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Phloem pp 109–122Cite as

Immunohistochemical Localization of Proteins in the Phloem: Problems and Solutions

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2014))

Abstract

Immunolocalization of proteins in differentiated phloem cells is a challenging task given their special anatomy, organellar infrastructure, and the phloem tissue’s heterogeneity. Incorporation of specific wall components in the thickened cell walls of phloem cells is often the source of unspecific labeling, leading to erroneous localization. Therefore, special care is required regarding generation and purification of specific antibodies. In addition, tissue preservation of phloem cells, which contain a high osmotic pressure in their functional state, is a very challenging task prone to various pitfalls. This chapter provides practical advice for cautious tissue preparation and antibody purification. Furthermore, methods that can be used to verify immunohistochemical localization data, such as promoter-reporter studies or activity tests, are discussed. Such confirmation experiments are essential for unambiguous determination of protein location in cells of the phloem.

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References

  1. Botha CE (2013) A tale of two neglected systems-structure and function of the thin- and thick-walled sieve tubes in monocotyledonous leaves. Front Plant Sci 4:297. https://doi.org/10.3389/fpls.2013.00297

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Sjølund RD (1997) The phloem sieve element: a river runs through it. Plant Cell 9(7):1137–1146. https://doi.org/10.1105/tpc.9.7.1137

    Article  PubMed  PubMed Central  Google Scholar 

  3. van Bel A, Knoblauch M (2000) Sieve element and companion cell: the story of the comatose patient and the hyperactive nurse. Funct Plant Biol 27(6):477–487. https://doi.org/10.1071/PP99172

    Article  Google Scholar 

  4. van Bel AJE, Ehlers K, Knoblauch M (2002) Sieve elements caught in the act. Trends Plant Sci 7(3):126–132

    Article  PubMed  Google Scholar 

  5. Turgeon R, Wolf S (2009) Phloem transport: cellular pathways and molecular trafficking. Annu Rev Plant Biol 60:207–221. https://doi.org/10.1146/annurev.arplant.043008.092045

    Article  CAS  PubMed  Google Scholar 

  6. Knoblauch AM, Divall MJ, Owuor M, Musunka G, Pascall A, Nduna K, Ng’uni H, Utzinger J, Winkler MS (2018) Selected indicators and determinants of women’s health in the vicinity of a copper mine development in northwestern Zambia. BMC Womens Health 18(1):62. https://doi.org/10.1186/s12905-018-0547-7

    Article  PubMed  PubMed Central  Google Scholar 

  7. Lin MK, Lee YJ, Lough TJ, Phinney BS, Lucas WJ (2009) Analysis of the pumpkin phloem proteome provides insights into angiosperm sieve tube function. Mol Cell Proteomics 8(2):343–356. https://doi.org/10.1074/mcp.M800420-MCP200

    Article  CAS  PubMed  Google Scholar 

  8. Zhang C, Yu X, Ayre BG, Turgeon R (2012) The origin and composition of cucurbit “phloem” exudate. Plant Physiol 158(4):1873–1882. https://doi.org/10.1104/pp.112.194431

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Zhang B, Tolstikov V, Turnbull C, Hicks LM, Fiehn O (2010) Divergent metabolome and proteome suggest functional independence of dual phloem transport systems in cucurbits. Proc Natl Acad Sci U S A 107(30):13532–13537. https://doi.org/10.1073/pnas.0910558107

    Article  PubMed  PubMed Central  Google Scholar 

  10. Hu C, Ham BK, El-Shabrawi HM, Alexander D, Zhang D, Ryals J, Lucas WJ (2016) Proteomics and metabolomics analyses reveal the cucurbit sieve tube system as a complex metabolic space. Plant J 87(5):442–454. https://doi.org/10.1111/tpj.13209

    Article  CAS  PubMed  Google Scholar 

  11. Knoblauch M, Peters WS, Bell K, Ross-Elliott TJ, Oparka KJ (2018) Sieve-element differentiation and phloem sap contamination. Curr Opin Plant Biol 43:43–49. https://doi.org/10.1016/j.pbi.2017.12.008

    Article  PubMed  Google Scholar 

  12. Paultre DS, Gustin MP, Molnar A, Oparka KJ (2016) Lost in transit: long-distance trafficking and phloem unloading of protein signals in Arabidopsis homografts. Plant Cell 28(9):2016–2025. https://doi.org/10.1105/tpc.16.00249

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Schulz A (2017) Long-distance trafficking: lost in transit or stopped at the gate? Plant Cell 29(3):426–430. https://doi.org/10.1105/tpc.16.00895

    Article  PubMed  PubMed Central  Google Scholar 

  14. Chou M, Krause KH, Campbell KP, Jensen KG, Sjølund RD (1989) Antibodies against the calcium-binding protein: calsequestrin from Streptanthus tortuosus (Brassicaceae). Plant Physiol 91(4):1259–1261

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Khan JA, Wang Q, Sjølund 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. https://doi.org/10.1104/pp.106.092296

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Meyer S, Lauterbach C, Niedermeier M, Barth I, Sjølund RD, Sauer N (2004) Wounding enhances expression of AtSUC3, a sucrose transporter from Arabidopsis sieve elements and sink tissues. Plant Physiol 134(2):684–693. https://doi.org/10.1104/pp.103.033399

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Wang Q, Monroe J, Sjølund RD (1995) Identification and characterization of a phloem-specific beta-amylase. Plant Physiol 109(3):743–750

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 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

    Article  PubMed  Google Scholar 

  19. Kühn C (2011) Sucrose transporters and plant development. In: Geisler M, Venema K (eds) Transporters and pumps in plant signaling. Springer, Heidelberg, pp 225–251

    Chapter  Google Scholar 

  20. He H, Chincinska I, Hackel A, Grimm B, Kühn C (2008) Phloem mobility and stability of sucrose transporter transcripts. Open Plant Sci J 2:1–14

    Article  CAS  Google Scholar 

  21. Vaughn MW, Harrington GN, Bush DR (2002) Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem. Proc Natl Acad Sci U S A 99(16):10876–10880. https://doi.org/10.1073/pnas.172198599

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Corbesier L, Vincent C, Jang S, Fornara F, Fan Q, Searle I, Giakountis A, Farrona S, Gissot L, Turnbull C, Coupland G (2007) FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis. Science 316(5827):1030–1033. https://doi.org/10.1126/science.1141752

    Article  CAS  PubMed  Google Scholar 

  23. Mathieu J, Yant LJ, Murdter F, Kuttner F, Schmid M (2009) Repression of flowering by the miR172 target SMZ. PLoS Biol 7(7):e1000148. https://doi.org/10.1371/journal.pbio.1000148

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Thompson MV, Wolniak SM (2008) A plasma membrane-anchored fluorescent protein fusion illuminates sieve element plasma membranes in Arabidopsis and tobacco. Plant Physiol 146(4):1599–1610. https://doi.org/10.1104/pp.107.113274

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 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. https://doi.org/10.1104/pp.106.085803

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Cerio R, Belter S, MacDonald DM (1987) The effect of fixation on monoclonal antibody labelling of cell surface antigens in cutaneous tissue. Clin Exp Dermatol 12:79–82

    Article  CAS  PubMed  Google Scholar 

  27. Milne RJ, Perroux JM, Rae AL, Reinders A, Ward JM, Offler CE, Patrick JW, Grof CP (2017) Sucrose transporter localization and function in phloem unloading in developing stems. Plant Physiol 173(2):1330–1341. https://doi.org/10.1104/pp.16.01594

    Article  CAS  PubMed  Google Scholar 

  28. Sauer N, Stadler R (1993) A sink-specific H+/monosaccharide co-transporter from Nicotiana tabacum: cloning and heterologous expression in baker’s yeast. Plant J 4(4):601–610

    Article  CAS  PubMed  Google Scholar 

  29. Baker RF, Leach KA, Boyer NR, Swyers MJ, Benitez-Alfonso Y, Skopelitis T, Luo A, Sylvester A, Jackson D, Braun DM (2016) Sucrose transporter ZmSut1 expression and localization uncover new insights into sucrose phloem loading. Plant Physiol 172(3):1876–1898. https://doi.org/10.1104/pp.16.00884

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kühn C, Quick WP, Schulz A, Riesmeier JW, Sonnewald U, Frommer WB (1996) Companion cell-specific inhibition of the potato sucrose transporter SUT1. Plant Cell Environ 19(10):1115–1123. https://doi.org/10.1111/j.1365-3040.1996.tb00426.x

    Article  Google Scholar 

  31. Weise A, Lalonde S, Kühn C, Frommer WB, Ward JM (2008) Introns control expression of sucrose transporter LeSUT1 in trichomes, companion cells and in guard cells. Plant Mol Biol 68(3):251–262. https://doi.org/10.1007/s11103-008-9366-9

    Article  CAS  PubMed  Google Scholar 

  32. Hafke JB, Holl SR, Kühn C, van Bel AJ (2013) Electrophysiological approach to determine kinetic parameters of sucrose uptake by single sieve elements or phloem parenchyma cells in intact Vicia faba plants. Front Plant Sci 4:274. https://doi.org/10.3389/fpls.2013.00274

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Schulz A, Kühn C, Riesmeier JW, Frommer WB (1998) Ultrastructural effects in potato leaves due to antisense-inhibition of the sucrose transporter indicate an apoplasmic mode of phloem loading. Planta 206:533–543

    Article  CAS  Google Scholar 

  34. Cronshaw J (1980) ATPase in mature and differentiating phloem and xylem. J Histochem Cytochem 28(4):375–377. https://doi.org/10.1177/28.4.6445380

    Article  CAS  PubMed  Google Scholar 

  35. Krügel U, He HX, Gier K, Reins J, Chincinska I, Grimm B, Schulze WX, Kühn C (2012) The potato sucrose transporter StSUT1 interacts with a DRM-associated protein disulfide isomerase. Mol Plant 5(1):43–62

    Article  PubMed  Google Scholar 

  36. Krügel U, Veenhoff LM, Langbein J, Wiederhold E, Liesche J, Friedrich T, Grimm B, Martinoia E, Poolman B, Kühn C (2008) Transport and sorting of the Solanum tuberosum sucrose transporter SUT1 is affected by posttranslational modification. Plant Cell 20(9):2497–2513. Correction: Plant Cell 2421 (2009) 4059

    Article  PubMed  PubMed Central  Google Scholar 

  37. Liesche J, Schulz A, Krügel U, Grimm B, Kühn C (2008) Dimerization and endocytosis of the sucrose transporter StSUT1 in mature sieve elements. Plant Signal Behav 3(12):1136–1137

    Article  PubMed  PubMed Central  Google Scholar 

  38. Liesche J, He HX, Grimm B, Schulz A, Kuhn C (2010) Recycling of Solanum sucrose transporters expressed in yeast, tobacco, and in mature phloem sieve elements. Mol Plant 3(6):1064–1074. https://doi.org/10.1093/mp/ssq059

    Article  CAS  PubMed  Google Scholar 

  39. Satiat-Jeunemaitre B (1992) Spatial and temporal regulations in helicoidal extracellular matrices: comparison between plant and animal systems. Tissue Cell 24(3):315–334

    Article  CAS  PubMed  Google Scholar 

  40. Ross-Elliott TJ, Jensen KH, Haaning KS, Wager BM, Knoblauch J, Howell AH, Mullendore DL, Monteith AG, Paultre D, Yan D, Otero S, Bourdon M, Sager R, Lee JY, Helariutta Y, Knoblauch M, Oparka KJ (2017) Phloem unloading in Arabidopsis roots is convective and regulated by the phloem-pole pericycle. elife 6:e24125. https://doi.org/10.7554/eLife.24125

    Article  PubMed  PubMed Central  Google Scholar 

  41. Milne RJ, Offler CE, Patrick JW, Grof CPL (2015) Cellular pathways of source leaf phloem loading and phloem unloading in developing stems of Sorghum bicolor in relation to stem sucrose storage. Funct Plant Biol 42(10):957–970. https://doi.org/10.1071/FP15133

    Article  CAS  PubMed  Google Scholar 

  42. Chincinska IA, Liesche J, Krügel U, Michalska J, Geigenberger P, Grimm B, Kühn C (2008) Sucrose transporter StSUT4 from potato affects flowering, tuberization, and shade avoidance response. Plant Physiol 146(2):515–528

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

Critical proof-reading by Alexander Schulz, Johannes Liesche and Pawel Brzezowski was greatly appreciated.

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Authors and Affiliations

  1. Department of Plant Physiology, Institute of Biology, Humboldt University Berlin, Berlin, Germany

    Christina Kühn

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  1. Christina Kühn
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Correspondence to Christina Kühn .

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Editors and Affiliations

  1. College of Life Sciences, Northwest A&F University, Yangling, China

    Johannes Liesche

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Kühn, C. (2019). Immunohistochemical Localization of Proteins in the Phloem: Problems and Solutions. In: Liesche, J. (eds) Phloem. Methods in Molecular Biology, vol 2014. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9562-2_9

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  • DOI: https://doi.org/10.1007/978-1-4939-9562-2_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9561-5

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