15N-labelled Nitrate Research Articles

A mycoheterotrophic orchid uses very limited soil inorganic nitrogen in its natural habitat

Mycoheterotrophic plants acquire nitrogen (N) directly from the soil and through their symbiotic fungi. The fungi-derived N has received considerable attention, but the contribution of soil-derived N has been largely overlooked. We investigated how the leafless, rootless, and almost mycoheterotrophic orchid Cymbidium macrorhizon obtains soil N by applying 15N-labeled ammonium nitrate in its natural habitat, and tracking metabolite accumulation and mycorrhizal fungal association after N application. The decline of N in the rhizome from flowering to fruiting indicated a transfer of N from the rhizome to fruits. At current dose of N application (0.6 g NH4NO3 each plant), only 1.5% of the plant's N was derived from fertilizer, resulting in a low nitrogen use efficiency of 0.27%. The majority of those newly absorbed N (88.89%) was found sank in the rhizome. Amino acids (or their derivatives) and alkaloids were predominant differentially accumulated nitrogenous metabolites after N application, with amino acids occurring in both fruits and the rhizome, and alkaloids primarily in the fruits. The addition of N did not alter the richness of mycorrhizal fungi, but did affect their relative abundance. Our findings suggest that Cymbidium macrorhizon uses very limited soil inorganic nitrogen in its natural habitat, and the root-like rhizome primarily stores N rather than absorbs its inorganic forms, offering new insights into how mycoheterotrophic plants utilize soil N, and the influence of nutrient availability on the orchid-fungi association.

Skeletal Muscle, Skin, and Bone as Three Major Nitrate Reservoirs in Mammals: Chemiluminescence and 15N-Tracer Studies in Yorkshire Pigs.

In mammals, nitric oxide (NO) is generated either by the nitric oxide synthase (NOS) enzymes from arginine or by the reduction of nitrate to nitrite by tissue xanthine oxidoreductase (XOR) and the microbiome and further reducing nitrite to NO by XOR or several heme proteins. Previously, we reported that skeletal muscle acts as a large nitrate reservoir in mammals, and this nitrate reservoir is systemically, as well as locally, used to generate nitrite and NO. Here, we report identifying two additional nitrate storage organs-bone and skin. We used bolus of ingested 15N-labeled nitrate to trace its short-term fluxes and distribution among organs. At baseline conditions, the nitrate concentration in femur bone samples was 96 ± 63 nmol/g, scalp skin 56 ± 22 nmol/g, with gluteus muscle at 57 ± 39 nmol/g. In comparison, plasma and liver contained 34 ± 19 nmol/g and 15 ± 5 nmol/g of nitrate, respectively. Three hours after 15N-nitrate ingestion, its concentration significantly increased in all organs, exceeding the baseline levels in plasma, skin, bone, skeletal muscle, and in liver 5-, 2.4-, 2.4-, 2.1-, and 2-fold, respectively. As expected, nitrate reduction into nitrite was highest in liver but also substantial in skin and skeletal muscle, followed by the distribution of 15N-labeled nitrite. We believe that these results underline the major roles played by skeletal muscle, skin, and bone, the three largest organs in mammals, in maintaining NO homeostasis, especially via the nitrate-nitrite-NO pathway.

Dietary nitrate flows and accumulation in pig organs:15N-tracer study

Nitric oxide (NO) is a free radical with multiple functions in virtually all cells. In mammals, NO is generated by nitric oxide synthase from arginine and by reduction of nitrate to nitrite by tissue xanthine oxidoreductase (XOR) and microbiome. Nitrite is then available for reduction to NO by XOR or several heme proteins. Previously we reported that 1) skeletal muscle acts as a body nitrate reservoir, and 2) stored nitrate is systemically, as well as locally used to generate nitrite and NO. Recently, we identified two additional nitrate storage organs - skeleton and skin. At baseline conditions, nitrate concentration in skeleton was 96±63 nmol/g, skeletal muscle 57±39 nmol/g and skin 56±22 nmol/g. For comparison, plasma and liver contained 34±19 nmol/g and 15±5 nmol/g of nitrate, respectively. We used 15N-labeled dose of dietary nitrate to trace its distribution among organs in time. Three hours after nitrate bolus ingestion, total nitrate levels significantly increased in all organs, reaching 221±91 nmol/g in plasma, 126±55 nmol/g in skin, 119±38 nmol/g in skeleton, 100±79 mmol/g in skeletal muscle, and 21±5 nmol/g in liver. As shown by MS, 15N-labeled nitrate distributes in non-uniform way, accumulating largely in skin. As expected, nitrate reductase activity is highest in liver, but also substantial in skin and skeletal muscle, as seen by presence of 15N-labeled nitrite. With consideration of skeletal muscle, skin and skeleton as three largest organs in mammals, these results underline their major role in maintaining homeostasis of the nitrate-nitrite-nitric oxide pathway. Also, important from therapeutic point of view, all three organs are well responding to dietary nitrate interventions by increasing their respective nitrate levels, which leads to general increase of systemic and local NO and perhaps could be used as one of the ways to increase NO availability. Work supported by NIH internal grant to ANS. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Fertilizer Application Method Provides an Environmental-Friendly Nitrogen Management Option for Sugarcane

PurposeNitrogen fertilizer management is an important agricultural tool that must be optimized to promote sustainable practices since the nitrogen-fertilizer recovery by plants (NRP) is low, leading to nitrogen losses to the environment. In sugarcane, N-fertilization has been investigated over the years but little attention has been given to N-fertilizer application methods. Sugarcane crop production and environmental impact regarding N-fertilizer application methods (i.e., applied onto the sugarcane straw layer and incorporated into the soil) were investigated in the present study aiming to achieve an environmental-friendly cropping system.MethodsSugarcane yield and NRP, N2O emissions, relevant components of the soil microbiological community and N-fertilizer retention in soil layers were quantified. The experiment was carried out in field conditions where N-fertilizer application methods using 15N-labelled ammonium nitrate (15NH415NO3) were compared to a control treatment with no N-fertilization.ResultsIncorporation of N-fertilizer into the soil increased the sugarcane yield by 17% (two-year average) compared to N-fertilizer applied onto the sugarcane straw layer, which was similar to control treatment. There was an increase in NRP-fertilizer of 79% due to the application of N-fertilizer incorporated into the soil. Furthermore, soil incorporation of N-fertilizer decreased N2O emission by 22% with the fertilizer N emission factor reduced four-fold. The N2O emissions were mostly associated with ammonium-oxidizing bacteria (AOB).ConclusionsOur results show that application of N-fertilizer incorporated into the soil is an environmental-friendly N-fertilization management which will improve agricultural sustainability and reduce environmental impacts.Graphical

Impact of liming and maize residues on N2O and N2 fluxes in agricultural soils: an incubation study

AbstractSince it is known that nitrous oxide (N2O) production and consumption pathways are affected by soil pH, optimising the pH of agricultural soils can be an important approach to reduce N2O emissions. Because liming effects on N2O reduction had not been studied under ambient atmosphere and typical bulk density of arable soils, we conducted mesoscale incubation experiments with soils from two liming trials to investigate the impact of long-term pH management and fresh liming on N transformations and N2O production. Soils differed in texture and covered a range of pH levels (3.8–6.7), consisting of non-limed controls, long-term field-limed calcite and dolomite treatments, and freshly limed soils. Both soils were amended with 15N-labelled potassium nitrate (KNO3) and incubated with and without incorporated maize litter. Packed soil mesocosms were cycled through four phases of alternating temperatures and soil moistures for at least 40 days. Emissions of N2O and dinitrogen (N2) as well as the product ratio of denitrification N2O/(N2O + N2), referred to as N2Oi were measured with the 15N gas flux method in N2-reduced atmosphere. Emissions of N2O increased in response to typical denitrifying conditions (high moisture and presence of litter). Increased temperature and soil moisture stimulated microbial activity and triggered denitrification as judged from 15NO3− pool derived N2O + N2 emissions. Fresh liming increased denitrification in the sandy soil up to 3-fold but reduced denitrification in the loamy soil by 80%. N2Oi decreased throughout the incubation in response to fresh liming from 0.5–0.8 to 0.3–0.4, while field-limed soils had smaller N2Oi (0.1–0.3) than unlimed controls (0.9) irrespective of incubation conditions. Our study shows that the denitrification response (i.e., N2O + N2 production) to liming is soil dependent, whereas liming effects on N2Oi are consistent for both long- and short-term pH management. This extends previous results from anoxic slurry incubation studies by showing that soil pH management by liming has a good mitigation potential for agricultural N2O emissions from denitrification under wet conditions outside of cropping season.

Dietary Nitrate Metabolism in Porcine Ocular Tissues Determined Using 15N-Labeled Sodium Nitrate Supplementation.

Nitrate (NO3-) obtained from the diet is converted to nitrite (NO2-) and subsequently to nitric oxide (NO) within the body. Previously, we showed that porcine eye components contain substantial amounts of nitrate and nitrite that are similar to those in blood. Notably, cornea and sclera exhibited the capability to reduce nitrate to nitrite. To gain deeper insights into nitrate metabolism in porcine eyes, our current study involved feeding pigs either NaCl or Na15NO3 and assessing the levels of total and 15N-labeled NO3-/NO2- in various ocular tissues. Three hours after Na15NO3 ingestion, a marked increase in 15NO3- and 15NO2- was observed in all parts of the eye; in particular, the aqueous and vitreous humor showed a high 15NO3- enrichment (77.5 and 74.5%, respectively), similar to that of plasma (77.1%) and showed an even higher 15NO2- enrichment (39.9 and 35.3%, respectively) than that of plasma (19.8%). The total amounts of NO3- and NO2- exhibited patterns consistent with those observed in 15N analysis. Next, to investigate whether nitrate or nitrite accumulate proportionally after multiple nitrate treatments, we measured nitrate and nitrite contents after supplementing pigs with Na15NO3 for five consecutive days. In both 15N-labeled and total nitrate and nitrite analysis, we did not observe further accumulation of these ions after multiple treatments, compared to a single treatment. These findings suggest that dietary nitrate supplementation exerts a significant influence on nitrate and nitrite levels and potentially NO levels in the eye and opens up the possibility for the therapeutic use of dietary nitrate/nitrite to enhance or restore NO levels in ocular tissues.

The differential assimilation of nitrogen fertilizer compounds by soil microorganisms.

The differential soil microbial assimilation of common nitrogen (N) fertilizer compounds into the soil organic N pool is revealed using novel compound-specific amino acid (AA) 15N-stable isotope probing. The incorporation of fertilizer 15N into individual AAs reflected the known biochemistry of N assimilation-e.g. 15N-labelled ammonium (15NH4+) was assimilated most quickly and to the greatest extent into glutamate. A maximum of 12.9% of applied 15NH4+, or 11.7% of 'retained' 15NH4+ (remaining in the soil) was assimilated into the total hydrolysable AA pool in the Rowden Moor soil. Incorporation was lowest in the Rowden Moor 15N-labelled nitrate (15NO3-) treatment, at 1.7% of applied 15N or 1.6% of retained 15N. Incorporation in the 15NH4+ and 15NO3- treatments in the Winterbourne Abbas soil, and the 15N-urea treatment in both soils was between 4.4% and 6.5% of applied 15N or 5.2% and 6.4% of retained 15N. This represents a key step in greater comprehension of the microbially mediated transformations of fertilizer N to organic N and contributes to a more complete picture of soil N-cycling. The approach also mechanistically links theoretical/pure culture derived biochemical expectations and bulk level fertilizer immobilization studies, bridging these different scales of understanding.

Root Development and Subsoil 15N-labelled N Uptake in Soybean (Glycine max (L.) Merr.)

The aim of this study was to investigate fertiliser-derived N uptake of soybean from different depths of the soil under field conditions. In addition, soybean root growth in sandy and loess soil was evaluated to understand the impact of site and soybean variety characteristics on soybean N uptake under continental conditions in Central Europe. Root analysis to determine rooting depth and root length density (RLD) was carried out using the profile wall method at three growth stages and two soybean cultivars (Glycine max (L.) Merr. cvs. Merlin and Sultana) in three consecutive years at two locations in eastern Germany. Fertiliser-derived N uptake of soybean from the soil surface and the subsoil was determined at 0.3 and 0.6 m depths using 15N-labelled nitrate N. Root studies showed that soybean roots grew up to 1.4 m on sandy and loess soil sites. Root length densities of up to 2.4 cm cm−3 were documented in the topsoil. By means of 15N application, soybean was shown to take up 15% of the surface-applied nitrogen in the dry growing season and 67 % in high rainfall years, between 19 and 77 % of the nitrogen placed at 0.3 m soil depth, and between 2 and 64 % of the nitrogen placed at 0.6 m soil depth by flowering. The field trials showed that soybeans can absorb a high proportion of the nitrogen placed in the subsoil by flowering time. Due to a well-developed root system reaching deep into the soil, soybeans are able to cover their N demand from soil-borne sources and secure yield formation during dry periods by water uptake from the subsoil.

Uptake of Fertilizer Nitrogen and Soil Nitrogen by Sorghum Sudangrass (Sorghum bicolor × Sorghum sudanense) in a Greenhouse Experiment with 15N-Labelled Ammonium Nitrate

A greenhouse experiment with sorghum sudangrass (Sorghum bicolor × Sorghum sudanense) and maize (Zea mays) was conducted to assess information on differences in their nitrogen and fertilizer utilization when used as energy crops. The aim was to contribute to the scarce data on sorghum sudangrass as an energy crop with regards to nitrogen derived from fertilizer (NdfF) in the plant’s biomass and fertilizer nitrogen utilization (FNU). Sorghum sudangrass and maize were each grown in eight bags of 45 L volume and harvested at maturity after 154 days. Each crop treatment was further divided in a control treatment (four bags each) that did not receive N fertilization and a fertilization treatment (four bags each) that received 1.76 g N, applying a 15N-labelled liquid ammonium nitrate fertilizer. Fertilization took place at the start of the experiment. After harvest, the whole plant was divided in the fractions “aboveground biomass” (ABM) and “stubble + rootstock” (S + R). Weight, N content and 15N content were recorded for each fraction. In addition, N content and 15N content were assessed in the soil before sowing and after harvest. The experiment showed that FNU of sorghum sudangrass (65%) was significantly higher than that of maize (49%). Both crops accumulated more soil N than fertilizer N. The share of fertilizer N on total N uptake was also higher with sorghum sudangrass (NdfF = 38%) compared to maize (NdfF = 34%). The observations made with our control plant (maize), showed that the results are plausible and comparable to other 15N studies on maize regarding yields, NdfF, and FNU, leading to the assumption that results on sorghum sudangrass are plausible as well. We therefore conclude that the results of our study can be used for the preliminary parametrization of sorghum sudangrass in soil organic matter (SOM) balance at field level.

Moisture effects on microbial protein biosynthesis from ammonium and nitrate in an unfertilised grassland

Incorporation of nitrogen (N) into soil microbial protein is central to the soil N cycle to mitigate N losses and support plant N supply. However, the effect of factors, such as water filled pore space (WFPS), which influence inorganic N transformations and losses, and thus microbial incorporation, are only poorly understood. This work aimed to bridge this gap, using compound-specific 15N-stable isotope probing to quantify microbial assimilation into the largest defined soil organic N pool, protein-N. This approach applied differentially 15N-labelled ammonium nitrate (NH4NO3) to an unfertilised UK grassland in a soil mesocosm study over 10 days. The soil microbial community showed a strong preference for NH4+ over NO3−, which varied with WFPS (85% > 55% > 70%). This preference decreased for amino acids further in biosynthetic proximity to the transamination step in amino acid biosynthesis. Combined incorporation of NH4+ and NO3− increased total hydrolysable amino acid-N concentration linked to WFPS (55% ∼ 85% > 70%). Incorporation rates of applied 15N showed the same trend as NH4+ preference with WFPS (85% > 55% > 70%), which is related to microbial activity and nutrient mobility. Despite differences in incorporation, when normalised to soil available N, incorporation was comparable in the short-term. Mechanistic control of WFPS via assimilation into the largest soil organic N pool is important to mitigate potential positive feedbacks to N losses and support N supply to plants.

15N tracers and microbial analyses reveal in situ N2O sources in contrasting water regimes of a drained peatland forest

Managed peatlands are a significant source of nitrous oxide (N2O), a powerful greenhouse gas and stratospheric ozone depleter. Due to the complexity and diversity of microbial N2O processes, different methods such as tracer, isotopomer, and microbiological technologies are required to understand these processes. The combined application of different methods helps to precisely estimate these processes, which is crucial for the future management of drained peatlands, and to mitigate soil degradation and negative atmospheric impact. In this study, we investigated N2O sources by combining tracer, isotopomer, and microbial analysis in a drained peatland forest under flooded and drained treatments. On average, the nitrification genes showed higher abundances in the drained treatment, and the denitrification genes showed higher abundances in the flooded treatment. This is consistent with the underlying chemistry, as nitrification requires oxygen while denitrification is anaerobic. We observed significant differences in labelled N2O fluxes between the drained and flooded treatments. The emissions of N2O from the flooded treatment were nearly negligible, whereas the N2O evolved from the nitrogen-15 (15N)-labelled ammonium (15NH4+) in the drained treatment peaked at 147 μg 15N m-2 h-1. This initially suggested nitrification as the driving mechanism behind N2O fluxes in drained peatlands, but based on the genetic data, isotopic analysis, and N2O mass enrichment, we conclude that hybrid N2O formation involving ammonia oxidation was the main source of N2O emissions in the drained treatment. Based on the 15N-labelled nitrate (15NO3-) tracer addition and gene copy numbers, the low N2O emissions in the flooded treatment came possibly from complete denitrification producing inert dinitrogen. At atomic level, we observed selective enrichment of mass 45 of N2O molecule under 15NH4+ amendment in the drained treatment and enrichment of both masses 45 and 46 under 15NO3- amendment in the flooded treatment. The selective enrichment of mass 45 in the drained treatment indicated the presence of hybrid N2O formation, which was also supported by the high abundances of archaeal genes.

Analysis of dietary nitrate incorporation in porcine ocular tissues after supplementing 15N-labeled sodium nitrate

The mechanisms for the generation of nitric oxide (NO) include reduction of nitrate (NO 3 - ) and nitrite (NO 2 - ) as well as the classic nitric oxide synthase (NOS) pathways. In the reductive pathways, ingested nitrate is reduced to nitrite, mainly by oral bacteria and to some extent by tissue enzymes, then these nitrite ions can be reduced to NO by several endogenous proteins or, in stomach, by acid disproportionation. Dietary nitrate, rich in green leafy vegetables and beets, has beneficial effects in lowering blood pressure and improving vascular health by enhancing NO bioavailability. Previously, we showed that porcine eye components contain substantial amounts of nitrate and nitrite ions that are similar to those in blood. More importantly, cornea and sclera exhibited a nitrate reduction capability. To extend our knowledge on nitrate metabolism in the eye, in the present study, we fed pigs (Yorkshire domestic cross swine weighing 35-40kg, housed in an AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care)-accredited facility with environmental enrichment) with either NaCl or Na 15 NO 3 and assessed the amount of 15 NO 3 - and 15 NO 2 - in various parts of the eye by LC-MS/MS at 3 or 24h after supplementation. We also measured the total concentrations of nitrate and nitrite ions using a standard chemiluminescence nitric oxide analyzer. At 3h, there was a marked increase in both 15 NO 3 - and 15 NO 2 - in all parts of the eye; in particular, aqueous and vitreous humor showed very high 15 NO 3 - contents (77.5 and 74.5% respectively), similar to plasma (77.1%) and even higher 15 NO 2 - contents (39.9% and 35.3% respectively) than plasma (19.8%). Cornea (64.4%) and sclera (63.4%) also showed notable increases in 15 NO 3 - content at 3h, then retina (48.8%) and lens (44.8%) followed. In 15 NO 2 - percent analysis at 3h, except aqueous and vitreous humor, other eye parts showed similar levels to plasma. At 24h, 15 NO 3 - contents decreased significantly compared to the 3h point, but it was still higher than the baseline in all ocular components. The 15 NO 2 - contents at 24h were almost back to the baseline. Absolute values of nitrate and nitrite ions in these tissues show similar patterns as shown in 15 N analysis. These results indicate that dietary nitrate supplementation has a vast impact on the levels of nitrate and nitrite and likely NO as well in the eye. NO generation via the nitrate-nitrite reductive pathway may contribute to various physiological and pathophysiological processes in the eye and may be useful therapeutically. This work was supported by intramural NIDDK/NIH grant ZIA DK 025104-15 to Alan N. Schechter. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Distribution of dietary nitrate and its metabolites in rat tissues after 15N-labeled nitrate administration

The reduction pathway of nitrate (NO3−) and nitrite (NO2−) to nitric oxide (NO) contributes to regulating many physiological processes. To examine the rate and extent of dietary nitrate absorption and its reduction to nitrite, we supplemented rat diets with Na15NO3-containing water (1 g/L) and collected plasma, urine and several tissue samples. We found that plasma and urine showed 8.8- and 11.7-fold increases respectively in total nitrate concentrations in 1-day supplementation group compared to control. In tissue samples—gluteus, liver and eyes—we found 1.7-, 2.4- and 4.2-fold increases respectively in 1-day supplementation group. These increases remained similar in 3-day supplementation group. LC–MS/MS analysis showed that the augmented nitrate concentrations were primarily from the exogenously provided 15N-nitrate. Overall nitrite concentrations and percent of 15N-nitrite were also greatly increased in all samples after nitrate supplementation; eye homogenates showed larger increases compared to gluteus and liver. Moreover, genes related to nitrate transport and reduction (Sialin, CLC and XOR) were upregulated after nitrate supplementation for 3 days in muscle (Sialin 2.3-, CLC1 1.3-, CLC3 2.1-, XOR 2.4-fold) and eye (XOR 1.7-fold) homogenates. These results demonstrate that dietary nitrate is quickly absorbed into circulation and tissues, and it can be reduced to nitrite in tissues (and likely to NO) suggesting that nitrate-enriched diets can be an efficient intervention to enhance nitrite and NO bioavailability.

N Absorption, Transport, and Recycling in Nodulated Soybean Plants by Split-Root Experiment Using 15N-Labeled Nitrate

Nitrate concentration is variable in soils, so the absorbed N from roots in a high-nitrate site is recycled from shoots to the root parts in N-poor niche. In this report, the absorption, transport, and recycling of N derived from 15N-labeled nitrate were investigated with split-root systems of nodulated soybean. The NO3− accumulated in the root in 5 mM NO3− solution; however, it was not detected in the roots and nodules in an N-free pot, indicating that NO3− itself is not recycled from leaves to underground parts. The total amount of 15NO3− absorption from 2 to 4 days of the plant with the N-free opposite half-root accelerated by 40% compared with both half-roots that received NO3−. This result might be due to the compensation for the N demand under one half-root could absorb NO3−. About 2–3% of the absorbed 15N was recycled to the opposite half-root, irrespective of N-free or NO3− solution, suggesting that N recycling from leaves to the roots was not affected by the presence or absence of NO3−. Concentrations of asparagine increased in the half-roots supplied with NO3− but not in N-free half-roots, suggesting that asparagine may not be a systemic signal for N status.

Effects of Irrigation and N Fertilization on 15N Fertilizer Utilization by Vitis vinifera L. Cabernet Sauvignon in China

This study investigated the interactions between different irrigation and nitrogen (N) fertilization rates and their effects on the 15N fertilizer absorption and utilization of Cabernet Sauvignon grapevines in a vineyard near Xinjiang, China. The fertilizer treatments consisted of three 15N-labeled urea nitrate fertilizer applications (191.4, 254.4, and 317.4 kg/ha). The irrigation treatments included two fractions (0.75 and 1.0) of estimated vineyard water use (ETc). The results showed that: (i) the residual amount of 15N fertilizer in the soil was mainly in the surface layer (0–20 cm), the residual fertilizer N in the surface layer accounted for 68–87% of the total residual N, the residual 15N fertilizer at different depths differed significantly, and the residual 15N was positively correlated with the amount of fertilizer applied (p < 0.05); (ii) The absorption of fertilizer N by grapevines accounted for only 12–17% of the total N absorption, and the proportion absorbed from soil N was as high as 82–87%. There was no significant difference in the amount of N absorbed between different water and fertilizer treatments; (iii) The 15N uptake under the different water and fertilizer treatments differed and was significantly higher in the roots than in other organs, followed by the fruit, the leaves, and finally, the stems. Our results provide a reference for improving the soil environment and encouraging a sustainable development of the grape industry. According to the experimental results, it was recommended that farmers adopt irrigation levels of 1.0 ETc and fertilizer application of 254.4 kg N/ha.

Urea uptake by spruce tree roots in permafrost-affected soils

Biomass productivity of black spruce trees is strongly limited by soil nitrogen (N) in shallow active layer on permafrost. Trees and mycorrhizal roots are known to absorb amino acids to bypass slow N mineralization in N-limited boreal forest soils. However, amino acid uptake strategy of tree roots cannot fully explain their advantages in competition for soil N with other plants and microbes. We investigated the potentials of tree roots to absorb urea as well as amino acids and inorganic N, using tracer experiments of 13C, 15N-labelled glutamic acid and urea and 15N-labelled ammonium nitrate in black spruce forests growing on permafrost-affected soils with different permafrost table depths in the eastern edge of the Mackenzie river delta (northwest Canada). We demonstrated that black spruce roots have potentials to absorb intact urea but only in soils with shallow permafrost, where urea accumulates due to limited microbial mineralization activity. This contrasts with soils with deep permafrost table, where roots absorb amino acids and inorganic N. Allocation of fine roots to colder subsoil above permafrost provides advantages for trees monopolizing urea-N. Despite lower energy efficiency of urea utilization compared to inorganic N and amino acids, urea uptake is one of N acquisition strategies for spruce growing on N-limited subarctic soil.

Preferences for different nitrogen forms in three dominant plants in a semi-arid grassland under different grazing intensities

Plant species generally absorb different nitrogen (N) forms from soil in ways that minimize N niche overlap, but whether and how grazing affects a plant’s preferred form of N remains unclear. Using the in-situ stable N isotope-labeling technique, we explored the preference of plants in taking up N of different chemical forms along an experimental gradient of grazing intensity in a semi-arid grassland in Inner Mongolia. 15N-labelled nitrate (NO3−), ammonium (NH4+) and glycine solutions were injected into the soil under nil (G0, as a control), light (G1) and heavy grazing (G2) grassland plots in June and August. Uptake of these three N forms was determined for three dominant plants, Leymus chinensis, Stipa grandis and Cleistogenes squarrosa. L. chinensis had the highest N acquisition capacity. Regardless of species, N absorption was higher in August than in June. While all three plant species absorbed glycine-N, they preferentially absorbed N in its inorganic forms (NO3−-N + NH4+-N), with NO3−-N having the highest uptake. Irrespective of grazing intensity, during peak growth (August) all three plant species preferred to absorb NO3−-N (the dominant N form) than NH4+-N or glycine-N. In contrast, in June plants differed in their preferred form of N uptake depending on the grazing treatment. Under heavy grazing (G2), all plants preferentially absorbed NO3−-N. Under light grazing (G1), all plants absorbing all three N chemical forms with no significant difference. Under no grazing (G0), L. chinensis preferentially absorbed NO3−-N but otherwise there was no significant effect of grazing intensity on the preferred form of N. Our results indicate that the three species of plants that we observed altered their N-uptake preference in response to grazing intensity differently in the early growth and vigorous growth stages. Plant N niche differentiation in time, space and chemical N forms needs to be explored systematically in future studies to aid understand of grazing effects on N processes in grassland ecosystem.

Nitrogen use efficiency, partitioning, and storage in cool climate potted Pinot Noir vines

Optimal timing of nitrogen (N) fertilizer to promote N use efficiency (NUE) in cool climate wine regions is largely unknown. One year-old potted Vitis vinifera L. (Vitaceae) cv. Pinot Noir vines were subjected to soil applied 15N-labelled calcium nitrate (5.5 atom%) fertilizer at four application timings (budbreak to fruit set, fruit set to veraison, veraison to harvest, or spread evenly across the growing season) over two growing seasons (2017/18 and 2018/19) at a rate of 12 g N vine−1. Annual vine parts (leaves and clusters) were collected at commercial harvest in both 2018 and 2019 and the remaining vine was destructively harvested during winter dormancy. Vines were separated into organs (leaves, clusters, shoot, trunk, and roots) and organ dry matter was determined before samples were ground into a fine powder to determine total N percentage and 15N atom percentage using flash combustion isotope ratio mass spectrometry (varioPYRO cube coupled to Isoprime100 mass spectrometer). Compared to the average values obtained from the other treatments, the veraison to harvest treatment significantly reduced whole vine dry matter by 39.1% and vine NUE by 42.1%. Yield was also reduced by 12.9% compared to the budbreak to fruit set treatment. NDF, defined here as the proportion of N within the vine (or vine organ) that was derived from fertilizer N, and overall vine total N content was lower in the veraison to harvest treatment due to the reduction of root and shoot NDF content. This suggests that for young, actively growing vines in cool climates, limiting N applications until veraison is inefficient and ineffective. No significant differences in NUE, N partitioning or N storage were observed between the other fertilizer N application timings.

Nitrate-dependent anaerobic methane oxidation and chemolithotrophic denitrification in a temperate eutrophic lake.

While the emissions of methane (CH4) by natural systems have been widely investigated, CH4 aquatic sinks are still poorly constrained. Here, we investigated the CH4 cycle and its interactions with nitrogen (N), iron (Fe) and manganese (Mn) cycles in the oxic-anoxic interface and deep anoxic waters of a small, meromictic and eutrophic lake, during two summertime sampling campaigns. Anaerobic CH4 oxidation (AOM) was measured from the temporal decrease of CH4 concentrations, with the addition of three potential electron acceptors (NO3-, iron oxides (Fe(OH)3) and manganese oxides (MnO2)). Experiments with the addition of either 15N-labeled nitrate (15N-NO3-) or 15N-NO3- combined with sulfide (H2S), to measure denitrification, chemolithotrophic denitrification and anaerobic ammonium oxidation (anammox) rates, were also performed. Measurements showed AOM rates up to 3.8µmol CH4 L-1 d-1 that strongly increased with the addition of NO3- and moderately increased with the addition of Fe(OH)3. No stimulation was observed with MnO2 added. Potential denitrification and anammox rates up to 63 and 0.27µmol N2 L-1 d-1, respectively, were measured when only 15N-NO3- was added. When H2S was added, both denitrification and anammox rates increased. Altogether, these results suggest that prokaryote communities in the redoxcline are able to efficiently use the most available substrates.

Nitrate and nitrite absorption, recycling and retention in tissues of sheep

Dietary nitrate is of increasing interest both for the pharmacological effects of its metabolites as well as its capacity to inhibit methanogenesis in the gut. A sequence of three experiments was conducted to investigate the absorption, metabolism and excretion of nitrate and nitrite through the gastrointestinal tract (GIT) of sheep, and to determine the fate of nitrate and nitrite in body fluids, tissues and faeces after intravenous dosing with 15N-labeled potassium nitrate (K15NO3) and with 15N-labeled sodium nitrite (Na15NO2). In Experiment 1, twelve female Merino sheep were assigned to one of two dietary treatments and adapted to the experimental diet over two weeks. Six sheep were fed a control diet of wheaten chaff mixture (600 g wheaten chaff plus 200 g wheat grain, CON) and six sheep were fed the CON diet with the inclusion of 18 g nitrate/kg DM (Nitrate). After acclimation to the diets, all sheep received a single intravenous dose of K15NO3 and were placed in metabolic cages for daily collection of total faeces and urine over 6 days. Experiment 2 studied movement of an intravenous dose of 15N in body fluids and tissues. Two sheep not adapted to dietary nitrate were dosed intravenously with K15NO3 or Na15NO2 and body fluids and tissue samples were collected 60 min after dosing. Finally, Experiment 3 was conducted to identify and quantify the major sites of nitrate and nitrite transfer within the body, focusing on absorption, partitioning and secretion into the GIT of anaesthetised sheep. A single dose of sodium nitrate (NaNO3) or sodium nitrite (NaNO2) was introduced into the rumen or abomasum or small intestine, and changes in nitrate and nitrite concentrations in other pools, including plasma, urine and saliva, were determined. Results from Experiment 1 showed that urinary recovery of 15N dose in urea after 46 h and total urinary recovery of 15N 141 h after dosing were greater in sheep fed the Nitrate diet relative to CON (P < 0.05). Recoveries of 15N in tissues indicated that nitrate and nitrite principally accumulated in the skin and muscle of sheep (Experiment 2). Finally, Experiment 3 indicated that nitrate and nitrite were rapidly absorbed from the rumen, abomasum and small intestine into the bloodstream. Nitrite was oxidized in plasma and the resultant nitrate was concentrated and recycled via saliva. Appearance of 15N in urinary urea confirmed the passage of plasma nitrate to the digestive tract, via saliva or transruminal flow to be reduced by gut biota to ammonia.