EARLY NODULIN93 acts via cytochrome c oxidase to alter respiratory ATP production and root growth in plants.

EARLY NODULIN 93 (ENOD93) has been genetically associated with biological nitrogen fixation in legumes and nitrogen use efficiency in cereals, but its precise function is unknown. We show that hidden Markov models define ENOD93 as a homolog of the N-terminal domain of RESPIRATORY SUPERCOMPLEX FACTOR 2 (RCF2). RCF2 regulates cytochrome oxidase (CIV), influencing the generation of a mitochondrial proton motive force in yeast (Saccharomyces cerevisiae).

Mitochondrial atp1 mRNA knockdown by a custom-designed pentatricopeptide repeat protein alters ATP synthase.

Spontaneous mutations are rare in mitochondria and the lack of mitochondrial transformation methods has hindered genetic analyses. We show that a custom-designed RNA-binding pentatricopeptide repeat (PPR) protein binds and specifically induces cleavage of ATP synthase subunit1 (atp1) mRNA in mitochondria, significantly decreasing the abundance of the Atp1 protein and the assembled F1Fo ATP synthase in Arabidopsis (Arabidopsis thaliana). The transformed plants are characterized by delayed vegetative growth and reduced fertility.

Growth Developmental Defects of Mitochondrial Iron Transporter 1 and 2 Mutants in Arabidopsis in Iron Sufficient Conditions.

Iron is the most abundant micronutrient in plant mitochondria, and it has a crucial role in biochemical reactions involving electron transfer. It has been described in that () is an essential gene and that knockdown mutant rice plants have a decreased amount of iron in their mitochondria, strongly suggesting that OsMIT is involved in mitochondrial iron uptake. In , two genes encode MIT homologues.

Defects in autophagy lead to selective in vivo changes in turnover of cytosolic and organelle proteins in Arabidopsis.

Identification of autophagic protein cargo in plants in autophagy-related genes (ATG) mutants is complicated by changes in protein synthesis and protein degradation. To detect autophagic cargo, we measured protein degradation rate in shoots and roots of Arabidopsis (Arabidopsis thaliana) atg5 and atg11 mutants. These data show that less than a quarter of proteins changing in abundance are probable cargo and revealed roles of ATG11 and ATG5 in degradation of specific glycolytic enzymes and of other cytosol, chloroplast, and ER-resident proteins, and a specialized role for ATG11 in degradation of proteins from mitochondria and chloroplasts.

Metabolic evidence for distinct pyruvate pools inside plant mitochondria.

The majority of the pyruvate inside plant mitochondria is either transported into the matrix from the cytosol via the mitochondria pyruvate carrier (MPC) or synthesized in the matrix by alanine aminotransferase (AlaAT) or NAD-malic enzyme (NAD-ME). Pyruvate from these origins could mix into a single pool in the matrix and contribute indistinguishably to respiration via the pyruvate dehydrogenase complex (PDC), or these molecules could maintain a degree of independence in metabolic regulation. Here we demonstrate that feeding isolated mitochondria with uniformly labelled C-pyruvate and unlabelled malate enables the assessment of pyruvate contribution from different sources to intermediate production in the tricarboxylic acid cycle.

Enzymes degraded under high light maintain proteostasis by transcriptional regulation in .

Photoinhibitory high light stress in Arabidopsis leads to increases in markers of protein degradation and transcriptional up-regulation of proteases and proteolytic machinery, but proteostasis is largely maintained. We find significant increases in the in vivo degradation rate for specific molecular chaperones, nitrate reductase, glyceraldehyde-3 phosphate dehydrogenase, and phosphoglycerate kinase and other plastid, mitochondrial, peroxisomal, and cytosolic enzymes involved in redox shuttles. Coupled analysis of protein degradation rates, mRNA levels, and protein abundance reveal that 57% of the nuclear-encoded enzymes with higher degradation rates also had high light–induced transcriptional responses to maintain proteostasis.

Assessing the Kinetics of Metabolite Uptake and Utilization by Isolated Mitochondria Using Selective Reaction Monitoring Mass Spectrometry (SRM-MS).

Transport of tricarboxylic acid (TCA) cycle substrates across mitochondrial membranes and their subsequent oxidative decarboxylation in the matrix provide reductants for respiration-coupled ATP synthesis. These processes are typically assessed together through the ability of mitochondria to consume oxygen or release carbon dioxide, however, this approach fails to assess or separate the complexity of transport and the subsequent metabolism of substrates and products. In this chapter, we provide a strategy for simultaneously measuring substrate transport and utilization by isolated mitochondria using a mass spectrometry-based technique.

The mitochondrial pyruvate carrier (MPC) complex mediates one of three pyruvate-supplying pathways that sustain Arabidopsis respiratory metabolism.

Malate oxidation by plant mitochondria enables the generation of both oxaloacetate and pyruvate for tricarboxylic acid (TCA) cycle function, potentially eliminating the need for pyruvate transport into mitochondria in plants. Here, we show that the absence of the mitochondrial pyruvate carrier 1 (MPC1) causes the co-commitment loss of its putative orthologs, MPC3/MPC4, and eliminates pyruvate transport into Arabidopsis thaliana mitochondria, proving it is essential for MPC complex function. While the loss of either MPC or mitochondrial pyruvate-generating NAD-malic enzyme (NAD-ME) did not cause vegetative phenotypes, the lack of both reduced plant growth and caused an increase in cellular pyruvate levels, indicating a block in respiratory metabolism, and elevated the levels of branched-chain amino acids at night, a sign of alterative substrate provision for respiration.

Increased expression of ANAC017 primes for accelerated senescence.

Recent studies in Arabidopsis (Arabidopsis thaliana) have reported conflicting roles for NAC DOMAIN CONTAINING PROTEIN 17 (ANAC017), a transcription factor regulating mitochondria-to-nuclear signaling, and its closest paralog NAC DOMAIN CONTAINING PROTEIN 16 (ANAC016), in leaf senescence. By synchronizing senescence in individually darkened leaves of knockout and overexpressing mutants from these contrasting studies, we demonstrate that elevated ANAC017 expression consistently causes accelerated senescence and cell death. A time-resolved transcriptome analysis revealed that senescence-associated pathways such as autophagy are not constitutively activated in ANAC017 overexpression lines, but require a senescence-stimulus to trigger accelerated induction.

Metabolite Regulatory Interactions Control Plant Respiratory Metabolism via Target of Rapamycin (TOR) Kinase Activation.

Respiration rate measurements provide an important readout of energy expenditure and mitochondrial activity in plant cells during the night. As plants inhabit a changing environment, regulatory mechanisms must ensure that respiratory metabolism rapidly and effectively adjusts to the metabolic and environmental conditions of the cell. Using a high-throughput approach, we have directly identified specific metabolites that exert transcriptional, translational, and posttranslational control over the nighttime O consumption rate (R) in mature leaves of Arabidopsis ().

Variation in Leaf Respiration Rates at Night Correlates with Carbohydrate and Amino Acid Supply.

Plant respiration can theoretically be fueled by and dependent upon an array of central metabolism components; however, which ones are responsible for the quantitative variation found in respiratory rates is unknown. Here, large-scale screens revealed 2-fold variation in nighttime leaf respiration rate (R) among mature leaves from an Arabidopsis () natural accession collection grown under common favorable conditions. R variation was mostly maintained in the absence of genetic variation, which emphasized the low heritability of R and its plasticity toward relatively small environmental differences within the sampling regime.

Nitrite Protects Mitochondrial Structure and Function under Hypoxia.

Oxygen deprivation leads to changes in mitochondrial morphology and impaired flow of reducing equivalents through the electron transport chain. The extent of these changes depends on the duration and severity of the treatment as well as on the species and cell type. Nitrate is known to ameliorate these effects in some instances, but it is possible that it is nitrite, rather than nitrate, that is the key to the mechanism.

MSL1 is a mechanosensitive ion channel that dissipates mitochondrial membrane potential and maintains redox homeostasis in mitochondria during abiotic stress.

Mitochondria must maintain tight control over the electrochemical gradient across their inner membrane to allow ATP synthesis while maintaining a redox-balanced electron transport chain and avoiding excessive reactive oxygen species production. However, there is a scarcity of knowledge about the ion transporters in the inner mitochondrial membrane that contribute to control of membrane potential. We show that loss of MSL1, a member of a family of mechanosensitive ion channels related to the bacterial channel MscS, leads to increased membrane potential of Arabidopsis mitochondria under specific bioenergetic states.

The Plant Mitochondrial Transportome: Balancing Metabolic Demands with Energetic Constraints.

In plants, mitochondrial function is associated with hundreds of metabolic reactions. To facilitate these reactions, charged substrates and cofactors move across the charge-impermeable inner mitochondrial membrane via specialized transporters and must work cooperatively with the electrochemical gradient which is essential for mitochondrial function. The regulatory framework for mitochondrial metabolite transport is expected to be more complex in plants than in mammals owing to the close metabolic association between mitochondrial, plastids, and peroxisome metabolism, as well as to the major diurnal fluctuations in plant metabolic function.

Activity assay for plant mitochondrial enzymes.

Mitochondria are sites for respiration to produce chemical energy via oxidative phosphorylation. Their primary role has been viewed as the oxidation of organic acids via the tricarboxylic acid (TCA) cycle and the synthesis of ATP coupled to the transfer of electrons to O2. TCA cycle enzymes are essential for plant carbon metabolism and provide the reductant for the electron transport chain (ETC) enzymes that in turn drives ATP synthesis.

An Arabidopsis stomatin-like protein affects mitochondrial respiratory supercomplex organization.

Stomatins belong to the band-7 protein family, a diverse group of conserved eukaryotic and prokaryotic membrane proteins involved in the formation of large protein complexes as protein-lipid scaffolds. The Arabidopsis (Arabidopsis thaliana) genome contains two paralogous genes encoding stomatin-like proteins (SLPs; AtSLP1 and AtSLP2) that are phylogenetically related to human SLP2, a protein involved in mitochondrial fusion and protein complex formation in the mitochondrial inner membrane. We used reverse genetics in combination with biochemical methods to investigate the function of AtSLPs.

Evidence for several cysteine transport mechanisms in the mitochondrial membranes of Arabidopsis thaliana.

Cysteine is essential for many mitochondrial processes in plants, including translation, iron-sulfur cluster biogenesis and cyanide detoxification. Its biosynthesis is carried out by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) which can be found in the cytosol, plastids and mitochondria. Mutants lacking one compartment-specific OAS-TL isoform show viable phenotypes, leading to the hypothesis that the organellar membranes are permeable to substrates and products of the cysteine biosynthetic pathway.

Recent advances in the composition and heterogeneity of the Arabidopsis mitochondrial proteome.

Mitochondria are important organelles for providing the ATP and carbon skeletons required to sustain cell growth. While these organelles also participate in other key metabolic functions across species, they have a specialized role in plants of optimizing photosynthesis through participating in photorespiration. It is therefore critical to map the protein composition of mitochondria in plants to gain a better understanding of their regulation and define the uniqueness of their metabolic networks.

Mitochondrial composition, function and stress response in plants.

The primary function of mitochondria is respiration, where catabolism of substrates is coupled to ATP synthesis via oxidative phosphorylation. In plants, mitochondrial composition is relatively complex and flexible and has specific pathways to support photosynthetic processes in illuminated leaves. This review begins with outlining current models of mitochondrial composition in plant cells, with an emphasis upon the assembly of the complexes of the classical electron transport chain (ETC).

Mitochondrial cysteine synthase complex regulates O-acetylserine biosynthesis in plants.

Cysteine synthesis is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) in the cytosol, plastids, and mitochondria of plants. Biochemical analyses of recombinant plant SAT and OAS-TL indicate that the reversible association of the proteins in the cysteine synthase complex (CSC) controls cellular sulfur homeostasis. However, the relevance of CSC formation in each compartment for flux control of cysteine synthesis remains controversial.

Mitochondrial proteome heterogeneity between tissues from the vegetative and reproductive stages of Arabidopsis thaliana development.

Specialization of the mitochondrial proteome in Arabidopsis has the potential to underlie the roles of these organelles at different developmental time points and in specific organs; however, most research to date has been limited to studies of mitochondrial composition from a few vegetative tissue types. To provide further insight into the extent of mitochondrial heterogeneity in Arabidopsis, mitochondria isolated from six organ/cell types, leaf, root, cell culture, flower, bolt stem, and silique, were analyzed. Of the 286 protein spots on a 2-D gel of the mitochondrial proteome, the abundance of 237 spots was significantly varied between different samples.

Combining proteomics of root and shoot mitochondria and transcript analysis to define constitutive and variable components in plant mitochondria.

Mitochondria undertake respiration in plant cells, but through metabolic plasticity utilize differ proportions of substrates and deliver different proportions of products to cellular metabolic and biosynthetic pathways. In Arabidopsis the mitochondrial proteome from shoots and cell culture have been reported, but there has been little information on mitochondria in roots. We compare the root mitochondrial proteome with mitochondria isolated from photosynthetic shoots to define the role of protein abundance in these differences.

TCP transcription factors link the regulation of genes encoding mitochondrial proteins with the circadian clock in Arabidopsis thaliana.

Diurnal regulation of transcripts encoding proteins located in mitochondria, plastids, and peroxisomes is important for adaptation of organelle biogenesis and metabolism to meet cellular requirements. We show this regulation is related to diurnal changes in promoter activities and the presence of specific cis-acting regulatory elements in the proximal promoter region [TGGGC(C/T)], previously defined as site II elements, and leads to diurnal changes in organelle protein abundances. These site II elements can act both as activators or repressors of transcription, depending on the night/day period and on the number and arrangement of site II elements in the promoter tested.

Mitochondrial malate dehydrogenase lowers leaf respiration and alters photorespiration and plant growth in Arabidopsis.

Malate dehydrogenase (MDH) catalyzes a reversible NAD(+)-dependent-dehydrogenase reaction involved in central metabolism and redox homeostasis between organelle compartments. To explore the role of mitochondrial MDH (mMDH) in Arabidopsis (Arabidopsis thaliana), knockout single and double mutants for the highly expressed mMDH1 and lower expressed mMDH2 isoforms were constructed and analyzed. A mmdh1mmdh2 mutant has no detectable mMDH activity but is viable, albeit small and slow growing.