Factors affecting determination of superoxide anion generated by mitochondria from barley roots after anaerobiosis

During the post-hypoxic period, symptoms of oxidative stress and activation of enzymatic and non-enzymatic antioxidant systems were observed in several plant tissues. In the roots, mitochondrial respiratory chain is the main source of ROS. Superoxide anion radical is formed in the mitochondrial electron-transport chain at the level of Complexes I and III. The purpose of this work was to estimate superoxide anion production by the mitochondria isolated after a period of hypoxic treatment. Seedlings of barley (Hordeum vulgare L.) were grown on a nutrient medium flushed for 5 d with air (control) or nitrogen (hypoxia) and then transferred for 24 h to aerated medium (post-hypoxia). Production of superoxide anion by the mitochondria was measured by SOD-inhibitable oxidation of adrenaline to adrenochrome with NADH as a respiratory substrate. Hypoxic treatment increased mitochondrial activity but decreased mitochondrial superoxide anion appearance outside the mitochondrial membrane as compared to the mitochondria isolated from the roots continuously grown on aerated medium. The result of lower superoxide anion determination is attributed to increased antioxidants concentration during hypoxia. This was confirmed by inhibition of O₂⁻ production by exogenous GSH and stimulation by addition of 1-chloro-2,4-dinitrobenzene (CDNB), which depleted endogenous mitochondrial GSH.     

Nitrite reduction and superoxide-dependent nitric oxide degradation by Arabidopsis mitochondria: Influence of external NAD(P)H dehydrogenases and alternative oxidase in the control of nitric oxide levels

Mitochondria recently have emerged as important sites in controlling NO levels within the cell. In this study, the synthesis of nitric oxide (NO) from nitrite and its degradation by mitochondria isolated from Arabidopsis thaliana were examined. Oxygen and NO concentrations in the reaction medium were measured with specific electrodes. Nitrite inhibited the respiration of isolated A. thaliana mitochondria, in competition with oxygen, an effect that was abolished or potentiated when electron flow occurred via alternative oxidase (AOX) or cytochrome c oxidase (COX), respectively. The production of NO from nitrite was detected electrochemically only under anaerobiosis because of a superoxide-dependent process of NO degradation. Electron leakage from external NAD(P)H dehydrogenases contributed the most to NO degradation as higher rates of Amplex Red-detected H₂O₂ production and NO consumption were observed in NAD(P)H-energized mitochondria. Conversely, the NO-insensitive AOX diminished electron leakage from the respiratory chain, allowing the increase of NO half-life without interrupting oxygen consumption. These results show that the accumulation of nitric oxide derived from nitrite reduction and the superoxide-dependent mechanism of NO degradation in isolated A. thaliana mitochondria are influenced by the external NAD(P)H dehydrogenases and AOX, revealing a role for these alternative proteins of the mitochondrial respiratory chain in the control of NO levels in plant cells.     

Regulation of alternative oxidase activity during phosphate deficiency in bean roots (Phaseolus vulgaris)

Cyanide-resistant respiration was studied in mitochondria isolated from the roots of bean plants ( Phaseolus vulgaris L. cv. Złota Saxa) grown hydroponically up to 16 days on a phosphate-sufficient (+P, control) or phosphate-deficient (−P) medium. Western blotting indicated that the alternative oxidase (AOX) was present only in its reduced (active) form, both in phosphate-sufficient and phosphate-deficient roots, but in the latter, the amount of AOX protein was greater. Addition of pyruvate to the isolation, washing and reaction media made mitochondria from +P roots cyanide-insensitive, similar to mitochondria from −P roots. The doubled activity of NAD-malic enzyme (NAD-ME) in −P compared with +P root mitochondria may suggest increased pyruvate production in −P mitochondria. Lower cytochrome c oxidase (COX) activity and no uncoupler effect on respiration indicated limited cytochrome chain activity in −P mitochondria. In −P mitochondria, the oxygen uptake decreased and the level of Q reduction increased from 60 to 80%. With no pyruvate present (AOX not fully activated), inhibition of the cytochrome pathway resulted in an increased level of the ratio of reduced ubiquinone (Qr) to total ubiquinone (Qt) (Qr/Qt) in +P mitochondria, but did not change Qr/Qt in −P mitochondria. When pyruvate was present, the kinetics for AOX were similar in mitochondria from −P and +P roots. It is suggested that AOX participation in −P respiration may provide an acclimation to phosphate deficiency. Stabilization of the ubiquinone reduction level by AOX might prevent the harmful effect of an increased formation of reactive oxygen species.     

Functional expression of plant alternative oxidase decreases antimycin A-induced reactive oxygen species production in human cells

Alternative oxidase (AOX) plays a pivotal role in cyanide-resistance respiration in the mitochondria of plants, fungi and some protists. Here we show that AOX from thermogenic skunk cabbage successfully conferred cyanide resistance to human cells. In galactose medium, HeLa cells with mitochondria-targeted AOX proteins were found to have significantly less reactive oxygen species production in response to antimycin-A exposure, a specific inhibitor of respiratory complex III. These results suggest that skunk cabbage AOX can be used to create an alternative respiration pathway, which might be important for therapy against various mitochondrial diseases.     

The effect of phosphate deficiency on mitochondrial activity and adenylate levels in bean roots

Bean plants ( Phaseolus vulgaris ) were grown for 16–20 days with or without phosphate in Knop nutrient medium. It was found in previous experiments that for roots grown on a P_i-deficient medium respiration is mainly carried out by the cyanide-insensitive pathway. Mitochondria isolated from—P_i, roots had poor respiratory control and their respiration exhibited 62% inhibition by cyanide and was inhibited (30%) by salicylhydroxamic acid (SHAM). In contrast, mitochondria obtained with control (+P_i) roots had respiratory control and ADP/O ratios typical for succinate as the substrate; their respiration was inhibited to 95% by cyanide and insensitive to SHAM. The integrity of mitochondrial membranes was similar in both types of mitochondria. Cytochrome oxidase activity, however, was about 20% lower in -P_i mitochondria, but the cytochrome composition was the same in both types of mitochondria. The cytochrorae pathway was not operating at full capacity in mitochondria isolated from—P_i roots but the alternative oxidation pathway participated in a great part in mitochondrial respiration, similar to in vivo whole roots. The participation of the non-phosphorylating., alternative pathway decreased the respiratory control ratio in mitochondria and had an effect on the total adenine nucleotide pool and energy charge values which were lower (16 and 13% respectively) in -P_i roots. About 50% lower ADP and 20% lower ATP levels were observed whereas AMP levels were several times higher.     

Properties of mitochondria isolated from cyanide-sensitive and cyanide-stimulated cultures of Acanthamoeba castellanii

1. Mitochondria isolated from cultures of Acanthamoeba castellanii exhibit respiratory control and oxidize alpha-oxoglutarate, succinate and NADH with ADP:O ratios of about 2.4, 1.4 and 1.25 respectively. 2. Mitochondria from cultures of which the respiration was stimulated up to 50% by 1mm-cyanide (type-A mitochondria) and from cyanide-sensitive cultures (type-B mitochondria) had similar respiratory-control ratios and ADP:O ratios. 3. State-3 rates of respiration were generally more cyanide-sensitive than State-4 rates, and the respiration of type-A mitochondria was more cyanide-resistant than that of type-B mitochondria. 4. Salicylhydroxamic acid alone had little effect on respiratory activities of either type of mitochondria, but when added together with cyanide, irrespective of the order of addition, inhibition was almost complete. 5. Oxidation of externally added NADH by type-A mitochondria was mainly via an oxidase with a low affinity for oxygen (K(m)[unk]15mum), which was largely cyanide-sensitive and partially antimycin A-sensitive; this electron-transport pathway was inhibited by ADP. 6. Cyanide-insensitive but salicylhydroxamic acid-sensitive respiration was stimulated by AMP and ADP, and by ATP after incubation in the presence of MgCl(2). 7. Addition of rotenone to mitochondria oxidizing alpha-oxoglutarate lowered the ADP:O ratios by about one-third and rendered inhibition by cyanide more complete. 8. The results suggest that mitochondria of A. castellanii possess branched pathways of electron transport which terminate in three separate oxidases; the proportions of electron fluxes via these pathways vary at different stages of growth.     

The Alternative Respiratory Pathway of Euglena Mitochondria

Mitochondria, isolated from heterotrophic Euglena gracilis , have cyanide-resistant alternative oxidase (AOX) in their respiratory chain. Cells cultured under a variety of oxidative stress conditions (exposure to cyanide, cold, or H2O2) increased the AOX capacity in mitochondria and cells, although it was significant only under cold stress; AOX sensitivity to inhibitors was also increased by cold and cyanide stress. The value of AOX maximal activity reached 50% of total respiration below 20 degrees C, whereas AOX full activity was only 10-30% of total respiration above 20 degrees C. The optimum pH for AOX activity was 6.5 and for the cytochrome pathway was 7.3. GMP, AMP, pyruvate, or DTT did not alter AOX activity. The reduction level of the quinone pool was higher in mitochondria from cold-stressed than from control cells; furthermore, the content of reduced glutathione was lower in cold-stressed cells. Growth in the presence of an AOX inhibitor was not affected in control cells, whereas in cold-stressed cells, growth was diminished by 50%. Cyanide diminished growth in control cells by 50%, but in cold-stressed cells this inhibitor was ineffective. The data suggest that AOX activity is part of the cellular response to oxidative stress in Euglena .     

Inhibition of aconitase by nitric oxide leads to induction of the alternative oxidase and to a shift of metabolism towards biosynthesis of amino acids

Nitric oxide (NO) is a free radical molecule involved in signalling and in hypoxic metabolism. This work used the nitrate reductase double mutant of Arabidopsis thaliana (nia) and studied metabolic profiles, aconitase activity, and alternative oxidase (AOX) capacity and expression under normoxia and hypoxia (1% oxygen) in wild-type and nia plants. The roots of nia plants accumulated very little NO as compared to wild-type plants which exhibited ～20-fold increase in NO emission under low oxygen conditions. These data suggest that nitrate reductase is involved in NO production either directly or by supplying nitrite to other sites of NO production (e.g. mitochondria). Various studies revealed that NO can induce AOX in mitochondria, but the mechanism has not been established yet. This study demonstrates that the NO produced in roots of wild-type plants inhibits aconitase which in turn leads to a marked increase in citrate levels. The accumulating citrate enhances AOX capacity, expression, and protein abundance. In contrast to wild-type plants, the nia double mutant failed to show AOX induction. The overall induction of AOX in wild-type roots correlated with accumulation of glycine, serine, leucine, lysine, and other amino acids. The findings show that NO inhibits aconitase under hypoxia which results in accumulation of citrate, the latter in turn inducing AOX and causing a shift of metabolism towards amino acid biosynthesis.     

Reduction of superoxide production by mitochondria oxidizing NADH in the presence of organic acids

Effects of multiple substrates on oxygen uptake and superoxide production by mitochondria isolated from the pericarp tissue of green bell pepper (Capsicum annuum L.) were studied. Mitochondria isolated from peppers stored at 4 °C for 5 and 6 days had higher rates of oxygen uptake and were less sensitive to cyanide than mitochondria isolated from freshly harvested peppers. Succinate enhanced state 2 and state 4 rates of oxygen uptake with exogenous NADH in the absence of cytochrome path inhibitors, but not state 3 rates by mitochondria isolated from either freshly harvested or cold-stored bell peppers. The sensitivity of NADH oxidation to cyanide was reduced by both malate and succinate in mitochondria from cold-stored bell peppers, whereas only succinate was effective in mitochondria from freshly harvested peppers.Mitochondria isolated from both freshly harvested peppers and those stored at 4 °C for 5 and 6 days produced superoxide in the absence of exogenous substrates. Superoxide production by mitochondria from freshly harvested bell peppers increased when the mitochondria were supplied with malate, succinate or NADH, but only NADH enhanced superoxide production by mitochondria from cold-stored peppers. Both succinate and malate reduced the production of superoxide by mitochondria isolated from cold-stored bell peppers. Succinate and malate as second substrates also reduced the production of superoxide with NADH by mitochondria from both freshly harvested and cold-stored bell peppers. Malonate, a competitive inhibitor of succinate dehydrogenase, was inhibitory to oxygen uptake and to superoxide production.Mitochondria isolated from cold-stored bell peppers converted succinate to pyruvate at 25 °C at considerably higher rates than those of mitochondria from freshly harvested bell peppers. Since pyruvate has been shown to activate the alternative oxidase and the presence of pyruvate is essential for continued alternative oxidase activity, we suggest that pyruvate limits superoxide production by enhancing the flow of electrons through the alternative path. A direct scavenging of superoxide by succinate, malate and pyruvate, however, cannot be ruled out.     

Cyanide-resistant, ATP-synthesis-sustained, and uncoupling-protein-sustained respiration during postharvest ripening of tomato fruit

Tomato (Lycopersicon esculentum) mitochondria contain both alternative oxidase (AOX) and uncoupling protein as energy-dissipating systems that can decrease the efficiency of oxidative phosphorylation. We followed the cyanide (CN)-resistant, ATP-synthesis-sustained, and uncoupling-protein-sustained respiration of isolated mitochondria, as well as the immunologically detectable levels of uncoupling protein and AOX, during tomato fruit ripening from the mature green stage to the red stage. The AOX protein level and CN-resistant respiration of isolated mitochondria decreased with ripening from the green to the red stage. The ATP-synthesis-sustained respiration followed the same behavior. In contrast, the level of uncoupling protein and the total uncoupling-protein-sustained respiration of isolated mitochondria decreased from only the yellow stage on. We observed an acute inhibition of the CN-resistant respiration by linoleic acid in the micromolar range. These results suggest that the two energy-dissipating systems could have different roles during the ripening process.     

Characteristics of External NADH Oxidation by Beetroot Mitochondria

Mitochondria isolated from fresh red beetroot (Beta vulgaris L.) tissue do not oxidize external NADH with O₂ as the electron acceptor. These mitochondria have a rotenone- and antimycin-insensitive pathway of NADH oxidation associated with the outer membrane and are capable of reducing cytochrome c or potassium ferricyanide. They are also capable of oxidizing internal NADH via the inner membrane electron transport chain with normal rotenone and antimycin sensitivity and ADP/O ratios. They differ from other plant mitochondria in the apparent lack of the NADH dehydrogenase located on the outer surface of the inner membrane. It is shown that this activity develops during the aging of red beetroot slices in aerated dilute CaSO₄ solutions, and is present in the mitochondria isolated from aged tissue.     

Cyanide-Resistant Respiration in Suspension Cultured Cells of Nicotiana glutinosa L

The respiration of dark-grown Nicotiana glutinosa L. cells in liquid suspension culture was found to be highly cyanide resistant and salicylhydroxamic acid (SHAM) sensitive, indicative of an active alternative respiratory pathway. This was especially true during the lag and logarithmic phases of the 14-day growth cycle. Mitochondria isolated from logarithmically growing cells exhibited active oxidation of malate, succinate, and exogenous NADH. Oxidation of all three substrates had an optimum pH of 6.5 and all were highly resistant to inhibited by cyanide and sensitive to SHAM. Respiratory control was exhibited by all three substrates but only if SHAM was present to block the alternative pathway and divert electrons to the phosphorylating cytochrome pathway. The cyanide-resistant oxidation of exogenous NADH has previously only been associated with Arum spadix mitochondria. Coemergence during evolution of the alternative respiratory pathway and the exogenous NADH dehydrogenase in plant mitochondria as a possible mechanism for removal of cytoplasmic NADH is proposed. Evidence is presented which suggests that mitochondrial assays should be performed at pH 6.5.     

Superoxide production by mitochondria isolated from green bell pepper fruit

Evidence is increasing to suggest that a wide range of environmentally induced plant disorders, including chilling injury, is mediated by reactive oxygen species produced during stress or upon relief from stress. Mitochondria were isolated from pericarp tissue of chilling-sensitive bell pepper fruit and their respiratory activity and ability to produce superoxide when supplied with NADH, succinate or malate-pyruvate were determined. Oxygen uptake rates were greater and less sensitive to cyanide with succinate than with NADH; rates increased and sensitivity to cyanide and respiratory control ratios (RCRs) decreased in fruit stored at 2°C. Disrupting mitochondrial membranes led to increased oxygen consumption with NADH and decreased consumption with succinate. resulting in RCRs of approximately 1 with both substrates. Superoxide production was greater with NADH than with either succinate or malatepyruvate. Superoxide dismutase and cyanide inhibited superoxide production almost completely. Antimycin A did not inhibit superoxide production with NADH, but did partially with succinate, especially in mitochondria sensitive to cyanide. Disrupting mitochondrial membranes enhanced superoxide production with NADH. Superoxide production by mitochondria isolated from fruit stored at 2°C increased with NADH and decreased with succinate. Results provide evidence that mitochondria may be a major source of superoxide in chilling-sensitive plant tissues exposed to low temperatures.     

Changes in oxidative properties of Kalanchoe blossfeldiana leaf mitochondria during development of Crassulacean acid metabolism

Kalanchoe blossfeldiana plants grown under long days (16 h light) exhibit a C₃-type photosynthetic metabolism. Switching to short days (9 h light) leads to a gradual development of Crassulacean acid metabolism (CAM). Under the latter conditions, dark CO₂ fixation produces large amounts of malate. During the first hours of the day, malate is rapidly decarboxylated into pyruvate through the action of a cytosolic NADP⁺-or a mitochondrial NAD⁺-dependent malic enzyme. Mitochondria were isolated from leaves of plants grown under long days or after treatment by an increasing number of short days. Tricarboxylic acid cycle intermediates as well as exogenous NADH and NADPH were readily oxidized by mitochondria isolated from the two types of plants. Glycine, known to be oxidized by C₃-plant mitochondria, was still oxidized after CAM establishment. The experiments showed a marked parallelism in the increase of CAM level and the increase in substrate-oxidation capacity of the isolated mitochondria, particularly the capacity to oxidize malate in the presence of cyanide. These simultaneous variations in CAM level and in mitochondrial properties indicate that the mitochondrial NAD⁺-malic enzyme could account at least for a part of the oxidation of malate. The studies of whole-leaf respiration establish that mitochondria are implicated in malate degradation in vivo. Moreover, an increase in cyanide resistance of the leaf respiration has been observed during the first daylight hours, when malate was oxidized to pyruvate by cytosolic and mitochondrial malic enzymes.Abbreviations CAM Crassulacean acid metabolism - MDH malate dehydrogenase - ME malic enzyme     

Light intensity affects chlorophyll synthesis during greening process by metabolite signal from mitochondrial alternative oxidase in Arabidopsis

Although mitochondrial alternative oxidase (AOX) has been proposed to play essential roles in high light stress tolerance, the effects of AOX on chlorophyll synthesis are unclear. Previous studies indicated that during greening, chlorophyll accumulation was largely delayed in plants whose mitochondrial cyanide‐resistant respiration was inhibited by knocking out nuclear encoded AOX gene. Here, we showed that this delay of chlorophyll accumulation was more significant under high light condition. Inhibition of cyanide‐resistant respiration was also accompanied by the increase of plastid NADPH/NADP⁺ ratio, especially under high light treatment which subsequently blocked the import of multiple plastidial proteins, such as some components of the photosynthetic electron transport chain, the Calvin–Benson cycle enzymes and malate/oxaloacetate shuttle components. Overexpression of AOX1a rescued the aox1a mutant phenotype, including the chlorophyll accumulation during greening and plastidial protein import. It thus suggests that light intensity affects chlorophyll synthesis during greening process by a metabolic signal, the AOX‐derived plastidial NADPH/NADP⁺ ratio change. Further, our results thus revealed a molecular mechanism of chloroplast–mitochondria interactions.     

Stress-Induced Changes in Ubiquinone Concentration and Alternative Oxidase in Plant Mitochondria

We have investigated the influence of stress conditions such as incubation at 4°C and incubation in hyperoxygen atmosphere, on plant tissues. The ubiquinone (Q) content and respiratory activity of purified mitochondria was studied. The rate of respiration of mitochondria isolated from cold-treated green bell peppers (Capsicum annuum L) exceeds that of controls, but this is not so for mitochondria isolated from cold-treated cauliflower (Brassica oleracea L). Treatment with high oxygen does not alter respiration rates of cauliflower mitochondria. Analysis of kinetic data relating oxygen uptake with Q reduction in mitochondria isolated from tissue incubated at 4°C (bell peppers and cauliflowers) and at high oxygen levels (cauliflowers) reveals an increase in the total amount of Q and in the percentage of inoxidizable QH₂. The effects are not invariably accompanied by an induction of the alternative oxidase (AOX). In those mitochondria where the AOX is induced (cold-treated bell pepper and cauliflower treated with high oxygen) superoxide production is lower than in the control. The role of reduced Q accumulation and AOX induction in the defense against oxidative damage is discussed.     

Effect of pH on CN-resistant respiratory activity and regulation on Vigna uniguiculata mitochondria

Mitochondria from Vigna unguiculata cv. Vita 5 have a cyanide insensitive alternative oxidase cross-reacting with a monoclonal antibody raised against the alternative oxidase of Sauromatum guttatum. In the presence of NADH as substrate and dithiothreitol, the CN-resistant respiration of V. unguiculata intact mitochondria was found to be significantly influenced by assay pH with an optimum value of 6.25. This effect was still observed when nigericin, known to abolish ΔpH between the matrix and the intermembrane space, was present in the reaction medium. This pH effect was shown to be reversible. Alternative oxidase activation by pyruvate, also appeared to be pH dependent with an optimum pH value of 7.25. This modulation of the alternative oxidase activity by pH has been tentatively attributed to a new protonated-deprotonated status of this protein.     

Balance between Cyanide-Sensitive and -Insensitive Respiration in Wheat Root Mitochondria, as Influenced by Salt Concentration in the Plant Growth Medium

Mitochondria were isolated from 7-day-old wheat roots (Triticum vulgare Vill. cv. Svenno Spring Wheat) grown in either a full-strength culture medium (100%) or in the same medium diluted 100 times (1%). Outer membrane integrity was assayed using the cytochrome c reduction assay. This indicated about 20% damage. Using an oxygen electrode the respiration of the mitochondria was measured with either malate or succinate as the substrate (both 40 mM). KCN (3 mM) and salicylhydroxamic acid (SHAM, 1 mM) were used as inhibitors. The properties of the isolated mitochondria (STATE 3 rate, ADP/O ratio, and KCN-sensitivity) depend upon the ionic concentration of the growth medium of the roots. In the mitochondria isolated from roots grown in the 1% medium (1% mitochondria) there is a synergistic effect of KCN and SHAM. This means that electrons can be shifted from one pathway to the other when only one of the inhibitors is added. This flexibility between the electron pathways is almost nil in the mitochondria isolated from roots grown in the 100% medium (100% mitochondria). The maximal capacity of the alternative electron pathway (= rate in the presence of KCN) is higher in 1% (40 nmol O₂ min^?1 (mg protein)^?1) than in 100% mitochondria (20 nmol O₂ min^?1 (mg protein)^?1. In 100% mitochondria the alternative pathway seems to be operating at maximal capacity in the absence of KCN with both substrates and in both STATES 3 and 4. In 1% mitochondria the alternative pathway functions at >50% of its capacity in the absence of KCN.