Increase in Cellulose Accumulation and Improvement of Saccharification by Overexpression of Arabinofuranosidase in Rice

Cellulosic biomass is available for the production of biofuel, with saccharification of the cell wall being a key process. We investigated whether alteration of arabinoxylan, a major hemicellulose in monocots, causes an increase in saccharification efficiency. Arabinoxylans have β-1,4-D-xylopyranosyl backbones and 1,3- or 1,4-α-l-arabinofuranosyl residues linked to O-2 and/or O-3 of xylopyranosyl residues as side chains. Arabinose side chains interrupt the hydrogen bond between arabinoxylan and cellulose and carry an ester-linked feruloyl substituent. Arabinose side chains are the base point for diferuloyl cross-links and lignification. We analyzed rice plants overexpressing arabinofuranosidase (ARAF) to study the role of arabinose residues in the cell wall and their effects on saccharification. Arabinose content in the cell wall of transgenic rice plants overexpressing individual ARAF full-length cDNA (OsARAF1-FOX and OsARAF3-FOX) decreased 25% and 20% compared to the control and the amount of glucose increased by 28.2% and 34.2%, respectively. We studied modifications of cell wall polysaccharides at the cellular level by comparing histochemical cellulose staining patterns and immunolocalization patterns using antibodies raised against α-(1,5)-linked l-Ara (LM6) and β-(1,4)-linked d-Xyl (LM10 and LM11) residues. However, they showed no visible phenotype. Our results suggest that the balance between arabinoxylan and cellulose might maintain the cell wall network. Moreover, ARAF overexpression in rice effectively leads to an increase in cellulose accumulation and saccharification efficiency, which can be used to produce bioethanol

Increase in Cellulose Accumulation and Improvement of Saccharification by Overexpression of Arabinofuranosidase in Rice

<div><p>Cellulosic biomass is available for the production of biofuel, with saccharification of the cell wall being a key process. We investigated whether alteration of arabinoxylan, a major hemicellulose in monocots, causes an increase in saccharification efficiency. Arabinoxylans have β-1,4-D-xylopyranosyl backbones and 1,3- or 1,4-α-l-arabinofuranosyl residues linked to <i>O</i>-2 and/or <i>O</i>-3 of xylopyranosyl residues as side chains. Arabinose side chains interrupt the hydrogen bond between arabinoxylan and cellulose and carry an ester-linked feruloyl substituent. Arabinose side chains are the base point for diferuloyl cross-links and lignification. We analyzed rice plants overexpressing arabinofuranosidase (ARAF) to study the role of arabinose residues in the cell wall and their effects on saccharification. Arabinose content in the cell wall of transgenic rice plants overexpressing individual ARAF full-length cDNA (<i>OsARAF1</i>-FOX and <i>OsARAF3</i>-FOX) decreased 25% and 20% compared to the control and the amount of glucose increased by 28.2% and 34.2%, respectively. We studied modifications of cell wall polysaccharides at the cellular level by comparing histochemical cellulose staining patterns and immunolocalization patterns using antibodies raised against α-(1,5)-linked l-Ara (LM6) and β-(1,4)-linked d-Xyl (LM10 and LM11) residues. However, they showed no visible phenotype. Our results suggest that the balance between arabinoxylan and cellulose might maintain the cell wall network. Moreover, ARAF overexpression in rice effectively leads to an increase in cellulose accumulation and saccharification efficiency, which can be used to produce bioethanol.</p></div

Characteristics of <i>Oryza sativa ARAF1</i> and <i>ARAF3</i>.

<p>(A) Phylogenetic tree of putative ARAFs and xylosidases (members of the GH families 51 and 3) in <i>Oryza sativa</i>, Arabidopsis <i>thaliana</i> (<i>AtARAF1</i>: <i>At3g10740</i>, <i>AtARAF2</i>: <i>At5g26120</i>, <i>XLY1</i>: <i>At5g49360</i>, <i>XYL3</i>: <i>At5g09730</i>) and <i>Hordeum vulgare</i> (<i>AXHAI</i> and <i>AXAHII</i>). Phylogenetic trees were constructed by the neighbor-joining method in ClustalX. (B) The expression patterns of <i>OsARAF1</i> and <i>OsARAF3</i>. RT-PCR analysis was performed using total RNA isolated from different organs of 14-day-old seedlings and 60-day-old mature plants. The numbers in parentheses indicate the numbers of PCR cycles. These experiments were performed at least twice with similar results.</p

Characteristics of the <i>OsARAF1</i>-FOX and <i>OsARAF3</i>-FOX lines.

<p>(A) RT-PCR analysis of transcripts in mature leaves from the control, <i>OsARAF1</i>-FOX and <i>OsARAF3</i>-FOX lines. The levels of <i>OsARAF1</i> and <i>OsARAF3</i> transcripts were higher in each FOX line. 17S Ribosomal RNA-specific primers were used as controls. The numbers in parentheses indicate the numbers of PCR cycles. These experiments were performed at least twice with similar results. (B) Relative ARAF activities in <i>OsARAF1</i>-FOX and <i>OsARAF3</i>-FOX leaves determined using 4-nitrophenyl-α-l-arabinofuranide as a substrate. Activity is expressed as a ratio of the activity in each FOX line to that in the control leaves. Error bars indicate the SD (<i>n</i> = 3). Letters in each panel indicate significant differences at <i>P</i><0.05 (Tukey's test). Black, white and gray columns indicate the control, <i>OsARAF1</i>-FOX and <i>OsARAF3</i>-FOX lines, respectively. (C) Relative xylosidase activities in <i>OsARAF1</i>-FOX and <i>OsARAF3</i>-FOX leaves determined using 4-nitrophenyl-β-d-xylopyranoside as a substrate. Activity is expressed as a ratio of the activity in each FOX line to that in the control leaves. Error bars indicate the SD (<i>n</i> = 3). Letters in each panel indicate significant differences at <i>P</i><0.05 (Tukey's test). Black, white and gray columns indicate the control, <i>OsARAF1</i>-FOX and <i>OsARAF3</i>-FOX lines, respectively.</p

Mechanical properties of leaves of the control, <i>OsARAF1</i>-FOX and <i>OsARAF3</i>-FOX lines.

<p>The break force is expressed per leaf width. The horizontal axis shows the extension length. Black, white and gray symbols indicate the control, <i>OsARAF1</i>-FOX and <i>OsARAF3</i>-FOX, respectively. Error bars indicate the SD (<i>n</i> = 8).</p

Monosaccharide compositions of TFA-insoluble fractions.

<p>The monosaccharide compositions of alcohol-insoluble residues (AIRs) in mature leaves of the control, <i>OsARAF1</i>-FOX and <i>OsARAF3</i>-FOX were determined by GC. The values are means ± SD (<i>n</i> = 12). Different letters within the same column indicate significant differences among means (<i>P</i><0.05) as determined by Tukey's test. N.D. means “not detected”.</p

Cell wall component analysis of the control, <i>OsARAF1</i>-FOX and <i>OsARAF3</i>-FOX.

<p>The amounts of sugar in the acetic/nitric acid-insoluble fraction (A) and the amounts of lignin (B) and phenolic acids released by mild alkaline hydrolysis (C) from AIRs in the control, <i>OsARAF1</i>-FOX and <i>OsARAF3</i>-FOX lines are shown Black, white and gray symbols indicate the control, <i>OsARAF1</i>-FOX and <i>OsARAF3</i>-FOX, respectively. Error bars indicate the SD (<i>n</i> = 4). Different letters in each panel indicate significant differences at <i>P</i><0.05 (Tukey's test).</p

Immunofluorescent labeling of the control and FOX lines with arabinoxylan related antiibodies.

<p>Immunohistochemistry on sections of agar-embedded mature leaves of the control (D, G, J and M), <i>OsARAF1</i>-FOX (E, H, K and N) and <i>OsARAF3</i>-FOX (F, I, L and O) line were observed. The sections were labeled with the monoclonal antibodies LM6 (D–F), LM11 (G–l) and LM10 (J–L). Sections were observed under bright-field illumination (A–C). The micrographs (M–O) show the negative control performed without the first antibody step. All experiments were performed at least twice with similar results. Bars  = 100 µm.</p