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A rapid method for chloroplast isolation from the green alga Chlamydomonas reinhardtii

Nature Protocols volume 1, pages 2227–2230 (2006)Cite this article

Abstract

This method has been developed to yield highly purified intact chloroplasts from Chlamydomonas reinhardtii. This procedure involves breaking cell-wall-deficient cells by passage through a narrow-bore syringe needle and purifying the intact chloroplasts by differential centrifugation and Percoll gradient centrifugation. This procedure can be completed in less than 3 h and is capable of generating relatively high yields of chloroplasts that should be useful for researchers studying the biochemistry and cell biology of C. reinhardtii chloroplasts.

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Figure 1: Pictures of an unbroken cell and a field of intact chloroplasts.
Figure 2: Transmission electron micrographs showing

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References

  1. Harris, E.H. The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use (Academic Press, San Diego, 1989).

    Google Scholar 

  2. Spreitzer, R.J., Al-Abed, S.R. & Huether, M.J. Temperature sensitive photosynthesis-deficient mutants of Chlamydomonas reinhardtii. Plant Physiol. 86, 773–777 (1988).

    Article  CAS  Google Scholar 

  3. Sultemeyer, D.F., Klock, G., Kreuzberg, K. & Fock, H. Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations. Planta 176, 256–260 (1988).

    Article  CAS  Google Scholar 

  4. Klein, U., Chen, C., Gibbs, M. & Platt-Aloia, K.A. Cellular fractionation of Chlamydomonas reinhardtii with emphasis on the isolation of the chloroplast. Plant Physiol. 72, 481–487 (1983).

    Article  CAS  Google Scholar 

  5. Klein, U., Chen, C. & Gibbs, M. Photosynthetic properties of chloroplasts from Chlamydomonas reinhardtii. Plant Physiol. 72, 488–491 (1983).

    Article  CAS  Google Scholar 

  6. Göhre, V., Ossenbühl, F., Crèvecoeur, M., Eichacker, L.A. & Rochaix, J.D. One of two alb3 proteins is essential for the assembly of the photosystems and for cell survival in Chlamydomonas. Plant Cell 18, 1454–1466 (2006).

    Article  Google Scholar 

  7. Togasaki, R.K., Brunke, K., Kitayama, M. & Griffith, M. Isolation of intact chloroplasts from Chlamydomonas reinhardtii with the Beckman centrifugal elutriation system. in Progress in Photosynthesis Research Vol. 3 (ed. Biggins, J.) pp 499–505 (Martinus Nijhoff, Dordrecht, 1987).

    Chapter  Google Scholar 

  8. Mendiola-Morgenthaler, L., Leu, S. & Boschetti, A. Isolation of biochemically active chloroplasts from Chlamydomonas. Plant Sci. 38, 33–39 (1985).

    Article  CAS  Google Scholar 

  9. Mendiola-Morgenthaler, L., Eichenberger, W. & Boschetti, A. Isolation of chloroplast envelopes from Chlamydomonas. Lipid and polypeptide composition. Plant Sci. 41, 97–104 (1985).

    Article  CAS  Google Scholar 

  10. Moroney, J.V., Kitayama, M., Togasaki, R.K. & Tolbert, N.E. Evidence for inorganic carbon transport by intact chloroplasts of Chlamydomonas reinhardtii. Plant Physiol. 83, 460–463 (1987).

    Article  CAS  Google Scholar 

  11. Mason, C.B., Manuel, L.J. & Moroney, J.V. A new chloroplast protein is required for growth on low CO2 in Chlamydomonas reinhardtii. Plant Physiol. 93, 833–836 (1990).

    Article  CAS  Google Scholar 

  12. Goldschmidt-Clermont, M., Malone, P. & Rochaix, J.D. Preparation of Chlamydomonas chloroplasts for the in vitro impact of polypeptide precursors. Plant Physiol. 89, 15–18 (1989).

    Article  CAS  Google Scholar 

  13. Price, C.A. & Reardon, E.M. Isolation of chloroplasts for protein synthesis from spinach and Euglena gracilis by centrifugation in silica sols. in Methods in Chloroplast Molecular Biology (eds. Edelman. M., Hallick, R.B. & Chua, N.H.) pp 189–209 (Elsevier, Amsterdam, 1982).

    Google Scholar 

  14. Mason, C.B., Matthews, S., Bricker, T.M. & Moroney, J.V. Simplified procedure for the isolation of intact chloroplasts from Chlamydomonas reinhardtii. Plant Physiol. 97, 1576–1580 (1991).

    Article  CAS  Google Scholar 

  15. Lilley, R.Mc.C., Fitzgerald, M.P., Rienits, K.G. & Walker, D.A. Criteria of intactness and the photosynthetic activity of spinach chloroplast preparations. New Phytol. 75, 1–10 (1975).

    Article  CAS  Google Scholar 

  16. Chen, Z.Y., Lavigne, L.L., Mason, C.B. & Moroney, J.V. Cloning and overexpression of two cDNAs encoding the low-CO2-inducible chloroplast envelope protein LIP-36 from Chlamydomonas reinhardtii. Plant Physiol. 114, 265–273 (1997).

    Article  CAS  Google Scholar 

  17. Rexach, J., Fernandez, E. & Galvan, A. The Chlamydomonas reinhartii NAR1 encodes a chloroplast membrane protein involved in nitrite transport. Plant Cell 12, 1441–1453 (2000).

    Article  CAS  Google Scholar 

  18. Chen, H.C. & Melis, A. Localization and function of SulP, a nuclear-encoded sulfate permease in Chlamydomonas reinhardtii. Planta 220, 198–210 (2004).

    Article  CAS  Google Scholar 

  19. Blouin, A., Lavezzi, T. & Moore, T.S. Membrane lipid biosynthesis in Chlamydomonas reinhardtii. Partial characterization of CDP-diacylgycerol: myo-inositol 3-phosphatidyltransferase. Plant Physiol. Biochem. 41, 11–16 (2003).

    Article  CAS  Google Scholar 

  20. Jabusch, T.W. & Swackhamer, D.L. Subcellular accumulation of polychlorinated biphenyls in the green alga Chlamydomonas reinhardtii. Environ. Toxicol. Chem. 23, 2823–2830 (2004).

    Article  Google Scholar 

  21. Sueoka, N. Mitotic replication of deoxyribonucleic acids in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 46, 83–91 (1960).

    Article  CAS  Google Scholar 

  22. Lane, M.D., Maruyama, H. & Easterday, R.L. Phosphoenolpyruvate carboxylase from peanut cotyledons. Methods Enzymol. 13, 277–283 (1969).

    Article  CAS  Google Scholar 

  23. Raphael, J. Cytochrome c oxidase. in Methods of Enzymatic Analysis Vol. 3 (ed. Bergmeyer, HU) 266–273 (Verlag Chemie, VCH Publishers, Wienheim, 1987).

    Google Scholar 

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Acknowledgements

This work was supported by awards from the National Science Foundation to JVM and TMB and a grant from the Department of Energy to TMB.

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Authors and Affiliations

  1. Department of Biological Sciences, Louisiana State University, Baton Rouge, 70803, Louisiana, USA

    Catherine B Mason, Terry M Bricker & James V Moroney

Authors
  1. Catherine B Mason
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  2. Terry M Bricker
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  3. James V Moroney
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Correspondence to James V Moroney.

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Mason, C., Bricker, T. & Moroney, J. A rapid method for chloroplast isolation from the green alga Chlamydomonas reinhardtii. Nat Protoc 1, 2227–2230 (2006). https://doi.org/10.1038/nprot.2006.348

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