Nitrate Efflux Research Articles

Inflammation-induced sialin mediates nitrate efflux in dysfunctional endothelium affecting NO bioavailability

AimThe mechanism of NO bioavailability in endothelial dysfunction, the trigger for atherogenesis is still unclear as exogenous nitrate therapy fails to alleviate endothelial dysfunction. Recently, sialin, a nitrate transporter, has been linked to affect tissue nitrate/nitrite levels. Hence, we investigated the role of sialin in NO bioavailability in endothelial dysfunction. MethodsSerum-starved HUVECs were stimulated with either TNFα or AT-2 for 24 h either alone or in the presence of autophagy inducer or autophagy inhibitor alone. Nitric oxide, nitrite, and nitrate levels were measured in cell supernatant and cell lysate. Quantitative real-time PCR, Annexin V-PI, and monocyte adhesion assays were performed. Immunofluorescence staining for sialin, vWF, and LC3 was performed. STRING database was used to create protein interacting partners for sialin. ResultsSialin is strongly expressed in activated EC in vitro and atherosclerotic plaque as well as tumor neo-vessel ECs. Sialin mediates nitrate ion efflux and is negatively regulated by autophagy via mTOR pathway. Blocking sialin enhances NO bioavailability, autophagy, cell survival, and eNOS expression while decreasing monocyte adhesion. PPI shows LGALS8 to directly interact with sialin and regulate autophagy, cell-cell adhesion, and apoptosis. ConclusionSialin is a potential novel therapeutic target for treating endothelial dysfunction in atherosclerosis and cancer.

Changes in isotope fractionation during nitrate assimilation by marine eukaryotic and prokaryotic algae under different pH and CO2 conditions

AbstractThe impact of environmental factors on nitrogen (N) and oxygen (O) isotope effects during algal nitrate assimilation causes uncertainty in the field application of sedimentary N isotope records and nitrate isotopes to understand the marine nitrogen cycle. Ocean acidification is predicted to change nitrogen cycling including nitrate assimilation, but how N and O isotope effects during algal nitrate assimilation vary in response to changes in seawater pH and partial pressure CO2 (pCO2) remains unknown. We measured N and O isotope effects during nitrate assimilation and physiological states of the marine diatom Thalassiosira weissflogii and Synechococcus under different pH (8.1 or 7.8) and pCO2 (400 or 800 μatm) conditions. Low pH and/or high pCO2 equally decreased N and O isotope effects during nitrate assimilation by diatoms possibly due to reducing cellular nitrate efflux/uptake ratio and decreased isotope effects for nitrate uptake, whereas they did not affect those by Synechococcus with low intracellular nitrate concentration and limited nitrate efflux. Our results provide compelling experimental evidence showing different changes in N and O isotope effects during nitrate assimilation by marine eukaryotic and prokaryotic phytoplankton at low pH and/or high pCO2. These findings suggest new insight into environmental controls on variability in the isotope effect during algal nitrate assimilation, and have implications for improving a predictive understanding of N and O isotope tools in acidified oceans.

CHLORIDE CHANNEL-b mediates vacuolar nitrate efflux to improve low nitrogen adaptation in Arabidopsis.

The vacuole is an important organelle for nitrate storage, and the reuse of vacuolar nitrate under nitrate starvation helps plants adapt to low-nitrate environments. CHLORIDE CHANNEL-b (CLC-b) in the vacuolar membrane is a nitrate transporter; however, its regulation and effects on nitrate efflux have not been established. Here, we evaluated CLC-b expression and its effects on physiological parameters under low nitrate conditions. CLC-b expression increased significantly in the roots of wild-type Arabidopsis (Arabidopsis thaliana) Col-0 under nitrate starvation. Under low nitrate, clcb mutants showed reductions in chlorophyll content and xylem sap nitrate concentration, shoot/root nitrate ratios, shoot/root total N ratios, and biomass. CLC-b-overexpression yielded opposite phenotypes and increased nitrogen use efficiency. CLC-b mutants showed elevated chlorate tolerance and an increased proportion of vacuolar nitrate relative to the total protoplast nitrate content as compared to the wild type. Yeast 1-hybrid, EMSA, and chromatin immunoprecipitation (ChIP) experiments showed that HRS1 HOMOLOG2 (HHO2), the expression of which is downregulated under low nitrate, binds directly to the promoter of CLC-b. clcb/hho2 double mutants and HHO2-overexpressing clcb plants had similar phenotypes under low nitrate to those of clcb single mutants. Thus, CLC-b mediates vacuolar nitrate efflux and is negatively regulated by HHO2, providing a theoretical basis for improving plant adaptability to low nitrate.

The cassava (Manihot-esculenta Crantz)'s nitrate transporter NPF4.5, expressed in seedling roots, involved in nitrate flux and osmotic stress

AtNPF4.5/AIT2, which was predicted to be a low-affinity transporter capable for nitrate uptake, was screened by ABA receptor complex in Arabidopsis ten years ago. However, the molecular and biochemical characterizations of AtNPF4.5 in plants remained largely unclear. In this study, the function of a plasma-membrane-localized and root-specifically-expressed gene MeNPF4.5 (Manihot-esculenta NITRATE TRANSPORTER 1 PTR FAMILY4.5), an ortholog of the Arabidopsis thaliana NPF4.5, was investigated in cassava roots as a nitrate efflux transporter on low nitrate medium and an influx transporter following exposure to high concentration of external nitrates. Moreover, RNA interference (RNAi) of MeNPF4.5 reduced the nitrate efflux capacity but the overexpressing cassava seedlings increased the ability of efflux from the elongation to the mature zone of root under low nitrate treatments. Besides, MeNPF4.5-RNAi expression reduced the nitrate influx capacity but enhanced nitrate absorption in parts of overexpressing plants from the meristem, elongation to mature zone of roots under high nitrate conditions. Furthermore, MeNPF4.5-RNAi seedlings survived owing to roots that could grow normally, but the MeNPF4.5-over-expressors showed adverse growth under 7% PEG6000 stress, suggesting that MeNPF4.5 negatively regulated the osmotic stress and was involved in nitrate flux through cassava seedlings.

Vacuolar nitrate efflux requires multiple functional redundant nitrate transporter in Arabidopsis thaliana.

Nitrate in plants is preferentially stored in vacuoles; however, how vacuolar nitrate is reallocated and to which biological process(es) it might contribute remain largely elusive. In this study, we functionally characterized three nitrate transporters NPF5.10, NPF5.14, and NPF8.5 that are tonoplast-localized. Ectopic expression in Xenopus laevis oocytes revealed that they mediate low-affinity nitrate transport. Histochemical analysis showed that these transporters were expressed preferentially in pericycle and xylem parenchyma cells. NPF5.10, NPF5.14, and NPF8.5 overexpression significantly decreased vacuolar nitrate contents and nitrate accumulation in Arabidopsis shoots. Further analysis showed that the sextuple mutant (npf5.10 npf5.14 npf8.5 npf5.11 npf5.12 npf5.16) had a higher 15NO3-uptake rate than the wild-type Col-0, but no significant difference was observed for nitrate accumulation between them. The septuple mutant (npf5.11 npf5.12 npf5.16 npf5.10 npf5.14 npf8.5 clca) generated by using CRISPR/Cas9 showed essentially decreased nitrate reallocation compared to wild type when exposed to nitrate starvation, though no further decrease was observed when compared to clca. Notably, NPF5.10, NPF5.14, and NPF8.5 as well as NPF5.11, NPF5.12, and NPF5.16 were consistently induced by mannitol, and more nitrate was detected in the sextuple mutant than in the wild type after mannitol treatment. These observations suggest that vacuolar nitrate efflux is regulated by several functional redundant nitrate transporters, and the reallocation might contribute to osmotic stress response other than mineral nutrition.

Shorebirds Affect Ecosystem Functioning on an Intertidal Mudflat

Ecosystem functioning and services have provided a rationale for conservation over the past decades. Intertidal muddy sediments, and the microphytobenthic biofilms that inhabit them, perform crucial ecosystem functions including erosion protection, nutrient cycling and carbon sequestration. It has been suggested that predation on sediment macrofauna by shorebirds may impact biofilms, and shorebirds are known to consume biofilm, potentially causing significant top-down effects on mudflat ecosystem functioning. We carried out an exclusion experiment on the Colne Estuary, Essex, to examine whether shorebird presence significantly affects sediment erodibility measured with a Cohesive Strength Meter (CSM) and microphytobenthos biomass measured using PAM fluorescence (Fo) and chlorophyll a content. We also tested for treatment effects on sediment-water nutrient fluxes [nitrate, nitrite, ammonia, phosphate and dissolved organic carbon (DOC)] during periods of both dark and light incubation. Excluding shorebirds caused statistically significant changes in regulating and provisioning ecosystem functions, including mudflat erodibility and nutrient fluxes. The presence of shorebirds lowered the sediment critical erosion threshold τcr, reduced nitrate fluxes into the sediment under illumination, lowered nitrate efflux, and reduced phosphate uptake, compared to sediments where birds were excluded. There were no significant differences in macrofauna community composition within the sediment between treatments after 45 days of bird exclusion, suggesting a direct link between shorebird presence or absence and the significant differences in biofilm-related variables. This study introduces previously unknown effects of shorebird presence on ecosystem functions within this system and highlights an area of shorebird science that could aid joint conservation and human provisioning action.

Solvent stress-induced changes in membrane fatty acid composition of denitrifying bacteria reduce the extent of nitrogen stable isotope fractionation during denitrification

Microcosm experiments with the well-studied denitrifier Thaurera aromatica show a link between a higher maximum membrane concentration (MMC) of the toxic organic solvents 1-octanol and 4-chlorophenol and a higher degree of saturation (DoS) of the fatty acids in the cell membrane. This coincides with less pronounced stable isotope fractionation during denitrification. We suggest that the change in cell membrane fluidity and the cell’s stress response leads to a decrease in nitrate transport across the cell membrane and/or an increase in the relative ratio of respiratory nitrate reduction rate versus efflux of unreacted nitrate. Both models show that the apparent kinetic isotope effect (AKIE) approach unity and thus reduce the extent of the resulting stable isotope enrichment factor ε15N-NO3− in dissolved nitrate during denitrification, as experimentally and mathematically shown in this study. This may lead to an underestimation of nitrate reduction determined by nitrate stable isotope analysis in aquatic habitats where various types of stresses may affect the physiology of the driving microorganisms.

The S-Type Anion Channel ZmSLAC1 Plays Essential Roles in Stomatal Closure by Mediating Nitrate Efflux in Maize.

Diverse stimuli induce stomatal closure by triggering the efflux of osmotic anions, which is mainly mediated by the main anion channel SLAC1 in plants, and the anion permeability and selectivity of SLAC1 channels from several plant species have been reported to be variable. However, the genetic identity as well as the anion permeability and selectivity of the main S-type anion channel ZmSLAC1 in maize are still unknown. In this study, we identified GRMZM2G106921 as the gene encoding ZmSLAC1 in maize, and the maize mutants zmslac1-1 and zmslac1-2 harboring a mutator (Mu) transposon in ZmSLAC1 exhibited strong insensitive phenotypes of stomatal closure in response to diverse stimuli. We further found that ZmSLAC1 functions as a nitrate-selective anion channel without obvious permeability to chloride, sulfate and malate, clearly different from SLAC1 channels of Arabidopsis thaliana, Brassica rapa ssp. chinensis and Solanum lycopersicum L. Further experimental data show that the expression of ZmSLAC1 successfully rescued the stomatal movement phenotypes of the Arabidopsis double mutant atslac1-3atslah3-2 by mainly restoring nitrate-carried anion channel currents of guard cells. Together, these findings demonstrate that ZmSLAC1 is involved in stomatal closure mainly by mediating the efflux of nitrate in maize.

OsNRT2.4 encodes a dual-affinity nitrate transporter and functions in nitrate-regulated root growth and nitrate distribution in rice

Plant NRT2 nitrate transporters commonly require a partner protein, NAR2, for transporting nitrate at low concentrations, but their role in plants is not well understood. In this study, we characterized the gene for one of these transporters in the rice genome, OsNRT2.4, in terms of its activity and roles in rice grown in environments with different N supply. In Xenopus oocytes, OsNRT2.4 alone without OsNAR2 co-expression facilitated nitrate uptake showing biphasic kinetics at a wide concentration range, with high- and low-affinity KM values of 0.15 and 4 mM, respectively. OsNRT2.4 did not have nitrate efflux or IAA influx activity. In rice roots, OsNRT2.4 was expressed mainly in the base of lateral root primordia. Knockout of OsNRT2.4 decreased lateral root number and length, and the total N uptake per plant at both 0.25 and 2.5 mM NO3- levels. In the shoots, OsNRT2.4 was expressed mainly in vascular tissues, and its knockout decreased the growth and NO3--N distribution. Knockout of OsNRT2.4, however, did not affect rice growth and N uptake under conditions without N or with only NH4+ supply. We conclude that OsNRT2.4 functions as a dual-affinity nitrate transporter and is required for nitrate-regulated root and shoot growth of rice.

Role of Micro‐Topographic Variability on the Distribution of Inorganic Soil‐Nitrogen Age in Intensively Managed Landscape

AbstractHow does the variability of topography structure the spatial heterogeneity of nutrient dynamics? In particular, what role does micro‐topographic depression play in the spatial and temporal dynamics of nitrate, ammonia, and ammonium? We explore these questions using the 3‐D simulation of their joint dynamics of concentration and age. To explicitly resolve micro‐topographic variability and its control on moisture, vegetation, and carbon‐nitrogen dynamics, we use a high‐resolution LiDAR data over an agricultural site under a corn‐soybean rotation in the Intensively Managed landscapes Critical Zone Observatory in the U.S. Midwest. We utilize a hybrid CPU‐GPU parallel computing architecture to reduce the computational cost associated with such high‐resolution simulations. Our results show that in areas that present closed topographic depressions, relatively lower nitrate concentration and age are observed compared to elsewhere. The periodic ponding in depressions increases the downward flux of water that carries more dissolved nitrate to the deeper soil layer. However, the variability in the depressions is relatively higher as a result of the episodic ponding pattern. When aggregate efflux from the soil domain at the bottom of the soil is considered, we find a gradual decrease in the age on the rising limb of nitrate efflux and a gradual increase on the falling limb. In addition, the age of the nitrate efflux ranges from 4 to 7 years. These are significantly higher as compared to the ages associated with a nonreactive tracer indicating that they provide an inaccurate estimate of residence time of a reactive constituent through the soil column.

Tonoplast-localized nitrate uptake transporters involved in vacuolar nitrate efflux and reallocation in Arabidopsis

A great proportion of nitrate taken up by plants is stored in vacuoles. Vacuolar nitrate accumulation and release is of great importance to nitrate reallocation and efficient utilization. However, how plants mediate nitrate efflux from vacuoles to cytoplasm is largely unknown. The current study identified NPF5.11, NPF5.12 and NPF5.16 as vacuolar nitrate efflux transporters in Arabidopsis. Histochemical analysis showed that NPF5.11, NPF5.12 and NPF5.16 were expressed preferentially in root pericycle cells and xylem parenchyma cells, and further analysis showed that these proteins were tonoplast-localized. Functional characterization using cRNA-injected Xenopus laevis oocytes showed that NPF5.11, NPF5.12 and NPF5.16 were low-affinity, pH-dependent nitrate uptake transporters. In npf5.11 npf5.12 npf5.16 triple mutant lines, more root-fed 15NO3− was translocated to shoots compared to the wild type control. In the NPF5.12 overexpression lines, proportionally less nitrate was maintained in roots. These data together suggested that NPF5.11, NPF5.12 and NPF5.16 might function to uptake nitrate from vacuoles into cytosol, thus serving as important players to modulate nitrate allocation between roots and shoots.

Seasonal benthic nitrogen cycling in a temperate shelf sea: the Celtic Sea

We undertook a seasonal study of benthic N-cycling on the Celtic Sea continental shelf in 2015, augmented by an earlier cruise in 2014. Two cruises in 2015 were centred before and after the Spring phytoplankton bloom and a further cruise was carried out in late summer. Five sites covering the mud to sand continuum were visited on all cruises, where we determined ammonium-oxidation, anammox and denitrification rates, expression of anammox and denitrification genes, N-nutrient fluxes and sediment porewater profiles of N-nutrients. Highest process rates were found during the post-bloom and late summer periods. The Celtic Sea was overwhelmingly a source of inorganic-N to the overlying water column. The efflux of nitrate was controlled by the magnitude of ammonium-oxidation. The latter accounted for 10–16% of total Oxygen consumption in cohesive sediments and 35–56% in sandy sediments. Ammonium oxidation rates in the range of 0.001–2.288 mmol m−2 days−1 were inversely correlated with sediment porosity and positively correlated with organic matter content (OM) which together explained 66% of the variance in rates. N-removal was dominated by anammox (0.003–0.636 mmol m−2 days−1), rather than denitrification (0.000–0.034 mmol m−2 days−1). This finding was supported by the corresponding gene expression data. The expression of hydrazine oxidoreductase (anammox) was significantly correlated with anammox and total N-removal rates. Anammox was positively correlated with porosity and OM, whilst denitrification was correlated with OM. The N-requirement of these processes was largely met through nitrification (ammonium-oxidation) rather than influx from the overlying water column. We estimated that N-removal via denitrification and anammox removed 6–9% of the organic-N deposited at the sea-floor from the overlying water column. The Celtic Sea system was thereby losing N which must be replenished on an annual basis in order to sustain productivity.

Effects of experimental warming and carbon addition on nitrate reduction and respiration in coastal sediments

Climate change may have differing effects on microbial processes that control coastal N availability. We conducted a microcosm experiment to explore effects of warming and carbon availability on nitrate reduction pathways in marine sediments. Sieved continental shelf sediments were incubated for 12 weeks under aerated seawater amended with nitrate (~50 μM), at winter (4 °C) or summer (17 °C) temperatures, with or without biweekly particulate organic C additions. Treatments increased diffusive oxygen consumption as expected, with somewhat higher effects of C addition compared to warming. Combined warming and C addition had the strongest effect on nitrate flux across the sediment water interface, with a complete switch early in the experiment from influx to sustained efflux. Supporting this result, vial incubations with added 15N-nitrate indicated that C addition stimulated potential rates of dissimilatory nitrate reduction to ammonium (DNRA), but not denitrification. Overall capacity for both denitrification and DNRA was reduced in warmed treatments, possibly reflecting C losses due to increased respiration with warming. Anammox potential rates were much lower than DNRA or denitrification, and were slightly negatively affected by warming or C addition. Overall, results indicate that warming and C addition increased ammonium production through remineralization and possibly DNRA. This stimulated nitrate production through nitrification, but without a comparable increase in nitrate consumption through denitrification. The response to C of potential DNRA rates over denitrification, along with a switch to nitrate efflux, raises the possibility that DNRA is an important and previously overlooked source of internal N cycling in shelf sediments.

H(+) -pyrophosphatase from Salicornia europaea confers tolerance to simultaneously occurring salt stress and nitrogen deficiency in Arabidopsis and wheat.

High salinity and nitrogen (N) deficiency in soil are two key factors limiting crop productivity, and they usually occur simultaneously. Here we firstly found that H(+) -PPase is involved in salt-stimulated NO3 (-) uptake in the euhalophyte Salicornia europaea. Then, two genes (named SeVP1 and SeVP2) encoding H(+) -PPase from S. europaea were characterized. The expression of SeVP1 and SeVP2 was induced by salt stress and N starvation. Both SeVP1 or SeVP2 transgenic Arabidopsis and wheat plants outperformed the wild types (WTs) when high salt and low N occur simultaneously. The transgenic Arabidopsis plants maintained higher K(+) /Na(+) ratio in leaves and exhibited increased NO3 (-) uptake, inorganic pyrophosphate-dependent vacuolar nitrate efflux and assimilation capacity under this double stresses. Furthermore, they had more soluble sugars in shoots and roots and less starch accumulation in shoots than WT. These performances can be explained by the up-regulated expression of ion, nitrate and sugar transporter genes in transgenic plants. Taken together, our results suggest that up-regulation of H(+) -PPase favours the transport of photosynthates to root, which could promote root growth and integrate N and carbon metabolism in plant. This work provides potential strategies for improving crop yields challenged by increasing soil salinization and shrinking farmland.

Coupled nitrate N and O stable isotope fractionation by a natural marine plankton consortium

The stable nitrogen (N) and oxygen (O) isotope ratios (15N/14N and 18O/16O, respectively) of nitrate (NO3-) were measured during incubations of freshly collected seawater to investigate the effect of light intensity on the isotope fractionation associated with nitrate assimilation and possible co-occurring regeneration and nitrification by in situ plankton communities. Surface seawater was collected off the coast of Vancouver, Canada, in late fall and in late summer and was incubated under different laboratory light conditions for 10 and 30 days, respectively. In the late summer experiments, parallel incubations were supplemented with 15NH4+ and H218O tracers to monitor co-occurring nitrification. Differences in irradiance in the fall incubations resulted in reduced nitrate consumption at low light, but had no distinguishable impact on the N-isotope isotope effect (15e) associated with NO3- assimilation, which ranged between 5 and 8‰. The late-summer community incubations, in contrast, showed reduced growth rates at low light and more elevated 15e of 11.9 ± 0.4‰, compared to 8.4 ± 0.3‰ at high-light conditions. The seasonal differences could reflect physiological adaptations of the fall plankton community to reduced irradiance, such that their incubation at low light did not elicit the increase in proportional cellular nitrate efflux required to raise the isotope effect. In both the fall and summer incubations, the ratio of the coincident rises in the δ15N and δ18O of NO3- was comparable to previous monoculture phytoplankton experiments, with a ∆δ18O:∆δ15N of ~1, regardless of light level. A decoupling of ∆δ18O:∆δ15N is expected if nitrification occurs concomitantly with nitrate assimilation. The lack of such decoupling is best explained by the absence of significant nitrification in any of our study’s treatments, an interpretation supported by our inability to identify any tracer 15N and 18O uptake into the NO3- pool in the late-summer community incubations.

Induction of nitrate uptake in Sauvignon Blanc and Chardonnay grapevines depends on the scion and is affected by the rootstock

Background and Aims Nitrate is a major form of inorganic nitrogen present in cultivated soils; however, information on the mechanisms responsible for uptake of the anion in grapevines is scarce. Methods and Results The response to external nitrate was studied in two clones of the cultivars Chardonnay and Sauvignon Blanc. Grapevines, own-rooted or grafted on the two rootstocks SO4 and K5BB, were grown in nutrient solution and, after a period of nitrogen (N)-deprivation, exposed to 0.2 or 1 mmol/L nitrate. The uptake was an inducible process, operated by high- and low-affinity transport systems. The magnitude and time dependence of the induction varied as a function of the clone. Uptake rate as a function of nitrate external concentration showed a multiphasic pattern. Although nitrate exposure caused a transient up-regulation of nitrate influx transporter genes, a clear correlation with the rise in nitrate uptake could not be found; in contrast, a negative relationship between changes in uptake rates and expression of a nitrate efflux transporter gene was observed. Conclusions In the grapevine, the response of low- and high-affinity transport systems to external nitrate was favoured by the presence of the rootstock and also dependent on the characteristics of the scion. Low-affinity uptake and efflux from root cells conceivably play an important role in the overall N-nitrate acquisition. Significance of the Study This study shows that activation of mechanisms involved in nitrate uptake in the grapevine is strongly affected by the scion–rootstock combination. Results may be significant for developing agronomic practices and selection programmes related to nitrogen use in the grapevine.

Anion channel SLAH3 functions in nitrate-dependent alleviation of ammonium toxicity in Arabidopsis.

Slow anion channels (SLAC/SLAH) are efflux channels previously shown to be critical for stomatal regulation. However, detailed analysis using the β-glucuronidase reporter gene showed that members of the SLAC/SLAH gene family are predominantly expressed in roots, in addition to stomatal guard cells, implicating distinct function(s) of SLAC/SLAH in the roots. Comprehensive mutant analyses of all slac/slah mutants indicated that slah3 plants showed a greater growth defect than wild-type plants when ammonium was supplied as the sole nitrogen source. Ammonium toxicity was mimicked by acidic pH in nitrogen-free external medium, suggesting that medium acidification by ammonium-fed plants may underlie ammonium toxicity. Interestingly, such toxicity was more severe in slah3 mutants and, particularly in wild-type plants, was alleviated by supplementing the media with micromolar levels of nitrate. These data thus provide evidence that SLAH3, a nitrate efflux channel, plays a role in nitrate-dependent alleviation of ammonium toxicity in plants.

The contributions of nitrate uptake and efflux to isotope fractionation during algal nitrate assimilation

In order to strengthen environmental application of nitrate N and O isotopes, we measured the N and O isotopic fractionation associated with cellular nitrate uptake and efflux in the nitrate-assimilating marine diatom Thalassiosira weissflogii. We isolated nitrate uptake and efflux from nitrate reduction by growing the cells in the presence of tungsten, which substitutes for molybdenum in assimilatory nitrate reductase, yielding an inactive enzyme. After growth on ammonium and then N starvation, cells were exposed to nitrate. Numerical models fit to the evolution of intracellular nitrate concentration and N and O isotopic composition yielded distinct N isotope effects (15ε) for nitrate uptake and nitrate efflux (2.0±0.3‰ and 1.2±0.4‰, respectively). The O isotope effects (18ε) for nitrate uptake and nitrate efflux were indistinguishable (2.8±0.6‰), yielding a ratio of O to N isotopic fractionation for uptake of 1.4±0.4 and for efflux of 2.3±0.9. The 15ε for nitrate uptake can account for at most 40% of the organism-level N isotope effect (15εorg) measured in laboratory studies of T. weissflogii and in the open ocean (typically 5‰ or greater). This observation supports previous evidence that most isotope fractionation during nitrate assimilation is due to intracellular nitrate reduction, with nitrate efflux allowing the signal to be communicated to the environment. An O to N fractionation ratio (18εorg:15εorg) of ∼1 has been measured for nitrate assimilation in algal cultures and linked to the N and O isotope effects of nitrate reductase. Our results suggest that the ratios of O to N fractionation for both nitrate uptake and efflux may be distinct from a ratio of 1, to a degree that could cause the net 18εorg:15εorg to rise appreciably above 1 when 15εorg is low (e.g., yielding a ratio of 1.1 when 15εorg is 5‰). However, field and culture studies have consistently measured nearly equivalent fractionation of N and O isotopes in association with low isotope effects, calling for isotopic studies of nitrate transport by other phytoplankton strains.

Vertical Distribution and Flux of Nutrients in the Sediments of the Mangrove Reclamation Region of Muara Angke Kapuk, Jakarta

The reclaimed mangrove estuary in Muara Angke Kapuk is a reclaimed area that has not evaded the impacted of pollution and waste in the areas surrounding Cengkareng, Jakarta. This is apparent from the fact that almost all sediments under the mangrove trees are buried under heaps of plastic trash. However, the reclaimed region still has variety of organism, which indicating that the region still has an internal carrying capacity, especially nutrients from sediment. The purpose of this research was to examine the condition of sediment nutrients in this mangrove reclamation region. The research was conducted by taking water samples using a modification of the stratified cup at a sediment depth of 0-15 cm with depth intervals of 2.5 cm, and taking sediment samples using the sediment ring. Pore water samples were measured for dissolved oxygen (DO) and concentrations of ammonia, nitrite, nitrate, and phosphate. Sediment samples were used to obtain porosity values. The data obtained is used to make vertical concentration profiles and analysis of vertical nutrient flux. Vertical nutrient flux analysis was performed with the aid of QUAL2K software version 2.11. The results showed different vertical distributions and flux of nutrients, where influx for ammonia and phosphate and an increase in line with increasing sediment depth, while nitrate efflux and a decreased concentration. The flux calculation of nitrite as transitory nutrient was not done, but the concentration decreased after a depth of 2.5 cm. This indicates that the high contamination on the surface does not prevent the natural chemical processes so the reclaimed region can still provide nutritional support for its organism.

Reduced isotope fractionation by denitrification under conditions relevant to the ocean

Experiments with two well-studied denitrifiers and one recently isolated marine suboxic zone denitrifier show that the cellular-level denitrification N isotope effect (15ε) is typically lower than the canonical value of ∼25‰ under many conditions prevalent in the ocean. Across all three strains, 15ε is 10–15‰ at cellular nitrate reduction rates that are more representative of the environment than the very high rates under which we and previous investigators measure 15ε to be 20–30‰. A sharp decrease in 15ε is also observed in individual nitrate drawdown assays as the extracellular nitrate concentrations approach 2–35μM and nitrate uptake becomes the rate-limiting step. On an apparently strain-specific basis, lower values of 15ε are observed under diverse conditions common in the natural environment: less reduced carbon sources, small inputs of oxygen, nutrient availability, agitation, and age of starter culture (i.e., initiation of assays with cells that had recently depleted a large previous nitrate amendment or were more recently in the exponential growth (“bloom”) phase). A conserved oxygen-to-nitrogen isotope relationship across the experiments for all three denitrifiers (18ε/15ε=0.93±0.06 (1SD)) supports the interpretation that fractionation is imparted solely by the internal respiratory nitrate reductase, with the amplitude of 15ε varying with the proportional importance of cellular nitrate efflux relative to uptake. Aspects of the 15ε variation are unexpected; nevertheless, the occurrence of lower 15ε is robust. It is uncertain if our lower 15ε estimates apply to oceanic water column denitrification because field studies have generally yielded 15εwc between 20–30‰, more similar to previous culture estimates and our estimates at high cell specific nitrate reduction rates. If denitrification in the ocean’s major suboxic zones does have an 15ε of ∼10–15‰, it would remove an apparent imbalance between global ocean N inputs and outputs previously suggested by fixed N isotope budgeting.