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Animals vary tremendously in their capacities to survive anoxia, and the mechanisms responsible are poorly understood. Adult Drosophila melanogaster are rapidly paralyzed and survive up to 12 h of anoxia, whereas larvae vigorously attempt escape but then die if anoxia exceeds 2 h. Here we use nuclear magnetic resonance methods to compare the metabolome of larvae and adult D. melanogaster under normoxic conditions and after various anoxic durations up to 1 h. Glucose increased during anoxia in both larvae and adults, so anoxic death by carbohydrate limitation is unlikely for either stage. Lactate and alanine were the primary anaerobic end products in both adults and larvae. During the first 30 min of anoxia, larvae accumulated anaerobic end products (predominately lactate) at a higher rate, suggesting that larvae may experience greater initial acid-base disruption during anoxic exposures. Adult Drosophila did not possess higher levels of putative protective metabolites; however, these increased during anoxia in adults and decreased in larvae. Metabolites that decreased during anoxia in larvae included mannitol, xylitol, glycerol, betaine, serine, and tyrosine, perhaps due to use as fuels, antioxidants, or binding to denatured proteins. Adults showed significant increases in glycine, taurine, and the polyols glycerol, mannitol, and xylitol, suggesting that adults upregulate protective metabolites to prevent damage. Our results suggest that lower initial metabolic demand due to paralytic hypometabolism and capacities to upregulate protective metabolites may assist the better anoxia tolerance of adult Drosophila. [ABSTRACT FROM AUTHOR]

The world is currently experiencing dramatic increases in flood events impacting on natural vegetation and crops. Flooding often results in low O2 status in root tissues during waterlogging, but sometimes also in shoot tissues when plants become completely submerged. Plants possess a suite of traits enabling tissue aeration and/or adjusted metabolism during hypoxia or even in the absence of O2. This special issue of Functional Plant Biology presents key papers for plant scientists on the quest to further address and improve flood tolerance of terrestrial plants. The papers address low O2 responses in roots, shoots or whole plants in controlled laboratory conditions or in the field situation using natural wetland plants as models as well as economically important crops, such as rice, wheat and barley. The studies advance our understanding of low O2 responses in plant tissues as caused by O2 shortage during flooding. However, in most instances, submergence not only leads to hypoxic or anoxic tissues, but inundation in water also results in accumulation of CO2 and the important plant hormone ethylene. Thus, carefully designed laboratory studies are often needed to unravel the mechanistic relationships between a combined decline in O2 followed by increases in CO2 and ethylene at tissue as well as on the cellular level. [ABSTRACT FROM AUTHOR]

The article presents community recommendations on terminology and procedures used in flooding and low oxygen stress research. This include flooding, anaerobiosis, anaerobic metabolism, anoxia, hypoxia, hyperoxia/superoxia, stress conditions and scoring survival. It also discusses the challenge of monitoring oxygen levels in plants.

Seagrasses grow submerged in aerated seawater but often in low O2 sediments. Elevated temperatures and low O2 are stress factors., Internal aeration was measured in two tropical seagrasses, Thalassia hemprichii and Enhalus acoroides, growing with extreme tides and diel temperature amplitudes. Temperature effects on net photosynthesis ( PN) and dark respiration ( RD) of leaves were evaluated., Daytime low tide was characterized by high pO2 (54 kPa), pH (8.8) and temperature (38°C) in shallow pools. As PN was maximum at 33°C (9.1 and 7.2 μmol O2 m−2 s−1 in T. hemprichii and E. acoroides, respectively), the high temperatures and reduced CO2 would have diminished PN, whereas RD increased (Q10 of 2.0-2.7) above that at 33°C (0.45 and 0.33 μmol O2 m−2 s−1, respectively). During night-time low tides, O2 declined resulting in shoot base anoxia in both species, but incoming water containing c. 20 kPa O2 relieved the anoxia. Shoots exposed to 40°C for 4 h showed recovery of PN and RD, whereas 45°C resulted in leaf damage., These seagrasses are 'living near the edge', tolerant of current diel O2 and temperature extremes, but if temperatures rise both species may be threatened in this habitat. [ABSTRACT FROM AUTHOR]

Ethylene is known to play an essential role in mediating hypoxic responses in plants. Here, we show that in addition to regulating hypoxic responses, ethylene also regulates cellular responses in the reoxygenation stage after anoxic treatment in Arabidopsis. We found that expression of several ethylene biosynthetic genes and ethylene-responsive factors, including ERF1 and ERF2, was induced during reoxygenation. Compared with the wild type, two ethylene-insensitive mutants ( ein2-5 and ein3eil1) were more sensitive to reoxygenation and displayed damaged phenotypes during reoxygenation. To characterize the role of ethylene, we applied microarray analysis to Col-0, ein2-5 and ein3eil1 under reoxygenation conditions. Our results showed that gene transcripts involved in reactive oxygen species ( ROS) detoxification, dehydration response and metabolic processes were regulated during reoxygenation. Moreover, ethylene signalling may participate in regulating these responses and maintaining the homeostasis of different phytohormones. Our work presents evidence that ethylene has distinct functions in recovery after anoxia and provides insight into the reoxygenation signalling network. [ABSTRACT FROM AUTHOR]

The physiological role of nitrate as a protective factor against anaerobic stress was studied in experiments with tolerant to anoxia sugarcane ( Saccharum officibarum L.) callus lines obtained by in vitro selection in the absence of exogenous carbohydrates. Original cell lines, which were not subjected to selection and therefore more sensitive to oxygen shortage, served as a control. In these lines, anaerobic stress was created in the presence or absence of nitrate in nutrient medium. The presence of nitrate in nutrient medium increased markedly tolerance to anaerobic stress of both lines differing in their sensitivity to anaerobiosis. However, the degree of tolerance differed substantially in compared lines. In the presence of exogenous nitrate, in tolerant cells there were no signs of mitochondrial membrane destruction or degradation even after 72 h of anoxia, whereas in control cells 48-h anaerobic incubation led to the complete degradation of mitochondrial membranes and membranes of other organelles. It is concluded that significant increase in the tolerance of S. officinarum cells in the process of in vitro selection most likely occurred due to induction and stimulation of not only the processes of glycolysis and fermentation, but also nitrate and maybe nitrite utilization. [ABSTRACT FROM AUTHOR]

• Pondweed (Potamogeton distinctus) turions can elongate in the absence of O2. Alcoholic fermentation serves to produce energy for anoxic elongation via the breakdown of starch stored in cells. However, the mechanism of cell growth during anoxic elongation is not fully understood. • Changes in pH, H+ equivalent and lactate content of the incubation medium were measured during anoxic elongation. The effects of fusicoccin (FC), indole-3- acetic acid (IAA), vanadate, erythrosine B and K+ channel blockers on anoxic elongation were examined. Cytoplasmic pH and vacuolar pH were measured by 31P nuclear magnetic resonance (NMR) spectroscopy. • Acidification of the incubation medium occurred during anoxic elongation. The contribution of CO2 and lactic acid was not sufficient to explain the acidification. FC and IAA enhanced the elongation of stem segments. Vanadate and erythrosine B inhibited anoxic elongation. Acid growth of notched segments was observed. The activity of plasma membrane H+-ATPase extracted from pondweed turions was increased slightly in anoxic conditions, but that from pea epicotyls sensitive to anoxic conditions was decreased by incubation in anoxic conditions. Both the cytoplasmic pH and vacuolar pH of pondweed turion cells chased by 32P NMR spectroscopy were stabilized during a short period < 3 h after anoxic conditions.• We propose that the enhancement of H+ extrusion by anoxic conditions induces acidification in the apoplast and may contribute to the stabilization of pH in the cytoplasm. [ABSTRACT FROM AUTHOR]

In this article the authors discuss several studies related to plants and flooding stress, which are published in the 2011 issue of the journal "New Phytologist." They say that flooding is a compound stress consists of changes inside the plant cells caused by the flood water surrounding the plant. They cite the study of D. Vashisht and colleagues on natural variation in flooding tolerance of Arabidopsis thaliana. They also mention a study on acclimation and genetic diversity in flooding.

The annual killifish Ausirofundulus limnaeus survives in ephemeral pond habitats by producing drought-tolerant diapausing embryos. These embryos probably experience oxygen deprivation as part of their normal developmental environment. We assessed the anoxia tolerance of A. limnaeus embryos across the duration of embryonic development. Embryos develop a substantial tolerance to anoxia during early development, which peaks during diapause II. This extreme tolerance of anoxia is retained during the first 4 days of post-diapause II development and is then lost. Metabolism during anoxia appears to be supported mainly by production of lactate, with alanine and succinate production contributing to a lesser degree. Anoxic embryos also accumulate large quantities of γ-arninobutyrate (GABA), a potential protector of neural function. It appears that the suite of characters associated with normal development and entry into diapause II in this species prepares the embryos for long-term survival in anoxia even while the embryos are exposed to aerobic conditions. This is the first report of such extreme anoxia tolerance in a vertebrate embryo, and introduces a new model for the study of anoxia tolerance in vertebrates. [ABSTRACT FROM AUTHOR]

This review is a logical development of a previous publication, which summarized the main results of the early period of the systematic and active studying of hypoxic and anoxic stresses in plants. These studies laid a foundation for a new scientific discipline in biology, the investigation relevant to plant anaerobic stress. This review considers a further development of this trend when the investigations embraced a wider set of topics and the discipline acquired an international recognition. The results obtained during last decades by physiologists, biochemists, and molecular biologists engaged in the problem of plant anaerobic stress confirmed the correctness of a concept of the two principal strategies of plant adaptation to hypoxia and anoxia conditions. They are “true” tolerance manifesting at the molecular level under conditions of oxygen deficiency or its absence and “apparent” tolerance, which is realized by avoidance of anaerobiosis due to the long-distance oxygen transport. Therefore, experimental material available now is considered and discussed in this review mainly in the light of these principal notions. Especial attention is paid to the role of stress proteins, which synthesis is induced under hypoxia and anoxia. The results of these experiments confirmed earlier conclusions about the key role of energy (glycolysis and alcoholic fermentation) and carbohydrate (mobilization and utilization of reserved carbohydrates) metabolism in plant adaptation to oxygen deficiency or its absence from the environment. The phenomenon of hypoxic acclimation and its role in plant adaptation to anoxia are also considered. Along with these topics, a further development of pH-stat theory is discussed. A special attention is paid to plant strategy realized by the formation of the net of air-filled spaces (aerenchyma) and long-distance oxygen transport from aerated plant parts to those located in anaerobic environment (apparent tolerance). Among other important aspects, we consider (1) post-anaerobic plant injury by free oxygen radicals; (2) the physiological role of alternative pathways of plant adaptation (nitrate reduction and lipid synthesis); (3) the phenomenon of the adaptation syndrome in plants and possible molecular mechanisms of its realization; and (4) some biotechnological advances in the field of genetic and cell engineering used for the creation of plants more tolerant to anaerobic stress. [ABSTRACT FROM AUTHOR]

This review attempted to follow the establishment of a novel branch of biology arisen at the interfaces between plant physiology, biochemistry, and molecular biology—plant anaerobic stress. Most attention was given to the early period of these investigations, the activity of the members of International Society for Plant Anaerobiosis in particular, and the contribution of Russian scientists, who played a significant role at that time in the establishment and international recognition of this new trend. In this connection, the following points are considered: (1) Crawford's metabolic theory, which could not withstand experimental verification but induced an active discussion, thus stimulating further investigations in this field; (2) a concept of two main strategies of plant adaptation to anaerobic stress (true and apparent adaptation), which was put forward based on the following experimental data: (a) a discovery of a paradoxical phenomenon of hyper-sensitivity, but not hyper-resistance to anoxia, of the flood-tolerant plant roots (“apparent” tolerance); (b) the elucidation of the physiological role of oxygen transported from aerated organs of flood-tolerant plants to the roots inhabiting anaerobic environment; (c) demonstration of the key role of both energy metabolism, and (d) substrate providing for glycolysis and ethanolic fermentation in plants manifesting “true” tolerance to oxygen deprivation; (3) the discovery of plant stress proteins; and finally (4) pH-stat theory put forward by Davies. [ABSTRACT FROM AUTHOR]

Glutathione S-transferases (GSTs) play critical roles in detoxification, response to oxidative stress, regeneration of S-thiolated proteins, and catalysis of reactions in nondetoxification metabolic pathways. Liver GSTs were purified from the anoxia-tolerant turtle, Trachemys scripta elegans. Purification separated a homodimeric (subunit relative molecular mass =34 kDa) and a heterodimeric (subunit relative molecular mass = 32.6 and 36.8 kDa) form of GST. The enzymes were purified 23–69-fold and 156–174-fold for homodimeric and heterodimeric GSTs, respectively. Kinetic data gathered using a variety of substrates and inhibitors suggested that both homodimeric and heterodimeric GSTs were of the α class although they showed significant differences in substrate affinities and responses to inhibitors. For example, homodimeric GST showed activity with known α class substrates, cumene hydroperoxide and p-nitrobenzylchloride, whereas heterodimeric GST showed no activity with cumene hydroperoxide. The specific activity of liver GSTs with chlorodinitrobenzene (CDNB) as the substrate was reduced by 2.6- and 8.7-fold for homodimeric and heterodimeric GSTs isolated from liver of anoxic turtles as compared with aerobic controls, suggesting an anoxia-responsive stable modification of the protein that may alter its function during natural anaerobiosis. [ABSTRACT FROM AUTHOR]

The tiger beetle, Phaeoxantha klugii Chaudoir, survives the annual inundation period in Central Amazonian floodplains as a third-instar larvae submerged in the soil at approximately 29°C for up to 3.5 months. Previous studies showed an exceptional anoxia resistance in these larvae and this study investigates whether they perform anaerobiosis. Larvae collected in the field were exposed to a pure nitrogen atmosphere for 0-9 days in the laboratory. The content of lactate, alanine, free sugars and glycogen is analysed in surviving larvae. Lactate and alanine contents rise during anoxia from around 1.5 and 7 to 6-14 and 15-22 µmol g-1 fresh mass, respectively, providing evidence for anaerobic metabolism. Both compounds show a steep increase during the first 12 h and a tendency to rise further with increasing duration of anoxic conditions, indicating a significant metabolic depression within the first day. Content of free sugars and glycogen varies greatly between individuals and ranges from 0.08-2.5 and 0.05-2.9 mg g-1 fresh mass, respectively. Whether glycogen is used as metabolic substrate for anaerobiosis could not be verified. The findings for free sugars indicate that larvae apparently maintain the ability to regulate the level of glucose and/or trehalose even after 9 days of anoxia. [ABSTRACT FROM AUTHOR]

Although grape berries have been classified as non-climacteric fruits, ongoing studies on grape ethylene signalling challenge the role of ethylene in their ripening. One of the significant molecular changes in berries is the up-regulation of ADH (alcohol dehydrogenase, EC 1.1.1.1) enzyme activity at the inception of fruit ripening and of VvADH2 transcript levels. This paper shows that the ethylene signal transduction pathway could be involved in the control of VvADH2 expression in grapevine berries and in cell suspensions. The induction of VvADH2 transcription, either in berries at the inception of ripening or in cell suspensions, was found to be partly inhibited by 1-methylcyclopropene (1-MCP), an inhibitor of ethylene receptors. Treatment of cell suspensions with 2-chloroethylphosphonic acid (2-CEPA), an ethylene-releasing compound, also resulted in a significant increase in ADH activity and VvADH2 transcription under anaerobiosis, showing that concomitant ethylene and anaerobic treatments in cell suspensions could result in changes in VvADH2 expression. All these results associated with the presence in the VvADH2 promoter of regulatory elements for ethylene and anaerobic response, suggest that the ethylene transduction pathway and anaerobic stress could be, in part, involved in the regulation of VvADH2 expression in ripening berries and cell suspensions. These data open new aspects of the expression control of a ripening-related gene in a non-climacteric fruit. [ABSTRACT FROM PUBLISHER]

The activity of alcohol dehydrogenase (ADH) was measured in corms and roots of the submerged freshwater macrophyte Isoetes alpinus Kirk. growing in situ, and related to its capacity for internal oxygen transport and to carbohydrate translocation. ADH activity was present in roots but not corms at uniform activity (0.15–0.35 × 10−6 mol g−1 fresh weight s−1) over the entire plant depth range (3–7 m depth), and was intermediate to that developed in excised roots after 1-week exposure to either dissolved oxygen at air-saturation or to anoxia. Responses of photosynthesis and root oxygen release to light intensity confirmed that shoot-to-root oxygen transport saturated at similar light intensities to photosynthetic oxygen evolution, but was positive in the dark and at irradiances below the compensation point for photosynthesis, due to contributions to transport by oxygen diffusion from the external medium. Transport of 14C-labelled photo-assimilates to roots nevertheless ceased when intact plants were exposed to a combination of leaf darkness and root external anoxia, even when high 14C concentrations were present in shoots, but remained high when the roots were provided with external oxygen. The lack of any control over permeability of the root surface to gases in this species suggested that ADH activity and reduced translocation is most likely caused by development of hypoxic tissues in the apical tissue. These results suggest that reductions in ambient light intensity may have indirect effects on I. alpinus viability by increasing the degree of root hypoxia and impairing carbon partitioning. [ABSTRACT FROM AUTHOR]

Terrestrial invertebrates experience hypoxia in many habitats and under a variety of physiological conditions. Some groups (at least insects) are much more capable of recovery from anoxia than most vertebrates, but there is still a tremendous unexplained variation in hypoxia/anoxia tolerance among terrestrial invertebrates. Crustaceans and arachnids may be less often confronted with hypoxic environments than insects and myriapods and also seem to be less hypoxia/anoxia tolerant. Tracheated groups, especially those that are able to ventilate their tracheal system like many insects, cope with lower critical PO2 than nontracheated groups. Modulation of oxygen carrier proteins is normally not important in hypoxia resistance. Recent application of genetic and cellular tools are revealing that many of the same pathways documented for mammals (e.g. HIF, nitric oxide) function to regulate morphological and biochemical responses to hypoxia/anoxia in insects. [Copyright &y& Elsevier]

Production of α-amylase during the germination of rice grains is thought to play an important role for tolerance to anoxia of these cereal grains. Under aerobic conditions α-amylases production is enhanced in response to gibberellins produced by the embryos, but the role of these hormones is less clear under anoxia. In this paper we analysed α-amylase gene expression in a rice mutant (Tan-ginbozu) severely impaired in gibberellin biosynthesis. Expression of α-amylase genes others than the gibberellin-induced Amy1A gene is observed. The expression of the Amy3D gene, which does dot require gibberellins to be induced, is high under anoxia in the Tan-ginbozu mutant suggesting that germination under anoxia can proceed thanks to the activity of the α-amylase isoform encoded by the Amy3D gene. Amy3D gene expression is repressed in the presence of high levels of soluble carbohydrates, indicating that the anaerobic expression of this gene can be triggered by a lower carbohydrate content of rice grains kept under anoxia. Germination under anoxia of Tan-ginbozu grains can proceed even in absence of exogenously-added gibberellic acid. Overall, results indicate that gibberellins are not required for the anaerobic germination of rice grains. [ABSTRACT FROM AUTHOR]

Under hyper-salinity stress from freshwater to 17 and 25 ppt seawater, red swamp crayfish Procambarus clarkii largely accumulated d- and l-alanine together with glycine, l-glutamine, and l-proline in both muscle and hepatopancreas. The increases of d- and l-alanine in muscle were the highest in all amino acids and reached 6.8- and 5.4-fold, respectively, from freshwater to 25 ppt seawater. These results indicate that both d- and l-alanine are the most potent osmolytes for intracellular isosmotic regulation in crayfish as well as other crustaceans thus far examined. Under anoxia stress below 0.1 mg/l dissolved oxygen for 12 h and subsequent recovery in normoxia for 12 h in freshwater, 17 and 25 ppt seawater, muscle ATP decreased dramatically in all salinity levels and almost depleted in seawater. Along with the decrease of muscle glycogen level, the significant increase of l-lactate was found in muscle, hepatopancreas, and hemolymph for each salinity level, suggesting the transport of l-lactate from muscle into hepatopancreas via hemolymph. Under anoxia, d- and l-alanine also largely increased in both muscle and hepatopancreas for each salinity level. The increase was much higher in seawater than in freshwater. Thus, both d- and l-alanine are possible to be anaerobic end products during prolonged anaerobiosis of this species. [Copyright &y& Elsevier]

Shoot elongation of arrowhead (Sagittaria pygmaea Miq.) tubers was stimulated in anaerobic conditions. The anaerobic elongation was attributed to stimulation of cell elongation in the middle of the shoots. The anaerobic elongation of the shoots was severely inhibited by ethylene glycol bis(β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). The EGTA inhibition was completely nullified by exogenous CaCl2, which acts as an enhancer of anaerobic elongation. Moreover, calcium channel blockers, varapamil, diltiazem and LaCl3, inhibited the anaerobic elongation enhanced by CaCl2. These results showed that calcium plays an important role in stimulating the elongation in anaerobic conditions. Incorporation of 45Ca into the shoot tissues was measured to determine the involvement of calcium uptake in anaerobic elongation. Incorporation of 45Ca into the cell sap, which was collected from frozen and thawed shoots after thorough washing with LaCl3, was significantly stimulated in anaerobic conditions. Verapamil and diltiazem prevented the stimulation of 45Ca incorporation in anaerobic conditions. These results suggest that calcium uptake from the medium serves to enhance shoot elongation of arrowhead tubers under anaerobic conditions. [ABSTRACT FROM AUTHOR]

Oxygen plays a crucial role in energetic metabolism of most eukaryotes. Yet adaptations to low-oxygen concentrations leading to anaerobiosis have independently arisen in many eukaryotic lineages, resulting in a broad spectrum of reduced and modified mitochondrion-related organelles (MROs). In this study, we present the discovery of two new class-level lineages of free-living marine anaerobic ciliates, Muranotrichea, cl. nov. and Parablepharismea, cl. nov., that, together with the class Armophorea, form a major clade of obligate anaerobes (APM ciliates) within the Spirotrichea, Armophorea, and Litostomatea (SAL) group. To deepen our understanding of the evolution of anaerobiosis in ciliates, we predicted the mitochondrial metabolism of cultured representatives from all three classes in the APM clade by using transcriptomic and metagenomic data and performed phylogenomic analyses to assess their evolutionary relationships. The predicted mitochondrial metabolism of representatives from the APM ciliates reveals functional adaptations of metabolic pathways that were present in their last common ancestor and likely led to the successful colonization and diversification of the group in various anoxic environments. Furthermore, we discuss the possible relationship of Parablepharismea to the uncultured deep-sea class Cariacotrichea on the basis of single-gene analyses. Like most anaerobic ciliates, all studied species of the APM clade host symbionts, which we propose to be a significant accelerating factor in the transitions to an obligately anaerobic lifestyle. Our results provide an insight into the evolutionary mechanisms of early transitions to anaerobiosis and shed light on fine-scale adaptations in MROs over a relatively short evolutionary time frame. • Discovery and cultivation of two new classes of marine ciliates thriving in anoxia • Phylogenomics reveals a major clade of obligate anaerobes in ciliates • Novel insights into evolution of mitochondrial metabolism in anaerobic eukaryotes • Transitions to obligate anaerobiosis might be facilitated by prokaryotic symbionts Numerous eukaryotes have switched to anaerobiosis, variously reducing their mitochondrial metabolism. Rotterová et al. describe two new classes of obligately anaerobic ciliates with prokaryotic symbionts and reduced mitochondria with unique pathways for energetic metabolism, introducing possible mechanisms of transitions to obligate anaerobiosis. [ABSTRACT FROM AUTHOR]