Impact of oxygen stress and energy availability on membrane stability of plant cells.
ABSTRACT: This article reviews the relationship between the energy status of plant cells under O(2) stress (e.g. waterlogging) and the maintenance of membrane intactness, using information largely derived from suspension cultures of anoxia-intolerant potato cells. Energy-related parameters measured were fermentation end-products (ethanol, lactate, alanine), respiratory rate, ATP, adenylate energy charge, nitrate reductase activity and biomass. ATP synthesis rates were calculated from the first four parameters. Reactive oxygen species were estimated from H(2)O(2) and superoxide levels, and the enzymatic detoxification potential from the activity levels of catalase and superoxide dismutase. Structure-related parameters were total fatty acids, free fatty acids (FFAs), lipid hydroperoxides, total phospholipids, N-acylphosphatidylethanolamine (NAPE) and cell viability. The following issues are addressed in this review: (1) what is the impact of anoxia on membrane lipids and how does this relate to energy status; (2) does O(2) per se play a role in these changes; (3) under which conditions and to what extent does lipid peroxidation occur upon re-aeration; and (4) can the effects of re-aeration be distinguished from those of anoxia? The emerging picture is a reappraisal of the relative contributions of anoxia and re-aeration. Two successive phases (pre-lytic and lytic) characterize potato cells under anoxia. They are connected by a threshold in ATP production rate, below which membrane lipids are hydrolysed to FFAs, and NAPE increases. Since lipid peroxidation occurs only when cells are reoxygenated during the lytic phase, its biological relevance in an already damaged system is questionable.
Project description:<h4>Background and aims</h4>Anoxia-tolerant plant tissues synthesize a number of proteins during anoxia, in addition to the 'classical anaerobic proteins' involved in glycolysis and fermentation. The present study used a model system of rice coleoptile tips to elucidate patterns of protein synthesis in this anoxia-tolerant plant tissue.<h4>Methods</h4>Coleoptile tips 7-11 mm long were excised from intact seedlings exposed to anoxia, or excised from hypoxically pre-treated seedlings and then exposed to anoxia for 72 h. Total proteins or 35S-labelled proteins were extracted, separated using two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis and analysed using mass spectrometry.<h4>Key results</h4>The coleoptile tips excised after intact seedlings had been exposed to anoxia for 72 h had a similar proteome to tips that were first excised and then exposed to anoxia. After 72 h anoxia, Bowman-Birk trypsin inhibitors and a glycine-rich RNA-binding protein decreased in abundance, whereas a nucleoside diphosphate kinase and several proteins with unknown functions were strongly enhanced. Using [35S]methionine as label, proteins synthesized at high levels in anoxia, and also in aeration, included a nucleoside diphosphate kinase, a glycine-rich RNA-binding protein, a putative elicitor-inducible protein and a putative actin-depolymerizing factor. Proteins synthesized predominately in anoxia included a pyruvate orthophosphate dikinase (PPDK), alcohol dehydrogenase 1 and 2, fructose 1,6-bisphosphate aldolase and a protein of unknown function.<h4>Conclusion</h4>The induction of PPDK in anoxic rice coleoptiles might, in combination with pyruvate kinase (PK), enable operation of a 'substrate cycle' producing PPi from ATP. Production of PPi would (a) direct energy to crucial transport processes across the tonoplast (i.e. the H+-PPiase); (b) be required for sucrose hydrolysis via sucrose synthase; and (c) enable acceleration of glycolysis, via pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) acting in parallel with phosphofructokinase (PFK), thus enhancing ATP production in anoxic rice coleoptiles; ATP production would need to be increased if there was a substantial requirement for PPi.
Project description:<i>Dinoroseobacter shibae</i> DFL 12<sup>T</sup> is a metabolically versatile member of the world-wide abundant Roseobacter clade. As an epibiont of dinoflagellates <i>D. shibae</i> is subjected to rigorous changes in oxygen availability. It has been shown that it loses up to 90% of its intracellular ATP when exposed to anoxic conditions. Yet, <i>D. shibae</i> regenerates its ATP level quickly when oxygen becomes available again. In the present study we focused on the bioenergetic aspects of the quick recovery and hypothesized that the proton-motive force decreases during anoxia and gets restored upon re-aeration. Therefore, we analyzed ?pH and the membrane potential (??) during the oxic-anoxic transitions. To visualize changes of ?? we used fluorescence microscopy and the carbocyanine dyes DiOC<sub>2</sub> (3; 3,3'-Diethyloxacarbocyanine Iodide) and JC-10. In control experiments the ??-decreasing effects of the chemiosmotic inhibitors CCCP (carbonyl cyanide m-chlorophenyl hydrazone), TCS (3,3',4',5<i>-</i>tetrachlorosalicylanilide) and gramicidin were tested on <i>D. shibae</i> and Gram-negative and -positive control bacteria (<i>Escherichia coli</i> and <i>Micrococcus luteus</i>). We found that ?pH is not affected by short-term anoxia and does not contribute to the quick ATP regeneration in <i>D. shibae.</i> By contrast, ?? was increased during anoxia, which was astonishing since none of the control organisms behaved that way. Our study shows physiological and bioenergetical aspects comparing to previous studies on transcriptomic responses to the transition from aerobic to nitrate respiration in <i>D. shibae</i>. For the lifestyle as an epibiont of a dinoflagellate, the ability to stand phases of temporary oxygen depletion is beneficial. With a boosted ??, the cells are able to give their ATP regeneration a flying start, once oxygen is available again.
Project description:The objective of this study was to evaluate the role of enzyme induction and aerenchyma formation in prolonged tolerance to soil flooding in a variety of underground clover (Trifolium subterraneum 'Park') previously selected for resistance. Seedlings were grown in hydroponic tanks, initially with aeration for 3 weeks and subsequently in the absence of aeration for up to 3 weeks. After 1 h in the absence of aeration, the oxygen concentration in the hydroponic medium had decreased to 1.5 %. During the 3 weeks of extreme oxygen deficiency, primary roots died and were replaced by considerable numbers of adventitious roots. Activities of many glycolytic and fermentative enzymes increased in adventitious roots. Excised adventitious roots were capable of immediate induction of ethanol in the absence of lactate production, in association with energy charge higher than that in excised roots of aerobically maintained controls. Energy charge was even higher when measured in adventitious roots in planta. Interestingly, haemoglobin protein could be correlated with energy charge. Aerenchyma was readily visualized in adventitious roots by optical microscopy of longitudinal and transverse sections. We conclude that avoidance of root anoxia via aerenchyma is the major mechanism for prolonged root tolerance in Trifolium subterraneum 'Park'.
Project description:ATP depletion and succinate accumulation during ischemia lead to oxidative damage to mammalian organs upon reperfusion. In contrast, freshwater turtles survive weeks of anoxia at low temperatures without suffering from oxidative damage upon reoxygenation, but the mechanisms are unclear. To determine how turtles survive prolonged anoxia, we measured ~80 metabolites in hearts from cold-acclimated (5?°C) turtles exposed to 9 days anoxia and compared the results with those for normoxic turtles (25?°C) and mouse hearts exposed to 30?min of ischemia. In turtles, ATP and ADP decreased to new steady-state levels during fasting and cold-acclimation and further with anoxia, but disappeared within 30?min of ischemia in mouse hearts. High NADH/NAD<sup>+</sup> ratios were associated with succinate accumulation in both anoxic turtles and ischemic mouse hearts. However, succinate concentrations and succinate/fumarate ratios were lower in turtle than in mouse heart, limiting the driving force for production of reactive oxygen species (ROS) upon reoxygenation in turtles. Furthermore, we show production of ROS from succinate is prevented by re-synthesis of ATP from ADP. Thus, maintenance of an ATP/ADP pool and low succinate accumulation likely protects turtle hearts from anoxia/reoxygenation injury and suggests metabolic interventions as a therapeutic approach to limit ischemia/reperfusion injury in mammals.
Project description:Oxygen deprivation limits the energy available for cellular processes and yet no comprehensive ATP budget has been reported for any plant species under O(2) deprivation, including Oryza sativa. Using 3-d-old coleoptiles of a cultivar of O. sativa tolerant to flooding at germination, (i) rates of ATP regeneration in coleoptiles grown under normoxia (aerated solution), hypoxia (3% O(2)), and anoxia (N(2)) and (ii) rates of synthesis of proteins, lipids, nucleic acids, and cell walls, as well as K(+) transport, were determined. Based on published bioenergetics data, the cost of synthesizing each class of polymer and the proportion of available ATP allocated to each process were then compared. Protein synthesis consumed the largest proportion of ATP synthesized under all three oxygen regimes, with the proportion of ATP allocated to protein synthesis in anoxia (52%) more than double that in normoxic coleoptiles (19%). Energy allocation to cell wall synthesis was undiminished in hypoxia, consistent with preferential elongation typical of submerged coleoptiles. Lipid synthesis was also conserved strongly in O(2) deficits, suggesting that membrane integrity was maintained under anoxia, thus allowing K(+) to be retained within coleoptile cells. Rates of protein synthesis in coleoptiles from rice cultivars with contrasting tolerance to oxygen deficits (including mutants deficient in fermentative enzymes) confirmed that synthesis and turnover of proteins always accounted for most of the ATP consumed under anoxia. It is concluded that successful establishment of rice seedlings under water is largely due to the capacity of coleoptiles to allocate energy to vital processes, particularly protein synthesis.
Project description:The concentrations of adenine nucleotides were determined in germinating lettuce (Lactuca sativa) seeds after transitions from air to hypoxic or anoxic atmospheres. The ratio ATP/ADP and the energy charge were rapidly lowered after the transitions and remained stable at low values for hours. The energy charge in anoxia stabilized at a value close to 0.3. After 24 h in anoxia the energy charge rose rapidly to high values (0.9) when N2 was replaced by air. The metabolic properties of lettuce seeds had then been conversed for hours at low energy charge. In hypoxia the O2 uptake was decreased and the energy charge was stabilized at values intermediate between that in air and that in anoxia. When the O2 partial pressures (pO2) were 5 and 2kPa, the values of O2 uptake were one-third and one-sixth of that in air, and the energy charges were 0.7 and 0.5. These results show that the energy charge is regulated over a wide range of values. The ratio ATP/ADP and the energy charge are indicators of the limitation of metabolic activity by hypoxia.
Project description:Studies with the isolated perfused working rat heart were carried out to investigate factors that may enable the heart to recover after periods of anoxia. It was found that the presence of glucose in the perfusion fluid during anoxia was essential for complete post-anoxic recovery and the presence of a high concentration of K(+) increased not only the rate of recovery but also the final extent of recovery. In an attempt to clarify the roles played by glucose and K(+) in aiding the survival and recovery of the anoxic myocardium the concentrations of parameters associated with energy liberation and anaerobic glycolysis (ATP, ADP, AMP, P(i), creatine phosphate, glycogen and lactate) were measured in the presence and absence of glucose during the anoxic phase. Determinations of these parameters were carried out during the working aerobic control period, the anoxic period (K(+) arrest) and the recovery period. The results demonstrated that glucose acted as an energy source during anoxia and thus maintained myocardial concentrations of high-energy phosphates, particularly ATP. These studies have also shown a direct relationship between the ability of the heart to recover and the concentration of myocardial ATP at the time of reoxygenation.
Project description:Our aim was to elucidate how plant tissues under a severe energy crisis cope with imposition of high NaCl, which greatly increases ion fluxes and hence energy demands. The energy requirements for ion regulation during combined salinity and anoxia were assessed to gain insights into ion transport processes in the anoxia-tolerant coleoptile of rice.We studied the combined effects of anoxia plus 50 or 100 mm NaCl on tissue ions and growth of submerged rice (Oryza sativa) seedlings. Excised coleoptiles allowed measurements in aerated or anoxic conditions of ion net fluxes and O2 consumption or ethanol formation and by inference energy production.Over 80?h of anoxia, coleoptiles of submerged intact seedlings grew at 100 mm NaCl, but excised coleoptiles, with 50 mm exogenous glucose, survived only at 50 mm NaCl, possibly due to lower energy production with glucose than for intact coleoptiles with sucrose as substrate. Rates of net uptake of Na+ and Cl- by coleoptiles in anoxia were about half those in aerated solution. Ethanol formation in anoxia and O2 uptake in aerobic solution were each increased by 13-15 % at 50 mm NaCl, i.e. ATP formation was stimulated. For acclimation to 50 mm NaCl, the anoxic tissues used only 25 % of the energy that was expended by aerobic tissues. Following return of coleoptiles to aerated non-saline solution, rates of net K+ uptake recovered to those in continuously aerated solution, demonstrating there was little injury during anoxia with 50 mm NaCl.Rice seedlings survive anoxia, without the coleoptile incurring significant injury, even with the additional energy demands imposed by NaCl (100 mm when intact, 50 mm when excised). Energy savings were achieved in saline anoxia by less coleoptile growth, reduced ion fluxes as compared to aerobic coleoptiles and apparent energy-economic ion transport systems.
Project description:HIGHLIGHTS: Sedimentation of fine sediment particles onto seagrass leaves severely hampers the plants' performance in both light and darkness, due to inadequate internal plant aeration and intrusion of phytotoxic H2S. Anthropogenic activities leading to sediment re-suspension can have adverse effects on adjacent seagrass meadows, owing to reduced light availability and the settling of suspended particles onto seagrass leaves potentially impeding gas exchange with the surrounding water. We used microsensors to determine O2 fluxes and diffusive boundary layer (DBL) thickness on leaves of the seagrass Zostera muelleri with and without fine sediment particles, and combined these laboratory measurements with in situ microsensor measurements of tissue O2 and H2S concentrations. Net photosynthesis rates in leaves with fine sediment particles were down to ~20% of controls without particles, and the compensation photon irradiance increased from a span of 20-53 to 109-145 ?mol photons m-2 s-1. An ~2.5-fold thicker DBL around leaves with fine sediment particles impeded O2 influx into the leaves during darkness. In situ leaf meristematic O2 concentrations of plants exposed to fine sediment particles were lower than in control plants and exhibited long time periods of complete meristematic anoxia during night-time. Insufficient internal aeration resulted in H2S intrusion into the leaf meristematic tissues when exposed to sediment resuspension even at relatively high night-time water-column O2 concentrations. Fine sediment particles that settle on seagrass leaves thus negatively affect internal tissue aeration and thereby the plants' resilience against H2S intrusion.
Project description:Uptake of external glucose and production of lactate were measured in freshly-excised bovine articular cartilage under O2 concentrations ranging from 21% (air) to zero (N2-bubbled). Anoxia (O2 concentration < 1% in the gas phase) severely inhibited both glucose uptake and lactate production. The decrease in lactate formation correlated closely with the decrease in glucose uptake, in a mole ratio of 2:1. This reduction in the rate of glycolysis in anoxic conditions is seen as evidence of a negative Pasteur effect in bovine articular cartilage. Anoxia also suppressed glycolysis in articular cartilage from horse, pig and sheep. Inhibitors acting on the glycolytic pathway (2-deoxy-D-glucose, iodoacetamide or fluoride) strongly decreased aerobic lactate production and ATP concentration, consistent with the belief that articular cartilage obtains its principal supply of ATP from substrate-level phosphorylation in glycolysis. Azide or cyanide lowered the ATP concentration in aerobic cartilage to approximately the same extent as did anoxia but, because glycolysis (lactate production) was also inhibited by these treatments, the importance of any mitochondrial ATP production could not be assessed. A negative Pasteur effect would make chondrocytes particularly liable to suffer a shortage of energy under anoxic conditions. Incorporation of [35S]sulphate into proteoglycan was severely curtailed by treatments, such as anoxia, which decreased the intracellular concentration of ATP.