Plant Science 179 (2010) 273–280 Contents lists available at ScienceDirect Plant Science journal homepage: www.elsevier.com/locate/plantsci Rice Os4BGlu12 is a wound-induced -glucosidase that hydrolyzes cell wall--glucan-derived oligosaccharides and glycosides Rodjana Opassiri a,∗ , Janjira Maneesan a , Takashi Akiyama b , Busarakum Pomthong c , Shigeki Jin d , Atsuo Kimura e , James R. Ketudat Cairns a a School of Biochemistry, Institute of Science, Suranaree University of Technology, Muang District, Nakhon Ratchasima 30000, Thailand b Department of Low-Temperature Science, National Agricultural Research Center for Hokkaido Region, Sapporo 062-8555, Japan c Faculty of Sciences and Technology, Nakhon Ratchasima Rajabhat University, Nakhon Ratchasima 30000, Thailand d Graduate School of Health Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan e Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan article info Article history: Received 25 March 2010 Received in revised form 24 May 2010 Accepted 27 May 2010 Available online 4 June 2010 Keywords: -Glucosidase Endo-(1,3 ;1,4)--glucanase Glycosides Rice Cell wall Wound abstract Rice Os4BGlu12 -glucosidase is a family 1 glycoside hydrolase, the transcript levels of which have pre- viously been found to be induced in response to herbivore attack and salinity stress. Here, high levels of Os4bglu12 transcripts were also detected in the shoot during germination, in the leaf sheath and stem of mature rice plants under normal growth conditions. The transcripts of this gene were up-regulated in response to wounding, methyl jasmonate and ethephon in 10-day-old rice seedlings. Os4BGlu12 expressed in recombinant Escherichia coli hydrolyzed -(1,3;1,4)-glucooligosaccharides generated by the wounding-induced rice endo-(1,3;1,4)--glucanase OsEGL1, suggesting that both enzymes may act in concert in remodeling of damaged cell wall. Among oligosaccharides tested, Os4BGlu12 hydrolyzed -(1,4)-linked glucooligosaccharides with highest catalytic efficiency (k cat /K m = 2.7–4.9 s -1 mM -1 ) when the degree of polymerization ranged from 3 to 6. It also hydrolyzed the -(1,3)-linked disaccharide lam- inaribiose with high catalytic efficiency (k cat /K m = 4.5 s -1 mM -1 ). Among the natural glycosides tested, Os4BGlu12 efficiently hydrolyzed deoxycorticosterone 21-glucoside (k cat /K m = 20 s -1 mM -1 ) and api- genin 7-O--d-glucoside (k cat /K m = 6.7 s -1 mM -1 ). The amino acid residues predicted to line the active site of Os4BGlu12 are more similar to those of cyanogenic and flavonoid -glucosidases than oligosaccharide hydrolases, and it may function in defense, as well as in cell wall-derived oligosaccharide break-down. © 2010 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Plant glycosyl hydrolase family 1 (GH1) -glucosidases (EC 3.2.1.21) hydrolyze the -O-glycosidic bond at the anomeric car- bon of glucose moieties at the nonreducing end of carbohydrate or glycoside molecules [1]. The glycones recognized by GH1 - glycosidases include glucose, galactose, fucose, mannose, xylose, 6-phospho-glucose and 6-phospho-galactose. The diversity of agly- Abbreviations: DP, degree of polymerization; ESI-MS, electrospray ionization- mass spectroscopy; GH1, glycosyl hydrolase family 1; GLC, gas liquid chromatog- raphy; GST-OsEGL1, glutathione-S-transferase-rice endoglucanase OsEGL1 fusion protein; MS, mass spectroscopy; PMAAs, partially methylated alditol acetates; pNP, p-nitrophenyl; TLC, thin layer chromatography; Trx-Os4BGlu12, thioredoxin-rice -glucosidase Os4BGlu12 fusion protein. ∗ Corresponding author. Tel.: +66 44 224598; fax: +66 44 224185. E-mail addresses: opassiri@sut.ac.th (R. Opassiri), jmaneesan@hotmail.com (J. Maneesan), takiyama@affrc.go.jp (T. Akiyama), busarakum p@yahoo.com (B. Pomthong), s-jin@hs.hokudai.ac.jp (S. Jin), kimura@abs.agr.hokudai.ac.jp (A. Kimura), cairns@sut.ac.th (J.R.K. Cairns). cones is higher, including monosaccharides, oligosaccharides and aryl or alkyl groups. The physiological functions of these enzymes in plants based on the activities of the aglycone moieties of sub- strate include (1) defense against pathogens and herbivores [2–4], (2) phytohormone activation [5,6], (3) lignification [7], (4) cell wall catabolism [8,9] and (5) release of active metabolic intermediate molecules [10]. The GH1 enzymes may hydrolyze substrates with a broad range of different glycones or aglycones with different specificities, but some enzymes may be specific for only one type of glycone or aglycone. The fundamental substrate specificity of these enzymes depends on the overall dimensions and geometry of the binding site and the distribution of the active site amino acids that are important for the substrate recognition and binding, which complement the structure of the aglycone and glycone moieties of the substrate [11,12]. Forty genes homologous to GH1 -glucosidases have been identified in rice genomic sequences, 34 of which appear to be functional in rice [13]. To date, only a few rice -glucosidase isoenzymes have been characterized for their possible function. Partially purified -glucosidases from rice were described that 0168-9452/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2010.05.013