Xylem Sap Proteomics

  • Thomas Dugé de Bernonville
  • Cécile Albenne
  • Matthieu Arlat
  • Laurent Hoffmann
  • Emmanuelle Lauber
  • Elisabeth Jamet
Part of the Methods in Molecular Biology book series (MIMB, volume 1072)


Proteomic analysis of xylem sap has recently become a major field of interest to understand several biological questions related to plant development and responses to environmental clues. The xylem sap appears as a dynamic fluid undergoing changes in its proteome upon abiotic and biotic stresses. Unlike cell compartments which are amenable to purification in sufficient amount prior to proteomic analysis, the xylem sap has to be collected in particular conditions to avoid contamination by intracellular proteins and to obtain enough material. A model plant like Arabidopsis thaliana is not suitable for such an analysis because efficient harvesting of xylem sap is difficult. The analysis of the xylem sap proteome also requires specific procedures to concentrate proteins and to focus on proteins predicted to be secreted. Indeed, xylem sap proteins appear to be synthesized and secreted in the root stele or to originate from dying differentiated xylem cells. This chapter describes protocols to collect xylem sap from Brassica species and to prepare total and N-glycoprotein extracts for identification of proteins by mass spectrometry analyses and bioinformatics.

Key words

Cell wall Glycoproteomics Xylem sap 



The authors are grateful to the Université Paul Sabatier-Toulouse 3, France, CNRS, and INRA for support. A grant for the experiments and the postdoctoral position of TDDB were provided by the French Agence Nationale de la Recherche (Grant ANR-08-BLAN-0193-01). The authors wish to thank Drs Benoît Valot and Michel Zivy at the Plateforme d'Analyse Protéomique de Paris Sud-Ouest (PAPPSO) for fruitful collaboration. Thibaut Douché is acknowledged for his contribution to proteomics developments in the lab.


  1. 1.
    Raven JA (1993) The evolution of vascular plants in relation to quantitative functioning of dead water-conducting cells and stomata. Biol Rev 68:337–363CrossRefGoogle Scholar
  2. 2.
    Sattelmacher B (2001) The apoplast and its significance for plant mineral nutrition. New Phytol 149:167–192CrossRefGoogle Scholar
  3. 3.
    Fukuda H (1996) Xylogenesis: initiation, progression, and cell death. Annu Rev Plant Physiol Plant Mol Biol 47:299–325CrossRefPubMedGoogle Scholar
  4. 4.
    Satoh S (2006) Organic substances in xylem sap delivered to above-ground organs by the roots. J Plant Res 119:179–187CrossRefPubMedGoogle Scholar
  5. 5.
    Buhtz A, Kolasa A, Arlt K et al (2004) Xylem sap protein composition is conserved among different plant species. Planta 219:610–618CrossRefPubMedGoogle Scholar
  6. 6.
    Kehr J, Buhtz A, Giavalisco P (2005) Analysis of xylem sap proteins from Brassica napus. BMC Plant Biol 5:11CrossRefPubMedGoogle Scholar
  7. 7.
    Ligat L, Lauber E, Albenne C et al (2011) Analysis of the xylem sap proteome of Brassica oleracea reveals a high content in secreted proteins. Proteomics 11:1798–1813CrossRefPubMedGoogle Scholar
  8. 8.
    Masuda S, Kamada H, Satoh S (2001) Chitinase in cucumber xylem sap. Biosci Biotechnol Biochem 65:1883–1885CrossRefPubMedGoogle Scholar
  9. 9.
    Djordjevic M, Oakes M, Li et al (2007) The Glycine max xylem sap and apoplast proteome. J Proteome Res 6:3771–3779CrossRefPubMedGoogle Scholar
  10. 10.
    Krishnan H, Natarajan S, Bennett J et al (2011) Protein and metabolite composition of xylem sap from field-grown soybeans (Glycine max). Planta 233:921–931CrossRefPubMedGoogle Scholar
  11. 11.
    Biles C, Abeles F (1991) Xylem sap proteins. Plant Physiol 96:597–601CrossRefPubMedGoogle Scholar
  12. 12.
    Aki T, Shigyo M, Nakano R et al (2008) Nano scale proteomics revealed the presence of regulatory proteins including three FT-like proteins in phloem and xylem saps from rice. Plant Cell Physiol 49:767–790CrossRefPubMedGoogle Scholar
  13. 13.
    Dafoe N, Constabel C (2009) Proteomic analysis of hybrid poplar xylem sap. Phytochemistry 70:856–863CrossRefPubMedGoogle Scholar
  14. 14.
    Aguero C, Thorne E, Ibanez A et al (2008) Xylem sap proteins from Vitis vinifera L. Chardonnay. Am J Enol Vitic 59:306–311Google Scholar
  15. 15.
    Basha S, Mazhar H, Vasanthaiah H (2010) Proteomics approach to identify unique xylem sap proteins in Pierce’s disease-tolerant Vitis species. Appl Biochem Biotechnol 160:932–944CrossRefPubMedGoogle Scholar
  16. 16.
    Alvarez S, Goodger J, Marsh E et al (2006) Characterization of the maize xylem sap proteome. J Proteome Res 5:963–972CrossRefPubMedGoogle Scholar
  17. 17.
    Fernandez-Garcia N, Hernandez M, Casado-Vela J et al (2011) Changes to the proteome and targeted metabolites of xylem sap in Brassica oleracea in response to salt stress. Plant Cell Environ 34:821–836CrossRefPubMedGoogle Scholar
  18. 18.
    Alvarez S, Marsh E, Schroeder S et al (2008) Metabolomic and proteomic changes in the xylem sap of maize under drought. Plant Cell Environ 31:325–340CrossRefPubMedGoogle Scholar
  19. 19.
    Rep M, Dekker H, Vossen J et al (2002) Mass spectrometric identification of isoforms of PR proteins in xylem sap of fungus-infected tomato. Plant Physiol 130:904–917CrossRefPubMedGoogle Scholar
  20. 20.
    Houterman P, Speijer D, Dekker H et al (2007) The mixed xylem sap proteome of Fusarium oxysporum-infected tomato plants. Mol Plant Pathol 8:215–221CrossRefPubMedGoogle Scholar
  21. 21.
    Li S, Hartman G, Lee B-S et al (2000) Identification of a stress-induced protein in stem exudates of soybean seedlings root-infected with Fusarium solani f. sp. glycines. Plant Physiol Biochem 38:803–809CrossRefGoogle Scholar
  22. 22.
    Subramanian S, Cho U-H, Keyes C et al (2009) Distinct changes in soybean xylem sap proteome in response to pathogenic and symbiotic microbe interactions. BMC Plant Biol 9:119CrossRefPubMedGoogle Scholar
  23. 23.
    Emanuelsson O, Brunak S, von Heijne G et al (2007) Locating proteins in the cell using TargetP, SignalP, and related tools. Nat Protoc 2:953–971CrossRefPubMedGoogle Scholar
  24. 24.
    Small I, Peters N, Legeai F et al (2004) Predotar: a tool for rapidly screening proteomes for N-terminal targeting sequences. Proteomics 4:1581–1590CrossRefPubMedGoogle Scholar
  25. 25.
    Fankhauser N, Mäser P (2005) Identification of GPI anchor attachment signals by a Kohonen self-organizing map. Bioinformatics 21:1846–1852CrossRefPubMedGoogle Scholar
  26. 26.
    Eisenhaber B, Wildpaner M, Schultz CJ et al (2003) Glycosylphosphatidylinositol lipid anchoring of plant proteins. Sensitive prediction from sequence- and genome-wide studies for Arabidopsis and rice. Plant Physiol 133:1691–1701CrossRefPubMedGoogle Scholar
  27. 27.
    Sigrist CJA, Cerutti L, de Castro E et al (2010) PROSITE, a protein domain database for functional characterization and annotation. Nucleic Acids Res 38(D):161–166CrossRefGoogle Scholar
  28. 28.
    Punta M, Coggill PC, Eberhardt RY et al (2012) The Pfam protein families database. Nucleic Acids Res 40(D):290–301CrossRefGoogle Scholar
  29. 29.
    McDowall J, Hunter S (2011) InterPro protein classification. Methods Mol Biol 694:37–47CrossRefPubMedGoogle Scholar
  30. 30.
    Altschul SF, Madden TL, Schäffer AA et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402CrossRefPubMedGoogle Scholar
  31. 31.
    Marchler-Bauer A, Lu S, Anderson JB et al (2011) CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res 39(D):225–229CrossRefGoogle Scholar
  32. 32.
    Albenne C, Canut H, Boudart G et al (2009) Plant cell wall proteomics: mass spectrometry data, a trove for research on protein structure/function relationships. Mol Plant 2:977–989CrossRefPubMedGoogle Scholar
  33. 33.
    Faye L, Boulaflous A, Benchabane M et al (2005) Protein modifications in the plant secretory pathway: current status and practical implications in molecular pharming. Vaccine 23:1770–1778CrossRefPubMedGoogle Scholar
  34. 34.
    Minic Z, Jamet E, Négroni L et al (2007) A sub-proteome of Arabidopsis thaliana mature stems trapped on Concanavalin A is enriched in cell wall glycoside hydrolases. J Exp Bot 58:2503–2512CrossRefPubMedGoogle Scholar
  35. 35.
    Shevchenko A, Wilm M, Vorm O et al (1996) Mass spectrometric sequencing of proteins from silver stained polyacrylamide gels. Anal Chem 68:850–858CrossRefPubMedGoogle Scholar
  36. 36.
    San Clemente H, Pont-Lezica R, Jamet E (2009) Bioinformatics as a tool for assessing the quality of sub-cellular proteomic strategies and inferring functions of proteins: plant cell wall proteomics as a test case. Bioinform Biol Insights 3:15–28Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Thomas Dugé de Bernonville
    • 1
  • Cécile Albenne
    • 2
  • Matthieu Arlat
    • 1
  • Laurent Hoffmann
    • 2
  • Emmanuelle Lauber
    • 3
  • Elisabeth Jamet
    • 2
  1. 1.LIPMUMR CNRS-INRACastanet-TolosanFrance
  2. 2.Pôle de Biotechnologie Végétale, LRSV, UMR 5546Université Paul SabatierToulouse 3/CNRS, AuzevilleCastanet-TolosanFrance
  3. 3.LRSV, UMR 5546 UPS/CNRSCastanet-TolosanFrance

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