Targeting the NAD7 subunit to mitochondria restores a functional complex I and a wild type phenotype in the Nicotiana sylvestris CMS II mutant lacking nad7.
The mitochondrial DNA of the Nicotiana sylvestris CMSII mutant carries a 72-kb deletion comprising the single copy nad7 gene that encodes the NAD7 subunit of the respiratory complex I (NADH-ubiquinone oxidoreductase). CMSII plants lack rotenone-sensitive complex I activity and are impaired in physiological and phenotypical traits. To check whether these changes directly result from the deletion of nad7, we constructed CMS transgenic plants (termed as CMSnad7) carrying an edited nad7 cDNA fused to the CAMV 35S promoter and to a mitochondrial targeting sequence. The nad7 sequence was transcribed and translated and the NAD7 protein directed to mitochondria in CMSnad7 transgenic plants, which recovered both wild type morphology and growth features. Blue-native/SDS gel electrophoresis and enzymatic assays showed that, whereas fully assembled complex I was absent from CMSII mitochondria, a functional complex was present in CMSnad7 mitochondria. Furthermore, a supercomplex involving complex I and complex III was present in CMSnad7 as in the wild type. Taken together, these data demonstrate that lack of complex I in CMSII was indeed the direct consequence of the absence of nad7. Hence, NAD7 is a key element for complex assembly in plants. These results also show that allotopic expression from the nucleus can fully complement the lack of a mitochondrial-encoded complex I gene.
Pineau
Bernard
B
Institut de Biotechnologie des Plantes, Laboratoire Mitochondries et Métabolisme Centre National de la Recherche Scientifique-Université Paris-Sud, Unite Mixte de Recherche 8618, 91405 Orsay, France.
Mathieu
Chantal
C
Gérard-Hirne
Catherine
C
De Paepe
Rosine
R
Chétrit
Philippe
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000378:metabolism; D042967:Electron Transport Complex I / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005560:Formate Dehydrogenases / Q000378:metabolism; D017353:Gene Deletion; D007425:Intracellular Membranes / Q000378:metabolism; D013058:Mass Spectrometry; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D009243:NAD / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D010940:Plant Proteins / Q000235:genetics* / Q000502:physiology*; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012402:Rotenone / Q000494:pharmacology; D011198:Solanum tuberosum / Q000378:metabolism; D014026:Nicotiana / Q000378:metabolism
D018076:DNA, Complementary; D004272:DNA, Mitochondrial; D024101:Mitochondrial Proteins; D010455:Peptides; D010940:Plant Proteins; D012402:Rotenone; D009243:NAD; D012313:RNA; D005560:Formate Dehydrogenases; D042967:Electron Transport Complex I