Directed chloroplast transformation in Chlamydomonas reinhardtii: insertional inactivation of the psaC gene encoding the iron sulfur protein destabilizes photosystem I

Article Article scientifique Article Directed chloroplast transformation in Chlamydomonas reinhardtii: insertional inactivation of the psaC gene encoding the iron sulfur protein destabilizes photosystem I eng 1991 author Takahashi Yuichiro 24543 author Goldschmidt-Clermont Michel P. 25594 author Soen Su Yin Bianca Maria 22957 author Franzen Lars Gunnar 39535 author Rochaix Jean-David 25289 2033-2040 10.1002/j.1460-2075.1991.tb07733.x 1712288 0261-4189 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 Southern blot analysis of total cell DNA of the transformants shows that the wild type psaC gene has been replaced by the interrupted psaC gene through homologous recombination.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. Amino acid sequence Base sequence Northern blotting Southern blotting Chlamydomonas/genetics Chloroplasts DNA/genetics DNA transposable elements Polyacrylamide gel electrophoresis Genes Iron-slfur proteins/genetics Kinetics Membrane proteins Molecular sequence data Phenotype Photosynthetic reaction center complex proteins/metabolism Photosystem I protein complex Proteins/genetics RNA/genetics Nucleic acid sequence homology Fluorescence spectrometry Genetic transformation Dewey 570 Dewey 580 Swiss National Science Foundation 31.26345.89 CH 43 Département de biologie moléculaire 161 45 Département des sciences végétales 163 false EMBO Journal 10 8 77b2bc51-c28e-433c-8ba8-e7019c849671 Article (Published version) Restricted emboj00106-0077.pdf 1789954 application/pdf restrictedAccess Faculté des sciences / Section de biologie / Département de biologie moléculaire Faculté des sciences / Section de biologie / Département des sciences végétales

Title	Published in	Year	Views	Downloads

Characterization of Chlorophyll a/b Proteins of Photosystem I from Chlamydomonas reinhardtii	Journal of Biological Chemistry	1992	168	117
Directed chloroplast transformation in Chlamydomonas reinhardtii: insertional inactivation of the psaC gene encoding the iron sulfur protein destabilizes photosystem I	EMBO Journal	1991	357	0

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Soen, Su Yin Bianca Maria