Plant Cell Reports

, 27:363 | Cite as

Proteomic analysis of reactive oxygen species (ROS)-related proteins in rice roots

  • Sang Gon Kim
  • Sun Tae Kim
  • Sun Young Kang
  • Yiming Wang
  • Wook Kim
  • Kyu Young KangEmail author
Biotic and Abiotic Stress


To investigate the rice root proteome, we applied the PEG fractionation technique combined with two-dimensional gel electrophoresis which rendered more well-separated protein spots. Out of the 295 chosen proteins, 93 were identified by MALDI-TOF mass spectrometry. The proteins were classified as relating to metabolism (38.7%), reactive oxygen species (ROS)-related proteins (22.5%), protein processing/degradation (8.6%), stress/defense (7.5%), energy (6.5%) and signal transduction (5.4%). The high percentage of ROS-related proteins found in rice root brings us to assess the roles of ROS on rice root growth. Treatment with ROS quenching chemicals such as reduced glutathione (GSH), diphenyleneiodonium (DPI) and ascorbate inhibited root growth dose-dependently. Forty-nine proteins identified were either up- or down-regulated by GSH treatment, of which 14 were ROS-related proteins, such noticeably modulated ones as glutathione-S-transferase (GST), superoxide dismutases (SOD) and l-ascorbate peroxidases. The protein levels of four GSTs (NS4, 8, 56 and 57), three APXs (NS46, 49 and 50) and MnSOD (NS45) were strongly reduced by GSH treatment but slightly reduced by ascorbate and DPI. Ascorbate and DPI strongly inhibited expression levels of a catalase A (NP23) and an APX (NS65) but did not affect APXs (NS46, 49 and 50) protein levels. Northern analysis demonstrated that changes in transcript levels of five genes––GST (NS4), GST (NS43), Mn-SOD (NS45), APX (NS50) and APX (NS46/49) in response to ROS quenching chemicals were coherent with patterns shown in two-dimensional electrophoresis analyses. Taken together, we suggest that these proteins may take part in an important role in maintaining cellular redox homeostasis during rice root growth.


MALDI-TOF Reactive oxygen species (ROS) ROS-related protein Proteomics Two-dimensional electrophoresis 



This work was supported by Grant No. CG1122 from the Crop Functional Genomic Center; by a grant from KOSEF/MOST to the Environmental Biotechnology National Core Research Center (to S.G. Kim and S.T. Kim); and by scholarships from the Brain Korea 21 Program, Ministry of Education and Human Resources Development, Korea (to S.G. Kim, Y. Wang).


  1. Azhiri-Sigari T, Yamauchi A, Kamoshita A, Wade LJ (2000) Genotypic variation in response of rainfed lowland rice to drought and rewatering. Plant Prod Sci 3:180–188CrossRefGoogle Scholar
  2. Bevan M, Bancroft I, Bent E, Love K, Goodman H, Dean C, Bergkamp R, Dirkse W, van Staveren M, Stiekema W, Drost L, Ridley P, Hudson SA, Patel K, Murphy G, Piffanelli P, Wedler H, Wedler E, Wambutt R, Weitzenegger T, Pohl TM, Terryn N, Gielen J, Villarroel R, De Clerck R, Van Montagu M, Lecharny A, Auborg S, Gy I, Kreis M, Lao N, Kavanagh T, Hempel S, Kotter P, Entian KD, Rieger M, Schaeffer M, Funk B, Mueller-Auer S, Silvey M, James R, Montfort A, Pons A, Puigdomenech P, Douka A, Voukelatou E, Milioni D, Hatzopoulos P, Piravandi E, Obermaier B, Hilbert H, Dusterhoft A, Moores T, Jones JDG, Eneva T, Palme K, Benes V, Rechman S, Ansorge W, Cooke R, Berger C, Delseny M, Voet M, Volckaert G, Mewes HW, Klosterman S, Schueller C, Chalwatzis N (1998) Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana. Nature 391:485–488PubMedCrossRefGoogle Scholar
  3. Blokhina O, Virolainen E, Fagerstedt KV (2003) Antioxidants, oxidative damage and oxygen deprivation stress: a review. Ann Bot (Lond) 91:179–194CrossRefGoogle Scholar
  4. Blum H, Beier H, Gross HJ (1987) Improved silver staining of plant proteins, RNA, and DNA in polyacrylamide gels. Electrophoresis 8:93–99CrossRefGoogle Scholar
  5. Edwards R, Dixon DP, Walbot V (2000) Plant glutathione S-transferase: enzymes with multiple functions in sickness and in health. Trends Plant Sci 5:193–198PubMedCrossRefGoogle Scholar
  6. Fang YZ, Yang S, Wu G (2002) Free radical, antioxidants, and nutrition. Nutrition 18:872–879PubMedCrossRefGoogle Scholar
  7. Foreman J, Demidchik V, Bothwell JHF, Mylona P, Miedema H, Torres MA, Linstead P, Costa S, Brownlee C, Jones JDG, Davies JM, Dolan L (2003) Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature 422:442–446PubMedCrossRefGoogle Scholar
  8. Gong H, Jiao Y, Hu WW, Pua EC (2005) Expression of glutathione-S-transferase and its role in plant growth and development in vivo and shoot morphogenesis in vitro. Plant Mol Biol 57:53–66PubMedCrossRefGoogle Scholar
  9. Halliwell B (2006) Reactive species and antioxidants. Redox biology is a fundamental theme of aerobic life. Plant Physiol 141:312–322PubMedCrossRefGoogle Scholar
  10. Herouatr D, Montagu MV, Inze D (1993) Redox-activated expression of the cytosolic copper/zinc superoxide dismutase gene in Nicotiana. Proc Natl Acad Sci 90:3108–3112CrossRefGoogle Scholar
  11. Hochholdinger F, Guo L, Schnable PS (2004) Lateral roots affect the proteome of the primary root of maize (Zea mays L.). Plant Mol Biol 2004:397–412CrossRefGoogle Scholar
  12. Hochholdinger F, Woll K, Guo L, Schnable PS (2005) The accumulation of abundant soluble proteins changes early in the development of the primary roots of maize (Zea mays L.). Proteomics 5:4885–4893PubMedCrossRefGoogle Scholar
  13. International rice genome sequencing project (2005) The map-based sequence of the rice genome. Nature 436:793–800CrossRefGoogle Scholar
  14. Jones AME, Thomas V, Truman B, Lilley K, Mansfield J, Grant M (2004) Specific changes in the Arabidopsis proteome in response to bacterial challenge: differentiating basal and R-gene mediated resistance. Phytochemistry 65:1805–1816PubMedCrossRefGoogle Scholar
  15. Joo JH, Bae YS, Lee JS (2001) Role of auxin-induced reactive oxygen species in root gravitropism. Plant Physiol 126:1055–1060PubMedCrossRefGoogle Scholar
  16. Kim ST, Cho KS, Jang YS, Kang KY (2001) Two-dimensional electrophoretic analysis of rice proteins by polyethylene glycol fractionation for protein arrays. Electrophoresis 22:2103–2109PubMedCrossRefGoogle Scholar
  17. Kim ST, Cho KS, Yu S, Kim SG, Hong JC, Han CD, Bae DW, Nam MH, Kang KY (2003a) Proteomic analysis of differentially expressed proteins induced by rice blast fungus and elicitor in suspension-cultured rice cells. Proteomics 3:2368–2378PubMedCrossRefGoogle Scholar
  18. Kim ST, Kim HS, Kim HJ, Kim SG, Kang SY, Lim DB, Kang KY (2003b) Prefractionation of protein samples for proteome analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mol Cells 16:316–322PubMedGoogle Scholar
  19. Kim ST, Kim SG, Hwang DH, Kang SY, Kim HJ, Lee BH, Lee JJ, Kang KY (2004) Proteomic analysis of pathogen-responsive proteins from rice leaves induced by rice blast fungus, Magnaporthe grisea. Proteomics 4:938–949Google Scholar
  20. Koller A, Washburn MP, Lange BM, Andon NL, Deciu C, Haynes PA, Hays L, Schieltz D, Ulaszek R, Wei J, Wolters D, Yates III JR (2002) Proteomic survey of metabolic pathways in rice. Proc Natl Acad Sci 99:11969–11974PubMedCrossRefGoogle Scholar
  21. Komatsu S, Konishi H (2005) Proteome analysis of rice root proteins regulated by gibberellin. Genomics Proteomics Bioinformatics 3:132–142PubMedGoogle Scholar
  22. Kwak JM, Nguyen V, Schroeder JI (2006) The role of reactive oxygen species in hormonal responses. Plant Physiol 141:323–329PubMedCrossRefGoogle Scholar
  23. Leamml UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophgae. Nature 227:680–685CrossRefGoogle Scholar
  24. Liszkay A, van der Zalm E, Schopfer P (2004) Production of reactive oxygen inermediates (O2 •−, H2O2, and OH) by maize roots and their role in wall loosening and elongation growth. Plant Physiol 136:3144–3123CrossRefGoogle Scholar
  25. Lopez-Bucio J, Cruz-Ramirez A, Herrera-Estrella L (2003) The role of nutrient availability in regulating root architecture. Curr Opin Plant Biol 6:280–287PubMedCrossRefGoogle Scholar
  26. Marrs KA (1996) The function and regulation of glutathione S-transferase in plants. Annu Rev Plant Physiol Plant Mol Biol 47:127–158PubMedCrossRefGoogle Scholar
  27. May MJ, Vernoux T, Leaver C, Montagu MV, Inze D (1998) Glutathione homeostasis in plants: implications for environmental sensing and plant development. J Exp Bot 49:649–667CrossRefGoogle Scholar
  28. Miller SS, Driscoll BT, Gregerson RG, Gantt JS, Vance CP (1998) Alfalfa malate dehydrogenase (MDH): molecular cloning and characterization of five different forms reveals a unique nodule-enhanced MDH. Plant J 15:173–184PubMedCrossRefGoogle Scholar
  29. Morita S, Suga T, Yamazaki K (1988) The relationship between root length density and yield in rice plants. Jpn J Crop Sci 57:438–443Google Scholar
  30. Neuefeind T, Huber R, Dasenbrock H, Prade L, Bieseler B (1997) Crystal structrure of herbicide-detoxifying maize glutathione S-transferase-I in complex with lactoylglutathione: evidence for an induced-fit mechanism. J Mol Biol 274:446–453PubMedCrossRefGoogle Scholar
  31. O’Donnell BV, Tew DG, Jones OT, England PJ (1993) Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase. Biochem J 290:41–49PubMedGoogle Scholar
  32. Ostergaard O, Melchior S, Roepstorff P, Svensson B (2002) Initial proteome analysis of mature barley seeds and malt. Proteomics 2:733–739PubMedCrossRefGoogle Scholar
  33. Pullar JM, Hampton MB (2002) Diphenyleneiodonium triggers the efflux of glutathione from cultured cells. J Biol Chem 277:19402–19407PubMedCrossRefGoogle Scholar
  34. Reinemer P, Prade L, Hof P, Neuefeind T, Huber R, Zettl R, Palme K, Schell J, Koelln I, Bartunik HD, Bieseler B (1996) Three-dimensional structure of glutathione S-transferase from Arabidopsis thaliana at 2.2 Å resolution: structural characterization of herbicide-conjugating plant glutathione S-transferases and a novel active site architecture. J Mol Biol 255:289–309PubMedCrossRefGoogle Scholar
  35. Renew S, Heyno E, Schopfer P, Liszkay A (2005) Sensitive detection and localization of hydroxyl radical production in cucumber roots and Arabidopsis seedlings by spin trapping electron paramagnetic resonance spectroscopy. Plant J 44:342–347PubMedCrossRefGoogle Scholar
  36. Rodriguez AA, Grunberg KA, Taleisnik EL (2002) Reactive oxygen species in the elongation zone of maize leaves are necessary for leaf extension. Plant Physiol 129:1627–1632PubMedCrossRefGoogle Scholar
  37. Ros-Barcelo A, Pomar F, Lopez-Serrano M, Martinez P, Pedreno MA (2002) Developmental regulation of the H2O2-producing system and of a basic peroxidase isoenzyme in the Zinnia elegans lifnifying xylem. Plant Physiol Biochem 40:325–332CrossRefGoogle Scholar
  38. Schopfer P, Plachy C, Frahry G (2001) Release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide, and hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light, gibberellin, and abscisic acid. Plant Physiol 125:1591–1602PubMedCrossRefGoogle Scholar
  39. Schroeder JI, Mori IC (2004) Reactive oxygen species activation of plant Ca2+ channels. A signaling mechanism in polar growth, hormone transduction, stress signaling, and hypothetically mechanotransduction. Plant Physiol 135:702–708PubMedCrossRefGoogle Scholar
  40. Sheffield J, Taylor N, Fauquet C, Chen S (2006) The cassava (Manihot esculenta Crantz) root proteome: protein identification and differential expression. Proteomics 6:1588–1598PubMedCrossRefGoogle Scholar
  41. Smirnoff N (1996) The function and metabolism of ascorbic acid in plants. Ann Bot 78:661–669CrossRefGoogle Scholar
  42. Smirnoff N (2000) Ascorbate acid: metabolism and functions of a multi-facetted molecule. Curr Opin Plant Biol 3:229–235PubMedGoogle Scholar
  43. Suzuki H, Matsumori A, Matoba Y, Kyu BS, Tanaka A, Funita J, Sasayama S (1993) Enhanced expression of superoxide dismutase messenger RNA in viral myocarditis: SH-dependant reduction of its expression and myocardial injury. J Clin Invest 6:2727–2733CrossRefGoogle Scholar
  44. Tanaka N, Konishi H, Khan MMK, Komatsu S (2004) Proteome analysis of rice tissues by two-dimensional electrophoresis: an approach to the investigation of gibberellin regulated proteins. Mol Genet Genomics 270:485–496PubMedCrossRefGoogle Scholar
  45. Tanaka N, Mitsui S, Nobori H, Yanagi K, Komatsu S (2005) Expression and function of proteins during development of the basal region in rice seedling. Mol Cell Proteomics 4:796–808PubMedCrossRefGoogle Scholar
  46. Teo YH, Beyrouty CA, Norman RJ, Gbur EE (1995) Nutrient uptake relationship to root characteristics of rice. Plant Soil 171:297–302CrossRefGoogle Scholar
  47. Terashima K, OgataT, Akita S (1994) Eco-physiological characteristics related with lodging tolerance of rice in direct sowing cultivation. Jpn J Crop Sci 63:34–41Google Scholar
  48. Ueda M, Matsui K, Ishiguro S, Sano R, Wada T, Paponov I, Palme K, Okada K (2004) The HALTED ROOT gene encoding the 26S proteasome subunit RPT2a is essential for the maintenance of Arabidopsis meristems. Development 131:2101–2111PubMedCrossRefGoogle Scholar
  49. Yang Y, Kwon HB, Peng HP, Shih MC (1993) Stress response and metabolic regulation of glyceraldehyde-3-phosphate dehydrogenase gene in Arabidopsis. Plant Physiol 101:209–216PubMedCrossRefGoogle Scholar
  50. Wingsle G, Karpinski G (1996) Differential redox regulation by glutathione of glutathione reductase and CuZn-superoxide dismutase gene expression in Pinus sylvestris L. needles. Planta 198:151–157PubMedCrossRefGoogle Scholar

Copyright information

© The Author(s) 2007

Authors and Affiliations

  • Sang Gon Kim
    • 1
    • 2
  • Sun Tae Kim
    • 2
  • Sun Young Kang
    • 1
  • Yiming Wang
    • 1
  • Wook Kim
    • 3
  • Kyu Young Kang
    • 1
    • 2
    Email author
  1. 1.Division of Applied Life Science (BK21 program)Plant Molecular Biology and Biotechnology Research Center JinjuSouth Korea
  2. 2.Environmental Biotechnology National Core Research CenterGyeongsang National UniversityJinjuSouth Korea
  3. 3.College of Life Sciences and Biotechnology, Division of BiotechnologyKorea UniversitySeoulSouth Korea

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