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PubMed2001

The state-of-the-art in the analysis of estrogenic mycotoxins in cereals.

The increasing public awareness of chemicals that mimic or otherwise interfere with the activity of natural hormones - so-called endocrine disrupters - has also led to greater study of mycotoxins with estrogenic potential. The purpose of this paper is to introduce the topic of estrogenic mycotoxins and to discuss the state-of-the-art in the analysis of these substances in cereals, with special emphasis on zearalenone (ZON) as its most relevant representative. Because the use of immunoaffinity columns (IAC) followed by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) and immunoassays are currently the most frequently used methods for the determination of ZON and its metabolites, these techniques are discussed in more detail. Recent papers, which have revealed the great potential of HPLC-MS(MS) for the simultaneous detection and identification of several estrogenic mycotoxins, are discussed. The performances of the state-of-the-art methods are finally compared by study of the results obtained in recent international intercomparison studies. On the one hand, these studies revealed the good performance of both chromatographic and antibody-based methods. On the other hand, the need for better means of external quality assurance measures, especially the availability of certified reference materials and certified standards, has clearly been demonstrated.

Chromatography, High Pressure Liquid↗
PubMed2001

The state-of-the-art in the analysis of type-A and -B trichothecene mycotoxins in cereals.

The aim of this review is to describe the state-of-the-art in the analysis of A- and B-trichothecene mycotoxins in cereals and to support knowledge and experience exchange between laboratories in the field of Fusarium mycotoxin analysis. Current screening tests and quantitative methods for the most prevalent type-A and -B trichothecenes, HT-2 and T-2-toxin, and deoxynivalenol (DON) are reviewed. This includes the extraction and clean-up procedures and chromatographic methods (TLC, HPLC, GC) applied and the immunochemical methods, especially enzyme-linked immunosorbent assay (ELISA), employed for the determination of these mycotoxins. Results from recent intercomparison studies of the determination of DON are also discussed. Experience gained during these intercomparisons clearly shows the need for further improvement in the determination of trichothecenes, to obtain more accurate and comparable results. This also indicates there is a strong need for the development of further certified reference materials (CRM) which would enable comparison of measurement results between different European laboratories for several A- and B-trichothecenes. For both A- and B-trichothecenes there is still a lack of simple and reliable screening methods enabling the rapid detection of these mycotoxins at low cost.

Chemistry Techniques, Analytical↗
PubMed2006

Comparison of bacteriocins produced by lactic-acid bacteria isolated from boza, a cereal-based fermented beverage from the Balkan Peninsula.

Boza is a low-pH and low-alcohol cereal-based beverage produced in the Balkan Peninsula. From a total population of 9 x 10(6) colony-forming units ml(-1), four isolates (JW3BZ, JW6BZ, JW11BZ, and JW15BZ) produced bacteriocins active against a broad spectrum of Gram-positive bacteria. Bacteriocin JW15BZ inhibited the growth of Klebsiella pneumoniae. The producer strains were identified as Lactobacillus plantarum (strains JW3BZ and JW6BZ) and L. fermentum (strains JW11BZ and JW15BZ). The spectrum of antimicrobial activity, characteristics, and mode of action of these bacteriocins were compared with bacteriocins previously described for lactic-acid bacteria isolated from boza.

Anti-Bacterial Agents↗
PubMed2003

Somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye ( Secale cereale L.).

A method for somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (Secale cereale L. cv. Auvinen) was developed. Fast-growing and friable embryogenic calli with a high regeneration capacity were induced from immature rye inflorescences using modified MS medium. These friable embryogenic calli were used for suspension culture initiation in liquid AA medium. A high yield of protoplasts was obtained from suspension cell clumps after 3-5 days of subculture. Isolated protoplasts were cultured in KM8p medium. The frequency of protoplast cell divisions and colony formations in liquid culture medium were similar to those on agarose-solidified medium. Compact embryogenic calli were developed from protoplast-derived microcalli in growth medium mMS. Approximately 7% of the transferred embryogenic calli produced green shoots on N6 regeneration medium. Of 33 green plants, 28 were fertile with normal flowering and seed set. The ratio of green and albino plantlets was 1:4. Rye protoplast-derived green plants showed normal diploid characters as determined by flow cytometer analysis and chromosome counting.

Cell Division↗
PubMed1999

Microdissection and microcloning of rye (Secale cereale L.) chromosome 1R.

Chromosome 1R was microdissected and collected from mitotic metaphase spreads of rye (Secale cereale L.) by using glass needles. The isolated chromosomes were amplified in vitro by Sau3A linker adaptor-mediated polymerase chain reaction (PCR). After amplification, the presence of rye-specific DNA was verified by Southern hybridization. The second-round PCR products from five 1R chromosomes were cloned into a plasmid vector to create a chromosome-specific library, which produced approximately 220,000 recombinant clones. Characterization of the microclone library showed that the 172 clones evaluated ranged in size from 300-1800 bp with an average size of 950 bp, of which approximately 42% were medium/high copy and 58% were low/unique copy clones. Chromosome in situ hybridization confirmed that the PCR products from microdissected chromosomes originated from chromosome 1R, indicating that many chromosome 1R-specific sequences were present in the library.

Base Sequence↗
PubMed2002

Increased capacity for synthesis of the D1 protein and of catalase at low temperature in leaves of cold-hardened winter rye (Secale cereale L.).

The effect of low temperature on protein synthesis, particularly the synthesis of the photolabile proteins D1 of photosystem II and catalase (EC 1.11.1.6), was compared in non-hardened leaves (NHL) and cold-hardened leaves (CHL) of winter rye (Secale cereale L.). At 4 degrees C, both the uptake of L-[(35)S]methionine into leaf sections and its incorporation into proteins were reduced, relative to 25 degrees C. However, much lower reductions were observed in CHL than in NHL. In particular, the proportion of the L-[(35)S]methionine uptake incorporated into membrane proteins at 4 degrees C was considerably higher in CHL than in NHL. At 25 degrees C, the incorporation of L-[(35)S]methionine into both the D1 protein and catalase was lower in CHL than in NHL, in accord with a slower light-induced turnover in CHL. At 4 degrees C, the incorporation into the D1 protein and catalase was, however, much higher in CHL than in NHL, indicating that their de novo synthesis was less suppressed by the low temperature. The results indicate that cold-acclimated leaves had an improved ability to repair the photolabile proteins D1 and catalase at low temperature, relative to NHL. mRNAs for the D1 protein and for leaf catalase were not increased in CHL, relative to NHL. The superior capacity of CHL for repair at low temperature must result from posttranscriptional mechanisms. The translational efficiency of the catalase mRNA was similarly increased in both NHL and CHL during 7-h exposures to high light at 4 degrees C, while the amounts of the catalase transcript declined under these conditions. However, during a recovery period at 22 degrees C, subsequent to an exposure of NHL to 4 degrees C and high light, transient increases of the D1 and catalase mRNAs were observed.

Adaptation, Physiological↗
PubMed2004

Changes in gene expression during dehardening of cold-hardened winter rye (Secale cereale L.) leaves and potential role of a peptide methionine sulfoxide reductase in cold-acclimation.

Suppression subtractive hybridization and differential display polymerase chain reactions were used to identify genes that were differentially expressed in cold-hardened and dehardened leaves of winter rye (Secale cereale L.). The transcripts of nine genes declined during dehardening at 22 degrees C of cold-hardened 4 degrees C-grown leaves, indicating some role in cold-acclimation. Among the genes that were strongly expressed in cold-hardened leaves were five genes of photosynthetic metabolism, the gene of the antioxidative enzyme peptide methionine sulfoxide reductase (PMSR) and three genes of RNA and protein metabolism. Four genes were identified that were more strongly expressed during dehardening of cold-hardened leaves at 22 degrees C. A full-length cDNA for a presumed cytosolic PMSR (EC 1.8.4.6) of rye leaves was identified. After heterologous expression in Escherichia coli, an antiserum against the ScPMSR was produced. The content of the ScPMSR protein, visualized by immunoblotting, was much higher in cold-hardened than in non-hardened leaves and declined during dehardening. In non-hardened leaves the mRNA of ScPMSR increased only slowly during exposures to 4 degrees C in light and was not affected by exposure to 4 degrees C in darkness. However, the ScPMSR mRNA was also induced by prolonged exposure (48 h) to high light at 22 degrees C, or by treatment with 2 muM paraquat. Consequently, the induction of cytosolic ScPMSR is a late response to prolonged photooxidative stress conditions, as expected during growth at low temperature in light. In cold-hardened leaves, PMSR may protect proteins from photodamage and thus prevent their degradation and the need for repair.

Acclimatization↗
PubMed2005

Cereal phosphate transporters associated with the mycorrhizal pathway of phosphate uptake into roots.

A very large number of plant species are capable of forming symbiotic associations with arbuscular mycorrhizal (AM) fungi. The roots of these plants are potentially capable of absorbing P from the soil solution both directly through root epidermis and root hairs, and via the AM fungal pathway that delivers P to the root cortex. A large number of phosphate (P) transporters have been identified in plants; tissue expression patterns and kinetic information supports the roles of some of these in the direct root uptake pathways. Recent work has identified additional P transporters in several unrelated species that are strongly induced, sometimes specifically, in AM roots. The primary aim of the work described in this paper was to determine how mycorrhizal colonisation by different species of AM fungi influenced the expression of members of the Pht1 gene families in the cereals Hordeum vulgare (barley), Triticum aestivum (wheat) and Zea mays (maize). RT-PCR and in-situ hybridisation, showed that the transporters HORvu;Pht1;8 (AY187023), TRIae;Pht1;myc (AJ830009) and ZEAma;Pht1;6 (AJ830010), had increased expression in roots colonised by the AM fungi Glomus intraradices,Glomus sp. WFVAM23 and Scutellospora calospora. These findings add to the increasing body of evidence indicating that plants that form AM associations with members of the Glomeromycota have evolved phosphate transporters that are either specifically or preferentially involved in scavenging phosphate from the apoplast between intracellular AM structures and root cortical cells. Operation of mycorrhiza-inducible P transporters in the AM P uptake pathway appears, at least partially, to replace uptake via different P transporters located in root epidermis and root hairs.

Cloning, Molecular↗
PubMed2006

Mode of translational activation of the catalase (cat1) mRNA of rye leaves (Secale cereale L.) and its control through blue light and reactive oxygen.

The enzyme catalase (EC 1.11.1.6) is inactivated by light and must be continuously replaced by new synthesis in order to maintain a constant enzyme activity in leaves. In winter rye leaves (Secale cereale L.) posttranscriptional mechanisms determine the rate of new catalase synthesis, including a light-controlled reversible modification of the catalase cat1 mRNA by methylation which greatly enhanced its translation efficiency. The specificity and regulation of this mRNA activation were further investigated. The translation efficiency of the rye cat1 mRNA was much more enhanced by N-7 methylation of the cap than that of an lhcb transcript. Investigations with truncated rye cat1 mRNAs indicated that the translational enhancement resulting from N-7 cap methylation did not require the presence of specific sequences of cat1 5'- and 3'-untranslated regions. Translational activation of the cat1 mRNA in rye leaves was independent of photosynthesis and most effectively induced by blue light. Peroxides (H(2)O(2), tertiary butyl hydroperoxide) and conditions enforcing an H(2)O(2) accumulation in the leaves (aminotriazole, paraquat) also caused an activation of the cat1 mRNA. A search for further signalling systems controlling the replenishment of inactivated catalase in light suggested that an inositol-1,4,5-triphosphate-mediated liberation of Ca(2+) from internal stores and a protein phosphatase played some role. However, these signalling systems did not affect the activation of the cat1 mRNA. After removal of Ca(2+) by EGTA the cat1 mRNA was rapidly degraded.

Catalase↗
PubMed2005

Studies on the function of TM20, a transmembrane protein present in cereal embryos.

The possible function of the maize transmembrane protein TM20 in hormone transport has been investigated using immunological methods and by microinjection of TM20 cRNA in Xenopus oocytes. The existence of a similar gene in rice indicates that the overall structure of the deduced protein is conserved between these two cereals. An antibody raised against a conserved motif, in a loop between two transmembrane domains, locates the protein (TM20) in differentiating provascular cells in maize embryo. The protein has a polarized distribution within the cell in the most differentiated stages of development. Xenopus laevis oocytes microinjected with TM20 appear to modify transport activities across the plasma membrane. These results are discussed in relation to other transport proteins that influence plant development.

Amino Acid Sequence↗
PubMed2002

Tetrad-FISH analysis reveals recombination suppression by interstitial heterochromatin sequences in rye (Secale cereale).

Tetrad analysis is a genetic method that can locate genes and centromeres on a linkage map with a high degree of precision. Despite its effectiveness and accuracy, application of this method is generally limited to fungi, algae and mosses. Here we demonstrate a new method of tetrad analysis that is applicable to other organisms. This combines tetrad analysis with fluorescence in situ hybridization (FISH), and is thus referred to as tetrad-FISH analysis. We demonstrate the effectiveness of this method using tetrads of rye, Secale cereale. The rye strain JNK contains interstitial heterochromatin in a region of Chromosome 2R. We have previously cloned the tandemly repeated sequence forming this heterochromatin in the plasmid pScJNK. We performed FISH using pScJNK as the probe on tetrads obtained from heterozygotes for the heterochromatin region. The frequency of tetrads demonstrating positive signals in two cells that are diagonally opposite one another must correspond to the frequency of recombination in the interval between the heterochromatin and the centromere. Comparison between the results of tetrad-FISH analysis and linkage maps based on RFLP markers clearly indicated that heterochromatin strongly suppresses recombination of whole chromosomal regions. We discuss the effectiveness of tetrad-FISH analysis, particularly for the localization of functional centromeres in linkage maps.

Genetic Linkage↗
PubMed2006

Synthesis of genomic and subgenomic RNAs by a membrane-bound RNA-dependent RNA polymerase isolated from oat plants infected with cereal yellow dwarf virus.

A membrane-bound RNA-dependent RNA polymerase (RdRp) complex was isolated by differential sedimentation from oat plants infected with cereal yellow dwarf virus (CYDV). When incubated with 32P-labelled UTP, unlabelled ATP, CTP and GTP, and Mg2+ ions, the RdRp preparation catalysed the synthesis of double-stranded (ds) RNAs corresponding in size to the virus genomic RNA (5.7 kbp) and two putative subgenomic RNAs (2.8 and 0.7 kbp). Hybridisation using strand-specific hybridization targets showed that the 5.7-kbp dsRNA was labelled mainly in the plus strand, whereas the 2.8- and 0.7-kbp dsRNAs were labelled only in the minus strand. Genomic-length single-stranded, plus-strand RNA of 5.7 kb and single-stranded, plus-strand subgenomic RNAs of 2.8 and 0.7 kbp were detected in RNA isolated from oat plants infected with CYDV. Mapping experiments were consistent with the genomic and subgenomic RNAs having common 3' ends, but different 5' ends, whether produced in vitro or in vivo. The RdRp-encoding region of the CYDV genome was cloned and expressed in Escherichia coli, and the purified protein was used to raise antibodies in a rabbit. In immunoblots, the antibodies detected a protein of about 68 kDa in RdRp preparations from CYDV-infected oat plants, but not from equivalent preparations from healthy oats. As far as we are aware, this is the first report of an in vitro RNA synthesis system for a phloem-limited virus.

Avena↗
PubMed2000

Complete nucleotide sequence of Northern cereal mosaic virus and its genome organization.

We determined the complete nucleotide sequence, 13, 222 nucleotides (nts) of the Northern cereal mosaic virus (NCMV). The genome had 273 nt 5' trailer sequence and 90nts 3' leader sequence. It formed a terminal complementarity in 25 nts of both terminal sequences. A characteristic intergenic sequence (consensus) separating genes, 3'-AUUCUUUUUGACUCUAGU-5' was presented. The genome had nine open reading frames (ORFs) on the viral complementary sequence. Five putative proteins of NCMV were postulated by its molecular weight or comparison of the similarities to other rhabdovirus proteins: nucleocapsid (N), non-structural protein or phosphoprotein (P), matrix protein (M), glycoprotein (G), and polymerase (L). A series of four small ORFs (genes 3 to 6) were also presented between P and M gene sequences. The proposed NCMV genome organization was 3'leader-N-P-3-4-5-6-M-G-L-5'trailer. N and L proteins of NCMV had low but distinct similarities to those of lettuce necrotic yellows virus and Sonchus yellow net virus, respectively.

Edible Grain↗
PubMed2004

The bioinformatics challenges in comparative analysis of cereal genomes-an overview.

Comparative genomic analysis is the cornerstone of in silico-based approaches to understanding biological systems and processes across cereal species, such as rice, wheat and barley, in order to identify genes of agronomic interest. The size of the genomic repositories is nearly doubling every year, and this has significant implications on the way bioinformatics analyses are carried out. In this overview the concepts and technology underpinning bioinformatics as applied to comparative genomic analysis are considered in the context of other manuscripts appearing in this issue of Functional and Integrative Genomics.

Computational Biology↗
PubMed2006

Is the cereal rust mite, Abacarus hystrix really a generalist? - Testing colonization performance on novel hosts.

The majority of eriophyoid mites are highly host specific and restricted to a narrow range of acceptable host plant species. The cereal rust mite, Abacarus hystrix was considered to be one of a few exceptions among them and has been found to be using a relatively wide host range. Since this species is a vagrant, inhabiting short-lived plants and aerially dispersing, it has commonly been considered to be a host generalist. Here the opposite hypothesis is tested, that host populations of A. hystrix are specialized on their local host plants and may represent host races. For this purpose, females from two host populations (quack grass, Agropyron repens and ryegrass, Lolium perenne) were transferred, and subsequently reared, on their normal (grass species from which females came from) and novel (other grass species) hosts. The female's fitness was assessed by survival and fecundity on the normal and novel host. Females of both populations had no success in the colonization of the novel host. They survived significantly better and had significantly higher fecundity on their normal host than on the novel one. These findings correspond with observations on host-dependent phenotype variability and host acceptance. The presence of locally specialized host populations in A. hystrix may be evidence for high host specificity among eriophyoids and the viruses they transmit. The main conclusion is that A. hystrix, which so far has been considered as a host generalist, in fact may be a complex species consisting of highly specialized host races.

Agropyron↗
PubMed2004

Vacuolar H+-translocating inorganic pyrophosphatase (Vpp1) marks partial aleurone cell fate in cereal endosperm development.

Cereal endosperm is a model system for cell fate determination in plants. In wild-type plants the outermost endosperm cells adopt aleurone cell fate, while all underlying cells display starchy endosperm cell fate. Mutant analysis showed that cell fate is determined by position rather than lineage. To further characterise the precise cell fate of the outermost cells, we performed a differential screen and isolated the novel marker gene Vpp1 . It encodes a vacuolar H+-translocating inorganic pyrophosphatase (V-PPase) and is mainly expressed in kernels, leaves and tassels. In kernels, its expression is restricted to the aleurone layer with the maximum of expression shifting from the adaxial to the abaxial side during early stages. Together with three other marker genes Vpp1 was then used to analyse the cell fate of the outermost cells in Dap3 , Dap7 , cr4 and dek1 mutants, all of which have aberrant aleurone layers. In the Dap3 and Dap7 mutants the Vpp1 and Ltp2 markers but not the A1 and Zein markers were expressed in patches without aleurone indicating that the outermost cells had some but not all features of aleurone cells and did not simply adopt starchy endosperm cell fate. A similar result was obtained in the cr4 mutant, although Ltp2 expression was less generalised. In other Dap7 patches characterised by multiple aleurone-like cell layers the expression of Vpp1 and Ltp2 confirmed the aleurone cell fate of the cells in the additional cell layers. The analysis of dek1 mutants confirmed the starchy endosperm cell fate of the majority but not all outermost cells. Based on these data we propose a model suggesting a stepwise commitment to aleurone cell fate. Sequential steps are marked by the expression of Vpp1 , the expression of Ltp2 , the acquisition of a regular shape and thick walls and finally pigmentation coupled with A1 expression.

Cell Lineage↗
PubMed2005

Wheat containing snowdrop lectin (GNA) does not affect infection of the cereal aphid Metopolophium dirhodum by the fungal natural enemy Pandora neoaphidis.

Studies were carried out to determine if susceptibility of the cereal aphid Metopolophium dirhodum to the fungus Pandora neoaphidis was affected by wheat expressing snowdrop lectin (GNA). Aphid infection did not differ significantly between the transgenic GNA and non-transformed lines (91 and 82%, respectively). Fecundity also did not differ between aphids on the two lines, and was ca. 18 nymphs adult(-1). Time to infection was ca. 5 days for M. dirhodum on both lines in two of three assays. Our results indicate that wheat expressing GNA would not compromise the efficacy of P. neoaphidis as a biocontrol agent.

Animals↗
PubMed1986

Determination of phosphorus in cereal lipids.

The effect of digestion methods on the determination of phosphorus in cereal lipids was reinvestigated. Samples were either digested with sulfuric acid or ashed in a muffle furnace at 600 degrees C. The standard deviation and the coefficient of variation were significantly higher for the acid-digested samples. Ashing gave more reliable results, especially when large amounts of lipid material had to be oxidized.

Edible Grain↗