External NADH Dehydrogenase Research Articles

Effect of increased gene expression of alternative external NADH dehydrogenase of mitochondria of Arabidopsis thaliana on the generation of reactive oxygen in Nicotiana tabacum tobacco leaves at low temperatures

Low temperature is an important factor limiting plant viability and productivity. Along with other stresses, low temperatures increase the generation of reactive oxygen species, which are signaling molecules that can damage cell components. As well as representing one of the main targets of oxidative damage during stress, mitochondria represent a significant source of reactive oxygen species. Plant mitochondria have a large number of enzymes providing alternative electron transport pathways, many of which are activated under stress. Our aim was to assess the effect of low positive temperatures and increased expression of the heterologous gene NDB2 (alternative external NADH dehydrogenase of mitochondria) on the generation of reactive oxygen species, which involve an alternative respiratory chain in mitochondria and the expression of stress proteins under lighting conditions in Nicotiana tabacum tobacco leaves. In the leaves of tobacco plants with increased expression of the Arabidopsis thaliana NDB2 (AtNDB2) gene, a decrease in reactive oxygen species production was observed under normal and low temperature conditions. The results indicate that the heterologous Arabidopsis thaliana NDB2 gene is involved in increasing the activity of the alternative electron transport chain in mitochondria, which reduces the level of reactive oxygen species generation and affects the content of stress proteins under normal and low-temperature exposure.

Effect of the overexpression of external alternative NADH dehydrogenase gene in Arabidopsis on the resistance of transformed tobacco plants to negative temperatures

The study aims to confirm the functional activity and localization of the At_NDB2 transgenic protein of Arabidopsis in tobacco cells and to evaluate the effect produced by the permanent expression of external alternative NADH dehydrogenase on the resistance of a heat-loving plant to low temperatures. Proteins and mitochondria were isolated from the leaves of tobacco plants grown at 25 °С (day/night) at the 7-leaf stage. At_NDB2 protein localization in mitochondria was determined via electrophoresis and immunoblotting. The functional activity of At_NDB2 was confirmed through the polarography of isolated mitochondria and the specific mitochondrial complex I inhibitor (rotenone). It was also found that the cyanide-resistant respiration rate and the activity of an alternative oxidase enzyme were significantly higher in transgenic plants than in wild-type plants. In order to determine the resistance to low temperatures, the parent and transgenic tobacco plants were grown in soil until the 2–3 and 6–7 leaf stages, after which they were kept at 3 to -3 °С for a day in the dark and left to regrow at 25 °С for seven days. Although it was previously shown that oxidative stress is reduced in transgenic plants at low temperatures as compared to wild-type plants, it was established that the tolerance of transgenic and nontransformed plants does not differ. Thus, alternative NADH dehydrogenase activity was found to reduce oxidative stress and increase alternative oxidase activity, without enhancing the resistance of Nicotiana tabacum to negative temperatures.

Development of an industrial yeast strain for efficient production of 2,3-butanediol

As part of the transition from a fossil resources-based economy to a bio-based economy, the production of platform chemicals by microbial cell factories has gained strong interest. 2,3-butanediol (2,3-BDO) has various industrial applications, but its production by microbial fermentation poses multiple challenges. We have engineered the bacterial 2,3-BDO synthesis pathway, composed of AlsS, AlsD and BdhA, in a pdc-negative version of an industrial Saccharomyces cerevisiae yeast strain. The high concentration of glycerol caused by the excess NADH produced in the pathway from glucose to 2,3-BDO was eliminated by overexpression of NoxE and also in a novel way by combined overexpression of NDE1, encoding mitochondrial external NADH dehydrogenase, and AOX1, encoding a heterologous alternative oxidase expressed inside the mitochondria. This was combined with strong downregulation of GPD1 and deletion of GPD2, to minimize glycerol production while maintaining osmotolerance. The HGS50 strain produced a 2,3-BDO titer of 121.04 g/L from 250 g/L glucose, the highest ever reported in batch fermentation, with a productivity of 1.57 g/L.h (0.08 g/L.h per gCDW) and a yield of 0.48 g/g glucose or with 96% the closest to the maximum theoretical yield ever reported. Expression of Lactococcus lactis NoxE, encoding a water-forming NADH oxidase, combined with similar genetic modifications, as well as expression of Candida albicans STL1, also minimized glycerol production while maintaining high osmotolerance. The HGS37 strain produced 130.64 g/L 2,3-BDO from 280 g/L glucose, with productivity of 1.58 g/L.h (0.11 g/L.h per gCDW). Both strains reach combined performance criteria adequate for industrial implementation.

AIFM2 Is Required for High-Intensity Aerobic Exercise in Promoting Glucose Utilization.

Skeletal muscle is a major regulator of glycemic control at rest, and glucose utilization increases drastically during exercise. Sustaining a high glucose utilization via glycolysis requires efficient replenishment of NAD+ in the cytosol. Apoptosis-inducing mitochondrion-associated factor 2 (AIFM2) was previously shown to be a NADH oxidoreductase domain-containing flavoprotein that promotes glycolysis for diet and cold-induced thermogenesis. Here, we find that AIFM2 is selectively and highly induced in glycolytic extensor digitorum longus (EDL) muscle during exercise. Overexpression (OE) of AIFM2 in myotubes is sufficient to elevate the NAD+-to-NADH ratio, increasing the glycolytic rate. Thus, OE of AIFM2 in skeletal muscle greatly increases exercise capacity, with increased glucose utilization. Conversely, muscle-specific Aifm2 depletion via in vivo transfection of hairpins against Aifm2 or tamoxifen-inducible haploinsufficiency of Aifm2 in muscles decreases exercise capacity and glucose utilization in mice. Moreover, muscle-specific introduction of NDE1, Saccharomyces cerevisiae external NADH dehydrogenase (NDE), ameliorates impairment in glucose utilization and exercise intolerance of the muscle-specific Aifm2 haploinsufficient mice. Together, we show a novel role for AIFM2 as a critical metabolic regulator for efficient utilization of glucose in glycolytic EDL muscles.

The effect of pesticides on the NADH-supported mitochondrial respiration of permeabilized potato mitochondria

Pesticides can seriously affect the respiratory chain of the mitochondria of many crops, reducing the intensity of plant growth and its yield. Studying the effect of pesticides on the bioenergetic parameters of intact plant mitochondria is a promising approach for assessing their toxicity. In this study, we investigated the effect of some pesticides on isolated potato mitochondria, which used exogenous NADH as a substrate for respiration. We showed that succinate is the most preferred substrate for phosphorylating respiration of intact potato tubers mitochondria. Potato mitochondria poorly oxidize exogenous NADH, despite of the presence of external NADH dehydrogenases. Permeabilization of the mitochondrial membrane with alamethicin increased the availability of exogenous NADH to complex I. However, the pathway of electrons through complex I to complex IV makes intact potato mitochondria susceptible to a number of pesticides such as difenoconazole, fenazaquin, pyridaben and tolfenpyrad, which strongly inhibit the rate of mitochondrial respiration. However, these pesticides only slightly inhibited the rate of oxygen consumption during succinate-supported respiration. Dithianon, the inhibitor of Complex II, is the only pesticide which significantly increased the respiratory rate of NADH-supported respiration of permeabilized mitochondria of potato. Thus, it can be assumed that the alternative NADH dehydrogenases for electron flow represent a factor responsible for plant resistance to xenobiotics, such as mitochondria-targeted pesticides.

Aifm2, a NADH Oxidase, Supports Robust Glycolysis and Is Required for Cold- and Diet-Induced Thermogenesis.

Brown adipose tissue (BAT) is highly metabolically active tissue that dissipates energy via UCP1 as heat, and BAT mass is correlated negatively with obesity. The presence of BAT/BAT-like tissue in humans renders BAT as an attractive target against obesity and insulin resistance. Here, we identify Aifm2, a NADH oxidoreductase domain containing flavoprotein, as a lipid droplet (LD)-associated protein highly enriched in BAT. Aifm2 is induced by cold as well as by diet. Upon cold or β-adrenergic stimulation, Aifm2 associates with the outer side of the mitochondrial inner membrane. As a unique BAT-specific first mammalian NDE (external NADH dehydrogenase)-like enzyme, Aifm2 oxidizes NADH to maintain high cytosolic NAD levels in supporting robust glycolysis and to transfer electrons to the electron transport chain (ETC) for fueling thermogenesis. Aifm2 in BAT and subcutaneous white adipose tissue (WAT) promotes oxygen consumption, uncoupled respiration, and heat production during cold- and diet-induced thermogenesis. Aifm2, thus, can ameliorate diet-induced obesity and insulin resistance.

AtNDB2 Is the Main External NADH Dehydrogenase in Mitochondria and Is Important for Tolerance to Environmental Stress.

In addition to the classical electron transport pathway coupled to ATP synthesis, plant mitochondria have an alternative pathway that involves type II NAD(P)H dehydrogenases (NDs) and alternative oxidase (AOX). This alternative pathway participates in thermogenesis in select organs of some species and is thought to help prevent cellular damage during exposure to environmental stress. Here, we investigated the function and role of one alternative path component, AtNDB2, using a transgenic approach in Arabidopsis (Arabidopsis thaliana). Disruption of AtNDB2 expression via T-DNA insertion led to a 90% decrease of external NADH oxidation in isolated mitochondria. Overexpression of AtNDB2 led to increased AtNDB2 protein abundance in mitochondria but did not enhance external NADH oxidation significantly unless AtAOX1A was concomitantly overexpressed and activated, demonstrating a functional link between these enzymes. Plants lacking either AtAOX1A or AtNDB2 were more sensitive to combined drought and elevated light treatments, whereas plants overexpressing these components showed increased tolerance and capacity for poststress recovery. We conclude that AtNDB2 is the predominant external NADH dehydrogenase in mitochondria and together with AtAOX1A forms a complete, functional, nonphosphorylating pathway of electron transport, whose operation enhances tolerance to environmental stress. This study demonstrates that at least one of the alternative NDs, as well as AOX, are important for the stress response.

Do angiosperms with highly divergent mitochondrial genomes have altered mitochondrial function?

Angiosperm mitochondrial (mt) genes are generally slow-evolving, but multiple lineages have undergone dramatic accelerations in rates of nucleotide substitution and extreme changes in mt genome structure. While molecular evolution in these lineages has been investigated, very little is known about their mt function. Some studies have suggested altered respiration in individual taxa, although there are several reasons why mt variation might be neutral in others. Here, we develop a new protocol to characterize respiration in isolated plant mitochondria and apply it to species of Silene with mt genomes that are rapidly evolving, highly fragmented, and exceptionally large (~11 Mbp). This protocol, complemented with traditional measures of plant fitness, cytochrome c oxidase activity assays, and fluorescence microscopy, was also used to characterize inter- and intraspecific variation in mt function. Contributions of the individual “classic” OXPHOS complexes, the alternative oxidase, and external NADH dehydrogenases to overall mt respiratory flux were found to be similar to previously studied angiosperms with more typical mt genomes. Some differences in mt function could be explained by inter- and intraspecific variation. This study suggests that Silene species with peculiar mt genomes still show relatively normal mt respiration. This may be due to strong purifying selection on mt variants, coevolutionary responses in the nucleus, or a combination of both. Future experiments should explore such questions using a comparative framework and investigating other lineages with unusual mitogenomes.

Involvement of the external mitochondrial NADH dehydrogenase Nde1 in glycerol metabolism by wild-type and engineered Saccharomyces cerevisiae strains.

Glycerol is an attractive substrate for microbial fermentations due to its higher degree of reduction compared to glucose. The replacement of the native FAD-dependent glycerol catabolic pathway in Saccharomyces cerevisiae by an artificial NADH-delivering dihydroxyacetone (DHA) pathway is supposed to facilitate the capturing of electrons in fermentation products. This requires that the electrons from the cytosolic NADH are not exclusively transferred to oxygen. However, the external NADH dehydrogenases (Nde1/2) and the L-glycerol 3-phosphate shuttle (composed of Gpd1/2 and Gut2), both coupled to the respiratory chain, are known to contribute to cytosolic NAD+ regeneration during growth on non-fermentable carbon sources. In order to evaluate the role of these mechanisms during growth on glycerol, we deleted GPD1/2, GUT2 as well as NDE1/2, separately and in combinations in both the glycerol-utilizing wild-type strain CBS 6412-13A and the corresponding engineered strain CBS DHA in which glycerol is catabolized by the DHA pathway. Particularly, the nde1Δ mutants showed a significant reduction in growth rate and the nde1∆ nde2∆ double deletion mutants did not grow at all in synthetic glycerol medium. The current work also demonstrates a positive impact of deleting NDE1 on the production of the fermentation product 1,2-propanediol in an accordingly engineered S. cerevisiae strain.

Expression of alternative NADH dehydrogenases (NDH-2) in the phytopathogenic fungus Ustilago maydis.

Type 2 alternative NADH dehydrogenases (NDH‐2) participate indirectly in the generation of the electrochemical proton gradient by transferring electrons from NADH and NADPH into the ubiquinone pool. Due to their structural simplicity, alternative NADH dehydrogenases have been proposed as useful tools for gene therapy of cells with defects in the respiratory complex I. In this work, we report the presence of three open reading frames, which correspond to NDH‐2 genes in the genome of Ustilago maydis. These three genes were constitutively transcribed in cells cultured in YPD and minimal medium with glucose, ethanol, or lactate as carbon sources. Proteomic analysis showed that only two of the three NDH‐2 were associated with isolated mitochondria in all culture media. Oxygen consumption by permeabilized cells using NADH or NADPH was different for each condition, opening the possibility of posttranslational regulation. We confirmed the presence of both external and internal NADH dehydrogenases, as well as an external NADPH dehydrogenase insensitive to calcium. Higher oxygen consumption rates were observed during the exponential growth phase, suggesting that the activity of NADH and NADPH dehydrogenases is coupled to the dynamics of cell growth.

Elevation of cytosolic Ca2+ in response to energy deficiency in plants: the general mechanism of adaptation to low oxygen stress.

Ca2+ can be released from cell compartments to the cytosol during stress conditions. We discuss here the causes of Ca2+ release under conditions of ATP concentration decline that result in the suppression of ATPases and activation of calcium ion channels. The main signaling and metabolic consequences of Ca2+ release are considered for stressed plant cells. The signaling function includes generation and spreading of calcium waves, while the metabolic function results in the activation of particular enzymes and genes. Ca2+ is involved in the activation of glutamate decarboxylase, initiating the γ-aminobutyric acid shunt and triggering the formation of alanine, processes which play a role, in particular, in pH regulation. Ca2+ activates the transcription of several genes, e.g. of plant hemoglobin (phytoglobin, Pgb) which scavenges nitric oxide and regulates redox and energy balance through the Pgb-nitric oxide cycle. This cycle involves NADH and NADPH oxidation from the cytosolic side of mitochondria, in which Ca2+- and low pH-activated external NADH and NADPH dehydrogenases participate. Ca2+ can also activate the genes of alcohol dehydrogenase and pyruvate decarboxylase stimulating hypoxic fermentation. It is concluded that calcium is a primary factor that causes the metabolic shift under conditions of oxygen deficiency.

Mitochondrial energy metabolism is required for lifespan extension by the spastic paraplegia-associated protein spartin.

Hereditary spastic paraplegias, a group of neurodegenerative disorders, can be caused by loss-of-function mutations in the protein spartin. However, the physiological role of spartin remains largely elusive. Here we show that heterologous expression of human or Drosophila spartin extends chronological lifespan of yeast, reducing age-associated ROS production, apoptosis, and necrosis. We demonstrate that spartin localizes to the proximity of mitochondria and physically interacts with proteins related to mitochondrial and respiratory metabolism. Interestingly, Nde1, the mitochondrial external NADH dehydrogenase, and Pda1, the core enzyme of the pyruvate dehydrogenase complex, are required for spartin-mediated cytoprotection. Furthermore, spartin interacts with the glycolysis enhancer phospo-fructo-kinase-2,6 (Pfk26) and is sufficient to complement for PFK26-deficiency at least in early aging. We conclude that mitochondria-related energy metabolism is crucial for spartin’s vital function during aging and uncover a network of specific interactors required for this function.

The role of flavin-containing enzymes in mitochondrial membrane hyperpolarization and ROS production in respiring Saccharomyces cerevisiae cells under heat-shock conditions

Heat shock is known to accelerate mitochondrial ROS production in Saccharomyces cerevisiae cells. But how yeast mitochondria produce ROS under heat-shock condition is not completely clear. Previously, it was shown that ROS production in heat-stressed fermenting yeast cells was accompanied by mitochondrial membrane potential (MMP) increase. In the current investigation the relationship between ROS production and MMP was studied in respiring yeast cells in stationary phase, using diphenyleneiodonium chloride (DPI), an inhibitor of flavin-containing proteins, as well as the mutants deleted for NDE1, NDE2 and NDI1 genes, encoding flavin-containing external and internal NADH dehydrogenases. It was shown that heat shock induced a transient burst in mitochondrial ROS production, which was paralleled by MMP rise. ROS production and MMP was significantly suppressed by DPI addition and deletion of NDE1. The effect of DPI on ROS production and MMP rise was specific for respiring cells. The results obtained suggest that the functioning of mitochondrial flavin-binding enzymes, Nde1p for instance, is required for the hyperpolarization of inner mitochondrial membrane and ROS production in respiring S. cerevisiae cells under heat-shock conditions.

The evolution of substrate specificity-associated residues and Ca(2+) -binding motifs in EF-hand-containing type II NAD(P)H dehydrogenases.

Most eukaryotic organisms, except some animal clades, have mitochondrial alternative electron transport enzymes that allow respiration to bypass the energy coupling in oxidative phosphorylation. The energy bypass enzymes in plants include the external type II NAD(P)H dehydrogenases (DHs) of the NDB family, which are characterized by an EF-hand domain for Ca(2+) binding. Here we investigate these plant enzymes by combining molecular modeling with evolutionary analysis. Molecular modeling of the Arabidopsis thaliana AtNDB1 with the yeast ScNDI1 as template revealed distinct similarities in the core catalytic parts, and highlighted the interaction between the pyridine nucleotide and residues correlating with NAD(P)H substrate specificity. The EF-hand domain of AtNDB1 has no counterpart in ScNDI1, and was instead modeled with Ca(2+) -binding signal transducer proteins. Combined models displayed a proximity of the AtNDB1 EF-hand domain to the substrate entrance side of the catalytic part. Evolutionary analysis of the eukaryotic NDB-type proteins revealed ancient and recent reversions between the motif observed in proteins specific for NADH (acidic type) and NADPH (non-acidic type), and that the clade of enzymes with acidic motifs in angiosperms derives from non-acidic-motif NDB-type proteins present in basal plants, fungi and protists. The results suggest that Ca(2+) -dependent external NADPH oxidation is an ancient process, indicating that it has a fundamental importance for eukaryotic cellular redox metabolism. In contrast, the external NADH DHs in plants are products of a recent expansion, mirroring the expansion of the alternative oxidase family.

Changes in the OXPHOS system in leaf and root mitochondria of Arabidopsis thaliana subjected to long-term sulphur deficiency

Long-term sulphur (S) deficiency in Arabidopsis thaliana affects the functioning of the mitochondrial oxidative phosphorylation system (OXPHOS) via alteration of the multisubunit NADH-ubiquinone oxidoreductase (Complex I; EC 1.6.5.3), which contains several iron–sulphur clusters. Densitometric analysis of bands of respiratory chain complexes after one-dimensional blue-native polyacrylamide gel electrophoresis (BN-PAGE) showed that levels and in-gel capacities of Complex I in leaf and root mitochondria were lower than those of the control. Two-dimensional BN/SDS-PAGE showed lower abundance of all Complex I subunits, but the qualitative structural composition (subunit expression and mobility) did not change. In mitochondria of S-deficient A. thaliana, impairment of Complex I could be compensated to some extent by additional type II NADH dehydrogenases that do not contain iron–sulphur clusters. The level and capacity of external NADH dehydrogenases in leaf and root mitochondria was higher under S deficiency, but that of internal NADH dehydrogenases did not differ from the control. The amount of COXII (mitochondrial-encoded subunit of cytochrome c oxidase in Complex IV; EC 1.9.3.1) and the capacity of Complex IV were lower under S deficiency, but levels of alternative oxidase, a bypass to Complex IV, did not change. We discuss S deficiency in A. thaliana in relation to the assembly and stability of Complex I and to a bypass of Complex I by external type II NADH dehydrogenases.

Debaryomyces Hansenii: Adaptation Mechanisms to Different Carbon Sources and Oxygen Concentrations

Debaryomyces hansenii is an oleaginous, mostly aerobic yeast. Its mitochondrial respiratory chain contains complejxes I, II, III and IV, an alternative oxidase, aln alternative external NADH dehydrogenase an a glycerol-phosphate dehydrogenase (Cabrera-Orefice et al, 2014, Biochim Biophys Acta 1837(1): 73-84; 2014, Biochimie 102: 124-136). Alternative dehydrogenases, seem to physiologically uncouple mitochondria in order to prevent ROS production (Guerrero-Castillo et al, 2011, Biochim Biophys Acta 1817(2): 353-62). In addition, the opening of a unspecific channel releases NAD+, thus inactivating complex I and decreasing the proton-pumping stoichiometry in the system. In regard to the carbon source, lactate or succinate led to the expression of AOX at all growth phases. In contrast, in the presence of glucose or galactose, AOX was only expressed at the late exponential phase. In contrast, in glycerol AOX was never expressed and biomass growth was larger. In addition, large lipidic production was observed. It is suggested that D. hansenii is useful to study cell adaptations to different growth conditions. Also its lipid production mechanisms might be interesting from a biotechnological point of view. The regulation of redox enzyme expression maintains a high respiratory activity even when ATP consumption is low, probably preventing overproduction of ROS.

Proton pumping complex I increases growth yield in Candida utilis

In living cells, growth is the result of coupling between substrate catabolism and multiple metabolic processes that take place during net biomass formation and cellular maintenance processes. A crucial parameter for growth evaluation is its yield, i.e. the efficiency of the transformation processes. The yeast Candida utilis is of peculiar interest since its mitochondria exhibit a complex I that is proposed to pump protons but also an external NADH dehydrogenase that do not pump protons. Here, we show that in C. utilis cells grown on non-fermentable media, growth yield is 30% higher as compared to that of Saccharomyces cerevisiae that do not exhibit a complex I. Moreover, ADP/O determination in C. utilis shows that electrons coming from internal NADH dehydrogenase go through proton pumping complex I, whereas electrons coming from external NADH dehydrogenases do not go through proton pumping complex I. Furthermore, we show that electron competition strictly depends on extra-mitochondrial NADH concentration, i.e. the higher the extra-mitochondrial NADH concentration, the higher the competition process with a right way for electrons coming from external NADH dehydrogenases. Such a complex regulation in C. utilis allows an increase in growth yield when cytosolic NADH is not plentiful but still favors the cytosolic NADH re-oxidation at high NADH, favoring biomass generation metabolic pathways.

Engineering cellular redox balance in Saccharomyces cerevisiae for improved production of L-lactic acid.

Owing to the growing market for the biodegradable and renewable polymer, polylactic acid, world demand for lactic acid is rapidly increasing. However, the very high concentrations desired for industrial production of the free lactic acid create toxicity and low pH concerns for manufacturers. Saccharomyces cerevisiae is the most well characterized eukaryote, a preferred microbial cell factory for the largest industrial biotechnology product (bioethanol), and a robust, commercially compatible workhorse to be exploited for the production of diverse chemicals. S. cerevisiae has also been explored as a host for lactic acid production because of its high acid tolerance. Here, we constructed an L-lactic acid-overproducing S. cerevisiae by redirecting cellular metabolic fluxes to the production of L-lactic acid. To this end, we deleted the S. cerevisiae genes encoding pyruvate decarboxylase 1 (PDC1), L-lactate cytochrome-c oxidoreductase (CYB2), and glycerol-3-phosphate dehydrogenase (GPD1), replacing them with a heterologous L-lactate dehydrogenase (LDH) gene. Two new target genes encoding isoenzymes of the external NADH dehydrogenase (NDE1 and NDE2), were also deleted from the genome to re-engineer the intracellular redox balance. The resulting strain was found to produce L-lactic acid more efficiently (32.6% increase in final L-lactic acid titer). When tested in a bioreactor in fed-batch mode, this engineered strain produced 117 g/L of L-lactic acid under low pH conditions. This result demonstrates that the redox balance engineering should be coupled with the metabolic engineering in the construction of L-lactic acid-overproducing S. cerevisiae.

Mitochondrial energy-dissipating systems (alternative oxidase, uncoupling proteins, and external NADH dehydrogenase) are involved in development of frost-resistance of winter wheat seedlings.

Gene expression, protein synthesis, and activities of alternative oxidase (AOX), uncoupling proteins (UCP), adenine nucleotide translocator (ANT), and non-coupled NAD(P)H dehydrogenases (NDex, NDPex, and NDin) were studied in shoots of etiolated winter wheat (Triticum aestivum L.) seedlings after exposure to hardening low positive (2°C for 7 days) and freezing (-2°C for 2 days) temperatures. The cold hardening efficiently increased frost-resistance of the seedlings and decreased the generation of reactive oxygen species (ROS) during further cold shock. Functioning of mitochondrial energy-dissipating systems can represent a mechanism responsible for the decrease in ROS under these conditions. These systems are different in their response to the action of the hardening low positive and freezing temperatures. The functioning of the first system causes induction of AOX and UCP synthesis associated with an increase in electron transfer via AOX in the mitochondrial respiratory chain and also with an increase in the sensitivity of mitochondrial non-phosphorylating respiration to linoleic and palmitic acids. The increase in electron transfer via AOX upon exposure of seedlings to hardening freezing temperature is associated with retention of a high activity of NDex. It seems that NDex but not the NDPex and NDin can play an important role in maintaining the functional state of mitochondria in heterotrophic tissues of plants under the influence of freezing temperatures. The involvement of the mitochondrial energy-dissipating systems and their possible physiological role in the adaptation of winter crops to cold and frost are discussed.

Nitric oxide inhibits succinate dehydrogenase-driven oxygen consumption in potato tuber mitochondria in an oxygen tension-independent manner

NO (nitric oxide) is described as an inhibitor of plant and mammalian respiratory chains owing to its high affinity for COX (cytochrome c oxidase), which hinders the reduction of oxygen to water. In the present study we show that in plant mitochondria NO may interfere with other respiratory complexes as well. We analysed oxygen consumption supported by complex I and/or complex II and/or external NADH dehydrogenase in Percoll-isolated potato tuber (Solanum tuberosum) mitochondria. When mitochondrial respiration was stimulated by succinate, adding the NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine) or DETA-NONOate caused a 70% reduction in oxygen consumption rate in state 3 (stimulated with 1mM of ADP). This inhibition was followed by a significant increase in the Km value of SDH (succinate dehydrogenase) for succinate (Km of 0.77±0.19 to 34.3±5.9mM, in the presence of NO). When mitochondrial respiration was stimulated by external NADH dehydrogenase or complex I, NO had no effect on respiration. NO itself and DETA-NONOate had similar effects to SNAP. No significant inhibition of respiration was observed in the absence of ADP. More importantly, SNAP inhibited PTM (potato tuber mitochondria) respiration independently of oxygen tensions, indicating a different kinetic mechanism from that observed in mammalian mitochondria. We also observed, in an FAD reduction assay, that SNAP blocked the intrinsic SDH electron flow in much the same way as TTFA (thenoyltrifluoroacetone), a non-competitive SDH inhibitor. We suggest that NO inhibits SDH in its ubiquinone site or its Fe-S centres. These data indicate that SDH has an alternative site of NO action in plant mitochondria.