Amino Acids

, Volume 38, Issue 3, pp 729–738 | Cite as

Identification of flooding stress responsible cascades in root and hypocotyl of soybean using proteome analysis

  • Setsuko Komatsu
  • Tetsuya Sugimoto
  • Tomoki Hoshino
  • Yohei Nanjo
  • Kiyoshi Furukawa
Original Article


Flooding inducible proteins were analyzed using a proteomic technique to understand the mechanism of soybean response to immersion in water. Soybeans were germinated for 2 days, and then subjected to flooding for 2 days. Proteins were extracted from root and hypocotyl, separated by two-dimensional polyacrylamide gel electrophoresis, stained by Coomassie brilliant blue, and analyzed by protein sequencing and mass spectrometry. Out of 803 proteins, 21 proteins were significantly up-regulated, and seven proteins were down-regulated by flooding stress. Of the total, 11 up-regulated proteins were classified as related to protein destination/storage and three proteins to energy, while four down-regulated proteins were related to protein destination/storage and three proteins to disease/defense. The expression of 22 proteins significantly changed within 1 day after flooding stress. The effects of flooding, nitrogen substitution without flooding, or flooding with aeration were analyzed for 1–4 days. The expression of alcohol dehydrogenase increased remarkably by nitrogen substitution compared to flooding. The expression of many proteins that changed due to flooding showed the same tendencies observed for nitrogen substitution; however, the expression of proteins classified into protein destination/storage did not.


Flooding Proteome Soybean Hypoxic response 



Two-dimensional polyacrylamide gel electrophoresis


Mass spectrometry


Coomassie brilliant blue


Isoelectric focusing


Immobilized pH gradient


Isoelectric point



This work was supported by grants from National Agriculture and Food Research Organization, Japan. The authors thank Dr. S. Kuroda for his kind support of our research. We also thank Dr. S. Shimamura, Dr. N. Nakayama, Dr. R. Yamamoto and Dr. T. Nakamura for their valuable discussion.


  1. Aghaei K, Ehsanpour AA, Shah AH, Komatsu S (2009) Proteome analysis of soybean hypocotyl and root under salt stress. Amino Acids 36:91–98. doi: 10.1007/s00726-008-0036-7 CrossRefPubMedGoogle Scholar
  2. Armstrong W (1979) Aeration in higher plants. Adv Bot Res 7:225–232. doi: 10.1016/S0065-2296(08)60089-0 CrossRefGoogle Scholar
  3. Bailey-Serres J, Voesenek LACJ (2008) Flooding stress: acclimations and genetic diversity. Annu Rev Plant Biol 16:313–339. doi: 10.1146/annurev.arplant.59.032607.092752 CrossRefGoogle Scholar
  4. Baxter-Burrell A, Yang Z, Springer PS, Bailey-Serres J (2002) RopGAP4-dependent Rop GTPase rheostat control of Arabidopsis oxygen deprivation tolerance. Science 296:2026–2028. doi: 10.1126/science.1071505 CrossRefPubMedGoogle Scholar
  5. Bevan M, Bancroft I, Bent E et al (1998) Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana. Nature 391:485–488. doi: 10.1038/35140 CrossRefPubMedGoogle Scholar
  6. Cho HT, Kende H (1997) Expansins and intermodal growth of deepwater rice. Plant Physiol 113:1145–1151. doi: 10.1104/pp.113.4.1137 CrossRefPubMedGoogle Scholar
  7. Cleveland DW, Fisher SG, Kirschner MW, Laemmli UK (1977) Peptide mapping proteolysis in sodium dodecyl sulphate and analysis by gel electrophoresis. J Biol Chem 252:1102–1106PubMedGoogle Scholar
  8. Coleman HD, Canam T, Kang K-Y, Ellis DD, Mansfield SD (2007) Over-expression of UDP-glucose pyrophosphorylase in hybrid poplar affects carbon allocation. J Exp Bot 58:4257–4268. doi: 10.1093/jxb/erm287 CrossRefPubMedGoogle Scholar
  9. Darley CP, Forrester AM, McQueen-Mason SJ (2001) The molecular basis of plant cell wall extension. Plant Mol Biol 47:179–195. doi: 10.1023/A:1010687600670 CrossRefPubMedGoogle Scholar
  10. Dixon MH, Hill SA, Jackson MB, Ratcliffe RC (2006) Physiological and metabolic adaptations of Patamogetom pectinatus L. tubers support rapid elongation of stem tissue in the absence of oxygen. Plant Cell Physiol 47:128–140. doi: 10.1093/pcp/pci229 CrossRefPubMedGoogle Scholar
  11. Dordas C, Hasinoff BB, Rivoal J, Hill RD (2004) Class-1 hemoglobins, nitrate and NO levels in anoxic maize cell-suspension cultures. Planta 219:66–72. doi: 10.1007/s00425-004-1212-y CrossRefPubMedGoogle Scholar
  12. Gonzali S, Loreti E, Novi G, Poggi A, Alpi A, Perata P (2005) The use of microarrays to study the anaerobic response in Arabidopsis. Ann Bot (Lond) 96:661–668. doi: 10.1093/aob/mci218 CrossRefGoogle Scholar
  13. Hirano H, Kawasaki H, Sassa H (2000) Two-dimensional gel electrophoresis using immobilized pH gradient tube gels. Electrophoresis 21:440–445. doi: 10.1002/(SICI)1522-2683(20000101)21:2<440::AID-ELPS440>3.0.CO;2-X CrossRefPubMedGoogle Scholar
  14. Huang S, Greenway H, Colmer TD, Millar AH (2005) Protein synthesis by rice coleoptiles during prolonged anoxia: implications for glycolysis, growth and energy utilization. Ann Bot (Lond) 96:661–668. doi: 10.1093/aob/mci218 CrossRefGoogle Scholar
  15. Hunt PW, Klok EJ, Trevaskis B, Watts RA, Ellis MH, Peacock WJ, Dennis ES (2002) Increased level of hemoglobin 1 enhances survival of hypoxic stress and promotes early growth in Arabidopsis thaliana. Proc Natl Acad Sci USA 99:17197–17202. doi: 10.1073/pnas.212648799 CrossRefPubMedGoogle Scholar
  16. Jackson MB, Colmer TD (2005) Response and adaptation by plants to flooding stress. Ann Bot (Lond) 96:501–505. doi: 10.1093/aob/mci205 CrossRefGoogle Scholar
  17. Kamauchi S, Wadahama K, Iwasaki K, Nakamoto Y, Nishizawa K, Ishimoto M, Kawada T, Urada R (2008) Molecular cloning and characterization of two soybean protein disulfide isomerases as molecular chaperones for seed storage proteins. FEBS J 275:2644–2658. doi: 10.1111/j.1742-4658.2008.06412.x CrossRefPubMedGoogle Scholar
  18. Kim TD (2006) Protein phosphatase inhibitor-1 (PPI-1) has protective activities in stress conditions in E. coli. Int J Biol Macromol 38:70–76. doi: 10.1016/j.ijbiomac.2006.01.001 CrossRefPubMedGoogle Scholar
  19. Komatsu S, Yano H (2006) Update and challenges on proteomics in rice. Proteomics 6:4057–4068. doi: 10.1002/pmic.200600012 CrossRefPubMedGoogle Scholar
  20. Komatsu S, Konishi H, Shen S, Yang G (2003) Rice proteomics: a step toward functional analysis of the rice genome. Mol Cell Proteomics 2:2–10. doi: 10.1074/mcp.R200008-MCP200 CrossRefPubMedGoogle Scholar
  21. Liu F, Vantoai T, Moy L, Bock G, Linford LD, Quackenbush J (2005) Global transcription profiling reveals novel insights into hypoxic response in Arabidopsis. Plant Physiol 137:1115–1129. doi: 10.1104/pp.104.055475 CrossRefPubMedGoogle Scholar
  22. Loreti E, Poggi A, Novi G, Alpi A, Perata P (2005) Genome-wide analysis of gene expression in Arabidopsis seedlings under anoxia. Plant Physiol 137:1130–1138. doi: 10.1104/pp.104.057299 CrossRefPubMedGoogle Scholar
  23. Matsumoto H (1998) Inhibition of proton transport activity of microsomal membrane vesicle of barley roots by aluminum. Soil Sci Plant Nutr 34:499–526Google Scholar
  24. Mattana M, Coraggio I, Bertani A, Reggiani R (1994) Expression of the enzymes of nitrate reduction during the anaerobic germination of rice. Plant Physiol 106:1605–1608PubMedGoogle Scholar
  25. Nakayama N, Hashimoto S, Shimada S, Takahashi M, Kim Y, Oya T, Arihara J (2004) The effect of flooding stress at the germination stage on the growth of soybean in relation to initial seed moisture content (Japanese). Jpn J Crop Sci 74:325–329. doi: 10.1626/jcs.74.325 CrossRefGoogle Scholar
  26. Navrot N, Collin V, Gualberto J, Gelhaye E, Hirasawa M, Rey P, Knaff DB, Issakidis E, Jacquot JP, Rouhier N (2006) Plant glutathione peroxidase are functional peroxiredixins distributed in several subcellular compartments and regulated during biotic and abiotic stresses. Plant Physiol 142:1364–1379. doi: 10.1104/pp.106.089458 CrossRefPubMedGoogle Scholar
  27. O’Farrell PH (1975) High resolution two-dimensional electrophoresis of protein. J Biol Chem 250:4007–4021PubMedGoogle Scholar
  28. Peumans WJ, van Damme EJM (1995) Lectins as plant defense proteins. Plant Physiol 109:347–352CrossRefPubMedGoogle Scholar
  29. Pezeshki SR (2001) Wetland plant responses to soil flooding. Environ Exp Bot 46:299–312CrossRefGoogle Scholar
  30. Probert ME, Keating BA (2000) What soil constraints should be included in crop and forest model? Agric Ecosyst Environ 82:273–281CrossRefGoogle Scholar
  31. Pulido P, Cazalis R, Cejudo FJ (2008) An antioxidant redox system in the nucleus of wheat seed cells suffering oxidative stress. Plant J (on line)Google Scholar
  32. Reggiani R (2006) A role for ethylene in low-oxygen signaling in rice roots. Amino Acids 30:299–301CrossRefPubMedGoogle Scholar
  33. Saab IN, Sachs MM (1996) A flooding-induced xyloglucan endo-transglycosylase homolog in maize is responsive to ethylene and associated with aerenchyma. Plant Physiol 112:385–391CrossRefPubMedGoogle Scholar
  34. 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
  35. Shi F, Yamamoto R, Shimamura S, Hiraga S, Nakayama N, Nakamura T, Yukawa K, Hachinohe M, Matsumoto H, Komatsu S (2008) Cytosolic ascorbate peroxidase 2 (cAPX 2) is involved in the soybean response to flooding. Phytochem 69:1295–1303CrossRefGoogle Scholar
  36. Tanaka N, Fihjita M, Handa H et al (2004) Proteomics of the rice cell: systematic identification of the protein populations in subcellular compartments. Mol Genet Genom 271:566–576CrossRefGoogle Scholar
  37. Voesenek LA, Colmer TD, Pierik R, Millenaar FF, Peeters AJ (2006) How plants cope with complete submergence. New Phytol 170:213–226CrossRefPubMedGoogle Scholar
  38. Wang K, Jiang Y (2007) Antioxidant responses of creeping bent-grass roots to water-logging. Crop Sci 47:232–238CrossRefGoogle Scholar
  39. Zhong Z, Karibe H, Komatsu S, Ichimura H, Nagamura Y, Sasaki T, Hirano H (1997) Screening of rice genes from a cDNA catalog based on the sequence data-file of proteins separated by two-dimensional electrophoresis. Breed Sci 47:245–251Google Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Setsuko Komatsu
    • 1
  • Tetsuya Sugimoto
    • 1
    • 2
  • Tomoki Hoshino
    • 1
  • Yohei Nanjo
    • 1
  • Kiyoshi Furukawa
    • 2
  1. 1.National Institute of Crop ScienceTsukubaJapan
  2. 2.Nagaoka University of TechnologyNagaokaJapan

Personalised recommendations