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.     

Nonvolatiles of commercial lime and grapefruit oils separated by high-speed countercurrent chromatography

The nonvolatile fractions of cold-pressed peel oils of Key and Persian lime as well as grapefruit were separated by high-speed countercurrent chromatography (HS-CCC). In addition to the isolation of the main coumarins, psoralens and polymethoxyflavones, a number of minor constituents were enriched and successfully characterized by GC-MS and HPLC-UV. 5,7,8-Trimethoxycoumarin and the cyclical acetals of oxypeucedanin hydrate with citral were determined as new nonvolatile trace constituents of lime oils and confirmed by NMR spectroscopy. The citral oxypeucedaninyl acetals were found particularly in Key lime oil type A, which as a result of the juice-oil contact, is exposed to acidic conditions during industrial processing. Some of the confirmed minor constituents, such as pabulenol, isooxypeucedanin, and oxypeucedanin methanolate in lime as well as auraptenol in grapefruit, may have been generated by hydrolysis-sensitive precursors during CCC separation or their respective industrial processing techniques.     

Bacterial community composition of sediments from a milkfish Chanos chanos Forsskål farm

Bacterial communities in sediments underneath milkfish cages and adjacent off‐cage areas in Bolinao, Lingayen Gulf, West Philippine Sea were characterized using PCR‐denaturing gradient gel electrophoresis to assess influence of the fish farming to the sediments. The sediments in the fish cage (FC) area showed anoxic conditions [redox potential of ?165 to ?213 mV and acid‐volatile sulphide‐sulphur (AVS‐S) of 0.55–1.88 mg g^?1 (dry weight)] compared to the relatively oxic off‐cage area [redox potential of 72–81 mV and AVS‐S of 0.09–0.14 mg g^?1 (dry weight)]. The composition of the microbial populations revealed influence of the fish feed sedimentation with higher dominance of putative sulphate‐reducing bacteria (SRB) in the FC area. Compositions of the bacterial taxa showed dominance of the Bacteroidetes group and Deltaproteobacteria. The results suggest that the fish feeds created a new suitable environment, in which reductive microorganisms such as SRB, ferric‐ion‐reducing bacteria and nitrate‐reducing bacteria can thrive. Organic load from the feed and quantity of its leftover constituents may be the key factors that influence the structure of the bacterial community in the sediments of the milkfish farms.     

Demonstration of the articular cartilage of the canine ulnar trochlear notch using high-field magnetic resonance imaging

The articular cartilage covering the ulnar trochlear notch (UTN) of both elbow joints from 25 adult canine cadavers was demonstrated by the use of high-field magnetic resonance imaging (MRI) with a fat-suppressed, three-dimensional, spoiled gradient echo sequence. The dogs used for this study were divided into five groups: giant, large, mid-sized, small, and chondrodystrophic breeds. The distribution of articular cartilage of the UTN was assessed on MR images. The joints were then opened, and the distribution of the articular cartilage of the UTN was verified. In 29/50 joints, there was good agreement between gross findings of the distribution of the articular cartilage of the UTN and MRI interpretation. Reasons for the poor results in small and chondrodystrophic breeds were decreased thickness of the articular cartilage, small size of the articular surface and of its structural features, and the frequent inability to visualize the joint space.     

Are survival and reproduction of Enchytraeus albidus (Annelida: Enchytraeidae) at risk by feeding on Bt-maize litter?

Enchytraeids are saprophagous soil organisms, appearing in high abundances and contributing to ecological processes within the soil. For decades they have been used as model species for biological research. In the framework of research on genetically modified plants, however, they have not been considered to date. Following the ISO/DIS guideline, survival and reproduction of Enchytraeus albidus, fed with diets containing Bt-maize (N4640Bt Cry1Ab, DKC5143Bt Cry3Bb1) leaf material were analysed. For comparison, diets with the corresponding untransformed near-isolines (N4640, DKC5143) were examined. Additionally a high quality control diet (oat flakes) was included. Survival and reproduction showed no significant differences between the Cry3Bb1 treatment and the treatment with the untransformed counterpart. For the Cry1Ab treatment survival was significantly higher than for the treatment with the corresponding near-isoline. In contrast, reproduction was significantly lower for the Cry1Ab treatment compared to that for the isoline. For the Cry3Bb1 treatment, no effect was shown on survival or reproduction. For the Cry1Ab variety and its untransformed counterpart, a contrasting result was detected, which is unlikely to be caused by the Bt-protein but rather by differences in other plant components. Overall survival and reproduction were highest for the control.     

Effect of source germplasm and season on the in vivo haploid induction rate in tropical maize

For in vivo production of doubled haploid (DH) lines in maize, the rate of haploid induction is of crucial importance. Maternal haploid induction depends primarily on the inducer used as a pollinator. However, the source germplasm used as a maternal parent and the environmental conditions for induction may also influence haploid induction and these aspects have not been examined in tropical maize so far. The objectives of our study were to (i) monitor the variation for haploid induction rate (HIR) among diverse source germplasm in tropical maize, (ii) determine the relative importance of general (GCA) and specific (SCA) combining abilities for HIR, and (iii) investigate the influence of summer and winter seasons and genotype × season interactions on this trait. Ten inbreds were mated in a half diallel design. The resulting 45 F₁ single crosses were pollinated with the haploid inducer hybrid RWS × UH400 during the summer 2008 and winter 2009 seasons in a lowland tropical environment in Mexico. HIR of the single crosses averaged over seasons ranged from 2.90 to 9.66% with an overall mean of 6.74%. Mean HIR was significantly (P < 0.01) higher during the winter (7.37%) than summer season (6.11%). Significant (P < 0.01) variation was observed due to GCA effects of parental inbreds of single crosses but not for SCA, GCA × season and SCA × season interactions. Our study underpins that a higher HIR in tropical maize can be obtained by selecting appropriate source germplasm and undertaking pollination under favorable environmental conditions.     

Polychlorinated biphenyls (PCBs) in bulk soil and particle size separates of soils in a rural community

In a rural community (Stephanskirchen, Southern Germany) situated near a waste incineration plant six A horizons were sampled to determine 14 polychlorinated biphenyls (PCBs) in bulk soil and particle size separates (clay, silt, fine and coarse sand) and to test the suitability of alkaline saponification for PCB extraction. The PCB concentrations were higher in alkaline saponification extracts than in Soxhlet extracts. However, the extraction efficiency of up to fourfold chlorinated PCBs was lower and more than sevenfold chlorinated PCBs were destroyed. Nevertheless, alkaline saponification was suitable to determine the indicator congeners 28, 52, 101, 138, 152, and 180. The sum of the PCB concentrations ranged from 8.4 to 59.5 μg kg^?1 which was as high as in soils of big cities. However, the concentrations did not reach the threshold value for agricultural purposes of 200 μg kg^?1 as proposed in literature for the sum of the six indicator congeners. The hexachlorinated PCBs 138 and 153 were the most abundant (together > 50% in all samples) whereas PCB 1 could not be detected in any sample. On the average, PCB concentrations increased as particle size decreased. When normalized to C_org, however, the fine sand fraction had the highest PCB concentrations. A slight decrease of lower chlorinated PCBs as particle size decreased indicated an increasing state of degradation. The PCB compositions in the emissions of a comparable waste incineration plant (Bamberg, Bavaria) and in Stephanskirchen soils differed markedly. Therefore, we did not get any indications that waste incineration was a main source of PCBs in the examined soils. Instead, higher chlorinated technical PCB mixtures such as Clophen A 60 or Aroclor 1260 may be the most important sources.     

A two-step modification treatment of solid wood by bulk modification and surface treatment

A combination of bulk and surface modification of wood could lead to a product that shows the advantages of both treatments. This study evaluates the penetration of melamine resin into acetylated and silylated cell walls and possible side effects of melamine impregnation on this bulk treatment in order to clarify the feasibility of a combination of the chosen bulk modification (acetylation or silylation) and surface treatments (hardening by melamine modification). UV microscopy confirmed that melamine resin penetrates into wood cell walls even after acetylation and silylation treatment. Energy dispersive X-ray analysis showed that the melamine treatment did not lead to substantial washing out of silyl groups with the silylation reagent used. The possibility of a combination of the selected bulk and surface modification methods is given.     

Development of locally-adapted faba bean cultivars for organic conditions in Germany through a participatory breeding approach

Organic farming requires cultivars that are specifically adapted to this low input cropping system. Hence, organic farmers and scientists joined in a participatory breeding approach to develop region-specific genotypes of spring faba bean for organic conditions in Germany. A set of 49 genotypes with contrasting degrees of heterozygosity and heterogeneity was used in field trials across five locations in Germany during 3 years 2004, 2005 and 2006. The material involved 18 inbred lines, their 18 polycross progenies, one blend of inbred lines, one blend of polycross progenies, one blend of hybrids and ten checks. Inbred lines are uniform, thus giving the option to be specifically adapted; whereas the polycross progenies and synthetics (Syn-1; predicted from the inbred lines and polycross progenies performance) are partly heterogeneous and heterozygous, thus giving the option to evolve. Agronomic performance was assessed and a “personal appreciation” score of the material was assigned to each genotype by each partner. This personal appreciation was strongly influenced by biotic and abiotic constraints faced by the crop in each location and by the expected grain yield of the genotypes. Uniformity was apparently appreciated by organic farmers. In all locations, the highest yielding inbred line yielded slightly better than the predicted highest yielding synthetic. However, this slight disadvantage of the synthetic is very likely to disappear if the synthetic (Syn-1) is propagated during successive generations.     

Effect of hardening on frost tolerance and fatty acid composition of leaves and stems of a set of faba bean (<Emphasis Type="Italic">Vicia faba</Emphasis> L.) genotypes

Frost tolerance is a main component of winter-hardiness and improving it would promote faba bean (Vicia faba L.) cropping in cool-temperate regions. In many species, leaf fatty acid composition was found to be related to frost tolerance. The objective of this study was to determine, in a representative sample of genotypes, the effect of hardening on leaf and stem (1) frost tolerance and (2) fatty acid composition, and to seek correlations between them. First leaf, second leaf and stem of 31 faba bean genotypes were analyzed after hardening and without hardening. High frost tolerance of known winter genotypes and several experimental lines was shown. Hardening had a significant, positive effect on frost tolerance of all three organs. Stems were on average more frost tolerant than leaves. Hardening induced significant changes in the fatty acid composition: oleic acid decreased significantly in leaves by 3.24% and in stems by 1.77%, whereas linolenic acid increased in leaves by 6.28% and in stems by 9.06%. In stems, correlations between frost tolerance and fatty acid composition were not significant. Correlation coefficients strongly indicated that non-hardened oleic acid content, changes in oleic acid and in linoleic plus linolenic acid content in leaves partly explained their frost tolerance; 0.347 (P < 0.1) < |r| < 0.543 (P < 0.01). The results corroborate the importance of using genetic differences in the fatty acid metabolism in breeding grain legumes for frost tolerance.