Alternative Oxidase Gene Research Articles

Engineering the central pathways in Lactococcus lactis: Functional expression of the phosphofructokinase (pfk) and alternative oxidase (aox1) genes from Aspergillus niger in Lactococcus lactis facilitates improved carbon conversion rates under oxidizing conditions

The present work describes a novel central pathway engineering method that has been designed with the aim to increase the carbon conversion rates under oxidizing conditions in L. lactis fermentations. The nisin producer L. lactis ATCC11454 strain has been genetically engineered by cloning a truncated version of the phosphofructokinase gene (pfk13), along with the pkaC, encoding for the catalytic subunit of cAMP-dependent protein kinase, and the alternative oxidase (aox1) genes of A. niger. Functional expression of the above genes resulted in enhanced PFK activity and the introduction of AOX activity and alternative respiration in the presence of a source of heme in the substrate, under fully aerobic growth conditions. The constructed strain is capable of fermenting high concentrations of glucose as was demonstrated in a series of glucostat fed-batch fermentations with glucose levels maintained at 55, 138 and 277mM. The high maximum specific uptake rate of glucose of 1.8mMs−1gCDW−1 at 277mM glucose is characteristic of the improved ability of the microorganism to handle elevated glucose concentrations under conditions otherwise causing severe reduction of PFK activity. The increased carbon flow through glycolysis led to increased protein synthesis that was reflected in increased biomass and nisin levels. The pfk13–pkaC–aox1-transformant strain's fermentation at 277mM glucose gave a final biomass concentration of 7.5g/l and nisin activity of 14,000IU/ml which is, compared to the parental strain's production levels at its optimal 55mM glucose, increased by a factor of 2.34 for biomass and 4.37 for nisin.

CHARACTERIZATION OF THE ALTERNATIVE OXIDASE GENE IN PORPHYRA YEZOENSIS (RHODOPHYTA) AND CYANIDE-RESISTANT RESPIRATION ANALYSIS(1).

The full-length cDNA of the alternative oxidase (AOX) gene from Porphyra yezoensis Ueda (PyAOX) [currently assigned as Pyropia yezoensis (Ueda) M. S. Hwang et H. G. Choi (http://www.algaebase.org)] an ancient member of the Rhodphyta, was cloned by electronic cloning, rapid amplification of cDNA ends (RACE), and reverse transcription PCR. The nucleotide sequence of PyAOX consists of 1,650 bp, including a 5' untranslated region (UTR) of 170 bp, a 3' UTR of 148 bp, and an open reading frame (ORF) of 1,332 bp that can be translated into a 443-amino-acid residue with a molecular mass of 47.33 kDa and a putative isoelectric point (pI) of 9.71. The putative amino acids had 50%-61% identity with AOX genes in Eukaryota and higher plants and had AOX-like characteristics. The expression of PyAOX mRNA in different stages of the life cycle, conchospores, filamentous thalli (conchocelis stage), and leafy thalli, was detected by real-time quantitative PCR (qPCR). The highest level of expression, which was observed in filamentous thalli, was three times higher than that observed in leafy thalli. The next highest level, which was observed in the conchospores, was twice as high as that observed in leafy thalli. We showed that an alternative respiration pathway existed in P. yezoensis with a noninvasive microsensing system. The contribution of the alternative pathway to total respiration in filamentous thalli was greater than that in leafy thalli. This result was consistent with the level of AOX gene expression observed in different stages of the life cycle.

Involvement of alternative oxidase (AOX) in adventitious rooting of Olea europaea L. microshoots is linked to adaptive phenylpropanoid and lignin metabolism

Alternative oxidase (AOX) has been proposed as a functional marker candidate in a number of events involving cell differentiation, including rooting efficiency in semi-hardwood shoot cuttings of olive (Olea europaea L.). To ascertain the general importance of AOX in olive rooting, the auxin-induced rooting process was studied in an in vitro system for microshoot propagation. Inhibition of AOX by salicylhydroxamic acid (SHAM) significantly reduced rooting efficiency. However, the inhibitor failed to exhibit any effect on the preceding calli stage. This makes the system appropriate for distinguishing dedifferentiation and de novo differentiation during root induction. Metabolite analyses of microshoots showed that total phenolics, total flavonoids and lignin contents were significantly reduced upon SHAM treatment. It was concluded that the influence of alternative respiration on root formation was associated to adaptive phenylpropanoid and lignin metabolism. Transcript profiles of two olive AOX genes (OeAOX1a and OeAOX2) were examined during the process of auxin-induced root induction. Both genes displayed stable transcript accumulation in semi-quantitative RT-PCR analysis during all experimental stages. In contrary, when the reverse primer for OeAOX2 was designed from the 3'-UTR instead of the ORF, differential transcript accumulation was observed suggesting posttranscriptional regulation of OeAOX2 during metabolic acclimation. This result confirms former observations in olive semi-hardwood shoot cuttings on differential OeAOX2 expression during root induction. It further points to the importance of future studies on the functional role of sequence and length polymorphisms in the 3'-UTR of this gene. The manuscript reports the general importance of AOX in olive adventitious rooting and the association of alternative respiration to adaptive phenylpropanoid and lignin metabolism.

The hemibiotrophic cacao pathogen Moniliophthora perniciosa depends on a mitochondrial alternative oxidase for biotrophic development

The tropical pathogen Moniliophthora perniciosa causes witches’ broom disease in cacao. As a hemibiotrophic fungus, it initially colonizes the living host tissues (biotrophic phase), and later grows over the dead plant (necrotrophic phase). Little is known about the mechanisms that promote these distinct fungal phases or mediate the transition between them.An alternative oxidase gene (Mp-aox) was identified in the M. perniciosa genome and its expression was analyzed througout the fungal life cycle. In addition, the effects of inhibitors of the cytochrome-dependent respiratory chain (CRC) and alternative oxidase (AOX) were evaluated on the in vitro development of M. perniciosa.Larger numbers of Mp-aox transcripts were observed in the biotrophic hyphae, which accordingly showed elevated sensitivity to AOX inhibitors. More importantly, the inhibition of CRC prevented the transition from the biotrophic to the necrotrophic phase, and the combined use of a CRC and AOX inhibitor completely halted fungal growth.On the basis of these results, a novel mechanism is presented in which AOX plays a role in the biotrophic development of M. perniciosa and regulates the transition to its necrotrophic stage. Strikingly, this model correlates well with the infection strategy of animal pathogens, particularly Trypanosoma brucei, which uses AOX as a strategy for pathogenicity.

CDNA cloning and expression analysis of a putative alternative oxidase HsAOX1 from wild barley (Hordeum spontaneum)

A novel alternative oxidase (AOX1) gene, designated HsAOX1 (GenBank accession number JF440341) was cloned by RT-PCR from wild barley (Hordeum spontaneum). The full length of HsAOX1 is 1115 bp with an open reading frame of 987 bp, encoding a protein of 328 amino acids with molecular weight of 36.89 kDa and a theoretical isoelectric point of 6.81. As found in other plant AOX1 proteins, sequence alignment showed that HsAOX1 had conserved metal binding and hydrophobic α-helix regions and had high homology to other AOX1 in plants. The expression analysis by semi-quantitative RT-PCR revealed that HsAOX1 was induced in response to cold stress, H2O2 treatment, SA, antimycin A and KCN. These results showed that HsAOX1 functions not only during inhibition of cytochrome electron transport but also during oxidative stresses, thus suggesting a role of HsAOX1 in preventing the generation of free radicals by the mitochondrial electron transport chain. The cloning and characterization of the HsAOX1 gene will be useful for further studies of biological roles of HsAOX1 in plants.

Visual expression analysis of the responses of the alternative oxidase gene (aox1) to heat shock, oxidative, and osmotic stresses in conidia of citric acid-producing Aspergillus niger

The citric acid-producing filamentous fungus Aspergillus niger WU-2223L shows cyanide-insensitive respiration catalyzed by alternative oxidase in addition to the cytochrome pathway. Sequence analysis of the 5' flanking region of the alternative oxidase gene (aox1) revealed a potential heat shock element (HSE) and a stress response element (STRE). We have previously confirmed aox1 expression in conidia. In this study, to confirm whether the upstream region of aox1 responds to various stresses, we used a visual expression analysis system for single-cell conidia of the A. niger strain AOXEGFP-1. This strain harbored a fusion gene comprising aox1 and egfp, which encodes the enhanced green fluorescent protein (EGFP). The fluorescence intensity of EGFP increased in conidia of A. niger AOXEGFP-1 that were subjected to heat shock at 35-45 °C, oxidative stress by exposure to 5mM paraquat or 1 mM t-butylhydroperoxide, or osmotic stresses by exposure to 0.5 M KCl or 1.0 M mannitol. These results indicate that the putative HSE and STRE in the upstream region of aox1 directly or indirectly respond to heat shock, oxidative, and osmotic stresses.

Modified expression of alternative oxidase in transgenic tomato and petunia affects the level of tomato spotted wilt virus resistance.

BackgroundTomato spotted wilt virus (TSWV) has a very wide host range, and is transmitted in a persistent manner by several species of thrips. These characteristics make this virus difficult to control. We show here that the over-expression of the mitochondrial alternative oxidase (AOX) in tomato and petunia is related to TSWV resistance.ResultsThe open reading frame and full-length sequence of the tomato AOX gene LeAox1au were cloned and introduced into tomato 'Healani' and petunia 'Sheer Madness' using Agrobacterium-mediated transformation. Highly expressed AOX transgenic tomato and petunia plants were selfed and transgenic R1 seedlings from 10 tomato lines and 12 petunia lines were used for bioassay. For each assayed line, 22 to 32 tomato R1 progeny in three replications and 39 to 128 petunia progeny in 13 replications were challenged with TSWV. Enzyme-Linked Immunosorbent Assays showed that the TSWV levels in transgenic tomato line FKT4-1 was significantly lower than that of wild-type controls after challenge with TSWV. In addition, transgenic petunia line FKP10 showed significantly less lesion number and smaller lesion size than non-transgenic controls after inoculation by TSWV.ConclusionIn all assayed transgenic tomato lines, a higher percentage of transgenic progeny had lower TSWV levels than non-transgenic plants after challenge with TSWV, and the significantly increased resistant levels of tomato and petunia lines identified in this study indicate that altered expression levels of AOX in tomato and petunia can affect the levels of TSWV resistance.

Identification of a gene for pyruvate-insensitive mitochondrial alternative oxidase expressed in the thermogenic appendices in Arum maculatum.

Heat production in thermogenic plants has been attributed to a large increase in the expression of the alternative oxidase (AOX). AOX acts as an alternative terminal oxidase in the mitochondrial respiratory chain, where it reduces molecular oxygen to water. In contrast to the mitochondrial terminal oxidase, cytochrome c oxidase, AOX is nonprotonmotive and thus allows the dramatic drop in free energy between ubiquinol and oxygen to be dissipated as heat. Using reverse transcription-polymerase chain reaction-based cloning, we reveal that, although at least seven cDNAs for AOX exist (AmAOX1a, -1b, -1c, -1d, -1e, -1f, and -1g) in Arum maculatum, the organ and developmental regulation for each is distinct. In particular, the expression of AmAOX1e transcripts appears to predominate in thermogenic appendices among the seven AmAOXs. Interestingly, the amino acid sequence of AmAOX1e indicates that the ENV element found in almost all other AOX sequences, including AmAOX1a, -1b, -1c, -1d, and -1f, is substituted by QNT. The existence of a QNT motif in AmAOX1e was confirmed by nano-liquid chromatography-tandem mass spectrometry analysis of mitochondrial proteins from thermogenic appendices. Further functional analyses with mitochondria prepared using a yeast heterologous expression system demonstrated that AmAOX1e is insensitive to stimulation by pyruvate. These data suggest that a QNT type of pyruvate-insensitive AOX, AmAOX1e, plays a crucial role in stage- and organ-specific heat production in the appendices of A. maculatum.

Cloning and functional expression of the mitochondrial alternative oxidase gene ( aox1) of Aspergillus niger in Lactococcus lactis and its induction by oxidizing conditions

Lactococcus lactis is a widely used food bacterium mainly known for its fermentation metabolism. An important, and for long time overlooked, trait of this species is its ability to perform respiratory metabolism in the presence of heme and under aerobic conditions. There is no evidence however for the presence of an alternative respiration pathway and AOX activity. In this study, a cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for alternative respiration, from a citric acid producing Aspergillus niger strain was cloned and expressed in L. lactis as a host strain. Expression of aox1 conferred on this organism cyanide-resistant and salicylhydroxamate-sensitive growth. Bioreactor cultures under fully aerobic conditions of the transformed L. lactis showed that the alternative respiratory pathway operates and improves significantly the microorganism's response to oxidizing stress conditions as it enhances biomass production, suppresses lactate formation, and leads to accumulation of large amounts of nisin.

Role of a Newly Cloned Alternative Oxidase Gene (BjAOX1a) in Turnip Mosaic Virus (TuMV) Resistance in Mustard

A new alternative oxidase (AOX) gene, designated as BjAOX1a, of mustard (Brassica juncea) was cloned by reverse transcription-polymerase chain reaction (RT-PCR). The full-length cDNA of BjAOX1a was 1,346 bp in size and containing an open reading frame (ORF) of 1,083 bp in size. The predicted amino acid sequence exhibited 82.17% homology to the alternative oxidase of Arabidopsis thaliana. Analysis of functional regions revealed that the BjAOX1a protein contained several metal-binding regions, α-helical regions, and cysteine residues, similar to other AOX1 proteins. Pretreatment of mustard plants with salicylhydroxamic acid (SHAM), an inhibitor of AOX protein, inhibited AOX activity and promoted systemic movement of the coat protein of Turnip mosaic virus (TuMV), whereas pretreatment of plants with antimycin A (AA), a cytochrome pathway inhibitor, inhibited the systemic movement of the coat protein of TuMV. Furthermore, H2O2 and O2− contents were enhanced following SHAM pretreatment. Thus, AOX1 might alleviate reactive oxygen species (ROS) and enhance resistance of mustard plants to TuMV, and could serve as a critical component in TuMV resistance.

Involvement of hydrogen peroxide, calcium, and ethylene in the induction of the alternative pathway in chilling-stressed Arabidopsis callus

The roles of ethylene, hydrogen peroxide (H(2)O(2)), and calcium in inducing the capacity of the alternative respiratory pathway (AP) under chilling temperature in Arabidopsis thaliana calli were investigated. Exposure of wild-type (WT) calli, but not the calli of ethylene-insensitive mutants, etr1-3 and ein2-1, to chilling led to a marked increase of the AP capacity and triggered a rapid ethylene emission and H(2)O(2) generation. Increasing ethylene emission by applying 1-aminocyclopropane-1-carboxylic (an ethylene precursor) markedly enhanced the AP capacity in WT calli, but not in etr1-3 and ein2-1 calli, whereas suppressing ethylene emission by applying aminooxyacetic acid (an ethylene biosynthesis inhibitor) abolished the chilling-induced AP capacity in WT calli. Furthermore, exogenous H(2)O(2) treatment increased the AP capacity in WT calli, but not in etr1-3 and ein2-1 calli, while both catalase (H(2)O(2) scavenger) and diphenylene iodonium (DPI, an inhibitor of NADPH oxidase) completely inhibited the chilling-induced H(2)O(2) generation and largely inhibited the chilling-induced AP capacity. Interestingly, the chilling-induced AP capacity was completely inhibited by DPI and EGTA (calcium chelator). Further investigation demonstrated that H(2)O(2) and calcium induced ethylene emission under chilling stress. Ethylene modulated the chilling-induced increase of pyruvate content and the expression of alternative oxidase genes (AOX1a and AOX1c). Taken together, these results indicate that H(2)O(2)-, calcium- and ethylene-dependent pathways are required for chilling-induced increase in AP capacity. However, only ethylene is indispensable for the activation of the AP capacity.

The transcription factor SNT2 is involved in fungal respiration and reactive oxidative stress in Fusarium oxysporum and Neurospora crassa

The Fusarium oxysporum f. sp. melonis (FOM) Snt2 transcription factor is related to pathogenesis. In FOM and Neurospora crassa Δsnt2 strains, hydrogen peroxide-induced SOD activity was impaired. The expression of fe,mn sod genes in the Δsnt2 mutants, was up-regulated following hydrogen peroxide induction, indicative of a mitochondrial function defect. Furthermore, cytochrome-dependent oxidation in FOM and N. crassa was affected, leading to reduced oxygen consumption in the mutants, linking snt2 to regulation of fungal aerobic respiration. This was accompanied by increased expression of an alternative oxidase gene, suggesting the presence of a compensatory mechanism or the potential presence of a direct link between snt2 and regulation of alternative oxidation.

The reduction of reactive oxygen species formation by mitochondrial alternative respiration in tomato basal defense against TMV infection

The role of mitochondrial alternative oxidase (AOX) and the relationship between systemic AOX induction, ROS formation, and systemic plant basal defense to Tobacco mosaic virus (TMV) were investigated in tomato plants. The results showed that TMV inoculation significantly increased the level of AOX gene transcripts, ubiquinone reduction levels, pyruvate content, and cyanide-resistant respiration (CN-resistant R) in upper, un-inoculated leaves. Pretreatment with potassium cyanide (KCN, a cytochrome pathway inhibitor) greatly increased CN-resistant R and reduced reactive oxygen species (ROS) formation, while application of salicylhydroxamic acid (SHAM, an AOX inhibitor) blocked the AOX activity and enhanced the production of ROS in the plants. Furthermore, TMV systemic infection was enhanced by SHAM and reduced by KCN pretreatment, as compared with the un-pretreated TMV counterpart. In addition, KCN application significantly diminished TMV-induced increase in antioxidant enzyme activities and dehydroascorbate/total ascorbate pool, while an opposite change was observed with SHAM-pretreated plants. These results suggest that the systemic induction of the mitochondrial AOX pathway plays a critical role in the reduction of ROS to enhance basal defenses. Additional antioxidant systems were also coordinately regulated in the maintenance of the cellular redox homeostasis.

Immunolocalization of an alternative respiratory chain in Antonospora (Paranosema) locustae spores: mitosomes retain their role in microsporidial energy metabolism.

Microsporidia are a group of fungus-related intracellular parasites with severely reduced metabolic machinery. They lack canonical mitochondria, a Krebs cycle, and a respiratory chain but possess genes encoding glycolysis enzymes, a glycerol phosphate shuttle, and ATP/ADP carriers to import host ATP. The recent finding of alternative oxidase genes in two clades suggests that microsporidial mitosomes may retain an alternative respiratory pathway. We expressed the fragments of mitochondrial chaperone Hsp70 (mitHsp70), mitochondrial glycerol-3-phosphate dehydrogenase (mitG3PDH), and alternative oxidase (AOX) from the microsporidium Antonospora (Paranosema) locustae in Escherichia coli. Immunoblotting with antibodies against recombinant polypeptides demonstrated specific accumulation of both metabolic enzymes in A. locustae spores. At the same time comparable amounts of mitochondrial Hsp70 were found in spores and in stages of intracellular development as well. Immunoelectron microscopy of ultrathin cryosections of spores confirmed mitosomal localization of the studied proteins. Small amounts of enzymes of an alternative respiratory chain in merogonial and early sporogonial stages, alongside their accumulation in mature spores, suggest conspicuous changes in components and functions of mitosomes during the life cycle of microsporidia and the important role of these organelles in parasite energy metabolism, at least at the final stages of sporogenesis.

Involvement of Ethylene and Hydrogen Peroxide in Induction of Alternative Respiratory Pathway in Salt-Treated Arabidopsis Calluses

The role of ethylene and hydrogen peroxide (H₂O₂) in the induction of the alternative respiratory pathway (AP) in calluses from wild-type (WT) Arabidopsis and ethylene-insensitive mutant etr1-3 under salt stress was investigated. The capacity and the contribution of the AP to the total respiration were significantly induced by 100 mM sodium chloride (NaCl) in WT calluses but only slightly induced in etr1-3 calluses. Ethylene emission was enhanced in WT calluses under salt stress. Application of 1-aminocyclopropane-1-carboxylic acid (an ethylene precursor) further increased the AP capacity in WT calluses but not in etr1-3 calluses under salt stress. Reduction of ethylene production by aminooxyacetic acid (AOA, an ethylene biosynthesis inhibitor) in WT calluses eliminated the NaCl-induced increase of ethylene emission and inhibited AP induction under salt stress, suggesting that ethylene is required for AP induction. H₂O₂ enhanced ethylene production while ethylene reduced H₂O₂ generation in WT calluses under salt stress. In addition, ethylene and H₂O₂ modulated NaCl-induced alternative oxidase gene (AOX1a) expression and the increase in pyruvate content in WT calluses. Inhibition of the AP by salicylhydroxamic acid in WT calluses under salt stress resulted in severe cellular damage as indicated by the high content of H₂O₂, malondialdehyde and more electrolyte leakage. Taken together, ethylene and H₂O₂ are involved in the salt-induced increase of the AP, which plays an important role in salt tolerance in WT calluses, and ethylene may be acting downstream of H₂O₂.

Effects of Hydrogen Sulfide on Alternative Pathway Respiration and Induction of Alternative Oxidase Gene Expression in Rice Suspension Cells

The toxic effects of H2S on plants are well documented. However, the molecular mechanisms reponsible for inhibition of plants by H2S are still not completely understood. We determined the effects of NaHS in the range of 0.5-10 mM on the growth of rice suspension culture cells, as well as on the expression of the alternative oxidase (AOX) gene. AOX is the terminal oxidase of the alternative pathway (AP) and exists in plant mitochondria. The results showed that H2S treatment enhanced the AP activity. During the process of H2S treatment for 4 h, the AP activity increased dramatically and achieved the peak value at a concentration of 2 mM NaHS. Then it declined at higher concentrations of NaHS (5-10 mM) and maintained a steady level. The AOX1 gene transcript level also showed a similar change as the AP activity. Interestingly, different NaHS concentrations seemed to have different effects on the expression of AOX1a, AOX1b, and AOX1c. The induction of AOX expression by low concentrations of NaHS was inferred through a reactive oxygen species (ROS)-independent pathway. At the same time, rice cells grown in culture were very sensitive to H2S, different H2S concentrations induced an increase in the cell viability. These results indicate that the H2S-induced AOX induction might play a role in inhibiting the ROS production and have an influence on cell viability.

The alternative oxidase (AOX) gene in Vibrio fischeri is controlled by NsrR and upregulated in response to nitric oxide

Alternative oxidase (AOX) is a respiratory oxidase found in certain eukaryotes and bacteria; however, its role in bacterial physiology is unclear. Exploiting the genetic tractability of the bacterium Vibrio fischeri, we explore the regulation of aox expression and AOX function. Using quantitative PCR and reporter assays, we demonstrate that aox expression is induced in the presence of nitric oxide (NO), and that the NO-responsive regulatory protein NsrR mediates the response. We have identified key amino acid residues important for NsrR function and experimentally confirmed a bioinformatically predicted NsrR binding site upstream of aox. Microrespirometry demonstrated that oxygen consumption by V. fischeri CydAB quinol oxidase is inhibited by NO treatment, whereas oxygen consumption by AOX is less sensitive to NO. NADH oxidation assays using inverted membrane vesicles confirmed that NO directly inhibits CydAB, and that AOX is resistant to NO inhibition. These results indicate a role for V. fischeri AOX in aerobic respiration during NO stress.

Alternative oxidase, a determinant of plant gametophyte fitness and fecundity

The alternative oxidase (AOX) is the terminal oxidase that comprises the cyanide-resistant respiratory pathway in plant mitochondria. While the role of AOX in plant thermogenesis is well established, its role in the reproductive development of non-thermogenic species is not well understood. AOX genes can be separated into two groups based on sequence homology, AOX1 and AOX2. Reverse genetic experiments carried out primarily in Arabidopsis and tobacco have largely focussed on examining the role of AOX1-type genes in stress responses. We recently reported a systematic characterisation of the reproductive phenotypes of three AOX2 antisense lines of soybean. This addendum summarises the key evidence in our recent paper that points to a role for AOX in the development and function of both male and female gametophytes. Furthermore, we discuss the relative importance of AOX in the reproductive biology of plant species examined to date and highlight practical implications of our findings to crop improvement research.

배추 alternative oxidase 합성 유전자와 연관된 분자마커 개발

The low and high temperature stress might affect the yield and quality of many crop species. Alternative oxidase (AOX) gene is known as factors related to stress resistance in plants. In order to develop molecular markers related to stress resistance in Chinese cabbage, fifteen ESTs sharing sequence similarity to arabidopsis AOX genes were found using Brassica rapa EST database from NCBI. The polymorphic DNA sequences using the ESTs were then screened between Chinese cabbage, `Chiifu` and `Kenshin`. We found four ESTs that have either insertion or deletion between the two cultivars. These polymorphic sites were then targeted for development of the four PCR based molecular markers. These molecular markers developed in this study could be useful for a test of their relationship with abiotic stress resistance in Chinese cabbage.

A broad distribution of the alternative oxidase in microsporidian parasites.

Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of iron-sulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX), a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1) as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosomes.