Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Koki Fujita Last modified date:2023.07.12

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

1. Diego Alonso Yoshikay‑Benitez, Kaori Ohira, Kasturi Banerjee, Koki Fujita, Jun Shigeto, Yuji Tsutsumi, The Populus alba cationic cell‑wall‑bound peroxidase (CWPO‑C) regulates plant growth, lignin content and composition in poplar, Journal of Wood Science,, 2023.03, Cationic cell-wall-bound peroxidase (CWPO-C) from Populus alba is the only Class III peroxidase that has been shown to be able to oxidize high molecular weight lignin polymers from sinapyl alcohol and previously, has been believed to be a lignin polymerization-specific peroxidase. However, using an Arabidopsis heterologous expression system, we showed recently that CWPO-C contributes to differentiation or early growth and is involved in auxin catabolism. In this study, to clarify the function of CWPO-C in poplar, we analyzed CWPO-C gene expression and phenotypic changes with CWPO-C overexpression and suppression. Real-time PCR and monitoring promoter activity of CWPO-C using β-glucuronidase (GUS) assay revealed that CWPO-C was strongly expressed in immature tissues, such as the upper stem, axillary buds, and young leaves, in addition to expression in developing xylem. In transgenic poplars in which the expression of CWPO-C was upregulated or suppressed, changes in stem growth, gravitropism bending time, lignin content and syringyl/guaiacyl (S/G) composition were observed. Overexpressing CWPO-C enhanced stem growth and gravitropic response (shorter bending time). With suppressed CWPO-C expression, the lignin content was reduced approximately 45% and the S/G ratio decreased by half. These results strongly suggest that CWPO-C plays a role in differentiation and early growth, as well as in lignin polymerization..
2. Diego Alonso Yoshikay-Benitez, Yusuke Yokoyama, Kaori Ohira, Koki Fujita, Azusa Tomie, Yoshio Kijidani, Jun Shigeto, Yuji Tsutsumi, Populus alba cationic cell-wall-bound peroxidase (CWPO-C) regulates the plant growth and affects auxin concentration in Arabidopsis thaliana, Physiology and Molecular Biology of Plants,, 2022.10, The poplar cationic cell-wall-bound peroxidase (CWPO-C) mediates the oxidative polymerization of lignin precursors, especially sinapyl alcohols, and high molecular weight compounds that cannot be oxidized by other plant peroxidases, including horseradish peroxidase C. Therefore, CWPO-C is believed to be a lignification-specific peroxidase, but direct evidence of its function is lacking. Thus, the CWPO-C expression pattern in Arabidopsis thaliana (Arabidopsis) was determined using the β-glucuronidase gene as a reporter. Our data indicated that CWPO-C was expressed in young organs, including the meristem, leaf, root, flower, and young xylem in the upper part of the stem. Compared with the wild-type control, transgenic Arabidopsis plants overexpressing CWPO-C had shorter stems. Approximately 60% of the plants in the transgenic line with the highest CWPO-C content had curled stems. These results indicate that CWPO-C plays a role in cell elongation. When plants were placed horizontally, induced CWPO-C expression was detected in the curved part of the stem during the gravitropic response. The stem curvature associated with gravitropism is controlled by auxin localization. The time needed for Arabidopsis plants overexpressing CWPO-C placed horizontally to bend by 90° was almost double the time required for the similarly treated wild-type controls. Moreover, the auxin content was significantly lower in the CWPO-C-overexpressing plants than in the wild-type plants. These results strongly suggest that CWPO-C has pleiotropic effects on plant growth and indole-3-acetic acid (IAA) accumulation. These effects may be mediated by altered IAA concentration due to oxidation..
3. Jun Shigeto, Hiroki Honjo, Koki Fujita, Yuji Tsutsumi, Generation of lignin polymer models via dehydrogenative polymerization of coniferyl alcohol and syringyl alcohol via several plant peroxidases involved in lignification and analysis of the resulting DHPs by MALDI-TOF analysis, Holzforschung, 10.1515/hf-2017-0125, 72, 4, 267-274, 2018.03, The mechanism of lignin dehydrogenative polymerization (DHP), made by means of horseradish peroxidase (HRP), was studied in comparison with other plant peroxidases. Interestingly, HRP is efficient for guaiacyl type polymer formation (G-DHPs), but is not efficient in the case of syringyl type DHPs (S-DHPs). It was previously demonstrated that lignification-related Arabidopsis thaliana peroxidases, AtPrx2, AtPrx25 and AtPrx71, and cationic cell-wall-bound peroxidase (CWPO-C) from Populus alba 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..
4. Analysis of volatile compounds and their seasonal changes in room using Sugi (Cryptomeria japonica) wood boards as interior materials.
5. Jun Shigeto, Yukie Ueda, Shinya Sasaki, Koki Fujita, Yuji Tsutsumi, Enzymatic activities for lignin monomer intermediates highlight the biosynthetic pathway of syringyl monomers in Robinia pseudoacacia, Journal of Plant Research, 10.1007/s10265-016-0882-4, 130, 1, 203-210, 2017.01, Most of the known 4-coumarate:coenzyme A ligase (4CL) isoforms lack CoA-ligation activity for sinapic acid. Therefore, there is some doubt as to whether sinapic acid contributes to sinapyl alcohol biosynthesis. In this study, we characterized the enzyme activity of a protein mixture extracted from the developing xylem of Robinia pseudoacacia. The crude protein mixture contained at least two 4CLs with sinapic acid 4-CoA ligation activity. The crude enzyme preparation displayed negligible sinapaldehyde dehydrogenase activity, but showed ferulic acid 5-hydroxylation activity and 5-hydroxyferulic acid O-methyltransferase activity; these activities were retained in the presence of competitive substrates (coniferaldehyde and 5-hydroxyconiferaldehyde, respectively). 5-Hydroxyferulic acid and sinapic acid accumulated in the developing xylem of R. pseudoacacia, suggesting, in part at least, sinapic acid is a sinapyl alcohol precursor in this species..
6. Koki Fujita, Ryo Kambe, Ransika de Alwis, Tatsuya Yagi, Yuji Tsutsumi, Airborne Monoterpenes Emitted from a Cupressus lusitanica Cell Culture Induce a Signaling Cascade that Produces β-Thujaplicin, Journal of Chemical Ecology, 10.1007/s10886-016-0739-2, 42, 8, 814-820, 2016.09, [URL], A cell culture of Cupressus lusitanica was used to investigate the reaction of a plant to certain airborne chemicals. Compared with laboratory and field methods using intact plants or tissues, a cell culture is advantageous because it is not affected by environmental factors and the experiments are easier to reproduce. When exposed to an elicitor, our cell line produces 10 olefin monoterpenes and beta-thujaplicin, which is a strong phytoalexin. These monoterpenes are emitted into the vapor phase and are expected to play a role in airborne signaling. In the present study, the cells were exposed to monoterpene vapors, and the volatiles present in the culture flasks were monitored. When the culture cells were exposed to low doses of sabinene, we detected beta-terpinene and p-cymene. After exposure to beta-terpinene, we found p-cymene and terpinolene, whereas p-cymene exposure resulted in terpinolene emission. By contrast, the other seven monoterpenes we investigated did not induce any emissions of other monoterpenes. These results strongly suggest that in C. lusitanica a signaling cascade exists that starts with the emission of sabinene and moves to beta-terpinene, p-cymene, and finally to terpinolene, which accelerates the production of the phytoalexin beta-thujaplicin..
7. Jun Shigeto, Yoshitaka Itoh, Sakie Hirao, Kaori Ohira, Koki Fujita, Yuji Tsutsumi, Simultaneously disrupting AtPrx2, AtPrx25 and AtPrx71 alters lignin content and structure in Arabidopsis stem, Journal of Integrative Plant Biology, 10.1111/jipb.12334, 57, 4, 349-356, 2015.01, Plant class III heme peroxidases catalyze lignin polymerization. Previous reports have shown that at least three Arabidopsis thaliana peroxidases, AtPrx2, AtPrx25 and AtPrx71, are involved in stem lignification using T-DNA insertion mutants, atprx2, atprx25, and atprx71. Here, we generated three double mutants, atprx2/atprx25, atprx2/atprx71, and atprx25/atprx71, and investigated the impact of the simultaneous deficiency of these peroxidases on lignins and plant growth. Stem tissue analysis using the acetyl bromide method and derivatization followed by reductive cleavage revealed improved lignin characteristics, such as lowered lignin content and increased arylglycerol-β-aryl (β-O-4) linkage type, especially β-O-4 linked syringyl units, in lignin, supporting the roles of these genes in lignin polymerization. In addition, none of the double mutants exhibited severe growth defects, such as shorter plant stature, dwarfing, or sterility, and their stems had improved cell wall degradability. This study will contribute to progress in lignin bioengineering to improve lignocellulosic biomass..
8. Takako Harada, Koki Fujita, Jun Shigeto, Yuji Tsutsumi, Stereo-selective oxidations of terpinolene by cytochrome P450 monooxygenases in the microsomal fraction of Cupressus lusitanica cultured cells, JOURNAL OF WOOD SCIENCE, 10.1007/s10086-014-1432-0, 60, 6, 446-452, 2014.12, We showed previously that in Cupressus lusitanica (Mexican cypress), the first two steps of terpinolene oxidation, beginning at hydroxylation at the aryl position and then forming epoxide at the double bond, were driven by cytochrome P450s. The significance of enantio differences, in general, has received attention because those enantiomers often have their own biological activities. We, therefore, investigated the stereo-specificity of the substrate and the enantio-selectivity of these reactions. The hydroxylation of terpinolene by cytochrome P450 in the microsomal fraction from C. lusitanica cells gave a single product with an S configuration of 5-isopropylidene-2-methylcyclohex-2-enol. Next, epoxidizing enzyme accepted only the S-configuration substrate and produced a single enantiomer product, (1R, 2S, 6S)-(+)-1,6-epoxy-4(8)-p-menthen-2-ol. No isomer was detected at our gas chromatography/mass spectrometry sensitivity; therefore, the calculated enantiomeric excess values were 100 %. These results indicate that the cytochrome P450s involved in terpinolene metabolism in C. lusitanica cells have very strict stereo-selective ability. Our findings may be helpful in the stereo-selective synthesis of fine chemicals, although the physiological meanings of these chiral products are still not unclear..
9. Jun Shigeto, Mariko Nagano, Koki Fujita, Yuji Tsutsumi, Catalytic Profile of Arabidopsis Peroxidases, AtPrx-2, 25 and 71, Contributing to Stem Lignification , PLOS one, DOI: 10.1371/journal.pone.0105332, 2014.08, Lignins are aromatic heteropolymers that arise from oxidative coupling of lignin precursors, including lignin monomers (p-coumaryl, coniferyl, and sinapyl alcohols), oligomers, and polymers. Whereas plant peroxidases have been shown to catalyze oxidative coupling of monolignols, the oxidation activity of well-studied plant peroxidases, such as horseradish peroxidase C (HRP-C) and AtPrx53, are quite low for sinapyl alcohol. This characteristic difference has led to controversy regarding the oxidation mechanism of sinapyl alcohol and lignin oligomers and polymers by plant peroxidases. The present study explored the oxidation activities of three plant peroxidases, AtPrx2, AtPrx25, and AtPrx71, which have been already shown to be involved in lignification in the Arabidopsis stem. Recombinant proteins of these peroxidases (rAtPrxs) were produced in Escherichia coli as inclusion bodies and successfully refolded to yield their active forms. rAtPrx2, rAtPrx25, and rAtPrx71 were found to oxidize two syringyl compounds (2,6-dimethoxyphenol and syringaldazine), which were employed here as model monolignol compounds, with higher specific activities than HRP-C and rAtPrx53. Interestingly, rAtPrx2 and rAtPrx71 oxidized syringyl compounds more efficiently than guaiacol. Moreover, assays with ferrocytochrome c as a substrate showed that AtPrx2, AtPrx25, and AtPrx71 possessed the ability to oxidize large molecules. This characteristic may originate in a protein radical. These results suggest that the plant peroxidases responsible for lignin polymerization are able to directly oxidize all lignin precursors..
10. Koki Fujita, Yasufumi Bunyu, Ken-ichi Kuroda, Tatsuya Ashitani, Jun Shigeto, Yuji Tsutsumi, A novel synthetic pathway for tropolone ring formation via the olefin monoterpene intermediate terpinolene in cultured Cupressus lusitanica cells, Journal of Plant Physiology, 10.1016/j.jplph.2013.12.016, 171, 8, 610-614, 2014.03, β-Thujaplicin is a wood monoterpene and tropolone compound with a unique conjugated 7-membered ring. Because of its strong antifungal and antitumor activities, β-thujaplicin is used in several fields. The biosynthesis pathway of β-thujaplicin has not yet been elucidated. Using Cupressus lusitanica cell cultures in a radioisotope feeding experiment, our group previously demonstrated that geranyl pyrophosphate (GPP) is the starting material of β-thujaplicin biosynthesis. The results of our previous terpene synthase assay suggested that terpinolene is the first olefin terpenoid intermediate from GPP to β-thujaplicin, although there was no experimental evidence of this at that time. In the present study, we fed deuterium-labeled terpinolene to cultured C. lusitanica cells to determine whether terpinolene is an intermediate metabolite of β-thujaplicin biosynthesis. A gas chromatography-mass spectroscopy analysis of the cell extracts from labeled terpinolene cultures revealed a peak of labeled β-thujaplicin that was not observed after treatment with non-labeled terpinolene. The identification of labeled β-thujaplicin was also performed by mass spectrum assignment. The outcome indicated that terpinolene is indeed an intermediate metabolite of β-thujaplicin biosynthesis. To the best of our knowledge, there has been no prior report that tropolone compounds are biosynthesized via a terpene biosynthesis system, and our results thus suggest the existence of a novel biosynthetic pathway that produces the conjugated 7-membered ring..
11. Tatsuya Ashitani, Norihisa Kusumoto, Annna-Karin Borg-Karlson, Koki Fujita, Koetsu Takahashi, Antitermite Activity of β-Caryophyllene Epoxide and Episulfide, Zeitschrift für Naturforschung C, 68C, 302-306, 2013.09, [URL], Caryophyllene-6,7-epoxide and caryophyllene-6,7-episulfide can be easily synthesized
from β-caryophyllene by autoxidation or episulfidation. The bioactivities of β-caryophyllene
and its derivatives were investigated against the subterranean termite Reticulitermes speratus
Kolbe. The antifeedant, feeding, and termiticidal activities of each compound were tested using
no-choice, dual-choice, and non-contact methods. Antitermitic activities were not shown
by β-caryophyllene, but were observed for the oxide and sulfi de derivatives. Caryophyllene-
6,7-episulfi de showed especially high antifeedant and termiticidal activities. Thus, naturally
abundant, non-bioactive β-caryophyllene can be easily converted into an antitermite
reagent via a non-biological process..
12. 重藤 潤, Yuko Kiyonaga, Koki Fujita, RYUICHIRO KONDO, Yuji Tsutsumi, Putative Cationic Cell-Wall-Bound Peroxidase Homologues in Arabidopsis, AtPrx2, AtPrx25, and AtPrx71, Are Involved in Lignification., Journal of Agriculture and Food Chemistry, 10.1021/jf400426g, 61, 16, 3781-3788, 2013.04, The final step of lignin biosynthesis, which is catalyzed by a plant peroxidase, is the oxidative coupling of the monolignols to growing lignin polymers. Cationic cell-wall-bound peroxidase (CWPO-C) from poplar callus is a unique enzyme that has oxidative activity for both monolignols and synthetic lignin polymers. This study shows that putative CWPO-C homologues in Arabidopsis, AtPrx2, AtPrx25, and AtPrx71, are involved in lignin biosynthesis. Analysis of stem tissue using the acetyl bromide method and derivatization followed by the reductive cleavage method revealed a significant decrease in the total lignin content of ATPRX2 and ATPRX25 deficient mutants and altered lignin structures in ATPRX2, ATPRX25, and ATPRX71 deficient mutants. Among Arabidopsis peroxidases, AtPrx2 and AtPrx25 conserve a tyrosine residue on the protein surface, and this tyrosine may act as a substrate oxidation site as in the case of CWPO-C. AtPrx71 has the highest amino acid identity with CWPO-C. The results suggest a role for CWPO-C and CWPO-C-like peroxidases in the lignification of vascular plant cell walls..
13. Takako Harada, Eriko Harada, Ryoko Sakamoto, Tatsuya Ashitani, Koki Fujita, Ken'ichi Kuroda, Regio- and Substrate-specific Oxidative Metabolism of Terpinolene by Cytochrome P450 Monooxygenases in Cupressus lusitanica Cultured Cells, American journal of plant sciences, 3, 268-275, 2012.02.
14. Kuroda, Tatsuya Ashitani, Koki Fujita, Tetramethylammonium hydroxide (TMAH) thermochemolysis of lignin: Formation of (E)-5-formyl-2,3,3´,4´-tetramethoxystilbene and its origins., Journal of Analytical and Applied Pyrolysis, 10.1016/j.jaap.2010.08.008, 89, 2, 233-238, 2010.09.
15. Ken-ichi Kuroda, Akiko Nakagawa-izumi , Tatsuya Ashitani , Koki Fujita, Tetramethylammonium hydroxide (TMAH) thermochemolysis of 2-arylcoumaran lignin model compounds, Journal of Analytical and Applied Pyrolysis, 86(1), 185-191, 2009.09.
16. Ransika De Alwis, Koki Fujita, Tatsuya Ashitani, Ken’ichi Kuroda, Volatile and non-volatile monoterpenes produced by elicitor-stimulated Cupressus lusitanica cultured cells, Journal of Plant Physiology, 166, 720-728, 2009.05.
17. Ransika De Alwis, Koki Fujita, Tatsuya Ashitani, Ken’ichi Kuroda, Induced monoterpene and lignin production in mechanically stressed and fungal elicited cultured Cupressus lusitanica cells, Plant Biotechnol Rep, 3, 57-65, 2009.03.
18. Ashitani T, Borg-Karlson AK, Fujita K, Nagahama S, Reaction mechanism of direct episulfidation of caryophyllene and humulene, NATURAL PRODUCT RESEARCH, 22: 495-498, 2008 , 2008.04.
19. Ken-ichi Kuroda, Tatsuya Ashitani, Koki Fujita, Takefumi Hattori, Thermal Behavior of β-1 Subunits in Lignin: Pyrolysis of 1,2-Diarylpropane-1,3-diol-type Lignin Model Compounds, Journal of Agricultural and Food Chemistry, 55,2770-2778, 2007.04.
20. Zhao J , Fujita K, Sakai K, Reactive oxygen species, nitric oxide, and their interactions play different roles in Cupressus lusitanica cell death and phytoalexin biosynthesis, NEW PHYTOLOGIST, 175 (2): 215-229 2007, 2007.02.
21. R. Tomoshige, T. Ashitani, Y. Kurimoto, K. Fujita. K. Kuroda, Combustion Synthesis of Composites Consisting of Ti-Si-C Ceramics from Sugi Bark and Alumina, Proceedings of the 23rd International Japan-Korea Seminar on Ceramics, 189-192, 2006.11.
22. Tatsuya ASHITANI, Koki FUJITA, Yasuji KURIMOTO, Preparation of Polyurethane Film from Conifer Tree Bark Liquefied by PEG-Bisulfite Method, Non-destructive evaluation for wood and woody materials for development new functional wood-based materials, Proceedings of JSPS Japan and Hungary Research Cooperative Program / Joint Seminar, 170-177, 2006.10.
23. Jian Zhao, Yoko Matsunaga, Koki Fujita, Kokki Sakai, Signal transduction and metabolic flux of beta-thujaplicin and monoterpene biosynthesis in elicited Cupressus lusitanica cell cultures, Metabolic Engineering, 8(1),14-29, 2006.01.
24. Tatsuya Ashitani, Ryuichi Tomoshige, Koki Fujita, Ken-ichi Kuroda, Combustion Synthesis of Ti-Si-C System Composites using Woody Waste Material, Proceedings of the 22nd International Japan-Korea Seminar on Ceramics, 91-94, 2005.08.
25. Zhao J, Fujita K, Sakai K, Oxidative stress in plant cell culture: A role in production of beta-thujaplicin by Cupresssus lusitanica suspension culture, Biotechnology and Bioengineering, 10.1002/bit.20465, 90, 5, 621-631, 90 (5): 621-631, 2005.06.
26. Zhao J, Zheng SH, Fujita K, Sakai K, Jasmonate and ethylene signalling and their interaction are integral parts of the elicitor signalling pathway leading to beta-thujaplicin biosynthesis in Cupressus lusitanica cell cultures, Journal of Experimental Botany, 10.1093/jxb/erh127, 55, 399, 1003-1012, 55(399): 1003-1012, 2004, 2004.05.
27. Zhao J, Guo YQ, Fujita K, Sakai K, Involvement of cAMP signaling in elicitor-induced phytoalexin accumulation in Cupressus lusitanica cell cultures, New Phytologist, 10.1046/j.1469-8137.2003.00976.x, 161, 3, 723-733, 161(3): 723-733, 2004, 2004.05.
28. Matsunaga Y, Fujita K, Yamada J, Ashitani T, Sakai K, Monoterpenes produced by Cupressus lusitanica cultured cells including a novel monoterpene (1S, 2S, 6S)-(+)-1,6-epoxy-4(8)-p-menthen-2-ol, Natural Product Research, 10.1080/1478641031000111525, 17, 6, 441-443, 17 (6): 441-443, 2003.12.
29. Effect of major inorganic nutrients on beta-thujaplicin production in a suspension culture of Cupressus lusitanica cells.
30. Yamada J, Fujita K, Eto K, Sakai K, Cell growth and nutrient uptake by cell suspensions of Cupressus lusitanica, Journal of Wood Science, 10.1007/s100860300001, 49, 1, 5-10, 49 (1): 5-10, 2003.02.
31. Yamda J, Fujita K, Sakai K, Feedback regulation of beta-thujaplicin production and formation of its methyl ether in a suspension culture of Cupressus lusitanica, Phytochemistry, 60, 5, 447-450, 60 (5): 447-450, 2002.07.
32. Zhao J, Fujita K, Sakai K, Production of beta-thujaplicin in Cupressus lusitanica suspension cultures fed with organic acids and monoterpenes, Bioscience Biotechnology and Biochemistry, 10.1271/bbb.65.1027, 65, 5, 1027-1032, 65 (5): 1027-1032, 2002.05.
33. Cell growth and beta-thujaplicin production with a Cupressus lusitanica culture in jar fermentors.
34. Zhao J, Fujita K, Yamada J, Sakai K, Improved beta-thujaplicin production in Cupressus lusitanica suspension cultures by fungal elicitor and methyl jasmonate, Applied Microbiologt and Biotechnology, 55, 3, 301-305, 55 (3): 301-305, 2001.04.
35. Kashino Y, Nishida T, Takahara Y, Fujita K, Kondo R, Sakai K, BIOMECHANICAL PULPING USING WHITE-ROT FUNGUS IZU-154, TAPPI J, 76, 12, 167-171, 76 (12): 167-171, 1993.12.
36. Yamaguchi T, Fujita K, Sakai K, Biological activity of extracts from Cupressus lusitanica cell culture, Journal of Wood Science, 10.1007/BF01192336, 45, 2, 170-173, Vol.45, No.2, 1994.04.
37. Fujita K, Yamaguchi T, Itose R, Sakai K, Biosynthetic pathway of beta-thujaplicin in the Cupressus lusitanica cell culture, Journal of Plant Physiology, 156, 4, 462-467, 156 (4): 462-467, 2000.04.
38. Fujita K, Kondo R, Sakai K, Kashino Y, Nishida T, Takahara Y, BIOBLEACHING OF SOFTWOOD KRAFT PULP WITH WHITE-ROT FUNGUS IZU-154, TAPPI J, 76, 1, 81-84, 76 (1): 81-84, 1993.01.
39. Fujita K, Kondo R, Sakai K, Kashino Y, Nishida T, Takahara Y, BIOBLEACHING OF KRAFT PULP USING WHITE-ROT FUNGUS IZU-154, TAPPI J, 74, 11, 123-127, 74 (11): 123-127, 1991.11.