MOESM2 of Development of a new heat tolerance assay system for rice spikelet sterility

Additional file 2: Fig. S2. The high temperature-induced spikelet sterility assay system before improvement in this study. A. The system had a fixed distance from lamp to panicle. Ten plants were planted in the same pot. B. Spikelet fertility of eight rice cultivars [19] was examined at 36 °C/30 °C for 3 days. Values are the mean ± SE of 3–5 plants

MOESM1 of Development of a new heat tolerance assay system for rice spikelet sterility

Additional file 1: Fig. S1. Time course of changes in set air temperature (T a : solid line), panicle temperatures 1 (T P1: closed circle) and 2 (T P2: opened circle), and water temperature (T W : closed triangle) in the artificial paddy field for 2 days recorded after inserting the fine thermocouples in two spikelets. Panicle temperatures were measured once the flowers were closed, as T P1 and T P2 started at 1 h after insertion of thermocouples. ‘+TC’ and ‘-TC’ indicate the time at insertion and removal of the sensors, respectively. Black bars indicate night. Note that the temperature of water in the paddy displayed diurnal changes according to the changes in air temperature and half of the lamps were turned off for 1 h at the beginning and end of 13 h of daytime, where T P1 and T P2 started to decline prior to the decline in T a

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 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

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

Saccharification of the control <i>OsARAF1</i>-FOX and <i>OsARAF3</i>-FOX lines in dry leaves.

<p>Time-dependent saccharification (A) and saccharification efficiency after 24 h (B) were measured. Error bars indicate the SD (<i>n</i> = 3). Black, white and gray columns indicate the control, <i>OsARAF1</i>-FOX and <i>OsARAF3</i>-FOX, respectively. Different letters in the same column indicate significant differences at <i>P</i><0.05 (Tukey's test).</p

Calcofluor white staining for cellulose of mature leaves of the control and FOX lines.

<p>The section of agar-embedded mature leaves of the control (D), <i>OsARAF1</i>-FOX (E) and <i>OsARAF3</i>-FOX (F). Sections were observed under bright-field illumination (A–C). The micrographs (G–I) show negative controls performed without calcofluor white. Bars  = 500 µm. All experiments were performed at least twice with similar results.</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

The phenotypes of the control (A), <i>OsARAF1</i>-FOX (B) and <i>OsARAF3</i>-FOX (C) in 60-day-old plant after sowing.

<p>The growth levels of FOX lines were very similar to those of the control, and all FOX lines had normal fertility. Bar  = 10 cm.</p