Live Microscopy Analysis of Endosomes and Vesicles in Tip-Growing Root Hairs

  • Miroslav OvečkaEmail author
  • Irene Lichtscheidl
  • Jozef Šamaj
Part of the Methods in Molecular Biology book series (MIMB, volume 1209)


Tip growth is one of the most preferable models in the study of plant cell polarity; cell wall deposition is restricted mainly to a certain area of the cell, and cell expansion at this specific area leads to the development of tubular outgrowth. Tip-growing root hairs are well-established systems for such studies, because their lateral position within the root makes them easily accessible for experimental approaches and microscopic observations. Fundamental structural and molecular processes driving tip growth are exocytosis, endocytosis, and all aspects of vesicular and endosomal dynamic trafficking, as related to targeted membrane flow. Study of vesicles and endosomes in living root hairs, however, is rather difficult, due to their small size and due to the resolution limits of conventional light microscopes. Here we present noninvasive approaches for visualizing vesicular and endosomal compartments in the tip of growing root hairs using electronic light microscopy, contrast-enhanced video light microscopy, and confocal laser scanning microscopy (CLSM). These methods allow utilizing the maximum resolution of the light microscope. Together with protocols for appropriate preparation of living plant samples, the described methods should help improve our understanding on how tiny vesicles and endosomes support the process of tip growth in root hairs.

Key words

Arabidopsis thalianaConfocal laser scanning microscopy Electronic light microscopy Endosomes Medicago sativaRoot hairs Tip growth Triticum aestivumVesicles Video microscopy 



This work was supported by grant LO1204 from the National Program of Sustainability I to the Centre of the Region Haná for Biotechnological and Agricultural Research in Olomouc and by the Austrian ŒAD/appear funded project BIOREM (Appear 43).


  1. 1.
    Baluška F, Salaj J, Mathur J, Braun M, Jasper F, Šamaj J, Chua NH, Barlow PW, Volkmann D (2000) Root hair formation: F-actin-dependent tip growth is initiated by local assembly of profilin-supported F-actin meshworks accumulated within expansin-enriched bulges. Dev Biol 227:618–632PubMedCrossRefGoogle Scholar
  2. 2.
    Hepler PK, Vidali L, Cheung AY (2001) Polarized cell growth in higher plants. Annu Rev Cell Dev Biol 17:159–187PubMedCrossRefGoogle Scholar
  3. 3.
    Šamaj J, Müller J, Beck M, Böhm N, Menzel D (2006) Vesicular trafficking, cytoskeleton and signalling in root hairs and pollen tubes. Trends Plant Sci 11:594–600PubMedCrossRefGoogle Scholar
  4. 4.
    Campanoni P, Blatt MR (2007) Membrane trafficking and polar growth in root hairs and pollen tubes. J Exp Bot 58:65–74PubMedCrossRefGoogle Scholar
  5. 5.
    Ketelaar T, Galway ME, Mulder BM, Emons AMC (2008) Rates of exocytosis and endocytosis in Arabidopsis root hairs and pollen tubes. J Microsc 231:265–273PubMedCrossRefGoogle Scholar
  6. 6.
    Galway ME, Heckman JW, Schiefelbein JW (1997) Growth and ultrastructure of Arabidopsis root hairs: the rhd3 mutation alters vacuole enlargement and tip growth. Planta 201:209–218PubMedCrossRefGoogle Scholar
  7. 7.
    Galway ME (2000) Root hair ultrastructure and tip growth. In: Ridge RW, Emons AMC (eds) Root hairs: cell and molecular biology. Springer, Tokyo, pp 1–17CrossRefGoogle Scholar
  8. 8.
    Ketelaar T, Faivre-Moskalenko C, Esseling JJ, de Ruijter NC, Grierson CS, Dogterom M, Emons AMC (2002) Positioning of nuclei in Arabidopsis root hairs: an actin-regulated process of tip growth. Plant Cell 14:2941–2955PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Robertson JG, Lyttleton P (1982) Coated and smooth vesicles in the biogenesis of cell walls, plasma membranes, infection threads and peribacteroid membranes in root hairs and nodules of white clover. J Cell Sci 58:63–78PubMedGoogle Scholar
  10. 10.
    Emons EMC, Traas JA (1986) Coated pits and coated vesicles on the plasma membrane of plant cells. Eur J Cell Biol 41:57–64Google Scholar
  11. 11.
    Ridge RW (1995) Micro-vesicles, pyriform vesicles and macrovesicles associated with the plasma membrane in the root hairs of Vicia hirsuta after freeze-substitution. J Plant Res 108:363–368CrossRefGoogle Scholar
  12. 12.
    Ovečka M, Lang I, Baluška F, Ismail A, Illeš P, Lichtscheidl IK (2005) Endocytosis and vesicle trafficking during tip growth of root hairs. Protoplasma 226:39–54PubMedCrossRefGoogle Scholar
  13. 13.
    Yoo C-M, Quan L, Cannon AE, Wen J, Blancaflor EB (2012) AGD1, a class 1 ARF-GAP, acts in common signaling pathways with phosphoinositide metabolism and the actin cytoskeleton in controlling Arabidopsis root hair polarity. Plant J 69:1064–1076PubMedCrossRefGoogle Scholar
  14. 14.
    Ovečka M, Berson T, Beck M, Derksen J, Šamaj J, Baluška F, Lichtscheidl IK (2010) Structural sterols are involved in both the initiation and tip growth of root hairs in Arabidopsis thaliana. Plant Cell 22:2999–3019PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Li R, Liu P, Wan Y, Chen T, Wang Q, Mettbach U, Baluška F, Šamaj J, Fang X, Lucas WJ, Lin J (2012) A membrane microdomain-associated protein, Arabidopsis Flot1, is involved in a clathrin-independent endocytic pathway and is required for seedling development. Plant Cell 24:2105–2122PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Nielsen E, Cheung AY, Ueda T (2008) The regulatory RAB and ARF GTPases for vesicular trafficking. Plant Physiol 147:1516–1526PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Stenmark H (2009) Rab GTPases as co-ordinators of vesicle traffic. Nat Rev Mol Cell Biol 10:513–525PubMedCrossRefGoogle Scholar
  18. 18.
    Lee Y, Bak G, Choi Y, Chuang W-I, Cho H-T, Lee Y (2008) Roles of phosphatidylinositol 3-kinase in root hair growth. Plant Physiol 147:624–635PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Voigt B, Timmers ACJ, Šamaj J, Hlavačka A, Ueda T, Preuss M, Nielsen E, Mathur J, Emans N, Stenmark H, Nakano A, Baluška F, Menzel D (2005) Actin-propelled motility of endosomes is tightly linked to polar tip-growth of root hairs. Eur J Cell Biol 84:609–621PubMedCrossRefGoogle Scholar
  20. 20.
    Žárský V, Cvrčková F, Potocký M, Hála M (2009) Exocytosis and cell polarity in plants – exocyst and recycling domains. New Phytol 183:255–272PubMedCrossRefGoogle Scholar
  21. 21.
    Richter S, Müller LM, Stierhof Y-D, Mayer U, Takada N, Kost B, Vieten A, Geldner N, Koncz C, Jürgens G (2012) Polarized cell growth in Arabidopsis requires endosomal recycling mediated by GBF1-related ARF exchange factors. Nat Cell Biol 14:80–87CrossRefGoogle Scholar
  22. 22.
    Bonnett HT, Newcomb EH (1966) Coated vesicles and other cytoplasmic components of growing root hairs of radish. Protoplasma 62:59–75CrossRefGoogle Scholar
  23. 23.
    Emons AMC (1987) The cytoskeleton and secretory vesicles in root hairs of Equisetum and Limnobium and cytoplasmic streaming in root hairs of Equisetum. Ann Bot 60:625–632Google Scholar
  24. 24.
    Galway ME, Lane DC, Schiefelbein JW (1999) Defective control of growth rate and cell diameter in tip-growing root hairs of the rhd4 mutant of Arabidopsis thaliana. Can J Bot 77:494–507Google Scholar
  25. 25.
    Ridge RW (1988) Freeze-substitution improves the ultrastructural preservation of legume root hairs. Bot Mag Tokyo 101:427–441CrossRefGoogle Scholar
  26. 26.
    Shotton DM (1988) Video-enhanced light microscopy and its application in cell biology. J Cell Sci 89:129–150PubMedGoogle Scholar
  27. 27.
    Allen RD, Allen NS, Travis JL (1981) Video-enhanced contrast, differential interference contrast (AVEC-DIC) microscopy: a new method capable of analyzing microtubule-related motility in the reticulopodial network of Allogromia laticollaris. Cell Motil 1:291–302PubMedCrossRefGoogle Scholar
  28. 28.
    Lichtscheidl IK (1995) An introduction to the video microscopy of plant cells: principles of modern light microscopical techniques and their application for the study of plant cells. Wiss Film (Wien) 47:11–40Google Scholar
  29. 29.
    Lichtscheidl IK, Foissner I (1996) Video microscopy of dynamic plant cell organelles: principles of the technique and practical application. J Microsc 181:117–128CrossRefGoogle Scholar
  30. 30.
    Lichtscheidl IK, Url WG (1987) Investigation of the protoplasm of Allium cepa inner epidermal cells using ultraviolet microscopy. Eur J Cell Biol 43:93–97Google Scholar
  31. 31.
    Šamaj J, Ovečka M, Hlavačka A, Lecourieux F, Meskiene I, Lichtscheidl I, Lenart P, Salaj J, Volkmann D, Bögre L, Baluška F, Hirt H (2002) Involvement of the mitogen-activated protein kinase SIMK in regulation of root hair tip-growth. EMBO J 21:3296–3306PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Ovečka M, Baluška F, Lichtscheidl IK (2008) Non-invasive microscopy of tip growing root hairs as a tool for study of dynamic, cytoskeleton-based processes. Cell Biol Int 32:549–553PubMedCrossRefGoogle Scholar
  33. 33.
    Volgger M, Lang I, Ovečka M, Lichtscheidl IK (2010) Plasmolysis and cell wall deposition in wheat root hairs under osmotic stress. Protoplasma 243:51–62PubMedCrossRefGoogle Scholar
  34. 34.
    Fahraeus G (1957) The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. J Gen Microbiol 16:374–381PubMedCrossRefGoogle Scholar
  35. 35.
    Sanderfoot AA, Assaad FF, Raikhel NV (2000) The Arabidopsis genome. An abundance of soluble N-ethylmaleimide-sensitive factor adaptor protein receptors. Plant Phys 124:1558–1569CrossRefGoogle Scholar
  36. 36.
    Geldner N, Dénervaud-Tendon V, Hyman DL, Mayer U, Stierhof Y-D, Chory J (2009) Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. Plant J 59:169–178PubMedCrossRefGoogle Scholar
  37. 37.
    Takáč T, Pechan T, Šamajová O, Ovečka M, Richter H, Eck C, Niehaus K, Šamaj J (2012) Wortmannin treatment induces changes in Arabidopsis root proteome and post-Golgi compartments. J Proteome Res 11:3127–3142PubMedCrossRefGoogle Scholar
  38. 38.
    Grebe M, Xu J, Möbius W, Ueda T, Nakano A, Geuze HJ, Rook MB, Scheres B (2003) Arabidopsis sterol endocytosis involves actin-mediated trafficking via ARA6-positive early endosomes. Curr Biol 13:1378–1387PubMedCrossRefGoogle Scholar
  39. 39.
    Geldner N, Anders N, Wolters H, Keicher J, Kornberger W, Muller P, Delbarre A, Ueda T, Nakano A, Jürgens G (2003) The Arabidopsis GNOM ARF-GEF mediates endosomal recycling, auxin transport, and auxin-dependent plant growth. Cell 112:219–230PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Miroslav Ovečka
    • 1
    Email author
  • Irene Lichtscheidl
    • 2
  • Jozef Šamaj
    • 1
  1. 1.Department of Cell Biology, Faculty of Science, Centre of the Region Haná for Biotechnological and Agricultural ResearchPalacký University of OlomoucOlomoucCzech Republic
  2. 2.Core Facility of Cell Imaging and Ultrastructure ResearchUniversity of ViennaViennaAustria

Personalised recommendations