Identification of Fatty Acid Glucose Esters as Os9BGlu31 Transglucosidase Substrates in Rice Flag Leaves

Rice Os9BGlu31 transglucosidase transfers glucosyl moieties between various carboxylic acids and alcohols, including phenolic acids and flavonoids, in vitro. The role of Os9BGlu31 transglucosidase in rice plant metabolism has only been suggested to date. Methanolic extracts of rice bran and leaves were found to contain oleic acid and linoleic acid to which Os9BGlu31 could transfer glucose from the 4-nitrophenyl β-d-glucoside (4NPGlc) donor to form 1-<i>O</i>-acyl glucose esters. Os9BGlu31 showed higher activity with oleic acid (18:1) and linoleic acid (18:2) than with stearic acid (18:0) and had both a higher <i>k</i>_cat and a higher <i>K</i>_m for linoleic than oleic acid in the presence of 8 mM 4NPGlc donor. <i>Os9BGlu31</i> knockout mutant rice lines were found to have significantly larger amounts of fatty acid glucose esters than wild-type control lines. Because the transglucosylation reaction is reversible, these data suggest that fatty acid glucose esters act as glucosyl donor substrates for Os9BGlu31 transglucosidase in rice

Interactions between XopN_KXO85 and OsVOZ2 and OsXNP.

<p><b>A</b>. Screening for interactors of XopN_KXO85 in rice using a yeast two-hybrid system. S (strong: pEXP ^TM32/Krev1 + pEXP ^TM22/RalGDS-wt), W (weak: pEXP ^TM32/Krev1 + pEXP ^TM22/RalGDS-m1), and A (absent: pEXP ^TM32/Krev1 + pEXP ^TM22/RalGDS-m2) indicate the strength of each interaction. Three independent and representative colonies are shown for each bait–prey combination. <b>B</b>. <i>In vivo</i> pull-down analysis of XopN_KXO85 and OsVOZ2 (left panel) and XopN_KXO85 and OsXNP (right panel). Total proteins from <i>N</i><i>. benthamiana</i> leaves co-expressing XopN_KXO85-6× His and Flag-OsVOZ2 or XopN_KXO85-6× His and OsXNP-Flag protein were purified by Ni⁺ affinity chromatography followed by Western blotting using anti-His and anti-Flag antibodies. The expected molecular weights were as follows: XopN_KXO85-6× His = 78.7 kDa; Flag-OsVOZ2 = 74.6 kDa; OsXNP-Flag = 40.1 kDa; +, protein expressed; and -, vector control. <b>C</b>. BiFC analysis of XopN_KXO85 -OsVOZ2, XopN_KXO85 -OsXNP, and XopN_KXO85 -OsVOZ1 interactions in <i>N</i><i>. benthamiana</i> leaves. Negative, pDEST-SCYNE(R)^GW + pDEST-SCYCE(R)^GW; positive, pEXP-SCYNE(R)-Cnx7 + pEXP-SCYCE(R)-Cnx6. Bars = 50 µm.</p

Virulence assay in wild-type Dongjin rice and the OsVOZ2 mutant line PFG_3A-07565.

<p><b>A</b>. Schematic representation of the T-DNA insertion in OsVOZ2 T₇ transgenic rice. <i>OsVOZ2</i> consists of four exons (orange boxes) and three introns (line between the orange boxes). The T-DNA was located in the second intron from the translational start site. F and R are the primers used for RT-PCR analysis, which showed the expected size of <i>OsVOZ2</i> in wild-type Dongjin but not in the <i>OsVOZ2</i> mutant rice PFG_3A-07565. Actin1 was used for normalization of the cDNA quantity. <b>B</b>. Virulence assay of the <i>xopN</i>_<i>KXO85</i> mutant in wild-type Dongjin rice and OsVOZ2 mutant rice. W, water; 85, KXO85; N, KXO85 <i>xopN</i>_<i>KXO85</i>::EZ-Tn<i>5</i>; and N^C, KXO85 <i>xopN</i>_<i>KXO85</i>::EZ-Tn<i>5</i> (pML122G2). Photographs were taken 21 days after inoculation. <b>C</b>. Measurement of disease severity in flag leaves of wild-type Dongjin rice (□) and OsVOZ2 mutant rice (▨). W, water; 85, KXO85; N, KXO85 <i>xopN</i>_<i>KXO85</i>::EZ-Tn<i>5</i>; and N^C, KXO85 <i>xopN</i>_<i>KXO85</i>::EZ-Tn<i>5</i> (pML122G2). Lesion lengths were determined 21 days after inoculation. Vertical error bars indicate the standard deviation (SD). The statistical significance was determined using a two-way ANOVA as compared to wild-type Dongjin rice with the post hoc Tukey HSD test (***, P<0.001). <b>D</b>. Growth patterns of the KXO85, <i>xopN</i>_<i>KXO85</i> mutant, and complemented <i>xopN</i>_<i>KXO85</i> mutant in the flag leaves of OsVOZ2 mutant rice (PFG_3A-07565). The data are the average values of three replicates; vertical bars indicate the error ranges (±SD). The bacterial populations were assessed every 3 days after inoculation. Different letters at day 21 indicate significant differences (P<0.05) as determined by a one-way ANOVA (P<0.001) followed by post hoc Tukey HSD analysis.</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

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 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

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

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

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