Research Institute for Biological science RIBS Okayama

Okayama-shi, Japan

Research Institute for Biological science RIBS Okayama

Okayama-shi, Japan

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Koshiba T.,Kyoto University | Koshiba T.,EARTHNOTE Co. | Yamamoto N.,Kyoto University | Yamamoto N.,International Rice Research Institute | And 11 more authors.
Plant Biotechnology | Year: 2017

Lignin encrusts lignocellulose polysaccharides, and has long been considered an obstacle for the efficient use of polysaccharides during processes such as pulping and bioethanol fermentation. However, lignin is also a potential feedstock for aromatic products and is an important by-product of polysaccharide utilization. Therefore, producing biomass plant species exhibiting enhanced lignin production is an important breeding objective. Herein, we describe the development of transgenic rice plants with increased lignin content. Five Arabidopsis thaliana (Arabidopsis) and one Oryza sativa (rice) MYB transcription factor genes that were implicated to be involved in lignin biosynthesis were transformed into rice (O. sativa L. ssp. japonica cv. Nipponbare). Among them, three Arabidopsis MYBs (AtMYB55, AtMYB61, and AtMYB63) in transgenic rice T1 lines resulted in culms with lignin content about 1.5-fold higher than that of control plants. Furthermore, lignin structures in AtMYB61-overexpressing rice plants were investigated by wet-chemistry and two-dimensional nuclear magnetic resonance spectroscopy approaches. Our data suggested that heterologous expression of AtMYB61 in rice increased lignin content mainly by enriching syringyl units as well as p-coumarate and tricin moieties in the lignin polymers. We contemplate that this strategy is also applicable to lignin upregulation in large-sized grass biomass plants, such as Sorghum, switchgrass, Miscanthus and Erianthus. © 2017 The Japanese Society for Plant Cell and Molecular Biology.


Fujishiro T.,Max Planck Institute For Terrestrische Mikrobiologie | Tamura H.,Max Planck Institute For Terrestrische Mikrobiologie | Tamura H.,Research Institute for Biological science RIBS Okayama | Schick M.,Max Planck Institute For Terrestrische Mikrobiologie | And 5 more authors.
Angewandte Chemie - International Edition | Year: 2013

[Fe]-Hydrogenase requires the iron guanylylpyridinol (FeGP) cofactor for activity. The function of HcgB, an enzyme in the biosynthesis of the FeGP cofactor, was predicted by structural genomics and confirmed by model reactions and various analytical methods: HcgB catalyzes the terminal guanylyltransferase reaction for the formation of guanylylpyridinol. GMP=guanosine monophosphate. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Tamura H.,Max Planck Institute For Terrestrische Mikrobiologie | Tamura H.,Research Institute for Biological science RIBS Okayama | Salomone-Stagni M.,EMBL Hamburg | Fujishiro T.,Max Planck Institute For Terrestrische Mikrobiologie | And 6 more authors.
Angewandte Chemie - International Edition | Year: 2013

Inhibition mechanism: Isocyanides strongly inhibit [Fe]-hydrogenase. X-ray crystallography and X-ray absorption spectroscopy revealed that the isocyanide binds to the trans position, versus the acyl carbon of the Fe center, and is covalently bound to the pyridinol hydroxy oxygen. These results also indicated that the hydroxy group is essential for H2 activation. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Arima J.,Tottori University | Ito H.,Tottori University | Hatanaka T.,Research Institute for Biological science RIBS Okayama | Mori N.,Tottori University
Biochimie | Year: 2011

From investigation of 2000 soil isolates, we identified two serine-type amidohydrolases that can hydrolyze d-aminoacyl derivatives from the culture supernatant of Streptomyces species 82F2 and 83D12. The enzymes, redesignated as 82F2-DAP and 83D12-DAP, were purified for homogeneity and characterized. Each enzyme had molecular mass of approximately 40 kDa, and each showed moderate stability with respect to temperature and pH. Among hydrolytic activities toward d-aminoacyl-pNAs, the enzymes showed strict specificity toward d-Phe-pNA, but showed broad specificity toward d-aminoacyl esters. The specific activity for d-Phe-pNA hydrolysis of 82F2-DAP was ten-fold higher than that of 83D12-DAP. As a second function, each enzyme showed peptide bond formation activity by its function of aminolysis reaction. Based on results of d-Phe-d-Phe synthesis under various conditions, we propose a reaction mechanism for d-Phe-d-Phe production. Furthermore, the enzymes exhibited peptide elongation activity, producing oligo homopeptide in a one-pot reaction. We cloned the genes encoding each enzyme, which revealed that the primary structure of each enzyme showed 30-60% identity with those of peptidases belonging to the clan SE, S12 peptidase family categorized as serine peptidase with d-stereospecificity. © 2011 Elsevier Masson SAS. All rights reserved.


Noda S.,Kyoto University | Noda S.,Research Institute for Biological science RIBS Okayama | Koshiba T.,Kyoto University | Hattori T.,Kyoto University | And 5 more authors.
Planta | Year: 2015

Main conclusion: A rice MYB transcription factor, OsMYB58/63, was found to directly upregulate the expression of a rice secondary wall-specific cellulose synthase gene,cellulose synthase A7(OsCesA7); in contrast, theArabidopsisputative orthologs AtMYB58 and AtMYB63 have been shown to specifically activate lignin biosynthesis. Although indirect evidence has shown that grass plants are similar to but partially different from dicotyledonous ones in transcriptional regulation of lignocellulose biosynthesis, little is known about the differences. This study showed that a rice MYB transcription factor, OsMYB58/63, directly upregulated the expression of a rice secondary wall-specific cellulose synthase gene, cellulose synthase A7 (OsCesA7). Gene co-expression analysis showed that, in rice, OsMYB58/63 and several rice MYB genes were co-expressed with genes encoding lignocellulose biosynthetic enzymes. The expression levels of OsMYB55/61, OsMYB55/61-L, OsMYB58/63, and OsMYB42/85 were commonly found to be high in culm internodes and nodes. All four MYB transcription factors functioned as transcriptional activators in yeast cells. OsMYB58/63 most strongly transactivated the expression of OsCesA7 in rice protoplasts. Moreover, recombinant OsMYB58/63 protein was bound to two distinct cis-regulatory elements, AC-II and SMRE3, in the OsCesA7 promoter. This is in sharp contrast to the role of Arabidopsis orthologs, AtMYB58 and AtMYB63, which had been reported to specifically activate lignin biosynthesis. The promoter analysis revealed that AC elements, which are the binding sites for MYB58 and MYB63, were lacking in cellulose and xylan biosynthetic genes in Arabidopsis, but present in cellulose, xylan, and lignin biosynthetic genes in rice, implying that the difference of transcriptional regulation between rice and Arabidopsis is due to the distinct composition of promoters. Our results provide a new insight into transcriptional regulation in grass lignocellulose biosynthesis. © 2015, Springer-Verlag Berlin Heidelberg.


Hatano-Iwasaki A.,Research Institute for Biological science RIBS Okayama | Ogawa K.,Research Institute for Biological science RIBS Okayama | Ogawa K.,Japan Science and Technology Agency
Plant and Cell Physiology | Year: 2012

Keeping imbibed seeds at low temperatures for a certain period, so-called seed vernalization (SV) treatment, promotes seed germination and subsequent flowering in various plants. Vernalization-promoting flowering requires GSH. However, we show here that increased GSH biosynthesis partially mimics SV treatment in Arabidopsis thaliana. SV treatment (keeping imbibed seeds at 4°C for 24 h) induced a specific pattern of gene expression and promoted subsequent flowering in WT A. thaliana. A similar pattern was observed at 22°C in transgenic (35S-GSH1) plants overexpressing the γ- glutamylcysteine synthetase gene GSH1, coding for an enzyme limiting GSH biosynthesis, under the control of the cauliflower mosaic virus 35S promoter. This pattern of gene expression was further strengthened at 4°C and indistinguishable from the WT pattern at 4°C. However, flowering in 35S-GSH1 plants was less responsive to SV treatment than in WT plants. There was a difference in the transcript behavior of the flowering repressor FLC between WT and 35S-GSH1 plants. Unlike other genes responsive to SV treatment, the SV-dependent decrease in FLC in WT plants was reversed in 35S-GSH1 plants. SV treatment increased the GSSG level in WT seeds while its level was high in 35S-GSH1 plants, even at a non-vernalizing temperature. Taking into consideration that low temperatures stimulate GSH biosynthesis and cause oxidative stress, GSSG is considered to trigger a low-temperature response, although enhanced GSH synthesis was not enough to completely mimic the SV treatment. © 2012 The Author.


Narusaka M.,Research Institute for Biological science RIBS Okayama | Shiraishi T.,Okayama University | Iwabuchi M.,Research Institute for Biological science RIBS Okayama | Narusaka Y.,Research Institute for Biological science RIBS Okayama
Plant Biotechnology | Year: 2010

The floral dip protocol mediated by Agrobacterium tumefaciens is the most widely used transformation method for Arabidopsis thaliana. The "floral dip" process in which A. thaliana flower buds are dipped in an Agrobacterium cell suspension requires large volumes of bacterial cultures grown in liquid media, large shakers and centrifuges, and experimental space for them. These factors limit the number of transformations that can occur at once. We established that A. thaliana can be transformed by inoculating 5m l of Agrobacterium cell suspension in flower buds, thus avoiding the use of large volumes of Agrobacterium culture. Using this modified protocol, we obtained 15-50 transgenic plants per transformation from each pot containing 3 A. thaliana plants. The protocol is satisfactory to be used for subsequent analyses. This simplified method, without floral dipping, which requires large volumes of Agrobacterim culture, offers as efficient a transformation as previously reported protocols. This method reduces the required workload, cost, time, and space. Furthermore, an important aspect of this modified protocol is that it allows many independent transformations to be performed at once. © 2010 The Japanese Society for Plant Cell and Molecular Biology.


PubMed | Research Institute for Biological science RIBS Okayama
Type: Journal Article | Journal: Plant & cell physiology | Year: 2012

Keeping imbibed seeds at low temperatures for a certain period, so-called seed vernalization (SV) treatment, promotes seed germination and subsequent flowering in various plants. Vernalization-promoting flowering requires GSH. However, we show here that increased GSH biosynthesis partially mimics SV treatment in Arabidopsis thaliana. SV treatment (keeping imbibed seeds at 4C for 24 h) induced a specific pattern of gene expression and promoted subsequent flowering in WT A. thaliana. A similar pattern was observed at 22C in transgenic (35S-GSH1) plants overexpressing the -glutamylcysteine synthetase gene GSH1, coding for an enzyme limiting GSH biosynthesis, under the control of the cauliflower mosaic virus 35S promoter. This pattern of gene expression was further strengthened at 4C and indistinguishable from the WT pattern at 4C. However, flowering in 35S-GSH1 plants was less responsive to SV treatment than in WT plants. There was a difference in the transcript behavior of the flowering repressor FLC between WT and 35S-GSH1 plants. Unlike other genes responsive to SV treatment, the SV-dependent decrease in FLC in WT plants was reversed in 35S-GSH1 plants. SV treatment increased the GSSG level in WT seeds while its level was high in 35S-GSH1 plants, even at a non-vernalizing temperature. Taking into consideration that low temperatures stimulate GSH biosynthesis and cause oxidative stress, GSSG is considered to trigger a low-temperature response, although enhanced GSH synthesis was not enough to completely mimic the SV treatment.

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