AOX1 Expression Research Articles

Genome-wide Scan Identifies Role for AOX1 in Prostate Cancer Survival

BackgroundMost men diagnosed with prostate cancer have low-risk cancers. How to predict prostate cancer progression at the time of diagnosis remains challenging. ObjectiveTo identify single nucleotide polymorphisms (SNPs) associated with death from prostate cancer. Design, setting, and participantsBlood samples from 11 506 men in Sweden were collected during 1991–1996. Of these, 1053 men were diagnosed with prostate cancer and 245 died from the disease. Stage and grade at diagnosis and outcome information were obtained, and DNA from all cases was genotyped. Outcome measurements and statistical analysisA total of 6 126 633 SNPs were tested for association with prostate-cancer-specific survival time using a Cox proportional hazard model, adjusted for age, stage, and grade at diagnosis. A value of 1×10−6 was used as suggestive significance threshold. Positive candidate SNPs were tested for association with gene expression using expression quantitative trait locus analysis. Results and limitationsWe found 12 SNPs at seven independent loci associated with prostate-cancer-specific survival time. One of 6 126 633 SNPs tested reached genome-wide significance (p<5×10−8) and replicated in an independent cohort: rs73055188 (p=5.27×10−9, per-allele hazard ratio [HR]=2.27, 95% confidence interval [CI] 1.72–2.98) in the AOX1 gene. A second SNP reached a suggestive level of significance (p<1×10−6) and replicated in an independent cohort: rs2702185 (p=7.1×10−7, per-allele HR=2.55, 95% CI=1.76–3.69) in the SMG7 gene. The SNP rs73055188 is correlated with AOX1 expression levels, which is associated with biochemical recurrence of prostate cancer in independent cohorts. This association is yet to be validated in other ethnic groups. ConclusionsThe SNP rs73055188 at the AOX1 locus is associated with prostate-cancer-specific survival time, and AOX1 gene expression level is correlated with biochemical recurrence of prostate cancer. Patient summaryWe identify two genetic markers that are associated with prostate-cancer-specific survival time.

Regulation of alternative oxidase 1 in Chlamydomonas reinhardtii during sulfur starvation

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

Using Phytochemicals to Investigate the Activation of Nicotine Detoxification via Upregulation of CYP2A6 in Animal Models Exposed Tobacco Smoke Condensate by Intratracheal Instillation.

This study examined the efficacy of standardized Smilax china L. root extract (SSCR) containing chlorogenic acid on detoxifying nicotine from tobacco smoke condensate (TSC) in vitro and in vivo. Chlorogenic acid is an identified bioactive component in SSCR by ultraperformance liquid chromatography/photodiode array/electrospray ionization/mass spectroscopy (UPLC/PDA/ESI/MS). HepG2 liver cells and A549 lung cells were carried for measuring ROS and antioxidant enzymes. Sprague-Dawley rats were treated with nicotine by intratracheal instillation (ITI). Cell viabilities by pretreatments of 5, 12.5, and 25, 50 μg SSCR/mL ranged from 41 to 76% in HepG2 and 65 to 95% in A549. Pretreatments of SSCR inhibited TSC-mediated production of reactive oxygen species (ROS) by 8 and 10% in HepG2 and A549 cells, respectively. However, the expression of CAT, SOD1, and AOX1 was downregulated by SSCR in the both cells. The highest conversion of cotinine was observed at 50 μg/mL of SSCR after 120 min of incubation. SSCR upregulated CYP2A6 3-fold in A549 cells regardless of TSC cotreatment. When Sprague-Dawley rats were treated with nicotine by ITI or subjected to SSCR administration for 14 days, the levels of cotinine in urine increased in SSCR treatment only. The cellular level of antioxidant capacity at 10 or 100 mg/kg body weight/day of SSCR treatment was 1.89 and 1.86 times higher than those of nicotine-control. Results suggest that the intake of SSCR can detoxify nicotine by elevating nicotine conversion to cotinine and antioxidant capacity.

Nitric oxide upregulates expression of alternative oxidase 1 in Chlamydomonas reinhardtii

The mitochondrial respiratory chain in plants, many fungi and some protists consists of the ATP-coupling cyanide-sensitive cytochrome pathway and the cyanide-resistant alternative respiratory pathway. The alternative pathway is mediated by alternative oxidase (AOX). In unicellular algae, AOXs are monomeric fungi-type proteins. Studies performed in the model plant Chlamydomonas reinhardtii showed that a range of stress factors lead to induction of its AOX1. However, signaling molecules that trigger upregulation of AOX1 have not yet been identified. Here, we were able to discriminate between two alternative oxidases of the alga. In this work, we demonstrated that exposure of C. reinhardtii to nitric oxide (NO) resulted in the up-regulation of AOX1 expression and an increased AOX1 protein. Furthermore, NO-treated C. reinhardtii cells displayed the enhanced AOX1 capacity. We also clearly demonstrated that AOX1 can function in C. reinhardtii when the cytochrome oxidase became inhibited by NO. Although the pathway(s) that leads to increased AOX1 levels and activity upon NO treatment is yet unknown, it is now clear that NO serves as the signal to trigger this regulatory process in C. reinhardtii.

The mitochondrial alternative oxidase Aox1 is needed to cope with respiratory stress but dispensable for pathogenic development in Ustilago maydis.

The mitochondrial alternative oxidase is an important enzyme that allows respiratory activity and the functioning of the Krebs cycle upon disturbance of the respiration chain. It works as a security valve in transferring excessive electrons to oxygen, thereby preventing potential damage by the generation of harmful radicals. A clear biological function, besides the stress response, has so far convincingly only been shown for plants that use the alternative oxidase to generate heat to distribute volatiles. In fungi it was described that the alternative oxidase is needed for pathogenicity. Here, we investigate expression and function of the alternative oxidase at different stages of the life cycle of the corn pathogen Ustilago maydis (Aox1). Interestingly, expression of Aox1 is specifically induced during the stationary phase suggesting a role at high cell density when nutrients become limiting. Studying deletion strains as well as overexpressing strains revealed that Aox1 is dispensable for normal growth, for cell morphology, for response to temperature stress as well as for filamentous growth and plant pathogenicity. However, during conditions eliciting respiratory stress yeast-like growth as well as hyphal growth is strongly affected. We conclude that Aox1 is dispensable for the normal biology of the fungus but specifically needed to cope with respiratory stress.

Transcription factor Mxr1 promotes the expression of Aox1 by repressing glycerol transporter 1 in Pichia pastoris.

In the methylotrophic yeast Pichia pastoris (P. pastoris), the efficient promoter of alcohol oxidase (PAox1) is induced by methanol and repressed by glycerol, but the molecular mechanism is not clear. In this study, the relationship between alcohol oxidase 1 (aox1), methanol expression regulator 1 (mxr1) and glycerol transporter 1 (gt1) was studied. By RT-PCR, it was found that the overexpression of gt1 could increase the glycerol content in cells and repress the expression of mxr1 and aox1, and the deletion of gt1 reduced the glycerol content in cells and promoted the expression of aox1. The overexpression of mxr1 could repress the expression of gt1, and the deletion of mxr1 could promote the expression of gt1 to some extent. By EMSA, Mxr1 binding sites were found in the promoter of gt1 (PGt1) (-141 to -138, CCCC), and Mxr1 could regulate the expression of gt1 by binding to PGt1. The relationships among aox1, mxr1 and gt1 revealed here provide a reference for the understanding of the mechanism of glycerol repression of PAox1.

Implications of terminal oxidase function in regulation of salicylic acid on soybean seedling photosynthetic performance under water stress

The aim of this study is to investigate whether exogenous application of salicylic acid (SA) could modulate the photosynthetic capacity of soybean seedlings in water stress tolerance, and to clarify the potential functions of terminal oxidase (plastid terminal oxidase (PTOX) and alternative oxidase (AOX)) in SA′ s regulation on photosynthesis. The effects of SA and water stress on gas exchange, pigment contents, chlorophyll fluorescence, enzymes (guaiacol peroxidase (POD; EC 1.11.1.7), superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11) and NADP-malate dehydrogenase (NADP-MDH; EC1.1.1.82)) activity and transcript levels of PTOX, AOX1, AOX2a, AOX2b were examined in a hydroponic cultivation system. Results indicate that water stress significantly decreased the photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (E), pigment contents (Chla + b, Chla/b, Car), maximum quantum yield of PSⅡphotochemistry (Fv/Fm), efficiency of excitation capture of open PSⅡcenter (Fv’/Fm’), quantum efficiency of PSⅡphotochemistry (ΦPSⅡ), photochemical quenching (qP), and increased malondialdehyde (MDA) content and the activity of all the enzymes. SA pretreatment led to significant decreases in Ci and MDA content, and increases in Pn, Gs, E, pigment contents, Fv/Fm, Fv’/Fm’, ΦPSⅡ, qP, and the activity of all the enzymes. SA treatment and water stress alone significantly up-regulated the expression of PTOX, AOX1 and AOX2b. SA pretreatment further increased the transcript levels of PTOX and AOX2b of soybean seedling under water stress. These results indicate that SA application alleviates the water stress-induced decrease in photosynthesis may mainly through maintaining a lower reactive oxygen species (ROS) level, a greater PSⅡefficiency, and an enhanced alternative respiration and chlororespiration. PTOX and AOX may play important roles in SA-mediated resistance to water stress.

The Chlamydomonas alternative oxidase 1 is regulated by cadmium stress: New insights into control of expression

Although mitochondrial alternative oxidase (AOX) has been proposed to play essential roles in stress tolerance of plants, the effects of cadmium (Cd), an important environmental pollutant, on AOX are largely unknown. The green alga Chlamydomonas reinhardtii reacts to Cd stress conditions with the induced expression of many genes. In this work, we demonstrated a sharp increase in AOX1 transcript and protein abundance under Cd stress. Furthermore, C. reinhardtii cells displayed a large increase in alternative respiration in response to Cd treatment. Cd-induced AOX1 production and alternative pathway capacity were lower in illuminated cells than in dark-adapted cells. Moreover, in dark-adapted unstressed cells, AOX1 was significantly expressed. The results clearly indicate that the dark-mediated signals function in an additive manner with Cd-mediated signals, and thus likely act in parallel pathways to regulate the expression of AOX1.

Characterization of harpinXoo induced hypersensitive responses in non host plant, tobacco

Harpin proteins encoded by hrp genes are bacterial protein elicitors that can stimulate hypersensitive response (HR) in non-host plants. HR-related pathogen resistance involves a complex form of programmed cell death (PCD). It is increasingly viewed as a key component of the hypersensitive disease response of plants. Currently, the evidence of harpin proteins-induced PCD is deficient though it exhibits phenotypic parallels with HR, and the mechanism of harpin proteins-induced PCD is not well understood. In this study, we demonstrate that harpinXoo protein from Xanthomonas oryzae pv. oryzae of rice bacterial blight expressed and isolated from bacterial cells acted as an agent to induce PCD in infiltrated tobacco plants. Treatment of tobacco leaves with harpinXoo induced typical PCD-related morphological and biochemical changes including cell shrinkage and nuclear DNA degradation. We further analyzed the expression of several genes in signal transduction pathway of PCD in tobacco plants by real-time qRT-PCR analysis using EF-1α as an endogenous control. Our results showed that the expression of NtDAD1 was down-regulated and the expression of BI-1, tpa1 and aox1 was up-regulated following the infiltration of harpinXoo into tobacco leaves. Our data suggest that harpinXoo can induce PCD with the coordination of PCD-related genes in infiltrated tobacco leaves, providing evidence to further investigate the signal transduction pathways of HR and PCD.

The Pichia pastoris transmembrane protein GT1 is a glycerol transporter and relieves the repression of glycerol on AOX1 expression.

Promoter of alcohol oxidase I (PAOX1) is the most efficient promoter involved in the regulation of recombinant protein expression in Pichia pastoris (P. pastoris). PAOX1 is tightly repressed by the presence of glycerol in the culture medium; thus, glycerol must be exhausted before methanol can be taken up by P. pastoris and the expression of the heterologous protein can be induced. In this study, a candidate glycerol transporter (GT1, GeneID: 8197545) was identified, and its role was confirmed by further studies (e.g. bioinformatics analysis, heterologous complementation in Schizosaccharomyces pombe (S. pombe)). When GT1 is co-expressed with enhanced green fluorescent protein (EGFP), it localizes to the membrane and S. pombe carrying gt1 but not the wild-type strain can grow on medium containing glycerol as the sole carbon source. The present study is the first to report that AOX1 in the X-33Δgt1 mutant can achieve constitutive expression in medium containing glycerol; thus, knocking down gt1 can eliminate the glycerol repression of PAOX1 in P. pastoris These results suggest that the glycerol transporter may participate in the process of PAOX1 inhibition in glycerol medium.

The Chlamydomonas reinhardtii alternative oxidase 1 is regulated by heat stress

The alternative oxidase (AOX) is a non-energy conserving terminal oxidase that has emerged as an important mitochondrial component of the cell stress responses. Although the most studied abiotic condition in relation to Chlamydomonas reinhardtii is high temperature, changes in AOX capacity of the alga were studied only under oxidative stress and cold. To examine whether elevated temperatures affected AOX1 expression, we applied quantitative real-time PCR and pharmaceutical approaches. In this work, we demonstrated a sharp increase in AOX1 transcript and protein abundance under heat stress. Furthermore, C. reinhardtii cells displayed a large increase in alternative respiration in response to high temperature. Feeding with the protein kinase inhibitor staurosporine strongly retarded the AOX1 transcription. Finally, the addition of the calcium chelator EGTA prevented heat-induced AOX1 expression. Together, our results imply that heat-inducible Ca2+ influx and protein kinase(s) may mediate AOX1 expression at elevated temperatures. Characterization of heat-induced AOX1 regulation in the green alga C. reinhardtii provides a framework for a more complete understanding of the function of this conserved protein.

Involvement of AOX and UCP pathways in the post-harvest ripening of papaya fruits

Enhanced respiration during ripening in climacteric fruits is sometimes associated with an uncoupling between the ATP synthesis and the mitochondrial electron transport chain. While the participation of two energy-dissipating systems, one of which is mediated by the alternative oxidase (AOX) and the other mediated by the uncoupling protein (UCP), has been linked to fruit ripening, the relation between the activation of both mitochondrial uncoupling systems with the transient increase of ethylene synthesis (ethylene peak) remains unclear. To elucidate this question, ethylene emission and the two uncoupling (AOX and UCP) pathways were monitored in harvested papaya fruit during the ripening, from green to fully yellow skin. The results confirmed the typical climacteric behavior for papaya fruit: an initial increase in endogenous ethylene emission which reaches a maximum (peak) in the intermediate ripening stage, before finally declining to a basal level in ripe fruit. Respiration of intact fruit also increased and achieved higher levels at the end of ripening. On the other hand, in purified mitochondria extracted from fruit pulp the total respiration and respiratory control decrease while an increase in the participation of AOX and UCP pathways was markedly evident during papaya ripening. There was an increase in the AOX capacity during the transition from green fruit to the intermediate stage that accompanied the transient ethylene peak, while the O2 consumption triggered by UCP activation increased by 80% from the beginning to end stage of fruit ripening. Expression analyses of AOX (AOX1 and 2) and UCP (UCP1–5) genes revealed that the increases in the AOX and UCP capacities were linked to a higher expression of AOX1 and UCP (mainly UCP1) genes, respectively. In silico promoter analyses of both genes showed the presence of ethylene-responsive cis-elements in UCP1 and UCP2 genes. Overall, the data suggest a differential activation of AOX and UCP pathways in regulation related to the ethylene peak and induction of specific genes such as AOX1 and UCP1.

FABP4 reversed the regulation of leptin on mitochondrial fatty acid oxidation in mice adipocytes.

Fatty acid binding protein 4 (FABP4), plays key role in fatty acid transportation and oxidation, and increases with leptin synergistically during adipose inflammation process. However, the regulation mechanism between FABP4 and leptin on mitochondrial fatty acid oxidation remains unclear. In this study, we found that FABP4 reduced the expression of leptin, CPT-1 and AOX1 in mice adipocytes. Conversely, FABP4 was down-regulated in a time-dependent manner by leptin treatment. Additionally, forced expression of FABP4 attenuated the expression of PGC1-α, UCP2, CPT-1, AOX1 and COX2 compared with leptin incubation. Moreover, mitochondrial membrane potential, fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase (MCAD), long-chain acyl-CoA dehydrogenase (LCAD) and Cyt C levels were reduced in response to the overexpression of FABP4. These reductions correspond well with the reduced release of free fatty acid and the inactivation of mitochondrial complexes I and III by FABP4 overexpression. Furthermore, addition of the Akt/mTOR pathway-specific inhibitor (MK2206) blocked the mitochondrial fatty acid oxidation and respiration factors, whereas interference of FABP4 overcame these effects. Taken together, FABP4 could reverse the activation of the leptin-induced mitochondrial fatty acid oxidation, and the inhibition of Akt/mTOR signal pathway played a key role in this process.

The alternative respiratory pathway is involved in brassinosteroid-induced environmental stress tolerance in Nicotiana benthamiana.

Brassinosteroids (BRs), plant steroid hormones, play essential roles in modulating cell elongation, vascular differentiation, senescence, and stress responses. However, the mechanisms by which BRs regulate plant mitochondria and resistance to abiotic stress remain largely unclear. Mitochondrial alternative oxidase (AOX) is involved in the plant response to a variety of environmental stresses. In this report, the role of AOX in BR-induced tolerance against cold, polyethylene glycol (PEG), and high-light stresses was investigated. Exogenous applied brassinolide (BL, the most active BR) induced, while brassinazole (BRZ, a BR biosynthesis inhibitor) reduced alternative respiration and AOX1 expression in Nicotiana benthamiana. Chemical scavenging of H2O2 and virus-induced gene silencing (VIGS) of NbRBOHB compromised the BR-induced alternative respiratory pathway, and this result was further confirmed by NbAOX1 promoter analysis. Furthermore, inhibition of AOX activity by chemical treatment or a VIGS-based approach decreased plant resistance to environmental stresses and compromised BR-induced stress tolerance. Taken together, our results indicate that BR-induced AOX capability might contribute to the avoidance of superfluous reactive oxygen species accumulation and the protection of photosystems under stress conditions in N. benthamiana.

ALTERNATIVE OXIDASE1a modulates the oxidative challenge during moderate Cd exposure in Arabidopsis thaliana leaves.

This study aims to unravel the functional significance of alternative oxidase1a (AOX1a) induction in Arabidopsis thaliana leaves exposed to cadmium (Cd) by comparing wild-type (WT) plants and aox1a knockout mutants. In the absence of AOX1a, differences in stress-responsive transcript and glutathione levels suggest an increased oxidative challenge during moderate (5 µM) and prolonged (72h) Cd exposure. Nevertheless, aox1a knockout leaves showed lower hydrogen peroxide (H2O2) accumulation as compared to the WT due to both acute (24h) and prolonged (72h) exposure to 5 µM Cd, but not to 10 µM Cd. Taken together, we propose a working model where AOX1a acts early in the response to Cd and activates or maintains a mitochondrial signalling pathway impacting on cellular antioxidative defence at the post-transcriptional level. This fine-tuning pathway is suggested to function during moderate (5 µM) Cd exposure while being overwhelmed during more severe (10 µM) Cd stress. Within this framework, ethylene is required - either directly or indirectly via NADPH oxidase isoform C - to fully induce AOX1 expression. In addition, reciprocal crosstalk between these components was demonstrated in leaves of A. thaliana plants exposed to Cd.

Classification of serous ovarian tumors based on microarray data using multicategory support vector machines.

Ovarian cancer, the most fatal of reproductive cancers, is the fifth leading cause of death in women in the United States. Serous borderline ovarian tumors (SBOTs) are considered to be earlier or less malignant forms of serous ovarian carcinomas (SOCs). SBOTs are asymptomatic and progression to advanced stages is common. Using DNA microarray technology, we designed multicategory classification models to discriminate ovarian cancer subclasses. To develop multicategory classification models with optimal parameters and features, we systematically evaluated three machine learning algorithms and three feature selection methods using five-fold cross validation and a grid search. The study included 22 subjects with normal ovarian surface epithelial cells, 12 with SBOTs, and 79 with SOCs according to microarray data with 54,675 probe sets obtained from the National Center for Biotechnology Information gene expression omnibus repository. Application of the optimal model of support vector machines one-versus-rest with signal-to-noise as a feature selection method gave an accuracy of 97.3%, relative classifier information of 0.916, and a kappa index of 0.941. In addition, 5 features, including the expression of putative biomarkers SNTN and AOX1, were selected to differentiate between normal, SBOT, and SOC groups. An accurate diagnosis of ovarian tumor subclasses by application of multicategory machine learning would be cost-effective and simple to perform, and would ensure more effective subclass-targeted therapy.

Identification of New Genes Downregulated in Prostate Cancer and Investigation of Their Effects on Prognosis

Prostate cancer is the most common noncutaneous malignant neoplasm in men in the Western countries. It is well established that genetic and epigenetic alterations are common events in prostate cancer, which may lead to aberrant expression of critical genes. Most of the studies are focused on the overexpressed or duplicated genes in prostate cancer. However, it is known that some of the differentially expressed genes in prostate cancer are downregulated. Since the inventory of downregulated genes is incomplete, we performed in silico approaches to reveal the novel prostate cancer downregulated genes. Moreover, we also investigated for a possible link between the expression of the downregulated genes and tumor grade, recurrence, metastasis, or survival status in prostate cancer. Our results showed that the expression of GSTP1 and AOX1 are downregulated in prostate cancer, in concordance with previous reports. Moreover, we showed that TPM2, CLU, and COL4A6 mRNA levels are downregulated in prostate cancer. Further, we found a significant negative correlation between the expression of the above-mentioned genes and the prognosis of prostate cancer.

Влияние источника азота на экспрессию генов, контролирующих первые этапы утилизации метанола у дрожжейPichia pastoris

Pichia pastoris is a methylotrophic yeast that is widely used for the expression of heterologous proteins. Here, we have investigated the dependence of AOX1 and CAT1 expression on the source of nitrogen. It has shown that the expression of AOX1 and CAT1 depends on the source of nitrogen. In the presence of a rich nitrogen source, such as glutamine or ammonium sulfate, the AOX1 and CAT1 expression was dramatically induced. With proline as the nitrogen source AOX1 and CATI expression was reduced by more than threefold. The expression of these genes is controlled at the level of transcription. We have shown the impact of the nitrogen source on the growth rate in the presence of methanol as a sole carbon source. The specific growth rate was highest on media that contained proline as a nitrogen source compared to media with a rich nitrogen source.

Overexpression of Alternative Oxidase Gene Confers Aluminum Tolerance by Altering the Respiratory Capacity and the Response to Oxidative Stress in Tobacco Cells

Aluminum (Al) stress represses mitochondrial respiration and produces reactive oxygen species (ROS) in plants. Mitochondrial alternative oxidase (AOX) uncouples respiration from mitochondrial ATP production and may improve plant performance under Al stress by preventing excess accumulation of ROS. We tested respiratory changes and ROS production in isolated mitochondria and whole cell of tobacco (SL, ALT 301) under Al stress. Higher capacities of AOX pathways relative to cytochrome pathways were observed in both isolated mitochondria and whole cells of ALT301 under Al stress. AOX1 when studied showed higher AOX1 expression in ALT 301 than SL cells under stress. In order to study the function of tobacco AOX gene under Al stress, we produced transformed tobacco cell lines by introducing NtAOX1 expressed under the control of the cauliflower mosaic virus (CaMV) 35 S promoter in sensitive (SL) Nicotiana tabacum L. cell lines. The enhancement of endogenous AOX1 expression and AOX protein with or without Al stress was in the order of transformed tobacco cell lines>ALT301>wild type (SL). A decreased respiratory inhibition and reduced ROS production with a better growth capability were the significant features that characterized AOX1 transformed cell lines under Al stress. These results demonstrated that AOX plays a critical role in Al stress tolerance with an enhanced respiratory capacity, reducing mitochondrial oxidative stress burden and improving the growth capability in tobacco cells.

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.