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Plant Cell Physiol. 48(9): 1291–1298 (2007)
doi:10.1093/pcp/pcm095, available online at www.pcp.oxfordjournals.org
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Blue Light-Dependent Nuclear Positioning in Arabidopsis thaliana Leaf Cells
Kosei Iwabuchi
1,
*, Tatsuya Sakai
2
and Shingo Takagi
1
1
Department of Biological Sciences, Graduate School of Science, Osaka University, Machikaneyama-cho 1-1, Toyonaka, Osaka, 560-0043
Japan
2
RIKEN Plant Science Center, Suehiro-cho 1-7-22, Tsurumi, Yokohama, Kanagawa, 230-0045 Japan
The plant nucleus changes its intracellular position not
only upon cell division and cell growth but also in response to
environmental stimuli such as light. We found that the nucleus
takes different intracellular positions depending on blue light
in Arabidopsis thaliana leaf cells. Under dark conditions,
nuclei in mesophyll cells were positioned at the center of the
bottom of cells (dark position). Under blue light at 100 kmol
m2 s1, in contrast, nuclei were located along the anticlinal
walls (light position). The nuclear positioning from the dark
position to the light position was fully induced within a few
hours of blue light illumination, and it was a reversible
response. The response was also observed in epidermal cells,
which have no chloroplasts, suggesting that the nucleus has
the potential actively to change its position without chloroplasts. Light-dependent nuclear positioning was induced
specifically by blue light at `50 kmol m2 s1. Furthermore,
the response to blue light was induced in phot1 but not in
phot2 and phot1phot2 mutants. Unexpectedly, we also found
that nuclei as well as chloroplasts in phot2 and phot1phot2
mutants took unusual intracellular positions under both dark
and light conditions. The lack of the response and the unusual
positioning of nuclei and chloroplasts in the phot2 mutant
were recovered by externally introducing the PHOT2 gene
into the mutant. These results indicate that phot2 mediates the
blue light-dependent nuclear positioning and the proper
positioning of nuclei and chloroplasts. This is the first
characterization of light-dependent nuclear positioning in
spermatophytes.
Keywords: Arabidopsis thaliana — Blue light — Epidermal
cell — Mesophyll cell — Nuclear positioning —
Phototropin.
Abbreviation: BL, blue light; CaMV, cauliflower mosaic
virus; Col, Columbia; LED, light-emitting diode; Ler, Landsberg
erecta; WS, Wassilewskija.
Introduction
Nuclear movement in plant cells is a well-known
event generally seen upon cell division. For example, before
asymmetrical division that leads to the formation of root
hair cells and guard mother cells, the nucleus migrates to
the prospective division plane (for a review, see Britz 1979).
In wounded tissues, nuclei in cells adjacent to the wound
exhibit a characteristic pattern of movement before they
enter a division cycle (for a review, see Nagai 1993). In the
moss Physcomitrella patens, when subapical cells in
caulonemal filaments are induced to form a side branch
after a brief exposure to light, the nucleus migrates from the
middle of the cell toward the cross wall separating the
subapical cell from the apical cell (Doonan et al. 1986). The
nuclear migration can be seen in cultured cells such as
tobacco BY-2 cells. In pre-mitotic BY-2 cells, the nucleus
that was originally positioned at the periphery in interphase
migrates to the central region of the cell (Katsuta and
Shibaoka 1988, Katsuta et al. 1990). Nuclear movement is
also essential for the growth of tip-growing cells such as
root hair cells. In elongating root hair cells of Arabidopsis
thaliana, the position of the nucleus is always maintained at
a fixed distance from the apex (Ketelaar et al. 2002).
All the examples described above indicate that nuclear
movement plays pivotal roles in various responses involved
in plant development. On the other hand, more physiological aspects of nuclear movement have been reported in the
fern Adiantum capillus-veneris. Kagawa and Wada (1993,
1995) found that nuclei in prothallial cells of A. capillusveneris take different intracellular positions depending upon
dark and light conditions. In darkness, nuclei are located
along the anticlinal walls. In the light, in contrast, nuclei are
located along the outer periclinal walls. Since the lightdependent nuclear positioning is induced irrespective of cell
growth and cell division, it may play some physiologically
important role in plants exposed to light. However, the
physiological significance as well as photoreceptor and
motile systems involved in the response have not been
completely elucidated.
Chloroplast relocation is a most extensively analyzed
organelle movement in a variety of plant species including
algae, mosses, ferns and angiosperms (for reviews, see Britz
1979, Nagai 1993, Wada et al. 2003). In general, under low
fluence rate light, chloroplasts are distributed along the
periclinal walls perpendicular to the direction of incident
light, presumably to optimize photosynthesis. This is called
the chloroplast accumulation response. On the other hand,
*Corresponding author: E-mail, [email protected]; Fax, þ81-6-6850-6776.
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1292
Light-dependent nuclear positioning in Arabidopsis
under high fluence rate light, chloroplasts are distributed
along the anticlinal walls parallel to the direction of incident
light in order to avoid surplus light (Kasahara et al. 2002).
This is called the avoidance response. These responses are
induced specifically by blue light, and it has been revealed
that phototropin, the blue light receptor, mediates the
responses in A. thaliana. There are two phototropins,
termed phot1 and phot2, in A. thaliana. The accumulation
response is mediated by phot1 and phot2 that act redundantly, whereas the avoidance response is mediated by
phot2 alone (Jarillo et al. 2001, Kagawa et al. 2001, Sakai
et al. 2001). Recently, several components involved in
chloroplast relocation, which function downstream of
phototropins, have been identified and partially characterized in A. thaliana (Oikawa et al. 2003, DeBlasio et al. 2005,
Suetsugu et al. 2005, Luesse et al. 2006). Chloroplast
relocation appears to be regulated mainly by actin in many
kinds of plants (Takagi 2003, Wada and Suetsugu 2004).
Other than chloroplast relocation, phototropins control a wide range of responses, including phototropism
(Liscum and Briggs 1995, Sakai et al. 2001), stomatal
opening (Kinoshita et al. 2001), cotyledon/leaf expansion
(Sakamoto and Briggs 2002, Ohgishi et al. 2004), leaf
movement (Inoue et al. 2005), hypocotyl growth inhibition
(Folta and Spalding 2001), plant growth promotion
(Takemiya et al. 2005), the cytoplasmic level of Ca2þ
(Harada et al. 2003) and mRNA destabilization (Folta and
Kaufman 2003). In general, phot1 is more sensitive to blue
light than phot2; phot1 is mainly responsible for low fluence
rate responses, while phot2 is responsible for high fluence
rate responses. In A. thaliana, both phot1 and phot2 are
localized to the plasma membrane region in darkness,
while under blue light they seem to be re-localized from
the plasma membrane into the cytoplasm (Sakamoto
and Briggs 2002, Kong et al. 2006). Intriguingly, phot2 is
co-localized with the Golgi apparatus, forming punctate
structures in the cytoplasm under blue light (Kong et al.
2006). However, relationships between the localization of
phototropins and the modes of regulation of phototropinmediated responses have not been fully understood to date.
In contrast to chloroplast relocation, little is known
about light-dependent nuclear positioning in plant cells.
In this study, we found that the nucleus in leaf cells of
A. thaliana takes different intracellular positions depending
upon dark and light conditions. From the analysis using
phototropin mutants, we have concluded that the lightdependent nuclear positioning is a phot2-mediated organelle movement in plant cells.
Results
Positioning of nuclei in leaf cells of Arabidopsis thaliana
First, we observed the intracellular distribution of
nuclei in mesophyll cells underlying the adaxial epidermis
of wild-type Columbia (Col) leaves under dark and light
conditions. After dark treatment for 16 h, almost all nuclei
seemed to be located at the center of the cells (Fig. 1A–C).
To determine the position of nuclei more correctly, transverse sections of the leaves were made. We clearly observed
that the nucleus was located at the center of the bottom of
each cell (Fig. 1D). For convenience, this position is termed
the ‘dark position’. Chloroplasts were distributed along the
inner peliclinal wall and the lower half of the anticlinal
walls. On the other hand, after irradiation of dark-adapted
leaves from the adaxial side with blue light (BL) at 100 mmol
m2 s1 for 5 h, nuclei were located along the anticlinal
walls (Fig.1 E–G). From precise observations of transverse
sections of the leaves, we were assured that the nucleus was
located along the anticlinal wall (Fig. 1H). This position is
termed the ‘light position’. Chloroplasts were exclusively
distributed along the anticlinal walls, which is known as
an avoidance response of chloroplasts (Kagawa and Wada
2000). As far as we could determine, nuclei were never
located along the outer periclinal wall facing the adaxial
epidermis under either dark or light conditions.
Since both nuclei and chloroplasts were located along
the anticlinal walls after BL irradiation, nuclei might have
passively changed their positions with the chloroplasts,
performing the avoidance response. Therefore, we examined
the positioning of nuclei in epidermal cells that have no
chloroplasts. In epidermal cells of dark-adapted leaves,
nuclei were also located at the center of the bottom of cells
(Fig. 1I –L). After irradiation with BL at 100 mmol m2 s1
for 5 h, nuclei were located along the anticlinal walls as was
the case in the mesophyll cells (Fig. 1M–P). These findings
suggest that the nucleus has the potential actively to change
intracellular position without any interaction with chloroplasts. We noticed that after staining with Hoechst 33342,
nuclei exhibited various shapes, such as round (Fig. 1B) and
spindle-like (Fig. 1J), in both mesophyll and epidermal cells.
The nuclei in the light position appeared to be much more
slender than those in the dark position (compare Fig. 1B, C,
J, K with Fig. 1F, G, N, O).
Time course of light-dependent nuclear positioning
Next, the time course of nuclear positioning in mesophyll cells (Col) induced by BL was investigated (Fig. 2A).
Under a fluorescence microscope, we defined nuclei in the
light position as follows: nuclei located in the vicinity of or
along the anticlinal walls with the spindle-like shapes as
shown in Fig. 1F, G. Nuclei in other positions were defined
as being in the dark position, which means that the nuclei
were on the bottom of the cell. Before BL irradiation, 95%
of nuclei took the dark position (Fig. 2A). Within 1 h of BL
irradiation at 100 mmol m2 s1, 450% of nuclei took the
light position, and the response reached 80% within a few
hours of BL irradiation (Fig. 2A).
Light-dependent nuclear positioning in Arabidopsis
B
Mesophyll cells
Dark
A
1293
C
D
E
F
G
BL
H
J
K
Dark
I
Epidermal cells
L
N
O
BL
M
P
Fig. 1 Distribution of nuclei in leaf cells of Arabidopsis thaliana in darkness and under blue light. After dark treatment for 16 h (Dark),
leaves of wild-type plants (Columbia) were irradiated with blue light (470 nm, 100 mmol m2 s1) for 5 h (BL) (A–H, mesophyll cells; I–P,
epidermal cells). Nuclei were stained with Hoechst 33342 and observed with a fluorescence microscope (B, F, J, N). Bright-field and
fluorescence images are shown as merged pictures (C, G, K, O). Transverse sections of the adaxial part of the leaves were stained with
0.5% toluidine blue (D, H, L, P). Each arrow indicates the position of the nucleus. Bars represent 20 mm.
To examine further whether light-dependent nuclear
positioning is a reversible response, the time course of
nuclear positioning in mesophyll cells after transfer from
light conditions to dark conditions was determined.
As shown in Fig. 2B, nuclei re-changed their positions
from the light position to the dark position within several
hours of dark treatment.
Dependence of nuclear positioning on wavelength and fluence
rate of light
To determine the wavelength dependence of the
nuclear positioning, dark-adapted mesophyll cells (Col)
were continuously irradiated with blue (470 nm), green
(510–560 nm), red (660 nm) or far-red light (730 nm) at
100 mmol m2 s1 up to 5 h. Fig. 3A clearly shows that
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Light-dependent nuclear positioning in Arabidopsis
A
Dark
A
BL
100
100
Response (%)
Response (%)
75
75
50
50
25
25
0
0
0
1
2
3
Time (h)
4
470
510–560
5
660
730
Wavelength (nm)
B
B
Dark
BL
100
100
Response (%)
Response (%)
75
75
50
50
25
25
0
0
0
2
4
6
8
10
Time (h)
12
14
16
Fig. 2 Time courses of nuclear positioning in Arabidopsis thaliana
mesophyll cells. In (A), after dark treatment for 16 h (Dark),
mesophyll cells of wild-type plants (Columbia) were continuously
irradiated with blue light (BL; 470 nm, 100 mmol m2 s1).
The abscissa shows the irradiation time. The ordinate shows the
ratio of the number of cells that have nuclei located in the light
position to the total number of cells observed. In (B), mesophyll
cells of wild-type plants (Columbia), which had been irradiated
with blue light for 5 h (BL), were transferred to dark conditions
(Dark). The abscissa shows the time of dark treatment. The
ordinate is the same as in (A). Five different leaves were examined
for each time course. More than 100 cells were observed in each
leaf. Each plot shows the mean SE.
nuclear positioning from the dark position to the light
position was induced specifically by BL.
To examine the effective fluence rate of light to induce
the nuclear positioning, dark-adapted mesophyll cells (Col)
were continuously irradiated with either BL or red light at
a fluence rate ranging from 0.1 to 100 mmol m2 s1 for 5 h
(Fig. 3B). The nuclear positioning was remarkably induced
by BL at fluence rates over 50 mmol m2 s1 but not under
10 mmol m2 s1. Under BL at 0.1–10 mmol m2 s1, most
nuclei remained in the dark position, whereas chloroplasts
0.1
1
10
Fluence rate (µmol
100
m−2 s−1)
Fig. 3 Dependence of nuclear positioning on the wavelength and
fluence rate of light in Arabidopsis thaliana mesophyll cells. In (A),
mesophyll cells of wild-type plants (Columbia) were continuously
irradiated with light of 470, 510–560, 660 or 730 nm at 100 mmol
m2 s1 for 5 h. The ordinate is the same as in Fig. 2. In (B),
mesophyll cells of wild-type plants (Columbia) were continuously
irradiated with blue (470 nm, filled circles) or red (660 nm, filled
triangles) light at different fluence rates of 0.1, 1, 10, 50 and
100 mmol m2 s1 for 5 h. The ordinate is the same as in Fig. 2.
The abscissa shows the fluence rate of monochromatic light.
Five different leaves were examined for each experiment. More
than 100 cells were observed in each leaf. Each plot shows the
mean SE.
were distributed along the outer periclinal walls (data not
shown), which is known as an accumulation response of
chloroplasts (Kagawa and Wada 2000). Under red light,
in contrast, nuclei remained in the dark position irrespective
of the fluence rate (Fig. 3B).
Nuclear positioning in mesophyll cells of phototropin mutants
Since nuclear positioning to the light position was
induced specifically by BL of high fluence rates (Fig. 3),
Light-dependent nuclear positioning in Arabidopsis
B
phot1
Dark
A
D
F
BL
E
phot2
Dark
J
K
L
N
BL
M
O
P
R
Dark
Q
phot1phot2
G
H
I
S
T
U
BL
C
V
W
X
Fig. 4 Distribution of nuclei in mesophyll cells of Arabidopsis
thaliana phototropin mutants in darkness and under blue light.
After dark treatment for 16 h (Dark), leaves of each phototropin
mutant, phot1 (Ler), phot2 (WS) and phot1phot2, were irradiated
with blue light (470 nm, 100 mmol m2 s1) for 5 h (BL) (A–H,
phot1; I–P, phot2; Q–X, phot1phot2). Nuclei were stained with
Hoechst 33342 and observed with a fluorescence microscope
1295
we anticipated that phototropin, the BL receptor responsible for the light-induced chloroplast relocation (Wada
et al. 2003), may be involved in the response. Fig. 4
shows representative images of the distribution of nuclei in
phot1, phot2 and phot1phot2 mutants under dark and light
conditions. Under a fluorescence microscope, nuclei in
dark-adapted mesophyll cells of each mutant were located
at the center of the bottom of the cells, as was the case
in the wild-type plants (Fig. 4A–D, I–L, Q–T). After BL
irradiation at 100 mmol m2 s1 for 5 h, nuclei were located
in the light position in phot1, but not in phot2 and
phot1phot2 mutants (Fig. 4E–H, M–P, U–X). The lack
of response to BL was also observed in epidermal cells of
phot2 and phot1phot2 mutants but not in those of the phot1
mutant (data not shown).
Responses of nuclei to BL in mesophyll cells of wildtype plants and phototropin mutants were quantitatively demonstrated in Fig. 5. Clearly, nuclei in phot2
and phot1phot2 mutants exhibited no response to BL.
Unexpectedly, from the observations of transverse sections
of the leaves, we found that nuclei in phot2 and phot1phot2
but not phot1 mutants took unusual intracellular positions.
A considerable number of nuclei took the light position
even under dark conditions, and some nuclei were located
along the outer periclinal walls under either dark or light
conditions (data not shown). In a transformant expressing
the PHOT2 gene driven under a cauliflower mosaic virus
(CaMV) 35S promoter in the phot2 mutant, we have confirmed that the response of nuclei as well as chloroplasts
to BL was recovered in both mesophyll cells and epidermal
cells of at least three independent lines (data not shown).
In addition, the unusual positioning of nuclei and chloroplasts under dark and light conditions was also rescued
and both organelles exhibited a normal distribution as
observed in wild-type plants (data not shown). These results
indicate that phot2 is a genuine photoreceptor for the
BL-dependent nuclear positioning and also plays a crucial
role for the proper positioning of nuclei and chloroplasts.
Discussion
Nuclear positioning in plant cells
Nuclear movement is frequently associated with cell
division (for a review, see Britz 1979; Doonan et al. 1986,
Katsuta and Shibaoka 1988, Katsuta et al. 1990) or cell
elongation (Ketelaar et al. 2002). On the other hand, the
nucleus can move in response to environmental stimuli such
(B, F, J, N, R, V). Bright-field and fluorescence images are shown
as merged pictures (C, G, K, O, S, W). Transverse sections of the
adaxial part of the leaves were stained with 0.5% toluidine blue
(D, H, L, P, T, X). Each arrow indicates the position of the
nucleus. Bars represent 20 mm.
1296
Light-dependent nuclear positioning in Arabidopsis
100
Response (%)
75
50
25
0 Dark BL Dark BL Dark BL Dark BL Dark BL
WT
(Ler)
WT
(WS)
phot1
(Ler)
phot2
(WS)
phot1phot2
Fig. 5 Effects of blue light on nuclear positioning in phototropin
mutants of Arabidopsis thaliana. Dark-adapted mesophyll cells
(Dark) of wild-type plants (Ler, WS), phot1 (Ler), phot2 (WS) and
phot1phot2 mutants were continuously irradiated with blue light
(BL; 470 nm, 100 mmol m2 s1) for 5 h. The ordinate is the same as
in Fig. 2. Five different leaves were examined for each experiment.
More than 100 cells were observed in each leaf. Each plot shows
the mean SE.
as light. Kagawa and Wada (1993, 1995) reported for the
first time that nuclei in prothallial cells of the pteridophyte
A. capillus-veneris take different positions depending on
dark and light conditions. However, study of lightdependent nuclear positioning has not progressed very
much compared with that of light-induced chloroplast
relocation, mainly because of the difficulty in observing
transparent nuclei in living cells. In the present study, we
addressed the light-dependent nuclear positioning in the
spermatophyte A. thaliana and revealed for the first time
that nuclei in leaf cells change their intracellular positions
in response to BL (Figs. 1–3). Furthermore, using phototropin mutants, we clarified that the BL-dependent nuclear
positioning is mediated by phot2 (Figs. 4, 5). Consequently,
it now becomes evident that the nuclear positioning is a
phot2-mediated organelle movement in plant cells.
Under dark conditions, nuclei in the mesophyll cells
were positioned at the center of the bottom of cells (Fig. 1).
With the nucleus, a large number of chloroplasts were
also distributed in the cell bottom. Nevertheless, almost all
the nuclei were always positioned at the center of the cell
bottom. This was also the case when light-adapted leaves
were transferred to dark conditions (Fig. 2B). Consequently, the dark position of nuclei must be strictly determined through an unknown mechanism. In prothallial cells
of A. capillus-veneris, nuclei were positioned along the anticlinal walls in darkness and along the outer periclinal walls
in the light (Kagawa and Wada 1993). In the present study,
since most nuclei remained in the dark position under BL of
low fluence rates (Fig. 3B), nuclear positioning along the
outer periclinal wall is unlikely in A. thaliana or, if it does
occur, it requires illumination for 45 h. Since the pattern of
nuclear positioning is quite different between Arabidopsis
and Adiantum, the physiological significance of the response
would also be different between them. Nevertheless, we
presume that light-dependent nuclear positioning may be
conserved in plants.
At present, the physiological significance of BLdependent nuclear positioning is unknown. However, the
responsiveness of mesophyll cells to BL is very high,
i.e. almost all the nuclei take the light position within
a few hours of BL irradiation (Fig. 2A). Moreover, the
nuclear positioning is observed not only in mesophyll cells
but also in epidermal cells (Fig. 1). Therefore, the nuclear
positioning may play some important role in plants exposed
to BL of high fluence rate. As the avoidance response of
chloroplasts reduces photodamage caused by surplus light
(Kasahara et al. 2002), it is possible that the BL-dependent
nuclear positioning is one of the defense responses, for
example for reducing DNA damage caused by surplus light
including the ultraviolet region.
Involvement of phot2 in BL-dependent nuclear positioning
It is well established that the avoidance response of
chloroplasts is mediated by phot2 alone and induced by BL
at fluence rates of 32 mmol m2 s1 or higher (Sakai et al.
2001). Intriguingly, there are many similarities between the
nuclear positioning and the chloroplast avoidance response
with regard to the effective wavelength, fluence rate dependence and direction of movement. We can assume that
plant cells use an identical machinery to regulate both the
nuclear positioning and chloroplast avoidance response.
However, in a chloroplast unusual positioning1 (chup1)
mutant of A. thaliana, which is deficient in proper lightinduced chloroplast relocation, chloroplasts are aberrantly
aggregated on the cell bottom, whereas the distribution
pattern of other organelles, namely nuclei, mitochondria
and peroxisomes, is not very different from that in wild-type
plants (Oikawa et al. 2003). This indicates that although
many similarities are present between the nuclear positioning and the chloroplast avoidance response, some components involved in the machinery may be specific to each
organelle. This assumption may be supported by the fact
that the nuclear positioning was observed in epidermal cells
possessing no chloroplasts (Fig. 1).
In phot2 and phot1phot2 but not in phot1 mutants, an
unusual distribution of nuclei was observed; namely, some
nuclei were located in the light position even in darkness or
along the outer periclinal walls under both dark and light
conditions (data not shown). Chloroplasts also showed an
unusual distribution under dark and light conditions; they
Light-dependent nuclear positioning in Arabidopsis
were dispersed along whole periclinal and anticlinal walls
(Fig. 4L, P, T, X). An unusual positioning of chloroplasts
has also been reported in another mutant of A. thaliana,
J-domain protein required for chloroplast accumulation
response 1 (jac1), which is deficient in the chloroplast accumulation response (Suetsugu et al. 2005). The distribution
pattern of chloroplasts in a jac1 mutant was similar to that
in phot2 and phot1phot2 mutants (Suetsugu et al. 2005).
Thus, since phot2 and JAC1 are necessary for the proper
positioning of chloroplasts, it will be interesting to test
whether JAC1 is also involved in the positioning of nuclei.
These factors might send some signal to establish proper
positioning of organelles. Alternatively, phot2 and JAC1
might be involved in the determination of ‘cell polarity’,
which is indispensable to maintain the intracellular distribution of the organelles. If the ‘cell polarity’ is under
the control of photoreceptors such as phot2, it should be
influenced by changes in light conditions. Actually, it has
been reported that palisade cells of A. thaliana grow to
a round shape under low fluence rate light, while they
exhibit columnar shapes as a result of polarized growth
in the leaf thickness direction under high fluence rate
light (Tsukaya 2005). Thus, the ‘cell polarity’ of leaf cells
seems to be rearranged in response to changes in light
conditions.
Possible motile system for nuclear positioning
Nuclear migration in the moss Physcomitrella patens
and tobacco BY-2 cells is mediated by microtubules
(Doonan et al. 1986, Katsuta et al. 1990), whereas that in
root hair cells of A. thaliana is regulated by actin filaments
(Chytilova et al. 2000, Ketelaar et al. 2002). Furthermore,
Kandasamy and Meagher (1999) suggested a possible
interaction of actin filaments with the nucleus as well
as with chloroplasts in A. thaliana mesophyll cells. They
visualized that a basket composed of thin actin bundles
surrounded the nucleus, and thicker actin bundles
seemed to be connected directly to the nucleus or to the
nuclear basket. Therefore, actin is presumably involved
in the BL-dependent nuclear positioning, as is the case in
chloroplast relocation. We are currently examining this
possibility.
Chytilova et al. (2000) revealed that nuclei in root
hair cells exhibited astonishingly dynamic properties such
as polymorphic shape changes and a rapid, long-distance
intracellular movement using transgenic A. thaliana expressing a chimeric protein comprising the green fluorescent protein fused to the nuclear localization signal and
b-glucronidase from Escherichia coli. In the present
study, we could not follow nuclear movement in living
cells, but various morphological patterns of nuclei were
observed under a fluorescence microscope, for example
round, depressed and spindle-like (Fig. 1). Thus, a motile
1297
feature and morphological flexibility may be the nature
of the plant nucleus. Real-time observation of nuclei
is indispensable for better understanding of the BLdependent nuclear positioning and the nature of the plant
nucleus.
Materials and Methods
Plant materials
Wild-type A. thaliana (L) Heynh. ecotypes Columbia (Col),
Landsberg erecta (Ler) and Wassilewskija, and the phototropin
mutants phot1-101 (Ler background), phot2-5 (WS background)
and phot1-101phot2-5 (Sakai et al. 2001), were used. For the complementation analysis of the phot2 mutant, the PHOT2 cDNA was
cloned downstream of a CaMV 35S promoter in the pBI121 binary
vector and transformed into phot2-5 by the vacuum infiltration
method mediated by Agrobacterium tumefaciens (Bechtold et al.
1993). Transgenic plants were selected on kanamycin-containing
agar medium, as described previously (Harada et al. 2003), and the
T2 plants were used for the complementation analysis. Seeds were
sown on compost and grown under cycles of 16 h of white light
(80 mmol m2 s1) and 8 h of dark at 228C for 4–5 weeks. Rosette
leaves taken from 4- to 5-week-old plants were used.
Dark adaptation and light irradiation
For dark adaptation, leaves were detached from the plants at
the petioles, floated on deionized water in a Petri dish, and then
kept in darkness for 16 h. The dark-adapted leaves were irradiated
with blue (470 nm), red (660 nm) and far-red (730 nm) light using a
light-emitting diode (LED) light source system (MIL-C1000T for
a light source controller and MIL-U200 for a light source frame;
SMS, Osaka, Japan). LEDs used in this study were MIL-B18
(blue), MIL-R18 (red) and MIL-IF18 (far-red). Green light (510–
560 nm) was obtained from a light path of the epi-illumination
system (EFD2; Nikon, Tokyo, Japan) of a fluorescence microscope
(Optiphot-2; Nikon, Tokyo, Japan). The light source was a mercury lamp (USH-102DH; Ushio Inc., Tokyo, Japan). The intensity
of blue, green and red light was measured with a quantum sensor
and data logger (LI-1400; LI-COR Inc., Lincoln, NE, USA). The
intensity of far-red light was measured with a silicon photodiode
(S1337-1010BQ; Hamamatsu Photonics, Hamamatsu, Japan).
Staining of nuclei with Hoechst 33342
In a desiccator, leaves were fixed with 3.7% formaldehyde
in a buffer solution [137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4
and 8 mM NaH2PO4 (pH 7.2)] for 60 min under evacuation. After
fixation, leaves were stained with 5 mg ml1 Hoechist 33342
(Calbiochem, Darmstadt, Germany) þ 0.03% Triton X-100 in the
buffer solution for 1.5 h and then with a Hoechst 33342 solution
without Triton X-100 overnight. Then, specimens were observed
and photographed using a fluorescence microscope (BX-50;
Olympus, Tokyo, Japan).
Transverse sections of rosette leaves
Leaves were fixed with 2.5% glutaraldehyde in 25 mM
sodium cacodylate buffer (pH 7.2), then with 2% OsO4, dehydrated with acetone and embedded in Spurr’s resin (Spurr 1969).
Transverse sections of 0.8 mm thickness were prepared with an
ultramicrotome (Reichert Ultracut S; Leica, Vienna, Austria) and
stained with 0.5% toluidine blue. The sections were observed
and photographed using a light microscope (BX-50; Olympus,
Tokyo, Japan).
1298
Light-dependent nuclear positioning in Arabidopsis
Acknowledgements
This work was partly supported by Grants-in-Aid for
Scientific Research no. 16570033 from the Japanese Society
for the Promotion of Science and by the 21st Century Center of
Excellence (COE) Program.
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(Received March 26, 2007; Accepted July 18, 2007)
Note added in proof
Very recently, photoreceptors involved in the light-dependent
nuclear positing in the fern Adiantum capillus-veneris have been
elucidated. (Tsuboi, H., Suetsugu, N., Kawai-Toyooka, H. and
Wada, M. (2007) Phototropins and neochrome1 mediate nuclear
movement in the fern Adiantum capillus-veneris. Plant Cell Physiol.
48: 892–896).