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Phenotypic characterization, genetic analysis and gene-mapping for a brittle mutant in rice Jian-Di Xu1,2,3*, Quan-Fang Zhang 1,2,3*, Tao Zhang 1,4, Hong-Yu Zhang 1,3, Pei-Zhou Xu1,3, Xu-Dong Wang 1,3 and Xian-Jun Wu 1,3** (1. Rice Research Institute, Sichuan Agricultural University, Wenjiang, Sichuan 611130, China; 2. Shandong Rice Research Institute, Jining, Shandong, 272100, China; 3. Key Laboratory of Crop Gene Resources and Improvement, Ministry of Education of China, Sichuan Agricultural University, Yaan, Sichuan 625014, China; 4. Rice and Sorghum Research Institute, Sichuan Academy of Agricultural Sichuan Sciences, Luzhou, 646100, China) Received: 2 Nov. 2006 Accepted: 12 Mar. 2007 Handling editor: Wei-Cai Yang Abstract: Plant mechanical strength is an important agronomic trait of rice. An EMS-induced rice mutant, fragile plant 2 (fp2), showed morphological changes and reduced mechanical strength. Genetic analysis indicated that the brittle of fp2 was controlled by a recessive gene. The fp2 gene was mapped on chromosome 10. Anatomical analyses showed that the fp2 mutation caused the reduction of cell length and cell wall thickness, increasing of cell width, and the alteration of cell wall structure as well as the vessel elements. And the consequence was the global alteration in plant morphology. Chemical analyses indicated that the contents of cellulose and lignin 1 decreased, hemicelluloses and silicon increased in fp2. These results were different from the other mutants reported in rice. Thus, fp2 might affect the deposition and patterning of microfibrils, the biosynthesis and deposition of cell wall components, wSupported by the program for Changjiang Scholars and Innovative Research Team in University (IRT0453) * These authors contributed equally to the work. ** Author for correspondence. Tel: (+86)028 8272 9305; Fax: (+86)028 8272 6875; Email: [email protected] hich influences the formation of primary and secondary cell walls, the thickness of cell wall, cell elongation and expansion, plant morphology and plant strength in rice. Key words: fragile plant (fp)2; phenotype; cell wall; gene mapping; rice (Oryza sativa L.) Plant mechanical strength is an important agronomic trait. The plant cell wall, as the major component of mechanical support to cells, tissues, and the entire plant body, can be grouped, into three basic types: parenchyma, collenchyma and sclerenchma, according to their wall thickening. Both parenchyma and collenchyma cells, consisting of the primary wall, provide main mechanism support in growing regions of the plant body, sclerenchyma cells having both primary wall and thick secondary wall, provide the major mechanical support in non-elongating regions of the plant body (Carpita and McCann, 2000). Cell walls delimit the boundaries of individual cell, the shapes of individual cell walls determine cell morphology and whole plant morphology (David et al., 2001). Cell wall contains different substances to suited its function, for example, cellulose usually constitutes 20%~30% of the dry weight of the primary wall and 2 40%~90% of secondary cell wall (Neil et al., 1999), lignin and hemicellulose are other two important contents in cell wall. The mechanism involved in the mechanical strength of the plant and the biosynthesis of plant cell walls are still not fully understood. Some mutants defecting in plant strength have been isolated and characterized. The barley brittle culm showed reduced mechanical strength and cellulose content (Kokubo et al., 1989, 1991), indicating a correlation between the cellulose content and the plant mechanical strength (Li et al., 2003). Similarly, in Arabidopsis, some mutants with reduced mechanical strength and alteration of some cells shape and cell elongation have been illustrated, the corresponding genes have been cloned and characterized. The irregular xylem mutants (irx1 to irx3) showed cellulose synthesis defects in secondary walls, and the decreased stiffness of mature stems (Turer and Somerville, 1997; Jones et al., 2001). Moreover, in the fra4 mutant, the RHD3 gene caused an alteration in cell wall composition which resulted in a dramatic reduction in the mechanical strength of stems and wall thickness (Hu et al., 2003). The change crystalline cellulose synthetic biosynthesis caused changes in the shape of rsw1 mutants, indicating the direct role of cellulose in maintaining cell morphology (Arioli et al., 1998); and the kor1 mutation caused abnormal cell wall structure and aberrant cell plate formation. Cells in the kor1 mutants cannot elongate normally, resulting in an extremely dwarf phenotype (Stéphanie Robert et al., 2001). In rice, several brittle culm mutations have been genetically identified and characterized (Nagato and Yoshimura, 1998). For example, bc1 mutation was characterized by a reduction in cell wall thickness and cellulose 3 content and an increase in lignin level. These findings suggested that the biosynthesis and/or modifications of cell walls are essential for plant mechanical strength and normal cell morphology. In this study, we described a new mutant with fragile plant (fp2) in rice and mapped the fp2 gene on chromosome 10. The fp2 mutation affected the deposition and patterning of microfibrils, the biosynthesis and deposition of cell wall components, and resulted in the changes in plant morphology and plant strength. Results The source and identification of fp2 The mutant was derived from an M1 population of rice cultivar E-you 532 after treated by 0.1% EMS. Several brittle mutants were found in a plot which was derived from an M0 plant. The mutants showed brittle in root, culm, leaf, sheath and seed. Brittle trait showed no separation after planted for three years, and then the mutant was named as fragile plant 2 (fp2). The mechanical property of fp2 mutant The mutant has a reduction in mechanical strength compared with the wild type. The culm, leaf, sheath could be broken easily (Fig. 1A, B), root and seed were brittler than that of wild type too. To accurately describe this phenotype, we quantitatively compared the breaking forces of fp2 and the wild type, which define the forces required to break the segments of culms and the elongation ratios of the culm, which reflect the elasticity of plant tissues. The forces required to break the mutant culms was decreased to 70% of that required for the wild type (Fig. 1C); the elongation ratio of the mutant culm was reduced by ~65% compared with that of the wild type (Fig. 1D). 4 These showed that the fp2 affected the mechanical strength and the elasticity in the plant. The fp2 mutant has an alteration in plant morphology The fp2 plants exhibited a semi-dwarf with shorter culm, shorter drooping leaf and a disperse phenotype compared with the wild type (Fig. 2A). The height of the main culm of mature mutant plant reached only ~65% of the height of the wild-type (Fig. 2A, Table 1). Measurement of internode number and length showed no change in internode number but had a dramatic reduction in internode length. The length of the leaves and roots were shorter than those of the wild type, too (Fig. 2B, Table 1). It suggested that the decrease in plant height in fp2 was caused by a reduction in internode length. The width of fp2 leaf blades and culm diameter were wider than those of the wide type (Table 1). The alteration of the culm length was resulted from the decrease of cell length (Fig. 2C, D). Longitudinal sections of mature culm showed an alteration in cells length and width in fp2 (Fig. 2C). In the wild type, cells were long and narrow (Fig. 2D); however, cells in fp2 were much shorter and wider than those in wild type (Fig. 2C, 2D). The length of mature leaf in fp2 reached only ~70% of that in wild type (Table1). The reduction in organ length was also obvious in young seedlings. Two-week-old fp2 seedlings had much shorter hypocotyls and roots than those of wild-type (Fig. 2B), and until maturation. These results indicated that the fp2 influenced the normal cell expansion and elongation process, and altered the phenotype. fp2 was defective in cell walls thickness and alteration in structure 5 Reduction in the mechanical strength of plant may reflect alterations in cell wall structure, composition, or fiber length (Li et al., 2003). Therefore, cell walls shapes were examined by SEM. In wild-type plant, several layers of the mechanical tissues, especially under the epidermal layer in culms, sheath and leaf veins, provided the main mechanical support for the plants. SEM observations revealed that the wild-type sclerenchyma cell walls were much more thickened in culm, sheath and leaf (Fig. 3A, C, E), in striking contrast to sclerenchyma cell walls in fp2 which were very thinner (Fig. 3B, D, F). The sclerenchyma of fp2 had changed into hollow reticulate structure which was different from those solid in wild-type, the vascular cell walls altered too. The cell walls of fp2 became thinner. To understand the changes in cell walls structure, cell walls were examined by TEM. TEM revealed that while the walls of all mechanical tissues cell were thinner than those of the wild type (Fig. 4A, B), occasionally, a few cells had deformed and thin walls (Fig. 4B). In addition to the reduced wall thickness in mechanical tissues cells, the fp2 mutant also exhibited reduced wall thickness and an alteration of structure of vessel elements (Fig. 4C, D). The primary cell wall and the secondary cell wall of the wild type arrayed in order, however, the cell walls structure of fp2 became immethodical (Fig. 4D). These results indicated that the fp2 mutation affects the formation of mechanical tissues cells wall and vessel cells wall. So, the fp2 caused a reduction in the wall thickness and alteration of the cell wall structure, which influenced the mechanism strength and plant morphology. Chemical analysis of cell wall composition 6 To determine whether the cellular phenotype and the reduced mechanical strength in fp2 mutant plant result from altered cell wall compositions biosynthesis, we analyzed cell wall compositions of mature plant. Because cell walls constituted a major fraction of total wall materials in mature plants, it was expected that any significant change in cell wall composition should be detected by analyzing the cell wall compositions of plants, as demonstrated in irx, fra2 and bc1 mutants (Turner and Somerville, 1997; Burk et al., 2001, 2002; Li et al., 2003). Cellulose is the major component of plant cell walls, so we compared the crystalline cellulose contents of mutant and wild type. The crystalline cellulose amount in the fp2 mutant was reduced to ~75% of that in the wild type (Fig. 5A), suggesting that the mutation of fp2 may directly or indirectly play an important role in cellulose biosynthesis. Lignin is also the important component in cell wall which contributes to mechanism strength, therefore, lignin was assayed too. As shown in Fig. 5B, the lignin of the fp2 culms reduced ~68% compared with that of the wild-type culms. The rice plant has a mechanism which regulated the balance of the contents in cell wall to protect the cell (Shen et al., 2004), so we assayed other compositions in culm segments to look for the composition increasing in fp2 plant. We could find that hemicellulose and silicon content increased to ~31% and ~30% respectively compared with the wild type (Fig. 5C, D). These data suggested that the fp2 had an alteration in the biosynthesis of cell wall compositions, the reduction of the cellulose and lignin content may be the main reasons for the decline of the mechanical strength and the change of plant morphology. Genetic analysis and genetic mapping of the brittle mutant fp2 7 Genetic analysis of the gene for the fp2 trait To determine the genetic control of fp2, fp2 was crossed with the indica rice 93-11, R527 and the japonica rice Nipponbare. The individual plants in the F1 and F2 progenies from the above crosses were investigated. In the three F1 progenies, all plants exhibited wild-type phenotype, suggesting that the mutant trait was recessive. In the three F2 populations, all the segregation ratios of wild-types (WT) and fp2 plants fit to 3:1. Therefore, the brittle trait is controlled by one recessive gene. Chromosomal mapping of fp2 gene locus The F2 population from the cross of Nipponbare/fp2 was used as fp2 gene mapping. The polymorphisms between fp2 and Nipponbare were examined with 355 SSR makers distributed evenly on 12 chromosomes. Among of which, 98 polymorphic markers were using for surveying a total of 1149 F2 individuals. The result showed that an SSR marker RM311 located on chromosome 10 was obviously linked to the fp2. Then other markers on the chromosome 10 were employed to survey the same F2 population, and four SSR makers (RM467, RM271, RM258 and RM1375) were also linked with fp2. Based on the segregation data, a local linkage map around fp2 was constructed. It showed that fp2 was located between the molecular markers RM271 and RM258, at the genetic distance of 1.4cM and 11.4cM respectively, on chromosome 10 (Fig. 6). Discussion fp2 and other brittle mutants in rice We presented here a new brittle mutant fp2, named after the fp1 (Qian et al., 2001). 8 In rice, at least six brittle culm mutants (bc1 to bc6) have been reported. These brittle mutants have been genetically identified in rice and mapped onto genetic map as follows: bc1 on chromosome 3, bc2 on chromosome5, bc3 and bc5 on chromosome 2, bc4 on chromosome 6 (Nagato and Yoshimura, 1998), bc5 is a brittle node gene and bc6 is a dominant gene. However, except bc1, the genes associated with these mutations have not been isolated. The fp2 was mapped on chromosome 10, this gene genetic location was difference to those brittle mutants in rice. In addition, the phynotype of fp2 was different from the other brittle mutants found in rice. Other mutants showed one same character: a normal morphology like the wild-type plant (Katsuyuki Tanakaet et al., 2003), for example, the change between the bc1 mutant and its wild type only in the mechanism strength. The fp2 mutation caused a defect in the overall cell wall synthesis, especially in the mechanical tissues. The obvious distinctness between the fp2 and other brittle rice mutants was that the fp2 had a change in plant morphology and the alteration of cell wall structure. The fp2 exhibited a semi-dwarf with shorter culm, shorter leaf and a disperse phenotype compared with the wild type (Fig. 2A). As description above, the reduced length of cells induced the morphology of fp2. In rice, only bc1 was studied particular. The diversifications of fp2 were obvious distinguished with bc1 comparing with each wild type plant. Three CesAs have been identified in rice through isolation of relative mutants which induced by the Tos17. These genes are shown to encode three distinct CESAs, OsCESA4, OsCESA7, and OsCESA9 (Katsuyuki Tanaka et al., 2003). The morphological characteristic of these mutants were likely to the fp2. These genes 9 might be involved in the synthesis of cellulose or noncellulosic components in secondary cell walls. The fp2 gene mightly does not correspond to the causativegenes for the brittle culm mutations found previously. In rice, only BC1 has been cloned which was presumed to encode a cobra-like protein, causing a reduction in cell wall thickness, cellulose content and an increase in lignin level. From those evidences, we concluded that the fp2 was probably a novel brittle mutant in rice. Function of the fp2 gene The mechanism that regulates the mechanical strength of the plant body and the biosynthesis of plant cell wall are very complex. Some genes for brittle culm mutants in rice have been mapped on different chromosomes, which indicated the complex of the brittle mechanism too. It has been known that deficiency in material in the primary cell walls often leads to a globally altered morphology (Arioli et al., 1998). By contrast, plants with mutations that affect the formation of the secondary cell walls usually grow relatively normally (Taylor et al., 1999; 2000), for example, the bc1 with mutation which affect the formation of the secondary cell wall, it only altered in mechanical strengthen. The plant morphology of fp2 was different to the wild type, the structure and sizes of sclerenchyma and parenchyma cells of fp2 were differ to the wild type, the material of cell walls in fp2 were changed much compared to wild type, too. All of those suggested that the fp2 have mutations to affect the formation of the primary cell walls and the secondary cell walls all, so the fp2 gene is very likely to play a role in regulating the biosynthesis of the material in cell walls, and the formation of the 10 primary and second cell walls. The cellulose in cell wall provides not only the necessary strength to resist the turgor pressure in plant cells but also has a distinct role in maintaining the size, shape and division/differentiation potential of most plant cells and ultimately the direction of plant growth. The deposition of cellulose microfibrils in a specific orientation determined the direction of plant cell elongation. It was generally accepted that the orientation of cellulose microfibrils synthesized from the cellulose synthase complex is regulated by underlying microtubules beneath the plasma membranes. The trafficking of vesicles containing noncellulosic materials synthesized in the Golgi body also may be guided by microtubules. Thus, it is conceivable that alteration of the normal dynamic changes in cortical microtubules might result in an inability of microtubules to guide efficiently the deposition of cell wall materials, thereby leading to a delay of cell wall biosynthesis. In Arabidopsis, PROCUSTE1 (PRC1) locus showed decrease cell elongation, cell elongation defects are correlated with a cellulose deficiency and the presence of gapped walls (Mathilde et al., 2000). RHD3 gene in the fra4 mutant played an essential role in cell wall biosynthesis and actin organization, which was important to cell expansion (Hu et al., 2003). From Fig. 4, we concluded that some problems were occurring in the process of the cells wall biosynthesis, especially in the biosynthesis and deposition of the cellulose microfibre. The alteration of the cellulose microfibre deposition leaded to the alteration of the cells wall structure. The decrease of cellulose reduced the change of the entire cells wall material amount in turn. In conclusion, we have showed that the fp2 mutation resulted in a reduction in cellulose, 11 lignin, cell length and mechanical strength; an increase in hemicellulose, silicon and cell width; an alteration in structure and thickness of cell wall and plant morphology. The fp2 gene might play a role in the regulation of dynamic changes of microfibrils during the all stages of establishment of the microfibril or other noncellulosic components deposition and array as well as during cell elongation, which in turn influences cell morphogenesis, the biosynthesis and deposition of cell wall materials, the formation of primary and secondary cell wall, mechanism strength and plant morphology. We can explain the phenomena according Fig. 4, but may be different to that in Arabidopsis. The mechanism about the decline of mechanical strength and the alteration of plant morphology in fp2 is complex. It was needed further elucidation the function of fp2 by gene cloning. Materials and Methods Plant materials The brittle mutant fp2 was discovered in an M1 treated by 0.1% EMS for a cultivar E-you 532 (Oryza sativa L.). The mutant and wild type were planted in Sichuan and Hainan alternatively. Genetic analysis populations were planted in Sichuan, China. Measurements of physical properties The breaking force and elongation ratio of rice culms were measured with a universal strength testing device (mode 7001; ZhongKai, China). To avoid inaccuracies from sampling, the first internodes of culms were used for measurement. The elongation ratio (%) was defined by the formula 100×(L1-L2)/L2, where L1 represents the length of the culm segments at breaking and L2 stands for the original 12 length of the culm segments. Culms dried at 30℃ for three days, ten culms were used to measure. Paraffin sectioning The third internodes of culm were fixed in FAA (formalin: acetic acid: 70% ethanol [5:5:90,v/v]), for approximately 16 hr at 4℃, dehydrated in a graded ethanol series, substituted with a xylene series, and embedded in paraffin. Samples were sectioned at 12µm and then stained with phloroglucin solution (2% in ethanol: water [95:5, v/v]), images were taken under Olympus light microscopes. Scanning electron microscopy (SEM) Samples were prepared as described previously (Mou et al., 2000) with some modifications. Briefly, rice tissues were excised with a razor and immediately placed in 3% (v/v) glutaraldehyde in phosphate buffer (50mM, pH 7.2) for 24h, dehydrated in a series of ethanol-water and incubated in an ethanol-isoamyl acetate mixture for 1h. Samples were critical point dried, sputter-coated with gold in a Model E-1010/E-1020 Hitachi Ion Sputter Jeol, and observed with KYKY- IO00B SEM at an acceleration voltage of 25 kV. Transmission electron microscopy (TEM) Samples were fixed in 3% (v/v) glutaraldehyde in phosphate buffer (50mM, pH7.2) at 4℃ overnight. After fixation, samples were post-fixed in 2% (v/v) OsO4 for 2 hr. After being washed in phosphate buffer, tissues were dehydrated in ethanol, infiltrated with Araldite/Embed 812 resin, and finally polymerized in Araldite/Embed resin. For electron microscopy, 90-nm-thick sections were cut with a Reichert-Jung ultrathin 13 microtome (C. Reichert Optische Werke AG, Vienna, Austria), mounted on formvarcoated gold slot grids, and post-stained with uranyl acetate and lead citrate. Cell wall structure was visualized with a Zeiss EM902A electron microscope. Measurements of cellulose, lignin, hemicellulose and silicon assay The second culm internodes of rice were freeze dried at _20°C for 1 d and 4°C for 2 d. The freeze-dried materials were ground into fine powder in liquid nitrogen. The powder was suspended in 50 mm Tris-HCl (pH 7.0), incubated for 5min 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 _-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). The dried material was hydrolyzed with 67% (v/v) H2SO4 for 1 h at room temperature, and cellulose content was determined on the appropriate dilution of the hydrolyzed sample by the phenol-sulfic acid method, as described by Dubois et al. (1956). To measure lignin content, the second internodes of culms were ground into fine powder and extracted four times with methanol. After vacuum drying, lignin content was quantified 14 according to the method described by Kirk and Obst (1988). The second internode were ground into powder and extracted twice with 70% ethanol at 70℃ for 1 hr. After vacuum drying, cell wall materials were used for assays of hemicellulose and silicon according to Van. Soest (Van. Soest et al, 1985, 1991). Molecular mapping Extraction of genomic DNA and simple sequence repeat analysis Genomic DNA of rice leaf was extracted as described by McCouch et al. (1998). Simple sequence repeat (SSR) analysis and PCR protocols were performed according to the methods described by Akagi et al. (2001). The PCR products were separated on a 3.0%-5.0% agarose gel according to the length of the amplified fragments and stained using ethidium bromide. Construction of mapping population and molecular mapping of target gene The F2 mapping populations were derived from Nipponbare /fp2, including a total of 1149 F2 plants with 297 fp2 plants was used for gene mapping. The leaf of each selected individual was used for DNA extraction and SSR analysis. A linkage map was constructed with Mapmaker/Exp version 3.0b according to the linkage data of the fp2 loci and polymorphic SSR markers in the F2 mapping population. 15 References Akagi H, Yokozeki Y,Inagaki A, Mori K, Fujimura T (2001). Micron, a microsatellite-targeting transposable element in the rice genome. Mol Genet Genomics 266,471-480 Arioli, Liangcai Peng, Andreas S. Betznet et al. (1998). 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Methods Enzymol. 161, 87–101. Li YH, Qian Q, et al. (2003). BRITTLE CULM1, which encodes a COBRA-Like protein affets the mechanical properties of rice plants. Plant Cell 15, 2020–2031. McCouch S R, Kochert G, Yu Z H, et al. (1988). Molecular mapping of rice chromosomes. Theor Appl Genet 76, 815–820 Mathilde Fagard, Thierry Desnos, Thierry Desprez et al. (2000). PROCUSTE1 encodes a cellulose synthase required for normal cell elongation specifically in roots and dark-grown hypocotyls of Arabidopsis. Plant Cell 12, 2409-2423. Mou ZL, He YK, Dai Y, Liu XF, Li J (2000). Deficiency in fatty acid synthase leads to premature cell death and dramatic alteration in plant morphology. Plant Cell 12, 405–417. 17 Nagato Y, Yoshimura A (1998). Report of the committee on gene symbolization, nomenclature and linkage groups. Rice Genet Newslett 15, 13–74. Neil G. Taylor, Wolf-Rüdiger Scheible, Sean Cutler, Chris R. Somerville, Simon R. Turner (1999). The irregular xylem3 Locus of Arabidopsis encodes a cellulose synthase required for secondary cell wall synthesis. Plant Celll 11, 769–779. Qian Q, Li YH et al. (2001). Isolation and genetic characterization of a fragile plant mutant in rice. Chin. Sci. Bull. 46, 2082–2085. Shen HS, Chen JC, Zeng DL, Tu JF, Tang BS, Teng S (2004). Dynamic analysis on composition of cell wall for low-fiber mutation rice. Science Agricultura Sinica 37, 943-947. Stéphanie Robert, Adeline Bicheta,, Olivier Grandjean et al. (2005). An Arabidopsis endo-1, 4-ß-D-Glucanase involved in cellulose synthesis undergoes regulated intracellular cycling. Plant cell 17, 3378-3389 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. Taylor, N.G., Scheible, W., Cutler, S., Somerville, C.R., 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. Turner, S.R., and Somerville, C.R. (1997). Collapsed xylem phenotype of Arabidopsis in the secondary cell wall. Plant Cell 9, 689–701. Van Soest P J, Robertson J B, Lewis B A (1991). Method for dietary fiber ,neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Sciences 74, 3583-3597. 18 Van Soest P J, Robertson J B (1985). Analysis of forages and fibrous food. A laboratory Manual for Animal Science 613. Cornell University, Ithaca, New York 74-75, 80-82. Figure 1. Phenotypes and physical properties of wild-type (WT) and fp2 mutant plants. A. Leaf mechanical strength of mutant and wild-type. B. Culm mechanical strength of mutant and wild-type. C. The force needed to break the culm of wild-type and the mutant. D. The elongation ratios of culm between the wild-type and the mutant Figure 2. The morphology of wild-type plant and the fp2 mutant plants. A. Phnotype of the wild-type and the mutant, the main culm of fp2 much shorter than that of the wild-type. B. Seedling of the wild-type and the mutant at 2-week-old, the length of leaves and roots are shorter than the wild-type. C. Longitudinal sections of the third internode of the wild-type culm, the length and width of the parenchyma cells were longer and narrower than these of the fp2. (20×) D. Longitudinal sections of the third internode of fp2 culm, the length and width of the parenchyma were shorter and wider than these in the fp2. (20×) pc: parenchyma cell Figure 3. Scanning electron micrographs showing the differences in structure between wildtype and fp2. A. Cross-section of a wild-type culm. (1450×) B. Cross-section of a fp2 culm.(1450×) C. Cross-section of a wild-type sheath.(950×) D. Cross-section of a fp2 sheath.(950×) E. Cross-section of a wild-type leaf.(1000×) F. Cross-section of a fp2 leaf.(1000×) Sc, sclerenchyma cells 19 Figure 4. Transmission electron micrographs showing the differences between the mechanical tissues cells wall and vessels cells wall between the wild type and fp2 plants. A. The cross section of the mechanical tissues cell wall of the wild type. (20000×) B. The cross section of the mechanical tissues cell wall of the fp2, note a few cells with extremely thin walls (arrow). (20000×) C. The cross section of the vessel cell wall of the wild type. (20000×) D. The cross section of the vessel cell wall of the fp2. (20000×) Figure 5. Changes in the contents of the culm segments between the wild type (WT) and fp2. A. Cellulose content of the culm segments from wild-type (WT) and fp2 plants. B. Lignin content of the culm segments from wild-type (WT) and fp2 plants. C. Hemicellulose content of the culm segments from wild-type (WT) and fp2 plants. D. Silicon content of the culm segments from wild-type (WT) and fp2 plants. Figure 6. The location of fp2 gene in the molecular linkage map on the chromosome 10 Figure 7. Segregation of SSR marker RM467 in F2 population. P1: fp2 P2: Nipponbare. M: DNA maker Figure 8. Segregation of SSR marker RM271 in F2 population. P1: fp2 P2: Nipponbare. M: DNA maker 20 Table 1 Morphology of culms, leaves and roots of the wild-type plant and the fp2 plant Morphology wild type fp2 Main culm Height (cm) ~115.2 ± 2.8 ~75.4 ± 2.4 Diameter (cm) ~0.55 ± 0.035 ~0.6 ± 0.026 Internode number 5 5 The first leaf Length (cm) ~50.6 ± 4.6 ~35.5 ± 5.4 Width (cm) ~2.3 ±0.05 ~2.2 ± 0.04 ~10.5 ± 0.36 ~7.5 ± 0.28 ~37.7 ± 1.2 ~31 ± 1.4 Length of root (2-week-old) (cm) Length of parenchyma Cell (µm) a. The third internodes was used for measurement of the culm diameter b. A total of 20 of plants were used for measurement of all the data. Table 2 Segregation of F2 population from the crosses between fp2 and three varieties Cross WT † Mutant † † Total χc2(3;1) P 9311/fp2 944 333 1277 0.79 R527/fp2 952 324 1256 0.43 0.90~0.80 Nipponbare/fp2 852 297 1149 0.44 0.90~0.80 †, 0.70~0.50 number of wild type plants; † †, number of brittle mutant plants. 21 fp2 fp2 WT WT B A Elongation ratio(%) Breaking force(kg) culm 16 6 12 5 10 4 8 3 6 4 2 2 1 0 0 C culm 7 14 wt fp2 D WT fp2 Figure 1 22 MT MT Root fp2 A fp2 B C pc D pc Figure 2. 23 A sc B C sc sc D sc E sc sc F Figure 3. 24 A C B D Figure 4. 25 cellulose contnet(mg/g) lignin content(mg/g) 400 140 350 120 300 100 250 80 200 60 150 40 100 20 50 0 0 WT A fp2 B hemicellulose content(mg/g) 350 WT fp2 silicon content(mg/g) 9 8 300 7 250 6 200 5 150 4 3 100 2 50 1 0 C 0 WT fp2 D WT fp2 Figure 5. Figure 6. 26 fp2 plants WT plants P 2 P1 M Figure 7. P2 P 1 M fp2 plants Figure 8. 27