Journal of Biosciences

, Volume 35, Issue 1, pp 49–62 | Cite as

Proteome analysis of soybean roots under waterlogging stress at an early vegetative stage

  • Iftekhar Alam
  • Dong-Gi Lee
  • Kyung-Hee Kim
  • Choong-Hoon Park
  • Shamima Akhtar Sharmin
  • Hyoshin Lee
  • Ki-Won Oh
  • Byung-Wook Yun
  • Byung-Hyun Lee


To gain better insight into how soybean roots respond to waterlogging stress, we carried out proteomic profiling combined with physiological analysis at two time points for soybean seedlings in their early vegetative stage. Seedlings at the V2 stage were subjected to 3 and 7 days of waterlogging treatments. Waterlogging stress resulted in a gradual increase of lipid peroxidation and in vivo H2O2 level in roots. Total proteins were extracted from root samples and separated by two-dimensional gel electrophoresis (2-DE). A total of 24 reproducibly resolved, differentially expressed protein spots visualized by Coomassie brilliant blue (CBB) staining were identified by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis. Of these, 14 proteins were upregulated; 5 proteins were decreased; and 5 were newly induced in waterlogged roots. The identified proteins include well-known classical anaerobically induced proteins as well as novel waterlogging-responsive proteins that were not known previously as being waterlogging responsive. The novel proteins are involved in several processes, i.e. signal transduction, programmed cell death, RNA processing, redox homeostasis and metabolisms of energy. An increase in abundance of several typical anaerobically induced proteins, such as glycolysis and fermentation pathway enzymes, suggests that plants meet energy requirement via the fermentation pathway due to lack of oxygen. Additionally, the impact of waterlogging on the several programmed cell death- and signal transduction-related proteins suggest that they have a role to play during stress. RNA gel blot analysis for three programmed cell death-related genes also revealed a differential mRNA level but did not correlate well with the protein level. These results demonstrate that the soybean plant can cope with waterlogging through the management of carbohydrate consumption and by regulating programmed cell death. The identification of novel proteins such as a translation initiation factor, apyrase, auxin-amidohydrolase and coproporphyrinogen oxidase in response to waterlogging stress may provide new insight into the molecular basis of the waterlogging-stress response of soybean.


Abiotic stress programmed cell death proteomics, soybean root waterlogging 

Abbreviations used


two-dimensional gel electrophoresis




alcohol dehydrogenase


anaerobic polypeptide


Coomassie brilliant blue


electrospray ionization tandem mass spectrometry


glutamine synthetase




isoflavone reductase


matrix assisted laser desorption ionization time-of-flight


nitrous oxide


programmed cell death




peptide mass fingerprinting


inorganic pyrophosphate


reactive oxygen species


S-adenosyl-L-methionine synthetase


sodium dodecylsulphate polyacrylamide gel electrophoresis


thiobarbituric acid reactive substance




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  1. Aggarwal P K, Kalra N, Chander S and Pathak H 2006 InfoCrop: a dynamic simulation model for the assessment of crop yields, losses due to pests, and environmental impact of agroecosystems in tropical environments. I. Model description; Agric. Syst. 89 1–25CrossRefGoogle Scholar
  2. Ahsan N, Lee D-G, Lee S-H, Lee K-W, Bahk J-D and Lee B-H 2007a A proteomic screen and identification of waterloggingregulated proteins in tomato roots; Plant Soil 295 37–51CrossRefGoogle Scholar
  3. Ahsan N, Lee D-G, Lee S-H, Kang K Y, Bahk J D, Choi M S, Lee JJ, Renaut J and Lee B-H 2007b Comparative proteomic analysis of tomato leaves in response to waterlogging stress; Physiol. Plant 131 555–570CrossRefPubMedGoogle Scholar
  4. Amarante L and Sodek L 2006 Water logging effect on xylem sap glutamine of nodulated soybean; Biol. Plant. 50 405–410CrossRefGoogle Scholar
  5. Amillet J M, Buisson N and Labbe-Bois R 1996 Characterization of an upstream activation sequence and two Rox1p-responsive sites controlling the induction of the yeast HEM13 gene by oxygen and heme deficiency; J. Biol. Chem. 271 24425–24432CrossRefPubMedGoogle Scholar
  6. Araki H 2006 Water uptake of soybean (Glycine max L. Merr.) during exposure to O2 deficiency and field level CO2 concentration in the root zone; Field Crops Res. 96 98–105CrossRefGoogle Scholar
  7. Bailey-Serres J and Freeling M 1990 Hypoxic stress-induced changes in ribosomes of maize seedling roots; Plant Physiol. 94 1237–1243CrossRefPubMedGoogle Scholar
  8. Blanco M, Becerra M, González-Siso M I and Cerdán M E 2005 Functional characterization of KlHEM13, a hypoxic gene of Kluyveromyces lactis; Can. J. Microbiol. 51 241–249CrossRefPubMedGoogle Scholar
  9. Boru G, Vantoai T, Alves J, Hua D and Knee M 2003 Responses of soybean to oxygen deficiency and elevated root-zone carbon dioxide concentration; Ann. Bot. 91 447–453CrossRefPubMedGoogle Scholar
  10. Chang W W P, Huang L, Shen M, Webster C, Burlingame A M and Roberts J K M 2000 Patterns of protein synthesis and tolerance of anoxia in root tips of maize seedlings acclimated to a lowoxygen environment, and identification of proteins by mass spectrometry; Plant Physiol. 122 295–318CrossRefPubMedGoogle Scholar
  11. Chivasa S, Ndimba B K, Simon W J, Lindsey K and Slabasc A R 2005 Extracellular ATP functions as an endogenous external metabolite regulating plant cell viability; Plant Cell 17 3019–3034CrossRefPubMedGoogle Scholar
  12. Dakora F D and Phillips D A 1996 Diverse functions of isoflavonoids in legumes transcend anti-microbial definitions of phytoalexins; Physiol. Mol. Plant Pathol. 49 1–20CrossRefGoogle Scholar
  13. Dennis E S, Dolferus R, Ellis M, Rahman M, Wu Y, Hoeren F U, Grover A, Ismond K P, Good A G and Peacock W J 2000 Molecular strategies for improving waterlogging tolerance in plants; J. Exp. Bot. 51 89–97CrossRefPubMedGoogle Scholar
  14. Drew M C 1997 Oxygen deficiency and root metabolism: injury and acclimation under hypoxia and anoxia; Annu. Rev. Plant Physiol. Plant Mol. Biol. 48 223–250CrossRefPubMedGoogle Scholar
  15. Dubey H, Bhatia G, Pasha S and Grover A 2003 Proteome maps of flood-tolerant FR 13A and flood-sensitive IR 54 rice types depicting proteins associated with O2 deprivation stress and recovery regimes; Curr. Sci. 84 83–89Google Scholar
  16. FAO 2002 Statistical database of Food and Agriculture Organization; URL:
  17. Githiri S M, Watanabe S, Harada K and Takahashi R 2006 QTL analysis of flooding tolerance in soybean at an early vegetative growth stage; Plant Breeding 125 613–618CrossRefGoogle Scholar
  18. Haffani Y Z, Silva N F and Goring D R 2004 Receptor kinase signaling in plants; Can. J. Bot. 85 1–15CrossRefGoogle Scholar
  19. Heath R L and Packer L 1968 Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation; Arch. Biochem. Biophys. 125 189–198CrossRefPubMedGoogle Scholar
  20. Huang S, Greenway H, Colmer T D and Millar A H 2005 Protein synthesis by rice coleoptiles during prolonged anoxia: implications for glycolysis, growth and energy utilization; Ann. Bot. 96 703–715CrossRefPubMedGoogle Scholar
  21. Huynh L N, Toai T V, Streeter J and Banowetz G 2005 Regulation of flooding tolerance of SAG12: ipt Arabidopsis plants by cytokinin; J. Exp. Bot. 56 1397–1407CrossRefGoogle Scholar
  22. Ismond K P, Dolferus R, Pauw M D, Dennis E S and Good A G 2003 Enhanced low oxygen survival in Arabidopsis through increased metabolic flux in the fermentative pathway; Plant Physiol. 132 1292–1302CrossRefPubMedGoogle Scholar
  23. Ito J, Heazlewood J L and Millar A H 2007 The plant mitochondrial proteome and the challenge of defining the posttranslational modifications responsible for signaling and stress effects on respiratory functions; Physiol. Plant. 129 207–224CrossRefGoogle Scholar
  24. Jain M, Nijhawan A, Arora R, Agarwal P, Ray S, Sharma P, Kapoor S, Tyagi A K and Khurana J P 2007 F-Box proteins in rice. Genome-wide analysis, classification, temporal and spatial gene expression during panicle and seed development, and regulation by light and abiotic stress; Plant Physiol. 143 1467–1483CrossRefPubMedGoogle Scholar
  25. Jambunathan N and Mahalingam R 2006 Analysis of Arabidopsis growth factor gene 1 (GFG1) encoding a nudix hydrolase during oxidative signaling; Planta 224 1–11CrossRefPubMedGoogle Scholar
  26. Jao D L E and Chen K Y 2006 Tandem affinity purification revealed the hypusine-dependent binding of eukaryotic initiation factor 5A to the translating 80S ribosomal complex; J. Cell. Biochem. 97 583–598CrossRefPubMedGoogle Scholar
  27. Jensen O N 2004 Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry; Curr. Opin. Chem. Biol. 8 33–41CrossRefPubMedGoogle Scholar
  28. Jeter C R, Tang W, Henaff E, Butterfield T and Roux S J 2004 Evidence of a novel cell signaling role for extra cellular adenosine triphosphates and diphosphates in Arabidopsis; Plant Cell 16 2652–2664CrossRefPubMedGoogle Scholar
  29. Kim S T, Yu S, Kim S G, Kim H J, Kang S Y, Hwang D H, Jang Y S and Kang K Y 2004 Proteome analysis of rice blast fungus (Magnaporthe grisea) proteome during aspersorium formation; Proteomics 4 3579–3587CrossRefPubMedGoogle Scholar
  30. Kim S Y, Sivaguru M and Stacey G 2006 Extracellular ATP in plants: visualization, localization, and analysis of physiological significance in growth and signaling; Plant Physiol. 142 984–992CrossRefPubMedGoogle Scholar
  31. Klok E J, Wilson I W, Wilson D, Chapman S C, Ewing R M, Somerville S C, Peacock W J, Dolferus R and Dennis E S 2002 Expression profile analysis of the low-oxygen response in Arabidopsis root cultures; Plant Cell 14 2481–2494CrossRefPubMedGoogle Scholar
  32. Laemmli U K 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T 4; Nature (London) 227 680–685CrossRefGoogle Scholar
  33. Lee D-G, Ahsan N, Lee S-H, Kang K Y, Bahk J-D, Lee I J and Lee B-H 2007a A proteomic approach in analyzing heat-responsive proteins in rice leaves; Proteomics 7 3369–3383CrossRefPubMedGoogle Scholar
  34. Lee D-G, Ahsan N, Lee S-H, Kang K Y, Lee J-J and Lee B-H 2007b An approach to identify cold-induced low-abundant proteins in rice leaf; C. R. Biologies 330 215–225CrossRefPubMedGoogle Scholar
  35. Lers A, Burd S, Lomaniec E, Droby, S and Chalutz E 1998 The expression of a grapefruit gene encoding an isoflavone reductase-like protein is induced in response to UV irradiation; Plant Mol. Biol. 36 847–856CrossRefPubMedGoogle Scholar
  36. Lin K H, Weng C C, Lo H F and Chen J T 2004 Study of the root antioxidative system of tomatoes and eggplant under waterlogged conditions; Plant Sci. 167 355–365CrossRefGoogle Scholar
  37. Liu F, VanToai T, Moy L P, Bock G, Linford L D and Quackenbush J 2005 Global transcription profiling reveals comprehensive insights into hypoxic response in Arabidopsis; Plant Physiol. 137 1115–1129CrossRefPubMedGoogle Scholar
  38. Lowry O H, Rosebrough N J, Farr A L and Randall R J 1951 Protein measurement with the folin phenol reagent; J. Biol. Chem. 193 265–275PubMedGoogle Scholar
  39. Manac’h-Little N, Igamberdiev A U and Hill R D 2005 Hemoglobin expression affects ethylene production in maize cell cultures; Plant Physiol. Biochem. 43 485–489CrossRefPubMedGoogle Scholar
  40. McLennan A G 2006 The Nudix hydrolase superfamily; Cell Mol. Life Sci. 63 123–143CrossRefPubMedGoogle Scholar
  41. Mustroph A, Albrecht G, Hajirezaei M, Grimm B and Biemelt S 2005 Low levels of pyrophosphate in transgenic potato plants expressing E. coli pyrophosphatase lead to decreased vitality under oxygen deficiency; Ann. Bot. 96 717–726CrossRefPubMedGoogle Scholar
  42. Nandi D, Tahiliani P, Kumar A and Chandu D 2006 The ubiquitinproteasome system; J. Biosci. 31 137–155CrossRefPubMedGoogle Scholar
  43. Oosterhuis D M, Scott H D, Hampton R E and Wullschleger SD 1990 Physiological responses of two soybean [Glycine max L. Merri.] cultivar to short term flooding; Environ. Exp. Bot. 30 85–92CrossRefGoogle Scholar
  44. Petrucco S, Bolchi A, Foroni C, Percudani R, Rossi G L and Ottonello S 1996 A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation; Plant Cell 8 69–80CrossRefPubMedGoogle Scholar
  45. Reggiani R, Nebuloni M, Mattana M and Brambilla I 2000 Anaerobic accumulation of amino acids in rice roots: role of the glutamine synthetase/glutamate synthase cycle; Amino Acids 18 207–217CrossRefPubMedGoogle Scholar
  46. Roux S J and Steinebrunner I 2007 Extracellular ATP: an unexpected role as a signaler in plants; Trends Plant Sci. 12 522–527CrossRefPubMedGoogle Scholar
  47. Sachs M M, Subbaiah C C and Saab I N 1996 Anaerobic gene expression and flooding tolerance in maize; J. Exp. Bot. 47 1–15CrossRefGoogle Scholar
  48. Sairam R K, Kumutha D, Ezhilmathi K, Deshmukh P S and Srivastava G C 2008 Physiology and biochemistry of waterlogging tolerance in plants; Biol. Plant. 52 401–412CrossRefGoogle Scholar
  49. Sakata K, Ohyanagi H, Nobori H, Nakamura T, Hashiguchi A, Nanjo Y, Mikami Y, Yunokawa H and Komatsu S 2009 Soybean proteome database: a data resource for plant differential omics; J. Proteome Res. 8 3539–3548CrossRefPubMedGoogle Scholar
  50. Serrano M, Parra S, Alcaraz L D and Guzma P 2006 The ATL gene family from Arabidopsis thaliana and Oryza sativa comprises a large number of putative ubiquitin ligases of the RING-H2 type; J. Mol. Evol. 62 434–445CrossRefPubMedGoogle Scholar
  51. Shi F, Yamamoto R, Shimamura S, Hiraga S, Nakayama N, Nakamura T, Yukawa K, Hachinohe M, Matsumoto H and Komatsu S 2008 Cytosolic ascorbate peroxidase 2 (cAPX 2) is involved in the soybean response to flooding; Phytochemistry 69 1295–1303CrossRefPubMedGoogle Scholar
  52. Stone S L, Hauksdottir H, Troy A, Herschleb J, Kraft E and Callis J 2005 Functional analysis of the RING-type ubiquitin ligase family of Arabidopsis; Plant Physiol. 137 13–30CrossRefPubMedGoogle Scholar
  53. Subbaiah C C and Sachs M M 2003 Molecular and cellular adaptations of maize to flooding stress; Ann. Bot. 91 119–127CrossRefPubMedGoogle Scholar
  54. Thompson J E, Hopkins M T, Taylor C and Wang T-W 2004 Regulation of senescence by eukaryotic translation initiation factor 5A: implications for plant growth and development; Trends Plant Sci. 9 174–179CrossRefPubMedGoogle Scholar
  55. Torii K U and Clark S E 2000 Receptor-like kinases in plant development; in Plant protein kinases, advances in botanical research (eds) M Kreis and J C Walker (thematic volume) (London: Academic Press) pp 270–298Google Scholar
  56. Watts R A, Hunt P W, Hvitved, A N, Hargrove M S, Peacock W J and Dennis E S 2001 A hemoglobin from plants homologous to truncated hemoglobins of microorganisms; Proc. Natl. Acad. Sci. USA 98 10119–10124CrossRefPubMedGoogle Scholar
  57. Wang T W, Lu L, Wang D and Thompson J E 2001 Isolation and characterization of senescence induced cDNAs encoding deoxyhypusine synthase and eukaryotic translation initiation factor 5A from tomato; J. Biol. Chem. 276 17541–17549CrossRefPubMedGoogle Scholar
  58. Weger H G and Turpin D H 1989 Mitochondrial respiration can support NO3/− and NO2/− reduction during photosynthesis. Interactions between photosynthesis, respiration and N assimilation in the N-limited green alga Selenastrum minutum; Plant Physiol. 89 409–415CrossRefPubMedGoogle Scholar
  59. Woodward A W and Bartel B 2005 Auxin: regulation, action, and interaction; Ann. Bot. 95 707–735CrossRefPubMedGoogle Scholar
  60. Xie J and Guo Q 2006 Apoptosis antagonizing transcription factor protects renal tubule cells against oxidative damage and apoptosis induced by ischemia-reperfusion; J. Am. Soc. Nephrol. 17 3336–3346CrossRefPubMedGoogle Scholar
  61. Yan B, Dai Q, Liu X, Huang S and Wang Z 1996 Flooding induced membrane damage, lipid oxidation and active oxygen generation in corn leaves; Plant Soil 179 261–268CrossRefGoogle Scholar

Copyright information

© Indian Academy of Sciences 2010

Authors and Affiliations

  • Iftekhar Alam
    • 1
  • Dong-Gi Lee
    • 1
  • Kyung-Hee Kim
    • 1
  • Choong-Hoon Park
    • 1
  • Shamima Akhtar Sharmin
    • 1
  • Hyoshin Lee
    • 2
  • Ki-Won Oh
    • 3
  • Byung-Wook Yun
    • 4
  • Byung-Hyun Lee
    • 1
  1. 1.Division of Applied Life Science (BK21 Program), IALS, PMBBRCGyeongsang National UniversityJinjuKorea
  2. 2.Biotechnology DivisionKorea Forest Research InstituteSuwonKorea
  3. 3.Research Policy BureauRDASuwonKorea
  4. 4.Institute of Molecular Plant SciencesUniversity of EdinburghEdinburghUK

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