Complete Mitochondrial Complex I Deficiency Induces an Up-Regulation of Respiratory Fluxes That Is Abolished by Traces of Functional Complex I

Abstract

Complex I (NADH:ubiquinone oxidoreductase) is central to cellular NAD + recycling and accounts for approximately 40% of mitochondrial ATP production. To understand how complex I function impacts respiration and plant development, we isolated Arabidopsis (Arabidopsis thaliana) lines that lack complex I activity due to the absence of the catalytic subunit NDUFV1 (for NADH:ubiquinone oxidoreductase flavoprotein1) and compared these plants with ndufs4 (for NADH:ubiquinone oxidoreductase Fe-S protein4) 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 and ATP synthesis 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 tricarboxylic acid cycle were dramatically increased compared with ndufs4, which showed near wild-type-like fluxes. This indicates that the strong growth defects seen for plants lacking complex I originate from a switch in the metabolic mode of mitochondria and 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.In most eukaryotic organisms, energy is mainly provided by cellular respiration, which is composed of three main pathways. Glycolysis in the cytosol, and additionally in the plastids of plants, degrades sugars into pyruvate. The tricarboxylic acid (TCA) cycle, which largely resides in the mitochondrial matrix, further dissimilates pyruvate into CO 2 . These two pathways generate reduced cofactors, mostly NADH. The oxidative phosphorylation (OXPHOS) system couples cofactor recycling with ATP production. The electron transfer chain (ETC) located in the mitochondrial inner membrane (IM) uses the redox energy of the reduced cofactors to create an electrochemical gradient across the IM. This gradient is then used by the ATP synthase to convert ADP to ATP, which is subsequently exported from the mitochondria to fuel cellular metabolism and sustain housekeeping functions and growth.The ETC is composed of four large multiprotein complexes. The first of these complexes is the NADHubiquinone oxidoreductase, also called complex I. It plays a crucial role in recycling NAD + for the TCA cycle, and its activity is responsible for about 40% of the total proton pumping across the IM (Wikström, 1984;Galkin et al., 2006). In plants, additional NADH dehydrogenases located on both sides of the IM are present (for review, see Rasmusson et al., 2008). These enzymes can recycle NAD + for use in glycolysis and the TCA cyc...

Photoperiod Affects the Phenotype of Mitochondrial Complex I Mutants

“…In the N. sylvestris mutants, limited carbon supply resulting from higher photorespiration is likely to contribute to the slowgrowth phenotype (Priault et al, 2006b). Although similar metabolic alterations were observed in N. sylvestris (Dutilleul et al, 2005;Szal et al, 2008;Djebbar et al, 2012) and Arabidopsis CI mutants (Meyer et al, 2009;Keren et al, 2012) as increased levels of amino acids, ATP, and NAD(H), noteworthy differences were reported recently between Arabidopsis mutants (Kühn et al, 2015). Moreover, an increase in total reactive oxygen species (ROS) content was reported for many Arabidopsis CI mutants (Meyer et al, 2009;Keren et al, 2012;Soto et al, 2015) but not in the CMSII mutant (Dutilleul et al, 2003b).…”

“…Plant CI mutants have various phenotypes, from mild to severe or even near lethal (Kühn et al, 2015); in addition to a slow-growing phenotype, morphological distortions and male sterility were reported in maize NCS2 (Karpova and Newton, 1999) and in N. sylvestris NMS1 and CMSII (De Paepe et al, 1990). Seedlings of Arabidopsis nMat1/4 (Keren et al, 2012;Cohen et al, 2014) and ndufv1 (Kühn et al, 2015) mutants barely survive unless grown in vitro with Suc supplementation.…”

“…Seedlings of Arabidopsis nMat1/4 (Keren et al, 2012;Cohen et al, 2014) and ndufv1 (Kühn et al, 2015) mutants barely survive unless grown in vitro with Suc supplementation. Differential impact of the mutations also was observed at the physiological and metabolic levels.…”

“…Mutations in genes controlling NAD cis/trans-processing have been described in N. sylvestris, maize, and Arabidopsis (Arabidopsis thaliana; for review, see Colas des Francs-Small and Small, 2014), and insertion mutants lacking nucleus-encoded subunits have been characterized in Arabidopsis (Meyer et al, 2009;Kühn et al, 2015;Soto et al, 2015). Impaired holocomplex assembly/activity are common features of all plant mutants characterized so far, but putative intermediate assembly forms have been observed in several cases (Gutierres et al, 1997;Karpova and Newton, 1999;Perales et al, 2005;Pineau et al, 2008;Meyer et al, 2009Meyer et al, , 2011Kühn et al, 2015). A general respiratory impairment, measured as either oxygen consumption of leaf discs or CO 2 emission by attached leaves in the dark (dark respiration), has not been observed, and in organello experiments showed the induction of nonphosphorylating NAD(P)H dehydrogenases (Rasmusson et al, 2008) in all cases analyzed (Marienfeld and Newton, 1994;Sabar et al, 2000;Meyer et al, 2009;Keren et al, 2012).…”

“…Mutants devoid of NAD subunits were characterized in maize (Zea mays; ncs2, deleted for NAD4; Marienfeld and Newton, 1994) and in Nicotiana sylvestris (CMSI and CMSII, deleted for NAD7; Pla et al, 1995). Mutations in genes controlling NAD cis/trans-processing have been described in N. sylvestris, maize, and Arabidopsis (Arabidopsis thaliana; for review, see Colas des Francs-Small and Small, 2014), and insertion mutants lacking nucleus-encoded subunits have been characterized in Arabidopsis (Meyer et al, 2009;Kühn et al, 2015;Soto et al, 2015). Impaired holocomplex assembly/activity are common features of all plant mutants characterized so far, but putative intermediate assembly forms have been observed in several cases (Gutierres et al, 1997;Karpova and Newton, 1999;Perales et al, 2005;Pineau et al, 2008;Meyer et al, 2009Meyer et al, , 2011Kühn et al, 2015).…”