Stress-induced protein CSP 310: a third uncoupling system in plants

Addition of the cold-stress-related protein CSP 310 to mitochondria isolated from winter wheat ( Triticum aestivum L. cv. Zalarinka), winter rye ( Secale cereale L. cv. Dymka), maize ( Zea mays L. cv. VIR 36) and pea ( Pisum sativum L. cv. Marat) caused an increase in non-phosphorylative respiration. This increase was inhibited by KCN, indicating that the protein is not a CN-resistant alternative oxidase. Unlike plant mitochondrial uncoupling proteins such as PUMP, the uncoupling action of CSP 310 did not depend on the presence of free fatty acids in the incubation medium. We propose that the mechanism of the uncoupling action of CSP 310 differs from that of other known plant uncoupling systems and that the CSP 310 uncoupling system is a third uncoupling system in cereals.     

Characterization of oxidative phosphorylation in the colorless chlorophyte Polytomella sp. Its mitochondrial respiratory chain lacks a plant-like alternative oxidase

The presence of an alternative oxidase (AOX) in Polytomella sp., a colorless relative of Chlamydomonas reinhardtii, was explored. Oxygen uptake in Polytomella sp. mitochondria was inhibited by KCN (94%) or antimycin (96%), and the remaining cyanide-resistant respiration was not blocked by the AOX inhibitors salicylhydroxamic acid (SHAM) or n-propylgallate. No stimulation of an AOX activity was found upon addition of either pyruvate, alpha-ketoglutarate, or AMP, or by treatment with DTT. An antibody raised against C. reinhardtii AOX did not recognized any polypeptide band of Polytomella sp. mitochondria in Western blots. Also, PCR experiments and Southern blot analysis failed to identify an Aox gene in this colorless alga. Finally, KCN exposure of cell cultures failed to stimulate an AOX activity. Nevertheless, KCN exposure of Polytomella sp. cells induced diminished mitochondrial respiration (20%) and apparent changes in cytochrome c oxidase affinity towards cyanide. KCN-adapted cells exhibited a significant increase of a-type cytochromes, suggesting accumulation of inactive forms of cytochrome c oxidase. Another effect of KCN exposure was the reduction of the protein/fatty acid ratio of mitochondrial membranes, which may affect the observed respiratory activity. We conclude that Polytomella lacks a plant-like AOX, and that its corresponding gene was probably lost during the divergence of this colorless genus from its close photosynthetic relatives.     

Up-regulation of mitochondrial alternative oxidase concomitant with chloroplast over-reduction by excess light

Alternative oxidase (AOX), the unique terminal oxidase in plant mitochondria, catalyzes the energy-wasteful cyanide (CN)-resistant respiration. Although it has been suggested that AOX might prevent chloroplast over-reduction through the efficient dissipation of excess reducing equivalents, direct evidence for this in the physiological context has been lacking. In this study, we examined the mitochondrial respiratory properties, especially AOX, connected to the accumulation of reducing equivalents in the chloroplasts and the activities of enzymes needed to transport the reducing equivalents. We used Arabidopsis thaliana mutants defective in cyclic electron flow around PSI, in which the reducing equivalents accumulate in the chloroplast stroma due to an unbalanced ATP/NADPH production ratio. These mutants showed higher activities of the enzymes needed to transport the reducing equivalents even in low-light growth conditions. The amounts of AOX protein and CN-resistant respiration in the mutants were also higher than those in the wild type. After high-light treatment, AOX, even in the wild type, was preferentially up-regulated concomitant with the accumulation of reducing equivalents in the chloroplasts and an increase in the activities of enzymes needed to transport reducing equivalents. These results indicate that AOX can dissipate the excess reducing equivalents, which are transported from the chloroplasts, and serve in efficient photosynthesis.     

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.     

Inhibition of specific electron transport pathways leads to oxidative stress and decreased Candida albicans proliferation

Candida parapsilosis mitochondria contain three respiratory chains: the classical respiratory chain (CRC), a secondary parallel chain (PAR) and an “alternative” oxidative pathway (AOX). We report here the existence of similar pathways in C. albicans. To observe the capacity of each pathway to sustain yeast growth, C. albicans cells were cultured in the presence of inhibitors of these pathways. Antimycin A and KCN totally abrogated yeast growth, while rotenone did not prevent proliferation. Furthermore, rotenone promoted only partial respiratory inhibition. Lower concentrations of KCN that promote partial inhibition of respiration did not inhibit yeast growth, while partial inhibition of respiration with antimycin A did. Similarly, AOX inhibitor BHAM decreased O₂ consumption slightly but completely stunted cell growth. Reactive oxygen species production and oxidized glutathione levels were enhanced in cells treated with antimycin A or BHAM, but not rotenone or KCN. These findings suggest that oxidative stress prevents C. albicans growth.     

Influence of chloroplastic photo-oxidative stress on mitochondrial alternative oxidase capacity and respiratory properties: a case study with Arabidopsis yellow variegated 2

Mitochondrial alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, catalyzes the energy-wasteful cyanide (CN)-resistant respiration. Although it has been demonstrated that leaf AOX is up-regulated under high-light (HL) conditions, the in vivo mechanism of AOX up-regulation by light is still unknown. In the present study, we examined whether the photo-oxidative stress in the chloroplast modulates mitochondrial respiratory properties, especially the AOX capacity, using Arabidopsis leaf-variegated mutant yellow variegated 2 (var2) and exposing plants to HL. var2 mutants lack FtsH2 metalloprotease required for the repair of damaged PSII. Indeed, var2-1 suffered from photo-oxidative stress even before the HL treatments. While the activities of tricarboxylic acid cycle enzymes and cytochrome c oxidase in var2-1 were almost identical to those in the wild type, the amount of AOX protein and the CN-resistant respiration rate were higher in var2-1. Real-time PCR analysis revealed that HL treatment induced the expression of some energy-dissipating respiratory genes, including AOX1a, NDB2 and UCP5, more strongly in var2-1. Western blotting using var2-1 leaf extracts specific to green or white sectors, containing functional or non-functional photosynthetic apparatus, respectively, revealed that more AOX protein was induced in the green sectors by the HL treatment. These results indicate that photo-oxidative stress by excess light is involved in the regulation of respiratory gene expression and the modulation of respiratory properties, especially the AOX up-regulation.     

Reversion from light-induced inhibition of seed germination by respiratory inhibitors

Treating of the dark-imbibed lettuce (Lactuca sativa L.) seeds prior to light irradiation with 1 millimolar KCN or NaN₃ in the dark for 3 hours prevented blue light and far-red light-induced inhibitions of phytochrome-mediated germination. Similarly, salicylhydroxamic acid (SHAM) at 10 millimolar counteracted the blue and far-red light inhibitions, the combined application of KCN and SHAM being more effective than KCN or SHAM alone in some experiments. These respiratory inhibitors slightly inhibited phytochrome-mediated lettuce seed germination. These results indicate that both CN-sensitive, conventional cytochrome oxidase and CN-resistant (SHAM-sensitive), alternative respiration may be involved in the light inhibition or that an appropriate balance of both may be necessary for the light inhibition.     

Characterization of oxidative phosphorylation in the colorless chlorophyte Polytomella sp.: Its mitochondrial respiratory chain lacks a plant-like alternative oxidase

The presence of an alternative oxidase (AOX) in Polytomella sp., a colorless relative of Chlamydomonas reinhardtii, was explored. Oxygen uptake in Polytomella sp. mitochondria was inhibited by KCN (94%) or antimycin (96%), and the remaining cyanide-resistant respiration was not blocked by the AOX inhibitors salicylhydroxamic acid (SHAM) or n-propylgallate. No stimulation of an AOX activity was found upon addition of either pyruvate, α-ketoglutarate, or AMP, or by treatment with DTT. An antibody raised against C. reinhardtii AOX did not recognized any polypeptide band of Polytomella sp. mitochondria in Western blots. Also, PCR experiments and Southern blot analysis failed to identify an Aox gene in this colorless alga. Finally, KCN exposure of cell cultures failed to stimulate an AOX activity. Nevertheless, KCN exposure of Polytomella sp. cells induced diminished mitochondrial respiration (20%) and apparent changes in cytochrome c oxidase affinity towards cyanide. KCN-adapted cells exhibited a significant increase of a-type cytochromes, suggesting accumulation of inactive forms of cytochrome c oxidase. Another effect of KCN exposure was the reduction of the protein/fatty acid ratio of mitochondrial membranes, which may affect the observed respiratory activity. We conclude that Polytomella lacks a plant-like AOX, and that its corresponding gene was probably lost during the divergence of this colorless genus from its close photosynthetic relatives.     

A novel role for cyanide in the control of cucumber (Cucumis sativus L.) seedlings response to environmental stress

The effects of potassium cyanide (KCN) pretreatment on the response of cucumber (Cucumis sativus L.) plants to salt, polyethylene glycol (PEG) and cold stress were investigated in the present study. Here, we found that KCN pretreatment improved cucumber seedlings tolerance to stress conditions with maximum efficiency at a concentration of 20 µM. The results showed that pretreatment with 20 µM KCN alleviated stress‐induced oxidative damage in plant cells and clearly induced the activity of alternative oxidase (AOX) and the ethylene production. Furthermore, the structures of thylakoids and mitochondria in the KCN‐pretreated seedlings were less damaged by the stress conditions, which maintained higher total chlorophyll content, photosynthetic rate and photosystem II (PSII) proteins levels than the control. Importantly, the addition of the AOX inhibitor salicylhydroxamic acid (1 mm ; SHAM) decreased plant resistance to environmental stress and even compromised the cyanide (CN)‐enhanced stress tolerance. Therefore, our findings provide a novel role of CN in plant against environmental stress and indicate that the CN‐enhanced AOX might contribute to the reactive oxygen species (ROS) scavenging and the protection of photosystem by maintaining energy charge homoeostasis from chloroplast to mitochondria.     

Maintenance of growth rate at low temperature in rice and wheat cultivars with a high degree of respiratory homeostasis is associated with a high efficiency of respiratory ATP production

Some plants have the ability to maintain similar respiratory rates (measured at the growth temperature) when grown at different temperatures. This phenomenon is referred to as respiratory homeostasis. Using wheat and rice cultivars with different degrees of respiratory homeostasis (H), we previously demonstrated that high-H cultivars maintained shoot and root growth at low temperature [Kurimoto et al. (2004) Plant Cell Environ., 27: 853]. Here, we assess the relationship between respiratory homeostasis and the efficiency of respiratory ATP production, by measuring the levels of alternative oxidase (AOX) and uncoupling protein (UCP), which have the potential to decrease respiratory ATP production per unit of oxygen consumed. We also measured SHAM- and CN-resistant respiration of intact roots, and the capacity of the cytochrome pathway (CP) and AOX in isolated mitochondria. Irrespective of H, SHAM-resistant respiration of intact roots and CP capacity of isolated root mitochondria were larger when plants were grown at low temperature, and the maximal activity and relative amounts of cytochrome c oxidase showed a similar trend. In contrast, CN-resistant respiration of intact roots and relative amounts of AOX protein in mitochondria isolated from those roots, were lower in high-H plants grown at low temperature. In the roots of low-H cultivars, relative amounts of AOX protein were higher at low growth temperature. Relative amounts of UCP protein showed similar trends to AOX. We conclude that maintenance of growth rate in high-H plants grown at low temperature is associated with both respiratory homeostasis and a high efficiency of respiratory ATP production.     

An alternative respiratory pathway on Candida krusei: implications on susceptibility profile and oxidative stress

Our aim was to detect the presence of an alternative oxidase (AOX) in Candida krusei clinical strains and its influence on fluconazole susceptibility and in reactive oxygen species (ROS) production. Candida krusei clinical isolates were tested to evaluate the presence of AOX. Debaromyces hansenii 2968 (AOX positive) and Saccharomyces cerevisiae BY4742 (AOX negative) were used as control strains. Measurements of oxygen consumption were performed in the presence of 1?mM KCN, an inhibitor of the classical respiratory chain, and 5?mM salicylhydroxamic acid (SHAM). AOX expression was monitored by Western blotting using an AOX monoclonal antibody. Interactions between fluconazole and SHAM were performed using checkerboard assay. ROS production was evaluated in the presence of SHAM plus fluconazole, H(2) O(2) , menadione, or plumbagin. AOX was present in all C.?krusei tested. The combination of fluconazole with SHAM resulted in an indifferent effect. In the presence of SHAM, the treatment with ROS inductors or fluconazole increased ROS production, except in the AOX-negative strain. An alternative respiratory pathway resistant to cyanide is described for the first time as a characteristic of C.?krusei species. This AOX is unrelated to fluconazole resistance; however, it protects C.?krusei from oxidative stress.     

H2O2对热带假丝酵母醇氧化酶的影响及其作用机理分析

考察了H2 O2 对热带假丝酵母代谢烷烃生产二元酸过程中醇氧化酶 (fattyalcoholoxidase ,AOX)的影响。研究表明 :2mmol L和 5mmol L的H2 O2 对AOX的比酶活有明显的促进作用 ,分别比对照提高了 68 2 %和 82 9%。研究还发现 ,H2 O2 是以超氧阴离子自由基等形式在胞内存在 ,并对H2 O2 在代谢过程中的作用机理进行了分析。     

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.     

Mitochondrial alternative oxidase acts to dampen the generation of active oxygen species during a period of rapid respiration induced to support a high rate of nutrient uptake

When wild type (wt) tobacco ( Nicotiana tabacum L. cv. Petit Havana SR1) suspension cells were grown under phosphate (P) limitation, they contained large amounts of mitochondrial alternative oxidase (AOX). When these cells were resupplied with P, there was a large, immediate and sustained stimulation of respiration to support a period of rapid P uptake. Two lines of evidence suggest that the abundant level of AOX present in wt cells contributed to this stimulated rate of respiration. First, when P-limited transgenic antisense tobacco cells (AS8) lacking AOX were resupplied with P, the stimulation of respiration was much less dramatic even though these cells displayed similar rates of P uptake. Second, while the stimulated rate of respiration in AS8 cells was insensitive (as expected) to the AOX inhibitor n -propyl gallate (nPG), much of the stimulated rate of respiration in wt cells could be inhibited by nPG. Given the non-phosphorylating nature of AOX respiration, wt cells required higher rates of electron transport to O₂ than AS8 cells to support similar rates of P uptake. The utilization of AOX by wt cells during P uptake was apparently not occurring because the cytochrome (Cyt) pathway alone could not fully support the rate of P uptake, as the respiration of cells lacking AOX (either untreated AS8 cells or wt cells treated with nPG) supported similar rates of P uptake as wt cells with abundant AOX. Rather, we provide in vivo evidence that the utilization of AOX during the period of high respiration supporting P uptake was to dampen the mitochondrial generation of active oxygen species (AOS).     

Signals Regulating the Expression of the Nuclear Gene Encoding Alternative Oxidase of Plant Mitochondria

Suspension cells of tobacco (Nicotiana tabacum L. cv Bright Yellow) were used to investigate signals regulating the expression of the nuclear gene Aox1 encoding the mitochondrial alternative oxidase (AOX) protein responsible for cyanide-resistant respiration in plants. We found that an increase in the tricarboxylic acid cycle intermediate citrate (either after its exogenous supply to cells or after inhibition of aconitase by monofluoroacetate) caused a rapid and dramatic increase in the steady-state level of Aox1 mRNA and AOX protein. This led to a large increase in the capacity for AOX respiration, defined as the amount of salicylhydroxamic acid-sensitive O2 uptake by cells in the presence of potassium cyanide. The results indicate that citrate may be an important signal metabolite regulating Aox1 gene expression. A number of other treatments were also identified that rapidly induced the level of Aox1 mRNA and AOX capacity. These included short-term incubation of cells with 10 mM acetate, 2 [mu]M antimycin A, 5 mM H2O2, or 1 mM cysteine. For some of these treatments, induction of AOX occurred without an increase in cellular citrate level, indicating that other signals (possibly related to oxidative stress conditions) are also important in regulating Aox1 gene expression. The signals influencing Aox1 gene expression are discussed with regard to the potential function(s) of AOX to modulate tricarboxylic acid cycle metabolism and/or to prevent the generation of active oxygen species by the mitochondrial electron transport chain.