In situ quantification of mitochondrial respiration in permeabilized fibers of a marine invertebrate with low aerobic capacity

The aim of this study was to design a protocol to allow the assessment of normal and alternative pathways for electron transport in mitochondria using an in situ approach (on permeabilized fibers) in high-resolution respirometry. We measured the oxygen consumption of permeabilized fibers from Nereis (Neanthes) virens with different substrates and the presence of ADP. To estimate the alternative oxidase (AOX) activity, antimycin A was introduced in order to inhibit complex III. Moreover, the apparent complex IV (COX) excess capacity was evaluated using different substrates to assess the implication of this complex in the partitioning of electrons during its progressive inhibition. Our in situ method enabled to quantify the activity of the normal COX pathway as well as the AOX pathway when different substrates were oxidized by either complex I, complex II or both. Using this approach, we confirmed that according to the substrates used, each pathway has a different role and consequently is otherwise involved in the partitioning of electrons through the electron transport system, and suggested that the AOX activity is triggered not only by the redox state of the cell but also by the type of substrates provided to mitochondria.     

烟草叶片中呼吸电子传递途径在缓解叶绿体PSⅡ光抑制中的作用

前期研究发现线粒体交替氧化酶(AOX)呼吸途径对叶绿体光系统II(PSII)的光抑制有明显的缓解作用。线粒体内另一条呼吸途径——细胞色素氧化酶(COX)呼吸途径是否也具有光保护作用尚不清楚。该文通过荧光快速诱导动力学和荧光淬灭分析,解析了烟草(Nicotiana tabacum)叶片中COX途径对PSII光保护的贡献及其与AOX途径的关系。结果表明,强光处理后PSII活性在所有叶片中均下降。AOX途径受抑明显加速了叶片PSII活性的下降。而当COX途径受抑后,叶片PSII活性的下降与水处理的对照叶片无明显差异。当AOX途径与COX途径同时受抑时,叶片PSII活性的下降比单独抑制AOX途径时更严重。此外,呼吸电子传递受抑均导致叶片非光化学淬灭(NPQ)增加,AOX途径受抑导致的NPQ上调比COX途径受抑时更明显,AOX和COX途径同时受抑时NPQ的增幅最大。上述结果表明,烟草叶片中COX途径和AOX途径均参与PSⅡ的光保护。当COX途径受抑时,其光保护功能可以被AOX途径和NPQ补偿,而AOX途径的光保护作用不能被COX途径和NPQ完全补偿。     

Characteristics of external and internal NAD(P)H dehydrogenases in Hoya carnosa mitochondria

This study aims at characterizing NAD(P)H dehydrogenases on the inside and outside of the inner membrane of mitochondria of one phosphoenolpyruvate carboxykinase??crassulacean acid metabolism plant, Hoya carnosa. In crassulacean acid metabolism plants, NADH is produced by malate decarboxylation inside and outside mitochondria. The relative importance of mitochondrial alternative NADH dehydrogenases and their association was determined in intact??and alamethicin??permeabilized mitochondria of H. carnosa to discriminate between internal and external activities. The major findings in H. carnosa mitochondria are: (i) external NADPH oxidation is totally inhibited by DPI and totally dependent on Ca²⁺, (ii) external NADH oxidation is partially inhibited by DPI and mainly dependent on Ca²⁺, (iii) total NADH oxidation measured in permeabilized mitochondria is partially inhibited by rotenone and also by DPI, (iv) total NADPH oxidation measured in permeabilized mitochondria is partially dependent on Ca²⁺ and totally inhibited by DPI. The results suggest that complex I, external NAD(P)H dehydrogenases, and internal NAD(P)H dehydrogenases are all linked to the electron transport chain. Also, the total measurable NAD(P)H dehydrogenases activity was less than the total measurable complex I activity, and both of these enzymes could donate their electrons not only to the cytochrome pathway but also to the alternative pathway. The finding indicated that the H. carnosa mitochondrial electron transport chain is operating in a classical way, partitioning to both Complex I and alternative Alt. NAD(P)H dehydrogenases.     

Molecular Genetic Alteration of Plant Respiration (Silencing and Overexpression of Alternative Oxidase in Transgenic Tobacco)

The alternative oxidase (AOX) of plant mitochondria is encoded by the nuclear gene Aox1. Sense and antisense DNA constructs of Nicotiana tabacum Aox1 were introduced into tobacco, and transgenic plants with both increased and decreased levels of mitochondrial AOX protein were identified. Suspension cells derived from wild-type and transgenic plants were grown in heterotrophic batch culture. Transgenic cells with increased AOX protein had an increased capacity for cyanide-resistant, salicylhydroxamic acid-sensitive respiration compared to wild-type cells, whereas transgenic cells with decreased AOX protein had a decreased capacity for such respiration. Thus, genetic alteration of the level of AOX protein was sufficient to alter the capacity for electron transport through the alternative pathway. Under our standard growth conditions, "antisense" cells with dramatically reduced levels of AOX protein had growth and respiration rates similar to the wild type. However, whereas wild-type cells were able to grow under conditions that severely suppressed cytochrome pathway activity, antisense cells could not survive this treatment. This suggests that a critical function of AOX may be to support respiration when the cytochrome pathway is impaired. The much higher level of AOX protein in "sense" cells compared to the wild type did not appreciably alter the steady-state partitioning of electrons between the cytochrome path and the alternative pathway in vivo, suggesting that this partitioning may be subject to additional regulatory factors.     

Sensitivities of the alternative respiratory components of potato tuber mitochondria to thiol reagents and Ca2+.

Plant mitochondria differ from those of mammals, since they incorporate an alternative electron transport pathway, which branches at ubiquinol to an alternative oxidase (AOX), characteristically inhibited by salicylhydroxamic acid (SHAM). Another feature of plant mitochondria is that besides complex I (EC 1.6.5.3) they possess alternative NAD(P)H-dehydrogenases insensitive to rotenone. Many stress conditions are known to alter the expression of the alternative electron transport pathway in plant mitochondria. In the present study we investigated the effects of some thiol reagents and Ca(2+) on potato mitochondrial respiratory chain presenting different activities of the alternative respiratory components AOX and external NADH dehydrogenase, a condition induced by previous treatment of potato tubers (Solanum tuberosum L., cv. Bintje) to cold stress. The results showed that Ca(2+) presented an inhibitory effect on AOX pathway in potato mitochondria energized with NADH or succinate, which was only now observed when the cytochrome pathway was inhibited by cyanide. When the cytochrome pathway was functional, Ca(2+) stimulated the external NADH dehydrogenase. Diamide was a potent AOX inhibitor and this effect was only now observed when the cytochrome pathway was inactive, as was the case for the calcium ion. Mersalyl inhibited the externally located NADH dehydrogenase and had no effect on AOX activity. The results may represent an important function of Ca(2+) on the alternative mitochondrial enzymes NADH-DH(ext) and AOX.     

Partitioning of respiratory electrons in the dark in leaves of transgenic tobacco with modified levels of alternative oxidase

This study examined tobacco ( Nicotiana tabacum cv. Petit Havana SR1) leaf respiration in the dark, utilizing both wild-type plants and transgenic plants with increased or decreased levels of alternative oxidase (AOX) protein. AOX represents a non-energy-conserving branch in mitochondrial electron transport. Inhibitor studies showed that the maximum possible flux of electrons to AOX (AOX capacity) correlated with the level of AOX protein present in the different plant lines. A comparison of the plants using online ¹⁸O isotope discrimination was done to determine whether AOX protein level would impact the actual steady-state partitioning of electrons to AOX (AOX engagement). Under a range of pretreatment and measurement conditions, there was little if any effect of AOX protein level on the degree of engagement. This suggests that the metabolic conditions inherent to a particular growth condition and/or the biochemical regulatory properties of AOX itself are the critical factors that control partitioning. Interestingly, we found that measurement temperature and water status are parameters that may have some influence over AOX engagement.     

Seasonal variation of leaf respiration and the alternative pathway in field-grown Populus × canadensis

The temperature response of plant respiration varies between species and can acclimate to changing temperatures. Mitochondrial respiration in plants has two terminal oxidases: the cytochrome c oxidase (COX) and the cyanide-resistant alternative oxidase (AOX). In Populus × canadensis var. italica, a deciduous tree species, we investigated the temperature response of leaf respiration via the alternative and cytochrome pathways, as well as seasonal changes in these pathways, using the oxygen isotope fractionation technique. The electron partitioning through the alternative pathway (τ(a) ) increased from 0 to 30-40% with measurement temperatures from 6 to 30°C at all times measured throughout the growing season. τ(a) at the growth temperature (the average temperature during 3 days prior to sampling) increased from 12 to 29% from spring until late summer and decreased thereafter. Total respiration declined throughout the growing season by 50%, concomitantly with decreases in both AOX (64%) and COX (32%) protein abundances. Our results provide new insight into the natural variability of AOX protein abundances and alternative respiration electron partitioning over immediate and seasonal timescales.     

The gene for alternative oxidase-2 (AOX2) from Arabidopsis thaliana consists of five exons unlike other AOX genes and is transcribed at an early stage during germination.

We investigated the expressions of genes for alternative oxidase (AOX1a, AOX1b, AOX1c and AOX2) and genes for cytochrome c oxidase (COX5b and COX6b) during germination of Arabidopsis thaliana, and examined oxygen uptakes of the alternative respiration and the cytochrome respiration in imbibed Arabidopsis seeds. A Northern blot analysis showed that AOX2 mRNA has already accumulated in dry seeds and subsequently decreased, whereas accumulation ofAOX1a mRNA was less abundant from 0 hours to 48 hours after imbibition and then increased. The increase of the capacity of the alternative pathway appeared to be dependent on the expressions of both AOX2 and AOX1a. On the other hand, steady-state mRNA levels of COX5b and COX6b were gradually increased during germination, and the capacity of the cytochrome pathway was correlated with the increase of expressions of the COX genes. Antimycin A, the respiratory inhibitor, strongly increased the expression of AOX1a but had no effect on the expression of AOX2. A 5'RACE analysis showed that AOX2 consists of five exons, which is different from the case of most AOX genes identified so far. Analysis of subcellular localization of AOX2 using green fluorescent protein indicated that the AOX2 protein is imported into the mitochondria.     

The Regulation of Electron Partitioning between the Cytochrome and Alternative Pathways in Soybean Cotyledon and Root Mitochondria

The regulation of electron partitioning between the cytochrome (Cyt) and alternative pathways in soybean (Glycine max L. cv Ransom) mitochondria in the absence of added inhibitors has been studied using the oxygen isotope fractionation technique. This regulation can depend on several factors, including the amount of alternative oxidase protein, the redox status of the alternative oxidase regulatory sulfhydryl-disulfide system, the degree of activation by [alpha]-keto acids, and the concentration and redox state of the ubiquinone pool. We studied electron partitioning onto the alternative pathway in mitochondria isolated from etiolated and light-grown cotyledons and roots to ascertain how these factors interact in different tissues. In light-grown cotyledon mitochondria there is some partitioning to the alternative pathway in state 4, which is increased dramatically by either pyruvate or dithiothreitol. In etiolated cotyledon mitochondria, the alternative pathway shows little ability to compete for electrons with the Cyt pathway under any circumstances. In root mitochondria, control of alternative pathway activity is exercised by both the ubiquinone pool and the regulatory sulfhydryl-disulfide system. In addition, oxygen isotope fractionation by the Cyt and alternative pathways in mitochondria were identical to the fractionation for the respective pathways seen in intact tissue, suggesting that residual respiration is not present in the absence of inhibitors.     

Leaf respiration and alternative oxidase in field-grown alpine grasses respond to natural changes in temperature and light

? We report the first investigation of changes in electron partitioning via the alternative respiratory pathway (AP) and alternative oxidase (AOX) protein abundance in field-grown plants and their role in seasonal acclimation of respiration. ? We sampled two alpine grasses native to New Zealand, Chionochloa rubra and Chionochloa pallens, from field sites of different altitudes, over 1 yr and also intensively over a 2-wk period. ? In both species, respiration acclimated to seasonal changes in temperature through changes in basal capacity (R??) but not temperature sensitivity (E?). In C. pallens, acclimation of respiration may be associated with a higher AOX : cytochrome c oxidase (COX) protein abundance ratio. Oxygen isotope discrimination (D), which reflects relative changes in AP electron partitioning, correlated positively with daily integrated photosynthetically active radiation (PAR) in both species over seasonal timescales. Respiratory parameters, the AOX : COX protein ratio and D were stable over a 2-wk period, during which significant temperature changes were experienced in the field. ? We conclude that respiration in Chionochloa spp. acclimates strongly to seasonal, but not to short-term, temperature variation. Alternative oxidase appears to be involved in the plant response to both seasonal changes in temperature and daily changes in light, highlighting the complexity of the function of AOX in the field.     

Analysis of Respiratory Chain Regulation in Roots of Soybean Seedlings

Changes in the respiratory rate and the contribution of the cytochrome (Cyt) c oxidase and alternative oxidase (COX and AOX, respectively) were investigated in soybean (Glycine max L. cv Stevens) root seedlings using the ¹⁸O-discrimination method. In 4-d-old roots respiration proceeded almost entirely via COX, but by d 17 more than 50% of the flux occurred via AOX. During this period the capacity of COX, the theoretical yield of ATP synthesis, and the root relative growth rate all decreased substantially. In extracts from whole roots of different ages, the ubiquinone pool was maintained at 50% to 60% reduction, whereas pyruvate content fluctuated without a consistent trend. In whole-root immunoblots, AOX protein was largely in the reduced, active form at 7 and 17 d but was partially oxidized at 4 d. In isolated mitochondria, Cyt pathway and succinate dehydrogenase capacities and COX I protein abundance decreased with root age, whereas both AOX capacity and protein abundance remained unchanged. The amount of mitochondrial protein on a dry-mass basis did not vary significantly with root age. It is concluded that decreases in whole-root respiration during growth of soybean seedlings can be largely explained by decreases in maximal rates of electron transport via COX. Flux via AOX is increased so that the ubiquinone pool is maintained in a moderately reduced state.     

Electron Partitioning between the Cytochrome and Alternative Pathways in Plant Mitochondria

The contribution of the cyanide-resistant, alternative pathway to plant mitochondrial electron transport has been studied using a modified aqueous phase on-line mass spectrometry-gas chromatography system. This technique permits direct measurement of the partitioning of electrons between the cytochrome and alternative pathways in the absence of added inhibitors. We demonstrate that in mitochondria isolated from soybean (Glycine max L. cv Ransom) cotyledons, the alternative pathway contributes significantly to oxygen uptake under state 4 conditions, when succinate is used as a substrate. However, when NADH is the substrate, addition of pyruvate, an allosteric activator of the alternative pathway, is required to achieve the same level of alternative pathway activity. Under state 3 conditions, when the reduction state of the ubiquinone pool is low, the addition of pyruvate allows the alternative pathway to compete with the cytochrome pathway for electrons from the ubiquinone pool when the cytochrome pathway is not saturated. These results provide direct experimental verification of the kinetics consequences of pyruvate addition on the partitioning of electron flow between the two respiratory pathways. This distribution of electrons between the two unsaturated pathways could not be measured using conventional oxygen electrode methods and illustrates a clear advantage of the mass spectrometry technique. These results have significant ramifications for studies of plant respiration using the oxygen electrode, particularly those studies involving intact tissues.     

Identification and characterization of an alternative oxidase in the entomopathogenic fungus Metarhizium anisopliae

Mitochondria of Metarhizium anisopliae contain an alternative oxidase (AOX), which reduces oxygen to water by accepting electrons directly from ubiquinol. AOX activity is demonstrated in situ as a constitutive enzyme. Greatest activity of AOX appears at the beginning and at the end of the fungal developmental cycle, germination of aerial conidia and the formation of submerged conidia, respectively. Changes in nutritional conditions, e.g., the presence of host insect cuticle or nutrient starvation had no effect on the induction of AOX activity. Antimycin A, an electron transport chain inhibitor, induced AOX activity. Cloning of the AOX DNA and the alignment of the deduced amino acid sequence of a segment of the AOX gene from M. anisopliae shows structural similarities with other AOX sequences with differing levels of variation when compared with homologous sequences from plants, yeasts, and filamentous fungi. Alternative oxidase in entomopathogenic fungi may have a positive contribution to ecological fitness.     

Lack of respiratory chain complex I impairs alternative oxidase engagement and modulates redox signaling during elicitor-induced cell death in tobacco

Alternative oxidase (AOX) functions in stress resistance by preventing accumulation of reactive oxygen species (ROS), but little is known about in vivo partitioning of electron flow between AOX and the cytochrome pathway. We investigated the relationships between AOX expression and in vivo activity in Nicotiana sylvestris and the complex I-deficient CMSII mutant in response to a cell death elicitor. While a specific AOX1 isoform in the active reduced state was constitutively overexpressed in CMSII, partitioning through the alternative pathway was similar to the wild type. Lack of correlation between AOX content and activity indicates severe metabolic constraints in nonstressed mutant leaves. The bacterial elicitor harpin N(Ea) induced similar timing and extent of cell death and a twofold respiratory burst in both genotypes with little change in AOX amounts. However, partitioning to AOX was increased twofold in the wild type but remained unchanged in CMSII. Oxidative phosphorylation modeling indicated a twofold ATP increase in both genotypes. By contrast, mitochondrial superoxide dismutase activity and reduced forms of ascorbate and glutathione were higher in CMSII than in the wild type. These results demonstrate genetically programmed flexibility of plant respiratory routes and antioxidants in response to elicitors and suggest that sustained ATP production, rather than AOX activity by itself or mitochondrial ROS, might be important for in planta cell death.     

The alternative oxidase of Yarrowia lipolytica mitochondria is unable to compete with the cytochrome pathway for electrons

The activity of the cyanide-resistant alternative oxidase (pathway) of Y. lipolytica mitochondria was studied as a function of the activity of the major, cyanide-sensitive, cytochrome pathway. The contribution of the alternative oxidase to the total respiration of mitochondria was evaluated by measuring the rate of oxygen consumption in the presence of cyanide (an inhibitor of the cytochrome pathway). The potential activity of the cytochrome pathway was evaluated spectrophotometrically, by measuring the oxidation rate of cytochrome c by ferricyanide, which accepts electrons from complex III (cytochrome c) of this pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was accompanied by oxygen consumption due to the transfer of electrons through the alternative pathway. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation in the cytochrome pathway) completely inhibited the consumption of oxygen by the mitochondria. Under these conditions, the inhibition of the alternative pathway by benzohydroxamic acid failed to affect the transfer of electrons from cytochrome c to ferricyanide. Benzohydroxamic acid did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These findings indicate that the alternative pathway is unable to compete with the cytochrome respiratory chain for electrons. The alternative pathway transfers only electrons that are superfluous for the cytochrome chain.     

The mitochondrial respiratory chain of Ustilago maydis

Ustilago maydis mitochondria contain the four classical components of the electron transport chain (complexes I, II, III, and IV), a glycerol phosphate dehydrogenase, and two alternative elements: an external rotenone-insensitive flavone-sensitive NADH dehydrogenase (NDH-2) and an alternative oxidase (AOX). The external NDH-2 contributes as much as complex I to the NADH-dependent respiratory activity, and is not modulated by Ca²⁺, a regulatory mechanism described for plant NDH-2, and presumed to be a unique characteristic of the external isozyme. The AOX accounts for the 20% residual respiratory activity after inhibition of complex IV by cyanide. This residual activity depends on growth conditions, since cells grown in the presence of cyanide or antimycin A increase its proportion to about 75% of the uninhibited rate. The effect of AMP, pyruvate and DTT on AOX was studied. The activity of AOX in U. maydis cells was sensitive to AMP but not to pyruvate, which agrees with the regulatory characteristics of a fungal AOX. Interestingly, the presence of DTT during cell permeabilisation protected the enzyme against inactivation.The pathways of quinone reduction and quinol oxidation lack an additive behavior. This is consistent with the competition of the respiratory components of each pathway for the quinol/quinone pool.     

Mitochondrial complex I impairment and differential carbon monoxide sensitivity of cytochrome c oxidase in wild type and CMS II mutants of Nicotiana sylvestris

Plant mitochondria unlike their animal counterpart have some unique features with highly branched respiratory chain. The present work was undertaken in order to investigate the effect of loss/dysfunction of plant mitochondrial complex I on the relative flux of electrons through alternative oxidase (AOX) and cytochrome oxidase. Loss of a major subunit of mitochondrial complex I in cytoplasmic male sterile II (CMS II) mutant of Nicotiana sylvestris caused respiratory redox perturbations, as evident from the differential CO sensitivity of cytochrome oxidase. The leaf segments of CMS II mutant when exposed to CO under dark aerobic condition were insensitive to the inhibition of cytochrome oxidase, as against the wild type (WT). The differential CO response of WT and CMS II mutants appeared to be due to differences in the redox state of cytochrome a3 (cyt a3), the terminal electron acceptor during in situ respiration. Cyt a3 appeared to be more in its oxidized form in CMS II and hence unable to form cyt a3-CO complex. Pre-treatment of CMS II leaves with 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation increased the CO response. The slight increase in rotenone-insensitive respiration of CMS II could be attributed partly to enhanced flux of electrons through cytochrome pathway to compensate for the loss of phosphorylation site and partly through AOX, which was induced by nitrate.     

An Off-Line Implementation of the Stable Isotope Technique for Measurements of Alternative Respiratory Pathway Activities

In situ measurements of alternative respiratory pathway activity are needed to provide insight into the energy efficiency of plant metabolism under various conditions in the field. The only reliable method at present to measure alternative oxidase (AOX) activity is through measurement of changes in delta(18)O(O(2)), which to date has only been used in laboratory environments. We have developed a cuvette system to measure partitioning of electrons to AOX that is suitable for off-line use and for field experiments. Plant samples are enclosed in airtight cuvettes and O(2) consumption is monitored. Gas samples from the cuvette are stored in evacuated gas containers until measurement of delta(18)O(O(2)). We have validated this method using differing plant material to assess AOX activity. Fractionation factors were calculated from delta(18)O(O(2)) measurements, which could be measured with an accuracy and precision to 0.1 per thousand and 0.3 per thousand, respectively. Potential sources of error are discussed and quantified. Our method provides results similar to those obtained with laboratory incubations on-line to a mass spectrometer but greatly increases the potential for adoption of the stable isotope method.