Directed chloroplast transformation in Chlamydomonas reinhardtii: insertional inactivation of the psaC gene encoding the iron sulfur protein destabilizes photosystem I.
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 Southern blot analysis of total cell DNA of the transformants shows that the wild type psaC gene has been replaced by the interrupted psaC gene through homologous recombination.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.
Takahashi
Y
Y
Department of Molecular Biology, University of Geneva, Switzerland.
Goldschmidt-Clermont
M
M
Soen
S Y
SY
Franzén
L G
LG
Rochaix
J D
JD
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002696:Chlamydomonas / Q000235:genetics*; D002736:Chloroplasts; D004247:DNA / Q000235:genetics; D004251:DNA Transposable Elements; D004591:Electrophoresis, Polyacrylamide Gel; D005796:Genes; D007506:Iron-Sulfur Proteins / Q000235:genetics*; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D010641:Phenotype; D045322:Photosynthetic Reaction Center Complex Proteins / Q000378:metabolism*; D045331:Photosystem I Protein Complex; D011506:Proteins / Q000235:genetics*; D012313:RNA / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013050:Spectrometry, Fluorescence; D014170:Transformation, Genetic
D004251:DNA Transposable Elements; D007506:Iron-Sulfur Proteins; D008565:Membrane Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D011506:Proteins; C089909:photosystem I, psaB subunit; D012313:RNA; D004247:DNA