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Plant Molecular Biology

, Volume 59, Issue 5, pp 697–711 | Cite as

Controlled Expression of Recombinant Proteins in Physcomitrella patens by a Conditional Heat-shock Promoter: a Tool for Plant Research and Biotechnology

  • Younousse Saidi
  • Andrija Finka
  • Mickhail Chakhporanian
  • Jean-Pierre Zrÿd
  • Didier G. Schaefer
  • Pierre GoloubinoffEmail author
Article

Abstract

The ability to express tightly controlled amounts of endogenous and recombinant proteins in plant cells is an essential tool for research and biotechnology. Here, the inducibility of the soybean heat-shock Gmhsp17.3B promoter was addressed in the moss Physcomitrella patens, using β-glucuronidase (GUS) and an F-actin marker (GFP-talin) as reporter proteins. In stably transformed moss lines, Gmhsp17.3B-driven GUS expression was extremely low at 25 °C. In contrast, a short non-damaging heat-treatment at 38 °C rapidly induced reporter expression over three orders of magnitude, enabling GUS accumulation and the labelling of F-actin cytoskeleton in all cell types and tissues. Induction levels were tightly proportional to the temperature and duration of the heat treatment, allowing fine-tuning of protein expression. Repeated heating/cooling cycles led to the massive GUS accumulation, up to 2.3% of the total soluble proteins. The anti-inflammatory drug acetyl salicylic acid (ASA) and the membrane-fluidiser benzyl alcohol (BA) also induced GUS expression at 25 °C, allowing the production of recombinant proteins without heat-treatment. The Gmhsp17.3B promoter thus provides a reliable versatile conditional promoter for the controlled expression of recombinant proteins in the moss P. patens.

Keywords

acetyl salicylic acid actin cytoskeleton benzyl alcohol GFP-talin β-glucuronidase Gmhsp17.3B promoter inducible gene-expression system 

Abbreviations

ASA

acetyl salicylic acid

BA

benzyl alcohol

GFP

green fluorescent protein

GT

GFP-talin

GUS

β-glucuronidase

Fv/Fm

the maximal photochemical efficiency of PSII

MU

4-methylumbelliferone

MUG

4-methylumbelliferyl-β-D-glucuronide

PSII

photosystem II

SA

specific activity

sHSP

small heat-shock protein

TSP

total soluble proteins

Ubi

ubiquitin

X-Gluc

5-bromo-4-chloro-3-indolyl-β-D-glucuronide

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References

  1. Ainley, W.M., Key, J.L. 1990Development of a heat shock inducible expression cassette for plants: characterization of parameters for its use in transient expression assaysPlant Mol. Biol14949967CrossRefPubMedGoogle Scholar
  2. Amici, C., Rossi, A., Santoro, M.G. 1995Aspirin enhances thermotolerance in human erythroleukemic cells: an effect associated with the modulation of the heat shock responseCancer Res5544524457PubMedGoogle Scholar
  3. Aoyama, T., Chua, N.-H. 1997A glucocorticoid-mediated transcriptional induction system in transgenic plantsPlant J11605611CrossRefPubMedGoogle Scholar
  4. Ashton, N.W., Grimsley, N.H., Cove, D.J. 1979Analysis of gametophytic development in the moss Physcomitrella patens, using auxin and cytokinin resistant mutantsPlanta144427435CrossRefGoogle Scholar
  5. Baniwal, S.K., Bharti, K., Chan, K.Y., Fauth, M., Ganguli, A., Kotak, S., Mishra, S.K., Nover, L., Port, M., Scharf, K.D., Tripp, J., Weber, C., Zielinski, D., Koskull-Doring, P. 2004Heat stress response in plants: a complex game with chaperones and more than twenty heat stress transcription factorsJ. Biosci2947187PubMedGoogle Scholar
  6. Bierfreund, N.M., Reski, R., Decker, E.L. 2003Use of an inducible reporter gene system for the analysis of auxin distribution in the moss Physcomitrella patens.Plant Cell Rep1211431152CrossRefGoogle Scholar
  7. Bonneville, J.M., Sanfaçon, H., Fütterer, J., Hohn, T. 1989Posttranscriptionnal trans-activation in Cauliflower Mosaic VirusCell5911351143CrossRefPubMedGoogle Scholar
  8. Caddick, M.X., Greenland, A.J., Jepson, I., Krause, K.P., Qu, N., Riddel, K.V., Salter, M.G., Schuch, W., Sonnewald, U., Tomsett, A.B. 1998An ethanol inducible gene switch for plants used to manipulate carbon metabolismNature Biotechnol.16177180CrossRefGoogle Scholar
  9. Chakhparonian, M. 2001. Développement d’outils de la mutagenèse ciblée par recombinaison homologue chez Physcomitrella patens. Université de Lausanne, Switzerland “www.unil.ch/lpc/docs/”Google Scholar
  10. Cove, D.J., Knight, C.D., Lamparter, T. 1997Mosses as model systemsTrends in Plant Sci.299105CrossRefGoogle Scholar
  11. Jaeger, G., Scheffer, S., Jacobs, A., Zambre, M., Zobell, O., Goossens, A., Depicker, A., Angenon, G. 2002Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequencesNature Biotechnol2012651268CrossRefGoogle Scholar
  12. Decker, E.L., Reski, R. 2004The moss bioreactorCurr. Opin. Plant Biol716670CrossRefPubMedGoogle Scholar
  13. Diamant, S., Ben-Zvi, A.P., Bukau, B., Goloubinoff, P. 2000Size-dependent disaggregation of stable protein aggregates by the DnaK chaperone machineryJ. Biol. Chem2752110721113CrossRefPubMedGoogle Scholar
  14. Donovan, R.S., Robinson, C.W., Glick, B.R. 1996Optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoterJ. Ind. Microbiol16145154CrossRefPubMedGoogle Scholar
  15. Doonan, J.H., Duckett, J.D. 1988The bryophyte cytoskeleton: experimental and immunofluorescence studies of morphogenesisAdv. Bryol3131Google Scholar
  16. Fischer, R., Stoger, E., Schillberg, S., Christou, P., Twyman, R.M. 2004Plant-based production of biopharmaceuticalsCurr. Opin. Plant Biol7152158CrossRefPubMedGoogle Scholar
  17. Gatz, C. 1997Chemical control of gene expressionAnn. Rev. Plant Physiol. Plant Mol. Biol4889108CrossRefGoogle Scholar
  18. Havaux, M. 1992Stress tolerance of photosystem II in vivoPlant Physiol100424432Google Scholar
  19. Horstmann, V., Huether, C.M., Jost, W., Reski, R., Decker, E.L. 2004Quantitative promoter analysis in Physcomitrella patens: a set of plant vectors activating gene expression within three orders of magnitudeBMC Biotechnol4113CrossRefPubMedGoogle Scholar
  20. Jefferson, R.A., Kavanagh, T.A., Bevan, M.W. 1987GUS fusions: ß-glucuronidase as a sensitive and versatile gene fusion marker in higher plantsEMBO J639013907PubMedGoogle Scholar
  21. Johnston, M. 1987A model fungal gene regulatory mechanism: the GAL genes of Saccharomyces cerevisiaeMicrobiol. Rev.51458476PubMedGoogle Scholar
  22. Jost, W., Link, S., Horstmann, V., Decker, E.L., Reski, R., Gorr, G. 2005Isolation and characterisation of three moss-derived beta-tubulin promoters suitable for recombinant expressionCurr. Genet47111120CrossRefPubMedGoogle Scholar
  23. Jurivich, D.A., Sistonen, L., Kroes, R.A., Morimoto, R.I. 1992Effect of sodium salicylate on the human heat shock responseScience25512431245PubMedGoogle Scholar
  24. Kilby, N.J., Fyvie, M.J., Sessions, R.A., Davies, G.J., Murray, J.A. 2000Controlled induction of GUS marked clonal sectors in ArabidopsisJ. Exp. Bot51853863CrossRefPubMedGoogle Scholar
  25. Knight, C.D., Sehgal, A., Atwal, K., Wallace, J.C., Cove, D.J., Coates, D., Quatrano, R.S., Bahadur, S., Stockley, P.G., Cuming, A.C. 1995Molecular responses to abscisic acid and stress are conserved between moss and cerealsPlant Cell7499506CrossRefPubMedGoogle Scholar
  26. Koprivova, A., Stemmer, C., Altmann, F., Hoffmann, A., Kopriva, S., Gorr, G., Reski, R., Decker, E.L. 2004Targeted knockouts of Physcomitrella lacking plant-specific immunogenic N-glycansPlant Biotechnol. J2517523CrossRefGoogle Scholar
  27. Kost, B., Spielhofer, P., Chua, N.H. 1998A GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualises the actin cytoskeleton in growing pollen tubesPlant J16393401CrossRefPubMedGoogle Scholar
  28. Krueger, G.H.J., Tsimilli-Michael, M., Strasser, R.J. 1997Light stress provokes plastic and elastic modifications in structure and function of photosystem II in camellia leavesPhysiol. Plantarum101256277Google Scholar
  29. Lu, C., Qiu, N., Wang, B., Zhang, J. 2003Salinity treatment shows no effects on photosystem II photochemistry, but increases the resistance of photosystem II to heat stress in halophyte Suaeda salsaJ. Exp. Bot54851860CrossRefPubMedGoogle Scholar
  30. Lyznik, L.A., Hirayama, L., Rao, K.V., Abad, A., Hodges, T.K. 1995Heat-inducible expression of FLP gene in maize cellsPlant J8177186CrossRefPubMedGoogle Scholar
  31. Masclaux, F., Charpenteau, M., Takahashi, T., Pont-Lezica, R., Galaud, J.P. 2004Gene silencing using a heat-inducible RNAi system in ArabidopsisBiochem. Biophys. Res. Commun321364369CrossRefPubMedGoogle Scholar
  32. Mathur, J., Spielhofer, P., Kost, B., Chua, N. 1999The actin cytoskeleton is required to elaborate and maintain spatial patterning during trichome cell morphogenesis in Arabidopsis thalianaDevelopment12655595568PubMedGoogle Scholar
  33. Maundrell, K. 1993Thiamine-repressible expression vectors pREP and pRIP for fission yeastGene123127130CrossRefPubMedGoogle Scholar
  34. McElroy, D., Blowers, A.D., Jenes, B., Wu, R. 1991Construction of expression vectors based on the rice actin 1 (Act1) 5′ region for the use in monocot transformationMol. Gen. Genet231150160CrossRefPubMedGoogle Scholar
  35. Merquiol, E., Pneuli, , L., , Cohen, M., Simovitch, M., Rachmilevitch, S., Goloubinoff, P., Kaplan, A., Mittler, R. 2002Seasonal and diurnal variations in gene expression in the desert legume Retama raetamPlant Cell Environ2516271638CrossRefGoogle Scholar
  36. Molina, A., Hervas-Stubbs, S., Daniell, H., Mingo-Castel, A. M., Veramendi, J. 2004High-yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplastsPlant Biotechnol. J2141153CrossRefGoogle Scholar
  37. Nishal, B., Tantikanjana, T., Sundaresan, V. 2005An inducible targeted tagging system for localized saturation mutagenesis in ArabidopsisPlant Physiol137312CrossRefPubMedGoogle Scholar
  38. Nishiyama, T., Fujita, T., Shin, I.T., Seki, M., Nishide, H., Uchiyama, I., Kamiya, A., Carninci, P., Hayashizaki, Y., Shinozaki, K., Kohara, Y., Hasebe, M. 2003Comparative genomics of Physcomitrella patens gametophytic transcriptome and Arabidopsis thaliana: implication for land plant evolutionProc. Natl. Acad. Sci. USA10080078012CrossRefPubMedGoogle Scholar
  39. Nishiyama, T., Hiwatashi, Y., Sakakibara, I., Kato, M., Hasebe, M. 2000Tagged mutagenesis and gene-trap in the moss, Physcomitrella patens by shuttle mutagenesisDNA Res7917CrossRefPubMedGoogle Scholar
  40. Nover, L., Bharti, K., Doring, P., Mishra, S.K., Ganguli, A., Scharf, K.D. 2001Arabidopsis and the heat stress transcription factor world: how many heat stress transcription factors do we need?Cell Stress Chaperones6177189PubMedGoogle Scholar
  41. Padidam, M. 2003Chemically regulated gene expression in plantsCurr. Opin. Plant Biol6169177CrossRefPubMedGoogle Scholar
  42. Prandl, R., Kloske, E., Schöffl, F. 1995Developmental regulation and tissue-specific differences of heat shock gene expression in transgenic tobacco and Arabidopsis plantsPlant Mol. Biol287382CrossRefPubMedGoogle Scholar
  43. Rensing, S.A., Rombauts, S., Peer, Y., Reski, R. 2002Moss transcriptome and beyondTrends Plant Sci7535538CrossRefPubMedGoogle Scholar
  44. Reski, R. 1998Development, genetics and molecular biology of mossesBot. Acta111115Google Scholar
  45. Rieping, M., Schoffl, F. 1992Synergistic effect of upstream sequences, CCAAT box elements, and HSE sequences for enhanced expression of chimaeric heat shock genes in transgenic tobaccoMol. Gen. Genet231226232PubMedGoogle Scholar
  46. Rizhsky, L., Liang, H.J., Shuman, J., Shulaev, V., Davletova, S., Mittler, R. 2004When desense pathways collide. The response of Arabidopsis to a combination of drought and heat stressPlant Physiol13416831696CrossRefPubMedGoogle Scholar
  47. Roslan, H.A., Salter, M.G., Wood, C.D., White, M.R., Croft, K.P., Robson, F., Coupland, G., Doonan, J., Laufs, P., Tomsett, A.B., Caddick, M.X. 2001Characterisation of an ethanol-inducible alc gene-expression system in Arabidopsis thalianaPlant J28225235CrossRefPubMedGoogle Scholar
  48. Schaefer, D.G. 2002A new moss genetics: targeted mutagenesis in Physcomitrella patensAnnu. Rev. Plant Biol53477501CrossRefPubMedGoogle Scholar
  49. Schaefer, D.G., Zryd, J.P. 1997Efficient gene targeting in the moss Physcomitrella patensPlant J1111951206CrossRefPubMedGoogle Scholar
  50. Schaefer, D.G., Zryd, J.P. 2001The moss Physcomitrella patens, now and thenPlant Physiol12714301438CrossRefPubMedGoogle Scholar
  51. Schaefer, D.G., Zryd, J.P. 2004Principles of targeted mutagenesis in the moss Physcomitrella patensWood, A.J.Oliver, M.J.Cove, D. eds. New Frontiers in BryologyKluwer Academic PublishersDordrecht, Boston, London3749Google Scholar
  52. Schöffl, F., Raschke, E., Nagao, R.T. 1984The DNA sequence analysis of soybean heat-shock genes and identification of possible regulatory promoter elementsThe EMBO J324912497Google Scholar
  53. Schöffl, F., Rieping, M., Baumann, G., Bevan, M., Angermuller, S. 1989The function of plant heat shock promoter elements in the regulated expression of chimaeric genes in transgenic tobaccoMol. Gen. Genet217246253PubMedGoogle Scholar
  54. Schweizer, P., Pokorny, J., Abderhalden, O., Dudler, R. 1999A transient assay system for the functional assessment of defense-related genes in wheatMol. Plant Microbe Interact12647654Google Scholar
  55. Severin, K., Schoffl, F. 1990Heat-inducible hygromycin resistance in transgenic tobaccoPlant Mol. Biol15827833CrossRefPubMedGoogle Scholar
  56. Shigapova, N., Török, S., Balogh, G., Goloubinoff, P., Vigh, L., Horvath, I. 2005Membrane fluidization triggers membrane remodeling which affects the thermotolerance in Escherichia coliBiochem. Biophys. Res. Commun32812161223CrossRefPubMedGoogle Scholar
  57. Srivastava, A., Guissé, B., Greppin, H., Strasser, R.J. 1997Regulation of antenna structure and electron transport in photosystem II of Pisum sativum under elevated temperature probed by the fast polyphasic chlorophyll a fluorescence transient: OKJIPBiochemica et Biophysica acta132095106CrossRefGoogle Scholar
  58. Strasser, R.J., Srivastava, A., Govindjee,  1995Polyphasic chlorophyll a fluorescence transient in plants and cyanobacteriaPhytochem. photobiol613242Google Scholar
  59. Sun, W., Montagu, M., Verbruggen, N. 2002Small heat shock proteins and stress tolerance in plantsBiochim. Biophys. Acta157719PubMedGoogle Scholar
  60. Vierling, E., Kimpel, J.A. 1992Plant responses to environmental stressCurr. Opin. Biotechnol3164170CrossRefPubMedGoogle Scholar
  61. Vigh, L., Maresca, B., Harwood, J.L. 1998Does the membrane’s physical state control the expression of heat shock and other genes?Trends Biochem. Sci23369374CrossRefPubMedGoogle Scholar
  62. Yoshida, K., Kasai, T., Garcia, M.R., Sawada, S., Shoji, T., Shimizu, S., Yamazaki, K., Komeda, Y., Shinmyo, A. 1995Heat-inducible expression system for a foreign gene in cultured tobacco cells using the HSP18.2 promoter of Arabidopsis thalianaAppl. Microbiol. Biotechnol44466472CrossRefPubMedGoogle Scholar
  63. Zeidler, M., Gatz, C., Hartmann, E., Hughes, J. 1996Tetracycline-regulated reporter gene expression in the moss Physcomitrella patensPlant Mol. Biol30199205CrossRefPubMedGoogle Scholar
  64. Zeidler, M., Hartmann, E., Hughes, J. 1999Transgene expression in the moss Ceratodon purpureusJ. Plant Physiol154641650Google Scholar
  65. Zuo, J., Niu, Q.-W., Chua, N.-H. 2000An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plantsPlant J24265273CrossRefPubMedGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Younousse Saidi
    • 1
  • Andrija Finka
    • 1
  • Mickhail Chakhporanian
    • 1
  • Jean-Pierre Zrÿd
    • 1
  • Didier G. Schaefer
    • 1
  • Pierre Goloubinoff
    • 1
    Email author
  1. 1.Department of Plant Molecular BiologyLausanne UniversityLausanneSwitzerland

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