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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.

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.

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.

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.