The effects of the different incubation times and substrate concentrations on the transglycosylation activity of Os1BGlu4.

<p>TLC analysis of products are shown. The standards are marked as M: marker, including <i>p</i>NPGlc (pG), <i>p</i>NP-<i>β</i>-cellobioside (pC2). <i>p</i>NP marks the position of <i>p</i>-nitrophenol. For the reactions, con is a control reaction without rOs1BGlu4, and 0.5–40, stand for reactions including 0.5 mM <i>p</i>NPGlc, 5 mM <i>p</i>NPGlc, 10 mM <i>p</i>NPGlc, 20 mM <i>p</i>NPGlc, and 40 mM <i>p</i>NPGlc, respectively, while 1, 2 and 3 hours are the incubation times. A: TLC plate visualized by the carbohydrate staining method. B: TLC plate visualized by UV light.</p

TLC of hydrolysis products of rOs1BGlu4 with cello-oligosaccharides and laminari-oligosaccharides.

<p>In each 50 µl reaction, 0.125 µg rOs1BGlu4 was incubated with 1 mM oligosaccharide in 50 mM sodium phosphate, pH 6.5, at 30 °C for 20 min. Samples were incubated with (+) and without (−) enzyme. Then, 2 µl of the reaction was spotted onto the TLC plate. Standards and substrates are: G, glucose; C2, cellobiose; C3, cellotriose; C4, cellotetraose; C5, cellopentaose; C6, cellohexaose; L2, laminaribiose; L3, laminaritriose; L4, laminaritetraose and L5, laminaripentaose.</p

Recombinant Expression and Characterization of the Cytoplasmic Rice β-Glucosidase Os1BGlu4

<div><p>The Os1BGlu4 β-glucosidase is the only glycoside hydrolase family 1 member in rice that is predicted to be localized in the cytoplasm. To characterize the biochemical function of rice Os1BGlu4, the <i>Os</i>1<i>bglu</i>4 cDNA was cloned and used to express a thioredoxin fusion protein in <i>Escherichia coli</i>. After removal of the tag, the purified recombinant Os1BGlu4 (rOs1BGlu4) exhibited an optimum pH of 6.5, which is consistent with Os1BGlu4's cytoplasmic localization. Fluorescence microscopy of maize protoplasts and tobacco leaf cells expressing green fluorescent protein-tagged Os1BGlu4 confirmed the cytoplasmic localization. Purified rOs1BGlu4 can hydrolyze <i>p</i>-nitrophenyl (<i>p</i>NP)-<i>β</i>-d-glucoside (<i>p</i>NPGlc) efficiently (<i>k</i>_cat/<i>K</i>_m  =  17.9 mM⁻¹·s⁻¹), and hydrolyzes <i>p</i>NP-<i>β</i>-d-fucopyranoside with about 50% the efficiency of the <i>p</i>NPGlc. Among natural substrates tested, rOs1BGlu4 efficiently hydrolyzed β-(1,3)-linked oligosaccharides of degree of polymerization (DP) 2–3, and β-(1,4)-linked oligosaccharide of DP 3–4, and hydrolysis of salicin, esculin and <i>p</i>-coumaryl alcohol was also detected. Analysis of the hydrolysis of <i>p</i>NP-<i>β</i>-cellobioside showed that the initial hydrolysis was between the two glucose molecules, and suggested rOs1BGlu4 transglucosylates this substrate. At 10 mM <i>p</i>NPGlc concentration, rOs1BGlu4 can transfer the glucosyl group of <i>p</i>NPGlc to ethanol and <i>p</i>NPGlc. This transglycosylation activity suggests the potential use of Os1BGlu4 for <i>p</i>NP-oligosaccharide and alkyl glycosides synthesis.</p></div

The pH optimum and pH stability of rOs1BGlu4 hydrolysis activity.

<p>A. pH optimum determination: rOs1BGlu4 (0.25 µg) was assayed with 1 mM <i>p</i>NPGlc in different 50 mM pH buffers (formate, pH 4.0; sodium acetate, pH 4.5–5.5; sodium phosphate, pH 6.0–7.5; Tris, pH 8.0–9.5; CAPS, pH 10.0–11.0) at 30°C for 10 min. B. pH stability evaluation: rOs1BGlu4 (20 µg) was incubated in the buffers described above for 10 min, 1, 3, 6, 12 and 24 h, then diluted 40-fold in 50 mM phosphate buffer, pH 6.5, and the activity was determined. The data are provided as mean + SE.</p

Evaluation of laminaritriose hydrolysis.

<p>A. Thin layer chromatographic evaluation of products of laminaritriose hydrolysis at different time points. rOs1BGlu4 (0.125 µg) was incubated with 1 mM laminaritriose in 50 mM sodium phosphate, pH 6.5, at 30 °C from 5 to 30 min (5 m–30 m). Samples were incubated with (+) and without (-) enzyme, then evaluated by silica gel TLC with sulfuric acid staining. The positions of glucose (G); laminaribiose (L2); and laminaritriose (L3) are marked. B: Kinetic data for laminaritriose hydrolysis. The Michaelis–Menten curve and inset Lineweaver-Burk plot are shown, along with the derived kinetic parameters and standard errors.</p

Kinetic parameters of rOs1BGlu4 in the hydrolysis of <i>p</i>NP glycosides and oligosaccharides.

<p>Kinetic parameters of rOs1BGlu4 in the hydrolysis of <i>p</i>NP glycosides and oligosaccharides.</p

The subcellular localization of Os1BGlu4-GFP and GFP-Os1BGlu4.

<p>Subcellular localization of Os1BGlu4-GFP (A–C) and GFP- Os1BGlu4 (D-F) fusion proteins in maize protoplasts. Fluorescent GFP signals (A, D), chlorophyll autofluorescence (B, E) and merged images (C, F) are shown. C, chloroplast; V, vacuole. The bar in the merged images represents 5 µm.</p

The relative expression of <i>Os1bglu4</i> under wounding stress.

<p>The stressed <i>Os1bglu4</i> expression was determined by quantitative real-time RT-PCR relative to untreated rice with actin as a control gene at various numbers of minutes (min) after wounding of 10 day old rice seedling shoots. The data are given as mean + SE.</p

SDS-PAGE profiles of recombinant Os1BGlu4 expressed in Origami B(DE3).

<p>Lane 1, standard protein marker (Bio-RAD); Lane 2, crude Trx-His₆-rOs1BGlu4; lane 3, purified Trx-His₆-rOs1BGlu4; lane 4, Trx-His₆-rOs1BGlu4 cleaved by enterokinase; lane 5, purified rOs1BGlu4. The numbers 45 and 66 indicate the molecular weights in kDa of the most relevant protein standards. Each lane was loaded with 8 µl of the sample mixed with 2 µl 5× sample dye.</p

Protein sequence-based phylogenetic tree of rice and Arabidopsis glycoside hydrolase family GH1 enzymes.

<p>The phylogenetic clusters described by Opassiri et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096712#pone.0096712-Opassiri1" target="_blank">[3]</a> are separated by red lines and labeled At/Os1-8, At I and At II. Os9BGlu36 was used to root the tree, so the Arabidopsis member of At/Os8, SFR2, is not shown. The Arabidopsis sequences are named as described by Xu et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096712#pone.0096712-Xu1" target="_blank">[37]</a>, while the rice members are named as described by Opassiri et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096712#pone.0096712-Opassiri1" target="_blank">[3]</a>. The pine coniferin β-glucosidase (PC AAC69619) and rubber tree β-glucosidase (HV AAP51059), which are mentioned in the text, are shown labeled by the first letters of their genus and species and Genbank accession numbers. The tree was developed with the neighbor-joining method based on the protein sequence alignment made with the MUSCLE algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096712#pone.0096712-Edgar1" target="_blank">[38]</a> implemented in MEGA 5.2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096712#pone.0096712-Tamura1" target="_blank">[39]</a>. Cluster At/Os4 is emphasized with a larger underlined label, since it is the subject of the current work.</p