pH regulation in anoxic plants

BACKGROUND: pH regulation is the result of a complex interaction of ion transport, H+ buffering, H+-consuming and H+-producing reactions. Cells under anoxia experience an energy crisis; an early response thereof (in most tissues) is a rapid cytoplasmic acidification of roughly half a pH unit. Depending on the degree of anoxia tolerance, this pH remains relatively stable for some time, but then drops further due to an energy shortage, which, in concert with a general breakdown of transmembrane gradients, finally leads to cell death unless the plant finds access to an energy source. SCOPE: In this review the much-debated origin of the initial pH change and its regulation under anoxia is discussed, as well as the problem of how tissues deal with the energy crisis and to what extent pH regulation and membrane transport from and into the vacuole and the apoplast is a part thereof. CONCLUSIONS: It is postulated that, because a foremost goal of cells under anoxia must be energy production (having an anaerobic machinery that produces insufficient amounts of ATP), a new pH is set to ensure a proper functioning of the involved enzymes. Thus, the anoxic pH is not experienced as an error signal and is therefore not reversed to the aerobic level. Although acclimated and anoxia-tolerant tissues may display higher cytoplasmic pH than non-acclimated or anoxia-intolerant tissues, evidence for an impeded pH-regulation is missing even in the anoxia-intolerant tissues. For sufficient energy production, residual H+ pumping is vital to cope with anoxia by importing energy-rich compounds; however it is not vital for pH-regulation. Whereas the initial acidification is not due to energy shortage, subsequent uncontrolled acidosis occurring in concert with a general gradient breakdown damages the cell but may not be the primary event.     

Winter anoxic layer in Lake Hibara

The concentration of dissolved oxygen in waters 0.5–0.6 m above the bottom of Lake Hibara, a dimictic lake, was zero in early spring of 1994 and 1997. The concentrations in early spring of 1992, 1993, 1995, 1996, and 1998 ranged from 3.75 to 10.1 mg l⁻¹. The depth profiles of water temperature suggest that water had not circulated prior to the sample collections of 1994 and 1997, but it had done so in the cases of the other years, suggesting that winter conditions were well preserved in the former years. On the other hand, the dissolved oxygen in the same strata decreased severely in summer. However, more or less titratable amounts of dissolved oxygen still remained (0.01–0.73 mg l⁻¹) at the final stages of summer stratification from 1992 to 1998. These facts show that a completely anoxic condition is not formed in this lake in summer but is sometimes formed in winter. It is interesting to note that in spite of unfavorable winter conditions for oxygen consumption, i.e., shorter duration and lower water temperature, oxygen is exhausted. These facts suggest that ventilation to the depths is much greater in summer than in winter. Received: March 5, 1999 / Accepted: October 18, 1999     

Metabolic suppression in anoxic frog muscle

Microcalorimetry is the only direct method for measuring moment-to-moment changes in whole-cell metabolism (as heat output) during anoxia. We have adapted this methodology, in conjunction with standard muscle isolation techniques, to monitor metabolic transitions in isolated frog (Rana temporaria) sartorius muscle during anoxia and recovery (reoxygenation). Anoxia (sustained 1 h, following 2 h progressive hypoxia) suppressed muscle heat output to 20% of the stable normoxic level. This effect was fully reversible upon reoxygenation. Metabolite profiles were consistent with other anoxia-tolerant vertebrates – most notably, adenosine triphosphate (ATP) content during anoxia and reoxygenation remained unchanged from normoxia (pre-anoxic control). In addition, the concentration of K⁺ ions ([K⁺]) in interstitial dialysates remained stable (2–3 mM) throughout anoxia and recovery. Interstitial [lactate⁻] increased slightly, in accord with anaerobiosis supporting suppressed metabolic rates during anoxia. The degree of anoxic suppression of metabolism observed is similar to other vertebrate models of anoxia tolerance. Furthermore, stable ATP concentrations and interstitial [K⁺] in the isolated tissue suggests that intrinsic mechanisms suppress metabolism in a manner that coordinates ATP supply and demand and avoids the severe ion imbalances that are characteristic of hypoxia-sensitive systems. Accepted: 15 January 1998     

Activated sludge transformation in anoxic condition

Summary Substantial increase of methanogens and obligate hydrogen producing acetogens is occurs when activated sludges are allowed to remain under anoxic conditions at either 25°C and 35°C, without oxygen free atmosphere or any external addition of reductants. Acetate addition to activated sludge stimulates anoxic growth of all anaerobes except for butyrate users. During this period, the sludge volume index decrease.     

Different effects of simple anoxic lactic acidosis and simulated in vivo anoxic acidosis on turtle heart.

We compared responses of turtle heart at 20 degrees C to an anoxic lactic acidosis solution (LA) containing 35 mM lactic acid in an otherwise normal turtle Ringers equilibrated with 3% CO2/97% N2 at pH 7.0) to a solution simulating in vivo anoxic acidosis (VA), with elevated concentrations of lactate, Ca2+, Mg2+, and K+, and decreased Cl-, equilibrated with 10.8% CO2/89.2% N2 at pH 7.0. We examined mechanical properties on cardiac muscle strips and determined intracellular pH (pHi) and high energy phosphates on perfused hearts using 31P-NMR. Maximum active force (Fmax) and the maximum rate of force development (dF/dtmax) of muscle strips were significantly higher during VA than during LA superfusion. An elevation of Ca2+ alone (to 6 mM) in LA significantly increased both Fmax and dF/dtmax but the effects diminished toward the end of the exposure; however, hypercapnic anoxic lactic acidosis (addition of 20 mM HCO3- to LA, equilibrated with 10.8% CO2/89.2% N2, pH 7.0) did not significantly affect Fmax or dF/dtmax. During VA perfusion, pHi (6.73 +/- 0.01) was significantly higher than that during LA perfusion (pHi 6.69 +/- 0.013), but the difference is probably too small to have physiological significance. ATP, creatine phosphate, and inorganic phosphate were not significantly different in the two anoxic solutions. We conclude that the reduction of cardiac mechanical function in vivo is minimized by the integrated effects of changes of ionic concentrations, but the observed changes in Ca2+ and pHi cannot fully explain the effect.     

Biodegradation of cresol isomers in anoxic aquifers

The biodegradation of o-, m-, and p-cresol was examined in material obtained from a shallow anaerobic alluvial sand aquifer. The cresol isomers were preferentially metabolized, with p-cresol being the most easily degraded. m-Cresol was more persistent than the para-isomer, and o-cresol persisted for over 90 days. Biodegradation of cresol isomers was favored under sulfate-reducing conditions (SRC) compared with that under methanogenic conditions (MC). Slurries that were acclimated to p-cresol metabolism transformed this substrate at 18 and 330 nmol/h per g (dry weight) for MC and SRC, respectively. Inhibition of electron flow to sulfate reduction with 2.0 mM molybdate reduced p-cresol metabolism in incubations containing sulfate. When methanogenesis was blocked with 5 mM bromoethanesulfonic acid in incubations lacking sulfate, p-cresol catabolism was retarded. Under SRC 3.4 mol of sulfate was consumed per mol of p-cresol metabolized. The addition of sulfate to methanogenic incubations stimulated p-cresol degradation. Simultaneous adaptation studies in combination with spectrophotometric and chromatographic analysis of metabolites indicated that p-cresol was oxidized under SRC to p-hydroxybenzoate via the corresponding alcohol and aldehyde. This series of reactions was inhibited under sulfate-limited or aerobic conditions. Therefore, the primary catabolic event for p-cresol decomposition under SRC appears to involve the hydroxylation of the aryl methyl group.     

Photochemical synthesis of biomolecules under anoxic conditions

We report the long-wavelength UV anoxic photosynthesis of uracil, various sugars (including deoxyribose and glycoaldehyde), amino acids, and other organic photoproducts. These reactions occur in mixtures of water, calcium carbonate, formaldehyde and hydrazine. Our data demonstrate that under several sets of conditions biomolecules can be formed in variety and abundance from reduced compounds (formaldehyde and hydrazine) derived from anoxic dinitrogen/carbon dioxide environments. The formaldehyde concentrations were varied from 10 mM to 0.005 mM, and the hydrazine concentrations were varied from 1 mM to 0.01 mM. The highest of these reactant concentrations were 500 and 6 times greater than those reported for earlier experiments upon the synthesis of these precursors from CO₂ or N₂, while the lowest of reactant concentrations employed here were 0.5 (formaldehyde) and 0.006 (hydrazine). Product yields were greatest when the hydrazine/formaldehyde ratio was 1, and when the reactant concentrations were low. These data suggest that organic products can be formed in variety from those amounts of formaldehyde and hydrazine precursors which are themselves formed under anoxic UV photochemical conditions. Hence these various reactions would seem to have prebiotic relevance. The UV 254 nm photon flux employed was 100 times higher than unattenuated solar flux. Durations of UV exposure were 24 hrs and 72 hrs. No experiments have been addressed to the possibility of UV flux dependency.     

Accelerated phospholipid degradation in anoxic rat hepatocytes

Accelerated degradation of membrane phospholipids characterizes the reaction of rat liver and myocardial cells to ischemia. A similar disturbance in phospholipid metabolism was sought in anoxic hepatocytes. Primary cultures of adult rat hepatocytes were made anoxic by evacuation of the CO₂O₂ atmosphere with N₂. The resulting loss of ATP was reversible upon reoxygenation after periods of anoxia up to 2 h. With 3–4 h of anoxia, the cells were incapable of regenerating ATP levels. Loss of viability was also indicated by the inability of over 90% of the cells after 3–4 h to exclude trypan blue. The baseline rate of turnover of [¹⁴C]-ethanolamine or glycerol prelabeled phospholipids was then established. A constant rate of turnover was found for, at least, the first 3 days the cells were in culture. No loss of total phospholipid occurred during this time. Anoxia induced very significant differences in the fate of prelabeled phospholipids. With [¹⁴C]-ethanolamine there was a 30% loss of total cellular radioactivity within 4 h. Total phospholipids determined as lipid phosphate decreased by 20%. This depletion of cellular phospholipids was paralleled by an accumulation of hydrophilic degradation products in the culture medium. Phosphorylethanolamine accounted for 50% of these, with equal amounts of glycerophosphorylethanolamine and ethanolamine the other 50%. A similar accumulation in the medium occurred with [¹⁴C]-glycerol- and [¹⁴C]choline-prelabeled phospholipids. The accelerated degradation of phospholipid was accompanied by evidence of membrane dysfunction as shown by the loss of 50% of the glucose 6-phosphatase activity in whole cell homogenates. The results of these studies establish that anoxia induces in cultured rat hepatocytes a similar disturbance to phospholipid metabolism as does ischemia of the same cells in the intact animal. This implies that the deprivation of oxygen per se determines the characteristic reaction of cells to ischemia. This conclusion allows further analysis of the effects of O₂ deprivation on cultured hepatocytes as a new experimental model with which to further explore the effects of ischemia on cells.     

Digastric muscle activities in anoxic infant rats

The digastric muscle acts for both feeding (including mastication and swallowing) and respiration. In this study, we examined whether or not the muscle activity is detectable during anoxia in developing rats. Rats at 4 different ages, days 5, 10, 16, and 24, were exposed to 100% N2 under pentobarbital or ketamine-xylazine anesthesia, and the electromyograms of digastric muscles (dEMG) and the diaphragm (diaEMG) were examined simultaneously. Prior to the anoxic exposure, at all ages, the dEMG was similar to or less apparent than the diaEMG, which was detected at each inspiratory movement. In anoxia, we first observed dEMG activity, mostly sporadic (days 5 and 10) or mostly tonic (days 16 and 24), when diaEMG activity was temporarily suppressed (we termed it Phase 1). Second, synchronous phasic or tonic dEMG and phasic diaEMG were recorded temporarily before terminal apnea (we termed it Phase 2). These phenomena were also obtained in vagotomized rats (all ages) or in rats injected with the N-methyl-D-aspartate receptor antagonist MK-801 (dizocilpine (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate) (days 16 and 24). In conclusion, our results suggest that in anoxia, dEMG activity is detectable during diaEMG suppression in early anoxia, irrespective of the developmental age, the anesthetic (pentobarbital or ketamine-xylazine), vagotomy, or MK-801 injections.     

Phylogenetic diversity of transition and anoxic zone bacterial communities within a near‐shore anoxic basin: Nitinat Lake

At two stations surveyed in Nitinat Lake, a ～200‐m‐deep anoxic tidal fjord, sulfide was detected as close as 15 m from the surface. Biological characterization, determined from small subunit ribosomal RNA gene sequencing, of the chemocline and anaerobic zone revealed many sequences related to sulfur‐oxidizing bacteria, suggesting that sulfur cycling is a dominant process. γ‐ and ε‐Proteobacteria related to thiotrophic symbionts, as well as Chlorobium sp., dominated the transition zone. These are expected to play a role in dark and phototrophic CO₂ fixation, respectively. ε‐Proteobacteria phylotype abundance increased with depth, eventually comprising 69–97% of all sequences recovered from the anoxic zone. The vast majority (74%) of these phylotypes were affiliated with a novel Acrobacter sp. group (NITEP5). Quantification of NITEP5 revealed that up to 2.8 × 10⁵ cells ml^?1 were present in the anoxic zone. Surprisingly, although sequences related to known sulfate‐reducing bacteria were recovered from the transition zone, quantification of the dsr gene and ³⁵SO₄^2? uptake tests suggest that sulfate‐reduction within the water column is negligible. Overall, sequence diversity between different vertical zones was high, although the spatial segregation of γ‐Proteobacteria, Chlorobi, and ε‐Proteobacteria did not appear to vary significantly between seasons.     

Developmental regulation of anoxic stress tolerance in maize

Anoxia associated with flooding stress is detrimental to plant growth and productivity. When maize seedlings 2 to 7 d old were exposed to anoxic stress, 3-d-old seedlings were found to have much lower tolerance than 2-d-old seedlings. Ninety per cent of 2-d-old seedlings survived 72 h of anoxic stress compared with 0% of the 3-d-old seedlings. Since 2-d-old isolated root tips survived anoxic stress better than 3-d-old tips, the anoxic tolerance of 2-d-old seedlings was independent of the translocation of nutrient reserves from the endosperm to the root. The addition of glucose to the medium improved the anoxia tolerance of 2-d-old seedlings by 25% but had no effect on 3-d-old seedlings. Acclimation by pre-cxposure to 4% oxygen and pre-treatment with 100mmol m^?1 abscisic acid (ABA) improved the anoxia tolerance of 3-d-old seedlings by 2- and 4-fold, respectively. However, acclimation and ABA treatment had no effect on 2-d-old seedlings. The results indicate that anoxia tolerance in maize is develop-mentally regulated. The mechanism of anoxia tolerance innate to 2-d-old seedlings was inducible in 3-d-old seedlings by acclimation or treatment with ABA.     

Responses of intertidal nematodes to short-term anoxic events

We investigated tolerance to, and activity of, nematodes in hypoxic and anoxic/partially sulphidic conditions and their ability for recovery after reoxygenation of anoxic sediment. To this end, sediments from an intertidal flat were incubated under oxic, suboxic and anoxic/partially sulphidic conditions for a 14-day period and the final density of nematodes, as a group, and of the most abundant species were assessed. In one treatment, oxygen was restored after anoxic incubation. The incorporation of ¹³C, originating from labeled algae added on top of the sediment, was taken as an indication of nematode activity.Short-term suboxic and anoxic/partially sulphidic conditions had similar structuring impact on the nematode community, reducing total densities by about one third. Survival in suboxic and anoxic/partially sulphidic conditions was species-specific. Daptonema setosum, D. tenuispiculum and Chromadora macrolaima, dominant in the oxic incubation, disappeared when the oxygen level was reduced. The density of the other dominant species was slightly reduced (Sabatieria pulchra), similar (Terschellingia communis) or even increased in the suboxic and anoxic conditions (Metachromadora vivipara). The activity level of these three species was, however, reduced under oxygen limitation. Our results are discussed in terms of the life-history strategies of these species.     

Geomorphology modification and its impact to anoxic lagoons

Anoxia constitutes an important environmental problem, affecting coastal systems around the world. The physicochemical alterations on the water column of anoxic basins, caused by morphological modifications, were studied.Deepening on the connecting sill between the permanent anoxic Aitoliko lagoon and Messolonghi lagoon was accomplished on May 2006. Seasonal variations of parameters like temperature, salinity and dissolved oxygen along the lagoon's water column were recorded and studied in a net of 14 stations, after sill's dredging. Wind speed and wind direction time series were used to estimate the wind's contribution to the hydrographical changes.The water fluxes between the two environments increased, due to the sill's cross section increase. Salty water inflow into Aitoliko lagoon was recorded during the sampling period and was correlated with monimolimnion oxygenation throughout winter months. The meteorological conditions that prevailed during the sampling period could not create strong water inflows into the Aitoliko lagoon, and consequently it was not the reason for the recorded alterations in the lagoon's water body anoxia.The limited deepening of the sill created a mild increase of water flow into the anoxic lagoon. This inflow of the saltier water resulted in a weak mixing of the water column, introducing oxygen into the bottom water for the first time in 55 years, without destroying the stratification.