Simplified procedure for the isolation of intact chloroplasts from Chlamydomonas reinhardtii

A simple procedure that yields highly purified intact chloroplasts from Chlamydomonas reinhardtii is described. This procedure involves breakage of cell wall-deficient cells by passing them through a narrow bore syringe needle. The intact chloroplasts are then purified from the crude homogenate by d...

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Bibliographic Details
Published in:Plant physiology (Bethesda) 1991-12, Vol.97 (4), p.1576-1580
Main Authors: MASON, C. B, MATTHEWS, S, BRICKER, T. M, MORONEY, J. V
Format: Article
Language:English
Subjects:
Acetates
anatomia de la planta
anatomie vegetale
Biological and medical sciences
Carbon dioxide
Cell growth
Cell physiology
cell structure
Cell walls
Centrifugation
Chlamydomonas reinhardtii
chlorophyceae
Chlorophyta
chloroplaste
Chloroplasts
cloroplasto
Communications
Electron microscopy
Enzymes
estructura celular
Freshwater
Fundamental and applied biological sciences. Psychology
General aspects, methods
isolation techniques
Metabolism and Enzymology
plant anatomy
Plant cells
Plant physiology and development
Plants
structure cellulaire
technique de l' isolement
tecnicas de aislamiento
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Summary:A simple procedure that yields highly purified intact chloroplasts from Chlamydomonas reinhardtii is described. This procedure involves breakage of cell wall-deficient cells by passing them through a narrow bore syringe needle. The intact chloroplasts are then purified from the crude homogenate by differential centrifugation and Percoll gradient centrifugation. This procedure generates relatively high yields of chloroplasts capable of CO2 fixation. These chloroplasts were characterized by electron microscopy, marker enzyme analysis, and ferricyanide exclusion. Transmission electron microscopy indicates that these chloroplasts retain their pyrenoids and eyespots. Scanning electron microscopy confirms that the characteristic cup shape of C. reinhardtii chloroplasts persists in vitro. This rapid, inexpensive procedure produces chloroplasts that should be useful for researchers studying the biochemistry and cell biology of C. reinhardtii chloroplasts.
ISSN:0032-0889
1532-2548
DOI:10.1104/pp.97.4.1576