Seed Storage Protein Composition Research Articles

Genetic variation for seed storage protein composition in rapeseed (Brassica napus) and development of near‐infrared reflectance spectroscopy calibration equations

AbstractRapeseed protein consists mainly of the seed storage protein cruciferin and napin. Cruciferin and napin have different nutritional values and techno‐functional properties. Shifting the cruciferin/napin ratio towards either more napin or more cruciferin could allow diversified applications. The objective of this study was to investigate the genetic variation of cruciferin and napin in modern winter rapeseed cultivars. Cruciferin and napin contents were analysed by SDS‐PAGE. Genetic variation for both protein fractions was highly significant. Heritabilities were high ranging from 74% for cruciferin to 82% for napin. Napin was positively correlated with glucosinolate (rS = .52**) and seed protein content (rS = .48**). Additional plant material with much larger trait variation was included to develop near‐infrared reflectance spectroscopical calibrations. The Near‐infrared reflectance spectroscopy (NIRS) equations showed high fractions of explained variance in cross and independent validation of around .9 for all traits, indicating that the NIRS equations can be applied in routine screening of plant material.

Effect of nitrogen and sulphur nutrition on storage protein quality in soybean [Glycine max (L.) Merrill

The relative availabilities of nitrogen and sulphur can modulate seed storage protein composition in grain legumes and cereals. Soybean contains two major seed storage proteins, glycinins and ?-conglycinins that account for approximately 70% of total protein and their composition is affected by nitrogen and sulphur supplies. The present study demonstrated the effect of sulphur (Gypsum @ 20 kg S ha-1 ) and recommended a dose of nitrogen (Urea @ 31.25 kg N ha-1 ) alone or in combination on accumulation patterns of various protein fractions and protein quality in soybean seeds under agro-climatic conditions of Punjab. Application of nitrogen or sulphur alone decreased the relative proportion of ?-subunit of purified ?-conglycinin fraction than control. The acidic polypeptides of glycinin fraction of globulin and 11S/7S ratio were increased in all the treatments in comparison to control, and the maximum increase was reported in the combined application of gypsum with the recommended dose of urea. The lower proportion of total 7S, increased acidic polypeptides and improved 11S:7S ratio by combined treatment of nitrogen and sulphur suggests that gypsum @ 20 kg S ha-1 can be beneficial when applied along the recommended nitrogen dose to improve soybean protein quality.

Changes in the Subunit Composition of Seed Storage Proteins by Controlling Nitrogen and Sulfur Metabolism I

Changes in the Subunit Composition of Seed Storage Proteins by Controlling Nitrogen and Sulfur Metabolism I

Amino Acid Composition, Protein Fractions and Electrophoretic Analysis of Seed Storage Proteins in Lupins

Amino Acid Composition, Protein Fractions and Electrophoretic Analysis of Seed Storage Proteins in Lupins

Transgenic sorghum with improved digestibility of storage proteins obtained by Agrobacterium-mediated transformation

Development of transgenic plants with modified seed storage protein composition and increased nutritive value is one of the most promising areas of genetic engineering. This task is especially important for sorghum—a unique drought tolerant cereal crop that is characterized, however, by a relatively poor nutritive value in comparison with other cereals. It is considered that one of the reasons of the low nutritive value of the sorghum grain is the resistance of one of its seed storage proteins, γ-kafirin, located in the outer layer of endosperm protein bodies, to protease digestion. Using Agrobacterium-mediated genetic transformation, we obtained transgenic sorghum plants (Sorghum bicolor (L.) Moench) harboring a genetic construct for RNAi silencing of the γ-kafirin gene. In the T1 generation, the plants with almost floury or modified endosperm texture of kernels were found. In these kernels, the vitreous endosperm layer has been reduced and/or covered by a thin layer of floury endosperm. In vitro protein digestibility (IVPD) analysis showed that the amount of undigested protein in transgenic plants from the T3 generation was reduced by 2.9–3.2 times, in comparison with the original non-transgenic line, and the digestibility index reached 85–88% (in comparison with 59% in the original line). In T2 families, the plants combining high IVPD with vitreous endosperm type were found. In the electrophoretic spectra of endosperm proteins of transgenic plants with increased digestibility, the proportion of 20 kD protein that is encoded by the γ-kafirin gene, was significantly reduced, in comparison with the original non-transgenic line. HPLC analysis showed total amino acid content in two out of the three studied transgenic plants from the T2 generation was reduced in comparison with the original non-transgenic line, while the lysine proportion increased by 1.6–1.7 times. The mechanisms conditioning improved digestibility of storage proteins in transgenic plants are discussed. The results of experiments demonstrate that it is feasible to develop sorghum lines combining high protein digestibility and vitreous endosperm that has a high breeding value.

Deep sequencing transcriptional fingerprinting of rice kernels for dissecting grain quality traits

BackgroundRice represents one the most important foods all over the world. In Europe, Italy is the first rice producer and Italian production is driven by tradition and quality. All main rice grain quality traits, like cooking properties, texture, gelatinization temperature, chalkiness and yield, are related to the content and composition of starch and seed-storage proteins in the endosperm and to grain shape. In addition, a number of nutraceutical compounds and allergens are known to have a significant effect on grain quality determination. To investigate the genetic bases underlying the qualitative differences that characterize traditional Italian rice cultivars, a comparative RNA-Seq-based transcriptomic analysis of developing caryopsis was conducted at 14 days after flowering on six popular Italian varieties (Carnaroli, Arborio, Balilla, Vialone Nano, Gigante Vercelli and Volano) phenotypically differing for qualitative grain-related traits.ResultsCo-regulation analyses of differentially expressed genes showing the same expression patterns in the six genotypes highlighted clusters of loci up or down-regulated in specific varieties, with respect to the others. Among them, we detected loci involved in cell wall biosynthesis, protein metabolism and redox homeostasis, classes of genes affecting in chalkiness determination. Moreover, loci encoding for seed-storage proteins, allergens or involved in the biosynthesis of specific nutraceutical compounds were also present and specifically regulated in the different clusters. A wider investigation of all the DEGs detected in pair-wise comparisons revealed transcriptional variation, among the six genotypes, for quality-related loci involved in starch biosynthesis (e.g. GBSSI, starch synthases and AGPase), genes encoding for transcription factors, additional seed storage proteins, allergens or belonging to additional nutraceutical compounds biosynthetic pathways and loci affecting grain size. Putative functional SNPs associated to amylose content in starch, gelatinization temperature and grain size were also identified.ConclusionsThe present work represents a more extended phenotypic characterization of a set of rice accessions that present a wider genetic variability than described nowadays in literature. The results provide the first transcriptional picture for several of the grain quality differences observed among the Italian rice varieties analyzed and reveal that each variety is characterized by the over-expression of a peculiar set of loci affecting grain appearance and quality. A list of candidates and SNPs affecting specific grain properties has been identified offering a starting point for further works aimed to characterize genes and molecular markers for breeding programs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2321-7) contains supplementary material, which is available to authorized users.

Differential response to sulfur nutrition of two common bean genotypes differing in storage protein composition.

It has been hypothesized that the relatively low concentration of sulfur amino acids in legume seeds might be an ecological adaptation to nutrient poor, marginal soils. SARC1 and SMARC1N-PN1 are genetically related lines of common bean (dry bean, Phaseolus vulgaris) differing in seed storage protein composition. In SMARC1N-PN1, the lack of phaseolin and major lectins is compensated by increased levels of sulfur-rich proteins, resulting in an enhanced concentration of cysteine and methionine, mostly at the expense of the abundant non-protein amino acid, S-methylcysteine. To identify potential effects associated with an increased concentration of sulfur amino acids in the protein pool, the response of the two genotypes to low and high sulfur nutrition was evaluated under controlled conditions. Seed yield was increased by the high sulfate treatment in SMARC1N-PN1. The seed concentrations of sulfur, sulfate, and S-methylcysteine were altered by the sulfur treatment in both genotypes. The concentration of total cysteine and extractible globulins was increased specifically in SMARC1N-PN1. Proteomic analysis identified arcelin-like protein 4, lipoxygenase-3, albumin-2, and alpha amylase inhibitor beta chain as having increased levels under high sulfur conditions. Lipoxygenase-3 accumulation was sensitive to sulfur nutrition only in SMARC1N-PN1. Under field conditions, both SARC1 and SMARC1N-PN1 exhibited a slight increase in yield in response to sulfur treatment, typical for common bean.

Evidence for Proteomic and Metabolic Adaptations Associated with Alterations of Seed Yield and Quality in Sulfur-limited Brassica napus L

In Brassica napus, seed yield and quality are related to sulfate availability, but the seed metabolic changes in response to sulfate limitation remain largely unknown. To address this question, proteomics and biochemical studies were carried out on mature seeds obtained from plants grown under low sulfate applied at the bolting (LS32), early flowering (LS53), or start of pod filling (LS70) stage. The protein quality of all low-sulfate seeds was reduced and associated with a reduction of S-rich seed storage protein accumulation (as Cruciferin Cru4) and an increase of S-poor seed storage protein (as Cruciferin BnC1). This compensation allowed the protein content to be maintained in LS70 and LS53 seeds but was not sufficient to maintain the protein content in LS32 seeds. The lipid content and quality of LS53 and LS32 seeds were also affected, and these effects were primarily associated with a reduction of C18-derivative accumulation. Proteomics changes related to lipid storage, carbohydrate metabolism, and energy (reduction of caleosins, phosphoglycerate kinase, malate synthase, ATP-synthase β-subunit, and thiazole biosynthetic enzyme THI1 and accumulation of β-glucosidase and citrate synthase) provide insights into processes that may contribute to decreased oil content and altered lipid composition (in favor of long-chain fatty acids in LS53 and LS32 seeds). These data indicate that metabolic changes associated with S limitation responses affect seed storage protein composition and lipid quality. Proteins involved in plant stress response, such as dehydroascorbate reductase and Cu/Zn-superoxide dismutase, were also accumulated in LS53 and LS32 seeds, and this might be a consequence of reduced glutathione content under low S availability. LS32 treatment also resulted in (i) reduced germination vigor, as evidenced by lower germination indexes, (ii) reduced seed germination capacity, related to a lower seed viability, and (iii) a strong decrease of glyoxysomal malate synthase, which is essential for the use of fatty acids during seedling establishment.

Effects of Reduced Prolamin on Seed Storage Protein Composition and the Nutritional Quality of Rice

Rice seed storage proteins accumulate in two types of protein body (PB-I and PB-II) that are nutrient sources for animals. PB-I is indigestible and negatively affects rice protein quality. To improve the nutritional value of rice seeds we are aiming to engineer the composition and accumulation of endogenous seed storage proteins. In this study we generated transgenic rice plants in which 13 kD prolamin genes were suppressed by RNA interference (13 kD pro-RNAi). Analysis based on qRT-PCR confirmed that the targeted 13 kD prolamins were markedly suppressed, and were compensated for by an increase in other storage proteins including 10 kD prolamin, glutelins, and chaperone proteins. The storage protein profiles further revealed that the levels of 13 kD prolamins were significantly reduced, while that of the glutelin precursor was slightly increased and the remaining storage proteins did not change. Amino acid analysis showed that the reduction of 13 kD prolamins resulted in a 28% increase in the lysine content relative to the wild type, indicating that the 13 kD pro-RNAi rice seeds are more nutritious. Furthermore, a reduction in the levels of 13 kD prolamins resulted in abnormal formation of PB-I, which was small and had no lamellar structure. These results suggest that alteration of prolamins can contribute to improving the nutritional quality of rice.

Alteration of Seed Storage Protein Composition in Soybean [Glycine max (L.) Merrill] Mutant Lines Induced by γ-Irradiation Mutagenesis

This study investigated the alteration of seed storage proteins in soybean mutants induced by γ-irradiation. Five soybean cultivars and four landraces were irradiated with 250 Gy of γ rays to induce variability. The seed storage protein profiles of 414 genetic fixed mutants (M(12)-M(20)) having excellent agricultural traits were analyzed by SDS-PAGE. Among the 414 mutants, 58 were identifed as lacking lipoxygenase, 89 lacking the α' subunit, 113 lacking the α subunit, and 40 with an altered β subunit. One hundred and forty-nine mutants lacked the A(3) subunit of glycinin. Fifty-four mutants showed higher trypsin inhibitor (TIA) activity, whereas 139 showed lower TIA activity compared to their original cultivars. The selected mutants with low amounts of antinutritional factors such as trypsin inhibitor, lipoxygenase, and α subunit will constitute genetic resources for improving soybean protein quality.

Positive or negative effects on dough strength in large-scale group-1 chromosome deletion lines of common wheat (Triticum aestivum L.)

We studied the seed storage protein composition and dough strength of chromosome deletion (CD) lines involving group-1 chromosomes. The presence or absence of genes and protein bands corresponding to glutenin and gliadin was assessed by using locus-specific DNA markers, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and acid-polyacrylamide gel electrophoresis (A-PAGE). In this study, we were able to map the physical positions of several glutenin and gliadin genes in detail. Dough strength was evaluated by SDS sedimentation volume and protein content. We found that protein composition affected dough strength. The absence of chromosome arm 1AL, which carries the truncated glutenin gene Glu-A1c, significantly increased dough strength, although the protein composition did not change when the size of the deleted chromosome region was varied. In contrast, the presence of chromosome arm 1DL, which carries Glu-D1a (the gene for glutenin subunits 2 and 12), significantly increased dough strength. We did not find any known seed storage protein loci in any of the other chromosomal regions that significantly affected dough strength.

Effect of Nutrient Solution Concentration at the Heading Time on the Growth, Development, and Seed Storage Protein Content of Rice Plants in a Controlled Environment

Genetically modified (GM) rice varieties that store up pharmaceutical proteins in seeds have recently been developed. Since the pharmaceutical protein-encoding genes of these varieties are connected with the promoter of seed storage protein-encoding genes, the pharmaceutical protein content increases as the seed protein content increases. Rice varieties were grown in hydroponic culture at different nutrient solution concentrations at the heading time in a controlled environment. Emergence of non-productive stems was inhibited through an early reduction in the nutrient solution concentration due to accelerated translocation from the leaf blade, leaf sheath, and stem to the seed. Further, the seed storage protein composition was altered by regulating the concentration of the nutrient solution. When the nutrient solution concentration was increased just before heading occurred, translocation to the seeds as well as the generation of non-productive stems decreased. The total protein concentration of rice seeds was not increased by the treatments of nutrient solution concentration, however, it was greater in the plants grown in the present study than in plants grown in field cultivation. Seeds in which high concentrations of total protein and seed storage protein accumulate can be produced when the nutritional content is kept constant in hydroponic culture.

Dissecting the proteome of pea mature seeds reveals the phenotypic plasticity of seed protein composition.

Pea (Pisum sativum L.) is the most cultivated European pulse crop and the pea seeds mainly serve as a protein source for monogastric animals. Because the seed protein composition impacts on seed nutritional value, we aimed at identifying the determinants of its variability. This paper presents the first pea mature seed proteome reference map, which includes 156 identified proteins (http://www.inra.fr/legumbase/peaseedmap/). This map provides a fine dissection of the pea seed storage protein composition revealing a large diversity of storage proteins resulting both from gene diversity and post-translational processing. It gives new insights into the pea storage protein processing (especially 7S globulins) as a possible adaptation towards progressive mobilization of the proteins during germination. The nonstorage seed proteome revealed the presence of proteins involved in seed defense together with proteins preparing germination. The plasticity of the seed proteome was revealed for seeds produced in three successive years of cultivation, and 30% of the spots were affected by environmental variations. This work pinpoints seed proteins most affected by environment, highlighting new targets to stabilize storage protein composition that should be further analyzed.

Seed storage protein composition of Hellenic bread wheat cultivars

AbstractThe allelic diversity in seed storage proteins of 25 bread wheat cultivars grown in Hellas was investigated. In total, 15–20 seeds per cultivar were used for the determination of the alleles present at the loci coding for high‐molecular‐weight glutenin subunits and gliadins. For this purpose, acid polyacrylamide gel electrophoresis for gliadins and SDS‐electrophoresis for glutenins were employed. Analysis of the electrophoretic patterns revealed that intravarietal selections obtained from the cultivar ‘Nestos’, together with the cultivar ‘Eurydice’ which was selected from the cultivar ‘Nestos’, were identical to their original cultivar and to the cultivar ‘Dodoni’. The cultivars ‘Penios’, ‘Siette Cerros E’, ‘Gorgona’ and ‘Louros’, although recorded to differ in descent, were found to be identical at all the loci examined. Finally, it was revealed that four of the Hellenic cultivars carry the wheat‐rye 1BL/1RS translocation. These data could be beneficial for a better understanding of the existing differences in quality and stress‐resistance between the cultivars examined.

Characterisation and Variation of Morphological Traits and Storage Proteins in Spanish Emmer Wheat Germplasm (Triticum Dicoccon)

Emmer wheat is hulled wheat that was wide cultivated in Spain at the past. Actually, the most of this germplasm is conserved in Germplasm Banks, and only two small populations have been found in Asturias (North of Spain) in a recent collecting mission. In this work, a collection of 31 Spanish emmer lines developed from identical number of accessions of two Germplasm Banks was analysed for morphological spike traits and seed storage protein composition. Up to seven different botanical varieties were detected, which suggest the presence of a wide diversity, although lower than the historically described 10 botanical varieties. At level of seed storage proteins, the lines showed a high diversity, although the new alleles were present with low frequency in materials with scarce agronomic interest for the farmers (var. atratum, var. lagascae or var. pycnurum). This last circumstance could translate in a lost of variability by genetic drift.

Polymorphism and Genetic Diversity for the Seed Storage Proteins in Spanish Cultivated Einkorn Wheat (Triticum monococcum L. ssp. monococcum)

The seed storage protein composition of one collection of cultivated einkorn wheat (Triticum monococcum L. ssp. monococcum) of Spanish origin has been analysed by SDS-PAGE and A-PAGE. Three allelic variants were detected for the Glu-A1 m , whereas up to six alleles were detected for the Glu-A3 m . For the gliadins, 7 and 14 alleles for the Gli-A1 m and Gli-A2 m were found between the evaluated accessions. Internal variability was detected in some of these materials, which could be related to the landrace nature of them. Up to 48 different genotypes based on the origin and seed storage protein composition have been identified. Further researches on these materials must be carried out for determining the variability degree in morphological traits that could complement the evaluation for their safeguard.

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.

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.

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

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.