Skip to main content
SpringerLink
Log in

Rapid Isolation of Arabidopsis Chloroplasts and Their Use for In Vitro Protein Import Assays

  • Protocol
  • First Online:
Chloroplast Research in Arabidopsis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 774))

Abstract

In vitro chloroplast protein import assays have been performed since the late 1970s, initially with plant species (e.g., pea and spinach) that readily provide an abundant source of starting material and also, subsequently, a good yield of chloroplasts for import assays. However, the sequencing of the Arabidopsis genome paved the way for an additional model system that is more amenable to genetic analysis, as a complement to the more biochemically orientated models such as pea and spinach. A prerequisite for this change was an efficient and reliable protocol for the isolation of chloroplasts for use in protein import assays, enabling biochemical approaches to be combined with the genetic potential of the plant. The method described here was developed as a rapid and low-cost procedure that can be accessed by everyone due to its simplicity. Despite its rapidity and simplicity, the method yields highly pure chloroplasts, and in addition works well with mutant plants that exhibit pale or chlorotic phenotypes. The protocol is also optimized for work with material from young plants (10–14 days old), when protein import is believed to be at its peak, and so plant growth can be conducted in vitro on Murashige and Skoog medium. The isolation method has been used not only for protein import assays, but also for proteomic analysis and further subfractionation studies.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Blobel, G., and Sabatini, D. D. (1971) Ribosome-membrane interactions in eukaryotic cells. In, Biomembranes, Vol. 2 (Manson, L. A., ed.) Plenum, New York, USA, pp. 193–195.

    Google Scholar 

  2. Blobel, G., and Dobberstein, B. (1975) Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J. Cell Biol. 67, 835–851.

    Article  PubMed  CAS  Google Scholar 

  3. Blobel, G., and Dobberstein, B. (1975) Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J. Cell Biol. 67, 852–862.

    Article  PubMed  CAS  Google Scholar 

  4. Sandelius, A. S., and Aronsson, H. (2009) The Chloroplast - Interactions with the Environment. Plant Cell Monographs, Vol. 13. Springer-Verlag, Berlin, Germany.

    Google Scholar 

  5. Abdallah, F., Salamini, F., and Leister, D. (2000) A prediction of the size and evolutionary origin of the proteome of chloroplasts of Arabidopsis. Trends Plant Sci. 5, 141–142.

    Article  PubMed  CAS  Google Scholar 

  6. Miras, S., Salvi, D., Ferro, M., Grunwald, D., Garin, J., Joyard, J., and Rolland, N. (2002) Non-canonical transit peptide for import into the chloroplast. J. Biol. Chem. 277, 47770–47778.

    Article  PubMed  CAS  Google Scholar 

  7. Nada, A., and Soll, J. (2004) Inner envelope protein 32 is imported into chloroplasts by a novel pathway. J. Cell Sci. 117, 3975–3982.

    Article  PubMed  CAS  Google Scholar 

  8. Miras, S., Salvi, D., Piette, L., Seigneurin-Berny, D., Grunwald, D., Reinbothe, C., Joyard, J., Reinbothe, S., and Rolland, N. (2007) Toc159- and Toc75-independent import of a transit sequence-less precursor into the inner envelope of chloroplasts. J. Biol. Chem. 282, 29482–29492.

    Article  PubMed  CAS  Google Scholar 

  9. Villarejo, A., Buren, S., Larsson, S., Dejardin, A., Monne, M., Rudhe, C., Karlsson, J., Jansson, S., Lerouge, P., Rolland, N., von Heijne, G., Grebe, M., Bako, L., and Samuelsson, G. (2005) Evidence for a protein transported through the secretory pathway en route to the higher plant chloroplast. Nat. Cell Biol. 7, 1224–1231.

    Article  PubMed  Google Scholar 

  10. Nanjo, Y., Oka, H., Ikarashi, N., Kaneko, K., Kitajima, A., Mitsui, T., Munoz, F. J., Rodriguez-Lopez, M., Baroja-Fernandez, E., and Pozueta-Romero, J. (2006) Rice plastidial N-glycosylated nucleotide pyrophosphatase/phosphodiesterase is transported from the ER-golgi to the chloroplast through the secretory pathway. Plant Cell 18, 2582–2592.

    Article  PubMed  CAS  Google Scholar 

  11. Aronsson, H., and Jarvis, P. (2009) The chloroplast protein import apparatus, its components, and their roles. In, The Chloroplast - Interactions with the Environment (Sandelius, A. S., and Aronsson, H., eds.) Springer-Verlag, Berlin, Germany, pp. 89–124.

    Google Scholar 

  12. Chua, N. H., and Schmidt, G. W. (1978) Post-translational transport into intact chloroplasts of a precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase. Proc. Natl. Acad. Sci. USA 75, 6110–6114.

    Article  PubMed  CAS  Google Scholar 

  13. Highfield, P. E., and Ellis, R. J. (1978) Synthesis and transport of the small subunit of chloroplast ribulose bisphosphate carboxylase. Nature 271, 420–424.

    Article  CAS  Google Scholar 

  14. Aronsson, H., and Jarvis, P. (2002) A simple method for isolating import-competent Arabidopsis chloroplasts. FEBS Lett. 529, 215–220.

    Article  PubMed  CAS  Google Scholar 

  15. Boij, P., Patel, R., Garcia, C., Jarvis, P., and Aronsson, H. (2009) In vivo studies on the roles of Tic55-related proteins in chloroplast protein import in Arabidopsis thaliana. Mol. Plant 2, 1397–1409.

    Article  PubMed  CAS  Google Scholar 

  16. Jarvis, P., Chen, L. J., Li, H., Peto, C. A., Fankhauser, C., and Chory, J. (1998) An Arabidopsis mutant defective in the plastid general protein import apparatus. Science 282, 100–103.

    Article  PubMed  CAS  Google Scholar 

  17. Somerville, C. R., Somerville, S. C., and Ogren, W. L. (1981) Isolation of photosynthetically active protoplasts and chloroplasts from Arabidopsis thaliana. Plant Sci. Lett. 21, 89–96.

    Article  CAS  Google Scholar 

  18. Fitzpatrick, L. M., and Keegstra, K. (2001) A method for isolating a high yield of Arabidopsis chloroplasts capable of efficient import of precursor proteins. Plant J. 27, 59–65.

    Article  PubMed  CAS  Google Scholar 

  19. Dahlin, C., and Cline, K. (1991) Developmental regulation of the plastid protein import apparatus. Plant Cell 3, 1131–1140.

    Article  PubMed  CAS  Google Scholar 

  20. Kovacheva, S., Bedard, J., Patel, R., Dudley, P., Twell, D., Rios, G., Koncz, C., and Jarvis, P. (2005) In vivo studies on the roles of Tic110, Tic40 and Hsp93 during chloroplast protein import. Plant J. 41, 412–428.

    Article  PubMed  CAS  Google Scholar 

  21. Kovacheva, S., Bedard, J., Wardle, A., Patel, R., and Jarvis, P. (2007) Further in vivo studies on the role of the molecular chaperone, Hsp93, in plastid protein import. Plant J. 50, 364–379.

    Article  PubMed  CAS  Google Scholar 

  22. Kubis, S., Baldwin, A., Patel, R., Razzaq, A., Dupree, P., Lilley, K., Kurth, J., Leister, D., and Jarvis, P. (2003) The Arabidopsis ppi1 mutant is specifically defective in the expression, chloroplast import, and accumulation of photosynthetic proteins. Plant Cell 15, 1859–1871.

    Article  PubMed  CAS  Google Scholar 

  23. Kubis, S., Patel, R., Combe, J., Bedard, J., Kovacheva, S., Lilley, K., Biehl, A., Leister, D., Rios, G., Koncz, C., and Jarvis, P. (2004) Functional specialization amongst the Arabidopsis Toc159 family of chloroplast protein import receptors. Plant Cell 16, 2059–2077.

    Article  PubMed  CAS  Google Scholar 

  24. Sjögren, L. L., MacDonald, T. M., Sutinen, S., and Clarke, A. K. (2004) Inactivation of the clpC1 gene encoding a chloroplast Hsp100 molecular chaperone causes growth retardation, leaf chlorosis, lower photosynthetic activity, and a specific reduction in photosystem content. Plant Physiol 136, 4114–4126.

    Article  PubMed  Google Scholar 

  25. Aronsson, H., Boij, P., Patel, R., Wardle, A., Töpel, M., and Jarvis, P. (2007) Toc64/OEP64 is not essential for the efficient import of proteins into chloroplasts in Arabidopsis thaliana. Plant J. 52, 53–68.

    Article  PubMed  CAS  Google Scholar 

  26. Aronsson, H., Combe, J., Patel, R., Agne, B., Martin, M., Kessler, F., and Jarvis, P. (2010) Nucleotide binding and dimerization at the chloroplast pre-protein import receptor, atToc33, are not essential in vivo but do increase import efficiency. Plant J. 63, 297–311.

    Article  PubMed  CAS  Google Scholar 

  27. Kubis, S. E., Lilley, K. S., and Jarvis, P. (2008) Isolation and preparation of chloroplasts from Arabidopsis thaliana plants. Methods Mol. Biol. 425, 171–486.

    Article  PubMed  Google Scholar 

  28. Garcia, C., Khan, N. Z., Nannmark, U., and Aronsson, H. (2010) The chloroplast protein CPSAR1, dually localized in the stroma and the inner envelope membrane, is involved in thylakoid biogenesis. Plant J. 63, 73–85.

    PubMed  CAS  Google Scholar 

  29. Park, S., and Rodermel, S. R. (2004) Mutations in ClpC2/Hsp100 suppress the requirement for FtsH in thylakoid membrane biogenesis. Proc. Natl. Acad. Sci. USA 101, 12765–12770.

    Article  PubMed  CAS  Google Scholar 

  30. Sinvany-Villalobo, G., Davydov, O., Ben-Ari, G., Zaltsman, A., Raskind, A., and Adam, Z. (2004) Expression in multigene families. Analysis of chloroplast and mitochondrial proteases. Plant Physiol. 135, 1336–1345.

    CAS  Google Scholar 

  31. Koo, A. J., Fulda, M., Browse, J., and Ohlrogge, J. B. (2005) Identification of a plastid acyl-acyl carrier protein synthetase in Arabidopsis and its role in the activation and elongation of exogenous fatty acids. Plant J. 44, 620–632.

    Article  PubMed  CAS  Google Scholar 

  32. Flores-Perez, U., Sauret-Gueto, S., Gas, E., Jarvis, P., and Rodriguez-Concepcion, M. (2008) A mutant impaired in the production of plastome-encoded proteins uncovers a mechanism for the homeostasis of isoprenoid biosynthetic enzymes in Arabidopsis plastids. Plant Cell 20, 1303–1315.

    Article  PubMed  CAS  Google Scholar 

  33. Aronsson, H., Schottler, M. A., Kelly, A. A., Sundqvist, C., Dörmann, P., Karim, S., and Jarvis, P. (2008) Monogalactosyldiacylglycerol deficiency in Arabidopsis affects pigment composition in the prolamellar body and impairs thylakoid membrane energization and photoprotection in leaves. Plant Physiol. 148, 580–592.

    Article  PubMed  CAS  Google Scholar 

  34. Kozak, M. (2002) Pushing the limits of the scanning mechanism for initiation of translation. Gene 299, 1–34.

    Article  PubMed  CAS  Google Scholar 

  35. Robinson, S. P. (1985) Osmotic adjustment by intact isolated chloroplasts in response to osmotic stress and its effect on photosynthesis and chloroplast volume. Plant Physiol. 79, 996–1002.

    Article  PubMed  CAS  Google Scholar 

  36. Rathnam, C. K. M., and Edwards, G. E. (1976) Protoplasts as a tool for isolating functional chloroplasts from leaves. Plant Cell Physiol. 17, 177–186.

    CAS  Google Scholar 

  37. Sirevag, R., Buchanan, B. B., Berry, J. A., and Troughton, J. H. (1977) Mechanisms of CO2 fixation in bacterial photosynthesis studied by the carbon isotope fractionation technique. Arch. Microbiol. 112, 35–38.

    Article  PubMed  CAS  Google Scholar 

  38. Shaul, O. (2002) Magnesium transport and function in plants: the tip of the iceberg. Biometals 15, 309–323.

    Article  PubMed  CAS  Google Scholar 

  39. Neuhaus, H. E., and Wagner, R. (2000) Solute pores, ion channels, and metabolite transporters in the outer and inner envelope membranes of higher plant plastids. Biochim. Biophys. Acta 1465, 307–323.

    Article  PubMed  CAS  Google Scholar 

  40. Schulz, A., Knoetzel, J., Scheller, H. V., and Mant, A. (2004) Uptake of a fluorescent dye as a swift and simple indicator of organelle intactness: import-competent chloroplasts from soil-grown Arabidopsis. J. Histochem. Cytochem. 52, 701–704.

    Article  PubMed  CAS  Google Scholar 

  41. Cowan, J. A. (2002) Structural and catalytic chemistry of magnesium-dependent enzymes. Biometals 15, 225–235.

    Article  PubMed  CAS  Google Scholar 

  42. Melis, A., and Homann, P. H. (1978) A selective effect of Mg2+ on the photochemistry at one type of reaction center in photosystem II of chloroplasts. Arch Biochem Biophys 190, 523–530.

    Article  PubMed  CAS  Google Scholar 

  43. Porra, R. J., Thompson, W. A., and Kriedemann, P. E. (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta 975, 384–394.

    Article  CAS  Google Scholar 

  44. Heimpel, S., Basset, G., Odoy, S., and Klingenberg, M. (2001) Expression of the mitochondrial ADP/ATP carrier in Escherichia coli. Renaturation, reconstitution, and the effect of mutations on 10 positive residues. J. Biol. Chem. 276, 11499–11506.

    Article  PubMed  CAS  Google Scholar 

  45. Chinopoulos, C., Vajda, S., Csanady, L., Mandi, M., Mathe, K., and Adam-Vizi, V. (2009) A novel kinetic assay of mitochondrial ATP-ADP exchange rate mediated by the ANT. Biophys. J. 96, 2490–2504.

    Article  PubMed  CAS  Google Scholar 

  46. Morgenthaler, J. J., Marsden, M. P., and Price, C. A. (1975) Factors affecting the separation of photosynthetically competent chloroplasts in gradients of silica sols. Arch. Biochem. Biophys. 168, 289–301.

    Article  PubMed  CAS  Google Scholar 

  47. Zemell, R., Burstein, Y., and Schechter, I. (1978) Initiator methionine residues at the NH2-termini of the two precursors of MOPC-41 immunoglobulin light chain. Studies with the initiator and internal tRNAMet species. Eur. J. Biochem. 89, 187–193.

    CAS  Google Scholar 

  48. Pain, D., and Blobel, G. (1987) Protein import into chloroplasts requires a chloroplast ATPase. Proc. Natl. Acad. Sci. USA 84, 3288–3292.

    Article  PubMed  CAS  Google Scholar 

  49. Theg, S. M., Bauerle, C., Olsen, L. J., Selman, B. R., and Keegstra, K. (1989) Internal ATP is the only energy requirement for the translocation of precursor proteins across chloroplastic membranes. J. Biol. Chem. 264, 6730–6736.

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

Thanks to Dr. Sazzad Karim and Nadir Zaman Khan for valuable comments on the manuscript. This work was supported by the Swedish Research Council VR (H.A.), and the Royal Society and the Biotechnology and Biological Sciences Research Council (BBSRC) (P.J.).

Author information

Authors and Affiliations

  1. Department of Plant and Environmental Sciences, University of Gothenburg, Gothenburg, Sweden

    Henrik Aronsson

  2. Department of Biology, University of Leicester, Leicester, UK

    R. Paul Jarvis

Authors
  1. Henrik Aronsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R. Paul Jarvis
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Henrik Aronsson .

Editor information

Editors and Affiliations

  1. , Department of Biology, University of Leicester, University Road, Leicester, LE1 7RH, United Kingdom

    R. Paul Jarvis

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Aronsson, H., Jarvis, R.P. (2011). Rapid Isolation of Arabidopsis Chloroplasts and Their Use for In Vitro Protein Import Assays. In: Jarvis, R. (eds) Chloroplast Research in Arabidopsis. Methods in Molecular Biology, vol 774. Humana Press. https://doi.org/10.1007/978-1-61779-234-2_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-234-2_17

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-233-5

  • Online ISBN: 978-1-61779-234-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation