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.     

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 concomitant expression and availability of conventional and alternative, cyanide-insensitive, respiratory pathways in Candida albicans

The opportunistic oral pathogen Candida albicans expresses a cyanide-insensitive alternative oxidase (AOX) upon exposure to respiratory inhibitors that act downstream from coenzyme Q, and upon ageing of cells. To investigate whether the conventional pathway is retained when the alternative pathway is induced, cells were grown in the presence of sodium cyanide, a reversible inhibitor of cytochrome oxidase. AOX expression was monitored by Western blotting and the presence of cytochromes associated with complexes III and IV of the conventional pathway was monitored by recording spectra between 500 and 650 nm at 77K. The activities of complexes III and IV were determined in polarographic and enzyme-kinetic experiments using specific respiratory substrates and inhibitors. Results indicated that complexes III and IV are constitutively expressed and are functional in cells expressing AOX. Furthermore, the enzymatic activities of complexes III and IV were similar in mitochondrial preparations from cells grown with or without cyanide. We next investigated whether both pathways are simultaneously available for electron transfer from the Q pool to molecular oxygen. Respiration was virtually completely inhibited by the combination of cyanide and salicyl hydroxamic acid (SHAM) or antimycin A and SHAM, but only partly inhibited by either of these inhibitors alone. This indicates that electrons can in principle flow either through the conventional or the alternative respiratory pathway. The availability of two electron pathways in C. albicans and the potential use of either pathway endows this pleomorphic fungus with another level at which it can rapidly adjust to altered environmental conditions.     

Responses of spinach leaf mitochondria to low N availability

Low N availability induces carbohydrate accumulation in leaf cells, which often causes suppression of photosynthesis. Under low N supply, excess carbohydrates would be preferentially respired by the non-phosphorylating pathways, such as the alternative oxidase (AOX) and uncoupling protein (UCP), which would suppress the excessive increase in the ratio of C to N (C/N ratio). In leaves, however, responses of these pathways to the low N stress are still unknown. We examined the mitochondrial respiratory pathways in spinach leaves grown at three different N availabilities to clarify whether the respiratory pathways change depending on the N availabilities. With the decrease in N availability, leaf respiratory rates per leaf area decreased, but the rates on the leaf N basis were comparable. Using fumarase activities of whole leaf extracts and isolated mitochondria, we estimated mitochondrial protein contents per leaf N. The contents increased with the decrease in the N availability, that is, at the low N availability, N was preferentially invested into mitochondria. On the mitochondrial protein basis, capacities of cytochrome pathway (CP) and cytochrome c oxidase (COX) were comparable regardless of the N availabilities, whereas both AOX capacity and the amounts of AOX protein increased with the decrease in the N availability. Some enzymes of tricarboxylic acid (TCA) cycle, especially NAD-dependent malic enzyme (NAD-ME), showed higher capacities under lower N. On the other hand, amounts of UCP did not differ amongst the N availabilities. These results indicated that, under low N stress, AOX will be preferentially up-regulated and will efficiently consume excess carbohydrates, which leads to suppressing the rise in the C/N ratio to a moderate level.     

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。     

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.     

Interaction between nitric oxide and ethylene in the induction of alternative oxidase in ozone-treated tobacco plants

The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain, an alternative pathway that terminates with a single homodimeric protein, the alternative oxidase (AOX). We recorded temporary inhibition of cytochrome capacity respiration and activation of AOX pathway capacity in tobacco plants (Nicotiana tabacum L. cv BelW3) fumigated with ozone (O(3)). The AOX1a gene was used as a molecular probe to investigate its regulation by signal molecules such as hydrogen peroxide, nitric oxide (NO), ethylene (ET), salicylic acid, and jasmonic acid (JA), all of them reported to be involved in the O(3) response. Fumigation leads to accumulation of hydrogen peroxide in mitochondria and early accumulation of NO in leaf tissues. Although ET accumulation was high in leaf tissues 5 h after the start of O(3) fumigation, it declined during the recovery period. There were no differences in the JA and 12-oxo-phytodienoic acid levels of treated and untreated plants. NO, JA, and ET induced AOX1a mRNA accumulation. Using pharmacological inhibition of ET and NO, we demonstrate that both NO- and ET-dependent pathways are required for O(3)-induced up-regulation of AOX1a. However, only NO is indispensable for the activation of AOX1a gene expression.     

Regulation of alternative oxidase 1 in Chlamydomonas reinhardtii during sulfur starvation

The mitochondrial respiratory chain in plants, some protists and many fungi consists of the ATP-coupling cyanide-sensitive cytochrome pathway and the cyanide-resistant alternative respiratory pathway. The alternative pathway is mediated by alternative oxidase (AOX). Although AOX has been proposed to play essential roles in nutrient stress tolerance of plants and protists, the effects of sulfur (S) deprivation, on AOX are largely unknown. The unicellular green alga Chlamydomonas reinhardtii reacts to S limitation conditions with the induced expression of many genes. In this work, we demonstrated that exposure of C. reinhardtii to S deprivation results in the up-regulation of AOX1 expression and an increased AOX1 protein. Furthermore, S-deprived C. reinhardtii cells display the enhanced AOX1 capacity. Moreover, nitrate assimilation regulatory protein (NIT2) is involved in the control of the AOX1 gene expression in the absence of S. Together, the results clearly indicate that AOX1 relates to S limitation stress responses and is regulated in a NIT2-dependent manner, probably together with yet-unknown regulatory factor(s).     

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.     

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.     

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.     

The physiologic role of alternative oxidase in Ustilago maydis

Alternative oxidase (AOX) is a ubiquitous respiratory enzyme found in plants, fungi, protists and some bacterial species. One of the major questions about this enzyme is related to its metabolic role(s) in cellular physiology, due to its capacity to bypass the proton-pumping cytochrome pathway, and as a consequence it has great energy-wasting potential. In this study, the physiological role and regulatory mechanisms of AOX in the fungal phytopathogen Ustilago maydis were studied. We found evidence for at least two metabolic functions for AOX in this organism, as a major part of the oxidative stress-handling machinery, a well-described issue, and as part of the mechanisms that increase the metabolic plasticity of the cell, a role that might be valuable for organisms exposed to variations in temperature, nutrient source and availability, and biotic or abiotic factors that limit the activity of the cytochrome pathway. Experiments under different culture conditions of ecological significance for this organism revealed that AOX activity is modified by the growth stage of the culture, amino acid availability and growth temperature. In addition, nucleotide content, stimulation of AOX by AMP and respiratory rates obtained after inhibition of the cytochrome pathway showed that fungal/protist AOX is activated under low-energy conditions, in contrast to plant AOX, which is activated under high-energy conditions. An estimation of the contribution of AOX to cell respiration was performed by comparing the steady-state concentration of adenine nucleotides, the mitochondrial membrane potential, and the respiratory rate.