Os03g0416200

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Annotated Information

Mutation

• Compared to the cortical Wber cell walls of the wild-type Kinmaze internodes showing uniform thickness with a smooth surface on the cytoplasmic side (Fig. 1c), the sclerenchyma secondary cell wall of the mutant internodes had uneven thickness with a rough surface on the cytoplasmic side, resulting in a mixture of thin layers and irregular architecture (Fig. 1c). Therefore, we deWned this novel mutant as the cell wall architecture1 (cwa1) mutant. The cwa1 mutant plant had similar growth to the wildtypeKinmaze plant (Fig. 1a) and showed a morphologicallynormal tissue structure (Fig. 1b). However, the mutantexhibited a brittle culm phenotype with dramaticallyreduced mechanical strength in its tissue (supplementalFigure S1), implying improper formation of the secondarycell wall.

Fig. 1 Phenotypes of the wild-type and cwa1 mutant plants. a Mature wild-type (left) and cwa1 (right) plants showing almost the same height. b Cross-sections of the wild-type (left) and cwa1 (right) internodes showing no diVerence in morphology. c Under high magniWcation, the cwa1 cortical Wbers (right) show amorphous secondary cell walls. CF cortical Wber, LVB large vascular bundle, Pa fundamental parenchyma, SVB small vascular bundle. Bars 20 cm (a), 50 μm (b), 5 μm (c) .

• To examine at which point the amorphous cell wall of the cwa1 mutant develops, we compared the cortical Wber cell wall in the wild-type and cwa1 internodes at diVerent developmental stages. Just after cessation of internode elongation, slightly thickened secondary cell walls of wildtype cortical Wbers were observed by light microscopy, suggesting that secondary cell wall formation had already started (Fig. 2a); similar patterns were observed in cwa1 internodes at this stage. Then, the wild-type secondary cell wall began to thicken more uniformly during secondary cell wall synthesis (Fig. 2b). At almost the same stage, slight lesser extent than that for the wild-type (Fig. 2b). Notably, abnormal and irregularly thick regions were also observed in the same cwa1 cell wall (Fig. 2b, arrowheads), resulting in the formation of an amorphous secondary cell wall with uneven thickness. Such uneven cell wall structure in the cwa1 internodes was more prominent at the late stage of cell wall formation (Fig. 2c). In longitudinal sections of cwa1 internodes, both irregularly thick and thin regions were also clearly seen within the same sclerenchyma cell (Fig. 2d, e), although no diVerences in length or width were found between the cwa1 and wild-type sclerenchyma cells. These observations indicate marked disruption of secondary cell wall thickening of cwa1 sclerenchyma following normal cell expansion and elongation.

Fig. 2 Light microscopic images of abnormal secondary cell walls of the cwa1 internodes. .

• The cwa1 secondary cell walls were thinner than those of wild-type (Fig. 3a, c). Moreover, the inner surfaces of most cwa1 cortical Wbers had amorphous bulky structures (Fig. 3d–f), unlike the smooth and uniform inner surfaces observed in wild-type cortical Wbers (Fig. 3b). These amorphous bulky structures randomly adhered to the inner surface of the thin cell wall layers, presenting an irregular architecture as observed by light microscopy. The abnormal sclerenchyma cell walls in cwa1 internodes were stained with potassium permanganate, a general electron-dense staining agent for lignin (Hepler et al. 1970; Bland et al. 1971), for further examination by TEM. The cwa1 cortical Wber secondary cell wall (Fig. 4c, d) consisted of layers that were thinner than those of wild-type (Fig. 4a, b). Furthermore, amorphous bulky structures were irregularly deposited in the sclerenchyma cell wall throughout the cwa1 cortical Wbers (Fig. 4c–f); similar patterns were also seen in longitudinal sections (Fig. 4g, h). These abnormal, amorphous deposits occasionally raided the pit regions in the cwa1 cell wall, inhibiting pit formation (Fig. 4d, h, arrowheads). No distinct boundaries occurred between the thin cell wall layer and amorphous bulky structures (Figs. 3, 4). On the other hand, the outermost cell wall layer of cwa1 cortical Wbers appeared to form normally (Fig. 4e, f, h, asterisks) as in wild-type, suggesting no disruption in primary cell wall formation of the cwa1 mutant. Taken together, these amorphous bulky structures are indeed in the secondary cell wall, indicating defective cell wall formation speciWcally in secondary cell walls of cwa1 sclerenchyma cells.

Fig. 3 SEM results of crosssections of cortical Wbers from internodes at maturity. .

Fig. 4 TEM results of cortical Wbers from internodes in mature plants. .

• During secondary cell wall formation, wild-type cortical Wbers contained dense deposits of phenolic components in the middle lamellae and cell corners (Fig. 5a, b, shown as dark gray) and less dense deposits in the cell wall region (Fig. 5a, b, shown as light gray). Its phenolic component deposition was uniform in each region of the sclerenchyma cell walls, which is consistent with the TEM observations. In contrast, dense deposits were found in the middle lamellae and amorphous structures at the surface of the cytoplasm side of the cwa1 sclerenchyma cell wall (Fig. 5c, d), which corresponded to the structures observed by TEM. These dense deposits continued to expand throughout the cortical cell wall at the late stage of secondary cell wall formation (Fig. 5e, f), indicating that uneven deposition of phenolic components was more prominent at the later stage. In longitudinal sections, disruption of phenolic component deposition was also observed as dense deposits in the middle lamella and cell wall layers as well as amorphous structures on the cytoplasm surface (Fig. 5g, h). Therefore, mutation of the CWA1 gene appears to aVect normal deposition and assembly of phenolic components in the cortical Wber secondary cell walls in the internodes.

Fig. 5 UV micrographs of cross-sections of cortical Wbers from internodes. .

• The researchers analyzed the alteration of the sclerenchyma cell wall composition in the internodes at maturity (Table 1). Taken together, mutation of the CWA1 gene results in reduced cellulose and increased hemicellulose and phenolic components in the internodes.

Table 1 Comparison of cell wall composition between the wild-type and cwa1 internodes. .

Labs working on this gene

• Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei 184-8588, Japan
• College of Bioresource Science, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan
• Forestry Research Institute, Oji Paper Co. Ltd, 24-9 Nobono-cho, Kameyama, Mie 519-0212, Japan
• Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu 183-8538, Japan
• Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
• Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan