Alternative Oxidase Expression Research Articles

Molecular basis for thermogenesis and volatile production in the titan arum.

The titan arum (Amorphophallus titanum), commonly known as the corpse flower, produces the largest unbranched inflorescence in the world. Its rare blooms last only a few days and are notable both for their burst of thermogenic activity and for the odor of rotting flesh by which they attract pollinators. Studies on the titan arum can therefor lend insight into the mechanisms underlying thermogenesis as well as the production of sulfur-based volatiles, about which little is known in plants. Here, we made use of transcriptome and metabolite analyses to uncover underlying mechanisms that enable thermogenesis and volatile production in the titan arum. The ability to perform thermogenesis correlated with the expression of genes involved in bypass steps for the mitochondrial electron transport chain, in particular alternative oxidase expression, and through our analysis is placed within the context of sugar transport and metabolism. The major odorants produced by the titan arum are dimethyl disulfide and dimethyl trisulfide, and we identified pathways for sulfur transport and metabolism that culminate in the production of methionine, which our analysis identifies as the amino acid substrate for production of these odorants. Putrescine, derived from arginine, was identified as an additional and previously unrecognized component of the titan arum's odor. Levels of free methionine and putrescine were rapidly depleted during thermogenesis, consistent with roles in production of the titan arum's odor. Models for how tissues of the titan arum contribute to thermogenesis and volatile production are proposed.

Effect of alternative oxidase (AOX) expression on mouse cerebral mitochondria bioenergetics

Alternative oxidase (AOX) is an enzyme that transfers electrons from reduced quinone directly to oxygen without proton translocation. When AOX from Ciona intestinalis is xenotopically expressed in mice, it can substitute the combined electron-transferring activity of mitochondrial complexes III/IV. Here, we used brain mitochondria from AOX-expressing mice with such a chimeric respiratory chain to study respiratory control bioenergetic mechanisms.AOX expression did not compromise the function of the mammalian respiratory chain at physiological conditions, however the complex IV inhibitor cyanide only partially blocked respiration by AOX-containing mitochondria. The relative fraction of cyanide-insensitive respiration increased at lower temperatures, indicative of a temperature-controlled attenuation of mammalian respiratory enzyme activity.As AOX does not translocate protons, the mitochondrial transmembrane potential in AOX-containing mitochondria was more sensitive to cyanide during succinate oxidation than during malate/pyruvate-supported respiration. High concentrations of cyanide fully collapsed membrane potential during oxidation of either succinate or glycerol 3-phosphate, but not during malate/pyruvate-supported respiration. This confirms AOX's electroneutral redox activity and indicates differences in the proton-translocating capacity of dehydrogenases upstream of the ubiquinone pool. Our respiration data refutes previous proposals for quinone partitioning within the supercomplexes of the respiratory chain, instead supporting the concept of a single homogeneous, freely diffusing quinone pool.Respiration with either succinate or glycerol 3-phosphate promotes reverse electron transfer (RET) towards complex I. AOX expression significantly decreased RET-induced ROS generation, with the effect more pronounced at low temperatures. Inhibitor-sensitivity analysis showed that the AOX-induced decrease in H2O2 release is due to the lower contribution of complex I to net ROS production during RET.Overall, our findings provide new insights into the role of temperature as a mechanism to control respiration and highlight the utility of AOX as a genetic tool to characterize both the distinct pathways of oxygen reduction and the role of redox control in RET.

Alternative Oxidase Expression Restores Kidney Progenitor Differentiation in Mice with Mitochondrial Complex lll Disruption

Alternative Oxidase Expression Restores Kidney Progenitor Differentiation in Mice with Mitochondrial Complex lll Disruption

Gene expression and metabolite levels converge in the thermogenic spadix of skunk cabbage.

The inflorescence (spadix) of skunk cabbage (Symplocarpus renifolius) is strongly thermogenic and can regulate its temperature at around 23 °C even when the ambient temperature drops below freezing. To elucidate the mechanisms underlying developmentally controlled thermogenesis and thermoregulation in skunk cabbage, we conducted a comprehensive transcriptome and metabolome analysis across 3 developmental stages of spadix development. Our RNA-seq analysis revealed distinct groups of expressed genes, with selenium-binding protein 1/methanethiol oxidase (SBP1/MTO) exhibiting the highest levels in thermogenic florets. Notably, the expression of alternative oxidase (AOX) was consistently high from the prethermogenic stage through the thermogenic stage in the florets. Metabolome analysis showed that alterations in nucleotide levels correspond with the developmentally controlled and tissue-specific thermogenesis of skunk cabbage, evident by a substantial increase in AMP levels in thermogenic florets. Our study also reveals that hydrogen sulfide, a product of SBP1/MTO, inhibits cytochrome c oxidase (COX)-mediated mitochondrial respiration, while AOX-mediated respiration remains relatively unaffected. Specifically, at lower temperatures, the inhibitory effect of hydrogen sulfide on COX-mediated respiration increases, promoting a shift toward the dominance of AOX-mediated respiration. Finally, despite the differential regulation of genes and metabolites throughout spadix development, we observed a convergence of gene expression and metabolite accumulation patterns during thermogenesis. This synchrony may play a key role in developmentally regulated thermogenesis. Moreover, such convergence during the thermogenic stage in the spadix may provide a solid molecular basis for thermoregulation in skunk cabbage.

Non-thermal plasma enhances rice seed germination, seedling development, and root growth under low-temperature stress

Recently, non-thermal plasma (NTP) technologies have found widespread application across diverse fields, including plant growth, medical science, and biological and environmental research. Rice (Oryza sativa L.) is exceptionally sensitive to temperature changes. Notably, low-temperature stress primarily affects the germination and reproductive stages of rice, often leading to reduced crop yield. This study aimed to identify optimal conditions for enhancing rice seed germination and seedling growth under low temperatures using NTP technology. Our research indicated that NTP treatment at 15.0 kV for 30 s optimally promotes rice seed germination and growth under low-temperature stress. Furthermore, NTP treatment increases the activity and expression of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), under low-temperature conditions. Moreover, it downregulates the expression of β-ketoacyl-[acyl carrier protein] synthase I (KASI) and cis-epoxy carotenoid dioxygenase 3 (NCED3) and upregulates the expression of alternative oxidase (AOX1B), BREVIS RADIX-like homologous gene (BRXL2), WRKY transcription factor 29 (WRKY29), and EREBP transcription factor 2 (EREBP2) in roots after tandem 7 days low-temperature (16 ℃) and 7 days room-temperature (28 ℃) treatments. Transcriptomic analysis revealed the involvement of various key genes in phosphotransferase activity, phosphate-containing compound metabolic processes, and defense responses. These analyses provide comprehensive information on gene expression at the transcriptional level, offering new insights for a deeper understanding of candidate genes required for root growth in rice.

Alternative oxidase causes cell type- and tissue-specific responses in mutator mice.

Energetic insufficiency, excess production of reactive oxygen species (ROS), and aberrant signaling partially account for the diverse pathology of mitochondrial diseases. Whether interventions affecting ROS, a regulator of stem cell pools, could modify somatic stem cell homeostasis remains unknown. Previous data from mitochondrial DNA mutator mice showed that increased ROS leads to oxidative damage in erythroid progenitors, causing lifespan-limiting anemia. Also unclear is how ROS-targeted interventions affect terminally differentiated tissues. Here, we set out to test in mitochondrial DNA mutator mice how ubiquitous expression of the Ciona intestinalis alternative oxidase (AOX), which attenuates ROS production, affects murine stem cell pools. We found that AOX does not affect neural stem cells but delays the progression of mutator-driven anemia. Furthermore, when combined with the mutator, AOX potentiates mitochondrial stress and inflammatory responses in skeletal muscle. These differential cell type-specific findings demonstrate that AOX expression is not a global panacea for curing mitochondrial dysfunction. ROS attenuation must be carefully studied regarding specific underlying defects before AOX can be safely used in therapy.

Alternaria alternata stimulates blackhead disease development of ‘Korla’ fragrant pear (Pyrus bretschneideri Rehd) by regulating energy status and respiratory metabolism

Blackhead disease of ‘Korla’ fragrant pear (Pyrus bretschneideri Rehd), caused by the fungus Alternaria alternata (A. alternata), limits postharvest pear fruit storage and transportation. Herein, we explored the effects of A. alternata infection on respiratory metabolism and energy status of pear fruit, and their correlations with disease development. Compared with control fruit, A. alternata-inoculated fruit displayed a higher respiration rate and disease index. Infection increased the gene expression and enzyme activities of phosphohexose isomerase (PGI), alternative oxidase (AOX), succinate dehydrogenase (SDH) and cytochrome C oxidase (CCO) in pear fruit, and decreased those of 6-phosphogluconate dehydrogenase (6PGDH) and glucose-6-phosphate dehydrogenase (G6PDH) in the late storage period. Infection reduced nicotinamide adenine dinucleotide kinase (NADK) activity and expression level in fruit, inhibited NADP accumulation, and increased NAD, NADH, and NADPH contents in the early storage period. Infection also lowered adenosine triphosphate (ATP), adenosine diphosphate (ADP) and energy charge (EC) levels as well as ATPase activity, and enhanced adenosine monophosphate (AMP) content and PbAtpB expression levels, resulting in decreased fruit energy status. All these results suggest that disease development may be connected to the enhancement of the Embden-Meyerhof-Parnas (EMP) pathway, the tricarboxylic acid (TCA) cycle, the cytochrome pathway (CCP), the alternative pathway (AP), the increased then decrease in the pentose phosphate pathway (PPP), and reduced energy status in pear fruit.

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.

Metabolic remodeling caused by heat hardening in the Mediterranean mussel Mytilus galloprovincialis.

Organisms can modify and increase their thermal tolerance faster and more efficiently after a brief exposure to sublethal thermal stress. This response is called 'heat hardening' as it leads to the generation of phenotypes with increased heat tolerance. The aim of this study was to investigate the impact of heat hardening on the metabolomic profile of Mytilus galloprovincialis in order to identify the associated adjustments of biochemical pathways that might benefit the mussels' thermal tolerance. Thus, mussels were exposed sequentially to two different phases (heat hardening and acclimation phases). To gain further insight into the possible mechanisms underlying the metabolic response of the heat-hardened M. galloprovincialis, metabolomics analysis was complemented by the estimation of mRNA expression of phosphoenolpyruvate carboxykinase (PEPCK), pyruvate kinase (PK) and alternative oxidase (AOX) implicated in the metabolic pathways of gluconeogenesis, glycolysis and redox homeostasis, respectively. Heat-hardened mussels showed evidence of higher activity of the tricarboxylic acid (TCA) cycle and diversification of upregulated metabolic pathways, possibly as a mechanism to increase ATP production and extend survival under heat stress. Moreover, formate and taurine accumulation provide an antioxidant and cytoprotective role in mussels during hypoxia and thermal stress. Overall, the metabolic responses in non-heat-hardened and heat-hardened mussels underline the upper thermal limits of M. galloprovincialis, set at 26°C, and are in accordance with the OCLTT concept. The ability of heat-hardened mussels to undergo a rapid gain and slow loss of heat tolerance may be an advantageous strategy for coping with intermittent and often extreme temperatures.

Xenotopic expression of alternative oxidase (AOX) to study mechanisms of mitochondrial disease

The mitochondrial respiratory chain or electron transport chain (ETC) facilitates redox reactions which ultimately lead to the reduction of oxygen to water (respiration). Energy released by this process is used to establish a proton electrochemical gradient which drives ATP formation (oxidative phosphorylation, OXPHOS). It also plays an important role in vital processes beyond ATP formation and cellular metabolism, such as heat production, redox and ion homeostasis. Dysfunction of the ETC can thus impair cellular and organismal viability and is thought to be the underlying cause of a heterogeneous group of so-called mitochondrial diseases. Plants, yeasts, and many lower organisms, but not insects and vertebrates, possess an enzymatic mechanism that confers resistance to respiratory stress conditions, i.e., the alternative oxidase (AOX). Even in cells that naturally lack AOX, it is autonomously imported into the mitochondrial compartment upon xenotopic expression, where it refolds and becomes catalytically engaged when the cytochrome segment of the ETC is blocked. AOX was therefore proposed as a tool to study disease etiologies. To this end, AOX has been xenotopically expressed in mammalian cells and disease models of the fruit fly and mouse. Surprisingly, AOX showed remarkable rescue effects in some cases, whilst in others it had no effect or even exacerbated a condition. Here we summarize what has been learnt from the use of AOX in various disease models and discuss issues which still need to be addressed in order to understand the role of the ETC in health and disease.

Novel Insights into Floral Thermogenesis: In Vivo Analyses of Mitochondrial Dynamics in Nelumbo nucifera Flowers.

Animal-like thermogenic (TM) activities in flowers have been reported in several families of seed plants. While an association of mitochondria with floral thermogenesis has been described, how mitochondrial dynamics are involved in the regulation of floral thermogenesis is unclear. In this study, the morphological and functional dynamics of mitochondria in vivo were assessed in Nelumbo nucifera Gaertn. flowers during floral thermogenesis. The results showed that mitochondrial biogenesis increased considerably in N. nucifera flowers during thermogenesis, accompanied by notable morphological changes in the mitochondria, including long elliptical, rod-shaped, and dumbbell-shaped morphologies, as well as increased mitochondrial reactive oxygen species (ROS) levels in TM cells. An increase in the expression of alternative oxidase (AOX) during the thermogenesis of N. nucifera flowers was also observed. These observations suggested the rapid change in mitochondrial morphology and increased density during thermogenesis implied activation of mitochondrial fission, which combined with elevated levels of mitochondrial ROS trigger a substantial increase in AOX within the respiratory pathway of TM N. nucifera.

The Role of Alternative Oxidase in the Interplay between Nitric Oxide, Reactive Oxygen Species, and Ethylene in Tobacco (Nicotiana tabacum L.) Plants Incubated under Normoxic and Hypoxic Conditions.

The transgenic tobacco (Nicotiana tabacum L.) plants with the modified levels of alternative oxidase (AOX) were used to evaluate the physiological roles of AOX in regulating nitro-oxidative stress and metabolic changes after exposing plants to hypoxia for 6 h. Under normoxia, AOX expression resulted in the decrease of nitric oxide (NO) levels and of the rate of protein S-nitrosylation, while under hypoxia, AOX overexpressors exhibited higher NO and S-nitrosylation levels than knockdowns. AOX expression was essential in avoiding hypoxia-induced superoxide and H2O2 levels, and this was achieved via higher activities of catalase and glutathione reductase and the reduced expression of respiratory burst oxidase homolog (Rboh) in overexpressors as compared to knockdowns. The AOX overexpressing lines accumulated less pyruvate and exhibited the increased transcript and activity levels of pyruvate decarboxylase and alcohol dehydrogenase under hypoxia. This suggests that AOX contributes to the energy state of hypoxic tissues by stimulating the increase of pyruvate flow into fermentation pathways. Ethylene biosynthesis genes encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC oxidase, and ethylene-responsive factors (ERFs) were induced during hypoxia and correlated with AOX and NO levels. We conclude that AOX controls the interaction of NO, reactive oxygen species, and ethylene, triggering a coordinated downstream defensive response against hypoxia.

Mitochondrial electron transport chain is necessary for NLRP3 inflammasome activation

The NLRP3 inflammasome is linked to sterile and pathogen-dependent inflammation, and its dysregulation underlies many chronic diseases. Mitochondria have been implicated as regulators of the NLRP3 inflammasome through several mechanisms including generation of mitochondrial reactive oxygen species (ROS). Here, we report that mitochondrial electron transport chain (ETC) complex I, II, III and V inhibitors all prevent NLRP3 inflammasome activation. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI1) or Ciona intestinalis alternative oxidase, which can complement the functional loss of mitochondrial complex I or III, respectively, without generation of ROS, rescued NLRP3 inflammasome activation in the absence of endogenous mitochondrial complex I or complex III function. Metabolomics revealed phosphocreatine (PCr), which can sustain ATP levels, as a common metabolite that is diminished by mitochondrial ETC inhibitors. PCr depletion decreased ATP levels and NLRP3 inflammasome activation. Thus, the mitochondrial ETC sustains NLRP3 inflammasome activation through PCr-dependent generation of ATP, but via a ROS-independent mechanism.

Alternative oxidase plays a role in minimizing ROS and RNS produced under salinity stress in Arabidopsis thaliana.

Under stress conditions, the overproduction of different reactive oxygen species (ROS) and reactive nitrogen species (RNS) causes imbalance in the redox homeostasis of the cell leading to nitro-oxidative stress in plants. Alternative oxidase (AOX) is a conserving terminal oxidase of the mitochondrial electron transport chain, which can minimize the ROS. Still, the role of AOX in the regulation of RNS during nitro-oxidative stress imposed by salinity stress is not known. Here, we investigated the role of AOX in minimizing ROS and RNS induced by 150 mM NaCl in Arabidopsis using transgenic plants overexpressing (AOX OE) and antisense lines (AOX AS) of AOX. Imposing NaCl treatment leads to a 4-fold enhanced expression of AOX accompanied by enhanced AOX capacity in WT Col-0. Further AOX-OE seedlings displayed enhanced growth compared with the AOX-AS line under stress. Examination of NO levels by DAF-FM fluorescence and chemiluminescence revealed that AOX overexpression leads to reduced levels of NO. The total NR activity was elevated under NaCl, but no significant change was observed in wild-type (WT), AOX OE, and AS lines. The total ROS, superoxide, H2 O2 levels, and lipid peroxidation were higher in the AOX-AS line than in WT and AOX-OE lines. The peroxynitrite levels were also higher in the AOX-AS line than in WT and AOX-OE lines; further, the expression of antioxidant genes was elevated in AOX-AS. Taken together, our results suggest that AOX plays an important role in the mitigation of ROS and RNS levels and enhances plant growth, thus providing tolerance against nitro-oxidative stress exerted by NaCl.

Diversity Control in Early Transcribed ROS/RNS-balancing Genes: A Common Mechanism for Healthy Resilience?

Diversity Control in Early Transcribed ROS/RNS-balancing Genes: A Common Mechanism for Healthy Resilience?

Adaptive Reprogramming During Early Seed Germination Requires Temporarily Enhanced Fermentation-A Critical Role for Alternative Oxidase Regulation That Concerns Also Microbiota Effectiveness.

Plants respond to environmental cues via adaptive cell reprogramming that can affect whole plant and ecosystem functionality. Microbiota constitutes part of the inner and outer environment of the plant. This Umwelt underlies steady dynamics, due to complex local and global biotic and abiotic changes. Hence, adaptive plant holobiont responses are crucial for continuous metabolic adjustment at the systems level. Plants require oxygen-dependent respiration for energy-dependent adaptive morphology, such as germination, root and shoot growth, and formation of adventitious, clonal, and reproductive organs, fruits, and seeds. Fermentative paths can help in acclimation and, to our view, the role of alternative oxidase (AOX) in coordinating complex metabolic and physiological adjustments is underestimated. Cellular levels of sucrose are an important sensor of environmental stress. We explored the role of exogenous sucrose and its interplay with AOX during early seed germination. We found that sucrose-dependent initiation of fermentation during the first 12 h after imbibition (HAI) was beneficial to germination. However, parallel upregulated AOX expression was essential to control negative effects by prolonged sucrose treatment. Early downregulated AOX activity until 12 HAI improved germination efficiency in the absence of sucrose but suppressed early germination in its presence. The results also suggest that seeds inoculated with arbuscular mycorrhizal fungi (AMF) can buffer sucrose stress during germination to restore normal respiration more efficiently. Following this approach, we propose a simple method to identify organic seeds and low-cost on-farm perspectives for early identifying disease tolerance, predicting plant holobiont behavior, and improving germination. Furthermore, the research strengthens the view that AOX can serve as a powerful functional marker source for seed hologenomes.

The tardigrade Hypsibius exemplaris has the active mitochondrial alternative oxidase that could be studied at animal organismal level.

Mitochondrial alternative oxidase (AOX) is predicted to be present in mitochondria of several invertebrate taxa including tardigrades. Independently of the reason concerning the enzyme occurrence in animal mitochondria, expression of AOX in human mitochondria is regarded as a potential therapeutic strategy. Till now, relevant data were obtained due to heterologous AOX expression in cells and animals without natively expressed AOX. Application of animals natively expressing AOX could importantly contribute to the research. Thus, we decided to investigate AOX activity in intact specimens of the tardigrade Hypsibius exemplaris. We observed that H. exemplaris specimens' tolerance to the blockage of the mitochondrial respiratory chain (MRC) cytochrome pathway was diminished in the presence of AOX inhibitor and the inhibitor-sensitive respiration enabled the tardigrade respiration under condition of the blockage. Importantly, these observations correlated with relevant changes of the mitochondrial inner membrane potential (Δψ) detected in intact animals. Moreover, detection of AOX at protein level required the MRC cytochrome pathway blockage. Overall, we demonstrated that AOX activity in tardigrades can be monitored by the animals' behavior observation as well as by measurement of intact specimens' whole-body respiration and Δψ. Furthermore, it is also possible to check the impact of the MRC cytochrome pathway blockage on AOX level as well as AOX inhibition in the absence of the blockage on animal functioning. Thus, H. exemplaris could be consider as a whole-animal model suitable to study AOX.

Kinetic characterisation and inhibitor sensitivity of Candida albicans and Candida auris recombinant AOX expressed in a self-assembled proteoliposome system

Candidemia caused by Candida spp. is a serious threat in hospital settings being a major cause of acquired infection and death and a possible contributor to Covid-19 mortality. Candidemia incidence has been rising worldwide following increases in fungicide-resistant pathogens highlighting the need for more effective antifungal agents with novel modes of action. The membrane-bound enzyme alternative oxidase (AOX) promotes fungicide resistance and is absent in humans making it a desirable therapeutic target. However, the lipophilic nature of the AOX substrate (ubiquinol-10) has hindered its kinetic characterisation in physiologically-relevant conditions. Here, we present the purification and expression of recombinant AOXs from C. albicans and C. auris in a self-assembled proteoliposome (PL) system. Kinetic parameters (Km and Vmax) with respect to ubiquinol-10 have been determined. The PL system has also been employed in dose–response assays with novel AOX inhibitors. Such information is critical for the future development of novel treatments for Candidemia.

Transcriptome Responses of Ripe Cherry Tomato Fruit Exposed to Chilling and Rewarming Identify Reversible and Irreversible Gene Expression Changes.

Tomato fruit stored below 12°C lose quality and can develop chilling injury upon subsequent transfer to a shelf temperature of 20°C. The more severe symptoms of altered fruit softening, uneven ripening and susceptibility to rots can cause postharvest losses. We compared the effects of exposure to mild (10°C) and severe chilling (4°C) on the fruit quality and transcriptome of ‘Angelle’, a cherry-type tomato, harvested at the red ripe stage. Storage at 4°C (but not at 10°C) for 27 days plus an additional 6 days at 20°C caused accelerated softening and the development of mealiness, both of which are commonly related to cell wall metabolism. Transcriptome analysis using RNA-Seq identified a range of transcripts encoding enzymes putatively involved in cell wall disassembly whose expression was strongly down-regulated at both 10 and 4°C, suggesting that accelerated softening at 4°C was due to factors unrelated to cell wall disassembly, such as reductions in turgor. In fruit exposed to severe chilling, the reduced transcript abundances of genes related to cell wall modification were predominantly irreversible and only partially restored upon rewarming of the fruit. Within 1 day of exposure to 4°C, large increases occurred in the expression of alternative oxidase, superoxide dismutase and several glutathione S-transferases, enzymes that protect cell contents from oxidative damage. Numerous heat shock proteins and chaperonins also showed large increases in expression, with genes showing peak transcript accumulation after different times of chilling exposure. These changes in transcript abundance were not induced at 10°C, and were reversible upon transfer of the fruit from 4 to 20°C. The data show that genes involved in cell wall modification and cellular protection have differential sensitivity to chilling temperatures, and exhibit different capacities for recovery upon rewarming of the fruit.

Production and scavenging of reactive oxygen species both affect reproductive success in male and female Drosophila melanogaster

Sperm aging is accelerated by the buildup of reactive oxygen species (ROS), which cause oxidative damage to various cellular components. Aging can be slowed by limiting the production of mitochondrial ROS and by increasing the production of antioxidants, both of which can be generated in the sperm cell itself or in the surrounding somatic tissues of the male and female reproductive tracts. However, few studies have compared the separate contributions of ROS production and ROS scavenging to sperm aging, or to cellular aging in general. We measured reproductive fitness in two lines of Drosophila melanogaster genetically engineered to (1) produce fewer ROS via expression of alternative oxidase (AOX), an alternative respiratory pathway; or (2) scavenge fewer ROS due to a loss-of-function mutation in the antioxidant gene dj-1β. Wild-type females mated to AOX males had increased fecundity and longer fertility durations, consistent with slower aging in AOX sperm. Contrary to expectations, fitness was not reduced in wild-type females mated to dj-1β males. Fecundity and fertility duration were increased in AOX and decreased in dj-1β females, indicating that female ROS levels may affect aging rates in stored sperm and/or eggs. Finally, we found evidence that accelerated aging in dj-1β sperm may have selected for more frequent mating. Our results help to clarify the relative roles of ROS production and ROS scavenging in the male and female reproductive systems.