Abstract
The so-called “complex” plastids from diatoms possessing four envelope membranes are a typical feature of algae that arose from secondary endosymbiosis. Studying isolated plastids from these algae may allow answering a number of fundamental questions regarding diatom photosynthesis and plastid functionality. Due to their complex architecture and their integration into the cellular endoplasmic reticulum (ER) system, their isolation though is still challenging. In this work, we report a reliable isolation technique that is applicable for the two model diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum. The resulting plastid-enriched fractions are of homogenous quality, almost free from cellular contaminants, and feature structurally intact thylakoids that are capable to perform oxygenic photosynthesis, though in most cases they seem to lack most of the stromal components as well as plastid envelopes.
Key words
- Diatom organelles
- Thylakoids
- Complex plastid fractionation
- Chloroplast
This is a preview of subscription content, access via your institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Walker DA (1964) Improved rates of carbon dioxide fixation by illuminated chloroplasts. Biochem J 92(3):22C–23C. https://doi.org/10.1042/bj0920022C
Jensen RG, Bassham JA (1966) Photosynthesis by isolated chloroplasts. Proc Natl Acad Sci U S A 56(4):1095–1101
Stankovic ZS, Walker DA (1977) Photosynthesis by isolated pea chloroplasts: some effects of adenylates and inorganic pyrophosphate. Plant Physiol 59(3):428–432. https://doi.org/10.1104/pp.59.3.428
Lyttleton JW (1962) Isolation of ribosomes from spinach chloroplasts. Exp Cell Res 26(2):312–317. https://doi.org/10.1016/0014-4827(62)90183-0
Edwards GE, Robinson SP, Tyler NJ et al (1978) Photosynthesis by isolated protoplasts, protoplast extracts, and chloroplasts of wheat: influence of orthophosphate, pyrophosphate, and adenylates. Plant Physiol 62(2):313–319. https://doi.org/10.1104/pp.62.2.313
Pollock SV, Colombo SL, Prout DL Jr et al (2003) Rubisco activase is required for optimal photosynthesis in the green alga Chlamydomonas reinhardtii in a low-CO(2) atmosphere. Plant Physiol 133(4):1854–1861. https://doi.org/10.1104/pp.103.032078
Schnell RA, Lefebvre PA (1993) Isolation of the Chlamydomonas regulatory gene NIT2 by transposon tagging. Genetics 134(3):737–747
Mendiola-Morgenthaler L, Leu S, Boschetti A (1985) Isolation of biochemically active chloroplasts from chlamydomonas. Plant Sci 38(1):33–39. https://doi.org/10.1016/0168-9452(85)90076-7
Mason CB, Bricker TM, Moroney JV (2006) A rapid method for chloroplast isolation from the green alga Chlamydomonas reinhardtii. Nat Protocols 1(5):2227–2230. https://doi.org/10.1038/nprot.2006.348
Round FE, Crawford RM, Mann DG (2007) Diatoms: biology and morphology of the genera. Plastids. Cambridge University Press, Cambridge, p 59
Wittpoth C, Kroth PG, Weyrauch K et al (1998) Functional characterization of isolated plastids from two marine diatoms. Planta 206(1):79–85. https://doi.org/10.1007/s004250050376
Guillard RL (1975) Culture of phytoplankton for feeding marine invertebrates. In: Smith W, Chanley M (eds) Culture of marine invertebrate animals. Springer, New York, pp 29–60. https://doi.org/10.1007/978-1-4615-8714-9_3
Flori S, Jouneau P-H, Bailleul B et al (2017) Plastid thylakoid architecture optimizes photosynthesis in diatoms. Nat Commun 8:15885. https://doi.org/10.1038/ncomms15885
Acknowledgments
We would like to thank Doris Ballert for the help with the cultivation of T. pseudonana and P. tricornutum and the BioImaging Center at the University of Konstanz for the assistance during fluorescence microscopy. This work was supported by a stipend of the Graduate School Chemical Biology (KoRS-CB) and by the University of Konstanz to AFS. CRB further wishes to acknowledge the German Research Foundation (DFG, grant KR 1661/6-1) and the Gordon and Betty Moore Foundation GBMF 4966 (grant DiaEdit). SF and GF acknowledge funds from the Marie Curie Initial Training Network Accliphot (FP7-PEOPLE-2012-ITN, 316427) and from the DRF impulsion FIB-Bio program.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Schober, A.F., Flori, S., Finazzi, G., Kroth, P.G., Bártulos, C.R. (2018). Isolation of Plastid Fractions from the Diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum. In: Maréchal, E. (eds) Plastids. Methods in Molecular Biology, vol 1829. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8654-5_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8654-5_13
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8653-8
Online ISBN: 978-1-4939-8654-5
eBook Packages: Springer Protocols