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Vacuolar Staining Methods in Plant Cells

  • David Scheuring
  • Maria Schöller
  • Jürgen Kleine-VehnEmail author
  • Christian LöfkeEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1242)

Abstract

Commercially available fluorescent dyes enable the fast and specific visualization of plant vacuoles, allowing for investigation of membrane dynamics and vacuolar biogenesis in living cells. Here, we describe different approaches tinting the tonoplast or the vacuolar lumen with a range of dyes, and illustrate its utilization with established fluorescent-tagged marker lines.

Key words

Tonoplast Vacuole Membrane staining BCECF-AM FM4-64 MDY-64 VAMP711 PIN-FORMED (PIN) Arabidopsis thaliana Bright Yellow-2 (BY-2) 

Notes

Acknowledgement

We are grateful to N. Geldner and C. Luschnig for providing material, D. Whittaker for help in preparing the manuscript, and the BOKU-VIBT Imaging Center for access and expertise. This work was supported by the Vienna Science and Technology Fund (WWTF) (to J.K.-V.) and the Deutsche Forschungsgemeinschaft (DFG) (personal postdoctoral research grant to C.L. and D.S.).

References

  1. 1.
    Wink M (1993) The plant vacuole: a multifunctional compartment. J Exp Bot 44:231–246Google Scholar
  2. 2.
    Marty F (1999) Plant vacuoles. Plant Cell 11(4):587–600PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Herman EM, Larkins BA (1999) Protein storage bodies and vacuoles. Plant Cell 11(4):601–614PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Löfke C, Luschnig C, Kleine-Vehn J (2013) Posttranslational modification and trafficking of PIN auxin efflux carriers. Mech Dev 130(1):82–94PubMedCrossRefGoogle Scholar
  5. 5.
    Ozkan P, Mutharasan R (2002) A rapid method for measuring intracellular pH using BCECF-AM. Biochim Biophys Acta 1572(1):143–148PubMedCrossRefGoogle Scholar
  6. 6.
    Jelinkova A et al (2010) Probing plant membranes with FM dyes: tracking, dragging or blocking? Plant J 61(5):883–892PubMedCrossRefGoogle Scholar
  7. 7.
    Löfke C, Dünser K, Kleine-Vehn J (2013) Epidermal patterning genes impose non-cell autonomous cell size determination and have additional roles in root meristem size control. J Integr Plant Biol 55(9):864–875PubMedCrossRefGoogle Scholar
  8. 8.
    Cole L, Orlovich DA, Ashford AE (1998) Structure, function, and motility of vacuoles in filamentous fungi. Fungal Genet Biol 24(1–2):86–100PubMedCrossRefGoogle Scholar
  9. 9.
    Wiltshire EJ, Collings DA (2009) New dynamics in an old friend: dynamic tubular vacuoles radiate through the cortical cytoplasm of red onion epidermal cells. Plant Cell Physiol 50(10):1826–1839PubMedCrossRefGoogle Scholar
  10. 10.
    Abrahams S et al (2003) The Arabidopsis TDS4 gene encodes leucoanthocyanidin dioxygenase (LDOX) and is essential for proanthocyanidin synthesis and vacuole development. Plant J 35(5):624–636PubMedCrossRefGoogle Scholar
  11. 11.
    Jackson MA et al (2007) A bioinformatic approach to the identification of a conserved domain in a sugarcane legumain that directs GFP to the lytic vacuole. Funct Plant Biol 34(7):633–644CrossRefGoogle Scholar
  12. 12.
    Schwab B, Hulskamp M (2010) Neutral red staining for plant vacuoles. Cold Spring Harb Protoc 2010(6):pdb prot4953Google Scholar
  13. 13.
    Hillmer S, Quader H, Robert-Nicoud M, Robinson DG (1989) Lucifer yellow uptake in cells and protoplasts of Daucas carota visualized by laser scanning microscopy. J Exp Bot 40(4):417–423CrossRefGoogle Scholar
  14. 14.
    Gao XQ et al (2005) The dynamic changes of tonoplasts in guard cells are important for stomatal movement in Vicia faba. Plant Physiol 139(3):1207–1216PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Löfke C et al (2013) Asymmetric gibberellin signaling regulates vacuolar trafficking of PIN auxin transporters during root gravitropism. Proc Natl Acad Sci U S A 110(9):3627–3632PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Geldner N et al (2009) Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. Plant J 59(1):169–178PubMedCrossRefGoogle Scholar
  17. 17.
    Abas L et al (2006) Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism. Nat Cell Biol 8(3):249–256PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Applied Genetics and Cell BiologyUniversity of Natural Resources and Life Sciences, ViennaViennaAustria
  2. 2.Institute of Applied Genetics and Cell Biology (IAGZ)ViennaAustria

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