Nitrogen and phosphorus cycling in relation to stand age of Eucalyptus regnans F. Muell

Lupins, canola, ryegrass and wheat fertilized with Na₂ ³⁵SO₄ and either ¹⁵NH₄Cl or K¹⁵NO₃(N:S=10:1), were grown in the field in unconfined microplots, and the sources of N and S (fertilizer, soil, atmosphere, seed) in plant tops during crop development were estimated. Modelled estimates of the proportion of lupin N derived from the atmosphere, which were obtained independently of reference plants, were used to calculate the proportion of lupin N derived from the soil. Total uptake of N and S and uptake of labelled N and S increased during crop development. Total uptake of S by canola was higher than lupins, but labelled S uptake by lupins exceeded uptake by canola. The form of N applied had no effect on uptake of labelled and unlabelled forms of N or S. Ratios of labelled to unlabelled S and ratios of labelled to unlabelled N derived from soil sources decreased during growth, and were less for S than for N for each crop at each sampling time. Although ratios of labelled to unlabelled soil-derived N were similar between crops at 155, 176 and 190 days after sowing, ratios of labelled to unlabelled S for lupins were higher than for the reference crops and declined during this period. The ratios of labelled to unlabelled S in lupins and the reference plants therefore bore no relationship either to ratios of labelled to unlabelled soil-derived N in the plants, or to total S uptake by the plants. Therefore the hypothesis that equal ratios of labelled N to unlabelled soil-derived N in legumes (Rleg) and reference plants (Rref) would be indicated by equal ratios of labelled to unlabelled S was not supported by the data. The results therefore show that the accuracy of reference plant-derived values of Rleg cannot be evaluated by labelling with ³⁵S.     

Nuclear-accumulation kinetics of p9 CksHs1 and p9 CksHs2 in live plant cells correlate with immunochemical characteristics

Summary The two human homologues of the fission yeast cell cycle protein p13 ^suc1 displayed structural characteristics consistent with their existing in solution as differently folded monomers despite 81% identity with respect to their primary structures and both being capable of fulfilling the functions of their homologues in fission and budding yeasts. Carboxyfluorescein-labelled p9 ^CksHs1 and p9 ^CksHs2 retained their native structures. When microinjected into live stamen hair cells ofTradescantia virginiana, the labelled proteins accumulated in the nuclei of the cells. Markedly different nuclearaccumulation kinetics indicated that the human proteins interact differently with other cellular constituents, which supports the proposition that they may have different roles in cellular regulation.Abbreviations Cdk cyclin-dependent kinase - tris tris(hydroxymethyl)aminomethane - Hepes N-(2-hydroxyethyl)piperazine-N-(3-ethanesulphonic acid) - CF 5(6)-carboxyfluorescein-N-hydroxysuccinamide ester - SDS-PAGE sodium dodecyl sulphatepolyacrylamide gel electrophoresis - IEF isoelectric focusing - DEAE Sephacel diethylaminoethyl Sephacel - ELISA enzyme-linked immunosorbent assay - IgG immunoglobulin     

Utilization of organic nitrogen at two different substrate pH by different ectomycorrhizal fungi growing in symbiosis with Pinus sylvestris seedlings

The aim of this study was to test the potential of four isolates of ectomycorrhizal (EM) fungi to utilize organic nitrogen (N) at two different substrate pHs. The organic N source (¹⁵N labelled lyophilised fungal mycelium) was mixed with either untreated peat/sand mixture (pH 4.9) or peat/sand mixture limed to a pH of 5.9 and put in cylindrical containers added to each pot. The content of the containers was separated from the roots of Pinus sylvestris seedlings by a nylon mesh and a 2 mm air gap to reduce diffusion of labelled N to the roots. The mycorrhizal plants (except those colonized by Suillus variegatus 2) took up significantly more ¹⁵N from the labelled mycelium than uncolonized seedlings. Liming significantly reduced the uptake of ¹⁵N by one of the EM fungi (unidentified) but not the other tested species (Paxillus involutus and two isolates of S. variegatus). The EM fungal isolates differed in their influence on the bacterial activity of the soil. This was reduced with P. involutus at both pH levels and increased with one of the two S. variegatus isolates at the high pH and with the other S. variegatus isolate at the low pH level. Liming the soil generally increased bacterial activity. The influence of liming on the proportion of organic N uptake in relation to inorganic N uptake by ectomycorrhizal trees is discussed.     

A simple technique for producing 13C or 14C-labelled fronds of Lemna gibba and their use in soil incubation investigations

The duckweed Lemna gibba required light and a suitable energy source such as sucrose, glucose or fructose, for maximum growth in culture. The requirement for light was relatively unimportant and the plants grew well in a photon flux density of only 52 μmol m^-2s^-1 PAR. The uptake and incorporation of uniformly labelled ¹⁴C-glucose into fronds was related only to the concentration of the sugar. When incubated with soil, labelled L. gibba behaved in a manner similar to that of labelled ryegrass roots which had been produced by a more elaborate technique using a ¹⁴CO₂ labelled atmosphere. During incubation with soil for 224 days the L. gibba material (specific activity 6133 Bq mg^-1 d. wt) lost 64% of its radioactivity as ¹⁴CO₂ and ryegrass (specific activity 6634 Bq mg^-1 d. wt) lost 49%. Alkaline extracted humic and fulvic acids from soil had specific activities for the L. gibba incubation of 3409 and 407 Bq mg^-1 solid and for ryegrass roots of 4609 and 546 Bq mg^-1 solid respectively. The production of ¹³C or ¹⁴C-labelled L. gibba can be undertaken using only simple equipment producing material the specific radioactivity of which can be controlled by adjusting the activity of the sugar energy source.     

Nitrogen assimilation in field-grown winter wheat: Direct measurements of nitrate reduction in roots using15N

Summary Nitrate fertiliser labelled with¹⁵N was applied to a field grown crop of winter wheat. Uptake and assimilation of fertiliser nitrate was studied by monitoring the appearance of labelled nitrate and labelled amino acids in the xylem sap. Shortly after applying¹⁵N-nitrate to the soil about 30 per cent of recently absorbed¹⁵N was in the reduced form, indicating that roots of cereal crops can make a substantial contribution in reducing nitrate. Seasonal changes in crop growth andin vivo NRA are also described.     

Cell migration and differentiation during wound healing and regeneration in Microstomum lineare (Turbellaria)

Using transmission electron microscopy and serial sections with light-microscopic autoradiography, I have investigated the ultrastructure of wound healing, the distribution of cells preparing for proliferation, and the fates of cells labelled with exogenous tritiated thymidine ([³H]T) in Microstomum lineare undergoing wound healing and regeneration. Immediately after decapitation the open wound was reduced to a minimum by strong contraction of circular muscle fibers. The wound epidermis was cellular, consisting of thin parts of epidermal cells from the epidermis around the wound. These epidermal cells maintained close adhesive contact with one another through zonulae adherentes and septate junctions. No proliferating cells were found in the old epidermis. The only cells taking up [³H]T were mesenchymal and gastrodermal neoblasts which proliferated and migrated towards the surface. The final epidermis was formed by conjunction of the wound epidermis and newly differentiated epidermal cells. Regeneration in Microstomum, in contrast to that of planarians, occurs mainly by morphallaxis, without the formation of a regeneration blastema, but also through continuous cell proliferation, migration, and differentiation.     

Nutrient acquisition from different soil depths by pedunculate oak

Eight oak trees (Quercus robur L.) received ³²P at a soil depth of 50 cm and ³³P at a soil depth of 15 cm at the end of June 2002 through plastic tubes inserted into the mineral soil. The phosphorus uptake from different soil depths was estimated by analysing the concentration of ³²P and ³³P in the foliage of oak growing in a mixed stand in southern Sweden. ³²P and ³³P were recovered in the leaves/needles after 21 and 39 days. The recovery of labelled P in oak was higher from 15 cm soil depth than from 50 cm, however, more than 4% of the total amount of labelled P was taken up from 50 cm. This indicates that oak can utilize deep soil layers for nutrient uptake. A study on the uptake of Cs (as an analogue to K) and ¹⁵N into the leaves was performed on the same trees and detectable amounts of ¹⁵N and Cs were recovered in leaves and buds. This indicates that ¹⁵N and Cs can be used to study nutrient uptake of mature trees from the mineral soil.     

Changes in the expression pattern of the plasma membrane H<Superscript>+</Superscript>-ATPase in developing<Emphasis Type="Italic"> Ricinus communis</Emphasis> cotyledons undergoing the sink/source transition

The plasma membrane (PM) H⁺-ATPase is thought to play a key role in generating the proton motive force used to drive the uptake and accumulation of solutes in plant cells. Changes in its expression pattern were studied in the Ricinus communis L. cotyledon as it changed from a sink to a source organ. Expression was monitored in 3-, 10- and 14-day-old cotyledons using an antibody to the maize PM H⁺-ATPase. The antibody labelled a 100-kDa protein in membrane fractions prepared from cotyledons and this protein occurred at higher levels in the PM-enriched fractions compared to those enriched in intracellular membranes. Immunostaining of tissue sections of 3-day-old Ricinus cotyledons (sinks) with this antibody demonstrated that the PM H⁺-ATPase was highly expressed in the lower epidermal cells and also in the vascular bundles, particularly the phloem. The high expression in the epidermis suggests that these cells may be important in the initial active uptake of solutes from the endosperm. A similar distribution was observed in the 10-day-old seedlings but, in addition, larger, more spherical cells (idioblasts) had developed in the lower and upper epidermal layers and these were also labelled. In 14-day-old seedlings the cotyledons are no longer reliant on nutrients from the endosperm (which has totally degraded) and they are functioning as source organs. This is reflected in a decrease in PM H⁺-ATPase expression in the lower epidermal cells, apart from idioblasts and stomatal guard cells. The latter were also observed in the upper epidermis. Expression remained high in the vascular bundles of 14-day-old seedlings with strong staining in the phloem.Abbreviation PM Plasma membrane     

Can cells extruded from denervated skeletal muscle become circulating potential myoblasts?

Summary The hypothesis that satellite cells which leave denervated skeletal muscle might become circulating potential myoblasts which could participate in myogenesis in distant sites in the body has been tested.Sixteen mice had one hindlimb denervated and were given 7 daily injections of ³H-thymidine (³H-Tdr). One day later extensor digitorum longus muscle isografts from unlabelled mice were inserted into each hindlimb. As controls, the procedure was repeated in 6 non-denervated labelled mice. Fourteen days after their insertion, isografts in denervated mice contained many labelled myotubes with a labelling index of 55±4% (mean±SEM). In the control isografts in non-denervated mice, 38±4% of myotube nuclei were labelled. The results show that either labelled cells, or ³H-Tdr, had transferred from the host to isografts in both cases. The probability of ³H-Tdr reutilization was demonstrated in regenerating livers of 8 similarly labelled mice, where 34±3% hepatocytes adjacent to crush lesions were labelled after 14 days. This conclusion was reached because only 2–3% of normal hepatocytes incorporate ³H-Tdr under these conditions and this population is inadequate to provide sufficient labelled precursor cells for the large numbers of labelled regenerated hepatocytes. Therefore, it was concluded that ³H-Tdr reutilization is the most likely explanation for labelled myotube nuclei in the muscle isografts (rather than movement of labelled precursor cells), and that additional label for reutilization had been derived from breakdown of labelled cells in denervated muscle. The data do not support the hypothesis of a circulating precursor for skeletal muscle cells.     

The efficiency of (Na + K)-ATPase in tumorigenic cells

The efficiency of (Na⁺ + K⁺)-ATPase (i.e. the amount of K⁺ pumped per ATP hydrolyzed) in intact tumorigenic cells was estimated in this study. This was accomplished by simultaneously measuring the rate of ouabain-sensitive K⁺ uptake and oxygen consumption in tumorigenic cell suspensions during the reintroduction of K⁺ to K⁺-depleted cells. The ATP turnover was then estimated by assuming 5.6–6 ATP/O₂ as the stoichiometry of NADH-linked respiration in these cells. In the three cell lines tested (hamster and chick embryo cells transformed with Rous sarcoma virus and Ehrlich ascites cells), the K⁺/ATP ratio was approximately 2, the same value as that found in normal tissues. Furthermore, only 20% of the total ATP production of these cells was used by (Na⁺ + K⁺)-ATPase.     

The fate of Corydalis cava elaiosomes within an ant colony of Myrmica rubra: elaiosomes are preferentially fed to larvae

Summary. The diaspores of many plant species are adapted to dispersal by ants (myrmecochory). The ants carry the diaspores into their nests where the appendages of the diaspores (elaiosomes) are consumed. Little is known, however, about the fate of diaspores in the colony, i.e., whether elaiosomes are consumed only by the larvae or also by the workers. In this study, the distribution of Corydalis cava (Fumariaceae) elaiosomes was monitored between the larvae and the working caste of laboratory colonies of Myrmica rubra (Formicidae, Myrmicinae). In the first part of the study, ¹⁵N labelled elaiosomes were found to be mainly consumed by larvae. To find out whether elaiosomes are a more attractive food source than an artificial, nutritionally optimized diet for ants (Bhatkar diet), a combined ¹³C and ¹⁵N labelling experiment was conducted. Ants were offered ¹⁵N labelled elaiosomes for two days (pulse labelling), while being continuously fed with ¹³C labelled Bhatkar diet over 19 days. Under the given laboratory conditions, elaiosomes proved to be a far more attractive diet for the larvae than Bhatkar diet, contributing 87 ± 2% of the daily nitrogen and 79 ± 3% of daily carbon incorporation of larvae during the first four days of the experiment. The mean elaiosome incorporation met 73 ± 5% of nitrogen and 63 ± 6% of carbon demand of workers during the first four days of the experiment. Generally, incorporation rates in workers (per dry body mass) were lower both for carbon and nitrogen during the experiment – by a factor of 6.8 for nitrogen and by a factor of 6.2 for carbon compared to larvae. On a colony basis, workers received 39% and larvae 61% of the elaiosome nitrogen. The results indicate that elaiosomes are a major food source for growing larvae and thus support the hypothesis that elaiosomes play an important role in the life cycle of temperate ants.Received 22 December 2003; revised 16 June 2004; accepted 22 June 2004.     

Validation of the design of feeding experiments involving [(14)C]substrates used to monitor metabolic flux in higher plants

The aim of this study was to examine whether flux through the pathways of carbohydrate oxidation is accurately reflected in the pattern of ¹⁴CO₂ release from positionally labelled [¹⁴C]substrates in conventional radiolabel feeding studies. Heterotrophic cell suspension cultures of Arabidopsis thaliana were used for this work. The presence of an alkaline trap to capture metabolically generated ¹⁴CO₂ had no significant effect on the ratio of ¹⁴CO₂ release from specifically labelled [¹⁴C]substrates, or on the metabolism of [U-¹⁴C]glucose by the cells. Although the amount of ¹⁴CO₂ captured in a conventional time-course study was only about half of that released from a sample acidified at an equivalent time point, the ratios of ¹⁴CO₂ released from different positionally labelled [¹⁴C]glucose and [1-¹⁴C]gluconate were the same in untreated and acidified samples. Less than 5% of radioactivity supplied to the growth medium as [¹⁴C]bicarbonate was incorporated into acid-stable compounds, and there was no evidence for appreciable reassimilation of ¹⁴CO₂ generated intracellularly during oxidation of [1-¹⁴C]gluconate by the cells. It is concluded that the ratio of label captured from specifically labelled [¹⁴C]glucose is a valid and convenient measure of the relative rates of oxidation of the different positional carbon atoms within the supplied respiratory substrate. However, it is argued that failure to compensate for the incomplete absorption of ¹⁴CO₂ by an alkaline trap may distort estimates of respiration that rely on an absolute measure of the amount of ¹⁴CO₂ generated by metabolism.     

Estimation of biological nitrogen fixation associated withBrachiaria andPaspalum grasses using15N labelled organic matter and fertilizer

Summary Six pasture grasses,Paspalum notatum cv batatais,P. notatum cv pensacola,Brachiaria radicans, B. ruziziensis, B. decumbens andB. humidicola, were grown in concrete cylinders (60 cm diameter) in the field for 31 months. The soil was amended with either a single addition of¹⁵N labelled organic matter or frequent small (2 kg N. ha^–1) additions of¹⁵N enriched (NH₄)₂SO₄. In the labelled fertilizer treatment soil analysis revealed that there was a very drastic change in¹⁵N enrichment in plant-available nitrogen (NO ₃ ^– +NH ₄ ⁺ ) with depth. The different grass cultivars recovered different quantities of applied labelled N, and evidence was obtained to suggest that the roots exploited the soil to different depths thus obtaining different¹⁵N enrichments in soil derived N. This invalidated the application of the isotope dilution technique to estimate the contribution of nitrogen fixation to the grass cultivars in this treatment. In the labelled organic matter treatment the¹⁵N label in the plant-available N declined at a decreasing rate during the experiment until in the last 12 months the decrease was only from 0.274 to 0.222 atom % excess. There was little change in¹⁵N enrichment of available N with depth, hence it was concluded that although the grasses recovered different quantities of labelled N, they all obtained virtually the same¹⁵N enrichment in soil derived N. Data from the final harvests of this treatment indicated thatB. humidicola andB. decumbens obtained 30 and 40% respectively of their nitrogen from N₂ fixation amounting to an input of 30 and 45 kg N.ha^–1 year^–1 respectively.     

Electrophoretic analysis of newly synthesized and stored maternal RNA during oogenesis ofCalliphora erythrocephala (Dipt.)

Summary Ovaries ofC. erythrocephala synthesize large amounts of poly(A)⁺ and poly(A)^– RNA during early and middle stages of oogenesis as shown by labelling with³H-uridine in vivo. After incubation for 1 h, a striking difference in the electrophoretic pattern of newly synthesized labelled poly(A)⁺ RNA and the poly(A)⁺ RNA present in sufficient amounts for optical density measurements (steady state poly(A)⁺ RNA) was observed. During early and mid-oogenesis, in the poly(A)^– RNA fraction, 4S predominantly mature rRNA, 5S RNA and tRNA were labelled. These fractions were no longer synthesized during late oogenesis, whereas poly(A)⁺ RNA was labelled continously During oogenesis stage specific differences in the size distribution of newly synthesized and steady state poly(A)⁺ RNA were not obvious. However, different sizes of labelled poly(A)⁺ RNA species were detected in 0–2h old preblastoderm embryos, after injection of³H-uridine into females either 3–4 days (stage 3–4 of oogenesis) or 24 h before oviposition (stage 5–6 of oogenesis). This difference in RNA synthesis was related to the presence of active nurse cell nuclei. The poly(A)⁺ RNA fraction represents about 2–3% of the total RNA in both ovaries and freshly laid eggs as judged by measurements of optical density and radioactivity bound to oligo(dT). The length of poly(A)-segments in ovarian poly(A)⁺ RNA varied from about 30 to 200 nucleotides.     

A re-investigation of the path of carbon in photosynthesis utilizing GC/MS methodology. Unequivocal verification of the participation of octulose phosphates in the pathway

A GC/EIMS/SIM methodology has been developed to re-examine the path of carbon in photosynthesis. Exposing isolated spinach chloroplasts to ¹³CO₂ on a solid support for a defined period followed by quenching and work-up provided a mixture of labelled sugar phosphates. After enzymatic dephosphorylation and derivatization, the Mox-TMS sugars were analysed using the above method. The purpose of the study was to try to calculate the atom% enrichment of ¹³C in as many of the individual carbons in each of the derivatized sugars as was practical using diagnostic fragment ions. In the event, only one 45 s experiment provided sufficient data to enable a range of enrichment values to be calculated. This confirmed that D-glycero-D-altro-octulose phosphate was present in the chloroplasts and was heavily labelled in the C4, C5 and C6 positions, in keeping with the hypothesis that it had an inclusive role and a labelling pattern consistent with a new modified pathway of carbon in photosynthesis.     

Polyglucose synthesis in Chloroflexus aurantiacus studied by 13C-NMR

Chloroflexus aurantiacus OK-70 fl was grown photoautotrophically with hydrogen as electron source. The cultures were subjected to long term labelling experments with ¹³C-labelled acetate or alanine in the presence of sodium fluoroacetate. The presence of fluoroacetate caused the cells to accumulate large amounts of polyglucose which was hydrolysed and analysed by NMR. The labelling patterns of glucose were symmetric and in agreement with carbohydrate synthesis from acetate and CO₂ via pyruvate synthase. The content of carbon derived from added acetate was highest in C2 and C5 of glucose, at least 20% higher than in C1 and C6. About one third of the glucose carbon was derived from added acetate, the rest being from CO₂. Contrary to expectations, in glucose formed in the presence of C1-labelled acetate C1 and C6 contained more label than C2 and C5, and with C2-labelled acetate as the tracer glucose was mainly labelled in C2 and C5. Labelled CO₂ was formed from acetate labelled at either position. The labelling data indicate a new metabolic pathway in C. aurantiacus. It is suggested that the cells form C1-labelled acetyl-CoA from C2-labelled acetyl-CoA and vice versa by a cyclic mechanism involving concomitant CO₂ fixation and that this cycle is the part of the autotrophic CO₂ fixation pathways in C. aurantiacus in which acetyl-CoA is formed from CO₂.The polyglucose of C. aurantiacus appears to have predominantly (1–4)-linked structure with about 10% (1–6)-linkages as revealed by ¹³C-NMR.     

Assimilation of nod gene inducer 14C-naringenin and the incorporation of labelled carbon atoms into the acyl side chain of a host-specific Nod factor produced by Rhizobium leguminosarum bv. viciae

The fate of ¹⁴C-naringenin during its specific activation of nod genes in Rhizobium leguminosarum bv. viciae was examined. After incubation with either strain RBL5560 or its pSym-cured derivative in a medium supplemented with ¹⁴C-naringenin at nod gene-inducing concentrations of 2 nM (ca. 12.5 kBq) plus cold acetate (0.5 M), a radiocarbon inventory for the cells and supernatant extracts was obtained. The level of ¹⁴C-label incorporation was also determined in the fractionated cellular components. Using ¹⁴C-acetate at 0.5 M (1036 kBq) and cold naringenin (2 nM) in incubations with strain RBL5560 as a separate treatment, the Nod metabolites were detected by thin layer and high performance liquid chromatographic methods and the data provided the basis for identification of the Nod factors from the supernatant obtained from ¹⁴C-naringenin treatments. Subsequent radio-biochemical and chemical analyses revealed that RBL5560 cells assimilated ¹⁴C-naringenin during the activation of nod genes. Our analysis also showed that labelled carbon atoms from the ¹⁴C-naringenin were incorporated into the acyl moiety of a lipo-oligosaccharide Nod factor, NodRlv IV, present in the culture supernatants of RBL5560. The pSym-cured derivative failed to synthesize any Nod metabolites in a ¹⁴C-naringenin supplemented medium. The tracing of flavonoid-derived carbon atoms to the acyl chain of a host-specific Nod factor, a moiety that defines host specificity for this Rhizobium, adds a new dimension to the signalling function of flavonoids in legume-Rhizobium interactions.Abbreviations Ac acyl chain - ca calculated approximately - dpm disintegrations per minute - HPLC High Performance Liquid Chromatography - pSym symbiotic plasmid - R. Rhizobium - TLC Thin Layer Chromatography     

Compartmentation and Fluxes of Sugars in Roots of Phaseolus Vulgaris Under Phosphate Deficiency

The influence of phosphate deficiency on the sugar accumulation and sugar partitioning in the root cells of bean (Phaseolus vulgaris L.) was studied. Bean plants were cultured 17 - 19 d on a phosphate-sufficient and phosphate-deficient nutrient medium. Phosphate deficit in the growth medium resulted in increased sugar concentration for about 30 % in the apoplastic and cytoplasmic compartments as well as in the vacuoles of root cells. However, the distribution of sugars between apoplast and cytoplasm compartment and vacuole was not affected by decreased phosphate concentration. About 20 % of sugars were found in the apoplast and cytoplasm, about 80 % in the vacuole. Low phosphate concentration enhanced influx of exogenous ¹⁴C-sucrose into meristematic and elongation zones of root. The ¹⁴C-labelled sugar content in the root tips increased for about 60 % as compared to control plants. Phosphate deficiency increased also ¹⁴C-glucose uptake and content in the root tips. However, the amount of ¹⁴CO₂ liberated during respiration of P-deficient roots (after feeding with uniformly labelled ¹⁴C-glucose) was lower than ¹⁴CO₂ respired by control plants, thus a large part of accumulated sugars seems to be metabolically inactive. This revised version was published online in July 2006 with corrections to the Cover Date.     

Site-dependent N uptake from N-form mixtures by arctic plants,soil microbes and ectomycorrhizal fungi

Soil microbes constitute an important control on nitrogen (N) turnover and retention in arctic ecosystems where N availability is the main constraint on primary production. Ectomycorrhizal (ECM) symbioses may facilitate plant competition for the specific N pools available in various arctic ecosystems. We report here our study on the N uptake patterns of coexisting plants and microbes at two tundra sites with contrasting dominance of the circumpolar ECM shrub Betula nana. We added equimolar mixtures of glycine-N, NH₄⁺–N and NO₃⁻–N, with one N form labelled with ¹⁵N at a time, and in the case of glycine, also labelled with ¹³C, either directly to the soil or to ECM fungal ingrowth bags. After 2 days, the vegetation contained 5.6, 7.7 and 9.1% (heath tundra) and 7.1, 14.3 and 12.5% (shrub tundra) of the glycine-, NH₄⁺- and NO₃⁻–¹⁵N, respectively, recovered in the plant–soil system, and the major part of ¹⁵N in the soil was immobilized by microbes (chloroform fumigation-extraction). In the subsequent 24 days, microbial N turnover transferred about half of the immobilized ¹⁵N to the non-extractable soil organic N pool, demonstrating that soil microbes played a major role in N turnover and retention in both tundra types. The ECM mycelial communities at the two tundras differed in N-form preferences, with a higher contribution of glycine to total N uptake at the heath tundra; however, the ECM mycelial communities at both sites strongly discriminated against NO₃⁻. Betula nana did not directly reflect ECM mycelial N uptake, and we conclude that N uptake by ECM plants is modulated by the N uptake patterns of both fungal and plant components of the symbiosis and by competitive interactions in the soil. Our field study furthermore showed that intact free amino acids are potentially important N sources for arctic ECM fungi and plants as well as for soil microorganisms. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

3D 13C/1H NMR-based assignments for side-chain resonances of Lactobacillus casei dihydrofolate reductase. Evidence for similarities between the solution and crystal structures of the enzyme

Summary ¹³C-based three-dimensional ¹H–¹H correlation experiments have been used to determine essentially complete ¹³C and ¹H resonance assignments for the amino acid side chains of uniformly ¹³C/¹⁵N labelled L. casei dihydrofolate reductase in a complex with the drug methotrexate. Excellent agreement is observed between these assignments and an earlier set of partial assignments made on the basis of correlating nuclear Overhauser effect and crystal structure data, indicating that the tertiary structure of the enzyme is similar in solution and in the crystal state.To whom correspondence should be addressed.