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.     

Effect of Localized Anoxia on Phaseolus vulgaris L. Root Growth

Heterogeneity within the root environment results in differentialgrowth within root systems. The response of five Phaseolus vulgarisL. cultivars to non-uniform root aeration was evaluated. Threetreatments were applied to a split root system for a periodof 72 h. Treatments consisted of an aerated control, a non-aeratedcontrol (both halves non-aerated, using N₂). and localized anoxia(one-half the root system aerated and the remaining half subjectedto N₂). Shoot and root growth were reduced in the anoxic controlbut not in the aerated control or localized anoxia treatment. Root growth was greatest in the aerated portion of the localizedanoxia treatment for all genotypes. Contributions of the rootcomponents to the compensatory responses differed dependingon the plant cultivar examined. The growth of branched and lateralroots present before the treatment period increased by 65% inline 31908. A 50% increase in the growth of lateral roots whichemerged during the treatment period occurred in another line(Swan Valley). Other genotypes responded in an intermediatemanner. These observations indicate differences in cultivarresponses to localized soil stress. Key words: Phaseolus vulgaris L., Anoxia, Root growth     

Mitochondrial ultrastructure in roots of mesophyte and hydrophyte at anoxia and after glucose feeding

Summary In order to investigate the nature of the tolerance of mesophytes and hydrophytes to root anaerobiosis, changes in the mitochondrial ultrastructure of excised roots (with and without added glucose under anoxia) were studied in plants from two ecologically opposite types-pumpkin and rice.A 12-hour exposure to anoxia led to mitochondrial degradation in roots of adult rice and pumpkin plants. The addition of glucose preserved cell ultrastructure for up to 72–96 hours. During this period mitochondrial ultrastructure changed. In rice roots this primarily involved an increased number of cristae and a change in their arrangement into parallel rows. Cells of pumpkin roots displayed long mitochondria (up to 55 m) of different profiles which fused to form a complex mitochondrial network that was in close association with parts of the endoplasmic reticulum carrying a large number of ribosomes. This may be regarded as an adaptive development that facilitates the transport of glycolytic energy along mitochondrial membranes to the sites of protein synthesis.It is concluded that root cells of a hydrophyte are not more tolerant to anoxia than mesophyte. Thus, the ability of hydrophytes to grow on anaerobic soils should be attributed not so much to peculiar features of the roots' metabolism but to the ability of these plants to perform an easy transport of O₂ from leaves to roots. With respect to mesophytes it is stressed that the supply of assimilates is important for the resistance of roots to soil anaerobiosis.     

Soil O2 and CO2 Effects on Apparent Cell Viability for Roots of Desert Succulents

Roots of desert succulents occupy the upper layers of porous,well-aerated soils. However, roots of Agave deserti, Ferocactusacanthodes, and Opuntia ficus-indica all tolerated many daysof soil anoxia; 0% O₂ in the soil gas phase for 30 d reducedthe fraction of cells taking up the vital stain neutral red,an average of only 18% for the cortex and 6% for parenchymacells within the stele of perennial established roots. Ephemeralrain roots, induced by watering as branches on the establishedroots, were more susceptible to 0% O₂ in the soil gas phase;19 d abolished stain uptake for cortical cells and 32 d forstelar parenchyma cells. Soil CO₂ levels above the 0.1% observedin the root zone in the field rapidly reduced uptake of neutralred; the fraction of cortical cells taking up the stain decreased30% in 10 h at 0.5% CO₂ and was abolished in 9 h at 2% and 7h at 10% CO₂ averaged for the three species. Rain roots weresomewhat more susceptible than established roots to elevatedsoil CO₂ levels, and stelar parenchyma cells were much lesssusceptible than were cortical cells. When uptake of the vitalstain was abolished by elevated soil CO₂, no anatomical evidenceof cellular damage was observed. For A. deserti exposed to 2%CO₂, the pH of macerated root tissue decreased about 0.35 pHunit over 10 h; CO₂ apparently entered the cells, lowered theintracellular and/or cell wall pH, and prevented the accumulationof neutral red. Elevated soil CO₂ also inhibits root respirationfor the three desert succulents considered. Hence, the restrictionof such species to porous soils may reflect the relatively rapidinhibiting effects of elevated soil CO₂ levels rather than arequirement for high soil O₂ levels, consistent with the observationthat desert soils tend to have low gas-phase CO₂ levels near0.1% compared with 1% or more in the root zone of non-desertspecies. Key words: Agave deserti, Ferocactus acanthodes, neutral red, Opuntia ficus-indica, pH     

Sugar Accumulation in Barley and Rice Grown in Solutions with low Concentrations of Oxygen

Barley and rice, at the early tillering stage, were grown inaerated nutrient solutions (> 7 mg O₂ l^–1) and transferredto solutions of low O₂ concentrations (< 0.5 mg l ^–1). For barley, low O₂ concentrations during the first 5 days severelyinhibited growth of seminal roots had less effect on nodal roots,and did not reduce shoot growth. Longer exposure to low O₂ concentrationsreduced shoot as well as root growth. Sugar concentrations inroots and shoots increased within 7 h after transfer of plantsto low O₂ concentrations. After 5 days at low O₂ concentrationssugar concentrations were very high in fast growing nodal rootsand in shoots, as well as in the slower growing seminal roots. In rice, low O₂ concentrations increased sugar levels of rootsduring summer, but not during winter. In summer, the highersugar levels at low O₂ concentrations persisted throughout adiurnal cycle. In root apices, sugar concentrations were increasedby low O₂ concentrations, even though the experiment was donein winter and the bulk of the root system showed no differencein sugar levels. The data indicate that sugar accumulation, at low O₂ concentrations,is caused by reduced growth and also that even apices of rootsgrown at low O₂ concentrations have sufficient substrates forrespiration. Hordeum vulgare L, barley, Oryza sativa L, rice, sugar accumulation, oxygen concentration     

Regulation of Pyruvate Decarboxylase In Vitro and In Vivo

Results presented in this paper strongly support the view thatregulation of the key enzyme of alcoholic fermentation, pyruvatedecarboxylase (PDC), is achieved in a number of ways, all associatedwith possible lowering of the cytoplasmic pH during anoxia.These mechanisms include not only the well-known acid pH optimumof PDC, but also long-term, reversible changes in characteristicsof the enzyme established both in vitro and in vivo. Following transfer of desalted extracts from pH 6.0 to 7.4,maximal activity of PDC was decreased, while there was a considerableincrease in the lag before maximal activity was reached. Similarchanges in enzyme characteristics were observed when wheat (Triticumaestivum L. cv. Gamenya) roots and rice (Oryza sativa L. cv.Calrose) coleoptiles were transferred from anoxic to aerobicsolutions, provided PDC was assayed within 10 min of the startof maceration. All of the above changes were usually readilyreversible when extracts were returned to pH 6.0, or when plantswere returned to anoxic solutions. Additional regulation of PDC would be achieved by the S_0.5 forpyruvate which is 0.75 mol m^–3 at pH 6.0, 1.0 mol m^–3at pH 6.8, and 2.5 mol m^–3 at pH 7.4; the latter is wellabove estimates for pyruvate concentrations in the cytoplasmof aerated tissues. We assess that the combined effects of the acid pH optimum,the high S_0.5 at pH 7.4 and the long-term decreases in activityobserved during incubation at pH 7.4 would reduce PDC activityin aerobic cells to at most 7% of the activity in anoxic cells.Possible additional controls for the pathway of alcoholic fermentationare briefly considered. Key words: PDC, regulation, anoxia     

The Response of Phaseolus vulgaris L. to Root-zone Anaerobiosis, Waterlogging and High Sodium Chloride

Phaseolus vulgaris L. grown at a range of external concentrationsof NaCl (0 to 80 mM) responded differently to gaseous anaerobiosis(N₂ gas) in nutrient solution or stagnant waterlogging of theroot-zone. With similar patterns of distribution of Na⁺ andCl^- occurring in the plants with comparable NaCl treatments,and similar final concentrations of Na⁺ and Cl^- in plants grownunder both root-zone conditions, rates of uptake of Na⁺ andCl^- were much higher in plants with the stagnant waterloggedrootzones. After 72 h stagnant waterlogging, plant tops fromplants grown at 40 mM NaCl contained 1.42 per cent Na⁺ and 3.44per cent Cl^- (d. wt basis) while after 9 days exposure to NaClwith gaseous anaerobiosis, leaf tissue contained 1.49 per centNa⁺ and 4.28 per cen Cl^- (d. wt basis). Plants exposed to 40mM external NaCl were severely damaged within 72 h when grownwith stagnant waterlogged root-zones; those grown with N₂ anaerobiosiscontinued growth and development over the 9 d period. Plantsgrown in nutrient solution showed changes in distribution andconcentration of Na⁺ and Cl^- when oxygen concentration was reducedbelow 21 per cent O₂ (full aeration). Phaseolus vulgaris. L., bean, mineral salt distribution, anaerobiosis, salinity, waterlogging     

Changes in Nitrogenase Activity and Nodule Diffusion Resistance of Subterranean Clover in Response to pO2

Diffusion resistance to oxygen within nodules was calculatedusing the respiratory quotient (RQ) of nodules from intact plantsof subterranean clover (Trifolium subterraneum L.) cv. SeatonPark nodulated by Rhizobiun trifolii WU95. From 21 to 52% O₂,the RQ remained between 0.94 and 1.04, whereas at 10% O₂, theRQ was 1.65. When nodulated roots of intact plants were exposedto sub-ambient pO₂ in a continuous flow-through system, respirationdeclined immediately, followed by a partial recovery within30 min. The magnitude of the final respiration rate was dependentupon the pO₂ in the gas stream. Initial rates of respirationwere re-established after 24 h at sub-ambient pO₂ as a resultof changes in the resistance of the variable barrier to oxygendiffusion within the nodules. Nitrogenase activity also decreasedlinearly with decreasing pO₂ in the gas stream, but partialrecovery occurred after 24 h incubation at sub-ambient pO₂.Maximum rates of nitrogenase activity occurred at rhizosphereoxygen concentrations between 21% and 36% O₂. Resistance tothe diffusion of oxygen within the nodules increased at supra-ambientpO₂ and at oxygen concentrations above 36% O₂, resulted in adecrease in both nitrogenase activity and nodulated root respiration.The diffusion resistance of nodules to oxygen increased rapidlyin the presence of either supra-ambient pO₂ or saturating pC₂H₂.Reductions in nodule diffusion resistance either during recoveryfrom exposure to 10% acetylene or to sub-ambient pO₂ occurredmore slowly. It is concluded that subterranean clover is welladapted for maximum nitrogen fixation at ambient pO₂. Key words: Nitrogenase activity, oxygen, subterranean clover, diffusion resistance     

A microsensor for direct measurement of O2 partial pressure within plant tissues3

Widespread use of O₂ microsensors to measure O₂ partial pressure(pO₂) in plant tissues has been limited in part because of difficultyof construction and other technical obstacles. By modifyingpublished techniques, an O₂ microsensor was constructed thatcombined the advantages of Clark-type microsensors with lesscomplicated construction techniques. The specifications andsome performance characteristics of the microsensor are: tipdiameter 1–5 µm; sensitivity 7.5–25 pA kPa^–1;negligible stir-induced current; response time 540 ms. The microsensorcan be used in air or solution, and each sensor can be usedfor several experiments. The sensitivity of the microsensorwas unchanged during measurements over the physiological rangeof pO₂ in intact, growing maize (Zea mays L.) primary roots,and was thus unaffected by cellular fluids and turgor pressure.Use of the microsensor to compare pO₂ profiles in vermiculite-and solution-grown roots is described. The O₂ microsensor couldfind application in studies in which information on tissue pO₂is needed, but for which conventional O₂ probes are too large. Key words: Oxygen microsensor, Zea mays L., roots, oxygen partial pressure     

Effect of Oxygen on Photosynthetic 14CO2 Fixation in Chroomonas sp. (Cryptophyta) II. Effects of Inhibitors, Uncouplers and an Artificial Electron Mediator on the Inhibition of 14CO2 Fixation by Anaerobiosis

In "air-grown" Chroomonas sp. cells, low concentrations of DCMU(less than 0.1 µM) could prevent the inhibition of ¹⁴CO₂fixation by anaerobiosis under light-saturating conditions (morethan 40 W.m^–2), with phenazine methosulfate showing asimilar effect. Antimycin A, carbonyl cyanide m-chlorophenylhydrazone(CCCP), and N,N'-dicyclohexylcarbodiimide strongly inhibitedanaerobic photosynthesis at concentrations which did not significantlyinhibit the rate under 2% O₂ at high light intensity (200 W.m^–2),although 0.2 µM CCCP stimulated the rate under 2% O₂ tosome extent. On the other hand, KCN inhibited the rate muchmore strongly under 2% O₂ than N₂, although it inhibited therate very strongly at concentrations above 5 µM both underN₂ and 2% O₂. These results suggest that the inhibition of photosynthetic¹⁴CO₂ fixation by anaerobiosis in this alga result from ATPdeficiency caused by over-reduction of electron carriers ofthe cyclic electron flow and that oxygen can prevent the over-reduction.Cyclic electron flow seems to be necessary to provide additionalATP for CO₂ reduction under anaerobic conditions, although itseems to be less necessary under aerobic conditions. (Received July 21, 1983; Accepted January 23, 1984)     

Studies on the change of the respiratory system in roots in relation to the growth of plants II. Activity of iron-containing oxidases in roots of cereal crops with the aging of plants

Distribution of iron-containing oxidases in aging nodal rootsof rice and wheat was studied. Activities of cytochrome c oxidase(1.9.3.1 [EC] , cytochrome c : O₂ oxidoreductase), catalase (1.11.1.6 [EC] ,H₂O₂: H₂O₂ oxidoreductase) and peroxidase (1.11.1.7 [EC] , donor:H₂O₂ oxidoreductase) in wheat roots were comparatively higherthan were those in rice roots at corresponding stages. Cytochromec oxidase in roots remained active throughout the lives of therice and wheat crops. In rice roots, catalase seemed to playa distinct role around the panicle formation stage. Decay ofcatalase activity took place earlier than did that of peroxidaseand cytochrome c oxidase activities. In wheat roots similarenzyme activity changes were not observed. Data may suggestthat the high activity of iron containing oxidases at the panicleformation stage (I) may be chiefly due to catalase activityin rice roots. ¹Paper presented at the 14th Annual Meeting of the Society ofthe Science of Soil and Manure, Japan (1968). (Received November 21, 1968; )     

Supply and partitioning of assimilates to roots of Medicago sativa L. and Lotus corniculatus L. under anoxia

Abstract A current explanation of the mechanism of flooding injury to roots suggests that oxygen deficiency depresses the supply of respirable carbohydrates sufficiently to inhibit fermentation. However, even though it has been shown that phloem transport of assimilate is sharply reduced to anaerobic roots, inhibition of assimilate metabolism has also been suggested to be an important factor. This study examines these hypotheses by relating assimilate supply and metabolic activity in anoxic roots of alfalfa (Medicago sativa L.), a flood-intolerant species, and birdsfoot trefoil (Lotus corniculatus L.), a flood-tolerant plant. Roots were made anoxic (severe O₂ deficiency) for 2, 4 or 6 d and shoots were labelled with ¹⁴CO₂. Assimilate transport to the roots and metabolism to structural components were significantly decreased in both species in response to anoxia. Trefoil exhibited significantly greater ¹⁴C incorporation into the residue fraction at 4 d anoxia than did alfalfa, and this was consistent with the greater flooding tolerance of trefoil. When assimilate supply to O₂-deficient roots was decreased by shoot shading, shoot fresh weight was reduced by both anoxia and light treatments. Root-soluble sugars were significantly decreased by shading but were greatly increased in response to anoxia. Root starch concentration also increased under anoxia. Root K⁺ concentration was reduced by anoxia only. The energy status (ATP/ADP) of roots was significantly decreased by shading; however, anoxia reduced the energy status only in unshaded plants. The data indicate that carbohydrate supply to anaerobic roots does not appear to be a limiting factor in the metabolic response of alfalfa roots. Alternatively, metabolism of assimilate in anoxic roots may be an important determinant of survival.     

Effects of a Range of O2 Concentrations on Porosity of Barley Roots and on their Sugar and Protein Concentrations

Barley was grown at a range of oxygen concentrations (0.5–9mg l^–1), in nutrient solutions. Growth of both shootsand seminal roots was restricted by O₂ concentrations lowerthan 2–3 mg l^–1) but nodal root growth was not. Root porosities were increased even at those O₂ concentrationswhich did not restrict growth, and were inversely proportionalto the protein levels of the roots. Sugar concentrations increasedappreciably only at those O₂ concentrations which also restrictedgrowth. Hordeum vulgare L., barley, root porosity, sugar, protein, oxygen concentration     

Wavelength options for monitoring leghaemoglobin oxygenation gradients in intact legume root nodules

Nodule oximetry, based on spectrophotometric measurements ofleghaemoglobin (Lb) oxygenation in intact nodules, has providednumerous insights into legume nodule physiology. Fractionaloxygenation of Lb (FOL) has been monitored at various wavelengths,but comparisons among wavelengths have not been published previously.Changes in transmittance were monitored simultaneously at 660nm and either 560 or 580 nm as FOL was manipulated by changingthe O₂ concentration around nodules of Medicago sativa L. orLotus comiculatus L. Video microscopy at 580 nm was used togenerate two-dimensional maps of FOL gradients in intact nodules.In general, all three wavelengths gave similar results. Smalldiscrepancies between 660 and 580 nm, sometimes seen in noduleswith high O₂ permeability, may indicate interference by theferric Lb peak at 625 nm. A slightly longer wavelength, forexample 670 nm, might be preferable. No significant discrepanciesamong wavelengths were seen in nodules whose O₂ permeabilityhad been reduced by a 48 h exposure to 10 mM nitrate. Minorgradients in FOL were seen in nodules of M. sativa and Trifoliumrepens L. under air and steeper gradients could be induced byvarious treatments. The existence of these gradients indicatesat least some restriction of longrange O₂ diffusion within theinfected zone. The FOL maps do not have enough spatial resolutionto measure gradients within infected cells. Key words: Leghaernoglobin oxygenation, nodules, spectrophotometry, nodule oximetry     

Relationship between Coleoptile Elongation and Alcoholic Fermentation in Rice Exposed to Anoxia. II. Cultivar Differences

The relationship between coleoptile elongation and survivalvs. alcoholic fermentation of rice under anoxia is examinedusing eight cultivars differing in submergence tolerance. Anoxiawas imposed on either 1 or 4 d aerated seeds either by N₂ flushingsubmerged tissues or by incubating tissues in stagnant deoxygenatedagar at 0·1% w/v; the latter simulated the stagnant conditionsof waterlogged soil. Two cultivars that were most tolerant tosubmergence also had the greatest tolerance to anoxia, whilea submergence intolerant cultivar was also intolerant to anoxia. Coleoptile growth under anoxia was related to rates of ethanolsynthesis (R_E), however differences between growth during anoxiaand survival after anoxia indicated that post-anoxic injurymay also be important in rice seeds exposed to anoxia. The correlationbetween coleoptile growth and R_E measured on a tissue basisusing intact seeds was r² = 0·67 among six varietiesover 0-3 d anoxia. This correlation improved to about r² = 0·85using R_E of (embryos plus coleoptiles) over 0-3 d, or coleoptilesat 3 d after anoxia. Coleoptile growth of individual seeds wasusually poorly correlated to R_E in these cultivars at 2-3 dafter anoxia. When coleoptiles of similar lengths were obtainedfrom different cultivars using 4 d aerated seeds, there weredifferences in R_E and coleoptile growth which were related tocoleoptile growth during 3 or 5 d anoxia, either on a tissue(r² = 0·85) or a fresh weight basis (r² = 0·70-0·97respectively). Results are discussed in relation to factorswhich may limit ethanol synthesis in rice exposed to anoxiaand the importance of growth to the survival of seeds and matureplants during submergence in the natural environment.Copyright1994, 1999 Academic Press Anoxia, ethanol, alcoholic fermentation, Oryza sativa L., rice, submergence     

Demonstration of plant adaptation syndrome in plants and possible molecular mechanisms of its realization under conditions of anaerobic stress

Electron-microscopic examination of mitochondrial membrane ultrastructure in detached leaves of four-day-old wheat (Triticum aestivum L.) seedlings incubated under conditions of strict anoxia in the presence of exogenous glucose and cycloheximide or in the absence of these compounds revealed a paradoxical phenomenon: in the absence of exogenous glucose and cycloheximide, even a short-term (15–30 min) anaerobiosis resulted in a pathological destruction of mitochondria (swelling and the loss of cristae); however, a longer uninterrupted anaerobiosis (3–4 h) did not induce further mitochondria degradation but, in contrast, resulted in the recovery of their initial ultrastructure. Irreversible mitochondria degradation was observed only during subsequent still longer leaf anaerobic treatment (24–48 h). When, under conditions of strict anoxia, leaves were fed with glucose to stimulate glycolysis and ethanolic fermentation, we did not observe any signs of early destruction of mitochondrial ultrastructure and their swelling. Blockage of anaerobic protein synthesis with cycloheximide resulted in early destruction and subsequent irreversible degradation of mitochondria without any indications of their structural recovery. Based on the results of the experiments, we concluded that cell energy metabolism controlled byboth the presence of utilizable carbohydrates and also by the induction of anaerobic protein synthesis played a key role during early mitochondria destruction under extreme conditions of anaerobic stress, their subsequent recovery, and irreversible degradation during continuous long-term strict anoxia.     

Glucosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein O-GlcNAc and increased mitochondrial Bcl-2

We have previously reported that glucosamine protected neonatal rat ventricular myocytes against ischemia-reperfusion (I/R) injury, and this was associated with an increase in protein O-linked-N-acetylglucosamine (O-GlcNAc) levels. However, the protective effect of glucosamine could be mediated via pathways other that O-GlcNAc formation; thus the initial goal of the present study was to determine whether increasing O-GlcNAc transferase (OGT) expression, which catalyzes the formation of O-GlcNAc, had a protective effect similar to that of glucosamine. To better understand the potential mechanism underlying O-GlcNAc-mediated cytoprotection, we examined whether increased O-GlcNAc levels altered the expression and translocation of members of the Bcl-2 protein family. Both glucosamine (5 mM) and OGT overexpression increased basal and I/R-induced O-GlcNAc levels, significantly decreased cellular injury, and attenuated loss of cytochrome c. Both interventions also attenuated the loss of mitochondrial membrane potential induced by H₂O₂ and were also associated with an increase in mitochondrial Bcl-2 levels but had no effect on Bad or Bax levels. Compared with glucosamine and OGT overexpression, NButGT (100 µM), an inhibitor of O-GlcNAcase, was less protective against I/R and H₂O₂ and did not affect Bcl-2 expression, despite a 5- to 10-fold greater increase in overall O-GlcNAc levels. Decreased OGT expression resulted in lower basal O-GlcNAc levels, prevented the I/R-induced increase in O-GlcNAc and mitochondrial Bcl-2, and increased cellular injury. These results demonstrate that the protective effects of glucosamine are mediated via increased formation of O-GlcNAc and suggest that this is due, in part, to enhanced mitochondrial Bcl-2 translocation. mitochondria; apoptosis; necrosis, O-linked-N-acetylglucosamine     

Anaerobiosis-induced differentiation of Acanthamoeba castellanii

Acanthamoeba castellanii is a free living amoeba ubiquitous in soil and also commonly found in aquatic environments. In waterlogged soils, anoxia is quickly established as the dissolved oxygen is consumed by the organisms present. We were interested in the effects of anoxic conditions upon this organism. Batch cultures degassed with N₂ during mid-exponential growth, induced encystation within 12 h of anoxia, and mature cysts were formed within 2–3 days. Excystation (99%) was achieved by subsequent aeration of these cultures after 3–6 days. Anoxia-induced cysts, maintained in anoxic conditions for up to four months, remained viable. Difference spectra, during anaerobiosis, revealed that cytochromes were not lost, suggesting that the organism retains its respiratory components. The growth rate of trophozoites, grown in a chemostat, was dependent on the concentration of O₂ in the head space and glucose uptake increased at lower dissolved O₂ tensions. The results obtained suggest that A. castellanii has a complex adaptive strategy enabling it to cope with microaerobic and anoxic conditions which may be experienced in the environment.     

The Response of Sugarcane (Saccharum officinarum) Cultured Cells to Anoxia and the Selection of a Tolerant Cell Line

The effect of anoxia on the sugarcane (Saccharum officinarum L.) cultured cells was studied in order to elaborate a technique for in vitro selection of cell lines, which would be tolerant to anaerobic stress. Inhibitory and lethal doses of anaerobic incubation were established from the state of the mitochondrial ultrastructure during the anaerobic incubation of cells either with or without exogenous glucose, as well as from the pattern of the post-anaerobic callus growth. An intact state of the mitochondrial ultrastructure and the viability of some cells in the presence of 3% glucose were shown to be maintained for at least 14 days of anaerobic incubation, while the index of post-anaerobic growth decreased by almost 50% even after 72-hour-long anaerobiosis. In the absence of exogenous glucose, a marked destruction of mitochondria and a twofold decrease in the callus growth index were observed as early as after six-hour-long anaerobic stress. A 48-hour-long incubation under these conditions resulted in the maintenance of the intact ultrastructure only in 7–10% of cells, while a 96-hour-long anaerobiosis brought about the complete degradation of the subcellular structure and cell death. A 48-hour-long anaerobiosis without exogenous glucose was chosen for selecting the anoxia-tolerant cell lines. After three cycles of selection, the anoxia tolerance of the selected cell line exceeded the respective index of the initial callus several-fold. In selected line, about 50% of cells retained viability and could resume growth even after 96-hour-long anaerobic incubation. The experimental results obtained were used to determine the possible causes of the heterogeneity of callus cells as regards their anoxia resistance.