Adaptative response of Vitis root to anoxia

The effect of anoxia on the energy economy of root cells was studied by measuring heat production, ethanol and ATP production, K(+) fluxes and electrical activity in two Vitis species, V. riparia and V. rupestris, that differ in their tolerance to anoxia. Anoxia triggered a marked decrease of metabolic activity (measured by microcalorimetry) and of ATP levels in both species. In V. riparia after the first 2 h of anoxia, the decrease in the rate of heat production was not associated with a further significant decrease in ATP content, whereas in V. rupestris the ATP level continued to decrease until very low values were reached. The concomitant increase in the rate of ethanol production did not compensate for the decreased aerobic ATP supply. In V. rupestris, anoxia typically led to energy deficit and ATP imbalance, together with the subsequent disruption of ion homeostasis and cell death. In V. riparia, the strong decrease in K(+) membrane permeability together with the fast down-regulation of the electrical signals allowed the cells to avoid severe ion imbalances during prolonged anoxic episodes.     

Characterisation of the oxygen fluxes in the division, elongation and mature zones of Vitis roots: influence of oxygen availability

Oxygen fluxes into and from root cells of Vitis rupestris (flooding sensitive), V. riparia (flooding tolerant) and V. vinifera (medium tolerance to flooding) were measured under different levels of O2 availability using a recently developed polarographic O2-selective, vibrating-microelectrode system. The system enables fluxes to be measured with a spatial resolution of 2-3 microm and a temporal resolution of 10 s. No difference in root porosity was found among the genotypes when grown for 30 days in an aerated solution. Under normoxic conditions, O2 influx was characterised by two distinct peaks, one in the division zone and the other in the elongation zone of the roots. This pattern was found in all three species studied, although the fluxes showed a different magnitude. The peak in the elongation zone coincided with maximum relative elemental growth rates. When the energetics of the cell was disturbed by cyanide, both growth and oxygen O2 influxes ceased at the same time. Under hypoxic conditions, V. riparia plants showed a precise strategy directed toward the maintenance of enough O2 for the respiratory needs of mitosis in the apical meristem of the roots. Thus, whereas in the division zone of V. rupestris and V. vinifera, at bulk O2 concentrations of 0.094 mol x m(-3), the O2 influx was reduced by 70.5 and 38.5%, respectively, for V. riparia no variation in the O2 influx was detected down to bulk O2 concentrations of 0.078 mol x m(-3). Moreover, in accordance with the different tolerances of the plants, the Vitis genotypes were found to differ in their radial O2 loss from the adventitious roots when in an O2-free environment. The results are discussed in terms of possible mechanisms of response to anoxia in Vitis species with different tolerances to flooding.     

Ethanolic fermentation and anoxia tolerance in four rice cultivars

The relationship between coleoptile elongation and ethanolic fermentation was investigated in rice (Oryza sativa L.) coleoptiles of four cultivars subjected to a 48-h anoxic stress. The coleoptile elongation of all cultivars was suppressed by anoxic stress; however, the elongation of cvs Yukihikari and Nipponbare was much greater than that of cvs Leulikelash and Asahimochi. The stress did not significantly increase lactate dehydrogenase (LDH) activity or lactate concentration, but increased alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) activities, as well as ethanol concentration in the coleoptiles of all cultivars. The elevated ADH and PDC activities and ethanol concentration in cvs Yukihikari and Nipponbare were much greater than those of cvs Leulikelash and Asahimochi, suggesting that ethanolic fermentation is likely more active in cvs Yukihikari and Nipponbare than in cvs Leulikelash and Asahimochi. ATP concentration in cvs Yukihikari and Nipponbare in anoxia was also greater than that in cvs Leulikelash and Asahimochi in anoxia. The ethanol concentration in the coleoptiles was correlated with anoxia tolerance with respect to the ATP concentration and coleoptile elongation. These results suggest that the ability to increase ethanolic fermentation may be one of the determinants in anoxia tolerance of rice coleoptiles.     

Quantifying ATP turnover in anoxic coleoptiles of rice (Oryza sativa) demonstrates preferential allocation of energy to protein synthesis

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.     

Opening mitoKATP increases superoxide generation from complex I of the electron transport chain

Opening the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)) increases levels of reactive oxygen species (ROS) in cardiomyocytes. This increase in ROS is necessary for cardioprotection against ischemia-reperfusion injury; however, the mechanism of mitoK(ATP)-dependent stimulation of ROS production is unknown. We examined ROS production in suspensions of isolated rat heart and liver mitochondria, using fluorescent probes that are sensitive to hydrogen peroxide. When mitochondria were treated with the K(ATP) channel openers diazoxide or cromakalim, their ROS production increased by 40-50%, and this effect was blocked by 5-hydroxydecanoate. ROS production exhibited a biphasic dependence on valinomycin concentration, with peak production occurring at valinomycin concentrations that catalyze about the same K(+) influx as K(ATP) channel openers. ROS production decreased with higher concentrations of valinomycin and with all concentrations of a classical protonophoretic uncoupler. Our studies show that the increase in ROS is due specifically to K(+) influx into the matrix and is mediated by the attendant matrix alkalinization. Myxothiazol stimulated mitoK(ATP)-dependent ROS production, whereas rotenone had no effect. This indicates that the superoxide originates in complex I (NADH:ubiquinone oxidoreductase) of the electron transport chain.     

Nucleotides and Mg2+ ions differentially regulate K+ channels and non-selective cation channels present in cells forming the stomatal complex

Voltage-dependent inward-rectifying (K(in)) and outward-rectifying (K(out)) K(+) channels are capable of mediating K(+) fluxes across the plasma membrane. Previous studies on guard cells or heterologously expressed K(+) channels provided evidence for the requirement of ATP to maintain K(+) channel activity. Here, the nucleotide and Mg(2+) dependencies of time-dependent K(in) and K(out) channels from maize subsidiary cells were examined, showing that MgATP as well as MgADP function as channel activators. In addition to K(out) channels, these studies revealed the presence of another outward-rectifying channel type (MgC) in the plasma membrane that however gates in a nucleotide-independent manner. MgC represents a new channel type distinguished from K(out) channels by fast activation kinetics, inhibition by elevated intracellular Mg(2+) concentration, permeability for K(+) as well as for Na(+) and insensitivity towards TEA(+). Similar observations made for guard cells from Zea mays and Vicia faba suggest a conserved regulation of channel-mediated K(+) and Na(+) transport in both cell types and species.     

Anoxia tolerance in tobacco roots: effect of overexpression of pyruvate decarboxylase

Plant survival during flooding relies on ethanolic fermentation for energy production. The available literature indicates that the first enzyme of the ethanolic fermentation pathway, pyruvate decarboxylase (PDC), is expressed at very low levels and is likely to be rate-limiting during oxygen deprivation. The authors expressed high levels of bacterial PDC in tobacco to study the modulation of PDC activity in vivo, and assess its impact on the physiology of ethanolic fermentation and survival under oxygen stress. In contrast to leaves, wild-type normoxic roots contained considerable PDC activity, and overexpression of the bacterial PDC caused only a moderate increase in acetaldehyde and ethanol production under anoxia compared to wild-type roots. No significant lactate production could be measured at any time, making it unlikely that lactate-induced acidification (LDH/PDC pH-stat) triggers the onset of ethanol synthesis. Instead, the authors favour a model in which the flux through the pathway is regulated by substrate availability. The increased ethanolic flux in the transgenics compared to the wild-type did not enhance anoxia tolerance. On the contrary, rapid utilisation of carbohydrate reserves enhanced premature cell death in the transgenics while replenishment of carbohydrates improved survival under anoxia.     

Plasma membrane depolarization reduces nitric oxide (NO) production in P388D.1 macrophage-like cells during Leishmania major infection

In the present study, we compare changes in host cell plasma membrane potential (V(m)), K(+) fluxes, and NO production during K(+) channel blockade with those changes that occur during infection with Leishmania major. Infection of P388D.1 cells with L. major promastigotes or treatment with K(+) channel blockers (either 1mM 4-AP, 10mM TEA, or 200 microM quinine) suppressed NO production. Inhibition of NO production correlated with depolarization of the P388D.1 cell V(m). Infection of P388D.1 cells with L. major increased the unidirectional influx of rubidium (86Rb), a tracer for K(+) flux, that was comparable to that induced by K(+) channel blockade by 1mM 4-AP. The similar effects of K(+) channel blockers and L. major on NO production, K(+) influx, and V(m) suggest that K(+) channel activity and the maintenance of V(m) is important for NO production in these cells. We suggest that intracellular parasites employ a strategy to inhibit NO production by disrupting V(m) during the invasion/infection process by altering host cell K(+) channel activity.     

Anoxic survival of the isolated cerebellum of the turtle Pseudemis scripta elegans

Intact turtle brain provides a useful model for the study of anoxia and potential survival strategies, since this tissue maintains transmembrane ion gradients and ATP levels during prolonged anoxia and recovers functional activity afterwards. Since isolated tissues offer experimental advantages, the present study sought to determine effects of anoxia on the isolated turtle cerebellum and to define relationships between anoxia survival and glucose supply. In normoxia, the extracellular potassium ([K+]o) activity and evoked potentials were maintained with 5 mM glucose, but 20 mM glucose was required to maintain adenosine triphosphate (ATP) levels and prevent significant increases in [K+]o during anoxia. Inhibition of glycolysis by iodoacetic acid (IAA) during anoxia provoked large increases in [K+]o at all glucose levels. These results demonstrate the usefulness of the isolated turtle cerebellum for studies of anoxic survival since this tissue can maintain ATP levels and [K+]o during prolonged anoxia with 20 mM glucose in the artificial cerebrospinal fluid medium. They also suggest the presence of a Pasteur effect at least during the transition to a hypometabolic state.     

The pyruvate decarboxylase1 gene of Arabidopsis is required during anoxia but not other environmental stresses

Ethanolic fermentation is classically associated with flooding tolerance when plant cells switch from respiration to anaerobic fermentation. However, recent studies have suggested that fermentation also has important functions in the presence of oxygen, mainly in germinating pollen and during abiotic stress. Pyruvate decarboxylase (PDC), which catalyzes the first step in this pathway, is thought to be the main regulatory enzyme. Here, we characterize the PDC gene family in Arabidopsis. PDC is encoded by four closely related genes. By using real-time quantitative polymerase chain reaction, we determined the expression levels of each individual gene in different tissues, under normal growth conditions, and when the plants were subjected to anoxia or other environmental stress conditions. We show that PDC1 is the only gene induced under oxygen limitation among the PDC1 gene family and that a pdc1 null mutant is comprised in anoxia tolerance but not other environmental stresses. We also characterize the expression of the aldehyde dehydrogenase (ALDH) gene family. None of the three genes is induced by anoxia but ALDH2B7 reacts strongly to ABA application and dehydration, suggesting that ALDH may play a role in aerobic detoxification of acetaldehyde. We discuss the possible role of ethanolic fermentation as a robust back-up energy production pathway under adverse conditions when mitochondrial function is disturbed.     

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     

Stress preconditioning of spreading depression in the locust CNS

Cortical spreading depression (CSD) is closely associated with important pathologies including stroke, seizures and migraine. The mechanisms underlying SD in its various forms are still incompletely understood. Here we describe SD-like events in an invertebrate model, the ventilatory central pattern generator (CPG) of locusts. Using K(+) -sensitive microelectrodes, we measured extracellular K(+) concentration ([K(+)](o)) in the metathoracic neuropile of the CPG while monitoring CPG output electromyographically from muscle 161 in the second abdominal segment to investigate the role K(+) in failure of neural circuit operation induced by various stressors. Failure of ventilation in response to different stressors (hyperthermia, anoxia, ATP depletion, Na(+)/K(+) ATPase impairment, K(+) injection) was associated with a disturbance of CNS ion homeostasis that shares the characteristics of CSD and SD-like events in vertebrates. Hyperthermic failure was preconditioned by prior heat shock (3 h, 45 degrees C) and induced-thermotolerance was associated with an increase in the rate of clearance of extracellular K(+) that was not linked to changes in ATP levels or total Na(+)/K(+) ATPase activity. Our findings suggest that SD-like events in locusts are adaptive to terminate neural network operation and conserve energy during stress and that they can be preconditioned by experience. We propose that they share mechanisms with CSD in mammals suggesting a common evolutionary origin.     

Regulation of alcoholic fermentation in coleoptiles of two rice cultivars differing in tolerance to anoxia

To investigate regulation of anaerobic carbohydrate catabolism in anoxia-tolerant plant tissue, rate of alcoholic fermentation and maximum catalytic activities of four key enzymes were assessed in coleoptiles of two rice cultivars that differ in tolerance to anoxia. The enzymes were ATP-dependent phosphofructokinase (PFK), pyrophosphate-dependent phosphofructokinase (PFP), pyruvate decarboxylase (PDC), and alcohol dehydrogenase (ADH). During anoxia, rates of coleoptile elongation and ethanol synthesis were faster in the more tolerant variety Calrose than in IR22. Calrose coleoptiles, in contrast to IR22, also showed a sustained Pasteur effect, with the estimated rate of glycolysis during anoxia being 1.4-1.7-fold faster than that of aerobic coleoptiles. In Calrose after 5 d anoxia, maximum catalytic activities of crude enzyme extracts were (in mumol substrate g-1 fresh weight min.-1) 170-240 for ADH, 4-6 for PDC and PFP and 0.4-0.7 for PFK. During anoxia, activity per coleoptile of all four enzymes increased 3-5.5-fold, suggesting that PFK, and PFP, like PDC and ADH, are synthesised in anoxic rice coleoptiles. Enzyme activities, on a fresh weight basis, were lower in IR22 than in Calrose. In vivo activities of PDC and PFK in anoxic coleoptiles from both cultivars were calculated using in vitro activities, estimated substrate levels, cytoplasmic pH, and S0.5 (the substrate level at which 0.5Vmax is reached, without inferring Michaelis-Menten kinetics). Data indicated that potential carbon flux through PFK, rather than through PDC, more closely approximated rates of alcoholic fermentation. That PFK is an important site of regulation was supported further for Calrose coleoptiles by a decrease in the concentration of its substrate pool (F-6-P + G-6-P) following the onset of anoxia. By contrast, in IR22, there was little evidence for control by PFK, consistent with recent evidence that suggests substrate supply limits alcoholic fermentation in this cultivar.     

The role of glucose in the survival and `recovery'' of the anoxic isolated perfused rat heart

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.