15N Studies on the In Vivo Assay of Nitrate Reductase in Leaves: Occurrence of Underestimation of the Activity Due to Dark Assimilation of Nitrate and Nitrite

The reduction of nitrate and nitrite in leaf disks from sevendi- and two monocotyledonous species under in vivo nitrate reductaseassay conditions was studied using 15N-labeled substrates. Significantreduction of both nitrate and nitrite into ammonia and aminoacids was detected under aerobic conditions (in an atmosphereof air): in some cases, the amount of nitrate-N reduced to ammoniaand amino acids was more than that remaining as nitrite. Anaerobicincubation (under an atmosphere of N₂ gas) enhanced the accumulationof nitrite, but the subsequent reduction to the basic nitrogencompounds was 40 to 180% of the aerobic rates. The present examinationindicates that in vivo assays of nitrate reductase under aerobicconditions may give greatly underestimated results due to nitritereduction and that exclusion of oxygen from the in vivo assaymixture is desirable in terms of the quantity of nitrite formed.n-Propanal treatment increased nitrite accumulation under aerobicbut not under anaerobic conditions, and depressed the incorporationof nitrate-N into basic fractions under both conditions. Therefore,addition of n-propanol may be desirable for assays under aerobicconditions. No significant difference in the reduction of nitratesupplied as sodium and potassium salts was observed on the nitriteformation and on the incorporation of nitrate-N into basic fractions. ¹⁵N experiments on dark assimilation of nitrate, nitrite andammonia into amino acids in wheat leaves showed that these threenitrogen sources were assimilated through the same route andthat the glutamine synthetase/glutamate synthase pathway wasthe major route. With anaerobic treatment, the incorporationof nitrogen into alanine and serine remained at relatively high,but the incorporation into aspartate and asparagine was muchlower than in the cases of aerobic treatment. (Received July 11, 1981; Accepted October 3, 1981)     

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     

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.     

Exogenous Nitrate as a Terminal Acceptor of Electrons in Rice (Oryza sativa) Coleoptiles and Wheat (Triticum aestivum) Roots under Strict Anoxia

To elucidate the physiological role of exogenous nitrate under anaerobic conditions, we studied the effect of 10 mM KNO₃ on the mitochondrial ultrastructure in rice (Oryza sativa L.) coleoptiles and in wheat (Triticum aestivum L.) roots, detached from four-day-old seedlings, under strict anoxia. In wheat roots, following 6-h-long anoxia in the absence of exogenous nitrate, the mitochondrial membranes were partially degraded and, after 9 h under anoxia, the mitochondrial membranes and the membranes of other organelles were completely destroyed. In rice coleoptiles, the partial membrane degradation was observed only after 24 h and their complete breakdown after 48 h of anaerobiosis. In the presence of exogenous nitrate, no membrane destruction was noticed even after 9 and 48 h of anaerobiosis in wheat roots and rice coleoptiles, respectively. These results indicate that exogenous nitrate exerts protective action as a terminal electron acceptor, alternative to the molecular oxygen. Our findings are compared with the results of other researchers concerning the adverse or favorable nitrate action on plant growth, metabolism, and energy status under anaerobic stress.     

The role of nitrate reduction in the anoxic metabolism of roots II. Anoxic metabolism of tobacco roots with or without nitrate reductase activity

The effects of root anoxia on a tobacco (Nicotiana tabacum) wild type (WT) and a transformant (LNR-H) lacking root nitrate reductase were compared. LNR-H plants were visibly more sensitive to oxygen deprivation than WT, showing rapid and heavy wilting symptoms. LNR-H roots also produced substantially more ethanol and lactate than WT roots under anoxia, and their sugar and sugar-P content, as well as their ATP levels, remained higher. The fermentation rates of WT and LNR-H roots were unaffected by sugar feeding and the higher fermentation rate in the LNR-H roots was associated with a greater acidification of the cytoplasm under anoxia. From these observations it is concluded: (i) that the absence of NR activity in the LNR-H roots does not necessarily limit NADH recycling; and (ii) that nitrate reduction in the WT roots results in a more acidifying metabolism. It is the higher metabolic rate in the LNR-H roots that leads to the greater cytoplasmic acidification under anoxia despite the absence of a contribution from the metabolism of nitrate. Competition for NADH cannot explain this difference in metabolic rate, and it remains unclear why the NR-free LNR-H, and tungstate-treated WT roots, had much higher fermentation rates than WT roots. The difference in anaerobic metabolism could still be due to the presence or absence of nitrate reductase and the possibility that this could occur through the production of nitric oxide is discussed.     

Control of nitrate and nitrite assimilation by carbohydrate reserves,adenosine nucleotides and pyridine nucleotides in leaves of Zea mays L. under dark conditions

The rate of in-vivo nitrate reduction by leaf segments of Zea mays L. was found to decline during the second hour of dark anaerobic treatment. On transfer to oxygen the capacity to reduce nitrate under dark conditions was restored. These observations led to the proposal that nitrate reductase is a regulatory enzyme with ADP acting as a negative effector. The effect of ADP on the invitro activity of nitrate reductase and the changes in the in-vivo adenylate pool under dark-N₂ and dark-O₂ were investigated. It was found that ADP inhibited the activity of partially purified nitrate reductase. Similarly, the in-vivo anaerobic inhibition of nitrate reduction was associated with a build-up of ADP in the leaf tissue. Under anaerobic conditions nitrite accumulated and on transfer to oxygen the accumulated nitrite was reduced. To explain this phenomenon the following hypothesis was proposed and tested. Under anaerobic conditions the supply of reducing equivalents for nitrite reduction in the plastid becomes restricted and nitrite accumulates as a consequence. On transfer to oxygen this restriction is removed and nitrite disappears. This capacity to reduce accumulated nitrite was found to be dependent on the carbohydrate status of the leaf tissue.     

Plant anaerobic stress as a novel trend in ecological physiology, biochemistry, and molecular biology: 2. Further development of the problem

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.     

On the Physiological Role of Anaerobically Synthesized Lipids in <Emphasis Type="Italic">Oryza sativa</Emphasis> Seedlings

The objective of this work was to elucidate a possible adaptive role of lipid biosynthesis and unsaturated fatty acids (FAs), esterified to lipids, as terminal acceptors of electrons, alternative to molecular oxygen, in the shoots of rice seedlings (Oryza sativa L.) under conditions of strict anoxia. Biosynthesis of lipids and their accumulation, as well as the reduction of double bonds in unsaturated FAs, were studied by electron microscopic observation of the accumulation of lipid bodies in the cytoplasm and by the biochemical analysis of FAs in shoot lipids before and after anaerobic incubation of the shoots. The experiments were carried out with intact coleoptiles after 5 and 8 days of anaerobic germination of seeds (primary anoxia) and with detached shoots, preliminarily grown in air and then subjected to anoxia in the presence of 2% glucose for 48 h (secondary anoxia). In these experiments, lipid bodies did not accumulate in the cytoplasm under anoxic conditions. Lipid bodies appeared only during 48-h anaerobic incubation of detached coleoptiles in the absence of exogenous glucose, when mitochondria degraded. There was no change either in the double bond index of FAs, or in the qualitative and quantitative composition of FAs during shoot anaerobic incubation. We conclude that neither lipids synthesized under anaerobic conditions nor esterified unsaturated FAs are involved in plant adaptation to anaerobiosis as terminal acceptors of electrons, alternative to molecular oxygen. Lipid biosynthesis under anoxic conditions, which was demonstrated for anoxia-tolerant seedlings of Oryza sativa and Echinochloa phyllopogon in experiments with radioactive precursors, ¹⁴C-acetate and ³H-glycerol, is only the manifestation of a turnover of saturated FAs and various classes of lipids, which stabilizes cell membranes under adverse conditions of strict anoxia.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 540–548.Original Russian Text Copyright © 2005 by Generosova, Vartapetian.     

Effect of aerobic and anaerobic conditions on the in vivo nitrate reductase assay in spinach leaves

¹⁵N-labelled nitrate was used to show that nitrate reduction by leaf discs in darkness was suppressed by oxygen, whereas nitrite present within the cell could be reduced under aerobic dark conditions. In other experiments, unlabelled nitrite, allowed to accumulate in the tissue during the dark anaerobic reduction of nitrate was shown by chemical analysis to be metabolised during a subsequent dark aerobic period. Leaves of intact plants resembled incubated leaf discs in accumulating nitrite under anaerobic conditions. Nitrate, n-propanol and several respiratory inhibitors or uncouplers partly reversed the inhibitory effect of oxygen on nitrate reduction in leaf discs in the dark. Of these nitrate and propanol acted synergistically. Reversal was usually associated with inhibition of respiration but some concentrations of 2,4-dinitrophenol (DNP) and ioxynil reversed inhibition without affecting respiratory rates. Respiratory inhibitors and uncouplers stimulated nitrate reduction in the anaerobic in vivo assay i.e. in conditions where the respiratory process is non-functional. Freezing and thawing leaf discs diminished but did not eliminate the sensitivity of nitrate reduction to oxygen inhibition.Abbreviations DNP 2,4-dinitrophenol - HOQNO 8-hydroxyquinoline-N-oxide - DCPIP 2,6-dichlorophenolindophenol - CCCP Carbonyl cyanide m-chlorophenylhydrazone - TES N-tris(hydroxymethyl)methyl-2-amino ethanesulphonic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Nitrate reductase regulation in tomato roots by exogenous nitrate: a possible role in tolerance to long-term root anoxia

The mechanism of nitrate reductase (NR) regulation under long-term anoxia in roots of whole plants and the putative role of nitrate in anoxia tolerance have been addressed. NR activity in tomato roots increased significantly after 24 h of anaerobiosis and increased further by 48 h, with a concomitant release of nitrite into the culture medium. Anoxia promoted NR activation through dissociation of the 14-3-3 protein inhibitor and NR dephosphorylation. After 24 h of anoxia, the total amount of NR increased slightly up to 48 h. However, NR-mRNA levels remained constant between 0 h and 24 h of root anoxia and decreased after 48 h. This is probably due to the inhibition of NR degradation and the accumulation of its native form. NR was slightly dephosphorylated in the absence of oxygen and nitrate. Under anoxia, NR dephosphorylation was modulated by nitrate-controlled NR activity. In addition, the presence of nitrate prevents anoxic symptoms on leaves and delays wilting by 48 h during root anoxia. In the absence of nitrate, plants withered within 24 h, as they did with tungstate treatment, an inhibitor of NR activity. Thus, anoxia tolerance of tomato roots could be enhanced by nitrate reduction.     

Genetic and biochemical analysis of anaerobically-induced enzymes during seed germination of Echinochloa crus-galli varieties tolerant and intolerant of anoxia

To compare the regulation of anaerobic metabolism during germination in anoxia-tolerant and intolerant plants, enzymes associated with anaerobic metabolism such as sucrose synthase, aldolase, enolase, pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and aldehyde dehydrogenase (ALDH) were assayed in two varieties of Echinochloa crus-galli, formosensis (tolerant) and praticola (intolerant). The initial and intervening enzymes of the pathway (sucrose synthase and aldolase) and enzymes in the last part of the pathway (PDC, ADH and ALDH) revealed similar changing patterns in activities during germination. This implies that each group of enzymes may be controlled by an identical regulatory mechanism. During anoxia, activities of all enzymes increased 1.5-30-fold in both varieties compared to their activities under aerobic conditions. Activities of sucrose synthase, enolase and ADH exhibited the same induction patterns under anoxia in formosensis and praticola. However, the activities of aldolase, ALDH and PDC were more strongly induced in formosensis under anoxia (1.2-2-fold) than in praticola. These enzymes were also assayed in F(3) families which varied in their anaerobic germinability. For PDC, activities under anoxia in anoxia-tolerant families were similar to those of an anoxia-intolerant family during the whole period although the family did not exhibit anaerobic germinability. This suggests that there is no correlation between PDC activity and anaerobic germinability. For ALDH, activities were more strongly induced under anoxia in anoxia-tolerant families than in anoxia-intolerant families, a trend also exhibited by the parents. This indicates that ALDH may play a role in detoxifying acetaldehyde formed through alcoholic fermentation during anaerobic germination.     

Physiological role of nitrate under anaerobic stress in Saccharum officinarum callus cells tolerant and sensitive to anoxia

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.     

Physiological roles for two periplasmic nitrate reductases in Rhodobacter sphaeroides 2.4.3 (ATCC 17025)

The metabolically versatile purple bacterium Rhodobacter sphaeroides 2.4.3 is a denitrifier whose genome contains two periplasmic nitrate reductase-encoding gene clusters. This work demonstrates nonredundant physiological roles for these two enzymes. One cluster is expressed aerobically and repressed under low oxygen while the second is maximally expressed under low oxygen. Insertional inactivation of the aerobically expressed nitrate reductase eliminated aerobic nitrate reduction, but cells of this strain could still respire nitrate anaerobically. In contrast, when the anaerobic nitrate reductase was absent, aerobic nitrate reduction was detectable, but anaerobic nitrate reduction was impaired. The aerobic nitrate reductase was expressed but not utilized in liquid culture but was utilized during growth on solid medium. Growth on a variety of carbon sources, with the exception of malate, the most oxidized substrate used, resulted in nitrite production on solid medium. This is consistent with a role for the aerobic nitrate reductase in redox homeostasis. These results show that one of the nitrate reductases is specific for respiration and denitrification while the other likely plays a role in redox homeostasis during aerobic growth.     

The rice coleoptile: an example of anaerobic nitrate assimilation

Nitrate present in rice caryopses can be reduced to ammonium and the ammonium subsequently assimilated by the coleoptile during anaerobic germination. All the enzymes of nitrate reduction and ammonia assimilation are present in the coleoptile. The supply of ¹⁵NO₃ confirms that the nitrate nitrogen is anaerobically incorporated into amino acids. Under anoxia, nitrate and nitrite reductase activities are increased in the coleoptile by exogenous nitrate. The importance of nitrate utilization during the anaerobic germination of rice caryopses is discussed.     

Enhancement of Denitrifying Activity in Cells of Roseobacter denitrificans Grown Aerobically in the Light

The effects of light on denitrifying activity during growthwere studied in an aerobic photosynthetic bacterium, Roseobacterdenitrificans (formerly Erythrobacter sp. OCh 114). When aerobicallygrown cells were transferred to anaerobic conditions in thepresence of nitrate, this bacterium exhibited denitrifying activity,with either succinate or malate serving as an electron donorin addition to endogenous substrates. The final product of denitrificationwas identified as nitrous oxide (N₂O), a result that confirmsthe presence of nitrate and nitrite reductases, but not N₂Oreductase, in these cells. Illumination during aerobic growthcaused a marked enhancement of the denitrifying activity. Theactivity increased with increasing intensity of light up to40 mW cm^–2 and was over 20 times that in dark-grown cells.Enhancement of denitrifying activity in illuminated cells wasclosely related to increases in levels of components that areinvolved in the denitrifying pathway, namely, nitrate and nitritereductases. Development of a denitrifying system under aerobicconditions and the enhancement of denitrifying ability by lightin Roseobacter denitrificans are unique characteristics, unlikethose of other known denitrifying bacteria. (Received October 29, 1990; Accepted January 17, 1991)     

Plant Growth and Survival under Strict Anaerobiosis

Anaerobic incubation of seedlings and rhizomes reveals interspecificdifferences in the ability of seedlings and rhizomes of higherplants to survive under prolonged and strict anaerobiosis. Rhizomesof several species were killed by an anaerobic incubation at22 °C for 7 d while others survived and showed normal shootextension on return to aerobic conditions. A third group ofspecies showed healthy and geotropically normal bud and shootextension while their rhizomes were in the oxygen free environment.A detailed comparison of bud and shoot growth rates was madewith Scirpus maritimus under aerobic and anaerobic conditionsover a 14 d period. Under similar conditions four species of grass seedlings weresubjected to anaerobic conditions for 2–8 d at 5°C,15 °C and 25 °C. Seedling mortality was highest at 25°C in all four grasses. Interspecific differences were evident.The wetland species were more tolerant of anoxia than the grassesfrom drier habitats.     

Anaerobic nitrate reductase (narGHJI) activity of Mycobacterium bovis BCG in vitro and its contribution to virulence in immunodeficient mice

Mycobacterium tuberculosis and Mycobacterium bovis cause tuberculosis, which is responsible for the deaths of more people each year than any other bacterial infectious disease. Disseminated disease with Mycobacterium bovis BCG, the only currently available vaccine against tuberculosis, occurs in immunocompetent and immunodeficient individuals. Although mycobacteria are obligate aerobes, they are thought to face an anaerobic environment during infection, notably inside abscesses and granulomas. The purpose of this study was to define a metabolic pathway that could allow mycobacteria to exist under these conditions. Recently, the complete genome of M. tuberculosis has been sequenced, and genes homologous to an anaerobic nitrate reductase (narGHJI), an enzyme allowing nitrate respiration when oxygen is absent, were found. Here, we show that the narGHJI cluster of M. tuberculosis is functional as it conferred anaerobic nitrate reductase activity to Mycobacterium smegmatis. A narG mutant of M. bovis BCG was generated by targeted gene deletion. The mutant lacked the ability to reduce nitrate under anaerobic conditions. Both mutant and M. bovis BCG wild type grew equally well under aerobic conditions in vitro. Histology of immunodeficient mice (SCID) infected with M. bovis BCG wild type revealed large granulomas teeming with acid-fast bacilli; all mice showed signs of clinical disease after 50 days and succumbed after 80 days. In contrast, mice infected with the mutant had smaller granulomas containing fewer bacteria; these mice showed no signs of clinical disease after more than 200 days. Thus, it seems that nitrate respiration contributes significantly to virulence of M. bovis BCG in immunodeficient SCID mice.     

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     

A Denitrifying Activity in an Aerobic Photosynthetic Bacterium, Erythrobacter sp. Strain OCh 114

An aerobic photosynthetic bacterium, Erythrobacter sp. strainOCh 114, was capable of growth under anaerobic conditions inthe dark with nitrate as a terminal electron acceptor. The optimalnitrate concentration was about 6 mM for anaerobic growth, althougha wide range of concentrations from 1 to 400 mM were effective.A large amount of N₂O gas was released during this anaerobicgrowth, indicating a denitrifying activity in this bacterium.Light had no stimulating or inhibiting effect on the rates ofanaerobic growth and gas release. The enzymes responsible forthe denitrifying activity, dissimilatory nitrate and nitritereductases, were present in aerobically grown cells. (Received February 19, 1988; Accepted May 16, 1988)     

Mitochondrial Ultrastructure of Three Hygrophyte Species at Anoxia and in Anoxic Glucose-Supplemented Medium

Mitochondrial ultrastructure of excised roots of Alisma plantago-aqisaticaL., Lycopus europaeus L and Glyceria fluitans L. were electron-microscopicallystudied at anoxia and in anoxic glucose- supplemented mediumin order to find Out if the roots of the three hygrophytes growingon water-logged anaerobic soils have an increased resistanceto anoxia. Irreversible destruction of mitochondrial membranes and othersubcellular structures was shown to occur in the above plants'roots after 24 h at anoxia or in anoxic glucose-supplementedmedium. Only in roots of Glyceria had exogenous glucose a protectiveaction, yet in this case, too, a 48 h anacrobic exposure resultedin a deep-going degradation of cell ultrastructure. It is concluded that though the plants in question grow on soilsdevoid of O₂ their roots avoid anaerobiosis through translocationof O₂ from aerated parts, which appears to explain why theseplants have not developed a biochemical mechanism of adaptationto anaerobiosis in the process of evolution. Key words: Anaerobiosis, hygrophytes, mitochondrial ultrastructure