Metabolic labeling of plant cell cultures with K15NO3 as a tool for quantitative analysis of proteins and metabolites

Strategies for robust quantitative comparison between different biological samples are of high importance in experiments that address biological questions beyond the establishment of protein lists. Here, we propose the use of ¹⁵N-KNO₃ as the only nitrogen source in Arabidopsis cell cultures in order to achieve a metabolically fully labeled cell population. Proteins from such metabolically labeled culture are distinguishable from unlabeled protein populations by a characteristic mass shift that depends on the amino acid composition of the tryptic peptide analyzed. In addition, the metabolically labeled cell extracts are also suitable for comparative quantitative analysis of nitrogen-containing cellular metabolic complement. Protein extracts from unlabeled and from standardized ¹⁵N-labeled cells were combined into one sample for joined analytical processing. This has the advantage of (i) reduced experimental variability and (ii) immediate relative quantitation at the level of single extracted peptide and metabolite spectra. Together ease and accuracy of relative quantitation for profiling experiments is substantially improved. The metabolic labeling strategy has been validated by mixtures of protein extracts and metabolite extracts from the same cell cultures in known ratios of labeled to unlabeled extracts (1:1, 1:4, and 4:1). We conclude that saturating metabolic ¹⁵N-labeling provides a robust and affordable integrative strategy to answer questions in quantitative proteomics and nitrogen focused metabolomics.     

Spatial distribution of soil heavy metal concentrations as indicator of pollution sources at Mount Križna (Great Fatra,central Slovakia)

The objective of this study was to test the suitability of a simple approach to identify the direction from where airborne heavy metals reach the study area as indication of their sources. We examined the distribution of heavy metals in soil profiles and along differently exposed transects. Samples were taken from 10 soils derived from the same parent material along N-, S-, and SE-exposed transects at 0—10, 10—20, and 20—40 cm depth and analyzed for total Al, Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn concentrations. The heavy metal concentrations at 0—10 cm were larger than background concentrations in German arable soils except for Cr (Cd: 0.6—1.8 mg kg^—1; Cr: 39—67; Cu: 40—77; Ni: 87—156; Pb: 48—94; Zn: 71—129; Fe: 26—34 g kg^—1; Mn: 1.1—2.4). Decreasing Cd, Cu, Mn, and Pb concentrations with increasing soil depth pointed at atmospheric inputs. Aluminum and Ni concentrations increased with soil depth. Those of Fe, Cr, and Zn did not change with depth indicating that inputs at most equalled leaching losses. The Pb accumulation in the surface layer (i.e. the ratio between the Pb concentrations at 0—10 to those at 20—40 cm depth) was most pronounced at N-exposed sites; Pb obviously reached Mount Križna mainly by long-range transport from N where several industrial agglomerations are located. Substantial Cd, Cu, and Mn accumulations at the S- and SE-exposed sites indicated local sources such as mining near to the study area which probably are also the reason for slight Cr and Zn accumulations in the SE-exposed soils. Based on a principal component analysis of the total concentrations in the topsoils four metal groups may be distinguished: 1. Cr, Ni, Zn; 2. Mn, Cd; 3. Pb (positive loading), Cu (negative loading); 4. Al, Fe, indicating common sources and distribution patterns. The results demonstrate that the spatial distribution of soil heavy metal concentrations can be used as indication of the location of pollution sources.     

Inherent interreplicate variability during small-scale rainfall simulations

Purpose

The validity of soil erosion data is often questioned because of the variation between replicates. This paper aims to evaluate the relevance of interreplicate variability to soil and soil organic carbon (SOC) erosion over prolonged rainfall.

Materials and methods

Two silty loams were subjected to simulated rainfall of 30 mm h^?1 for 360 min. The entire rainfall event was repeated ten times to enable statistical analysis of the variability of the runoff and soil erosion rates.

Results and discussion

The results show that, as selective removal of depositional particles and crust formation progressively stabilized the soil surface, the interreplicate variability of runoff and soil erosion rates declined considerably over rainfall time. Yet, even after the maximum runoff and erosion rates were reached, the interreplicate variability still remained between 15 and 39 %, indicating the existence of significant inherent variability in soil erosion experiments.

Conclusions

Great caution must be paid when applying soil and SOC erosion data after averaging from a small number of replicates. While not readily applicable to other soil types or rainfall conditions, the great interreplicate variability observed in this study suggests that a large number of replicates is highly recommended to ensure the validity of average values, especially when extrapolating them to assess soil and SOC erosion risk in the field.

     

Transformation of organic rhizodepositions by rhizosphere bacteria and its influence on the availability of tertiary calcium phosphate

This study assesses the influence of saccharides in the rhizodeposition on the phosphate solubilizing ability of rhizosphere bacteria. Water‐soluble rhizodeposits were analyzed of ¹⁴C‐labeled pea plants (Pisum sativum, cv. ‘Grapis’) which were grown at two different levels of P‐nutrition. The sugars produced were fed in vitro either as single compounds or as synthetic mixtures to three bacterial strains and the ability of the bacteria to mobilize Ca₃(PO₄)₂ was measured. The relative glucose proportion of pea exudates decreased under P deficiency while the content of galactose, ribose, xylose and fucose increased. In vitro feeding of single sugars and sugar mixtures showed that the ability of Pseudomonas fluorescens (PsIA12) to dissolve tertiary calcium phosphate was lower with pentoses and the mixed sugars of the P‐deficient plants than with glucose. On the other hand, the shifted sugar pattern observed under P deficiency increased the phosphate mobilization ability of Pantoea agglomerans (D5/23) and Azospirillum sp. (CC 322) considerably. This observation can only partly be explained by the acidification of the nutrient medium. Bacteria also produced different carboxylic anions depending on sugar supply. In addition to low‐molecular mono‐, di‐, and tricarboxylic acids which are known as P‐solubilizing substances, sugar acids also played an important role in cultures D 5/23 and CC 322.     

The influence of nitrate and ammonium fertilization on N2O release and CH4 uptake of a well-drained topsoil demonstrated by a soil microcosm experiment

The effects of the application of KNO₃ and NH₄Cl (100 kg N ha^?1) on N₂O release and CH₄ uptake by a well-aerated topsoil (porosity: 55%, water-filled pore space: 67% of the total pore space) were studied in a laboratory incubation experiment over 50 days using a soil microcosm system with an automated registration of N₂O and CH₄ fluxes. The total N₂O-N losses over 50 days were low for all treatments and amounted to 0.9 mg m^?2 for the control, 1.2 mg m^?2 for the soil columns fertilized with KNO3, and 7.3 mg m^?2 for the soil columns fertilized with NH₄Cl. The slightly elevated N₂O release after the application Of NH₄Cl was associated with the nitrification of NH₄⁺ added. Only ?0.06% of the fertilized NH₄?N was lost as N₂O. This nitrogen fertilization reduced the CH₄ uptake of the soil columns by 43% (NH₄Cl) and 21% (KNO₃), respectively.     

Contents and bioavailability of rare earth elements in agricultural soils in Hesse (Germany)

With increasing agricultural and industrial use of rare earth elements (REEs), input of REEs into the pedosphere has risen in parallel. Since total REE budgets for soils in Germany are mainly unknown, this pilot study investigates the concentrations of REEs in three loess soil profiles under agricultural land use in the Wetterau region, Germany. There were no significant REE applications in the past, so the presented data can be considered as background concentrations of REEs in soils. In addition to the total REE contents (aqua regia digestion), potentially plant‐available (EDTA) and at actual conditions available, mobile (NH₄NO₃) REEs have been determined for each horizon. Interactions between REE availability and properties of soils are explained after assessing several chemical and physical properties. The results reveal a wide range of total REE contents, ranging from 222 to 423 mg kg^–1. With 10.1% of total REE amounts, the potentially plant‐available proportions of REEs are generally low. In contrast, yttrium, which showed high available proportion of 24.8%, was found to be comparable with Cd. Cerium is the most abundant REE in aqua regia digests, whereas only small amounts in the potentially plant‐available fraction were found. Thus negative cerium anomalies could be concluded concerning its potential availability. Generally, bioavailability of REE had significant correlations between clay contents and Fe and Mn oxides for the majority of REEs. Due to moderately high pH (CaCl₂) in soils (average 6.6) the mobile fraction for most of the elements was not detectible.     

Contamination of highly weathered urban soils in Uberlândia,Brazil

We determined heavy metal, polycyclic aromatic hydrocarbon (PAH), and polychlorinated biphenyl (PCB) concentrations in 18 topsoils of Uberlândia (420,000 inhabitants, Brazil) and in 3 rural topsoils. Concentrations of Al (11—124 g kg^—1) and Fe (13—109 g kg^—1) are large because of desilification. Concentrations of Cd (0.1—0.5 mg kg^—1), Cr (13—72), Cu (6—154), Mn (28—974), Ni (4—29), Pb (3—26), Zn (4—107), the sum of 20 PAHs (=Σ20PAHs:7—390 μg kg^—1), and the sum of 14 PCBs (=Σ14PCBs:0.05—1.25) are comparable to or below background concentrations in temperate soils except for Cu at two sites. More than 67% of the metals are strongly bound in Fe oxides and silicates; metals are more bioavailable in the urban than in the rural soils. The most abundant PAHs in the urban soils, on average, are naphthalene (19.0 ± 13.4% of Σ20PAHs) and the benzo(b+j+k)fluoranthenes (11.4 ± 6.7%); the most abundant PCBs are nos. 138 (23.3 ± 11.0% of Σ14PCBs) and 153 (14.3 ± 6.4%). The rural soils contain larger percentages of low molecular PAHs and up to tetra‒chlorinated PCBs than the urban soils. The different pollutant concentrations and patterns in the studied tropical compared with many temperate soils indicate different sources and fate.     

Einfluss assoziativer rhizosphärenbakterien auf die äHrstoffaufnahme und den Ertrag von Mais

In mehrjährigen Feldversuchen (1990–1995) in Nordostdeutschland wurde der Einfluß selektierter assoziativer Rhizosphärenbakterien auf den Ertrag und die Nährstoffaufnahme bei Mais untersucht.

Der Trockenmasse‐ und Kolbenertrag zur Siloreife bzw. der Samenertrag wurde durch die Bakterienstämme Pseudomonas fluorescens (PsIA12), Agrobacterium rhizogenes (A1A4), Rhizobium trifolii (R39) und z.T. durch Stenotrophomonas maltophilia (PsIB2) in den Jahren 1990 bis 1995 auf lehmigen Sandböden wiederholt signifikant, auf sandigem Lehm zum Teil signifikant erhöht. Maissorten können unterschiedlich auf die Bakterieninokulation reagieren. Die Bakterien stimulierten insbesondere bei Jungpflanzen die Wurzelentwicklung und die Nährstoffaufnahme (N, P, K) aus dem Boden. Sie bildeten Phytohormone (Auxine und z.T. Cytokinine) und überlebten, auch unter Feldbedingungen, im Rhizosphärenraum von Mais während der gesamten Vegetation.