Download Culm strenth of a rice brittle mutant

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
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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
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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
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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).
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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
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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
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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
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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).
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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
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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
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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,
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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
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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
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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
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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.
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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
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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
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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.
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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
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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.
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