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First published online August 7, 2003; 10.1104/pp.103.022442 Plant Physiology 133:73-83 (2003) © 2003 American Society of Plant Biologists Three Distinct Rice Cellulose Synthase Catalytic Subunit Genes Required for Cellulose Synthesis in the Secondary Wall1Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305-0854, Japan (K.T., K.M., K.O.); and Molecular Genetics Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan (M.Y., A.M., H.H.)
Several brittle culm mutations of rice (Oryza sativa) causing fragility of plant tissues have been identified genetically but not characterized at a molecular level. We show here that the genes responsible for three distinct brittle mutations of rice, induced by the insertion of the endogenous retrotransposon Tos17, correspond to CesA (cellulose synthase catalytic subunit) genes, OsCesA4, OsCesA7 and OsCesA9. Three CesA genes were expressed in seedlings, culms, premature panicles, and roots but not in mature leaves, and the expression profiles were almost identical among the three genes. Cellulose contents were dramatically decreased (8.9%-25.5% of the wild-type level) in the culms of null mutants of the three genes, indicating that these genes are not functionally redundant. Consistent with these results, cell walls in the cortical fiber cells were shown to be thinner in all the mutants than in wild-type plants. Based on these observations, the structure of a cellulose-synthesizing complex involved in the synthesis of the secondary cell wall is discussed.
Cellulose is a homogenous polymer of  -1,4-glucan synthesized from UDP-Glc (Delmer and Amor, 1995 ). Plant CesA (cellulose synthase catalytic subunit) genes were first isolated by random sequencing of cotton (Gossypium hirsutum) expressed sequence tags (Pear et al., 1996). The first evidence that the CesA gene encodes the enzyme responsible for cellulose synthesis was obtained from analysis of a CesA mutant of Arabidopsis (Arioli et al., 1998). Another important piece of evidence rests on the structural analysis of cellulose synthesis complexes. The rosette terminal cellulose-synthesizing complexes, displaying 6-fold symmetry, are known to associate with cellulose microfibril impressions in the plasma membrane (Mueller and Brown, 1980), and CESA protein has been immunolocalized to the rosette complexes (Kimura et al., 1999a). In addition, the korrigan gene encoding a endo-1,4--glucanase has been shown to be required for cellulose synthesis (Zuo et al., 2000; Lane et al., 2001; Sato et al., 2001) and proposed to function in the cleavage of sitosterol--glucoside from the growing cellulose polymer chain (Peng et al., 2002).
Ten CesA genes have been found in the completed genomic sequence of Arabidopsis (http://cellwall.stanford.edu). Based on mutant analyses, characterization of six CesA genes of Arabidopsis has been reported. The rsw1 mutation in AtCesA1 causes a reduction of cellulose synthesis, when grown at the nonpermissive temperature, resulting in disassembly of the rosette complexes, widespread morphological abnormalities, and the accumulation of noncrystalline
The CesA gene family in monocot crop plants such as maize (Zea mays), barley (Hordeum vulgare), and rice (Oryza sativa) was also identified by analyses of cDNA, expressed sequence tags, and genome sequencing (Holland et al., 2000
We produced about 50,000 mutant lines of rice induced by the insertion of the endogenous retrotransposon Tos17 (A. Miyao, K. Tanaka, K. Murata, H. Sawaki, S. Takeda, K. Abe, Y. Shinozuka, K. Onosato, and H. Hirochika, unpublished data). Tos17 is inactive in normally propagated plants but becomes active in tissue culture (Hirochika et al., 1996
Screening of the Brittle Culm Mutants Induced by the Endogenous Retrotransposon Tos17 To screen for the mutants displaying the brittle phenotype, the next generations (R1) of about 3,500 regenerated rice lines were observed in the paddy field. In these populations, four regenerated lines, called NC0259, ND2395, ND8759, and NE1031, exhibited similar mutant phenotypes; a dwarfed growth habit (Fig. 1A, 2-5) and easy fracturing of leaf and culm by stressing between fingers, the so-called brittle culm phenotype (Fig. 1B, 2-5). These four mutants also exhibited other similar characteristic phenotypes, such as small leaves, thin culms, and withering of leaf apex (data not shown). In addition, fertility of the four mutants was low (0%-32.3%). The semidwarfed mutant of NF1011 (Fig. 1A, 6) also showed a brittle culm phenotype (Fig. 1B, 6). Although this mutant had normal-sized leaves, most of the mature leaves of the mutant plants growing in the paddy field were fractured in the middle region by wind pressure (data not shown). The semidwarfed mutant of NF1011 also had a thin culm, but it was thicker than those of the other four mutants (data not shown).
To examine whether these mutations were induced by the insertion of Tos17 and to identify the specific transposed Tos17 copies responsible for the mutations, cosegregation of Tos17 with the observed phenotypes was examined by genomic Southern analysis of the R1 populations of the five lines as described previously (Agrawal et al., 2001
To identify the causative brittle culm genes, the genomic fragments from the region flanking Tos17 were amplified by thermal asymmetric interlaced (TAIL)-PCR and suppression PCR methods. Using genomic DNA from the mutant of NC0259, TAIL-PCR was performed with a Tos17-specific primer and the degenerate primer called AD7. Several DNA fragments were amplified, but genomic Southern analysis with these fragments as probes showed cosegregation of the fragment NC0259_14_701_1A with brittle culm phenotype (data not shown). The amino acid sequence deduced from the DNA sequence of NC0259_14_701_1A showed high similarity to those of plant CESAs (data not shown). Because the brittle phenotype can be explained by a CesA mutation, it is likely that the CesA gene is the gene responsible. This was further confirmed by the reduction of cellulose content in the mutant (see the following section). Using NC0259_14_701_1A as a probe, the locus was mapped onto the rice linkage map. The disrupted CesA gene of NC0259 was mapped at 44.0 cM on chromosome 10 (data not shown), and the RFLP marker clone R2825, which shows high similarity to plant CESAs (data not shown), was located at the identical position. The clone R2825 was shown to be a partial cDNA clone of the disrupted gene. The genomic sequence of the locus at 44.0 cM is published by the International Rice Genome Sequencing Project, and the disrupted gene of NC0259, encoding CESA, is annotated as OsCesA7 (CESA sequence database at http://cellwall.stanford.edu). OsCesA7 consists of nine exons and eight introns (Fig. 2) and encodes a protein of 1,063 amino acids (Fig. 3). In NC0259, Tos17 was inserted in the fifth exon of OsCesA7 (Fig. 2). In ND8759, Tos17 was inserted into the seventh exon of OsCesA7 (Fig. 2), and the homozygous mutant of ND8759 also exhibited a similar brittle culm phenotype, as shown in Figure 1B, 3. Based on these results, it is concluded that the OsC-esA7 gene is a causative gene for the brittle culm mutation.
The DNA fragments carrying putative brittle culm genes of NE1031, ND2395, and NF1011 were amplified by the suppression PCR method. The amplified DNA fragments T12748T, ND2395_30, and NF1011-7_2 also showed high similarity to CesA genes and cosegregated with the brittle culm phenotype (data not shown). The disrupted gene of NE1031, encoding CESA, was mapped at 129.6 cM on chromosome 1 (data not shown), and this locus corresponds to RFLP marker R2417, whose clone also shows high similarity to plant CESAs (data not shown). R2417 was shown to be a partial cDNA of the disrupted gene of NE1031, whose genomic sequence was determined by International Rice Genome Sequencing Project and shown to be OsCesA4. OsCesA4 consists of 13 exons and 12 introns (Fig. 2) and encodes a protein of 990 amino acids (Fig. 3). The insertion site of Tos17 in NE1031 was found in the sixth exon (Fig. 2). To isolate allelic mutants of OsCesA4, PCR screening was carried out using DNA pools from mutant lines induced by the insertion of Tos17 (Hirochika, 2001 The disrupted gene of ND2395 was mapped at 158.9 cM on chromosome 1 (data not shown), but the corresponding sequence was not found in the published genomic sequence. Therefore, using the Monsanto Rice Genome Sequence Database (http://www.rice-research.org/), the genomic sequence of the brittle culm gene of ND2395, encoding CESA, was determined. This CesA gene was shown to be OsCesA9, which consists of 11 exons and 10 introns (Fig. 2) and encodes a protein of 1,055 amino acids (Fig. 3). Part of the genomic sequence of OsCesA9 was identical to that of NF1011-7_2 and shown to cosegregate with the mutant phenotype of NF1011 by the genomic Southern analysis (data not shown). Tos17 was inserted into the 6th exon in ND2395 and the ninth intron in NF1011 (Fig. 2). Based on the results described in this section, it is concluded that the genes responsible for the three brittle mutations are three different CesA genes.
To characterize the three rice CesA genes, the deduced amino acid sequences were compared (Fig. 3). There is a high degree of sequence similarity among the deduced CESA proteins. Furthermore, the proteins possess four motifs that have been identified as being conserved in CESAs and all processive glucosyl transferase (Saxena et al. 1995
Based on the sequences shown in Figure 3, phylogenetic relationships of CesA genes from higher plants were examined (Fig. 4). Three rice CesA genes were considered to function to synthesize cellulose in the secondary cell walls responsible for the overall strength of the plant, judging from the brittle culm phenotype (Fig. 1B) exhibited by their respective mutants (Fig. 2). In Arabidopsis, three CesA genes contributing to cellulose synthesis in the secondary cell walls, AtCesA4, -7, and -8, have been found by mutant analyses (Taylor et al., 1999
The expression patterns for OsCesA4, OsCesA7, and OsCesA9 were investigated in culms, mature leaves, roots and immature panicles, and 2-week old seedlings. Signals corresponding to these genes could be detected by northern-blot analyses (Fig. 5). Transcripts of the three CesA genes were found in seedlings, culms, immature panicles and roots, but not in mature leaves, and the expression patterns were almost identical among three genes. Therefore, it is concluded that the three rice CesA genes are expressed coordinately in seedlings and three organs, although it is uncertain whether these genes are co-expressed in the same cell.
Cellulose contents in the second culm internodes of the mutants andthe wild-type plant were investigated. As summarized in Table I, cellulose contents were decreased in all the mutants, but some differences were observed among the mutants. The brittle culm phenotype of the mutants can be readily explained by these reductions in cellulose. The OsCesA4 and -7 mutants exhibiting the same phenotype showed almost the same reductions, and these Tos17 insertion sites are found in the exon of the gene (Fig. 2). This result indicates that the mutations of OsCesA4 and -7 affected the gene function, possibly leading to complete loss of function. On the other hand, the cellulose contents were different in two OsCesA9 mutants exhibiting different phenotypes, and the Tos17 insertion sites are one in an exon and the other in an intron (Fig. 2). The mild phenotype (semidwarf, Fig. 1A) exhibited by the NF1011 line could be explained by its higher cellulose content. One possible explanation for the partial loss-of-function phenotype of NF1011 line is incomplete splicing out of the Tos17-containing intron. Analysis of reverse transcription (RT)-PCR products showed that incomplete splicing out did occur (data not shown). One important finding here is that complete loss of function of any one of the three CesA genes leads to a dramatic reduction in cellulose contents (8.9%-25.5% of the wild-type level), suggesting that these genes are not functionally redundant to each other.
To investigate structural changes in the culms of the five brittle culm mutants, cross sections of the second culm internodes from the wild-type plant and five mutants were observed by scanning electron micrograph (Fig. 6). Notably thinner cell walls were found in the cortical fiber cells in all the mutants. Similar results were obtained by the analysis of the third internodes (data not shown). The significant differences in the cell walls between the wild-type plant and five mutants can be explained by a decrease in cellulose content of the cell walls of the mutants (Table I). In three Arabidopsis mutants of the CesA genes required for cellulose synthesis in the secondary cell walls, collapsed xylem cells have been found (Taylor et al., 1999
Brittle culm mutants have been isolated in several monocot species (Jones, 1933 ; Takahashi et al., 1953, 1966, 1968; Nagano and Takahashi, 1963; Briggs and Robert, 1966), and some of the mutants have been studied in relation to cellulose synthesis (Kimura et al., 1999b; Kokubo et al., 1989, 1991). However, the genes affected in these mutations were still not known. Here, isolation of the causative genes for three brittle culm mutations induced by the retrotransposon Tos17 is reported. These genes are shown to encode three distinct CESAs, OsCESA4, OsCESA7, and OsCESA9.
The plant CESAs are generally considered to be associated with rosette complexes, the cellulose-synthesizing units in the plasma membrane (Kimura et al., 1999a The phylogenetic analysis shows that the secondary wall-forming enzymes in Arabidopsis are classified into three subgroups (Fig. 4). Interestingly, the secondary wall-forming enzymes in rice are also classified into these three subgroups. This suggests that a functional CESA unit would consist of three different CESA subunits in higher plants. This suggestion agrees with the model of a functional CESA unit (Doblin et al., 2002), proposing that three different types of subunits might be required for rosette assembly.
All the plant CESA proteins possess not only the conserved three Asp residues and QxxRW motif that are critical for the function of CESA but also the RING finger motif in the N-terminal region of the proteins (Delmer, 1999 Two OsCesA9 allelic mutants, ND2395 and NF1011, although both showing the brittle culm phenotype (Fig. 1B, 5 and 6) and thinning of cortical fiber cell walls in the culm (Fig. 6), exhibited different growth habits (Fig. 1A, 5 and 6) and different cellulose contents (Table I). The mild mutant phenotype exhibited by the NF1011 line is likely due to partial loss of function. The partial loss of function is considered to result from partial inhibition of splicing of the Tos17-containing intron because the Tos17 insertion site is found in the intron (Fig. 2). Data supporting this hypothesis were obtained by RT-PCR analysis.
The complete loss-of-function mutants of OsCesA4, -7, and -9 exhibited morphological abnormality such as dwarf (Fig. 1A). In Arabidopsis, the collapsed xylem elements have been observed in the mutants of AtCesA4, -7, and -8, the members of the secondary wall-forming genes, but these mutants did not exhibit morphological abnormalities (Turner and Somerville, 1997
The mutant of AtCesA3, one of the members of the primary wall-forming genes, has been reported to overproduce jasmonate and ethylene, which are induced by a number of biotic and abiotic stresses such as wounding and pathogen attack (Ellis et al., 2002
Several brittle culm mutations have been genetically identified in rice and mapped onto the linkage map as follows; bc1 on chromosome 3, bc2 on chromosome 5, bc3 on chromosome 2, bc4 on chromosome 6, and bc5 on chromosome 2 (Nagato and Yoshimura, 1998 In the present study, three causative genes for brittle culm mutations were isolated after screening only 3,500 mutant lines induced by the Tos17. Further screening should contribute to the isolation of new genes involved in cell wall synthesis.
Plant Materials
Rice (Oryza sativa) mutant lines induced by insertion of the endogenous retrotransposon Tos17 were grown in the paddy field. For various analyses, such as observation by scanning electron microscopy, the culm internode was collected after harvesting and fixed in Nawaschin Claf type III (3:2:1:4 [v/v] 1% chromic acid:10% acetic acid:35% formalin:water; Miksche, 1976
Rice genomic DNA was prepared from mature leaves by the cetyltrimethyl-ammonium bromide method (Murray and Thompson, 1980
TAIL-PCR (Liu and Whittier, 1995
A modified method of suppression PCR was used, as described elsewhere (Miyao et al., 1998
Total RNA was isolated from elongating culms, mature leaves, panicles at the meiotic stage, roots, and 2-week-old seedlings with the Isogen kit (Nippon Gene, Toyama, Japan), and 10 µg of total RNA was electrophoresed on 0.8% (w/v) denatured agarose gels. The gel was blotted on Hybond N+ membrane (Amersham, Little Chalfont, UK), and the membrane was hybridized with the same buffer as Southern analysis. The probes for OsCesA4, -7, and -9 were prepared with the DNA fragments re-amplified from the RT-PCR products by PCR with the gene-specific primers designed from the nested region of the sequence of the primers used in the first PCR. RT-PCR was carried out using a ReverTra Dash kit (Toyobo, Osaka) with the gene-specific primers according to the manufacturer's recommendation. The forward and reverse primers used for primary and secondary reactions were as follows: for OsCesA4, 5'-GACAGATGCATATGAGCCAC-3' and 5'-CTTTCTTGTGGTGTTGGTAC-3', and 5'-CTCGGATCATTCCTATATCG-3' and 5'-CTCGAAACATAGACTAGCCG-3'; for OsCesA7, 5'-GCGGCTTCCCGGTGTGCAAG-3' and 5'-GCTGGACCTTGTCCTTGAGG-3', and 5'-TGCTACGAGTACGAGCGCAG-3' and 5'-GTCGAAGAGCAGCATGGACGCGC-3'; and for OsCesA9, 5'-CATGCTCAACTTGGATTGCG-3' and 5'-TCCACTTGAAGGCGTAGAGC-3', and 5'-TCAACAACAGCAAGGCCATC-3' and 5'-CGGTGAAGTTGGTGTCGATC-3'.
The DNA sequencing analysis was performed using the dideoxy chain termination method (Sanger et al., 1977
The loci of the rice brittle genes were mapped on the Nipponbare/Kasalath high-density linkage map constructed by Harushima et al. (1998
The second culm internodes of rice were freeze dried at -20°C for 1 d and 4°C for 2 d with Unitop HL and Freezemobile 12EL systems (Virtis, Gardiner, NY). The freeze-dried materials were homogenized at 2,400 rpm for 20 min with Multi-Beads Shocker MB301 (Yasui Kikai, Osaka). The homogenate was suspended in 50 mM Tris-HCl (pH 7.0), incubated for 5 min in boiled water, and then cooled at room temperature. The suspension was treated for 2 h at 37°C with 20 units mL-1 porcine pancreatic
The fixed culm internodes were substituted in water overnight. The substituted material was cut perpendicularly to culm axis with a microtome blade. The specimens were examined directly with a scanning electron microscope (S-2380N, Hitachi, Tokyo).
We thank M. Harada, N. Kojima, Y. Machida, M. Tanabe, and Y. Yamashita (Institute of Society for Techno-Innovation of Agriculture, Forestry and Fisheries, Tsukuba, Japan) for experimental and technical assistance during this study. We thank T. Ando, A. Shomura (Institute of Society for Techno-Innovation of Agriculture, Forestry, and Fisheries, Tsukuba, Japan), and Dr. M. Yano (National Institute of Society for Techno-Innovation of Agriculture, Forestry, and Fisheries, and National Institute of Agrobiological Sciences, Ibaraki, Japan) for supporting the experimental procedure of gene mapping. We thank Drs. H. Nakagawa and H. Takatsuji (National Institute of Agrobiological Sciences) for performing the scanning electron microscopy. We thank Prof. N. Sakurai (Hiroshima University, Japan) for insightful advice regarding the cellulose content measurement. Received February 19, 2003; returned for revision March 24, 2003; accepted May 20, 2003.
1 This work was supported by the Program for Promotion of Basic Research Activities for Innovation Biosciences. 
2 Present address: Advanced Technology Research Laboratory, Oji Paper Co., Ltd., Koto-ku 135-8558, Tokyo, Japan.
3 Present address: Toyama Agricultural Research Center, Toyama 939-8153, Toyama, Japan.
4 Present address: HITEC Yokohama R&D Center, Yokohama 220-0004, Kanagawa, Japan. * Corresponding author; e-mail hirohiko{at}nias.affrc.go.jp; fax 81-298-38-7020.
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RESULTS
3)(1
-amylase (Sigma, St. Louis) and centrifuged at 1,000g for 10 min. The resulting precipitate was resuspended in 17.5% (w/v) NaOH/0.02% (w/v) NaBH4. The suspension was centrifuged, and the precipitate was resuspended in the alkaline solution as described above and incubated at room temperature for 24 h. The incubated suspension was centrifuged, and the precipitate was washed once with the same alkaline solution. After alkaline treatment, the precipitate was washed three times with 0.03 M acetic acid, three times with ethanol, three times with ethanol/ether (1:1 [v/v]), and three times with ether. These procedures were modified from the method of Kokubo et al. (1991
