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In vivo chlorophyll fluorescence screening allows the isolation of a Chlamydomonas mutant defective for NDUFAF 3, an assembly factor involved in mitochondrial complex I assembly
Author(s) -
Massoz Simon,
Hanikenne Marc,
Bailleul Benjamin,
Coosemans Nadine,
Radoux Michèle,
MirandaAstudillo Hector,
Cardol Pierre,
Larosa Véronique,
Remacle Claire
Publication year - 2017
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.13677
Subject(s) - chlamydomonas , mutant , fluorescence , microbiology and biotechnology , isolation (microbiology) , in vivo , chemistry , biology , biochemistry , genetics , gene , physics , quantum mechanics
SUMMARY The qualitative screening method used to select complex I mutants in the microalga Chlamydomonas , based on reduced growth under heterotrophic conditions, is not suitable 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 the wild type by their photosystem II efficiency under the conditions tested. We next performed insertional mutagenesis on the pgrl1 mutant. Out of about 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 NDUFAF 3 , a homolog of the human NDUFAF 3 gene, encoding for an assembly factor involved in complex I assembly. Complemented strains showed restored complex I activity and assembly. Overall, we describe here a screening method which is fast and particularly suited for the identification of Chlamydomonas complex I mutants.