László Tamás

Bánkúti 1201--an old Hungarian wheat variety with special storage protein composition.

Abstract Bánkúti 1201, an old Hungarian wheat variety with special quality traits, was analysed to determine the relationships between its storage protein composition and superior quality-attributes for breadmaking. Based on the storage protein composition, the variety appears to have the nature of a population, containing several genotypes with different gluten protein alleles. Using molecular markers, a new mutant x-type HMW glutenin allele was identified, containing an extra cysteine residue and showing a moderate, positive-effect on gluten properties. In lines possessing subunits Bx7+By8 the overexpression of the Bx-type subunit could be detected, resulting in a higher unextractable polymeric protein (UPP) content and increased dough strength. It was found that the presence or absence of subunit Bx7 has an equilibrating effect on the dough extensibility, which is generally characteristic of the Bánkúti 1201 population. The complex good bread-making quality of the variety, which has strong but highly extensible dough, is probably due to the balance between lines which express subunit Bx7 and those which do not.

Expression of a synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of Escherichia coli heat labile enterotoxin in rice endosperm.

Epitopes often require co-delivery with adjuvant and targeting proteins to enable recognition by the immune system, and this approach may also increase the efficacy of the antigen. In this study, we assess and describe the ability of transgenic rice plants to express a fusion protein consisting of the B-subunit of the Escherichia coli heat-labile enterotoxin (LTB) and a synthetic core-neutralizing epitope (COE) of porcine epidemic diarrhea virus (PEDV), inducing an enteric disease that is seen most predominantly in piglets. Both components of the fusion proteins were detected with Western blot analysis. The fusion protein was determined to assemble into pentamers, as was evidenced by its ability to bind to GM1 gangliosides, and evidenced an average level of expression in a trangenic rice endosperm. This indicates that the expression system of the plant is capable of generating a sizable amount of antigen, possibly allowing for the successful development of an edible vaccine.

Expression of cholera toxin B subunit in transgenic rice endosperm.

The synthetic cholera toxin B subunit (CTB) gene, modified according to the optimized codon usage of plant genes, was introduced into a plant expression vector and expressed under the control of the Bx17 HMW (high molecular weight) wheat endosperm-specific promoter containing an intron of the rice act1. The recombinant vector was transformed into rice plants using a biolistic-mediated transformation method. Stable integration of the synthetic CTB gene into the chromosomal DNA was confirmed by PCR amplification analysis. A high level of CTB (2.1% of total soluble protein) was expressed in the endosperm tissue of the transgenic rice plants. The synthetic CTB produced only in the rice endosperm demonstrated strong affinity for GM1-ganglioside, thereby suggesting that the CTB subunits formed an active pentamer. The successful expression of CTB genes in transgenic plants makes it a powerful tool for the development of a plant-derived edible vaccine.

Transgenic approach to improve wheat (Triticum aestivum L.) nutritional quality

An amaranth (Amaranthus hypochondriacus) albumin gene, encoding the 35-kDa AmA1 protein of the seed, with a high content of essential amino acids, was used in the biolistic transformation of bread wheat (Triticum aestivum L.) variety Cadenza. The transformation cassette carried the ama1 gene under the control of a powerful wheat endosperm-specific promoter (1Bx17 HMW-GS). Southern-blot analysis of T1 lines confirmed the integration of the foreign gene, while RT-PCR and Western-blot analyses of the samples confirmed the transcription and translation of the transgene. The effects of the extra albumin protein on the properties of flour, produced from bulked T2 seeds, were calculated using total protein and essential amino acid content analysis, polymeric/monomeric protein and HMW/LMW glutenin subunit ratio measurements. The results indicated that not only can essential amino acid content be increased, but some parameters associated with functional quality may also be improved because of the expression of the AmA1 protein.

Structure, assembly and targeting of wheat storage proteins

The functional properties of wheat flour in various food systems (most notably for breadmaking) are determined largely by the structures and interactions of the prolamin storage proteins, termed gliadins and glutenins. It is therefore important to understand the structures of the individual proteins, their pathways and mechanisms of synthesis and deposition, and their interactions with other flour components (for example, starch and lipids) in order to manipulate the quality of wheat for food and other uses, Me are using a range of biophysical and biochemical approaches to determine the structures of individual proteins, using fractions purified from flour or expressed in heterologous systems. We are also isolating cDNAs for proteins involved in protein folding and assembly in the endoplasmic reticulum (protein disulphide isomerase, BiP and peptidyl-proly cis-trans isomerase) and studying their interactions with gluten proteins synthesised in E. coli. This work is complemented by immunocytochemical studies of gluten protein deposition in the leaves and seeds of transgenic tobacco plants, using wild-type proteins and mutants in which specific transit-targeting sequences have been added or deleted. Taken together these results are giving a detailed picture of the structure, synthesis, assembly and deposition of wheat gluten proteins.

Stability of sunflower 2S albumins and LTP to physiologically relevant in vitro gastrointestinal digestion

In order for a protein to elicit a systemic allergic response it must reach the circulatory system through the intestinal mucosa as a sufficiently large fragment with adequate structural integrity. Sunflower LTP and 2S albumins (SFA8 and three mixed fractions of Alb1 and Alb2) were digested in simulated gastric fluid (SGF) for 2h and the conditions were then changed to mimic the intestinal environment for a further 2h digestion. The effects of phosphatidylcholine (PC) and emulsification on the digestibility of the proteins were investigated. PC protected all of the proteins studied against both gastric and intestinal digestive enzymes but to different extents. Emulsification of SFA8 resulted in strong protection against digestion, which was further enhanced by the presence of PC in the SGF. These results highlight the importance of considering real food structures such as emulsified systems and also the gastrointestinal environment that proteins are exposed to once consumed when assessing allergenicity.

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.

Structural stability and surface activity of sunflower 2S albumins and nonspecific lipid transfer protein

The structural and interfacial properties of five different fractions of sunflower ( Helianthus annuus L.) seed storage proteins were studied. The fractions comprised lipid transfer protein (LTP), the methionine-rich 2S albumin SFA8 (sunflower albumin 8), and three mixtures of non-methionine-rich 2S albumins called Alb1 and Alb2 proteins (sunflower albumins 1 and 2). Heating affected all of the proteins studied, with SFA8 and LTP becoming more surface active than the native proteins after heating and cooling. LTP appeared to be less thermostable than homologous LTPs from other plant species. SFA8 generated the greatest elastic modulus and formed the most stable emulsions, whereas LTP showed poorer emulsification properties. The mixed 2S albumin fractions showed moderate levels of surface activity but had the poorest emulsification properties among the proteins studied.

Housekeeping gene selection in poplar plants under Cd-stress: comparative study for real-time PCR normalisation

Real-time RT-PCR is currently the most sensitive, specific and precise approach to analyse gene expression changes in plant stress studies. The determination of biologically meaningful transcript quantities requires accurate normalisation of the raw data. During relative quantification the reliability of the results depends on the stable expression of the endogenous control genes across the experimental samples. Four widely used internal control genes (cyclophilin, elongation factor 1α, polyubiquitin, tubulin β-chain) and two potential candidates (serine/threonine-protein phosphatase 2A and ubiquitin-conjugating enzyme) genes were assessed under Cd-stress and at different developmental stages in leaves of Populus jacquemontiana D. var. glauca H. Complementary DNA (RiboGreen) based quantification method revealed variations in the expression level of reference genes. The variability was more pronounced under severe stress conditions. Less variation was observed in the case of ef-1α, pp2a and ubc10. Transcript level changes of a target gene, psa-h, was also evaluated by two independent normalisation strategies, by the RiboGreen method or by using multiple references. The impact of variability of reference gene on the target gene evaluation was demonstrated. It was proved that in the absence of suitable housekeeping genes, for example under severe stress, RiboGreen method is convenient tool for transcript normalisation.

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.