Inorganic Nitrogen Metabolism in Barley Roots Under Poorly Aerated Conditions

Nitrate reduction, nitrite reduction, and ammonium assimilationwere measured over c. 24 h in excised sterile barley roots,in air or under low oxygen tensions. Partial anoxia had relativelylittle effect, but the pathway of nitrogen assimilation wasseverely inhibited during complete anoxia, when the uptake ofnitrate ceased. Much of the nitrate which was present in theroots at the time of excision was apparently unavailable forassimilation. None of the reactions of the pathway served inplace of oxygen as an electron acceptor under anaerobic conditions.The concentration of nitrate in the external solution duringgrowth and during the experimental treatments had no directeffect on anaerobic ethanol formation, although an indirecteffect was noted which was due to variations in the carbohydratecontent of the tissue.     

Anaerobic Amino Acid Metabolism

Anoxic stress induces a strong change in sugar, protein, and amino acid metabolism in higher plants. Sugars are rapidly consumed through the anaerobic glycolysis to sustain energy production. Protein degradation under anoxia is a mechanism to release free amino acids contributing in this way to maintaining the osmotic potential of the tissue under stress. Among free amino acids, a particular role is played by glutamic acid, being a precursor of some characteristic compounds of the anaerobic metabolism (alanine, -aminobutyric acid, and putrescine). The glutamine synthetase/glutamate synthase cycle contributes to ammonia reassimilation and primary assimilation of nitrate, and resynthesizes constantly glutamate for the synthesis of other compounds. Some polypeptides involved in these pathways are expressed under anoxia. The importance of amino acid metabolism for the response to anaerobic stress is discussed.     

Metabolic Acclimation to Anoxia Induced by Low (2-4 kPa Partial Pressure) Oxygen Pretreatment (Hypoxia) in Root Tips of Zea mays

Young intact plants of maize (Zea mays L. cv INRA 508) were exposed to 2 to 4 kilopascals partial pressure oxygen (hypoxic pretreatment) for 18 hours before excision of the 5 millimeter root apex and treatment with strictly anaerobic conditions (anoxia). Hypoxic acclimation gave rise to larger amounts of ATP, to larger ATP/ADP and adenylate energy charge ratios, and to higher rates of ethanol production when excised root tips were subsequently made anaerobic, compared with root tips transferred directly from aerobic to anaerobic media. Improved energy metabolism following hypoxic pretreatment was associated with increased activity of alcohol dehydrogenase (ADH), and induction of ADH-2 isozymes. Roots of Adh1⁻ mutant plants lacked constitutive ADH and only slowly produced ethanol when made anaerobic. Those that were hypoxically pretreated acclimated to anoxia with induction of ADH2 and a higher energy metabolism, and a rate of ethanol production comparable to that of nonmutants. All these responses were insensitive to the presence or absence of NO₃⁻. Additionally, the rate of ethanol production was about 50 times greater than the rate of reduction of NO₃⁻ to NO₂⁻. These results indicate that nitrate reductase does not compete effectively with ADH for NADH, or contribute to energy metabolism during anaerobic respiration in this tissue through nitrate reduction. Unacclimated root tips of wild type and Adhl⁻ mutants appeared not to survive more than 8 to 9 hours in strict anoxia; when hypoxically pretreated they tolerated periods under anoxia in excess of 22 hours.     

The Effects of Anoxia and Carbohydrates on the Growth and Viability of Rice, Pea and Pumpkin Roots

Intact or excised roots, immersed in anaerobic aqueous agarmedia with or without additions of sugar, were made anoxic byexposing the shoots (or cut-ends) to oxygen-free nitrogen. Polarographicmonitoring of the internal oxygen status showed that roots rapidlybecame anoxic; also extension growth immediately declined andwas soon halted. Growth was re-started only by re-aeration and,in sugar deficient media, apices survived only if the periodof anoxia had not exceeded 4 h (rice), 6 h (pea) and 12 h (pumpkin). Utilizable carbohydrate supplied exogenously could reduce therate of decline in growth rate (rice and pumpkin) but couldnot of itself indefinitely sustain or induce growth; it enhancedviability, however, and rice became the most tolerant of anoxia(c. 44 h). Oxygen was essential both to sustain and initiateroot extension but growth could cease and apical death ensuefrom an insufficiency of carbohydrate even in the presence ofoxygen. It is concluded that the normal response of roots toanoxia is a hypersensitive one arising from sugar deficiency. The discussion relates the results to recent reports concerningcarbohydrate and energy levels in anoxic and aerated roots,to work on ultrastructural change under anoxia, and to the subjectof flood tolerance. The results are not thought to accord witha metabolic theory of flood-tolerance based upon differencesin the accumulation and phytotoxicities of ethanol and otherby-products of anaerobic respiration. They are considered tobe more in keeping with the view that flood-tolerance in rootsis chiefly a property of internal aeration and the potentialfor producing well-ventilated roots in response to soil wetness. Key words: Anoxia, Carbohydrates, Growth, Roots     

Catalase Activity and Post-anoxic Injury in Monocotyledonous Species

Three anoxia-intolerant species, Glyceria maxima, Juncus effususand Iris germanica (var. Quechei), and three anoxia-tolerantspecies Schoenoplectus lacustris, Acorus calamus and Iris pseudacoruswere chosen for investigation. Rhizomes of anoxia-intolerantspecies show increased catalase activities when returned toair after periods of prolonged anoxia. Levels of catalase remainedfairly constant in anoxia-tolerant species under the same conditions.In the anoxia intolerant G. maxima, the post-anoxic increasein catalase activity was reduced by circulating the anaerobicatmosphere. This treatment also reduced the ethanol contentof the tissue under incubation, and increased the survival ofthe rhizomes as seen in their ability to resume growth in thepost-anoxic phase. Exposure of anaerobic G. maxima rhizomesto ethanol vapour increased post-anoxic levels of catalase activityand when this produced a 5-fold increase always resulted indeath of the rhizomes. Acetaldehyde vapour applied in the sameway gave rise to increases in catalase activity followed byrapid death of the rhizomes. It is suggested that post-anoxic oxidation of anaerobicallyaccumulated ethanol may result in a surge of acetaldehyde production,which could exert a toxic effect on the recovering tissues.The possible role of catalase in an ethanol-oxidation reaction,which is well documented in animals, is discussed in the lightof the association between the natural accumulation of largeconcentrations of ethanol and subsequent post-anoxic death insome plant tissues. Key words: Catalase, post-anoxia, ethanol     

Anoxic Seed Germination of Erythrina caffra: Ethanol Fermentation and Response to Metabolic Inhibitors

Erythrina caffra seeds were shown to be true anaerobic germinators.They exhibit a Pasteur effect, high alcohol dehydrogenase activityand produce high levels of ethanol under anoxia, in which situationgermination starts to be suppressed by as little as 0.1% externallyapplied ethanol. Toxic levels of ethanol production appear tobe prevented by a decrease in the rate of ethanol accumulation.Carbon monoxide does not inhibit germination. Cyanide, SHAM,iodoacetate, pyrazole, and 4-methylpyrazole are more inhibitoryto anoxic than aerobic germination whereas azide, arsenate,and fluoride inhibit both. Azide, pyrazole, 4-methylpyrazoleand a low concentration of cyanide and SHAM tend to stimulateethanol production in air. At 10 mol m^–3, 4-methylpyrazolestimulates anaerobic ethanol production. At higher concentrationsthis compound and all other inhibitors used suppress anaerobicethanol production initially. Inhibition of ethanol productionby 10 mol m^–3 cyanide is paralleled by an accumulationof acetaldehyde. Azide and cyanide appear to exert their inhibitoryeffect at different loci. Key words: Erythrina caffra, anoxic germination, fermentation, metabolic inhibitors     

Tolerance of Anoxia and Ethanol Toxicity in Chickpea seedlings (Cicer arietinum L.)

Controversy exists as to whether ethanol ever accumulates totoxic levels in anaerobic tissues of higher plants. In orderto manipulate the internal concentrations of ethanol and relatethese to anaerobic injury, seedlings of chickpea (Cicer arietinumL.) were incubated under strict anoxia in vessels in which theanaerobic atmosphere either remained static or else was circulatedwith that of a large anaerobic incubator. Incubation with acirculating, as compared with a static, anaerobic atmospheredoubled the time that the seedlings could be kept under anoxiaand emerge in subsequent survival testing in the glasshouse.Circulating the anaerobic atmosphere gave a 13-fold reductionin the accumulation of ethanol in the seedlings. Parallel experimentswhich varied the ratio of head space relative to seedling numberconfirmed that the dilution of the volatile products of anoxia.increasedsurvival. These products included carbon dioxide, ethanol andtraces of acetaldehyde. While carbon dioxide may play a rolein modifying glycolytic activity under anoxia, it is suggestedthat it is not directly toxic and that it is the reduction inethanol concentration in the seedlings as a result of head spacedilution that contributes to their increased longevity in circulatinganaerobic atmospheres. Key words: Cicer arietinum L., Ethanol, Anaerobic conditions     

Chilling slows anaerobic metabolism to improve anoxia tolerance of insects

Background

Insects are renowned for their ability to survive anoxia. Anoxia tolerance may be enhanced during chilling through metabolic suppression.

Aims

Here, the metabolomic response of insects to anoxia, both with and without chilling, for different durations (12–36 h) was examined to assess the potential cross-tolerance mechanisms.

Results

Chilling during anoxia (cold anoxia) significantly improved survival relative to anoxia at warmer temperatures. Reduced intermediate metabolites and increased lactic acid, indicating a switch to anaerobic metabolism, were characteristic of larvae in anoxia.

Conclusions

Anoxia tolerance was correlated survival improvements after cold anoxia were correlated with a reduction in anaerobic metabolism.

     

Carbohydrate metabolism in germinating caryopses of <Emphasis Type="Italic">Oryza sativa</Emphasis> L. exposed to prolonged anoxia

Anoxia tolerance can be evaluated not only in terms of growth or survival of plant organs during oxygen deprivation, but also in relation to carbohydrate utilization in the context of a well-modulated fermentative metabolism. Rice (Oryza spp.) is unique among cereals, in that it has the distinctive ability to germinate under complete anaerobiosis by using the starchy reserves in its seeds to fuel the anaerobic metabolism. The aim of the present study was to evaluate the ability of germinating rice seedlings to survive a long-term oxygen deficiency [40 days after sowing (DAS)] and the effects on sugar metabolism, focusing on starch degradation as well as soluble sugars transport and storage under anoxia. No significant decline in vitality occurred until 30 DAS though no recovery was detected following longer anoxic treatments. Growth arrest was observed following anoxic treatments longer that 20 DAS, in concomitance with considerably lower ethanol production. Amylolytic activity in embryos and endosperms had similar responses to anoxia, reaching maximum content 30 days after the onset of stress, following which the levels declined for the remainder of the experiment. Under anoxia, average amylolytic activity was twofold higher in embryos than endosperms. Efficient starch degradation was observed in rice under anoxia at the onset of the treatment but it decreased over time and did not lead to a complete depletion. Our analysis of α-amylase activity did not support the hypothesis that starch degradation plays a critical role in explaining differences in vitality and coleoptile growth under prolonged oxygen deprivation.     

Organ specific analysis of the anaerobic primary metabolism in rice and wheat seedlings II: Light exposure reduces needs for fermentation and extends survival during anaerobiosis

Low oxygen stress in plants can occur during flooding and compromise the availability and utilization of carbohydrates in root and shoot tissues. Low-oxygen-tolerant rice and -sensitive wheat plants were analyzed under anaerobiosis in light to evaluate main factors of the primary metabolism that affect sensitivity against oxygen deprivation: activity of glycolysis and the rate of photosynthesis. Relatively stable ATP contents (93 and 58% of aerated control levels after 24 h anaerobiosis) in illuminated shoot tissues account for enhanced tolerance of rice and wheat seedlings to anaerobiosis upon light exposure in comparison to anoxia in darkness. Although the photosynthetic process was inhibited during low oxygen stress, which was partly due to CO₂ deficiency, more light-exposed than dark-incubated seedlings survived. Illuminated plants could tolerate a 70% lower anaerobic ethanol production in shoots in comparison to darkness, although still an 18-times higher ethanol production rate was determined in rice than in wheat leaves. In conclusion, light-exposed plants grown under anaerobiosis may recycle low amounts of generated oxygen between photosynthesis and dissimilation and generate additional energy not only from substrate phosphorylation during glycolysis but also from other sources like cyclic electron transport.     

Key role of PLC-gamma in EGF protection of epithelial barrier against iNOS upregulation and F-actin nitration and disassembly

Upregulation of inducible nitric oxide synthase (iNOS) is key to oxidant-induced disruption of intestinal (Caco-2) monolayer barrier, and EGF protects against this disruption by stabilizing the cytoskeleton. PLC- appears to be essential for monolayer integrity. We thus hypothesized that PLC- activation is essential in EGF protection against iNOS upregulation and the consequent cytoskeletal oxidation and disarray and monolayer disruption. Intestinal cells were transfected to stably overexpress PLC- or to inhibit its activation and were then pretreated with EGF ± oxidant (H₂O₂). Wild-type (WT) intestinal cells were treated similarly. Relative to WT monolayers exposed to oxidant, pretreatment with EGF protected monolayers by: increasing native PLC- activity; decreasing six iNOS-related variables (iNOS activity/protein, NO levels, oxidative stress, actin oxidation/nitration); increasing stable F-actin; maintaining actin stability; and enhancing barrier integrity. Relative to WT cells exposed to oxidant, transfected monolayers overexpressing PLC- (+2.3-fold) were protected, as indicated by decreases in all measures of iNOS-driven pathway and enhanced actin and barrier integrity. Overexpression-induced inhibition of iNOS was potentiated by low doses of EGF. Stable inhibition of PLC- prevented all measures of EGF protection against iNOS upregulation. We conclude that 1) EGF protects against oxidative stress disruption of intestinal barrier by stabilizing F-Actin, largely through the activation of PLC- and downregulation of iNOS pathway; 2) activation of PLC- is by itself essential for cellular protection against oxidative stress of iNOS; and 3) the ability to suppress iNOS-driven reactions and cytoskeletal oxidation and disassembly is a novel mechanism not previously attributed to the PLC family of isoforms. actin cytoskeleton; gut barrier; growth factors; oxidative stress; nitration and carbonylation; reactive nitrogen metabolites; phospholipase C isoform; inflammatory bowel disease; Caco-2 cells     

The influence of anoxia and ethanol on barley seed death

Survival of deteriorated barley seeds in wet soil was improved by exposure to 70% oxygen compared with that in air and decreased by the imposition of anoxia. Deteriorated seeds were more sensitive to anoxia than non-deteriorated seeds, the former died within 4 days, while the latter survived more than 7 days. Ethanol accumulated in non-deteriorated seeds in wet soil conditions during the first 24 h and thereafter declined, while it increased to higher concentrations over 3–4 days in deteriorated seeds. High concentrations of ethanol were recorded in seeds in anaerobic conditions regardless of the level of deterioration. It was concluded that oxygen diffusion to the seeds was severely restricted in wet soil resulting in a switch to anaerobic respiration, the products of which, indicated by ethanol content, accumulated within the tissues. Non-deteriorated seeds tolerated higher concentrations of ethanol than deteriorated seeds and the coleorhiza extruded through the covering layers more rapidly facilitating gaseous exchange, while in the latter, anaerobic products accumulated to toxic levels.     

Role of water and electrolyte influxes in anoxic plasma membrane disruption

The role of water and electrolyte influxes in anoxia-inducedplasma membrane disruption was investigated using rabbit proximal tubule suspension. The results indicated that normal proximal tubule(PT) cells have a great capacity for expanding cell volume in responseto water influx, whereas anoxia increases the susceptibility to waterinflux-induced disruption, and this was attenuated by glycine. However,resistance of anoxic plasma membranes to water influx-induced stress isnot lost, although their mechanical strength was diminished, comparedwith normoxic membranes. Anoxic membranes did not disrupt under anintra-to-extracellular osmotic difference as great as 150 mosM.Potentiating or attenuating water influx by incubating PT cells inhypotonic or hypertonic medium, respectively, during anoxia, did notaffect anoxia-induced membrane disruption. After the transmembraneelectrolyte concentration gradient was eliminated by a"intracellular" buffer or by permeabilizing the plasma membraneto molecules <4 kDa using -toxin, anoxia still caused furthermembrane disruption that was prevented by glycine or low pH. Theseresults demonstrate that 1) water ornet electrolyte influxes are probably not a primary cause foranoxia-induced membrane disruption and2) glycine could prevent the plasmamembrane disruption during anoxia independently from its effect ontransmembrane electrolyte or water influxes. The present data support abiochemical rather than a mechanical alteration of the plasma membraneas the underlying cause of membrane disruption during anoxia.

     

Anaerobic metabolism in aquatic insect larvae: studies onChironomus thummi andCulex pipiens

Summary The metabolic effect of hypoxia and anoxia on the larvae ofChironomus thummi andCulex pipiens was investigated. InC. thummi anoxia resulted in a characteristic decrease of ATP and P-arginine concentrations and in an accumulation of alanine and lactate within 60 minutes. These changes continued during prolonged incubation but at lower rates. Ethanol, the major product during long-term anoxia, was largely excreted into the ambient water.A significant accumulation of these metabolites occurred only at a of 7 Torr. However, the proportion of anaerobic energy production even at this low amounted to less than 5% of the total energy consumption measured during experimental anoxia. Thus the chironomid larvae exhibited a remarkable capacity for utilizing very low levels of oxygen to maintain an aerobic metabolism. Complete anaerobiosis was observed only under anoxic conditions.Recovery from prior anoxia began with the reestablishment of normal ATP, P-arginine and succinate concentrations, whereas removal of the accumulated alanine and lactate and replenishment of the normally high level of malate required several hours. Culex larvae were shown to have a very low anaerobic capacity and a high rate of lactate accumulation.The significance of the results is discussed with particular emphasis on comparative aspects.     

Anaerobic energy-metabolism of goldfish,Carassius auratus (L.)

Summary Goldfish, acclimated to 20°C and normal = 130 mmHg) and low ( = 19 mmHg) oxygen levels, were exposed to different periods of hypoxia and anoxia. Experiments were carried out at night. ATP, ADP, AMP, IMP, CrP, glycogen and lactate were determined in red muscle, white muscle and liver. Acclimation to hypoxia resulted in a marked increase of the energy charge of liver and red muscle and of the glycogen content of red and white muscle, indicating an increased anaerobic capacity. Short exposures to anoxia, up to 1 h, had little influence on the value of the measured parameters. Long-term exposures (12 h) to anoxia caused a significant decrease of CrP and glycogen levels in all tissues examined. The energy-charge of red and white muscle was hardly affected by a 12 h exposure to anoxia, but in liver tissue the energy charge decreased from 0.60 to 0.32. It is concluded that during anoxia muscle tissues are able to maintain high energy-charges, probably by means of a yet unknown anaerobic energy-producing system.Abbreviations CrP creatine phosphate - EC energy charge - IMP inosine monophosphate - I.U. International Unit (mole/min)     

The Enzymic Decarboxylation of {gamma}-methyleneglutamic Acid by Plant Extracts

-Methyleneglutamic acid, an acidic amino-acid isolated fromgroundnut plants, was decarboxylated by enzymes present in extractsof Capsicum fruits, barley roots, and tulip leaves, and alsoby intact cells of Clostridium welchii S.R. I2. The amino-acidwas attacked in a similar manner to, but in all cases at a slowerrate than, l-glutamic acid. The nature of the enzyme responsiblefor the decarboxylation of -methyleneglutamic acid was furtherinvestigated using preparations from barley roots (which donot contain the amino-acid) and from tulip leaves (in whichthe amino-acid is normally present, together with larger amountsof its amide form, -methyleneglutamine). The effects of pH,inhibitors, and partial heat denaturation upon the enzyme systemspresent in the barley and tulip extracts indicated that a singleenzyme was responsible for the decarboxylation of both l-glutamicacid and -methyleneglutamic acid. Although the Cl. welchii rapidlydeamidated and then decarboxylated l-glutamine, -methyleneglutaminewas not attacked by the organism.     

Intragenic Hill-Robertson interference influences selection intensity on synonymous mutations in Drosophila

Natural selection influences synonymous mutations and synonymouscodon usage in many eukaryotes to improve the efficiency oftranslation in highly expressed genes. Recent studies of genecomposition in eukaryotes have shown that codon usage also variesindependently of expression levels, both among genes and atthe intragenic level. Here, we investigate rates of evolution(Ks) and intensity of selection (_s) on synonymous mutationsin two groups of genes that differ greatly in the length oftheir exons, but with equivalent levels of gene expression andrates of crossing-over in Drosophila melanogaster. We estimate_s using patterns of divergence and polymorphism in 50 Drosophilagenes (100 kb of coding sequence) to take into account possiblevariation in mutation trends across the genome, among genesor among codons. We show that genes with long exons exhibithigher Ks and reduced _s compared to genes with short exons.We also show that Ks and _s vary significantly across long exons,with higher Ks and reduced _s in the central region comparedto flanking regions of the same exons, hence indicating thatthe difference between genes with short and long exons can bemostly attributed to the central region of these long exons.Although amino acid composition can also play a significantrole when estimating Ks and _s, our analyses show that the differencesin Ks and _s between genes with short and long exons and acrosslong exons cannot be explained by differences in protein composition.All these results are consistent with the Interference Selection(IS) model that proposes that the Hill-Robertson (HR) effectcaused by many weakly selected mutations has detectable evolutionaryconsequences at the intragenic level in genomes with recombination.Under the IS model, exon size and exon-intron structure influencethe effectiveness of selection, with long exons showing reducedeffectiveness of selection when compared to small exons andthe central region of long exons showing reduced intensity ofselection compared to flanking coding regions. Finally, ourresults further stress the need to consider selection on synonymousmutations and its variation—among and across genes andexons—in studies of protein evolution.