, Volume 250, Issue 5, pp 1731–1741 | Cite as

Isolation of plastids and mitochondria from Chromera velia

  • Abdoallah SharafEmail author
  • Zoltán Füssy
  • Aleš Tomčala
  • Jitka Richtová
  • Miroslav OborníkEmail author
Original Article


Main conclusion

We present an easy and effective procedure to purify plastids and mitochondria from Chromera velia. Our method enables downstream analyses of protein and metabolite content of the organelles.


Chromerids are alveolate algae that are the closest known phototrophic relatives to apicomplexan parasites such as Plasmodium or Toxoplasma. While genomic and transcriptomic resources for chromerids are in place, tools and experimental conditions for proteomic studies have not been developed yet. Here we describe a rapid and efficient protocol for simultaneous isolation of plastids and mitochondria from the chromerid alga Chromera velia. This procedure involves enzymatic treatment and breakage of cells, followed by differential centrifugation. While plastids sediment in the first centrifugation step, mitochondria remain in the supernatant. Subsequently, plastids can be purified from the crude pellet by centrifugation on a discontinuous 60%/70% sucrose density gradient, while mitochondria can be obtained by centrifugation on a discontinuous 33%/80% Percoll density gradient. Isolated plastids are autofluorescent, and their multi-membrane structure was confirmed by transmission electron microscopy. Fluorescent optical microscopy was used to identify isolated mitochondria stained with MitoTrackerTM green, while their intactness and membrane potential were confirmed by staining with MitoTrackerTM orange CMTMRos. Total proteins were extracted from isolated organellar fractions, and the purity of isolated organelles was confirmed using immunoblotting. Antibodies against the beta subunit of the mitochondrial ATP synthase and the plastid protochlorophyllide oxidoreductase did not cross-react on immunoblots, suggesting that each organellar fraction is free of the residues of the other. The presented protocol represents an essential step for further proteomic, organellar, and cell biological studies of C. velia and can be employed, with minor optimizations, in other thick-walled unicellular algae.


Chromerids Isolation Microalgae Mitochondrion Plastid 



Breaking buffer


Protochlorophyllide oxidoreductase


Storage buffer



This work was supported by the Czech Science Foundation (15-17643S and 16-24027S) and ERDF/ESF Centre for research of pathogenicity and virulence of parasites (No.CZ.02.1.01/0.0/0.0/16_019/0000759). Antibodies were kindly provided by Dr. Alena Panicucci-Zíková (anti-β-ATPase) and Dr. Roman Sobotka (anti-POR). Moreover, the authors would like to thank Dr. Ansgar Gruber for his helpful advice during the electron microscopy samples preparation.Also, the laboratory of electron microscopy in Biology Centre CAS, supported by the MEYS CR (LM2015062 Czech- BioImaging) for providing the electron microscopy facilities.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

425_2019_3259_MOESM1_ESM.pdf (79 kb)
Supplementary material 1 (PDF 78 kb)


  1. Angelova A, Park SH, Kyndt J et al (2014) Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for Illumina genome sequencing. J Appl Phycol 26:209–218. CrossRefGoogle Scholar
  2. Botte CY, Yamaryo-Botte Y, Rupasinghe TWT et al (2013) A typical lipid composition in the purified relict plastid (apicoplast) of malaria parasites. Proc Natl Acad Sci USA 110:7506–7511. CrossRefPubMedGoogle Scholar
  3. Calvayrac R, Laval-Martin D, Briand J, Farineau J (1981) Paramylon synthesis by Euglena gracilis photoheterotrophically grown under low O2 pressure. Description of a mitochloroplast complex. Planta 153:6–13. CrossRefPubMedGoogle Scholar
  4. Choo K, Tan T, Ranganathan S (2009) A comprehensive assessment of N-terminal signal peptides prediction methods. BMC Bioinform 10:S2. CrossRefGoogle Scholar
  5. Flegontov P, Michálek J, Janouškovec J et al (2015) Divergent mitochondrial respiratory chains in phototrophic relatives of apicomplexan parasites. Mol Biol Evol 32:1115–1131. CrossRefPubMedGoogle Scholar
  6. Füssy Z, Oborník M (2017) Chromerids and their plastids. Adv Bot Res 84:187–218CrossRefGoogle Scholar
  7. Füssy Z, Faitová T, Oborník M (2019) Subcellular compartments interplay for carbon and nitrogen allocation in Chromera velia and Vitrella brassicaformis. Genome Biol Evol 11(7):1765–1779. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Gilmore K, Wilson M (1999) The use of chloromethyl-X-rosamine (mitotracker red) to measure loss of mitochondrial membrane potential in apoptotic cells is incompatible with cell fixation. Cytometry 36:355–358.;2-9 CrossRefPubMedGoogle Scholar
  9. Goodman CD, Pasaje CFA, Kennedy K et al (2016) Targeting protein translation in organelles of the Apicomplexa. Trends Parasitol 32:953–965CrossRefGoogle Scholar
  10. Gruber A, Rocap G, Kroth PG et al (2015) Plastid proteome prediction for diatoms and other algae with secondary plastids of the red lineage. Plant J 81:519–528. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Hikosaka K, Kita K, Tanabe K (2013) Diversity of mitochondrial genome structure in the phylum apicomplexa. Mol Biochem Parasitol 188:26–33. CrossRefPubMedGoogle Scholar
  12. Hopkins J, Fowler R, Krishna S et al (1999) The plastid in Plasmodium falciparum asexual blood stages: a three-dimensional ultrastructural analysis. Protist 150:283–295. CrossRefPubMedGoogle Scholar
  13. Islam MS, Takagi S (2010) Co-localization of mitochondria with chloroplasts is a light-dependent reversible response. Plant Signal Behav 5:146–147. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Janouškovec J, Horák A, Oborník M, Lukeš J, Keeling PJ (2010) A common red algal origin of the apicomplexan, dinoflagellate, and heterokont plastids. Proc Natl Acad Sci USA 107:10949–10954. CrossRefPubMedGoogle Scholar
  15. Janouškovec J, Horák A, Barott KL et al (2012) Global analysis of plastid diversity reveals apicomplexan-related lineages in coral reefs. Curr Biol. CrossRefPubMedGoogle Scholar
  16. Janouškovec J, Horák A, Barott KL et al (2013) Environmental distribution of coral-associated relatives of apicomplexan parasites. ISME J 7:444–447. CrossRefPubMedGoogle Scholar
  17. Janouškovec J, Tikhonenkov DV, Burki F et al (2015) Factors mediating plastid dependency and the origins of parasitism in apicomplexans and their close relatives. Proc Natl Acad Sci USA 112:10200–10207. CrossRefPubMedGoogle Scholar
  18. Kopečná J, Sobotka R, Komenda J (2013) Inhibition of chlorophyll biosynthesis at the protochlorophyllide reduction step results in the parallel depletion of photosystem I and photosystem II in the cyanobacterium Synechocystis PCC 6803. Planta 237:497–508. CrossRefPubMedGoogle Scholar
  19. Lang EGE, Mueller SJ, Hoernstein SNW et al (2011) Simultaneous isolation of pure and intact chloroplasts and mitochondria from moss as the basis for sub-cellular proteomics. Plant Cell Rep 30:205–215. CrossRefPubMedGoogle Scholar
  20. Logan DC, Millar AH, Sweetlove LJ, Hill SA, Leaver CJ (2001) Mitochondrial biogenesis during germination in maize embryos. Plant Physiol 125:662–672. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Mason CB, Bricker TM, Moroney JV (2006) A rapid method for chloroplast isolation from the green alga Chlamydomonas reinhardtii. Nat Protoc 1:2227–2230. CrossRefPubMedGoogle Scholar
  22. Métivier D, Dallaporta B, Zamzami N et al (1998) Cytofluorometric detection of mitochondrial alterations in early CD95/Fas/APO-1-triggered apoptosis of Jurkat T lymphoma cells. Comparison of seven mitochondrion-specific fluorochromes. Immunol Lett 61:157–163. CrossRefPubMedGoogle Scholar
  23. Molloy MP, Herbert BR, Walsh BJ et al (1998) Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis 19:837–844. CrossRefPubMedGoogle Scholar
  24. Moore RB, Oborník M, Janouškovec J et al (2008) A photosynthetic alveolate closely related to apicomplexan parasites. Nature 452:959–963. CrossRefGoogle Scholar
  25. Moreno-Rojas JM, Moreno-Ortega A, Ordóñez JL et al (2018) Development and validation of UHPLC-HRMS methodology for the determination of flavonoids, amino acids and organosulfur compounds in black onion, a novel derived product from fresh shallot onions (Allium cepa var. aggregatum). LWT 97:376–383. CrossRefGoogle Scholar
  26. Nash EA, Nisbet RER, Barbrook AC, Howe CJ (2008) Dinoflagellates: a mitochondrial genome all at sea. Trends Genet 24:328–335CrossRefGoogle Scholar
  27. Oborník M, Lukeš J (2015) The organellar genomes of Chromera and Vitrella, the phototrophic relatives of apicomplexan parasites. Annu Rev Microbiol 69:129–144. CrossRefPubMedGoogle Scholar
  28. Oborník M, Janouškovec J, Chrudimský T, Lukeš J (2009) Evolution of the apicoplast and its hosts: from heterotrophy to autotrophy and back again. Int J Parasitol 39:1–12CrossRefGoogle Scholar
  29. Oborník M, Vancová M, Lai DH et al (2011) Morphology and ultrastructure of multiple life cycle stages of the photosynthetic relative of apicomplexa, Chromera velia. Protist 162:115–130. CrossRefPubMedGoogle Scholar
  30. Oborník M, Modrý D, Lukeš M et al (2012) Morphology, ultrastructure and life cycle of Vitrella brassicaformis n. sp., n. gen., a novel chromerid from the great barrier reef. Protist 163:306–323. CrossRefPubMedGoogle Scholar
  31. Pan H, Šlapeta J, Carter D, Chen M (2013) Isolation of complete chloroplasts from Chromera Velia—the photosynthetic relative of parasitic apicomplexa. Photosynthesis research for food, fuel and the future. Advanced topics in science and technology in China. Springer, Berlin, pp 436–439CrossRefGoogle Scholar
  32. Pietruszka M, Lewicka S (2007) Effect of temperature on plant elongation and cell wall extensibility. Gen Physiol Biophys 26:40–47PubMedGoogle Scholar
  33. Poot M, Zhang YZ, Krämer JA et al (1996) Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. J Histochem Cytochem 44:1363–1372. CrossRefPubMedGoogle Scholar
  34. Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208–212. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Satori CP, Kostal V, Arriaga EA (2012) Review on recent advances in the analysis of isolated organelles. Anal Chim Acta 753:8–18CrossRefGoogle Scholar
  36. Schober AF, Río Bártulos C, Bischoff A et al (2019) Organelle studies and proteome analyses on mitochondria and plastids fractions from the diatom Thalassiosira pseudonana. Plant Cell Physiol. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Segui-Simarro JM, Coronado MJ, Staehelin LA (2008) The mitochondrial cycle of Arabidopsis shoot apical meristem and leaf primordium meristematic cells Is defined by a perinuclear tentaculate/cage-like mitochondrion. Plant Physiol 148:1380–1393. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Slamovits CH, Saldarriaga JF, Larocque A, Keeling PJ (2007) The highly reduced and fragmented mitochondrial genome of the early-branching dinoflagellate Oxyrrhis marina shares characteristics with both Apicomplexan and Dinoflagellate mitochondrial genomes. J Mol Biol 372:356–368. CrossRefPubMedGoogle Scholar
  39. Sobotka R, Esson HJ, Koník P et al (2017) Extensive gain and loss of photosystem i subunits in chromerid algae, photosynthetic relatives of apicomplexans. Sci Rep 7:13214. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Sperschneider J, Catanzariti AM, Deboer K et al (2017) LOCALIZER: subcellular localization prediction of both plant and effector proteins in the plant cell. Sci Rep 7:44598. CrossRefPubMedPubMedCentralGoogle Scholar
  41. Šubrtová K, Panicucci B, Zíková A (2015) ATPaseTb2, a unique membrane-bound FoF1-ATPase component, is essential in bloodstream and dyskinetoplastic trypanosomes. PLoS Pathog 11:e1004660. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Takishita K, Yamaguchi H, Maruyama T, Inagaki Y (2009) A hypothesis for the evolution of nuclear-encoded, plastid-targeted glyceraldehyde-3-phosphate dehydrogenase genes in “chromalveolate” members. PLoS One 4:e4737. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Tomčala A, Kyselová V, Schneedorferová I et al (2017) Separation and identification of lipids in the photosynthetic cousins of apicomplexa Chromera velia and Vitrella brassicaformis. J Sep Sci 40:3402–3413. CrossRefPubMedGoogle Scholar
  44. Vazač J, Füssy Z, Hladová I et al (2018) Ploidy and number of chromosomes in the alveolate alga Chromera velia. Protist 169:53–63. CrossRefPubMedGoogle Scholar
  45. Waller RF, Jackson CJ (2009) Dinoflagellate mitochondrial genomes: stretching the rules of molecular biology. Bioessays 31:237–245. CrossRefPubMedGoogle Scholar
  46. Waller RF, Kořený L (2017) Plastid complexity in dinoflagellates: a picture of gains, losses, replacements and revisions. Adv Bot Res 84:105–143CrossRefGoogle Scholar
  47. Woo YH, Ansari H, Otto TD et al (2015) Chromerid genomes reveal the evolutionary path from photosynthetic algae to obligate intracellular parasites. Elife 4:1–41. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of ParasitologyBiology Centre CASČeské BudějoviceCzech Republic
  2. 2.Genetic Department, Faculty of AgricultureAin Shams UniversityCairoEgypt
  3. 3.Faculty of ScienceUniversity of South BohemiaČeské BudějoviceCzech Republic

Personalised recommendations