Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Salinity is an abiotic stress factor that reduces yield and threatens food security in the world’s arid and semi-arid regions. The development of salt-tolerant genotypes is critical for mitigating yield losses, and this journey begins with the identification of sensitive and tolerant plants. Numerous physiologic and molecular markers for detecting salt-tolerant wheat genotypes have been developed. One of them is proline, which has been used for a long time but has received little information about proline-related genes in wheat genotypes. In this study, proline content and the expression levels of proline-related genes (TaPTF1, TaDHN, TaSRG, TaSC, TaPIMP1, TaMIP, TaHKT1;4, TaGSK, TaP5CS, and TaMYB) were examined in sensitive, moderate, and tolerant genotypes under salt stress (0, 50, 150, and 250 mM NaCl) for 0, 12, and 24 h. Our results show that salt stress increased the proline content in all genotypes, but it was found higher in salt-tolerant genotypes than in moderate and sensitive genotypes. The salinity stress increased gene expression levels in salt-tolerant and moderate genotypes. While salt-stress exposure for 12 and 24 h had a substantial effect on gene expression in wheat, TaPTF1, TaPIMP1, TaMIP, TaHKT1;4, and TaMYB genes were considerably upregulated in 24 h. The salt-tolerant genotypes showed a higher positive interaction than a negative interaction. The TaPTF1, TaP5CS, TaGSK1, and TaSRG genes were found to be more selective than the other analyzed genes under salt-stress conditions. Despite each gene’s specific function, increasing proline biosynthesis functioned as a common mechanism for separating salt tolerance from sensitivity.

DOI： 10.3390/plants11233401

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Here, for the first time, we aimed to identify in rice the key mechanisms and processes underlying tolerance to high-temperature (HT) or salt stress (SS) alone, the co-occurrence of both stresses, and recovery using physiological and biochemical measurements and gene expression analysis. We also investigated whether recovery from the two stressors depended on the relative intensities/relief of each stressor. Wild type (‘Yukinkomai’) rice plants were found to be more susceptible to salinity or heat applied individually. SS leads to a depletion of cellular water content, higher accumulation of Na+, and alterations in photosynthetic pigments. The stress-tolerant cultivar ‘YNU31-2-4’ (YNU) displayed a lower Na+/K+ ratio, higher water content in cells and improved photosynthetic traits, antioxidant system, and expression of defence genes. Strikingly, the SS + HT combination provided a significant level of protection to rice plants from the effects of SS alone. The expression pattern of a selected set of genes showed a specific response and dedicated pathways in plants subjected to each of the different stresses, while other genes were explicitly activated when the stresses were combined. Aquaporin genes were activated by SS, while stress-related (P5CS, MSD1, HSPs, and ions transporters) genes were shaped by HT. Hierarchical clustering and principal component analyses showed that several traits exhibited a gradually aggravating effect as plants were exposed to the combined stresses and identified heat as a mitigating factor, clearly separating heat + salt-stressed from salt-non-heat-stressed plants. Furthermore, seedling recovery was far more dependent on the relative intensities of stressors and cultivars, demonstrating the influence of one stressor over another upon stress-release. Taken together, our data show the uniqueness and complexity of the physiological and molecular network modules used by rice plants to respond to single and combined stresses and recovery.

DOI： 10.3390/plants11040501

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Language：English   Publishing type：Research paper (scientific journal)  

The present study aimed to determine the effects of biostimulants on the physicochemical parameters of the agricultural soil of quinoa under two water regimes and to understand the mode of action of the biostimulants on quinoa for drought adaptation. We investigated the impact of two doses of vermicompost (5 and 10 t/ha) and arbuscular mycorrhizal fungi applied individually, or in joint application, on attenuating the negative impacts of water shortage and improving the agro-physiological and biochemical traits of quinoa, as well as soil fertility, under two water regimes (well-watered and drought stress) in open field conditions. Exposure to drought decreased biomass, leaf water potential, and stomatal conductance, and increased malondialdehyde and hydrogen peroxide content. Mycorrhiza and/or vermicompost promoted plant growth by activating photosynthesis machinery and nutrient assimilation, leading to increased total soluble sugars, proteins, and antioxidant enzyme activities in the leaf and root. After the experiment, the soil's total organic matter, phosphorus, nitrogen, calcium, and soil glomalin content improved by the single or combined application of mycorrhiza and vermicompost. This knowledge suggests that the combination of mycorrhiza and vermicompost regulates the physiological and biochemical processes employed by quinoa in coping with drought and improves the understanding of soil-plant interaction.

DOI： 10.3390/plants11030393

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Language：English   Publishing type：Research paper (scientific journal)  

Modern agriculture is facing multiple and complex challenges and has to produce more food and fiber to feed a growing population. Increasingly volatile weather and more extreme events such as droughts can reduce crop productivity. This implies the need for significant increases in production and the adoption of more efficient and sustainable production methods and adaptation to climate change. A new technological and environment-friendly management technique to improve the tolerance of quinoa grown to maturity is proposed using native microbial biostimulants (arbuscular mycorrhizal fungi; AMF) alone, in the consortium, or in combination with compost (Comp) as an organic matter source under two water treatments (normal irrigation and drought stress (DS)). Compared with controls, growth, grain yield, and all physiological traits under DS were significantly decreased while hydrogen peroxide, malondialdehyde, and antioxidative enzymatic functions were significantly increased. Under DS, biofertilizer application reverted physiological activities to normal levels and potentially strengthened quinoa's adaptability to water shortage as compared to untreated plants. The dual combination yielded a 97% improvement in grain dry weight. Moreover, the effectiveness of microbial and compost biostimulants as a biological tool improves grain quality and limits soil degradation under DS. Elemental concentrations, particularly macronutrients, antioxidant potential (1,1-diphenyl-2-picrylhydrazyl radical scavenging activity), and bioactive compounds (phenol and flavonoid content), were accumulated at higher levels in biofertilizer-treated quinoa grain than in untreated controls. The effects of AMF + Comp on post-harvest soil fertility traits were the most positive, with significant increases in total phosphorus (47%) and organic matter (200%) content under drought conditions. Taken together, our data demonstrate that drought stress strongly influences the physiological traits, yield, and quality of quinoa. Microbial and compost biostimulation could be an effective alternative to ensure greater recovery capability, thereby maintaining relatively high levels of grain production. Our study shows that aboveground stress responses in quinoa can be modulated by signals from the microbial/compost-treated root. Further, quinoa grains are generally of higher nutritive quality when amended and inoculated with AMF as compared to non-inoculated and compost-free plants.

DOI： 10.3389/fpls.2022.860484

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Language：English   Publishing type：Research paper (scientific journal)  

In the current study, an eco-friendly management technology to improve young carob (Ceratonia siliqua L.) tree tolerance to water deficit was set up by using single or combined treatments of arbuscular mycorrhizal fungi (AMF) and/or compost (C). Two groups of young carob have been installed: (i) carob cultivated under well-watered conditions (WW; 70% field capacity (FC)) and (ii) where the plants were drought-stressed (DS; 35% FC) during 2, 4, 6, and 8 months. The effect of used biofertilizers on the course of growth, physiological (photosynthetic traits, water status, osmolytes, and mineral content), and biochemical (hydrogen peroxide (H2O2), oxidative damage to lipids (malondialdehyde (MDA), and membrane stability (MS)) traits in response to short- and long-term droughts were assessed. The dual application of AMF and C (C + AMF) boosted growth, physiological and biochemical parameters, and nutrient uptake in carob under WW and DS. After eight months, C + AMF significantly enhanced stomatal conductance by 20%, maximum photochemical efficiency of PSII by 7%, leaf water potential by 23%, chlorophyll and carotenoid by 40%, plant uptake of mineral nutrients (P by 75%, N by 46%, K+ by 35%, and Ca2+ by 40%), concentrations of soluble sugar by 40%, and protein content by 44% than controls under DS conditions. Notably, C + AMF reduced the accumulation of H2O2 and MDA content to a greater degree and increased MS. In contrast, enzyme activities (superoxide dismutase, catalase, peroxidase, and polyphenoloxidase) significantly increased in C + AMF plants under DS. Overall, our findings suggest that the pairing of C + AMF can mediate superior drought tolerance in young carob trees by increasing leaf stomatal conductance, cellular water content, higher solute concentration, and defense response against oxidative damage during the prolonged period of DS.

DOI： 10.1038/s41598-021-02018-3

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.envexpbot.2021.104648

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.plaphy.2021.05.041

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Publishing type：Research paper (scientific journal)   Publisher：Crop Science Society of Japan  

DOI： 10.1626/jcs.90.269

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Publishing type：Research paper (scientific journal)   Publisher：Asian Journal of Agriculture and Biology  

DOI： 10.35495/ajab.2020.04.255

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Language：English   Publisher：MDPI  

C and N are the most important essential elements constituting organic compounds in plants. The shoots and roots depend on each other by exchanging C and N through the xylem and phloem transport systems. Complex mechanisms regulate C and N metabolism to optimize plant growth, agricultural crop production, and maintenance of the agroecosystem. In this paper, we cover the recent advances in understanding C and N metabolism, regulation, and transport in plants, as well as their underlying molecular mechanisms. Special emphasis is given to the mechanisms of starch metabolism in plastids and the changes in responses to environmental stress that were previously overlooked, since these changes provide an essential store of C that fuels plant metabolism and growth. We present general insights into the system biology approaches that have expanded our understanding of core biological questions related to C and N metabolism. Finally, this review synthesizes recent advances in our understanding of the trade-off concept that links C and N status to the plant's response to microorganisms.

DOI： 10.3390/ijms22010318

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

Salinity is one of the devastating abiotic stresses that cause reductions in agricultural production. The increased salinization affects alfalfa growth, metabolism, and rhizobium capacity for symbiotic N-2 fixation negatively. This study was undertaken to investigate the efficiency of green compost (C; made from green waste), arbuscular mycorrhizal fungi (M; field-sourced native consortium), and/or rhizobium (R; a salt-tolerant rhizobium strain) individually or in combination as an effective strategy to improve alfalfa productivity under non-saline and high-saline (120 mM NaCl) conditions. In addition, we aimed to understand the agro-physiological and metabolic basis as well as glomalin content in the soil of biofertilizers-induced salt tolerance in alfalfa. Here, we show that mycorrhizal infection was enhanced after MR inoculation, while C application decreased it significantly. Salinity reduced growth, physiological functioning, and protein concentration, but the antioxidant system has been activated. Application of the selected biofertilizers, especially C alone or combined with M and/or R improved alfalfa tolerance. The tri-combination CMR mitigated the negative effects of high salinity by stimulating plant growth, roots and nodules dry matters, mineral uptake (P, N, and K), antioxidant system, synthesis of compatible solutes, and soil glomalin content, sustaining photosynthesis-related performance and decreasing Na+ and Cl- accumulation, lipid peroxidation, H2O2 content, and electrolyte leakage.

DOI： 10.3390/microorganisms8111695

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS MEDIA SA  

Rainfall regimes are expected to shift on a regional scale as the water cycle intensifies in a warmer climate, resulting in greater extremes in dry versus wet conditions. Such changes are having a strong impact on the agro-physiological functioning of plants that scale up to influence interactions between plants and microorganisms and hence ecosystems. In (semi)-arid ecosystems, the date palm (Phoenix dactylifera L.) -an irreplaceable tree- plays important socio-economic roles. In the current study, we implemeted an adapted management program to improve date palm development and its tolerance to water deficit by using single or multiple combinations of exotic and native arbuscular mycorrhizal fungi (AMF1 and AMF2 respectively), and/or selected consortia of plant growth-promoting rhizobacteria (PGPR: B1 and B2), and/or composts from grasses and green waste (C1 and C2, respectively). We analyzed the potential for physiological functioning (photosynthesis, water status, osmolytes, mineral nutrition) to evolve in response to drought since this will be a key indicator of plant resilience in future environments. As result, under water deficit, the selected biofertilizers enhanced plant growth, leaf water potential, and electrical conductivity parameters. Further, the dual-inoculation of AMF/PGPR amended with composts alone or in combination boosted the biomass under water deficit conditions to a greater extent than in non-inoculated and/or non-amended plants. Both single and dual biofertilizers improved physiological parameters by elevating stomatal conductance, photosynthetic pigments (chlorophyll and carotenoids content), and photosynthetic efficiency. The dual inoculation and compost significantly enhanced, especially under drought stress, the concentrations of sugar and protein content, and antioxidant enzymes (polyphenoloxidase and peroxidase) activities as a defense strategy as compared with controls. Under water stress, we demonstrated that phosphorus was improved in the inoculated and amended plants alone or in combination in leaves (AMF2: 807%, AMF1+B2: 657%, AMF2+C1+B2: 500%, AMF2+C2: 478%, AMF1: 423%) and soil (AMF2: 397%, AMF1+B2: 322%, AMF2+C1+B2: 303%, AMF1: 190%, C1: 188%) in comparison with controls under severe water stress conditions. We summarize the extent to which the dual and multiple combinations of microorganisms can overcome challenges related to drought by enhancing plant physiological responses.

DOI： 10.3389/fpls.2020.516818

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS MEDIA SA  

The date palm is a commercially important woody crop and is a good target plant for improving agricultural yields in extreme environments. However, salinity has been the primary abiotic stress complicating its cultivation and damaging its production worldwide. This study investigated the effect of alleviating salt stress on date palm growth and development by using arbuscular mycorrhizal fungi (AMF) and/or compost. The experiment was arranged in a completely randomized design with eight treatments. The treatments comprised control without inoculation or amendment and application of compost (made from green waste) and AMF (an autochthonous consortium) individually or in combination under non-saline (0 mM NaCl) or saline (240 mM NaCl) conditions. Growth, physiological characteristics, nutrient uptake, chlorophyll content, oxidative stress markers, and antioxidant enzyme activities were assessed. Salt stress increased sodium (Na+) and chlorine (Cl-) content, lipid peroxidation and proline, soluble sugar, and H2O2 content. However, it reduced growth parameters, AMF colonization, leaf water potential, nitrogen (N), phosphorus (P), potassium (K+), calcium (Ca2+), and chlorophyll content. The application of AMF and compost separately or in combination mitigated the deleterious effects induced by salinity. AMF inoculation contributed to plant salt tolerance through strategies such as increased nutrient uptake (particularly P and Ca2+), chlorophyll content, relative water content, stomatal conductance, antioxidant enzymatic activities (superoxide dismutase, ascorbate peroxidase, catalase) and by decreasing lipid peroxidation and H2O2 content. Plants grown in soil amended with compost under salt stress showed an improvement particularly in K+ and proline content and a decrease in H2O2 concentration compared to controls under the saline condition. In the presence of NaCl stress, the dual application of the compost and AMF consortium maximized plant growth, stomatal conductance, leaf water potential, all antioxidant enzyme activities and P, K+, N, and Ca2+ uptake as well as proline and soluble sugar content. However, it reduced Na(+)and Cl(-)uptake and oxidative stress marker content. In conclusion, our study suggests that the application of AMF with compost has the potential to improve the tolerance of date palm seedlings to salt stress more than AMF or compost applied separately.

DOI： 10.3389/fsufs.2020.00131

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Language：English   Publisher：FRONTIERS MEDIA SA  

Photosynthesis is the major process leading to primary production in the Biosphere. There is a total of 7000bn tons of CO(2)in the atmosphere and photosynthesis fixes more than 100bn tons annually. The CO(2)assimilated by the photosynthetic apparatus is the basis of crop production and, therefore, of animal and human food. This has led to a renewed interest in photosynthesis as a target to increase plant production and there is now increasing evidence showing that the strategy of improving photosynthetic traits can increase plant yield. However, photosynthesis and the photosynthetic apparatus are both conditioned by environmental variables such as water availability, temperature, [CO2], salinity, and ozone. The "omics" revolution has allowed a better understanding of the genetic mechanisms regulating stress responses including the identification of genes and proteins involved in the regulation, acclimation, and adaptation of processes that impact photosynthesis. The development of novel non-destructive high-throughput phenotyping techniques has been important to monitor crop photosynthetic responses to changing environmental conditions. This wealth of data is being incorporated into new modeling algorithms to predict plant growth and development under specific environmental constraints. This review gives a multi-perspective description of the impact of changing environmental conditions on photosynthetic performance and consequently plant growth by briefly highlighting how major technological advances including omics, high-throughput photosynthetic measurements, metabolic engineering, and whole plant photosynthetic modeling have helped to improve our understanding of how the photosynthetic machinery can be modified by different abiotic stresses and thus impact crop production.

DOI： 10.3389/fpls.2020.00882

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Background and objectives Brown rice grains are not considered suitable as table rice despite its high nutritional value. In previous studies, we have shown that red onion solution accelerated rice seed germination. In this study, we investigated the effect of germination in red onion solution on high-quality brown rice, black rice, and giant embryo rice, examining biofunctional components, physical properties, and palatability after cooking. Findings Germination in red onion solution increased the antioxidant capacity and gamma-aminobutyric acid content in rice, even when cooked, compared with germination in water. Moreover, the cooked rice after germination in red onion solution was slightly softer and stickier than that germinated in water. Conclusions We found that germination of brown rice in red onion solution produced a palatable and biofunctional product. Acceleration of germination by the red onion solution may be due to an increase in phytohormones through stimulation of signal transduction. Significance and novelty Palatability and biofunctionality were improved by germinating three kinds of brown rice in the red onion solution. The hypothesis for the acceleration of germination by the red onion was proposed that the phytohormone and protein kinase take important roles from the results of proteomic analysis.

DOI： 10.1002/cche.10305

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

The long-chain acyl-CoA synthetases (LACSs) are involved in lipid synthesis, fatty acid catabolism, and the transport of fatty acids between subcellular compartments. These enzymes catalyze the critical reaction of fatty acyl chains to fatty acyl-CoAs for the triacylglycerol biosynthesis used as carbon and energy reserves. In Arabidopsis, LACSs are encoded by a family of nine genes, with LACS9 being the only member located in the chloroplast envelope membrane. However, the comprehensive role of LACS9 and its contribution to plant metabolism have not been explored thoroughly. In this study, we report on the identification and characterization of LACS9 mutants in rice plants. Our results indicate that the loss-of-function mutations in OsLACS9 affect the architecture of internodes resulting in dwarf plants with large starch granules in the chloroplast, showing the suppression of starch degradation. Moreover, the plastid localization of alpha-amylase I-1 (AmyI-1)-a key enzyme involved in starch breakdown in plastids-was suppressed in the lacs9 mutant line. Immunological and confocal laser scanning microscopy analyses showed that OsLACS9-GFP is located in the chloroplast envelope in green tissue. Microscopic analysis showed that OsLACS9s interact with each other in the plastid envelope membrane. Furthermore, OsLACS9 is also one of the proteins transported to plastids without a transit peptide or involvement of the Toc/Tic complex system. To identify the plastid-targeting signal of OsLACS9, the transient expression and localization of a series of N-terminal truncated OsLACS9-green fluorescent protein (GFP) fusion proteins were examined. Truncation analyses identified the N-terminal 30 amino acid residues to be required for OsLACS9 plastid localization. Overall, the data in this study provide an advanced understanding of the function of OsLACS9 and its role in starch degradation and plant growth.

DOI： 10.3390/ijms21062223

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

Irregular precipitation and drought caused an increase in tree mortality rates in multiple forest biomes with alterations in both ecosystem services and carbon balance. Carob (Ceratonia siliqua) growth and production in arid and semi-arid ecosystems are likely affected by climate change-induced droughts. Understanding the physiological responses of drought-induced early-stage tree death and strategies to enhance drought tolerance and optimize growth will help tree improvement programs. Mycorrhizal inoculation has a pronounced impact on plant growth, water absorption, mineral nutrition, and protection from abiotic stresses. However, a better understanding of these complex interconnected cellular processes and arbuscular mycorrhizal fungi (AMF)-mediated mechanisms regulating drought tolerance in plants will enhance its potential application as an efficient approach for bio-amelioration of stresses. The objectives of this work were to elucidate the different effects of autochthone AMF on inorganic solute and water content uptakes, organic adjustments (sugar and proteins content), leaf gas exchange (stomatal conductance and efficiency of photosystems I and II), and oxidative damage of two contrasting ecotypes of carob seedlings: coastal (southern ecotype (SE)) and in-land (northern ecotype (NE)) under control (C), drought (by cessation of irrigation for 15 days (15D)), and recovery (R) conditions. Our findings showed that AMF promoted growth, nutrient content, and physiological and biochemical parameters in plants of both ecotypes during C, 15D, and R conditions. After four days of recovery, stomatal conductance (g(s)), the maximum photochemical efficiency of PSII (F-v/F-m), water content, and plant uptake of mineral nutrients (P, K, Na, and Ca) were significantly higher in shoots of mycorrhizal (AM) than non-mycorrhizal (NM) control plants. Consequently, AMF reduced to a greater degree the accumulation of hydrogen peroxide (H2O2) and oxidative damage to lipid (malondialdehyde (MDA)) content in AM than NM plants in NE and SE, after recovery. Altogether, our findings suggest that AMF can play a role in drought resistance of carob trees at an early stage by increasing the inorganic solutes (P, K, Na, and Ca), water content uptake, organic solutes (soluble sugars and protein content), stomatal conductance, and defense response against oxidative damage during re-watering after drought stress.

DOI： 10.3390/plants9010080

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Publishing type：Part of collection (book)   Publisher：Springer International Publishing  

DOI： 10.1007/978-3-030-37510-2_20

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Publishing type：Part of collection (book)  

© Springer Science+Business Media, LLC, part of Springer Nature 2020. Cereal proteins have formed the basis of human diet worldwide, and their level of consumption is expected to increase. The knowledge of the protein composition and variation of the cereal grains is helpful for characterizing cereal varieties and to identify biomarkers for tolerance mechanisms. Grains produce a wide array of proteins, differing under conditions. Quantitative proteomics is a powerful approach allowing the identification of proteins expressed under defined conditions that may contribute understanding the complex biological systems of grains. Isobaric tags for relative and absolute quantitation (iTRAQ) is a mass spectrometry–based quantitative approach allowing, simultaneously, for protein identification and quantification from multiple samples with high coverage. One of the challenges in identifying grains proteins is their relatively high content (~90–95%) of carbohydrate (starch) and low protein (~4–10%) and lipid (~1%) fractions. In this chapter, we present a robust workflow to carry out iTRAQ quantification of the starchy rice grains.

DOI： 10.1007/978-1-0716-0528-8_29

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Language：English   Publishing type：Research paper (scientific journal)  

Autophagy plays crucial roles in the recycling of metabolites, and is involved in many developmental processes. Rice mutants defective in autophagy are male sterile due to immature pollens, indicating its critical role in pollen development. However, physiological roles of autophagy during seed maturation had remained unknown. We here found that seeds of the rice autophagy-deficient mutant Osatg7-1, that produces seeds at a very low frequency in paddy fields, are smaller and show chalky appearance and lower starch content in the endosperm at the mature stage under normal growth condition. We comprehensively analyzed the effects of disruption of autophagy on biochemical properties, proteome and seed quality, and found an abnormal activation of starch degradation pathways including accumulation of α-amylases in the endosperm during seed maturation in Osatg7-1. These results indicate critical involvement of autophagy in metabolic regulation in the endosperm of rice, and provide insights into novel autophagy-mediated regulation of starch metabolism during seed maturation.

DOI： 10.1038/s41598-019-54361-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The Human Proteome Project (HPP) has made great efforts to clarify the existing evidence of human proteins since 2012. However, according to the recent release of neXtProt (2019-1), approximately 10% of all human genes still have inadequate or no experimental evidence of their translation at the protein level. They were categorized as missing proteins (PE2-PE4). To further the goal of HPP, we developed a two-step bioinformatic strategy addressing the utilization of the SRMAtlas synthetic peptides corresponding to the missing proteins as an exclusive reference in order to explore their natural counterparts within GPM. In the first step, we searched the GPM for the non-nested SRMAtlas peptides corresponding to the missing proteins, taking under consideration only those detected via >= 2 non-nested unitypic/proteotypic peptides "Stranded peptides" with length >= 9 amino acids in the same proteomic study. As a result, 51 missing proteins were newly detected in 35 different proteomic studies. In the second step, we validated these newly detected missing proteins based on matching the spectra of their synthetic and natural peptides in SRMAtIas and GPM, respectively. The results showed that 23 of the missing proteins with >= 2 non-nested peptides were validated by careful spectral matching.

DOI： 10.1021/acs.jproteome.9b00355

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Microorganisms produce volatile compounds (VCs) that promote plant growth and photosynthesis through complex mechanisms involving cytokinin (CK) and abscisic acid (ABA). We hypothesized that plants' responses to microbial VCs involve posttranslational modifications of the thiol redox proteome through action of plastidial NADPH-dependent thioredoxin reductase C (NTRC), which regulates chloroplast redox status via its functional relationship with 2-Cys peroxiredoxins. To test this hypothesis, we analysed developmental, metabolic, hormonal, genetic, and redox proteomic responses of wild-type (WT) plants and a NTRC knockout mutant (ntrc) to VCs emitted by the phytopathogen Alternaria alternata. Fungal VC-promoted growth, changes in root architecture, shifts in expression of VC-responsive CK- and ABA-regulated genes, and increases in photosynthetic capacity were substantially weaker in ntrc plants than in WT plants. As in WT plants, fungal VCs strongly promoted growth, chlorophyll accumulation, and photosynthesis in ntrc-Delta 2cp plants with reduced 2-Cys peroxiredoxin expression. OxiTRAQ-based quantitative and site-specific redox proteomic analyses revealed that VCs promote global reduction of the thiol redox proteome (especially of photosynthesis-related proteins) of WT leaves but its oxidation in ntrc leaves. Our findings show that NTRC is an important mediator of plant responses to microbial VCs through mechanisms involving global thiol redox proteome changes that affect photosynthesis.

DOI： 10.1111/pce.13601

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Next-generation sequencing (NGS) is a revolutionary advancement allowing large-scale discovery of functional molecular markers that has many applications, including plant breeding. High-quality genomic DNA (gDNA) is a prerequisite for successful NGS library preparation and sequencing; however, few reliable protocols to obtain such plant gDNA exist. A previously reported nuclear pellet (NP) method enables extraction of high-yielding gDNA from fresh leaf tissue of maize (Zea mays L.), but the quality does not meet the stringent requirements of NGS. In this study, we optimized the NP method for whole-genome sequencing of rice (Oryza sativa L.) through the integration of simple purification steps. The optimized NP method relied on initial nucleus enrichment, cell lysis, extraction, and subsequent gDNA purification buffers. The purification steps used proteinase K, RNase A, phenol/chloroform/isoamyl alcohol (25:24:1), and chloroform/isoamyl alcohol (24:1) treatments for protein digestion and RNA, protein, and phenol removal, respectively. Our data suggest that this optimized NP method allowed extraction of consistently high-yielding and high-quality undegraded gDNA without contamination by protein and RNA. Moreover, the extracted gDNA fulfilled the quality metrics of NGS library preparation for the Illumina HiSeq X Ten platform by the TruSeq DNA PCR-Free Library Prep Kit (Illumina). We provide a reliable step-by-step guide to the extraction of high-quality gDNA from fresh leaf tissues of rice for molecular biologists with limited resources.

DOI： 10.3390/mps2020054

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Language：English   Publishing type：Research paper (scientific journal)  

Salinity critically limits rice metabolism, growth, and productivity worldwide. Improvement of the salt resistance of locally grown high-yielding cultivars is a slow process. The objective of this study was to develop a new salt-tolerant rice germplasm using speed-breeding. Here, we precisely introgressed the hst1 gene, transferring salinity tolerance from "Kaijin" into high-yielding "Yukinko-mai" (WT) rice through single nucleotide polymorphism (SNP) marker-assisted selection. Using a biotron speed-breeding technique, we developed a BC3F3 population, named "YNU31-2-4", in six generations and 17 months. High-resolution genotyping by whole-genome sequencing revealed that the BC3F2 genome had 93.5% similarity to the WT and fixed only 2.7% of donor parent alleles. Functional annotation of BC3F2 variants along with field assessment data indicated that "YNU31-2-4" plants carrying the hst1 gene had similar agronomic traits to the WT under normal growth condition. "YNU31-2-4" seedlings subjected to salt stress (125 mM NaCl) had a significantly higher survival rate and increased shoot and root biomasses than the WT. At the tissue level, quantitative and electron probe microanalyzer studies indicated that "YNU31-2-4" seedlings avoided Na+ accumulation in shoots under salt stress. The "YNU31-2-4" plants showed an improved phenotype with significantly higher net CO2 assimilation and lower yield decline than WT under salt stress at the reproductive stage. "YNU31-2-4" is a potential candidate for a new rice cultivar that is highly tolerant to salt stress at the seedling and reproductive stages, and which might maintain yields under a changing global climate.

DOI： 10.3390/ijms20102585

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

Rice nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) catalyzes the hydrolytic breakdown of the pyrophosphate and phosphodiester bonds of a number of nucleotides including ADP-glucose and ATP. Under high temperature and elevated CO2 conditions (HT + ECO2), the npp1 knockout rice mutant displayed rapid growth and high starch content phenotypes, indicating that NPP1 exerts a negative effect on starch accumulation and growth. To gain further insight into the mechanisms involved in the NPP1 downregulation induced starch overaccumulation, in this study we conducted photosynthesis, leaf proteomic, and chloroplast phosphoproteomic analyses of wild-type (WT) and npp1 plants cultured under HT + ECO2. Photosynthesis in npp1 leaves was significantly higher than in WT. Additionally, npp1 leaves accumulated higher levels of sucrose than WT. The proteomic analyses revealed upregulation of proteins related to carbohydrate metabolism and the protein synthesis system in npp1 plants. Further, our data indicate the induction of 14-3-3 proteins in npp1 plants. Our finding demonstrates a higher level of protein phosphorylation in npp1 chloroplasts, which may play an important role in carbohydrate accumulation. Together, these results offer novel targets and provide additional insights into carbohydrate metabolism regulation under ambient and adverse conditions.

DOI： 10.3390/ijms19092655

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Walter de Gruyter GmbH  

<title>Abstract</title>
α-Amylases have been of interest in diverse fields for many years because of their importance in basic biology, agriculture, and industry. Starch hydrolysis in plants has been studied extensively in germinating cereal seeds. It is generally accepted that α-amylases are secretory enzymes with a pivotal role in the breakdown of starch reserves in the endosperm. Intriguingly, however, recent investigations reveal that some α-amylases degrade starch in the plastids of living cells. The recent solving of the crystal structure of rice AmyI-1 isoform shows that the binding pocket of starch binding site 1 situated outside of the active site cleft interacts with the substances other than oligosaccharides. These findings provided novel insights into structural and cell biological aspects of α-amylase functions in intracellular transport, organelle targeting, and organ-specific actions. Under global warming, abnormal high temperatures during rice grain filling increase grain chalkiness, resulting in yield loss. Intensive “omics” analyses of developing caryopses and mature grains grown under heat stress showed the downregulation of starch synthesis enzymes and the upregulation of α-amylases. Transgenic studies using ectopic overexpression and suppression of α-amylase revealed that α-amylase is a key factor in grain chalkiness. Here we discuss unique new functions of α-amylase in rice cells.

DOI： 10.1515/amylase-2018-0004

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Other Link： http://www.degruyter.com/view/j/amylase.2018.2.issue-1/amylase-2018-0004/amylase-2018-0004.pdf

Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS MEDIA SA  

Chloroplasts, which perform photosynthesis, are one of the most important organelles in green plants and algae. Chloroplasts maintain an independent genome that includes important genes encoding their photosynthetic machinery and various housekeeping functions. Owing to its non-recombinant nature, low mutation rates, and uniparental inheritance, the chloroplast genome (plastome) can give insights into plant evolution and ecology and in the development of biotechnological and breeding applications. However, efficient methods to obtain high-quality chloroplast DNA (cpDNA) are currently not available, impeding powerful sequencing and further functional genomics research. To investigate effects on rice chloroplast genome quality, we compared cpDNA extraction by three extraction protocols: liquid nitrogen coupled with sucrose density gradient centrifugation, high-salt buffer, and Percoll gradient centrifugation. The liquid nitrogen-sucrose gradient method gave a high yield of high-quality cpDNA with reliable purity. The cpDNA isolated by this technique was evaluated, resequenced, and assembled de novo to build a robust framework for genomic and genetic studies. Comparison of this high-purity cpDNA with total DNAs revealed the read coverage of the sequenced regions; next-generation sequencing data showed that the high-quality cpDNA eliminated noise derived from contamination by nuclear and mitochondrial DNA, which frequently occurs in total DNA. The assembly process produced highly accurate, long contigs. We summarize the extent to which this improved method of isolating cpDNA from rice can provide practical progress in overcoming challenges related to chloroplast genomes and in further exploring the development of new sequencing technologies.

DOI： 10.3389/fpls.2018.00266

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Language：English   Publishing type：Part of collection (book)   Publisher：Humana Press Inc.  

The Golgi apparatus is an endomembrane system organelle and has roles in glycosylation, sorting, and secretion of proteins in the secretory pathway. It has a central function in living organism and is also essential for plant growth. Proteomic approaches to identify the Golgi membrane proteins have been performed in cell suspension cultures and many Golgi membrane-associated proteins were found, whereas it has well established in rice seedling yet. In this chapter, our recent improving published methods for isolated rice Golgi membranes by floating through a discontinuous sucrose density gradient are provided in detail with proteomic analyses.

DOI： 10.1007/978-1-4939-7411-5_6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

In an attempt to complete human proteome project (HPP), Chromosome-Centric Human Proteome Project (C-HPP) launched the journey of missing protein (MP) investigation in 2012. However, 2579 and 572 protein entries in the neXtProt (2017-1) are still considered as missing and uncertain proteins, respectively. Thus, in this study, we proposed a pipeline to analyze, identify, and validate human missing and uncertain proteins in open-access transcriptomics and proteomics databases. Analysis of RNA expression pattern for missing proteins in Human protein Atlas showed that 28% of them, such as Olfactory receptor 1I1 (060431), had no RNA expression, suggesting the necessity to consider uncommon tissues for transcriptomic and proteomic studies. Interestingly, 21% had elevated expression level in a particular tissue (tissue-enriched proteins), indicating the importance of targeting such proteins in their elevated tissues. Additionally, the analysis of RNA expression level for missing proteins showed that 95% had no or low expression level (0-10 transcripts per million), indicating that low abundance is one of the major obstacles facing the detection of missing proteins. Moreover, missing proteins are predicted to generate fewer predicted unique tryptic peptides than the identified proteins. Searching for these predicted unique tryptic peptides that correspond to missing and uncertain proteins in the experimental peptide list of open-access MS-based databases (PA, GPM) resulted in the detection of 402 missing and 19 uncertain proteins with at least two unique peptides (>= 9 aa) at <(5 X 10(-4))% FDR Finally, matching the native spectra for the experimentally detected peptides with their SRMAtlas synthetic counterparts at three transition sources (QQQ, QTOF, QTRAP) gave us an opportunity to validate 41 missing proteins by >= 2 proteotypic peptides.

DOI： 10.1021/acs.jproteome.7b00423

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Language：Japanese   Publisher：CROP SCIENCE SOCIETY OF JAPAN  

DOI： 10.14829/jcsproc.244.0_125

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press  

Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1-NPP6. Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)-Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2-GFP and NPP6-GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER-Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/ mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs.

DOI： 10.1093/pcp/pcw089

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1-NPP6. Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)-Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2-GFP and NPP6-GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER-Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs.

DOI： 10.1093/pcp/pcw089

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Background: Global climate models predict an increase in global mean temperature and a higher frequency of intense heat spikes during this century. Cereals such as rice (Oryza sativa L.) are more susceptible to heat stress, mainly during the gametogenesis and flowering stages. During periods of high temperatures, grain filling often causes serious damage to the grain quality of rice and, therefore, yield losses. While the genes encoding enzymes involved in carbohydrate metabolism of chalky grains have been established, a significant knowledge gap exists in the proteomic and glycomic responses to warm temperatures in situ. Here, we studied the translucent and opaque characters of high temperature stressed chalky grains of 2009 and 2010 (ripening temperatures: 24.4 and 28.0 degrees C, respectively).
Results: Appearance of chalky grains of both years showed some resemblance, and the high-temperature stress of 2010 remarkably extended the chalking of grain. Scanning electron microscopic observation showed that round-shaped starch granules with numerous small pits were loosely packed in the opaque part of the chalky grains. Proteomic analyzes of rice chalky grains revealed deregulations in the expression of multiple proteins implicated in diverse metabolic and physiological functions, such as protein synthesis, redox homeostasis, lipid metabolism, and starch biosynthesis and degradation. The glycomic profiling has shown slight differences in chain-length distributions of starches in the grains of 2009 to 2010. However, no significant changes were observed in the chain-length distributions between the translucent and opaque parts of perfect and chalky grains in both years. The glucose and soluble starch contents in opaque parts were increased by the high-temperature stress of 2010, though those in perfect grains were not different regardless of the environmental changes of 2009 to 2010.
Conclusion: Together with previous findings on the increased expression of alpha-amylases in the endosperm, these results suggested that unusual starch degradation rather than starch synthesis is involved in occurring of chalky grains of rice under the high-temperature stress during grain filling period.

DOI： 10.1186/s12284-016-0100-y

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Language：English   Publishing type：Research paper (bulletin of university, research institution)  

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Other Link： http://hdl.handle.net/10191/39815

Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

High-temperature stress during grain filling hastens the growth rate of endosperm and causes grain chalkiness. Scanning microscopy of chalky areas reveals loosely packed, rounded starch granules with occasional small pits. Intensive investigation of the transcriptome, proteome, and metabolome in developing caryopses under high-temperature stress revealed the downregulation of starch synthesis enzymes and the upregulation of alpha-amylases. High-temperature ripening may unbalance the synthesis and degradation of starch in the developing endosperm cells. In addition to starches, storage proteins are synthesized, assembled, and stored in developing seeds. Several lines of evidence suggest that redox regulation affects seed maturation, including the accumulation of storage starches and proteins, and thus grain quality. A heat-tolerant cultivar of rice shows a characteristic high expression of superoxide dismutase (SOD). H2O2 produced by SOD under high-temperature stress possibly acts as a signal that rapidly can promote the expression of stress-response proteins. Herein, we will discuss the possible molecular physiology of grain chalking under high-temperature stress.

DOI： 10.1080/1343943X.2015.1128112

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

The diversity of protein targeting pathways to plastids and their regulation in response to developmental and metabolic status is a key issue in the regulation of cellular function in plants. The general import pathways that target proteins into and across the plastid envelope with changes in gene expression are critical for plant development by regulating the response to physiological and metabolic changes within the cell. Glycoprotein targeting to complex plastids involves routing through the secretory pathway, among others. However, the mechanisms of trafficking via this system remain poorly understood. The present article discusses our results in site-specific N-glycosylation of nucleotide pyrophosphatase/phosphodiesterases (NPPs) glycoproteins and highlights protein delivery in Golgi/plastid pathway via the secretory pathway. Furthermore, we outline the hypotheses that explain the mechanism for importing vesicles trafficking with nucleus-encoded proteins into plastids.

DOI： 10.1080/15592324.2016.1221558

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Language：Japanese   Publisher：CROP SCIENCE SOCIETY OF JAPAN  

DOI： 10.14829/jcsproc.241.0_114

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Superoxide dismutase (SOD) is widely assumed to play a role in the detoxification of reactive oxygen species caused by environmental stresses. We found a characteristic expression of manganese SOD 1 (MSD1) in a heat-stress-tolerant cultivar of rice (Oryza sativa). The deduced amino acid sequence contains a signal sequence and an N-glycosylation site. Confocal imaging analysis of rice and onion cells transiently expressing MSD1-YFP showed MSD1-YFP in the Golgi apparatus and plastids, indicating that MSD1 is a unique Golgi/plastid-type SOD. To evaluate the involvement of MSD1 in heat-stress tolerance, we generated transgenic rice plants with either constitutive high expression or suppression of MSD1. The grain quality of rice with constitutive high expression of MSD1 grown at 33/28 degrees C, 12/12 h, was significantly better than that of the wild type. In contrast, MSD1-knock-down rice was markedly susceptible to heat stress. Quantitative shotgun proteomic analysis indicated that the overexpression of MSD1 up-regulated reactive oxygen scavenging, chaperone and quality control systems in rice grains under heat stress. We propose that the Golgi/plastid MSD1 plays an important role in adaptation to heat stress.

DOI： 10.1111/pbi.12314

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We investigated the effects of soaking temperature and duration on the germinability of seeds of rice (Oryza sativa L., cv. Koganemochi, Gohyakumangoku, and Koshihikari) that had been stored for a long period. The germinability of the seeds soaked at 5 degrees C for 5 d was markedly lower than that of seeds soaked at 12 degrees C for 5 d. The germinability of the seeds soaked at 5 degrees C for 24 hr was not increased by subsequent soaking at 12 degrees C for 4 d. On the other hand, the germinability of the seeds soaked either at 12 degrees C for 24 hr or at 30 degrees C for 80 min was similar to that of seeds soaked at 12 degrees C for 5 d, even when followed by treatment at 5 degrees C. Thus, the soaking temperature during the first 24 hr was most important for the germination of rice seeds that had been stored for a long period. Western blotting analysis revealed characteristic expression patterns of alpha-amylase isoforms in cultivars correlating with the germinability after soaking at a low-temperature.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

DOI： 10.1038/NPLANTS2015.57

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Life on earth relies upon photosynthesis, which consumes carbon dioxide and generates oxygen and carbohydrates. Photosynthesis is sustained by a dynamic environment within the plant cell involving numerous organelles with cytoplasmic streaming. Physiological studies of chloroplasts, mitochondria and peroxisomes show that these organelles actively communicate during photorespiration, a process by which by-products produced by photosynthesis are salvaged. Nevertheless, the mechanisms enabling efficient exchange of metabolites have not been clearly defined. We found that peroxisomes along chloroplasts changed shape from spherical to elliptical and their interaction area increased during photorespiration. We applied a recent femtosecond laser technology to analyse adhesion between the organelles inside palisade mesophyll cells of Arabidopsis leaves and succeeded in estimating their physical interactions under different environmental conditions. This is the first application of this estimation method within living cells. Our findings suggest that photosynthetic-dependent interactions play a critical role in ensuring efficient metabolite flow during photorespiration.

DOI： 10.1038/NPLANTS.2015.35

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Society for Crop Science and Plant Breeding of Hokuriku  

DOI： 10.19016/hokurikucs.50.0_32

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

We previously developed a molecularly uniform rice-based oral cholera vaccine (MucoRice-CTB) by using an overexpression system for modified cholera toxin B-subunit, CTB (N4Q) with RNAi to suppress production of the major rice endogenous storage proteins. To establish MucoRice-CTB for human use, here we developed hygromycin phosphotransferase selection marker-free MucoRice-CTB by using two different Agrobacterium tumefaciens, each carrying a distinct T-DNA for co-transformation. In the marker-free candidates from co-transformants by segregation in the seed progeny, we selected a line with high CTB expression, line 51A, which we advanced to the T6 generation by self-pollination to obtain a homozygous line without down-regulation of CTB expression. Southern blot analyses showed that three copies of the CTB gene, but not the backbone of the T-DNA binary vector, were inserted into the rice genome of MucoRice-CTB line 51A. By whole genome resequencing, we showed that the transgenes in this line were inserted into intergenic regions in chromosome 3 and chromosome 12. We determined that two full-length copies, each containing the CTB and RNAi expression cassettes, were inserted in a tandem reverted orientation into chromosome 3. An additional copy of the CTB over-expression cassette with a truncated RNAi cassette was inserted into chromosome 12. These findings provide useful information for the establishment of a seed bank of marker-free MucoRice-CTB for human use.

DOI： 10.1007/s11240-014-0575-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

We investigated the proteome of a female Crested Ibis (Nipponia nippon, ID#162) that died on March 10, 2010 at the Sado Japanese Crested Ibis Conservation Center. Protein preparations from the brain, trachea, liver, heart, lung, proventriculus, muscular stomach, small intestine, duodenum, ovary and neck muscle were subjected to in-solution shotgun mass spectrometry (MS)/MS analyses using an LTQ Orbitrap XL mass spectrometer. A search of the National Center for Biotechnology Information Gallus gallus databases revealed 4253 GI (GenInfo Identifier) numbers with the sum of the same 11 tissues examined in the Crested Ibis. To interpret the obtained proteomics data, it was verified in detail with the data obtained from the brain of the Crested Ibis. It has been reported that drebrin A is specifically expressed in adult chicken brain. In the shotgun proteomic analyses of the Crested Ibis, we identified drebrin A as a brain-specific protein. Furthermore, Western blotting analysis of the protein preparations from 10 tissues of the Crested Ibis and 150-day-old hens using anti-drebrin antibodies showed intensive expression of approximately 110 kDa polypeptides of drebrin in both brains. We believe firmly that the present data will contribute to initial and fundamental steps toward understanding the Crested Ibis proteome.

DOI： 10.1111/asj.12214

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

In leaves, it is widely assumed that starch is the end-product of a metabolic pathway exclusively taking place in the chloroplast that (a) involves plastidic phosphoglucomutase (pPGM), ADPglucose (ADPG) pyrophosphorylase (AGP) and starch synthase (SS), and (b) is linked to the Calvin-Benson cycle by means of the plastidic phosphoglucose isomerase (pPGI). This view also implies that AGP is the sole enzyme producing the starch precursor molecule, ADPG. However, mounting evidence has been compiled pointing to the occurrence of important sources, other than the pPGI-pPGM-AGP pathway, of ADPG. To further explore this possibility, in this work two independent laboratories have carried out HPLC-MS/MS analyses of ADPG content in leaves of the near-starchless pgm and aps1 mutants impaired in pPGM and AGP, respectively, and in leaves of double aps1/pgm mutants grown under two different culture conditions. We also measured the ADPG content in wild type (WT) and aps1 leaves expressing in the plastid two different ADPG cleaving enzymes, and in aps1 leaves expressing in the plastid GlgC, a bacterial AGP. Furthermore, we measured the ADPG content in ss3/ss4/aps1 mutants impaired in starch granule initiation and chloroplastic ADPG synthesis. We found that, irrespective of their starch contents, pgm and aps1 leaves, WT and aps1 leaves expressing in the plastid ADPG cleaving enzymes, and aps1 leaves expressing in the plastid GlgC accumulate WT ADPG content. In clear contrast, ss3/ss4/aps1 leaves accumulated ca. 300 fold-more ADPG than WT leaves. The overall data showed that, in Arabidopsis leaves, (a) there are important ADPG biosynthetic pathways, other than the pPGI-pPGM-AGP pathway, (b) pPGM and AGP are not major determinants of intracellular ADPG content, and (c) the contribution of the chloroplastic ADPG pool to the total ADPG pool is low.

DOI： 10.1371/journal.pone.0104997

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The involvement of salicylic acid (SA) in the regulation of stress-induced flowering in the short-day plant pharbitis (also called Japanese morning glory) Ipomoea nil (formerly Pharbitis nil) was studied. Pharbitis cv. Violet was induced to flower when grown in 1/100-strength mineral nutrient solution under non-inductive long-day conditions. All fully expanded true leaves were removed from seedlings, leaving only the cotyledons, and flowering was induced under poor-nutrition stress conditions. This indicates that cotyledons can play a role in the regulation of poor-nutrition stress-induced flowering. The expression of the pharbitis homolog of PHENYLALANINE AMMONIA-LYASE, the enzyme activity of phenylalanine ammonia-lyase (PAL; E.C. 4.3.1.5) and the content of SA in the cotyledons were all up-regulated by the stress treatment. The Violet was also induced to flower by low-temperature stress, DNA demethylation and short-day treatment. Low-temperature stress enhanced PAL activity, whereas non-stress factors such as DNA demethylation and short-day treatment decreased the activity. The PAL enzyme activity was also examined in another cultivar, Tendan, obtaining similar results to Viole

DOI： 10.1016/j.jplph.2014.03.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER GMBH, URBAN & FISCHER VERLAG  

The involvement of salicylic acid (SA) in the regulation of stress-induced flowering in the short-day plant pharbitis (also called Japanese morning glory) Ipomoea nil (formerly Pharbitis nil) was studied. Pharbitis cv. Violet was induced to flower when grown in 1/100-strength mineral nutrient solution under non-inductive long-day conditions. All fully expanded true leaves were removed from seedlings, leaving only the cotyledons, and flowering was induced under poor-nutrition stress conditions. This indicates that cotyledons can play a role in the regulation of poor-nutrition stress-induced flowering. The expression of the pharbitis homolog of PHENYLALANINE AMMONIA-LYASE, the enzyme activity of phenylalanine ammonia-lyase (PAL; E. C. 4.3.1.5) and the content of SA in the cotyledons were all up-regulated by the stress treatment. The Violet was also induced to flower by low-temperature stress, DNA demethylation and short-day treatment. Low-temperature stress enhanced PAL activity, whereas non-stress factors such as DNA demethylation and short-day treatment decreased the activity. The PAL enzyme activity was also examined in another cultivar, Tendan, obtaining similar results to Violet. The exogenously applied SA did not induce flowering under non-stress conditions but did promote flowering under weak stress conditions in both cultivars. These results suggest that stress-induced flowering in pharbitis is induced, at least partly, by SA, and the synthesis of SA is promoted by PAL. (C) 2014 Elsevier GmbH. All rights reserved.

DOI： 10.1016/j.jplph.2014.03.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

AmyI-1 is an alpha-amylase from Oryza sativa (rice) and plays a crucial role in degrading starch in various tissues and at various growth stages. This enzyme is a glycoprotein with an N-glycosylated carbohydrate chain, a unique characteristic among plant alpha-amylases. In this study, we report the first crystal structure of AmyI-1 at 2.2-angstrom resolution. The structure consists of a typical (beta/alpha)(8)-barrel, which is well-conserved among most alpha-amylases in the glycoside hydrolase family-13. Structural superimposition indicated small variations in the catalytic domain and carbohydrate-binding sites between AmyI-1 and barley alpha-amylases. By contrast, regions around the N-linked glycosylation sites displayed lower conservation of amino acid residues, including Asn-263, Asn-265, Thr-307, Asn-342, Pro-373, and Ala-374 in AmyI-1, which are not conserved in barley alpha-amylases, suggesting that these residues may contribute to the construction of the structure of glycosylated AmyI-1. These results increase the depths of our understanding of the biological functions of AmyI-1.

DOI： 10.1080/09168451.2014.917261

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Background and Objective: Porphyromonas gingivalis is a major etiological agent in the development and progression of periodontal diseases. In this study, we isolated a cell growth inhibitor against P. gingivalis species from rice protein extract.
Material and Methods: The cell growth inhibitor active against P. gingivalis was purified from polished rice extract using a six-step column chromatography process. Its antimicrobial properties were investigated through microscope analysis, spectrum of activity and general structure.
Results: The inhibitor was identified as AmyI-1, an alpha-amylase, and showed significant cell growth inhibitory activity against P. gingivalis species. Scanning electron microscopy micrograph analysis and bactericidal assay indicated an intriguing possibility that the inhibitor compromises the cell membrane structure of the bacterial cells and leads to cell death. Moreover, alpha-amylases from human saliva and porcine pancreas showed inhibitory activity similar to that of AmyI-1.
Conclusions: This is the first study to report that alpha-amylases cause cell death of periodontal pathogenic bacteria. This finding highlights the potential importance and therapeutic potential of alpha-amylases in treating periodontal diseases.

DOI： 10.1111/jre.12079

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Nucleotide pyrophosphatase/phosphodiesterase (NPP) is a widely distributed enzymatic activity occurring in both plants and mammals that catalyzes the hydrolytic breakdown of the pyrophosphate and phosphodiester bonds of a number of nucleotides. Unlike mammalian NPPs, the physiological function of plant NPPs remains largely unknown. Using a complete rice NPP1-encoding cDNA as a probe, in this work we have screened a rice shoot cDNA library and obtained complete cDNAs corresponding to six NPP genes (NPP1-NPP6). As a first step to clarify the role of NPPs, recombinant NPP1, NPP2 and NPP6 were purified from transgenic rice cells constitutively expressing NPP1, NPP2 and NPP6, respectively, and their enzymatic properties were characterized. NPP1 and NPP6 exhibited hydrolytic activities toward ATP, UDP-glucose and the starch precursor molecule, ADP-glucose, whereas NPP2 did not recognize nucleotide sugars as substrates, but hydrolyzed UDP, ADP and adenosine 5'-phosphosulfate. To gain insight into the physiological function of rice NPP1, an npp1 knockout mutant was characterized. The ADP-glucose hydrolytic activities in shoots of npp1 rice seedlings were 8% of those of the wild type (WT), thus indicating that NPP1 is a major determinant of ADP-glucose hydrolytic activity in rice shoots. Importantly, when seedlings were cultured at 160 Pa CO2 under a 28 degrees C/23 degrees C (12h light/12 h dark) regime, npp1 shoots and roots were larger than those of wild-type (WT) seedlings. Furthermore, the starch content in the npp1 shoots was higher than that of WT shoots. Growth and starch accumulation were also enhanced under an atmospheric CO2 concentration (40 Pa) when plants were cultured under a 33 degrees C/28 degrees C regime. The overall data strongly indicate that NPP1 exerts a negative effect on plant growth and starch accumulation in shoots, especially under high CO2 concentration and high temperature conditions.

DOI： 10.1093/pcp/pct139

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Nucleotide pyrophosphatase/phosphodiesterase (NPP) is a widely distributed enzymatic activity occurring in both plants and mammals that catalyzes the hydrolytic breakdown of the pyrophosphate and phosphodiester bonds of a number of nucleotides. Unlike mammalian NPPs, the physiological function of plant NPPs remains largely unknown. Using a complete rice NPP1-encoding cDNA as a probe, in this work we have screened a rice shoot cDNA library and obtained complete cDNAs corresponding to six NPP genes (NPP1-NPP6). As a first step to clarify the role of NPPs, recombinant NPP1, NPP2 and NPP6 were purified from transgenic rice cells constitutively expressing NPP1, NPP2 and NPP6, respectively, and their enzymatic properties were characterized. NPP1 and NPP6 exhibited hydrolytic activities toward ATP, UDP-glucose and the starch precursor molecule, ADP-glucose, whereas NPP2 did not recognize nucleotide sugars as substrates, but hydrolyzed UDP, ADP and adenosine 5′-phosphosulfate. To gain insight into the physiological function of rice NPP1, an npp1 knockout mutant was characterized. The ADP-glucose hydrolytic activities in shoots of npp1 rice seedlings were 8% of those of the wild type (WT), thus indicating that NPP1 is a major determinant of ADP-glucose hydrolytic activity in rice shoots. Importantly, when seedlings were cultured at 160 Pa CO2 under a 28°C/23°C (12 h light/12 h dark) regime, npp1 shoots and roots were larger than those of wild-type (WT) seedlings. Furthermore, the starch content in the npp1 shoots was higher than that of WT shoots. Growth and starch accumulation were also enhanced under an atmospheric CO2 concentration (40 Pa) when plants were cultured under a 33°C/28°C regime. The overall data strongly indicate that NPP1 exerts a negative effect on plant growth and starch accumulation in shoots, especially under high CO 2 concentration and high temperature conditions. © 2013 The Author 2013.

DOI： 10.1093/pcp/pct139

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Humana Press Inc.  

Plastids are specialized cell organelles in plant cells that are differentiated into various forms including chloroplasts, chromoplasts, and amyloplasts, and fulfill important functions in maintaining the overall cell metabolism and sensing environmental factors such as sunlight. It is therefore important to grasp the mechanisms of differentiation and functional changes of plastids in order to enhance the understanding of vegetality. In this chapter, details of a method for the extraction of intact plastids that makes analysis possible while maintaining the plastid functions are provided
in addition, a quantitative shotgun method for analyzing the composition and changes in the content of proteins in plastids as a result of environmental impacts is described. © 2014 Springer Science+Business Media, LLC.

DOI： 10.1007/978-1-62703-631-3_32

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Blackwell Publishing  

We investigated the proteome of a female Crested Ibis (Nipponia nippon,ID#162) that died on March 10, 2010 at the Sado Japanese Crested Ibis Conservation Center. Protein preparations from the brain, trachea, liver, heart, lung, proventriculus, muscular stomach, small intestine, duodenum, ovary and neck muscle were subjected to in-solution shotgun mass spectrometry (MS)/MS analyses using an LTQ Orbitrap XL mass spectrometer. A search of the National Center for Biotechnology Information Gallus gallus databases revealed 4253 GI (GenInfo Identifier) numbers with the sum of the same 11 tissues examined in the Crested Ibis. To interpret the obtained proteomics data, it was verified in detail with the data obtained from the brain of the Crested Ibis. It has been reported that drebrin A is specifically expressed in adult chicken brain. In the shotgun proteomic analyses of the Crested Ibis, we identified drebrin A as a brain-specific protein. Furthermore, Western blotting analysis of the protein preparations from 10 tissues of the Crested Ibis and 150-day-old hens using anti-drebrin antibodies showed intensive expression of approximately 110kDa polypeptides of drebrin in both brains. We believe firmly that the present data will contribute to initial and fundamental steps toward understanding the Crested Ibis proteome. © 2014 Japanese Society of Animal Science.

DOI： 10.1111/asj.12214

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Humana Press Inc.  

Glycoprotein is a major element in higher organisms including mammalians and plants. It is widely accepted that variation in cellular N-glycome is related to modulation in dynamic cellular mechanisms such as cell-cell adhesion, cell activation, and malignant alterations in mammalian cells. However, the physiological importance of glycan modification of glycoproteins in plant cells is still a matter of dispute. Therefore, a comprehensive and high-throughput analysis of N-glycome in plant glycoproteins is needed. Here, an application of the glycoblotting-mass spectrometry technique to plant glycoprotein research is described. © 2014 Springer Science+Business Media, LLC.

DOI： 10.1007/978-1-62703-631-3_44

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press  

In plants, environmental stresses cause an increase in the intracellular level of reactive oxygen species (ROS), leading to tissue injury. To obtain biochemical insights into this damage process, we investigated the protein carbonyls formed by ROS or by the lipid peroxide-derived α,β- unsaturated aldehydes and ketones (i.e. reactive carbonyl species, or RCS) in the leaves of Arabidopsis thaliana under salt stress. A. thaliana Col-0 plants that we treated with 300 mM NaCl for 72 h under continuous illumination suffered irreversible leaf damage. Several RCS such as 4-hydroxy-(E)-2-nonenal (HNE) were increased within 12 h of this salt treatment. Immunoblotting using distinct antibodies against five different RCS, i.e. HNE, 4-hydroxy-(E)-2-hexenal, acrolein, crotonaldehyde and malondialdehyde, revealed that RCS-modified proteins accumulated in leaves with the progress of the salt stress treatment. The band pattern of Western blotting suggested that these different RCS targeted a common set of proteins. To identify the RCS targets, we collected HNE-modified proteins via an anti-HNE antiserum affinity trap and performed an isobaric tag for relative and absolute quantitation, as a quantitative proteomics approach. Seventeen types of protein, modified by 2-fold more in the stressed plants than in the non-stressed plants, were identified as sensitive RCS targets. With aldehyde-reactive probe-based affinity trapping, we collected the oxidized proteins and identified 22 additional types of protein as sensitive ROS targets. These RCS and ROS target proteins were distributed in the cytosol and apoplast, as well as in the ROS-generating organelles the peroxisome, chloroplast and mitochondrion, suggesting the participation of plasma membrane oxidation in the cellular injury. Possible mechanisms by which these modified targets cause cell death are discussed. © 2014 The Author 2014.

DOI： 10.1093/pcp/pcu072

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY  

In plant cells, glycerol 3-phosphate (G3P) is apparently able to permeate the plastid envelope, but no specific transporter has been characterized so far. The Arabidopsis five G3Pp proteins have been predicted as putative G3P permeases because of their high homologies with the prokaryotic G3P/phosphate antiporter GlpT. In the present study, G3Pp4 was characterized in detail utilizing reverse genetic approaches. Promoter analysis using GUS expression revealed that G3Pp4 was expressed strongly throughout the embryos during late developmental stages, and the seed lipid contents decreased in two g3pp4 knockout mutants. An enhanced yellow fluorescent protein-fused G3Pp4 was localized in the plastids, functioned physiologically in A. thaliana, and had G3P-transport activity in E. coli. These results suggest that Arabidopsis G3Pp4 is a plastid envelope-localized G3P transporter and involved in accumulation of storage lipids in late embryogenesis.

DOI： 10.5511/plantbiotechnology.14.0222a

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Society of Applied Glycoscience  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS RESEARCH FOUNDATION  

Recent proteomic analyses revealed dynamic changes of metabolisms during rice grain development. Interestingly, proteins involved in glycolysis, citric acid cycle, lipid metabolism, and proteolysis were accumulated at higher levels in mature grain than those of developing stages. High temperature (HT) stress in rice ripening period causes damaged (chalky) grains which have loosely packed round shape starch granules. The HT stress response on protein expression is complicated, and the molecular mechanism of the chalking of grain is obscure yet. Here, the current state on the proteomics research of rice grain grown under HT stress is briefly overviewed.

DOI： 10.3389/fpls.2013.00036

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Language：English   Publishing type：Part of collection (book)   Publisher：Elsevier Inc.  

DOI： 10.1016/B978-0-08-092615-5.50017-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Genes that promote DNA methylation and demethylation in plants have been characterized mainly in Arabidopsis. Arabidopsis DNA demethylation is mediated by bi-functional DNA enzymes with glycosylase activity that removes 5-methylcytosine and lyase activity that nicks double-stranded DNA at an abasic site. Homologous recombination-promoted knock-in targeting of the ROS1a gene, the longest of six putative DNA demethylase genes in the rice genome, by fusing its endogenous promoter to the GUS reporter gene, led to reproducibly disrupted ROS1a in primary (T0) transgenic plants in the heterozygous condition. These T0 plants exhibited no overt morphological phenotypes during the vegetative phase, and GUS staining showed ROS1a expression in pollen, unfertilized ovules and meristematic cells. Interestingly, neither the maternal nor paternal knock-in null allele, ros1a-GUS1, was virtually detected in the progeny; such an intransmittable null mutation is difficult to isolate by conventional mutagenesis techniques that are usually used to identify and isolate mutants in the progeny population. Even in the presence of the wild-type paternal ROS1a allele, the maternal ros1a-GUS1 allele caused failure of early-stage endosperm development, resulting in incomplete embryo development, with embryogenesis producing irregular but viable embryos that failed to complete seed dormancy, implying non-equivalent maternal and paternal contribution of ROS1a in endosperm development. The paternal ros1a-GUS1 allele was not transmitted to progeny, presumably because of a male gametophytic defect(s) prior to fertilization. Thus, ROS1a is indispensable in both male and female gametophytes, and DNA demethylation must plays important roles in both gametophytes.

DOI： 10.1111/j.1365-313X.2012.05009.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The Cry2Aa3 gene was introduced into asporogenic Bacillus thuringiensis, and the synthesized protoxin killed Bombyx mori and Lymantria dispar larvae. Chymotrypsin hydrolyzed the linkages between 49Tyr/Va150 and 145Lys/Ser146 in the protoxin, and 50- and 58-kDa fragments were generated, respectively. Both peptides killed the larvae of both insects.

DOI： 10.1128/AEM.07123-11

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The viability of the estrogen-receptor (ER)-based chemically inducible gene expression system LexA-VP16-ER (XVE) in combination with the site-specific DNA recombination Cre/loxP system in rice was examined using transgenic plants introduced with a plasmid vector pUH-GFP2 that controls the expression of a green fluorescent protein (GFP) gene. β-estradiol applied to the germinating seeds of the transgenic plants, successfully induced the mRNA expression of the GFP gene. Inducible gene suppression was also tested by replacing the GFP gene by an RNAi cassette
this cassette targeted OsSPS1, a gene encoding sucrose phosphate synthase. When the RNAi plants were treated with the inducer, the transcript levels of OsSPS1 decreased. Concomitantly, the plant length became shorter or the sucrose/starch molar ratio in the leaf blades decreased, suggesting the successful suppression of the target gene. Finally, the utility and remaining problems of this inducible expression system are discussed.

DOI： 10.1626/pps.15.73

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society for Food Science and Technology  

Rice grains contain starch degrading enzymes, including α-glucosidases, α-amylases, β-amylases, pullulanase and isoamylases. To investigate the elution behavior of these enzymes from rice grains into cooking water during rice cooking, we separated cooking water from rice grains after soaking (20°C) and when the temperature reached 40°C and 60°C during cooking. Immunological detection of these enzymes in rice grains and cooking water was carried out with SDS-PAGE and immunoblot. Bands corresponding to pullulanase, α-glucosidase, isoamylase 1, and α-amylase E were detected in both rice grains and cooking water at each temperature. Bands corresponding to α-amylases A + B and H were not detected in rice grains, but were detected in the cooking water at each temperature. The β-amylase band was detected in rice grains but was not detected in cooking water. These results suggest that the amount of enzyme eluted into cooking water depends on enzyme localization and quantity in rice grains. © 2012 Food Sci. Technol. Res.

DOI： 10.3136/fstr.18.659

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Language：Japanese   Publisher：Japanese Proteomics Society (Japan Human Proteome Organisation)  

DOI： 10.14889/jhupo.2012.0.152.0

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DOI： 10.14889/jhupo.2012.0.61.0

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DOI： 10.14889/jhupo.2012.0.118.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

SecA is an ATP-driven motor for protein translocation in bacteria and plants. Mycobacteria and listeria were recently found to possess two functionally distinct secA genes. In this study, we found that Cyanidioschyzon merolae, a unicellular red alga, possessed two distinct secA-homologous genes; one encoded in the cell nucleus and the other in the plastid genome. We found that the plastid-encoded SecA homolog showed significant ATPase activity at low temperature, and that the ATPase activity of the nuclear-encoded SecA homolog showed significant activity at high temperature. We propose that the two SecA homologs play different roles in protein translocation.

DOI： 10.1271/bbb.110338

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Midgut juice of Plutella xylostella strain PXR which is resistant to Cry1Ac was biochemically characterized relative to the susceptible PXS strain. The midgut juice of PXR (PXR-Juice) was shown to process Cry1Ac protoxin to 60 kDa active toxin with the same processing pattern as that of juice from PXS (PXS-Juice) in SOS-PAGE. PXS larvae which were given the Cry1Ac toxin pre-processed with PXR-Juice were killed with the same rate as that with Cry1Ac pre-activated by trypsin. PXR-Juice was found to contain three times larger amount of 66 kDa protein (P66) than PXS-Juice and the N-terminal amino acid sequence of P66 was matched to that of glucosinolate sulfatase in data base search. The protein band of P66 was coincided with the band of p-nitro phenyl sulfatase activity in zymogram. P66 purified to homogeneity in SOS-PAGE bound to Cry1Ac and soybean agglutinin, and K-D for Cry1Ac was estimated to be 718 nM with surface plasmon resonance analysis. Using purified sulfatase, K-m and V-max were estimated and involvement of the enzyme in the PXR resistance was discussed. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.pestbp.2011.09.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

We previously demonstrated that the rice kinesin K16, which belongs to the kinesin-7 subfamily, has unique enzymatic properties and atomic structure within key functional regions. In this study, we focused on a novel rice plant kinesin, K23, which also belongs to the kinesin-7 subfamily. The biochemical characterization of the K23 motor domain (K23MD) was studied and compared with the rice kinesin K16 and other related kinesins. K23 exhibits similar to 45-fold (1.3 Pi mol(-1)site mol(-1)s(-1)) lower microtubule-dependent ATPase activity than conventional kinesins, whereas its affinity for microtubules is comparable with conventional kinesins. MgADP-free K23 is unstable compared with the unusually stable MgADP-free K16MD. The enzymatic properties of K23MD are somewhat different from those of K16. We used a fluorescent ATP analogue 2'(3')-O-(N'-methylanthraniloyl)-ATP (mant-ATP) for the kinetic characterization of K23. The fluorescence of mant-ATP was not significantly altered during its hydrolysis by K23. However, significant fluorescence resonance energy transfer (FRET) between mant-ATP and W21 in the motor domain was observed. The kinetic study using FRET revealed that K23 has unique kinetic characteristics when compared with other kinesins.

DOI： 10.1093/jb/mvr012

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Other Link： http://agriknowledge.affrc.go.jp/RN/2010811615

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Other Link： http://hdl.handle.net/10191/18614

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

The Rv3377c and Rv3378c genes from Mycobacterium tuberculosis are specifically found in the virulent Mycobacterium species, but not in the avirulent species. The Rv3378c-encoded enzyme produced tuberculosinol 2 (5(6), 13(14)-halimadiene-15-ol), 13R-5a and 13S-isotuberculosinol 5b (5(6), 14(15)-halimadiene-13-ol) as its enzymatic products from tuberculosinyl diphosphate 3, indicating that the Rv3378c enzyme catalyzed the nucleophilic addition of a water molecule after the release of a diphosphate moiety. The three enzymatic products 2, 5a, and 5b were produced irrespective of the N- and C-terminal His-tagged Rv3378c enzymes, and of the maltose-binding protein fusion enzyme; the product distribution ratio was identical between the enzymes as 1:1 for 2:5, and 1:3 for 5a:5b. The successful separation of 5a and 5b by a chiral HPLC column provided the first complete assignments of H-1- and C-13-NMR data for 5a and 5b. The enzymatic mechanism for producing 2, 5a, and 5b is proposed here, and the optimal catalytic conditions and kinetic parameters, in addition to the divalent metal effects, are described. Site-directed mutagenesis of Asp into Asn, targeted at the DDXXD motif, resulted in significantly decreased enzymatic activity.

DOI： 10.1271/bbb.100570

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Genomic analysis predicted that the rice (Oryza sativa var. japonica) genome encodes at least 41 kinesin-like proteins including the novel kinesin O12, which is classified as a kinesin-14 family member. O12 has a calponin homology (CH) domain that is known as an actin-binding domain. In this study, we expressed the functional domains of O12 in Escherichia coli and determined its enzymatic characteristics compared with other kinesins. The microtubule-dependent ATPase activity of recombinant O12 containing the motor and CH domains was significantly reduced in the presence of actin. Interestingly, microtubule-dependent ATPase activity of the motor domain was also affected by actin in the absence of the CH domain. Our findings suggest that the motor activity of the rice plant-specific kinesin O12 may be regulated by actin.

DOI： 10.1093/jb/mvq122

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT CELL & MOLECULAR BIOL  

Our previous investigation demonstrated that ADP-glucose hydrolytic nucleotide pyrophosphatase/phosphodiesterase (NPP) 1 is transported from the endoplasmic reticulum (ER)-Golgi system to the plastids via a secretory pathway in rice cells [Nanjo et al. (2006) Plant Cell 18: 2582-2592]. In this study, we analyzed the enzymatic characteristics and subcellular localization of its isozyme, NPP3. Unlike NPP1, NPP3 exhibited no hydrolytic activity toward ADP-glucose and no plastid-targeting ability. Furthermore, there was a clear difference between their N-terminal proteolytic processing schemes to form mature enzyme proteins. NPP1 is matured to a 70-kDa protein by two-step proteolytic processing. We detected the 72 kDa form of NPP1 in the microsomes of rice cells in addition to the 70 kDa mature protein, strongly suggesting that proprotein processing occurs post-translationally in the ER-Golgi system. To clarify the existence of the plastid-targeting signal of NPP1, the plastid localization of a series of carboxy-terminal truncated NPP1 proteins fused with green fluorescence protein was tested in rice cells. The results showed that NPP1 cannot be delivered to the plastid by the N-terminal region, including the ER signal sequence and the proprotein processing site, and that the peptide region, from 308 to 478 amino acid residues, is probably important for the transport of NPP1 into plastids in rice cells.

DOI： 10.5511/plantbiotechnology.10.1228a

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Language：Japanese   Publisher：Japanese Proteomics Society (Japan Human Proteome Organisation)  

DOI： 10.14889/jhupo.2011.0.214.0

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DOI： 10.14889/jhupo.2011.0.188.0

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2011.0.27.0

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Other Link： http://id.ndl.go.jp/bib/10916561

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We established an in situ hybridization (ISH) technique by modification of hybridization probes with locked nucleic acids (LNAs) and demonstrated isoform-specific localization of transcripts of Brassica rapanitrilase (BrNIT-T) genes in clubroot tissue infected with Plasmodiophora brassicae. Chimeric oligo DNA probes containing LNAs demonstrated highly improved specificities and could discriminate between BrNIT-T1 and BrNIT-T2. These LNA-containing probes were applied to ISH. BrNIT-T1 was strongly expressed in cells containing expanding secondary plasmodia of P. brassicae, but not in cells containing resting spores. On the other hand, BrNIT-T2 transcripts were localized in noninfected cells rather than infected cells during the clubroot growth phase but coexisted with mature resting spores at a later phase of clubroot development. Immunostaining for indole-3-acetic acid (IAA) revealed IAA accumulation in cells containing growing plasmodia. IAA immunostaining in infected cells was reduced as the pathogen formed resting spores, but the signal was again enhanced in cells containing mature resting spores at a later phase of infection, suggesting that IAA is involved in both the early growth and the latest maturation phase of clubroot development. Expression of BrNIT-T1 and BrNIT-T2 in turnip roots was upregulated by exogenous treatment with cytokinin and jasmonic acid, respectively. Thus, these two phytohormones are possible triggers of abnormal IAA production in clubroot tissue via induction of the respective nitrilase. Given these results, we propose a model for isoform-specific roles of B. rapa nitrilases in auxin biosynthesis involved in phytohormone crosstalk during development of clubroot disease. © Springer Science+Business Media, LLC 2009.

DOI： 10.1007/s00344-009-9131-6

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DOI： 10.11541/jsag.2010.0.33.0

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DOI： 10.11541/jsag.2010.0.47.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

Secretory proteins and extracellular glycans are transported to the extracellular space during cell growth. These materials are carried in secretory vesicles generated at the trans-Golgi network (TGN). Analysis of the mammalian post-Golgi secretory pathway demonstrated the movement of separated secretory vesicles in the cell. Using secretory carrier membrane protein 2 (SCAMP2) as a marker for secretory vesicles and tobacco (Nicotiana tabacum) BY-2 cell as a model cell, we characterized the transport machinery in plant cells. A combination of analyses, including electron microscopy of quick-frozen cells and four-dimensional analysis of cells expressing fluorescent-tagged SCAMP2, enabled the identification of a clustered structure of secretory vesicles generated from TGN that moves in the cell and eventually fuses with plasma membrane. This structure was termed the secretory vesicle cluster (SVC). The SVC was also found in Arabidopsis thaliana and rice (Oryza sativa) cells and moved to the cell plate in dividing tobacco cells. Thus, the SVC is a motile structure involved in mass transport from the Golgi to the plasma membrane and cell plate in plant cells.

DOI： 10.1105/tpc.108.058933

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DOI： 10.11541/jsag.2008.0.87.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER  

The hydrolytic enzyme v.-amylase (EC 3.2.1.1) is produced mainly in aleurone cells of germinating cereals, and the phytohormone gibberellin (GA) is essential for its induction. However, in rice (Oryza sativa L.), sulfuric acid (H2SO4) induces alpha-amylase production in aleurone tissue even in the absence of GA. Here, the pre-treatment of rice aleurone cells with H2SO4 and incubation in water induced a-amylase activity, as if the cells had been incubated in GA solution. (C) 2007 Elsevier Masson SAS. All rights reserved.

DOI： 10.1016/j.plaphy.2007.09.004

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We found that appropriate treatment with a highly potent and long-lasting abscisic acid analog enhanced the tissue expansion of scutellum during early seedling development of rice, accompanied by increases of protein and starch accumulation in the tissue. A comparative display of the protein expression patterns in the abscisic acid analog-treated and non-treated tissues on two dimensional gel electrophoretogram indicated that approximately 30% of the scutellar proteins were induced by abscisic acid. The abscisic acid-induced proteins included sucrose metabolizing, glycolytic, and ATP-producing enzymes. Most of these enzyme proteins also increased during the seedling growth. In addition, the expression of some isoforms of UDP-glucose pyrophosphorylase, 3-phosphoglycerate kinase, and mitochondrial ATP synthase beta chain was stimulated in the scutellum, with suppressed expression of α-amylase. We concluded that abscisic acid directly and indirectly stimulates the expression of numerous proteins, including carbohydrate metabolic enzymes, in scutellar tissues.

DOI： 10.1271/bbb.60675

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A nucleotide pyrophosphatase/phosphodiesterase (NPP) activity that catalyzes the hydrolytic breakdown of ADP-glucose (ADPG) has been shown to occur in the plastidial compartment of both mono- and dicotyledonous plants. To learn more about this enzyme, we purified two NPPs from rice (Oryza sativa) and barley (Hordeum vulgare) seedlings. Both enzymes are glycosylated, since they bind to concanavalin A, stain with periodic acid-Schiff reagent, and are digested by Endo-H. A complete rice NPP cDNA, designated as NPP1, was isolated, characterized, and overexpressed in transgenic plants displaying high ADPG hydrolytic activity. Databank searches revealed that NPP1 belongs to a functionally divergent group of plant nucleotide hydrolases. NPP1 contains numerous N-glycosylation sites and a cleavable hydrophobic signal sequence that does not match with the N-terminal part of the mature protein. Both immunocytochemical analyses and confocal fluorescence microscopy of rice cells expressing NPP1 fused with green fluorescent protein (GFP) revealed that NPP1-GFP occurs in the plastidial compartment. Brefeldin A treatment of NPP1-GFP-expressing cells prevented NPP1-GFP accumulation in the chloroplasts. Endo-H digestibility studies revealed that both NPP1 and NPP1-GFP in the chloroplast are glycosylated. Collectively, these data demonstrate the trafficking of glycosylated proteins from the endoplasmic reticulum-Golgi system to the chloroplast in higher plants. © 2006 American Society of Plant Biologists.

DOI： 10.1105/tpc.105.039891

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We evaluated the ability of Cry1Aa9, Cry1Ab4, and Cry1Ac1 insecticidal toxins from Bacillus thuringiensis to destroy liposomes. Cry1A toxins are thought to form pores in midgut apical cell membranes (BBMV), thereby disrupting midgut cells. Liposomes containing fluorescent calcein were prepared using phosphatidylcholine (PC) and phosphatidylserine (PS) (PC/PS-Liposomes) or PC alone (PC-Liposomes). Cry1Ab (1.4 μM), but not Cry1Aa or Cry1Ac, disrupted PC/PS-Liposomes and PC-Liposomes. PC/PS-Liposomes containing cholesterol and oligosaccharylceramide from Plutella xylostella midgut were damaged even more extensively by Cry1Ab, but the inclusion of either lipid alone had no effect. The initial velocity of Cry1Ab-mediated liposome disruption increased 17-fold when liposomes were prepared with Triton X-100-soluble proteins from Bombyx mori BBMV and PC (PC/Proteo-Liposomes), and Cry1Aa and Cry1Ac also caused slight disruption. These data suggest that Cry1Ab achieves higher penetration into PC/PS-Liposomes, PC-Liposomes, and PC/Proteo-Liposomes compared with Cry1Aa or Cry1Ac and that Cry1Ab may interact with membrane proteins. © 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.pestbp.2005.02.001

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The Golgi membranes labeled with cis-Golgi marker GFP-SYP31 were isolated from suspension-cultured cells of rice transformed with 35S::GFP-SYP31 by floating through a discontinuous sucrose density gradient in the presence of 5 mM MgCl2. The specific fluorescence intensity of final membrane preparation increased to approximately 150-fold in comparison with that of the post-nucleus soluble fraction. Specific activity of membrane-bound α-mannosidase (cis-Golgi) markedly increased, but NDPase (medial-/trans-Golgi) and NADPH-cytochrome c reductase endoplasmic reticulum were weakly detected in the membrane fraction. The other organelle marker enzymes, cytochrome c oxidase (mitochondria), alkaline pyrophosphatase (plastid), and catalase (peroxisome) were not detectable. Comparative display of protein spots in GFP-SYP31-labeled membranes, microsomal membranes and soluble fraction on the two dimensional gels showed that some proteins are markedably concentrated in the cis-Golgi membrane fraction. Furthermore, the mass spectrometric analysis of proteins separated by SDS-polyacrylamide gel electrophoresis indicated that the highly purified Golgi membranes contained several membrane traffic-related proteins and ER resident proteins. These findings indicated a close relationship between the ER and the cis-Golgi membranes. Copyright © 2006 The Japanese Society for Plant Cell and Molecular Biology.

DOI： 10.5511/plantbiotechnology.23.475

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S350_3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Applied Entomology & Zoology  

DOI： 10.1303/aez.2005.125

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The region containing reactive cysteines, Cys 707 (SH1)-Cys 697 (SH2), of skeletal muscle myosin is thought to play a key role in the conformational changes of the myosin head during force generation coupled to ATP hydrolysis. In the present study, we synthesized a photochromic crosslinker, 4,4'-azobenzene-dimaleimide (ABDM), that undergoes reversible cis-trans isomerization upon ultra violet (UV) and visible (VIS) light irradiation resulting in a change in the crosslinking length from 5 to 17 A. The reactive cysteines, SH1 and SH2, of myosin subfragment 1 (S1) were crosslinked with ABDM, yielding an ABDM-S1 complex. The changes in absorbance induced by UV/VIS light irradiation of the complex were similar to those of free ABDM indicating that the incorporation of ABDM at the SH1 and SH2 sites did not disrupt the isomerization of crosslinked ABDM. Small-angle synchrotron X-ray scattering analysis of the ABDM-S1 complex in solution suggested that the localized conformational changes resulting from the cis to trans isomerization on ABDM crosslinking of SH1 and SH2 induced a small but significant swing in the lever arm portion of S1 in the opposite direction from that induced by ATP.

DOI： 10.1093/jb/mvh194

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER  

Hormonal regulation of expression of alpha-amylase II-4 that lacks the gibberellin-response cis-element (GARE) in the promoter region of the gene was studied in germinating rice (Oryza sativa L.) seeds. Temporal and spatial expression of alpha-amylase II-4 in the aleurone layer were essentially identical to those of alpha-amylase I-1 whose gene contains GARE, although these were distinguishable in the embryo tissues at the early stage of germination. The gibberellin-responsible expression of a-amylase II-4 was also similar to that of alpha-amylase I-1. However, the level of a-amylase II-4 mRNA was not increased by gibberellin, indicating that the transcriptional enhancement of a-amylase II-4 expression did not occur in the aleurone. Gibberellin stimulated the accumulation of Ca-45(2+) into the intracellular secretory membrane system. In addition, several inhibitors for Ca2+ signaling, such as EGTA, neomycin, ruthenium red (RuR), and W-7 prevented the gibberellin-induced expression of a-amylase II-4 effectively. While the gibberellin-induced expression of a-amylase II-4 occurred normally in the aleurone layer of a rice dwarf mutant d1 which is defective in the alpha subunit of the heterotrimeric G protein. Based on these results, it was concluded that the posttranscriptional regulation of a-amylase II-4 expression by gibberellin operates in the aleurone layer of germinating rice seed, which is mediated by Ca2+ but not the G protein. (C) 2004 Elsevier SAS. All rights reserved.

DOI： 10.1016/j.plaphy.2004.04.005

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CiNii Article

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Effects of phosphate on the Ca2 uptake and the sucrose-controlled secretion of ±-amylase molecules in cultured rice cells were investigated. Phosphate markedly stimulated Ca2, uptake into rice cells, particularly at the outer cell layer of the cell cluster. Phosphate also increased the synthesis and extracellular liberation of a - amylase II-4 in sucrose-supplemented cells. The distribution pattern of enzyme in rice cell clusters induced by phosphate was similar to that of Ca2+ uptake. Phosphate did not increase the level of mRNA of ±-amylase II-4, indicating that phosphate stimulates the translation and posttranslational secretory processes of ±-amylase II-4 in the presence of sucrose. Furthermore, phosphate enhanced both the Ca2r uptake and α-amylase II-4 synthesis in the microsomes. These results strongly suggested that the ratio of phosphate to sugar is important for regulating the Ca2+ uptake, and that phosphate and sugar precisely coordinate the Ca2- mediated synthesis and extracellular liberation of α-amylase II-4. © 2003, Japanese Society for Plant Cell and Molecular Biology. All rights reserved.

DOI： 10.5511/plantbiotechnology.20.187

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Indole acetic acid (IAA) level and nitrilase activity were measured in infected and healthy turnips, Brassica campestris L., subsp. Rapa, over a time course to confirm increases in IAA levels upon infection by Plasmodiophora brassicae and to investigate underlying mechanisms. Healthy and infected seedlings were assayed from 20 to 90 days after sowing. IAA levels in both roots fluctuated similarly over days 20-35. By day 45, IAA in infected roots increased to five-fold over healthy roots then decreased to the level of healthy roots by day 90. Nitrilase activity was negligible on days 20 and 25 but increased thereafter in infected and healthy roots. However, activity in healthy roots decreased substantially by day 40 while infected roots showed a continued increase to day 45 then decreased to a low level. These findings suggest that IAA concentration increases after P. brassicae infection, possibly due to IAA synthesis via pathways involving nitrilase. © 2003, Japanese Society for Plant Cell and Molecular Biology. All rights reserved.

DOI： 10.5511/plantbiotechnology.20.215

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Language：English   Publishing type：Research paper (bulletin of university, research institution)   Publisher：Niigata University  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

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Cytosolic and membrane-associated proteins prepared from rice cells were separated and compared by two different 2D-PAGE methods, isoelectric focusing (IEF)/SDS-PAGE and nonequilibrium pH gradient electrophoresis (NEPHGE)/SDS-PAGE. Although IEF/SDS-PAGE of the cytosolic proteins showed sufficient resolution, some mitochondrial and basic microsomal membrane-associated proteins were weakly or hardly detectable on the 2D gel. High-quality and -quantity separation of the organelle membrane-associated proteins was accomplished by NEPHGE/SDS-PAGE, the advantage of this method being more critical in tightly membrane-bound proteins that were unwashable with NaCl. These results indicate that NEPHGE/SDS-PAGE is a useful tool for the proteomic analysis of rice membrane-associated proteins. © 2002, Taylor &amp
Francis Group, LLC. All rights reserved.

DOI： 10.1271/bbb.66.1170

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Publisher：The Japanese Society of Sericultural Science  

CHAPS-soluble proteins from BBMV of Plutella xylostella which were highly resistant and susceptible to Cry1Ac of Bacillus thuringiensis were used to determine the capability of binding with Cry1A toxins. Both ligand and western blot analyses with the proteins and Cry1A toxins showed that major three 120, 110 and 100 kDa proteins bound with Cry1Aa and Ac, but not with Cry1Ab. Furthermore, the proteins were positive with anti aminopeptidase N antibody and the intensity and pattern of the binding were almost the same, respectively in the both straihs. Cry1Aa and Ac were shown to bind with the proteins at about 0.7 ng toxin/mm<SUP>2</SUP> membrane/10 min in both the strains using plasmon resonance. Their binding curves were almost the same each other. Cry1Ab, however, was not shown to bind with the BBMV proteins in both the strains.

DOI： 10.11416/jsss.jsss72.0.66.0

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We studied the separation of distinct compartments of the Golgi complex in rice and tobacco employing the discontinuous sucrose density gradient centrifugation technique with EDTA or MgCl2. In the presence of EDTA, rice Golgi marker enzymes, nucleoside diphosphatase and N-acetylglucosaminyltransferase were fractionated in the 28 and 33% sucrose fractions, respectively. Peanut lectin-recognized glycoproteins and reversibly glycosylated polypeptide-1 that are shown to be located at Golgi complex were broadly distributed around a 31.5% sucrose density. Under a MgCl2-supplemented condition, the peak of nucleoside diphosphatase was shifted to 29 and 37% sucrose and N-acetylglucosaminyltransferase was distributed in the 37% sucrose fraction. The distribution of peanut lectin-recognized glycoproteins and reversibly glycosylated polypeptide-1 were also shifted to the 34.5% sucrose fraction. Furthermore, glucan synthase-I and UDP-glucose pyrophosphorylase were fractionated in the 26.5% sucrose fraction, regardless of the presence or absence of MgCl2. These results indicate that there exist at least four distinct compartments of the rice Golgi complex. The tobacco Golgi marker enzymes and Golgi-localized proteins were distributed in the 30-31% sucrose fractions in the presence of EDTA and slightly shifted to the 32-33% sucrose fractions under a MgCl2-supplemented condition. These results suggest that the sedimentation behavior of compartments of the rice Golgi complex in the gradients is specific for rice cells. © 2001 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0168-9452(01)00455-1

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α-Mannosidase (EC 3.2.1.24) from rice dry seeds was purified to homogeneity. Optimum pH and Km for pNP-α-Man hydrolysis were pH 4.3-4.5 and 1.04 mM, respectively. The enzyme digested mannobioses such as Manα-1,2Man, Manα1,6Man, Manα-1,3Man but Manα-1,4Man. Zn2+ ion was required for the activity, whereas EDTA and swainsonine inhibited the activity by 80 and 96%, respectively. The rice storage protein, glutelin was prepared and its basic subunits were shown to have high mannose-type sugar chains by two-dimensional mapping using NH2-P and C18 silica columns. They were Man9GlcNAc2, Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2 and Man5GlcNAc2. All these oligosaccharides were digested by the purified α-mannosidase, and ManGlcNAc2 and mannose were formed. Glycopeptides, having these high mannose-type sugar chains, could also be digested by the α-mannosidase. Subunits were prepared from glutelin basic subunit and the richest subunit among them, subunit 2 (isoform 2), was digested by the α-mannosidase. Isoform 2 was digested by V8 protease only partially and slowly. However, isoform 2, pre-treated with the α-mannosidase, was rapidly and completely digested by V8 protease.

DOI： 10.1034/j.1399-3054.2001.1120103.x

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Plutella xylostella (PX) that were 130 000-fold more resistant to Cry1Ac were selected from the susceptible strain and maintained in the laboratory. The LC50 of the susceptible strain (PXS) was 0.38 μg toxin/g diet, whereas that of the resistant strain (PXR) was 4800 μg toxin/g diet. Brush border membrane vesicles (BBMV) were prepared from both PXS and PXR. In ligand blot analysis, Cry1Ac bound to a 120-kDa protein of BBMV
however, the intensity of the band was almost equal in both strains of insect. Hence, we analyzed the lipid components of BBMV from PXS and PXR. BBMV lipids were fractionated into non-polar lipid, phospholipid, neutral glycolipid and acidic glycolipid. Neutral glycolipid content was substantially lower in the BBMV of PXR than of PXS. The same trend was observed when lipids were extracted from whole midgut instead of BBMV. Thin layer chromatography of midgut neutral glycolipids revealed the presence of more than seven components. Among the midgut neutral glycolipids, a possible hexasaccharylceramide and a possible trisaccharylceramide of PXR were less than half the level found in PXS. The other lipid fractions in PXR and PXS were similar to each other. © 2001 Published by Elsevier Science Inc.

DOI： 10.1016/S1096-4959(01)00327-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society for Plant Cell and Molecular Biology  

Inosine diphosphatase (IDPase) isoforms associated with Golgi membranes were studied in sycamore cell culture. These enzyme isoforms were solubilized with Triton X-100 and purified by chromatography using DEAE-Toyopearl and SOURCE-S columns. The isoforms were separated into two distinguishable fractions (peak 1 and 2) by SOURCE-S column chromatography. Furthermore the peak 1 contained at least two isoform bands detected by native- PAGE analysis. The apparent molecular sizes of these three isoforms were estimated by both gel filtration and SDS- PAGE to be 50 kDa, indicating that the Golgi membrane - bound IDPase has a monomeric structure. These IDPase isoforms required divalent cations (Ca2+, Mg2+, Co2+, Mn2+) for their hydrolyzing activity, and were inhibited by ATP. IDP, UDP, and GDP were effective substrates for these enzymes. It is clearly indicated that the sycamore Golgi membrane-bound IDPase is a nucleoside diphosphatase.

DOI： 10.5511/plantbiotechnology.18.259

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society for Plant Cell and Molecular Biology  

The calluses were induced from turnips, Brassicae campestris L. which were susceptible and resistant to Plasmodiophora brassicae in Murashige-Skoog's agar medium supplemented with 1.0 ppm 6-benzylaminopurine and 0.5 ppm α - naphthalene acetic acid. When a 10 μ1 water suspension containing 104 resting spores of P. brassicae was placed on the surface of the calluses, about 3 × 105 zoosporangium-like spheroids (SLS) were recovered from the susceptible calluses after 24 h of the treatment but no SLS was found in the resistant callus. On 6th day after the treatment, the SLS increased to about 4 × 106 in the susceptible callus. Upon inoculation of resistant callus with 104 resting spores, the phenylalanine ammonia lyase (PAL) activity increased about 4-fold after 20 h, however, such increase was not observed in the susceptible callus during the same period. The constitutive PAL activity of susceptible callus was roughly one 6th of that of resistant callus.

DOI： 10.5511/plantbiotechnology.18.267

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We have studied the effects of neomycin, a potent inhibitor of inositol phospholipid-specific phospholipase C (PLC), on the germination of rice seed and the gibberellin-induced expression of α-amylase in the aleurone layer and the scutellar tissues. It was shown that, in the absence of exogenous Ca2+, neomycin markedly reduced the germination speed and seedling growth of rice seeds and inhibited the gibberellin-induced expression of α-amylase in both secretory tissues. In addition, neomycin decreased the formation of inositol 1,4,5-trisphosphate (IP3) in the gibberellin-treated aleurone layer and the scutellar tissues. However, the inhibitory effects on the germination speed and the expression of α-amylase activity were overcome by supplementation of Ca2+. In addition, gibberellin elevated the level of IP3, and ABA prevented the gibberellin-induced formation of IP3, although ABA alone did not alter the IP3 level. The expression of a membrane-bound PLC molecule in rice aleurone layer was shown to be induced by gibberellin, and the gibberellin-induced expression of PLC was markedly delayed by treatment with ABA. These results strongly suggest that the phosphoinositide-Ca2+ signal transduction pathway may play an important role in the gibberellin-induced expression of α-amylase molecules closely related to the germination processes of rice seed.

DOI： 10.1093/pcp/41.4.399

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society for Plant Cell and Molecular Biology  

The basic subunit of glutelin from rice dry seeds were shown to have N-linked sugar chains by lectin staining with PHA-E4, WGA and Con A. NaIO4 and N-glycanase treatments also provided the evidences of N-linked sugar chains. The purified basic subunit was applied to NEPHGE and eight isoforms of it were separated. The fractionated eight isoforms were shown to be nearly homogeneous by C18-silica column chromatography. Each was subject to lectin staining. The eight isoforms were hybridized with PNA, PHA-E4 and Con A. This lectin staining indicated that each of the isoforms of the basic subunit had N-linked sugar chain. This is the first report showing the occurrence of N-linked sugar chain in isoforms of glutelin basic subunit.

DOI： 10.5511/plantbiotechnology.17.217

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Crop Science Society of Japan  

The effects of various constant temperatures (4, 9, 14, 19, 24, 29, 34, and 38°C) on the germination of winter wheat seed (Triticum aestivum L. cv. 'Koyuki') in a dark condition were studied. The maximum germination percentage was 98% at 24°C. The speed of germination was fastest at 29°C. These results indicate that the most suitable temperature for germination was in the range of 24 to 29°C. α-Amylase expression during germination was also high at higher temperature, and maximum expression occurred at 29°C, although a high temperature of 38°C prevented the synthesis of α-amylase. The close correlation between germination and α-amylase activity at various temperatures indicates that α-amylase is an essential factor for the temperature-dependent germination of wheat seed. In contrast, accumulation of proline increased at a lower temperature, and was the highest at 4°C. We also studied the effects of gibberellin (GA3) and proline, a compatible osmotica in alleviating the effect of low and high temperature stresses. Pre-soaking treatment with GA3 and proline was effective in promoting germination and increasing α-amylase expression at a low (4°C) and high (38°C) temperature. These results suggest that GA3 and proline exhibit positive effects on stress alleviation through the stimulation of α-amylase expression.

DOI： 10.1626/pps.3.232

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Characterization was done on the sugar chains of glutelin, a major storage protein in rice seeds. The basic subunits of glutelin were shown to be glycoproteins by staining with PAS and the lectins concanavalin A (Con A) and peanut agglutinin (PNA). The binding of PNA to the basic subunits was substantially reduced by treatment of the glutelin with NaIO4, NaOH, or O-glycanase. The sugar chains of the subunits, obtained by hydrazinolysis and O-glycanase, were pyridylaminated and subjected to 2-dimensional HPLC analysis using C-18 and acrylamide-derivatized columns. It was found that the Gal beta-1,3GalNAc disaccharide, which was previously identified as a core 1 structure of mucin-type sugar chains, is conjugated to the glutelin subunits. Furthermore, amino acid sequencing of an 11-kDa peptide of the subunits, recognized by both PNA and Con A, suggested that both N-linked and O-linked glycosylation occurs in the carboxy-terminal region of these subunits. (C) 1999 Academic Press.

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Sugar starvation-induced synthesis and extracellular liberation of α-amylase molecules in suspension-cultured cells of rice (Oryza sativa L.) required Ca2+, although the level of translatable α-amylase mRNA was not affected in the presence of Ca2+. Sugar depletion markedly stimulated Ca2+ uptake by rice cells and sucrose supplementation reduced it. Immunohistochemical and electron probe microanalyzer studies indicated an apparent resemblance between the distribution pattern of Ca2+ and that of α-amylase molecules induced in the sugar-depleted cells. Ca2+ uptake was reduced by sucrose, maltose, fructose, and glucose similarly at more than 5 mM, but was unaffected by mannitol (88 mM), 6-deoxy-D-glucose (10 mM), and 3-O-methyl-D-glucose (10 mM). Furthermore, an effective Ca2+ channel blocker, La3+ significantly inhibited the Ca2+ uptake and the synthesis and extracellular liberation of α-amylase molecules in the absence of sucrose, while a general P-type ATPase inhibitor, vanadate greatly stimulated both in the presence of sucrose. We concluded that, by controlling the Ca2+ uptake, metabolic sugars regulate the protein synthesis and posttranslational secretory processes of α-amylase molecules in rice cells.

DOI： 10.1093/oxfordjournals.pcp.a029618

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Plant Physiologists  

We studied the sucrose-controlled intracellular transport and turnover of α-amylase molecules in suspension-cultured cells of rice (Oryza sativa L.) employing pulse labeling techniques with [35S] amino acids. The secretion of two classes of α-amylase isoforms, α-amylase I-1 (encoded by RAmy1A) and II-4 (encoded by RAmy3D) was differentially controlled by sucrose. In rice cells labeled with [35S] amino acids under different sucrose-supplemented conditions, sucrose preferentially prevented the extracellular liberation of [35S]-labeled α-amylase H-4 molecules from rice cells at around 2 mM, whereas the de novo protein synthesis still occurred at this concentration. Pulse-chase experiments showed that sucrose regulates the intracellular transport of [35S]α-amylase II-4 molecules and stimulates the protein turnover. However, cydoheximide, a protein synthesis inhibitor, was induced the extracellular liberation and reduced the turnover of [35S]α-amylase II-4 molecules in the presence of sucrose. These results strongly suggested that newly synthesized sucrose-induced proteins are involved in the posttranslational regulation on sucrose-controlled a-amylase secretion in rice cells.

DOI： 10.1093/oxfordjournals.pcp.a029605

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Language：English   Publishing type：Research paper (bulletin of university, research institution)   Publisher：Niigata University  

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DOI： 10.1271/nogeikagaku1924.71.sup_1

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An 8′,8′,8′-trideuterated analog of abscisic acid (ABA) was diastereoselectively synthesized as a new analog of ABA that is resistant to 8′-hydroxyIation, the first metabolic reaction of ABA, owing to the primary kinetic isotope effect. (+)-8′,8′,8′-Trideutero-ABA showed long-term activity in the rice elongation assay. The rate of metabolism of this analog in rice cell suspension culture was about two fold slower than that of (+)-ABA. The concentration of 8′,8′-dideuterophaseic acid produced was about 1/3 that of phaseic acid converted from (+)-ABA. This result indicated that the long-lasting activity of the (+)-trideutero-ABA in the rice assay was the result of the delayed 8′-hydroxylation as expected. © 1997, Taylor &amp
Francis Group, LLC. All rights reserved.

DOI： 10.1271/bbb.61.1872

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Ltd  

Many α-amylase genes have been cloned from various cereals, and sequence analysis reveals that these fall into two major classes and three subfamilies. In parallel studies, many α-amylase isoforms have been characterized and their corresponding genes identified. Understanding of the regulatory mechanisms that operate to control expression of the α-amylase multigene family has also advanced in recent years. In the light of the improved understanding of α-amylase activity, we have addressed a classical argument over the 'scutellum versus aleurone' concept. This disagreement concerns the site of synthesis and secretion of α-amylase. We have focused our attention on this by comparing gene expression at the transcriptional and post-transcriptional level.

DOI： 10.1016/S1360-1385(97)86347-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT PHYSIOLOGISTS  

We have identified, purified, and characterized 10 alpha-amylase isoforms from suspension-cultured rice (Oryza sativa L.) cells having different isoelectric point values. They had distinguishable optimum temperatures for enzymatic activity and molecular sizes. The results of immunoblotting indicated that polyclonal anti-A + B antibodies bound well to isoforms A, B, Y, and Z but weakly or not at all to E, F, G, H, I, and J. However, the anti-A + B antibodies inhibited the enzyme activities of only isoforms A and B. Polyclonal anti-H antibodies strongly bound to isoforms F, G, H, I, and J, whereas polyclonal anti-E antibodies preferentially recognized isoform E. A monoclonal antibody against isoform H (H-G49) inhibited the activities of isoforms E, G, H, I, and J, whereas it did not inhibit those of isoforms A, B, Y, and Z. Judging from their physicochemical and serological properties, we classified the rice cu-amylase isoforms into two major classes, class I (A, B, Y,and Z) and class II (E, F, G, H, I, and J), and into four subgroups, group 1 (A and B), group 2 (Y and Z), group 3 (E), and group 4 (F, G, H, I, and J). Partial amino acid sequences for isoforms A, E, G, and H were also determined. In addition, the recombinant alpha-amylases expressed by plasmid pEno/103 containing the rice alpha-amylase gene RAmy1A in yeast were identified as both isoforms A and B. These analyses indicated that isoforms A and B were encoded by the gene RAmy1A, isoforms G and H were encoded by the gene RAmy3D, and isoform E was encoded by RAmy3E. The results strongly suggest that some isoforms within subgroups are formed by posttranslational modifications.

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Phosphoinositol kinase was purified from suspension-cultured rice (Oryza sativa L,) cells, The apparent molecular mass of the rice enzyme was estimated to be 58 kDa by SDS-PAGE and 234 kDa by Toyopearl HW-55S gel filtration, indicating that it is a tetrameric enzyme. The enzyme absolutely required Mg2+ for the activity, but Mn2+ and Ca2+ did not affect it. In addition, this kinase phosphorylated inositol monophosphate to phytate, Judging from these data, rice phosphoinositol kinase was concluded to be a different enzyme distinguished from the other plant phosphoinositol kinases.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Q(10) values of the protein phosphatases that can dephosphorylate the regulatory light chain of smooth muscle myosin were determined. Six phosphatases were examined, i.e. skeletal muscle protein phosphatase 1c; protein phosphatase 2Ac; smooth muscle phosphatases (SMP) I, II, and IV; and myosin-associated protein phosphatase (MAP phosphatase). Among them, SMP-IV and MAP phosphatase, which can dephosphorylate intact smooth muscle myosin, showed extremely high Q(10) values (5.3 and 5.2, respectively). On the other hand, the Q(10) values of other tested phosphatases were within the range of the normal enzyme reaction (Q(10) = 2.0). The rate of dephosphorylation of the myosin light chain in alpha-toxin-skinned strips was measured at different temperatures. The results provided a Q(10) of 5.1, which was quite similar to those values obtained for SMP-IV and MAP phosphatase. These results suggest that the physiological myosin light chain phosphatases are SMP-IV and/or MAP phosphatase, i.e. type 1 protein phosphatases. The temperature dependence of maximum force, the steady-state extent of myosin light chain phosphorylation, and the relaxation rate of alpha-toxin-permeabilized rabbit postal vein smooth muscle strips were measured. Both maximum force and the extent of myosin light chain phosphorylation were significantly higher at lower temperature (15 degrees C) than at higher temperature (25 degrees C) under all pCa conditions tested, i.e. &gt;8, 6.3, and 5. The temperature dependence of the relaxation rate was much steeper (decreased 4 times by lowering the temperature from 25 to 15 degrees C) than that of the initial rate of increase in force development (decreased 1.4 times by lowering the temperature from 25 to 15 degrees C). These results are consistent with the and to values of myosin light chain phosphatases (Q(10) = 5) and myosin light chain kinase (Q(10) = 1.7) and further show that the smooth muscle type 1 phosphatases are responsible for the dephosphorylation of smooth muscle myosin in situ.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society of Plant Biologists  

DOI： 10.1104/pp.106.1.119

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：GAUTHIER-VILLARS  

A major alpha-amylase isozyme H synthesized in suspension-cultured cells of rice (Oryza sativa L. cv. Nipponkai) was purified and characterized. The molecular mass of isozyme H was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 44,000 Da. Isozyme H had an isoelectric point of pH 5.7. Isozyme H was stained with periodic acid-Schiff reagents and concanavalin A-peroxidase, indicating that isozyme H was a glycoprotein. The carbohydrate content of isozyme H was determined by phenol-sulfuric acid reagents to be 2% (w/w). Specific polyclonal antibodies against isozyme H did not bind to the isozyme A + B, while anti-isozyme A + B antibodies did not recognise isozyme H. Isozyme H required greater than 80 nM Ca2+ for activity and was maximally activated by 500 nM Ca2+. The activity of isozyme H exhibited a stringent divalent cation dependency on Ca2+, Sr2+ and Ba2+. These results support the conclusion that isozyme H is a novel rice alpha-amylase isozyme.

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The structure and synthesis of the saccharide chains of Golgi membrane glycoproteins in suspension-cultured rice (Oryza sativa L.) cells were studied. Peanut lectin (PNA) and Ulex europaeus lectin-I (UEA-I) have high affinity for typical O-linked saccharide chains and both recognized the saccharide chains of rice Golgi membrane glycoproteins. These glycoproteins were also sensitive to alkali and to O-glycanase. These results indicate that the Golgi membrane glycoproteins have O-linked saccharide chains. Brefeldin A, a specific inhibitor of Golgi-mediated secretion, induced morphological changes in Golgi complexes and prevented the synthesis of the saccharide chains of the membrane glycoproteins that could be recognized by PNA and UEA-I. These glycoproteins were typically localized in all compartments of the Golgi complex. Monensin can arrest the transport of secretory proteins from medial to trans Golgi compartments but did not affect the formation and localization of the Golgi membrane glycoproteins. Tunicamycin, an inhibitor of the synthesis of N-linked saccharide chains, did not inhibit the synthesis of the saccharide chains of these Golgi membrane glycoproteins. These results strongly suggest that the synthesis of O-linked saccharide chains of Golgi membrane glycoproteins is initiated in the cis Golgi compartment.

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A membrane-bound inositol phospholipid-specific phospholipase C was solubilized from rice (Oryza sativa L.) microsomal membranes and purified to apparent homogeneity using a series of chromatographic separations. The apparent molecular mass of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 42,000 D, and the isoelectric point was 5.1. The optimum pH for the enzyme activity was approximately 6.5, and the enzyme was activated by both Ca2+ and Sr2+. The chemical and catalytic properties of the purified membrane-bound phospholipase C differed from those of the soluble enzyme reported previously (K. Yotsushima, K. Nakamura, T. Mitsui, 1. Igaue [1992] Biosci Biotech Biochem 56: 1247-1251). In addition, we found a regulatory factor for the phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolyzing activity of phospholipase C from rice cells. The regulatory factor was dissociated from the catalytic subunit of phospholipase C during the purification. The regulatory factor was necessary to induce PIP2-hydrolyzing activity of both membrane-bound and -soluble phospholipase C; these purified enzymes had no activity alone. Because the plasma membranes isolated from rice cells could also act as a regulatory factor, the regulatory factor seems to be localized in the plasma membranes. Regulation of inositol phospholipid turnover in rice cells is discussed.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

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UDP-glucose pyrophosphorylase was purified from the soluble fraction of suspension-cultured rice cells. The molecular weight of the rice enzyme was estimated by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis to be 54,000 Da and 55,000 Da, respectively, indicating that the enzyme is composed of a single polypeptide. The rice enzyme had an isoelectric point at pH 4.6. The pyrophosphorylase exhibited a high degree of specificity toward UDP-glucose and did not react significantly with the other UDP-sugars. The UDP-glucose pyrophosphorylase was preferentially localized in the scutellum among the rice seedings. Furthermore, immunocytochemical and biochemical studies using antibodies against UDP-glucose pyrophosphorylase strongly suggested that UDP-glucose pyrophosphorylase was localized in amyloplasts and Golgi membranes. The physiological meaning of this tissue specific localization of UDP-glucose pyrophosphorylase and possible roles of the enzyme in these organelles are discussed.

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Phosphatidylinositol-specific phospholipase C was purified from the soluble fraction of suspension-cultured rice cells. The apparent molecular weight of rice enzyme was estimated to be 50,000 by both Sephadex G-100 gel filtration and SDS-polyacrylamide gel electrophoresis, indicating that the enzyme is composed of a single polypeptide. The enzyme had an isoelectric point of 6.3. The soluble phospholipase C had a high degree of specificity toward phosphatidylinositol and a weak activity toward phosphatidyl-inositol monophosphate, while the enzyme did not hydrolyze the other phospholipids or p-nitro-phenylphosphorylcholine. V(max) and K(m) values were 5.0-mu-mol/min/mg protein and 0.3 mm, respectively. The pH dependency of the enzyme activity was sharp with an optimum of 5.2. In addition, the phospholipase C was a Ca2+-dependent enzyme. The marked activation of enzyme was observed in the presence of 10 to 250-mu-M Ca2+ and higher Ca2+ concentrations than 1 mm had a strong inhibitory effect. A possible regulation of the phospholipase C activity by pH and Ca2+ Concentrations in the rice cells is discussed.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Myosin light chain phosphatase associated with smooth muscle myosin (MAPP) was isolated from chicken gizzard. The MAPP was tightly associated with myosin and was not dissociated from myosin under the physiological ionic conditions. The phosphatase was dissociated from myosin in the presence of high MgCl2, i.e. 80 mM MgCl2. The binding site of the enzyme on the myosin molecule was the subfragment-2 region, since the enzyme did bind to the myosin rod and heavy meromyosin but not to the subfragment-1 affinity column. MAPP was purified with a heparin-Sepharose 6B column, and two activity peaks were obtained, i.e. MAPP I and MAPP II. The major activity peak, MAPP I, was further purified to homogeneity by thiophosphorylated myosin light chain-Sepharose 4B column chromatography. MAPP I was a tetramer composed of four 34-kDa subunits. The enzyme preferentially dephosphorylated the beta-subunit of phosphorylase kinase and was strongly inhibited by the heat- and acid-stable protein phosphatase inhibitor-1, whereas it was partially inhibited by the inhibitor-2. The IC50 (concentration of inhibitor giving 50% inhibition) value for the inhibition of the enzyme by okadaic acid was 70 nM which was about eight times higher than skeletal muscle type-1 and 390 times higher than type-2 protein phosphatase. These results demonstrate that the MAPP I is a type-1-like protein phosphatase, although the properties are not the same as type-I phosphatase. The properties of the myosin-associated phosphatase were distinct from the phosphatases reported previously, although some properties were similar to smooth muscle phosphatase-IV. Therefore, it is concluded that MAPP I is a novel smooth muscle protein phosphatase. Since it strongly associated with smooth muscle myosin, it is likely that MAPP I is responsible for the dephosphorylation of smooth muscle myosin in situ.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT PHYSIOLOGISTS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society for Plant Cell and Molecular Biology  

Plant mesophyll protoplasts with a endo-polygalacturonase produced by <i>Trichosporon penicillatum</i> SNO-3 was isolated. The following mesophyll tissues of shoot were used for the isolation: <i>Tagetes minuta</i>, <i>Brassica rapa</i>, <i>Raphanus sativus</i>, <i>Lactuca sativa</i>, <i>Triticum aestivum</i>, <i>Secale cereale</i>, <i>Hordeum vulgare</i>, <i>Panicum crusgalli</i>, <i>Avena sativa</i>, <i>Zeamays</i> and <i>Oryza sativa</i>. Pectinase SE-150, a partially purified endo-polygalacturonase, combined with cellulase ONOZUKA RS liberated protoplasts from all mesophyll tissues tested.<br>The protoplast yields were 1-4×10⁶ per gram fresh weight. Cell division and colony formation from lettuce protoplasts obtained with a pectinase SE-150 enzyme system were also observed. The percentage of colony formation on the 14th day after incubation in a culturing medium was estimated to be approximately 18%.

DOI： 10.5511/plantbiotechnology1984.7.14

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DOI： 10.1038/NPLANTS2015.57

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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Small ubiquitin-related modifier (SUMO) is a type of ubiquitin-like proteins and regulates various protein functions through post-translational modification. The SUMO precursor proteins are processed by C-terminal cleavage reaction at the end of the di-glycine(GG) motif, and then activated and bind to substrate proteins by a series of enzymatic reaction. We cloned SUMO1-3 genes in rice, and analyzed the transient expression and intracellular localization of the DsRed fusion proteins, DsRed:SUMO1, 2, and 3 in onion epidermal cells by using confocal laser scanning microscopy. The DsRed signals from DsRed:SUMO1 and DsRed:SUMO2 were detected both in nuclei and cytoplasmic location, but not in nucleoli. In the case of DsRed:SUMO3, the DsRed signal was detected mainly in nucleus, and formed sub-nuclear domain like structure. We also tested effect of the GG motif on intracellular localization of SUMO proteins. The GG deletion mutant vectors, pDsRed:SUMO1ΔGG, pDsRed:SUMO2ΔGG, and pDsRed:SUMO3ΔGG were constructed and transiently expressed in onion epidermal cells. Result showed that the deletion mutation of GG motif suppressed the accumulation of the DsRed:SUMOΔGG proteins in the nucleus. These results indicated that the C-terminal processing of OsSUMO precursor proteins are necessary for OsSUMO localization to nucleus in onion epidermal cells.Small-ubiquitin related modifier (SUMO)はユビキチン様タンパク質の一種であり、翻訳後修飾により多様なタンパク質の機能を調節する。SUMO 前駆体タンパク質はＣ末側に存在するジグリシン（GG）モチーフの末端でプロセシングされ、一連の酵素反応により活性化され基質タンパク質に結合する。私たちはイネSUMO1-3遺伝子を単離し、DsRed 融合タンパク質であるDsRed:SUMO1, ２、および３をタマネギ表皮細胞において一過的に発現させ、その細胞局在性を共焦点レーザー顕微鏡により解析した。DsRed:SUMO1およびDsRed:SUMO2のシグナルは核と細胞質にも分布したが、核小体には観察されなかった。DsRed:SUMO3の場合は、主に核に局在し、そのシグナルは細胞核ドメイン様構造を形成した。私たちはまた、GGモチーフ欠失変異細胞内局在性に関わる影響をテストした。GG モチーフ欠失変異を持つベクター、pDsRed:SUMO1ΔGG、pDsRed:SUMO2Δ GG、およびpDsRed:SUMO3ΔGG を構築し、タマネギ表皮細胞において発現させた。GG モチーフの欠失変異はDsRed:SUMO Δ GG タンパク質の核への集積を抑制した。これらの結果から、OsSUMO 前駆体タンパク質のＣ末端のプロセシングがタマネギ表皮細胞におけるOsSUMO タンパク質の核への局在性に必要であることが示唆された。

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Language：Japanese   Publisher：日本応用糖質科学会  

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Language：Japanese   Publisher：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2011.0.139.0

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Language：Japanese   Publisher：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2011.0.65.0

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DOI： 10.14841/jspp.2006.0.260.0

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Language：English   Publishing type：Book review, literature introduction, etc.  

Abstract Since the first report of Bacillus sotto by Ishiwata in 1901, thousands of related papers about Bacillus thuringiensis have been documented. In the field of biocontrol of insect pests by this bacterium, after the initial discovery of several B. thuringiensis isolates specific for lepidopteran insects, the isolation of B. thuringiensis israelensis, specific to dipteran larvae by Goldberg and Margalit, and B. thuringiensis tenebrionis, specific to some group of coleopteran insects by Krieg et al. were epoch making advances. In 1992, Ohba et al. isolated B. thuringiensis ja ponensis strain Buibui, which was specific to only scarabaeid larvae. This isolate is the main target of our discussion in this review. These discoveries by which not only B. thuringiensis sciences, but also applied biological control strategies have been enriched, which inspirit us to screen novel isolates. On the other hand, the fields of molecular biology and biochemistry studies on the structural elucidation of toxin proteins and mechanism of action have also tremendously progressed. But the complete mechanism has yet to be solved. For instance, interaction between receptor proteins locating on the plasma membrane of insect midgut epithelial cells and insecticidal proteins has not been fully sketched- In a way, this field is still in chaos. Addressing these exciting and enigmatic subjects will eventually lead to the construction of sustainable agriculture in the 21st century. © 2017 Wiley. All rights reserved.

DOI： 10.1111/j.1744-7917.2000.tb00235.x

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Language：English   Publisher：Japanese Society of Plant Physiologists  

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DOI： 10.1271/nogeikagaku1924.73.1273

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