Download Different Distribution of Cellulose Synthesizing Complexes in Brittle

Plant Cell Physiol. 40(3): 335-338 (1999)
JSPP © 1999
Short Communication
Different Distribution of Cellulose Synthesizing Complexes in Brittle and
Non-Brittle Strains of Barley
Satoshi Kimura', Naoki Sakurai2 and Takao Itoh '•3
1
2
Wood Research Institute, Kyoto University, Uji, Kyoto, 611-0011 Japan
Faculty of Integrated Arts & Sciences, Hiroshima University, Higashi-Hiroshima, 739-8521 Japan
The cause of low cellulose content in brittle mutants of
barley was studied. No differences were found in the dimension of crystalline cellulose microfibrils among mutant
and normal strains by X-ray diffraction analysis. By contrast, the number of cellulose synthesizing terminal complexes (TCs) in a selected brittle mutant, Kobinkatagi 4,
decreased to one fifth of that in the normal strain. These
findings suggest that the low cellulose content in brittle
mutants of barley is caused by the decrease in the number of TCs on its plasma membrane.
normal strains of barley (Kokubo et al. 1991). Therefore, it
is necessary to clarify the relation between the low cellulose content and the width and number of cellulose microfibrils. We examined whether the physical and chemical nature of the mutant and normal strains in barley
mentioned above is due to the difference in the width of
microfibrils or in the cellulose synthesizing ability.
We have reexamined the thickness of cell walls in
different tissues of mutant and normal strains of Kobinkatagi 4 by scanning electron microscopy. The cell wall
thickness in outer layer cells (four cells width including
epidermis and collenchyma), parenchyma cells and the cells
in vascular bundle showed significant differences between
mutant and normal strains (Fig. 1). The cell walls in outer
layer cells in the normal strain was most distinct in their
thickness compared to that in other regions. We did the
statistical analysis of the cell wall thickness in three
different regions (Table 1). The cell wall in the outer layer
cells of normal strains was 3.6±0.4yum thick, being two
times thicker than that of mutant strains (1.7±0.3//m).
The thickness of the parenchyma cell wall of normal strains
was also greater, 1.5 ± 0.2/im compared to 1.0±0.1//m, of
mutant strains. Cells in vascular bundle also showed significant differences in cell wall thickness in both normal
and mutant strains. It should be noted that the culm
strength is dependent on the cell wall thickness of the outer
cell layer in barley.
Key words: Barley — Brittle mutant — Cellulose microfibrils — TCs — X-ray diffraction.
Single-gene brittle mutants of barley (Hordeum valgare L., cv. Ohichi [fs2], cv. Shiroseto [fs2], and cv.
Kobinkatagi 4 [fs3] are spontaneous mutants and produce about 20% of cellulose in the cell walls of corresponding non-brittle barley strains that were isogenic with
the corresponding mutant strains, except for the brittle
gene (Kokubo et al. 1991). The genetic background of
Ohichi is different from that of Shiroseto. The maximum
bending stress of culms of brittle mutant strains is less than
half of that of non-brittle normal strains. The amounts of
lignin, pectin and non-cellulosic polysaccharides in the cell
walls did not show any significant differences between
mutant and normal strains. Thus, the culm brittleness of
the mutant strains was attributed to the lower cellulose
content of the cell walls.
Cellulose microfibrils are composed of bundling of
single glucan chains. Thus, either the shortening of glucan
chains or narrowing of the bundling of glucan chains may
affect the low cellulose content in the brittle mutants of
barley. This means that the low cellulose content in the
mutants are dependent on either the molecular weight of
cellulose, that is the degree of polymerization (DP) of cellulose, or the length of cellulose crystals. However, the DP
of cellulose has been reported to be similar in mutants and
There are three possible reasons for the lower cellulose content in the mutants; firstly, the width of microfibrils and the number of glucan chains within single microfibrils are the same, but the length of microfibrils is
shorter in the mutant than in the normal strains; secondly, the length and number of microfibrils are the same,
but the number of glucan chains within a single microfibril is smaller in the mutant than in the normal strains;
thirdly, the length and width of single microfibrils are the
same, but the number of microfibrils is smaller in the
mutant than in the normal strains. Kokubo et al. (1991)
reported that the length of each cellulose molecule is
almost the same between the normal and corresponding
mutant strains. Therefore, the first hypothesis should be
discarded. If the width of cellulose crystals is smaller in the
mutants than in normal strains, the lower cellulose content
3
To whom correspondence should be addressed. Tel, + 81-77438-3631; Fax, +81-774-3635; E-mail, [email protected]
335
336
TC distribution in brittle and non-brittle barley
Table 2 Crystalline width of cellulose microfibril by Xray diffraction analysis
Strain
m
Fig. 1 Scanning electron micrographs of cross section in the 4th
internode of normal (a) and mutant (b) strain of Kobinkatagi 4.
The thickness of outer layer cell is particularly different between
normal and mutant strains.
results from the small width of cellulose crystals. Alternatively, if the width of cellulose crystals in mutants is similar to that in the normal strains, then the number of
cellulose microfibrils and thus cellulose-synthesizing complexes should be smaller in mutants.
The fourth internode of the three mutant and normal
strains of barley, cv. Ohichi, Shiroseto, and Kobinkatagi 4,
was investigated by X-ray diffraction analysis. The crystal
width of cellulose microfibrils in the cell wall of this internode in these strains was between 5.2 and 5.4 nm (Table 2),
Table 1 Cell wall thickness in outer layer cell, parenchyma and cells in vascular bundle of Kobinkatagi 4
Cell wall thickness
Normal
Mutant
Outer layer cell
3.6±0.4
1.7±0.3
Parenchyma cell
1.5±0.2
1.0±0.1
Cells in vascular bundle
1.7±0.3
l.l±0.2
Ohichi
Crystalline width (nm)
Kobinkatagi 4
Shiroseto
Normal
5.3
5.3
5.4
Mutant
5.4
5.4
5.2
for all three mutant and normal strains, no distinct difference in the crystal width being found among these strains.
Clearly, the number of cellulose molecules per single microfibril is the same in the mutant and normal strains of
barley. This indicates that the brittleness is due to the fewer
cellulose microfibrils in the cell walls of mutants, resulting
in the reduction of culm strength.
Some modification must have occurred in the pathway of cellulose biosynthesis in the mutants such as the
level of UDP-Glc, breakdown of cellulose synthesizing enzyme complexes, so-called terminal complexes (TCs), activity, of individual TCs and number of TCs. Among all,
the most probable candidate causing the small number of
microfibrils and low cellulose content is the difference in
the number of TCs per unit area of plasma membrane in
the mutant cells. Finally, we investigated the distribution
of TCs in the plasma membrane of cortical and/or parenchyma cells in the mutant and normal strains of Kobinkatagi 4. The TCs in the normal strain showed frequent
distribution in the plasma membrane of collenchyma and/
or parenchyma cells (Fig. 2a), although the number of TCs
per unit area was variable in individual cells, possibly because the cellulose synthesizing activity of TCs is different from cell to cell even in the same tissue or different
among cell types such as epidermal, collenchyma, and parenchyma cells. We found TCs in the mutant strain with a
very low frequency of distribution (Fig. 2b). In order to
prove these observations, we made a statistical analysis
on the distribution of TCs by measuring the number of
rosettes per unit area as shown in Figure 3. The data were
obtained from the observation of 19 cells of the normal
and 13 cells of the mutant strain of Kobinkatagi 4. It is
evident that the number of rosettes in the normal strain
shows a broad range compared to that of the mutant strain
which show a distribution of rosettes in a narrow range
mostly inclined toward a small number of less than five per
unit area. The distribution of TCs in the normal strain is
significantly different from that of the mutant at the \%
level by Kolmogorov-Smirnov analysis. The average number of rosettes in the normal strain (11.8 ±1.9) was significantly higher than that in the mutant (2.5±0.4) per 200
fxm2, which means that the distribution of rosettes in the
normal strain is five times higher than that in the mutant.
TC distribution in brittle and non-brittle barley
337
Fig. 2 Freeze fracture images of P-fracture face in the 1st internode of normal (a) and mutant (b) strain of Kobinkatagi 4. The frequency of TCs (encircled) is decreased in the plasma membrane of the mutant.
The evidence is in good coincidence with the previous result
of Kokubo et al. (1991) that the sugar content of cellulose fraction was conspicuously lower in mutants than in
normal strains, ranging from 17.5 to 20.3%.
Arioli et al. (1998b) demonstrated that the Arabidopsis Rswl mutant makes non-crystalline cellulose with
disassembly of TCs upon exposure at a high temperature. They also showed that the defective gene of Rswl is a
homolog of the cotton CelA genes (Pear et al. 1996) which
encode the catalytic subunit of cellulose synthase. Thus,
direct evidence was provided that TC contains the catalytic
subunits for cellulose synthesis. We demonstrated that the
amount of cellulose microfibrils decreased corresponding
to the number of TCs in the barley mutant, supporting
their view that cellulose production is closely related to the
number of TCs; however, there are some differences between the Rswl mutant of Arabidopsis and barley mutant.
In barley mutant, there are no irregular TCs and clustered
membrane particles which are observed in the Rswl mutant
of Arabidopsis. Non-crystalline microfibrils were not de-
TC distribution in brittle and non-brittle barley
338
•
m
2
8
Ia
Kobinkatagi 4 Normal (19cells) 1
Kobinkatagi 4 Mutant (13cells) 1
78
3
r
34
• 56
0
1.
11
10
15 17
20 22
27
33
11 II 1 ....I.
5
10
15
20
25
30
35
Number of rosettes (Number / 200\im2)
Fig. 3 Frequency distribution of number of TCs in the 1st internode of normal and mutant strain of Kobinkatagi 4. The frequency of TCs in the mutant is little with narrow distribution
range. By contrast, that of the normal strain shows wide range
distribution.
tected in mutant strains of barley. These findings suggest
that the decrease in TCs in the barley mutant is caused by
the decreased secretion of TCs, not by the disassembly of
TCs. The function of the defective gene may be different in
Arabidopsis Rswl and barley mutants. The defective
gene of barley mutant may encode one of the regulator
genes involved in the secretion of TCs for plasma membrane.
Recently, Arabidopsis has been reported to contain
several homologs of cotton CelA (Csl= cellulose synthase-like gene) by the analysis of expressed sequence tags
from the database (Cutler and Somerville 1997, Arioli et al.
1998a). It is presumed that more than one gene controls the
secretion of rosettes and the expression of these genes is
differently controlled in the respective tissues and in the
stage of development. In another Arabidopsis mutant,
cellulose deposition is decreased in secondary walls of xylem cell, as a result of tissue-specific cellulose deficiency
(Turner and Somerville 1997). This mutant (Irx) has a
normal appearance, but shows a decrease in stiffness in
stem. The Irx mutant, therefore, in Arabidopsis is similar to the barley mutant. The characterization and comparison of the Irx and the defective gene of barley mutant
is prerequisite for future work.
This work is supported by Grant-in-Aid for "Research for the
Future" Program (nos. JSPS-RFTF 96L00605) from the Ministry
of Education, Sciences, Sports and Culture of Japan.
References
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many cellulose synthase-like gene products actually make cellulose?
Trends Plant Sci. 3: 164-165.
Arioli, T., Peng, L., Betzner, S., Burn, J., Wittke, W., Herth, W.,
Camilleri, C , Hofte, H., Plazinski, J., Birch, R., Cork, A., Glover, J.,
Rehmond, J. and Williamson, R.E.(1998b) Molecular analysis of cellulose biosynthesis in Arabidopsis. Science 279: 717-720.
Cutler, S. and Somerville, C. (1997) Cellulose biosynthesis: cloning in
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brittleness of barley (Hordeum vulgare L.) mutants is caused by smaller
number of cellulose molecules in cell wall. Plant Physiol. 97: 509-514.
Pear, J.R., Kawagoe, Y., Scheckengost, W.E., Delmer, D.P. and Stalker,
D.M. (1996) Higher plants contain homologues of the bacterial celA
genes encoding the catalytic subunit of cellulose synthase. Proc. Natl.
Acad. Sci. USA 93: 12637-12642.
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.
(Received August 1, 1998; Accepted December 21, 1998)