Alternative Oxidase Activity Research Articles

Glyoxylic acid overcomes 1-MCP-induced blockage of fruit ripening in Pyrus communis L. var. \u2018D\u2019Anjou\u2019

1-methylcyclopropene (1-MCP) in an ethylene receptor antagonist that blocks ethylene perception and downstream ripening responses in climacteric fruit imparting a longer shelf life. However, in European pear, the application of 1-MCP irreversibly obstructs the onset of system 2 ethylene production resulting in perpetually unripe fruit with undesirable quality. Application of exogenous ethylene, carbon dioxide and treatment to high temperatures is not able to reverse the blockage in ripening. We recently reported that during cold conditioning, activation of alternative oxidase (AOX) occurs pre-climacterically. In this study, we report that activation of AOX via exposure of 1-MCP treated ‘D’Anjou’ pear fruit to glyoxylic acid triggers an accelerated ripening response. Time course physiological analysis revealed that ripening is evident from decreased fruit firmness and increased internal ethylene. Transcriptomic and functional enrichment analyses revealed genes and ontologies implicated in glyoxylic acid-mediated ripening, including AOX, TCA cycle, fatty acid metabolism, amino acid metabolism, organic acid metabolism, and ethylene-responsive pathways. These observations implicate the glyoxylate cycle as a biochemical hub linking multiple metabolic pathways to stimulate ripening through an alternate mechanism. The results provide information regarding how blockage caused by 1-MCP may be circumvented at the metabolic level, thus opening avenues for consistent ripening in pear and possibly other fruit.

The alternative oxidase pathway is involved in optimizing photosynthesis inMedicago truncatulainfected byFusarium oxysporumandRhizoctonia solani

Phytopathogen infection alters primary metabolism status and plant development. The alternative oxidase (AOX) has been hypothesized to increase under pathogen attack preventing reductions, thus optimizing photosynthesis and growth. In this study, two genotypes of Medicago truncatula, one relatively resistant (Jemalong A17) and one susceptible (TN1.11), were infected with Fusarium oxysporum and Rhizoctonia solani. The in vivo foliar respiratory activities of the cytochrome oxidase pathway (COP) and the alternative oxidase pathway (AOP) were measured using the oxygen isotope fractionation. Gas exchange and photosynthesis-related parameters were measured and calculated together with antioxidant enzymes activities and organic acids contents. Our results show that the in vivo activity of AOX (valt ) plays a role under fungal infection. When infected with R. solani, the increase of valt in A17 was concomitant to an increase in net assimilation, in mesophyll conductance, to an improvement in the maximum velocity of Rubisco carboxylation and to unchanged malate content. However, under F. oxysporum infection, the induced valt was accompanied by an enhancement in the antioxidant enzymes, superoxide dismutase (SOD; EC1.15.1.1), catalase (CAT; EC1.11.1.6) and guaiacol peroxidase (GPX; EC1.11.1.7), activities and to an unchanged tricarboxylic acid cycle intermediates. These results provide new insight into the role of the in vivo activity of AOX in coordinating primary metabolism interactions that, partly, modulate the relative resistance of M. truncatula to diseases caused by soil-borne pathogenic fungi.

Activity of Respiratory Pathways in Cultured Yam Cells under the Influence of Furostanol Glycosides

The role of furostanol glycosides (FGs) in the intensification of main metabolic processes in cultured yam (Dioscorea deltoidea Wall.) cells was shown. The effects of FGs were associated with changes in the complicated network of intracellular enzymatic reactions. This was evidenced by an increase in the activities of the enzymes from the glucose glycolytic oxidation pathway (hexokinase and fructose-1,6-difosphate aldolase) and the malate dehydrogenase complex (oxidoreductive—NADH/NAD-malate dehydrogenase and malic enzyme—decarboxylating NADP-malate dehydrogenase) as well as the stimulation of mitochondrial respiration accompanied by changes in activity of the alternative respiratory pathway. At the lag-phase of the growth cycle, the cell treatment with FGs was capable to guide glucose conversion through the glycolytic pathway. This was witnessed by a decrease in the activity of glucose-6-phosphate dehydrogenase and increase in that of hexokinase. Inhibition of NADPH oxidase afforded by diphenyliodonium chloride resulted in a decrease in activities of the key enzymes of glycolysis and the pentose phosphate cycle of glucose oxidation. Simultaneously, the aldolase activity increased and raised the content of its product glyceraldehyde 3-phosphate. The effect may explain the earlier-observed activation of the aldehyde-utilizing enzymes diminishing the initial POL level. The modulating action of FGs on activities of the enzymes of sugar glycolytic oxidation was shown. This was expressed as the activation of the tested enzymes at the lag- and exponential phases of the cellular growth and as the absence of the FG effect at the stationary phase. The short-term exposure of yam cells to FGs intensified the total mitochondrial respiration. An inhibitory analysis revealed the dominance of the cytochrome respiration pathway at the lag-phase, while its contribution became considerably lower at the later growth phases. FGs did not affect the rate of this respiration path but increased the potential activity of alternative oxidase (AO) during the lag- and exponential phases of the growth cycle. The FG-induced activation of AO, which may be intended to maintain a balance between carbohydrate metabolism and the rate of electron transport, is discussed.

The enhancement of salt stress tolerance by salicylic acid pretreatment in Arabidopsis thaliana

Salicylic acid (SA) is an important plant hormone involved in the activation of defense responses against environmental stresses. However, there are still large of unsolved mysteries about how SA pretreatment affects the establishment of plant stress tolerance. In this study, application of SA at different concentrations and different times were conducted to investigate their effects on the response of Arabidopsis seedlings to salt stress. The pretreatment with 10 or 20 μM SA for more than 6 h promoted Arabidopsis seedlings resistance to salt stress. On the other hand, pretreatment with 200 μM SA reduced Arabidopsis resistance to salt stress and aggravated oxidative damage to the seedlings. At all concentrations used, SA pretreatment inhibited the total respiration and promoted reactive oxygen species (ROS) generation. However, the ROS content in 10 or 20 μM SA pretreated seedlings decreased to the basal level within 6 h and high activities of antioxidant enzymes and alternative oxidase were maintained. Notably, the SA-enhanced salt stress resistance was significantly impaired by blocking alternative oxidase (AOX) pathway. Our findings indicate that SA-mediated salt stress response is in a dose- and time-dependent manner and that the effects were related to the induction of AOX capacity and antioxidant system.

Effects of Salicylic Acid on the Metabolism of Mitochondrial Reactive Oxygen Species in Plants.

Different abiotic and biotic stresses lead to the production and accumulation of reactive oxygen species (ROS) in various cell organelles such as in mitochondria, resulting in oxidative stress, inducing defense responses or programmed cell death (PCD) in plants. In response to oxidative stress, cells activate various cytoprotective responses, enhancing the antioxidant system, increasing the activity of alternative oxidase and degrading the oxidized proteins. Oxidative stress responses are orchestrated by several phytohormones such as salicylic acid (SA). The biomolecule SA is a key regulator in mitochondria-mediated defense signaling and PCD, but the mode of its action is not known in full detail. In this review, the current knowledge on the multifaceted role of SA in mitochondrial ROS metabolism is summarized to gain a better understanding of SA-regulated processes at the subcellular level in plant defense responses.

Post-Anoxia in Plants: Reasons, Consequences, and Possible Mechanisms

One of the adverse factors affecting plants is oxygen deficiency, which develops as a result of excessive waterlogging and flooding. In a natural habitat, following a period of oxygen deficiency, aerobic conditions are usually restored, resulting in exposure of plants to post-anoxic oxidation conditions. The endogenous reduced metabolites and soil toxins accumulated during oxygen deficiency begin to be oxidized by atmospheric oxygen, which leads to the generation of reactive oxygen species (ROS) and the development of damage that can cause the death of a plant even if it has survived anoxic conditions. Recent studies discussed in the review indicate that the main mechanisms of adaptation to the post-anoxic period are mainly the biosynthesis of antioxidants and the effective operation of antioxidant system, the activation of alternative oxidase and detoxification of anaerobic metabolites. Ethylene-dependent transcription factors ERF-VII, which are also involved in the regulation of adaptation directly to oxygen deficiency, as well as phytohormones, such as jasmonic and abscisic acids, play an important role in ensuring the regulation of adaptation to post-anoxic conditions. Post-anoxia should not be considered as an independent stressor, since it represents a complex effect (the effects of anoxia, oxidative stress, and desiccation together contribute to the development of damage), and adaptation mechanisms and their regulation have many common elements with adaptation to oxygen deficiency.

The Mediation of NO-Enhanced Chilling Tolerance by GSK-3 in Postharvest Peach Fruit

The role of glycogen synthase kinase-3 (GSK-3) in nitric oxide (NO)-enhanced chilling tolerance in postharvest peach fruit was investigated. The fruits were immersed in sodium nitroprusside (SNP; exogenous NO donor) and bikinin (GSK-3 inhibitor). Results showed that the chilling injury (CI) index declined following the exposure of the peach fruit to exogenous SNP. SNP treatment also induced GSK-3 expression. Furthermore, SNP treatment reduced malondialdehyde (MDA) content and electrolyte leakage in the peach fruit. In addition, SNP treatment induced the increase in alternative oxidase (AOX) activity and the upregulation of the gene expression of 18.1-kDa class I heat shock protein (HSP), WRKY2, and C-repeat binding factor (CBF). The effects of SNP treatment were partly weakened by the addition of bikinin. These findings indicate that GSK-3 mediated the reduction of MDA content and electrolyte leakage and the activation of AOX, 18.1-kDa class I HSP, WRKY2, and CBF by NO, thereby inducing chilling tolerance in peach fruit.

Increased reactive oxygen species production and maintenance of membrane potential in VDAC-less Neurospora crassa mitochondria.

The highly abundant voltage-dependent anion-selective channel (VDAC) allows transit of metabolites across the mitochondrial outer membrane. Previous studies in Neurospora crassa showed that the LoPo strain, expressing 50% of normal VDAC levels, is indistinguishable from wild-type (WT). In contrast, the absence of VDAC (ΔPor-1), or the expression of an N-terminally truncated variant VDAC (ΔN2-12porin), is associated with deficiencies in cytochromes b and aa3 of complexes III and IV and concomitantly increased alternative oxidase (AOX) activity. These observations led us to investigate complex I and complex II activities in these strains, and to explore their mitochondrial bioenergetics. The current study reveals that the total NADH dehydrogenase activity is similar in mitochondria from WT, LoPo, ΔPor-1 and ΔN2-12porin strains; however, in ΔPor-1 most of this activity is the product of rotenone-insensitive alternative NADH dehydrogenases. Unexpectedly, LoPo mitochondria have increased complex II activity. In all mitochondrial types analyzed, oxygen consumption is higher in the presence of the complex II substrate succinate, than with the NADH-linked (complex I) substrates glutamate and malate. When driven by a combination of complex I and II substrates, membrane potentials (Δψ) and oxygen consumption rates (OCR) under non-phosphorylating conditions are similar in all mitochondria. However, as expected, the induction of state 3 (phosphorylating) conditions in ΔPor-1 mitochondria is associated with smaller but significant increases in OCR and smaller decreases in Δψ than those seen in wild-type mitochondria. High ROS production, particularly in the presence of rotenone, was observed under non-phosphorylating conditions in the ΔPor-1 mitochondria. Thus, the absence of VDAC is associated with increased ROS production, in spite of AOX activity and wild-type OCR in ΔPor-1 mitochondria.

The Lack of Mitochondrial Thioredoxin TRXo1 Affects In Vivo Alternative Oxidase Activity and Carbon Metabolism under Different Light Conditions.

The alternative oxidase (AOX) constitutes a nonphosphorylating pathway of electron transport in the mitochondrial respiratory chain that provides flexibility to energy and carbon primary metabolism. Its activity is regulated in vitro by the mitochondrial thioredoxin (TRX) system which reduces conserved cysteines residues of AOX. However, in vivo evidence for redox regulation of the AOX activity is still scarce. In the present study, the redox state, protein levels and in vivo activity of the AOX in parallel to photosynthetic parameters were determined in Arabidopsis knockout mutants lacking mitochondrial trxo1 under moderate (ML) and high light (HL) conditions, known to induce in vivo AOX activity. In addition, 13C- and 14C-labeling experiments together with metabolite profiling were performed to better understand the metabolic coordination between energy and carbon metabolism in the trxo1 mutants. Our results show that the in vivo AOX activity is higher in the trxo1 mutants at ML while the AOX redox state is apparently unaltered. These results suggest that mitochondrial thiol redox systems are responsible for maintaining AOX in its reduced form rather than regulating its activity in vivo. Moreover, the negative regulation of the tricarboxylic acid cycle by the TRX system is coordinated with the increased input of electrons into the AOX pathway. Under HL conditions, while AOX and photosynthesis displayed similar patterns in the mutants, photorespiration is restricted at the level of glycine decarboxylation most likely as a consequence of redox imbalance.

The Role of IP3 in NO-Enhanced Chilling Tolerance in Peach Fruit.

The role of inositol 1,4,5-trisphosphate (IP3) in nitric oxide (NO)-reduced chilling injury (CI) in peach fruit was investigated. The fruit were immersed in sodium nitroprusside (SNP) (NO donor) and neomycin (IP3 inhibitor). Results showed that chilling tolerance was enhanced upon exogenous SNP in postharvest peach fruit. Further, GABA accumulation was stimulated by SNP. The increase in protein expression and activity for enzymes in GABA biosynthesis, including glutamate decarboxylase (GAD), polyamine oxidase (PAO), and amino aldehyde dehydrogenase (AMADH), upon SNP treatment was also observed. Also, the up-regulation of Δ1-pyrroline-5-carboxylate synthetase (P5CS) and ornithine d-aminotransferase (OAT) and the down-regulation of proline dehydrogenase (PDH) were induced by SNP treatment, thereby accelating proline production. Additionally, SNP treatment elevated protein expression and activity of alternative oxidase (AOX). The above effects induced upon SNP were partly weakened by neomycin. Therefore, IP3 mediated NO-activated GABA and proline accumulation as well as AOX, thus inducing chilling tolerance in postharvest peach fruit.

Sucrose treatment suppresses programmed cell death in broccoli florets by improving mitochondrial physiological properties

The effect of sucrose treatment on mitochondrial membrane potential (ΔΨm), contents of reactive oxygen species (ROS), cytochrome c oxidase (COX) activity, alternative oxidase (AOX) activity, mitochondrial Ca2+ content and cytochrome c release from mitochondria in broccoli florets during storage at 20 °C was investigated. The results showed that sucrose treatment effectively retarded the onset of programmed cell death (PCD) in broccoli florets by a mitochondria-dependent pathway. Sucrose treatment inhibited the increase of ROS and a decrease in ΔΨm, which was associated with the open state of permeability transition pore (PTP) and the ratio of cytochrome c/a. The decrease of COX activity and Ca2+ content in broccoli florets was suppressed under sucrose treatment. Sucrose treatment maintained higher AOX activity, and retarded the cytochrome c release from mitochondria. These results indicated that sucrose treatment delayed the appearance of PCD in broccoli florets through improving mitochondrial physiological properties.

Chitosan-induced enhanced expression and activation of alternative oxidase confer tolerance to salt stress in maize seedlings

This study aimed to investigate the possible alleviating effect of chitosan on salt-induced growth retardation and oxidative stress and to elucidate whether this effect is linked to activation of mitochondrial respiration on the basis of alternative respiration in maize seedlings. Salt stress significantly reduced root length and plant height in comparison to the control, whereas foliar application of chitosan ameliorated the adverse effect of salinity to a certain degree. Moreover, chitosan resulted in plant growth promotion as compared to unstressed seedlings. The separate applications of chitosan and salt had a stimulatory effect on the activities of antioxidant enzymes; however, combined application of chitosan and salt were more effective than that of chitosan or salt alone. Similarly, mitochondrial total respiration rate (Vt) and alternative respiration capacity (Valt) were increased by separate applications of chitosan and salt; however, the combination of chitosan and salt gave the highest values for these parameters. The highest values of Valt/Vt was recorded at seedlings treated with salt plus chitosan. Similarly, cytochrome respiration capacity was also increased by chitosan in both stress-free and stressed conditions. In addition, AOX1, encoding alternative oxidase, was significantly upregulated by chitosan and/or salt. The maximum transcript level was recorded at seedlings treated with salt plus chitosan. Chitosan also significantly decreased superoxide anion and hydrogen peroxide contents and lipid peroxidation level under normal and the stressed conditions. These results suggest that the mitigating effect of chitosan on salt stress is linked to activation of alternative respiration at biochemical and molecular level.

Application of abscisic acid prevents leaf senescence at decreased irradiance

Several lines of evidence suggest that plant hormone abscisic acid (ABA) and the activity of alternative respiratory pathway are somehow interrelated. In this paper, we tested how an application of ABA affects the alternative oxidase (AOX) pathway and leaf senescence in Arabidopsis thaliana (L.) Heynh. Different effects of ABA application on the AOX activity were observed under high irradiance (HI) and low irradiance (LI). The expression of AOX1D gene was stimulated, whereas the expression of senescence marker gene WRKY53 was decreased due to ABA treatment. We also noticed that changes of AOX genes AOX1A and AOX1D, as well as AOX regulatory gene ANAC017, were rather inversely correlated with irradiance-dependent changes in AOX activity. The expression of AOX1D and WRKY53 was diminished due to the application of H2O2 under LI. Our results are indicative that in some conditions an exogenous application of ABA may suppress leaf senescence. This action does not go via the induction of AOX activity, despite stimulation of AOX genes.

Alternative oxidase inhibitors: Mitochondrion-targeting as a strategy for new drugs against pathogenic parasites and fungi.

The alternative oxidase (AOX) is a ubiquitous terminal oxidase of plants and many fungi, catalyzing the four-electron reduction of oxygen to water alongside the cytochrome-based electron transfer chain. Unlike the classical electron transfer chain, however, the activity of AOX does not generate adenosine triphosphate but has functions such as thermogenesis and stress response. As it lacks a mammalian counterpart, it has been investigated intensely in pathogenic fungi. However, it is in African trypanosomes, which lack cytochrome-based respiration in their infective stages, that trypanosome alternative oxidase (TAO) plays the central and essential role in their energy metabolism. TAO was validated as a drug target decades ago and among the first inhibitors to be identified was salicylhydroxamic acid (SHAM), which produced the expected trypanocidal effects, especially when potentiated by coadministration with glycerol to inhibit anaerobic energy metabolism as well. However, the efficacy of this combination was too low to be of practical clinical use. The antibiotic ascofuranone (AF) proved a much stronger TAO inhibitor and was able to cure Trypanosoma vivax infections in mice without glycerol and at much lower doses, providing an important proof of concept milestone. Systematic efforts to improve the SHAM and AF scaffolds, aided with the elucidation of the TAO crystal structure, provided detailed structure-activity relationship information and reinvigorated the drug discovery effort. Recently, the coupling of mitochondrion-targeting lipophilic cations to TAO inhibitors has dramatically improved drug targeting and trypanocidal activity while retaining target protein potency. These developments appear to have finally signposted the way to preclinical development of TAO inhibitors.

Downregulation of mitochondrial alternative oxidase affects chloroplast function, redox status and stress response in a marine diatom.

Diatom dominance in contemporary aquatic environments indicates that they have developed unique and effective mechanisms to cope with the rapid and considerable fluctuations that characterize these environments. In view of their evolutionary history from a secondary endosymbiosis, inter-organellar regulation of biochemical activities may be of particular relevance. Diatom mitochondrial alternative oxidase (AOX) is believed to play a significant role in supplying chloroplasts with ATP produced in the mitochondria. Using the model diatom Phaeodactylum tricornutum we generated AOX knockdown lines, and followed sensitivity to stressors, photosynthesis and transcriptome and metabolome profiles of wild-type and knockdown lines. We show here that expression of the AOX gene is upregulated by various stresses including H2 O2 , heat, high light illumination, and iron or nitrogen limitation. AOX knockdown results in hypersensitivity to stress. Knockdown lines also show significantly reduced photosynthetic rates and their chloroplasts are more oxidized. Comparisons of transcriptome and metabolome profiles suggest a strong impact of AOX activity on gene expression, which is carried through to the level of the metabolome. Our data provide evidence for the involvement of mitochondrial AOX in processes central to the cell biology of diatoms, revealing that cross-talk between mitochondria and chloroplasts is crucial for maintaining sensitivity to changing environments.

Mitochondrial alternative oxidase is determinant for growth and sporulation in the early diverging fungus Blastocladiella emersonii

Blastocladiella emersonii is an early diverging fungus of the phylum Blastocladiomycota. During the life cycle of the fungus, mitochondrial morphology changes significantly, from a fragmented form in sessile vegetative cells to a fused network in motile zoospores. In this study, we visualize these morphological changes using a mitochondrial fluorescent probe and show that the respiratory capacity in zoospores is much higher than in vegetative cells, suggesting that mitochondrial morphology could be related to the differences in oxygen consumption. While studying the respiratory chain of the fungus, we observed an antimycin A and cyanide-insensitive, salicylhydroxamic (SHAM)-sensitive respiratory activity, indicative of a mitochondrial alternative oxidase (AOX) activity. The presence of AOX was confirmed by the finding of a B. emersonii cDNA encoding a putative AOX, and by detection of AOX protein in immunoblots. Inhibition of AOX activity by SHAM was found to significantly alter the capacity of the fungus to grow and sporulate, indicating that AOX participates in life cycle control in B. emersonii.

PGPR Reduce Root Respiration and Oxidative Stress Enhancing Spartina maritima Root Growth and Heavy Metal Rhizoaccumulation.

The present study aims to unravel ecophysiological mechanisms underlying plant-microbe interactions under natural abiotic stress conditions, specifically heavy metal pollution. Effect of plant growth promoting rhizobacteria (PGPR) bioaugmentation on Spartina maritima in vivo root respiration and oxidative stress was investigated. This autochthonous plant is a heavy metal hyperaccumulator cordgrass growing in one of the most polluted estuaries in the world. The association with native PGPR is being studied with a view to their biotechnological potential in environmental decontamination. As a novelty, the oxygen-isotope fractionation technique was used to study the in vivo activities of cytochrome oxidase (COX) and alternative oxidase (AOX) pathways. Inoculated plants showed decreased antioxidant enzymatic activities and in vivo root respiration rates. The reduction in respiratory carbon consumption and the stress alleviation may explain the increments observed in S. maritima root biomass and metal rhizoaccumulation after inoculation. For the first time, plant carbon balance and PGPR are interrelated to explain the effect of rhizobacteria under abiotic stress.

The alternative oxidase pathway is involved in the BR-induced salt resistance in mustard

In this study, the role of Brassinosteroids (BRs) and the relationship between the mitochondrial alternative oxidase (AOX) and ROS in the BR-induced defence response to salt stress was studied in mustard plants. Salt stress induced a significant activation of AOX. Exogenous BR significantly enhanced the capacity of the cyanide-resistant pathway, and reduced the damage of cell membrane. Pretreatment with brassinazole (Brz, an inhibitor of the BR biosynthesis pathway) significantly blocked the capacity of the cyanide-resistant pathway. BR could partly recover the AOX inactivation under salicylhydroxamic acid (SHAM, an inhibitor of the cyanide-resistant pathway) pretreatment. It was also found that BR could enhance the ROS accumulation and the antioxidant enzyme activities, while the AOX could eliminate the excessive ROS and enhance the antioxidant enzyme activities. Furthermore, the suppression of the cyanide-resistant pathway significantly increased the MDA content and the electrolyte leakage in mustard leaves, and the suppression of the BR biosynthesis had little effect on their recovering. Taken together, the cyanide-resistant pathway was involved in BR-induced salt tolerance and played an important role in maintaining the permeability of the cell membrane.

High oxygen tension increases itaconic acid accumulation, glucose consumption, and the expression and activity of alternative oxidase in Aspergillus terreus

Itaconic acid is a five-carbon dicarboxylic acid with an unsaturated alkene bond, frequently used as a building block for the industrial production of a variety of synthetic polymers. It is also one of the major products of fungal "overflow metabolism" which can be produced in submerged fermentations of the filamentous fungus Aspergillus terreus. At the present, molar yields of itaconate are lower than those obtained in citric acid production in Aspergillus niger. Here, we have studied the possibility that the yield may be limited by the oxygen supply during fermentation and hence tested the effect of the dissolved oxygen concentration on the itaconic acid formation rate and yield in lab-scale bioreactors. The data show that a dissolved oxygen concentration of 2% saturation was sufficient for maximal biomass formation. Raising it to 30% saturation had no effect on biomass formation or the growth rate, but the itaconate yield augmented substantially from 0.53 to 0.85mol itaconate/mol glucose. Furthermore, the volumetric and specific rates of itaconic acid formation ameliorated by as much as 150% concurrent with faster glucose consumption, shortening the fermentation time by 48h. Further increasing the dissolved oxygen concentration over 30% saturation had no effect. Moreover, we show that this increase in itaconic acid production coincides with an increase in alternative respiration, circumventing the formation of surplus ATP by the cytochrome electron transport chain, as well as with increased levels of alternative oxidase transcript. We conclude that high(er) itaconic acid accumulation requires a dissolved oxygen concentration that is much higher than that needed for maximal biomass formation, and postulate that the induction of alternative respiration allows the necessary NADH reoxidation ratio without surplus ATP production to increase the glucose consumption and the flux through overflow metabolism.

Alternative Oxidase Activity Reduces Stress in Vibrio fischeri Cells Exposed to Nitric Oxide.

Alternative oxidase (Aox) is a non-energy-conserving respiratory oxidase found in certain eukaryotes and bacteria, whose role in physiology is not entirely clear. Using the genetically tractable bacterium Vibrio fischeri as a model organism, I have identified a role for Aox to reduce levels of stress in cells exposed to oxygen and nitric oxide (NO). In V. fischeri lacking the NO-detoxifying enzyme flavohemoglobin (Hmp), deletion of aox in cells grown in the presence of oxygen and NO results in alterations to the transcriptome that include increases in transcripts mapping to stress-related genes. Using fluorescence-based reporters, I identified corresponding increases in intracellular reactive oxygen species and decreases in membrane integrity in cells lacking aox Under these growth conditions, activity of Aox is linked to a decrease in NADH levels, indicating coupling of Aox activity with NADH dehydrogenase activity. Taken together, these results suggest that Aox functions to indirectly limit production of ferrous iron and damaging hydroxyl radicals, effectively reducing cellular stress during NO exposure.IMPORTANCE Unlike typical respiratory oxidases, alternative oxidase (Aox) does not directly contribute to energy conservation, and its activity would presumably reduce the efficiency of respiration and associated ATP production. Aox has been identified in certain bacteria, a majority of which are marine associated. The presence of Aox in these bacteria poses the interesting question of how Aox function benefits bacterial growth and survival in the ocean. Using the genetically tractable marine bacterium Vibrio fischeri, I have identified a role for Aox in reduction of stress under conditions where electron flux through the aerobic respiratory pathway is inhibited. These results suggest that Aox activity could positively impact longer-term bacterial fitness and survival under stressful environmental conditions.