Superoxide Dismutase as an Anaerobic Polypeptide : A Key Factor in Recovery from Oxygen Deprivation in Iris pseudacorus?

The perennating organ, the rhizome, was chosen for examination of response to anoxia in the species Iris pseudacorus L., Iris germanica L. var Quechei, and Glyceria maxima (Hartm.) Holmberg. These monocots are known to differ in their tolerance of anoxia. Intact rhizomes were subjected to periods of prolonged anoxia of up to 28 days and superoxide dismutase (SOD) activity was determined in a 48 hour postanoxic recovery phase. Tests were performed to ensure the accuracy of the measured enzyme activities. In the most anoxia tolerant species, I. pseudacorus, SOD activity rose continuously during the period of imposed anoxia, and levels were maintained in the postanoxic recovery phases: 28 days brought about a 13-fold increase to 1576 U SOD per milligram protein. Small increases were found in the less anoxia tolerant I. germanica during anoxic/postanoxic phases, while a drop in activity was recorded in the least anoxia tolerant G. maxima. However, initial levels in G. maxima were more than twice as high as in the other two species. Experiments applying cycloheximide to anoxic rhizome slices of I. pseudacorus inhibited the increase in SOD activity. This indicates that SOD is, paradoxically, induced under anoxia and we suggest that in this species SOD is one of the enzymes identified as anaerobic polypeptides. The significance of the induction of an `oxygen-protecting' enzyme during complete oxygen deprivation is discussed with regard to a possible critical role during recovery from anoxic stress.     

Fermentation Rates and Ethanol Accumulation in Relation to Flooding Tolerance in Rhizomes of Monocotyledonous Species

Under anoxia an accumulation of ethanol took place in all species.Lactic fermentation was found to be of less importance in everycase. The amount of ethanol accumulated depended on the abilityof the rhizome to eliminate it from its tissues. Despite greatvariations in fermentative capacity as seen in the ADH measurementsthe wetland species did not accumulate ethanol to concentrationsgreater than 30 µmol g^–1 fresh weight. This representeda plateau above which the ethanol concentration did not riseeven with continued anaerobic incubation. No such plateau ofethanol accumulation was found in the dryland species Iris germanicawhich accumulated ethanol steadily reaching concentrations of70 µmol g^–1 fresh weight. If ethanol is toxic to higher plant tissues then the steadystate condition of low ethanol accumulation found in wetlandspecies will minimize this danger. Key words: Ethanol, Flooding, Rhizomes     

Comparison of the effect of natural and experimental anoxia on carbohydrate and energy metabolism in Iris pseudacorus rhizomes

Carbohydrate utilization in Iris pseudacorus rhizomes was investigated under natural conditions in the field for a period of one year. The area of study was under flood from mid-December to end of May and wet for the rest of the period of study. Rhizome samples were collected in the wild twice monthly. in order to determine glucose content and amount of reducing and non-reducing sugars in the rhizome core material. For comparison with experimental anoxia, rhizomes were washed and kept under strict anoxia in the dark at +20°C. Carbohydrates were measured in groups as above. The amount of ethanol and the levels, of ATP. ADP and AMP were also measured and adenylate energy charge computed.
Glucose content of the rhizomes was 10 mg g⁻¹ fresh weight in the autumn and decreased during winter to reach ca 5 mg g⁻¹ fresh weight in the spring. Total reducing sugars increased in w inter while the amount of non-reducing sugars increased during early autumn and decreased during winter and spring. Not all non-reducing sugar stores had been used up in the spring when growth was resumed. Under experimental anoxia adenylate energy charge remained relatively high for 30 days (near 0.6 or over) and then dropped quickly to bekm 0.3. indicating death of the tissues. After 30 days under anoxia the amount of ethanol still increased and that of total reducing sugars decreased. The level of non-reducing sugars decreased from 0 to 15 days of anoxia before an increase took place durine the next 20 days.     

Long-term anoxia tolerance in leaves of Acorus calamus L. and Iris pseudacorus L

Mature green leaves of Acorus calamus and Iris pseudacorus have been shown to survive at least 28 d of total anoxia in the dark during the growing season, increasing up to 75 d and 60 d in overwintering leaves in A. calamus and I. pseudacorus, respectively. During the period of anaerobic incubation the glycolytic rate is reduced, carbohydrate reserves are conserved and ethanol levels in the tissues reached an equilibrium. Prolonged anoxia significantly suppressed leaf capacity for respiration and photosynthesis. After 28 d of anoxia, respiratory capacity was reduced in A. calamus and I. pseudacorus by 80% and 90%, respectively. The photosynthetic capacity of leaves decreased by 83% in A. calamus and by 97% in I. pseudacorus after 28 d of anoxia. This reduction in photosynthetic capacity was accompanied by a modification of the chlorophyll fluorescence pattern indicating damage to the PSII reaction centre and subsequent electron transport. Chlorophyll content was only slightly reduced after 28 d under anoxia and darkness in A. calamus, whereas there was a 50% reduction in I. pseudacorus. On return to air A. calamus leaves that endured 28 d of anoxia recovered full photosynthetic activity within 7 d while those of I. pseudacorus had a lag phase of 3-10 d. This well-developed ability to endure prolonged periods of oxygen deprivation in both these species is associated with a down-regulation in metabolic activity in response to the imposition of anaerobiosis. It is suggested that when leaf damage eventually does take place in these species after protracted oxygen deprivation, it is anoxic rather than post-anoxic stress that is responsible.     

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     

Comparison of carbohydrate utilization and energy charge in the yellow flag iris (Iris pseudacorus) and garden iris (Iris germanica) under anoxia

Carbohydrate and energy metabolism of the flooding- and anoxia-tolerant Iris pseudacorus and the intolerant Iris germanica rhizomes were investigated under experimental anoxic conditions. Rhizomes of I. pseudacorus and I. Germanica were incubated in the absence of oxygen from 0 to 60 and 16 days, respectively. Amounts of glucose, total reducing sugars and non-reducing sugars (starch, fructan and oligosaccharides) in the rhizomes were measured. Ethanol concentration and adenylate energy charge were determined enzymatically. Glucose content of I. pseudacorus rhizomes decreased gradually during the first 30 days under anoxia and then increased at the same time as adenylate energy charge values started to decline. In I. germanica rhizomes the changes were more dramatic and the time scale was much shorter than in I. pseudacorus but the changes were similar. Non-reducing sugar content of I. pseudacorus rhizomes decreased rapidly during the first 15 days under oxygen deprivation and then increased again, to near starting levels at 35 days. In I. germanica the amount of non-reducing sugars decreased gradually during the anoxic incubation. Under aerobic control conditions, adenylate energy charge (AEC) of I. pseudacorus and I. germanica rhizome tissue was 0.87±0.01 and 0.81±0.01, respectively. In I. pseudacorus AEC remained high until 30 days under anoxia. In contrast, the energy charge of I. germanica rhizome tissue remained above 0.6 for 4 days only. Large amounts of ethanol were found in anoxic rhizome tissues of I. pseudacorus (up to 0.21 M ) and I. germanica (0.06 M ) after 45 days and 8 days, respectively. The results are discussed in relation to flooding tolerance of these species.     

The calanoid copepod Acartia italica Steuer, phenomenon in the small saline Lake Rogoznica (Eastern Adriatic coast)

Lake Rogoznica, near ibenik, Croatia, is a salt coastal lakewith a maximum depth of about 15 m. During most of the year,this small, naturally eutrophied lake is highly stratified,with hypoxia/anoxia occurring in the bottom layer. Total anoxia,with the presence of hydrogen sulphide, was recorded on September27, 1997. At that moment, massive mortality of all planktonicand benthic organisms was observed. After the anoxia, Acartiaitalica, the only planktonic copepod species of the lake, re-establishedquickly. Before anoxia, the population dynamics of the A.italicapopulation depended primarily on predator/prey relationships.However, in the post-anoxic period, nutrient–phytoplankton–copepodrelationships became more important, as there was no longerany predation pressure. From all accounts, it would appear thatonly A.italica is adapted to the extreme conditions which appearfrom time to time in the lake, and that this species has animportant role in the functioning of the lake ecosystem. Acartiaitalica was very important for the gradual normalization oflife in the lake after total anoxia.     

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     

Survival of Fungi in Hyperbaric Oxygen in Relation to Changes in Catalase Activity

Survival of replacement cultures of Mucor species in oxygenat 10 atm was markedly affected by the carbon source; culturesreplaced on glucose media survived for a much shorter periodthan cultures on water while 1 per cent ethanol increased thesurvival time. This effect of carbon source on survival wascorrelated with changes in catalase activity; in the presenceof glucose and other carbohydrates, catalase activity decreasedwhereas on 1 per cent ethanol a large increase in catalase activityoccurred which was maintained for several days. An increasein catalase activity also occurred on methanol, pyruvate, glycerol,and formate. Induced changes in peroxidase activity were similarto those shown by catalase but the activity of three other enzymesdecreased in hyperbaric oxygen on all media.     

The Role of Shikimic Acid in Waterlogged Roots and Rhizomes of Iris pseudacorus L.

Levels of shikimic acid in the roots and rhizomes of Iris pseudacorusgrowing under natural conditions were measured at monthly intervalsfor a period of one year. Seasonal fluctuations in the shikimatecontent suggest that the high levels during winter floodingand lower levels during the summer period of low water tableare related to a particular flood-tolerance metabolism in theroots and rhizomes. The suggested pathway, involving the condensationof phosphoenolpyruvate and crythrose 4-phosphato (both formedduring anaerobic carbohydrate breakdown) to 3-deoxy-D-arabino-heptulosouicacid 7-phosphate and the subsequent synthesis of shikimie acid,appears to function as a physiological adaptation to floodingin rhizomatous species such as I. pseuducorus. Other pathwaysof anaerobic respiration in waterlogged roots are discussedin relation to the proposed scheme in Iris.     

Oxygen deprivation stress in a changing environment

Past research into flooding tolerance and oxygen shortages inplants has been motivated largely by cultivation problems ofarable crops. Unfortunately, such species are unsuitable forinvestigating the physiological and biochemical basis of anoxia-toleranceas selection has reduced any tolerance of anaerobiosis and anaerobicsoil conditions that their wild ancestors might have possessed.Restoration of anoxia-tolerance to species that have lost thisproperty is served better by physiological and molecular studiesof the mechanisms that are employed in wild species that stillpossess long-term anoxia-tolerance. Case studies developingthese arguments are presented in relation to a selection ofcrop and wild species. The flooding sensitivity and metabolismof maize is compared in relation to rice in its capacity foranaerobic germination. The sensitivity of potato to floodingis related to its disturbed energy metabolism and inabilityto maintain functioning membranes under anoxia and postinoxia.By contrast, long-term anoxia-tolerance in the American cranberry(Vaccinium macrocarpon) and the arctic grass species Deschampsiaberingensis can be related to the provision and utilizationof carbohydrate reserves. Among temperate species, the sweetflag (Acorus calamus) shows a remarkable tolerance of anoxiain both shoots and roots and is also able to mobilize carbohydrateand maintain ATP levels during anoxia as well as preservingmembrane lipids against anoxic and post-anoxic injury. Phragmitesaustralis and Spartina alterniflora, although anoxia-tolerant,are both sulphide-sensitive species which can pre-dispose themto the phenomenon of die-back in stagnant, nutrient-rich water.Glyceria maxima adapts to flooding through phenological adaptationswith a seasonal metabolic tolerance of anoxia confined to winterand spring which, combined with a facility for root aerationand early spring growth, allows rapid colonization of siteswith only shallow flooding. The diversity of responses to floodingin wild plants suggests that, depending on the life strategyand habitat of the species, many different mechanisms may beinvolved in adapting plants to survive periods of inundationand no one mechanism on its own is adequate for ensuring survival. Key words: Anoxia, hypoxia, flooding, Zea mays, Solanum tuberosum, Oryza sativa, Acorus calamus, Phragmites australis, Glyceria maxima, cranberry     

Relationship between Coleoptile Elongation and Alcoholic Fermentation in Rice Exposed to Anoxia. I. Importance of Treatment Conditions and Different Tissues

The relationship between coleoptile elongation and alcoholicfermentation of rice under anoxia is examined using seeds either:(a) N₂ flushed during submergence, (b) incubated in stagnantdeoxygenated agar at 0·1% w/v to simulate the stagnantconditions of waterlogged soil, or (c) incubated in waterloggedsoil. Coleoptile elongation growth was greater for N₂ flushing> stagnant agar > soil; seed survival was also greatestin this order over 1-5 d. Ethanol concentrations in coleoptiles and intact seeds (cv.IR42) were approximately 300 and 100 mol m^-3 respectively whenseeds were grown 3 d in stagnant agar, however 92% of the ethanolin seeds diffused into the external medium when solutions weremixed for 5-10 s. Coleoptile growth under anoxia was relatedto rates of ethanol synthesis (R_E) in different treatments;there was greater coleoptile growth and R_E for seeds in N₂ flushedsolutions than in stagnant deoxygenated agar. Coleoptile growthof individual seeds was also related to the R_E of each seedat 2-3 d after anoxia (r² = 0·46). Analysis of different tissues was important in evaluating growthand metabolism of coleoptiles. Although the coleoptile onlyaccounted for 0·7% of seed dry weight at 3 d after anoxia,it contained 21% of the ethanol produced by rice seeds. Therewere also three-fold higher rates of R_E on a fresh weight basisin expanding tissues in the base of the coleoptile relativeto the elongated tissues at the apex. Results are discussedin terms of the importance of environmental conditions usedto impose anoxia, quantification of R_E in specific tissues andthe possibility that under stagnant conditions high ethanolconcentrations in tissues may limit R_E and coleoptile growth.Copyright1994, 1999 Academic Press Anoxia, ethanol, alcoholic fermentation, Oryza sativa L., rice, submergence     

Relationships between lipid peroxidation and anoxia tolerance in a range of species during post-anoxic reaeration

Peroxidation was studied in anoxically treated plant tissues and quantified as conjugated dienes/trienes in the total lipid fraction and as the production of thiobarbituric acid reactive substances (TBARS). Oxidative stress caused by re-exposure of plants to oxygen led to an increase of conjugated diene/triene formation in rhizomes of Iris germanica and roots of wheat ( Triticum aestivum L.) and oats ( Avena sativa L.), and after a long anoxic exposure (45 days) in the rhizomes of the very anoxia tolerant Iris pseudacorus . Second derivative (SD) spectrophotometry of the UV spectrum of lipid extracts confirmed the formation of dienes. However, determination of TBARS in Iris spp. showed no lipid peroxidation in the anoxia tolerant I. pseudacorus . In the rhizomes of the anoxia intolerant I. germanica , elevated levels of TBARS correlated positively with conjugated diene/triene formation. The results suggest that anoxic stress may induce qualitative changes in membrane lipids, as indicated by lipid peroxidation after restoration of aerobic conditions. The rate of lipid peroxidation correlated negatively with anoxic stress tolerance.     

The Effects of Oxygen, Carbon Dioxide and Ethylene on Ethylene Biosynthesis in Relation to Shoot Extension in Seedlings of Rice (Oryza sativa) and Barnyard Grass (Echinochloa oryzoides)

Exposing dark-grown seedlings for 3 d to oxygen deficiency (0or 5 kPa) or to additions of carbon dioxide (10 kPa) or ethylene(0·1 Pa) slowed shoot extension in Echinochloa oryzoides,while in rice it was promoted by these treatments, except that5 kPa oxygen was without effect. In E. oryzoides this was dueto reduced growth of the mesocotyl, and in rice to enhancedgrowth of the coleoptile. These responses to carbon dioxideand oxygen deficiency were not consequences of increased ethyleneproduction, since this remained unchanged by carbon dioxideand depressed by oxygen shortage in both species. Furthermore,exogenous ethylene and the ethylene action inhibitor 2,5-norbornadieneeach failed to influence extension in anoxic seedlings, indicatingno regulatory role for ethylene in the absence of oxygen. However,concentrations of the ethylene precursor 1 -aminocyclopropane-1-carboxylic acid (ACC) were increased by carbon dioxide and0 kPa or 5 kPa oxygen, although after 72 h without oxygen totalACC production (i.e. changes in ethylene + ACC + MACC) was suppressedin both species. There was little effect on bound ACC [putativemalonyl-ACC (MACC)] formation. Transferring anaerobic (0 kPa)seedlings to oxygenated conditions (21 kPa) resulted in abnormallyfast rates of ethylene formation, possibly due to the accumulationof ACC under anoxia. This post-anoxic ethylene may have contributedto the faster extension by rice coleoptiles and slower extensionby mesocotyls of E. oryzoides compared with those of seedlingsmaintained continuously in air. Echinochloa oryzoides [Ard.] Fritsch, barnyard grass, Oryza sativa L, rice, oxygen shortage, carbon dioxide, ethylene biosynthesis, shoot extension, 1-aminocyclopropane-1-carboxylic acid (ACC), malonyl-ACC, GC-MS     

Elevation of apoptotic potential by anoxia-hyperoxia shift in NIH3T3 cells

Apoptosis has been hypothesized to be mediated through the induction of free radicals via oxidative pathway. In this study, we demonstrated the induction of cellular apoptosis by anoxia-hyperoxia shift, but not by anoxia or hyperoxia alone in NIH3T3 cells. The decrement of ROS by anoxia thus appears to be an essential early event leading to apoptosis. G1 arrest was detected in anoxia-treated cells, and postanoxic oxygen recovery could reverse this effect, and induce apoptosis. On analysis of the binding activity of AP-1, we found biphasic induction of binding ability in cells undergoing anoxia-hyperoxia shift. In the early stage of anoxia, a transitional increase of AP-1 binding activity was detected, which was reduced to the minimal levels after 24 h of anoxia. During the period of postanoxic hyperoxia treatment, the binding activity of AP-1 was reinduced and increased remarkably with time up to 24 h. These results were in accordance with the expressions of c-jun and c-fos proteins. Enhancement of poly(ADP-ribosyl)ation activities, especially ADP-ribosylation of histone H1 was detected in post-anoxic hyperoxia-treated cells, and cleavage of PARP and activation of caspase 3 were also observed in post-anoxic hyperoxia (recovery) treated cells, but not in anoxia-treated cells. We propose that the differential induction of c-jun/c-fos (AP-1) gene expressions and sequential activation of PARP activity are essential in anoxia/hyperoxia-induced apoptosis     

Regulatory involvement of abscisic acid in potato tuber wound-healing

Rapid wound-healing is crucial in protecting potato tubers frominfection and dehydration. Wound-induced suberization and theaccumulation of hydrophobic barriers to reduce water vapourconductance/loss are principal protective wound-healing processes.However, little is known about the cognate mechanisms that effector regulate these processes. The objective of this researchwas to determine the involvement of abscisic acid (ABA) in theregulation of wound-induced suberization and tuber water vapourloss (dehydration). Analysis by liquid chromatography–massspectrometry showed that ABA concentrations varied little throughoutthe tuber, but were slightly higher near the periderm and lowestin the pith. ABA concentrations increase then decrease duringtuber storage. Tuber wounding induced changes in ABA content.ABA content in wound-healing tuber discs decreased after wounding,reached a minimum by 24 h, and then increased from the 3rd tothe 7th day after wounding. Wound-induced ABA accumulationswere reduced by fluridone (FLD); an inhibitor of de novo ABAbiosynthesis. Wound-induced phenylalanine ammonia lyase activitywas slightly reduced and the accumulation of suberin poly(phenolics)and poly(aliphatics) noticeably reduced in FLD-treated tissues.Addition of ABA to the FLD treatment restored phenylalanineammonia lyase activity and suberization, unequivocally indicatingthat endogenous ABA is involved in the regulation of these wound-healingprocesses. Similar experiments showed that endogenous ABA isinvolved in the regulation of water vapour loss, a process linkedto wax accumulation in wound-healing tubers. Rapid reductionof water vapour loss across the wound surface is essential inpreventing desiccation and death of cells at the wound site;live cells are required for suberization. These results unequivocallyshow that endogenous ABA is involved in the regulation of wound-inducedsuberization and the processes that protect surface cells fromwater vapour loss and death by dehydration. Key words: Abscisic acid, poly(aliphatic), poly(phenolic), potato, Solanum tuberosum L., suberin     

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.     

Antioxidant status of anoxia-tolerant and -intolerant plant species under anoxia and reaeration

The redox potential of the cell, as well as the antioxidant status of the tissue, are considered to be important regulatory constituents in an adaptive response in plants. Here the involvement of active antioxidants ascorbic acid (AA), reduced glutathione (GSH) and α - and β -tocopherols in reactive oxygen species scavenging, and the effect of anoxic stress on their reduction state were studied in 4 anoxia-tolerant and -intolerant plant species: Iris germanica L., Iris pseudacorus L., wheat ( Triticum aestivum L. cv. Leningradka) and rice ( Oryza sativa L. cv. VNIIR). The initial antioxidant content (both AA and GSH) was higher in the rhizomes of the more anoxia-tolerant Iris spp., as compared with that of the roots of the cereals. The predominant form of ascorbate was dehydroascorbic acid (DHA) in the cereals and AA in the Iris spp. Imposition of anoxia with subsequent reoxygenation resulted in an overall depletion of the reduced forms of antioxidants. No concurrent increase in oxidised forms (DHA and conjugated glutathione) was observed in anoxic samples. α -tocopherol content in Iris spp. was in the range 1–2 μg g⁻¹ fresh weight, while β -tocopherol content was higher in the anoxia-intolerant I. germanica (7.2 μg g⁻¹ fresh weight) as compared with the tolerant I. pseudacorus (1.5 μg g⁻¹ fresh weight). In I. pseudacorus , a significant decrease in α - and β -tocopherol levels was observed only after long-term (45 days) anoxia. The results suggested exclusion of AA and GSH from the redox cycling under prolonged anoxia, and a concomitant decrease in the redox state, as well as an anoxia-induced depletion of α - and β -tocopherols.     

Anoxia tolerance and anaerobic catabolism of aged beetroot storage tissues5

The relationship between anoxia tolerance and rates of anaerobiccatabolism was studied using aged storage tissues of red beetroot.This tissue has substantial experimental advantages over mostother tissues for studies on response to anoxia; even the aeratedtissues do not grow, thus allowing assesment of any possiblePasteur effect. Furthermore, loss of the endogeneous dye, betacyanin,serves as a marker for death of individual cells. A high tolerance to anoxia was achieved by adding glucose andby applying a hypoxic pretreatment for 48 h (02 at 0.003–0.015mol m^– Such tissues survived anoxia for at least 150 h. Alcoholic fermentation was the principal catabolic pathway inanoxic beetroot tissue; ethanol synthesized over 88.5 h of anoxiaaccounted for about 86% of the decrease In endogenous sugarcontent in hypoxically pretreated tissues. During the first24 h of anoxia, rates of alcoholic fermentation were stimulatedby high concentrations of endogenous glucose, supplying exogenousglucose and also by hypoxic pretreatment. In aerobically pretreatedtissues, alcoholic fermentation increased with time over thefirst 24 h of anoxia and these increases were correlated withincreases in activity of pyruvate decarboxylase (PDC). The mostrapid rates of glycolysis under anoxia were observed in thepresence of glucose, c. 50% above the rate in aerated tissues. When anoxia lasted longer than 24 h, the rate of alcoholic fermentationdeclined with time, in all treatments. Furthermore, decreasesin content of endogen ous substrates (sugars + starch), between0 and 88.5 h anoxia, indicated that, in hypoxically pretreatedtissues, glycolysis was 30% lower under anoxia than in air.These decreases in rate of alcoholic fermentation were not dueto injury, or decreases in activity of PDC. Reduced availabilityof endogenous sugar can not be excluded as a cause for the decreasein rate of glycolysis. However, we favour fine control, whichwould regulate glycolysis once requirements for ATP are reduced,after adaptation to anoxia has been completed. We also estimatethat maintenance requirement for ATP is 10–25 times lowerIn anoxic than in aerated tissues. Key words: Anoxia tolerance, alcoholic fermentation, maintenance requirement, storage tissues     

Importance of glucose-6-phosphate dehydrogenase activity in cell death

The intracellular redox potential plays an important role incell survival. The principal intracellular reductant NADPH is mainlyproduced by the pentose phosphate pathway by glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme, and by6-phosphogluconate dehydrogenase. Considering the importance of NADPH,we hypothesized that G6PDH plays a critical role in cell death. Ourresults show that 1) G6PDHinhibitors potentiatedH₂O₂-inducedcell death; 2) overexpression ofG6PDH increased resistance toH₂O₂-induced cell death; 3) serum deprivation, astimulator of cell death, was associated with decreased G6PDH activityand resulted in elevated reactive oxygen species (ROS);4) additions of substrates for G6PDHto serum-deprived cells almost completely abrogated the serumdeprivation-induced rise in ROS; 5)consequences of G6PDH inhibition included a significant increase inapoptosis, loss of protein thiols, and degradation of G6PDH; and6) G6PDH inhibition caused changesin mitogen-activated protein kinase phosphorylation that were similarto the changes seen withH₂O₂.We conclude that G6PDH plays a critical role in cell death by affectingthe redox potential.