Kyushu University Academic Staff Educational and Research Activities Database
List of Presentations
Yuji Tsutsumi Last modified date:2020.06.17

Professor / Sustainable Bioresources Science / Department of Agro-environmental Sciences / Faculty of Agriculture


Presentations
1. Hiroshima Shota,Kakoi Takumi,Hayashi Junya,Tsutsumi Yuji,Shimizu Kuniyoshi, Cytotoxicity of lignin-derived compounds isolated from bamboo (Phyllostachys pubescence) on cancer cell line, 1st International Lignin Symposium, 2019.09.
2. Diego A. Yoshikay,Yusuke Yokoyama ,Jun Shigeto, Yuji Tsutsumi , Regulation of differentiation and growth by plant peroxidase CWPO-C, 1st International Lignin Symposium, 2019.09.
3. Diego Yoshikay, Jun Shigeto, Yusuke Yokoyama, Yuji Tsutsumi, The contribution of CWPO-C to primary stage of plant growth and organogenesis, 5th Symposium of Biotechnology Applied to Lignocelluloses, 2018.08, Cationic cell-wall-bound peroxidase (CWPO-C) from poplar has been believed as a lignification-specific peroxidase, but direct evidence that demonstrates the role of CWPO-C remained unachieved. To promote better understanding about CWPO-C functions, transcriptional analysis of CWPO-C using laser microdissection, gene expression quantification and reporter gene techniques were performed. The results showed that CWPO-C expressed in the most of young tissues including xylem of upper stem, but scarcely expressed in interfascicular fiber and undifferentiated tissue such as apical meristem and cambium. Heterologous overexpression of CWPO-C in Arabidopsis inhibited plant growth and caused stem curvature. In addition, CWPO-C expressed in the outer site of the curved stem that was subjected to gravity stress. These results indicate that CWPO-C plays a role in cell elongation and differentiation; suggesting a new aspect to the role of CWPO-C. CWPO-C may contribute to catabolism of plant hormones such as auxin, involved in cell elongation, differentiation and geotropism..
4. Yuji Tsutsumi, Jun Shigeto, Hiroki Honjo, Generation of lignin polymer models via dehydrogenative polymerization of coniferyl alcohol and sinapyl alcohol via plant peroxydase involved in lignification and analysis of the resulting DHPs by MALDI-TOF analysis, 29th International Conference of Polyphenols, 2018.07, The mechanism of lignin dehydrogenative polymerization (DHP), made by mean of horseradish peroxidase (HRP), has been studied. Interestingly, HRP is efficient for guaiacyl type polymer formation (G-DHPs), but is not efficient in case of syringyl type DHPs (S-DHPs). It was previously demonstrate that lignification-related Arabidopsis peroxidases, AtPrx2, AtPrx25 and AtPrx71, and cationic cell-wall-bound peroxidase (CWPO-C) from the poplar are successful to oxidize syringyl- and guaiacyl-type monomers and larger lignin-like molecules. This is the reason why in the present study the DHP formation by means of these recombinant peroxidases was tested, and all these enzymes were successful for formation of both G-DHP and S-DHP in acceptable yields. CWPO-C led to S-DHP molecular size distribution similar to that of isolated lignins..
5. Diego Yoshikay, 重藤 潤, 堤 祐司, Analysis of poplar CWPO-C gene expression using laser micro-dissection and RT-qPCR, 第67回日本木材学会大会, 2017.03.
6. Ichiro Kamai, Eri Takata, Yuji Tsutsumi, Bamboo Refinery: Production of phenolic compounds from lignin by catalytic hydrogenation and fermentation of xylose, Lignobiotech IV, 2016.06.
7. Yuji Tsutsumi, Jun Shigeto, Hiroki Honjyo, In vitro polymerization of lignin monomer using the plant peroxidases involved in lignification, Oxizymes 2016, 2016.07.
8. Yuji Tsutsumi, Jun Shigeto, Hiroki Honjyo, In vitro evaluation of the lignin forming ability of plant peroxidases involved in lignification, Lignobiotech IV, 2016.06.
9. Diego Yoshikay, 大平香織, 重藤潤, 堤 祐司, Transcriptional analysis of Poplar CWPO-C in different cell types and organs, 第61回リグニン討論会, 2016.10.
10. Yuji Tsutsumi, Manami Takeuchi, Screening of monolignol transport protein in Arabidopsis thaliana, International Symposium on Wood Science and Technology 2015, 2015.03, Screening of monolignol transport protein in Arabidopsis thaliana

Monolignols are synthesized in the cytosol and transported to the cell wall to biosynthesize linign. It was reported that one of ATP-binding cassette (ABC) proteins, AtABCG29, is involved in the transportation of p-coumaryl alcohol, however, there have been no report on other monolignols for guaiacyl and syringyl lignin, which are major constitute units of angiosperm lignin. In this study, 9 genes including ABC proteins were selected as monoligol transport candidates among the genes those expressions were largely changed during the tracheary element differentiation of Arabidopsis cell culture, and T-DNA insert mutants of these genes were subjected to lignin analyses. There were no difference in both lignin content and S/G ratio between wild type and each knockout mutant. The expression analysis of each gene in the several organs did not show specificity in the stem unlike ABCG29. These results suggest that the candidate genes may not be involved in monolignol transportation in the Arabidopsis stem.
In the next screening we focus on evolutionally altered genes. Vascular plants have developed vascular bundle to conduct water and support their own body. Along with this evolution, lignin biosynthesis system had been also evolved. ABC transporter genes were compared between Arabidopsis thaliana as vascular plant and Physcomitrella patens as nonvascular plant, and 66 ABC proteins were found to be vascular plant specific. Further 28 ABCG subfamily genes among them were chosen as candidates, because some ABCG subfamily proteins have been reported to be related to secondary metabolism. The monolignol transport assay using transformed yeast with candidate gene is also performed to screen potential monolignol exporter.

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11. Eri Takata, Tatsushi Tsuruoka, Ken Tsutsumi, Kenji Tabata, Yuji Tsutsumi, Selective production of tetrahydrofurfuryl alcohol and xylitol from napier grass by a hydrothermal
process with phosphorus oxoacids followed by catalytic hydrogenation, Lignobiotech III, 2014.10, [URL].
12. Yuji Tsutsumi, Jun Shigeto, Kaori Ohira, Ryunosuke Takao, Masaaki Kamada, Expression and subcellular localization analysis of poplar cationic cell-wall-bound peroxidase and its Arabidopsis putative homologs involved in lignification, Lignobiotech III, 2014.10, [URL], Genetic engineering of gene which encodes peroxidase responsible for lignin polymerization is expected as an effective technique to change lignin content and structure in plant, but it is still far from the industrial application. This is because of the lack of fundamental knowledge about physiological functions of peroxidases. Limited number of peroxidases involved in lignification were identified in some plants, and little is known about their tissue-specific expression and subcellular localization pattern.
Cationic cell-wall-bound peroxidase (CWPO-C) from poplar has been verified to serve oxidation of both monolignols and lignin polymer, for the first time [1, 2]. This oxidation property is good as the enzyme responsible for lignin polymerization. In addition, AtPrx-2, 25 and 71 which have high amino acid similarity to CWPO-C were shown to contribute to stem lignification of Arabidopsis [3], and have CWPO-C-like oxidation property. In this study, tissue-specific expression and localization of CWPO-C and its Arabidopsis putative homologs, AtPrx-2, 25 and 71, were investigated. Promoter assay using transgenic Arabidopsis line carrying a CWPO-C promoter driven β-glucuronidase construct revealed that CWPO-C expressed in young developing tissues including protoxylem, except root tip. We also generated transgenic Arabidopsis T87 cultured cell overexpressed CWPO-C-EGFP fusion protein, and the fluorescence was observed in helical cell wall thickening of the cell differentiated into tracheary element. This presentation will discuss about the role of each peroxidase in the lignification process from a viewpoint of expression and subcellular localization patterns of CWPO-C, AtPrx-2, 25 and 71.

References
[1] Aoyama W, Sasaki S, Matsumura S, Hirai H, Tsutsumi Y, et al. (2002), J Wood Sci 48: 497–504.
[2] Sasaki S, Nishida T, Tsutsumi Y, Kondo R (2004), FEBS Lett 562: 197–201.
[3] Shigeto J, Kiyonaga Y, Fujita K, Kondo R, Tsutsumi Y (2013), J Agric Food Chem 61: 3781–3788. .
13. Jun Shigeto, Yuji Tsutsumi, Characterization of Plant Peroxidases which Involved in Arabiodpsis Stem Lignification, XXVIIth International Conference on Polyphenols & 8th Tannin Conference, 2014.09.
14. 堤 祐司, 重藤 潤, OXIDATION ACTIVITIES OF PLANT PEROXIDASES INVOLVED IN LIGNIFICATION, Oxizymes, 2014.07, Lignin is a main component of vascular plant cell walls and possesses a complex and irregular structure. In angiosperms, lignins consist mainly of two monolignols, coniferyl (4-hydroxy-3-methoxycinnamyl) and sinapyl (3,5-dimethoxy-4-hydroxycinnamyl) alcohols, which polymerize through at least five different linkage types and result in 4-hydroxy-3-methoxyphenyl (guaiacyl) and 3,5-dimethoxy-4-hydroxyphenyl (syringyl) units, respectively. Monolignols are supplied to the cell wall and polymerized to fill, together with hemicellulose, the spaces between cellulose microfibrils; this polymerization proceeds through oxidative coupling catalyzed by plant peroxidases. Based on the ‘‘End-wise’’ polymerization process, monolignol radicals can be coupled to a growing lignin polymer to produce a lignin macromolecule [1].
However, most known plant peroxidases, including horseradish peroxidase (HRP) and AtPrx53, can only oxidize p-coumaryl and coniferyl alcohols. This characteristic difference has led to controversy regarding the oxidation mechanism of sinapyl alcohol, lignin oligomers and polymers by plant peroxidases. Until now, CWPO-C, a peroxidase isozyme from Populus alba L., has only been verified to serve combined oxidation activity for sinapyl alcohol, lignin ligomers and polymer [2].The tyrosine 74 and tyrosine 177 located on the surface of CWPO-C, instead of the heme pocket, take a central role in the oxidation of substrates [3].
Previously, we has focused on seven Arabidopsis plant peroxidases selected using amino acid similarities to CWPO-C as a probe and found that AtPrx2 or AtPrx25 deficiency led both decreased total lignin content and altered lignin structure, including cell wall thinning in the stem. In addition, AtPrx71 deficiency led an altered stem lignin structure, although the lignin content is not decreased. These results provided in vivo evidence that AtPrx-2, 25, and 71 are involved in Arabidopsis stem lignification [4].
The present study explored the oxidation activities of three plant peroxidases, AtPrx-2, 25, and 71, which have been already shown to be involved in lignification Arabidopsis stem. Recombinant proteins of these peroxidases (rAtPrxs) were produced in Escherichia coli as inclusion bodies and successfully refolded to yield their active forms. The specific activities of rAtPrx-2, 25 and 71 toward two syringyl compounds (2,6-dimethoxyphenol and syringaldazine) were higher than that of HRP-C and rAtPrx53. Interestingly, rAtPrx2 and rAtPrx71 oxidized syringyl compounds more efficiently than guaiacol. Moreover, assays with ferrocytochrome c showed that AtPrx2, AtPrx25, and AtPrx71 possessed the ability to oxidize large molecules. This characteristic presumably originate in a protein radical. These results provide the evidence that the plant peroxidases responsible for lignin polymerization are able to directly oxidize all lignin precursors. We will also present an assay of dehydrogenative polymer formed from monolignols with rAtPrxs.


[1] Sarkanen KV (1971) Precursors and their polymerization. In: Sarkanen KV and Ludwig CH (eds) Lignins, Occurrence, Formation, Structure and Reactions. Wiley-Interscience, New York, pp. 95–163.
[2] Aoyama W, Sasaki S, Matsumura S, Hirai H, Tsutsumi Y, et al. (2002) Sinapyl alcohol-specific peroxidase isoenzyme catalyzes the formation of the dehydrogenative polymer from sinapyl alcohol. J. Wood Sci 48: 497–504.
[3] Shigeto J, Itoh Y, Tsutsumi Y, Kondo R (2012) Identification of Tyr74 and Tyr177 as substrate oxidation sites in cationic cell wall-bound peroxidase from Populus alba L. FEBS J 279: 348-357.
[4] Shigeto J, Kiyonaga Y, Fujita K, Kondo R, Tsutsumi Y (2013) Putative Cationic Cell-Wall-Bound Peroxidase Homologues in Arabidopsis , AtPrx2, AtPrx25, and AtPrx71, Are Involved in Lignification. J Agric Food Chem 61: 3781–3788
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15. Ryo Kanbe, Koki Fujita, Tatsuya Yagi, Ransika De Alwis, Tatuya Ashitani, Yuji Tsutsumi, Air born defense signal transduction cascade with monoterpenes on Cupressus lusitanica culture cells (poster), TERPNET2013, 2013.06.
16. Ryo Kanbe, Koki Fujita, Tatsuya Yagi, Ransika De Alwis, Tatuya Ashitani, Yuji Tsutsumi, Air born defense signal transduction cascade with monoterpenes on Cupressus lusitanica culture cells (poster), TERPNET2013, 2013.06.
17. Eri Takata, Ken Tsutsumi, Yuji Tsutsumi, Kenji Tabata, Effect of pretreatment on the production of monosaccharides from napier grass by hydrothermal reaction with acid catalyst, Lignobiotech II Symposium, 2012.10.
18. Jun Shigeto, Yuji Tsutsumi, Yoshitaka Itoh, Three putative CWPO-C homologs of Arabidopsis peroxidases, AtPrx2, AtPrx25 and AtPrx71, are involved in lignifications, Lignobiotech II Symposium, 2012.10.
19. Jun Shigeto, Mariko Nagano, Yuji Tsutsumi, Oxidation activity of three Arabidopsis peroxidases, AtPrx2, AtPrx25 and AtPrx71, for monolignol model compounds (poster), Lignobiotech II Symposium, 2012.10.
20. Yuji Tsutsumi, Eri Takata, Yuichi Murayama, Recycling of Bovine Meat and Bone Meal and Inactivation of BSE Prion by Sub-Critical Water Treatment., 10th International Symposium on Supercritical Fluids., 2012.05.
21. Syringyl ignin biodynthesis in poplar callus administered with sinapic acid.
Yuji Tsutsumi, Kutsuyoshi Hamada, Tomoko Nishiguchi, Kazuhiko Fukushima, Tomoaki Nishida, Ryuichiro Kondo
67th Annual Meeting of The Botanical Society of Japan
Sapporo, Japan.
22. Linginn biodynthesis and 4-coumarate:CoA ligase in black locust.
Yuji Tsutsumi, Kutsuyoshi Hamada, Tomoko Nishiguchi, Kazuhiko Fukushima, Tomoaki Nishida, Ryuichiro Kondo
67th Annual Meeting of The Botanical Society of Japan
September, 2003
Sapporo, Japan.
23. Mechanisms of dehydrogenative polymerization of lignin and ligninfication-specific peroxidase isoenzyme.
Yuji Tsutsumi
Symposium on the wood structure (2003)
Kyoto University (Uji), Japan.
24. Characterization and localization of cell wall peroxidase responsible for the dehydrogenative polymerization of lignin.
Shinya Sasaki, Naoto Ogawa, Wataru Aoyama, Tomoaki Nishida, Yuji Tsutsumi, Ryuichiro Kondo
43rd Symposium of the Japanese Society for Plant Physiologists
March, 2003
Kinki University (Nara), Jpan.
25. Cloning and expression of 4-coumarate:CoA ligases from black locust.
Tomoko Nishiguchi, Kazuhiko Fukushima, Tomoaki Nishida, Yuji Tsutsumi, Ryuichiro Kondo
53rd Annual Meeting of the Japan Wood Reseach Society
March, 2003
Kyushu Sangyo University (Fukuoka).
26. Salt tolerance mechanisms for white-rot fungus MG-60.
Tomonori Sugiyama, Yuji Tsutsumi, Ryuichiro Kondo
53rd Annual Meeting of the Japan Wood Reseach Society
March, 2003
Kyushu Sangyo University (Fukuoka).
27. Characterization and expression of manganese peroxidases produced by white-rot fungus MG-60.
Chieko Daikoku, Hiroto Suhara, Yuji Tsutsumi, Ryuichiro Kondo53rd Annual Meeting of the Japan Wood Reseach Society
March, 2003
Kyushu Sangyo University (Fukuoka).
28. Syringyl lignin specific peroxidase isoenzyme -Isolation of cDNA and localization of its protein in poplar tree-.
Shinya Sasaki, Wataru Aoyama, Naoto Ogawa, Tomoaki Nishida, Yuji Tsutsumi, Ryuichiro Kondo
53rd Annual Meeting of the Japan Wood Reseach Society
March, 2003
Kyushu Sangyo University (Fukuoka).