Journal of Plant Biology

, Volume 55, Issue 5, pp 390–397 | Cite as

Preparation of leaf mesophyll protoplasts for transient gene expression in Brachypodium distachyon

  • Shin-Young Hong
  • Pil Joon Seo
  • Shin-Hae Cho
  • Chung-Mo Park
Original Article

Abstract

Transient gene expression systems using protoplasts have been widely used for rapid functional characterization of genes in many plant species. Brachypodium distachyon has recently been employed as a model plant for studies on biofuel grass species and grass crops because of its small genome size, short life-span, and availability of efficient transformation systems. Here, we report the an efficient protocol for the preparation of leaf mesophyll protoplasts from Brachypodium seedlings. We also modified the polyethylene glycol (PEG)-mediated transformation procedure to optimize experimental conditions, such as duration of enzyme digestion, PEG incubation time, and plasmid DNA concentration and size. The green fluorescence protein (GFP)- and β-glucuronidase (GUS)-coding genes were used as reporters to evaluate the feasibility of this transient expression system. We found that the yield of viable protoplasts was highest after 3 h of enzymatic digestion. Viability of enzyme-digested protoplasts was moderately maintained up to 24 h in Mmg preincubation solution. In addition, the highest transient expression of reporter genes was obtained when protoplasts were transformed with 20 μg of plasmid DNA and incubated for 16 h. Together with the recent completion of the Brachypodium genome sequence, the Brachypodium transient expression system using leaf mesophyll protoplasts can be widely used for cellular, molecular, and biochemical studies of genes involved in carbon metabolism and signaling pathways mediating intrinsic and environmental cues.

Keywords

Brachypodium GUS PEG-mediated transformation Protoplast Transient gene expression 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Abel S, Theologis A (1994) Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression. Plant J 5:421–427PubMedCrossRefGoogle Scholar
  2. Abel S, Theologis A (1996) Early genes and auxin action. Plant Physiol 111:9–17PubMedCrossRefGoogle Scholar
  3. Bart R, Chern M, Park CJ, Bartley L, Ronald PC (2006) A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. Plant Methods 2:13–22PubMedCrossRefGoogle Scholar
  4. Binder KA, Wegner LH, Heidecker M, Zimmermann U (2003) Gating of Cl currents in protoplasts from the marine alga Valonia utricularis depends on the transmembrane Cl gradient and is affected by enzymatic cell wall degradation. J Membr Biol 191:165–178PubMedCrossRefGoogle Scholar
  5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  6. Brkljacic J, Grotewold E, Scholl R, Mockler T, Garvin DF, Vain P, Brutnell T, Sibout R, Bevan M, Budak H, Caicedo AL, Gao C, Gu Y, Hazen SP, Holt BF 3rd, Hong SY, Jordan M, Manzaneda AJ, Mitchell-Olds T, Mochida K, Mur LA, Park CM, Sedbrook J, Watt M, Zheng SJ, Vogel JP (2011) Brachypodium as a model for the grasses: today and the future. Plant Physiol 157:3–13PubMedCrossRefGoogle Scholar
  7. Bruce WB, Christensen AH, Klein T, Fromm M, Quail PH (1989) Photoregulation of a phytochrome gene promoter from oat transferred into rice by particle bombardment. Proc Natl Acad Sci USA 86:9692–9696PubMedCrossRefGoogle Scholar
  8. Chang CC, Sheen J, Bligny M, Niwa Y, Lerbs-Mache S, Stern DB (1999) Functional analysis of two maize cDNAs encoding T7-like RNA polymerases. Plant Cell 11:911–926PubMedGoogle Scholar
  9. Chen S, Tao L, Zeng L, Vega-Sanchez ME, Umemura K, Wang GL (2006) A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice. Mol Plant Pathol 7:417–427PubMedCrossRefGoogle Scholar
  10. Datla RS, Hammerlindl JK, Panchuk B, Pelcher LE, Keller W (1992) Modified binary plant transformation vectors with the wild-type gene encoding NPTII. Gene 122:383–384PubMedCrossRefGoogle Scholar
  11. Datta K, Datta SK (1999) Transformation of rice via PEG-mediated DNA uptake into protoplasts. Methods Mol Biol 111:335–347PubMedGoogle Scholar
  12. Davey MR, Anthony P, Power JB, Lowe KC (2004) Protoplast applications in biotechnology. In: Goodman RM (ed) Encyclopaedia of plant and crop science. Marcel Dekker Inc., New York, pp 1061–1064Google Scholar
  13. Davey MR, Anthony P, Power JB, Lowe KC (2005) Plant protoplasts: status and biotechnological perspectives. Biotechnol Adv 23:131–171PubMedCrossRefGoogle Scholar
  14. Davey MR, Anthony P, Power JB, Lowe KC (2006) Isolation, culture, and plant regeneration from leaf protoplasts of Passiflora. Methods Mol Biol 318:201–210PubMedGoogle Scholar
  15. Davey MR, Kumar A (1983) Higher plant protoplasts-retrospect and prospect. Int Rev Cytol Suppl 16:219–299Google Scholar
  16. De Domenico S, Bonsegna S, Lenucci MS, Poltronieri P, Di Sansebastiano GP, Santino A (2011) Localization of seed oil body proteins in tobacco protoplasts reveals specific mechanisms of protein targeting to leaf lipid droplets. J Integr Plant Biol 53:858–868PubMedCrossRefGoogle Scholar
  17. De Sutter V, Vanderhaeghen R, Tilleman S, Lammertyn F, Vanhoutte I, Karimi M, Inzé D, Goossens A, Hilson P (2005) Exploration of jasmonate signalling via automated and standardized transient expression assays in tobacco cells. Plant J 44:1065–1076PubMedCrossRefGoogle Scholar
  18. Draper J, Mur LA, Jenkins G, Ghosh-Biswas GC, Bablak P, Hasterok R, Routledge AP (2001) Brachypodium distachyon. A new model system for functional genomics in grasses. Plant Physiol 127:1539–1555PubMedCrossRefGoogle Scholar
  19. Elliott AR, Campbell JA, Dugdale B, Brettell, RS, Grof CL (1999) Green fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells. Plant Cell Rep 18:707–714CrossRefGoogle Scholar
  20. Evans DA, Bravo JE (1983) Protoplast isolation and culture. In: Evans DA, Sharp WR, Ammirato PV, Yamada Y (eds) Handbook of Plant Cell Culture. MacMillan, New York, pp 124–176Google Scholar
  21. Filiz E, Ozdemir BS, Budak F, Vogel JP, Tuna M, Budak H (2009) Molecular, morphological, and cytological analysis of diverse Brachypodium distachyon inbred lines. Genome 52:876–890PubMedCrossRefGoogle Scholar
  22. Fischer R, Hain R (1995) Tobacco protoplast transformation and use for functional analysis of newly isolated genes and gene constructs. Methods Cell Biol 50:401–410PubMedCrossRefGoogle Scholar
  23. Frohnmeyer H, Hahlbrock K, Schafer E (1994) A light responsive in vitro transcription system from evacuolated parsley protoplasts. Plant J 5:437–449PubMedCrossRefGoogle Scholar
  24. 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
  25. Hansen G, Wright MS (1999) Recent advances in the transformation of plants. Trends Plant Sci 4:226–231PubMedCrossRefGoogle Scholar
  26. Hayashimoto A, Li Z, Murai N (1990) A polyethylene glycol-mediated protoplast transformation system for production of fertile transgenic rice plants. Plant Physiol 93:857–863PubMedCrossRefGoogle Scholar
  27. Hoffman A, Halfter U, Morris PC (1994) Transient expression in leaf mesophyll protoplasts of Arabidopsis thaliana. Plant Cell Tiss Org Cult 36:53–58CrossRefGoogle Scholar
  28. Hong SY, Park JH, Cho SH, Yang MS, Park CM (2011) Phenological growth stages of Brachypodium distachyon: codification and description according to the BBCH scale. Weed Res 51:612–620CrossRefGoogle Scholar
  29. Hong SY, Seo PJ, Yang MS, Xiang F, Park CM (2008) Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR. BMC Plant Biol 8:112–122PubMedCrossRefGoogle Scholar
  30. Hrazdina G, Marx GA, Hoch HC (1982) Distribution of secondary plant metabolites and their biosynthetic enzymes in pea (Pisum sativum L.) leaves: anthocyanins and flavonol glycosides. Plant Physiol 70:745–748PubMedCrossRefGoogle Scholar
  31. Huang CN, Cornejo MJ, Bush DS, Jones RL (1986) Estimating viability of plant protoplasts using double and single staining. Protoplasma 135:80–87CrossRefGoogle Scholar
  32. Huo N, Gu Y, Lazo G, Vogel J, Coleman-Derr D, Luo M, Thilmony R, Garvin D, Anderson O (2006) Construction and characterization of two BAC libraries from Brachypodium distachyon, a new model for grass genomics. Genome 49:1099–1108PubMedCrossRefGoogle Scholar
  33. International Brachypodium Initiative (2010) Genome sequencing and analysis of the model grass Brachypodium distachyon. Nature 463:763–768CrossRefGoogle Scholar
  34. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedGoogle Scholar
  35. Jin JB, Kim YA, Kim SJ, Lee SH, Kim DH, Cheong GW, Hwang I (2001) A new dynamin-like protein, ADL6, is involved in trafficking from the trans-Golgi network to the central vacuole in Arabidopsis. Plant Cell 13:1511–1526PubMedGoogle Scholar
  36. Kim MJ, Baek K, Park CM (2009) Optimization of conditions for transient Agrobacterium-mediated gene expression assays in Arabidopsis. Plant Cell Rep 28:1159–1167PubMedCrossRefGoogle Scholar
  37. Koop HU, Steinmüller K, Wagner H, Rössler C, Eibl C, Sacher L (1996) Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation. Planta 199:193–201PubMedCrossRefGoogle Scholar
  38. Larkin PJ (1976) Purification and viability of plant protoplasts. Planta 128:213–216CrossRefGoogle Scholar
  39. Li JF, Nebenführ A (2010) FAST technique for Agrobacterium-mediated transient gene expression in seedlings of Arabidopsis and other plant species. Cold Spring Harb Protoc 5:5428–5431Google Scholar
  40. Liu CN, Li XQ, Gelvin SB (1992) Multiple copies of virG enhance the transient transformation of celery, carrot and rice tissues by Agrobacterium tumefaciens. Plant Mol Biol 20:1071–1087PubMedCrossRefGoogle Scholar
  41. Loake GJ, Choudhary AD, Harrison MJ, Mavandad M, Lamb CJ, Dixon RA (1991) Phenylpropanoid pathway intermediates regulate transient expression of a chalcone synthase gene promoter. Plant Cell 3:829–840PubMedGoogle Scholar
  42. Locatelli F, Vannini C, Magnani E, Coraggio I, Bracale M (2003) Efficiency of transient transformation in tobacco protoplasts is independent of plasmid amount. Plant Cell Rep 21:865–871PubMedGoogle Scholar
  43. Luehrsen KR, de Wet JR, Walbot V (1992) Transient expression analysis in plants using firefly luciferase reporter gene. Methods Enzymol 216:397–414PubMedCrossRefGoogle Scholar
  44. Maas C, Werr W (1989) Mechanism and optimized conditions for PEG mediated DNA transfection into plant protoplasts. Plant Cell Rep 8:148–151CrossRefGoogle Scholar
  45. Manavella PA, Chan RL (2009) Transient transformation of sunflower leaf discs via an Agrobacterium-mediated method: applications for gene expression and silencing studies. Nat Protoc 4:1699–1707PubMedCrossRefGoogle Scholar
  46. Massa AN, Wanjugi H, Deal KR, O’Brien K, You FM, Maiti R, Chan AP, Gu YQ, Luo MC, Anderson OD, Rabinowicz PD, Dvorak J, Devos KM (2011) Gene space dynamics during the evolution of Aegilops tauschii, Brachypodium distachyon, Oryza sativa, and Sorghum bicolor genomes. Mol Biol Evol 28:2537–2547PubMedCrossRefGoogle Scholar
  47. Mathur J, Koncz C (1998) PEG-mediated protoplast transformation with naked DNA. Methods Mol Biol 82:267–276PubMedGoogle Scholar
  48. Mazarei M, Al-Ahmad H, Rudis MR, Stewart CN Jr (2008) Protoplast isolation and transient gene expression in switchgrass, Panicum virgatum L. Biotechnol J 3:354–359PubMedCrossRefGoogle Scholar
  49. Mitsuhara I, Ugaki M, Hirochika H, Ohshima M, Murakami T, Gotoh Y, Katayose Y, Nakamura S, Honkura R, Nishimiya S, Ueno K, Mochizuki A, Tanimoto H, Tsugawa H, Otsuki Y, Ohashi Y (1996) Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol 37:49–59PubMedCrossRefGoogle Scholar
  50. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue culture. Physiol Plant 15:473–497CrossRefGoogle Scholar
  51. Nimchuk Z, Marois E, Kjemtrup S, Leister RT, Katagiri F, Dangl JL (2000) Eukaryotic fatty acylation drives plasma membrane targeting and enhances function of several type III effector proteins from Pseudomonas syringae. Cell 101:353–363PubMedCrossRefGoogle Scholar
  52. Nurnberger T, Nennstiel D, Jabs T, Sacks WR, Hahlbrock K, Scheel D (1994) High affinity binding of a fungal oligopeptide elicitor to parsley plasma membranes triggers multiple defense responses. Cell 78:449–460PubMedCrossRefGoogle Scholar
  53. Opanowicz M, Vain P, Draper J, Parker D, Doonan JH (2008) Brachypodium distachyon: making hay with a wild grass. Trends Plant Sci 13:172–177PubMedCrossRefGoogle Scholar
  54. Power JB, Cocking EC (1970) Isolation of leaf protoplasts: macromolecular uptake and growth substance response. J Exp Bot 21:64–70CrossRefGoogle Scholar
  55. Rao AQ, Bakhsh A, Kiani S, Shahzad K, Shahid AA, Husnain T, Riazuddin S (2009) The myth of plant transformation. Biotechnol Adv 27:753–763PubMedCrossRefGoogle Scholar
  56. Rasmussen JO, Rasmussen OS (1993) PEG mediated DNA uptake and transient GUS expression in carrot, rapeseed and soybean protoplasts. Plant Sci 89:199–207CrossRefGoogle Scholar
  57. Sheen J (1999) C4 gene expression. Ann Rev Plant Physiol Plant Mol Biol 50:187–217CrossRefGoogle Scholar
  58. Sheen J (2001) Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol 127:1466–1475PubMedCrossRefGoogle Scholar
  59. Subramaniam R, Desveaux D, Spickler C, Michnick SW, Brisson N (2001) Direct visualization of protein interactions in plant cells. Nat Biotechnol 19:769–772PubMedCrossRefGoogle Scholar
  60. Swanson SJ, Bethke PC, Jones RL (1998) Barley aleurone cells contain two types of vacuoles. Characterization of lytic organelles by use of fluorescent probes. Plant Cell 10:685–698Google Scholar
  61. Ueki S, Lacroix B, Krichevsky A, Lazarowitz SG, Citovsky V (2009) Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment. Nat Protoc 4:71–77PubMedCrossRefGoogle Scholar
  62. Ulmasov T, Hagen G, Guilfoyle TJ (1999) Activation and repression of transcription by auxin-response factors. Proc Natl Acad Sci USA 96:5844–5849PubMedCrossRefGoogle Scholar
  63. Unver T, Budak H (2009) Conserved microRNAs and their targets in model grass species Brachypodium distachyon. Planta 230:659–669PubMedCrossRefGoogle Scholar
  64. Vain P, Worland B, Thole V, McKenzie N, Alves SC, Opanowicz M, Fish LJ, Bevan MW, Snape JW (2008) Agrobacterium-mediated transformation of the temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional mutagenesis. Plant Biotechnol J 6:236–245PubMedCrossRefGoogle Scholar
  65. Vogel J, Bragg J (2009) Brachypodium distachyon, a new model for the Triticeae. In: Feuillet C, Muehlbauer GJ (eds) Genetics and Genomics of the Triticeae. Springer, New York, pp 427–449Google Scholar
  66. Vogel J, Hill T (2008) High-efficiency Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21-3. Plant Cell Rep 27:471–478PubMedCrossRefGoogle Scholar
  67. Vogel JP, Garvin DF, Leong OM, Hayden DM (2006) Agrobacterium-mediated transformation and inbred line development in the model grass Brachypodium distachyon. Plant Cell Tiss Org Cult 85:199–211Google Scholar
  68. Wagner GJ, Butcher IV WC, Siegelman NW (1978) The plant protoplast: A useful tool for plant research and student instruction. Bioscience 28:95–101CrossRefGoogle Scholar
  69. Widholm JM (1972) The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technol 47:189–194PubMedGoogle Scholar
  70. Worley CK, Zenser N, Ramos J, Rouse D, Leyser O, Theologis A, Callis J (2000) Degradation of Aux/IAA proteins is essential for normal auxin signalling. Plant J 21:553–562PubMedCrossRefGoogle Scholar
  71. Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572PubMedCrossRefGoogle Scholar
  72. Zhang Y, Su J, Duan S, Ao Y, Dai J, Liu J, Wang P, Li Y, Liu B, Feng D, Wang J, Wang H (2011) A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes. Plant Methods 7:30–44PubMedCrossRefGoogle Scholar

Copyright information

© Korean Society of Plant Biologists and Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Shin-Young Hong
    • 1
  • Pil Joon Seo
    • 1
  • Shin-Hae Cho
    • 1
  • Chung-Mo Park
    • 1
    • 2
  1. 1.Department of ChemistrySeoul National UniversitySeoulKorea
  2. 2.Plant Genomics and Breeding InstituteSeoul National UniversitySeoulKorea

Personalised recommendations