Kis molekulatömegű polipeptidek hatása a polimer glutenin polimerizációs fok szerinti eloszlására és a sikérkomplex reológiai sajátságaira = The effect of low molecular weight polypeptides on the polymerization degree distribution of polymeric glutenin

A búza sikéralkotó fehérjéinek a bioszintézisét tanulmányoztuk a búzaszem fejlődése során a virágzástól számított 12. naptól kezdve a szemek teljes beéréséig. Öt magyarországi termestésű búzafajtát vontunk be a kísérletbe és három évjárat termésének a vizsgálatára került sor. A búza termesztése részben kísérleti gazdaságokban (Martonvásár, Monor) , részben üzemi buzatermelőknél (Mezőterm, Lórév). Fontosabb megállapításainkat a következőkben foglaljk össze. Bár az egy szemre számított teljes fehérjetartalom növekedését általában szigmoid jellegű görbe jellemzi, a termelési feltéelek hatására jelentő eltérések léphetnek fel. Az egyes fehérjék szintézisének sorrendje és a transzláció sebessége jelentősen változik. A metabolikusan aktív fehérjék szintézisét követően már a korai fejlődési szkszban megindul a sikéralkotó polpeptidek szintézise gliadinok, LMW, HMW peptidek sorrendben. A glutenin alegységek polimerizációja csak egy kritikus koncentráció elérés után indul meg. A sikér kialakulás megkezdődésének még további feltétele a glutenin meghatározott átlagos polimerizációs fokának az elérése. A sikéroldatok relatív viszkozitásának a követése alkalmasnak bizonyult a polimerizációs folyamat követésér. A vizsgálati adatok megerősíették a glutenin polimer linearis jellegét. | The biosynthesis of gluten forming polypeptides was studied starting from 12-th day after anthesis till full ripeness of wheat kernels. Five wheat cultivars, grown in Hungary were used in experiments and three years crops were studied. The wheat was grown partly in experimental stations (Martonvásár, Monor) and partly in commercial wheat producing farms (Mezőterm, Lórév). The most important statements of research are the following ones: Although the total protein content (calculated on single kernel) may be characterized by a sigmoid curve, the growing conditions may sinificantly change its form. The sequence and rate of translation of individual proteins is diffrent. After synthesis of metabolically active proteins, even in early phase of kernel development starts the synthesis of gluten forming peptides, in an order: gliadins, LMW- and HMW polypeptides. The polymerization of glutenin subunits starts only after the concntraton of subunits reches a critical concentration. The prerequisite of gluten formation is n addition a critical average polymerization level of polymeric glutenin. The follow of the relative viscosity of gluten solution was suitable for the control of growing average polymerization degree of glutenin. The experimetal data of viscosity measurements and their evaluation confirmed the linear character of molecules of polymeric glutenin

Búza tartalékfehérjék funkcionális tulajdonságainak tanulmányozása modell rendszerben = Studying functional properties of wheat storage proteins in a model system

A pályázat célja egy olyan új modell rendszer kidolgozása volt, melyben tanulmányozni tudjuk a búza fehérjék kölcsönhatását a sikér kialakulás során, illetve a búza tartalékfehérjék hatását a tészta minőségére. A választott modell a rizs volt. A modell rendszerben történő vizsgálatnak két eleme volt: tészta rekonstrukciós kísérletek (in vitro), illetve a rizs genetikai transzformációja (in vivo). Kidolgoztunk, illetve adaptáltuk egy a rizsliszt fehérje-összetételének és a rizstészta reológiai paramétereinek meghatározására alkalmas módszert (z-arm mixer), valamit a búza tartalékfehérjék rizsben történő vizsgálatához szükséges redukciós/oxidációs módszert. Vitális glutént, valamit egyes tisztított tartalékfehérje frakciókat (LMW és HMW glutenin gazdag frakciókat), baktériumban expresszált és tisztított glutenin alegység fehérjéket vizsgáltuk in vitro adagolásával és inkorporációval. Az inkorporált fehérjék szignifikáns hatással voltak a rizstészta funkcionális tulajdonságaira. Létrehoztunk és analizáltunk egy HMW glutenin alegységet jelentős mennyiségben expresszáló transzgenikus rizs vonalat. Az így megváltozott fehérje-összetétel jelentős hatással volt a rizstészta funkcionális tulajdonságaira, a tészta erősségére és stabilitására. Az általunk kidolgozott modell újat jelent abban, hogy a búza tartalékfehérjék tulajdonságainak, a tészta szerkezetére gyakorolt hatásának olyan elemei is vizsgálhatók, melyekre korábban nem volt lehetőség az endogén búzafehérjék hatása miatt. | The aim of our work was to study, on one hand the role of the protein interactions during gluten formation and, on the other hand the effects of the wheat storage proteins on the dough quality by in vitro (dough reconstitution) and by in vivo (transformation) methods. To achieve this goal rice was used as a model system. The methods developed for the characterization of wheat dough (micro-z-arm mixer) were adopted to determine the mixing properties of rice flour. A reduction/oxidation procedure was developed to incorporate glutenin subunit proteins into the polymeric protein structure of rice dough. Wheat gluten or its components, such as HMW and LMW wheat glutenin rich fractions, were incorporated in in vitro studies. Results showed that the in vitro incorporated gluten had significant effects on the functional properties of rice dough. Transgenic rice lines were produced and analysed in vivo. Substantial amounts of wheat HMW glutenin subunit (HMW-GS) was expressed in the endosperm of the transgenic rice seed. This alteration had remarkable effects on the functional properties, including dough strength and stability, of the transgenic rice flour. Our results demonstrated the potential of rice flour as a model system in wheat storage proteins structure-function studies, to determine the mechanism and the specificity of disulphide bond formation, furthermore the role of the protein interactions in wheat gluten formation

Improved reactive aldehyde, salt and cadmium tolerance of transgenic barley due to the expression of aldo–keto reductase genes

Under various stress conditions, plant cells are exposed to oxidative damage which triggers lipid peroxidation. Lipid peroxide breakdown products include protein crosslinking reactive aldehydes. These are highly damaging to living cells. Stress-protective aldo–keto reductase (AKR) enzymes are able to recognise and modify these molecules, reducing their toxicity. AKRs not only modify reactive aldehydes but may synthesize osmoprotective sugar alcohols as well. The role of these mixed function enzymes was investigated under direct reactive aldehyde, heavy metal and salt stress conditions. Transgenic barley (Hordeum vulgare L.) plants constitutively expressing AKR enzymes derived from either thale cress (Arabidopsis thaliana) (AKR4C9) or alfalfa (Medicago sativa) (MsALR) were studied. Not only AKR4C9 but MsALR expressing plants were also found to produce more sorbitol than the non-transgenic (WT) barley. Salinity tolerance of genetically modified (GM) plants improved, presumably as a consequence of the enhanced sorbitol content. The MsALR enzyme expressing line (called 51) exhibited almost no symptoms of salt stress. Furthermore, both transgenes were shown to increase reactive aldehyde (glutaraldehyde) tolerance. Transgenic plants also exhibited better cadmium tolerance compared to WT, which was considered to be an effect of the reduction of reactive aldehyde molecules. Transgenic barley expressing either thale cress or alfalfa derived enzyme showed improved heavy metal and salt tolerance. Both can be explained by higher detoxifying and sugar alcohol producing activity. Based on the presented data, we consider AKRs as very effective stress-protective enzymes and their genes provide promising tools in the improvement of crops through gene technology

Nanotubes connecting B lymphocytes: High impact of differentiation-dependent lipid composition on their growth and mechanics

Nanotubes (NTs) are thin, long membranous structures forming novel, yet poorly known communication pathways between various cell types. Key mechanisms controlling their growth still remained poorly understood. Since NT-forming capacity of immature and mature B cells was found largely different, we investigated how lipid composition and molecular order of the membrane affect NT-formation. Screening B cell lines with various differentiation stages revealed that NT-growth linearly correlates with membrane ganglioside levels, while it shows maximum as a function of cholesterol level. NT-growth of B lymphocytes is promoted by raftophilic phosphatidylcholine and sphingomyelin species, various glycosphingolipids, and docosahexaenoic acid-containing inner leaflet lipids, through supporting membrane curvature, as demonstrated by comparative lipidomic analysis of mature versus immature B cell membranes. Targeted modification of membrane cholesterol and sphingolipid levels altered NT-forming capacity confirming these findings, and also highlighted that the actual lipid raft number may control NT-growth via defining the number of membrane-F-actin coupling sites. Atomic force microscopic mechano-manipulation experiments further proved that mechanical properties (elasticity or bending stiffness) of B cell NTs also depend on the actual membrane lipid composition. Data presented here highlight importance of the lipid side in controlling intercellular, nanotubular, regulatory communications in the immune system

Role of rice and added wheat protein in the mixing properties of different rice flours - (a preliminary study)

Structure and composition of wheat storage proteins and functional properties of wheat gluten are well studied; therefore several methods and instruments are available to determine these properties. The investigation of functional properties of rice proteins, depending on the different goal of utilisation, has not been well established, yet. In this study, the theological properties of four varieties of rice flour were studied using a 50 g Farinograph demonstrating the mixing properties of rice flours and the alterations of these properties caused by the supplementation of wheat proteins. The considerable differences identified on the mixing curves of different rice flours indicate that the investigation of mixing properties can be one of the useful approaches for the characterisation of functional properties of rice dough. The large effects of the addition of wheat gluten on the mixing properties of rice flours demonstrate the possibility of using rice flour and dough as a wheat protein free model system for the in vitro investigation of the functional roles of wheat storage proteins

Synthesis and assembly of Escherichia coli heat-labile enterotoxin B subunit in transgenic rice (Oryza sativa L.)

Escherichia coli heat-labile enterotoxin B subunit (LTB) can be used as a potent mucosal immunogen and immunoadjuvant for co-administered antigens. The synthetic LTB (sLTB) was modified based on plant optimized codon usage, and fused to a translation signal (the Kozak sequence) in the front of start codon and the ER retention signal, SEKDEL, in the c-terminus of sLTB gene. The sLTB and the wild-type LTB gene (wLTB) were located into plant expression vectors under the control of the wheat Bx17 HMW (High Molecular Weight) glutenin endosperm-specific promoter containing the first intron of the rice actin 1 gene. Both genes were introduced into rice cells (Oryza sativa L.) via particle bombardment mediated transformation. The integration of LTB gene into the chromosome of transgenic plants was confirmed by genomic DNA PCR amplification methods. The transcription and translation of the LTB genes were demonstrated by reverse-transcription PCR (RT-PCR) and Western blot analyses, respectively. The LTB proteins produced in the seed tissues of transgenic rice showed binding affinity for Gm, ganglioside, a receptor for biologically active LTB, suggesting the plant-produced LTB are capable of forming active pentamers. The expression level of sLTB was higher than wLTB in transgenic rice plants and was up to 2.7% of the total soluble proteins of the seed tissues. (C) KSBB

Characterization of rice storage proteins by SE-HPLC and micro z-arm mixer

While the effect of protein content and composition on the functional properties of wheat flour is well studied, our knowledge on the same properties of rice flour is limited. This work was conducted to study the relationship between the dough mixing properties of flour from different rice cultivars and protein content and composition. An efficient sonication-based two-step extraction procedure was applied to isolate rice flour proteins. The size-exclusion HPLC (SE-HPLC) method, originally developed for separating wheat proteins, was applied with some minor modifications in order to study the size distribution of rice flour proteins. Four fractions were distinguished on the SE-HPLC profile and were further characterized by SDS-PAGE. Fractions I-III consisted of glutelins, while fraction IV contained albumin, globulin and prolamin proteins. When rice dough was characterized on the basis of mixing parameters in a micro z-arm mixer, significant differences were observed depending on the protein composition of the flour. Statistical analysis results indicated that the functional properties of the flour from different rice cultivars were associated with the amount of polymeric proteins and their size distribution. (c) 2007 Elsevier Ltd. All rights reserved

Research Methods in Plant Biology

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