Occurrence of Fusarium T-2 and HT-2 toxins in oats from cultivar studies in Germany and degradation of the toxins during grain cleaning treatment and food processing.

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.

Accumulation of plastid lipid-associated proteins (fibrillin/CDSP34) upon oxidative stress, ageing and biotic stress in Solanaceae and in response to drought in other species.

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.

Biochemical and molecular characterisation of xyloglucan endotransglycosylase from ripe kiwifruit.

Xyloglucan endotransglycosylase (XET) from the core tissue of ripe kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var. deliciosa cv. Hayward) was purified 3000-fold to homogeneity. The enzyme has a molecular weight of 34 kDa, is N-glycosylated, and is active between pH 5.0 and 8.0, with an optimum between 5.5 and 5.8. The Km was 0.6 mg.mL-1 for kiwifruit xyloglucan and 100 microM for [3H]XXXG-ol, a reduced heptasaccharide derived from kiwifruit xyloglucan. Kiwifruit core XET was capable of depolymerising xyloglucan in the absence of [3H]XXXG-ol by hydrolysis, and in the presence of [3H]XXXG-ol by hydrolysis and endotransglycosylation. Six cDNA clones (AdXET1-6) with homology to other reported XETs were isolated from ripe kiwifruit mRNA. The six cDNA clones share 93-99% nucleotide identity and appear to belong to a family of closely related genes. Peptide sequencing indicated that ripe kiwifruit XET was encoded by AdXET6. Northern analysis indicated that expression of the AdXET1-6 gene family was induced in ripening kiwifruit when endogenous ethylene production could first be detected, and peaked in climacteric samples when fruit were soft. A full-length cDNA clone (AdXET5) was overexpressed in E. coli to produce a recombinant protein that showed endotransglycosylase activity when refolded.

Sucrose-phosphate synthase steady-state mRNA increases in ripening kiwifruit.

Early during fruit ripening in kiwifruit (Actinidia deliciosa var. deliciosa [A. Chev.], C.F. Liang and A.R. Ferguson cv. Hayward), starch is broken down to sucrose and hexose sugars. Concomitantly, sucrose-phosphate synthase (SPS, EC 2.3.1.14) activity measured with saturating substrate increased, suggesting that SPS is induced in response to a higher requirement for sucrose synthesis. A 2584 bp long partial cDNA clone encoding SPS was isolated from ripening kiwifruit. cDNA fragments encoding the 5' end were isolated by PCR, and sequencing revealed at least four closely related (> 96% identity) mRNAs expressed early in kiwifruit ripening. Southern hybridisations in a diploid relative of kiwifruit, Actinidia chinensis (Planch.) var. chinensis, were consistent with the presence of a small gene family. Western analysis indicated a 125 kDa SPS protein present in all tissues of A. chitensis at all stages of development. Steady-state levels of SPS mRNA in A. chinensis increased near fruit maturity as net starch degradation began on the vine, and increased again during ethylene treatment of fruit after harvest. After removal from ethylene SPS transcript levels decreased, only to increase again as fruit moved into the climacteric and starch breakdown was completed. Exposure to low temperatures also caused an increase in SPS transcript level. These results indicate that SPS mRNA increases in kiwifruit in response to the presence of new substrate sourced from starch degradation, in response to ethylene and in response to low temperature.

Molecular characterization of the plastidic glucose-6-phosphate dehydrogenase from potato in comparison to its cytosolic counterpart.

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.