Phloem pp 83-94 | Cite as

Super-Resolution Microscopy of Phloem Proteins

  • Ryan C. Stanfield
  • Alexander SchulzEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2014)


Super-resolution microscopy bridges the gap between light and electron microscopy and gives new opportunities for the study of proteins that contribute to phloem function. The established super-resolution techniques are derived from fluorescence microscopy and depend on fluorescent dyes, proteins tagged with GFP variants or fluorochrome-decorated antibodies. Compared with confocal microscopy they improve the resolution between 2.5 and 10 times and, thus, allow a much more precise (co-) localization of membranes, plasmodesmata, and structural proteins. However, they are limited to thin tissue slices rather than intact plant organs and can only show immobilized or only slowly moving targets. Accordingly, the first super-resolution micrographs of the phloem were recorded from fixed tissue which was sectioned using a vibratome or microtome. As with transmission electron microscopy, preparation of phloem tissue for super-resolution microscopy is challenged by the sudden pressures release when dissecting the functional tissue (see Chapter  2).

This chapter describes a protocol for investigation of proteins in the plasma membranes of sieve elements and companion cells. It illustrates how high-resolution fluorescence imaging can provide information that could not be obtained with confocal or electron microscopy. Further, a brief introduction outlines the theoretical background of super-resolution techniques suitable for phloem imaging and summarizes the findings of the first available super-resolution studies on the phloem. The protocol focusses on the crucial steps for super-resolution microscopy of immunolocalized phloem proteins, adjusted to the use of three-dimensional structured illumination microscopy (3D-SIM).

Key words

Aquaporins Early Nodulin Like 9 Plasma membrane domains Pore-plasmodesma units Sieve-element reticulum 


  1. 1.
    Behnke HD, Sjolund RD (eds) (1990) Sieve elements—comparative structure, induction and development. Springer, Berlin Heidelberg. Scholar
  2. 2.
    Schulz A (1986) Wound phloem in transition to bundle phloem in primary roots of Pisum sativum L. 1. Development of bundle-leaving wound-sieve tubes. Protoplasma 130(1):12–26. Scholar
  3. 3.
    Ernst AM, Jekat SB, Zielonka S, Muller B, Neumann U, Ruping B, Twyman RM, Krzyzanek V, Prufer D, Noll GA (2012) Sieve element occlusion (SEO) genes encode structural phloem proteins involved in wound sealing of the phloem. Proc Natl Acad Sci U S A 109(28):E1980–E1989. Scholar
  4. 4.
    Knoblauch M, Froelich DR, Pickard WF, Peters WS (2014) SEORious business: structural proteins in sieve tubes and their involvement in sieve element occlusion. J Exp Bot 65(7):1879–1893. Scholar
  5. 5.
    Anstead JA, Froelich DR, Knoblauch M, Thompson GA (2012) Arabidopsis P-protein filament formation requires both AtSEOR1 and AtSEOR2. Plant Cell Physiol 53(6):1033–1042. Scholar
  6. 6.
    Froelich DR, Mullendore DL, Jensen KH, Ross-Elliott TJ, Anstead JA, Thompson GA, Pélissier HC, Knoblauch M (2011) Phloem ultrastructure and pressure flow: sieve-element-occlusion-related agglomerations do not affect translocation. Plant Cell 23(12):4428–4445. Scholar
  7. 7.
    Leineweber K, Schulz A, Thompson GA (2000) Dynamic transitions in the translocated phloem filament protein. Aust J Plant Physiol 27(8–9):733–741. Scholar
  8. 8.
    Golecki B, Schulz A, Thompson GA (1999) Translocation of structural P proteins in the phloem. Plant Cell 11(1):127–140CrossRefGoogle Scholar
  9. 9.
    Golecki B, Aca S, Carstens-Behrens U, Kollmann R (1998) Evidence for graft transmission of structural phloem proteins or their precursors in heterografts of Cucurbitaceae. Planta 206(4):630–640. Scholar
  10. 10.
    Engleman EM (1965) Sieve element of impatiens Sultanii2. developmental aspects. Ann Bot London 29(1):103–104. Scholar
  11. 11.
    van Bel AJE, Knoblauch M (2000) Sieve element and companion cell: the story of the comatose patient and the hyperactive nurse. Aust J Plant Physiol 27(6):477–487. Scholar
  12. 12.
    Liesche J, Schulz A (2018) Phloem transport in gymnosperms: a question of pressure and resistance. Curr Opin Plant Biol 43:36–42. Scholar
  13. 13.
    Martens HJ, Roberts AG, Oparka KJ, Schulz A (2006) Quantification of plasmodesmatal endoplasmic reticulum coupling between sieve elements and companion cells using fluorescence redistribution after photobleaching. Plant Physiol 142(2):471–480. Scholar
  14. 14.
    Stanfield RC, Hacke UG, Laur J (2017) Are phloem sieve tubes leaky conduits supported by numerous aquaporins? Am J Bot 104(5):719–732. Scholar
  15. 15.
    Laur J, Hacke UG (2014) Exploring Picea glauca aquaporins in the context of needle water uptake and xylem refilling. New Phytol 203(2):388–400. Scholar
  16. 16.
    Almeida-Rodriguez AM, Hacke UG (2012) Cellular localization of aquaporin mRNA in hybrid poplar stems. Am J Bot 99(7):1249–1254. Scholar
  17. 17.
    Ziomkiewicz I, Sporring J, Pomorski TG, Schulz A (2015) Novel approach to measure the size of plasma-membrane nanodomains in single molecule localization microscopy. Cytometry A 87A(9):868–877. Scholar
  18. 18.
    Khan JA, Wang Q, Sjolund RD, Schulz A, Thompson GA (2007) An early nodulin-like protein accumulates in the sieve element plasma membrane of Arabidopsis. Plant Physiol 143(4):1576–1589. Scholar
  19. 19.
    Ivashikina N, Deeken R, Ache P, Kranz E, Pommerrenig B, Sauer N, Hedrich R (2003) Isolation of AtSUC2 promoter-GFP-marked companion cells for patch-clamp studies and expression profiling. Plant J 36(6):931–945CrossRefGoogle Scholar
  20. 20.
    DeWitt ND, Sussman MR (1995) Immunocytological localization of an epitope-tagged plasma membrane proton pump (H+-ATPase) in phloem companion cells. Plant Cell 7(12):2053–2067PubMedPubMedCentralGoogle Scholar
  21. 21.
    Kühn C, Franceschi VR, Schulz A, Lemoine R, Frommer WB (1997) Macromolecular trafficking indicated by localization and turnover of sucrose transporters in enucleate sieve elements. Science 275(5304):1298–1300. Scholar
  22. 22.
    Liesche J, He H-X, Grimm B, Schulz A, Kuehn C (2010) Recycling of Solanum sucrose transporters expressed in yeast, tobacco, and in mature phloem sieve elements. Mol Plant 3(6):1064–1074. Scholar
  23. 23.
    Bell K, Oparka K (2011) Imaging plasmodesmata. Protoplasma 248(1):9–25. Scholar
  24. 24.
    Klar TA, Jakobs S, Dyba M, Egner A, Hell SW (2000) Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc Natl Acad Sci U S A 97(15):8206–8210. Scholar
  25. 25.
    Gustafsson MG (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198(Pt 2):82–87CrossRefGoogle Scholar
  26. 26.
    Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF (2006) Imaging intracellular fluorescent proteins at Nanometer resolution. Science 313(5793):1642–1645. Scholar
  27. 27.
    Fitzgibbon J, Bell K, King E, Oparka K (2010) Super-resolution imaging of plasmodesmata using three-dimensional structured illumination microscopy. Plant Physiol 153(4):1453–1463. Scholar
  28. 28.
    Schermelleh L, Heintzmann R, Leonhardt H (2010) A guide to super-resolution fluorescence microscopy. J Cell Biol 190(2):165–175. Scholar
  29. 29.
    Huang B, Bates M, Zhuang XW (2009) Super-resolution fluorescence microscopy. Ann Rev Biochem 78:993–1016. Scholar
  30. 30.
    Gustafsson MG, Shao L, Carlton PM, Wang CJ, Golubovskaya IN, Cande WZ, Agard DA, Sedat JW (2008) Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys J 94(12):4957–4970. Scholar
  31. 31.
    Schermelleh L, Carlton PM, Haase S, Shao L, Winoto L, Kner P, Burke B, Cardoso MC, Agard DA, Gustafsson MGL, Leonhardt H, Sedat JW (2008) Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320(5881):1332–1336. Scholar
  32. 32.
    Lingwood D, Simons K (2010) Lipid rafts as a membrane-organizing principle. Science 327(5961):46–50. Scholar
  33. 33.
    Raffaele S, Mongrand S, Gamas P, Niebel A, Ott T (2007) Genome-wide annotation of remorins, a plant-specific protein family: evolutionary and functional perspectives. Plant Physiol 145(3):593–600. Scholar
  34. 34.
    Jarsch IK, Konrad SSA, Stratil TF, Urbanus SL, Szymanski W, Braun P, Braun KH, Ott T (2014) Plasma membranes are subcompartmentalized into a plethora of coexisting and diverse microdomains in Arabidopsis and Nicotiana benthamiana. Plant Cell 26(4):1698–1711. Scholar
  35. 35.
    Bell K, Oparka K, Knox K (2018) Preparation and imaging of specialized ER using super-resolution and TEM techniques. In: Hawes C, Kriechbaumer V (eds) The plant endoplasmic reticulum: methods and protocols. Springer, New York, NY, pp 33–42. Scholar
  36. 36.
    Gong HQ, Peng YB, Zou C, Wang DH, Xu ZH, Bai SN (2006) A simple treatment to significantly increase signal specificity in immunohistochemistry. Plant Mol Biol Rep 24(1):93–101. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Renewable ResourcesUniversity of AlbertaEdmontonCanada
  2. 2.Department of Plant and Environmental SciencesUniversity of CopenhagenFrederiksbergDenmark

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