Using Arabidopsis Protoplasts to Study Cellular Responses to Environmental Stress

  • Ana Confraria
  • Elena Baena-GonzálezEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1398)


Arabidopsis mesophyll protoplasts can be readily isolated and transfected in order to transiently express proteins of interest. As freshly isolated mesophyll protoplasts maintain essentially the same physiological characteristics of whole leaves, this cell-based transient expression system can be used to molecularly dissect the responses to various stress conditions. The response of stress-responsive promoters to specific stimuli can be accessed via reporter gene assays. Additionally, reporter systems can be easily engineered to address other levels of regulation, such as transcript and/or protein stability. Here we present a detailed protocol for using the Arabidopsis mesophyll protoplast system to study responses to environmental stress, including preparation of reporter and effector constructs, large scale DNA purification, protoplast isolation, transfection, treatment, and quantification of luciferase-based reporter gene activities.

Key words

Protoplasts Arabidopsis thaliana Mesophyll Transient expression system Stress Luciferase-based reporters Promoter activity 



We thank Vera Nunes for excellent plant care and all the members of the Baena-González lab for helping to establish various reporter assays in Arabidopsis mesophyll protoplasts. We also thank Jen Sheen (Harvard Medical School/Massachusetts General Hospital) for all the guidance and training regarding this technique and cell signaling. Ana Confraria is supported by a fellowship from Fundação para a Ciência e Tecnologia (FCT, SFRH/BPD/47280/2008). The Baena-González lab is supported by grants from the EMBO Installation program, the Marie Curie ITN program (“MERIT”, PITN-GA-2010-264474) and FCT (PTDC/BIA-PLA/3937/2012; Research unit GREEN-it "Bioresources for Sustainability” UID/Multi/04551/2013).


  1. 1.
    Brenner S, Dark FA, Gerhardt P, Jeynes MH, Kandler O, Kellenberger E, Klieneberger-Nobel E, McQuillen K, Rubio-Huertos M, Salton MRJ, Strange RE, Tomcsik J, Weibull C (1958) Bacterial protoplasts. Nature 181(4625):1713–1715CrossRefGoogle Scholar
  2. 2.
    Weibull C (1953) The isolation of protoplasts from Bacillus megaterium by controlled treatment with lysozyme. J Bacteriol 66(6):688–695PubMedCentralPubMedGoogle Scholar
  3. 3.
    Eddy AA, Williamson DH (1957) A method of isolating protoplasts from yeast. Nature 179(4572):1252–1253CrossRefGoogle Scholar
  4. 4.
    Bachmann BJ, Bonner DM (1959) Protoplasts from neurospora crassa. J Bacteriol 78:550–556PubMedCentralPubMedGoogle Scholar
  5. 5.
    Cocking EC (1960) A method for the isolation of plant protoplasts and vacuoles. Nature 187(4741):962–963CrossRefGoogle Scholar
  6. 6.
    Power JB, Cocking EC (1969) A simple method for the isolation of very large numbers of leaf protoplasts by using mixtures of cellulase and pectinase. Biochem J 111(5):33PPubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Krens FA, Molendijk L, Wullems GJ, Schilperoort RA (1982) In vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296(5852):72–74CrossRefGoogle Scholar
  8. 8.
    Fromm M, Taylor LP, Walbot V (1985) Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc Natl Acad Sci U S A 82(17):5824–5828PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Riggs CD, Bates GW (1986) Stable transformation of tobacco by electroporation: evidence for plasmid concatenation. Proc Natl Acad Sci U S A 83(15):5602–5606PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Negrutiu I, Shillito R, Potrykus I, Biasini G, Sala F (1987) Hybrid genes in the analysis of transformation conditions : I. Setting up a simple method for direct gene transfer in plant protoplasts. Plant Mol Biol 8(5):363–373. doi: 10.1007/BF00015814 CrossRefPubMedGoogle Scholar
  11. 11.
    Pandey S, Wang X-Q, Coursol SA, Assmann SM (2002) Preparation and applications of Arabidopsis thaliana guard cell protoplasts. New Phytol 153(3):517–526. doi: 10.1046/j.0028-646X.2001.00329.x CrossRefGoogle Scholar
  12. 12.
    Davey MR, Anthony P, Power JB, Lowe KC (2005) Plant protoplasts: status and biotechnological perspectives. Biotechnol Adv 23(2):131–171. doi: 10.1016/j.biotechadv.2004.09.008, S0734-9750(04)00096-5 [pii]CrossRefPubMedGoogle Scholar
  13. 13.
    Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2(7):1565–1572. doi: 10.1038/nprot.2007.199, doi:nprot.2007.199 [pii]CrossRefPubMedGoogle Scholar
  14. 14.
    Miao Y, Jiang L (2007) Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells. Nat Protoc 2(10):2348–2353. doi: 10.1038/nprot.2007.360, nprot.2007.360 [pii]CrossRefPubMedGoogle Scholar
  15. 15.
    Eeckhaut T, Lakshmanan PS, Deryckere D, Van Bockstaele E, Van Huylenbroeck J (2013) Progress in plant protoplast research. Planta 238(6):991–1003. doi: 10.1007/s00425-013-1936-7 CrossRefGoogle Scholar
  16. 16.
    Li JF, Zhang D, Sheen J (2014) Epitope-tagged protein-based artificial miRNA screens for optimized gene silencing in plants. Nat Protoc 9(4):939–949. doi: 10.1038/nprot.2014.061, nprot.2014.061 [pii]PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Tran LS, Nakashima K, Sakuma Y, Simpson SD, Fujita Y, Maruyama K, Fujita M, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2004) Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1 promoter. Plant Cell 16(9):2481–2498. doi: 10.1105/tpc.104.022699tpc.104.022699 [pii]PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    De Sutter V, Vanderhaeghen R, Tilleman S, Lammertyn F, Vanhoutte I, Karimi M, Inze D, Goossens A, Hilson P (2005) Exploration of jasmonate signalling via automated and standardized transient expression assays in tobacco cells. Plant J 44(6):1065–1076. doi: 10.1111/j.1365-313X.2005.02586.x, TPJ2586 [pii]CrossRefPubMedGoogle Scholar
  19. 19.
    Vanden Bossche R, Demedts B, Vanderhaeghen R, Goossens A (2013) Transient expression assays in tobacco protoplasts. Methods Mol Biol 1011:227–239. doi: 10.1007/978-1-62703-414-2_18 CrossRefPubMedGoogle Scholar
  20. 20.
    Kwiatkowska A, Zebrowski J, Oklejewicz B, Czarnik J, Halibart-Puzio J, Wnuk M (2014) The age-dependent epigenetic and physiological changes in an Arabidopsis T87 cell suspension culture during long-term cultivation. Biochem Biophys Res Commun 447(2):285–291. doi: 10.1016/j.bbrc.2014.03.141, S0006-291X(14)00601-9 [pii]CrossRefPubMedGoogle Scholar
  21. 21.
    Li JF, Chung HS, Niu Y, Bush J, McCormack M, Sheen J (2013) Comprehensive protein-based artificial microRNA screens for effective gene silencing in plants. Plant Cell 25(5):1507–1522. doi: 10.1105/tpc.113.112235, tpc.113.112235 [pii]PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Fujikawa Y, Kato N (2007) Split luciferase complementation assay to study protein-protein interactions in Arabidopsis protoplasts. Plant J 52(1):185–195. doi: 10.1111/j.1365-313X.2007.03214.x, TPJ3214 [pii]CrossRefPubMedGoogle Scholar
  23. 23.
    Li JF, Bush J, Xiong Y, Li L, McCormack M (2011) Large-scale protein-protein interaction analysis in Arabidopsis mesophyll protoplasts by split firefly luciferase complementation. PLoS One 6(11), e27364. doi: 10.1371/journal.pone.0027364PONE-D-11-13286 [pii]PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Ehlert A, Weltmeier F, Wang X, Mayer CS, Smeekens S, Vicente-Carbajosa J, Droge-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(5):890–900. doi: 10.1111/j.1365-313X.2006.02731.x, TPJ2731 [pii]CrossRefPubMedGoogle Scholar
  25. 25.
    Sheen J (2001) Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol 127(4):1466–1475PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Boudsocq M, Willmann MR, McCormack M, Lee H, Shan L, He P, Bush J, Cheng SH, Sheen J (2010) Differential innate immune signalling via Ca(2+) sensor protein kinases. Nature 464(7287):418–422. doi: 10.1038/nature08794, nature08794 [pii]PubMedCentralCrossRefPubMedGoogle Scholar
  27. 27.
    Rodrigues A, Adamo M, Crozet P, Margalha L, Confraria A, Martinho C, Elias A, Rabissi A, Lumbreras V, Gonzalez-Guzman M, Antoni R, Rodriguez PL, Baena-Gonzalez E (2013) ABI1 and PP2CA phosphatases are negative regulators of Snf1-related protein kinase1 signaling in Arabidopsis. Plant Cell 25(10):3871–3884. doi: 10.1105/tpc.113.114066, tpc.113.114066 [pii]PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Wehner N, Hartmann L, Ehlert A, Bottner S, Onate-Sanchez L, Droge-Laser W (2011) High-throughput protoplast transactivation (PTA) system for the analysis of Arabidopsis transcription factor function. Plant J 68(3):560–569. doi: 10.1111/j.1365-313X.2011.04704.x CrossRefPubMedGoogle Scholar
  29. 29.
    Wu S, Lu D, Kabbage M, Wei HL, Swingle B, Records AR, Dickman M, He P, Shan L (2011) Bacterial effector HopF2 suppresses Arabidopsis innate immunity at the plasma membrane. Mol Plant Microbe Interact 24(5):585–593. doi: 10.1094/MPMI-07-10-0150 PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Baena-Gonzalez E, Rolland F, Thevelein JM, Sheen J (2007) A central integrator of transcription networks in plant stress and energy signalling. Nature 448(7156):938–942CrossRefPubMedGoogle Scholar
  31. 31.
    Remy E, Cabrito TR, Batista RA, Hussein MA, Teixeira MC, Athanasiadis A, Sa-Correia I, Duque P (2014) Intron Retention in the 5′UTR of the Novel ZIF2 Transporter Enhances Translation to Promote Zinc Tolerance in Arabidopsis. PLoS Genet 10(5), e1004375. doi: 10.1371/journal.pgen.1004375PGENETICS-D-13-02254 [pii]PubMedCentralCrossRefPubMedGoogle Scholar
  32. 32.
    Martinho C, Confraria A, Elias A, Crozet P, Rubio-Somoza I, Weigel D, Baena-Gonzalez E (2015) Dissection of miRNA pathways using Arabidopsis mesophyll protoplasts. Mol Plant 8(2):261–275CrossRefPubMedGoogle Scholar
  33. 33.
    Babu M, Griffiths JS, Huang TS, Wang A (2008) Altered gene expression changes in Arabidopsis leaf tissues and protoplasts in response to Plum pox virus infection. BMC Genomics 9:325. doi: 10.1186/1471-2164-9-325, 1471-2164-9-325 [pii]PubMedCentralCrossRefPubMedGoogle Scholar
  34. 34.
    Leonhardt N, Kwak JM, Robert N, Waner D, Leonhardt G, Schroeder JI (2004) Microarray expression analyses of Arabidopsis guard cells and isolation of a recessive abscisic acid hypersensitive protein phosphatase 2C mutant. Plant Cell 16(3):596–615. doi: 10.1105/tpc.019000tpc.019000 [pii]PubMedCentralCrossRefPubMedGoogle Scholar
  35. 35.
    Chang YM, Liu WY, Shih AC, Shen MN, Lu CH, Lu MY, Yang HW, Wang TY, Chen SC, Chen SM, Li WH, Ku MS (2012) Characterizing regulatory and functional differentiation between maize mesophyll and bundle sheath cells by transcriptomic analysis. Plant Physiol 160(1):165–177. doi: 10.1104/pp.112.203810, 112.203810 [pii]PubMedCentralCrossRefPubMedGoogle Scholar
  36. 36.
    Obulareddy N, Panchal S, Melotto M (2013) Guard cell purification and RNA isolation suitable for high-throughput transcriptional analysis of cell-type responses to biotic stresses. Mol Plant Microbe Interact 26(8):844–849. doi: 10.1094/MPMI-03-13-0081-TA PubMedCentralCrossRefPubMedGoogle Scholar
  37. 37.
    Bargmann BO, Birnbaum KD (2010) Fluorescence activated cell sorting of plant protoplasts. J Vis Exp 36: doi:1673 [pii] 10.3791/1673Google Scholar
  38. 38.
    Dinneny JR, Long TA, Wang JY, Jung JW, Mace D, Pointer S, Barron C, Brady SM, Schiefelbein J, Benfey PN (2008) Cell identity mediates the response of Arabidopsis roots to abiotic stress. Science 320(5878):942–945. doi: 10.1126/science.1153795, 1153795 [pii]CrossRefPubMedGoogle Scholar
  39. 39.
    Galbraith DW, Janda J, Lambert GM (2011) Multiparametric analysis, sorting, and transcriptional profiling of plant protoplasts and nuclei according to cell type. Methods Mol Biol 699:407–429. doi: 10.1007/978-1-61737-950-5_20 CrossRefPubMedGoogle Scholar
  40. 40.
    Gifford ML, Dean A, Gutierrez RA, Coruzzi GM, Birnbaum KD (2008) Cell-specific nitrogen responses mediate developmental plasticity. Proc Natl Acad Sci U S A 105(2):803–808. doi: 10.1073/pnas.0709559105, 0709559105 [pii]PubMedCentralCrossRefPubMedGoogle Scholar
  41. 41.
    Yadav RK, Girke T, Pasala S, Xie M, Reddy GV (2009) Gene expression map of the Arabidopsis shoot apical meristem stem cell niche. Proc Natl Acad Sci U S A 106(12):4941–4946. doi: 10.1073/pnas.0900843106, 0900843106 [pii]PubMedCentralCrossRefPubMedGoogle Scholar
  42. 42.
    Yadav RK, Tavakkoli M, Xie M, Girke T, Reddy GV (2014) A high-resolution gene expression map of the Arabidopsis shoot meristem stem cell niche. Development 141(13):2735–2744. doi: 10.1242/dev.106104, 141/13/2735 [pii]CrossRefPubMedGoogle Scholar
  43. 43.
    Kim J, Somers DE (2010) Rapid assessment of gene function in the circadian clock using artificial microRNA in Arabidopsis mesophyll protoplasts. Plant Physiol 154(2):611–621. doi: 10.1104/pp.110.162271, pp.110.162271 [pii]PubMedCentralCrossRefPubMedGoogle Scholar
  44. 44.
    Kovtun Y, Chiu WL, Zeng W, Sheen J (1998) Suppression of auxin signal transduction by a MAPK cascade in higher plants. Nature 395(6703):716–720. doi: 10.1038/27240 CrossRefPubMedGoogle Scholar
  45. 45.
    Yanagisawa S, Yoo SD, Sheen J (2003) Differential regulation of EIN3 stability by glucose and ethylene signalling in plants. Nature 425(6957):521–525. doi: 10.1038/nature01984, nature01984 [pii]CrossRefPubMedGoogle Scholar
  46. 46.
    Sheen J (1996) Ca2+-dependent protein kinases and stress signal transduction in plants. Science 274(5294):1900–1902CrossRefPubMedGoogle Scholar
  47. 47.
    Confraria A, Martinho C, Elias A, Rubio-Somoza I, Baena-Gonzalez E (2013) miRNAs mediate SnRK1-dependent energy signaling in Arabidopsis. Front. Plant Sci 4:197. doi: 10.3389/fpls.2013.00197 Google Scholar
  48. 48.
    Kovtun Y, Chiu WL, Tena G, Sheen J (2000) Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc Natl Acad Sci U S A 97(6):2940–2945PubMedCentralCrossRefPubMedGoogle Scholar
  49. 49.
    Fujii H, Chinnusamy V, Rodrigues A, Rubio S, Antoni R, Park SY, Cutler SR, Sheen J, Rodriguez PL, Zhu JK (2009) In vitro reconstitution of an abscisic acid signalling pathway. Nature 462(7273):660–664. doi: 10.1038/nature08599, nature08599 [pii]PubMedCentralCrossRefPubMedGoogle Scholar
  50. 50.
    Xiang L, Le Roy K, Bolouri-Moghaddam MR, Vanhaecke M, Lammens W, Rolland F, Van den Ende W (2011) Exploring the neutral invertase-oxidative stress defence connection in Arabidopsis thaliana. J Exp Bot 62(11):3849–3862. doi: 10.1093/jxb/err069, err069 [pii]PubMedCentralCrossRefPubMedGoogle Scholar
  51. 51.
    Asai T, Tena G, Plotnikova J, Willmann MR, Chiu WL, Gomez-Gomez L, Boller T, Ausubel FM, Sheen J (2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415(6875):977–983CrossRefPubMedGoogle Scholar
  52. 52.
    Wang Y, Li J, Hou S, Wang X, Li Y, Ren D, Chen S, Tang X, Zhou JM (2010) A Pseudomonas syringae ADP-ribosyltransferase inhibits Arabidopsis mitogen-activated protein kinase kinases. Plant Cell 22(6):2033–2044. doi: 10.1105/tpc.110.075697, tpc.110.075697 [pii]PubMedCentralCrossRefPubMedGoogle Scholar
  53. 53.
    Liu T, Liu Z, Song C, Hu Y, Han Z, She J, Fan F, Wang J, Jin C, Chang J, Zhou JM, Chai J (2012) Chitin-induced dimerization activates a plant immune receptor. Science 336(6085):1160–1164. doi: 10.1126/science.1218867, 336/6085/1160 [pii]CrossRefPubMedGoogle Scholar
  54. 54.
    Li M, Berendzen KW, Schoffl F (2010) Promoter specificity and interactions between early and late Arabidopsis heat shock factors. Plant Mol Biol 73(4-5):559–567. doi: 10.1007/s11103-010-9643-2 PubMedCentralCrossRefPubMedGoogle Scholar
  55. 55.
    Niu Y, Sheen J (2012) Transient expression assays for quantifying signaling output. Methods Mol Biol 876:195–206. doi: 10.1007/978-1-61779-809-2_16 CrossRefPubMedGoogle Scholar
  56. 56.
    Xiang C, Han P, Lutziger I, Wang K, Oliver DJ (1999) A mini binary vector series for plant transformation. Plant Mol Biol 40(4):711–717CrossRefPubMedGoogle Scholar
  57. 57.
    Bush J, Jander G, Ausubel FM (2006) Prevention and control of pests and diseases. Methods Mol Biol 323:13–25. doi: 10.1385/1-59745-003-0:13 PubMedGoogle Scholar
  58. 58.
    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. doi: 10.1186/1746-4811-5-16, 1746-4811-5-16 [pii]PubMedCentralCrossRefPubMedGoogle Scholar
  59. 59.
    Sheen J (1990) Metabolic repression of transcription in higher plants. Plant Cell 2(10):1027–1038. doi: 10.1105/tpc.2.10.10272/10/1027 [pii]PubMedCentralCrossRefPubMedGoogle Scholar
  60. 60.
    Sheen J (1991) Molecular mechanisms underlying the differential expression of maize pyruvate, orthophosphate dikinase genes. Plant Cell 3(3):225–245. doi: 10.1105/tpc.3.3.225, 3/3/225 [pii]PubMedCentralCrossRefPubMedGoogle Scholar
  61. 61.
    Shen J, Fu J, Ma J, Wang X, Gao C, Zhuang C, Wan J, Jiang L (2014) Isolation, culture, and transient transformation of plant protoplasts. Curr Protoc Cell Biol 63:2.8.1–2.8.17. doi: 10.1002/0471143030.cb0208s63 CrossRefGoogle Scholar
  62. 62.
    Teige M, Scheikl E, Eulgem T, Doczi R, Ichimura K, Shinozaki K, Dangl JL, Hirt H (2004) The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis. Mol Cell 15(1):141–152. doi: 10.1016/j.molcel.2004.06.023S1097276504003491 [pii]CrossRefPubMedGoogle Scholar
  63. 63.
    Boudsocq M, Barbier-Brygoo H, Lauriere C (2004) Identification of nine sucrose nonfermenting 1-related protein kinases 2 activated by hyperosmotic and saline stresses in Arabidopsis thaliana. J Biol Chem 279(40):41758–41766. doi: 10.1074/jbc.M405259200M405259200 [pii]CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Plant Stress Signaling, Instituto Gulbenkian de CiênciaOeirasPortugal

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