Abstract
While chlorophyll has served as an excellent label for plastids in green tissue, the development of fluorescent proteins has allowed their ready visualization in all tissues of the plants, revealing new features of their morphology and motility. Gene regulatory sequences in plastid transgenes can be optimized through the use of fluorescent protein reporters. Fluorescent labeling of plastids simultaneously with other subcellular locations reveals dynamic interactions and mutant phenotypes. Transient expression of fluorescent protein fusions is particularly valuable to determine whether or not a protein of unknown function is targeted to the plastid. Particle bombardment and agroinfiltration methods described here are convenient for imaging fluorescent proteins in plant organelles. With proper selection of fluorophores for labeling the components of the plant cell, confocal microscopy can produce extremely informative images at high resolution at depths not feasible by standard epifluorescence microscopy.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Maxwell K, Johnson GN (2000) Chlorophyll fluorescence—a practical guide. J Exp Bot 51:659–668
Wada M (2013) Chloroplast movement. Plant Sci 210:177–182
Kwok EY, Hanson MR (2004) In vivo analysis of interactions between GFP-labeled microfilaments and plastid stromules. BMC Plant Biol 4:2
Kwok EY, Hanson MR (2004) Plastids and stromules interact with the nucleus and cell membrane in vascular plants. Plant Cell Rep 23:188–195
Bhat RA, Lahaye T, Panstruga R (2006) The visible touch: in planta visualization of protein-protein interactions by fluorophore-based methods. Plant Methods 2:12
Wong KA, O’Bryan JP (2011) Bimolecular fluorescence complementation. J Vis Exp 50:2643
Vothknecht UC, Soll J (2000) Protein import: the hitchhikers guide into chloroplasts. Biol Chem 381:887–897
Seki M, Iida A, Morikawa H (1998) Transient expression of foreign genes in tissues of Arabidopsis thaliana by bombardment-mediated transformation. Methods Mol Biol 82:219–225
Lee DW, Hwang I (2011) Transient expression and analysis of chloroplast proteins in Arabidopsis protoplasts. Methods Mol Biol 774:59–71
Fischer R, Vaquero-Martin C, Sack M, Drossard J, Emans N, Commandeur U (1999) Towards molecular farming in the future: transient protein expression in plants. Biotechnol Appl Biochem 30:113–116
Kapila J, De Rycke R, Van Montagu M, Angenon G (1997) An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci 122:101–108
Pawley JB (ed) (1995) Handbook of biological confocal microscopy. Plenum Press, New York
Benediktyova Z, Nedbal L (2009) Imaging of multi-color fluorescence emission from leaf tissues. Photosynth Res 102:169–175
Feijo JA, Moreno N (2004) Imaging plant cells by two-photon excitation. Protoplasma 223:1–32
Wada M, Kagawa T, Sato Y (2003) Chloroplast movement. Annu Rev Plant Biol 54:455–468
Franklin S, Ngo B, Efuet E, Mayfield SP (2002) Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast. Plant J 30:733–744
Reed ML, Wilson SK, Sutton CA, Hanson MR (2001) High-level expression of a synthetic red-shifted GFP coding region incorporated into transgenic chloroplasts. Plant J 27:257–265
Millwood RJ, Moon HS, Stewart CNJ (2008) Fluorescent proteins in transgenic plants. In: Geddes CD (ed) Reviews in fluorescence 2008. Springer, New York, pp 387–403
Primavesi LF, Wu H, Mudd EA, Day A, Jones HD (2008) Visualisation of plastids in endosperm, pollen and roots of transgenic wheat expressing modified GFP fused to transit peptides from wheat SSU RubisCO, rice FtsZ and maize ferredoxin III proteins. Transgenic Res 17:529–543
Pyke KA (2013) Divide and shape: an endosymbiont in action. Planta 237:381–387
Hanson MR, Sattarzadeh A (2008) Dynamic morphology of plastids and stromules in angiosperm plants. Plant Cell Environ 31:646–657
Kwok EY, Hanson MR (2004) Stromules and the dynamic nature of plastid morphology. J Microsc 214:124–137
Natesan SK, Sullivan JA, Gray JC (2005) Stromules: a characteristic cell-specific feature of plastid morphology. J Exp Bot 56:787–797
Kohler RH, Zipfel WR, Webb WW, Hanson MR (1997) The green fluorescent protein as a marker to visualize plant mitochondria in vivo. Plant J 11:613–621
Fang Y, Spector DL (2010) Live cell imaging of plants. Cold Spring Harb Protoc 2012, pdb top68
Groover A, Jackson D (2007) Live-cell imaging of GFP in plants. CSH protocols 2007, pdb ip31
Ross FWD (1995) Fluorescence microscopy, vol 2. Cambridge University Press, Cambridge, England
Berg RH, Beachy RN (2008) Fluorescent protein applications in plants. Methods Cell Biol 85:153–177
Geddes CD (ed) (2008/2010) Reviews in fluorescence 2008/2010, vol 2008/2010. Springer, New York
Mathur J (2007) The illuminated plant cell. Trends Plant Sci 12:506–513
Shaw SL, Ehrhardt DW (2013) Smaller, faster, brighter: advances in optical imaging of living plant cells. Annu Rev Plant Biol 64:351–375
Coleman AW (1979) Use of the fluorochrome 4′6-diamidino-2-phenylindole in genetic and developmental studies of chloroplast DNA. J Cell Biol 82:299–305
Lippincott-Schwartz J, Patterson GH (2003) Development and use of fluorescent protein markers in living cells. Science 300:87–91
Nienhaus GU, Nienhaus K, Holzle A, Ivanchenko S, Renzi F, Oswald F, Wolff M, Schmitt F, Rocker C, Vallone B, Weidemann W, Heilker R, Nar H, Wiedenmann J (2006) Photoconvertible fluorescent protein EosFP: biophysical properties and cell biology applications. Photochem Photobiol 82:351–358
Shaner NC, Patterson GH, Davidson MW (2007) Advances in fluorescent protein technology. J Cell Sci 120:4247–4260
Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909
Dixit R, Cyr R, Gilroy S (2006) Using intrinsically fluorescent proteins for plant cell imaging. Plant J 45:599–615
Lippincott-Schwartz J, Patterson GH (2008) Fluorescent proteins for photoactivation experiments. Methods Cell Biol 85:45–61
Lippincott-Schwartz J, Patterson GH (2009) Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. Trends Cell Biol 19:555–565
Shaner NC, Lin MZ, McKeown MR, Steinbach PA, Hazelwood KL, Davidson MW, Tsien RY (2008) Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods 5:545–551
Mohanty A, Yang Y, Luo A, Sylvester AW, Jackson D (2009) Methods for generation and analysis of fluorescent protein-tagged maize lines. Methods Mol Biol 526:71–89
Tsien RY (2009) Indicators based on fluorescence resonance energy transfer (FRET). Cold Spring Harb Protoc 2009, pdb top57
Ishikawa-Ankerhold HC, Ankerhold R, Drummen GP (2012) Advanced fluorescence microscopy techniques—FRAP, FLIP, FLAP, FRET and FLIM. Molecules 17:4047–4132
Padilla-Parra S, Tramier M (2012) FRET microscopy in the living cell: different approaches, strengths and weaknesses. BioEssays 34:369–376
Robitaille M, Heroux I, Baragli A, Hebert TE (2009) Novel tools for use in bioluminescence resonance energy transfer (BRET) assays. Methods Mol Biol 574:215–234
Subramanian C, Woo J, Cai X, Xu X, Servick S, Johnson CH, Nebenfuhr A, von Arnim AG (2006) A suite of tools and application notes for in vivo protein interaction assays using bioluminescence resonance energy transfer (BRET). Plant J 48:138–152
Subramanian C, Xu Y, Johnson CH, von Arnim AG (2004) In vivo detection of protein-protein interaction in plant cells using BRET. Methods Mol Biol 284:271–286
Xu X, Soutto M, Xie Q, Servick S, Subramanian C, von Arnim AG, Johnson CH (2007) Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues. Proc Natl Acad Sci U S A 104:10264–10269
Gremillon L, Kiessling J, Hause B, Decker EL, Reski R, Sarnighausen E (2007) Filamentous temperature-sensitive Z (FtsZ) isoforms specifically interact in the chloroplasts and in the cytosol of Physcomitrella patens. New Phytol 176:299–310
Seidel T, Kluge C, Hanitzsch M, Ross J, Sauer M, Dietz KJ, Golldack D (2004) Colocalization and FRET-analysis of subunits c and a of the vacuolar H + -ATPase in living plant cells. J Biotechnol 112:165–175
Wise AA, Liu Z, Binns AN (2006) Three methods for the introduction of foreign DNA into Agrobacterium. Methods Mol Biol 343:43–53
Behera S, Kudla J (2013) High-resolution imaging of cytoplasmic Ca2+ dynamics in Arabidopsis roots. Cold Spring Harb. Protoc 2013(7). pii: pdb.prot073023. doi: 10.1101/pdb.prot073023.
Swanson SJ, Gilroy S (2013) Imaging changes in cytoplasmic calcium using the Yellow Cameleon 3.6 biosensor and confocal microscopy. Methods Mol Biol 1009:291–302
Wanke D, Hohenstatt ML, Dynowski M, Bloss U, Hecker A, Elgass K, Hummel S, Hahn A, Caesar K, Schleifenbaum F, Harter K, Berendzen KW (2011) Alanine zipper-like coiled-coil domains are necessary for homotypic dimerization of plant GAGA-factors in the nucleus and nucleolus. PloS One 6:e16070
Seidel T, Seefeldt B, Sauer M, Dietz KJ (2010) In vivo analysis of the 2-Cys peroxiredoxin oligomeric state by two-step FRET. J Biotechnol 149:272–279
Bucherl C, Aker J, de Vries S, Borst JW (2010) Probing protein-protein Interactions with FRET-FLIM. Methods Mol Biol 655:389–399
Wolf H, Barisas BG, Dietz KJ, Seidel T (2013) Kaede for detection of protein oligomerization. Mol Plant. doi:10.1093/mp/sst039
Zhang M, Hu Y, Jia J, Gao H, He Y (2009) A plant MinD homologue rescues Escherichia coli HL1 mutant (DeltaMinDE) in the absence of MinE. BMC Microbiol 9:101
Citovsky V, Lee LY, Vyas S, Glick E, Chen MH, Vainstein A, Gafni Y, Gelvin SB, Tzfira T (2006) Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta. J Mol Biol 362:1120–1131
Citovsky V, Gafni Y, Tzfira T (2008) Localizing protein-protein interactions by bimolecular fluorescence complementation in planta. Methods 45:196–206
Krenz B, Windeisen V, Wege C, Jeske H, Kleinow T (2010) A plastid-targeted heat shock cognate 70 kDa protein interacts with the Abutilon mosaic virus movement protein. Virology 401:6–17
Frommer WB, Davidson MW, Campbell RE (2009) Genetically encoded biosensors based on engineered fluorescent proteins. Chem Soc Rev 38:2833–2841
Jones AM, Grossmann G, Danielson JA, Sosso D, Chen LQ, Ho CH, Frommer WB (2013) In vivo biochemistry: applications for small molecule biosensors in plant biology. Curr Opin Plant Biol 16:389–395
Lalonde S, Ehrhardt DW, Frommer WB (2005) Shining light on signaling and metabolic networks by genetically encoded biosensors. Curr Opin Plant Biol 8:574–581
Hanson GT, Aggeler R, Oglesbee D, Cannon M, Capaldi RA, Tsien RY, Remington SJ (2004) Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J Biol Chem 279:13044–13053
Jiang K, Schwarzer C, Lally E, Zhang S, Ruzin S, Machen T, Remington SJ, Feldman L (2006) Expression and characterization of a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein) in Arabidopsis. Plant Physiol 141:397–403
Lindbo JA (2007) High-efficiency protein expression in plants from agroinfection-compatible tobacco mosaic virus expression vectors. BMC Biotechnol 7:52
Lindbo JA (2007) TRBO: a high-efficiency tobacco mosaic virus RNA-based overexpression vector. Plant Physiol 145:1232–1240
Sainsbury F, Thuenemann EC, Lomonossoff GP (2009) pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J 7:682–693
Wroblewski T, Tomczak A, Michelmore R (2005) Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis. Plant Biotechnol J 3:259–273
Miao Y, Jiang L (2007) Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells. Nat Protoc 2:2348–2353
Wu FH, Shen SC, Lee LY, Lee SH, Chan MT, Lin CS (2009) Tape-Arabidopsis Sandwich—a simpler Arabidopsis protoplast isolation method. Plant Methods 5:16
Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572
Hellwege EM, Raap M, Gritscher D, Willmitzer L, Heyer AG (1998) Differences in chain length distribution of inulin from Cynara scolymus and Helianthus tuberosus are reflected in a transient plant expression system using the respective 1-FFT cDNAs. FEBS Lett 427:25–28
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 5:6
Johansen LK, Carrington JC (2001) Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. Plant Physiol 126:930–938
Lombardi R, Circelli P, Villani ME, Buriani G, Nardi L, Coppola V, Bianco L, Benvenuto E, Donini M, Marusic C (2009) High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus. BMC Biotechnol 9:96
Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33:949–956
Dhillon T, Chiera JM, Lindbo JA, Finer JJ (2009) Quantitative evaluation of six different viral suppressors of silencing using image analysis of transient GFP expression. Plant Cell Rep 28:639–647
Chiera JM, Lindbo JA, Finer JJ (2008) Quantification and extension of transient GFP expression by the co-introduction of a suppressor of silencing. Transgenic Res 17:1143–1154
Sattarzadeh A, Krahmer J, Germain AD, Hanson MR (2009) A myosin XI tail domain homologous to the yeast myosin vacuole-binding domain interacts with plastids and stromules in Nicotiana benthamiana. Mol Plant 2:1351–1358
Scott A, Wyatt S, Tsou PL, Robertson D, Allen NS (1999) Model system for plant cell biology: GFP imaging in living onion epidermal cells. Biotechniques 26(1125):1128–1132
Xiao YL, Redman JC, Monaghan EL, Zhuang J, Underwood BA, Moskal WA, Wang W, Wu HC, Town CD (2010) High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns. Plant Methods 6:18
Xu R, Li QQ (2008) Protocol: streamline cloning of genes into binary vectors in agrobacterium via the gateway(R) TOPO vector system. Plant Methods 4:4
Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133:462–469
Earley KW, Haag JR, Pontes O, Opper K, Juehne T, Song K, Pikaard CS (2006) Gateway-compatible vectors for plant functional genomics and proteomics. Plant J 45:616–629
Tzfira T, Tian GW, Lacroix B, Vyas S, Li J, 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 57:503–516
Kagale S, Uzuhashi S, Wigness M, Bender T, Yang W, Borhan MH, Rozwadowski K (2012) TMV-Gate vectors: gateway compatible tobacco mosaic virus based expression vectors for functional analysis of proteins. Sci Rep 2:874
Dubin MJ, Bowler C, Benvenuto G (2010) Overexpressing tagged proteins in plants using a modified gateway cloning strategy. Cold Spring Harb Protoc 2010, pdb prot5401. doi: 10.1101/pdb.prot5401.
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 51:1126–1136
Koncz C, Schel l 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 204:383–396
Wise AA, Liu Z, Binns AN (2006) Culture and maintenance of agrobacterium strains. Methods Mol Biol 343:3–13
Lee MW, Yang Y (2006) Transient expression assay by agroinfiltration of leaves. Methods Mol Biol 323:225–229
Haseloff J (1999) GFP variants for multispectral imaging of living cells. Methods Cell Biol 58:139–151
Haseloff J, Siemering KR, Prasher DC, Hodge S (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 94:2122–2127
Hawes C, Brandizzi F, Batoko H, Moore I (2001) Organelle motility in plant cells: imaging golgi and ER dynamics with GFP. Curr Protoc Cell Biol 13:13.3.1–13.3.10. doi:10.1002/0471143030.cb0107s19
Sheahan MB, Staiger CJ, Rose RJ, McCurdy DW (2004) A green fluorescent protein fusion to actin-binding domain 2 of Arabidopsis fimbrin highlights new features of a dynamic actin cytoskeleton in live plant cells. Plant Physiol 136:3968–3978
Hanson MR, Sattarzadeh A (2011) Stromules: recent insights into a long neglected feature of plastid morphology and function. Plant Physiol 155:1486–1492
Holzinger A, Buchner O, Lutz C, Hanson MR (2007) Temperature-sensitive formation of chloroplast protrusions and stromules in mesophyll cells of Arabidopsis thaliana. Protoplasma 230:23–30
Kohler RH, Cao J, Zipfel WR, Webb WW, Hanson MR (1997) Exchange of protein molecules through connections between higher plant plastids. Science 276:2039–2042
Sattarzadeh A, Fuller J, Moguel S, Wostrikoff K, Sato S, Covshoff S, Clemente T, Hanson M, Stern DB (2010) Transgenic maize lines with cell-type specific expression of fluorescent proteins in plastids. Plant Biotechnol J 8:112–125
Acknowledgments
This work was supported by grants from the Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Energy Sciences, Office of Science, US Department of Energy to M.R.H., including DE-89-ER14030 and DE–FG02–09ER16070.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Hanson, M.R., Sattarzadeh, A. (2014). Fluorescent Labeling and Confocal Microscopic Imaging of Chloroplasts and Non-green Plastids. In: Maliga, P. (eds) Chloroplast Biotechnology. Methods in Molecular Biology, vol 1132. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-995-6_7
Download citation
DOI: https://doi.org/10.1007/978-1-62703-995-6_7
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-994-9
Online ISBN: 978-1-62703-995-6
eBook Packages: Springer Protocols