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    ABSTRACT: Carbohydrates represent up to 25% of soil organic matter and derive from fresh plant input or organic matter transformation within the soil. Compound-specific isotope analysis (CSIA) of monosaccharides (sugars) extracted from soil provides a powerful tool to disentangle the dynamics of different carbohydrate pools of soils. The use of high-performance liquid chromatography/oxidation/isotope ratio mass spectrometry (HPLC/o/IRMS) allows isotope measurements without the need for derivatisation and thus increasing accuracy and precision of the isotopic measurement, compared with gas chromatography/combustion/isotope ratio mass spectrometry (GC/c/IRMS). The CSIA of soil carbohydrates was performed using a HPLC/o/IRMS system. The chromatographic and mass spectrometric subunits were coupled with a LC-Isolink interface. Soil sugars were extracted after mild hydrolysis using 4 M trifluoroacetic acid (TFA). Chromatographic separation of the sugars was achieved using a low strength 0.25 mM NaOH solution as mobile phase at a flow rate of 250 μL min(-1) at 10 °C. The chromatographic conditions allowed the baseline separation of the seven most abundant sugars in soil. Complete removal of TFA from the soil hydrolysate ensured chromatographic stability. The accuracy was better than 0.66 ‰ for amounts of >2.5 nM sugar on column. The sugars extracted from an agricultural soil appeared to be more enriched in (13) C than the soil organic carbon, and to have a similar isotopic signature to the soil microbial biomass. The proposed method proved to be suitable for the analysis of the common sugars in soil extracts and represents a precise tool for the study of carbohydrate dynamics. Copyright © 2013 John Wiley & Sons, Ltd.
    Rapid Communications in Mass Spectrometry 11/2013; 27(22):2546-2550.
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    ABSTRACT: Cadmium (Cd) is a phytotoxic heavy metal that causes rapid growth reduction. To investigate if Cd interferes with the metabolism of auxin, a major growth hormone in plants, poplars (Populus × canescens) expressing a heterologous GH3::GUS reporter gene were exposed to 50 μM Cd in hydroponic solutions. Growth, photosynthetic performance, lignification, peroxidase activity, auxin concentration, and GUS staining were determined in order to record the activities of GH3 enzymes in the stem apex, the elongation zone, wood in the zone of radial growth, and in roots. Cd-induced growth reductions were tissue-specific decreasing in the order: roots>wood>shoot elongation and leaf initiation, whereas Cd concentrations increased in the order: leaves<wood<roots. Cd almost abolished the GH3 signal in the stem apex but caused strong increases in the vascular system of roots as well as in parenchymatic cells in the xylem. These changes were accompanied by increases in lignin and peroxidase activities and decreases in auxin concentrations. Since GH3 enzymes remove auxin from the active pool by conjugation and act as mediators between growth and defence, our data suggest that Cd stress triggered increases in GH3 activities which, in turn, depleted auxin in wood and thereby shunted the metabolism to enhanced formation of lignin.
    Journal of Experimental Botany 12/2011; 63(3):1413-21.
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    ABSTRACT: Aquifers within agricultural catchments are characterised by high spatial heterogeneity of their denitrification potential. Therefore, simple but sophisticated methods for measuring denitrification rates within the groundwater are crucial for predicting and managing N-fluxes within these anthropogenic ecosystems. Here, a newly developed automated online (15)N-tracer system is presented for measuring (N(2)+N(2)O) production due to denitrification in aquifer samples. The system consists of a self-developed sampler which automatically supplies sample aliquots to a membrane-inlet mass spectrometer. The developed system has been evaluated by a (15)N-nitrate tracer incubation experiment using samples (sulphidic and non-sulphidic) from the aquifer of the Fuhrberger Feld in northern Germany. It is shown that the membrane-inlet mass spectrometry (MIMS) system successfully enabled nearly unattended measurement of (N(2)+N(2)O) production within a range of 10 to 3300 µg N L(-1) over 7 days of incubation. The automated online approach provided results in good agreement with simultaneous measurements obtained with the well-established offline approach using isotope ratio mass spectrometry (IRMS). In addition, three different (15)N-aided mathematical approaches have been evaluated for their suitability to analyse the MIMS raw data under the given experimental conditions. Two approaches, which rely on the measurement of (28)N(2), (29)N(2) and (30)N(2), exhibit the best reliability in the case of a clear (15) N enrichment of evolved denitrification gases. The third approach, which uses only the ratio of (29)N(2)/(28)N(2), overestimates the concentration of labelled denitrification products under these conditions. By contrast, at low (15)N enrichments and low fractions of denitrified gas, the latter approach is on a par with the other two approaches. Finally, it can be concluded that the newly developed system represents a comprehensive and simply applicable tool for the determination of denitrification in aquifers.
    Rapid Communications in Mass Spectrometry 07/2011; 25(14):1993-2006.
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    ABSTRACT: To investigate early salt acclimation mechanisms in a salt-tolerant poplar species (Populus euphratica), the kinetics of molecular, metabolic, and physiological changes during a 24-h salt exposure were measured. Three distinct phases of salt stress were identified by analyses of the osmotic pressure and the shoot water potential: dehydration, salt accumulation, and osmotic restoration associated with ionic stress. The duration and intensity of these phases differed between leaves and roots. Transcriptome analysis using P. euphratica-specific microarrays revealed clusters of coexpressed genes in these phases, with only 3% overlapping salt-responsive genes in leaves and roots. Acclimation of cellular metabolism to high salt concentrations involved remodeling of amino acid and protein biosynthesis and increased expression of molecular chaperones (dehydrins, osmotin). Leaves suffered initially from dehydration, which resulted in changes in transcript levels of mitochondrial and photosynthetic genes, indicating adjustment of energy metabolism. Initially, decreases in stress-related genes were found, whereas increases occurred only when leaves had restored the osmotic balance by salt accumulation. Comparative in silico analysis of the poplar stress regulon with Arabidopsis (Arabidopsis thaliana) orthologs was used as a strategy to reduce the number of candidate genes for functional analysis. Analysis of Arabidopsis knockout lines identified a lipocalin-like gene (AtTIL) and a gene encoding a protein with previously unknown functions (AtSIS) to play roles in salt tolerance. In conclusion, by dissecting the stress transcriptome of tolerant species, novel genes important for salt endurance can be identified.
    Plant physiology 10/2010; 154(4):1697-709.
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    ABSTRACT: Nitrogen deposition is projected to increase rapidly in tropical ecosystems, but changes in soil-N-cycling processes in tropical ecosystems under elevated N input are less well understood. We used N-addition experiments to achieve N-enriched conditions in mixed-species, lowland and montane forests in Panama. Our objectives were to (1) assess changes in soil mineral N production (gross rates of N mineralization and nitrification) and retention (microbial immobilization and rapid reactions to organic N) during 1- and 9-yr N additions in the lowland forest and during 1-yr N addition in the montane forest and (2) relate these changes to N leaching and N-oxide emissions. In the old-growth lowland forest located on an Inceptisol, with high base saturation and net primary production not limited by N, there was no immediate effect of first-year N addition on gross rates of mineral-N production and N-oxide emissions. Changes in soil-N processes were only apparent in chronic (9 yr) N-addition plots: gross N mineralization and nitrification rates, NO3- leaching, and N-oxide emissions increased, while microbial biomass and NH4+ immobilization rates decreased compared to the control. Increased mineral-N production under chronic N addition was paralleled by increased substrate quality (e.g., reduced C:N ratios of litterfall), while the decrease in microbial biomass was possibly due to an increase in soil acidity. An increase in N losses was reflected in the increase in 15N signatures of litterfall under chronic N addition. In contrast, the old-growth montane forest located on an Andisol, with low base saturation and aboveground net primary production limited by N, reacted to first-year N addition with increases in gross rates of mineral-N production, microbial biomass, NO3- leaching, and N-oxide emissions compared to the control. The increased N-oxide emissions were attributed to increased nitrification activity in the organic layer, and the high NO3- availability combined with the high rainfall on this sandy loam soil facilitated the instantaneous increase in NO3-leaching. These results suggest that soil type, presence of an organic layer, changes in soil-N cycling, and hydrological properties are more important indicators than vegetation as an N sink on how tropical forests respond to elevated N input.
    Ecology 06/2010; 91(6):1715-29.
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    ABSTRACT: The relationships between plant carbon resources, soil carbon and nitrogen content, and ectomycorrhizal fungal (EMF) diversity in a monospecific, old-growth beech (Fagus sylvatica) forest were investigated by manipulating carbon flux by girdling. We hypothesized that disruption of the carbon supply would not affect diversity and EMF species numbers if EM fungi can be supplied by plant internal carbohydrate resources or would result in selective disappearance of EMF taxa because of differences in carbon demand of different fungi. Tree carbohydrate status, root demography, EMF colonization, and EMF taxon abundance were measured repeatedly during 1 year after girdling. Girdling did not affect root colonization but decreased EMF species richness of an estimated 79 to 90 taxa to about 40 taxa. Cenococcum geophilum, Lactarius blennius, and Tomentella lapida were dominant, colonizing about 70% of the root tips, and remained unaffected by girdling. Mainly cryptic EMF species disappeared. Therefore, the Shannon-Wiener index (H') decreased but evenness was unaffected. H' was positively correlated with glucose, fructose, and starch concentrations of fine roots and also with the ratio of dissolved organic carbon to dissolved organic nitrogen (DOC/DON), suggesting that both H' and DOC/DON were governed by changes in belowground carbon allocation. Our results suggest that beech maintains numerous rare EMF species by recent photosynthate. These EM fungi may constitute biological insurance for adaptation to changing environmental conditions. The preservation of taxa previously not known to colonize beech may, thus, form an important reservoir for future forest development.
    Applied and Environmental Microbiology 03/2010; 76(6):1831-41.
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    ABSTRACT: Forest ecosystems play an important role in the global carbon (C) cycle, acting as a carbon sink. Data on soil organic matter (SOM) stocks and turnover rates in relation to vegetation properties are important to understand carbon storage distribution mechanisms and to upscale carbon stocks to the stand scale. The canopy structure may influence the spatial variation of carbon input, firstly, by governing the throughfall and litterfall onto the organic layer, and, secondly, by influencing the climatic factors in the organic layer such as temperature and water content dynamics. Those climatic factors in turn have an impact on carbon mineralisation. In this study we investigated the influence of the canopy structure of a 55-year-old pine stand on the spatial distribution of forest floor carbon stocks (FFCS). The results showed that the spatial variation of FFCS was relatively low (cv 25%). The spatial variation of FFCS was partly caused by the small variation in needle litterfall (cv 8.8%) as a result of the heterogeneous canopy structure. The needle litterfall was significantly higher (7.5%) directly under the tree crowns compared with the areas without canopy cover. Also, the nutrient concentrations of the rain throughfall were significantly higher and its pH values were significantly lower under the crowns compared with the areas under the canopy gaps. However, the abiotic factors (pH, ion concentrations, N content, and C/N ratio) of the organic material sampled from the forest floor did not show any significant differences with respect to areas under tree crowns or in canopy gaps. Additionally, neither the carbon mineralisation nor enzyme activities of various steps of the mineralisation processes were related to the canopy structure. A geostatistical analysis revealed that the ranges of the FFCS and the thickness of the F layer were similar to the ranges of the crown structure. We concluded, firstly, that the needle litterfall is the main factor causing differences in FFCS and systematically varied as a function of tree canopy structure. Secondly, the reason for the small variations in litterfall and in carbon stocks was the high cover fraction of about 76% of the relatively young pine stand. We hypothesise that the litterfall variation will increase with the thinning of the stand. Finally, for upscaling the FFCS from the sample to the forest stand scale, the canopy structure can be neglected for pine stands with a cover fraction larger than 70–80%.
    Geoderma. 01/2010;
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    ABSTRACT: To investigate whether the performance of Populus×canescens, a water-spending species, can be improved by Paxillus involutus ectomycorrhizas (EM) and soil amendment with water-retaining polymers (hydrogel), the pre-dawn water potential, growth performance and the hydraulic architecture of well-irrigated and drought-stressed plants were studied. Roots of well-irrigated plants were about 75% colonized with P. involutus regardless the presence of hydrogel. Drought stress resulted in an about 10% loss of EM. Non-inoculated plants showed marginal EM colonization (3%). EM poplar in hydrogel-amended soil maintained the highest pre-dawn water potential under drought stress, showed lowest biomass loss and kept almost full photosynthetic activity, whereas non-EM plants in soil without hydrogel exhibited severe leaf loss under these conditions and did not show net CO2 assimilation. Non-EM plants in hydrogel-amended soil and EM plants without hydrogel showed intermediate stress symptoms. The soluble carbohydrate content of roots was correlated with the pre-dawn potential indicating osmotic regulation under drought stress. EM plants displayed larger vessel cross-sectional areas and increased radial stem growth compared with non-EM plants. EM plants contained the lowest numbers of vessels (mm−2) but the largest vessel diameters, whereas non-EM drought-stressed plants in soil without hydrogel showed the smallest vessel diameters and highest vessel numbers. Vessel diameter and vessel numbers were strictly inversely correlated suggesting strong developmental coordination between these two parameters. The finding that neither vessel diameters nor vessel numbers were correlated with the pre-dawn water potential suggests that the coordination between these wood features may be achieved by signals acting independent of acute water stress. Overall, EM and hydrogel, especially in combination, help to avoid drought stress. Thereby, these soil amendments improve plant performance and may be useful for the establishment of plantations on marginal sites.
    Environmental and Experimental Botany - ENVIRON EXP BOT. 01/2010; 69(2):189-197.
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    ABSTRACT: Basidiomycetes inhabiting lignocellulose comprise an important class of filamentous fungi with ecological and biotechnological relevance. Extracellular enzymes of wood-degrading fungi such as laccases, manganese-dependent (or independent) peroxidases, cellulases and xylanases are of considerable interest for biotechnological applications. Still, proteomic studies of fungal secretomes based on 2-DE can be very challenging due to low protein concentrations and variable amounts of fungal metabolites. Comparison of different standard methods for protein precipitation has demonstrated their limited applicability to fungal secretomes. The extracellular metabolites impair standard methods for protein quantification and can result in a strong overestimation of total protein. We have developed an optimized protocol for the precipitation of extracellular proteins from liquid cultures of Coprinopsis cinerea growing in an exponential phase on a complex media. We found that a considerable amount of gelatinous material can be removed from the liquid culture supernatants by high-speed centrifugation. Fungal proteins can be effectively enriched by TCA precipitation and coprecipitated metabolites hampering 2-DE can be removed through the application of Tris/acetone. Following our protocol makes it possible to concentrate proteins from culture supernatants and to simultaneously remove most of the impeding compounds from a given sample. We have applied this procedure in the 2-DE of secretomes from the model organism C. cinerea as well as other basidiomycetes such as Pleurotus ostreatus, Phanerochaete chrysosporium, Polyporus brumalis and Schizophyllum commune.
    Electrophoresis 08/2009; 30(14):2431-41.
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    ABSTRACT: Molecular genetic markers have numerous potential applications in environmental forensics if DNA can be isolated from 'difficult' non-human biological material such as hairs, feathers, or wood. The identification of the origin of wood is particularly important in order to identify illegally harvested and traded timber and wood products. We describe success rates of DNA isolation from wood based on a simple, previously published extraction protocol. The protocol was used to isolate DNA from a total of 406 wood samples, mainly of the important tropical tree family Dipterocarpaceae. The reliability of the extraction method was confirmed by comparing fragment sizes and sequences after isolation of DNA from leaves and wood of the same trees. We observed the success of amplification of chloroplast DNA (cpDNA) fragments of different lengths by means of PCR, investigated key factors influencing PCR, and conducted inhibitor tests for a subset of the samples. The average rate of successful PCR amplification was 75.7%. Main factors influencing the success of PCR amplification were the size of the amplified fragment and the processing status of the wood. Short fragments and unprocessed wood resulted in higher success rates. The success rate was also dependent on the age (storage duration) of the wood probe and on the investigated species. Amplification success was higher if DNA was isolated from outer sapwood (without cambium) in comparison to DNA isolated from the transition zone between sapwood and heartwood and the inner heartwood. However, inhibitor tests also indicated more PCR inhibitory substances in the outer sapwood in comparison to transition wood and heartwood. The addition of polyvinylpyrolidone (PVP) to the lysis buffer proved to be highly efficient to improve the amplification success if inhibitory substances were present.
    Forensic Science International: Genetics 07/2009; 3(3):185-92.
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