Influence of ethanol on alternative oxidase in mitochondria from callus-forming potato tuber discs

Ethanol, when added to the incubation medium of callus-forming potato tuber discs, inhibits callus growth and causes an increase of the mitochondrial antimycin-A resistant respiration, expressed as a percentage of state III-respiration. This increase in resistance to antimycin-A is the result of a poor development of the cytochrome pathway in tissue discs treated with ethanol. The development of the antimycin-A resistant alternative oxidase sensitive to chelator is about the same for treated and untreated discs. The respiratory control (RC) ratio of the mitochondrial respiration increases after addition of a chelator, which inhibits the alternative pathway. The RC ratio of the uninhibited mitochondrial respiration appears to be inversely related to the capacity of the alternative pathway, when mitochondrial preparations with different capacities to transfer electrons via the alternative path are compared. From the experimentally observed relation between RC-ratio and alternative oxidase capacity, it was concluded that at least half of the capacity of the alternative path is used in uninhibited state IV respiration.     

Melatonin-related mitochondrial respiration responses are associated with growth promotion and cold tolerance in plants

Melatonin has the ability to improve plant growth and strengthened plant tolerance to environmental stresses; however, the effects of melatonin on mitochondrial respiration in plants and the underlying biochemical and molecular mechanisms are still unclear. The objective of the study is to determine possible effects of melatonin on mitochondrial respiration and energy efficiency in maize leaves grown under optimum temperature and cold stress and to reveal the relationship between melatonin-induced possible alterations in mitochondrial respiration and cold tolerance. Melatonin and cold stress, alone and in combination, caused significant increases in activities and gene expressions of pyruvate dehydrogenase, citrate synthase, and malate dehydrogenase, indicating an acceleration in the rate of tricarboxylic acid cycle. Total mitochondrial respiration rate, cytochrome pathway rate, and alternative respiration rate were increased by the application of melatonin and/or cold stress. Similarly, gene expression and protein levels of cytochrome oxidase and alternative oxidase were also enhanced by melatonin and/or cold stress. The highest values for all these parameters were obtained from the seedlings treated with the combined application of melatonin and cold stress. The activity and gene expression of ATP synthase and ATP concentration were augmented by melatonin under control and cold stress. On the other hand, cold stress reduced markedly plant growth parameters, including root length, plant height, leaf surface area, and chlorophyll content and increased the content of reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide and oxidative damage, including malondialdehyde content and electrolyte leakage level; however, melatonin significantly promoted the plant growth parameters and reduced ROS content and oxidative damage under control and cold stress. These data revealed that melatonin-induced growth promotion and cold tolerance in maize is associated with its modulating effect on mitochondrial respiration.     

Regulation of thermogenesis in flowering Araceae: the role of the alternative oxidase

The inflorescences of several members of the Arum lily family warm up during flowering and are able to maintain their temperature at a constant level, relatively independent of the ambient temperature. The heat is generated via a mitochondrial respiratory pathway that is distinct from the cytochrome chain and involves a cyanide-resistant alternative oxidase (AOX). In this paper we have used flux control analysis to investigate the influence of temperature on the rate of respiration through both cytochrome and alternative oxidases in mitochondria isolated from the appendices of intact thermogenic Arum maculatum inflorescences. Results are presented which indicate that at low temperatures, the dehydrogenases are almost in full control of respiration but as the temperature increases flux control shifts to the AOX. On the basis of these results a simple model of thermoregulation is presented that is applicable to all species of thermogenic plants. The model takes into account the temperature characteristics of the separate components of the plant mitochondrial respiratory chain and the control of each process. We propose that 1) in all aroid flowers AOX assumes almost complete control over respiration, 2) the temperature profile of AOX explains the reversed relationship between ambient temperature and respiration in thermoregulating Arum flowers, 3) the thermoregulation process is the same in all species and 4) variations in inflorescence temperatures can easily be explained by variations in AOX protein concentrations.     

Increased respiratory restriction during phosphate-limited growth in transgenic tobacco cells lacking alternative oxidase

We found that mitochondrial alternative oxidase (AOX) protein and the capacity for CN-resistant respiration are dramatically increased in wild-type tobacco (Nicotiana tabacum) suspension-cultured cells in response to growth under P limitation, and antisense (AS8) tobacco cells unable to induce AOX under these conditions have altered growth and metabolism. Specifically, we found that the respiration of AS8 cells was restricted during P-limited growth, when the potential for severe adenylate control of respiration (at the level of C supply to the mitochondrion and/or at the level of oxidative phosphorylation) is high due to the low cellular levels of ADP and/or inorganic P. As a result of this respiratory restriction, AS8 cells had altered growth, morphology, cellular composition, and patterns of respiratory C flow to amino acid synthesis compared with wild-type cells with abundant AOX protein. Also, AS8 cells under P limitation displayed high in vivo rates of generation of active oxygen species compared with wild-type cells. This difference could be abolished by an uncoupler of mitochondrial oxidative phosphorylation. Our results suggest that induction of non-phosphorylating AOX respiration (like induction of adenylate and inorganic P-independent pathways in glycolysis) is an important plant metabolic adaptation to P limitation. By preventing severe respiratory restriction, AOX acts to prevent both redirections in C metabolism and the excessive generation of harmful active oxygen species in the mitochondrion.     

Pyruvate accumulation during phosphate deficiency stress of bean roots

Culture of bean plants (Phaseolus vulgaris L. cv., Złota Saxa) for 16 d on phosphate-deficient nutrient medium resulted in an over twofold increase of pyruvate concentration in the root tissues. In a variety of plant tissues, the marked decline in cellular concentrations of adenylates and inorganic phosphate (Pi) influences the activity of pyruvate producing enzymes, which are dependent on the availability of ADP. In bean roots after 16 d of phosphate starvation pyruvate producing enzymes: phosphoenolpyruvate phosphatase (EC 3.1.3.2) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) had higher activities compared to those of control plants. The observed decrease of alanine and ethanol concentration and also alcohol dehydrogenase (EC 1.1.1.1) activity in phosphate-deficient roots may be the effect of the restrictions in pyruvate utilizing pathways. The increased activity of mitochondrial NAD-malic enzyme (EC 1.1.1.40) as well as the lower consumption of pyruvate during respiration of phosphate-deficient roots indicate that pyruvate concentration in mitochondria may be elevated. It is proposed that pyruvate accumulation in phosphate-deficient roots and alternative oxidase participation in respiration are important aspects of plant metabolic adaptations to Pi limitation, and may play a role in reducing oxidative stress induced by phosphate deficiency.     

Nitric oxide affects plant mitochondrial functionality in vivo

In this report, we show that nitric oxide affects mitochondrial functionality in plant cells and reduces total cell respiration due to strong inhibition of the cytochrome pathway. The residual respiration depends on the alternative pathway and novel synthesis of alternative oxidase occurs. These modifications are associated with depolarisation of the mitochondrial membrane potential and release of cytochrome c from mitochondria, suggesting a conserved signalling pathway in plants and animals. This signal cascade is triggered at the mitochondrial level and induces about 20% of cell death. In order to achieve a higher level of cell death, the addition of H(2)O(2) is necessary.     

Mitochondrial uncoupling proteins in unicellular eukaryotes

Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier protein family that are present in the mitochondrial inner membrane and mediate free fatty acid (FFA)-activated, purine nucleotide (PN)-inhibited proton conductance. Since 1999, the presence of UCPs has been demonstrated in some non-photosynthesising unicellular eukaryotes, including amoeboid and parasite protists, as well as in non-fermentative yeast and filamentous fungi. In the mitochondria of these organisms, UCP activity is revealed upon FFA-induced, PN-inhibited stimulation of resting respiration and a decrease in membrane potential, which are accompanied by a decrease in membranous ubiquinone (Q) reduction level. UCPs in unicellular eukaryotes are able to divert energy from oxidative phosphorylation and thus compete for a proton electrochemical gradient with ATP synthase. Our recent work indicates that membranous Q is a metabolic sensor that might utilise its redox state to release the PN inhibition of UCP-mediated mitochondrial uncoupling under conditions of phosphorylation and resting respiration. The action of reduced Q (QH₂) could allow higher or complete activation of UCP. As this regulatory feature was demonstrated for microorganism UCPs (A. castellanii UCP), plant and mammalian UCP1 analogues, and UCP1 in brown adipose tissue, the process could involve all UCPs. Here, we discuss the functional connection and physiological role of UCP and alternative oxidase, two main energy-dissipating systems in the plant-type mitochondrial respiratory chain of unicellular eukaryotes, including the control of cellular energy balance as well as preventive action against the production of reactive oxygen species.     

Regulation of Thermogenesis in Plants:The Interaction of Alternative Oxidase and Plant Uncoupling Mitochondrial Protein

Thermogenesis is a process of heat production in living organisms.It is rare in plants,but it does occur in some species of angiosperm.The heat is generated via plant mitochondrial respiration.As possible involvement in thermogenesis of mitochondrial factors,alternative ox-idases(AOXs) and plant uncoupling mitochondrial proteins(PUMPs) have been well studied.AOXs and PUMPs are ubiquitously present in the inner membrane of plant mitochondria.They serve as two major energy dissipation systems that balance mit...     

The plant uncoupling protein homologues: a new family of energy-dissipating proteins in plant mitochondria.

Uncoupling proteins (UCPs) form a subfamily within the mitochondrial carrier protein family, which catalyze a free fatty acid-mediated proton recycling and can modulate the tightness of coupling between mitochondrial respiration and ATP synthesis. As in mammalian tissues, UCPs are rather ubiquitous in the plant kingdom and widespread in plant tissues in which they could have various physiological roles, such as heat production or protection against free oxygen radicals. The simultaneous occurrence in plant mitochondria of two putative energy-dissipating systems, namely UCP which dissipates the proton motive force, and alternative oxidase (AOX) which dissipates the redox potential, raises the question of their functional interactions.     

Steps towards a mechanistic understanding of respiratory temperature responses

Temperature crucially affects the speed of metabolic processes in poikilotherm organisms, including plants. The instantaneous temperature responses of O(2)-reduction and CO(2)-release can be approximated by Arrhenius kinetics, even though respiratory gas exchange of plants is the net effect of many constituent biochemical processes. Nonetheless, the classical Arrhenius equation must be modified to account for a dynamic response to measurement temperatures. We show that this dynamic response is readily explained by combining Arrhenius and Michaelis-Menten kinetics, as part of a fresh appraisal of metabolic interpretations of instantaneous temperature responses. In combination with recent experimental findings, we argue that control of mitochondrial electron flow is shared among cytochrome oxidase and alternative oxidase under in vivo conditions, and is continuously coordinated. In this way, upstream carbohydrate metabolism and downstream electron transport appear to be optimized according to the demand of ATP, TCA-cycle intermediates and anabolic reducing power under differing metabolic states. We provide a link to the 'Growth and Maintenance Paradigm' of respiration and argue that respiratory temperature responses can be used as a tool to probe metabolic states of plant tissue, such that we can learn more about the mechanisms that govern longer-term acclimatization responses of plant metabolism.     

Functional expression of the plant alternative oxidase affects growth of the yeast Schizosaccharomyces pombe

We have investigated the extent to which functional expression of the plant alternative oxidase (from Sauromatum guttatum) in Schizosaccharomyces pombe affects yeast growth. When cells are cultured on glycerol, the maximum specific growth rate is decreased from 0.13 to 0.11 h-1 while growth yield is lowered by 20% (from 1. 14 x 10(8) to 9.12 x 10(7) cells ml-1). Kinetic studies suggest that the effect on growth is mitochondrial in origin. In isolated mitochondria we found that the alternative oxidase actively competes with the cytochrome pathway for reducing equivalents and contributes up to 24% to the overall respiratory activity. Metabolic control analysis reveals that the alternative oxidase exerts a considerable degree of control (22%) on total electron flux. Furthermore, the negative control exerted by the alternative oxidase on the flux ratio of electrons through the cytochrome and alternative pathways is comparable with the positive control exerted on this flux-ratio by the cytochrome pathway. To our knowledge, this is the first paper to report a phenotypic effect because of plant alternative oxidase expression. We suggest that the effect on growth is the result of high engagement of the non-protonmotive alternative oxidase in yeast respiration that, consequently, lowers the efficiency of energy conservation and hence growth.     

Regulation of thermogenesis in plants: the interaction of alternative oxidase and plant uncoupling mitochondrial protein

Thermogenesis is a process of heat production in living organisms. It is rare in plants, but it does occur in some species of angiosperm. The heat is generated via plant mitochondrial respiration. As possible involvement in thermogenesis of mitochondrial factors, alternative oxidases (AOXs) and plant uncoupling mitochondrial proteins (PUMPs) have been well studied. AOXs and PUMPs are ubiquitously present in the inner membrane of plant mitochondria. They serve as two major energy dissipation systems that balance mitochondrial respiration and uncoupled phosphorylation by dissipating the H+ redox energy and proton electrochemical gradient (ΔμH+) as heat, respectively. AOXs and PUMPs exert similar physiological functions during homeothermic heat production in thermogenic plants. AOXs have five isoforms, while PUMPs have six. Both AOXs and PUMPs are encoded by small nuclear multigene families. Multiple isoforms are expressed in different tissues or organs. Extensive studies have been done in the area of thermogenesis in higher plants. In this review, we focus on the involvement and regulation of AOXs and PUMPs in thermogenesis.     

Responses of the Mitochondrial Respiratory System to Low Temperature in Plants

Low-temperature (LT) stress induces significant changes to plant cells including perturbations of various physio-biochemical and metabolic processes, which impact primary metabolism, respiratory rate, and the ATP production for biosynthesis and growth. Mitochondria from LT-tolerant species respond to LT through remodeling their composition that changes the structural and functional properties of the organelles. In this review, we discuss physiological aspects of mitochondrial respiration rate that are affected by LT, as well as, changes in the abundance of respiratory components under LT. The latter includes components of the phosphorylating and non-phosphorylating pathways and adjustments of mitochondrial membrane composition. Our objective is to provide a detailed overview of the often-contrasting reports of mitochondrial-specific changes and responses to LT and look for consensus themes to explain changes and draw more generally applicable observations about the LT response of plant respiration.     

Inactivation of the mitochondrial carrier SLC25A25 (ATP-Mg2+/Pi transporter) reduces physical endurance and metabolic efficiency in mice

An ATP-Mg(2+/)P(i) inner mitochondrial membrane solute transporter (SLC25A25), which is induced during adaptation to cold stress in the skeletal muscle of mice with defective UCP1/brown adipose tissue thermogenesis, has been evaluated for its role in metabolic efficiency. SLC25A25 is thought to control ATP homeostasis by functioning as a Ca(2+)-regulated shuttle of ATP-Mg(2+) and P(i) across the inner mitochondrial membrane. Mice with an inactivated Slc25a25 gene have reduced metabolic efficiency as evidenced by enhanced resistance to diet-induced obesity and impaired exercise performance on a treadmill. Mouse embryo fibroblasts from Slc25a25(-/-) mice have reduced Ca(2+) flux across the endoplasmic reticulum, basal mitochondrial respiration, and ATP content. Although Slc25a25(-/-) mice are metabolically inefficient, the source of the inefficiency is not from a primary function in thermogenesis, because Slc25a25(-/-) mice maintain body temperature upon acute exposure to the cold (4 °C). Rather, the role of SLC25A25 in metabolic efficiency is most likely linked to muscle function as evidenced from the physical endurance test of mutant mice on a treadmill. Consequently, in the absence of SLC25A25 the efficiency of ATP production required for skeletal muscle function is diminished with secondary effects on adiposity. However, in the absence of UCP1-based thermogenesis, induction of Slc25a25 in mice with an intact gene may contribute to an alternative thermogenic pathway for the maintenance of body temperature during cold stress.     

Dynamic Adaptation of Liver Mitochondria to Chronic Alcohol Feeding in Mice: BIOGENESIS,REMODELING, AND FUNCTIONAL ALTERATIONS*

Liver mitochondria undergo dynamic alterations following chronic alcohol feeding to mice. Intragastric alcohol feeding to mice resulted in 1) increased state III respiration (109% compared with control) in isolated liver mitochondria, probably due to increased levels of complexes I, IV, and V being incorporated into the respiratory chain; 2) increased mitochondrial NAD⁺ and NADH levels (∼2-fold), with no change in the redox status; 3) alteration in mitochondrial morphology, with increased numbers of elongated mitochondria; and 4) enhanced mitochondrial biogenesis in the liver, which corresponded with an up-regulation of PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α). Oral alcohol feeding to mice, which is associated with less liver injury and steatosis, slightly enhanced respiration in isolated liver mitochondria (30.8% compared with control), lower than the striking increase caused by intragastric alcohol feeding. Mitochondrial respiration increased with both oral and intragastric alcohol feeding despite extensive N-acetylation of mitochondrial proteins. The alcohol-induced mitochondrial alterations are probably an adaptive response to enhance alcohol metabolism in the liver. Isolated liver mitochondria from alcohol-treated mice had a greater rate of acetaldehyde metabolism and respiration when treated with acetaldehyde than control. Aldehyde dehydrogenase-2 levels were unaltered in response to alcohol, suggesting that the greater acetaldehyde metabolism by isolated mitochondria from alcohol-treated mice was due to increased mitochondrial respiration that regenerated NAD⁺, the rate-limiting substrate in alcohol/acetaldehyde metabolism. Overall, our work suggests that mitochondrial plasticity in the liver may be an important adaptive response to the metabolic stress caused by alcohol intake and could potentially play a role in many other vital functions performed by the liver.     

Studies on energy status and mitochondrial respiration during growth and senescence of mung bean cotyledons

Several characteristics of mitochondrial respiration and energy status have been studied during growth and senescence of mung bean ( Phaseolus radiatus L.) cotyledons. The results showed that mitochondrial oxygen consumption, respiratory control, ADP:O ratios, and energy charge changed in the cotyledons during germination and growth of the seedlings. The respiration rate of intact cotyledons approximately reflected the trend of the oxidative activities of the isolated mitochondria. An increase was observed in both whole cotyledon respiration and mitochondrial oxygen uptake at the onset of senescence of mung bean cotyledons (day 3 after germination), which thereafter declined gradually. The capacity and activity of the alternative pathway increased markedly in mitochondria isolated from senescent cotyledons. After the onset of senescence, the mung bean cotyledon mitochondria exhibited a decrease both in the respiratory control ratios and ADP:O ratios, and the cotyledons exhibited a gradual decline in energy charge. All these results showed an irreversible deterioration of energy conservation in mung bean cotyledons. The role(s) of the alternative pathway in senescent mung bean cotyledons is discussed.     

Defects in resting metabolic rates and mitochondrial respiration in Kwashiorkor and dietary obese rats

Summary Resting metabolic rates have been measured and compared with hepatic mitochondrial respiration in Kwashiorkor and diet-induced obese weaned rats. In Kwashiorkor, resting metabolic rate was 21% lower than the value of controls, while that of the obese rats was 14% higher than in control animals. The resting metabolic rate for Kwashiorkor animals was 50% of the predicted basal metabolic rate (BMR), whereas that of the obese rats was 23% higher than the predicted BMR. The mitochondrial oxygen consumption patterns, using malate plus glutamate or succinate as respiratory substrates, revealed that the resting respiration (state 4) was 23.9% higher in Kwashiorkor and 29.1% higher in obese animals, while the active (state 3) respiration was 34.8% lower in Kwashiorkor and 43.3% lower in obese rats compared to controls. The respiratory control ratios (RCR) were 51.1% and 43.8% in Kwashiorkor and obese rats, respectively, relative to the values in control rats. It is concluded from these studies that Kwashiorkor disease and diet-induced obesity appear to interfere with oxygen utilization at the level of state 3 mitochondrial respiration, which is markedly decreased when compared to the values for control animals.Abbreviations BMR basal metabolic rate - CPD commercially produced diet - KWD low protein, high carbohydrate diet - OBD high fat diet - PEM protein-energy malnutrition - RCR respiratory control ratios Present address: Department of Biochemistry, University of Nairobi, P.O. Box 30197, Nairobi, Kenya