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Light-Dependent Intracellular Positioning of Mitochondria
in Arabidopsis thaliana Mesophyll Cells
Md. Sayeedul Islam1,2,∗, Yasuo Niwa3 and Shingo Takagi1
Regular Paper
1Department of Biological Sciences, Graduate School of Science, Osaka University, Machikaneyama-cho 1-1, Toyonaka, Osaka,
560-0043 Japan
2Department of Genetic Engineering and Biotechnology, Rajshahi University, Rajshahi-6205, Bangladesh
3Laboratory of Plant Cell Technology, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka,
52-1 Yada, Shizuoka, 422-8526 Japan
Mitochondria, the power house of the cell, are one of the
most dynamic cell organelles. Although there are several
reports on actin- or microtubule-dependent movement of
mitochondria in plant cells, intracellular positioning and
motility of mitochondria under different light conditions
remain open questions. Mitochondria were visualized in
living Arabidopsis thaliana leaf cells using green fluorescent
protein fused to a mitochondrion-targeting signal. In
darkness, mitochondria were distributed randomly in
palisade cells. In contrast, mitochondria accumulated
along the periclinal walls, similar to the accumulation
response of chloroplasts, when treated with weak blue
light (470 nm, 4 µmol m−2 s−1). Under strong blue light
(100 µmol m−2 s−1), mitochondria occupied the anticlinal
positions similar to the avoidance response of chloroplasts
and nuclei. While strong red light (660 nm, 100 µmol m−2 s−1)
induced the accumulation of mitochondria along the
inner periclinal walls, green light exhibited little effect on
the distribution of mitochondria. In addition, the mode of
movement of individual mitochondria along the outer
periclinal walls under different light conditions was
precisely analyzed by time-lapse fluorescence microscopy.
A gradual increase in the number of static mitochondria
located in the vicinity of chloroplasts with a time period
of blue light illumination clearly demonstrated the
accumulation response of mitochondria. Light-induced
co-localization of mitochondria with chloroplasts strongly
suggested their mutual metabolic interactions. This is the
first characterization of the light-dependent redistribution
of mitochondria in plant cells.
∗Corresponding
Keywords: Arabidopsis thaliana • Blue light • Green
fluorescent protein • Mesophyll cell • Mitochondria •
Organelle positioning.
Abbreviations: Col, Columbia; mt-GFP, mitochondriontargeted green fluorescent protein; NA, numerical aperture;
sBL, strong blue light; sGL, strong green light; sRL, strong red
light; wBL, weak blue light.
Introduction
One of the characteristic features of plant cells is rapid, continuous movement of cell organelles, which is essential for
the active growth and development of plant cells. This
applies not only to nuclei and chloroplasts, of which various
aspects of intracellular distribution and movement have
been intensively investigated for a long period of time (Britz
1979, Haupt and Scheuerlin 1990, Nagai 1993), but it has
been revealed recently that other cell organelles such as the
Golgi apparatus and peroxisomes are also motile (Wada and
Suetsugu 2004, Logan 2006). Although the functional significance of organelle movement in plant cells has not been
completely uncovered, its occurrence throughout the plant
kingdom suggests indispensable physiological roles. Organelle movement is often affected by various environmental
stimuli such as cold, drought, salinity, light, nutrient deficiency, temperature and physical stress (Britz 1979, Nagai
1993, Wada et al. 2003). Among all of these environmental
stimuli, light is probably the most important factor in terms
of regulation of plant organelle movement.
author: E-mail, [email protected]; Fax, +81-6-6850-5818.
Plant Cell Physiol. 50(6): 1032–1040 (2009) doi:10.1093/pcp/pcp054, available online at www.pcp.oxfordjournals.org
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Plant Cell Physiol. 50(6): 1032–1040 (2009) doi:10.1093/pcp/pcp054 © The Author 2009.
Mitochondrial positioning in Arabidopsis
Chloroplast photorelocation movement is the most
extensively analyzed organelle movement in a variety of
plant species including algae, mosses, ferns and angiosperms
(Haupt and Scheuerlin 1990, Wada et al. 2003). In general,
chloroplasts move towards low-intensity light, resulting in
accumulation along the periclinal walls (accumulation
response) to maximize light capture for photosynthesis
(Zurzycki 1955). In contrast, under high-intensity light, chloroplasts move to the anticlinal walls parallel to the direction
of incident light (avoidance response) in order to avoid surplus light (Zurzycki 1957, Kasahara et al. 2002). In different
types of plant cells, it has been reported that chloroplast
movement is driven mainly by the actin cytoskeleton (Takagi
2003, Wada and Suetsugu 2004). Recent genetic approaches
using Arabidopsis thaliana identified phototropin (phot) as
the photoreceptor involved in blue-light-dependent chloroplast movement. The accumulation response is mediated by
both phot1 and phot2 redundantly, whereas the avoidance
response is mediated solely by phot2 (Jarillo et al. 2001,
Kagawa et al. 2001, Sakai et al. 2001). Several other factors
assumed to function in the signal transduction pathway
have also been identified in A. thaliana (Oikawa et al. 2003,
DeBlasio et al. 2005, Suetsugu et al. 2005, Luesse et al. 2006).
Light-dependent nuclear relocation movement in nondividing mature plant cells was reported in prothallial cells
of the fern Adiantum capillus-veneris for the first time
(Kagawa and Wada 1993, Kagawa and Wada 1995). This
unique phenomenon has also been found recently in leaf
cells of A. thaliana (Iwabuchi et al. 2007). Although relevant
photoreceptors have been identified both in Adiantum
(Tsuboi et al. 2007) and in Arabidopsis (Iwabuchi et al. 2007),
the general occurrence and physiological significance of
those responses are still not well understood compared with
chloroplast photorelocation movement (Iwabuchi and
Takagi 2008).
Mitochondria have been envisaged to serve as cellular
power plants and play a key role in cellular energy metabolism as the site of many anabolic and catabolic pathways.
Plant mitochondria have evolved distinct strategies for
genome maintenance, genetic decoding, gene regulation
and organelle segregation (Logan 2003). Furthermore, recent
research has also revealed that plant mitochondria function
as fundamental elements for metabolic interactions with
chloroplasts, for example in nitrogen assimilation, photorespiration and dissipation of excess reducing equivalents
generated from the photochemical reactions in chloroplasts
(Raghavendra and Padmasree 2003, Noctor et al. 2007,
Noguchi and Yoshida 2008). The well-known intimate association or co-localization of mitochondria with chloroplasts
and peroxisomes may be a prerequisite for such interactions
(Frederick and Newcomb 1969, Logan and Leaver 2000).
On the other hand, mitochondria are highly dynamic
organelles. Numerous studies have been carried out on
mitochondrial morphology and movement in a variety of
cells (Yaffe 1999), and it has been known that environmental
stresses affect those features of mitochondria (Logan 2006).
The molecular mechanism of mitochondrial movement has
been extensively investigated in budding yeast, in which
mitochondria exhibit a linear, bi-directional movement in a
polarized fashion from the mother cell to the bud (Boldogh
et al 2005). Plant mitochondria have also been revealed to
change their shape and position within a few seconds (Logan
and Leaver 2000), and visualized in association with actin
microfilaments (Van Gestel et al. 2002, Doniwa et al. 2007)
and microtubules (Romagnoli et al. 2007). Furthermore,
mutant plants exhibiting abnormal distribution patterns of
mitochondria were screened and partially characterized
(Logan et al. 2003). However, many questions, such as the
mechanisms underlying mitochondrial movement, positioning and distribution, still remain unanswered. The fusion of
S65T-type (replacement of serine in position 65 with threonine) green fluorescent protein (GFP) with mitochondriontargeting sequences has revealed the localization of GFP
signals in the mitochondria (Niwa et al. 1999). Using
A. thaliana stably expressing mitochondria-targeted GFP
(mt-GFP), the present article aims to ask whether mitochondria in leaf mesophyll cells change their intracellular positions depending on different light conditions. We focused in
particular on the mode of movement of individual mitochondria and co-localization with chloroplasts during those
processes.
Results
Light-induced redistribution of mitochondria in
mesophyll cells of A. thaliana
In dark-adapted palisade mesophyll cells, when viewed from
the adaxial side, mitochondria along the outer periclinal
walls seemed to be distributed uniformly over the cytoplasm
(Fig. 1A). In transverse sections of the leaves, we confirmed
that mitochondria were randomly distributed in the whole
cytoplasm (Fig. 1B). Chloroplasts were distributed along the
inner periclinal walls and the lower half of the anticlinal
walls. After continuous illumination with weak blue light
(wBL; 470 nm, 4 µmol m−2 s−1) for 4 h, mitochondria seemed
to accumulate along the outer periclinal walls (Fig. 1C).
In transverse sections of the leaves, we confirmed their
positions on the outer and inner periclinal regions of the
cells (Fig. 1D). Chloroplasts were exclusively distributed
along the outer and inner periclinal walls, known as the
accumulation response of chloroplasts. In contrast, after
continuous illumination with strong blue light (sBL; 470 nm,
100 µmol m−2 s−1) for 4 h, mitochondria occupied their positions along the anticlinal walls (Fig. 1E, F). Chloroplasts were
also distributed along the anticlinal walls, known as the
avoidance response of chloroplasts.
Plant Cell Physiol. 50(6): 1032–1040 (2009) doi:10.1093/pcp/pcp054 © The Author 2009.
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Md. S. Islam et al.
Fig. 1 Redistribution of mitochondria in palisade cells of A. thaliana exposed to wBL and sBL. GFP signals in fixed palisade cells were visualized
with confocal microscopy after dark treatment for 16 h (A, B), illumination with wBL (470 nm, 4 µmol m−2 s−1) for 4 h (C, D) and with sBL (470 nm,
100 µmol m−2 s−1) for 4 h (E, F), respectively. In the left panels (A, C, E), cells were observed from the adaxial side of the leaf. In the right panels
(B, D, F), cells in transverse sections of the leaf were observed from the side. Green, GFP signals; red, chlorophyll autofluorescence. Bars = 50 µm.
Fig. 2 shows the distribution patterns of mitochondria
and chloroplasts observed in single cells on transverse sections of the leaves after exposure to monochromatic light of
different wavelengths for 4 h. The periclinal (Fig. 2A) and
anticlinal (Fig. 2B) distributions of both organelles are clearly
seen under wBL and sBL, respectively. On the other hand,
under strong green light (sGL; 510–560 nm, 100 µmol m−2 s−1)
(Fig. 2C) or strong red light (sRL; 660 nm, 100 µmol m−2 s−1)
(Fig. 2D), mitochondria seemed to be randomly distributed
in the cells, similar to the dark-adapted cells (Fig. 1B). Chloroplasts exhibit a typical distribution pattern as observed in
the dark-adapted cells (Fig. 1B).
Semi-quantitative analysis of light-induced
redistribution of mitochondria
To characterize light-dependent redistribution of mitochondria more precisely, the distribution patterns of mitochondria in individual cells were analyzed in a semi-quantitative
manner by image processing of GFP signals. In the darkadapted cells, approximately half of the GFP signals were
detected in the periclinal regions, and the other half in the
anticlinal regions (Fig. 3A, Dark). After exposure to wBL or
sBL for 4 h, >80% of GFP signals were detected in the periclinal or anticlinal regions, respectively (Fig. 3A, wBL and sBL).
In cells after exposure to sGL for 4 h, the distribution pattern
of GFP signals exhibited little change from that obtained in
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the dark-adapted cells (Fig. 3A, sGL). On the other hand,
after exposure to sRL for 4 h, the GFP signals detected in the
periclinal regions showed a small but significant increase
compared with those of the dark-adapted cells (Fig. 3A,
sRL). We further examined the distribution patterns of GFP
signals in the inner and outer periclinal regions separately
(Fig. 3B). It becomes evident that sRL induced an accumulation of GFP signals only in the inner periclinal region.
Fig. 4 is the time course for redistribution of mitochondria induced by wBL. A gradual accumulation of GFP signals
in the periclinal regions with illumination time was clearly
detected. The response seemed to reach a plateau at 3–4 h
of illumination. In the case of illumination with sBL, the
accumulation of GFP signals in the anticlinal regions proceeded much more rapidly (data not shown). The response
seemed to be saturated within 2 h of illumination. Thus we
have succeeded in demonstrating light-induced redistribution of mitochondria in plant cells in a discernible way for
the first time.
Motion analysis of mitochondria
Next, we asked how the light-induced accumulation of mitochondria into specific intracellular regions is brought about.
After the behavior of mitochondria along the outer periclinal walls was sequentially recorded under fluorescence
microscopy, movement of the individual mitochondria was
Plant Cell Physiol. 50(6): 1032–1040 (2009) doi:10.1093/pcp/pcp054 © The Author 2009.
Mitochondrial positioning in Arabidopsis
A 100
**
Pixel (%)
80
Periclinal
Anticlinal
**
60
**
40
10
0
Dark
wBL
sBL
sGL
sRL
Inner Periclinal
Outer Periclinal
B 35
**
30
analyzed. Mitochondria move vigorously throughout the
cytoplasm, probably together with the cytoplasmic matrix
conducting the cytoplasmic streaming, but often stop,
change their direction, and form mutual attachments and
detachments. Their movement ranged from small oscillations of <1 µm to a large-scale displacement of >10 µm s−1.
Typical examples obtained at the start (Fig. 5) and after 4 h
(Fig. 6) of illumination with wBL are presented. From such
recordings, the movement of each mitochondrion was
traced (Fig. 7). We noticed that the mode of movement
of mitochondria can be divided mainly into three classes.
The first is static mitochondria exhibiting no or only very
fine movement at a velocity of <0.5 µm s−1 (yellow arrows in
Figs. 5 and 6). The second is slow mitochondria exhibiting
jerky movement in random directions at a velocity of
0.5–2.5 µm s−1 (white arrows in Fig. 5). The last one is fast
mitochondria exhibiting rapid and linear movement at a
velocity of >2.5 µm s−1 (red arrows in Fig. 5).
We examined changes in the frequency distribution
of the three classes of mitochondria induced by different
light treatment. Immediately after the dark adaptation,
approximately two-thirds of mitochondria moved rapidly
(Table 1, 0 min). The static mitochondria were very few and
25
Pixel (%)
Fig. 2 Distribution of mitochondria in single palisade cells of
A. thaliana under different light conditions. Dark-adapted leaves were
continuously illuminated for 4 h with wBL (A), sBL (B), sGL (510–
560 nm, 100 µmol m−2 s−1) (C) and sRL (660 nm, 100 µmol m−2 s−1) (D),
respectively. GFP signals in fixed palisade cells on transverse sections
were visualized with confocal microscopy. Green, GFP signals; red,
chlorophyll autofluorescence. Bars = 10 µm.
20
15
10
5
0
Dark
sGL
sRL
Fig. 3 Semi-quantitative analysis of the distribution of mitochondria
in palisade cells of A. thaliana under different light conditions. The
relative distribution of GFP signals in the periclinal and anticlinal
regions of fixed palisade cells was analyzed by image processing after
dark adaptation (Dark) and 4 h illumination with wBL, sBL, sGL and
sRL (A). The distribution of GFP signals in the periclinal regions was
further examined after separation into the inner and outer periclinal
regions (B). Asterisks indicate that significant differences were detected
when compared with the value obtained after dark adaptation
(∗∗P < 0.01, Student’s t-test). Out of 10 different leaves, 10 cells were
examined for each light condition. Data shown are the means ± SE.
comprised about 4%. Under continuous illumination with
wBL, the number of static mitochondria gradually increased
and, instead, that of fast mitochondria decreased (Fig. 7;
Table 1, wBL). After 4 h of illumination, almost one-third of
mitochondria became static (Fig. 7C; Table 1, wBL). This
phenomenon ultimately resulted in the accumulation of
mitochondria on the outer periclinal regions. We noticed
Plant Cell Physiol. 50(6): 1032–1040 (2009) doi:10.1093/pcp/pcp054 © The Author 2009.
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Md. S. Islam et al.
Fig. 4 Time course for redistribution of mitochondria in palisade cells
of A. thaliana induced by wBL. The relative distribution of GFP signals
in the periclinal regions of fixed palisade cells was analyzed at 0, 1, 2, 3
and 4 h of illumination with wBL. Asterisks indicate that significant
differences were detected when compared with the value obtained at
0 h (∗∗P <0.01, Student’s t-test). Out of 10 different leaves, 10 cells were
examined for each time point. Data shown are the means ± SE.
that many of the static mitochondria, if not all, were in close
proximity to chloroplasts, as shown in Fig. 6. While sBL exhibited a similar effect to wBL (Table 1, sBL), sGL and sRL never
induced any increase in the number of static mitochondria
on the outer periclinal walls (Table 1, sGL and sRL).
Intriguingly, although the number of static mitochondria
increased under wBL, the average velocity of the slow and
fast mitochondria was accelerated in the first 30 min of illumination with wBL (Table 1, wBL). After 4 h exposure to
wBL, however, the average velocity returned to the initial
level. In the case of illumination with sBL, which induced the
accumulation of mitochondria in the anticlinal regions, the
average velocity of the slow and fast mitochondria was accelerated more rapidly and reached a more elevated level at
30 min (Table 1, sBL), which was significantly higher than the
level obtained at 30 min of wBL illumination (P < 0.01, Student’s t-test). Since the average velocity obtained at 30 min of
sBL illumination was significantly higher than those obtained
using sGL and sRL (Table 1), the effect is BL specific. Even
after 4 h of exposure to sBL, the average velocity exhibited a
significantly higher level compared with the case of wBL
(P < 0.05, Student’s t-test) and sRL (P < 0.05, Student’s t-test).
Discussion
Light-dependent intracellular positioning of
mitochondria
Intracellular positioning of mitochondria has not been characterized in detail in photosynthesizing plant cells because
of the difficulty in observing the phenomenon clearly. Using
A. thaliana stably expressing mt-GFP (Niwa et al. 1999),
1036
we revealed that mitochondria in leaf palisade cells occupy
different intracellular positions under different light conditions (Figs. 1–3). To our knowledge, this is the first characterization of light-induced mitochondrial redistribution in
plant cells. In the present study, we followed the movement
of individual mitochondria on the outer periclinal region
of the living palisade cells under a fluorescence microscope.
Mitochondria exhibit typically discrete, spherical to sausageshaped morphology regardless of whether movement
occurs, and seem to be distributed throughout the cytoplasm.
In the dark-adapted cells, most of the mitochondria exhibited rather vigorous movement at an average velocity of
2.3 µm s−1 (Table 1, 0 min). The velocity is comparable
with the previously reported values ranging from 1.4 to
2.6 µm s−1, such as in pollen tubes (Heslop-Harrison and HeslopHarrison 1987, Romagnoli et al. 2007) and A. thaliana leaf
epidermal cells (Doniwa et al. 2007). Although this state was
maintained for 15 min after the start of continuous illumination with wBL to induce the accumulation of mitochondria
in the periclinal regions, the mitochondrial motility increased
afterwards (Table 1, wBL). Thereafter, mitochondria gradually became sluggish (Table 1, wBL; Fig. 7), and many such
mitochondria were apparently in contact with chloroplasts,
which also accumulated along the periclinal walls as a result
of the accumulation response (Fig. 6). Consequently, we
concluded that the progressive loss of motility of mitochondria on the periclinal regions is the first cause of redistribution of mitochondria induced by wBL. Whether the loss of
motility is a direct effect of the interaction with chloroplasts
remains to be elucidated.
Assuming that mitochondria lose their motility when
associated with chloroplasts under illumination, we propose
that the sBL-induced accumulation of mitochondria in the
anticlinal regions is brought about through similar processes
to those demonstrated on the outer periclinal regions. Under
sBL, mitochondria may lose their motility on the anticlinal
regions where chloroplasts accumulated as a result of the
avoidance response. Since sBL accelerated the velocity of
mitochondria more rapidly and markedly compared with
wBL (Table 1), translocation of mitochondria from the periclinal regions into the anticlinal regions should proceed
efficiently. This may explain why the redistribution of mitochondria under sBL was completed more rapidly than
under wBL.
Photoreceptor systems mediating mitochondrial
positioning
Illumination with wBL or sBL caused an early acceleration
and the subsequent deceleration of the velocity of mitochondria (Table 1). This intriguing response is reminiscent
of the accumulation response of chloroplasts observed in an
aquatic angiosperm Vallisneria gigantea (Dong et al. 1996).
Plant Cell Physiol. 50(6): 1032–1040 (2009) doi:10.1093/pcp/pcp054 © The Author 2009.
Mitochondrial positioning in Arabidopsis
Fig. 5 Movement of mitochondria in a living palisade cell of A. thaliana at 0 min of wBL illumination. Fluorescence images of the same cell were
sequentially captured at 1 s time intervals. Movement of mitochondria during a 5 s observation time was demonstrated. The yellow, white and
red arrows indicate static, slow and fast mitochondria, respectively. Bar =10 µm.
Fig. 6 Movement of mitochondria in a living palisade cell of A. thaliana at 4 h of wBL illumination. Movement of mitochondria was demonstrated
as described in the legend of Fig. 5. The yellow arrows indicate static mitochondria co-localized with chloroplasts. Bar = 10 µm.
Plant Cell Physiol. 50(6): 1032–1040 (2009) doi:10.1093/pcp/pcp054 © The Author 2009.
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Md. S. Islam et al.
Fig. 7 Changes in the mode of movement of mitochondria along the outer periclinal walls of palisade cells of A. thaliana exposed to wBL. From
the sequential fluorescence images captured at 0 min (A), 30 min (B) and 4 h (C) of wBL illumination, movements of individual mitochondria
were traced. Eight representative results obtained at each time point are demonstrated at 1 s time intervals. Bar = 10 µm.
Table 1 The mode of movement of mitochondria along the outer periclinal walls of palisade cells of A. thaliana under
different light conditions.
Illumination time
wBL
sBL
sGL
sRL
Static (%) <0.5 µm s−1
Slow (%) 0.5–2.5 µm s−1
Fast (%) >2.5 µm s−1
Velocity (µm s−1)
0 min
4
29
67
2.27 ± 0.17
15 min
4
31
65
2.54 ± 0.17
30 min
7
33
60
3.82 ± 0.16
1h
16
38
46
2h
21
42
37
4h
29
50
21
2.26 ± 0.14
15 min
4
28
68
2.87 ± 0.22
30 min
6
31
63
4.50 ± 0.16
4h
25
45
30
2.72 ± 0.16
30 min
6
34
60
2.46 ± 0.15
4h
6
32
62
2.59 ± 0.18
30 min
5
31
64
2.35 ± 0.16
4h
6
34
60
2.26 ± 0.15
From recordings taken with time-laps fluorescence microscopy at given time points, we classified mitochondria into three populations, namely, static,
slow and fast, according to their velocity. We examined the frequency distribution of mitochondria with the different modes of movement as well as
the average velocity of slow and fast mitochondria. Velocities were determined from the distance moved by individual mitochondrion in 1 s and are
given as the means ± SE. At each time point, 100 mitochondria out of 25 different cells were examined.
In the leaf epidermal cells of this plant, red light of a low fluence rate produced a rapid acceleration and a delayed deceleration of the chloroplast movement. The former response
is under the control of phytochrome and the latter occurs
depending on the operation of photosynthesis. We have also
hypothesized that the acceleration and deceleration of
mitochondrial movement in A. thaliana palisade cells are
mediated by different photoreceptor systems.
Since the early acceleration of mitochondrial movement
is induced by BL specifically and seemed to be fluence rate
dependent (Table 1), we can assume that this process is
mediated by phototropins. In A. thaliana, Kagawa and Wada
(2004) demonstrated that the velocity of chloroplasts during
the avoidance response depends on the fluence rate of
1038
incident light, and proposed that the velocity is determined
by the amount of light-activated photoreceptor molecules,
namely phot2. A similar mechanism might be involved in
regulation of the velocity of mitochondria, though the mode
of movement is not unidirectional as seen in the avoidance
response of chloroplasts. Examination of light-induced
changes in the mode of mitochondrial movement in mutant
plants deficient in phototropin functions should provide a
critical answer.
Concerning the late deceleration of mitochondrial movement, as discussed above, we propose that mitochondria
lose their motility through co-localization with chloroplasts
involved in photosynthesis. If the movements of mitochondria are hampered at specific intracellular regions, they
Plant Cell Physiol. 50(6): 1032–1040 (2009) doi:10.1093/pcp/pcp054 © The Author 2009.
Mitochondrial positioning in Arabidopsis
should be detected as specific distribution patterns of mitochondria. Under sRL, mitochondria accumulated in the inner
periclinal region (Fig. 3B), where the majority of chloroplasts
were located (Fig. 2D). Although it was almost impossible to
analyze the behavior of mitochondria along the inner periclinal walls under the present optical conditions, and it involved
only 30% of the mitochondria present in the cell, we believe
that those mitochondria lost their motility. On the other
hand, in dark-adapted cells and cells exposed to sGL, we
could not detect any specific distribution pattern of mitochondria (Figs. 1–3). Mitochondria in those cells kept active
movement at least on the outer periclinal regions (Fig. 7A,
Table 1), and it may be true of mitochondria on the
other regions of those cells. Consequently, we presume that
the well-known co-localization of mitochondria with
chloroplasts in photosynthesizing cells (Frederick and Newcomb 1969, Logan and Leaver 2000, Takagi 2003) is a lightdependent phenomenon regulated by specific photoreceptor
systems.
Materials and Methods
Plant materials
A wild-type A. thaliana (L.) Heynh. ecotype Columbia (Col)
and a transgenic A. thaliana (Col background) expressing
mitochondrion-targeted, S65T-type GFP (mt-GFP) (Niwa
et al. 1999) were used. Seeds were sown on compost and
grown under 16 h of white light (80 µmol m−2 s−1) and 8 h of
darkness at 23°C. Mature plants used in the experiments
were 4–5 weeks old and had 3–4 rosette leaves.
Light treatment
Rosette leaves were cut from the plants at the petioles,
floated on deionized water and then kept in complete darkness for 16 h. The dark-adapted specimens were illuminated
from the adaxial side with blue (470 nm), green (510–560 nm)
and red (660 nm) light as described in Iwabuchi et al. (2007).
The intensity of light was measured using a quantum sensor
and data logger (LI-1400; LI-COR Inc., Lincoln, NE, USA). We
confirmed that the fluorescence intensity of GFP in leaves of
mt-GFP plants was normal even after continuous illumination with blue light (470 nm, 100 µmol m−2 s−1) for 24 h.
Distribution of mitochondria on transverse sections
of the leaf explants
Specimens after different light treatments were fixed with
2.0% formaldehyde in a buffer solution (10 mM EGTA, 5 mM
MgSO4, 50 mM PIPES, pH 7.0) for 30 min under evacuation.
After fixation, transverse sections were prepared by hand.
Confocal laser-scanning microscopy was performed using a
confocal microscope (Fluoview; Olympus, Tokyo, Japan) in a
fluorescent mode. The microscope was equipped with a × 40
oil-immersion objective with a numerical aperture (NA) of
1.0 (UPlanApo; Olympus). Two fluorescence detection channels were used, one for the mt-GFP signal (excited at 488 nm)
and the other for chlorophyll autofluorescence (excited at
568 nm). Confocal images were collected only from the cells
and/or tissues which appeared undamaged, resembling
intact ones. All the experiments were done on palisade cells.
The distribution of GFP signals on confocal images was
semi-quantitatively analyzed by image-processing procedures. From confocal images obtained under different light
conditions, we randomly selected single cells in which GFP
signals were clearly demonstrated over the whole cytoplasm.
After each confocal image was digitized, maximum intensity
projections of GFP signals were changed into a gray image.
For all the gray images, one optimal threshold level for the
brightness of pixels was selected. On each gray image, a single
cell was separated into four parts, namely upper and lower
periclinal regions and right and left anticlinal regions. Then
we classified all pixels on each gray image with values above
the selected threshold level as white, and all other pixels as
black. After binarization of the gray images, the numbers of
white pixels in periclinal and anticlinal regions were calculated, respectively. We excluded the results obtained on
extremely narrow or wide cells, because such cells exhibited
a large imbalance between the periclinal and anticlinal area.
Motion analysis of individual mitochondria
Each specimen after a defined light treatment was simply
mounted on a glass slide with distilled water and overlaid
with a coverslip. Living palisade cells were observed and photographed using a fluorescence microscope (BX-50; Olympus, Tokyo, Japan). To take time-lapse images, the specimen
was sequentially scanned 20 times on the same region at the
same focal plane with 1 s time intervals. Images were captured by a high-sensitivity camera (Retiga-2000RV; TILL Photonics, Munich, Germany). To avoid potential damage and
non-specific effects on the cells caused by low oxygen pressure (Logan 2006), microscopic observations of one sample
were completed within 1 min. The mode of movement of
mitochondria was analyzed using image-processing software
(Adobe Photoshop 8.0.1; Adobe Systems, Mountain View,
CA, USA). From stored time-lapse images, 10 serial images
were superimposed onto one image and the movement of
each mitochondrion was traced. The velocity of mitochondria was determined from the distance each mitochondrion
moved in 1 s using image-processing software (Image Pro
Plus; Media Cybernetics, Bethesda, MD, USA).
Funding
The Ministry of Education, Culture, Sports, Science and
Technology (MEXT) Grant-in-Aid for Scientific research on
Priority Areas (No. 19039020 to S.T.); MEXT to M.S.I.
Plant Cell Physiol. 50(6): 1032–1040 (2009) doi:10.1093/pcp/pcp054 © The Author 2009.
1039
Md. S. Islam et al.
Acknowledgments
We are grateful to Dr. Takashi Hotta for his excellent technical assistance and constructive discussion.
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Plant Cell Physiol. 50(6): 1032–1040 (2009) doi:10.1093/pcp/pcp054 © The Author 2009.
(Received March 5, 2009; Accepted April 9, 2009)