C and N allocation in soil under ryegrass and alfalfa estimated by 13C and 15N labelling

Background and Aims

Below-ground translocated carbon (C) released as rhizodeposits is an important driver for microbial mobilization of nitrogen (N) for plants. We investigated how a limited substrate supply due to reduced photoassimilation alters the allocation of recently assimilated C in plant and soil pools under legume and non-legume species.

Methods

A non-legume (Lolium perenne) and a legume (Medicago sativa) were labelled with ¹⁵N before the plants were clipped or shaded, and labelled twice with ¹³CO₂ thereafter. Ten days after clipping and shading, the ¹⁵N and ¹³C in shoots, roots, soil, dissolved organic nitrogen (DON) and carbon (DOC) and in microbial biomass, as well as the ¹³C in soil CO₂ were analyzed.

Results

After clipping, about 50 % more ¹³C was allocated to regrowing shoots, resulting in a lower translocation to roots compared to the unclipped control. Clipping also reduced the total soil CO₂ efflux under both species and the ¹³C recovery of soil CO₂ under L. perenne. The ¹⁵N recovery increased in the shoots of M. sativa after clipping, because storage compounds were remobilized from the roots and/or the N uptake from the soil increased. After shading, the assimilated ¹³C was preferentially retained in the shoots of both species. This caused a decreased ¹³C recovery in the roots of M. sativa. Similarly, the total soil CO₂ efflux under M. sativa decreased more than 50 % after shading. The ¹⁵N recovery in plant and soil pools showed that shading has no effect on the N uptake and N remobilization for L. perenne, but, the ¹⁵N recovery increased in the shoot of M. sativa.

Conclusions

The experiment showed that the dominating effect on C and N allocation after clipping is the need of C and N for shoot regrowth, whereas the dominating effect after shading is the reduced substrate supply for growth and respiration. Only slight differences could be observed between L. perenne and M. sativa in the C and N distribution after clipping or shading.     

Strategies of carbon and nitrogen acquisition by saprotrophic and ectomycorrhizal fungi in Finnish boreal Picea abies-dominated forests

We compared the δ¹³C and δ¹⁵N of forest material with an extensive sporocarp collection to elucidate the role of litter, wood and soil as fungal carbon and nitrogen sources in Finnish boreal Picea abies-dominated forests. Ectomycorrhizal Hydnum and Cortinarius had higher δ¹⁵N than other ectomycorrhizal fungi, suggesting use of ¹⁵N-enriched, deeper nitrogen. Russula had lower δ¹⁵N than other ectomycorrhizal fungi and resembled some litter decay genera, suggesting use of litter-derived nitrogen. There was little variation in δ¹⁵N among other genera of ectomycorrhizal fungi, indicating limited functional diversity in nitrogen use. Saprotrophic Leotia, Gymnopus, Hypholoma, Pholiota, Rhodocollybia and Calocera had δ¹⁵N values similar to ectomycorrhizal fungi, indicating overlap in use of older nitrogen from soil or roots or use of newly fixed nitrogen. Genera of litter and wood decay fungi varied up to 6‰ in δ¹³C and 10‰ in δ¹⁵N, suggesting large differences in carbon and nitrogen sources and processing. Similar δ¹³C between white and brown rot wood decay fungi also suggest that white rot fungi do not use lignin-derived carbon. Together, these δ¹³C and δ¹⁵N patterns of fungi from Finnish boreal forests enhance our knowledge of fungal functional diversity and indicate broad use of litter, wood and soil resources.     

Carbon and nitrogen acquisition strategies by wood decay fungi influence their isotopic signatures in Picea abies forests

We examined whether sporocarp carbon and nitrogen isotope ratios (δ¹³C and δ¹⁵N values) reflected different functional strategies in 15 species of wood decay fungi. In Finnish Picea abies forests, we compared sporocarp δ¹³C and δ¹⁵N against log diameter, proximity to ground, and three wood decay types, specifically brown rot, nonselective white rot, and selective white rot (targeting hemicellulose and lignin preferentially). In regression analysis (adjusted r² = 0.576), species accounted for 31% of variability in δ¹³C, with factors influencing wood δ¹³C accounting for the remainder. Brown rot fungi and three white rot fungi that selectively attacked hemicellulose (Heterobasidion parviporum, Phellopilus nigrolimitatus, and Trichaptum abietinum) were higher in δ¹³C than nonselective white rot fungi. This was attributed to greater assimilation of ¹³C-enriched pentoses from hemicellulose by these fungi. The pathogenic white rot fungus Heterobasidion parviporum had higher δ¹⁵N with proximity to ground and increasing log diameter. This suggested that ¹⁵N-enriched soil N contributed to decomposing logs and that Heterobasidion growing from a bigger resource base had increased access to soil N. These isotopic patterns accordingly reflected both functional diversity of wood decay fungi and site-specific factors.     

Estimating the contribution of nitrogen from legume cover crops to the nitrogen nutrition of grapevines using a 15N dilution technique

The aim of this controlled environment experiment was to quantify the distribution of leaf-fed-¹⁵N and canopy fed-¹³C within nodulating, non-nodulating or N fertilized non-nodulating Cicer arietinum L. and in their surrounding rhizosphere soil, excluding soil?+?root respiration. Nodulating chickpea partitioned 32% of its total N and 27% of its total recoverable C below-ground, of which only 50% of N and 36% of C were in the clean root fraction. Non-nodulating chickpea allocated equal recoverable C but slightly less N (28%) below-ground but lost less C from plant induced below-ground respiration. The importance of this below-ground partitioning for crop systems C and N balances is highlighted by their large (45% and 33%, for N and C, respectively) contribution to the total plant derived residue (recyclable) fraction. Recovered ¹⁵N and ¹³C were greater (P?<?0.05) in the outer-rhizosphere (459?µg ¹⁵N and 3.2 mg ¹³C core^?1) than in the inner-rhizosphere soil (detached from roots during freeze-drying; 18?µg ¹⁵N and 67?µg ¹³C core^?1) in relation with the relative size of these compartments. This highlights the significance of the outer-rhizosphere soil when estimating C and N budgets and quantifying rhizodeposition, and the benefit of a double (¹⁵N, ¹³C) isotope approach to determine this flow against large background soil C and N pools.     

Trophic position and growth stages of Caddisfly (Stenopsyche marmorata Navas) larvae in the Echi River, Japan

We investigated the trophic position of the larval filter-feeding caddisfly species—Stenopsyche marmorata Navas in the Echi River (Japan) using δ¹³C and δ¹⁵N measurements by instar growth stage. We also analyzed δ¹³C and δ¹⁵N of food sources (periphyton and suspended and benthic POM) along the course of the river. The larvae showed some degree of spatial variation in δ¹³C and δ¹⁵N values with a downstream increase in δ¹³C and δ¹⁵N values. Our results demonstrate that the trophic structure of S. marmorata of different larval stages were spatially varying, depending on food sources. Small-sized instars displayed a higher trophic position than larger instars, confirming that their non-selective feeding behavior leads to consumption of items with high δ¹⁵N values according to the spatial distribution of food sources in the riverine system.     

Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR

Local dynamics of interhelical loops in bacteriorhodopsin (bR), the extracellular BC, DE and FG, and cytoplasmic AB and CD loops, and helix B were determined on the basis of a variety of relaxation parameters for the resolved ¹³C and ¹⁵N signals of [1-¹³C]Tyr-, [¹⁵N]Pro- and [1-¹³C]Val-, [¹⁵N]Pro-labeled bR. Rotational echo double resonance (REDOR) filter experiments were used to assign [1-¹³C]Val-, [¹⁵N]Pro signals to the specific residues in bR. The previous assignments of [1-¹³C]Val-labeled peaks, 172.9 or 171.1 ppm, to Val69 were revised: the assignment of peak, 172.1 ppm, to Val69 was made in view of the additional information of conformation-dependent ¹⁵N chemical shifts of Pro bonded to Val in the presence of ¹³C-¹⁵N correlation, although no assignment of peak is feasible for ¹³C nuclei not bonded to Pro. ¹³C or ¹⁵N spin-lattice relaxation times (T₁), spin-spin relaxation times under the condition of CP-MAS (T₂), and cross relaxation times (T_CH and T_NH) for ¹³C and ¹⁵N nuclei and carbon or nitrogen-resolved, ¹H spin-lattice relaxation times in the rotating flame (¹H T_1ρ) for the assigned signals were measured in [1-¹³C]Val-, [¹⁵N]Pro-bR. It turned out that V69-P70 in the BC loop in the extracellular side has a rigid β-sheet in spite of longer loop and possesses large amplitude motions as revealed from ¹³C and ¹⁵N conformation-dependent chemical shifts and T₁, T₂, ¹H T_1ρ and cross relaxation times. In addition, breakage of the β-sheet structure in the BC loop was seen in bacterio-opsin (bO) in the absence of retinal.     

Trophic Relationships and Habitat Preferences of Delphinids from the Southeastern Brazilian Coast Determined by Carbon and Nitrogen Stable Isotope Composition

To investigate the foraging habitats of delphinids in southeastern Brazil, we analyzed stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotopes in muscle samples of the following 10 delphinid species: Sotalia guianensis, Stenella frontalis, Tursiops truncatus, Steno bredanensis, Pseudorca crassidens, Delphinus sp., Lagenodelphis hosei, Stenella attenuata, Stenella longirostris and Grampus griseus. We also compared the δ¹³C and δ¹⁵N values among four populations of S. guianensis. Variation in carbon isotope results from coast to ocean indicated that there was a significant decrease in δ¹³C values from estuarine dolphins to oceanic species. S. guianensis from Guanabara Bay had the highest mean δ¹³C value, while oceanic species showed significantly lower δ¹³C values. The highest δ¹⁵N values were observed for P. crassidens and T. truncatus, suggesting that these species occupy the highest trophic position among the delphinids studied here. The oceanic species S. attenuata, G. griseus and L. hosei had the lowest δ¹⁵N values. Stable isotope analysis showed that the three populations of S. guianensis in coastal bays had different δ¹³C values, but similar δ¹⁵N results. Guiana dolphins from Sepetiba and Ilha Grande bays had different foraging habitat, with specimens from Ilha Grande showing more negative δ¹³C values. This study provides further information on the feeding ecology of delphinids occurring in southeastern Brazil, with evidence of distinctive foraging habitats and the occupation of different ecological niches by these species in the study area.     

Carbon and nitrogen fluxes between beech and their ectomycorrhizal assemblage

To determine the exchange of nitrogen and carbon between ectomycorrhiza and host plant, young beech (Fagus sylvatica) trees from natural regeneration in intact soil cores were labelled for one growing season in a greenhouse with ¹³CO₂ and ¹⁵NO₃ ¹⁵NH₄. The specific enrichments of ¹⁵N and ¹³C were higher in ectomycorrhizas (EMs) than in any other tissue. The enrichments of ¹³C and ¹⁵N were also higher in the fine-root segments directly connected with the EM (mainly second-order roots) than that in bulk fine or coarse roots. A strict, positive correlation was found between the specific ¹⁵N enrichment in EM and the attached second-order roots. This finding indicates that strong N accumulators provide more N to their host than low N accumulators. A significant correlation was also found for the specific ¹³C enrichment in EM and the attached second-order roots. However, the specific enrichments for ¹⁵N and ¹³C in EM were unrelated showing that under long-term conditions, C and N exchange between host and EMs are uncoupled. These findings suggest that EM-mediated N flux to the plant is not the main control on carbon flux to the fungus, probably because EMs provide many different services to their hosts in addition to N provision in their natural assemblages.     

Relationship between mercury,organochlorine compounds and stable isotope ratios of carbon and nitrogen in yellowfin tuna (Thunnus albacares) taken from different regions of the Pacific and Indian Oceans

The aim of this study was to compare the δ¹⁵N, δ¹³C, Hg and organochlorines (PCBs, p,p′-DDE and trans-nonachlor) in the yellowfin tuna taken from several regions of the Pacific and Indian Oceans. The highest values of δ¹⁵N and δ¹³C were observed in the tuna taken from Sri Lanka and Maldives (Indian Ocean), while the lowest values were observed in the tuna taken from Hawaii. In contrast, the highest Hg concentration was observed in the tuna taken from Hawaii, while the lowest concentration was observed in the tuna taken from Sri Lanka and Maldives. These orders of δ¹⁵N, δ¹³C and Hg may reflect the geographical characteristics of those among nine regions. On the other hand, the highest concentration of each organochlorine was found in the tuna taken from New Zealand, while the lowest was found in the tuna sample from Hawaii, probably reflecting the anthropogenic contamination among eight regions. The inter-regional variations in organochlorines were markedly larger than those in Hg concentration. The Hg uniformly distributed in the area under the normal curve of Hg concentration against δ¹³C or δ¹⁵N value, while the organochlorines did not. This difference may be ascribed to the fact that Hg is of natural and anthropogenic origins, while the organochlorines are of anthropogenic origin. A principal compartment analysis of δ¹³C, δ¹⁵N, Hg and organochlorines apparently clarified the tuna taken from eight regions into 5 groups of Sri Lanka and Maldives, New Zealand, Australia, Hawaii and Japan.     

Preservation Methods Alter Carbon and Nitrogen Stable Isotope Values in Crickets (Orthoptera: Grylloidea)

Stable isotope analysis (SIA) is an important tool for investigation of animal dietary habits for determination of feeding niche. Ideally, fresh samples should be used for isotopic analysis, but logistics frequently demands preservation of organisms for analysis at a later time. The goal of this study was to establish the best methodology for preserving forest litter-dwelling crickets for later SIA analysis without altering results. We collected two cricket species, Phoremia sp. and Mellopsis doucasae, from which we prepared 70 samples per species, divided among seven treatments: (i) freshly processed (control); preserved in fuel ethanol for (ii) 15 and (iii) 60 days; preserved in commercial ethanol for (iv) 15 and (v) 60 days; fresh material frozen for (vi) 15 and (vii) 60 days. After oven drying, samples were analyzed for δ ¹⁵N, δ ¹³C values, N(%), C(%) and C/N atomic values using continuous flow isotope ratio mass spectrometry. All preservation methods tested, significantly impacted δ ¹³C and δ ¹⁵N and C/N atomic values. Chemical preservatives caused δ ¹³C enrichment as great as 1.5‰, and δ ¹⁵N enrichment as great as 0.9‰; the one exception was M. doucasae stored in ethanol for 15 days, which had δ ¹⁵N depletion up to 1.8‰. Freezing depleted δ ¹³C and δ ¹⁵N by up to 0.7 and 2.2‰, respectively. C/N atomic values decreased when stored in ethanol, and increased when frozen for 60 days for both cricket species. Our results indicate that all preservation methods tested in this study altered at least one of the tested isotope values when compared to fresh material (controls). We conclude that only freshly processed material provides adequate SIA results for litter-dwelling crickets.     

Effect of lipid removal on carbon and nitrogen stable isotope ratios in crustacean tissues

The analysis of tissue's naturally occurring stable carbon and nitrogen isotope ratios is a useful tool to delineate trophic relationships. However, the interpretation of δ¹³C and δ¹⁵N is complicated by the influence of multiple factors such as the tissue-specific lipid content. The aim of this work was to evaluate the effects of lipid extraction on δ¹³C and δ¹⁵N compositions in muscle, hepatopancreas and gonads of a marine decapod crustacean, the spider crab Maja brachydactyla. Samples were analyzed for stable isotopes before and after lipid removal, using a derived Soxhlet extraction method. Differences in δ¹³C and δ¹⁵N were measured among tissues before and after treatment. Lipid extraction of muscle did not have a significant effect on either δ¹³C or δ¹⁵N. By contrast, ecologically significant shifts for both carbon and nitrogen stable isotopes ratios (+ 2.9 ± 0.8‰ for δ¹³C, and + 1.2 ± 0.7‰ for δ¹⁵N) were noticed in the hepatopancreas. In regard to gonads, lipid extraction led to a shift only on δ¹³C (+ 1.3 ± 0.3‰). Finally, the derived Soxhlet extraction method removed the lipid influence for δ¹³C, and had an effect on δ¹⁵N composition for lipid-rich samples. We recommend this treatment for carbon stable isotope studies on decapod crustacean lipid-rich tissues.     

Depth variation in isotopic composition of benthic resources and assessment of sculpin feeding patterns in an oligotrophic Alaskan lake

Stable isotopes of carbon (C) and nitrogen (N) are commonly used to track resource flows through lake food webs. However, there remains a weak understanding of the spatial variation in isotopic composition of benthic resources and how this variation affects inference about energy flows among species. Boundary layers at the interface between benthic substrates and the overlying water column restrict diffusion of nutrients and carbon from the water column to benthic algae and may affect the isotopic composition of benthic algae as nutrient and CO₂ concentrations can become locally depleted in the benthic boundary layer. We quantified the variation in C and N stable isotope composition of benthic resources along a depth gradient in a large oligotrophic lake to assess the magnitude of change in stable isotope composition. Snails were increasingly depleted in ¹³C with depth, by about 10 ‰ from 0 to 20 m, while ¹⁵N in snails showed only subtle enrichment over this depth range. Sculpin (Cottas aleuticus) δ ¹³C and δ ¹⁵N signatures did not significantly change with depth and were more enriched in ¹⁵N than would be expected from consumption of snails alone. A comparison of δ ¹³C and δ ¹⁵N values from sculpins relative to shallow and deep snails, and alternative prey (marine-derived salmon resources), within a mixing model suggested sculpins feed selectively on deep grazers in this system in addition to marine-derived resources provided by migrating sockeye salmon. This study illustrates the importance of accounting for depth-related variation in isotope patterns when assessing benthic resource contributions to food webs using stable isotope data.     

Fractionation of stable isotopes in the Arctic marine copepod Calanus glacialis: Effects on the isotopic composition of marine particulate organic matter

Fractionation of δ¹³C and δ¹⁵N between food, consumer, and faecal pellets was studied in the Arctic marine copepod Calanus glacialis Jaschnov, fed with isotopically distinct algal monocultures. Temporal variations in δ¹³C and δ¹⁵N of copepods that were fed ice algae and phytoplankton followed those of a control group consisting of starved animals. There were no significant trends in the δ¹³C and δ¹⁵N values of copepods that were starved for 42 days, suggesting that the isotopic composition of non-lipid body tissues is unaffected by the metabolic processes during prolonged periods of starvation. The stable isotopic composition of starved copepods therefore seems to reflect food consumed during the previous period of feeding and growth. Faecal pellets produced by feeding copepods were depleted in ¹³C and ¹⁵N by 6.3-11.2‰ and 0.7-9.1‰, respectively, relative to the food ingested. These results indicate that faecal pellet production is an important pathway for the trophic fractionation of δ¹³C, whereas other fractionation pathways, such as excretion of ammonia, may be relatively more important for δ¹⁵N. The strong depletion of ¹³C in faecal pellets compared to the food suggests that grazing by herbivorous copepods on primary production adds to the variability of δ¹³C in marine particulate organic matter.     

Sulfur stable isotope signature identifies the source of reduced sulfur in benthic communities in macrophyte zones of Lake Biwa, Japan

Carbon, nitrogen and sulfur stable isotope ratios (δ¹³C, δ¹⁵N and δ³⁴S) were measured from water column sulfate, sediments, particulate organic matter (POM), macrophytes, periphyton, macroinvertebrates and fish, sampled from the littoral, open water and macrophyte zones of Lake Biwa. In most of the littoral zones, the δ¹³C and δ¹⁵N values of organisms indicated that POM and periphyton support the consumers. However, in the dysoxic interior macrophyte (IM) zone, the δ¹³C values of Sinotaia quadrata histrica, Propsilocerus akamusi and Anodonta woodiana were lower than that of all resources. The δ¹⁵N values of S. quadrata histrica were lower than those of P. akamusi and A. woodiana. δ¹³C and δ¹⁵N values thus failed to distinguish the foods of these consumers. The δ³⁴S values of sediment and S. quadrata histrica were lower than those of water column sulfate, suggesting that this consumer incorporated reduced sulfur derived from sulfate reduction in the sediment by ingesting detritus. In contrast, the δ³⁴S values of P. akamusi and A. woodiana were higher than that of S. quadrata histrica, suggesting that they incorporated sulfur derived from water column sulfate by ingesting POM. Consequently, δ¹³C, δ¹⁵N and δ³⁴S signatures provide complementary estimates of foods for consumers in this freshwater lake.     

Quality or quantity: is nutrient transfer driven more by symbiont identity and productivity than by symbiont abundance?

By forming symbiotic interactions with microbes, many animals and plants gain access to the products of novel metabolic pathways. We investigated the transfer of symbiont-derived carbon and nitrogen to the sponges Aplysina cauliformis, Aplysina fulva, Chondrilla caribensis, Neopetrosia subtriangularis and Xestospongia bocatorensis, all of which host abundant microbial populations, and Niphates erecta, which hosts a sparse symbiont community. We incubated sponges in light and dark bottles containing seawater spiked with ¹³C- and ¹⁵N-enriched inorganic compounds and then measured ¹³C and ¹⁵N enrichment in the microbial (nutrient assimilation) and sponge (nutrient transfer) fractions. Surprisingly, although most sponges hosting abundant microbial communities were more enriched in ¹³C than N. erecta, only N. subtriangularis was more enriched in ¹⁵N than N. erecta. Although photosymbiont abundance varied substantially across species, ¹³C and ¹⁵N enrichment was not significantly correlated with photosymbiont abundance. Enrichment was significantly correlated with the ratio of gross productivity to respiration (P:R), which varied across host species and symbiont phylotype. Because irradiance impacts P:R ratios, we also incubated A. cauliformis in ¹³C-enriched seawater under different irradiances to determine whether symbiont carbon fixation and transfer are dependent on irradiance. Carbon fixation and transfer to the sponge host occurred in all treatments, but was greatest at higher irradiances and was significantly correlated with P:R ratios. Taken together, these results demonstrate that nutrient transfer from microbial symbionts to host sponges is influenced more by host–symbiont identities and P:R ratios than by symbiont abundance.     

1H, 13C and 15N resonance assignments of S114A mutant of UVI31+ from Chlamydomonas reinhardtii

Almost complete sequence specific ¹H, ¹³C and ¹⁵N resonance assignments of S114A mutant of UVI31+ from Chlamydomonas reinhardtii are reported. The cDNA of S114A mutant of UVI31+ was cloned from a eukaryotic green algae (C. reinhardtii) and overexpressed in E.coli from where the protein was purified to homogeneity. The point mutation S114A in UVI31+ reduces its DNA endonuclease activity substantially as compared with its wild type. As a prelude to the structural characterization of S114A mutant of UVI31+, we report here complete sequence-specific ¹H, ¹³C and ¹⁵N NMR assignments.     

Comparison of the exploitation of methane-derived carbon by tubicolous and non-tubicolous chironomid larvae in a temperate eutrophic lake

Carbon and nitrogen stable isotope ratios (δ¹³C and δ¹⁵N) in three sympatric species of larval chironomids were analyzed in a temperate eutrophic shallow lake in Japan. Markedly lower δ¹³C values were reported in Chironomus plumosus (?51.2 ‰) and Tanypus sp. (?43.5 ‰) than those in photoautotrophic carbon sources [particulate organic matter (POM) and sediment]. There were positive correlations between δ¹³C and δ¹⁵N in the two chironomid species. These results indicated that they assimilated carbon derived from biogenic methane by exploiting methane-oxidizing bacteria (MOB). In contrast, Propsilocerus akamusi exhibited similar δ¹³C values to those of POM or sediment. A δ¹³C-based isotope mixing model was used to estimate the dietary contributions of MOB to each chironomid species. The mean contributions ranged from 11 to 15 % in C. plumosus, 13 to 19 % in Tanypus sp., but only up to 5 % in P. akamusi. In an aquarium, we observed that individuals of C. plumosus and Tanypus sp., which exhibited low δ¹³C values, built U-shaped larval tubes in the sediment, and an oxidized layer developed around these tubes. Propsilocerus akamusi did not exhibit this behavior. These results suggest that tube building may provide larval chironomids with greater access to methane-derived carbon through increased opportunities to feed on MOB.     

Assessing nature and dynamics of POM in transitional environment (the Curonian Lagoon,SE Baltic Sea) using a stable isotope approach

The nature of the particulate organic matter (POM) as well as its temporal and spatial distribution and dynamics in the Curonian Lagoon (south-eastern part of the Baltic Sea) were investigated. The organic matter was characterized by the organic carbon and nitrogen content, δ¹³C and δ¹⁵N signatures as well as POC/Chl-a and C/N ratios. Additionally, data on hydrological, chemical and biological parameters were used for better understanding the POM distribution and dynamics. The sampling was performed at 13 stations in the Curonian Lagoon and its outflow in the Baltic Sea during the 2012–2013 period. Samples were also collected at the Nemunas River mouth in order to test the riverine impact. Obtained results showed that isotopic values of carbon and nitrogen ranged from −36.1‰ to −25.2‰ and from −0.9‰ to 15.5‰, respectively. The isotopic composition, together with the low C/N molar (∼7) and POC/Chl-a ratios (<100) of the POM, suggested the dominance of living phytoplankton in POM throughout the year with the higher input of detrital material (C/N >10, POC/Chl-a ratios >100) in late autumn − winter.The results of multivariate analysis evidenced a spatial distinction of POM distribution in the northern-transitional and central confined areas and allowed us to distinguish the main driving factors. The seasonal variation of the δ¹³C and δ¹⁵N values in POM (towards higher δ¹³C and lower δ¹⁵N values in the summer − early autumn period and lower δ¹³C and higher δ¹⁵N values in the late autumn − spring period) was determined by combination of factors such as availability of inorganic carbon and nitrogen, the riverine discharge, seasonal phytoplankton succession and by the short-term saline water intrusion to the northern-transitional part of the lagoon.     

Detection and characterization of serine and threonine hydroxyl protons in Bacillus circulans xylanase by NMR spectroscopy

Hydroxyl protons on serine and threonine residues are not well characterized in protein structures determined by both NMR spectroscopy and X-ray crystallography. In the case of NMR spectroscopy, this is in large part because hydroxyl proton signals are usually hidden under crowded regions of ¹H-NMR spectra and remain undetected by conventional heteronuclear correlation approaches that rely on strong one-bond ¹H–¹⁵N or ¹H–¹³C couplings. However, by filtering against protons directly bonded to ¹³C or ¹⁵N nuclei, signals from slowly-exchanging hydroxyls can be observed in the ¹H-NMR spectrum of a uniformly ¹³C/¹⁵N-labeled protein. Here we demonstrate the use of a simple selective labeling scheme in combination with long-range heteronuclear scalar correlation experiments as an easy and relatively inexpensive way to detect and assign these hydroxyl proton signals. Using auxtrophic Escherichia coli strains, we produced Bacillus circulans xylanase (BcX) labeled with ¹³C/¹⁵N-serine or ¹³C/¹⁵N-threonine. Signals from two serine and three threonine hydroxyls in these protein samples were readily observed via ³J_C–OH couplings in long-range ¹³C-HSQC spectra. These scalar couplings (~5–7 Hz) were measured in a sample of uniformly ¹³C/¹⁵N-labeled BcX using a quantitative ¹³C/¹⁵N-filtered spin-echo difference experiment. In a similar approach, the threonine and serine hydroxyl hydrogen exchange kinetics were measured using a ¹³C/¹⁵N-filtered CLEANEX-PM pulse sequence. Collectively, these experiments provide insights into the structural and dynamic properties of several serine and threonine hydroxyls within this model protein.     

A nitrogen fertilization field study of carbon-13 and nitrogen-15 transfers in ectomycorrhizas of Pinus sabiniana

Ectomycorrhizal (EM) fungi form relationships with higher plants; plants transfer C to fungi, and fungi transfer nutrients to their host. While evidence indicates that this interaction is largely mutualistic, less is known about how nutrient supply and EM associates may alter C and nutrient exchanges, especially in intact plant-soil-microbe systems in the field. In a dual-labeling experiment with N fertilization, we used C and N stable isotopes to examine in situ transfers in EM pine trees in a Pinus sabiniana woodland in northern California. We added ¹⁵NH₄SO₂ and ¹³CO₂ to track ¹³C transfer from pine needles to EM roots and ¹⁵N transfer from soil to EM roots and pine needles. Transfers of ¹³C and ¹⁵N differed with EM morphotype and with N fertilization. The brown morphotype received the least C per unit of N transferred (5:1); in contrast red and gold morphotypes gained more C and transferred less N (17:1 and 25:1, respectively). N fertilization increased N retention by ectomycorrhizas (EMs) but did not increase N transfer from EMs to pine needles. Therefore N fertilization positively affected both nutrient and C gains by EMs, increasing net C flows and N retention in EMs. Our work on intact and native trees/EM associations thereby extends earlier conclusions based on pot studies with young plants and culturable EM fungi; our results support the concept that EM-host relationships depend on species-level differences as well as responses to soil resources such as N.