Elimination of zero-repeat subunit in allergenic seed protein 13S globulin using the novel allele GlbNB2 in common buckwheat (Fagopyrum esculentum Moench).

Common buckwheat (Fagopyrum esculentum Moench) seeds contain 13S globulin, the zero-repeat subunit of which is trypsin-resistant and allergenic. Here, its two novel alleles were analyzed for development of hypoallergenic plants. The GlbNC allele has a Miniature Inverted-repeat Transposable Element (MITE)-like insertion in the 4th exon. However, most of the insertion was spliced-out, resulting in accumulation of zero-repeat subunit in GlbNC homozygotes. Meanwhile, the GlbNB2 has a 164-bp insertion in the 3rd exon, resulting in no accumulation of zero-repeat subunit in GlbNB2 homozygotes (NB2_homo). Both the insertion sequences were predicted to form a hairpin-like structure, and that of GlbNB2 was more rigid than that of GlbNC. Trypsin digestion in NB2_homo showed that the α polypeptide of Met-rich subunit is also hard to digest, that is a next target to eliminate for hypoallergenic buckwheat development.

Deciphering proteomic mechanisms explaining the role of glutathione as an aid in improving plant fitness and tolerance against cadmium-toxicity in Brassica napus L.

Cadmium (Cd) hazard is a serious limitation to plants, soils and environments. Cd-toxicity causes stunted growth, chlorosis, necrosis, and plant yield loss. Thus, ecofriendly strategies with understanding of molecular mechanisms of Cd-tolerance in plants is highly demandable. The Cd-toxicity caused plant growth retardation, leaf chlorosis and cellular damages, where the glutathione (GSH) enhanced plant fitness and Cd-toxicity in Brassica through Cd accumulation and antioxidant defense. A high-throughput proteome approach screened 4947 proteins, wherein 370 were differently abundant, 164 were upregulated and 206 were downregulated. These proteins involved in energy and carbohydrate metabolism, CO2 assimilation and photosynthesis, signal transduction and protein metabolism, antioxidant defense response, heavy metal detoxification, cytoskeleton and cell wall structure, and plant development in Brassica. Interestingly, several key proteins including glutathione S-transferase F9 (A0A078GBY1), ATP sulfurylase 2 (A0A078GW82), cystine lyase CORI3 (A0A078FC13), ferredoxin-dependent glutamate synthase 1 (A0A078HXC0), glutaredoxin-C5 (A0A078ILU9), glutaredoxin-C2 (A0A078HHH4) actively involved in antioxidant defense and sulfur assimilation-mediated Cd detoxification process confirmed by their interactome analyses. These candidate proteins shared common gene networks associated with plant fitness, Cd-detoxification and tolerance in Brassica. The proteome insights may encourage breeders for enhancing multi-omics assisted Cd-tolerance in Brassica, and GSH-mediated hazard free oil seed crop production for global food security.

Proteome insights of citric acid-mediated cadmium toxicity tolerance in Brassica napus L.

Cadmium (Cd) is a toxic substance that is uptake by plants from soils, Cd easily transfers into the food chain. Considering global food security, eco-friendly, cost-effective, and metal detoxification strategies are highly demandable for sustainable food crop production. The purpose of this study was to investigate how citric acid (CA) alleviates or tolerates Cd toxicity in Brassica using a proteome approach. In this study, the global proteome level was significantly altered under Cd toxicity with or without CA supplementation in Brassica. A total of 4947 proteins were identified using the gel-free proteome approach. Out of these, 476 proteins showed differential abundance between the treatment groups, wherein 316 were upregulated and 160 were downregulated. The gene ontology analysis reveals that differentially abundant proteins were involved in different biological processes including energy and carbohydrate metabolism, CO2 assimilation and photosynthesis, signal transduction and protein metabolism, antioxidant defense, heavy metal detoxification, plant development, and cytoskeleton and cell wall structure in Brassica leaves. Interestingly, several candidate proteins such as superoxide dismutase (A0A078GZ68) L-ascorbate peroxidase 3 (A0A078HSG4), glutamine synthetase (A0A078HLB2), glutathione S-transferase DHAR1 (A0A078HPN8), glutamine synthetase (A0A078HLB2), cysteine synthase (A0A078GAD3), S-adenosylmethionine synthase 2 (A0A078JDL6), and thiosulfate/3-mercaptopyruvate sulfur transferase 2 (A0A078H905) were involved in antioxidant defense system and sulfur assimilation-involving Cd-detoxification process in Brassica. These findings provide new proteome insights into CA-mediated Cd-toxicity alleviation in Brassica, which might be useful to oilseed crop breeders for enhancing heavy metal tolerance in Brassica using the breeding program, with sustainable and smart Brassica production in a metal-toxic environment.

Genome sequencing reveals the genetic architecture of heterostyly and domestication history of common buckwheat.

Common buckwheat, Fagopyrum esculentum, is an orphan crop domesticated in southwest China that exhibits heterostylous self-incompatibility. Here we present chromosome-scale assemblies of a self-compatible F. esculentum accession and a self-compatible wild relative, Fagopyrum homotropicum, together with the resequencing of 104 wild and cultivated F. esculentum accessions. Using these genomic data, we report the roles of transposable elements and whole-genome duplications in the evolution of Fagopyrum. In addition, we show that (1) the breakdown of heterostyly occurs through the disruption of a hemizygous gene jointly regulating the style length and female compatibility and (2) southeast Tibet was involved in common buckwheat domestication. Moreover, we obtained mutants conferring the waxy phenotype for the first time in buckwheat. These findings demonstrate the utility of our F. esculentum assembly as a reference genome and promise to accelerate buckwheat research and breeding.

Insertion of ten amino acids into 13S globulin zero-repeat subunit improves trypsin digestibility in common buckwheat (Fagopyrum esculentum Moench) seeds.

The 13S globulin zero-repeat subunit is resistant to trypsin and may have higher allergenicity than the 1-6 tandem repeat subunits in common buckwheat (Fagopyrum esculentum Moench). To explore alleles useful for lowering allergenicity, amplicon deep sequencing targeting the zero-repeat subunit gene was conducted in bulked genomic DNA from eight cultivars and landraces. The analysis identified a unique allele encoding a zero-repeat subunit with 10 amino acid insertion (10aa) at a position equivalent to the tandem repeat insertion. Prediction of its 3-D structure suggested that 10aa changes the ß-hairpin structure in the non-10aa (native) subunit to a random coil, which is also found in 1- and 3- repeat subunits. Homozygotes of the 10aa allele were developed and showed that the 10aa subunit was more digestible than the native subunit. However, the 10aa subunit was still less digestible than the 1-6 repeat subunits, suggesting needs to explore unfunctional alleles.

2S albumin g13 polypeptide, less related to Fag e 2, can be eliminated in common buckwheat (Fagopyrum esculentum Moench) seeds.

2S albumin (g11, g13, g14, and g28) is an important allergen in common buckwheat (Fagopyrum esculentum). g13 is hydrophobic, rare in seeds, and may show distinct allergenicity from the others; therefore, we tried to eliminate this protein. Phylogenetic and property distance analyses indicated g13 is less related to g14 (Fag e 2) than g11/g28 is related to g14, particularly in the second domain containing the II and III α-helices. A null allele with a 531 bp insertion in the coding region was found for g13 at an allele frequency of 2 % in natural populations of common buckwheat. The g13_null allele homozygote accumulated no g13 protein. A BLAST search for the 531 bp insertion suggested the insert-like sequence resided frequently in the buckwheat genome, including the self-incompatibility responsible gene ELF3 in Fagopyrum tataricum. The g13_null insert-like sequence could, therefore, help in producing hypoallergenic cultivars, and expand the genetic diversity of buckwheat.

Characterization of 2S albumin allergenic proteins for anaphylaxis in common buckwheat.

2S albumin (2SA) is responsible for anaphylaxis following consumption of buckwheat in allergic individuals. To reduce allergen incidents, characterization of 2SA polypeptides is prerequisite, thus was analyzed in this study. Of the five 2S albumin genes (g03, g11, g13, g14, and g28), g03 was seemingly non-functional. The g14 content was 3- and 40-fold higher than that of g11/g28 and g13, respectively. The g11/g28 were more processed to a ∼8 kDa band from a 16 kDa band than g14 in seeds, agreeing with that g11/g28 have high similarity with Fag e 8kD. Meanwhile, anti-g13 produced only a single ∼10 kDa band. Modification of g13 and domain exchange between g13 and g14 suggested that the hydrophobicity of the first domain and the nature of some amino acids in g13 contributed, at least in part, to the lower apparent molecular weight of g13 than expected. Thus, g13 might be an unexplored and noteworthy allergen.

High source-sink ratio at and after sink capacity formation promotes green stem disorder in soybean.

Green stem disorder (GSD) of soybean is characterized by delayed leaf and stem maturation despite normal pod maturation. Previous studies have suggested that GSD occurrence is promoted by a high source-sink ratio, which is produced by thinning or shade removal at the R5 growth stage (the beginning of seed filling). Here the effects of different times and durations of shade removal after the R5 stage on GSD severity were analyzed. First, shade removal for more than 28 days after R5 increased GSD severity by more than 0.4 point in GSD score. Thinning treatment at R5 increased specific leaf weight by 23%, suppressed stem dry weight reduction, and upregulated 19 genes including those encoding vegetative storage proteins at R5 + 28d, indicating excess source ability relative to sink size. On the contrary, shade removal for 14 days after R5 decreased GSD severity by 0.5 point in GSD score. In this treatment, seed size was smaller, while seed number was significantly larger than control, suggesting that shortage of source ability relative to sink size. These results implied that soybean plants regulate GSD occurrences either positively or negatively according to a source-sink ratio during the R5 to R5 + 28d growth stages.

Proteome Changes Reveal the Protective Roles of Exogenous Citric Acid in Alleviating Cu Toxicity in Brassica napus L.

Citric acid (CA), as an organic chelator, plays a vital role in alleviating copper (Cu) stress-mediated oxidative damage, wherein a number of molecular mechanisms alter in plants. However, it remains largely unknown how CA regulates differentially abundant proteins (DAPs) in response to Cu stress in Brassica napus L. In the present study, we aimed to investigate the proteome changes in the leaves of B. L. seedlings in response to CA-mediated alleviation of Cu stress. Exposure of 21-day-old seedlings to Cu (25 and 50 µM) and CA (1.0 mM) for 7 days exhibited a dramatic inhibition of overall growth and considerable increase in the enzymatic activities (POD, SOD, CAT). Using a label-free proteome approach, a total of 6345 proteins were identified in differentially treated leaves, from which 426 proteins were differentially expressed among the treatment groups. Gene ontology (GO) and KEGG pathways analysis revealed that most of the differential abundance proteins were found to be involved in energy and carbohydrate metabolism, photosynthesis, protein metabolism, stress and defense, metal detoxification, and cell wall reorganization. Our results suggest that the downregulation of chlorophyll biosynthetic proteins involved in photosynthesis were consistent with reduced chlorophyll content. The increased abundance of proteins involved in stress and defense indicates that these DAPs might provide significant insights into the adaptation of Brassica seedlings to Cu stress. The abundances of key proteins were further verified by monitoring the mRNA expression level of the respective transcripts. Taken together, these findings provide a potential molecular mechanism towards Cu stress tolerance and open a new route in accelerating the phytoextraction of Cu through exogenous application of CA in B. napus.

Structure and diversity of 13S globulin zero-repeat subunit, the trypsin-resistant storage protein of common buckwheat (Fagopyrum esculentum M.) seeds.

The zero-repeat subunit of 13S globulin, which lacks tandem repeat inserts, is trypsin-resistant and suggested to show higher allergenicity than the other subunits in common buckwheat (Fagopyrum esculentum Moench). To evaluate allelic variations and find novel alleles, the diversity of the zero-repeat genes was examined for two Japanese elite cultivars and 15 Pakistani landraces. The results demonstrated that two new alleles GlbNA1 and GlbNC1, plus three additional new alleles GlbNA2, GlbNA3, and GlbND, were identified besides the already-known GlbNA, GlbNB, and GlbNC alleles. In the Pakistani landraces, GlbNA was the most dominant allele (0.60-0.88 of allele frequency) in all except one landrace, where GlbNB was the most dominant allele (0.50 of allele frequency). Similar to GlbNC, the alleles GlbNA2 and GlbNA3 had extra ~200 bp MITE-like sequences around the stop codon. Secondary structure predictions of a sense strand demonstrated that the extra ~200 bp sequences of GlbNC, GlbNA2, and GlbNA3 can form rigid hairpin structures with free energies of -78.95, -67.06, and -29.90 kcal/mol, respectively. These structures may affect proper transcription and/or translation. In the GlbNC homozygous line, no transcript of a zero-repeat gene was detected, suggesting the material would be useful for developing hypoallergenic buckwheat.

FLOURY ENDOSPERM11-2 encodes plastid HSP70-2 involved with the temperature-dependent chalkiness of rice (Oryza sativa L.) grains.

The frequent occurrence of chalky rice (Oryza sativa L.) grains becomes a serious problem as a result of climate change. The molecular mechanism underlying chalkiness is largely unknown, however. In this study, the temperature-sensitive floury endosperm11-2 (flo11-2) mutant was isolated from ion beam-irradiated rice of 1116 lines. The flo11-2 mutant showed significantly higher chalkiness than the wild type grown under a mean temperature of 28°C, but similar levels of chalkiness to the wild type grown under a mean temperature of 24°C. Whole-exome sequencing of the flo11-2 mutant showed three causal gene candidates, including Os12g0244100, which encodes the plastid-localized 70-kDa heat shock protein 2 (cpHSP70-2). The cpHSP70-2 of the flo11-2 mutant has an amino acid substitution on the 259th aspartic acid with valine (D259V) in the conserved Motif 5 of the ATPase domain. Transgenic flo11-2 mutants that express the wild-type cpHSP70-2 showed significantly lower chalkiness than the flo11-2 mutant. Moreover, the accumulation level of cpHSP70-2 was negatively correlated with the chalky ratio, indicating that cpHSP70-2 is a causal gene for the chalkiness of the flo11-2 mutant. The intrinsic ATPase activity of recombinant cpHSP70-2 was lower by 23% at Vmax for the flo11-2 mutant than for the wild type. The growth of DnaK-defective Escherichia coli cells complemented with DnaK with the D201V mutation (equivalent to the D259V mutation) was severely reduced at 37°C, but not in the wild-type DnaK. The results indicate that the lowered cpHSP70-2 function is involved with the chalkiness of the flo11-2 mutant.

Possible cleavage sites of glutelin partial degradation confirmed by immunological analysis in globulin-less mutants of rice (Oryza sativa L.).

Proteolytic cleavage or partial degradation of proteins is one of the important post-translational modifications for various biological processes, but it is difficult to analyze. Previously, we demonstrated that some subunits of the major rice (Oryza sativa L.) seed storage protein glutelin are partially degraded to produce newly identified polypeptides X1-X5 in mutants in which another major seed storage protein globulin is absent. In this study, the new polypeptides X3 and X4/X5 were immunologically confirmed to be derived from GluA3 and GluA1/GluA2 subunits, respectively. Additionally, the new polypeptides X1 and X2 were at least in part the α polypeptides of the GluB4 subunit partially degraded at the C-terminus. Simulated 2D-PAGE migration patterns of intact and partially degraded α polypeptides based on the calculation of their MWs and pIs enabled us to narrow or predict the possible locations of cleavage sites. The predicted cleavage sites were also verified by the comparison of 2D-PAGE patterns between seed-extracted and E. coli-expressed proteins of the intact and truncated α polypeptides. The results and methodologies demonstrated here would be useful for analyses of partial degradation of proteins and the structure-function relationships of rice seed protein bodies.

Assembly of the draft genome of buckwheat and its applications in identifying agronomically useful genes.

Buckwheat (Fagopyrum esculentum Moench; 2n = 2x = 16) is a nutritionally dense annual crop widely grown in temperate zones. To accelerate molecular breeding programmes of this important crop, we generated a draft assembly of the buckwheat genome using short reads obtained by next-generation sequencing (NGS), and constructed the Buckwheat Genome DataBase. After assembling short reads, we determined 387,594 scaffolds as the draft genome sequence (FES_r1.0). The total length of FES_r1.0 was 1,177,687,305 bp, and the N50 of the scaffolds was 25,109 bp. Gene prediction analysis revealed 286,768 coding sequences (CDSs; FES_r1.0_cds) including those related to transposable elements. The total length of FES_r1.0_cds was 212,917,911 bp, and the N50 was 1,101 bp. Of these, the functions of 35,816 CDSs excluding those for transposable elements were annotated by BLAST analysis. To demonstrate the utility of the database, we conducted several test analyses using BLAST and keyword searches. Furthermore, we used the draft genome as a reference sequence for NGS-based markers, and successfully identified novel candidate genes controlling heteromorphic self-incompatibility of buckwheat. The database and draft genome sequence provide a valuable resource that can be used in efforts to develop buckwheat cultivars with superior agronomic traits.

Diversification of 13S globulins, allergenic seed storage proteins, of common buckwheat.

The α polypeptide of the 13S globulin subunit of common buckwheat is the counterpart of the major allergenic ß polypeptide. Trypsin digestibility varies between variants of the α polypeptide with and without a tandem repeat insert. To evaluate the intra-species diversity of 13S globulin, the comprehensive screening of a genomic DNA library was performed, resulting in the isolation of 14 and 3 genes for Met-poor and Met-rich subunits, respectively. Although most tandem repeat units were 45 bp in length, the two-repeat gene Glb2B and all one-repeat genes contained an additional 3 bp. Secondary structure predictions and polyacrylamide gel electrophoresis demonstrated that the sense strand of Glb2B-CCG, the additional 3 bp-deletion clone of Glb2B, formed a more rigid secondary structure than that of the wild-type. Thus, the large intra-species variation of 13S globulin revealed in this study and its diversification might be attributable to the unique nature of the tandem repeat sequences.

Capillary electrophoresis of seed storage proteins: the separation and identification of microheterogeneous rice glutelin subunits.

Glutelin, the major seed storage protein of rice (Oryza sativa L.), consists of multiple polymeric and monomeric subunits. Each subunit is composed of an α and a ß polypeptide that are covalently linked by a disulfide bond. To analyze the microheterogeneous glutelin subunits using capillary electrophoresis (CE), the author identified the appropriate sample preparation procedures as well as optimal CE conditions. The glutelin was dissociated into its component α and ß polypeptides by denaturation and reduction with low urea and 2-mercaptoethanol for a long incubation time at room temperature. The molecular species of the completely dissociated α and ß polypeptides were identified and quantitatively analyzed by CE and SDS-PAGE using glutelin mutants. The measured CE migration times of the polypeptides correlated well with the calculated charge-to-size parameter values. Therefore, the rapid, simple, and precise separation and quantification of microheterogeneous proteins by CE required not only optimal CE conditions but also adequate protein pretreatment based on the molecular nature of the protein tested.

RNA targeting to a specific ER sub-domain is required for efficient transport and packaging of α-globulins to the protein storage vacuole in developing rice endosperm.

Studies focusing on the targeting of RNAs that encode rice storage proteins, prolamines and glutelins to specific sub-domains of the endoplasmic reticulum (ER), as well as mis-localization studies of other storage protein RNAs, indicate a close relationship between the ER site of RNA translation and the final site of protein deposition in the endomembrane system in developing rice endosperm. In addition to prolamine and glutelin, rice accumulates smaller amounts of α-globulins, which are deposited together with glutelin in the protein storage vacuole (PSV). In situ RT-PCR analysis revealed that α-globulin RNAs are not distributed to the cisternal ER as expected for a PSV-localized protein, but instead are targeted to the protein body-ER (PB-ER) by a regulated process requiring cis-sorting sequences. Sequence alignments with putative maize δ-zein cis-localization elements identified several candidate regulatory sequences that may be responsible for PB-ER targeting. Immunocytochemical analysis confirmed the presence of α-globulin on the periphery of the prolamine protein bodies and packaging in Golgi-associated dense vesicles, as well as deposition and storage within peripheral regions of the PSV. Mis-targeting of α-globulin RNAs to the cisternal ER dramatically alters the spatial arrangement of α-globulin and glutelin within the PSV, with the accompanying presence of numerous small α-globulin particles in the cytoplasm. These results indicate that α-globulin RNA targeting to the PB-ER sub-domain is essential for efficient transport of α-globulins to the PSV and its spatial arrangement in the PSV. Such RNA localization prevents potential deleterious protein-protein interactions, in addition to performing a role in protein targeting.

Capillary electrophoresis for analysis of microheterogeneous glutelin subunits in rice (Oryza sativa L.).

Glutelin, the major storage protein of rice seed, consists of microheterogenous subunits and partially exists in a macromolecular form that is polymerized by intersubunit disulfide bonds. In order to analyze the glutelin subunits using high-throughput CE, we first identified a sample preparation procedure suitable for CE. The polymerized glutelin treated with a reductant could not dissociate into its constituent monomer subunits when it was dissolved in an acidic solution. However, the glutelin dissociated into its subunits and component α and ß polypeptides when it was denatured and reduced by an appropriate amount of urea and 2-mercaptoethanol at a specific incubation time and temperature. The molecular species of the completely dissociated α and ß polypeptides were identified and quantitatively analyzed by CE using glutelin mutants. The CE analysis also demonstrated that the actual subunit variation in terms of the charge and/or size of the ß polypeptides is much smaller than predicted when compared with that of α polypeptides, even under denaturing and reducing condition. Thus, the combined analytical system described here will be useful for basic and applied research, such as the kinetic characterization of higher-order structure and the quantitative evaluation of glutelin in a large number of diverse rice varieties.

Identification and variation of glutelin alpha polypeptides in the genus Oryza assessed by two-dimensional electrophoresis and step-by-step immunodetection.

To obtain fundamental information for nutritional improvement of rice (Oryza sativa) seed proteins, the alpha polypeptides of the major storage protein glutelin varied over the genus Oryza were qualitatively and quantitatively characterized with unique methods. The polypeptides were maximally separated by two-dimensional electrophoresis (2D-PAGE) composed of nonequilibrium pH gradient gel electrophoresis (NEPHGE) and higher temperature SDS-PAGE. Then the subunit for each polypeptide spot was identified with the sequential immunodetection called a step-by-step detection method, making use of highly subunit-specific antibodies. The comparative analysis showed considerable variation in the accumulation level of A-type and B-type glutelin subunits and found unknown glutelin subunits that were unable to be identified with the antibodies used. Wild species accumulating a high amount of lysine-rich B-type glutelin subunits and unknown unique subunits were identified as they might play a crucial role in nutritional quality improvement of the cultivated rice.

The cytoplasmic-localized, cytoskeletal-associated RNA binding protein OsTudor-SN: evidence for an essential role in storage protein RNA transport and localization.

Previous studies have demonstrated that the major storage protein RNAs found in the rice endosperm are transported as particles via actomyosin to specific subdomains of the cortical endoplasmic reticulum. In this study, we examined the potential role of OsTudor-SN, a major cytoskeletal-associated RNA binding protein, in RNA transport and localization. OsTudor-SN molecules occur as high-molecular-weight forms, the integrity of which are sensitive to RNase. Immunoprecipitation followed by RT-PCR showed that OsTudor-SN binds prolamine and glutelin RNAs. Immunofluorescence studies using affinity-purified antibodies show that OsTudor-SNs exists as particles in the cytoplasm, and are distributed to both the protein body endoplasmic reticulum (ER) and cisternal ER. Examination of OsTudor-SN particles in transgenic rice plants expressing GFP-tagged prolamine RNA transport particles showed co-localization of OsTudor-SN and GFP, suggesting a role in RNA transport. Consistent with this view, GFP-tagged OsTudor-SN is observed in living endosperm sections as moving particles, a property inhibited by microfilament inhibitors. Downregulation of OsTudor-SN by antisense and RNAi resulted in a decrease in steady state prolamine RNA and protein levels, and a reduction in the number of prolamine protein bodies. Collectively, these results show that OsTudor-SN is a component of the RNA transport particle, and may control storage protein biosynthesis by regulating one or more processes leading to the transport, localization and anchoring of their RNAs to the cortical ER.

Diversity of rice glutelin polypeptides in wild species assessed by the higher-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subunit-specific antibodies.

In efforts to find genetic resources with high nutritional value of rice seed, we assessed the diversity of the major storage protein glutelin in 13 wild and 2 cultivated rice species by a unique SDS-PAGE method and subunit-specific antibodies. Maximum separation of microheterogeneous glutelin alpha-polypeptides, which is a prerequisite for the diversity evaluation, could be attained by SDS-PAGE performed at higher temperature (45 degrees C) than the generally employed temperatures (4-25 degrees C). Seven antipeptide antibodies were raised against subunit-specific epitope sequences designed at five sites from four variable regions spanning the glutelin alpha-polypeptides. High specificity of each antibody was confirmed using rice glutelin mutants, and demonstrated considerable variation in amino acid sequence and accumulation level of glutelin subunit in wild species, in combination with the higher-temperature SDS-PAGE. The degree of the variation was, however, changed according to the site of variable regions and the type of subunit. Some wild species accumulated nutritious GluB subunits more than cultivated rice. The wild species Oryza longiglumis and O. brachyantha had glutelin with low reactivity against most antibodies examined in this study, reflecting the significant divergence. Such wild species may hopefully serve as important genetic resources for nutritional improvement of cultivated rice.