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Matsumoto S.,Forestry and Fisheries Technology Research Center | Yoshikawa M.,Forestry and Fisheries Technology Research Center
Japanese Journal of Crop Science | Year: 2010

As a part of the study on the causes of the decrease in yield of black soybeans in converted fields in Kyoto prefecture, we examined the annual changes in the yield of continuous cropping of black soybean and the chemical properties of the soil in the converted gray lowland paddy field. The yield of grains increased in the third year after field conversion, and gradually decreased after the fourth year. The number of pods increased in the third year, and remained at the same level thereafter. The 2L size ratio of grains (weight ratio of grains larger than 10mm in diameter to total grains harvested) was still high for three years after conversion, and then decreased greatly after the fourth year. The number of nodules decreased gradually after conversion, and greatly after the fourth year. The decrease in the contents of total carbon and nitrogen in the paddy soil was not observed after conversion. The application of cattle manure composted with bark (CMCB) mitigated the decrease in the nodule formation, but not that in the 2L size ratio of grains. The amount of available soil nitrogen in the fifth year after conversion with CMCB application was higher than that in the non-converted paddy field without CMCB application.


Kurokawa S.,Tokyo Medical University | Kurokawa S.,Kyoto Prefectural University | Kuroda M.,Japan National Agriculture and Food Research Organization | Mejima M.,Tokyo Medical University | And 13 more authors.
Plant Cell Reports | Year: 2014

Key message: RNAi-mediated suppression of the endogenous storage proteins in MucoRice-CTB-RNAi seeds affects not only the levels of overexpressed CTB and RAG2 allergen, but also the localization of CTB and RAG2. A purification-free rice-based oral cholera vaccine (MucoRice-CTB) was previously developed by our laboratories using a cholera toxin B-subunit (CTB) overexpression system. Recently, an advanced version of MucoRice-CTB was developed (MucoRice-CTB-RNAi) through the use of RNAi to suppress the production of the endogenous storage proteins 13-kDa prolamin and glutelin, so as to increase CTB expression. The level of the α-amylase/trypsin inhibitor-like protein RAG2 (a major rice allergen) was reduced in MucoRice-CTB-RNAi seeds in comparison with wild-type (WT) rice. To investigate whether RNAi-mediated suppression of storage proteins affects the localization of overexpressed CTB and major rice allergens, we generated an RNAi line without CTB (MucoRice-RNAi) and investigated gene expression, and protein production and localization of two storage proteins, CTB, and five major allergens in MucoRice-CTB, MucoRice-CTB-RNAi, MucoRice-RNAi, and WT rice. In all lines, glyoxalase I was detected in the cytoplasm, and 52- and 63-kDa globulin-like proteins were found in the aleurone particles. In WT, RAG2 and 19-kDa globulin were localized mainly in protein bodies II (PB-II) of the endosperm cells. Knockdown of glutelin A led to a partial destruction of PB-II and was accompanied by RAG2 relocation to the plasma membrane/cell wall and cytoplasm. In MucoRice-CTB, CTB was localized in the cytoplasm and PB-II. In MucoRice-CTB-RNAi, CTB was produced at a level six times that in MucoRice-CTB and was localized, similar to RAG2, in the plasma membrane/cell wall and cytoplasm. Our findings indicate that the relocation of CTB in MucoRice-CTB-RNAi may contribute to down-regulation of RAG2. © 2013 Springer-Verlag Berlin Heidelberg.


Shigemitsu T.,Kyoto Prefectural University | Masumura T.,Kyoto Prefectural University | Masumura T.,Forestry and Fisheries Technology Research Center | Morita S.,Kyoto Prefectural University | And 3 more authors.
Plant Cell Reports | Year: 2013

Key message: 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. © 2012 Springer-Verlag Berlin Heidelberg.


Sasou A.,Kyoto Prefectural University | Shigemitsu T.,Kyoto Prefectural University | Saito Y.,Kyoto Prefectural University | Tanaka M.,Kyoto Prefectural University | And 4 more authors.
Plant Cell Reports | Year: 2016

Key message: Prolamin–GFP fusion proteins, expressed under the control of native prolamin promoters, were localized in specific layers of PB-Is. Prolamin–GFP fusion proteins were gradually digested from outside by pepsin digestion.Abstract: In rice seed endosperm, protein body type I (PB-I) has a layered structure consisting of prolamin species and is the resistant to digestive juices in the intestinal tract. We propose the utilization of PB-Is as an oral vaccine carrier to induce mucosal immune response effectively. If vaccine antigens are localized in a specific layer within PB-Is, they could be protected from gastric juice and be delivered intact to the small intestine. We observed the localization of GFP fluorescence in transgenic rice endosperm expressing prolamin–GFP fusion proteins with native prolamin promoters, and we confirmed that the foreign proteins were located in specific layers of PB-Is artificially. Each prolamin–GFP fusion protein was localized in specific layers of PB-Is, such as the outer-most layer, middle layer, and core region. Furthermore, to investigate the resistance of prolamin–GFP fusion proteins against pepsin digestion, we performed in vitro pepsin treatment. Prolamin–GFP fusion proteins were gradually digested from the peripheral region and the contours of PB-Is were made rough by in vitro pepsin treatment. These findings suggested that prolamin–GFP fusion proteins accumulating specific layers of PB-Is were gradually digested and exposed from the outside by pepsin digestion. © 2016 The Author(s)


Saito Y.,Kyoto Prefectural University | Shigemitsu T.,Kyoto Prefectural University | Yamasaki R.,Kyoto Prefectural University | Sasou A.,Kyoto Prefectural University | And 10 more authors.
Plant Journal | Year: 2012

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. © 2012 Blackwell Publishing Ltd.


Kurokawa S.,Tokyo Medical University | Kurokawa S.,Kyoto Prefectural University | Nakamura R.,Japan National Institute of Health Sciences | Mejima M.,Tokyo Medical University | And 13 more authors.
Journal of Proteome Research | Year: 2013

To develop a cold chain- and needle/syringe-free rice-based cholera vaccine (MucoRice-CTB) for human use, we previously advanced the MucoRice system by introducing antisense genes specific for endogenous rice storage proteins and produced a molecularly uniform, human-applicable, high-yield MucoRice-CTB devoid of plant-associated sugar. To maintain the cold chain-free property of this vaccine for clinical application, we wanted to use a polished rice powder preparation of MucoRice-CTB without further purification but wondered whether this might cause an unexpected increase in rice allergen protein expression levels in MucoRice-CTB and prompt safety concerns. Therefore, we used two-dimensional fluorescence difference gel electrophoresis and shotgun MS/MS proteomics to compare rice allergen protein expression levels in MucoRice-CTB and wild-type (WT) rice. Both proteomics analyses showed that the only notable change in the expression levels of rice allergen protein in MucoRice-CTB, compared with those in WT rice, was a decrease in the expression levels of α-amylase/trypsin inhibitor-like protein family such as the seed allergen protein RAG2. Real-time PCR analysis showed mRNA of RAG2 reduced in MucoRice-CTB seed. These results demonstrate that no known rice allergens appear to be up-reregulated by genetic modification of MucoRice-CTB, suggesting that MucoRice-CTB has potential as a safe oral cholera vaccine for clinical application. © 2013 American Chemical Society.


Masumura T.,Kyoto Prefectural University | Masumura T.,Forestry and Fisheries Technology Research Center | Shigemitsu T.,Kyoto Prefectural University | Morita S.,Kyoto Prefectural University | And 3 more authors.
Bioscience, Biotechnology and Biochemistry | Year: 2015

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 ERderived 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 κDa prolamin-deletion mutants fused to green fluorescent protein (GFP) in heterologous tissues such as yeast. The 13 κDa 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 κDa prolamin formed ER-derived PB-like structures, whereas the NH2-terminal region of 13 κDa prolamin did not form such structures. These results suggest that the middle and COOH-terminal regions of 13κDa prolamin can be retained and thus can induce ER-derived PB in yeast. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry.


Morita T.,Fisheries Research Agency | Niwa K.,Fisheries Research Agency | Fujimoto K.,Fisheries Research Agency | Kasai H.,Fisheries Research Agency | And 15 more authors.
Science of the Total Environment | Year: 2010

Iodine-131 (physical half-life: 8.04days) was detected in brown algae collected off the Japanese coast. Brown algae have been extensively used as bioindicators for radioiodine because of their ability to accumulate radionuclides in high concentration factors. The maximum measured specific activity of 131I in brown algae was 0.37±0.010Bq/kg-wet. Cesium-137 was also detected in all brown algal samples used in this study. There was no correlation between specific activities of 131I and 137Cs in these seaweeds. The specific activity of 137Cs ranged from 0.0034±0.00075 to 0.090±0.014Bq/kg-wet. Low specific activity and minimal variability of 137Cs in brown algae indicated that past nuclear weapon tests were the source of 137Cs. Although nuclear power stations and nuclear fuel reprocessing plants are known to be pollution sources of 131I, there was no relationship between the sites where 131I was detected and the locations of nuclear power facilities. Most of the sites where 131I was detected were near big cities with large populations. Iodine-131 is frequently used in diagnostic and therapeutic nuclear medicine. On the basis of the results, we suggest that the likely pollution source of 131I, detected in brown seaweeds, is not nuclear power facilities, but nuclear medicine procedures. © 2010 Elsevier B.V.


Shigemitsu T.,Kyoto Prefectural University | Ozaki S.,Kyoto Prefectural University | Saito Y.,Kyoto Prefectural University | Kuroda M.,Japan National Agricultural Research Center | And 6 more authors.
Plant Cell Reports | Year: 2012

Rice seeds are potentially useful hosts for the production of pharmaceutical proteins. However, low yields of recombinant proteins have been observed in many cases because recombinant proteins compete with endogenous storage proteins. Therefore, we attempt to suppress endogenous seed storage proteins by RNA interference (RNAi) to develop rice seeds as a more efficient protein expression system. In this study, human growth hormone (hGH) was expressed in transgenic rice seeds using an endosperm-specific promoter from a 10 kDa rice prolamin gene. In addition, an RNAi cassette for reduction of endogenous storage protein expressions was inserted into the hGH expression construct. Using this system, the expression levels of 13 kDa prolamin and glutelin were effectively suppressed and hGH polypeptides accumulated to 470 μg/g dry weight at the maximum level in transgenic rice seeds. These results suggest that the suppression of endogenous protein gene expression by RNAi could be of great utility for increasing transgene products. © 2011 Springer-Verlag.


Mimura Y.,Forestry and Fisheries Technology Research Center | Minamiyama Y.,Forestry and Fisheries Technology Research Center | Sano H.,Kyoto Prefectural University | Hirai M.,Forestry and Fisheries Technology Research Center | Hirai M.,Kyoto Prefectural University
Journal of the Japanese Society for Horticultural Science | Year: 2010

A quantitative trait locus (QTL) analysis of pepper growth traits was performed using a doubled haploid (DH) population derived from a cross between two Capsicum annuum genotypes, a bell-type cultivar 'California Wonder' and a Malaysian small-fruited cultivar 'LS2341'. Simple sequence repeats and amplified fragment length polymorphism markers were used to construct a genetic map for this population. The map spans 1,213 cM, and consists of 15 linkage groups (LGs). The axillary shooting, flowering date, primary axis length, number of leaves on primary axis and mean internode length were evaluated in 94 F 1DH families. Twelve QTLs were identified by interval analysis, and each QTL accounts for 14 to 34% of the phenotypic variation. Markers on chromosomes 2, 3, 12, and linkage group 8 (LG8) were associated with QTL for these traits. The present analysis revealed 2 loci for the growth traits in LG 8. One affected mainly axis length and had a minor effect on flowering, while the other had a large effect on flowering date and a smaller effect on axis length. © 2010 JSHS.

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