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Background and objectives Determination of different grain protein fractions in wheat cultivars is an important task in analyzing bread baking quality. In many laboratories, the Bradford assay is used to determine protein concentrations in solutions. In any protein assay (including Bradford), the ideal protein to use as a standard is the purified protein being assayed. In the absence of such an absolute reference, protein another protein must be selected as a relative standard such as bovine serum albumin (BSA) which is widely used. The aim of this work was to find conversion factors for BSA to determine correct albumin–globulin, gliadin, and glutenin concentrations, because these purified wheat grain protein fractions are mostly not available to be used for calibration purposes. Findings In case of BSA calibration, gluten concentration was underestimated (50%–54%) compared to calibration with the respective purified wheat proteins (65%–70%) in extracts of wheat grain samples. This result is explained with the different amino acid composition of BSA and wheat protein fractions leading to a more intense signal with BSA in the Bradford assay. Calibration of the Bradford assay using BSA as well as purified wheat protein fractions allowed to calculate the conversion factors of 2.11 for BSA/albumin–globulin, 4.25 for BSA/gliadin, and 3.42 for BSA/glutenin. Application of these conversion factors proved to accurately adjust protein concentrations of wheat fractions originating from ten cultivars, determined with BSA calibration of the Bradford assay. Conclusions BSA calibration of the Bradford assay in combination with the conversion factors can be used to determine protein concentration of wheat grain fractions. Significance and novelty Findings of this study make a contribution toward the correction of a common method, to provide a basis for better comparability of research results from different laboratories.

Antibiotic drugs are excreted to a large proportion by livestock. Thus, antibiotics are distributed on fields with slurry and can be taken up by plants. In the present study, hydroponic experiments were performed to reveal whether the widely administered chlortetracycline is taken up into wheat grain in a concentration-dependent manner. A further goal was to determine (chlor)tetracyclines in wheat and rye grain from agricultural practice.; Results: Increasing chlortetracycline deposition in wheat grain was observed with a rising chlortetracycline spiking level in the hydroponic solution. In 371 selected wheat and rye samples from three growing years of agricultural practice, the overall detection frequency was 21% for tetracyclines. In the most highly contaminated sample, tetracyclines occurred at 18.2 μg kg-1 . Tetracycline residues were also found in rye grain. Conversion and degradation products of (chlor)tetracycline such as tetracycline, doxycycline and demeclocycline were detected in grains from hydroponic experiments and from agricultural practice.; Conclusion: Concentrations of tetracyclines found in wheat and rye grains were of no concern with respect to toxicity regarding human consumption. However, antibiotic concentrations below the minimum inhibitory concentration can select for antibiotic resistance in bacteria. Thus, low levels of different tetracycline residues contained in food should be taken into account regarding risk assessment. © 2018 Society of Chemical Industry.; © 2018 Society of Chemical Industry.

A large source of antibiotic active compounds and resistant bacteria is livestock waste, distributed as fertilizer on fields, used for vegetable cultivation. Frequently consumed vegetable, contaminated by low levels of antibiotics, may contribute to the development of bacterial antibiotic resistance. The objective of this work was to estimate the possible role of red cabbage as reservoir and carrier of antibiotic contaminants into the food chain. Red cabbage was exposed to antibiotics in hydroponic cultures and farming conditions. Enrofloxacin (ENR), chlortetracycline (CTC), monensin (MON) and amoxicillin (AMO) were separately added to nutrient solutions. Analysed by sequential extraction and LC–MS/MS methods, the plants revealed a remarkable uptake of CTC and in particular ENR (old leaves 0.22 mg/kg fresh weight (fw) CTC, 6.0 mg/kg fw ENR; roots up to 215.0 mg/kg fw CTC, 14.6 mg/kg fw ENR). The uptake of MON was significantly lower and AMO was not detectable. Red cabbage was also grown on manure-fertilized plots. The pig manure contained defined amounts of CTC and ENR (50 and 150 mg/kg). At harvest the edible parts of cabbage had between 9.2 to 16.9 µg/kg fw ENR, while there was no evidence obtained for the uptake of CTC. Finally, production steps of canned red cabbage were examined for carry over effects. Traces of tetracycline (16.4–19.2 µg/kg fw) were detected in supplies of freshly harvested vegetable, grown conventionally, but not in the marketable final product.

Antibiotic drugs are excreted to a large proportion by livestock. Thus, antibiotics are distributed on fields with slurry and can be taken up by plants. In the present study, hydroponic experiments were performed to reveal whether the widely administered chlortetracycline is taken up into wheat grain in a concentration-dependent manner. A further goal was to determine (chlor)tetracyclines in wheat and rye grain from agricultural practice.Increasing chlortetracycline deposition in wheat grain was observed with a rising chlortetracycline spiking level in the hydroponic solution. In 371 selected wheat and rye samples from three growing years of agricultural practice, the overall detection frequency was 21% for tetracyclines. In the most highly contaminated sample, tetracyclines occurred at 18.2 μg kgConcentrations of tetracyclines found in wheat and rye grains were of no concern with respect to toxicity regarding human consumption. However, antibiotic concentrations below the minimum inhibitory concentration can select for antibiotic resistance in bacteria. Thus, low levels of different tetracycline residues contained in food should be taken into account regarding risk assessment. © 2018 Society of Chemical Industry.

BACKGROUND: Identification of biomarkers capable of distinguishing organic and conventional products would be highly welcome to improve the strength of food quality assurance. Metabolite profiling was used for biomarker search in organic and conventional wheat grain (Triticum aestivum L.) of 11 different old and new bread wheat cultivars grown in the DOK system comparison trial. Metabolites were extracted using methanol and analysed by gas chromatography-mass spectrometry. RESULTS: Altogether 48 metabolites and 245 non-identified metabolites (TAGs) were detected in the cultivar Runal. Principal component analysis showed a sample clustering according to farming systems and significant differences in peak areas between the farming systems for 10 Runal metabolites. Results obtained from all 11 cultivars indicated a greater influence of the cultivar than the farming system on metabolite concentrations. Nevertheless, a t-test on data of all cultivars still detected 5 metabolites and 11 TAGs with significant differences between the farming systems. CONCLUSION: Based on individual cultivars, metabolite profiling showed promising results for the categorization of organic and conventional wheat. Further investigations are necessary with wheat from more growing seasons and locations before definite conclusions can be drawn concerning the feasibility to evolve a combined set of biomarkers for organically grown wheat using metabolite profiles. © 2014 Society of Chemical Industry.

For the Fusarium trichothecene mycotoxins T-2 and HT-2, a combined (T-2 + HT-2) temporary tolerable daily intake (tTDI) of 0.06 microg kg(-1) body weight day(-1) was proposed at the European level in 2001 (Opinion of the Scientific Committee on Food). In the near future, maximum levels for these trichothecenes will be regulated by the European Commission as announced in EU (VO) 1881/2006. For the implementation of these maximum levels, more data on occurrence and behaviour of T-2 and HT-2 toxins in primary agricultural products as well as during cleaning treatment and food processing are needed. In the current work, we determined the T-2/HT-2 concentrations in four oat cultivars (Aragon, Dominik, Ivory, Pergamon) from ten different agricultural sites in Germany, grown in cultivar studies in 2007. The grains were de-hulled, oat meal was prepared, and bread with 20% oat meal and 80% wheat flour was baked. In the cereal-processing chain, samples were taken at various steps and subsequently analysed for their T-2/HT-2 content. We employed liquid chromatography-mass spectrometry (LC-MS) and an immunological screening method (enzyme-linked immunoabsorbent assay (ELISA)) for T-2/HT-2 determination. Detection limits were between 1 and 10 microg kg(-1) in different matrices. T-2/HT-2 concentrations determined by ELISA in oat samples from ten different agricultural sites in Germany were between 9 and 623 microg kg(-1). The median and 90th percentile were 48 and 191 microg kg(-1) T-2/HT-2, respectively. One site showed six times higher T-2/HT-2 levels than the other sites, where concentrations ranged from 322 to 623 microg kg(-1). In 80% of the samples the cultivars Pergamon and Ivory had the lowest concentration of T-2 and HT-2 toxins. Using LC-MS for T-2/HT-2 determination, cleaning of the raw material did not lead to significant reductions of T-2 and HT-2 levels, whereas de-hulling led to a reduction of over 90%. Boiling of oat meal produced from cleaned raw material to yield 'porridge' resulted in varying T-2/HT-2 levels in experimental replicates. No major reduction of T-2/HT-2 levels in cooked porridge was obtained. Standardized baking experiments using 20% oat meal showed that T-2 and HT-2 toxins are relatively stable during the baking process, probably due to their temperature stability.

Das Aufnahmepotential von Weiskohl und Porree fur einige verordnungsstarke Wirkstoffe aus der Nutztierhaltung wurde uber Dotierungsexperimente in Hydrokultur ermittelt. Exemplarisch wurden dazu die Wirkstoffe Sulfadiazin (SFD), Enrofloxazin (ENR), Tetracyclin (TC), Chlortetracyclin (CTC) und Monensin (MON) ausgewahlt. Auserdem sollten ruckstandsanalytische Daten uber die Belastungssituation von Gemuse gewonnen werden, das in landwirtschaftlicher Praxis angebaut wird. Die augenscheinlichen Wirkungen der Antibiotika auf die in Hydrokultur angezogenen Gemusepflanzen (Dotierungskonzentration der Nahrlosung: 5 μmol/l je Antibiotikum) waren sehr unterschiedlich: Sie reichten beim Porree von nicht erkennbaren Effekten (MON, SFD), schwach ausgepragtem Ausbleichen in jungen Blattabschnitten (CTC) bis zu starken Effekten durch den Einfluss von ENR. Die (phytotoxischen) Wirkungen der Antibiotika auf die Weiskohlpflanzen waren wesentlich ausgepragter. So zeigte der mit CTC dotierte Weiskohl gelbliche Verfarbungen der Leitbahnen. MON fuhrte an einigen Blattern zu Lasionen und schlieslich zum Welken. Bei Dotierung mit ENR war eine fast vollstandige Ausbleichung der jungen Blatter festzustellen. Mit LC-MS/MS-Methoden (niedrig- und hochauflosende MS) wurden die dotierten Antibiotika-Wirkstoffe sowie deren Umwandlungsprodukte und Metaboliten separat in den Pflanzenteilen von Porree (Wurzel, junge und alte Blattabschnitte) sowie Weiskohl (Wurzel, Stangel, junge und alte Blatter) identifiziert und quantifiziert. Je nach Wirkstoff, Gemuseart und Pflanzenteil umfassten die ermittelten Arzneistoffruckstande mehrere Grosenordnungen (μg/kg bis mg/kg Frischgewicht, FG). In den Wurzeln beider Gemusepflanzen erreichten CTC und TC in Weiskohl (ca. 10 mg/kg FG) und Porree (ca. 20 mg/kg FG), sowie ENR in Weiskohl (ca. 12 mg/kg FG) die hochsten Konzentrationen. ENR wurde zu geringen Anteilen zu Ciprofloxazin (CIP) metabolisiert. In alten Weiskohlblattern wurde ENR in ahnlich hohen Gehalten (ca. 7 mg/kg FG) wie in Wurzeln gefunden. In den Stangeln und Blattern (jung und alt) des Weiskohls sowie den jungen und alten Blattabschnitten des Porrees wurden alle dotierten Wirkstoffe nachgewiesen. Dabei dominierten ENR und CTC einschlieslich seiner Umwandlungsprodukte wie z. B. Demeclocyclin (DMC) und TC, wahrend SFD und MON in deutlich niedrigeren Konzentrationen vorlagen (

Spring wheat (Triticum aestivum L. cv. Triso) was grown in a free-air CO2 enrichment (FACE) field experiment in order to gain information on CO2-induced effects on grain composition and quality at maturity. A proteome analysis was performed using two-dimensional gel electrophoresis (2-DE) and protein identification was done with mass spectrometry (MALDI-TOF MS). In elevated CO2 (526 μl l−1), an increase of 13.5% in grain yield was observed relative to 375 μl l−1 at a low level of significance (P = 0.528). Total grain protein concentration was decreased by 3.5% at a high level of statistical significance. Most importantly, a number of statistically significant changes within the grain proteome were observed, as the levels of 32 proteins were affected by elevated CO2: 16 proteins were up-regulated and 16 were down-regulated. Our experiment demonstrates that high-CO2 can markedly affect the proteome of mature wheat grain. The potential role of the proteins, changed in response to CO2 enrichment, is discussed as some may affect grain quality. For the task of selecting cultivars resistant to CO2-induced quality loss, we propose to consider the proteins affected by elevated CO2 identified in this work here.

We have characterised the expression of a subfamily of genes that encode sucrose phosphate synthase (SPS), the plant enzyme responsible for sucrose synthesis. cDNA fragments from four genes were cloned from diploid Actinidia chinensis Planch. var. chinensis. Three genes are highly similar (94–96% nucleotide identity in the overlapping region of the reading frame) and all belong to Family A, with the fourth gene (67% nucleotide identity) belonging to Family B. The A2 gene encodes a longer open reading frame than the other two A family genes, with a C-terminal extension of 102 amino acids. We compared transcript abundance of these four genes using RT-PCR. The three A genes were expressed in all tissues, but only A1 was present at high abundance and responded to developmental and environmental stimuli. The A1 transcript increased in fruit in response to treatment with exogenous ethylene and again during the ripening climacteric, but was not affected by low temperature. A1 expression was also high in senescent leaves, stems and in flower buds. The B family transcript was detectable in roots, albeit at very low levels and in all other tissues examined. It also increased in low temperature and in fruit at the climacteric (but not with applied ethylene). This is the first demonstration of differential expression patterns for highly homologous SPS genes in any plant species.

We present phylogenetic analyses to demonstrate that there are three families of sucrose phosphate synthase (SPS) genes present in higher plants. Two data sets were examined, one consisting of full-length proteins and a second larger set that covered a highly conserved region including the 14-3-3 binding region and the UDPGlu active site. Analysis of both datasets showed a well supported separation of known genes into three families, designated A, B, and C. The genomic sequences of Arabidopsis thaliana include a member in each family: two genes on chromosome 5 belong to Family A, one gene on chromosome 1 to Family B, and one gene on chromosome 4 to Family C. Each of three Citrus genes belong to one of the three families. Intron/exon organization of the four Arabidopsis genes differed according to phylogenetic analysis, with members of the same family from different species having similar genomic organization of their SPS genes. The two Family A genes on Arabidopsis chromosome 5 appear to be due to a recent duplication. Analysis of published literature and ESTs indicated that functional differentiation of the families was not obvious, although B family members appear not to be expressed in roots. B family genes were cloned from two Actinidia species and southern analysis indicated the presence of a single gene family, which contrasts to the multiple members of Family A in Actinidia. Only two family C genes have been reported to date.

Motivation: The research area metabolomics achieved tremendous popularity and development in the last couple of years. Owing to its unique interdisciplinarity, it requires to combine knowledge from various scientific disciplines. Advances in the high-throughput technology and the consequently growing quality and quantity of data put new demands on applied analytical and computational methods. Exploration of finally generated and analyzed datasets furthermore relies on powerful tools for data mining and visualization. Results: To cover and keep up with these requirements, we have created MeltDB 2.0, a next-generation web application addressing storage, sharing, standardization, integration and analysis of metabolomics experiments. New features improve both efficiency and effectivity of the entire processing pipeline of chromatographic raw data from pre-processing to the derivation of new biological knowledge. First, the generation of high-quality metabolic datasets has been vastly simplified. Second, the new statistics tool box allows to investigate these datasets according to a wide spectrum of scientific and explorative questions. Availability: The system is publicly available at https://meltdb.cebitec.uni-bielefeld.de. A login is required but freely available. Contact: nkessler@cebitec.uni-bielefeld.de

We report on the cloning of a plastidic glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from higher plants. The complete sequence of the plastidic enzyme was obtained after rapid amplification of cDNA ends and comprises a putative plastidic transit peptide. Sequences amplified from leaf or root poly(A+) RNA are identical. In contrast to the cytosolic enzyme, the plastidic isoform is subject to redox modulation, i.e. thioredoxin-mediated inactivation by light. But when the plastidic enzyme is compared to a cyanobacterial homolog, none of the cysteine residues is conserved. The recombinant enzyme was used to raise antibodies in rabbits. Gene expression was studied in potato (Solanum tuberosum L.), at both the RNA and protein levels, revealing different patterns for the isoforms. The gene encoding the cytosolic enzyme was transcribed in all tissues tested, and the highest transcription was detected in tubers. In contrast, expression of the gene encoding the plastidic enzyme was confined to green tissues. Wounding of leaves resulted in a slight increase in the expression of the gene encoding the cytosolic isoform and a shutdown of the plastidic counterpart. Compared to the situation in soil, elevated transcription of the gene encoding the plastidic enzyme is found in roots of hydroponically grown potato plants, which is in agreement with the postulated role for this isoform in nitrite reduction.

In this work, wheat from two farming systems, organic and conventional, was analyzed. Organic agriculture is one of the fastest growing sectors in the food industry of Europe and the United States. It is an open question, whether organic or conventional agricultural management influences variables such as metabolism, nutrient supply, seed loading and metabolite composition of wheat. Our aim was to detect if organic or conventional farming systems would affect concentrations of metabolites and substances in developing ears and in corresponding matured grain. Therefore, broadband metabolite profiles together with lipids, A cations, starch and protein concentrations of wheat ears in the last phase of grain development and of matured grain from organic and conventional agriculture of a rigorously controlled field trial with two organic and two conventional systems were examined. It appears that seed metabolism and supply of developing ears differ in organic and conventional agriculture. However, the differences in 62 metabolite concentrations become marginal or disappear in the matured grains, indicating an adjustment of nutrients in the matured grain from organic agriculture. This result suggests a high degree of homeostasis in the final seed set independent of the growing regime.

Increasing prices for wheat products and fertilizers, as well as reduced sulfur (S) contributions from the atmosphere, call for an improvement of product quality and agricultural management. To detect the impact of a time-dependent S fertilization, the quantitative protein composition and the baking quality of two different wheat cultivars, Batis and Turkis, were evaluated. The glutathione concentration in grains serves as a reliable marker of the need for added S fertilizer. The quantitation of gliadins and glutenin subunits by reversed-phase high-performance liquid chromatography confirmed that S-rich proteins significantly increased with S fertilization, whereas the S-poor proteins significantly decreased. Proteome analysis by means of high-resolution protein profiles detected 55 and 37 proteins from Batis and Turkis changed by late S fertilization. A microscale baking test using wholemeal flour was implemented for the evaluation of baking quality, and late S fertilization was found to improve the composition of gluten proteins and baking quality.

Research comparing the biochemical composition of wheat grains from organic or conventional agriculture has used the targeted analytical approach. To obtain a more comprehensive record of the food's composition, we employed protein profiling techniques. Levels of 1049 proteins were recorded in wheat grains (Triticum aestivum L., cv. Titlis) of two growing seasons from a rigorously controlled field trial in Switzerland, containing organic and conventional plots. Levels of 25 proteins were different between organic and conventional wheat in both years. Storage proteins, enzymes of carbohydrate metabolism, a peroxidase, and proteins of unknown function were affected by the agricultural regime. Total protein content was lower in organic wheat. We consider these differences negligible with regard to nutrition in an average diet and propose that food quality of conventional and organic wheat grown in the field trial was equal. Applying various filters and calculations, one of which takes seasonal influences into account, 16 of the 25 proteins with different levels in organic and conventional wheat were retained. These 16 "diagnostic" proteins have the potential to afford a signature to prove authenticity of organic wheat.

In some European community countries up to 8% of the agricultural area is managed organically. The aim was to obtain a metabolite profile for wheat (Triticum aestivum L.) grains grown under comparable organic and conventional conditions. These conditions cannot be found in plant material originating from different farms or from products purchased in supermarkets. Wheat grains from a long-term biodynamic, bioorganic, and conventional farming system from the harvest 2003 from Switzerland were analyzed. The presented data show that using a high throughput GC-MS technique, it was possible to determine relative levels of a set of 52 different metabolites including amino acids, organic acids, sugars, sugar alcohols, sugar phosphates, and nucleotides from wheat grains. Within the metabolites from all field trials, there was at the most a 50% reduction comparing highest and lowest mean values. The statistical analysis of the data shows that the metabolite status of the wheat grain from organic and mineralic farming did not differ in concentrations of 44 metabolites. This result indicates no impact or a small impact of the different farming systems. In consequence, we did not detect extreme differences in metabolite composition and quality of wheat grains.

Plastid lipid-associated proteins, also termed fibrillin/CDSP34 proteins, are known to accumulate in fibrillar-type chromoplasts such as those of ripening pepper fruit, and in leaf chloroplasts from Solanaceae plants under abiotic stress conditions. It is shown here that treatments generating active oxygen species (high light combined with low temperature, gamma irradiation or methyl viologen treatment) result in potato CDSP34 gene induction and protein accumulation in leaves. Using transgenic tomato plants containing the pepper fibrillin promoter, a significant increase in promoter activity in leaves subjected to biotic stress, namely bacterial infections, was observed. In WT, a higher level of the endogenous fibrillin/CDSP34 protein is also observed after infection by E. chrysanthemi strain 3739. In addition to stress-related induction, a progressive increase in the fibrillin promoter activity is noticed during ageing in various tomato photosynthetic tissues and this increase correlates with a higher abundance of the endogenous protein in WT leaves. It is proposed that a mechanism related to oxidative events plays an essential role in the regulation of fibrillin/CDSP34 genes during stress and also during development. Using a biolistic transient expression assay, the pepper fibrillin promoter is found to be active in various dicot species, but not in monocots. Further, substantially increased levels of fibrillin/ CDSP34 proteins are shown in various dicotyledonous and monocotyledonous plants in response to water deficit.

Spelt (Triticum aestivum L. ssp. spelta) is experiencing a renaissance in Europe and North America, where it is used for baking, brewing, production of pasta, and self-supplied animal feed. One of the characteristics of spelt is that in comparison to modern wheat it is more resistant to harsh climatic and poor soil conditions. In contrast to wheat the hulls remain on the grain after threshing. Drawbacks are that spelt yields are quite low compared to modern wheat. The subject of the current study was to gain information about the composition of soluble sugars and their concentrations in spelt wholemeal and flour. High performance liquid chromatography (HPLC) was used for analysis. Concentrations of nine free sugars in spelt wholemeal and flour are reported. Flour cumulative free sugar concentrations were 63% lower than in wholemeal. For comparisons, we also analyzed wholemeal of wheat. The cumulative concentration of free sugars was 27% lower than in spelt wholemeal. However, when published data for sugar concentration ranges of wheat are taken into account, the total concentration of free sugar was not different between spelt and modern wheats. Low concentrations of xylose and stachyose were detected in spelt. Higher concentrations of fructans such as 1-kestose and kestotetraose were detected in spelt when compared with wheat. Generally, concentrations of free sugars in spelt were in the range of free sugar levels published for wheat, except for maltose which was higher in spelt.