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Takehiro Masumura - One of the best experts on this subject based on the ideXlab platform.
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identification of the region of rice 13 kda Prolamin essential for the formation of er derived protein bodies using a heterologous expression system
Bioscience Biotechnology and Biochemistry, 2015Co-Authors: Takehiro Masumura, Takanari Shigemitsu, Shigeto Morita, Shigeru SatohAbstract:Cereal Prolamins, which are alcohol-soluble seed storage proteins, can induce ER-derived protein bodies (PBs) in heterologous tissue. Like maize and wheat Prolamins, rice Prolamins can form ER-derived PBs, but the region of mature polypeptides that is essential for PB formation has not been identified. In this study, we examined the formation mechanisms of ER-derived PB-like structures by expressing rice 13 kDa Prolamin-deletion mutants fused to green fluorescent protein (GFP) in heterologous tissues such as yeast. The 13 kDa Prolamin–GFP fusion protein was stably accumulated in transgenic yeast and formed an ER-derived PB-like structure. In contrast, rice α-globulin–GFP fusion protein was transported to vacuoles. In addition, the middle and COOH-terminal regions of 13 kDa Prolamin formed ER-derived PB-like structures, whereas the NH2-terminal region of 13 kDa Prolamin did not form such structures. These results suggest that the middle and COOH-terminal regions of 13 kDa Prolamin can be retained and thus ...
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in vivo digestibility of rice Prolamin protein body i particle is decreased by cooking
Journal of Nutritional Science and Vitaminology, 2014Co-Authors: Masatoshi Kubota, Takehiro Masumura, Yuhi Saito, Reiko Watanabe, Shinobu Fujimura, Motoni KadowakiAbstract:Rice has storage proteins, e.g., glutelin, globulin and Prolamin, in the seeds, which are used as nitrogen sources during germination. Rice Prolamin has been reported to be an indigestible protein that decreases the nutritional value of rice. However, the causes for the indigestibility of Prolamin are currently not clear. The objective of this study was to determine if Prolamin is naturally indigestible or if cooking affects its digestibility. The gastrointestinal (GI) transit of rice 23 kDa glutelin (23G) and 13 kDa Prolamin (13P) in Wistar/ST rats fed raw rice (RR) and cooked rice (CR) diets was assessed using Western blot analysis. We also measured the excretion of these proteins in the feces of these rats. Additionally, morphological observation of the structure of type-I protein bodies in the feces was performed using electron microscopy. Assessment of GI transit revealed that 23G rapidly disappeared from the GI contents of both the RR and CR groups, but 13P accumulated in the cecum of the CR group. In the CR group, Prolamin, maintaining the structure of PB-I, was fully excreted in the feces. These results indicate that rice Prolamin is not indigestible by nature, but is rendered indigestible by cooking.
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accumulation of rice Prolamin gfp fusion proteins induces er derived protein bodies in transgenic rice calli
Plant Cell Reports, 2013Co-Authors: Takanari Shigemitsu, Takehiro Masumura, Shigeto Morita, Shigeru SatohAbstract:We showed that rice Prolamin polypeptides formed ER-derived PBs in transgenic rice calli, and that this heterologous transgene expression system is suitable for studying the mechanism of rice PB-I formation. Rice Prolamins, alcohol-soluble seed storage proteins, accumulate directly within the rough endoplasmic reticulum (ER) lumen, leading to the formation of ER-derived type I protein bodies (PB-Is) in rice seed. Because rice Prolamins do not possess a well-known ER retention signal such as K(H)DEL, or a unique sequence for retention in the ER such as a tandem repeat domain of maize and wheat Prolamins, the mechanisms of Prolamin accumulation in the ER and PB-I formation are poorly understood. In this study, we examined the formation mechanisms of PBs by expressing four types of rice Prolamin species fused to green fluorescent protein (GFP) in transgenic rice calli. Each Prolamin–GFP fusion protein was stably accumulated in rice calli and formed ER-derived PBs. In contrast, GFP fused with the signal peptide of Prolamin was secreted into the intercellular space in rice calli. In addition, each of the four types of Prolamin–GFP fusion proteins was co-localized with the ER chaperone binding protein. These results suggest that the mature polypeptide of Prolamin is capable of being retained in the ER and induce the formation of PBs in non-seed tissue, and that the rice callus heterologous transgene expression system is useful for studying the mechanisms of rice PB-I formation.
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A green fluorescent protein fused to rice Prolamin forms protein body-like structures in transgenic rice
Journal of experimental botany, 2009Co-Authors: Yuhi Saito, Kunisuke Tanaka, Shigeto Morita, Koichi Kishida, Shigeru Satoh, Kenji Takata, Hideyuki Takahashi, Takeaki Shimada, Takehiro MasumuraAbstract:Prolamins, a group of rice (Oryza sativa) seed storage proteins, are synthesized on the rough endoplasmic reticulum (ER) and deposited in ER-derived type I protein bodies (PB-Is) in rice endosperm cells. The accumulation mechanism of Prolamins, which do not possess the well-known ER retention signal, remains unclear. In order to elucidate whether the accumulation of Prolamin in the ER requires seed-specific factors, the subcellular localization of the constitutively expressed green fluorescent protein fused to Prolamin (Prolamin-GFP) was examined in seeds, leaves, and roots of transgenic rice plants. The Prolamin-GFP fusion proteins accumulated not only in the seeds but also in the leaves and roots. Microscopic observation of GFP fluorescence and immunocytochemical analysis revealed that Prolamin-GFP fusion proteins specifically accumulated in PB-Is in the endosperm, whereas they were deposited in the electron-dense structures in the leaves and roots. The ER chaperone BiP was detected in the structures in the leaves and roots. The results show that the aggregation of Prolamin-GFP fusion proteins does not depend on the tissues, suggesting that the Prolamin-GFP fusion proteins accumulate in the ER by forming into aggregates. The findings bear out the importance of the assembly of Prolamin molecules and the interaction of Prolamin with BiP in the formation of ER-derived PBs.
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amino acid sequencing and cdna cloning of rice seed storage proteins the 13kda Prolamins extracted from type i protein bodies
Plant Biotechnology, 1999Co-Authors: Norihiro Mitsukawa, Takehiro Masumura, Kunitomo Kidzu, Kozo Ohtsuki, Ryoichi Konishi, Kunisuke TanakaAbstract:The 13kDa Prolamins, one of the rice storage proteins, consist of complex mixtures of polypeptides encoded by multigene family that show heterogeneity both in size and solubility. Although many researchers have isolated Prolamin cDNA clones, it has not been possible to correlate most of these cDNA clones with individual 13kDa Prolamin mature polypeptides. We isolated three new Prolamin cDNA clones, λRM1, λRM4 and λRM9. Further more, we purified six 13kDa Prolamin polypeptides from rice type I protein bodies, and determined these amino acid sequences. Here we demonstrate a classification for the 13kDa Prolamin polypeptides which can be divided four classes, 13-I, 13-IIa, 13-IIb and 13-III. Cysteine labeling of the Prolamin polypeptides indicated that 13-I contains cysteine residues, but 13-IIa or 13-IIb have no Cysteine residues. The 13-I polypeptide was soluble in nonreducing solution when their cysteine residues form inteamolecular disulfide bonds, but not soluble at intermolecular bonding.
Shigeru Satoh - One of the best experts on this subject based on the ideXlab platform.
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identification of the region of rice 13 kda Prolamin essential for the formation of er derived protein bodies using a heterologous expression system
Bioscience Biotechnology and Biochemistry, 2015Co-Authors: Takehiro Masumura, Takanari Shigemitsu, Shigeto Morita, Shigeru SatohAbstract:Cereal Prolamins, which are alcohol-soluble seed storage proteins, can induce ER-derived protein bodies (PBs) in heterologous tissue. Like maize and wheat Prolamins, rice Prolamins can form ER-derived PBs, but the region of mature polypeptides that is essential for PB formation has not been identified. In this study, we examined the formation mechanisms of ER-derived PB-like structures by expressing rice 13 kDa Prolamin-deletion mutants fused to green fluorescent protein (GFP) in heterologous tissues such as yeast. The 13 kDa Prolamin–GFP fusion protein was stably accumulated in transgenic yeast and formed an ER-derived PB-like structure. In contrast, rice α-globulin–GFP fusion protein was transported to vacuoles. In addition, the middle and COOH-terminal regions of 13 kDa Prolamin formed ER-derived PB-like structures, whereas the NH2-terminal region of 13 kDa Prolamin did not form such structures. These results suggest that the middle and COOH-terminal regions of 13 kDa Prolamin can be retained and thus ...
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accumulation of rice Prolamin gfp fusion proteins induces er derived protein bodies in transgenic rice calli
Plant Cell Reports, 2013Co-Authors: Takanari Shigemitsu, Takehiro Masumura, Shigeto Morita, Shigeru SatohAbstract:We showed that rice Prolamin polypeptides formed ER-derived PBs in transgenic rice calli, and that this heterologous transgene expression system is suitable for studying the mechanism of rice PB-I formation. Rice Prolamins, alcohol-soluble seed storage proteins, accumulate directly within the rough endoplasmic reticulum (ER) lumen, leading to the formation of ER-derived type I protein bodies (PB-Is) in rice seed. Because rice Prolamins do not possess a well-known ER retention signal such as K(H)DEL, or a unique sequence for retention in the ER such as a tandem repeat domain of maize and wheat Prolamins, the mechanisms of Prolamin accumulation in the ER and PB-I formation are poorly understood. In this study, we examined the formation mechanisms of PBs by expressing four types of rice Prolamin species fused to green fluorescent protein (GFP) in transgenic rice calli. Each Prolamin–GFP fusion protein was stably accumulated in rice calli and formed ER-derived PBs. In contrast, GFP fused with the signal peptide of Prolamin was secreted into the intercellular space in rice calli. In addition, each of the four types of Prolamin–GFP fusion proteins was co-localized with the ER chaperone binding protein. These results suggest that the mature polypeptide of Prolamin is capable of being retained in the ER and induce the formation of PBs in non-seed tissue, and that the rice callus heterologous transgene expression system is useful for studying the mechanisms of rice PB-I formation.
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Formation mechanism of the internal structure of type I protein bodies in rice endosperm: relationship between the localization of Prolamin species and the expression of individual genes
The Plant journal : for cell and molecular biology, 2012Co-Authors: Yuhi Saito, Kunisuke Tanaka, Ai Sasou, Takanari Shigemitsu, Shigeto Morita, Ryuichi Yamasaki, Futami Goto, Koichi Kishida, Masaharu Kuroda, Shigeru SatohAbstract:Rice Prolamins, a group of seed storage proteins, are synthesized on the rough endoplasmic reticulum (ER) and form type I protein bodies (PB-Is) in endosperm cells. Rice Prolamins are encoded by a multigene family. In this study, the spatial accumulation patterns of various Prolamin species in rice endosperm cells were investigated to determine the mechanism of formation of the internal structure of PB-Is. Immunofluorescence microscopic analysis of mature endosperm cells showed that the 10 kDa Prolamin is mainly localized in the core of the PB-Is, the 13b Prolamin is localized in the inner layer surrounding the core and the outermost layer, and the 13a and 16 kDa Prolamins are localized in the middle layer. Real-time RT-PCR analysis showed that expression of the mRNA for 10 kDa Prolamin precedes expression of 13a, 13b-1 and 16 kDa Prolamin in the developing stages. mRNA expression for 13b-2 Prolamin occurred after that of the other Prolamin species. Immunoelectron microscopy of developing seeds showed that the 10 kDa Prolamin polypeptide initially accumulates in the ER, and then 13b, 13a, 16 kDa and 13b Prolamins are stacked in layers within the ER. Studies with transgenic rice seeds expressing Prolamin-GFP fusion proteins under the control of native and constitutive promoters indicated that the temporal expression pattern of Prolamin genes influenced the localization of Prolamin proteins within the PB-Is. These findings indicate that the control of gene expression of Prolamin species contributes to the internal structure of PB-Is.
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A green fluorescent protein fused to rice Prolamin forms protein body-like structures in transgenic rice
Journal of experimental botany, 2009Co-Authors: Yuhi Saito, Kunisuke Tanaka, Shigeto Morita, Koichi Kishida, Shigeru Satoh, Kenji Takata, Hideyuki Takahashi, Takeaki Shimada, Takehiro MasumuraAbstract:Prolamins, a group of rice (Oryza sativa) seed storage proteins, are synthesized on the rough endoplasmic reticulum (ER) and deposited in ER-derived type I protein bodies (PB-Is) in rice endosperm cells. The accumulation mechanism of Prolamins, which do not possess the well-known ER retention signal, remains unclear. In order to elucidate whether the accumulation of Prolamin in the ER requires seed-specific factors, the subcellular localization of the constitutively expressed green fluorescent protein fused to Prolamin (Prolamin-GFP) was examined in seeds, leaves, and roots of transgenic rice plants. The Prolamin-GFP fusion proteins accumulated not only in the seeds but also in the leaves and roots. Microscopic observation of GFP fluorescence and immunocytochemical analysis revealed that Prolamin-GFP fusion proteins specifically accumulated in PB-Is in the endosperm, whereas they were deposited in the electron-dense structures in the leaves and roots. The ER chaperone BiP was detected in the structures in the leaves and roots. The results show that the aggregation of Prolamin-GFP fusion proteins does not depend on the tissues, suggesting that the Prolamin-GFP fusion proteins accumulate in the ER by forming into aggregates. The findings bear out the importance of the assembly of Prolamin molecules and the interaction of Prolamin with BiP in the formation of ER-derived PBs.
Toshihiro Kumamaru - One of the best experts on this subject based on the ideXlab platform.
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Genetic analysis of cysteine-poor Prolamin polypeptides reduced in the endosperm of the rice esp1 mutant
Plant science : an international journal of experimental plant biology, 2011Co-Authors: Tomokazu Ushijima, Masahiro Ogawa, Hiroaki Matsusaka, Hiroyuki Jikuya, Hikaru Satoh, Toshihiro KumamaruAbstract:The esp1 mutant CM21 specifically exhibits reduced levels of cysteine-poor (CysP) Prolamin bands with pIs of 6.65, 6.95, 7.10, and 7.35 in rice seed. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis demonstrated that the bands with pIs 6.65, 6.95, and 7.35 are encoded by different structural genes. These results suggest that the Esp1 locus encodes a regulatory factor involved in the synthesis and/or accumulation of CysP Prolamin molecules. Isoelectric focusing (IEF) analysis of CysP Prolamins in chromosome substitution lines showed that structural genes for bands with pI values of 6.95, 7.10, and 7.35, which are reduced in esp1 mutant lines, are located as a gene cluster in the 44.2 cM region on chromosome 5.
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A role for the cysteine-rich 10 kDa Prolamin in protein body I formation in rice.
Plant & cell physiology, 2011Co-Authors: Ai Nagamine, Masahiro Ogawa, Thomas W. Okita, Tomokazu Ushijima, Hiroaki Matsusaka, Yasushi Kawagoe, Toshihiro KumamaruAbstract:The rice Prolamins consist of cysteine-rich 10 kDa (CysR10), 14 kDa (CysR14) and 16 kDa (CysR16) molecular species and a cysteine-poor 13 kDa (CysP13) polypeptide. These storage proteins form protein bodies (PBs) composed of single spherical intracisternal inclusions assembled within the lumen of the rough endoplasmic reticulum. Immunofluorescence and immunoelectron microscopy demonstrated that CysR10 and CysP13 were asymmetrically distributed within the PBs, with the former concentrated at the electron-dense center core region and the latter distributed mainly to the electron-lucent peripheral region. These results together with temporal expression data showed that the formation of Prolamin-containing PB-I in the wild-type endosperm was initiated by the accumulation of CysR10 to form the center core. In mutants deficient for cysteine-rich Prolamins, the typical PB-I structures containing the electron-dense center core were not observed, and instead were replaced by irregularly shaped, electron-lucent, hypertrophied PBs. Similar, deformed PBs were observed in a CysR10 RNA interference plant line. These results suggest that CysR10, through its formation of the central core and its possible interaction with other cysteine-rich Prolamins, is required for tight packaging of the proteins into a compact spherical structure.
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distinct roles of protein disulfide isomerase and p5 sulfhydryl oxidoreductases in multiple pathways for oxidation of structurally diverse storage proteins in rice
The Plant Cell, 2011Co-Authors: Yayoi Onda, Masahiro Ogawa, Toshihiro Kumamaru, Ai Nagamine, Mutsumi Sakurai, Yasushi KawagoeAbstract:In the rice (Oryza sativa) endosperm, storage proteins are synthesized on the rough endoplasmic reticulum (ER), in which Prolamins are sorted to protein bodies (PBs) called type-I PB (PB-I). Protein disulfide isomerase (PDI) family oxidoreductase PDIL2;3, an ortholog of human P5, contains a conserved structural disulfide in the redox-inactive thioredoxin-like (TRX) domain and was efficiently targeted to the surface of PB-I in a redox active site–dependent manner, whereas PDIL1;1, an ortholog of human PDI, was localized in the ER lumen. Complementation analyses using PDIL1;1 knockout esp2 mutant indicated that the a and a′ TRX domains of PDIL1;1 exhibited similar redox activities and that PDIL2;3 was unable to perform the PDIL1;1 functions. PDIL2;3 knockdown inhibited the accumulation of Cys-rich 10-kD Prolamin (crP10) in the core of PB-I. Conversely, crP10 knockdown dispersed PDIL2;3 into the ER lumen. Glutathione S-transferase-PDIL2;3 formed a stable tetramer when it was expressed in Escherichia coli, and the recombinant PDIL2;3 tetramer facilitated α-globulin(C79F) mutant protein to form nonnative intermolecular disulfide bonds in vitro. These results indicate that PDIL2;3 and PDIL1;1 are not functionally redundant in sulfhydryl oxidations of structurally diverse storage proteins and play distinct roles in PB development. We discuss PDIL2;3-dependent and PDIL2;3-independent oxidation pathways that sustain disulfide bonds of crP10 in PB-I.
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the rice mutant esp2 greatly accumulates the glutelin precursor and deletes the protein disulfide isomerase
Plant Physiology, 2002Co-Authors: Yoko Takemoto, Masahiro Ogawa, Thomas W. Okita, Hikaru Satoh, Sean J Coughlan, Toshihiro KumamaruAbstract:Rice (Oryza sativa) accumulates Prolamins and glutelins as storage proteins. The latter storage protein is synthesized on the endoplasmic reticulum (ER) as a 57-kD proglutelin precursor, which is then processed into acidic and basic subunits in the protein storage vacuole. Three esp2 mutants, CM1787, EM44, and EM747, contain larger amounts of the 57-kD polypeptide and corresponding lower levels of acidic and basic glutelin subunits than normal. Electron microscopic observation revealed that esp2 contained normal-appearing glutelin-containing protein bodies (PB-II), but lacked the normal Prolamin-containing PB (PB-I). Instead, numerous small ER-derived PBs of uniform size (0.5 μm in diameter) and low electron density were readily observed. Immunoblot analysis of purified subcellular fractions and immunocytochemistry at the electron microscopy level showed that these new PBs contained the 57-kD proglutelin precursor and Prolamin polypeptides. The 57-kD proglutelin was extracted with 1% (v/v) lactic acid solution only after removal of cysteine-rich Prolamin polypeptides, suggesting that these proteins form glutelin-Prolamin aggregates via interchain disulfide bonds within the ER lumen. The endosperm of esp2 mutants contains the lumenal chaperones, binding protein and calnexin, but lacks protein disulfide isomerase (PDI) at the protein and RNA levels. The transcript of PDI was expressed in the seed only during the early stage of seed development in the wild type. These results suggest that PDI plays an essential role in the segregation of proglutelin and Prolamin polypeptides within the ER lumen.
Fumio Takaiwa - One of the best experts on this subject based on the ideXlab platform.
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compensatory rebalancing of rice Prolamins by production of recombinant Prolamin bioactive peptide fusion proteins within er derived protein bodies
Plant Cell Reports, 2018Co-Authors: Fumio Takaiwa, Lijun Yang, Yuhya Wakasa, Kenjiro OzawaAbstract:Bioactive peptide was produced by fusion to rice Prolamins in transgenic rice seeds. Their accumulation levels were affected by their deposition sites and by compensatory rebalancing between Prolamins within PB-Is. Peptide immunotherapy using analogue peptide ligands (APLs) is one of promising treatments against autoimmune diseases. Use of seed storage protein as a fusion carrier is reasonable strategy for production of such small size bioactive peptides. In this study, to examine the efficacy of various rice Prolamins deposited in ER-derived protein bodies (PB-Is), the APL12 from the Glucose-6-phosphate isomerase (GPI325-339) was expressed by fusion to four types of representative Prolamins under the control of the individual native promoters. When the 14 and 16 kDa Cys-rich Prolamins, which were localized in middle layer of PB-Is, were used for production of the APL12, they highly accumulated in transgenic rice seeds (~ 200 µg/grain). By contrast, fusion to the 10 and 13 kDa Prolamins, which were localized in the core and outermost layer of PB-Is, resulted in lower levels of accumulation (~ 40 µg/grain). These results suggest that accumulation levels were highly affected by their deposition sites. Next, when different Prolamin/APL12 fusion proteins were co-expressed to increase accumulation levels, they could not be increased so much as their expected additive levels. High accumulation of one type Prolamin/APL12 led to reduction of other type(s) Prolamin/APL12 to maintain the limited amounts of Prolamins that can be deposited in PB-Is. Moreover, suppression of endogenous seed proteins by RNA interference also did not significantly enhance the accumulation levels of Prolamin/APL12. These findings suggest that there may be compensatory rebalancing mechanism that controls the accumulation levels of Prolamins deposited within PB-Is.
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reducing rice seed storage protein accumulation leads to changes in nutrient quality and storage organelle formation
Plant Physiology, 2010Co-Authors: Taiji Kawakatsu, Sakiko Hirose, Hiroshi Yasuda, Fumio TakaiwaAbstract: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.
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evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice
Plant Biotechnology Journal, 2004Co-Authors: Leqing Qu, Fumio TakaiwaAbstract:Summary Using stable transgenic rice plants, the promoters of 15 genes expressed in rice seed were analysed for their spatial and temporal expression pattern and their potential to promote the expression of recombinant proteins in seeds. The 15 genes included 10 seed storage protein genes and five genes for enzymes involved in carbohydrate and nitrogen metabolism. The promoters for the glutelins and the 13 kDa and 16 kDa Prolamins directed endosperm-specific expression, especially in the outer portion (peripheral region) of the endosperm, whilst the embryo globulin and 18 kDa oleosin promoters directed expression in the embryo and aleurone layer. Fusion of the GUS gene to the 26 kDa globulin promoter resulted in expression in the inner starchy endosperm tissue. It should be noted that the 10 kDa Prolamin gene was the only one tested that required both the 5′ and 3′ flanking regions for intrinsic endosperm-specific expression. The promoters from the pyruvate orthophosphate dikinase (PPDK) and ADP-glucose pyrophosphorylase (AGPase) small subunit genes were active not only in the seed, but also in the phloem of vegetative tissues. Within the seed, the expression from these two promoters differed in that the PPDK gene was only expressed in the endosperm, whereas the AGPase small subunit gene was expressed throughout the seed. The GUS reporter gene fused to the alanine aminotransferase (AlaAT) promoter was expressed in the inner portion of the starchy endosperm, whilst the starch branching enzyme (SBE1) and the glutamate synthase (GOGAT) genes were mainly expressed in the scutellum (between the endosperm and embryo). When promoter activities were examined during seed maturation, the glutelin GluB-4, 26 kDa globulin and 10 kDa and 16 kDa Prolamin promoters exhibited much higher activities than the others. The seed promoters analysed here exhibited a wide variety of activities and expression patterns, thus providing many choices suitable for various applications in plant biotechnology.
Young Mi Kim - One of the best experts on this subject based on the ideXlab platform.
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rna interference mediated simultaneous suppression of seed storage proteins in rice grains
Frontiers in Plant Science, 2016Co-Authors: Kyoungwon Cho, Jong-yeol Lee, Sun-hyung Lim, Hyejung Lee, Randeep Rakwal, Young Mi KimAbstract:Seed storage proteins (SSPs) such as glutelin, Prolamin and globulin are abundant components in some of the most widely consumed food cereals in the world. Synthesized in the rough endoplasmic reticulum (ER), SSPs are translocated to the protein bodies. Prolamins are located at the spherical protein body I derived from the ER, whereas glutelins and globulin are accumulated in the irregularly shaped protein bodies derived from vacuoles. Our previous studies have shown that the individual suppression of glutelins, 13-kDa Prolamins and globulin caused the compensative accumulation of other SSPs. Herein, to investigate the phenotypic and molecular features of SSP deficiency transgenic rice plants suppressing all glutelins, Prolamins and globulin were generated using RNA interference (RNAi). The results revealed that glutelin A, cysteine-rich 13-kDa Prolamin and globulin proteins were less accumulated but that glutelin B and ER chaperones, such as binding protein 1 (BiP1) and protein disulfide isomerase-like 1-1 (PDIL1-1), were highly accumulated at the transcript and protein levels in seeds of the transformants compared to those in the wild-type seeds. Further, the transcription of starch synthesis-related genes was reduced in immature seeds at two weeks after flowering, and the starch granules were loosely packaged with various sphere sizes in seed endosperms of the transformants, resulting in a floury phenotype. Interestingly, the rates of sprouting and reducing sugar accumulation during germination were found to be delayed in the transformants compared to the wild-type. In all, our results provide new insight into the role of SSPs in the formation of intracellular organelles and in germination.
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lack of globulin synthesis during seed development alters accumulation of seed storage proteins in rice
International Journal of Molecular Sciences, 2015Co-Authors: Hyejung Lee, Jong-yeol Lee, Sun-hyung Lim, Young Mi KimAbstract:The major seed storage proteins (SSPs) in rice seeds have been classified into three types, glutelins, Prolamins, and globulin, and the proportion of each SSP varies. It has been shown in rice mutants that when either glutelins or Prolamins are defective, the expression of another type of SSP is promoted to counterbalance the deficit. However, we observed reduced abundances of glutelins and Prolamins in dry seeds of a globulin-deficient rice mutant (Glb-RNAi), which was generated with RNA interference (RNAi)-induced suppression of globulin expression. The expression of the Prolamin and glutelin subfamily genes was reduced in the immature seeds of Glb-RNAi lines compared with those in wild type. A proteomic analysis of Glb-RNAi seeds showed that the reductions in glutelin and Prolamin were conserved at the protein level. The decreased pattern in glutelin was also significant in the presence of a reductant, suggesting that the polymerization of the glutelin proteins via intramolecular disulfide bonds could be interrupted in Glb-RNAi seeds. We also observed aberrant and loosely packed structures in the storage organelles of Glb-RNAi seeds, which may be attributable to the reductions in SSPs. In this study, we evaluated the role of rice globulin in seed development, showing that a deficiency in globulin could comprehensively reduce the expression of other SSPs.
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Effects of Reduced Prolamin on Seed Storage Protein Composition and the Nutritional Quality of Rice
International journal of molecular sciences, 2013Co-Authors: Hyun-jung Kim, Jong-yeol Lee, Ung-han Yoon, Sun-hyung Lim, Young Mi KimAbstract: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.
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Structural and expression analysis of Prolamin genes in Oryza sativa L.
Plant Biotechnology Reports, 2012Co-Authors: Ung-han Yoon, Young Mi Kim, Jeonghwa Lee, Jang-ho Hahn, Yeon-ki Kim, Gang-seob Lee, Chang-kug Kim, Jeong-hwan Mun, Tae-ho KimAbstract:Rice is a staple crop with a small genome of 389 Mb. Rice grain is a source of carbohydrates and proteins and has a relatively low protein content compared to other legume seeds. Glutelin and Prolamin are the major storage proteins in rice. Prolamins are characterized by high glutamine and proline content and are generally soluble only in strong alcohol solutions. In this study, we obtained a total of 51,383 expressed sequence tags (ESTs) from Ilpumbyeo ( Oryza sativa L.), of which 33,201 and 18,182 clones were obtained from immature and germinating seeds, respectively. From the EST clones, 15,148 unigenes were identified, and 2,590 genes were expressed in both immature and germinating seeds. Gene expression profiling of rice Prolamins indicated that Prolamin gene expression increased 5 days after heading and reached maximal expression after 30 days, suggesting a high demand for Prolamins during seed development and germination. Phylogenetic analysis grouped 33 Prolamin genes based on the abundance of sulfur-containing amino acids methionine and cysteine according to the deduced amino acid sequences. Our results enhance the understanding of the regulation of seed maturation and germination, which can result in improved agricultural traits for the seed industry.
