Alternative Oxidase Protein Research Articles

Alternative Oxidase: From Molecule and Function to Future Inhibitors.

In the respiratory chain of the majority of aerobic organisms, the enzyme alternative oxidase (AOX) functions as the terminal oxidase and has important roles in maintaining metabolic and signaling homeostasis in mitochondria. AOX endows the respiratory system with flexibility in the coupling among the carbon metabolism pathway, electron transport chain (ETC) activity, and ATP turnover. AOX allows electrons to bypass the main cytochrome pathway to restrict the generation of reactive oxygen species (ROS). The inhibition of AOX leads to oxidative damage and contributes to the loss of adaptability and viability in some pathogenic organisms. Although AOXs have recently been identified in several organisms, crystal structures and major functions still need to be explored. Recent work on the trypanosome alternative oxidase has provided a crystal structure of an AOX protein, which contributes to the structure-activity relationship of the inhibitors of AOX. Here, we review the current knowledge on the development, structure, and properties of AOXs, as well as their roles and mechanisms in plants, animals, algae, protists, fungi, and bacteria, with a special emphasis on the development of AOX inhibitors, which will improve the understanding of respiratory regulation in many organisms and provide references for subsequent studies of AOX-targeted inhibitors.

Biomolecular interaction of purified recombinant Arabidopsis thaliana's alternative oxidase 1A with TCA cycle metabolites: Biophysical and molecular docking studies

In higher plants, the mitochondrial alternative oxidase (AOX) pathway plays an essential role in maintaining the TCA cycle/cellular carbon and energy balance under various physiological and stress conditions. Though the activation of AOX pathway upon exogenous addition of α-ketoacids/TCA cycle metabolites [pyruvate, α-ketoglutarate (α-KG), oxaloacetic acid (OAA), succinate and malic acid] to isolated mitochondria is known, the molecular mechanism of interaction of these metabolites with AOX protein is limited. The present study is designed to understand the biomolecular interaction of pure recombinant Arabidopsis thaliana AOX1A with TCA cycle metabolites under in vitro conditions using various biophysical and molecular docking studies. The binding of α-KG, fumaric acid and OAA to rAtAOX1A caused conformational change in the microenvironment of tryptophan residues as evidenced by red shift in the synchronous fluorescence spectra (∆λ = 60 nm). Besides, a decrease in conventional fluorescence emission spectra, tyrosine specific synchronous fluorescence spectra (∆λ = 15 nm) and α-helical content of CD spectra revealed the conformation changes in rAtAOX1A structure associated with binding of various TCA cycle metabolites. Further, surface plasmon resonance (SPR) and microscale thermophoresis (MST) studies revealed the binding affinity, while docking studies identified binding pocket residues, respectively, for these metabolites on rAtAOX1A.

Alternative oxidase promotes high iron tolerance in Candida albicans.

Although crucial for cellular functioning, iron is toxic when present in excess. Iron overload can create a labile iron pool (LIP) that harms the mitochondrion, resulting in disruption of oxidative respiration. Fungal pathogen C. albicans possesses an unconventional terminal oxidase [alternative oxidase (AOX)] that can disrupt the electron transport chain to remove excess electrons, generated from various stressors, to shield the respiratory apparatus. Here, we identify a novel role for AOX under high iron, whereby it works as "safety valve" to protect the mitochondrion from high iron-induced reactive oxygen species (ROS). C. albicans showed pronounced metabolic flexibility to environmental iron, ranging from 1 to 500 µM. High iron significantly increased intracellular iron, LIP, and levels of cellular ROS. Expression of both AOX1/2 was induced by iron, and cells lacking Aox1/2 showed high levels of mitochondrial ROS as well as significant growth defect under high iron. Thus, Aox1/2 helps mitigate the negative effects of iron. Furthermore, iron-induced expression of AOX1/2 was intermittent, thus preventing ATP loss despite AOX being non-proton motive. Mitochondrial oxygen consumption rate (OCR) was significantly higher (62.8%) under high iron conditions. Contribution of Aox1/2 was greater toward this high OCR, compared to their contribution in low iron conditions. Lastly, enhanced virulence of C. albicans that is observed for wild-type cells under high iron was lost when fungal cells lacking AOX1/2 were used for murine infection. Thus, AOX is crucial for preserving mitochondrial function for survival in a high iron host.IMPORTANCEThe yeast C. albicans exhibits metabolic flexibility for adaptability to host niches with varying availability of nutrients including essential metals like iron. For example, blood is iron deplete, while the oral cavity and the intestinal lumen are considered iron replete. We show here that C. albicans can tolerate very high levels of environmental iron, despite an increase in high iron-induced reactive oxygen species (ROS) that it mitigates with the help of a unique oxidase, known as alternative oxidase (AOX). High iron induces AOX1/2 that limits mitochondrial accumulation of ROS. Genetic elimination of AOX1/2 resulted in diminished virulence during oropharyngeal candidiasis in high iron mice. Since human mitochondria lack AOX protein, it represents a unique target for treatment of fungal infections.

Alternative oxidase 1a and 1d enable metabolic flexibility during Ala catabolism in Arabidopsis.

Ala is a central metabolite in leaf cells whose abundance is related to pyruvate (Pyr) metabolism and nocturnal respiration rates. Exposure of Arabidopsis (Arabidopsis thaliana) leaf disks to certain exogenous amino acids including Ala led to substantial increases in nighttime respiration rates as well as increases in alternative oxidase (AOX) 1d transcript and protein levels. During Ala treatment, AOX1d accumulation, but not AOX1a accumulation, was dependent upon the catabolism of Ala. Complete loss of AOX expression in aox1a aox1d leaf disks did not significantly affect oxygen consumption rates (OCR) under Ala treatment, indicating that AOX capacity per se was not essential for respiratory stimulation by Ala. Rather, Ala treatments caused induction of select antioxidant mechanisms in leaf disks, including a large increase of the ascorbate pool, which was substantially more oxidized in aox1a aox1d leaf disks. Furthermore, we observed differences in the accumulation of a sequence of TCA cycle intermediates from Pyr to 2-oxoglutarate (2-OG) in wild type (WT) upon Ala treatment that did not occur in aox1a aox1d leaf disks. The results indicate that AOX induction during enhanced Ala catabolism in leaves mediates mitochondrial redox status, allowing greater metabolic flexibility in mitochondrial organic acid metabolism.

Inhibition of alternative oxidase disrupts the development and oviposition of Biomphalaria glabrata snails

BackgroundBiomphalaria glabrata is one of the main intermediate hosts of Schistosoma mansoni, the most widespread species of Schistosoma. Our previous studies proved that alternative oxidase (AOX), the terminal oxidase in the mitochondrial respiratory chain, widely exists in several species of intermediate host snails of Schistosoma. Meanwhile, inhibition of AOX activity in Oncomelania hupensis snails could dramatically enhance the molluscicidal effect of niclosamide. As a hermaphroditic aquatic mollusc, the high fecundity and population density of B. glabrata increase the difficulty of snail control, which is one of the critical strategies for schistosomiasis elimination. The present study aimed to investigate the possible role of AOX in the development and fecundity of B. glabrata snail, which could be manipulated more manageable than other species of intermediate host snails of Schistosoma.MethodsThe dynamic expression of the AOX gene was investigated in different developmental stages and tissues of B. glabrata, with morphological change and oviposition behaviour observed from juvenile to adult snails. Furtherly, dsRNA-mediated knockdown of BgAOX mRNA and the AOX protein activity inhibiting was performed to investigate the effect of AOX on the development and oviposition of snails.ResultsThe BgAOX gene expression profile is highly related to the development from late juveniles to adults, especially to the reproductive system of snails, with a positive correlation of 0.975 between egg production and BgAOX relative expression in ovotestis of snails. The inhibition of BgAOX at the transcriptional level and AOX activity could efficiently inhibit snail growth. However, the interference at the BgAOX protein activity level led to more severe tissue damage and more significant inhibition of oviposition than at the transcriptional level. This inhibition of growth and oviposition decreased gradually with the increase in the snail size.ConclusionsThe inhibition of AOX could efficiently disrupt the development and oviposition of B. glabrata snails, and the intervention targeting AOX at the juvenile stage is more effective for snails. This investigation explored the role of AOX in the growth and development of snails. It would benefit snail control in the future by providing a potential target while using molluscicides more efficiently.Graphical abstract

Exogenous Spermidine Alleviated Waterlogging Damages in Two Varieties of Camellia oleifera

The potential management of waterlogging-damaged plants can be through the promotion of aliphatic polyamine accumulation, such as spermidine (SPD), in non-accumulator and accumulator species under stress. Camellia oleifera, commonly called tea oil, is an evergreen shrub confronting waterlogged soils in Hainan forest plantations during the pluvial season. As far as we know, few studies focused on the responses of C. oleifera to abiotic stresses, such as waterlogging (WL), and the involvement of SPD in WL tolerance remains unclear. Therefore, two cultivars of C. oleifera (CoH1 and CoH2) were subjected to WL and exogenous SPD to shed light on the role of SPD on WL tolerance via the morphological and physio-biochemical responses of C. oleifera under stress. The results showed that the two varieties of C. oleifera were sensitive to WL stress, and spraying SPD enhanced WL tolerance via root activities, photosynthesis, redox-homeostasis, antioxidant machinery, and compatible solute components. Thus, exogenous SPD significantly reduced the damages caused by WL in C. oleifera seedlings. Moreover, the alternative oxidase (AOX) protein content was down regulated by WL in both varieties of C. oleifera, whereas exogenous SPD enhanced the AOX protein under stress. The two varieties of C. oleifera generally had similar morphological and physiological responses to WL. However, CoH2 demonstrated better photosynthesis compared to CoH1. The results of the present study provide a significant outlook to improve the accumulation of SPD in trees under abiotic stress, particularly via genome editing techniques.

Changes in flower volatiles and analysis of alternative oxidase and ultrastructure in Dendrobium huoshanense induced by snail (Bradybaena ravida) infestation

Flower volatiles, as a class of signals that plants communicate with the outside world, are of great significance to the biological control of pests and plant breeding. To explore the biological control methods of snails and accumulate data for the selection and breeding of snail-resistant Dendrobium huoshanense varieties, this study analyzed characteristic changes in D. huoshanense flower volatiles, mitochondrial alternative oxidase (AOX), and ultrastructure under snail (Bradybaena ravida) infestation stress. The results showed that the volatile components of flowers induced by B. ravida infestation changed significantly. Variation of volatile components was mainly caused by five compounds, dipropyl disulfide, 1,8-cineol, β-elemene, β-ocimene, and β-caryophyllene. These compounds had significant repellent or luring effects on snails. AOX activity and AOX protein content also increased significantly after B. ravida infestation. The transcript levels of the AOX1a and AOX1b genes were significantly higher than those of healthy flowers (more than 1.8 folds). Moreover, ultrastructural indicators such as the size of intercellular space of epidermal cells and depth of depression on the cell surface, stomata area, mitochondrial volume, and the number of mitochondria and osmiophilic globules increased significantly. Pearson correlation analysis showed that AOX and ultrastructure were highly correlated with the above five compounds. The study clarified the change characteristics of volatiles in D. Huoshanense flowers induced by B. ravida infestation and preliminarily revealed the relation between flower volatile emissions and changes in AOX and ultrastructure. In the future, we need to strengthen the research on the molecular mechanism of flower volatile signal transmission and transduction.

Gasotransmitter H2S accelerates seed germination via activating AOX mediated cyanide-resistant respiration pathway

Hydrogen sulfide (H2S) has been witnessed as a crucial gasotransmitter involving in various physiological processes in plants. H2S signaling has been reported to involve in regulating seed germination, but the underlying mechanism remains poorly understood. Here, we found that endogenous H2S production was activated in germinating Arabidopsis seeds, correlating with upregulated both the transcription and the activity of L-cysteine desulfhydrase (EC 4.4.1.28, LCD and DES1) responsible for H2S production. Moreover, seed germination could be significantly accelerated by exogenous NaHS (the H2S donor) fumigation and over-expressing DES1, while H2S-generation defective (lcd/des1) seeds exhibited decreased germination speed. We also confirmed that the alternative oxidase (AOX), a cyanide-insensitive terminal oxidase, can be stimulated by imbibition. Furthermore, exogenous H2S fumigation and over-expressing DES1 could significantly reinforced imbibition induced increase of both the AOX1A expression and AOX protein abundance, while this increase could be obviously weakened in lcd/des1. Additionally, exogenous H2S fumigation mediated post-translational modification to keep AOX in its reduced and active state, which might involve H2S induced improvement of the reduced GSH content and the cell reducing power. The promotive effect of H2S on germination was clearly impaired by inducing aox1a mutation, indicating that AOX acts downstream of H2S signaling to accelerate seed germination. Consequently, H2S signaling was activated during germination then acted as a trigger to induce AOX mediated cyanide-resistant respiration to accelerate seed germination. Our study correlates H2S signaling to cyanide-resistant respiration, providing evidence for more extensive studies of H2S signaling.

Structural and Biophysical Characterization of Purified Recombinant Arabidopsis thaliana's Alternative Oxidase 1A (rAtAOX1A): Interaction With Inhibitor(s) and Activator.

In higher plants, alternative oxidase (AOX) participates in a cyanide resistant and non-proton motive electron transport pathway of mitochondria, diverging from the ubiquinone pool. The physiological significance of AOX in biotic/abiotic stress tolerance is well-documented. However, its structural and biophysical properties are poorly understood as its crystal structure is not yet revealed in plants. Also, most of the AOX purification processes resulted in a low yield/inactive/unstable form of native AOX protein. The present study aims to characterize the purified rAtAOX1A protein and its interaction with inhibitors, such as salicylhydroxamic acid (SHAM) and n-propyl gallate (n-PG), as well as pyruvate (activator), using biophysical/in silico studies. The rAtAOX1A expressed in E. coli BL21(DE3) cells was functionally characterized by monitoring the respiratory and growth sensitivity of E. coli/pAtAOX1A and E. coli/pET28a to classical mitochondrial electron transport chain (mETC) inhibitors. The rAtAOX1A, which is purified through affinity chromatography and confirmed by western blotting and MALDI-TOF-TOF studies, showed an oxygen uptake activity of 3.86 μmol min−1 mg−1 protein, which is acceptable in non-thermogenic plants. Circular dichroism (CD) studies of purified rAtAOX1A revealed that >50% of the protein content was α-helical and retained its helical absorbance signal (ellipticity) at a wide range of temperature and pH conditions. Further, interaction with SHAM, n-PG, or pyruvate caused significant changes in its secondary structural elements while retaining its ellipticity. Surface plasmon resonance (SPR) studies revealed that both SHAM and n-PG bind reversibly to rAtAOX1A, while docking studies revealed that they bind to the same hydrophobic groove (Met191, Val192, Met195, Leu196, Phe251, and Phe255), to which Duroquinone (DQ) bind in the AtAOX1A. In contrast, pyruvate binds to a pocket consisting of Cys II (Arg174, Tyr175, Gly176, Cys177, Val232, Ala233, Asn294, and Leu313). Further, the mutational docking studies suggest that (i) the Met195 and Phe255 of AtAOX1A are the potential candidates to bind the inhibitor. Hence, this binding pocket could be a ‘potential gateway' for the oxidation-reduction process in AtAOX1A, and (ii) Arg174, Gly176, and Cys177 play an important role in binding to the organic acids like pyruvate.

The identification of alternative oxidase in intermediate host snails of Schistosoma and its potential role in protecting Oncomelania hupensis against niclosamide-induced stress

BackgroundSnail intermediate hosts are mandatory for the transmission of schistosomiasis, which has to date infected more than 200 million people worldwide. Our previous studies showed that niclosamide treatment caused the inhibition of aerobic respiration and oxidative phosphorylation, and the disruption of energy supply, in one of the intermediate hosts of schistosomiasis, Oncomelania hupensis, which eventually led to the death of the snails. Meanwhile, the terminal oxidase in the mitochondrial respiratory chain, alternative oxidase (AOX), was significantly up-regulated, which was thought to counterbalance the oxidative stress and maintain metabolic homeostasis in the snails. The aims of the present study are to identify the AOXs in several species of snails and investigate the potential activation of O. hupensis AOX (OhAOX) under niclosamide-induced stress, leading to enhanced survival of the snail when exposed to this molluscicide.MethodsThe complete complementary DNA was amplified from the AOXs of O. hupensis and three species of Biomphalaria; the sequence characteristics were analysed and the phylogenetics investigated. The dynamic expression and localisation of the AOX gene and protein in O. hupensis under niclosamide-induced stress were examined. In addition, the expression pattern of genes in the mitochondrial respiratory complex was determined and the production of reactive oxygen species (ROS) calculated. Finally, the molluscicidal effect of niclosamide was compared between snails with and without inhibition of AOX activity.ResultsAOXs containing the invertebrate AOX-specific motif NP-[YF]-XPG-[KQE] were identified from four species of snail, which phylogenetically clustered together into Gastropoda AOXs and further into Mollusca AOXs. After niclosamide treatment, the levels of OhAOX messenger RNA (mRNA) and OhAOX protein in the whole snail were 14.8 and 2.6 times those in untreated snails, respectively, but varied widely among tissues. Meanwhile, the level of cytochrome C reductase mRNA showed a significant decrease in the whole snail, and ROS production showed a significant decrease in the liver plus gonad (liver-gonad) of the snails. At 24 h post-treatment, the mortality of snails treated with 0.06–0.1 mg/L niclosamide and AOX inhibitor was 56.31–76.12% higher than that of snails treated with 0.1 mg/L niclosamide alone.ConclusionsAOX was found in all the snail intermediate hosts of Schistosoma examined here. AOX was significantly activated in O. hupensis under niclosamide-induced stress, which led to a reduction in oxidative stress in the snail. The inhibition of AOX activity in snails can dramatically enhance the molluscicidal effect of niclosamide. A potential target for the development of an environmentally safe snail control method, which acts by inhibiting the activity of AOX, was identified in this study.Graphical abstract

Responses of woody plant Dalbergia odorifera treated with glycine betaine to drought and cold stresses: involvement of the alternative oxidase

Woody plants faced multiple abiotic stresses in forest plantation that can influence their growth and development. Dalbergia odorifera T. Chen is a vulnerably endangered tree species, and references about its responses to abiotic stresses are very rare in literature. Furthermore, the mechanisms underlying the abiotic stress tolerance in plants induced by exogenous glycine betaine (GB) remains unclear. Indeed, the alternative oxidase (AOX) is one of the major components of antioxidant enzymatic machinery, and there are no studies that focused on the effects of GB on the amount of AOX protein in plants under drought or cold stresses. Thus, the aim of this study was to investigate the effect of exogenous GB on the phenotype, osmoprotectants, photosynthetic pigments, and antioxidant systems in D. odorifera under cold and drought stresses. The layout of the trial was a factorial experiment in a completely randomized design using two factors including abiotic stress (drought and cold) and GB. Moreover, the principal component analysis based on the measured parameters manifested how the selected ROS scavengers were dispersed throughout the treatments. The results showed huge beneficial impacts of GB on the phenotypic traits of D. odorifera; GB also influences positively the antioxidant machinery, photosynthetic pigments, redox-homeostasis, and water status in D. odorifera seedlings under both stresses. Moreover, exogenous GB affects more the AOX pathway in D. odorifera under cold stress than under drought stress.

De novo transcriptome assembly reveals three alternative oxidase encoding genes in Nymphaea alba L.

Water lilies are aquatic, ornamental and economically valuable plants classified under Nymphaea genus. Nymphaea alba L., white water lily, has a special focus since it is a member of basal angiosperms. Alternative oxidase (AOX) proteins are the terminal oxidases in the electron transport chain of plants. Identification of alternative oxidase encoding genes for basal angiosperms is important to increase the quality of phylogenetic studies. However, AOX encoding genes were yet to be discovered for N. alba. In this study, we aimed to identify alternative oxidase encoding genes in N. alba by performing transcriptome analysis. Annotation of 272,934 unigenes with Trinotate tool revealed 77 transcripts with AOX domains characterized in known alternative oxidases. Blast analysis of these 77 sequences with known AOX proteins revealed three distinct AOX genes (AOX1, AOX2 and AOX4) in N. alba. After in silico subcellular localization analysis of three identified AOX proteins, AOX1, AOX2 are predicted as mitochondrial while AOX4 is a plastidic alternative oxidase protein. Template-based structural modeling results showed that all identified proteins are statistically similar to known structure models of corresponding AOXs.

Isolation and Measurement of Respiration and Structural Studies of Purified Mitochondria from Heterotrophic Plant Tissues.

Mitochondria are the power houses of eukaryotic cells. These organelles contain various oxidoreductase complexes. Electron transfer from different reducing equivalents channeled via these complexes drives proton translocation across the inner mitochondrial membrane, leading to ATP generation. Plant mitochondria contain alternative NAD(P)H dehydrogenases, alternative oxidase, and uncoupling protein, and TCA cycle enzymes are located in their matrix. Apart from ATP production, mitochondria are also involved in synthesis of vitamins and cofactors and participate in fatty acid, nucleotide, photorespiratory, and antioxidant metabolism. Recent emerging evidence suggests that mitochondria play a role in redox signaling and generation of reactive oxygen and nitrogen species. For mitochondrial studies, it is essential to isolate physiologically active mitochondria with good structural integrity. In this article, we explain a detailed procedure for isolation of mitochondria from various heterotrophic tissues, such as germinating chickpea seeds, potato tubers, and cauliflower florets. This procedure requires discontinuous Percoll gradient centrifugation and can give a good yield of mitochondria, in the range of 4 to 8 mg per 50 g tissue with active respiratory capacity. After MitoTracker staining, isolated mitochondria can be visualized by using a confocal microscope. The structure of mitochondria can be monitored by scanning electron microscopy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation of mitochondria from germinating chickpea seeds, potato tubers, and cauliflower florets Basic Protocol 2: Quantification of mitochondrial protein concentration by Bradford assay Basic Protocol 3: Quantification of mitochondrial respiration using single-channel free-radical analyzer Basic Protocol 4: Staining of mitochondria and confocal imaging Basic Protocol 5: Visualization of isolated mitochondria under scanning electron microscope.

Differential modulation of photosynthesis, ROS and antioxidant enzyme activities in stress-sensitive and -tolerant rice cultivars during salinity and drought upon restriction of COX and AOX pathways of mitochondrial oxidative electron transport

Drought and salt stresses are two major abiotic stress factors that hamper crop growth and productivity. Three rice cultivars with different sensitivity and tolerance towards abiotic stress were used in the current study. While cultivar Aiswarya is salt- and drought-sensitive, cultivar Vyttila is salt-tolerant and cultivar Vaisakh is drought-tolerant. We compared the physiological and biochemical responses of these rice cultivars under salt and drought stress conditions after restricting their cytochrome oxidase (COX) and alternative oxidase (AOX) pathways using antimycin A and salicylhydroxamic acid treatment. Further, changes in their expression of AOX genes and corresponding protein levels were compared and analysed. The sensitive and tolerant rice cultivars subjected to drought and salt stress showed differential responses in physiological and biochemical traits. Whereas Aiswarya showed clear phenotypic differences, such as stunted growth, leaf curling, and loss of greening in leaf tissues, with increase in salt content and progressive drought stress, Vyttila and Vaisakh showed no remarkable changes. Moreover, the drought-tolerant cultivar rehydrated after 10 days of drought exposure, whereas the sensitive variety did not show any rehydration of leaf tissue. The leaves of the tolerant cultivars showed lower reactive oxygen species (ROS) production than that of the sensitive plants under drought and salt stress conditions because of the activation of a stronger antioxidant defence. Although, the restriction of COX and AOX pathways increased the susceptibility of sensitive cultivars, it affected the tolerant varieties moderately. Higher photosynthetic rates, an efficient antioxidant system comprising higher superoxide dismutase, ascorbate peroxidase, and catalase activity along with higher AOX1a gene expression levels during drought and salt stress were observed in tolerant cultivars. The results suggest that an efficient antioxidant system and increased transcription of the AOX1a gene along with higher AOX protein levels are important for tolerant rice cultivars to maintain higher photosynthesis rates, lower ROS, and stress tolerance. Restriction of COX and AOX pathways impact the photosynthesis, ROS, and antioxidant enzymes in both sensitive and tolerant cultivars. The restriction of COX and AOX pathways have a stronger impact on gas exchange and fluorescence parameters of the sensitive cultivar than on that of the tolerant cultivars owing to the higher photosynthetic rates in tolerant cultivars.

Responses of genes of DNA repair, alternative oxidase, and pro-/antioxidant state in Arabidopsis thaliana with altered expression of AOX1a to gamma irradiation

Purpose High doses of gamma (γ) irradiation cause oxidative stress and DNA damage. Alternative oxidase (AOX) catalyzes the energy-dissipating cyanide-resistant alternative pathway in plant mitochondria and is an important part of the cellular defense network under stress conditions. In this study, Arabidopsis thaliana plants with an altered expression of the AOX1a gene were exposed by high dose-rate ionizing radiation to assess the expression of genes of DNA repair and pro-/antioxidant states to elucidate the functional significance of AOX in plant stress response. Materials and methods Five-week-old A. thaliana plants, either with basal AOX1a gene expression (wild-type Colombia-0 (Col-0)), antisense silencing of AOX1a (AS-12), and overexpression of the gene (XX-2), were γ-irradiated at a dose of 200 Gy. Gene expression and biochemical analyses were performed 12 h after irradiation. Results Acute γ-irradiation caused different responses between the genotypes. XX-2 plants, either control or irradiated, showed the highest expression of AOX1a gene and AOX protein, and the lowest expression of DNA repair genes. Wild type and AS-12 plants exposed to γ-irradiation upregulated another stress-induced gene, AOX1d, and DNA repair genes. Furthermore, a higher activity of Mn-dependent superoxide dismutase (Mn-SOD) was observed in the irradiated AS-12 plants than in the untreated plants of this line. However, AS-12 plants were less effective than Col-0 plants in controlling the accumulation of the superoxide anion. XX-2 plants had the lowest reactive oxygen species (ROS) levels among the genotypes. Conclusions AS-12 plants display a compensatory mechanism by increasing the expression of AOX1d and the synthesis of the AOX protein, as well as by Mn-SOD activation. However, these were insufficient to maintain the background level of embryonic lethal mutations, and thereby the reproductive capacity. These results highlight the importance of AOX in the successful adaptation of plants to acute γ-irradiation, and indicate that AOX1a plays a key role in the regulation of the stress response.

Alternative Pathway Is Involved in Hydrogen Peroxide-Enhanced Cadmium Tolerance in Hulless Barley Roots.

Hulless barley, grown in the Qinghai Tibet Plateau, has a wide range of environmental stress tolerance. Alternative pathway (AP) and hydrogen peroxide (H2O2) are involved in enhancing plant tolerance to environmental stresses. However, the relationship between H2O2 and AP in hulless barley tolerance to cadmium (Cd) stress remains unclear. In the study, the role and relationship of AP and H2O2 under Cd stress were investigated in hulless barley (Kunlun14) and common barley (Ganpi6). Results showed that the expression level of alternative oxidase (AOX) genes (mainly AOX1a), AP capacity (Valt), and AOX protein were clearly induced more in Kunlun14 than in Ganpi 6 under Cd stress; moreover, these parameters were further enhanced by applying H2O2. Malondialdehyde (MDA) content, electrolyte leakage (EL) and NAD(P)H to NAD(P) ratio also increased in Cd-treated roots, especially in Kunlun 14, which can be markedly alleviated by exogenous H2O2. However, this mitigating effect was aggravated by salicylhydroxamic acid (SHAM, an AOX inhibitor), suggesting AP contributes to the H2O2-enhanced Cd tolerance. Further study demonstrated that the effect of SHAM on the antioxidant enzymes and antioxidants was minimal. Taken together, hulless barley has higher tolerance to Cd than common barley; and in the process, AP exerts an indispensable function in the H2O2-enhanced Cd tolerance. AP is mainly responsible for the decrease of ROS levels by dissipating excess reducing equivalents.

Phytoglobin-NO cycle and AOX pathway play a role in anaerobic germination and growth of deepwater rice.

An important and interesting feature of rice is that it can germinate under anoxic conditions. Though several biochemical adaptive mechanisms play an important role in the anaerobic germination of rice but the role of phytoglobin-nitric oxide cycle and alternative oxidase pathway is not known, therefore in this study we investigated the role of these pathways in anaerobic germination. Under anoxic conditions, deepwater rice germinated much higher and rapidly than aerobic condition and the anaerobic germination and growth were much higher in the presence of nitrite. The addition of nitrite stimulated NR activity and NO production. Important components of phytoglobin-NO cycle such as methaemoglobin reductase activity, expression of Phytoglobin1, NIA1 were elevated under anaerobic conditions in the presence of nitrite. The operation of phytoglobin-NO cycle also enhanced anaerobic ATP generation, LDH, ADH activities and in parallel ethylene levels were also enhanced. Interestingly nitrite suppressed the ROS production and lipid peroxidation. The reduction of ROS was accompanied by enhanced expression of mitochondrial alternative oxidase protein and its capacity. Application of AOX inhibitor SHAM inhibited the anoxic growth mediated by nitrite. In addition, nitrite improved the submergence tolerance of seedlings. Our study revealed that nitrite driven phytoglobin-NO cycle and AOX are crucial players in anaerobic germination and growth of deepwater rice.

DIA-Based Quantitative Proteomics Reveals the Protein Regulatory Networks of Floral Thermogenesis in Nelumbo nucifera.

The sacred lotus (Nelumbo nucifera) can maintain a stable floral chamber temperature between 30 and 35 °C when blooming despite fluctuations in ambient temperatures between about 8 and 45 °C, but the regulatory mechanism of floral thermogenesis remains unclear. Here, we obtained comprehensive protein profiles from receptacle tissue at five developmental stages using data-independent acquisition (DIA)-based quantitative proteomics technology to reveal the molecular basis of floral thermogenesis of N. nucifera. A total of 6913 proteins were identified and quantified, of which 3513 differentially abundant proteins (DAPs) were screened. Among them, 640 highly abundant proteins during the thermogenic stages were mainly involved in carbon metabolism processes such as the tricarboxylic acid (TCA) cycle. Citrate synthase was identified as the most connected protein in the protein-protein interaction (PPI) network. Next, the content of alternative oxidase (AOX) and plant uncoupling protein (pUCP) in different tissues indicated that AOX was specifically abundant in the receptacles. Subsequently, a protein module highly related to the thermogenic phenotype was identified by the weighted gene co-expression network analysis (WGCNA). In summary, the regulation mechanism of floral thermogenesis in N. nucifera involves complex regulatory networks, including TCA cycle metabolism, starch and sucrose metabolism, fatty acid degradation, and ubiquinone synthesis, etc.

Role of AOX in low-temperature conditioning induced chilling tolerance in sweetpotato roots

Chilling injury is a significant postharvest physiological disorder in sweetpotatoes during storage. This study investigated the role of alternative oxidase (AOX) in low-temperature conditioning (LTC) induced chilling tolerance in sweetpotato storage roots. Roots were pre-treated with a LTC treatment (12°C for 5 days) or with salicylhydroxamic acid (SHAM, AOX inhibitor) combined with LTC, followed by storage at 4°C for 35 days. Results showed that LTC pre-treatment significantly lowered root chilling injury symptoms, suppressed malondialdehyde levels and the accumulation of reactive oxygen species. However, SHAM reduced the impact triggered by LTC and resulted in lower antioxidant enzyme activities and free proline content, reduced glutathione and adenosine triphosphate contents compared with LTC alone. The results from gene expression analysis showed that LTC-induced higher transcript level of IbAOX1a and IbAOX1b during storage at 4°C. However, SHAM suppressed the effect of LTC. SHAM treatment not only reduced AOX activity but also resulted in a lower AOX transcript and protein abundance, which might contribute to the lower AOX activity observed in the SHAM treated roots. These results suggest that AOX participates in LTC-induced chilling tolerance of sweetpotato storage roots.

Alternative oxidase encoded by sequence-optimized and chemically-modified RNA transfected into mammalian cells is catalytically active

Plants and other organisms, but not insects or vertebrates, express the auxiliary respiratory enzyme alternative oxidase (AOX) that bypasses mitochondrial respiratory complexes III and/or IV when impaired. Persistent expression of AOX from Ciona intestinalis in mammalian models has previously been shown to be effective in alleviating some metabolic stresses produced by respiratory chain inhibition while exacerbating others. This implies that chronic AOX expression may modify or disrupt metabolic signaling processes necessary to orchestrate adaptive remodeling, suggesting that its potential therapeutic use may be confined to acute pathologies, where a single course of treatment would suffice. One possible route for administering AOX transiently is AOX-encoding nucleic acid constructs. Here we demonstrate that AOX-encoding chemically-modified RNA (cmRNA), sequence-optimized for expression in mammalian cells, was able to support AOX expression in immortalized mouse embryonic fibroblasts (iMEFs), human lung carcinoma cells (A549) and primary mouse pulmonary arterial smooth muscle cells (PASMCs). AOX protein was detectable as early as 3 h after transfection, had a half-life of ~4 days and was catalytically active, thus supporting respiration and protecting against respiratory inhibition. Our data demonstrate that AOX-encoding cmRNA optimized for use in mammalian cells represents a viable route to investigate and possibly treat mitochondrial respiratory disorders.