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Norimoto Murai - One of the best experts on this subject based on the ideXlab platform.

  • trimeric glycoProteins of bean Seed Storage Protein phaseolin were purified from baculovirus infected insect sf9 cells for use of structural study
    Plant Science, 2010
    Co-Authors: Kazuo Miyairi, Takeo Harada, Norimoto Murai
    Abstract:

    Abstract We demonstrated here insect Sf9 cells provide a useful source of a large quantity of homogeneous plant Protein to be used for further structural analysis by X-ray crystallography and other biophysical probes. The Seed Storage Protein phaseolin accumulates in common bean (Phaseolus vulgaris L.) as a trimeric glycoProtein in the vacuolar Protein bodies of developing cotyledon. Here we characterized the post-translational modifications of phaseolin, glycosylation and trimer formation, after expression in Sf9 cells of fall armyworm (Spodoptera frugiperda) infected by baculovirus. When a cDNA for the mature phaseolin Protein (without its own signal peptide) was placed under control of the signal peptide of viral Protein GP67 in baculovirus transfer vectors pAcGP67A, phaseolin accumulated within cells at a high level (40 μg/mL). To facilitate the Protein purification, six histidines were added to the carboxyl terminal of phaseolin coding sequence as a metal-chelating affinity tag. Phaseolin was extracted from Sf9 cells by 6.0 M guanidinium chloride or 4.0 M urea as a Protein solubilizing agent not as a denaturant, and purified by step-wise elution from a nickel column. Phaseolin was modified by a high-mannose glycan at two potential glycosylation-sties in insect Sf9 cells as demonstrated by digestion with endoglycosidase H or peptide N-glycosidase F. Asn228 and Asn317 of two potential glycosylation-sites were converted either singly or simultaneously to Glu by site-directed mutagenesis of the cDNA. Similar amounts of wild-type and glycosylation-minus mutants were purified from Sf9 cells. Analytical equilibrium centrifugation analysis demonstrated trimer formation of both wild-type and glycosylation-minus phaseolin. The results indicate that glycosylation is not required for the Protein stability or trimer formation of phaseolin. When phaseolin was expressed under control of its own signal peptide in a second transfection vector pAcSG2, phaseolin was accumulated within cells similarly to the first constructs. However, elimination of two but not one glycosilation-sites resulted in the endoproteolytic cleavage(s) of the mature Protein. Circular dichroism analysis indicated the proper secondary structure formation of phaseolin in insect Sf9 cells. Taken together, phaseolin was glycosylated, folded into the proper tertiary structure, and assembled into a trimer in insect Sf9 cells.

  • a novel basic region helix loop helix Protein binds to a g box motif cacgtg of the bean Seed Storage Protein β phaseolin gene
    Plant Science, 1996
    Co-Authors: Yasushi Kawagoe, Norimoto Murai
    Abstract:

    Expression of the bean Seed Storage Protein fl-phaseolin is under strict developmental control primarily at the level of transcription. A G-box motif CACGTG has been shown to be a major positive &-acting element of the /3-phuseolit1 gene and to be recognized by a DNA binding Protein from bean Seed nuclei. To understand the molecular nature of the G-box-binding Protein, we screened a cDNA expression library from immature bean Seed and identified a positive clone based on binding activity of the Proteins expressed in Escherichia coli to an oligodeoxyribonucleotide probe. DNA sequence analysis showed that the cDNA for the phaseolin G-box-binding Protein (PGl) encodes a basic regionhelix-loop-h&x (bHLH) domain hear the carboxyl terminus. Sequence comparison with other known plant bHLH Proteins such as maize R/B and Antirrhinum DEL suggests that PGl represents a new member of bHLH Protein family in plants. Electrophoresis mobility-shift and DNase I footprinting assays demonstrated that PGl Protein binds preferentially to the G-box (CACGTG) and not to other two phaseolin E-box motifs (CACCTG and CATATG). Expression of PGl RNA was detzble in all stages of Seed development as well as in flower, roxand leaf. Wediscuss the implications of the bHLH Protein for transcriptional regulation of the /3-phaseolin gene.

  • synergism between cacgtg g box and cacctg cis elements is required for activation of the bean Seed Storage Protein β phaseolin gene
    Plant Journal, 1994
    Co-Authors: Yasushi Kawagoe, Bruce R Campell, Norimoto Murai
    Abstract:

    Summary Expression of bean Seed Storage Protein phaseolin is under strict developmental control. Four distinct nuclear Proteins recognize in vitro the proximal, β-phaseolin} promoter (−295/+45) which confers spatial and temporal regulation of the native gene. Functional significance of these Protein-binding sites was evaluated by substitution mutation of the motifs in the promoter, which was fused to GUS reporter gene, and subsequent transient gene expression assay using protoplasts from developing bean cotyledons. DNA-binding Protein CAN binds three CANNTG motifs, CACGTG (−248/−243), CACCTG (−163/−158), and CATATG (−100/−95). Substitution mutation of the CACGTG motif, which is commonly known as G-box, reduced the −295 promoter activity by 75%, indicating that the G-box is a major positive cis-element. Mutation analyses also demonstrated that the CACCTG and CATATG motifs act as positive and negative cis-elements, respectively. Substitution mutation of all three CANNTG motifs essentially eliminated the −295 promoter activity. A construct containing the G-box and CACCTG motif resulted in a transcriptional level that is much greater than the sum of the transcriptional levels from the individual cis-elements, demonstrating that the G-box and CACCTG act synergistically. Substitution mutations of two AT-rich sequences, to which a nuclear Protein AG-1 binds, showed that these sites function as major negative (−376/−367, −356/−347) or positive (−191/−182) cis-elements, and that the effect of the two AG-1 binding sites was counteractive in the −391 promoter. These results indicate that the three CANNTG motifs and two AG-1-binding sites play critical roles in transcription of the {β-phaseolin} gene in cotyledons.

  • strategies for selecting mutation sites for methionine enhancement in the bean Seed Storage Protein phaseolin
    Journal of Protein Chemistry, 1993
    Co-Authors: John M Dyer, Jeffrey W Nelson, Norimoto Murai
    Abstract:

    The complete three-dimensional structure of the bean Seed Storage Protein phaseolin was generated from α-carbon coordinates by using molecular mechanic calculations. This structure was used as a template to simulate modifications aimed at increasing the methionine content of phaseolin. A hydrophilic, methionine-rich looping insert sequence was designed. Simulated mutagenesis shows that the insert might be accommodated in turn and loop regions of the Protein, but not within an α-helix. Methionine content was also increased by the replacement of hydrophobic amino acids with methionine in the central core β-barrels of the phaseolin Protein. Calculations indicated that methionine can effectively replace conserved or variant leucine, isolecuine, and valine residues. However, alanine residues were much more sensitive to substitution, and demonstrated high variability in the effects of methionine replacement. Introduction of multiple substitutions in the barrel interior demonstrated that the replaced residues could interact favorably to relieve local perturbations caused by individual substitutions. Molecular dynamics simulations were also utilized to study the structural organization of phaseolin. The calculations indicate that there are extensive packing interactions between the major domains of phaseolin, which have important implications for Protein folding and stability. Since the proposed mutant Proteins can be produced and studied, the results presented here provide an ideal test to determine if there is a correlation between the effects obtained by computer simulation and the effects of the mutations on the Protein structure expressedin vivo.

  • 5 distal and proximal cis acting regulator elements are required for developmental control of a rice Seed Storage Protein glutelin gene
    Plant Journal, 1993
    Co-Authors: Zhenwei Zheng, Yasushi Kawagoe, Shouhua Xiao, Thomas W Okita, Toan Lam Hau, Angy Lin, Norimoto Murai
    Abstract:

    Using a homologous transgenic rice system it is demonstrated that 5' distal and proximal cis-acting transcriptional regulatory elements are required for developmental control of a rice Seed Storage Protein glutelin gene. Analyses of gene expression of nine progressively truncated 5' promoter sequences in developing endosperm indicated the existence of at least one major positive element located from the -5.1 to -1.8 kb region. The functional importance of proximal elements in the context of 1.8 kb promoter was demonstrated by single substitution mutations in the TATA box (-28/-23), AACA motif (-73/-61), and Protein-binding boxes I (-103/-86), II (-124/-110), III (-175/-158) and IV (-200/-217). A simultaneous mutation of five Protein-binding sites (-410/-86) essentially eliminated the activity of the 1.8 kb promoter. Although temporal control of the Gt1 gene during endosperm development was retained in plants of constructs from -5.1 kb to -155 bp, spatial control of the glutelin gene was altered when the 5.1 kb promoter was deleted to -507 bp or -154 bp as the reporter gene activities of these constructs were detected in phloem of leaves, and in stems, sheaths and roots of plants.

Fumio Takaiwa - One of the best experts on this subject based on the ideXlab platform.

  • reduction of 13 kd prolamins increases recombinant Protein yield and recovery rate in rice endosperm
    Plant Signaling & Behavior, 2012
    Co-Authors: Taiji Kawakatsu, Fumio Takaiwa
    Abstract:

    RNA silencing inducible sequence (RSIS) causes post-transcriptional gene silencing (PTGS) of 5' or 3' flanking sequence-containing genes by inhibiting proper transcriptional termination. Exploiting this nature, 13 kD Pro-less, in which major Seed Storage Protein (SSP) 13 kD prolamins are reduced, has been generated. In 13 kD Pro-less, other SSPs, such as glutelins, are increased as a compensation effect to maintain amino acid pool. 7Crp is the seven-linked epitope peptide derived from major cedar pollen allergens Cry j 1 and Cry j 2. When 7Crp is expressed in 13 kD Pro-less endosperm, accumulation level of 7Crp increased. Furthermore, recovery rate of 7Crp without reducing reagent increased. These findings indicate that 13 kD Pro-less endosperm provides a good production platform for recombinant Proteins.

  • expression of er quality control related genes in response to changes in bip1 levels in developing rice endosperm
    Plant Journal, 2011
    Co-Authors: Yuhya Wakasa, Lijun Yang, Taiji Kawakatsu, Sakiko Hirose, Hiroshi Yasuda, Youko Oono, Hideyuki Takahashi, Shimpei Hayashi, Fumio Takaiwa
    Abstract:

    Binding Protein (BiP) is the key chaperone involved in folding of secretory Proteins such as Seed Storage Proteins in the ER lumen. To obtain functional information about BiP1, a gene that is predominantly expressed during rice Seed maturation, we generated several transgenic rice plants in which various levels of BiP1 Protein accumulated in an endosperm-specific manner. Severe suppression (BiP1 KD) or significant over-expression (BiP1 OEmax) of BiP1 not only altered Seed phenotype and the intracellular structure of endosperm cells, but also reduced Seed Storage Protein content, starch accumulation and grain weight. Microarray and RT-PCR analyses indicated that expression of many chaperone and co-chaperone genes was induced in transgenic plants, with more prominent expression in the BiP1 KD line than in the BiP1 OEmax line. Transcriptional induction of most chaperones was observed in calli treated with dithiothreitol or tunicamycin, treatments that trigger ER stress, indicating that induction of the chaperone genes in transgenic rice was caused by an ER stress response. In transient assays using rice protoplasts, the ortholog (Os06g0622700) of the AtbZIP60 transcription factor was shown to be involved in activation of some chaperone genes. Slight increases in the BiP1 level compared with wild-type, accompanied by increased levels of calnexin and Protein disulfide isomerase-like Proteins, resulted in significant enhancement of Seed Storage Protein content, without any change in intracellular structure or Seed phenotype. Judicious modification of BiP1 levels in transgenic rice can provide suitable conditions for the production of secretory Proteins by alleviating ER stress.

  • reducing rice Seed Storage Protein accumulation leads to changes in nutrient quality and Storage organelle formation
    Plant Physiology, 2010
    Co-Authors: Taiji Kawakatsu, Sakiko Hirose, Hiroshi Yasuda, Fumio Takaiwa
    Abstract:

    Rice (Oryza sativa) Seed Storage Proteins (SSPs) are synthesized and deposited in Storage organelles in the endosperm during Seed maturation as a nitrogen source for germinating Seedlings. We have generated glutelin, globulin, and prolamin knockdown lines and have examined their effects on Seed quality. A reduction of one or a few SSP(s) was compensated for by increases in other SSPs at both the mRNA and Protein levels. Especially, reduction of glutelins or sulfur-rich 10-kD prolamin levels was preferentially compensated by sulfur-poor or other sulfur-rich prolamins, respectively, indicating that sulfur-containing amino acids are involved in regulating SSP composition. Furthermore, a reduction in the levels of 13-kD prolamin resulted in enhancement of the total lysine content by 56% when compared with the wild type. This observation can be mainly accounted for by the increase in lysine-rich Proteins. Although reducing the level of glutelins slightly decreased Protein Storage vacuoles (PSVs), the simultaneous reduction of glutelin and globulin levels altered the inner structure of PSVs, implicating globulin in framing PSV formation. Knock down of 13-kD prolamins not only reduced the size of endoplasmic reticulum-derived Protein bodies (PBs) but also altered the rugged peripheral structure. In contrast, PBs became slightly smaller or unchanged by severe suppression of 10- or 16-kD prolamins, respectively, indicating that individual prolamins have distinct functions in the formation of PBs. Extreme increases or decreases in sulfur-poor prolamins resulted in the production of small PBs, suggesting that the ratio of individual prolamins is crucial for proper aggregation and folding of prolamins.

  • cereal Seed Storage Protein synthesis fundamental processes for recombinant Protein production in cereal grains
    Plant Biotechnology Journal, 2010
    Co-Authors: Taiji Kawakatsu, Fumio Takaiwa
    Abstract:

    Cereal Seeds provide an ideal production platform for high-value products such as pharmaceuticals and industrial materials because Seeds have ample and stable space for the deposition of recombinant products without loss of activity at room. Seed Storage Proteins (SSPs) are predominantly synthesized and stably accumulated in maturing endosperm tissue. Therefore, understanding the molecular mechanisms regulating SSP expression and accumulation is expected to provide valuable information for producing higher amounts of recombinant products. SSP levels are regulated by several steps at the transcriptional (promoters, transcription factors), translational and post-translational levels (modification, processing trafficking, and deposition). Our objective is to develop a Seed production platform capable of producing very high yields of recombinant product. Towards this goal, we review here the individual regulatory steps controlling SSP synthesis and accumulation.

  • a rice based edible vaccine expressing multiple t cell epitopes induces oral tolerance for inhibition of th2 mediated ige responses
    Proceedings of the National Academy of Sciences of the United States of America, 2005
    Co-Authors: Hidenori Takagi, Lijun Yang, Takachika Hiroi, Yoshikazu Yuki, Hiroshi Kiyono, Yoshifumi Tada, Kaoru Takamura, Ryotaro Ishimitsu, Hideyuki Kawauchi, Fumio Takaiwa
    Abstract:

    Peptide immunotherapy using multiple predominant allergen-specific T cell epitopes is a safe and promising strategy for the control of type I allergy. In this study, we developed transgenic rice plants expressing mouse dominant T cell epitope peptides of Cry j I and Cry j II allergens of Japanese cedar pollen as a fusion Protein with the soybean Seed Storage Protein glycinin. Under the control of the rice Seed Storage Protein glutelin GluB-1 promoter, the fusion Protein was specifically expressed and accumulated in Seeds at a level of 0.5% of the total Seed Protein. Oral feeding to mice of transgenic rice Seeds expressing the T cell epitope peptides of Cry j I and Cry j II before systemic challenge with total Protein of cedar pollen inhibited the development of allergen-specific serum IgE and IgG antibody and CD4+ T cell proliferative responses. The levels of allergen-specific CD4+ T cell-derived allergy-associated T helper 2 cytokine production of IL-4, IL-5, and IL-13 and histamine release in serum were significantly decreased. Moreover, the development of pollen-induced clinical symptoms was inhibited in our experimental sneezing mouse model. These results indicate the potential of transgenic rice Seeds in production and mucosal delivery of allergen-specific T cell epitope peptides for the induction of oral tolerance to pollen allergens.

Joachim Messing - One of the best experts on this subject based on the ideXlab platform.

  • mutation in the Seed Storage Protein kafirin creates a high value food trait in sorghum
    Nature Communications, 2013
    Co-Authors: Lingling Yuan, Xiaomei Guo, David R Holding, Joachim Messing
    Abstract:

    Sustainable food production for the earth's fast-growing population is a major challenge for breeding new high-yielding crops, but enhancing the nutritional quality of staple crops can potentially offset limitations associated with yield increases. Sorghum has immense value as a staple food item for humans in Africa, but it is poorly digested. Although a mutant exhibiting high-Protein digestibility and lysine content has market potential, the molecular nature of the mutation is previously unknown. Here, building on knowledge from maize mutants, we take a direct approach and find that the high-digestible sorghum phenotype is tightly linked to a single-point mutation, rendering the signal peptide of a Seed Storage Protein kafirin resistant to processing, indirectly reducing lysine-poor kafirins and thereby increasing lysine-rich Proteins in the Seeds. These findings indicate that a molecular marker can be used to accelerate introduction of this high nutrition and digestibility trait into different sorghum varieties.

  • mutation in the Seed Storage Protein kafirin creates a high value food trait in sorghum
    Nature Communications, 2013
    Co-Authors: Yongrui Wu, Lingling Yuan, David R Holding, Joachim Messing
    Abstract:

    Sorghum is an important crop species for the African continent but the grain is difficult to digest. The authors of this study examine a previously identified sorghum mutant known for improved digestion and find that the mutant is the result of a point mutation in the Seed-processing Protein kafirin.

Satoshi Naito - One of the best experts on this subject based on the ideXlab platform.

  • role of o acetyl l serine in the coordinated regulation of the expression of a soybean Seed Storage Protein gene by sulfur and nitrogen nutrition
    Planta, 1999
    Co-Authors: Hoyeun Kim, Satoshi Naito, Masami Yokota Hirai, Mitsuo Chino, Hiroaki Hayashi, Toru Fujiwara
    Abstract:

    The composition of Seed Storage Proteins is regulated by sulfur and nitrogen supplies. Under conditions of a low sulfur-to-nitrogen ratio, accumulation of the β-subunit of β-conglycinin, a sulfur-poor Seed Storage Protein of soybean (Glycine max [L.] Merr.), is elevated, whereas that of glycinin, a sulfur-rich Storage Protein, is reduced. Using transgenic Arabidopsis thaliana [L.] Heynh., it was found that the promoter from the gene encoding the β-subunit of β-conglycinin up-regulates gene expression under sulfur deficiency and down-regulates gene expression under nitrogen deficiency. To obtain an insight into the metabolic control of this regulation, the concentrations of metabolites related to the sulfur assimilation pathway were determined. Among the metabolites, O-acetyl-l-serine (OAS), one of the precursors of cysteine biosynthesis, accumulated to higher levels under low-sulfur and high-nitrogen conditions in siliques of transgenic A. thaliana. The pattern of OAS accumulation in response to various levels of sulfur and nitrogen was similar to that of gene expression driven by the β-subunit promoter. Elevated levels of OAS accumulation were also observed in soybean cotyledons cultured under sulfur deficiency. Moreover, OAS applied to in-vitro cultures of immature soybean cotyledons under normal sulfate conditions resulted in a high accumulation of the β-subunit mRNA and Protein, whereas the accumulation of glycinin was reduced. These changes were very similar to the responses observed under conditions of sulfur deficiency. Our results suggest that the level of free OAS mediates sulfur- and nitrogen-regulation of soybean Seed Storage-Protein composition.

  • expression of a soybean glycine max l merr Seed Storage Protein gene in transgenic arabidopsis thaliana and its response to nutritional stress and to abscisic acid mutations
    Plant Physiology, 1994
    Co-Authors: Satoshi Naito, Masami Yokota Hirai, Mitsuo Chino, Yoshibumi Komeda
    Abstract:

    Among the three subunits of [beta]-conglycinin, the 7S Seed Storage Protein of soybean (Glycine max [L.] Merr.), expression of the [beta] subunit gene is unique. Accumulation of the [beta] subunit is enhanced in sulfate-deficient soybean plants, and its mRNA levels increase when abscisic acid (ABA) is added to the in vitro cotyledon culture medium. Transgenic Arabidopsis thaliana lines carrying a gene encoding the [beta] subunit was constructed and grown under sulfate deficiency. Accumulation of both [beta] subunit mRNA and Protein were enhanced in developing A. thaliana Seeds. Accumulation of one of the A. thaliana Seed Storage Protein mRNAs was also enhanced by sulfate deficiency, although the response was weaker than that observed for the soybean [beta] subunit mRNA. When the aba1–1 or abi3–1 mutations were crossed into the transgenic A. thaliana line, accumulation of the [beta] subunit was significantly reduced, whereas accumulation of the A. thaliana Seed Storage Protein was not greatly affected. These results indicate that soybean and A. thaliana share a common mechanism for response to sulfate deficiency and to ABA, although the sensitivity is different between the species. The transgenic A. thaliana carrying the [beta] subunit gene of [beta]-conglycinin will be a good system to analyze these responses.

  • Effects of Sulfur Nutrition on Expression of the Soybean Seed Storage Protein Genes in Transgenic Petunia
    Plant physiology, 1992
    Co-Authors: Toru Fujiwara, Mitsuo Chino, Masami Yokota Hirai, Yoshibumi Komeda, Satoshi Naito
    Abstract:

    The 7S Seed Storage Protein (beta-conglycinin) of soybean (Glycine max [L]. Merr.) has three major subunits; alpha, alpha', and beta. Accumulation of the beta-subunit, but not the alpha- and alpha'-subunits, has been shown to be repressed by exogenously applied methionine to the immature cotyledon culture system (LP Holowach, JF Thompson, JT Madison [1984] Plant Physiol 74: 576-583) and to be enhanced under sulfate deficiency in soybean plants (KR Gayler, GE Sykes [1985] Plant Physiol 78: 582-585). Transgenic petunia (Petunia hybrida) harboring either the alpha'- or beta-subunit gene were constructed to test whether the patterns of differential expression were retained in petunia. Petunia regulates these genes in a similar way as soybean in response to sulfur nutritional stimuli, i.e. (a) expression of the beta-subunit gene is repressed by exogenous methionine in in vitro cultured Seeds, whereas the alpha'-subunit gene expression is not affected; and (b) accumulation of the beta-subunit is enhanced by sulfur deficiency. The pattern of accumulation of major Seed Storage Protein of petunia was not affected by these treatments. These results indicate that this mechanism of gene regulation in response to sulfur nutrition is conserved in petunia even though it is not used to regulate its own major Seed Storage Proteins.

  • effects of sulfur nutrition on expression of the soybean Seed Storage Protein genes in transgenic petunia
    Plant Physiology, 1992
    Co-Authors: Toru Fujiwara, Mitsuo Chino, Masami Yokota Hirai, Yoshibumi Komeda, Satoshi Naito
    Abstract:

    The 7S Seed Storage Protein (β-conglycinin) of soybean (Glycine max [L]. Merr.) has three major subunits; α, α′, and β. Accumulation of the β-subunit, but not the α- and α′-subunits, has been shown to be repressed by exogenously applied methionine to the immature cotyledon culture system (LP Holowach, JF Thompson, JT Madison [1984] Plant Physiol 74: 576-583) and to be enhanced under sulfate deficiency in soybean plants (KR Gayler, GE Sykes [1985] Plant Physiol 78: 582-585). Transgenic petunia (Petunia hybrida) harboring either the α′- or β-subunit gene were constructed to test whether the patterns of differential expression were retained in petunia. Petunia regulates these genes in a similar way as soybean in response to sulfur nutritional stimuli, i.e. (a) expression of the β-subunit gene is repressed by exogenous methionine in in vitro cultured Seeds, whereas the α′-subunit gene expression is not affected; and (b) accumulation of the β-subunit is enhanced by sulfur deficiency. The pattern of accumulation of major Seed Storage Protein of petunia was not affected by these treatments. These results indicate that this mechanism of gene regulation in response to sulfur nutrition is conserved in petunia even though it is not used to regulate its own major Seed Storage Proteins.

N Murai - One of the best experts on this subject based on the ideXlab platform.

  • the bean Seed Storage Protein beta phaseolin is synthesized processed and accumulated in the vacuolar type ii Protein bodies of transgenic rice endosperm
    Plant Physiology, 1995
    Co-Authors: Zhenwei Zheng, K Sumi, K Tanaka, N Murai
    Abstract:

    The Seed Storage Protein [beta]-phaseolin of the common bean (Phaseolus vulgaris L.) was expressed in the endosperm of transgenic rice (Oryza sativa L.) plants. The 5.1- or 1.8-kb promoter fragment of the rice Seed Storage Protein glutelin Gt1 gene was fused transcriptionally to either the genomic or cDNA coding sequence of the [beta]-phaseolin gene. The highest quantity of phaseolin estimated by enzyme-linked immunosorbent assay was 4.0% of the total endosperm Protein in the transgenic rice Seeds. The phaseolin trait was segregated as a single dominant trait with a positive gene dosage effect and was stably inherited through three successive generations. Both phaseolin genomic and cDNA coding sequences were used to synthesize four isoforms of mature phaseolin Protein with apparent molecular masses of 51, 48, 47, and 45 kD. Enzyme deglycosylation experiments indicated that the 51-kD form contains high-mannose N-glycans; the 48- and 47-kD forms have further modified N-glycans; and the 45-kD form is a nonglycosylated Protein. Immunolabeling studies using light and electron microscopy demonstrated that phaseolin accumulates primarily in the vacuolar type-II Protein bodies located at the periphery of the endosperm near the aleurone layer. We discuss the implications of these results on nutritional improvement of rice grains.