Directed chloroplast transformation in Chlamydomonas reinhardtii

Insertional inactivation of the psaC gene encoding the iron sulfur protein destabilizes photosystem 1

Yuichiro Takahashi, M. Goldschmidt-Clermont, S. Y. Soen, L. G. Franzén, J. D. Rochaix

Research output: Contribution to journalArticle

150 Citations (Scopus)

Abstract

The chloroplast gene psaC encoding the iron sulfur protein of photosystem I (PSI) from the green alga Chlamydomonas reinhardtii has been cloned and characterized. The deduced amino acid sequence is highly related to that of higher plants and cyanobacteria. Using a particle gun, wild type C. reinhardtii cells have been transformed with a plasmid carrying the psaC gene disrupted by an aadA gene cassette designed to express spectinomycin/streptomycin resistance in the chloroplast. Transformants selected on plates containing acetate as a reduced carbon source and spectinomycin are unable to grow on minimal medium lacking acetate and are deficient in PSI activity. Southern blot analysis of total cell DNA of the transformants shows that the wild type psaC gene has been replaced by the interrupted psaC gene through homologous recombination. While authentic transcripts of the psaC gene are no longer detected, aadA gives rise to a few transcripts in the transformants. Biochemical analysis indicates that neither PSI reaction center subunits nor the seven small subunits belonging to PSI accumulate stably in the thylakoid membranes of the transformants. Pulse-chase labeling of cell proteins shows that the PSI reaction center subunits are synthesized normally but turn over rapidly in the transformants. We conclude that the iron sulfur binding protein encoded by the psaC gene is an essential component, both for photochemical activity and for stable assembly of PSI. The present study suggests that any chloroplast gene encoding a component of the photosynthetic apparatus can be disrupted in C. reinhardtii using the strategy described.

Original languageEnglish
Pages (from-to)2033-2040
Number of pages8
JournalEMBO Journal
Volume10
Issue number8
Publication statusPublished - 1991
Externally publishedYes

Fingerprint

Iron-Sulfur Proteins
Photosystem I Protein Complex
Chlamydomonas reinhardtii
Gene encoding
Gene Silencing
Chloroplasts
Genes
Chloroplast Genes
Spectinomycin
Acetates
Iron-Binding Proteins
Chlorophyta
Thylakoids
Homologous Recombination
Essential Genes
Firearms
Cyanobacteria
Streptomycin
Algae
Southern Blotting

Keywords

  • Chlamydomonas reinhardtii
  • Chloroplast transformation
  • Photosystem I
  • PsaC gene

ASJC Scopus subject areas

  • Cell Biology
  • Genetics

Cite this

Directed chloroplast transformation in Chlamydomonas reinhardtii : Insertional inactivation of the psaC gene encoding the iron sulfur protein destabilizes photosystem 1. / Takahashi, Yuichiro; Goldschmidt-Clermont, M.; Soen, S. Y.; Franzén, L. G.; Rochaix, J. D.

In: EMBO Journal, Vol. 10, No. 8, 1991, p. 2033-2040.

Research output: Contribution to journalArticle

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