Disruption of a Nuclear Gene Encoding a Mitochondrial Gamma Carbonic Anhydrase Reduces Complex I and Supercomplex I+III2 Levels and Alters Mitochondrial Physiology in Arabidopsis

Institut für Angewandte Genetik, Universität Hannover, Herrenhäuser Str. 2, D-30419 Hannover, Germany.
Journal of Molecular Biology (Impact Factor: 4.33). 08/2005; 350(2):263-77. DOI: 10.1016/j.jmb.2005.04.062
Source: PubMed


Mitochondrial NADH dehydrogenase (complex I) of plants includes quite a number of plant-specific subunits, some of which exhibit sequence similarity to bacterial gamma-carbonic anhydrases. A homozygous Arabidopsis knockout mutant carrying a T-DNA insertion in a gene encoding one of these subunits (At1g47260) was generated to investigate its physiological role. Isolation of mitochondria and separation of mitochondrial protein complexes by Blue-native polyacrylamide gel electrophoresis or sucrose gradient ultracentrifugation revealed drastically reduced complex I levels. Furthermore, the mitochondrial I + III2 supercomplex was very much reduced in mutant plants. Remaining complex I had normal molecular mass, suggesting substitution of the At1g47260 protein by one or several of the structurally related subunits of this respiratory protein complex. Immune-blotting experiments using polyclonal antibodies directed against the At1g47260 protein indicated its presence within complex I, the I + III2 supercomplex and smaller protein complexes, which possibly represent subcomplexes of complex I. Changes within the mitochondrial proteome of mutant cells were systematically monitored by fluorescence difference gel electrophoresis using 2D Blue-native/SDS and 2D isoelectric focussing/SDS polyacrylamide gel electrophoresis. Complex I subunits are largely absent within the mitochondrial proteome. Further mitochondrial proteins are reduced in mutant plants, like mitochondrial ferredoxin, others are increased, like formate dehydrogenase. Development of mutant plants was normal under standard growth conditions. However, a suspension cell culture generated from mutant plants exhibited clearly reduced growth rates and respiration. In summary, At1g47260 is important for complex I assembly in plant mitochondria and respiration. A role of At1g47260 in mitochondrial one-carbon metabolism is supported by micro-array analyses.

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    • "The primers used are listed in Table S3. Western blotting was performed as previously described using anti-CA2 antiserum (Perales et al., 2005). "
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    ABSTRACT: The NADH-ubiquinone oxidoreductase complex (Complex I -CI- EC is the main entrance site of electrons into the respiratory chain. In a variety of eukaryotic organisms, except animal and fungi (Opisthokonta), it contains an extra domain composed of trimers of putative gamma carbonic anhydrases, named CA domain, which has been proposed to be essential for assembly of the complex I. However, its physiological role in plants is not fully understood. In this work, we report that Arabidopsis mutants defective in two CA subunits show a photorespiratory phenotype. Corresponding mutants grown in ambient air show growth retardation compared to wild type plants, a feature that is reverted by cultivating plants in a high carbon dioxide atmosphere. Moreover, under photorespiratory conditions, carbon assimilation is diminished and glycine accumulates, suggesting an imbalance with respect to photorespiration. Additionally, transcript levels of specific CA subunits are reduced in plants grown under non-photorespiratory conditions. Taken together, these results suggest that the CA domain of plant complex I contribute to sustain efficient photosynthesis at ambient (photorespiratory) conditions. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    The Plant Journal 07/2015; 83(5). DOI:10.1111/tpj.12930 · 5.97 Impact Factor
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    • "For immunodetection, separated proteins were transferred onto polyvinylidene difluoride membranes (Immobilon-P, Millipore) and incubated with the primary antibodies. The primary antibodies anti-CA (Perales et al., 2005), anti-51-kD (Peters et al., 2012), and anti-AtpB (AS05085, Agrisera) were used at a dilution of 1:5,000. Secondary antibodies linked to horseradish peroxidase were used to detect the signal by chemiluminescence using the ECL Prime Detection Kit (GE Healthcare). "
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    ABSTRACT: Complex I is central to cellular NAD+ recycling and accounts for 40% of mitochondrial ATP production. To understand how complex I function impacts on plant development, we isolated Arabidopsis thaliana lines that lack complex I activity due to the absence of the catalytic subunit NDUFV1 and compared these plants with ndufs4 mutants possessing trace amounts of complex I. Unlike ndufs4 plants, ndufv1 lines were largely unable to establish seedlings in the absence of externally supplied sucrose. Measurements of mitochondrial respiration revealed that compared with ndufv1, the complex I amounts retained by ndufs4 did not increase mitochondrial respiration and oxidative phosphorylation capacities. No major differences were seen in the mitochondrial proteomes, cellular metabolomes or transcriptomes between ndufv1 and ndufs4. The analysis of fluxes through the respiratory pathway revealed that in ndufv1, fluxes through glycolysis and the TCA cycle were dramatically increased compared with ndufs4. This indicates that the strong growth defects seen for plants lacking complex I originate from an up-regulation of respiratory fluxes. Partial reversion of these phenotypes when traces of active complex I are present suggests that complex I is essential for plant development and likely acts as a negative regulator of respiratory fluxes. Copyright © 2015, Plant Physiology.
    Plant physiology 07/2015; 168(4). DOI:10.1104/pp.15.00589 · 6.84 Impact Factor
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    • "The T-DNA insertion mutations of Arabidopsis genes encoding the gCA subunits have been reported previously, but none of those mutants showed visible phenotypic alterations, making it difficult to directly test the physiological function of the gCA subcomplex (Perales et al., 2005). For example, mutants impaired in the gCA2 or gCA3 genes exhibited morphologic phenotypes indistinguishable from that of the wild-type plants (Perales et al., 2005). A suspension culture derived from the gca2 mutant showed clearly reduced growth rate and respiration, but it remains unclear whether such a defect is dependent on light and how the phenotype of the suspension culture is directly related to specific aspects of the development of whole plants. "
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    ABSTRACT: Complex I (NADH:ubiquinone oxidoreductase) is the entry point for electrons into the respiratory electron transport chain, and it therefore plays a central role in cellular energy metabolism. Complex I from different organisms has a similar basic structure. However, an extra structural module, referred to as the γ-carbonic anhydrase (γCA) subcomplex, is found in the mitochondrial complex I of photoautotrophic eukaryotes, such as green alga and plants, but not in that of the heterotrophic eukaryotes, such as fungi and mammals. It has been proposed that the γCA subcomplex is required for light-dependent life style of photoautotrophic eukaryotes, but this hypothesis has not been successfully tested. We report here a genetic study of the genes, γCAL1 and γCAL2, that encode two subunits of the γCA subcomplex of mitochondrial complex I. We found that mutations of the γCAL1 and γCAL2 in Arabidopsis result in defective embryogenesis and non-germinating seeds, demonstrating the functional significance of the γCA subcomplex of mitochondrial complex I in plant development. Surprisingly, we also found that reduced expression of γCAL1 and γCAL2 genes altered photomorphogenic development. The γcal1 mutant plant expressing the RNAi construct of the γCAL2 gene showed a partial cop (constitutive photomorphogenic) phenotype in young seedlings, and a reduced photoperiodic sensitivity in adult plants. The involvement of γCA subcomplex of mitochondrial complex I in plant photomorphogenesis and the possible evolutionary significance of this plant-specific mitochondrial protein complex is discussed.
    Plant physiology 09/2012; 160(3). DOI:10.1104/pp.112.204339 · 6.84 Impact Factor
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