Storage Protein Composition Research Articles

The redistribution of protein sulfur in transgenic rice expressing a gene for a foreign, sulfur-rich protein.

Sulfur amino acid composition is an important determinant of seed protein quality. A chimeric gene encoding sunflower seed albumin (SSA), one of the most sulfur-rich seed storage proteins identified so far, was introduced into rice (Oryza sativa) in order to modify cysteine and methionine content of the seed. Analysis of a transgenic line expressing SSA at approximately 7% of total seed protein revealed that the mature grain showed little change in the total sulfur amino acid content compared to the parental genotype. This result indicated that the transgenic rice grain was unable to respond to the added demand for cysteine and methionine imposed by the production of SSA. Analysis of the protein composition of the transgenic grain showed changes in the relative levels of the major seed storage proteins, as well as some non-storage proteins, compared to non-transgenic controls. Changes observed at the protein level were concomitant with differences in mRNA accumulation but not always with the level of transcription. The limited sulfur reserves appeared to be re-allocated from endogenous proteins to the new sulfur sink in the transgenic grain. We hypothesize that this response is mediated by a signal transduction pathway that normally modulates seed storage protein composition in response to environmental fluctuations in sulfur availability, via both transcriptional and post-transcriptional control of gene expression.

Distribution of Storage Proteins in Low-Glutelin Rice Seed Determined Using a Fluorescent Antibody

To compare the distribution of storage proteins in low-glutelin rice seed with that in other cultivars having normal protein compositions, immunofluorescence labeling with specific antibodies was applied to visualize the distribution of storage proteins in endosperm tissues. The endosperm tissues from five cultivars were reacted with anti-prolamin and anti-glutelin antibodies, and then observed by light microscopy and confocal laser scanning microscopy (CLSM). In low-glutelin rice, using microscopic analysis, a large proportion of storage proteins was observed in the endosperm tissue of 70% polished rice. To determine the localization of two types of protein bodies in endosperm tissues, images of the distribution of the type I protein body (PB-I) and the type II protein body (PB-II) were obtained by CLSM. The CLSM images showed that, in low-glutelin rice, prolamin which accumulates in PB-I remains in the center of 70% polished rice grains despite the elimination of 30% of the outer layer of brown rice grains. However, the other cultivars mostly contained glutelin which accumulates in PB-II and is distributed throughout the endosperm tissues. This shows that low-glutelin rice differs from the other cultivars not only in the major storage protein composition but also in the distribution of storage proteins in endosperm tissues.

Expression and localization of human lysozyme in the endosperm of transgenic rice.

In order to understand the characteristics of recombinant protein expression and sublocalization in rice ( Oryza sativa L.) endosperm, we examined the expression level of human lysozyme protein and its subcellular location in transgenic rice seeds driven by rice glutelin and globulin promoters and signal peptides. A time course of human lysozyme expression during endosperm development was analyzed. The results showed that the expression profile of recombinant protein accumulation in endosperm paralleled that of the two storage proteins. Immunofluorescence microscopy revealed that human lysozyme and storage proteins co-localized to type-II protein bodies. Both promoter-signal peptide parings targeted recombinant protein to the protein bodies. In addition, a transgenic line with a higher lysozyme expression level exhibited morphologically different protein bodies with an unbalanced composition of lysozyme and native storage proteins. The high-level expression of recombinant protein distorted the trafficking and sorting of native storage proteins in rice endosperm and affected the expression of native storage protein.

Seasonal effects on storage proteins and gluten strength in four Swedish wheat cultivars

AbstractWeather variations in Sweden result in differences in the bread‐making quality of wheat. This study investigated whether the variation in bread‐making quality caused by yearly weather fluctuations could be explained by variation in protein composition, amount of storage proteins, protein subunits and protein groups, and relative amount and size distribution of polymeric proteins. Four spring wheat (Triticum aestivum L) cultivars grown in Sweden during three different years were investigated. Bread‐making quality and gluten strength were measured using baking and glutograph tests. SDS‐PAGE, SE‐HPLC, RP‐HPLC and ELISA were applied for investigation of protein composition, amount of storage proteins, protein subunits and protein groups, and relative amount and size distribution of polymeric proteins. The bread volume within cultivars varied depending on the cultivation year. The highest gluten strength was found in 1994 and the weakest in 1991. Variation in composition or relative amount of specific storage proteins, protein subunits or protein groups could not explain the variation in gluten strength between years. Instead, a significant relationship was found between the cultivation year and the percentage of unextractable polymeric protein in the total polymeric protein. The percentage of unextractable polymeric protein in the total polymeric protein was found to be highest during years when gluten strength was also high.© 2002 Society of Chemical Industry

Relationships Between Barley Hordeins and Malting Quality in a Mutant of cv. Triumph I. Genotype by Environment Interaction of Hordein Content

The genetic (G), environmental (E) and G×E effects on total grain protein and hordein fractions, have been studied in a mutant (TL43) and its parent cultivar, the malting barley Triumph, in various seasons at Dundee (E Scotland) and Lleida (NE Spain). The grain protein content of TL43 was consistently about 2% higher than that of Triumph across environments. Differences in hordein composition were shown by SDS-PAGE electrophoresis. TL-43 had three B-hordein bands not present in Triumph which had a further three bands of the same hordein group not present in TL43. TL43 showed higher B-hordein content than Triumph in Scotland but lower in Spain, whereas the mutant showed consistently higher C- and D-hordein content than Triumph across environments, i.e. there was crossover G×E interaction for B-hordein and non-crossover for C- and D-hordein content. There were also differences in grain ultra-structure between the two lines, as TL43 showed a more dense protein matrix than Triumph, together with thinner pericarp, testa and aleurone layers. It was concluded that the mutation in TL43 had a significant effect on storage protein composition.

Evaluation of chemical and technological characteristics of new lines of Triticum turgidum ssp dicoccum.

Triticum durum ssp dicoccum also called farro in Italy is gaining more and more interest from farmer due to its commercial potentiality. In this paper, thirty lines F6 of farro obtained by crossing the cvs. Triticum turgidum ssp durum Ofanto and Simeto and T. turgidum ssp dicoccum Molise population were studied. They were evaluated with special reference to chemical composition and technological characteristics in order to select genotypes suitable for the production of food commodities. The storage protein composition (High Molecular Weight and Low Molecular Weight Glutenin) was also considered by different electrophoretic techniques.

Effects of Wheat Cultivar and Nitrogen Application on Storage Protein Composition and Breadmaking Quality

ABSTRACTInfluences of cultivar and nitrogen application on protein concentration and composition, and amount and size‐distribution of different protein components, were investigated in 10 spring wheat cultivars (Triticum aestivum L.) with widely varying gluten strength, grown under four nitrogen fertilizer conditions. The results showed that cultivar differences in gluten strength were determined by storage protein composition, differences in total amount of HMW glutenin subunits, the glutenin‐to‐gliadin ratio, and the relationship between SDS‐soluble and SDS‐insoluble protein polymers. Negative correlations were found between protein parameters related to gluten strength and bread volume. No cultivar stability for gluten strength in relation to differences in nitrogen application was found. Thus, the gluten strength was influenced by the nitrogen application in all the investigated cultivars. Increased nitrogen supply correlated significantly to an increase in all protein components containing gliadins and glutenins, but not to those containing albumins and globulins. The increase in protein components containing gliadins and glutenins correlated significantly with an increase in protein concentration and bread volume.

Inheritance of Gliadin and Glutenin Proteins in Four Durum Wheat Crosses

The F2 progenies, from four crosses between landraces and commercial Portuguese durum wheat cultivars, were analysed by electrophoresis for storage protein composition and genetic segregation. The chromosomal location of genes was determined for gliadin and glutenin allelic variations at the Gli-A1, Glu-A3 and Glu-A1 on 1A; Gli-B1, Glu-B3, Gli-B3, Glu-B2 and Glu-B1 on 1B; Gli-A2 on 6A and Gli-B2 on 6B. The protein patterns of landraces were uncommon and four new alleles were identified at different loci in chromosome 1 and four in the chromosome 6B (Gli-B2). The presence of γ-gliadin bands encoded at the Gli-B2 locus was also found.

Seed storage protein composition of non-nodulating soybean ( Glycine max (L.) Merr.) and its influence on protein quality

A 53 kDa protein, which accumulates at low levels in non-nodulating Clark soybeans, was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyclonal antibodies were raised against the gel-purified protein. N-terminal sequence analysis identified the 53 kDa protein as the β-subunit of β-conglycinin. Results from Western blot analysis, using antibodies raised against the purified β-subunit of β-conglycinin, revealed that accumulation of this protein was enhanced in non-nodulating soybeans when the plants were supplemented with nitrogen. Results from Northern blot analysis indicate that non-nodulating soybeans in general had lower levels of mRNA for the major soybean seed proteins. A one-time application of nitrogen to non-nodulating soybeans enhanced the accumulation of the 1.6 kb β-conglycinin β-subunit mRNA. The mRNA levels of the 2.1 kb β-conglycinin α′-subunit and the 2.2 kb G4 glycinin in the non-nodulating soybeans were several-fold lower than in nodulating soybeans. Nitrogen application had no effect on the abundance of these RNA transcripts. The amount of RNA encoding an 8.5 kDa sulfur-rich protein was two-fold higher in non-nodulating soybeans when compared with nodulated soybeans. Nitrogen application reduced the abundance of this transcript to levels comparable with those of nodulated soybeans. Despite lower levels of the β-subunit of β-conglycinin, the methionine content of the total seed protein fraction was lower than that of nodulated soybeans. In contrast, non-nodulating soybeans contained more cysteine than nodulating soybeans.

Composition and concentration of proteins in Lithuanian wheat cultivars: relationships with bread-making quality

The concentration and composition of storage proteins in Lithuanian-grown wheat cultivars were investigated. The protein concentration did not explain the differences in bread-making quality between the cultivars. Zentos and Sirvinta were considered as good bread-making quality wheats according to the Lithuanian millers and bakers. These cultivars showed the highest Glu-1 quality scores, contained high-molecular-weight (HMW) glutenin subunits and D-zone omega gliadins correlated with high gluten strength as well as a high percentage of large unextractable polymeric proteins in total large polymeric proteins.

The high and low molecular weight glutenin subunits and ω‐gliadin composition of bread and durum wheats commonly grown in Portugal

AbstractA collection of 63 bread wheats (Triticum aestivum L.) and 21 durum wheats (Triticum durum Desf.) commonly grown in Portugal since 1982 were characterized for the composition of wheat storage proteins (WSP), high molecular weight glutenin subunits (HMW‐GS), low molecular weight glutenin subunits (LMW‐GS) and ω‐gliadins. The composition of HMW‐GS, LMW‐GS and &‐gliadins, encoded at loci Glu‐1, Glu‐3 and Gli‐1, respectively, was revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. WSP allelic compositions of bread and durum wheat patterns were given. In the bread wheats, a total of 24, 24 and 18 patterns were observed for HMW‐GS, LMW‐GS and ω‐gliadins, respectively. Forty‐two different alleles were identified for the nine loci studied, Glu‐A1 (3), Glu‐B1 (7), Glu‐D1 (4), Glu‐A3 (5), Glu‐B1 (7), Glu‐D3 (2), Gli‐A1 (2), Gli‐B1 (8) and Gli‐D1 (4). In the case of durum wheats, 19 alleles were identified: one allele at Glu‐A1, two at Glu‐B3, Glu‐B2 and Gli‐A1, three at Glu‐B1, four at Glu‐A3 and five at Gli‐B1. For HMW‐GS, LMW‐GS and ω‐gliadins, three, six and six different patterns were revealed, respectively. This study represents the first attempt to discriminate the bread and durum wheat varieties commonly grown in Portugal by the allelic variation of storage proteins. The database is useful for varietal identification and for plant breeders who seek to devise effective programmes aimed at improving wheat quality.

Present-day hulled wheats from Spain

Present-day Spanish hulled wheats, Triticum monococcum L., T. dicoccum (Schrank) Schulb. and T spelta L. largely collected from the Asturias mountains have been grown and conserved at the John Innes Centre, Norwich, UK. A number of morphological characters have been recorded, including height, glume colour and pubescence, awn colour and grain colour. A preliminary screen of storage protein composition was also carried out. The value of these hulled wheats as a genetic resource and their possible unique origin is briefly discussed.

Processing effect on soybean storage proteins and their relationship with tofu quality.

Tofu was prepared from 13 soybean varieties according to three different methods (bench, pilot, and production methods). Different soybean varieties showed significant differences in storage protein composition (glycinin and beta-conglycinin). The beta-conglycinin (7S) and glycinin (11S) contents were 7.3-9.9 and 14.1-22.9% on the dry matter basis, respectively. The 11S/7S protein ratio varied from 1.64 to 2.51 among the varieties. Glycinin content and 11S/7S protein ratio of the 13 varieties did not change significantly from soy milk to tofu for the production and pilot methods. Soybean 11S/7S protein ratio positively correlated with the 11S/7S ratio of soy milk and tofu (0.57 < or = r < or = 0.83, p < or = 0.01). The correlation coefficient depended on the processing method. Processing method affected 7S and 11S protein contents of tofu and their contribution to tofu hardness, yield, and sensory quality. This may explain in part the contradictory findings of the relationships between storage proteins and tofu quality because processing methods differed in various studies.

Allelic variation at the storage protein loci of 55 US‐grown white wheats

AbstractFifty soft white and hard white wheat cultivars (Triticum aestivum L.), and five club wheat cultivars (T. compactum L.) were partially characterized in terms of their storage protein compositions, i.e. gliadins, and high molecular weight and low molecular weight glutenin subunits (HMW‐GS and LMW‐GS, respectively). At the Glu‐1 loci, HMW‐GS composition 1,7 + 9,2+ 12 was found to be predominant, being expressed in 11 cultivars out of 55. The most common alleles at the loci coding for gliadins and LMW‐GS were found to be Gli‐A1/Glu‐A3a (43.6%), Gli‐B1/Glu‐B3b (36.4%), Gli‐D1a/Glu‐D3a (38.1%) and Gli‐Dli/Glu‐D3a (21.8%). Two‐dimensional fractionation (acid‐poly‐acrylamide gel electrophoresis (A‐PAGE) × sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE)) of reduced and alkylated glutenins revealed that the number and the relative mobility of LMW‐GS polypeptides were different from those reported for the corresponding Glu‐3 alleles of hard‐bread wheat cultivars. This result could account for the different technological properties of soft white wheats compared with hard‐bread wheat cultivars, owing to the major impact of LMW‐GS on dough quality.

Seed Proteins of Lupinus mutabilis

Lupinus mutabilis sweet seeds possess the highest levels of oil and protein of all domesticated lupin species. However, no information has been published concerning the structure of its storage proteins. An electrophoretic comparative study of the seed storage protein composition of L. albus cv. Multolupa, L. mutabilis cv. Potosi, and L. mutabilis cv. Inti is reported. Small differences were observed between the two L. mutabilis cultivars, but broad differences were detected when the two species were compared. L. mutabilis seeds were considerably richer in total nitrogen, total protein, and total globulins but were poorer in albumins than L. albus. Besides, L. mutabilis total globulins showed less electrophoretic mobility under nondenaturing conditions when compared with L. albus. Total albumins and total globulins produced very different electrophoretic patterns in the two Lupinus species when analyzed by denaturing, reducing, or nonreducing electrophoresis. When the three individual conglutins were considered, differential results were obtained. γ-Conglutin structure seems to be identical in all cultivars investigated. Subunits and polypeptide chains of α-conglutin showed some degree of heterogeneity among the three cultivars analyzed. β-Conglutin possessed the greatest structural variation, being composed of over 20 polypeptide chains in L. albus, covering a broad range of molecular masses (15−65 kDa). Six to seven major polypeptide chains in L. mutabilis β-conglutin were observed, covering a limited range of molecular masses (50−67 kDa). Keywords: Seed protein; conglutin; L. mutabilis; PAGE

Free Amino Acid and Storage Protein Composition of Soybean Fruit Explants and Isolated Cotyledons Cultured with and without Methionine

Thein vitroculture of immature soybean cotyledons (in direct contact with the medium) and immature fruit explants (stem dipping into the medium) on a defined medium containing glutamine and sulphate as sole sources of N and S for 7 d led to rates of growth and reserve protein accumulation close to, or greater than, those occurringin situ. Supplementation of the medium with 8.4 mMmethionine had little effect on growth and protein accumulation of the cotyledons in the explant system, but did result in significant increases in the isolated cotyledon system. Methionine suppressed the synthesis of the 7S β-subunit in both systems. The free amino pool of the cotyledons increased more than three-fold when methionine was present in the explant medium. In the isolated cotyledon system, the basal medium alone caused a large increase (over 30-fold) in the free amino acid fraction, but methionine resulted in an even greater increase (over 50-fold). In both systems the expansion involved a very large increase in the methionine pool, but many other amino acids also showed large increases. Specific effects of methionine on individual amino acids were more clear in the explant system, where its presence resulted in marked increases in serine, alanine and asparagine. The data show that an abnormal situation arises on feeding with methionine, a fact to be considered before attributing effects on growth and protein synthesis directly to methionine.

Free Amino Acid and Storage Protein Composition of Soybean Fruit Explants and Isolated Cotyledons Cultured with and without Methionine

Free Amino Acid and Storage Protein Composition of Soybean Fruit Explants and Isolated Cotyledons Cultured with and without Methionine

Nutritional Control of Soybean Seed Storage Protein

There is interest in increasing the protein concentration of soybean [Glycine max (L.) Merr.] seed. Potentially, this could change its protein composition and perhaps its value as a livestock feed. Our objective was to examine the change in protein composition of soybean seed with change in protein concentration as influenced by nitrogen source supplied. ‘Harper’ soybean was grown in hydroponic culture and supplied with different nitrogen sources (KNO3, NH4NO3N2, urea) during pod filling. Protein quality was monitored as a function of soybean seed storage subunit composition. With N2 fixation as sole N source, the concentration of total seed protein decreased about 6%, storage protein 5%. Each seed storage protein subunit decreased, but the concentration of [β‐conglycinin, especially the sulfur‐poor [β‐subunit, decreased more than glycinin. Seed storage protein concentration was increased up to 4% by substituting reduced‐N for NO−3. The increased protein concentration resulted from a disproportionate increase of the β‐subunit of β‐conglycinin. The relative abundance of α‐ and α′‐subunits was not changed by N form. Thus, N form seems to control, at least in part, the relative portion of the β‐subunit of [β‐conglycinin, and consequently affects the 11S/7S ratio of seed storage protein. This change in composition with increased concentration diminishes the protein quality because the [β‐submit lacks sulfur‐amino acids. It is noteworthy that the concentration of glycinin remained constant while the total seed protein concentration increased. We think these variations in seed storage protein composition result from the relative abundance of S‐ and N‐metabolites available to developing soybean seed.

Changes in wheat seed storage protein fingerprint due to soil mineral content

Wheat seed storage protein fingerprint is used to determine the gluten protein pattern in studies aimed at improving flour quality. Wild wheat with high seed protein content is used extensively in wheat breeding programs. Although the wild wheat growth and protein content may be influenced by environmental conditions, the gluten-protein pattern is generally considered as indicative of a genotype, without the superimposition of environmental influences. The effects of soil type, habitat, and deficiencies of N, P, K and S on seed storage protein composition were examined in nine accessions of wild wheat (Triticum turgidum var. dicoccoides) and three varieties (two T. aestivum and one T. durum). Soil from ten natural habitats of the wild wheat that had not previously received any fertilizers or manures was sampled and used to grow wheat in a greenhouse. Seed storage protein composition was characterized by SDS-PAGE. Although deficiencies in soil nutrient caused variations in the seed storage proteins, the genotype was the main factor determining the seed storage protein composition. Seed storage protein composition of genotypes varied when grown under different mineral nutrient conditions. Only one genotype was stable showing almost identical protein patterns under all growing conditions studied without any qualitative change in fingerprint pattern. In the other genotypes, as well as the cultivars, the seed storage protein was affected at least to some extent by the soil. The ‘soil effect’ is summarized in terms of three main quantitative changes in the seeds: 1 – the relative amounts of the high-molecular-weight proteins; 2 – the relative amounts of proteins in the range of 45 and 65 kD; 3 – the percentage distribution of the HMW glutenin and other groups of seed storage proteins. The soild induced also qualitative differences in the composition of seed storage proteins, mostly in those of 45–65 kD. These differences were observed whenever a deficiency of S, N, P, K or Mg was identified. Therefore, in breeding programs that use seed storage protein fingerprints of wild wheat germplasms should be exercise caution when the germplasms selected from wild habitats.

Effects of exogenous ABA application on sulfate and OAS concentrations, and on composition of seed storage proteins in in vitro cultured soybean immature cotyledons

Composition of seed storage proteins in in vitro cultured cotyledons is known to be affected by exogenous application of abscisic acid (ABA) and sulfur deficiency. In this paper, we analyzed effects of exogenous ABA on regulation of soybean seed storage protein accumulation in relation to that of sulfur deficiency. When exogenous ABA was applied at 10 μM and 100 μM, accumulation of the β subunit of β-conglycinin, a major seed storage protein, increased, whereas that of glycinin, another major seed storage protein, decreased. Free sulfate concentrations in cotyledons were not significantly affected by application of 10 μM ABA. but doubled by application of 100 μM ABA. As we have previously shown that concentrations of O-acetyl-L-serine (OAS), a precursor of cysteine biosynthesis, in in vitro cultured soybean cotyledons increased under sulfur deficiency, effects of exogenous ABA application on OAS concentration in soybean cotyledons were determined. The concentrations of OAS increased in accordance with the increase of exogenous ABA concentrations. These results suggested that exogenous application of ABA induces changes in soybean storage protein composition through the increase of OAS concentration.