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Protoplast isolation and transient gene expression in the single-cell C4 species, Bienertia sinuspersici

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Abstract

Although transient gene expression using reporters such as green fluorescent protein is a versatile tool for examining gene functions and intracellular protein trafficking, the establishment of a highly efficient gene manipulation method remains a challenge in many plant species. A reliable transformation protocol has not yet been established for the three single-cell C4 species, despite their potential of serving as model systems for their extraordinary C4 photosynthetic metabolism. We report the first protocol optimized for isolating a large-scale and homogenous population of protoplasts from chlorenchyma cells of the single-cell C4 species Bienertia sinuspersici. Cytochemical staining confirmed the preservation of the unusual subcellular compartmentation of organelles in chlorenchyma cells after cell wall digestion. Approximately 84% of isolated protoplasts expressed the reporter fluorescent protein following our optimized polyethylene glycol-mediated transfection procedures. Fluorescent fusion protein tagged with various intracellular sorting signals demonstrated potential use of the transient gene expression system in subcellular protein localization and organelle dynamics studies. Further applications of the current protoplast isolation and transfection techniques in understanding the novel single-cell C4 photosynthetic mechanism are discussed.

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References

  • Akhani H, Barroca J, Koteeva N, Voznesenskaya E, Franceschi V, Edwards G, Ghaffari SM, Ziegler H (2005) Bienertia sinuspersici (Chenopodiaceae): a new species from Southwest Asia and discovery of a third terrestrial C4 plant without Kranz anatomy. Syst Bot 30:290–301

    Article  Google Scholar 

  • Chen S, Tao L, Zeng L, Vega-Sanchez M, Umemura K, Wang G (2006) A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice. Mol Plant Pathol 7:417–427

    Article  PubMed  CAS  Google Scholar 

  • Chung SM, Frankman EL, Tzfira T (2005) A versatile vector system for multiple gene expression in plants. Trends Plant Sci 10:357–361

    Article  PubMed  CAS  Google Scholar 

  • Chuong SDX, Franceschi VR, Edwards GE (2006) The cytoskeleton maintains organelle partitioning required for single-cell C4 photosynthesis in Chenopodiaceae species. Plant Cell 18:2207–2223

    Article  PubMed  CAS  Google Scholar 

  • Cocking EC (1960) A method for the isolation of plant protoplasts and vacuoles. Nature 187:962–963

    Article  Google Scholar 

  • Davey MR, Anthony P, Power JB, Lowe KC (2005) Plant protoplasts: status and biotechnological perspectives. Biotechnol Adv 23:131–171

    Article  PubMed  CAS  Google Scholar 

  • Edwards GE, Huber SC (1981) The C4 pathway. In: Hatch MD, Boardman NK (eds) The biochemistry of plants: a comprehensive treatise. Photosynthesis vol 8. Academic Press, New York, pp 237–281

  • Edwards GE, Franceschi VR, Voznesenskaya EV (2004) Single-cell C4 photosynthesis versus the dual-cell (Kranz) paradigm. Annu Rev Plant Biol 55:173–196

    Article  PubMed  CAS  Google Scholar 

  • Ehlert A, Weltmeier F, Wang X, Mayer CS, Smeekens S, Vicente-Carbojosa J, Dröge-Laser W (2006) Two-hybrid protein-protein interaction analysis in Arabidopsis protoplasts: establishment of a heterodimerization map of group C and group S bZIP transcription factors. Plant J 46:890–900

    Article  PubMed  CAS  Google Scholar 

  • Emanuelsson O, Nielsen H, von Heijne G (1999) ChloroP a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8:978–984

    Article  PubMed  CAS  Google Scholar 

  • Fromm M, Taylor LP, Walbot V (1985) Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc Natl Acad Sci USA 82:5824–5828

    Article  PubMed  CAS  Google Scholar 

  • Gould SJ, Keller GA, Hosken N, Wilkinson J, Subramani S (1989) A conserved tripeptide sorts proteins to peroxisomes. J Cell Biol 108:1657–1664

    Article  PubMed  CAS  Google Scholar 

  • Hatch MD (1971) Mechanism and function of C4 photosynthesis. In: Hatch MD, Osmond CB, Slatyer RO (eds) Photosynthesis and photorespiration. Wiley-Interscience, New York, pp 139–152

    Google Scholar 

  • Hatch MD, Slack CR (1970) Photosynthetic CO2-fixation pathways. Annu Rev Plant Physiol 21:141–162

    Article  CAS  Google Scholar 

  • Ichikawa K, Chikahiro M, Megumi I, Masami S, Atsuhiko S, Ko K (2008) Ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit translation is regulated in a small subunit-independent manner in the expanded leaves of tobacco. Plant Cell Physiol 49:214–225

    Article  PubMed  CAS  Google Scholar 

  • Ishikawa S, Bang SW, Kaneko Y, Matsuzawa Y (2003) Production and characterization of intergeneric somatic hybrids between Moricandia arvensis and Brassica oleracea. Plant Breed 122:233–238

    Article  Google Scholar 

  • Koch KE (1996) Carbohydrate-modulated gene expression in plants. Annu Rev Plant Physiol Mol Biol 47:509–540

    Article  CAS  Google Scholar 

  • Kost B, Spielhofer P, Chua NH (1998) A GFP–mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes. Plant J 16:393–401

    Article  PubMed  CAS  Google Scholar 

  • Krens FA, Molendijk L, Wullems GJ, Schilperoort RA (1982) In vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296:72–74

    Article  CAS  Google Scholar 

  • Lara MV, Offermann S, Smith M, Okita TW, Andreo CS, Edwards GE (2008) Leaf development in the single-cell C4 system in Bienertia sinuspersici: expression of genes and peptides levels for C4 metabolism in relation to chlorenchyma structure under different light conditions. Plant Physiol 148:593–610

    Article  PubMed  CAS  Google Scholar 

  • Larkin PJ (1976) Purification and viability determinations of plant protoplasts. Planta 128:213–216

    Article  Google Scholar 

  • Majeran W, Cai Y, Sun Q, van Wijk KJ (2005) Functional differentiation of bundle sheath and mesophyll maize chloroplasts determined by comparative proteomics. Plant Cell 17:3111–3140

    Article  PubMed  CAS  Google Scholar 

  • Majeran W, Zybailov B, Ytterberg AJ, Dunsmore J, Sun Q, van Wijk KJ (2008) Consequences of C4 differentiation for chloroplasts membrane proteomes in maize mesophyll and bundle sheath cells. Mol Cell Proteomics 7:1609–1638

    Article  PubMed  CAS  Google Scholar 

  • Marc J, Granger CL, Brincat J, Fisher DD, Kao TH, McCubbin AG, Cyr RJ (1998) A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells. Plant Cell 10:1927–1940

    Article  PubMed  CAS  Google Scholar 

  • Neuhaus G, Spangenberg G (1990) Plant transformation by microinjection techniques. Physiol Plant 79:213–217

    Article  CAS  Google Scholar 

  • Ohno M, Fornerod M, Mattaj IW (1998) Nucleocytoplasmic transport: the last 200 nanometers. Cell 92:327–336

    Article  PubMed  CAS  Google Scholar 

  • Orczyk W, Przetakiewicz J, Nadolska-Orczyk A (2003) Somatic hybrids of Solanum tuberosum—application to genetics and breeding. Plant Cell Tissue Organ Cult 74:1–13

    Article  CAS  Google Scholar 

  • Park J, Knoblauch M, Okita TW, Edwards GE (2009) Structural changes in the vacuole and cytoskeleton are key to development of the two cytoplasmic domains supporting single-cell C4 photosynthesis in Bienertia sinuspersici. Planta 229:369–382

    Article  PubMed  CAS  Google Scholar 

  • Paszkowski J, Shillito RD, Saul M, Mandák V, Hohn T, Hohn B, Potrykus I (1984) Direct gene transfer to plant. EMBO J 3:2717–2722

    PubMed  CAS  Google Scholar 

  • Rahim G, Bischof S, Kessler F, Agne B (2009) In vivo interaction between atToc33 and atToc159 GTP-binding domains demonstrated in a plant split-ubiquitin system. J Exp Bot 60:257–267

    Article  PubMed  CAS  Google Scholar 

  • Sage R (2001) Environmental and evolutionary preconditions for the origin and diversification of the C4 photosynthetic syndrome. Plant Biol 3:202–213

    Article  CAS  Google Scholar 

  • Sheen J (1990) Metabolic repression of transcription in higher plants. Plant Cell 2:1027–1038

    Article  PubMed  CAS  Google Scholar 

  • Sheen J (2001) Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol 127:1466–1475

    Article  PubMed  CAS  Google Scholar 

  • Surridge C (2002) The rice squad. Nature 416:576–578

    Article  PubMed  CAS  Google Scholar 

  • Voznesenskaya EV, Franceschi VR, Kiirats O, Freitag H, Edwards GE (2001) Kranz anatomy is not essential for terrestrial C4 plant photosynthesis. Nature 414:543–546

    Article  PubMed  CAS  Google Scholar 

  • Voznesenskaya EV, Franceschi VR, Kiirats O, Artyusheva EG, Freitag H, Edwards GE (2002) Proof of C4 photosynthesis without Kranz anatomy in Bienertia cycloptera (Chenopodiaceae). Plant J 31:649–662

    Article  PubMed  CAS  Google Scholar 

  • Voznesenskaya EV, Koteyeva NK, Chuong SDX, Akhani H, Edwards GE, Franceschi VR (2005) Differentiation of cellular and biochemical features of the single-cell C4 syndrome during leaf development in Bienertia cycloptera (Chenopodiaceae). Amer J Bot 92:1784–1795

    Article  CAS  Google Scholar 

  • Yamada M, Kawasaki M, Sugiyama T, Miyake H, Taniguchi M (2009) Differential positioning of C4 mesophyll and bundle sheath chloroplasts: aggregative movement of C4 mesophyll chloroplasts in response to environmental stresses. Plant Cell Physiol 50:1736–1749

    Article  PubMed  CAS  Google Scholar 

  • Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This research was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada Research Grants and the University of Waterloo Start-Up Fund to SDXC. SCL received additional financial support from Ontario Graduate Scholarship (Government of Ontario, Canada) and President’s Graduate Scholarship (University of Waterloo, Canada). The authors gratefully acknowledge the following colleagues for providing GFP constructs: pSAT6-35S::EGFP-N1 and pSAT6-35S::EGFP-C1 (Tzvi Tzfira, University of Michigan), talin-GFP (Nam-Hai Chua, The Rockefeller University), and MAP4-GFP (Richard Cyr, Pennsylvania State University).

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Authors and Affiliations

  1. Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, ON, N2L 3G1, Canada

    Shiu-Cheung Lung, Makoto Yanagisawa & Simon D. X. Chuong

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  1. Shiu-Cheung Lung
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  2. Makoto Yanagisawa
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  3. Simon D. X. Chuong
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Corresponding author

Correspondence to Simon D. X. Chuong.

Additional information

Communicated by F. Brandizzi.

S.-C. Lung and M. Yanagisawa contributed equally to this work.

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Lung, SC., Yanagisawa, M. & Chuong, S.D.X. Protoplast isolation and transient gene expression in the single-cell C4 species, Bienertia sinuspersici . Plant Cell Rep 30, 473–484 (2011). https://doi.org/10.1007/s00299-010-0953-2

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