Applications of Fluorescent Marker Proteins in Plant Cell Biology

  • Michael R. Blatt
  • Christopher Grefen
Part of the Methods in Molecular Biology book series (MIMB, volume 1062)


Over the past decade, confocal microscopy and the ever-expanding toolchest of fluorescent protein (xFP) markers and technologies have become routine methods for the biological laboratory. A common use of xFP fluorophores is in localizing proteins and the subcellular structures with which they associate, including analyzing their distribution and dynamics and the interactions of proteins in vivo. Additionally, a number of so-called optical highlighters have proven especially useful in analyzing the kinetics of these processes in pulse-chase studies of protein relocation(s) following an experimental challenge. Here we focus on exemplary methods in transformation and live-cell imaging in plant cells, with the expectation that researchers will find these and the accompanying resources useful as a starting point in developing their own expertise.

Key words

Confocal fluorescence microscopy Fluorescent marker proteins Optical highlighter proteins Transient Arabidopsis transformation Quantitative ratiometric fluorescence 


  1. 1.
    Fehr M, Ehrhardt DW, Lalonde S, Frommer WB (2004) Minimally invasive dynamic imaging of ions and metabolites in living cells. Curr Opin Plant Biol 7:345–351PubMedCrossRefGoogle Scholar
  2. 2.
    Allen GJ, Kwak JM, Chu SP, Llopis J, Tsien RY, Harper JF, Schroeder JI (1999) Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells. Plant J 19:735–747PubMedCrossRefGoogle Scholar
  3. 3.
    Meyer AJ, Brach T, Marty L, Kreye S, Rouhier N, Jacquot JP, Hell R (2007) Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer. Plant J 52:973–986PubMedCrossRefGoogle Scholar
  4. 4.
    Costa A, Drago I, Behera S, Zottini M, Pizzo P, Schroeder JI, Pozzan T, Lo Schiavo F (2010) H2O2 in plant peroxisomes: an in vivo analysis uncovers a Ca2+-dependent scavenging system. Plant J 62:760–772PubMedCrossRefGoogle Scholar
  5. 5.
    Murata Y, Iwasaki H, Sasaki M, Inaba K, Okamura Y (2005) Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor. Nature 435:1239–1243PubMedCrossRefGoogle Scholar
  6. 6.
    Villalba-Galea CA, Miceli F, Taglialatela M, Bezanilla F (2009) Coupling between the voltage-sensing and phosphatase domains of Ci-VSP. J Gen Physiol 134:5–14PubMedCrossRefGoogle Scholar
  7. 7.
    Tian GW, Mohanty A, Chary SN, Li SJ, Paap B, Drakakaki G, Kopec CD, Li JX, Ehrhardt D, Jackson D, Rhee SY, Raikhel NV, Citovsky V (2004) High-throughput fluorescent tagging of full-length Arabidopsis gene products in planta. Plant Physiol 135:25–38PubMedCrossRefGoogle Scholar
  8. 8.
    Day RN, Davidson MW (2009) The fluorescent protein palette: tools for cellular imaging. Chem Soc Rev 38:2887–2921PubMedCrossRefGoogle Scholar
  9. 9.
    Fricker MD, Runions J, Moore I (2006) Quantitative fluorescence microscopy: from art to science. Annu Rev Plant Biol 57:79–107PubMedCrossRefGoogle Scholar
  10. 10.
    Lukyanov KA, Chudakov DM, Lukyanov S, Verkhusha V (2005) Photoactivatable fluorescent proteins. Nat Rev Mol Cell Biol 6:885–891PubMedCrossRefGoogle Scholar
  11. 11.
    Brandizzi F, Fricker M, Hawes C (2002) A greener world: the revolution in plant bioimaging. Nat Rev Mol Cell Biol 3:520–530PubMedCrossRefGoogle Scholar
  12. 12.
    Patterson GH, Lippincott-Schwartz J (2004) Selective photolabeling of proteins using photo-activatable GFP. Methods 32:445–450PubMedCrossRefGoogle Scholar
  13. 13.
    Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805PubMedCrossRefGoogle Scholar
  14. 14.
    Herrera-Estrella L, Depicker A, VanMontagu M, Schell J (1983) Expression of chimaeric genes transferred into plant cells using a Ti-plasmid-derived vector. Nature 303:209–213CrossRefGoogle Scholar
  15. 15.
    Campanoni P, Sutter J-U, Craig S, Littlejohn G, Blatt MR (2007) A generalized method for transfecting root epidermis uncovers endosomal dynamics in Arabidopsis root hairs. Plant J 51:322–330PubMedCrossRefGoogle Scholar
  16. 16.
    Li JF, Park E, Von Arnim AG, Nebenfuhr A (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 5:6Google Scholar
  17. 17.
    Grefen C, Donald N, Hashimoto K, Kudla J, Schumacher K, Blatt MR (2010) A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies. Plant J 64:355–365PubMedCrossRefGoogle Scholar
  18. 18.
    Goodin MM, Dietzgen RG, Schichnes D, Ruzin S, Jackson AO (2002) pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves. Plant J 31:375–383PubMedCrossRefGoogle Scholar
  19. 19.
    Runions J, Brach T, Kuhner S, Hawes C (2006) Photoactivation of GFP reveals protein dynamics within the endoplasmic reticulum membrane. J Exp Bot 57:43–50PubMedCrossRefGoogle Scholar
  20. 20.
    daSilva LLP, Snapp EL, Denecke J, Lippincott-Schwartz J, Hawes C, Brandizzi F (2004) Endoplasmic reticulum export sites and golgi bodies behave as single mobile secretory units in plant cells. Plant Cell 16:1753–1771PubMedCrossRefGoogle Scholar
  21. 21.
    Brandizzi F, Snapp EL, Roberts AG, Lippincott-Schwartz J, Hawes C (2002) Membrane protein transport between the endoplasmic reticulum and the golgi in tobacco leaves is energy dependent but cytoskeleton independent: evidence from selective photobleaching. Plant Cell 14:1293–1309PubMedCrossRefGoogle Scholar
  22. 22.
    Boevink P, Oparka K, Cruz SS, Martin B, Betteridge A, Hawes C (1998) Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER network. Plant J 15:441–447PubMedCrossRefGoogle Scholar
  23. 23.
    Batoko H, Zheng HQ, Hawes C, Moore I (2000) A Rab1 GTPase is required for transport between the endoplasmic reticulum and Golgi apparatus and for normal Golgi movement in plants. Plant Cell 12:2201–2217PubMedGoogle Scholar
  24. 24.
    Geelen D, Leyman B, Batoko H, Di Sansabastiano GP, Moore I, Blatt MR (2002) The abscisic acid-related SNARE homolog NtSyr1 contributes to secretion and growth: evidence from competition with its cytosolic domain. Plant Cell 14:387–406PubMedCrossRefGoogle Scholar
  25. 25.
    Sutter JU, Campanoni P, Tyrrell M, Blatt MR (2006) Selective mobility and sensitivity to SNAREs is exhibited by the Arabidopsis KAT1 K+ channel at the plasma membrane. Plant Cell 18:935–954PubMedCrossRefGoogle Scholar
  26. 26.
    Tyrrell M, Campanoni P, Sutter J-U, Pratelli R, Paneque-Corralles M, Blatt MR (2007) Selective targeting of plasma membrane and tonoplast traffic by inhibitory (dominant-negative) SNARE fragments. Plant J 51:1099–1115PubMedCrossRefGoogle Scholar
  27. 27.
    Samalova M, Fricker M, Moore I (2006) Ratiometric fluorescence-imaging assays of plant membrane traffic using polyproteins. Traffic 7:1701–1723PubMedCrossRefGoogle Scholar
  28. 28.
    Bracha-Drori K, Shichrur K, Katz A, Oliva M, Angelovici R, Yalovsky S, Ohad N (2004) Detection of protein–protein interactions in plants using bimolecular fluorescence complementation. Plant J 40:419–427PubMedCrossRefGoogle Scholar
  29. 29.
    Honsbein A, Sokolovski S, Grefen C, Campanoni P, Pratelli R, Paneque M, Chen ZH, Johansson I, Blatt MR (2009) A tripartite SNARE-K+ channel complex mediates in channel-dependent K+ nutrition in Arabidopsis. Plant Cell 21:2859–2877PubMedCrossRefGoogle Scholar
  30. 30.
    Foresti O, daSilva LLP, Denecke J (2006) Overexpression of the Arabidopsis syntaxin PEP12/SYP21 inhibits transport from the prevacuolar compartment to the lytic vacuole in vivo. Plant Cell 18:2275–2293PubMedCrossRefGoogle Scholar
  31. 31.
    Meckel T, Hurst AC, Thiel G, Homann U (2004) Endocytosis against high turgor: intact guard cells of Vicia faba constitutively endocytose fluorescently labelled plasma membrane and GFP-tagged K+-channel KAT1. Plant J 39:182–193PubMedCrossRefGoogle Scholar
  32. 32.
    Sutter JU, Sieben C, Hartel A, Eisenach C, Thiel G, Blatt MR (2007) Abscisic acid triggers the endocytosis of the Arabidopsis KAT1 K + channel and its recycling to the plasma membrane. Curr Biol 17:1396–1402PubMedCrossRefGoogle Scholar
  33. 33.
    Duby G, Hosy E, Fizames C, Alcon C, Costa A, Sentenac H, Thibaud JB (2008) AtKC1, a conditionally targeted Shaker-type subunit, regulates the activity of plant K+ channels. Plant J 53:115–123PubMedCrossRefGoogle Scholar
  34. 34.
    Chen ZH, Grefen C, Donald N, Hills A, Blatt MR (2011) A bicistronic, Ubiquitin-10 promoter-based vector cassette for transient transformation and functional analysis of membrane transport demonstrates the utility of quantitative voltage clamp studies on intact Arabidopsis root epidermis. Plant Cell Environ 34:554–564PubMedCrossRefGoogle Scholar
  35. 35.
    Nelson BK, Cai X, Nebenfuhr A (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 51:1126–1136PubMedCrossRefGoogle Scholar
  36. 36.
    Hunter PR, Craddock CP, Di Benedetto S, Roberts LM, Frigerio L (2007) Fluorescent reporter proteins for the tonoplast and the vacuolar lumen identify a single vacuolar compartment in Arabidopsis cells. Plant Physiol 145:1371–1382PubMedCrossRefGoogle Scholar
  37. 37.
    Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877PubMedCrossRefGoogle Scholar
  38. 38.
    McKinney SA, Murphy CS, Hazelwood KL, Davidson MW, Looger LL (2009) A bright and photostable photoconvertible fluorescent protein. Nat Methods 6:131–133PubMedCrossRefGoogle Scholar
  39. 39.
    Gurskaya NG, Verkhusha VV, Shcheglov AS, Staroverov DB, Chepurnykh TV, Fradkov AF, Lukyanov S, Lukyanov KA (2006) Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat Biotechnol 24:461–465PubMedCrossRefGoogle Scholar
  40. 40.
    Lummer M, Humpert F, Steuwe C, Caesar K, Schuttpelz M, Sauer M, Staiger D (2011) Reversible photoswitchable DRONPA-s monitors nucleocytoplasmic transport of an RNA-binding protein in transgenic plants. Traffic 12:693–702PubMedCrossRefGoogle Scholar
  41. 41.
    Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388:882–887PubMedCrossRefGoogle Scholar
  42. 42.
    Hu K, Carroll J, Fedorovich S, Rickman C, Sukhodub A, Davletov B (2002) Vesicular restriction of synaptobrevin suggests a role for calcium in membrane fusion. Nature 415:646–650PubMedCrossRefGoogle Scholar
  43. 43.
    Grefen C, Chen ZH, Honsbein A, Donald N, Hills A, Blatt MR (2010) A novel motif essential for SNARE interaction with the K+ channel KC1 and channel gating in Arabidopsis. Plant Cell 22:3076–3092PubMedCrossRefGoogle Scholar
  44. 44.
    Koncz C, Schell J (1986) The promoter of the TL-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–396CrossRefGoogle Scholar
  45. 45.
    Shaner NC, Campbell RE, Steinbach PA, Giepmans BNG, Palmer AE, Tsien RY (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp red fluorescent protein. Nat Biotechnol 22:1567–1572PubMedCrossRefGoogle Scholar
  46. 46.
    Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, Lukyanov SA (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol 17:969–973PubMedCrossRefGoogle Scholar
  47. 47.
    Merzlyak EM, Goedhart J, Shcherbo D, Bulina ME, Shcheglov AS, Fradkov AF, Gaintzeva A, Lukyanov KA, Lukyanov S, Gadella TWJ, Chudakov DM (2007) Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat Methods 4:555–557PubMedCrossRefGoogle Scholar
  48. 48.
    Ando R, Hama H, Yamamoto-Hino M, Mizuno H, Miyawaki A (2002) An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc Natl Acad Sci U S A 99:12651–12656PubMedCrossRefGoogle Scholar
  49. 49.
    Wiedenmann J, Ivanchenko S, Oswald F, Schmitt F, Rocker C, Salih A, Spindler KD, Nienhaus GU (2004) EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc Natl Acad Sci U S A 101:15905–15910PubMedCrossRefGoogle Scholar
  50. 50.
    Ando R, Mizuno H, Miyawaki A (2004) Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306:1370–1373PubMedCrossRefGoogle Scholar
  51. 51.
    Adam V, Lelimousin M, Boehme S, Desfonds G, Nienhaus K, Field MJ, Wiedenmann J, McSweeney S, Nienhaus GU, Bourgeois D (2008) Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations. Proc Natl Acad Sci U S A 105:18343–18348PubMedCrossRefGoogle Scholar
  52. 52.
    Boevink P, Santacruz S, Hawes C, Harris N, Oparka KJ (1996) Virus-mediated delivery of the green fluorescent protein to the endoplasmic reticulum of plant cells. Plant J 10:935–941CrossRefGoogle Scholar
  53. 53.
    Napier RM, Fowke LC, Hawes C, Lewis M, Pelham HRB (1992) Immunological evidence that plants use both HDEL and KDEL for targeting proteins to the endoplasmic reticulum. J Cell Sci 102:261–271PubMedGoogle Scholar
  54. 54.
    Saint-Jore CM, Evins J, Batoko H, Brandizzi F, Moore I, Hawes C (2002) Redistribution of membrane proteins between the Golgi apparatus and endoplasmic reticulum in plants is reversible and not dependent on cytoskeletal networks. Plant J 29:661–678PubMedCrossRefGoogle Scholar
  55. 55.
    Brandizzi F, Frangne N, Marc-Martin S, Hawes C, Neuhaus JM, Paris N (2002) The destination for single-pass membrane proteins is influenced markedly by the length of the hydrophobic domain. Plant Cell 14:1077–1092PubMedCrossRefGoogle Scholar
  56. 56.
    Paris N, Stanley CM, Jones RL, Rogers JC (1996) Plant cells contain 2 functionally distinct vacuolar compartments. Cell 85: 563–572PubMedCrossRefGoogle Scholar
  57. 57.
    Gattolin S, Sorieul M, Hunter PR, Khonsari RH, Frigerio L (2009) In vivo imaging of the tonoplast intrinsic protein family in Arabidopsis roots. BMC Plant Biol 9Google Scholar
  58. 58.
    Gattolin S, Sorieul M, Frigerio L (2011) Mapping of tonoplast intrinsic proteins in maturing and germinating Arabidopsis seeds reveals dual localization of embryonic TIPs to the tonoplast and plasma membrane. Mol Plant 4:180–189PubMedCrossRefGoogle Scholar
  59. 59.
    Logan DC, Leaver CJ (2000) Mitochondria-targeted GFP highlights the heterogeneity of mitochondrial shape, size and movement within living plant cells. J Exp Bot 51: 865–871PubMedCrossRefGoogle Scholar
  60. 60.
    Kwok EY, Hanson MR (2004) GFP-labelled Rubisco and aspartate aminotransferase are present in plastid stromules and traffic between plastids. J Exp Bot 55:595–604PubMedCrossRefGoogle Scholar
  61. 61.
    Leyman B, Geelen D, Quintero FJ, Blatt MR (1999) A tobacco syntaxin with a role in hormonal control of guard cell ion channels. Science 283:537–540PubMedCrossRefGoogle Scholar
  62. 62.
    Leyman B, Geelen D, Blatt MR (2000) Localization and control of expression of Nt-Syr1, a tobacco SNARE protein. Plant J 24:369–381PubMedCrossRefGoogle Scholar
  63. 63.
    Collins NC, Thordal-Christensen H, Lipka V, Bau S, Kombrink E, Qiu JL, Huckelhoven R, Stein M, Freialdenhoven A, Somerville SC, Schulze-Lefert P (2003) SNARE-protein-mediated disease resistance at the plant cell wall. Nature 425:973–977PubMedCrossRefGoogle Scholar
  64. 64.
    Lefebvre B, Batoko H, Duby G, Boutry M (2004) Targeting of a Nicotiana plumbaginifolia H+-ATPase to the plasma membrane is not by default and requires cytosolic structural determinants. Plant Cell 16:1772–1789PubMedCrossRefGoogle Scholar
  65. 65.
    Eisenach C, Chen Z, Grefen C, Blatt, MR (2012) The trafficking protein SYP121 of Arabidopsis connects programmed stomatal closure and K channel activity with vegetative growth. Plant J 69:241–251Google Scholar
  66. 66.
    Geldner N, Ervaud-Tendon V, Hyman DL, Mayer U, Stierhof YD, Chory J (2009) Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. Plant J 59:169–178PubMedCrossRefGoogle Scholar
  67. 67.
    Karimi M, Inze D, Depicker A (2002) GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195PubMedCrossRefGoogle Scholar
  68. 68.
    Karimi M, De Meyer B, Hilson P (2005) Modular cloning in plant cells. Trends Plant Sci 10:103–105PubMedCrossRefGoogle Scholar
  69. 69.
    Tzfira T, Tian GW, Lacroix B, Vyas S, Li JX, Leitner-Dagan Y, Krichevsky A, Taylor T, Vainstein A, Citovsky V (2005) pSAT vectors: a modular series of plasmids for autofluorescent protein tagging and expression of multiple genes in plants. Plant Mol Biol 57:503–516PubMedCrossRefGoogle Scholar
  70. 70.
    Earley KW, Haag JR, Pontes O, Opper K, Juehne T, Song KM, Pikaard CS (2006) Gateway-compatible vectors for plant functional genomics and proteomics. Plant J 45:616–629PubMedCrossRefGoogle Scholar
  71. 71.
    Nakagawa T, Suzuki T, Murata S, Nakamura S, Hino T, Maeo K, Tabata R, Kawai T, Tanaka K, Niwa Y, Watanabe Y, Nakamura K, Kimura T, Ishiguro S (2007) Improved gateway binary vectors: High-performance vectors for creation of fusion constructs in transgenic analysis of plants. Biosci Biotechnol Biochem 71:2095–2100PubMedCrossRefGoogle Scholar
  72. 72.
    Zhong SL, Lin ZF, Fray RG, Grierson D (2008) Improved plant transformation vectors for fluorescent protein tagging. Transgenic Res 17:985–989PubMedCrossRefGoogle Scholar
  73. 73.
    De Rybel B, van den Berg W, Lokerse A, Liao CY, van Mourik H, Moller B, Peris CL, Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants. Plant Physiol 156:1292–1299PubMedCrossRefGoogle Scholar
  74. 74.
    Grefen C, Blatt MR (2012) A 2in1 cloning system enables ratiometric bimolecular fluorescence complementation (rBiFC). Biotechniques 53:311-314PubMedCrossRefGoogle Scholar
  75. 75.
    Karnik R, Grefen C, Bayne R, Honsbein A, Köhler T, Kioumourtzoglou D, Williams M, Bryant N, Blatt MR (2013) Arabidopsis Sec1/Munc18 protein SEC11 is a competitive and dynamic modulator of SNARE binding and SYP121-dependent vesicle traffic. Plant Cell 25:1368-1382PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Michael R. Blatt
    • 1
  • Christopher Grefen
    • 2
  1. 1.Laboratory of Plant Physiology and BiophysicsUniversity of GlasgowGlasgowUK
  2. 2.Centre for Molecular Biology of Plants (ZMBP), Developmental GeneticsUniversity of TübingenTübingenGermany

Personalised recommendations