Antigenicity of Wheat Prolamins: Detailed Epitope Analysis using a Panel of Monoclonal Antibodies

Putative continuous epitopes, recognised by five panels of monoclonal antibodies (MAb) with differing specificities for gliadins and glutenin subunits, were identified using overlapping nonapeptides. These peptides corresponded to the entire sequence of an α/β-gliadin, a γ-gliadin, an ω-prolamin (homologous to ω-gliadin), a low molecular weight glutenin subunit (L M_rGS) and several high molecular weight glutenin subunits (HM_r GS). Antibodies that bound to γ- or ω-gliadins, L M_rGS or HM_r GS bound to the peptides at similar concentrations used normally in direct ELISA, but little binding to the peptides was seen for several antibodies that bound specifically to small groups of α/β-gliadins. Epitopes for these antibodies in α/β-gliadin may be discontinuous (i.e. derived from amino acid residues that are brought together by folding of the polypeptide chain or by juxtaposition of two polypeptide chains), since binding of these antibodies to gliadins was greatly decreased following the reduction of intra-molecular disulphide bonds. While some regions in particular subunits were immunodominant, such as the cysteine–cysteine containing peptide found in the central domain of many prolamins, a diversity of reaction patterns was found. Cross-reaction of antibody with peptides from other prolamin families was often due to binding to a peptide having significant sequence homology, but in some cases no homology was obvious. Some major trends were as follows. Antibodies which bound to most or all H M_rGS recognised the central repeat region, while those that were selective for one or two subunits bound to epitopes in the unique N- and/or C-terminal domains. A high proportion of the epitopes recognised by MAb to α-, β-, ω-gliadins and L M_rGS contained cysteine; these MAb may be useful in detecting covalent binding sites within or between subunits. Although a number of MAb bound a wide range of gliadins and GS, several of these recognised single (and differing) epitopes in the target proteins. However, comparatively few MAb recognised epitopes from either the N- or C-terminal regions of the target proteins. Several explanations are possible; either these regions are buried in the immunogen and not accessible for antibody production or alternatively the repeat sequences are immunodominant.     

Detection of y-type Subunit at theGlu-A1Locus in Some Swedish Bread Wheat Lines

Electrophoretic and reversed phase high performance liquid chromatographic (RP–HPLC) analyses were performed on gluten proteins extracted from flours milled from two different Swedish bread wheat lines; these lines have been reported to possess a novel highM_rglutenin subunit controlled by a gene at theGlu-A1locus, referred to as 21*. Although RP–HPLC indicated that subunit 21* has a surface hydrophobocity similar to that of the commonly occurring allelic subunits 1 or 2*, it differs from them in isoelectric point, being more basic when analysed by two dimensional gel electrophoresis (IEF/SDS–PAGE). RP–HPLC separations of highM_rglutenin subunits showed the presence of an additional peak, the behaviour of which was similar to that of y-type subunits encoded by genes at theGlu-A1ylocus and present only in wild wheatsT. urartu(AA) orT. dicoccoides(AABB). Based on chromatographic results and on the tight linkage observed with subunit 21*, it is suggested that the additional component (indicated as 21*y), present in the breeding lines analysed, corresponds to the y-type subunit encoded at theGlu-A1locus. Genes encoding the subunits 21* and 21*y were also analysed by polymerase chain reaction (PCR). Contrary to what was observed for the polypeptide itself, the gene corresponding to subunit 21* was similar in size to that encoding subunit 2* and shorter than that corresponding to subunit 1. Moreover, the amplification product corresponding to the active 21*y gene was shorter than that of the allelic inactive gene present in the bread wheat cultivar Cheyenne. As reported for other highM_rglutenin subunits, gene size differences observed were due to a different length of the repetitive region. Because cultivated polyploid wheats have been shown to have only the x-type subunit at theGlu-A1locus, it is speculated that the new combination, with both x- and y-type subunits expressed, might have been introgressed during breeding processes from the wild wheat progenitorsT. urartuorT. dicoccoides, which have genotypes expressing both types of subunits.     

Functional Properties of Low Mr Wheat Proteins. I. Isolation, Characterization and Comparison with Other Reported Low Mr wheat Proteins

A group of low M_r wheat proteins with characteristic extractability behavior was isolated using two different isolation procedures. The proteins were extractable with water, salt solution and 70% (v/v) ethanol. After water extraction of flour and separation of gluten, a substantial proportion of these proteins was still extractable from gluten using 70% (v/v) ethanol. Based in their amino acid compositions, M_rs and IEF patterns, the isolated proteins resemble closely most of the alpha -amylase/protease inhibitors described in the literature. This was confirmed by enzyme inhibition studies in which it was shown that they inhibited mammalian, but not wheat, bacterial and fungal alpha-amylases. All proteases tested were inhibited by the low M_r proteins. Their M_rs and their high cysteine contents (6·5-8·1 mol%) indicated that the proteins contain four to five disulphide bonds. Free thiol groups were not detected in the proteins. Upon reduction, the M_r increased from 7-8000 to 14-19000. Furthermore, the disulphide bonds were highly reactive as determined by their reaction with the thiol-specific label monobromobimane. This suggests that the low M_r wheat proteins may play a role in thiol group/disulphide bond exchange in wheat proteins.     

部分栽培稻和野生稻种子谷蛋白的电泳分析

 供试的不同属、不同种及不同生态型的水稻材料的种子各蛋白经SDS-PAGE电泳后可分辨为3类多肽，分子量分别为l8～23 kDa．30～36 kDa和49～100 kDa。不同属的材料各蛋白带型明显不同； 同属内基因组相同或相近种的各蛋白有特征多肽，AA基因组的有约Mt.50 kDa的多肽2～3条，CCDD基因组的有约Mt.23 kDa的多肽一条；籼型和粳型稻间带型存在差异，而爪哇稻、粳稻、籼稻间，前二者略近，并以粳型陆稻与爪哇稻最近似。同一生态型的各品种，除细弱带偶尔有差异外，带型基本一致。由此可见，水稻各蛋白电泳分析作为方法之一， 对于属间和稻属内各种及种内分类是有意义的。     

Relationships Between Chromosome 1B-encoded Glutenin Subunit Compositions and Bread-making Quality Characteristics of Some Durum Wheat (Triticum turgidum) Cultivars

The high and low M_r glutenin subunit compositions (controlled by the Glu-1 loci and the Glu-B3 locus, respectively) and the bread-making quality characteristics of 26 durum wheat (Triticum turgidum) genotypes were determined. The relationships between quality parameters and Glu-B1 and Glu-B3 controlled glutenin subunit composition were also investigated. The Glu-A1-controlled null allele was present in all the genotypes. High M_r subunits 20, 6 + 8 and 7 + 8 occurred in similar proportions in the cultivars analysed. The Glu-B3 low M_r allelic variants, LMW-1 and LMW-2, were both represented, with LMW-1 being present in lower proportion. Flour protein, SDS-sedimentation volume, dough strength (Alveograph W value), dough mixing time and bread loaf volume varied among the genotypes. Most samples had high Alveograph tenacity/extensibility (P/G) ratios, typical of tenacious gluten character. SDS-sedimentation volume, dough strength, dough mixing time and bread loaf volume were all interrelated. An association with flour protein content was observed only for mixing time, while the Alveograph tenacity/extensibility ratio was not correlated with the other parameters. Comparisons within the Glu-B1 and Glu-B3 loci indicated that the high M_r subunit 7 + 8 and the low M_r subunit LMW-2 had significantly greater beneficial effects on gluten strength and bread-making quality than the high M_r subunits 6 + 8 or 20 and the low M_r subunit LMW-1, respectively. High M_r subunit 6 + 8 had greater beneficial effects on quality than subunit 20.     

Detection, Chromosomal Location and Evaluation of the Functional Value of a Novel High Mr Glutenin Subunit Found in Swedish Wheats

A high M_r glutenin subunit, which has not been described previously, was found in several Swedish wheat (Triticum aestivum L.) breeding lines. The electrophoretic mobility (sodium dodecyl sulphate polyacrylamide gel electrophoresis) of this band was close to the mobility of the subunit that has been referred to as band 21 encoded on chromosome 1B. Reciprocal crosses between wheat materials with and without this band have shown that the synthesis of this subunit is controlled by the locus on chromosome 1A. The new band, called 21^*, is thus allelic to bands 1 and 2^*. The relevance of the novel-subunit to breadmaking quality was investigated by partial-least-square regression analysis. Using this method, the relationship between the electrophoretic patterns of high M_r glutenin subunits and the specific Zeleny volume was determined. The novel glutenin subunit was found in cultivars with a high specific Zeleny volume. Further investigations are needed before it is possible to determined the influence of the new glutenin subunit on baking quality.     

Nutritional potential ofVigna minima (Roxb.) Ohwi and Ohashi:

Quantitative variation in different fractions of seed proteins and their amino acid levels in populations ofVigna minima (Roxb.) Ohwi and Ohashi and inV. umbellata cv. IC 1568 — the rice bean — were investigated. Globulin I fraction, together with globulin II, constitutes 38 to 54 per cent of the total seed protein. The alkali soluble (glutelin) fraction is the second largest fraction. Both these fractions show broad range of variation, suggesting a broad genetic base. The profiles are population specific; the coastal population, which contains higher seed protein also possesses maximum levels of globulin I and glutelin fraction suggesting its potentiality for breeding lines with high protein content, high nutritive value, and salt tolerance. Protein content is positively correlated with globulin I and glutelin fractions, which are in turn positively correlated with each other. The amino acid profiles are specific not only to the fractions but also to the populations. The range of variation in the levels of all amino acids in different fractions is broad suggesting substantial genetic diversity. The average levels of lysine and sulphur amino acids are high in globulin I and glutelin fractions.     

Extractability and chromatographic separation of rice endosperm proteins

The molecular weight (MW) distribution of proteins extracted with different solvents from defatted rice endosperm was examined by size exclusion-high performance liquid chromatography (SE-HPLC) with 2.0% sodium dodecyl sulfate (SDS) (w/v) as mobile phase. The resulting protein peaks were further characterized by SDS-PAGE. Under the experimental conditions, 2.0% SDS extracted 64% of the proteins. Adding 6.0 M urea resulted in a 15% increase in extractability (up to 79%). With using 20–100 mM NaOH, 70–81% of the proteins were extractable. Maximum extractability was reached with 2.0% SDS, 6.0 M urea and 0.5–1.5% dithiothreitol (DTT). Apparent MW profiles of rice endosperm proteins allowed classification into six fractions of decreasing apparent MW. Fraction VI contained the low MW albumin, globulin, and prolamin protein material. Fractions IV and V originated from α and β glutelin subunits, respectively. The polypeptides of fraction III consisted of an α and a β subunit linked by an intermolecular disulfide bond. The polypeptides of fractions I and II were dimers, trimers or more highly polymerized forms of the (α–β) glutelin subunit dimer in fraction III. While the work confirmed that rice glutelin is composed of polymers of α and β subunits, remarkably, higher MW glutelin aggregates (fractions I–III) only partly dissociated on reduction. Low MW protein material (fraction VI) was entrapped in the aggregated protein network and was released on reduction. The rapid and reproducible SE-HPLC method developed for rice protein separation allows a more quantitative approach than SDS-PAGE.     

Immunocytochemical Reactions to Intact Protein Bodies in Rice (Oryza sativaL.): Using Antibodies to Purified Fractions of Some Rice Polypeptides

Rice flour and commercial rice protein concentrate (enzymatically destarched rice flour) from mature rice grains (Oryza sativaL., cv. Lemont) were embedded and sectioned for use in electron-microscopical immunocytochemistry. Polyclonal antibodies were raised in rabbits against the following purified rice protein fractions: 10 and 13 kDa prolamins, 23–25 and 38 kDa glutelins, ‘total prolamins’, ‘total glutelins’, and two insoluble particulate waste products derived from protein concentrate. Western blot procedures showed that the antibodies produced were specific for their particular fractions, and that antibodies to particulate waste fractions reacted with bands at positions of approximately 12 and 28 kDa, normally not evident as strong bands in SDS-PAGE gel preparations of rice. Immunogold procedures, using Protein A and electron microscopy, significantly labelled both types of rice protein body (PB-I and PB-II) in the embedded substrates for each antibody. This varies from other reported research when rice proteins have been fractionated on gels, or when antibodies to total glutelins or total prolamins have been tested on developing rice grains. Estimates of labelling efficiencies show that each protein body type contains a substantial number of antigenic determinants for all the species of polypeptide studied.     

Seed protein fractions and amino acid composition in gram (Cicer arietinum)

Six chickpea strains were analysed for their protein content and various protein fractions. The protein content ranged from 20.9–25.27%. Albumin, globulin, prolamin and glutelin contents ranged from 8.39–12.31%; 53.44–60.29%; 3.12–6.89% and 19.38–24.40% respectively. Salt soluble proteins (albumin + globulin) and globulins resolved into 19–23 bands whereas albumin proteins resolved into 30–34 bands. The molecular weights of various polypeptides ranged from 10–91 kD. Amino acid analysis of total proteins revealed that glutamic acid was present in maximum concentration followed by aspartic acid and arginine. Just like other pulse proteins, chick pea proteins were also found deficient in sulphur containing amino acids.     

Functional Properties of LowMrWheat Proteins.III. Effects on Composition of the Glutenin Macropolymer During Dough Mixing and Resting

The effect of lowM_rwheat protein addition on the amount and composition of the glutenin macropolymer (GMP) of dough was investigated for the three wheat cultivars Obelisk (weak), Camp Remy (medium strong) and Rektor (strong). During mixing, the amounts of high and lowM_rglutenin subunit classes, and of the individual subunits decreased. The proportion of highM_rglutenin subunits decreased and that of lowM_rglutenin subunits increased, indicating an inhomogeneous distribution of the two subunit classes within the polymers present in GMP. During resting, the amounts of the glutenin subunit classes and of individual subunits increased. Meanwhile, the proportion of highM_rglutenin subunits in GMP increased. LowM_rwheat protein addition retarded re-polymerisation in that the amounts of glutenin subunit classes and of individual highM_rglutenin subunits in GMP increased less than without addition. The proportion of highM_rglutenin subunits in GMP directly after mixing was also decreased by lowM_rwheat protein addition, and the proportion increased faster during dough resting, compared with the GMP in dough without lowM_rwheat protein addition. Eventually, after 90 or 135 min resting, no differences existed in the proportions in GMP from doughs with and without lowM_rwheat protein addition. LowM_rwheat protein addition had no specific effect on individual highM_rglutenin subunits, nor on the x-type/y-type subunit ratio in the GMP. In contrast, with increasing lowM_rwheat protein addition, a highly significant reduction in the subunit 10 or 12/subunit 9 ratio in GMP was observed. This finding is in line with the decrease in this ratio directly after mixing in GMP of the dough without lowM_rwheat protein addition. Since no specific effects were observed, it can be concluded that the lowM_rwheat protein acts rather unspecifically on the GMP of dough.     

Preparative Separation of High and Low Molecular Weight Subunits of Glutenin from Wheat

Wheat flour was washed with Tris-HCl buffer containing 4% Triton X114 before extracting the residual gluten with 70% ethanol. The glutenin extraction with 50% ethanol was performed at various ratios of DTT/protein; a minimum ratio of 0·1 g/g was needed to solubilise the maximum amount of glutenin. An experimental design was used to optimise the extraction conditions to obtain the best yield and purity of lowM_rand highM_rglutenin subunits. The purity of each glutenin subunit fraction was measured by RP-HPLC analysis after reduction and alkylation. Both temperature and protein concentration had an effect on the preparation of these fractions. An increase in the protein concentration enhanced the yield of the highM_rglutenin fraction and simultaneously decreased that of the lowM_rglutenin. Using the Deringer desirability function, conditions giving the optimum separation were determined. The procedure was scaled up and permitted the preparation of 0·96 g of highM_rand 1·64 g of lowM_rglutenin subunits from 5 g of gluten. The purities of these fractions, determined by RP-HPLC, were 90% and 95%, respectively, and their amino acid compositions were similar to those of high and lowM_rsubunits separated by RP-HPLC.     

The location of disulphide bonds in α-type gliadins

Gliadin prepared from gluten of the cultivar Rektor by extraction with 70% (v/v) aqueous ethanol adjusted to pH 5.5 was separated by RP-HPLC. Amongst 23 components obtained, two α-type gliadins (α3- and α8-gliadin) were selected for the determination of disulphide bonds. After both proteins were digested with thermolysin, differential RP-HPLC (chromatography prior to and after reduction of disulphide bonds) was used for the detection of cystine peptides. Two cystine peptides from α3-gliadin and three cystine peptides from α8-gliadin were isolated by RP-HPLC. The resulting peptides were reduced and alkylated with 4-vinylpyridine, separated by RP-HPLC and their amino acid sequences determined. The cystine peptides from both α-type gliadins had similar structures, and the corresponding fragments had homologous sequences. One cystine peptide of each gliadin was composed of three fragments linked by two disulphide bonds. The second cystine peptide consisted of two fragments linked by one disulphide bond. The third cystine peptide derived from α8-gliadin was different from the second peptide in one position of the sequences (glutamic acid instead of glutamine). Comparing complete sequences of α-type gliadins described in the literature, the cystine peptides from α3- and α8-gliadins were identical with corresponding sequences of clones A1235 and A212, respectively¹¹. The structures of the cystine peptides analysed indicate one intramolecular disulphide bond within domain III of α-type gliadins and two disulphide bonds between domains III and V. The linkages found correspond to homologous linkages determined for low M_r subunits of glutenin and glutenin-bound γ-type gliadins⁶. Obviously, these intramolecular disulphide bonds are not linked randomly, but are strongly directed.     

Subunit composition of proteins from seeds of Lupinus albus

All the main globulins in the seeds ofLupinus Albus are oligomeric glycoproteins. Legumins (33%) consist of two similar protein molecules which contain protomers linked by disulphide bridges. They result from a partial proteolytic breakdown of an original polypeptide chain. Vicilins (44%) consist of four similar protein molecules with several protomers linked together by non-covalent bonds. Globulin 1 (6%) has a native M.W. of 199 kd and is formed by four 45.0 kd subunits consisting of two smaller protomers (28.0 and 16.0 kd) linked by -S-S- bonds. Globulin 9b (12.5%) has the lowest M.W. (44.0 kd) and is made up of three protomers, two of which are linked by disulphide bonds.     

Purification and Characterisation of HighMrGlutenin Subunit 20 and its Linked y-type Subunit from Durum Wheat

HighM_rglutenin subunit 20 and its linked y-type subunit, present in the durum wheat cultivar Lira, were purified by preparative reversed-phase high-performance liquid chromatography (RP–HPLC). Amino acid and N-terminal sequence analysis of subunit 20y confirmed that it corresponded to a y-type subunit. Moreover, the number and position of the cysteine residues in subunit 20 were determined by alkylation with the fluorogenic reagent 7-fluoro-4-sulfamoyl-2,1,3,-benzoxadiazole (ABD-F) and subsequent enzymic digestion with trypsin. N-terminal amino acid sequence analysis of the fluorescent peptides showed that subunit 20 had only two cysteine residues, one in the N-terminal region and the other in the C-terminal domain.     

Seed globulins of Cajanus cajan

Seed globulins ofCajanus cajan a widely cultivated legume were purified and characterised. About 78% of the seed proteins were salt soluble, out of which 61% were globulins which were further separated into three fractions. The fraction was insoluble at pH 4.7 and consisted of two subfractions. Fraction and were soluble at pH 4.7. All the fractions were characterised as glycoproteins by cesium chloride centrifugation. The proteins consisted of subunits which were not held together by covalent disulphide linkages. Amino acid analysis of the different globulin fractions showed that the fraction was comparatively rich in sulphur amino acids.     

RP–HPLC and Capillary Electrophoresis of Subunits from Glutenin Isolated by SDS and Osborne Fractionation

The polypeptide subunits present in SDS-unextractable glutenin, the glutenin macropolymer (GMP) and the 70% (v/v) ethanol unextractable protein, the Osborne glutenin fraction, of various cultivars were separated by RP–HPLC and capillary electrophoresis (CE) under denaturing (urea and SDS, respectively) and reducing conditions. In addition, the SDS-extractable protein was separated by CE. HighM_rglutenin subunits were well separated by CE, while the separation of lowM_rglutenin subunits was better by RP–HPLC. HighM_rglutenin subunits separated by RP–HPLC were collected and separated by CE. The subunits were identified unequivocally using the combined information from these two techniques and from SDS–PAGE patterns using the cvs. Spring and Troy Spring. By both RP–HPLC and CE it could be demonstrated for flour from three wheat cvs. (Camp Remy, Obelisk and Rektor) and a blend of flour from two of those cvs. (Camp Remy/Obelisk) that the highM_rglutenin subunit content of the GMP was 29–31%. In contrast, the SDS-extractable protein consisted of 4–6% highM_rglutenin subunits, which accounted for 14–23% of the highM_rglutenin subunits in flour. Interestingly, the SDS-extractable highM_rglutenin subunits consisted mainly (90–96%) of x-type subunits whereas, in the GMP, only 70–75% of the highM_rsubunits were x-type subunits. Although the SDS extractable protein was not separated by RP–HPLC, results similar to those obtained by CE could be inferred from the subtraction of the contents of glutenin subunits of the GMP from the contents in the Osborne glutenin fraction. The results suggest that x- and y-type highM_rglutenin subunits may have a different role in the structure (size and composition) of glutenin polymers.     

Quality-related Epitopes of High Mr Subunits of Wheat Glutenin

Three hundred and eighty four immobilised overlapping nonapeptides, corresponding to the full amino acid sequences of three high M_r subunits of glutenin from bread wheat (Triticum aestivum) grain, were used to determine the linear epitopes recognised by four monoclonal antibodies. These antibodies were selected on the basis of significant and positive correlations between their binding to wheat flour extracts in a two-site ('sandwich') enzyme immunoassay and rheological measures of dough strength, an important aspect of bread wheat quality. The antibodies did not bind to a single, specific sequence but bound a series of related peptides in each high M_r glutenin subunit examined. The sequences recognised were not identical for the four antibodies, but in each case were in the central repeating domain of the high M_r glutenin subunits, and usually comprised regions that overlapped the degenerate repeat nonamer and hexamer sequences. High M_r glutenin subunits that have been associated with greater dough strength, such as the D-genome allelic products 1Dx5 and 1Dy10, displayed an increased number of the epitope sequences. The location of the epitopes in sequences of overlapping β-turns in the repetitive region supports the hypothesis that dough elasticity arises partly from β-turn-forming secondary structure in the repeat regions of the M_r glutenin subunits. Additional β-turn within high M_r subunits may extend their structure to allow increased interaction between the glutenin subunits and with the other proteins of the gluten complex, thus improving dough strength.     

Antioxidative properties of partially purified barley hordein, rice bran protein fractions and their hydrolysates

Antioxidative properties of proteins from barley and rice bran and their hydrolysates were examined. Three major hordein fractions of barley, B, C and D hordeins, were partially purified by gel filtration. Albumin, globulin, prolamin and glutelin fractions of rice bran were fractioned by the Osborne method. Hydrolysates of these protein fractions were prepared by digesting with pepsin followed by trypsin. Antioxidant properties in terms of antioxidative activity against linoleic acid peroxidation and reducing activity without the lipid adjuvant were investigated. The globulin fraction from rice bran protein revealed the strongest antioxidative activity throughout the incubation time of 7 days (p ≤ 0.05). The albumin fraction of rice bran protein showed the highest reducing activity (6964 μmol of Fe²⁺) followed by globulin, prolamin, glutelin and hordein fractions with activities of 2904, 2017, 1809 and 1333 μmol of Fe²⁺, respectively (p ≤ 0.05). Partially purified C hordein exhibited the highest reducing activity compared with B and D hordeins. Protein hydrolysates obtained after digestion with pepsin and trypsin exhibited much greater antioxidative, as well as reducing, activities than those from before digestion.     

Identification of novel glutelin subunits and a comparison of glutelin composition between japonica and indica rice (Oryza sativa L.)

Glutelin, a major protein in rice grains, is encoded by a multigene family. However, its protein composition is not well characterised. Here, we identified and characterised two novel glutelin subunits, GluBX and GluC. The individual glutelin subunits of japonica cv. Nipponbare and indica cv. 93-11 rice were analysed using 2-dimensional gel electrophoresis, LC–MS/MS, and Western blotting. Comparison of the glutelin profiles between three japonica and three indica cultivars indicated two distinct subunits (GluA-1 and GluA-3 isomers) and a distinction in the subunit composition (notably GluA-3 and Lys-rich GluB-1 components) of these two subspecies. Sequence alignment revealed different nutritional (Lys residues) and functional (Cys residues) characteristics between the type-A and type-B glutelin subfamilies. We also analysed amino acid and total protein contents of the grains in thirty-five cultivars, and we demonstrated that the Lys-rich glutelin composition of indica cultivars is superior to that of japonica cultivars. The Lys-rich and Cys-poor GluBX subunit is a native protein and is a high nutritional protein in grains. Our combined approaches for the identification of glutelin subunits have revealed the nutritional characteristics of individual subunits in rice, and this knowledge will provide new insights for improving grain quality during rice breeding.