Overexpression of Poplar Cellulase Accelerates Growth
and Disturbs the Closing Movements of Leaves
in Sengon1[OA]
Sri Hartati, Enny Sudarmonowati, Yong Woo Park, Tomomi Kaku, Rumi Kaida,
Kei’ichi Baba, and Takahisa Hayashi*
Research Centre for Biotechnology, LIPI, Cibinong 16911, Indonesia (S.H., E.S.); and Kyoto University,
RISH, Uji 611–0011, Japan (Y.W.P., T.K., R.K., K.B., T.H.)
In this study, poplar (Populus alba) cellulase (PaPopCel1) was overexpressed in a tropical Leguminosae tree, sengon (Paraserianthes
falcataria), by the Agrobacterium tumefaciens method. PaPopCel1 overexpression increased the length and width of stems with larger
leaves, which showed a moderately higher density of green color than leaves of the wild type. The pairs of leaves on the
transgenic plants closed more slowly during sunset than those on the wild-type plants. When main veins from each genotype
were excised and placed on a paper towel, however, the leaves of the transgenic plants closed more rapidly than those of the wildtype plant. Based on carbohydrate analyses of cell walls, the leaves of the transgenic plants contained less wall-bound xyloglucan
than those of the wild-type plants. In situ xyloglucan endotransglucosylase activity showed that the incorporation of whole
xyloglucan, potentially for wall tightening, occurred in the parenchyma cells (motor cells) of the petiolule pulvinus attached to the
main vein, although the transgenic plant incorporated less whole xyloglucan than the wild-type plant. These observations
support the hypothesis that the paracrystalline sites of cellulose microfibrils are attacked by poplar cellulase, which loosens
xyloglucan intercalation, resulting in an irreversible wall modification. This process could be the reason why the overexpression
of poplar cellulase both promotes plant growth and disturbs the biological clock of the plant by altering the closing movements of
the leaves of the plant.
Overexpression of plant cellulase in plants does not
lead to a lack of cellulose; rather, it modifies the cell
walls by trimming off disordered Glc chains from the
microfibrils. This action has been demonstrated in
Arabidopsis (Arabidopsis thaliana) overexpressing poplar (Populus alba) cellulase (Park et al., 2003). Transgenic poplar overexpressing Arabidopsis cellulase
(cel1) had longer internodes and longer fiber cells
(Shani et al., 2004). Overexpression does not increase
xyloglucan depolymerization (Harpster et al., 2002),
because the reaction efficiency of plant cellulase for
xyloglucan is very low compared with that for amorphous 1,4-b-glucans (the amorphous regions of cellulose microfibrils). This is confirmed by the fact that
the enzyme has high reactive efficiency for artificial
substrates such as carboxymethylcellulose, phospho1
This work was supported by the Program for the Promotion of
Basic Research Activities for Innovative Biosciences and by JSPS
KAKENHI (grants nos. 19208016 and 19405030). This work is also
part of the outcome of the JSPS Global COE Program (E–04): In
Search of Sustainable Humanosphere in Asia and Africa.
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Takahisa Hayashi ([email protected]).
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.108.116970
552
swollen cellulose, and (1/3),(1/4)-b-glucan (Nakamura
and Hayashi, 1993). Nevertheless, the trimming of
microfibrils by cellulase might solubilize some xyloglucan that would otherwise have been intercalated
within the disordered paracrystalline domains of the
microfibrils (Hayashi, 1989). This kind of cell wall
modification in Arabidopsis causes an increase in the
size of cells in the petioles and blades of rosette leaves
and stems (Park et al., 2003). Based on relative load
extension curves, a cross-linking component appeared
to be reduced in the walls of the transgenic plants
compared with those of the wild-type plants. The
decreased cross-linking component is attributable to a
decreased amount of tethering xyloglucan and could
in turn accelerate growth by increasing plastic extensibility under turgor pressure. Thus, the overexpression of cellulase could affect wall dynamics,
particularly the turgor-related movements of plant
organs such as the opening and closing movements of
leaves and stomata, et cetera.
Poplar cellulase cDNA with 35S promoter has been
used in sengon (Paraserianthes falcataria) because the
overexpression of poplar cellulase in Arabidopsis and
poplar produced more visible effects in their leaves
(Ohmiya et al., 2003; Park et al., 2003). In these species,
leaf length and width were increased to the same extent
as the length of the blade and petiole. The question,
therefore, is whether Leguminosae plants that overexpress cellulase (in this case, sengon) show any phenotype
for leaf movements related to the walls of motor cells.
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Overexpression of Poplar Cellulose
Sengon belongs to the subfamily Mimosoideae of
Leguminosae and is native to Haiti, Indonesia, and
Papua New Guinea. The sengon variety used for
reforestation is the fastest growing tree in industrial
forests. It even thrives in marginal land, where it
grows symbiotically with nitrogen-fixing Rhizobium
and phosphorus-promoting mycorrhizal fungi. Therefore, it is a suitable species for industrial timber estates
in southeast Asian countries (Binkley et al., 2003;
Shively et al., 2004; Kurinobu et al., 2007; Siregar
et al., 2007). The sengon tree typically gains 7 m in
height per year and reaches a mean height of 25.5 m
and a bole diameter of 17 cm after 6 years. After 15
years, it reaches 39 m in height and 63.5 cm in
diameter. The tree is useful not only for timber material but also for pulp and paper. Due to its soft timber
and leaves that can be used as animal feed (Merkel
et al., 2000), the tree has a wider range of end uses than
Acacia species (Otsamo, 1998). Therefore, it is expected
to be one of the most useful tree species for industrial
forests. However, despite attempts with tree cuttings,
tissue culture techniques with multiple propagations,
and stable gene transfer using Agrobacterium tumefaciens, attempts to propagate the tree clonally have
failed. In this article, we demonstrate, to our knowledge for the first time, the production of transgenic
sengon.
Cellulose is an important component of plants and
serves as the most abundant biopolymer on earth, with
about 100 billion tons produced annually. In addition,
it is a significant biological sink for CO2. It has been
suggested that cellulases may have originally been
involved in either the repair or arrangement of cellulose microfibrils during their biosynthesis, rather than
in cellulose degradation (Hayashi et al., 2005). It has
also been reported that membrane-bound cellulase
(Korrigan) is required for cellulose biosynthesis (Nicol
et al., 1998), but nothing is known about its role in
cellulose biosynthesis.
In this study, we used the overexpression of poplar
cellulase in the sengon tree in order to increase its
growth rate. We hope, thereby, to increase the plant’s
production of raw material, not only for timber, pulp,
and paper, but also for use as a biofuel (Ragaukas et al.,
2006). Ultimately, experiments like this that result in an
increased deposition of cellulose in the stem could
produce the fastest growing tree in the world.
RESULTS
Transformation
Two-week-old hypocotyls germinated from seeds
were cut into 2- to 4-mm-long stems, the explants of
which were used for transformation in the same
manner as leaf discs for the Agrobacterium-mediated
transformation. The explants from the cut hypocotyls
formed a callus-like tissue, which was followed by
green color and shoot formation on Murashige and
Skoog (MS) medium containing 4 mM benzylaminopurine. The transformants were selected in MS medium containing kanamycin.
During several transfers of explants to fresh medium each month, shoots were induced by the addition of benzylaminopurine (Fig. 1A) under light (4,000
lux). Benzylaminopurine alone was used as a plant
hormone to induce shoots because auxin did not affect
the formation of callus tissue or shoots in the presence
of benzyladenine (Bon et al., 1998).
During direct adventitious shoot formation, parts
of the explants turned green and produced green
nodular structures to form adventitious buds at the
apical ends. The adventitious shoots (5 mm long) were
transplanted into the medium in the absence of
benzylaminopurine. After the shoots elongated to 3 cm
(Fig. 1B), they were again transplanted into fresh MS
medium in the absence of any plant hormone for the
induction of roots (Fig. 1C). No plant hormone was
used for rooting because auxin and cytokinin prevented
the induction of roots (Bon et al., 1998).
Pinnate leaflets were formed during shoot elongation and root formation, although young trees and
shoot apical meristems in adult trees form pinnate
leaves. About 30 shoots were regenerated from 400
cocultivated explants; 10 of these shoots produced
roots. Ultimately, seven independent seedlings were
obtained. Shoots and roots were also induced from the
young shoots of wild-type plants in the presence and
absence of benzylaminopurine, respectively (Fig. 1D).
It should be noted that it took about 5 to 6 months to
produce transgenic seedlings and about 2 to 3 months
to produce wild-type seedlings.
Cellulase Expression
To study the effects of cellulase on cell wall structure
and growth, we generated transgenic sengon plants
that expressed poplar cellulase (PaPopCel1) under
the control of a constitutive promoter. To assay the
expression of the transgene, we performed reverse
transcription-PCR Southern-blot analysis of mRNAs
derived from small sections excised from the petiolule pulvinus (Fig. 2A). PopCel1 mRNA was accumulated in transgenic lines 1 to 7 (trg1–trg7), and weak
signals were detected in trg4 to trg7. Also, we used an
antibody against a 15-amino acid sequence (163CWERPEDMDTPRNVY-167) for the PaPopCel1 gene
product (Ohmiya et al., 2003).
In each transgenic line (trg1–trg7), the antibody
recognized a single, 50-kD band on a western blot,
present in the petiolule pulvinus, running at a position
corresponding to the expected and actual size of the
mature cellulase (Fig. 2B). No signal was detected in
the wild-type plants.
In the pulvinus attached to the veins of the transgenic plants, cell wall fractions showed cellulase activity that was approximately 1.05- to 5.25-fold higher
than that of the wild type (Fig. 2C). The activity of
cellulase was also assessed by measuring soluble cello-
Plant Physiol. Vol. 147, 2008
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Hartati et al.
oligosaccharides, which are presumably released by
the enzyme (Fig. 2D). These oligosaccharides accumulated in the transgenic plants, as all of the transgenic
plants were found to contain far more oligosaccharides than the wild-type plants contained. The amount
of oligosaccharides was closely related to cellulase
activity levels in each of the seven nic lines. Thus, the
levels of expression and activity varied among the
transgenic plants: they were relatively high in trg1 to
trg3 and relatively low in trg7, although a trace of
cello-oligosaccharides were detected even in trg7. This
was probably because the poplar cellulase expressed
was discretely localized in the cellulose microfibrils of
the apoplastic spaces.
Based on the carbohydrate analyses of cell walls, it
appears that the petiolule pulvinus and the main vein
in the transgenic plants contained less wall-bound
xyloglucan than those in the wild-type plants (Table I).
Increased cellulase activity in the wall did not decrease
the levels of cellulose; rather, cellulose content per
plant increased with plant growth (i.e. cellulose per
milligram dry weight was not changed). The methylated sugars due to the minor components consisted
of 4-linked Xyl, 4-linked Gal, and 4-linked Man at a
constant proportion in both the transgenic and the
wild-type plants (data not shown). These methylated
sugars are probably derived from xylan, galactan, and
mannan at a ratio of 4.7:3.2:1. Therefore, the transgenic
plants differ from the wild-type plants only in the
amount of xyloglucan present in the cell walls.
Admittedly, there is no correlation between cellulase
activity (Fig. 2B) and the extent of xyloglucan solubilization (Table I). Nevertheless, the levels of soluble
cello-oligosaccharides, which could be related to in
vivo cellulase activity, were closely related to cellulase
activity levels across the seven transgenic lines and
were consistently higher in the transgenic plants than
in the wild-type plants, which again corresponds to
cellulase activity.
Growth Response of Transgenic Plants
Figure 1. Regenerating shoots and roots in transgenic and wild-type
plants. A, Regenerating shoots of the transgenic plants. Arrows indicate
adventitious buds. Bar 5 3 mm. B, Growth of regenerated transgenic
shoots. The shoots had pinnate leaflets during elongation. Bar 5 2.0
cm. C, Regenerating transgenic roots. The plantlets producing roots had
pinnate leaves. Bar 5 1 cm. D, Regenerated plantlets of wild-type
plants. Bar 5 1 cm.
We generated seven independent transgenic sengon
lines, four of which (trg1–trg4) grew significantly
better than the wild type, although the overall morphology of these transgenic plants was similar to that
of the wild type (Fig. 3). Two other transgenic lines
(trg5 and trg6) grew slightly better than the wild type,
and one (trg7) grew about as well as the wild type.
Based on the expression of the transgene, the four lines
that showed a high growth rate (approximately 20-cm
stem length) were selected for further analysis.
The transgenic sengon plants (trg1–trg4) grew faster
than the wild-type plants, although the young seedlings (less than 30 cm in height) of both types sometimes grew at the same rate. The stems of the transgenic
plants elongated faster than those of the wild-type
plants and had larger diameters (Fig. 3). The overall
morphology of the transgenic plants was similar to
that of the wild type (Fig. 4A). As with stem growth,
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Overexpression of Poplar Cellulose
Figure 2. Analysis of PaPopCel1 expression in the main vein with
petiolule pulvinus. A, Reverse transcription-PCR Southern-blot analysis of PaPopCel1 mRNA. The relative amounts of mRNAs by reverse
transcription-PCR analysis at 15 cycles are shown. B, Western-blot
analysis of cell wall proteins. Five micrograms of protein was used for
each. C, Level of cellulase activity. D, Level of cello-oligosaccharides.
Three separate main veins for each plant were used for the determination.
the leaves of the transgenic plants were greener and
larger than those of the wild type (Fig. 4B). In both
types, the length and width of the leaves increased to
the same extent as the length of the main and minor
veins, and this increase in size was even distributed
among all leaves of the plant. Parenchyma cells in
leaves of both types were identified in the central part
of the petioles. Finally, both palisade and epidermal
cells were a little larger in the leaves of the transgenic
plants than in the leaves of the wild-type plants (data
not shown).
Figure 5 shows the times at which the transgenic
and wild-type plants started leaf opening before sunrise and completed leaf closure during sunset. There
was no difference between transgenic and wild-type
plants in the starting time of leaf opening (Fig. 5A);
the leaves in both types of plants started opening
around midnight and completed opening by 5:25 AM.
In contrast, the leaf pairs closed more slowly in the
transgenic plants than in the wild-type plants. This
difference was visible in the upper, middle, and lower
parts of the petioles (Fig. 5B). In the transgenic plants,
older leaves located at the middle and bottom part of
the stems started closing 30 min later than the corresponding leaves in the wild-type plants. Likewise, in
the transgenic plants, leaves at the bottom part of the
stem completed closing more than 1 h later than the
corresponding wild-type leaves. However, both the transgenic and wild-type plants closed their leaves within a
few minutes when they were placed in darkness at
noon (data not shown). In spite of this change to their
normal conditions, they also started opening their
leaves at almost the same time (midnight) and finished
opening completely by 5:25 AM (Fig. 5A).
Interestingly, the transgene had the opposite effect
on excised main veins. When the main vein with
leaves was excised and placed on a paper towel, the
pairs of leaves from the transgenic plants closed faster
than those of the wild-type plants (Fig. 6). The younger
leaves in the upper part of each plant started closing
immediately; leaves in the middle part completed
closing more than 1 h later; and leaves in the lower
part completed closing 2 h later. The older leaves in the
middle and lower parts of the transgenic plants completed closing more than 30 min earlier than those of
the wild-type plants. Thus, when the main vein was
excised, closing was faster in the leaves of the transgenic plants than in those of the wild-type plants,
whereas in vivo, closing was slower in the transgenic
leaves.
Xyloglucan endotransglucosylase activity was detected in situ on the transverse sections of the petiolule
pulvinus attached to the main vein using either fluorescent whole xyloglucan (50 kD) or fluorescent xyloglucan heptasaccharide (XXXG; Takeda et al., 2002).
Whole xyloglucan was incorporated into the parenchyma cells (motor cells) of the pulvinus in both
genotypes, although the transgenic plants incorporated less xyloglucan than the wild-type plants (Fig. 7).
The incorporation of whole xyloglucan into the vascular bundle of the main vein was also greater in the
wild type than in the transgenic plants, although it was
incorporated into the connection between the petiolule
pulvinus and the main vein at the same rate in both
genotypes.
In contrast, the incorporation of XXXG, either into
the parenchyma cells of the pulvinus or into the
vascular bundle of the main vein, was not observed
Table I. Xyloglucan and cellulose content in cell walls
Xyloglucan was determined from 24% KOH-soluble fractions. Three
separate main veins for each plant were used for the determination. SE
values were calculated from three samples per line.
Petiolule Pulvinus
Main Vein
Plant
Xyloglucan
Cellulose
mg mg
Wild type
trg1
trg2
trg3
trg4
trg5
trg6
trg7
41.1
9.1
8.3
8.8
10.4
11.6
10.1
12.0
6
6
6
6
6
6
6
6
2.2
1.4
1.3
1.8
1.5
1.8
2.1
2.8
370
371
372
378
377
374
385
366
Plant Physiol. Vol. 147, 2008
6
6
6
6
6
6
6
6
21
32
18
20
21
28
32
38
24
Xyloglucan
Cellulose
dry weight
16.0
8.1
9.1
8.2
8.7
8.9
7.9
8.2
6
6
6
6
6
6
6
6
2.2
0.9
1.2
0.8
1.6
0.9
1.3
1.4
454
460
455
461
451
452
460
458
6
6
6
6
6
6
6
6
23
55
32
31
43
53
47
44
555
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Hartati et al.
MS medium in both the presence and absence of 4 mM
benzylaminopurine. The shoots always formed from
the callus-like tissues of explants from the cut hypocotyls, although the shoots and roots formed directly
from the explants of hypocotyls in the case of Acacia
sinuate (Vengadesan et al., 2006). Now sengon can be
Figure 3. Effects of PaPopCel1 transgenes on stem growth. A, Increase
in stem length. B, Increase in stem diameter. Black circles, trg1; white
circles, trg2; black squares, trg3; white squares, trg4; white triangles,
wild type.
in either genotype. These results show that the walls of
the parenchyma cells (motor cells) can incorporate
whole xyloglucan but not XXXG. The level of incorporation was higher in the wild-type plants than in the
transgenic plants, probably because the walls of the
transgenic plants contain less endogenous xyloglucan
molecules to act as donors. Another possible explanation is that the increased cellulase in the transgenic
plants cleaves the glucan chains to which xyloglucan
binds, so that the glucan chains are washed out,
making it more difficult for xyloglucan molecules to
attach firmly to the wall.
DISCUSSION
We have succeeded in producing transgenic sengon
plants for the first time. Seven independent shoots
regenerated from 400 cocultivated explants were demonstrated to be transgenic; this represents a transformation frequency of 1.75%. So far, we have not
succeeded in either multiple propagation of the transgenic shoots or clonal propagation by cutting their
stems. Nevertheless, shoots and roots were induced in
Figure 4. Wild-type and transgenic (trg1) plants. The wild-type and
transgenic plants are shown on the left and right, respectively, at 390 d
after adventitious shoot formation. A, Whole plants. Bar 5 10 cm. B,
Leaves. Bar 5 1.5 cm.
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Overexpression of Poplar Cellulose
Figure 5. Leaf movements of upper, middle, and
lower parts of petioles in the wild-type and transgenic
plants. A, Opening. All of the leaves are open (left).
Bar 5 4 cm. B, Closing. Closing leaves are distinguishable as yellow and white leaves (left). Bar 5 4
cm. The lower part of the petiole was defined as the
second petiole from the bottom, the middle part as the
fifth or sixth petiole from the bottom, and the upper
part as the ninth or tenth (or newest) petiole. All of the
leaves in the petiole were observed to determine the
opening and closing times of leaf pairs from start to
finish. SE values were calculated from four lines of
trg1, trg2, trg3, and trg4.
genetically modified to exhibit desirable traits, such as
easy degradation of cellulose microfibrils as well as
pathogen and insect resistance, qualities that are difficult to achieve by traditional breeding. It would also
be expected to increase the transformation frequency,
particularly the induction of roots from shoots. The
increased frequency of root induction should accelerate the biotechnological application of sengon as a
biomass resource. It should be noted that wild-type
sengon is already one of the fastest growing tree
species in the world; the transgenic sengon developed
in this study grows even faster and may ultimately be
the fastest growing tree in the world.
Transgenic sengon overexpressing poplar cellulase
(PaPopCel1) increased the size of leaves by increasing
cell volume, as other authors have demonstrated in
Arabidopsis leaves (Park et al., 2003). This phenomenon has been attributed to an increase in the specific
activity of cellulase, similar to the increase observed in
transgenic Arabidopsis and poplar (Park et al., 2003;
Shani et al., 2004). Nevertheless, no bulk degradation
of cellulose was confirmed, because the amount of
cellulose per dry weight was nearly the same in the
transgenic and the wild-type plants. Therefore, we
believe that cellulase promotes increased growth in
transgenic sengon by trimming off disordered Glc
chains from cellulose microfibrils, where some xyloglucan would otherwise be solubilized. Residual xyloglucan can adhere tightly to cellulose microfibrils,
perhaps as a monolayer coating the surface, but the
transgenic plants contained less xyloglucan bound to
cellulose microfibrils. This agrees with a previous
finding that a decrease in xyloglucan tethers accelerates cell elongation (Takeda et al., 2002).
We have found that leaf movements are somehow
disturbed by the transgene expression. The transgenic
plants opened their leaf pairs at the same time as the
wild-type plants (midnight) but started closing their
leaves 30 min later and completed closing them more
than 1 h later (Fig. 5). Nevertheless, transgenic sengon
still retains a type of circadian rhythm in the opening
and closing movements of leaves, although sterilized
sengon (in vitro culture) did not show closing movements at night, even if the plant was placed in darkness. The opening and closing movements of leaves
occur in the leaf bases (petiolule pulvinus) and are
caused by the expansion and contraction of the motor
cells. Motor cells occupy most of the space in the
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Hartati et al.
Figure 6. Closing movements of leaves whose main
vein was excised. Leaves in the vein at the upper,
middle, and lower parts of petioles are shown from left
to right. SE values were calculated from four lines of
trg1, trg2, trg3, and trg4. Bar 5 2 cm.
pulvinus, surrounding the central ring of the vascular
bundle. The expansion and contraction of these cells is
believed to result from changes in their turgor, which
is regulated in turn by the flow of K1 ions across the
cells’ thin walls.
In the case of transgenic sengon that overexpresses
poplar cellulase, an increase in wall plasticity may
cause changes in normal leaf movements, because the
movements correspond to the balance between turgor
pressure and wall pressure. Therefore, the turgor
pressure in transgenic sengon motor cells might increase during the day and decrease at night, while the
wall pressure remains constant. In the case of pea
(Pisum sativum) hypocotyls, the wall pressure in growing cells is decreased during elongation, while the
turgor pressure remains constant. Since the walls of
the motor cells in the pulvinus incorporated whole
xyloglucan but not XXXG, wall tightening rather than
loosening could be required to prevent the expansion
of the cells at a cut surface (Takeda et al., 2002).
In this case, the xyloglucan endotransglucosylase
activity could occur between high-molecular-size xyloglucans in the cell walls, where the enzyme has both
enzyme-donor and enzyme-acceptor complexes. Ueda
and Nakamura (2007) suggested that leaf movements
are controlled by the balance of the concentrations of
chemical substances involving an aglycon or a gluco-
side, which induce opening and closing, respectively.
Nakanishi et al. (2005) showed that in excised leaves of
Oxalis corymbosa, the opening movement was sensitive
to blue light but not to red light. It should be noted that
cellulase tends to affect the down-regulation of leaf
movements, whereas chemical substances and light
tend to affect the up-regulation of leaf movements.
Darwin (1880) postulated that the nyctinastic movements of plants occurred in order to hide the upper
surface of the leaves at night, because the upper leaf
epidermis was believed to be a sense organ for adaptive responses. Bunning and Moser (1969), on the
contrary, suggested that adaptive leaf movements
occurred to protect the plant’s photoperiodic rhythm
against radiation from moonlight rather than against
radiation from the leaf surfaces into the sky. This
cannot be considered a satisfactory explanation in the
case of sengon, however, because pairs of sengon
leaves start to open at night. It should be noted that
light does not induce the opening movements during
normal growth.
It is possible that the transgenic sengon plants have
slightly higher photosynthetic capability than the
wild-type plants, since the leaf pairs of the transgenic
plants close more slowly than those of the wild-type
plants. The sun always sets in Indonesia by 6:40 PM,
while the transgenic plants keep their leaf pairs open
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Overexpression of Poplar Cellulose
RNA Isolation and Reverse Transcription-PCR Analysis
Figure 7. In situ xyloglucan endotransglucosylase activity incorporating green fluorescent whole xyloglucan for 15 min on the transverse
section (200 mm) of the petiolule pulvinus attached to the main vein
of trg1. The tissues of the pulvinus and vein are shown in the upper
and lower areas, respectively, in the images of the wild-type (A) and
transgenic (B) plants. The arrows indicate the incorporated whole
xyloglucan in the parenchyma motor cells. The red color is due to the
autofluorescence of chloroplasts. Bars 5 0.5 mm.
for about 1 h after the normal sunset time. We are
performing detailed analyses of the plant’s leaf movements to determine their photosynthetic efficiency.
Total RNA was isolated from the main vein with petiolule pulvinus
(Ohmiya et al., 2000). The first-strand cDNA was synthesized using 5 mg of
total RNA at 42°C for 1 h using oligo(dT) (n 5 20) and SuperScript II reverse
transcriptase (Gibco BRL). PCR was performed with final volumes of 20 mL
containing 0.5 unit of cDNA polymerase mix (Clontech), 0.2 mM dNTPs, 3.5
mM Mg(OAc)2, and 0.4 mM gene-specific primers. The forward primer was
5#-CACCACGCAATGTGTACAAAGTAACCATC-3# (nucleotide positions 512–
540), and the reverse primer was 5#-GGGTGTATATTGGGCCTGGAAACTAGAAGTT-3# (nucleotide positions 993–1,023) with the first-strand cDNA. The
PCR was initially denatured at 94°C for 5 min and in the subsequent cycles at
94°C for 30 s. Annealing and elongation cycles were both performed for 3 min
at 68°C. PCR products were size separated by electrophoresis on a 0.9%
agarose gel and blotted onto nylon membranes (Hybond-N1; AmershamPharmacia Biotech).
Membranes were hybridized in 53 SSC, with 1.0% blocking reagent, 0.1%
lauroylsarcosine, and 0.02% SDS at 42°C, to digoxigenin-dUTP-labeled
probes. Probes were labeled using the DIG-DNA labeling kit (Roche Diagnostics) and were synthesized from PopCel1 cDNA by gene-specific primers.
Following hybridization, the membranes were washed in 23 SSC for 5 min
at room temperature and then two times in 0.13 SSC with 0.1% SDS at 68°C
for 15 min each time. The washed membranes were developed using the DIGDNA detection kit (Roche Diagnostics) for chemiluminescent detection.
Western-Blot Analysis
After the leaves were removed, the main vein with petiolule pulvinus in
the middle part of the petiole was homogenized in 20 mM sodium phosphate
buffer (pH 6.2) in a mortar, and the wall residue was washed three times. The
wall-bound proteins were extracted from the wall residue with a buffer
containing 1 M NaCl. The proteins were then subjected to electrophoresis with
10% SDS-PAGE, electrotransferred to Hybond-C Extra (Amersham), and
probed with an antibody against the PopCel sequence, followed by a second
antibody using the Toyobo ABC High-HRP immunostaining kit. Seven lines of
transgenic plants were assayed.
MATERIALS AND METHODS
Cellulase Activity Assay
Transgenic Constructs
The PaPopCel1 cDNA fragment was excised from pBluescript SK1 by
digestion with BamHI and KpnI (Nakamura et al., 1995). The GUS-coding
sequence of pBE2113 was removed from the fragment by digestion with
BamHI and SacI, and the cDNA fragment was inserted into the pBE vector
between the cauliflower mosaic virus 35S promoter and the Agrobacterium
tumefaciens NOS transcription terminator. The chimeric construct was introduced into the disarmed Agrobacterium strain LBA4404.
Plant Transformation
Agrobacterium carrying plasmid-harboring poplar (Populus alba) cellulase
cDNA (PaPopCel1) and selectable marker NPTII genes were cultured in YES
medium (0.1% yeast extract, 0.5% polypeptone, 0.5% Suc, and 0.0246%
MgSO4) containing 50 mg mL21 kanamycin. The bacterial suspension was
pelleted and resuspended with sterilized water. Seeds were germinated for
2 weeks to produce hypocotyls elongating 1 to 2 cm in length.
Pieces of sengon (Paraserianthes falcataria) stems (2–4 mm in length) excised
from their hypocotyls were dipped in diluted Agrobacterium solution (optical
density at 600 nm 5 0.1) for 5 to 10 min and put on sterile filter paper, then
cocultivated for 1 d on half-strength hormone-free MS medium. Next, the
pieces of stems were placed on MS agar medium containing 600 mg mL21
kanamycin for 2 weeks, after which they were washed with a water solution
containing 400 mg mL21 Claforan. The stems were cultured several times by
transplantation on MS agar medium containing 600 mg mL21 kanamycin and
4 mM benzylaminopurine for 2 to 4 months, under 14-h-day (4,000 lux)/10-hnight cycles, after which they were placed on a medium containing 300 mg
mL21 kanamycin for 2 weeks. Shoots 5 mm in length were excised from the
medium and cultured again on a medium containing 300 mg mL21 kanamycin in the absence of plant hormone. Roots were then formed in the
medium for 2 to 4 weeks, and the plantlets were further cultured for growth
in the medium for 2 months. Plantlets approximately 10 to 15 cm long were
planted in soil.
Each enzyme preparation was obtained from the wall residue of the main
vein with petiolule pulvinus in the middle part of the petiole with a buffer
containing 1 M NaCl, and its activity was assayed viscometrically at 35°C for
2 h, using 0.1 mL of the enzyme preparation plus 0.9 mL of 10 mM sodium
phosphate buffer (pH 6.2) containing 0.65% (w/v) carboxymethylcellulose in
semimicroviscometers from Cannon Instruments. One unit of activity is
defined as the amount of enzyme required to cause 0.1% loss in viscosity in
2 h under such conditions (Ohmiya et al., 1995). Protein was determined using
the Coomassie Plus protein assay reagent (Pierce), according to the method
described by Bradford (1976).
Determination of Cello-Oligosaccharides
After the leaves were removed, the main vein with petiolule pulvinus in
the middle part of the petiole was homogenized in 20 mM sodium phosphate
buffer (pH 6.2) in a mortar. The soluble extract was boiled for 5 min and left at
room temperature for 24 h to equilibrate the anomer configuration between
a- and b-types. After centrifugation, the amount of cello-oligosaccharides was
determined by cellobiose dehydrogenase purified from conidia spores of
Phanerochaete chrysosporium (Tominaga et al., 1999). The reaction mixture
contained 90 mU (10 mL) of cellobiose dehydrogenase, 50 mM cytochrome c (10
mL), and sample solution (70 mL) in 100 mM sodium acetate buffer, with a pH
of 4.2. After incubation for 5 min at room temperature, the A550 was determined. A linear standard curve was obtained with a standard cellobiose
solution, and an absorbance of 0.5% corresponded to approximately 270 ng
per 100 mL of reaction mixture for cellobiose.
Fractionation and Measurement of Wall Components
The main vein and petiolule pulvinus in the middle part of the petiole were
separated after the leaves had been removed. Each sample was ground in
liquid nitrogen and freeze-dried before its dry weight was determined. The
Plant Physiol. Vol. 147, 2008
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Copyright © 2008 American Society of Plant Biologists. All rights reserved.
Hartati et al.
sample was successively extracted six times with 10 mM sodium phosphate
buffer (pH 7.0) and three times with 24% KOH containing 0.1% NaBH4 at less
than 45°C for 3 h in an ultrasonic bath. The insoluble wall residue (the
cellulose fraction) was washed with water and solubilized with ice-cold 72%
sulfuric acid. Total sugar in each fraction was determined by the phenol/
sulfuric acid method (Dubois et al., 1956). The xyloglucan level was determined by the iodine/sodium sulfate method (Kooiman, 1961).
Growth Measurements
The growth response of the transgenic plants was monitored after they
were transplanted in soil and habituated for 3 weeks under nonsterile
conditions. Each stem (around 15 cm) was marked at a height of 5 cm, which
was used as a reference point for measuring the height, diameter, and number
of internodes every third day. The length of the stem was determined from the
top to the reference point. Dry weight was determined after freeze drying the
samples.
The timing of the leaf movement was determined by observing the movements every day for 20 d during both the rainy (January) and dry (May)
seasons. The closing movements of the leaves with the base of their main vein
excised occurred between 10 and 11 AM during both the rainy and dry seasons.
The veins with leaves attached were placed on paper towels immediately after
excision.
Four transgenic lines, trg1, trg2, trg3, and trg4, were used to observe the
opening and closing movements of leaves. These plants had nine or 10 petioles
each, all of which were more than 10 cm long. The lower part of the petiole
was defined as the second petiole from the bottom, the middle part as the fifth
or sixth petiole from the bottom, and the upper part as the ninth or tenth (or
newest) petiole. All of the pairs of leaves attached to the main vein in the
petiole were observed to determine the opening and closing movements.
Fluorescent Xyloglucans
Fluorescent xyloglucan (Takeda et al., 2002) was prepared by dissolving 10
mg of cyanogen bromide and 20 mg of pea (Pisum sativum) xyloglucan (50 kD)
in 1 mL of water and adjusting the pH to 11.0 by adding NaOH. The activated
polysaccharide was incubated with 4 mg of fluoresceinamine overnight at
room temperature. The fluorescein-labeled xyloglucan was purified by gel
filtration on a Sephadex G-50 column (Amersham-Pharmacia Biotech). Calculations showed that 1 mmol of fluorescein incorporated into 110 mmol of
sugar residues, corresponding to a substitution rate of 3.7 mol of fluorescein
per mole of xyloglucan. To prepare fluorescent XXXG (Fry, 1997), 40 mg of
XXXG was aminated with 2 mL of 0.4 M sodium cyanotrihydroborate in 2.0 M
ammonium chloride at 100°C for 120 min. The aminated oligosaccharide was
purified by gel filtration on Bio-Gel P-2 and incubated with 50 mg of
fluorescein isothiocyanate in sodium carbonate bicarbonate buffer (pH 9.0)
for 2 h at room temperature. The oligosaccharide-fluorescein isothiocyanate
conjugate was purified by gel filtration on Bio-Gel P-2.
In Situ Xyloglucan Endotransglucosylase Activity
Transverse sections (200 mm) of the main vein including petiolule pulvinus
with fluorescent derivatives were incubated for 15 min in 300 mL of 2 mM
MES/KOH buffer (pH 6.2) containing 0.2 mM fluorescent whole xyloglucan or
9 mM fluorescent XXXG while being shaken in darkness at 23°C. The sections
that were incubated with whole xyloglucan were washed three times in 0.01 M
NaOH for 30 min. Those incubated with XXXG were washed with 5% formic
acid in 90% ethanol for 5 min followed by 5% formic acid for 5 min (Takeda
et al., 2002). The sections were washed twice with water and examined using a
Zeiss Axioscope microscope equipped with epifluorescence illumination.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession number D32166 (PopCel1 cDNA).
ACKNOWLEDGMENTS
We thank Takahide Tsuchiya and Nobuyuki Kanzawa (Department of
Chemistry, Sophia University) for valuable discussions during the final
preparation of this article.
Received February 4, 2008; accepted March 11, 2008; published April 16, 2008.
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