Subcellular Localization of Transiently Expressed Fluorescent Fusion Proteins

  • David A. Collings
Part of the Methods in Molecular Biology book series (MIMB, volume 1069)


The recent and massive expansion in plant genomics data has generated a large number of gene sequences for which two seemingly simple questions need to be answered: where do the proteins encoded by these genes localize in cells, and what do they do? One widespread approach to answering the localization question has been to use particle bombardment to transiently express unknown proteins tagged with green fluorescent protein (GFP) or its numerous derivatives. Confocal fluorescence microscopy is then used to monitor the localization of the fluorescent protein as it hitches a ride through the cell. The subcellular localization of the fusion protein, if not immediately apparent, can then be determined by comparison to localizations generated by fluorescent protein fusions to known signalling sequences and proteins, or by direct comparison with fluorescent dyes. This review aims to be a tour guide for researchers wanting to travel this hitch-hiker’s path, and for reviewers and readers who wish to understand their travel reports. It will describe some of the technology available for visualizing protein localizations, and some of the experimental approaches for optimizing and confirming localizations generated by particle bombardment in onion epidermal cells, the most commonly used experimental system. As the non-conservation of signal sequences in heterologous expression systems such as onion, and consequent mis-targeting of fusion proteins, is always a potential problem, the epidermal cells of the Argenteum mutant of pea are proposed as a model system.

Key words

Argenteum mutant Confocal microscopy Fluorescence microscopy Fluorescent fusion proteins Gene gun Green fluorescent protein Live cell imaging Onion epidermis Particle bombardment Tracer dyes Transient gene expression 



This work was supported by funding from the University of Canterbury and the Biomolecular Interaction Centre. The author thanks numerous colleagues for their donations of plasmids, Philippa Barrell and David Goulden (Plant & Food Research, Lincoln, New Zealand) for Argenteum pea plants, and Rosemary White (CSIRO Plant Industry, Canberra, Australia), John Harper (Charles Sturt University, Wagga Wagga, Australia), and Krithika Yogeeswaran (University of Canterbury) for comments on the manuscript and discussions about confocal microscopy.


  1. 1.
    Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909PubMedCrossRefGoogle Scholar
  2. 2.
    Chudakov DM, Matz MV, Lukyanov S et al (2010) Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev 90:1103–1163PubMedCrossRefGoogle Scholar
  3. 3.
    Baulcombe DC, Chapman S, Santa CS (1995) Jellyfish green fluorescent protein as a reporter for virus infections. Plant J 7:1045–1053PubMedCrossRefGoogle Scholar
  4. 4.
    Sheen J, Hwang S, Niwa Y et al (1995) Green-fluorescent protein as a new vital marker in plant cells. Plant J 8:777–784PubMedCrossRefGoogle Scholar
  5. 5.
    Sparkes IA, Runions J, Kearns A et al (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc 1:2019–2025PubMedCrossRefGoogle Scholar
  6. 6.
    Marion J, Bach L, Bellec Y et al (2008) Systematic analysis of protein subcellular localizations and interaction using high-throughput transient transformation of Arabidopsis seedlings. Plant J 56:169–179PubMedCrossRefGoogle Scholar
  7. 7.
    Li J-F, Park E, von Arnim AG et al (2009) The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. Plant Methods 5, doi:10.1186/1746-4811-5-6Google Scholar
  8. 8.
    van Loock B, Markakis MN, Verbelen J-P et al (2010) High-throughput transient transformation of Arabidopsis roots enables systematic colocalization analysis of GFP-tagged proteins. Plant Signal Behav 5:261–263PubMedCrossRefGoogle Scholar
  9. 9.
    Haseloff J, Siemering KR, Prasher DC et al (1997) Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci U S A 94:2122–2127PubMedCrossRefGoogle Scholar
  10. 10.
    Scott A, Wyatt S, Tsou P-L et al (1999) Model system for plant cell biology: GFP imaging in living onion epidermal cells. Biotechniques 26:1125–1132PubMedGoogle Scholar
  11. 11.
    Collings DA, Carter CN, Rink JC et al (2000) Plant nuclei can contain extensive grooves and invaginations. Plant Cell 12:2425–2439PubMedGoogle Scholar
  12. 12.
    North AJ (2006) Seeing is believing? A beginners’ guide to practical pitfalls in image acquisition. J Cell Biol 172:9–18PubMedCrossRefGoogle Scholar
  13. 13.
    Pearson H (2007) The good, the bad and the ugly. Nature 440:138–140CrossRefGoogle Scholar
  14. 14.
    Conchello J-A, Lichtman JW (2005) Optical sectioning microscopy. Nat Methods 2: 920–931PubMedCrossRefGoogle Scholar
  15. 15.
    Lichtman JW, Conchello J-A (2005) Fluorescence microscopy. Nat Methods 2: 910–919PubMedCrossRefGoogle Scholar
  16. 16.
    Dixit R, Cyr R, Gilroy S (2006) Using intrinsically fluorescent proteins for plant cell imaging. Plant J 45:599–615PubMedCrossRefGoogle Scholar
  17. 17.
    Chudakov DM, Lukyanov S, Lukyanov KA (2005) Fluorescent proteins as a toolkit for in vivo imaging. Trends Biochem Sci 23:605–613Google Scholar
  18. 18.
    Matz MV, Fradkov AF, Labas YA et al (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol 17:969–973PubMedCrossRefGoogle Scholar
  19. 19.
    Campbell RE, Tour O, Palmer AE et al (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci U S A 99:7877–7882PubMedCrossRefGoogle Scholar
  20. 20.
    Shaner NC, Campbell RC, Steinbach PA et al (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567–1572PubMedCrossRefGoogle Scholar
  21. 21.
    Chudakov DM, Lukyanov S, Lukyanov KA (2007) Using photoactivatable fluorescent protein Dendra2 to track protein movement. Biotechniques 42:553–558PubMedCrossRefGoogle Scholar
  22. 22.
    Wu S, Koizumi K, Macrae-Crerar A et al (2011) Assessing the utility of photoswitchable fluorescent proteins for tracking intercellular protein movement in the Arabidopsis root. PLoS One 6:e27536PubMedCrossRefGoogle Scholar
  23. 23.
    Mathur J, Radhamony R, Sinclair AM et al (2010) mEosFP-based green-to-red photoconvertable subcellular probes for plants. Plant Physiol 154:1573–1587PubMedCrossRefGoogle Scholar
  24. 24.
    Ghosh I, Hamilton AD, Regan L (2000) Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. J Am Chem Soc 122:5658–5659CrossRefGoogle Scholar
  25. 25.
    Hu C-D, Chinenov Y, Kerpppola TK (2002) Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9:789–798PubMedCrossRefGoogle Scholar
  26. 26.
    Kodama Y, Hu C-D (2012) Bimolecular fluorescence complementation (BiFC): a 5-year update and future persepctives. Biotechniques 53:285–294PubMedCrossRefGoogle Scholar
  27. 27.
    Diaz I, Martinez M, Isabel-LaMoneda I et al (2005) The DOF protein, SAD, interacts with GAMYB in plant nuclei and activates transcription of endosperm-specific genes during barley seed development. Plant J 42:652–662PubMedCrossRefGoogle Scholar
  28. 28.
    Ohad N, Shichur K, Yalovsky S (2007) The analysis of protein-protein interactions in plants by bimolecular fluorescence complementation. Plant Physiol 145:1090–1099PubMedCrossRefGoogle Scholar
  29. 29.
    Kodama Y (2011) A bright green-colored bimolecular fluorescence complementation assay in living plant cells. Plant Biotechnol 28:95–98CrossRefGoogle Scholar
  30. 30.
    Klein TM, Wold ED, Wu R et al (1987) High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 327:70–74CrossRefGoogle Scholar
  31. 31.
    Silverstone AL, Ciampaglio CN, Sun T-P (1998) The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway. Plant Cell 10:155–169PubMedGoogle Scholar
  32. 32.
    von Arnim AG, Deng X-W, Stacey MG (1998) Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants. Gene 221:35–43CrossRefGoogle Scholar
  33. 33.
    Kikkert JR, Vidal JR, Reisch BI (2004) Stable transformation of plant cells by particel bomabrdment/biolistics. Methods Mol Biol 286:61–78Google Scholar
  34. 34.
    Finer JJ, Vain P, Jones MW et al (1992) Development of the particle inflow gun for DNA delivery to plant cells. Plant Cell Rep 11:323–328CrossRefGoogle Scholar
  35. 35.
    Zhang Y, Su J, Duan S et al (2011) A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes. Plant Methods 7:30PubMedCrossRefGoogle Scholar
  36. 36.
    Marc J, Granger CL, Brincat J et al (1998) A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells. Plant Cell 10:1927–1939PubMedGoogle Scholar
  37. 37.
    Hoch HC, Pratt C, Marx GA (1980) Subepidermal air spaces: basis for the phenotypic expression of the Argenteum mutant of Pisum. Am J Bot 67:905–911CrossRefGoogle Scholar
  38. 38.
    Marx GA (1982) Argenteum (Arg) mutant of Pisum. J Hered 73:413–420Google Scholar
  39. 39.
    Elzenga JTM, Staal M, Prins HBA (1997) Calcium-calmodulin signalling is involved in light-induced acidification by epidermal leaf cells of pea, Pisum sativum L. J Exp Bot 48:2055–2060Google Scholar
  40. 40.
    Jewer PC, Incoll LD, Shaw J (1982) Stomatal responses of Argenteum—a mutant of Pisum sativum L. with readily detachable leaf epidermis. Planta 155:146–153CrossRefGoogle Scholar
  41. 41.
    Dhanoa PK, Sinclair AM, Mullen RT et al (2006) Illuminating subcellular structures and dynamics in plants: a fluorescent protein toolbox. Can J Bot 84:515–522CrossRefGoogle Scholar
  42. 42.
    Cutler SR, Ehrhardt DW, Griffitts JS et al (2000) Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proc Natl Acad Sci U S A 97:3718–3723PubMedCrossRefGoogle Scholar
  43. 43.
    Rossner M, Yamada KM (2004) What’s in a picture? The temptation of image manipulation. J Cell Biol 166:11–15PubMedCrossRefGoogle Scholar
  44. 44.
    Staehelin LA (1997) The plant ER: a dynamic organelle composed of a large number of discrete functional domains. Plant J 11:1151–1165PubMedCrossRefGoogle Scholar
  45. 45.
    Natesan SK, Sullivan JA, Gray JC (2005) Stromules: a characterisatic cell-specific feature of plastid morphology. J Exp Bot 56:787–797PubMedCrossRefGoogle Scholar
  46. 46.
    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 50:1826–1839PubMedCrossRefGoogle Scholar
  47. 47.
    Nebenführ A, Ritzenthaler C, Robinson DG (2002) Brefeldin A: deciphering an enigmatic inhibitor of secretion. Plant Physiol 130:1102–1108PubMedCrossRefGoogle Scholar
  48. 48.
    Tse YC, Mo B, Hillmer S et al (2004) Identification of multivesicular bodies as prevacuolar compartments in Nicotiana tabacum BY-2 cells. Plant Cell 16:672–693PubMedCrossRefGoogle Scholar
  49. 49.
    Delhaize E, Gruber BD, Pittman JK et al (2007) A role for the AtMTP11 gene of Arabidopsis in manganese transport and tolerance. Plant J 51:198–210PubMedCrossRefGoogle Scholar
  50. 50.
    Christensen NM, Nicolaisen M, Hansen M et al (2004) Distribution of phytoplasmas in infected plants as revealed by real-time PCR and bioimaging. Mol Plant-Microbe Int 17:1175–1184CrossRefGoogle Scholar
  51. 51.
    Pagano RE (1989) A fluorescent derivative of ceramide: physical properties and use in studying the Golgi apparatus of animal cells. Methods Cell Biol 29:78–85Google Scholar
  52. 52.
    Bolte S, Talbot C, Boutte T et al (2004) FM-dyes as experimental probes for dissecting vesicle trafficking in living plant cells. J Microsc 214:159–173PubMedCrossRefGoogle Scholar
  53. 53.
    Collings DA, Collins PP, Chia XR et al (2012) A users guide to transient protein localisations in Allium epidermal cells. In preparationGoogle Scholar
  54. 54.
    Baskin TI, Betzner AS, Hoggart R et al (1992) Root morphology mutants in Arabidopsis thaliana. Aust J Plant Physiol 19:427–437CrossRefGoogle Scholar
  55. 55.
    Wu F-S (1987) Localization of mitochondria in plant cells by vital staining with rhodamine 123. Planta 171:346–357CrossRefGoogle Scholar
  56. 56.
    Landrum M, Smertenko A, Edwards R et al (2010) BODIPY probes to study peroxisome dynamics in vivo. Plant J 62:529–538PubMedCrossRefGoogle Scholar
  57. 57.
    Strader LC, Wheeler DL, Christensen SE et al (2011) Multiple factes of Arabidopsis seedling development require indole-3-butyric acid-derived auxin. Plant Cell 23:984–999PubMedCrossRefGoogle Scholar
  58. 58.
    Quader H, Schnepf E (1986) Endoplasmic reticulum and cytoplasmic streaming: fluorescence microscopical observations in adaxial epidermis cells of onion bulb scales. Protoplasma 131:250–252CrossRefGoogle Scholar
  59. 59.
    Villarejo A, Burén S, Larsson S et al (2005) Evidence for a protein transported through the secretory pathway en route to the higher plant chloroplast. Nat Cell Biol 7:1224–1231PubMedCrossRefGoogle Scholar
  60. 60.
    Patel S, Brkljacic J, Gindullis F et al (2005) The plant nuclear envelope protein MAF1 has an additional location at the Golgi and binds to a novel Golgi-associated coiled-coil protein. Planta 222:1028–1040PubMedCrossRefGoogle Scholar
  61. 61.
    Verbelen J-P, Tao W (1998) Mobile arrays of vacuole ripples are common in plant cells. Plant Cell Rep 17:917–920CrossRefGoogle Scholar
  62. 62.
    Greenspan P, Mayer EP, Fowler SD (1985) Nile Red: a selective fluorescent stain for intracellular lipid-droplets. J Cell Biol 100:965–973PubMedCrossRefGoogle Scholar
  63. 63.
    Rounds CM, Lubeck E, Winship LJ et al (2011) Propidium iodide competes with Ca2+ to label pectin in pollen tubes and arabidopsis root hairs. Plant Physiol 157:175–187PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • David A. Collings
    • 1
  1. 1.Biomolecular Interaction Centre, School of Biological SciencesThe University of CanterburyChristchurchNew Zealand

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