Green manuring combined with zeolite reduced nitrous oxide emissions in maize field by targeting microbial nitrogen transformations.

Green manure is a crucial strategy for increasing cereal yield and mitigating environmental burden while reducing chemical N fertilizer. To effectively tackle climate change, finding ways to reduce nitrous oxide (N2O) emissions from green manuring systems is vital. Herein, field and 15N labeled microcosm experiments were arranged to investigate the effect and mechanisms of green manuring and zeolite application on N2O emission. Both experiments comprised four treatments: conventional chemical N (N100), 70 % chemical N (N70), N70 with green manure (N70 + CV), and N70 + CV combined with zeolite (N70 + CV + Z). Compared with N100, both N70 + CV and N70 + CV + Z maintained maize yield, cumulative N2O emissions decreased by 37.7 % and 34.9 % in N70 + CV + Z in 2022-yr and 2023-yr, and by 12.8 % in N70 + CV in 2022-yr. Moreover, the reduction of N2O emission primarily occurred after incorporating green manure. The N100 and N70 + CV demonstrated a similar transformed proportion of chemical N to N2O (i.e., 4.9 % and 4.7 %) while reducing it to 2.7 % in N70 + CV + Z. Additionally, a mere 0.7 % of green manure N was transformed to N2O in both N70 + CV and N70 + CV + Z treatments. Compared with N100, both N70 + CV and N70 + CV + Z decreased the relative abundances of ammonia oxidation microbes, increased denitrifier and the ratios of (nirK + nirS)/nosZ and norBC/nosZ. Furthermore, compared with N70 + CV, N70 + CV + Z decreased the relative abundances of N2O-producer and the ratios of (nirK + nirS)/nosZ and norBC/nosZ in denitrification. These findings revealed that the reduction of N2O emissions resulting from green manure replaced chemical N was mainly due to weakened nitrification, while zeolite reduced N2O emissions attributed to enhanced conversion of N2O to N2. Moreover, certain key N-cycling functional bacteria, such as Phycisphaerae, Rubrobacteria, and Thermoflexia, were positively correlated with N2O emission. In contrast, Dehalococcoidia, Gammaproteobacteria, and Betaproteobacteria were negatively correlated with N2O emission. This investigation uncovered the underlying mechanisms for effectively reducing N2O emissions through green manuring combined with zeolite.

Straw returning and nitrogen reduction: Strategies for sustainable maize production in the dryland.

Implementing continue straw returning practices and optimizing nitrogen application can mitigate nitrogen losses and enhance nitrogen use efficiency (NUE) in dryland. 15N-labeled technique offers a robust approach for tracking fertilizer nitrogen fate and assessing nitrogen use efficiency. Based on the continue (>6 yr) experiment, we conducted a two-year experiment (2020 and 2021) to evaluate the effects of straw returning and nitrogen management under plastic film mulching on 15N recovery rates, N2O emissions and maize yield with three treatments: no straw returning with 225 kg N·ha-1 under plastic film mulching (RP-N225), straw returning with 225 kg N·ha-1 under plastic film mulching (RPS-N225), and straw returning with 20% nitrogen reduction (180 kg N·ha-1) under plastic film mulching (RPS-N180). After six years, both continue straw returning with plastic film mulching increased uptake of fertilizer nitrogen, had higher 15N recovery rates than RP-N225, leading to increased 15N accumulation in grain and aboveground biomass, ultimately enhancing yield. The RPS-N225 treatment exhibited the highest spring maize yield and nitrogen harvest index. The RPS-N180 treatment significantly increased maize yield more than RP-N225 and had the highest NUE, partial factor productivity of nitrogen fertilizer, and nitrogen uptake efficiency, with improvements ranging from 1.7 to 2.4%, 19.3-29.6%, and 17.3-27.5%, respectively, compared to the other treatments. Moreover, RPS-N225 resulted in significantly higher cumulative N2O emissions and yield-scaled N2O emissions than the other treatments, whereas the RPS-N180 treatment significantly decreased yield-scaled N2O emissions compared to RP-N225. Hence, combining continue straw returning with appropriate nitrogen reduction can effectively increase maize yield and yield-scaled N2O emissions. By offering insights into optimizing nitrogen fertilizer management after continue maize straw return, this study is contributed to widespread adoption of straw return practices and sustainable agricultural development in semi-arid areas.

Leaching sources and mechanisms of different nitrogen species from bioretention systems.

At present, nitrogen (N) leaching from bioretention systems (BRSs) has become a key issue, imposing constraints on their application, a consequence of N dynamics of both inflow and legacy N at different time scales. In this study, the distinct sources (IL: immediate leaching, FL: fast leaching, SL: slow leaching) and the principal transformation processes of different N species (i.e., NH4+, NO3- and DON) leaching originating from inflow and legacy of BRSs were firstly unveiled by various 15N species labeling (i.e., 15N-NH4+, 15N-NO3- and 15N-DON). Results indicate that: NH4+ leaching was primarily caused by FL from influent organic N and SL from influent NH4+, with mineralization being the main transformation process influencing NH4+ leaching; NO3- leaching primarily originated from SL, with the major proportion attributed to the influent organic N in SL, autotrophic and heterotrophic nitrification were the main influencing factors; DON leaching primarily originated from SL, with similar proportions coming from influent organic N, NH4+, and NO3-, inorganic N assimilation was the principal transformation process affecting DON leaching. This study provides an effective framework for apportioning the leaching sources of different N species, providing valuable insights for the implementation of both inflow and legacy N leaching control measures.

Does long-term drought or repeated defoliation affect seasonal leaf N cycling in young beech trees?

Forest trees adopt effective strategies to optimize nitrogen (N) use through internal N recycling. In the context of more recurrent environmental stresses due to climate change, the question remains of whether increased frequency of drought or defoliation threatens this internal N recycling strategy. We submitted 8-year-old beech trees to 2 years of either severe drought (Dro) or manual defoliation (Def) to create a state of N starvation. At the end of the second year before leaf senescence, we labeled the foliage of the Dro and Def trees, as well as that of control (Co) trees, with 15N-urea. Leaf N resorption, winter tree N storage (total N, 15N, amino acids, soluble proteins) and N remobilization in spring were evaluated for the three treatments. Defoliation and drought did not significantly impact foliar N resorption or N concentrations in organs in winter. Total N amounts in Def tree remained close to those in Co tree, but winter N was stored more in the branches than in the trunk and roots. Total N amount in Dro trees was drastically reduced (-55%), especially at the trunk level, but soluble protein concentrations increased in the trunk and fine roots compared with Co trees. During spring, 15N was mobilized from the trunk, branches and twigs of both Co and Def trees to support leaf growth. It was only provided through twig 15N remobilization in the Dro trees, thus resulting in extremely reduced Dro leaf N amounts. Our results suggest that stress-induced changes occur in N metabolism but with varying severity depending on the constraints: within-tree 15N transport and storage strategy changed in response to defoliation, whereas a soil water deficit induced a drastic reduction of the N amounts in all the tree organs. Consequently, N dysfunction could be involved in drought-induced beech tree mortality under the future climate.

Nitrogen Application Timing and Levels Affect the Fate and Budget of Fertilizer Nitrogen in the Apple-Soil System.

This study aimed to determine the effects of the nitrogen (N) application period and level on the fate of fertilizer N and the contribution of N absorption and translocation to apple organ N. Two N application periods (labeled by the 15N tracer technique in spring and summer, represented by SP and SU, respectively) and three N levels (N0, MN, and HN) were used to determine the physiological indexes and aboveground, root, and soil 15N content of 4-year-old dwarf ('Red Fuji'/M9T337) and arborized ('Red Fuji'/Malus hupehensis Rehd.) apple trees. The results showed that HN led to shoot overgrowth, which was not conducive to the growth of the apple root system (root length, root tips, root surface area, and root volume) or the improvement of root activity. The contribution of soil N to apple organ N accounted for more than 50%, and the contribution of N application in summer to fruit N was higher than that in spring. Under HN treatment, the proportion of soil N absorbed by trees decreased, while that of fertilizer N increased; however, the highest proportion was still less than 50%, so apple trees were highly dependent on soil N. Under MN treatment, fertilizer N residue was similar to soil N consumption, and soil N fertility maintained a basic balance. Under HN treatment, fertilizer N residue was significantly higher than soil N consumption, indicating that excessive N application increased fertilizer N residue in the soil. Overall, the 15N utilization rate of arborized trees (17.33-22.38%) was higher than that of dwarf trees (12.89-16.91%). A total of 12.89-22.38% of fertilizer 15N was absorbed by trees, 30.37-35.41% of fertilizer 15N remained in the soil, and 44.65-54.46% of fertilizer 15N was lost. The 15N utilization rate and 15N residual rate of summer N application were higher than those of spring N application, and the 15N loss rate was lower than that of spring N application. High microbial biomass N (MBN) may be one of the reasons for the high N utilization rate and the low loss rate of N application in summer.

Species-specific responses of C and N allocation to N addition: evidence from dual 13C and 15N labeling in three tree species.

Soil nitrogen (N) availability affects plant carbon (C) utilization. However, it is unclear how various tree functional types respond to N addition in terms of C assimilation, allocation, and storage. Here, a microcosm experiment with dual 13C and 15N labeling was conducted to study the effects of N addition (i.e., control, 0 g N kg-1; moderate N addition, 1.68 g N kg-1; and high N addition, 3.36 g N kg-1 soil) on morphological traits, on changes in nonstructural carbohydrates (NSC) in different organs, as well as on C and N uptake and allocation in three European temperate forest tree species (i.e., Acer pseudoplatanus, Picea abies and Abies alba). Our results demonstrated that root N uptake rates of the three tree species increased by N addition. In A. pseudoplatanus, N uptake by roots, N allocation to aboveground organs, and aboveground biomass allocation significantly improved by moderate and high N addition. In A. alba, only the high N addition treatment considerably raised aboveground N and C allocation. In contrast, biomass as well as C and N allocation between above and belowground tissues were not altered by N addition in P. abies. Meanwhile, NSC content as well as C and N coupling (represented by the ratio of relative 13C and 15N allocation rates in organs) were affected by N addition in A. pseudoplantanus and P. abies but not in A. alba. Overall, A. pseudoplatanus displayed the highest sensitivity to N addition and the highest N requirement among the three species, while P. abies had a lower N demand than A. alba. Our findings highlight that the responses of C and N allocation to soil N availability are species-specific and vary with the amount of N addition.

Foliar nitrogen uptake in broadleaf evergreen Mediterranean forests: Fertilisation experiment with labelled nitrogen.

Atmospheric nitrogen (N) deposition in Mediterranean sclerophyllous forests of Holm oak (Quercus rotundifolia, Q. ilex) in Spain often exceeds empirical critical loads established for ecosystem conservation. There are still uncertainties on the capacity of canopy retention and uptake of the atmospheric N deposited of these forests. Studying and analysing all the forest nitrogen-cycle processes is essential to understand the potential effect of N deposition in these ecosystems. This study conducted a year-long short-term fertilisation experiment with labelled ammonium (15N-NH4) and nitrate (15N-NO3) to estimate foliar N absorption rates and assess the influence of leaf phenology and meteorological seasonal variations. Fertilising solutions were prepared to simulate low and high wet N deposition concentration, based on data reported from previous studies. Additionally, ecophysiological and meteorological measurements were collected to explore potential relationships between absorption rates, plant activity, and weather conditions. The results showed that Holm oak leaves were able to absorb both oxidised and reduced N compounds, with higher rates of NH4+ absorption. N recovery of both NH4+ and NO3- was higher in the low concentration treatments, suggesting reduced effectiveness of absorption as concentration increases. Foliar absorption rates were leaf-age dependent, with the highest values observed in young developing leaves. Foliar uptake showed seasonal changes with a clear reduction during the summer, linked to drought and dry weather conditions, and showing also smaller leaf net assimilation and stomatal conductance. During the rest of the year, foliar N absorption was not clearly associated to plant physiological activity but with environmental conditions. Our findings suggest that Holm oak canopies could absorb an important part of the incoming N deposition, but this process is compound, season and leaf phenology dependent. Further research is therefore needed to better understand and model this part of the N cycle.

Nitrogen acquisition strategy and its effects on invasiveness of a subtropical invasive plant.

Introduction: Preference and plasticity in nitrogen (N) form uptake are the main strategies with which plants absorb soil N. However, little effort has been made to explore effects of N form acquisition strategies, especially the plasticity, on invasiveness of exotic plants, although many studies have determined the effects of N levels (e.g. N deposition). Methods: To address this problem, we studied the differences in N form acquisition strategies between the invasive plant Solidago canadensis and its co-occurring native plant Artemisia lavandulaefolia, effects of soil N environments, and the relationship between N form acquisition strategy of S. canadensis and its invasiveness using a 15N-labeling technique in three habitats at four field sites. Results: Total biomass, root biomass, and the uptakes of soil dissolved inorganic N (DIN) per quadrat were higher for the invasive relative to the native species in all three habitats. The invader always preferred dominant soil N forms: NH4 + in habitats with NH4 + as the dominant DIN and NO3 - in habitats with NO3 - as the dominant DIN, while A. lavandulaefolia consistently preferred NO3 - in all habitats. Plasticity in N form uptake was higher in the invasive relative to the native species, especially in the farmland. Plant N form acquisition strategy was influenced by both DIN levels and the proportions of different N forms (NO3 -/NH4 +) as judged by their negative effects on the proportional contributions of NH4 + to plant N (f NH4 +) and the preference for NH4 + (ß NH4 +). In addition, total biomass was positively associated with f NH4 + or ß NH4 + for S. canadensis, while negatively for A. lavandulaefolia. Interestingly, the species may prefer to absorb NH4 + when soil DIN and/or NO3 -/NH4 + ratio were low, and root to shoot ratio may be affected by plant nutrient status per se, rather than by soil nutrient availability. Discussion: Our results indicate that the superior N form acquisition strategy of the invader contributes to its higher N uptake, and therefore to its invasiveness in different habitats, improving our understanding of invasiveness of exotic plants in diverse habitats in terms of utilization of different N forms.

Silicon Supply Improves Nodulation and Dinitrogen Fixation and Promotes Growth in Trifolium incarnatum Subjected to a Long-Term Sulfur Deprivation.

In many crops species, sulfur (S) deprivation negatively affects growth, seed yield quality and plant health. Furthermore, silicon (Si) is known to alleviate many nutritional stresses but the effects of Si supply on plants subjected to S deficiency remain unclear and poorly documented. The objective of this study was to evaluate whether Si supply would alleviate the negative effects of S deprivation on root nodulation and atmospheric dinitrogen (N2) fixation capacity in Trifolium incarnatum subjected (or not) to long-term S deficiency. For this, plants were grown for 63 days in hydroponic conditions with (500 µM) or without S and supplied (1.7 mM) or not with Si. The effects of Si on growth, root nodulation and N2 fixation and nitrogenase abundance in nodules have been measured. The most important beneficial effect of Si was observed after 63 days. Indeed, at this harvest time, a Si supply increased growth, the nitrogenase abundance in nodules and N2 fixation in S-fed and S-deprived plants while a beneficial effect on the number and total biomass of nodules was only observed in S-deprived plants. This study shows clearly for the first time that a Si supply alleviates negative effects of S deprivation in Trifolium incarnatum.

From Soil to River: Revealing the Mechanisms Underlying the High Riverine Nitrate Levels in a Forest Dominated Catchment.

Elevated riverine nitrate (NO3-) levels have led to increased eutrophication and other ecological implications. While high riverine NO3- levels were generally ascribed to anthropogenic activities, high NO3- levels in some pristine or minimally disturbed rivers were reported. The drivers of these unexpectedly high NO3- levels remain unclear. This study combined natural abundance isotopes, 15N-labeling techniques, and molecular techniques to reveal the processes driving the high NO3- levels in a sparsely populated forest river. The natural abundance isotopes revealed that the NO3- was mainly from soil sources and that NO3- removal processes were insignificant. The 15N-labeling experiments also quantitatively showed that the biological NO3- removal processes, i.e., denitrification, dissimilatory NO3- reduction to ammonium (DNRA), and anaerobic ammonia oxidation (anammox), in the soils and sediments were weak relative to nitrification in summer. While nitrification was minor in winter, the NO3- removal was insignificant relative to the large NO3- stock in the catchment. Stepwise multiple regression analyses and structural equation models revealed that in summer, nitrification in the soils was regulated by the amoA-AOB gene abundances and NH4+-N contents. Low temperature constrained nitrification in winter. Denitrification was largely controlled by moisture content in both seasons, and anammox and DNRA could be explained by the competition with nitrification and denitrification on their substrate (nitrite-NO2-). We also revealed the strong hydrological control on the transport of soil NO3- to the river. This study effectively revealed the mechanisms underlying the high NO3- levels in a nearly pristine river, which has implications for the understanding of riverine NO3- levels worldwide.

Threshold responses of soil gross nitrogen transformation rates to aridity gradient.

The responses of soil nitrogen (N) transformations to climate change are crucial for biome productivity prediction under global change. However, little is known about the responses of soil gross N transformation rates to drought gradient. Along an aridity gradient across the 2700 km transect of drylands on the Qinghai-Tibetan Plateau, this study measured three main soil gross N transformation rates in both topsoil (0-10 cm) and subsoil (20-30 cm) using the laboratorial 15 N labeling. The relevant soil abiotic and biotic variables were also determined. The results showed that gross N mineralization and nitrification rates steeply decreased with increasing aridity when aridity was less than 0.5 but just slightly decreased with increasing aridity when aridity was larger than 0.5 at both soil layers. In topsoil, the decreases of the two gross rates were accompanied by the similar decreased patterns of soil total N content and microbial biomass carbon with increasing aridity (p < .05). In subsoil, although the decreased pattern of soil total N with increasing aridity was still similar to the decreases of the two gross rates (p < .05), microbial biomass carbon did not change (p > .05). Instead, bacteria and ammonia oxidizing archaea abundances decreased with increasing aridity when aridity was larger than 0.5 (p < .05). With an aridity threshold of 0.6, gross N immobilization rate increased with increasing aridity in wetter region (aridity < 0.6) accompanied with an increased bacteria/fungi ratio, but decreased with increasing aridity in drier region (aridity > 0.6) where mineral N and microbial biomass N also decreased at both soil layers (p < .05). This study provided new insight to understand the differential responses of soil N transformation to drought gradient. The threshold responses of the gross N transformation rates to aridity gradient should be noted in biogeochemical models to better predict N cycling and manage land in the context of global change.

15N-labelling of Leaves Combined with GC-MS Analysis as a Tool for Monitoring the Dynamics of Nitrogen Incorporation into Amino Acids.

Labeling plant material such as detached leaves with 15NH4+ is a very instrumental method for the characterization of metabolic pathways of mineral nitrogen assimilation and incorporation into amino acids. A procedure of labeling, followed by amino acid extraction, purification, and derivatization for gas chromatography coupled to mass spectrometry (GC/MS) analysis, is presented. The rationale of heavy isotope abundance calculations and amino acid quantification is detailed. This method is adaptable to various plant species and various kinds of investigations, such as elucidating physiological changes occurring as a result of gene mutations (overexpression or inhibition) in natural variants or genetically modified crops, or characterization of metabolic fluxes in genotypes exhibiting contrasted physiological or developmental adaptive responses to biotic and/or abiotic environmental stresses. Furthermore, the benefit of working on detached organs or pieces of organs is to investigate finely the metabolism of species that are not amenable to laboratory work, such as plants growing in natural environments or under agricultural conditions in the field.

Fungus Pichia kudriavzevii XTY1 and heterotrophic nitrifying bacterium Enterobacter asburiae GS2 cannot efficiently transform organic nitrogen via hydroxylamine and nitrite.

Heterotrophic nitrification is a process of organic nitrogen degradation completed by the participation of heterotrophic nitrifying microorganisms, which can accelerate the nitrogen transformation process. However, the current research mainly focuses on heterotrophic nitrifying bacteria and their ammonium degradation capacities. And there is little accumulation of research on fungi, the main force of heterotrophic nitrification, and their capacities to transform organic nitrogen. In this study, novel heterotrophic nitrifying fungus (XTY1) and bacterium (GS2) were screened and isolated from upland soil, and the strains were identified and registered through GenBank comparison. After 24 h single nitrogen source tests and 15N labeling tests, we compared and preliminarily determined the heterotrophic nitrification capacities and pathways of the two strains. The results showed that XTY1 and GS2 had different transformation capacities to different nitrogen substrates and could efficiently transform organic nitrogen. However, the transformation capacity of XTY1 to ammonium was much lower than that of GS2. The two strains did not pass through NH2OH and NO2 - during the heterotrophic nitrification of organic nitrogen, and mainly generated intracellular nitrogen and low N2O. Other novel organic nitrogen metabolism pathways may be existed, but they remain to be further validated.

Arbuscular Mycorrhiza and Nitrification: Disentangling Processes and Players by Using Synthetic Nitrification Inhibitors.

Both plants and their associated arbuscular mycorrhizal (AM) fungi require nitrogen (N) for their metabolism and growth. This can result in both positive and negative effects of AM symbiosis on plant N nutrition. Either way, the demand for and efficiency of uptake of mineral N from the soil by mycorrhizal plants are often higher than those of nonmycorrhizal plants. In consequence, the symbiosis of plants with AM fungi exerts important feedbacks on soil processes in general and N cycling in particular. Here, we investigated the role of the AM symbiosis in N uptake by Andropogon gerardii from an organic source (15N-labeled plant litter) that was provided beyond the direct reach of roots. In addition, we tested if pathways of 15N uptake from litter by mycorrhizal hyphae were affected by amendment with different synthetic nitrification inhibitors (dicyandiamide [DCD], nitrapyrin, or 3,4-dimethylpyrazole phosphate [DMPP]). We observed efficient acquisition of 15N by mycorrhizal plants through the mycorrhizal pathway, independent of nitrification inhibitors. These results were in stark contrast to 15N uptake by nonmycorrhizal plants, which generally took up much less 15N, and the uptake was further suppressed by nitrapyrin or DMPP amendments. Quantitative real-time PCR analyses showed that bacteria involved in the rate-limiting step of nitrification, ammonia oxidation, were suppressed similarly by the presence of AM fungi and by nitrapyrin or DMPP (but not DCD) amendments. On the other hand, abundances of ammonia-oxidizing archaea were not strongly affected by either the AM fungi or the nitrification inhibitors. IMPORTANCE Nitrogen is one of the most important elements for all life on Earth. In soil, N is present in various chemical forms and is fiercely competed for by various microorganisms as well as plants. Here, we address competition for reduced N (ammonia) between ammonia-oxidizing prokaryotes and arbuscular mycorrhizal fungi. These two functionally important groups of soil microorganisms, participating in nitrification and plant mineral nutrient acquisition, respectively, have often been studied in separation in the past. Here, we showed, using various biochemical and molecular approaches, that the fungi systematically suppress ammonia-oxidizing bacteria to an extent similar to that of some widely used synthetic nitrification inhibitors, whereas they have only a limited impact on abundance of ammonia-oxidizing archaea. Competition for free ammonium is a plausible explanation here, but it is also possible that the fungi produce some compounds acting as so-called biological nitrification inhibitors.

Metabolic15N labeling of the N-glycosylated immunoglobulin G1 Fc with an engineered Saccharomyces cerevisiae strain.

The predominant protein expression host for NMR spectroscopy is Escherichia coli, however, it does not synthesize appropriate post-translation modifications required for mammalian protein function and is not ideal for expressing naturally secreted proteins that occupy an oxidative environment. Mammalian expression platforms can address these limitations; however, these are not amenable to cost-effective uniform 15 N labeling resulting from highly complex growth media requirements. Yeast expression platforms combine the simplicity of bacterial expression with the capabilities of mammalian platforms, however yeasts require optimization prior to isotope labeling. Yeast expression will benefit from methods to boost protein expression levels and developing labeling conditions to facilitate growth and high isotope incorporation within the target protein. In this work, we describe a novel platform based on the yeast Saccharomyces cerevisiae that simultaneously expresses the Kar2p chaperone and protein disulfide isomerase in the ER to facilitate the expression of secreted proteins. Furthermore, we developed a growth medium for uniform 15 N labeling. We recovered 2.2 mg/L of uniformly 15 N-labeled human immunoglobulin (Ig)G1 Fc domain with 90.6% 15 N labeling. NMR spectroscopy revealed a high degree of similarity between the yeast and mammalian-expressed IgG1 Fc domains. Furthermore, we were able to map the binding interaction between IgG1 Fc and the Z domain through chemical shift perturbations. This platform represents a novel cost-effective strategy for 15 N-labeled immunoglobulin fragments.

Sample Preparation, Data Acquisition, and Data Analysis for 15N-Labeled and Nonlabeled Monoterpene Indole Alkaloids in Catharanthus roseus.

Recent approaches developed in metabolomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) enabled us to assign a part of specialized metabolites in plants. However, the approaches are not good enough for the rest of the metabolites, which are still unknown. To characterize the unknown metabolites, more appropriate and precise approaches need to be developed. Here, a procedure to analyze 15N-labeled and nonlabeled LC-MS/MS data for identification of monoterpene indole alkaloids was developed.

Deepened snow loosens temporal coupling between plant and microbial N utilization and induces ecosystem N losses.

Seasonal differences in plant and microbial nitrogen (N) acquisition are believed to be a major mechanism that maximizes ecosystem N retention. There is also a concern that climate change may interrupt the delicate balance in N allocation between plants and microbes. Yet, convincing experimental evidence is still lacking. Using a 15 N tracer, we assessed how deepened snow affects the temporal coupling between plant and microbial N utilization in a temperate Mongolian grassland. We found that microbial 15 N recovery peaked in winter, accounting for 22% of the total ecosystem 15 N recovery, and then rapidly declined during the spring thaw. By stimulating N loss via N2 O emission and leaching, deepened snow reduced the total ecosystem 15 N recovery by 42% during the spring thaw. As the growing season progresses, the 15 N released from microbial biomass was taken up by plants, and the competitive advantage for N shifted from microbes to plants. Plant 15 N recovery reached its peak in August, accounting for 17% of the total ecosystem 15 N recovery. The Granger causality test showed that the temporal dynamics of plant 15 N recovery can be predicted by microbial 15 N recovery under ambient snow but not under deepened snow. In addition, plant 15 N recovery in August was positively correlated with and best explained by microbial 15 N recovery in March. The lower microbial 15 N recovery under deepened snow in March reduced plant 15 N recovery by 73% in August. Together, our results provide direct evidence of seasonal differences in plant and microbial N utilization that are conducive to ecosystem N retention; however, deepened snow disrupted the temporal coupling between plant-microbial N use and turnover. These findings suggest that changes in snowfall patterns may significantly alter ecosystem N cycling and N-based greenhouse gas emissions under future climate change. We highlight the importance of better representing winter processes and their response to winter climate change in biogeochemical models when assessing N cycling under global change.

Post-anthesis Relationships Between Nitrogen Isotope Discrimination and Yield of Spring Wheat Under Different Nitrogen Levels.

Wheat grain yield and nitrogen (N) content are influenced by the amount of N remobilized to the grain, together with pre-anthesis and post-anthesis N uptake. Isotopic techniques in farmed areas may provide insight into the mechanism underlying the N cycle. 15N-labeled urea was applied to microplots within five different fertilized treatments 0 kg ha-1 (N1), 52.5 kg ha-1 (N2), 105 kg ha-1 (N3), 157.5 kg ha-1 (N4), and 210 kg ha-1 (N5) of a long-term field trial (2003-2021) in a rainfed wheat field in the semi-arid loess Plateau, China, to determine post-anthesis N uptake and remobilization into the grain, as well as the variability of 15N enrichment in aboveground parts. Total N uptake was between 7.88 and 29.27 kg ha-1 for straw and 41.85 and 95.27 kg ha-1 for grain. In comparison to N1, N fertilization increased straw and grain N uptake by 73.1 and 56.1%, respectively. Nitrogen use efficiency (NUE) and harvest index were altered by N application rates. The average NUE at maturity was 19.9% in 2020 and 20.01% in 2021; however, it was usually higher under the control and low N conditions. The amount of 15N excess increased as the N rate increased: N5 had the highest 15N excess at the maturity stage in the upper (2.28 ± 0.36%), the middle (1.77 ± 0.28%), and the lower portion (1.68 ± 1.01%). Compared to N1, N fertilization (N2-N5) increased 15N excess in the various shoot portions by 50, 38, and 35% at maturity for upper, middle, and lower portions, respectively. At maturity, the 15N excess remobilized to the grain under N1-N5 was between 5 and 8%. Our findings revealed that N had a significant impact on yield and N isotope discrimination in spring wheat that these two parameters can interact, and that future research on the relationship between yield and N isotope discrimination in spring wheat should take these factors into account.

Sources of nitrous oxide emissions from hydroponic tomato cultivation: Evidence from stable isotope analyses.

Introduction: Hydroponic vegetable cultivation is characterized by high intensity and frequent nitrogen fertilizer application, which is related to greenhouse gas emissions, especially in the form of nitrous oxide (N2O). So far, there is little knowledge about the sources of N2O emissions from hydroponic systems, with the few studies indicating that denitrification could play a major role. Methods: Here, we use evidence from an experiment with tomato plants (Solanum lycopersicum) grown in a hydroponic greenhouse setup to further shed light into the process of N2O production based on the N2O isotopocule method and the 15N tracing approach. Gas samples from the headspace of rock wool substrate were collected prior to and after 15N labeling at two occasions using the closed chamber method and analyzed by gas chromatography and stable isotope ratio mass spectrometry. Results: The isotopocule analyses revealed that either heterotrophic bacterial denitrification (bD) or nitrifier denitrification (nD) was the major source of N2O emissions, when a typical nutrient solution with a low ammonium concentration (1-6 mg L-1) was applied. Furthermore, the isotopic shift in 15N site preference and in δ18O values indicated that approximately 80-90% of the N2O produced were already reduced to N2 by denitrifiers inside the rock wool substrate. Despite higher concentrations of ammonium present during the 15N labeling (30-60 mg L-1), results from the 15N tracing approach showed that N2O mainly originated from bD. Both, 15N label supplied in the form of ammonium and 15N label supplied in the form of nitrate, increased the 15N enrichment of N2O. This pointed to the contribution of other processes than bD. Nitrification activity was indicated by the conversion of small amounts of 15N-labeled ammonium into nitrate. Discussion/Conclusion: Comparing the results from N2O isotopocule analyses and the 15N tracing approach, likely a combination of bD, nD, and coupled nitrification and denitrification (cND) was responsible for the vast part of N2O emissions observed in this study. Overall, our findings help to better understand the processes underlying N2O and N2 emissions from hydroponic tomato cultivation, and thereby facilitate the development of targeted N2O mitigation measures.

Tracking Animal-Dispersed Seedlings Using 15N Xylem Injection Method.

Although various seed-marking methods have been developed for seed dispersal, it remains difficult to track the actual patterns of seed dispersal and seedling recruitment. Thus, new labeling methods that accurately track seedling establishment along with seed movement would help us better understand seed dispersal. Here, we developed a new nondestructive method using 15N xylem injection to track seed dispersal and seedling recruitment based on the enriched isotopic signals in the mature seeds. Our results first showed that xylem injection of 15N successfully enriched 15N both in the acorns and seedlings of Quercus variabilis. By marking acorns and seedlings with 15N stable isotopes, we successfully tracked seedlings established from acorns dispersed by seed-eating animals in the field. Our xylem 15N injection caused little alteration to seeds and showed no significant effects on seed selection by seed-eating animals as well as seed germination and seedling establishment, verifying the validity of the 15N xylem injection method to track seedling establishment. Our xylem 15N injection method is expected to be a powerful tool for tracking seed dispersal and seedling recruitment mediated by seed-eating animals in seed dispersal ecology.