Protein Extraction from Xylem and Phloem Sap

  • Julia Kehr
  • Martijn Rep
Part of the Methods in Molecular Biology book series (MIMB, volume 355)


It is well known that phloem and xylem vessels transport small nutrient molecules over long distances in higher plants. The finding that proteins also occur in both transport fluids was unexpected, and the function of most of these proteins is not yet well understood. This chapter outlines how proteins can be obtained and purified from xylem and phloem saps to perform subsequent proteomic analyses.

Key words

Xylem sap phloem sap protein precipitation proteomics 


  1. 1.
    Rep, M., Dekker, H. L., Vossen, J. H., et al. (2003). A tomato xylem sap protein represents a new family of small cysteine-rich proteins with structural similarity to lipid transfer proteins. FEBS Lett. 534, 82–86.CrossRefPubMedGoogle Scholar
  2. 2.
    Rep, M., Dekker, H. L., Vossen, J. H., et al.. (2002). Mass spectrometric identification of isoforms of PR proteins in xylem sap of fungus-infected tomato. Plant Physiol. 130, 904–917.CrossRefPubMedGoogle Scholar
  3. 3.
    Sakuta, C. and Satoh, S. (2000). Vascular tissue-specific gene expression of xylem sap glycine-rich proteins in root and their localization in the walls of metaxylem vessels in cucumber. Plant Cell Physiol. 41, 627–638.PubMedGoogle Scholar
  4. 4.
    Buhtz, A., Kolasa, A., Arlt, K., Walz, C., Kehr, J. (2004). Xylem sap protein composition is conserved among different plant species. Planta 219, 610–618.CrossRefPubMedGoogle Scholar
  5. 5.
    Kehr, J., Buhtz, A. and Giavalisco, P. (2005). Analysis of xylem sap proteins from Brassica napus. BMC Plant Biol. 5, 11.CrossRefPubMedGoogle Scholar
  6. 6.
    Fukuda, A., Okada, Y., Suzui, N., Fujiwara, T., Yoneyama, T., and Hayashi, H. (2004). Cloning and characterization of the gene for a phloem-specific glutathione S-transferase from rice leaves. Physiol. Plant. 120, 595–602.CrossRefPubMedGoogle Scholar
  7. 7.
    Giavalisco, P., Kapitza, K., Kolasa, A., Buhtz, A., and Kehr, J. (2006). Towards the proteome of Brassica napus phloem sap. Proteomics 6, 896–909.CrossRefPubMedGoogle Scholar
  8. 8.
    Walz, C., Giavalisco, P., Schad, M., Juenger, M., Klose, J., and Kehr, J. (2004). Proteomics of curcurbit phloem exudate reveals a network of defence proteins. Phytochemistry 65, 1795–1804.CrossRefPubMedGoogle Scholar
  9. 9.
    Haebel, S. and Kehr, J. (2001). Matrix-assisted laser desorption/ionization time of flight mass spectrometry peptide mass fingerprints and post source decay: a tool for the identification and analysis of phloem proteins from Cucurbita maxima Duch. separated by two dimensional polyacrylamide gel electrophoresis. Planta 213, 586–593.CrossRefPubMedGoogle Scholar
  10. 10.
    Barnes, A., Bale, J., Constantinidou, C., Ashton, P., Jones, A., and Pritchard, J. (2004). Determining protein identity from sieve element sap in Ricinus communis L. by quadrupole time of flight (Q-TOF) mass spectrometry. J. Exp. Bot. 55, 1473–1481.CrossRefPubMedGoogle Scholar
  11. 11.
    Hayashi, H., Fukuda, A., Suzui, N., and Fujimaki, S. (2000). Proteins in the sieve element-companion cell complexes: their detection, localization and possible functions. Aust. J. Plant Physiol. 27, 489–496.Google Scholar
  12. 12.
    Lucas, W. J. (1999). Plasmodesmata and the cell-to-cell transport of proteins and nucleoprotein complexes. J. Exp. Bot. 50, 979–987.CrossRefGoogle Scholar
  13. 13.
    Kehr, J., Haebel, S., Blechschmidt-Schneider, S., Willmitzer, L., Steup, M., and Fisahn, J. (1999). Analysis of phloem protein patterns from different organs of Cucurbita maxima Duch. by matrix-assisted laser desorption/ionisation time of flight mass spectroscopy combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Planta 207, 612–619.CrossRefPubMedGoogle Scholar
  14. 14.
    Yoo, B.-C., Lee, J.-Y., and Lucas, W. J. (2002). Analysis of the complexity of protein kinases within the phloem sieve tube system. Characterization of Cucurbita maxima calmodulin-like domain protein kinase 1. J. Biol. Chem. 277, 15,325–15,332.CrossRefPubMedGoogle Scholar
  15. 15.
    Iwai, H., Usui, M., Hoshino, H., et al. (2003). Analysis of Sugars in squash xylem sap. Plant Cell Physiol. 44, 582–587.CrossRefPubMedGoogle Scholar
  16. 16.
    Heizmann, U., Kreuzwieser, J., Schnitzler, J.-P., Brüggemann, N., and Rennenberg, H. (2001). Assimilate transport in the xylem sap of pedunculate oak (Quercus robur) saplings. Plant Biol. 3, 132–138.CrossRefGoogle Scholar
  17. 17.
    Lopez-Millan, A. F., Morales, F., Abad’a, A., and Abad’a, J. (2000). Effects of iron deficiency on the composition of the leaf apoplastic fluid and xylem sap in sugar beet. implications for iron and carbon transport. Plant Physiol. 124, 873–884.CrossRefPubMedGoogle Scholar
  18. 18.
    Alosi, M. C., Melroy, D. L., and Park, R. B. (1988). The regulation of gelation of phloem exudate from Cucurbita fruit by dilution, glutathione, and glutathione reductase. Plant Physiol. 86, 1089–1094.CrossRefPubMedGoogle Scholar
  19. 19.
    King, R. and Zeevaart, J. (1974). Enhancement of phloem exudation from cut petioles by chelating agents. Plant Physiol. 53, 96–103.CrossRefPubMedGoogle Scholar
  20. 20.
    Hoffmann-Benning, S., Gage, D. A., McIntosh, L., Kende, H., and Zeevaart, J. A. D. (2002). Comparison of peptides in the phloem sap of flowering and nonflowering Perilla and Lupine plants using microbore HPLC followed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Planta 216, 140–147.CrossRefPubMedGoogle Scholar
  21. 21.
    Marentes, E. and Grusak, M. A. (1998). Mass determination of low-molecular-weight proteins in phloem sap using matrix-assisted laser desorption/ionization time of flight mass spectrometry. J. Exp. Bot. 49, 903–911.CrossRefGoogle Scholar
  22. 22.
    Kennedy, J. and Mittler, T. (1953). A method of obtaining phloem sap via the mouth parts of aphids. Nature 171, 528.CrossRefGoogle Scholar
  23. 23.
    Ishiwatari, Y., Honda, C., Kawashima, I., et al. (1995). Thioredoxin h is one of the major proteins in rice phloem sap. Planta 195, 456–463.CrossRefPubMedGoogle Scholar
  24. 24.
    Clark, A. M., Jacobsen, K. R., Bostwick, D. E., Dannenhoffer, J. M., Skaggs, M. I., and Thompson, G. A. (1997). Molecular characterization of a phloem-specific gene encoding the filament protein, Phloem Protein 1 (PP1), from Cucurbita maxima. Plant J. 12, 49–61.CrossRefPubMedGoogle Scholar
  25. 25.
    Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.CrossRefPubMedGoogle Scholar
  26. 26.
    Read, S. M. and Northcote, D. H. (1983). Chemical and immunological similarities between the phloem proteins of three genera of the Cucurbitaceae. Planta 158, 119–127.CrossRefGoogle Scholar
  27. 27.
    Read, S. M. and Northcote, D. H. (1983). Subunit structure and interactions of the phloem proteins of Cucurbita maxima (pumpkin). Eur. J. Biochem. 134, 561–569.CrossRefPubMedGoogle Scholar
  28. 28.
    Christeller, J. T., Farley, P. C., Ramsay, R. J., Sullivan, P. A., and Laing, W. A. (1998). Purification, characterization and cloning of an aspartic proteinase inhibitor from squash phloem exudate. Eur. J. Biochem. 254, 160–167.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Julia Kehr
    • 1
  • Martijn Rep
    • 2
  1. 1.Max-Planck-Institute of Molecular Plant PhysiologyPotsdamGermany
  2. 2.Plant Pathology, Swammerdam Institute for Life SciencesUniversity of AmsterdamAmsterdamThe Netherlands

Personalised recommendations