Gentiobiose, a β-1,6-linked glycosyl-disaccharide, accumulates abundantly in Gentianaceae and is involved in aspects of plant development, such as fruits ripening and release of bud dormancy. However, the mechanisms regulating the amount of gentio-oligosaccharide accumulation in plants remain obscure. The present study aimed to identify an enzyme that modulates gentio-oligosaccharide amount in gentian (Gentiana triflora). A protein responsible for gentiobiose hydrolysis, GtGen3A, was identified by partial purification and its peptide sequence analysis. The enzyme had a molecular mass of ∼67 kDa without a secretory signal peptide sequence. Sequence analysis revealed that GtGen3A could be a β-glucosidase member belonging to glycoside hydrolase family 3 (GH3). GtGen3A showed a homology to GH3 β-glucan exohydrolases, ExoI of Hordeum vulgare, and ExgI from Zea mays, which preferentially hydrolyzed β-1,3- and β-1,4-linked oligosaccharides. The purified recombinant GtGen3A (rGtGen3A) produced in Escherichia coli showed optimal reaction at pH 6.5 and 20°C. The rGtGen3A liberated glucose from β-1,2-, β-1,3-, β-1,4-, and β-1,6-linked oligosaccharides, and showed the highest activity toward gentiotriose among the substrates tested. Kinetic analysis also revealed that rGtGen3A preferentially hydrolyzed gentiotriose. Virus-induced gene silencing of Gtgen3A in gentian plantlets resulted in predominant accumulation of gentiotriose rather than gentiobiose. Furthermore, the expression level of Gtgen3A was almost similar to the amount of gentiobiose in field-grown gentians. These findings suggest that the main function of GtGen3A is the hydrolysis of gentiotriose to gentiobiose, and that GtGen3A plays a role in modulating gentiobiose amounts in gentian.
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April 2018
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DAPI staining of Candida tropicalis cell chromatin, imaged by laser scanning confocal microscopy. In this issue of the Biochemical Journal, Li et al. discuss the potential to induce apoptosis when treating Candida tropicalis with CGA-N12; for details see pages 1385–1396.
Research Article|
April 16 2018
Gtgen3A, a novel plant GH3 β-glucosidase, modulates gentio-oligosaccharide metabolism in Gentiana
Hideyuki Takahashi;
1Iwate Biotechnology Research Center, Kitakami, Iwate 024-0003, Japan
Correspondence: Hideyuki Takahashi ([email protected])
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Sayaka Kikuchi-Fujisaki;
Sayaka Kikuchi-Fujisaki
1Iwate Biotechnology Research Center, Kitakami, Iwate 024-0003, Japan
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Chiharu Yoshida;
Chiharu Yoshida
1Iwate Biotechnology Research Center, Kitakami, Iwate 024-0003, Japan
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Hidetoshi Yamada;
Hidetoshi Yamada
1Iwate Biotechnology Research Center, Kitakami, Iwate 024-0003, Japan
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Tetsuro Yamashita;
Tetsuro Yamashita
2Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan
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Naotake Konno;
Naotake Konno
3Department of Applied Biological Chemistry, Utsunomiya University, 350 Mine-machi, Utsunomiya, Tochigi 321-8505, Japan
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Takumi Takeda
Takumi Takeda
1Iwate Biotechnology Research Center, Kitakami, Iwate 024-0003, Japan
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Publisher: Portland Press Ltd
Received:
November 14 2017
Revision Received:
March 15 2018
Accepted:
March 26 2018
Accepted Manuscript online:
March 26 2018
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society
2018
Biochem J (2018) 475 (7): 1309–1322.
Article history
Received:
November 14 2017
Revision Received:
March 15 2018
Accepted:
March 26 2018
Accepted Manuscript online:
March 26 2018
Citation
Hideyuki Takahashi, Sayaka Kikuchi-Fujisaki, Chiharu Yoshida, Hidetoshi Yamada, Tetsuro Yamashita, Naotake Konno, Takumi Takeda; Gtgen3A, a novel plant GH3 β-glucosidase, modulates gentio-oligosaccharide metabolism in Gentiana. Biochem J 16 April 2018; 475 (7): 1309–1322. doi: https://doi.org/10.1042/BCJ20170866
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