Intracellular NO3 Research Articles

Metabolic preference of nitrate over oxygen as an electron acceptor in foraminifera from the Peruvian oxygen minimum zone

Benthic foraminifera populate a diverse range of marine habitats. Their ability to use alternative electron acceptors-nitrate (NO3-) or oxygen (O2)-makes them important mediators of benthic nitrogen cycling. Nevertheless, the metabolic scaling of the two alternative respiration pathways and the environmental determinants of foraminiferal denitrification rates are yet unknown. We measured denitrification and O2 respiration rates for 10 benthic foraminifer species sampled in the Peruvian oxygen minimum zone (OMZ). Denitrification and O2 respiration rates significantly scale sublinearly with the cell volume. The scaling is lower for O2 respiration than for denitrification, indicating that NO3- metabolism during denitrification is more efficient than O2 metabolism during aerobic respiration in foraminifera from the Peruvian OMZ. The negative correlation of the O2 respiration rate with the surface/volume ratio is steeper than for the denitrification rate. This is likely explained by the presence of an intracellular NO3- storage in denitrifying foraminifera. Furthermore, we observe an increasing mean cell volume of the Peruvian foraminifera, under higher NO3- availability. This suggests that the cell size of denitrifying foraminifera is not limited by O2 but rather by NO3- availability. Based on our findings, we develop a mathematical formulation of foraminiferal cell volume as a predictor of respiration and denitrification rates, which can further constrain foraminiferal biogeochemical cycling in biogeochemical models. Our findings show that NO3- is the preferred electron acceptor in foraminifera from the OMZ, where the foraminiferal contribution to denitrification is governed by the ratio between NO3- and O2.

Physiological and metabolic profiles of common reed provide insights into plant adaptation to low nitrogen conditions

One of the most distinct features of the common reed (Phragmites australis) is its ability to survive under extremely low nitrogen conditions. To explore the regulation mechanisms of reed to adapt to nitrogen deficiency, we treated reed seedlings under long-term extremely low nitrogen conditions and profiled the physiological and metabolic features of photosynthesis, metabolism, growth, nutrient balance, and enzyme activities. Unexpectedly, the photosynthesis, biomass and carbon content were still maintained at high levels in reed under N-deficient conditions regardless of the decreased content of chlorophyll and nitrogenous compounds. Using mass spectrometry, we profiled metabolism of 627 metabolites and found the concentrations of lactic acid and galactinol were accumulated under the treatment. The development of underground organs and nutrient accumulation (B, P, Zn and Na) were also enhanced under the condition. Unlike the positive correlation of nitrate reductases and N levels in other plants, we found the catalytic activities of nitrate reductases were dramatically elevated in roots under the N-deficient condition, which may increase the intracellular NO3− and NH4+ levels. Our experiments characterized the unique features of reed under extreme nitrogen deficiency conditions and also provided valuable information for other corps to develop the cultivars with high yield under low nitrogen input.

Anion Recognition Triggered Nanoribbon-Like Self-Assembly: A Fluorescent Chemosensor for Nitrate in Acidic Aqueous Solution and Living Cells.

A water-soluble π-conjugated bispyridinium phenylenevinylene-based fluorogenic probe has been developed as a novel fluorescent chemosensor for highly selective, sensitive, and rapid detection of NO3(-) anion in acidic aqueous media. This system self-assembles to a nanoribbon as a result of ionic interaction. The positively charged chemosensor generates a nearly instantaneous significant fluorescence signal (475 vs 605 nm) in response to NO3(-) in the green/yellow spectral region, with a large Stokes shift (130 nm). The fluorescence changes can be attributed to the self-aggregation of the sensor triggered by ionic interaction, which occurs as a consequence of the subtle cooperation of electrostatic ionic bonding, van der Waals forces, and π-stacking of the π-conjugated aromatic moieties. Importantly, this chemosensor has been employed for the first time for the fluorescence detection of intracellular NO3(-) anion in cultured cells.

Benthic mineralization and solute exchange on a Celtic Sea sand-bank (Jones Bank)

Benthic carbon mineralization and solute exchange was investigated on a Celtic Sea sandbank during July 2008. The sediment on the top of the bank consisted of consolidated sand, characterized by advective porewater transport and was difficult to sample, this to some extent compromised the investigations at this site. However, intact sediment cores were sampled at 4 stations at the slopes, base of the bank and off the bank (reference site). This sediment was used to assess rates and pathways for benthic diagenesis. Total sediment O2 uptake (TOU) ranged from 5.8 to 9.0mmolm−2d−1 and total sediment release rates of dissolved inorganic carbon (DIC) ranged between 8.60 and 13.8mmolm−2d−1. Microbial denitrification and sulfate reduction accounted for <2% and 12–28% of the total benthic carbon mineralization, respectively. The remaining mineralization is ascribed to O2 and Fe/Mn respiration, respectively. Activity profiles of unsupported 210Pb from all stations indicated deep mixing, presumably caused by intense trawling activity in the area. Calculation based on satellite tracking of fishing vessels suggest that on average 33% of the sediment is affected by trawling activity every year. This is presumed to facilitate high metal respiration rates by continuously oxidizing reduced Fe that would otherwise accumulate in the sediment. Extracted porewater profiles reflected elevated NO3- levels as compared to microsensor determined NO3- profiles that were measured in parallel. We suggest that this reflects NO3- leakage from meiofauna – and that the intracellular NO3- pool in the top 5cm of the sediment exceeds the porewater pool up to a factor of 45. However, this intracellular pool is presumably turned over at very slow rates, compared to the porewater pool subject to microbial denitrification.

Interactive effect of salicylic acid on some physiological features and antioxidant enzymes activity in ginger (Zingiber officinale Roscoe).

The effect of foliar salicylic acid (SA) applications (10−3 and 10−5 M) on activities of nitrate reductase, guaiacol peroxidase (POD), superoxide dismutases (SOD), catalase (CAT) and proline enzymes and physiological parameters was evaluated in two ginger varieties (Halia Bentong and Halia Bara) under greenhouse conditions. In both varieties, tested treatments generally enhanced photosynthetic rate and total dry weight. Photosynthetic rate increases were generally accompanied by increased or unchanged stomatal conductance levels, although intercellular CO2 concentrations of treated plants were typically lower than in controls. Lower SA concentrations were generally more effective in enhancing photosynthetic rate and plant growth. Exogenous application of SA increased antioxidant enzyme activities and proline content; the greatest responses were obtained in plants sprayed with 10–5 M SA, with significant increases observed in CAT (20.1%), POD (45.2%), SOD (44.1%) and proline (43.1%) activities. Increased CAT activity in leaves is naturally expected to increase photosynthetic efficiency and thus net photosynthesis by maintaining a constant CO2 supply. Our results support the idea that low SA concentrations (10–5 M) may induce nitrite reductase synthesis by mobilizing intracellular NO3− and can provide protection to nitrite reductase degradation in vivo in the absence of NO3–. Observed positive correlations among proline, SOD, CAT and POD activities in the studied varieties suggest that increased SOD activity was accompanied by increases in CAT and POD activities because of the high demands of H2O2 quenching.

Diatoms respire nitrate to survive dark and anoxic conditions

Diatoms survive in dark, anoxic sediment layers for months to decades. Our investigation reveals a correlation between the dark survival potential of marine diatoms and their ability to accumulate NO(3)(-) intracellularly. Axenic strains of benthic and pelagic diatoms that stored 11-274 mM NO(3)(-) in their cells survived for 6-28 wk. After sudden shifts to dark, anoxic conditions, the benthic diatom Amphora coffeaeformis consumed 84-87% of its intracellular NO(3)(-) pool within 1 d. A stable-isotope labeling experiment proved that (15)NO(3)(-) consumption was accompanied by the production and release of (15)NH(4)(+), indicating dissimilatory nitrate reduction to ammonium (DNRA). DNRA is an anaerobic respiration process that is known mainly from prokaryotic organisms, and here shown as dissimilatory nitrate reduction pathway used by a eukaryotic phototroph. Similar to large sulfur bacteria and benthic foraminifera, diatoms may respire intracellular NO(3)(-) in sediment layers without O(2) and NO(3)(-). The rapid depletion of the intracellular NO(3)(-) storage, however, implies that diatoms use DNRA to enter a resting stage for long-term survival. Assuming that pelagic diatoms are also capable of DNRA, senescing diatoms that sink through oxygen-deficient water layers may be a significant NH(4)(+) source for anammox, the prevalent nitrogen loss pathway of oceanic oxygen minimum zones.

Nitrogen cycling in a deep ocean margin sediment (Sagami Bay, Japan)

On the basis of in situ NO3−1 microprofiles and chamber incubations complemented by laboratory‐based assessments of anammox and denitrification we evaluate the nitrogen turnover of an ocean margin sediment at 1450‐m water depth. In situ NO3−1 profiles horizontally separated by 12 mm reflected highly variable NO3−1 penetration depths, NO3−1 consumption rates, and nitrification. On average the turnover time of the pore‐water NO3−1 pool was ~0.2 d. Net release of NH4+ during mineralization (0.95 mmol m−2 d−1) sustained a net efflux of ammonia (53%), nitrification (24%), and anammox activity (23%). The sediment had a relatively high in situ net influx of NO3−1 (1.44 mmol m−2 d−1) that balanced the N2 production as assessed by onboard tracer experiments. N2 production was attributed to prokaryotic denitrification (59%), anammox (37%), and foraminifera‐based denitrification (4%). Anammox thereby represented an important nutrient sink, but the N2 production was dominated by denitrification. Despite the fact that NO3−1 stored inside foraminifera represented ~80% of the total benthic NO3−1 pool, the slow intracellular NO3−1 turnover that, on average, sustained foraminifera metabolism for 12‐52 d, contributed only to a minor extent to the overall N2 production. The microbial activity in the surface sediment is a net nutrient sink of ~1.1 mmol N m−2 d−1, which aligns with many studies performed in coastal and shelf environments. Continental margin areas can act as significant N sinks and play an important role in regional N budgets.

Predicting Copper Toxicity with Its Intracellular or Subcellular Concentration and the Thiol Synthesis in a Marine Diatom

The accumulation and subcellular distribution of copper (Cu) in a marine diatom Thalassiosira weissflogii were examined under different nutrient conditions [nitrogen- (-N) and phosphorus-starved (-P) conditions as well as nutrient-enriched conditions with nitrate (+NO3-) and ammonium (+NH4+) as the nitrogen source]. Cu accumulation was induced in the NO3--exposed (+N03- and -P) cells, suggesting that Cu may be directly or indirectly required for intracellular NO3- reduction. The relationships between the response of the cell-specific growth rate or photosynthetic system II maximum quantum yield ((DM) to Cu exposure in different nutrient-conditioned cells and the free copper ion concentration,the intracellular Cu concentration (intra-Cu), and the distribution of Cu in different subcellular compartments were also examined. We found that the intra-Cu was the best Cu toxicity predictor, as it accounted for most of the Cu-induced PhiM response between different nutrient-conditioned cells. The synthesis of intracellular cysteine and five low molecular weight (LMW) thiols was not notably induced at high Cu levels possibly because of the existence of some other Cu detoxification mechanisms. This was further demonstrated by the much lower cysteine and LMW thiol contents in the -N cells with a similar Cu tolerance to the nutrient-enriched cells. Overall, our results suggest that Cu toxicity could be better predicted with the intra-Cu and its subcellular distribution as compared with the currently used free ion activity model and biotic ligand model. However, the LMW thiols had unexpectedly little contribution to Cu detoxification in T. weissflogii.

Ecology of Thioploca spp.: nitrate and sulfur storage in relation to chemical microgradients and influence of Thioploca spp. on the sedimentary nitrogen cycle.

Microsensors, including a recently developed NO3(-) biosensor, were applied to measure O(2) and NO3(-) profiles in marine sediments from the upwelling area off central Chile and to investigate the influence of Thioploca spp. on the sedimentary nitrogen metabolism. The studies were performed in undisturbed sediment cores incubated in a small laboratory flume to simulate the environmental conditions of low O(2), high NO3(-), and bottom water current. On addition of NO3(-) and NO2(-), Thioploca spp. exhibited positive chemotaxis and stretched out of the sediment into the flume water. In a core densely populated with Thioploca, the penetration depth of NO3(-) was only 0.5 mm and a sharp maximum of NO3(-) uptake was observed 0.5 mm above the sediment surface. In sediments with only few Thioploca spp., NO3(-) was detectable down to a depth of 2 mm and the maximum consumption rates were observed within the sediment. No chemotaxis toward nitrous oxide (N2O) was observed, which is consistent with the observation that Thioploca does not denitrify but reduces intracellular NO3(-) to NH(4)(+). Measurements of the intracellular NO3(-) and S(0) pools in Thioploca filaments from various depths in the sediment gave insights into possible differences in the migration behavior between the different species. Living filaments containing significant amounts of intracellular NO3(-) were found to a depth of at least 13 cm, providing final proof for the vertical shuttling of Thioploca spp. and nitrate transport into the sediment.

NO3--induced pH changes in mammalian cells. Evidence for an NO3--H+ cotransporter.

The effect of NO3- on intracellular pH (pHi) was assessed microfluorimetrically in mammalian cells in culture. In cells of human, hamster, and murine origin addition of extracellular NO3- induced an intracellular acidification. This acidification was eliminated when the cytosolic pH was clamped using ionophores or by perfusing the cytosol with highly buffered solutions using patch-pipettes, ruling out spectroscopic artifacts. The NO3-- induced pH change was not due to modulation of Na+/H+ exchange, since it was also observed in Na+/H+ antiport-deficient mutants. Though NO3- is known to inhibit vacuolar-type (V) H+-ATPases, this effect was not responsible for the acidification since it persisted in the presence of the potent V-ATPase inhibitor bafilomycin A1. NO3-/HCO3- exchange as the underlying mechanism was ruled out because acidification occurred despite nominal removal of HCO3-, despite inhibition of the anion exchanger with disulfonic stilbenes and in HEK 293 cells, which seemingly lack anion exchangers (Lee, B. S., R.B. Gunn, and R.R. Kopito. 1991. J. Biol. Chem. 266:11448- 11454). Accumulation of intracellular NO3-, measured by the Greiss method after reduction to NO2-, indicated that the anion is translocated into the cells along with the movement of acid equivalents. The simplest model to explain these observations is the cotransport of NO3- with H+ (or the equivalent counter-transport of NO3- for OH-). The transporter appears to be bi-directional, operating in the forward as well as reverse directions. A rough estimate of the fluxes of NO3- and acid equivalents suggests a one-to-one stoichiometry. Accordingly, the rate of transport was unaffected by sizable changes in transmembrane potential. The cytosolic acidification was a saturable function of the extracellular concentration of NO3- and was accentuated by acidification of the extracellular space. The putative NO3--H+ cotransport was inhibited markedly by ethacrynic acid and by alpha-cyano-4-hydroxycinnamate, but only marginally by 4, 4'-diisothiocyanostilbene-2,2' disulfonate or by p-chloromercuribenzene sulfonate. The transporter responsible for NO3--induced pH changes in mammalian cells may be related, though not identical, to the NO3--H+ cotransporter described in Arabidopsis and Aspergillus. The mammalian cotransporter may be important in eliminating the products of NO metabolism, particularly in cells that generate vast amounts of this messenger. By cotransporting NO3- with H+ the cells would additionally eliminate acid equivalents from activated cells that are metabolizing actively, without added energetic investment and with minimal disruption of the transmembrane potential, inasmuch as the cotransporter is likely electroneutral.

CHEMICAL COMPOSITION AND PHOTOSYNTHETIC CHARACTERISTICS OF ETHMODISCUS REX (BACILLARIOPHYCEAE): EVIDENCE FOR VERTICAL MIGRATION1

ABSTRACT Ethmodiscus rex (Rattray) Wiseman and Hendey cells from near surface net tows in the Southwest Atlantic Ocean and Caribbean Sea were examined for chemical composition, internal nutrient pool concentrations, and oxygen evolution characteristics. Elemental ratios indicated nitrogen limitation with C:N:P ratios of 125:9:1 (atoms), and carbon: chlorophyll (chl) ratios of 129:1 (weight). However, internal nitrate pools (1.4–27.1 mM) suggested that cells were not N‐limited. Intracellular NO3− accounted for up to 54% (range = 3–54%) of the total N quota in some samples. Photosynthetic parameters were consistent with a high‐light‐adapted population and suggested an instantaneous maximum chl‐specific photosynthetic rate (PBmax) of 4.8–12.4 nmol O2·μg chl−1· h−1. Respiration rates varied ten‐fold and were inversely related to PBmax Ethmodiscus chemical composition and buoyancy characteristics are similar to vertically migrating Rhizosolenia mats and the non‐motile dinoflagellate Pyrocystis noctiluca Murray (Schuett). The presence of internal NO3− pools in Ethmodiscus suggests that this genus is also vertically migrating to exploit sub‐surface nitrogen pools. Such behavior may be widespread in large, non‐motile oceanic phytoplankton. Based on ascent rate data, chemical composition, and photosynthetic rates, we estimate that the entire division–migration cycle for Ethmodiscus requires at least 7–12 days.

Nitrate Fluxes and Nitrate Reductase Activity of Suspension-Cultured Tobacco Cells (Effects of Internal and External Nitrate Concentrations).

Cell suspensions of tobacco (Nicotiana tabacum L., cv KY14) were used to determine the responses of NO3- uptake and NO3- reductase activity (NRA) to exogenous NO3- levels in the absence of long-distance NO3- transport. Tobacco cells grown with complete Murashige and Skoog medium for 7 d were subcultured for 3 d with NH4+-free media containing 0, 5, 10, 20, 30, and 40 mM NO3-. Cell NO3-, in vitro NRA, NO3- influx, and efflux of cell NO3- were determined. The NRA increased as cell NO3- increased. Cell NO3- efflux values increased as cell NO3- level increased. Cells with low intracellular NO3- had greater NO3- influx than cells with high intracellular NO3-. Woolf-Augustinsson-Hofstee transformations of the NO3- influx kinetic data revealed patterns characteristic of a high- and low-affinity two-component NO3- uptake system. Apparent Vmax values generally decreased and Km values increased as cell NO3- concentration increased. The NRA of cells supplied with 10 and 20 mM NO3- after 3-d growth in N- free medium increased about 5-fold within 2 h and then remained constant for the next 2 h, whereas NRA of cells supplied with 5 mM NO3- increased only 2-fold during the 4-h period. Intracellular NO3- and other N metabolites associated with cell NO3- levels exerted differential effects on the NO3- influx activity and NRA of tobacco cells cultured in suspension. Expression of high NRA was correlated with both high external and intracellular NO3-, whereas maximum NO3- influx activity required a low (depleted) level of cell NO3-.

Inorganic nitrogen metabolism in Ulva rigida illuminated with blue light

Inorganic nitrogen metabolism in blue light was studied for the green algaUlva rigida C. Agardh collected in the south of Spain (Punta Carnero, Algeciras) in the winter of 1987. NH4+ has been reported to inhibit NO3- uptake; however,U. rigida showed a net NO3- uptake even when the NH4+ concentration of the external medium was three or four times greater than the concentration of NO3-. NO3- uptake rates were similar in both darkness and in blue light of various photon fluence rates (PFR) ranging from 17 to 160 μmol m-2 s-1. Since NO3- uptake is an active mechanism involving the consumption of ATP, respiratory metabolism can provide enough ATP to maintain the energetic requirement of NO3- transport even in darkness. In contrast, NO3- reduction inU. rigida was highly dependent on the net photosynthetic rate. After 7 h in blue light, intracellular NO3- concentrations ([NO3-] i ) were higher in specimens exposed to intensities below the light compensation point (LCP) than in those incubated at a PFR above the LCP. When PFR is below the light compensation point, NO3- reduction is low, probably because all the NADH produced by the cells is oxidized in the respiratory chain in order to produce ATP to maintain a steady NO3- transport rate. The total nitrogen (TN) and carbon (TC) contents decreased from darkness to 33 μmol m-2 s-1 in blue light. In this range, catabolic processes prevailed over anabolic ones. In contrast, increases in TN and TC contents were observed above the light compensation point. The C : N ratio increased with light intensity, reaching a stable value of 17 at 78 μmol m-2 s-1 in blue light. Intracellular NO3- concentration and NO3- reduction appear to be directly controlled by light intensity. This external control of [NO3-]i and the small capacity ofU. rigida to retain incorporated NO3-, NO2- and NH4+ ions may explain its nitrophilic character.

OSMOTIC ADJUSTMENT IN MARINE EUKARYOTIC ALGAE: THE ROLE OF INORGANIC IONS, QUATERNARY AMMONIUM, TERTIARY SULPHONIUM AND CARBOHYDRATE SOLUTES: I. DIATOMS AND A RHODOPHYTE.

The unicellular marine algae, Phaeodactylum tricornutum Bohlin, Cyclotella cryptica Reimann and Cyclotella meneghiniana Kützing (Bacillariophyceae) and Porphyridium aerugineum Geitler (Rhodophyceae) synthesized and accumulated glycine betaine and proline in response to increases of the NaCl concentration (150 to 1000 mol m-3 NaCl) of the growth medium. C. cryptica and C. meneghiniana also synthesized and accumulated homarine (N-methyl picolinic acid betaine). Both P. tricornutum and P. aerugineum synthesized increasing amounts of intracellular glycerol and P. aerugineum also formed the heteroside, floridoside [O-α-D-galactopyranosyl (1 → 2)-glycerol], in response to the elevated salinities. No major low molecular weight carbohydrates were found in Cyclotella. Sucrose was not detected in the algal extracts. Only P. tricornutum synthesized the tertiary sulphonium compound, β-dimethylsulphoniopropionate (DMSP), and the quantity of this solute in the alga was dependent on the amount of NaCl in the medium. Intracellular K+ concentrations in the algae were three to six times greater than those of Na+ . Increases of the salinity of the media led to the uptake and accumulation of K+ by the cells, and smaller increases of Na+ and Cl-1 and loss of intracellular NO3 - . The inorganic cations Na+ and K+ , with their accompanying anions, and the estimated organic solutes could largely account for the osmotic balance of P. tricornutum and P. aerugineum.

Nitrate uptake and assimilation in Thalassiosira weissflogii and Phaeodactylum tricornutum: interactions with photosynthesis and with the uptake of other ions

Interactions of the nitrate, phosphate, and ammonium uptake systems and the interactions of these systems with photosynthesis were investigated for Thalassiosira weissflogii and Phaeodactylum tricornutum preconditioned in continuous culture. The cultures were supplied with NO-3and PO=4in an N:P atomic ratio of 15:1, and residual concentrations of both nutrients in the growth chamber were very low. The rate of NO-3uptake was reduced by the addition of NH+4or PO=4. The rate of PO=4uptake by T. weissflogii was reduced by the addition of NH+4. The rate of carbon fixation was reduced by NO-3additions and slightly reduced by the addition of PO=4. There were two components of NO-3uptake, one light-dependent and one light-independent. Uptake inhibition by added PO=4acted on the light-independent component. The change in the C fixation rate due to added NO-3was equal to the rate of NO-3uptake by the light-dependent component on a molar basis. Nitrate assimilation (reduction) rates were calculated from the time course of extracellular and intracellular NO-3concentrations. The light-dark change in the assimilation rate was similar to the light-dark change in the uptake rate, suggesting close coupling between the light-dependent components of uptake and assimilation. The assimilation rate dropped upon exhaustion of extracellular NO-3, implying that an uptake-coupled component of assimilation is unavailable for the assimilation of intracellular NO-3. The reduction in the C fixation rate due to NO-3was temporally associated with uptake rather than assimilation, but may reflect interaction with either the light-dependent uptake step or the closely coupled assimilation. Phosphate additions reduced the rate of NO-3uptake, while the rate of assimilation was unaffected.