In vivo chlorophyll fluorescence screening allows the isolation of a Chlamydomonas mutant defective for NDUFAF3, an assembly factor involved in mitochondrial complex I assembly
[en] The qualitative screening method to select complex I mutants in the microalga Chlamydomonas, based on reduced growth under heterotrophic condition, is not suited for high throughput screening. In order to develop a fast screening method based on measurements of chlorophyll fluorescence, we first demonstrated that complex I mutants displayed decreased photosystem II efficiency in the genetic background of a photosynthetic mutation leading to reduced formation of the electrochemical proton gradient in the chloroplast (pgrl1 mutation). In contrast, single mutants (complex I and pgrl1 mutants) could not be distinguished from wild type by their photosystem II efficiency in the tested conditions. We next performed an insertional mutagenesis on the pgrl1 mutant. Out of ~3000 hygromycin-resistant insertional transformants, 46 had decreased photosystem II efficiency and three were complex I mutants. One of the mutants was tagged and whole genome sequencing identified the resistance cassette in NDUFAF3, a homolog of the human NDUFAF3 gene, encoding for an assembly factor involved in complex I assembly. Complemented strains showed restored complex I activity and assembly. Overall, we described here a screening method which is fast and particularly suited for identification of Chlamydomonas complex I mutants.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Massoz, Simon; Université de Liège - ULiège > Département des sciences de la vie > Génétique et physiologie des microalgues
Hanikenne, Marc ; Université de Liège - ULiège > Département des sciences de la vie > Génomique fonctionnelle et imagerie moléculaire végétale
Bailleul, Benjamin; Université Pierre et Marie Currie - Paris 6 - UPMC > Institut de Biologie Physico-Chimique (IBPC)
Coosemans, Nadine ; Université de Liège - ULiège > Département des sciences de la vie > Génétique et physiologie des microalgues
Radoux, Michèle ; Université de Liège - ULiège > Département des sciences de la vie > Génétique et physiologie des microalgues
Miranda Astudillo, Héctor Vicente ; Université de Liège - ULiège > Département des sciences de la vie > Génétique et physiologie des microalgues
Cardol, Pierre ; Université de Liège - ULiège > Département des sciences de la vie > Génétique et physiologie des microalgues
Remacle, Claire ; Université de Liège - ULiège > Département des sciences de la vie > Génétique et physiologie des microalgues
Language :
English
Title :
In vivo chlorophyll fluorescence screening allows the isolation of a Chlamydomonas mutant defective for NDUFAF3, an assembly factor involved in mitochondrial complex I assembly
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