Angéla Juhász

Smoke-water-induced changes of expression pattern in Grand Rapids lettuce achenes.

Aerosol smoke and smoke-water can break dormancy and promote seed germination of many plant species. In this study we investigated changes in gene expression after imbibition of light-sensitive Lactuca sativa L. cv. ‘Grand Rapids’ achenes with dilute smoke-water compared to water control samples kept in the dark or continuous light, using the fluorescent differential display technique. Although no difference was detected in the smoke-water versus water control samples germinated in light, smoke-water treatment resulted in the differential display of several expressed sequence tags (ESTs) when compared to water control samples kept in the dark. The most pronounced fragments isolated correspond to known genes related to germination, with functions in cell wall expansion, regulation of translation, the cell division cycle, carbohydrate metabolism and abscisic acid (ABA) regulation. Real-time polymerase chain reaction (PCR) validation revealed that the transcript abundance of the genes, HVA22 , short-chain dehydrogenase/reductase and late embryogenesis abundant protein, are upregulated after smoke treatment when compared to control achenes kept in the light. The results indicate that smoke has a dual effect. On the one hand, the smoke can induce genes that may be linked to ABA action, whereas, on the other hand, it elicits a faster germination rate by inducing a similar pattern in gene expression as light treatment. Smoke effects could be manifested mainly through the induction of the cell division cycle, cell wall extension and storage mobilization.

Role of Conserved Non-Coding Regulatory Elements in LMW Glutenin Gene Expression

Transcriptional regulation of LMW glutenin genes were investigated in-silico, using publicly available gene sequences and expression data. Genes were grouped into different LMW glutenin types and their promoter profiles were determined using cis-acting regulatory elements databases and published results. The various cis-acting elements belong to some conserved non-coding regulatory regions (CREs) and might act in two different ways. There are elements, such as GCN4 motifs found in the long endosperm box that could serve as key factors in tissue-specific expression. Some other elements, such as the AACA/TA motifs or the individual prolamin box variants, might modulate the level of expression. Based on the promoter sequences and expression characteristic LMW glutenin genes might be transcribed following two different mechanisms. Most of the s- and i-type genes show a continuously increasing expression pattern. The m-type genes, however, demonstrate normal distribution in their expression profiles. Differences observed in their expression could be related to the differences found in their promoter sequences. Polymorphisms in the number and combination of cis-acting elements in their promoter regions can be of crucial importance in the diverse levels of production of single LMW glutenin gene types.

Identification, Cloning and Characterisation of a HMW-Glutenin Gene from an Old Hungarian Wheat Variety, Bánkúti 1201

Despite its good functional properties, the varietyBánkúti 1201 has been found to possess 2 + 12 or 3 + 12 allelecomposition on chromosome 1D. In order to determine the reason for itsquality traits a gene-specific PCR technique was applied in preliminaryexperiments to examine the HMW glutenin allele composition of thevariety. In the course of the analysis a fragment characteristic ofBánkúti 1201 was identified and the nucleotide sequence wasdetermined. This showed the presence of a 1Ax2* gene variantwhich, despite near homology, differed from the original 1Ax2* geneat one important point. At 1181 bp of the 1Ax2* sequencenucleotide exchange was observed which is the middle nucleotide of theTCT–TGT base triplet, involving the exchange of serine for cysteine. Thegene was designated 1Ax2*B. The presence of an extrasulphydryl group, like that of the extra cysteine in the 1Dx5 gene,facilitates the formation of further disulphide bonds, might lead to animprovement in gluten quality characters.

The epitopes in wheat proteins for defining toxic units relevant to human health

Wheat-related disorders are well-studied health problems. Knowledge of the composition and amounts of epitopes present in a single wheat sample represents a significant gap, and the detailed wheat proteome datasets now available can provide the necessary information to carry out an estimation of allergen prediction for a single cultivar. The combined use of genome sequence and allergen databases, prediction methodology, and cereal chemistry results in better understanding of the level of toxicity present in the end-products produced from wheat flour. The workflow presented in this review provides information about the number and distribution of epitopes at single protein, or protein fraction, levels. In addition, epitopes present in the highest frequency and harmful proteins expressed in the highest amount can be identified. The “epitope toxicity” value obtained in this way is a significant research output from the analysis of large datasets that can be applied to the food industry.

ProPepper: a curated database for identification and analysis of peptide and immune-responsive epitope composition of cereal grain protein families.

ProPepper is a database that contains prolamin proteins identified from true grasses (Poaceae), their peptides obtained with single- and multi-enzyme in silico digestions as well as linear T- and B-cell-specific epitopes that are responsible for wheat-related food disorders. The integrated database and analysis platform contains datasets that are collected from multiple public databases (UniprotKB, IEDB, NCBI GenBank), manually curated and annotated, and interpreted in three main data tables: Protein-, Peptide- and Epitope list views that are cross-connected by unique identifications. Altogether 21 genera and 80 different species are represented. Currently, the database contains 2146 unique and complete protein sequences related to 2618 GenBank entries and 35 657 unique peptide sequences that are a result of 575 110 unique digestion events obtained by in silico digestion methods involving six proteolytic enzymes and their combinations. The interface allows advanced global and parametric search functions along with a download option, with direct connections to the relevant public databases. Database URL: https://propepper.net

Celiac disease-specific prolamin peptide content of wheat relatives and wild species determined by ELISA assays and bioinformatics analyses

Enzyme-linked immunosorbent assays (ELISAs) are widely used to determine gluten contamination in gluten-free and low gluten food samples. ELISA assays developed using monoclonal antibodies against known toxic peptides have an advantage in the identification of toxic prolamin content in protein extracts of different food samples, as well as raw materials. R5 and G12 monoclonal antibodies specific for two known toxic peptides used in commercially available gluten ELISA assays were applied to test toxic peptide contents in wheat relatives and wild wheat species with different genome composition and complexity. Although the R5 peptide content showed some correlation with ploidy levels in Triticum species, there was a high variance among Aegilops species. Some of the analysed diploid Aegilops species showed extremely high R5 peptide contents. Based on the bioinformatics analyses, the R5 peptide was present in most of the sulphur rich prolamins in all the analysed species, whereas the G12 epitope was exclusivel...

Brachypodium distachyon as a model for defining the allergen potential of non-prolamin proteins

Epitope databases and the protein sequences of published plant genomes are suitable to identify some of the proteins causing food allergies and sensitivities. Brachypodium distachyon, a diploid wild grass with a sequenced genome and low prolamin content, is the closest relative of the allergen cereals, such as wheat or barley. Using the Brachypodium genome sequence, a workflow has been developed to identify potentially harmful proteins which may cause either celiac disease or wheat allergy-related symptoms. Seed tissue-specific expression of the potential allergens has been determined, and intact epitopes following an in silico digestion with several endopeptidases have been identified. Molecular function of allergen proteins has been evaluated using Gene Ontology terms. Biologically overrepresented proteins and potentially allergen protein families have been identified.

Impact of mid-season sulphur deficiency on wheat nitrogen metabolism and biosynthesis of grain protein

Wheat (Triticum aestivum) quality is mainly determined by grain storage protein compositions. Sulphur availability is essential for the biosynthesis of the main wheat storage proteins. In this study, the impact of different sulphur fertilizer regimes on a range of agronomically important traits and associated gene networks was studied. High-performance liquid chromatography was used to analyse the protein compositions of grains grown under four different sulphur treatments. Results revealed that sulphur supplementation had a significant effect on grain yield, harvest index, and storage protein compositions. Consequently, two comparative sulphur fertilizer treatments (0 and 30 kg ha−1 sulphur, with 50 kg ha−1 nitrogen) at seven days post-anthesis were selected for a transcriptomics analysis to screen for differentially expressed genes (DEGs) involved in the regulation of sulphur metabolic pathways. The International Wheat Genome Sequencing Consortium chromosome survey sequence was used as reference. Higher sulphur supply led to one up-regulated DEG and sixty-three down-regulated DEGs. Gene ontology enrichment showed that four down-regulated DEGs were significantly enriched in nitrogen metabolic pathway related annotation, three of which were annotated as glutamine synthetase. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment identified three significantly enriched pathways involved in nitrogen and amino acid metabolism.

Multiple elements controlling the expression of wheat high molecular weight glutenin paralogs.

Analysis of gene expression data generated by high-throughput microarray transcript profiling experiments coupled with cis-regulatory elements enrichment study and cluster analysis can be used to define modular gene programs and regulatory networks. Unfortunately, the high molecular weight glutenin subunits of wheat (Triticum aestivum) are more similar than microarray data alone would allow to distinguish between the three homoeologous gene pairs. However, combining complementary DNA (cDNA) expression libraries with microarray data, a co-expressional network was built that highlighted the hidden differences between these highly similar genes. Duplex clusters of cis-regulatory elements were used to focus the co-expressional network of transcription factors to the putative regulatory network of Glu-1 genes. The focused network helped to identify several transcriptional gene programs in the endosperm. Many of these programs demonstrated a conserved temporal pattern across the studied genotypes; however, few others showed variance. Based on this network, transient gene expression assays were performed with mutated promoters to inspect the control of tissue specificity. Results indicated that the interactions of the ABRE│CBF cluster with distal promoter regions may have a dual role in regulation by both recruiting the transcription complex as well as suppressing it in non-endosperm tissue. A putative model of regulation is discussed.

Distinct regulatory modules identified in the promoters of wheat Glu-1 genes suggest different regulatory mechanisms

High molecular weight glutenin subunits of wheat are economically important seed storage proteins. They are coded by paralog pairs of the Glu-1 gene on each of the three genomes in the hexaploid wheat. Their expressions are under both temporal and spatial control. Many factors have been identified that influence the activity of Glu-1 genes, but the underlying regulatory mechanisms are still unclear. In order to identify motifs and motif clusters responsible for quantitative regulation of Glu-1 gene expressions, promoter profiles and transcription dynamics of the genes were analysed. It was found that promoter motif compositions of homoeolog Glu-1 genes are conserved. Our results demonstrated that while promoter profiles explain the differences of expression between homoeologs and between paralogs, it does not explain the variation of activity between alleles. Interestingly, our analyses revealed that the promoters of Glu-1 genes are divided into six cis-regulatory modules that are either locally overrepresented by binding sites belonging to unique but distinct transcription factor (TF) families or have conserved motif clusters. Moreover, our analyses demonstrated that the varying expression dynamics of TFs across genotypes is likely to be the primary contributor of the allelic variation of Glu-1 gene expressions. Thus, the six putative cis-regulatory modules in the Glu-1 gene promoters bound by the differentially expressed TFs are suggested to play a key role in the quantitative and tissue specific regulation of these genes.