RESUMO
Alternative oxidase (AOX) is a terminal oxidase present in the electron transport system of all plants examined to date that plays an important role in the responses to abiotic and biotic stresses. Due to recent advances in cell and tissue culture, genetic engineering, and bioinformatic resources for nonmodel plants, it is now possible to study AOX in a broader diversity of species to investigate the full taxonomic distribution of AOX in plants. Additional functions of AOX should be investigated in thermogenic, carnivorous, and parasitic plants with atypical life histories. Recent methodological improvements in oxygen sensing, clustered regularly interspaced short palindromic repeats technology, and protein biochemistry will allow for considerable advancement on questions that have been long standing in the field due to experimental limitations. The role of AOX in secondary metabolism and mitochondrial metabolic pathways should also be examined due to recent discoveries in analogous systems in other organelles and fungi.
Assuntos
Proteínas de Plantas , Plantas , Plantas/genética , Plantas/metabolismo , Proteínas de Plantas/metabolismo , Oxirredutases/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismoRESUMO
Following the parasitic juvenile phase of their life cycle, sea lamprey (Petromyzon marinus) mature into a reproductive but rapidly aging and deteriorating adult, and typically die shortly after spawning in May or June. However, pre-spawning upstream migrant sea lamprey can be maintained for several months beyond their natural lifespan when held in cold water (â¼4-8 °C) under laboratory conditions. We exploited this feature to investigate the interactions between senescence, oxidative stress, and metabolic function in this phylogenetically ancient fish. We investigated how life history traits and mitochondria condition, as indicated by markers of oxidative stress (catalase activity, lipid peroxidation) and aerobic capacity (citrate synthase activity), changed in adult sea lamprey from June to December after capture during their upstream spawning migration. Body mass but not liver mass declined with age, resulting in an increase in hepatosomatic index. Both effects were most pronounced in males, which also tended to have larger livers than females. Lamprey experienced greater oxidative stress with age, as reflected by increasing activity of the antioxidant enzyme catalase and increasing levels of lipid peroxidation in liver mitochondrial isolates over time. Surprisingly, the activity of citrate synthase also increased with age in both sexes. These observations implicate mitochondrial dysfunction and oxidative stress in the senescence of sea lamprey. Due to their unique evolutionary position and the technical advantage of easily delaying the onset of senescence in lampreys using cold water, these animals could represent an evolutionary unique and tractable model to investigate senescence in vertebrates.
Assuntos
Petromyzon , Masculino , Feminino , Animais , Petromyzon/metabolismo , Catalase/metabolismo , Citrato (si)-Sintase/metabolismo , Estágios do Ciclo de Vida , Estresse OxidativoRESUMO
Invasive sea lampreys in the Laurentian Great Lakes are controlled by applying TFM (3-trifluoromethyl-4-nitrophenol) and niclosamide to streams infested with their larvae. Both agents uncouple oxidative phosphorylation in the mitochondria, but TFM specifically targets lampreys, which have a lower capacity to detoxify the lampricide. Niclosamide lacks specificity and is more potent than TFM. However, its greater potency is poorly understood. We tested the hypothesis that niclosamide is a stronger uncoupler of mitochondrial oxidative phosphorylation than TFM by measuring oxygen consumption rates in isolated liver mitochondria exposed to physiologically relevant concentrations of TFM, niclosamide, or their mixture (100 TFM:1 niclosamide) at environmentally relevant temperatures (7, 13, and 25 °C). Niclosamide increased State 4 respiration and decreased the respiratory control ratio (RCR) at much lower concentrations than TFM. Calculations of the relative EC50 values, the amount of TFM or niclosamide required to decrease the RCR by 50%, indicated that niclosamide was 40-60 times more potent than TFM. Warmer temperature did not appear to decrease the sensitivity of mitochondria to niclosamide or TFM, as observed in the intact sea lamprey exposed to TFM in warmer waters. We conclude that the extreme sensitivity of mitochondria to niclosamide contributes to its greater in vivo toxicity in the whole animal.
Assuntos
Petromyzon , Animais , Substâncias Perigosas , Lagos , Mitocôndrias , Niclosamida/farmacologia , RespiraçãoRESUMO
Deoxyribonucleoside triphosphates (dNTPs) are vital for the biosynthesis and repair of DNA. Their cellular concentration peaks during the S phase of the cell cycle. In non-proliferating cells, dNTP concentrations are low, making their reliable quantification from tissue samples of heterogeneous cellular composition challenging. Partly because of this, the current knowledge related to the regulation of and disturbances in cellular dNTP concentrations derive mostly from cell culture experiments with little corroboration at the tissue or organismal level. Here, we fill the methodological gap by presenting a simple non-radioactive microplate assay for the quantification of dNTPs with a minimum requirement of 4-12 mg of biopsy material. In contrast to published assays, this assay is based on long synthetic single-stranded DNA templates (50-200 nucleotides), an inhibitor-resistant high-fidelity DNA polymerase, and the double-stranded-DNA-binding EvaGreen dye. The assay quantified reliably less than 50 fmol of each of the four dNTPs and discriminated well against ribonucleotides. Additionally, thermostable RNAse HII-mediated nicking of the reaction products and a subsequent shift in their melting temperature allowed near-complete elimination of the interfering ribonucleotide signal, if present. Importantly, the assay allowed measurement of minute dNTP concentrations in mouse liver, heart and skeletal muscle.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Desoxirribonucleotídeos/isolamento & purificação , Oligonucleotídeos/genética , Animais , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/química , Desoxirribonucleotídeos/genética , Camundongos , Inibidores da Síntese de Ácido Nucleico/química , Oligonucleotídeos/síntese química , Ribonuclease H/genéticaRESUMO
Alternative oxidase (AOX) is a terminal oxidase within the inner mitochondrial membrane (IMM) present in many organisms where it functions in the electron transport system (ETS). AOX directly accepts electrons from ubiquinol and is therefore capable of bypassing ETS Complexes III and IV. The human genome does not contain a gene coding for AOX, so AOX expression has been suggested as a gene therapy for a range of human mitochondrial diseases caused by genetic mutations that render Complex III and/or IV dysfunctional. An effective means of screening mutations amenable to AOX treatment remains to be devised. We have generated such a tool by heterologously expressing AOX from the Pacific oyster (Crassostrea gigas) in the yeast Saccharomyces cerevisiae under the control of a galactose promoter. Our results show that this animal AOX is monomeric and is correctly targeted to yeast mitochondria. Moreover, when expressed in yeast, Pacific oyster AOX is a functional quinol oxidase, conferring cyanide-resistant growth and myxothiazol-resistant oxygen consumption to yeast cells and isolated mitochondria. This system represents a high-throughput screening tool for determining which Complex III and IV genetic mutations in yeast will be amenable to AOX gene therapy. As many human genes are orthologous to those found in yeast, our invention represents an efficient and cost-effective way to evaluate viable research avenues. In addition, this system provides the opportunity to learn more about the localization, structure, and regulation of AOXs from animals that are not easily reared or manipulated in the lab.
Assuntos
Crassostrea/enzimologia , Proteínas Mitocondriais/genética , Oxirredutases/genética , Proteínas de Plantas/genética , Saccharomyces cerevisiae/enzimologia , Animais , Crassostrea/genética , Transporte de Elétrons , Técnicas de Transferência de Genes , Terapia Genética/métodos , Humanos , Doenças Mitocondriais/terapia , Membranas Mitocondriais/química , Membranas Mitocondriais/enzimologia , Mutação , Saccharomyces cerevisiae/genéticaRESUMO
The alternative oxidase (AOX) is a terminal oxidase in the electron transport system that plays a role in mitochondrial bioenergetics. The past 20 years of research shows AOX has a wide yet patchy distribution across the tree of life. AOX has been suggested to have a role in stress tolerance, growth, and development in plants, but less is known about its function in other groups, including animals. In this study, we analyzed the taxonomic distribution of AOX across >2800 species representatives from prokaryotes and eukaryotes and developed a standardized workflow for finding and verifying the authenticity of AOX sequences. We found that AOX is limited to proteobacteria among prokaryotes, but is widely distributed in eukaryotes, with the highest prevalence in plants, fungi, and protists. AOX is present in many invertebrates, but is absent in others including most arthropods, and is absent from vertebrates. We found aberrant AOX sequences associated with some animal groups. Some of these aberrant AOXs were contaminants, but we also found putative cases of lateral gene transfer of AOX from fungi and protists to nematodes, springtails, fungus gnats, and rotifers. Our findings provide a robust and detailed analysis of the distribution of AOX and a method for identifying and verifying putative AOX sequences, which will be useful as more sequence data becomes available on public repositories.
Assuntos
Transferência Genética Horizontal , Proteínas de Plantas , Animais , Proteínas de Plantas/genética , Oxirredutases/genética , Mitocôndrias/genética , Plantas , Eucariotos/genéticaRESUMO
Oxygenic photosynthesis depends on a highly conserved electron transport system, which must be particularly dynamic in its response to environmental and physiological changes, in order to avoid an excess of excitation energy and subsequent oxidative damage. Apart from cyclic electron flow around PSII and around PSI, several alternative electron transport pathways exist including a plastoquinol terminal oxidase (PTOX) that mediates electron flow from plastoquinol to O(2). The existence of PTOX was first hypothesized in 1982 and this was verified years later based on the discovery of a non-heme, di-iron carboxylate protein localized to thylakoid membranes that displayed sequence similarity to the mitochondrial alternative oxidase. The absence of this protein renders higher plants susceptible to excitation pressure dependant variegation combined with impaired carotenoid synthesis. Chloroplasts, as well as other plastids (i.e. etioplasts, amyloplasts and chromoplasts), fail to assemble organized internal membrane structures correctly, when exposed to high excitation pressure early in development. While the role of PTOX in plastid development is established, its physiological role under stress conditions remains equivocal and we postulate that it serves as an alternative electron sink under conditions where the acceptor side of PSI is limited. The aim of this review is to provide an overview of the past achievements in this field and to offer directions for future investigative efforts. Plastoquinol terminal oxidase (PTOX) is involved in an alternative electron transport pathway that mediates electron flow from plastoquinol to O(2). This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.
Assuntos
Cloroplastos/enzimologia , Complexo Citocromos b6f/metabolismo , Fluorocarbonos/metabolismo , Fotossíntese/fisiologia , Proteínas de Plantas/metabolismo , Plantas/enzimologia , Plastoquinona/análogos & derivados , Transporte de Elétrons/fisiologia , Hidrocarbonetos Bromados , Plastoquinona/metabolismoRESUMO
The finding that alternative oxidase (AOX) is present in most kingdoms of life has resulted in a large number of AOX sequences that are available for analyses. Multiple sequence alignments of AOX proteins from evolutionarily divergent organisms represent a valuable tool and can be used to identify amino acids and domains that may play a role in catalysis, membrane association and post-translational regulation, especially when these data are coupled with the structural model for the enzyme. I validate the use of this approach by demonstrating that it detects the conserved glutamate and histidine residues in AOX that initially led to its identification as a di-iron carboxylate protein and the generation of a structural model for the protein. A comparative analysis using a larger dataset identified 35 additional amino acids that are conserved in all AOXs examined, 30 of which have not been investigated to date. I hypothesize that these residues will be involved in the quinol terminal oxidase activity or membrane association of AOX. Major differences in AOX protein sequences between kingdoms are revealed, and it is hypothesized that two angiosperm-specific domains may be responsible for the non-covalent dimerization of AOX, whereas two indels in the aplastidic AOXs may play a role in their post-translational regulation. A scheme for predicting whether a particular AOX protein will be recognized by the alternative oxidase monoclonal antibody generated against the AOX of Sauromatum guttatum (Voodoo lily) is presented. The number of functional sites in AOX is greater than expected, and determining the structure of AOX will prove extremely valuable to future research.
Assuntos
Oxirredutases/química , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Aminoácidos/química , Anticorpos Monoclonais/imunologia , Membrana Celular/enzimologia , Sequência Conservada , Proteínas Mitocondriais , Biologia Molecular , Dados de Sequência Molecular , Oxirredutases/imunologia , Proteínas de Plantas , Processamento de Proteína Pós-Traducional , Reprodutibilidade dos Testes , Alinhamento de SequênciaRESUMO
The gymnosperm Pinus pinea L. (stone pine) is a typical Mediterranean pine used for nuts and timber production, and as an ornamental around the world. Pine genomes are large in comparison to other species. The hypothesis that retrotransposons, such as gymny, made a large contribution to this alteration in genome size was recently confirmed. However, P. pinea is unique in other various aspects. P. pinea demonstrates a different pattern of gymny organization than other Pinus subgenera. Additionally, P. pinea has a highly recalcitrant behaviour in relation to standard conifer protocols for the induction of somatic embryogenesis or rooting. Because such types of cell reprogramming can be explained as a reaction of plant cells to external stress, it is of special interest to study sequence peculiarities in stress-inducible genes, such as the alternative oxidase (AOX). This is the first report containing molecular evidence for the existence of AOX in gymnosperms at the genetic level. P. pinea AOXs were isolated by a polymerase chain reaction (PCR) approach and three genes were identified. Two of the genes belong to the AOX1 subfamily and one belongs to the AOX2 subfamily. The existence of both AOX subfamilies in gymnosperms is reported here for the first time. This discovery supports the hypothesis that AOX1 and AOX2 subfamilies arose prior to the separation of gymnosperms and angiosperms, and indicates that the AOX2 is absent in monocots because of subsequent gene loss events. Polymorphic P. pinea AOX1 sequences from a selected genetic clone are presented indicating non-allelic, non-synonymous and synonymous translation products.
Assuntos
Genes de Plantas , Família Multigênica/genética , Oxirredutases/genética , Pinus/enzimologia , Pinus/genética , Sequência de Aminoácidos , Sequência Conservada , Evolução Molecular , Proteínas Mitocondriais , Dados de Sequência Molecular , Oxirredutases/química , Filogenia , Proteínas de Plantas , Polimorfismo de Nucleotídeo Único/genética , Biossíntese de Proteínas , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
The electron transport systems in mitochondria of many organisms contain alternative respiratory enzymes distinct from those of the canonical respiratory system depicted in textbooks. Two of these enzymes, the alternative NADH dehydrogenase and the alternative oxidase, were of interest to a limited circle of researchers until they were envisioned as gene therapy tools for mitochondrial disease treatment. Recently, these enzymes were discovered in several animals. Here, we analyse the functioning of alternative NADH dehydrogenases and oxidases in different organisms. We propose that both enzymes ensure bioenergetic and metabolic flexibility during environmental transitions or other conditions which may compromise the operation of the canonical respiratory system.
Assuntos
Metabolismo Energético , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , NADPH Desidrogenase/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , AnimaisRESUMO
In addition to the typical electron transport system (ETS) in animal mitochondria responsible for oxidative phosphorylation, in some species there exists an alternative oxidase (AOX) pathway capable of catalyzing the oxidation of ubiquinol and the reduction of oxygen to water. The discovery of AOX in animals is recent and further investigations into its expression, regulation, and physiological role have been hampered by the lack of a tractable experimental model organism. Our recent DNA database searches using bioinformatics revealed an AOX sequence in several marine copepods including Tigriopus californicus. This species lives in tidepools along the west coast of North America and is subject to a wide variety of daily environmental stresses. Here we verify the presence of the AOX gene in T. californicus and the expression of AOX mRNA and AOX protein in various life stages of the animal. We demonstrate that levels of the AOX protein increase in T. californicus in response to cold and heat stress compared to normal rearing temperature. We predict that a functional AOX pathway is present in T. californicus, propose that this species will be a useful model organism for the study of AOX in animals, and discuss future directions for animal AOX research.
Assuntos
Proteínas de Artrópodes , Copépodes , Regulação Enzimológica da Expressão Gênica/fisiologia , Resposta ao Choque Térmico/fisiologia , Oxirredutases , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/genética , Temperatura Baixa , Copépodes/enzimologia , Copépodes/genética , Oxirredutases/biossíntese , Oxirredutases/genéticaRESUMO
The study of glycolysis, the TCA cycle, and oxidative phosphorylation in animals has yielded a wealth of information about bioenergetics. Less is known about how animals use fuels other than glucose and less characterized enzymes that are also used to provide electrons to the electron transport system. It has become clear that bioenergetic flexibility is employed by a wide variety of animals in order to successfully grow, maintain cells, and reproduce, and has contributed to the exploitation of new environments and ecological niches through evolution. In most cases, the discovery of these "alternative" fuels and non-classical pathways is relatively recent, but is starting to call into question long believed paradigms about the diversity of animal bioenergetics. We present several specific examples of these "alternatives" and the animals that use them and present some implications for animal mitochondrial physiology research.
Assuntos
Ciclo do Ácido Cítrico/fisiologia , Glicólise/fisiologia , Mitocôndrias/fisiologia , Fosforilação Oxidativa , Animais , Transporte de Elétrons/fisiologia , HumanosRESUMO
Despite intense research on the in vitro characterization of regulatory factors modulating the alternative oxidase (AOX) pathway, the regulation of its activity in vivo is still not fully understood. Advances concerning in vivo regulation of AOX based on the oxygen-isotope fractionation technique are reviewed, and regulatory factors that merit future research are highlighted. In addition, we review and discuss the main biological functions assigned to the plant AOX, and suggest future experiments involving in vivo activity measurements to test different hypothesized physiological roles.
Assuntos
Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Plantas/enzimologia , Plantas/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Alternative oxidase (AOX) represents a non-energy conserving branch in mitochondrial electron transport while plastoquinol terminal oxidase (PTOX) represents a potential branch in photosynthetic electron transport. Using a metagenomics dataset, we have uncovered numerous and diverse AOX and PTOX genes from the Sargasso Sea. Sequence similarity, synteny and phylogenetic analyses indicate that the large majority of these genes are from prokaryotes. AOX appears to be widely distributed among marine Eubacteria while PTOX is widespread among strains of cyanobacteria closely related to the high-light adapted Prochlorococcus marinus MED4, as well as Synechococcus. The wide distribution of AOX and PTOX in marine prokaryotes may have important implications for productivity in the world's oceans.
Assuntos
Oxirredutases/análise , Plastoquinona/análogos & derivados , Plastoquinona/análise , Células Procarióticas/enzimologia , Água do Mar/microbiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Bactérias/enzimologia , Sequência Conservada , Cianobactérias/enzimologia , DNA Bacteriano/genética , Evolução Molecular , Proteínas Mitocondriais , Dados de Sequência Molecular , Oceanos e Mares , Filogenia , Proteínas de Plantas , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Synechococcus/enzimologia , SinteniaRESUMO
The alternative oxidase is a non-proton motive 'alternative' to electron transport through the cytochrome pathway. Despite its wasteful nature in terms of energy conservation, the pathway is likely present throughout the plant kingdom and appears to be expressed in most plant tissues. A small alternative oxidase gene family exists, the members of which are differentially expressed in response to environmental, developmental and other cell signals. The alternative oxidase enzyme possesses tight biochemical regulatory properties that determine its ability to compete with the cytochrome pathway for electrons. Studies show that alternative oxidase can be a prominent component of total respiration in important crop species. All these characteristics suggest this pathway plays an important role in metabolism and/or other aspects of cell physiology. This brief review is an introduction to experimental methods and approaches applicable to different areas of alternative oxidase research. We hope it provides a framework for further investigation of this fascinating component of primary plant metabolism.
RESUMO
A classification scheme based on protein phylogenies and sequence harmony method was used to clarify the taxonomic distribution and evolutionary history of the alternative oxidase (AOX) in angiosperms. A large data set analyses showed that AOX1 and AOX2 subfamilies were distributed into 4 phylogenetic clades: AOX1a-c/1e, AOX1d, AOX2a-c and AOX2d. High diversity in AOX family compositions was found. While the AOX2 subfamily was not detected in monocots, the AOX1 subfamily has expanded (AOX1a-e) in the large majority of these plants. In addition, Poales AOX1b and 1d were orthologous to eudicots AOX1d and then renamed as AOX1d1 and 1d2. AOX1 or AOX2 losses were detected in some eudicot plants. Several AOX2 duplications (AOX2a-c) were identified in eudicot species, mainly in the asterids. The AOX2b originally identified in eudicots in the Fabales order (soybean, cowpea) was divergent from AOX2a-c showing some specific amino acids with AOX1d and then it was renamed as AOX2d. AOX1d and AOX2d seem to be stress-responsive, facultative and mutually exclusive among species suggesting a complementary role with an AOX1(a) in stress conditions. Based on the data collected, we present a model for the evolutionary history of AOX in angiosperms and highlight specific areas where further research would be most beneficial.
Assuntos
Evolução Molecular , Magnoliopsida/enzimologia , Magnoliopsida/genética , Proteínas Mitocondriais/classificação , Proteínas Mitocondriais/genética , Oxirredutases/classificação , Oxirredutases/genética , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Biologia Computacional , Variação Genética , Magnoliopsida/classificaçãoRESUMO
Alternative oxidase (AOX) is a terminal ubiquinol oxidase present in the respiratory chain of all angiosperms investigated to date, but AOX distribution in other members of the Viridiplantae is less clear. We assessed the taxonomic distribution of AOX using bioinformatics. Multiple sequence alignments compared AOX proteins and examined amino acid residues involved in AOX catalytic function and post-translational regulation. Novel AOX sequences were found in both Chlorophytes and Streptophytes and we conclude that AOX is widespread in the Viridiplantae. AOX multigene families are common in non-angiosperm plants and the appearance of AOX1 and AOX2 subtypes pre-dates the divergence of the Coniferophyta and Magnoliophyta. Residues involved in AOX catalytic function are highly conserved between Chlorophytes and Streptophytes, while AOX post-translational regulation likely differs in these two lineages. We demonstrate experimentally that an AOX gene is present in the moss Physcomitrella patens and that the gene is transcribed. Our findings suggest that AOX will likely exert an influence on plant respiration and carbon metabolism in non-angiosperms such as green algae, bryophytes, liverworts, lycopods, ferns, gnetophytes, and gymnosperms and that further research in these systems is required.
Assuntos
Proteínas Mitocondriais/genética , Oxirredutases/genética , Proteínas de Plantas/genética , Plantas/classificação , Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Biologia Computacional , Bases de Dados Genéticas , Evolução Molecular , Ferro/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Oxirredutases/química , Oxirredutases/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas/enzimologia , Ligação Proteica , Alinhamento de Sequência , Viridiplantae/classificação , Viridiplantae/enzimologia , Viridiplantae/genéticaRESUMO
Alternative oxidase (AOX), a ubiquinol oxidase, introduces a branch point into the respiratory electron transport chain, bypassing complexes III and IV and resulting in cyanide-resistant respiration. Previously, AOX was thought to be limited to plants and some fungi and protists but recent work has demonstrated the presence of AOX in most kingdoms of life, including animals. In the present study we identified AOX in 28 animal species representing nine phyla. This expands the known taxonomic distribution of AOX in animals by 10 species and two phyla. Using bioinformatics we found AOX gene sequences in members of the animal phyla Porifera, Placozoa, Cnidaria, Mollusca, Annelida, Nematoda, Echinodermata, Hemichordata and Chordata. Using reverse-transcriptase polymerase chain reaction (RT-PCR) with degenerate primers designed to recognize conserved regions of animal AOX, we demonstrated that AOX genes are transcribed in several animals from different phyla. An analysis of full-length AOX sequences revealed an amino acid motif in the C-terminal region of the protein that is unique to animal AOXs. Animal AOX also lacks an N-terminal cysteine residue that is known to be important for AOX enzyme regulation in plants. We conclude that the presence of AOX is the ancestral state in animals and hypothesize that its absence in some lineages, including vertebrates, is due to gene loss events.
Assuntos
Invertebrados/enzimologia , Oxirredutases/metabolismo , Animais , Primers do DNA , Transporte de Elétrons , Eucariotos/enzimologia , Evolução Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/enzimologia , Membranas Mitocondriais/enzimologia , Proteínas Mitocondriais , Oxirredutases/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/enzimologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Alternative oxidase (AOX) is a terminal quinol oxidase located in the respiratory electron transport chain that catalyses the oxidation of quinol and the reduction of oxygen to water. However, unlike the cytochrome c oxidase respiratory pathway, the AOX pathway moves fewer protons across the inner mitochondrial membrane to generate a proton motive force that can be used to synthesise ATP. The energy passed to AOX is dissipated as heat. This appears to be very wasteful from an energetic perspective and it is likely that AOX fulfils some physiological function(s) that makes up for its apparent energetic shortcomings. An examination of the known taxonomic distribution of AOX and the specific organisms in which AOX has been studied has been used to explore themes pertaining to AOX function and regulation. A comparative approach was used to examine AOX function as it relates to the biochemical function of the enzyme as a quinol oxidase and associated topics, such as enzyme structure, catalysis and transcriptional expression and post-translational regulation. Hypotheses that have been put forward about the physiological function(s) of AOX were explored in light of some recent discoveries made with regard to species that contain AOX. Fruitful areas of research for the AOX community in the future have been highlighted.
RESUMO
We find that an expression system widely used to chemically induce transgenes of interest in tobacco (Nicotiana tabacum Petit Havana SR1) can cause severe growth defects in this species. This gene expression system has been shown to cause non-specific effects (including growth retardation) in other plant species, but has until now been largely accepted to be a relatively problem-free system for use in tobacco. The expression system is based on the ability of the glucocorticoid dexamethasone (DEX) to activate a non-plant chimeric transcription factor (GVG), which then activates expression of a transgene of interest. The aberrant growth phenotype only manifests itself after DEX application and only occurs in plants in which the constitutive levels of GVG expression are higher than average. We found that approximately 30% of all transgenic plants produced showed some level of growth retardation under our standard growth conditions. However, by modulating irradiance levels following DEX application, we also showed that the manifestation and severity of the aberrant phenotype is highly dependent upon growth conditions, highlighting that such conditions are a critical parameter to consider during all stages of using this gene expression system. We also identified an increase in ACC oxidase gene expression as an early, sensitive and robust molecular marker for the aberrant phenotype. This molecular marker should be valuable to investigators wishing to readily identify transgenic plants in which GVG expression levels are beyond a threshold that begins to produce non-specific effects of the gene expression system under a defined set of growth conditions.