRefPubMedGoogle Scholar
  70. Zhu JK (2001) Plant salt tolerance. Trends Plant Sci 6:66–67CrossRefPubMedGoogle Scholar
  71. Zottini M, Formentin E, Scattolin M, Carimi F, Lo Schiavo F, Terzi M (2002) Nitric oxide affects plant mitochondrial functionality in vivo. FEBS Lett 515:75–78CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Felipe Karam Teixeira
    • 1
  • Larissa Menezes-Benavente
    • 1
    • 4
  • Vinícius Costa Galvão
    • 1
  • Rogério Margis
    • 2
  • Márcia Margis-Pinheiro
    • 1
    • 3
  1. 1.Laboratório de Genética Molecular Vegetal, Departamento de GenéticaUniversidade Federal do Rio de JaneiroRio de JaneiroBrasil
  2. 2.Departamento de BioquímicaUniversidade Federal do Rio Grande do SulRio Grande do SulBrasil
  3. 3.Laboratório de Genética Vegetal, Departamento de GenéticaUniversidade Federal do Rio Grande do Sul. Av. Bento GonçalvesPorto AlegreBrasil
  4. 4.Department of GeneticsNorth Carolina State UniversityRaleighUSA

Personalised recommendations