Skip to main content
NCBI home page
Plant and Cell Physiology logoLink to Plant and Cell Physiology
. 2025 Aug 21;66(10):1444–1453. doi: 10.1093/pcp/pcaf096

The rice BRITTLE CULM 4 gene encodes a membrane protein affecting cellulose synthesis in the secondary cell wall

Masatoshi Yamaguchi 1, Ami Sato 2, Daisuke Takahashi 3, Kazuhisa Mori 4, Ryota Fujimoto 5, Atsuko Miyagi 6,7, Eriko Sato 8, Toshiki Ishikawa 9, Ryosuke Sano 10, Tetsuya Kurata 11,12, Shiro Suzuki 13, Yasuko Kaneko 14,15, Maki Kawai-Yamada 16, Toshihisa Kotake 17,18,✉
PMCID: PMC12540249  PMID: 40838742

Abstract

The formation of secondary cell walls, which provide mechanical strength to the plant body, depends on numerous factors. Studies on rice brittle culm (bc) mutants allow us to identify these factors and gain insights into the mechanisms of secondary cell wall formation. Rice bc4 is a recessive bc mutant with fragile culms and leaves, similar to other bc mutants. We found that the bc4 mutant exhibited reduced cellulose content in the culm cell walls compared to the japonica cultivar Taichung 65 and the indica cultivar Kasalath, while hemicellulose content remained unchanged. Transmission electron microscopy revealed reduced cell wall thickness in the sclerenchyma cells of the bc4 culm, indicating that BC4 contributes to normal cellulose synthesis or deposition in secondary cell walls. Positional cloning and subsequent genome sequencing revealed that the BC4 gene encodes a four α-helical transmembrane protein with 205 amino acids, and that the bc4 mutation results in a premature termination codon in this gene. Four bc4 mutants generated from the japonica cultivar Nipponbare, using genome editing with the CRISPR/Cas9 system, exhibited reduced cellulose content along with bc phenotypes. Gene clustering analysis based on expression patterns and metabolomic analysis suggested that BC4 functions independently from secondary cell wall cellulose synthase catalytic subunits and COBRA-like protein. These results suggest that the BC4 protein is a newly identified factor involved in cellulose synthesis or deposition in the secondary cell walls of rice.

Keywords: brittle culm, cellulose synthesis, genome editing, membrane protein, rice (Oryza sativa), secondary cell wall

Introduction

Secondary cell walls, generally rich in cellulose, xylan, and lignin, are deposited inside primary cell walls after cell growth and furnish the plant body with mechanical strength. As the synthesis and deposition of these cell wall components are developmentally controlled and require the interplay of many factors cooperating in various roles, the mechanism for secondary cell wall formation is still elusive.

To date, many genes necessary for secondary cell wall formation have been identified in studies on Arabidopsis (Arabidopsis thaliana) cell wall mutants. Arabidopsis irregular xylem (irx) mutants show collapsed morphology in xylem tissues that normally have thickened cell walls. The irx1, irx3, and irx5 mutants appear to have defects in secondary cell wall-specific cellulose synthase catalytic subunits (CesAs) (Turner and Somerville 1997, Taylor et al. 1999, 2000, 2003). Glycosyltransferase genes involved in the synthesis of secondary cell wall xylan have also been cloned from Arabidopsis irx7, irx8, irx9, irx10, and irx14 mutants (Brown et al. 2007, Peña et al. 2007, Persson et al. 2007, Wu et al. 2009). On the other hand, a series of studies on Arabidopsis fragile fiber (fra) mutants with reduced mechanical strength of the inflorescence stem has identified other factors including katanin, kinesin-like protein, and phosphatidylinositol phosphatase, together with CesAs, as factors necessary for normal secondary cell wall formation (Burk et al. 2001, Zhong et al. 2002, 2003, 2004, Zhang et al. 2009).

Along with mutant analysis, candidate factors important for cellulose synthesis and deposition in primary and secondary cell walls have been identified through co-expression analysis (Brown et al. 2005, Persson et al. 2005). Companions of cellulose synthase proteins (At5g42860 and At1g45688), highly co-expressed with primary cell wall CesAs, have been shown to interact with CesAs and contribute to microtubule organization and localization of the cellulose synthase complex (CSC) on the plasma membrane in Arabidopsis (Endler et al. 2015). TONNEAU1 (At2g45900), highly co-expressed with a secondary cell wall CesA gene, CesA7/IRX5, was later shown to be a microtubule-associated protein that localizes to cortical microtubules in Arabidopsis (Drevensek et al. 2012). On the other hand, a membrane protein (At4g27435, AtUNKA) belonging to the domain with unknown function (DUF) 1218 family has not yet been characterized, although this gene is also co-expressed with secondary cell wall-specific CesA genes (Ubeda-Tomas et al. 2007). Other DUF1218 proteins, MODIFYING WALL LIGNIN 1 (MWL1, At1g31720) and MWL2 (At4g19370), are presumed to have roles in secondary cell wall formation, based on the observation that an Arabidopsis mwl1 mwl2 double mutant exhibited slightly reduced lignin content in the inflorescence stem (Mewalal et al. 2016).

The brittle culm (bc) mutants of Poaceae plants exhibit reduced mechanical strength, especially in culms and leaves (Kokubo et al. 1989, 1991, Ching et al. 2006, Sindhu et al. 2007). Because many mutants in this class are morphologically normal, they have been expected to have defects in the secondary cell walls rather than the primary cell walls. The brittle stalk (bs), culm easily fragile (cef), and fragile culm (fc) mutants of Poaceae plants also show bc phenotypes, and it has been demonstrated that they have defects in secondary cell walls as well (Li et al. 2003, Ching et al. 2006, Ye et al. 2015, Zhang et al. 2020). At least ten bc, two cef, and three fc mutants have been characterized so far in rice (Li et al. 2003, Yan et al. 2007, Zhou et al. 2009, Hirano et al. 2010, Kotake et al. 2011, Zhang et al. 2012, Wu et al. 2012, Sakai et al. 2013, Ye et al. 2015, Zhang et al. 2020, Ma et al. 2021, Li et al. 2022, Jiang et al. 2022, Ruan et al. 2022, Dang et al. 2023) (Supplementary Table S1). Among these mutants, a decrease in the cellulose content is commonly observed, suggesting that cellulose synthesis and deposition in the secondary cell wall are central events for the mechanical strength of the plant body. This is supported by the fact that secondary cell wall-specific CesAs have been identified as causal genes for several bc mutants. On the other hand, the proper formation of secondary cell walls requires many factors other than CesAs. Interestingly, the factors identified through the analysis of rice mutants are not necessarily the same as those in Arabidopsis. Rice bc mutants thus remain interesting as materials for gaining new insights into the mechanisms of secondary cell wall formation.

Rice bc4 is one of the bc mutants yet to be studied (Khush et al. 1984, Khush 1995, Librojo and Khush 1986). In the present study, we characterized bc4 mutants through cell wall fractionation followed by sugar composition analysis and transmission electron microscopy (TEM) observation. The BC4 gene has been mapped to chromosome 6 on the classical linkage map. Through positional cloning and genome sequencing, we identified a premature stop mutation in a gene encoding a membrane protein with four α-helices. Based on the clustering of expression patterns, we suggest that the BC4 gene contributes to normal cellulose synthesis and/or deposition through an unknown function, distinct from those of the CesA and COBRA-like genes.

Results

Reduced cellulose content in the bc4 mutant

The rice bc4 mutant has been known for >40 years, but has remained uncharacterized (Khush et al. 1984, Librojo and Khush 1986). The bc4 mutant exhibited a weak dwarf phenotype compared to other indica cultivars such as Kasalath (Kas) (data not shown). Similar to other bc mutants, the plant body of the bc4 mutant lacks mechanical strength, which is presumed to be caused by defects in the secondary cell walls (Fig. 1A). However, the bc phenotypes of bc4 mutants are sometimes less pronounced compared to other bc mutants such as bc1 and Bc6, suggesting that the functions of BC4 are different from those of COBRA-like and CesA proteins, which are impaired in rice bc1 and Bc6 mutants, respectively (Li et al. 2003, Sato et al. 2010, Kotake et al. 2011).

Figure 1.

Figure 1

Open in a new tab

The bc phenotype and reduced cellulose content. (A) the bc phenotype observed in the uppermost culm. (B) Amount of cell wall fractions. Sugar amounts of cell wall fractions extracted from culms were measured two weeks after heading. The proportions of fractions in total cell wall sugar are shown in Supplementary Fig. S1. (C) Sugar composition of alkali fraction. The sugar composition of the alkali fraction was determined by HPAEC-PAD. The left and central bars, and the right bars indicate data for WT T65, Kas, and the bc4 mutant, respectively. Data are mean values with SD (n = 4 biological replicates). The asterisk indicates significant difference from Kas plants (Student’s t test, **, P < 0.01).

To address the changes in secondary cell walls in the bc4 mutant, we first fractionated cell wall polysaccharides of the culm into a hot water (HW) fraction, an ethylenediaminetetraacetic acid (EDTA) fraction containing pectin, an alkali fraction mainly containing arabinoxylan, and a cellulose fraction by sequential extraction and compared the content of these fractions with an indica cultivar, Kas, and a japonica cultivar, Taichung 65 (T65). We used these two cultivars for comparison because we could not obtain the normal line with the same genetic background as bc4. Consistent with our previous studies on rice cell walls (Aohara et al. 2009, Hirano et al. 2010, Kotake et al. 2011), the culms mainly consisted of alkali and cellulose fractions. The bc4 mutant had a significantly lower cellulose fraction content than T65 and Kas (Fig. 1B, Supplementary Fig. S1). On the other hand, the content and sugar composition of the alkali fraction rich in arabinoxylan were comparable to those in normal cultivars (Fig. 1C), indicating that only cellulose content was changed in the culm of the bc4 mutant.

Tissue morphology and cell wall thickness of sclerenchyma cells

In Arabidopsis, defects in secondary cell walls result in altered tissue organization, such as collapsed xylem (Turner and Somerville 1997, Taylor et al. 2000, 2003). To address the influence of the bc4 mutation on tissue morphology, microscopic observation of transverse sections of bc4 culms stained with Safranin O and Fast Green FCF was conducted. The bc4 mutant did not show any collapsed xylem phenotype at all (Fig. 2A-C). However, tissue staining with Safranin O showed weaker red coloration in the bc4 mutant compared to T65 and Kas. Since Safranin O is known to stain lignified cell walls (Bond et al. 2008), transverse sections were treated with phloroglucinol to selectively stain lignin-rich cell walls. However, we did not observe a clear difference in bc4 from Kas and T65 in phloroglucinol staining (Supplementary Fig. S2A-C). Quantitative analysis using the Klason method did not reveal a significant difference in lignin content (Kirk and Obst 1988) (Supplementary Fig. S2D).

Figure 2.

Figure 2

Open in a new tab

Tissue morphology and cell walls in bc4 culms. Transverse sections of uppermost culms of T65 (A), Kas (B), and bc4 (C) were stained with safranin O and fast Green FCF. Sclerenchyma cell walls of T65 (D, G) and Kas (E, H) were compared with the bc4 culm (F, I) cell wall by TEM observation. The boxed areas in D-F are magnified in G-I. Cell wall thickness measurements are in Supplementary Fig. S3.

To address the changes in cell walls in the bc4 mutant, TEM of culm transverse sections was also conducted. Although we did not observe obvious changes in tissue morphology, the cell wall thickness of sclerenchyma cells of the bc4 culm decreased compared with T65 and Kas (Fig. 2D-I, Supplementary Fig. S3). These results suggest that the bc4 mutant is impaired in cellulose synthesis or deposition in the culm.

Arabinoxylan deposition in the bc4 mutant was also compared by immunostaining with LM10 and LM28 specific to xylan with another normal japonica cultivar, Nipponbare (NP). Xylan deposition in sclerenchyma and vascular cells was similar in the culms of the bc4 mutant and NP (Fig. 3).

Figure 3.

Figure 3

Open in a new tab

Accumulation of xylan in bc4 culms. Xylan in the cross sections from the top internode of NP (A, C) and bc4 mutant (B, D) was detected and compared with LM10 specific to heteroxylan and LM28 specific to glucuronoxylan. For comparison, AGP was also detected with LM2 specific to D-glucuronosyl residues of AGP in the NP (E) and the bc4 mutant (F). Merged images of Alexa Fluor 488 and Calcofluor white are shown. The bars indicate 50 μm.

Cloning of the bc4 gene

The bc4 locus was previously mapped to chromosome 3 in the rice classical linkage map through trisomic tests (Librojo and Khush 1986), but the locus has been changed to chromosome 6 (https://shigen.nig.ac.jp/rice/oryzabase/marker/mapCirn/6). We first performed rough mapping of the bc4 gene using the F2 population generated by crossing the bc4 mutant with an indica cultivar, co39. We confirmed that the bc4 gene locates between the 2842-Indel and 3510-Indel DNA markers in chromosome 6 (Fig. 4A). In the following fine mapping with six DNA markers designed between these two markers, the bc4 locus was narrowed down to a 66 kb region corresponding to the bacterial artificial chromosome (BAC) clone AP001389 (P1-derived artificial chromosome, P0541H01) in chromosome 6 (Fig. 4A).

Figure 4.

Figure 4

Open in a new tab

Cloning of BC4. (A) Positional cloning of the BC4 gene. The bc4 locus in chromosome 6 was confirmed by rough mapping using an F2 population generated by crossing bc4 mutant and co39, and then with 620 F2 plants delimited to a 66 kb region in a BAC clone, AP001389 (P0541H01). The numbers of recombinants among the F2 individuals analyzed are shown. The primers for DNA markers are listed in Supplementary Table S2. (B) the bc4 mutation in ORF Os06g0114700. Genome sequencing identified a mutation in Os06g0114700 resulting in a premature stop codon, deleting a C-terminal region of 87 amino acids (underlined).

To identify the mutation that causes the bc phenotype, we obtained the whole genome sequence of the bc4 mutant by next-generation sequencing. By comparison of the genome sequence in the delimited 66 kb region between the bc4 mutant and NP, we found one point mutation from C to T, affecting molecular function, in an open reading frame, Os06g0114700 (Fig. 4B). Other than this mutation, there was no mutation that changed amino acid sequences in this region. This gene encoded a four α-helical transmembrane protein with 205 amino acids belonging to the DUF1218 family (Krogh et al. 2001) (Supplementary Fig. S4), and the mutation resulted in a premature stop codon deleting the C-terminal region of 87 amino acids (Fig. 4B). In Arabidopsis, a DUF 1218 gene (At4g27435) is co-expressed with secondary cell wall CesA genes and two DUF1218 genes (At1g31720 and At4g19370), which have been shown to be involved in the lignin deposition by analysis of the double mutant (Brown et al. 2005, Persson et al. 2005, Mewalal et al. 2016), although the amino acid sequences of these Arabidopsis proteins were not highly related to Os06g0114700. This is the first time that a loss-of-function mutation of DUF1218 has been characterized in Poaceae plants.

bc4 mutants generated by genome editing

In order to confirm that the bc4 mutation is a loss-of-function mutation in Os06g0114700, other alleles of bc4 mutants were generated from the japonica cultivar NP by genome editing using CRISPR-Cas9 technology (Mikami et al. 2015) (Fig. 5A). We obtained four independent mutant lines, c#1 to 4, with different mutations. In the c#1, 2, and 4 lines, base-insertion resulted in a frame-shift and a premature stop codon, while in the c#3 line, the targeted region was replaced with an unknown sequence including a stop codon in the frame. These genome-edited (GE) lines all encoded proteins much shorter than Os06g0114700 (Fig. 5B).

Figure 5.

Figure 5

Open in a new tab

The bc4 mutants generated by genome editing. (A) Mutations introduced in the mutant lines c#1–4. The genomic sequences of c#1–4 were aligned and compared with the NP sequence. The nucleotides of c#1–4 differing from NP are highlighted in black. The premature stop codons are underlined. (B) Schematic diagram of gene products from the bc4 mutant and lines c#1–4. The region identical to NP is shown in black, and that changed from NP due to the frame shift or the insertion of unknown fragments is shown in gray.

Consistent with the phenotype of bc4 mutant, GE lines c#1 to 4 had bc phenotypes and reduced cellulose content in cell walls (Fig. 6). In addition, semi-dwarf phenotypes were also observed in c#3 and 4 (Supplementary Fig. S5). These results confirmed that the gene encoding a DUF1218 protein is the causal gene of the rice bc4 mutant.

Figure 6.

Figure 6

Open in a new tab

Cell wall properties of GE lines. (A) Growth of the GE lines. From left to right, 52-day-old specimens of NP, bc4, c#3, and c#4. Bar = 10 cm. The fragile phenotype of GE line c#3 is shown in the inset (bar = 1 cm). (B) Cell wall fractions of lines c#1–4 compared to bc4 and NP. As in the results shown in Fig. 1, the cell walls of line c#1–4 culms were fractionated and quantified. Note that the proportions of fractions are shown here because fresh weight differed in biological replicates. Data are mean values with SD (n = 3 biological replicates). GE, genome-edited line. The asterisk indicates a significant difference from the NP plant (Student’s t test, *, P < 0.05, **, P < 0.01).

Discussion

Reduced cellulose content in the bc4 mutant

Cellulose synthesis and deposition are essential processes that provide mechanical strength to the rice plant body. Consistent with other bc mutants, the bc4 mutant showed a significantly lower cellulose content in the culm compared to T65 and Kas, a finding also confirmed in GE lines of NP (Fig. 6; Supplementary Fig. S1). However, the effects on cell wall properties aside from cellulose are not identical across rice bc mutants. For instance, in the bc3 mutant, the alkali fraction, which mainly contains arabinoxylan, remained unchanged (Hirano et al. 2010), whereas in the Bc6 mutant, this fraction significantly increased, likely as a compensatory response (Kotake et al. 2011). Although we could not perform a detailed cell wall analysis on the GE lines of NP, bc4 did not show a significant difference in the sugar composition of the alkali fraction or in lignin content compared with the japonica cultivar T65, which likely shares a highly related genetic background with NP. (Ichida et al. 2023, Guo et al. 2025). These results suggest that the impact of the bc4 mutation on other cell wall components differs from that of the Bc6 mutation in the CesA9 gene (Fig. 1).

Functions of BC4 in secondary cell wall formation

To gain insights into BC4 function, we performed hierarchical clustering analysis based on expression patterns with other bc genes (Brown et al. 2005, Persson et al. 2005, Miki et al. 2019). The bc genes clustered into at least three distinct groups. One group included cellulose synthesis-related genes, such as BC1 (COBRA-like 4), BC6 (CesA9), BC7/BC19 (CesA4), and CEF1 (Myb103-like), while another clade included genes involved in xylan synthesis, represented by FC18 (UDP-Xyl synthase) and FC19 (IRX10) (Li et al. 2003, Roudier et al. 2005, Liu et al. 2013, Ruan et al. 2022, Dang et al. 2023) (Supplementary Fig. S6). BC4 clustered within a membrane trafficking/cytoskeleton-related group, alongside BC3 (dynamin-related protein DRP2B), BC12 (kinesin-4), and CEF3 (membrane-trafficking protein SCD2). The co-expressed genes were also confirmed in the Rice FREND database (https://ricefrend.dna.affrc.go.jp/) (Sato et al. 2013). The secondary cell wall CesA genes were not included in a list of genes co-expressed with BC4, whereas BC6 is co-expressed with BC1 (COBRA-like 4) and OsCesA7, which encodes another secondary cell wall CesA (Supplementary Figs S7 and S8). These findings suggest that BC4 may contribute to secondary cell wall formation in conjunction with membrane trafficking genes, such as BC3, BC12, and CEF3 (Hirano et al. 2010, Zhang et al. 2012, Jiang et al. 2022).

Since secondary cell wall formation requires large amounts of nucleotide sugars such as UDP-D-glucose (UDP-Glc) and UDP-D-xylose (UDP-Xyl), disruptions in cell wall formation in bc mutants may be related to altered sugar metabolism. In our previous study, we compared the metabolites in the bc4 mutant with those in other bc mutants (Miyagi et al. 2022); however, due to differences in genetic backgrounds, clear distinctions in the bc4 mutant from the japonica cultivar T65 or indica cultivar IR68 were not observed. In this study, we identified metabolites that significantly increased or decreased in the culms of c#2 compared to NP (Supplementary Fig. S9). Notably, levels of UDP-D-glucuronic acid (UDP-GlcA)/UDP-D-galacturonic acid (UDP-GalA) were significantly elevated. Although not statistically significant, UDP-Glc/UDP-D-galactose (UDP-Gal) also tended to increase in c#2. It is probable that impaired cellulose synthesis increased the levels of UDP-Glc and UDP-GlcA, which is formed from UDP-Glc in the UDP-sugar pathway (Kotake et al. 2016). On the other hand, these shifts in UDP-sugars contrast with patterns in the bc1 and the Bc6 mutants, suggesting that the BC4 gene is involved in an unknown process distinct from cellulose synthesis (Miyagi et al. 2022).

Relationship of the BC4 protein with other DUF1218 proteins

Amino acid sequences with high similarity to BC4 are found in various land plants, including lycophytes and mosses, suggesting that this type of protein is widely distributed among plants. However, based on phylogenetic analysis (Supplementary Fig. S10), closely related homologs of BC4 are not highly conserved in land plants. In addition, in Arabidopsis, no cell wall defects have been reported in the loss-of-function mutant of BC4’s closest homolog (At5G17210). These facts suggest that DUF1218 proteins are redundant or not important for secondary cell wall formation in Arabidopsis.

The 3D structure of the plant DUF1218 protein has not yet been characterized experimentally. The 3D structure predictions generated using AlphaFold2 (Google Colab) and the Phyre2 server suggest that the BC4 protein shares structural similarity with mammalian claudin proteins, which are also transmembrane proteins composed of four α-helices. (Supplementary Fig. S3B) (Kelley et al. 2015, Jumper et al. 2021, Mirdita et al. 2022). Mammalian claudin proteins are known to function in tight junctions, acting as impermeable barriers for fluid on the plasma membrane (Nguyen et al. 2024). However, because plants have cell walls, it is unlikely that BC4 would seal intercellular spaces in the same manner. In addition, the different topological arrangement (clockwise versus anticlockwise) of α-helices and the loss of a relatively large domain between first and second α-helices suggest that BC4 protein functions differently from mammalian claudin.

The intracellular localization of the BC4 protein was predicted using the WoLF PSORT program, with comparisons to other proteins that share features with BC4 (Horton et al. 2007). Among the 14 proteins listed for BC4, nine are predicted to localize to the vacuole. By contrast, the closest homolog of BC4 in Arabidopsis (At5G17210) is a plasma membrane protein. While it remains possible that BC4 has a specific role in cellulose synthesis, it is more likely that BC4 serves a general function, such as membrane protein transport or the regulation of plasma membrane properties. Further studies are needed to clarify the molecular functions of BC4.

Materials and Methods

Plant materials

Seeds of bc4 mutant (RGS number 293) and co39 were provided by the International Rice Research Institute (IRRI, Los Baños, Laguna, Philippines) (Librojo and Khush 1986, Khush 1995). Seeds of T65 and Kas were obtained from the National Genetic Institute (Mishima, Shizuoka, Japan) and Genebank of the National Institute of Agrobiological Sciences (NIAS, Tsukuba, Ibaraki, Japan), respectively. T65 and Kas were mainly used to represent wild-type (WT) controls. Rice plants were grown under field conditions at NIAS or indoors at 28°C under a 12 h light/12 h dark photoperiod condition.

Analysis of cell wall polysaccharides

Cell wall polysaccharides were fractionated into HW, EDTA, alkali, and cellulose fractions as described (Kotake et al. 2011). Plant tissues were first crushed to powder using a mortar and pestle in liquid nitrogen and then homogenized in water. The homogenates were washed twice with water, suspended in 80% (v/v) ethanol, and heated at 100°C for 15 min, and then treated with 100 units of α-amylase from porcine pancreas (Sigma-Aldrich, St. Louis MO, USA) in 50 mM sodium 3-morpholinopropanesulfonic acid buffer (pH 6.5) at 37°C for 4 h. Released starch was removed by centrifugation at 1500 g. Cell wall polysaccharides were sequentially extracted at 100°C for 10 min with water thrice, 50 mM EDTA (pH 6.8) thrice, and 17.5% (w/v) NaOH containing 0.04% (w/v) NaBH4 thrice. The residual precipitate was washed with water, ethanol, and diethyl ether, and collected as the cellulose fraction. Hemicellulose was neutralized with acetic acid, dialyzed against water at 4°C for 1 day, and lyophilized. The cellulose fraction was hydrolyzed with 72% (v/v) sulfuric acid at 25°C for 1 h, followed by 8% (v/v) sulfuric acid at 100°C for 4 h (Sawake et al. 2015). The sugar content in each fraction was determined by the phenol-sulfuric acid method (Dubois et al. 1956) using Glc as the standard.

Determination of sugar composition

The dialyzed alkali fraction was hydrolyzed with 72% (v/v) sulfuric acid (0.2 ml) at 25°C and then 8% (v/v) sulfuric acid at 100°C as described above. The hydrolysate was neutralized with barium carbonate and desalted with Dowex 50 W (H+) resin. The content of each monosaccharide was determined by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using a Dionex DX-500 liquid chromatograph equipped with a CarboPac PA-1 column and a pulsed amperometric detector (Thermo Fisher Scientific, Waltham, MA, USA) as described previously (Ishikawa et al. 2000, Sawake et al. 2015). In this study, the proportion of L-arabinose (Ara), L-fucose (Fuc), Gal, GalA, Glc, GlcA, D-mannose (Man), L-rhamnose (Rha), and Xyl was determined.

Histology

For the observation of cross sections, the uppermost internode was used. Fixation, dehydration, embedding, and sectioning were performed as described (Kotake et al. 2011). The sections (thickness, 20 μm) were stained with 0.1% (w/v) Safranin O (Nacalai Tesque, Kyoto, Japan) for 24 h, washed in a series of ethanol/water solutions (50%–95% ethanol, v/v), stained with 0.05% (w/v) Fast Green FCF (Wako, Tokyo, Japan) for 45 s, and washed with ethanol. The sections were observed under a light microscope (Eclipse E400, Nikon, Tokyo, Japan).

The xylan and arabinogalactan-protein (AGP) in the culms of bc4 mutant and NP plants grown indoors were observed. Sections excised from the uppermost internode were fixed, dehydrated through a graded ethanol series, and embedded in Technovit 7100 (Nisshin EM, Tokyo, Japan) using a graded series of ethanol and Technovit 7100 mixtures. Then, the sections were solidified with Technovit hardener (Nisshin EM) at 60°C for 12 h. Sections were sliced to a thickness of 5 μm using a microtome (RM2125RT; Leica, Tokyo, Japan) equipped with a disposable steel blade (C35; Feather, Osaka, Japan).

The acetyl groups of glucomannan and xylan were removed in advance by treatment with 50 mM sodium hydroxide at 25°C for 30 min. The sections were washed with phosphate-buffered saline (PBS) containing 137 mM NaCl, 10 mM disodium hydrogenphosphate, 2.7 mM KCl, and 1.8 mM potassium dihydrogenphosphate (pH 7.4) and blocked in PBS containing 0.3% (w/v) blocking milk (Sigma-Aldrich, Tokyo, Japan) for 30 min. Then, the sections were incubated for 1.5 h with antibodies against AGP (LM2, Kerafast, Shirley, MA; dilution 1:5) or xylan (LM10, Kerafast; dilution 1:5; LM28, Kerafast; dilution 1:5) in PBS with blocking milk. After washing three times with PBS, the sections were incubated for 1 h with Alexa Fluor 488-conjugated anti-mouse IgM (Abcam, Cambridge, UK; dilution 1:100) for LM2 and LM28 or Alexa Fluor 488-conjugated anti-mouse IgG (Abcam; dilution 1:100) for LM10 in PBS with blocking milk. After washing three times with PBS, the cross sections were also stained with 0.025%(w/v) Calcofluor White in PBS for 5 min. The cross sections were imaged with a microscope (FV1000D, Olympus, Tokyo, Japan) at 405 nm for Calcofluor White and at 488 nm for Alexa Fluor 488.

Transmission electron microscopy

Observation of sclerenchyma cells in culms by TEM was performed as described previously (Hirano et al. 2010, Kotake et al. 2011). Briefly, pieces excised from uppermost culms (1–2 mm) were fixed in 2% (v/v) glutaraldehyde in 50 mM potassium phosphate buffer (pH 7.0) at room temperature for 2 h and then at 4°C overnight. After rinsing with the same buffer, tissues were postfixed in 2% (w/v) OsO4 in the buffer. After dehydration in a graded series of acetone, the tissues were embedded in Spurr’s resin, which was polymerized by incubation at 70°C overnight. Ultra-thin sections of 90–100 nm thickness were cut with a diamond knife and stained with 2% (w/v) uranyl acetate for 15 min, followed by lead citrate for 2 min. The sections were observed with a Hitachi H-7500 electron microscope (Hitachi Science Systems, Ibaraki, Japan) at an accelerating voltage of 100 kV.

Positional cloning

For positional cloning of the BC4 gene, bc4 was crossed with the indica variety co39. The resulting F2 population was analyzed for bc phenotype and genotype. For the rough mapping of BC4, the genotypes of 43 plants were analyzed with Indel DNA markers, 2842-Indel and 3510-Indel (Supplementary Table S2). Then fine-mapping was performed by analyzing the genotypes of 620 plants using Indel, cleaved amplified polymorphic sequence, and SNP DNA markers, 1552-Indel, 1389-SalI, 1389-SNP, 2838-SNP, 2542-SNP, and 0399-HindIII (Supplementary Table S2). These DNA markers were generated by direct sequencing of 1–2 kb PCR DNA fragments amplified by genomic PCR of bc4 mutant and co39.

Genome sequencing

Genome DNA of the bc4 mutant was extracted with a FavorPrep Plant Genome DNA Extraction Mini Kit (Favorgen, https://www.favorgen.com/en/product.php). The library for genome sequencing was prepared according to Uchida et al. (2011). The library was sequenced by Illumina Genome Analyzer IIx (Illumina, https://illumina.com). Obtained single end reads (ca. 140 million, 75 bp) were mapped onto Os-Nipponbare-Reference-IRGSP-1.0 (https://rapdb.dna.affrc.go.jp), and effective SNPs on gene function were analyzed by using Strand NGS (Strand Life Sciences, USA) according to Yamauchi et al. (2016).

Generation of bc4 mutants by genome editing

To obtain the double-strand target sequence, oligo DNA fragments (for c#1 and c#2; 5′- GTTGGGCGGAGGGCACACGCGTCA-3′ and 5’-AAACTGACGCGTGTGCCCTCCGCC-3′, and for c#3 and c#4; 5’-GTTGCCAGTCTATTATAAATACAG-3′ and 5’-AAACCTGTATTTATAATAGACTGG-3′) were incubated at 95°C for 5 min, and then at room temperature for 20 min to be annealed. The double-strand target sequence was ligated to pU6gRNA-oligo (Mikami et al. 2015) linearized with BbsI. The obtained plasmids, which contained the guide RNA driven by OsU6 promoter, were digested with PacI and AscI, and ligated with PacI and AscI sites of pZH_OsU3gYSA_MMCas9 vector (Mikami et al. 2015). The resultant vectors were used for generation of transgenic rice (NP) plants according to Toki et al. (2006). The resulting bc4 mutants c#1–4 were grown indoors.

Metabolomic analysis

The internodes (2 weeks after heading) of bc4 mutant c#4 and NP grown indoors were used. The metabolomic analysis was performed as described previously (Miyagi et al. 2010, 2019, 2022). Briefly, metabolites were extracted with 50% methanol containing 50 μM piperazine-1,4-bis(2-ethanesulfonic acid) and 50 μM methionine sulfone as internal standards and measured by capillary electrophoresis-triple quadrupole mass spectrometry (CE, 7100; MS, G6420A; Agilent Technologies, Santa Clara, CA, USA) or liquid chromatography-triple quadrupole-mass spectrometry (LC-MS-8030, Shimadzu GLC, Kyoto, Japan) with multi-reaction monitoring.

Supplementary Material

pcp-2024-e-00271-File009_pcaf096
pcp-2024-e-00271-file009_pcaf096.pdf (1.2MB, pdf)
pcp-2024-e-00271-File008_pcaf096
pcp-2024-e-00271-file008_pcaf096.pdf (319KB, pdf)

Acknowledgments

We are grateful to Dr. Gurdev Singh Khush of IRRI for providing the bc4 mutant (RGS number 293) and co39, and to Prof. Seiichi Toki and Dr. Masaki Endo of National Agriculture and Food Research Organization for providing pU6gRNA-oligo and pZH_OsU3gYSA_MMCas9 vectors. We are also grateful to Prof. Emeritus Toshiaki Umezawa of Kyoto University of Kyoto University, Dr. Shinji Kawasaki of the National Institute of Agrobiological Sciences, and Dr. Koki Yoshida for their technical advice and valuable discussion.

Contributor Information

Masatoshi Yamaguchi, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.

Ami Sato, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.

Daisuke Takahashi, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.

Kazuhisa Mori, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.

Ryota Fujimoto, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.

Atsuko Miyagi, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan; Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka, Yamagata 997-8555, Japan.

Eriko Sato, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.

Toshiki Ishikawa, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.

Ryosuke Sano, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan.

Tetsuya Kurata, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan; Integrated Initiative for Designing Future Society, Kyushu University, Fukuoka, 819-0395 Japan.

Shiro Suzuki, Faculty of Applied Biological Sciences, Graduate School of Natural Science and Technology, and the United Graduate School of Agricultural Science, Gifu University, 1-1 Yamagido, Gifu 501-1193, Japan.

Yasuko Kaneko, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan; Faculty of Education, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.

Maki Kawai-Yamada, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.

Toshihisa Kotake, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan; Green Bioscience Research Center, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.

Conflict of Interest

The authors have no conflict of interest to declare.

Funding

This work was supported by MEXT KAKENHI Grant-in-Aid for Scientific Research to T.K. (no. 18H05495, no. 23H02134, and no. 23H04302), to D.T. (no. 20K15494, no. 23K05144, and no. 24KK0273), and M.Y. (no. 24K08823), and by Kato Memorial Bioscience Foundation to D.T.

Data Availability

The data for genome sequencing have been deposited in DDBJ [https://ddbj.nig.ac.jp/search/entry/bioproject/PRJDB19637] with accession number PRJDB19637.

References

  1. Aohara, T., Kotake, T., Kaneko, Y., Takatsuji, H., Tsumuraya, Y. and Kawasaki, S. (2009) Rice BRITTLE CULM 5 (BRITTLE NODE) is involved in secondary cell wall formation in the sclerenchyma tissue of nodes. Plant Cell Physiol.  50: 1886–1897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bond, J., Donaldson, L., Hill, S. and Hitchcock, K. (2008) Safranine fluorescent staining of wood cell walls. Biotech. Histochem.  83: 161–171. [DOI] [PubMed] [Google Scholar]
  3. Brown, D.M., Zeef, L.A.H., Ellis, J., Goodacre, R. and Turner, S.R. (2005) Identification of novel genes in Arabidopsis involved in secondary cell wall formation using expression profiling and reverse genetics. Plant Cell  17: 2281–2295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown, D.M., Goubet, F., Wong, V.W., Goodacre, R., Stephens, E., Dupree, P., et al. (2007) Comparison of five xylan synthesis mutants reveals new insight into the mechanisms of xylan synthesis. Plant J.  52: 1154–1168. [DOI] [PubMed] [Google Scholar]
  5. Burk, D.H., Liu, B., Zhong, R., Morrison, W.H. and Ye, Z.-H. (2001) A katanin-like protein regulates normal cell wall biosynthesis and cell elongation. Plant Cell  13: 807–827. [PMC free article] [PubMed] [Google Scholar]
  6. Ching, A., Dhugga, K.S., Appenzeller, L., Meeley, R., Bourett, T.M., Howard, R.J., et al. (2006) Brittle stalk 2 encodes a putative glycosylphosphatidylinositol-anchored protein that affects mechanical strength of maize tissues by altering the composition and structure of secondary cell walls. Planta  224: 1174–1184. [DOI] [PubMed] [Google Scholar]
  7. Dang, Z., Wang, Y., Wang, M., Cao, L., Ruan, N., Huang, Y., et al. (2023) The fragile culm19 (FC19) mutation largely improves plant lodging resistance, biomass saccharification, and cadmium resistance by remodeling cell walls in rice. J. Hazard. Materials  458: 132020. [DOI] [PubMed] [Google Scholar]
  8. Drevensek, S., Goussot, M., Duroc, Y., Christodoulidou, A., Steyaert, S., Schaefer, E., et al. (2012) The Arabidopsis TRM1-TON1 interaction reveals a recruitment network common to plant cortical microtubule arrays and eukaryotic centrosomes. Plant Cell  24: 178–191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F. (1956) Colorimetric method for determination of sugars and related substances. Anal. Chem.  28: 350–356. [Google Scholar]
  10. Endler, A., Kesten, C., Schneider, R., Zhang, Y., Ivakov, A., Froehlich, A., et al. (2015) A mechanism for sustained cellulose synthesis during salt stress. Cell  162: 1353–1364. [DOI] [PubMed] [Google Scholar]
  11. Guo, D., Li, Y., Lu, H., Zhao, Y., Kurata, N., Wei, X., et al. (2025) A pangenome reference of wild and cultivated rice. Nature.  642: 662–671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hirano, K., Kotake, T., Kamihara, K., Tsuna, K., Aohara, T., Kaneko, Y., et al. (2010) Rice BRITTLE CULM 3 (BC3) encodes a classical dynamin OsDRP2B essential for proper secondary cell wall synthesis. Planta  232: 95–108. [DOI] [PubMed] [Google Scholar]
  13. Horton, P., Park, K.J., Obayashi, T., Fujita, N., Harada, H., Adams-Collier, C.J., et al. (2007) WoLF PSORT: protein localization predictor. Nuc. Acids Res.  35: W585–W587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ichida, H., Kazama, T., Arimura, S.I. and Toriyama, K. (2023) The mitochondrial and plastid genomes of Oryza sativa L. cv. Taichung 65. Plant Biotechnol. (Tokyo).  40: 109–112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ishikawa, M., Kuroyama, H., Takeuchi, Y. and Tsumuraya, Y. (2000) Characterization of pectin methyltransferase from soybean hypocotyls. Planta  210: 782–791. [DOI] [PubMed] [Google Scholar]
  16. Jiang, H., Ren, Y., Guo, J., Yang, H., Zhu, X., Li, W., et al. (2022) CEF3 is involved in membrane trafficking and essential for secondary cell wall biosynthesis and its mutation enhanced biomass enzymatic saccharification in rice. Biotechnol. Biofuels Bioprod.  15: 111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., et al. (2021) Highly accurate protein structure prediction with AlphaFold. Nature  596: 583–589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kelley, L.A., Mezulis, S., Yates, C.M., Wass, M.N. and Sternberg, M.J. (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc.  10: 845–858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Khush, G.S. (1995) Marker gene stocks maintained in IRRI. Rice Genet. Newslett.  12: 24–30. [Google Scholar]
  20. Khush, G.S., Singh, R.J., Sur, S.C. and Librojo, A.L. (1984) Primary trisomics of rice. Origin, morphology, cytology, and use in linkage mapping. Genetics  107: 141–163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kirk, T.K. and Obst, J.R. (1988) Lignin determination. Methods Enzymol.  161: 89–101. [Google Scholar]
  22. Kokubo, A., Kuraishi, S. and Sakurai, N. (1989) Culm strength of barley: correlation among maximum bending stress, cell wall dimensions, and cellulose content. Plant Physiol.  91: 876–882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kokubo, A., Sakurai, N., Kuraishi, S. and Takeda, K. (1991) Culm brittleness of barley (Hordeum vulgare L.) mutants is caused by smaller number of cellulose molecules in cell wall. Plant Physiol.  97: 509–514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kotake, T., Aohara, T., Hirano, K., Sato, A., Kaneko, Y., Tsumuraya, Y., et al. (2011) Rice brittle culm 6 encodes a dominant-negative form of CesA protein that perturbs cellulose synthesis in secondary cell walls. J. Exp. Bot.  62: 2053–2062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kotake, T., Yamanashi, Y., Imaizumi, C. and Tsumuraya, Y. (2016) Metabolism of L-arabinose in plants. J. Plant Res.  129: 781–792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Krogh, A., Larsson, B., von  Heijne, G. and Sonnhammer, E.L. (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol.  305: 567–580. [DOI] [PubMed] [Google Scholar]
  27. Li, Y., Qian, Q., Zhou, Y., Yan, M., Sun, L., Zhang, M., et al. (2003) BRITTLE CULM1, which encodes a COBRA-like protein, affects the mechanical properties of rice plants. Plant Cell  15: 2020–2031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Li, G., Zeng, X., Li, Y., Li, J., Huang, X. and Zhao, D. (2022) BRITTLE CULM17, a novel allele of TAC4, affects the mechanical properties of rice plants. Int. J. Mol. Sci.  23: 5305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Librojo, A.L. and Khush, G.S. (1986) Chromosomal location of some mutant genes through the use of primary trisomics in rice. In Rice Genetics I., edited by Banta, S.J., pp. 249–255. Manila, Philippines, IRRI. [Google Scholar]
  30. Liu, L., Shang-Guan, K., Zhang, B., Liu, X., Yan, M., Zhang, L., et al. (2013) Brittle Culm1, a COBRA-like protein, functions in cellulose assembly through binding cellulose microfibrils. PLoS Genet.  9: e1003704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ma, X., Li, C., Huang, R., Zhang, K., Wang, Q., Fu, C., et al. (2021) Rice brittle Culm19 encoding cellulose synthase subunit CESA4 causes dominant brittle phenotype but has no distinct influence on growth and grain yield. Rice (N Y)  14: 95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mewalal, R., Mizrachi, E., Coetzee, B., Mansfield, S.D. and Myburg, A.A. (2016) The Arabidopsis domain of unknown function 1218 (DUF1218) containing proteins, MODIFYING WALL LIGNIN-1 and 2 (At1g31720/MWL-1 and At4g19370/MWL-2) function redundantly to alter secondary cell wall lignin content. PLoS One  11: e0150254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mikami, M., Toki, S. and Endo, M. (2015) Comparison of CRISPR/Cas9 expression constructs for efficient target mutagenesis in rice. Plant Mol. Biol.  88: 561–572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Miki, Y., Takahashi, D., Kawamura, Y. and Uemura, M. (2019) Temporal proteomics of Arabidopsis plasma membrane during cold- and de-acclimation. J. Proteome  197: 71–81. [DOI] [PubMed] [Google Scholar]
  35. Mirdita, M., Schütze, K., Moriwaki, Y., Heo, L., Ovchinnikov, S. and Steinegger, M. (2022) ColabFold: making protein folding accessible to all. Nat. Methods  19: 679–682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Miyagi, A., Takahashi, H., Takahara, K., Hirabayashi, T., Nishimura, Y., Tezuka, T., et al. (2010) Principal component and hierarchical clustering analysis of metabolites in destructive weeds; polygonaceous plants. Metabolomics  6: 146–155. [Google Scholar]
  37. Miyagi, A., Noguchi, K., Tokida, T., Usui, Y., Nakamura, H., Sakai, H., et al. (2019) Oxalate contents in leaves of two rice cultivars grown at a free-air CO2 enrichment (FACE) site. Plant Prod. Sci.  22: 407–411. [Google Scholar]
  38. Miyagi, A., Mori, K., Ishikawa, T., Ohkubo, S., Adachi, S., Yamaguchi, M., et al. (2022) Metabolomic analysis of rice brittle culm mutants reveals each mutant- specific metabolic pattern in each organ. Metabolomics  18: 95. [DOI] [PubMed] [Google Scholar]
  39. Nguyen, T.P., Otani, T., Tsutsumi, M., Kinoshita, N., Fujiwara, S., Nemoto, T., et al. (2024) Tight junction membrane proteins regulate the mechanical resistance of the apical junctional complex. J. Cell Biol.  223: e202307104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Peña, M.J., Zhong, R., Zhou, G.K., Richardson, E.A., O'Neill, M.A., Darvill, A.G., et al. (2007) Arabidopsis irregular xylem8 and irregular xylem9: implications for the complexity of glucuronoxylan biosynthesis. Plant Cell  19: 549–563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Persson, S., Wei, H., Milne, J., Page, G.P. and Somerville, C.R. (2005) Identification of genes required for cellulose synthesis by regression analysis of public microarray data sets. Proc. Natl. Acad. Sci. USA  102: 8633–8638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Persson, S., Caffall, K.H., Freshour, G., Hilley, M.T., Bauer, S., Poindexter, P., et al. (2007) Arabidopsis irregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan, which are essential for secondary cell wall integrity. Plant Cell  19: 237–255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Roudier, F., Fernandez, A.G., Fujita, M., Himmelspach, R., Borner, G.H.H., Schindelman, G., et al. (2005) COBRA, an Arabidopsis extracellular glycosyl-phosphatidyl inositol-anchored protein, specifically controls highly anisotropic expansion through its involvement in cellulose microfibril orientation. Plant Cell  17: 1749–1763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ruan, N., Dang, Z., Wang, M., Cao, L., Wang, Y., Liu, S., et al. (2022) FRAGILE CULM 18 encodes a UDP-glucuronic acid decarboxylase required for xylan biosynthesis and plant growth in rice. J. Exp. Bot.  73: 2320–2335. [DOI] [PubMed] [Google Scholar]
  45. Sakai, H., Lee, S.S., Tanaka, T., Numa, H., Kim, J., Kawahara, Y., et al. (2013) Rice annotation project database (RAP-DB): an integrative and interactive database for rice genomics. Plant Cell Physiol.  54: e6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sato, K., Suzuki, R., Nishikubo, N., Takenouchi, S., Ito, S., Nakano, Y., et al. (2010) Isolation of a novel cell wall architecture mutant of rice with defective Arabidopsis COBL4 ortholog BC1 required for regulated deposition of secondary cell wall components. Planta  232: 257–270. [DOI] [PubMed] [Google Scholar]
  47. Sato, Y., Namiki, N., Takehisa, H., Kamatsuki, K., Minami, H., Ikawa, H., et al. (2013) RiceFREND: a platform for retrieving coexpressed gene networks in rice. Nuc. Acids Res.  41: D1214–D1221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sawake, S., Tajima, N., Mortimer, J.C., Lao, J., Ishikawa, T., Yu, X., et al. (2015) KONJAC1 and 2 are key factors for GDP-mannose generation and affect L-ascorbic acid and glucomannan biosynthesis in Arabidopsis. Plant Cell  27: 3397–3409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sindhu, A., Langewisch, T., Olek, A., Multani, D.S., McCann, M.C., Vermerris, W., et al. (2007) Maize brittle stalk2 encodes a COBRA-like protein expressed in early organ development but required for tissue flexibility at maturity. Plant Physiol.  145: 1444–1459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Taylor, N.G., Scheible, W.-R., Cutler, S., Somerville, C.R. and Turner, S.R. (1999) The irregular xylem3 locus of Arabidopsis encodes a cellulose synthase required for secondary cell wall synthesis. Plant Cell  11: 769–779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Taylor, N.G., Laurie, S. and Turner, S.R. (2000) Multiple cellulose synthase catalytic subunits are required for cellulose synthesis in Arabidopsis. Plant Cell  12: 2529–2539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Taylor, N.G., Howells, R.M., Huttly, A.K., Vickers, K. and Turner, S.R. (2003) Interactions among three distinct CesA proteins essential for cellulose synthesis. Proc. Natl. Acad. Sci. USA  100: 1450–1455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Toki, S., Hara, N., Ono, K., Onodera, H., Tagiri, A., Oka, S., et al. (2006) Early infection of scutellum tissue with agrobacterium allows high-speed transformation of rice. Plant J.  47: 969–976. [DOI] [PubMed] [Google Scholar]
  54. Turner, S.R. and Somerville, C.R. (1997) Collapsed xylem phenotype of Arabidopsis identifies mutants deficient in cellulose deposition in the secondary cell wall. Plant Cell  9: 689–701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Ubeda-Tomas, S., Edvardsson, E., Eland, C., Singh, S.K., Zadik, D., Aspeborg, H., et al. (2007) Genomic-assisted identification of genes involved in secondary growth in Arabidopsis utilising transcript profiling of poplar wood-forming tissues. Physiol. Plant.  129: 415–428. [Google Scholar]
  56. Uchida, N., Sakamoto, T., Kurata, T. and Tasaka, M. (2011) Identification of EMS-induced causal mutations in a non-reference Arabidopsis thaliana accession by whole genome sequencing. Plant Cell Physiol.  52: 716–722. [DOI] [PubMed] [Google Scholar]
  57. Wu, A.M., Rihouey, C., Seveno, M., Hörnblad, E., Singh, S.K., Matsunaga, T., et al. (2009) The Arabidopsis IRX10 and IRX10-LIKE glycosyltransferases are critical for glucuronoxylan biosynthesis during secondary cell wall formation. Plant J.  57: 718–731. [DOI] [PubMed] [Google Scholar]
  58. Wu, B., Zhang, B., Dai, Y., Zhang, L., Shang-Guan, K., Peng, Y., et al. (2012) Brittle culm 15 encodes a membrane-associated chitinase-like protein required for cellulose biosynthesis in rice. Plant Physiol.  159: 1440–1452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Yamauchi, S., Takemiya, A., Sakamoto, T., Kurata, T., Tsutsumi, T., Kinoshita, T., et al. (2016) The plasma membrane H+–ATPase AHA1 plays a major role in stomatal opening in response to blue light. Plant Physiol.  171: 2731–2743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Yan, C., Yan, S., Zeng, X., Zhang, Z. and Gu, M. (2007) Fine mapping and isolation of Bc7(t), allelic to OsCesA4. J. Gene. Genom.  34: 1019–1027. [DOI] [PubMed] [Google Scholar]
  61. Ye, Y., Liu, B., Zhao, M., Wu, K., Cheng, W., Chen, X., et al. (2015) CEF1/OsMYB103L is involved in GA-mediated regulation of secondary wall biosynthesis in rice. Plant Mol. Biol.  89: 385–401. [DOI] [PubMed] [Google Scholar]
  62. Zhang, B., Deng, L., Qian, Q., Xiong, G., Zeng, D., Li, R., et al. (2009) A missense mutation in the transmembrane domain of CESA4 affects protein abundance in the plasma membrane and results in abnormal cell wall biosynthesis in rice. Plant Mol. Biol.  71: 509–524. [DOI] [PubMed] [Google Scholar]
  63. Zhang, M., Zhang, B., Qian, Q., Yu, Y., Li, R., Zhang, J., et al. (2012) Brittle culm 12, a dual-targeting kinesin-4 protein, controls cell-cycle progression and wall properties in rice. Plant J.  63: 312–328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Zhang, R., Hu, H., Wang, Y., Hu, Z., Ren, S., Li, J., et al. (2020) A novel rice fragile culm 24 mutant encodes a UDP-glucose epimerase that affects cell wall properties and photosynthesis. J. Exp. Bot.  71: 2956–2969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Zhong, R., Burk, D.H., Morrison, W.H.  III and Ye, Z.-H. (2002) A kinesin-like protein is essential for oriented deposition of cellulose microfibrils and cell wall strength. Plant Cell  14: 3101–3117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Zhong, R., Morrison, W.H.  III, Freshour, G.D., Hahn, M.G. and Ye, Z.-H. (2003) Expression of a mutant form of cellulose synthase AtCesA7 causes dominant negative effect on cellulose biosynthesis. Plant Physiol.  132: 786–795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Zhong, R., Burk, D.H., Morrison, W.H.  III and Ye, Z.-H. (2004) FRAGILE FIBER3, an Arabidopsis gene encoding a type II inositol polyphosphate 5-phosphatase, is required for secondary wall synthesis and actin organization in FIBER cells. Plant Cell  16: 3242–3259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Zhou, Y., Li, S., Qian, Q., Zeng, D., Zhang, M., Guo, L., et al. (2009) BC10, a DUF266-containing and Golgi-located type II membrane protein, is required for cell-wall biosynthesis in rice (Oryza sativa L.). Plant J.  57: 446–462. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

pcp-2024-e-00271-File009_pcaf096
pcp-2024-e-00271-file009_pcaf096.pdf (1.2MB, pdf)
pcp-2024-e-00271-File008_pcaf096
pcp-2024-e-00271-file008_pcaf096.pdf (319KB, pdf)

Data Availability Statement

The data for genome sequencing have been deposited in DDBJ [https://ddbj.nig.ac.jp/search/entry/bioproject/PRJDB19637] with accession number PRJDB19637.

Articles from Plant and Cell Physiology are provided here courtesy of Oxford University Press

RESOURCES