patens
We first checked whether the CO2-tactic movement
occurred in the leaves of P. patens using the double-chamber
system in the gas | gas mode (Figure 1c). One-sided
CO2 supply for 2 h in the light induced the periclinal
chloroplast positioning on the CO2-supplied side (Figure
3a). This periclinal positioning to CO2-supplied side
was observed irrespective of the CO2 supply side was
adaxial or abaxial. These results suggest that positive
CO2-tactic movement occurred regardless of the cell
polarity. This one-sided periclinal arrangement was observed in both
blue and red light (Figure 4). However, strong blue light prohibited the
chloroplasts from taking the periclinal positions suggesting that the
strong blue light induced avoidance response, which masked the
CO2-tactic movement. In the dark,
CO2-tactic movement was not observed and the
chloroplasts remained at the anticlinal positions (Figure 4).
3.2 Effect of DCMU on chloroplast CO2 relocation
Effects of inhibition of photosynthetic electron transport on
chloroplast CO2 relocation were examined with
double-chamber system in the gas | gas mode. As shown in Figure
5, the CO2-tactic movement in blue light was not
inhibited by DCMU, while that in red light was substantially inhibited.
These indicate that the CO2-tactic movement occurred
depending on at least two pathways, blue-light pathway that would be
independent of photosynthesis and the other pathway that would at least
partly depend on photosynthesis.
On the other hand, to obtain some insight into roles of ROS, we
conducted a gas | gas mode experiment using
O2-free gases containing 700 ppm and 0 ppm
CO2 and observed no inhibitory effect of the
O2 elimination on CO2-tactic movement
(Figure 6a). We also checked effects of O2-containing
and O2-free gases. There was no indication of
O2-tactic chloroplast movements (Figure 6b).
3.3 Motility system of chloroplast CO2 relocation
To assess involvement of the MFs and/or MTs in
CO2-induced chloroplast relocation, we examined the
effects of oryzalin and/or latrunculin A (Figure 7). The leaves showing
anticlinal chloroplast arrangement in the dark were treated with the
inhibitor(s) and subject to the one-sided CO2 supply in
blue light. The simultaneous treatment with oryzalin and latrunculin A
prevented chloroplasts from relocating from their dark positions.
Oryzalin-treated leaves showed CO2-dependent one-sided
periclinal chloroplast arrangement, implying the contribution of MF to
CO2 relocation. The one-sidedness of periclinal
arrangement of these leaves was greater than that of control leaves
treated with DMSO. On the other hand, interestingly, chloroplasts in the
latrunculin A-treated leaves showed the periclinal arrangement like the
light-accumulation response but the one-sided movement towards the
CO2-supplied side was absent, viz . the MT-driven
movement was not responsible for CO2 relocation in this
moss.
3.4 Time course of chloroplast CO2 relocation and effect
of CO2 concentration
In the previous observations of CO2-tactic chloroplast
movements (Senn, 1908), the duration of the one-sided
CO2 gas treatment or how much difference in
CO2 concentration needed for the one-sided
CO2 relocation was not described. Here we addressed
these problems. Starting from the anticlinal dark arrangement,
chloroplasts moved to periclinal positions one-sidedly up to 2 h after
the onset of the one-sided CO2 supply. The longer
treatment for 4 h, instead, increased the chloroplasts in the
CO2 free side (Figure 8a). When the CO2concentrations of two half-chambers were 700 ppm | 0 ppm or 400
ppm | 0 ppm, chloroplasts moved towards
CO2-supplied sides. When the CO2concentrations were 200 ppm | 0 ppm, chloroplasts were still
attracted to the CO2 side, but the proportion of
periclinal position was less biased. At the CO2concentrations of 400 ppm | 200 ppm and 700 ppm | 400
ppm, one-sided chloroplast relocation to high CO2 side
was observed. However, one-sidedness indexes decreased considerably
(Figure 8b, see one-sidedness index e in Table 1). These
observations indicate that one-sided chloroplast relocation occurred
when the CO2 concentrations between the two sides
differed, and the one-sidedness would decrease with the decrease in the
CO2 concentration difference and/or the increase in the
absolute CO2 level.
3.5 Effects of the CO2 concentration and PFD level of
blue light supplied to both sides on chloroplast relocation
For the CO2-tactic movement to occur, it might be
possible that the CO2 elimination inhibits the
chloroplasts from taking the periclinal positions, while the
CO2 supply attracts chloroplasts in the periclinal
positions. Thus, we examined the effect of the same CO2concentration supply to both sides to check the effect of the absolute
CO2 concentration on the chloroplast positioning (Figure
9). At a given PFD level of blue light, CO2 enhanced the
periclinal positioning of chloroplasts and increased the index f ,
but the effect of PFD level was also marked (Table 2). At low
CO2 concentrations, there were substantial number of
chloroplasts showed periclinal positionings. However, with the increase
in the PFD level of the blue light such chloroplasts decreased. There
was also a clear tendency that the number of chloroplasts in the central
area of periclinal cell surface decreased with the increase in the PFD
level and/or the decrease in the CO2 concentration
(Figure 9).
3.6 CO2-dependent chloroplast movement in the gas
| gel mode and inhibitory effect of DCMU
The chloroplast arrangement reported for the moss leaf placed on the gel
by Senn (1908) would be caused by the CO2 gradient
caused by photosynthetic CO2 consumption by the
chloroplasts. The lower diffusivity of CO2 in the liquid
phase than in the air would result in a decrease in CO2concentrations around the chloroplasts due to their CO2fixation in the light. The window of the double chamber was covered with
a transparent gellan gum sheet, on which P. patens leaf samples
were placed (Figure 10c). The same gas was introduced to both
half-chambers. In blue light, chloroplasts showed periclinal arrangement
on the air side in a CO2-dependent manner (Figure 10a).
In the leaf samples treated with DCMU, however, one-sided chloroplast
relocation was not observed (Figure 10b).
4 Discussion
4.1 A possible explanation for light-dependent
CO2-tactic chloroplast movement
In the present study, we revealed basic characteristics of
CO2 relocation, and the light dependence would be one of
the most important ones. The first question would be how the
CO2 relocation is dependent on light conditions. It was
reported that CO2 changed the light intensity threshold
between the low- and high-light responses in Mougeotia sp.
(Mosebach, 1958). The idea of the threshold shift of photorelocation by
CO2 is an attractive explanation for the light dependent
CO2-tactic movement. If the increase in the
CO2 concentration increases the threshold PFD level for
light-avoidance response, the one-sided CO2 supply
induces the chloroplasts in the lower CO2 side to show
the light-avoidance response while chloroplasts in higher
CO2 side to show the light-accumulation response, which
results in a CO2-dependent one-sided periclinal
arrangement. Indeed, the threshold shift by CO2 may
explain clearly not only the present result in Figure 9 and Table 2 but
also all the other data obtained in the present study. However, it is
interesting to point out that, in Mougeotia sp., the
CO2 elimination increased the threshold light intensity
of chloroplast orientation change (Mosebach, 1958): the threshold shift
occurred in the direction contrary to the direction observed in the
present study.
The experiments using DCMU inferred the presence of a
photosynthesis-dependent CO2 relocation mechanism, in
addition to the photosynthesis-independent mechanism (Figure 5). The
red-light dependence of the chloroplast CO2 relocation
would be at least partly owing to photosynthesis driven by red light.
Since it was known that the phytochrome-dependent chloroplast
photorelocation was inhibited by far-red light and DCMU inVallisneria gigantea , an aquatic angiosperm (Dong et al., 1995),
and that the red-light response in P. patens was dependent on
phytochrome (Mittmann et al., 2004), it is necessary to examine
involvement of phytochrome in the photosynthesis-dependent
CO2 relocation pathway in P. patens leaves.
Preceding studies on chloroplast relocation of P. patens have
been carried out using the protonemata. When P. patensprotonemata were grown in weak red light, red light became effective in
inducing chloroplast photorelocation, while, in white-light-grown
protonemata, the red-light response was not observed (Kadota et al.,
2000). In the present report, using the leaves of P. patens grown
in white light, we revealed that not only blue light, but also red light
induced chloroplast CO2 relocation (Figure 4). The PFD
threshold between the blue-light-accumulation and -avoidance responses
in land plants ranges from 10 to 50 µmol m-2s-1(Zurzycki, 1962; Zurzycki, 1967; Yatsuhashi &
Wada, 1990; Trojan & Gabrys, 1996), but the threshold in protonemata
of P. patens was reported to be above 360 µmol
m-2 s-1 (Kadota et al., 2000, Sato
et al., 2001). In the present study using leaves of P. patens , we
found that the threshold was around 60 ~ 70 µmol
m-2 s-1 (Figure 9). The critical
difference in the present report and the previous studies remains
unanswered. We also note that the light avoidance overrode the one-sided
periclinal arrangement induced by CO2 (Figure 4). We
suppose this observation can be compared to the fact that the light
avoidance suppressed the epistrophe in Arabidopsis thalianaleaves (Tholen et al., 2008).
4.2 Motility system of CO2 relocation
The blue-light response of chloroplast relocation in P. patensprotonemata is known to rely on the MF and MT motility systems (Sato et
al., 2001). We also observed both the oryzalin-insensitive MF-based
movement and latrunculin A-insensitive MT-based chloroplast movement.
More importantly, the former was responsible for the
CO2-tactic movement, while the latter was not (Figure
7). Furthermore, the oryzalin-treated leaves showed more active
CO2-tactic movement than the control leaves, indicating
that the oryzalin-sensitive MT-system competitively suppressed the
CO2-tactic movement (see the difference in the eindex in Figure 7). This may also be related to the smaller effect of
CO2 supplied to both sides at the same concentration
than the effect of light irradiation (Figure 9 and Table 2b). In this
experiment as well, the CO2 effect on chloroplast
movement would be suppressed by the CO2-insensitive MT
system.
4.3 Ecological aspect of CO2 relocation
We observed the CO2-tactic chloroplast movement in the
gas | gas mode experiments, in which two sides of the leaves
were supplied with the gases of different CO2concentrations. In nature, the heterogeneity in CO2distribution within a plant cell is thought to be mainly created by the
photosynthetic consumption of CO2 by chloroplasts. In
particular, it was noteworthy that the biased chloroplast arrangement
observed in the gas | gel mode experiments was lost by
inhibiting photosynthesis by DCMU even in blue light (Figure 10). This
observation would confirm that the heterogeneity of CO2concentration in the cell was caused by photosynthesis. Obeying Fick’s
law, CO2 diffuses from the ambient air to the
chloroplasts along the concentration gradient. Since the
CO2 diffusion coefficient in the gas phase is greater
than that in the liquid phase by four orders of magnitude (Terashima, et
al., 2006) and the CO2 concentration is determined by
the CO2 supply and the CO2 assimilation
rate of each chloroplast at its position (Warren et al., 2007; Tholen &
Zhu 2011), the CO2 concentration in the cell steeply
decreases near the gas-liquid boundary while it is rather homogenous in
the places distant from such boundary (Ho et al., 2012). Thus, for the
CO2-tactic movement to occur, there should be the
gas-liquid boundary around the photosynthetic cells. In terrestrial
bryophytes and other land plants with gaseous intercellular spaces, such
interfaces commonly exist in photosynthetic tissues, while there are no
such interfaces in aquatic algae. In particular, in plants like mosses,
which have photosynthetic tissues exposed to the external environment,
water drops can cover the cell surface and change the
CO2 supply (Williams & Flanagan, 1996). This situation
would result in the conditional CO2 heterogeneity, and
chloroplasts would show the CO2-tactic movements towards
the water-free surfaces, which would be effective not only in increasing
the CO2 supply to the chloroplasts but also in avoiding
photoinhibition of the chloroplasts. The cost-benefit analysis of
CO2-relocation will require the discussion in context of
the cytoskeletal energetics (Hill & Kirschner, 1982; Okamoto &
Lightfoot, 1992; Leighton & Sivak, 2022) and evaluation of the cellular
CO2 conductance (Mizokami, et al., 2022).
Concluding remarks
In the present study, we observed the CO2-tactic
chloroplast movement in P. patens moss leaves by utilizing the
double side chamber with which we controlled CO2concentrations in the gases separately supplied to the adaxial and
abaxial surfaces. The chloroplast relocation occurred either in the
blue- or red-light, and not in the dark. There would be the
photosynthesis-dependent and -independent CO2 relocation
mechanisms, and the photosynthesis-dependent relocation occurred only in
the red light. We also identified MF system as the motility system
responsible for the CO2 relocation in the blue light,
while MT system would be suppressive (Figure 11). The photorelocation of
the land plant is broadly reported to be based on MF system, while,
among Streptophyta, MT-based relocation is unique to Bryophyte likeP. patens (Sato et al., 2003). Until now, the chloroplast
CO2-tactic movement has only been reported for two
species Bryophyta sensu strict . One is F. hygrometrica(Senn, 1908) and the second is P. patens by the present study.
However, our on-going extensive examinations have already revealed
CO2 relocation in some other land plant phyla:
CO2-tactic chloroplast movement would be a more general
phenomenon than it has been thought.
5 Acknowledgement
We thank Prof. K. Sakakibara (Rikkyo University) for kindly providing
the plant and training its culture, and Dr. Y. Sakai (Kobe University),
and Prof. E. Goto (Kyushu University) for advising about the observation
of chloroplast movements. The double chamber was made by Mr. S. Otsuka
(Univ. Tokyo). T. Sugiyama thanks for a scholarship from the
World-leading Innovative Graduate Study Program for Life Science and
Technology (WINGS-LST) of the University of Tokyo. The authors declare
that there is no conflict of interests.
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Tables
Table 1.
Effects of CO2 concentration on the e andf indexes of CO2-tactic chloroplast movement
shown in Figure 8b
The Kruskal-Wallis test’s p < 0.001 (N > 4
samples × 5 cells, df = 5) for both indexes, e and f . The
alphabets are the same of shown in Figure 8b, which indicate the
significant differences respectively for e and f .
Table 2.
Effects of the same CO2 concentration treatment on thef index for Figure 9
(a) f values are given as Mean±SD and Median (range) with cell
number (N). The Kruskal-Wallis test’s p < 0.001 (N
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CO2 concentration (p < 0.001. N =
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Figure legends
Figure 1.
The double-chamber system and its two experimental modes. A schematic
diagram of the double-chamber system showing the gas flows in half
chambers (a). Spectra of monochromatic blue and red LEDs (b). Two
experimental modes: the gas | gas mode (c) and the gas
| gel mode (d). In the gas | gas mode, 4
~ 5 leaves were attached to the rectangular windows
opened in a piece of aluminum foil. Two agar gel blocks (*) placed to
cover apical and basal ends of the leaves were used as water reservoirs
(bar: 1 mm) (e).
Figure 2.
The gas flow diagram of the double-chamber system. The leaves placed
over the windows open in an aluminum foil piece or those placed on a gel
sheet placed on the foil window, was set in the double chamber. The
chamber was illuminated from both side by monochromatic LED light via
the optic fibers. The chamber and thus the leaves were placed
vertically. The mixture of N2 and O2 was
humidified and then mixed with 1% CO2 in
N2. Two gas mixtures were prepared and introduced into
the respective half-chambers. The CO2 and
O2 concentrations were measured before the experiments
(a). Optical fibers hold by the brass blocks delivered light from LEDs
to illuminate the double chamber from both sides (blue arrows), and gas
mixtures were introduced to both half chambers (red block arrows) (bar:
5 cm) (b). In practice, a piece of tracing paper was inserted between
the fiber-end and the chamber on each side.
Figure 3.
Chloroplast relocation to the high CO2 concentration
side. The cross-sectional views of one-sided chloroplast arrangement are
shown (a). The gas containing 700 ppm CO2 and 21%
O2 was supplied to the adaxial/abaxial side and that
containing 0 ppm CO2 and 21% O2 gas was
to the abaxial/adaxial side for 2 h at 50 µmol m-2s-1 blue light irradiation (25 µmol
m-2 s-1 each side) (a-1/a-2). The
upper side is the adaxial surface. The bar denotes 50 µm. A cell in a
single cell-layer tissue (b inset), chloroplast positions in a cell were
categorized into ad, ad’, ant, ab’, and ab as shown in (b). Sequential
photographs were taken from the adaxial end (c-1) to the abaxial end
(c-7) of a leaf treated under the same conditions as (a-1). The boundary
of the focusing cell is shown in (c-8). The chloroplasts were
categorized into the ad (blue circles), ad’ (blue-outlined circles)
(c-2), ant (open circles) (c-3, 4, 5), ab’ (red-outlined circle), and ab
(red circle) (c-7). Bar indicates 20 µm (c).
Figure 4.
Effects of light on chloroplast CO2-relocation. The
proportion of the number of chloroplasts in each positional category to
total number of chloroplasts was calculated for each cell in the leaves
before and after the gas | gas mode experiment for 2 h. The
mean proportion is shown with a bar indicating ± S.E. in a cumulative
column. The CO2 concentrations were 0 ppm (shown as -)
| 700 ppm (shown as CO2) and the
O2 concentration was 21%. PFD levels were 50 µmol
m-2 s-1 (25 µmol
m-2 s-1 from each side) for
moderate-blue-light, 50 µmol m-2 s-1(25 µmol m-2 s-1 from each side) for
red-light irradiation, 120 µmol m-2s-1 (60 µmol m-2s-1 from each side) for high-blue-light, and 0 µmol
m-2 s-1 for dark condition.
The Kruskal-Wallis test’s p < 0.001 for both indexes,e and f . Different uppercase (for e ) /lowercase
(for f ) letters beside cumulative bars indicate significant
differences by the post-hoc DSCF pairwise comparisons, p< 0.01 or interpreted as p < 0.02 with the
Bonferroni correction for concurrent analyses in e and f .
Figure 5.
Effects of DCMU treatment on CO2 relocation. The
proportion of the number of chloroplasts in each positional category
after the gas | gas mode experiment for 2 h is shown as in
Figure 4. Leaves were treated either with 100 µM DCMU or 1% DMSO.
CO2 concentrations were 700 ppm | 0 ppm for the
adaxial and abaxial sides. O2 was 21%. The PFD level
was 40 µmol m-2 s-1 (20 µmol
m-2 s-1 from each side) for both
blue and red light. According to Kruskal-Wallis test, p< 0.001 for the differences in e index. Different
lowercase letters beside the cumulative bars indicate the significant
differences in e by the post-hoc DSCF comparisons
(p < 0.01).
Figure 6.
Effect of O2 on chloroplast movement. The proportion of
the number of chloroplasts in each positional category after the gas
| gas mode experiment for 2 h was shown (a) as in Figure 4. The
CO2 concentrations were 0 ppm (shown as -) |
700 ppm (shown as CO2). Leaves were illuminated with 40
µmol m-2 s-1 (20 µmol
m-2 s-1 from each side) blue light.
The difference in index e was significant (p <
0.001, Mann-Whitney U test) (a). In (b), the O2 was
supplied one-sidedly for 2 h. The O2 concentrations were
0% (shown in figure as -) | 21% (shown in figure as
O2). The CO2 concentration was 0 ppm.
Samples were illuminated with blue light at 40 µmol
m-2 s-1 (20 µmol
m-2 s-1 from each side). The
difference in index e between the two O2treatment was not significant (p = 0.957, Mann-Whitney U test).
Figure 7.
Effect of inhibition of cytoskeleton systems on CO2relocation. The proportion of the number of chloroplasts in each
positional category after the gas | gas mode experiment for 2 h
was shown as in Figure 4. The concentrations of the cytoskeleton
inhibitors were 50 µM for oryzalin and 10 µM for latrunculin A. The
CO2 concentrations were 0 ppm (shown in figure as -)
| 700 ppm (shown in figure as CO2).
O2 was 21%. Leaves were illuminated with blue-light at
40 µmol m-2 s-1 (20 µmol
m-2 s-1 from each side). Differences
in e and f were both statistically significant
respectively (p < 0.001, Kruskal-Wallis test). The
different alphabets besides the columns indicate significant differences
as in Figure 4. The photographs of the adaxial surfaces of leaves
treated with 700 ppm CO2 on adaxial side and 0 ppm
CO2 on abaxial side are shown (b). Bar denotes 20 µm.
Figure 8.
The time course of CO2 relocation (a) and the effect of
CO2 concentration (b). The proportion of the number of
chloroplasts in each positional category after the gas | gas
mode experiment for 1 to 4 h was shown (a) as in Figure 4. The
CO2 concentrations were 700 ppm | 0 ppm for
abaxial side. O2 was 21%. Samples were illuminated with
60 µmol m-2 s-1 (30 µmol
m-2 s-1 from each side) blue light
(a). In (b), the effect of CO2 concentration for gas
| gas experiment was closely inspected. The respective
CO2 concentration were shown in the panel.
O2 was 21%. Samples were illuminated with 50 µmol
m-2 s-1 (25 µmol
m-2 s-1 from each side) blue light
for 2 h (b).
Respectively, in both (a) and (b), the differences in indexes, eand f were statistically significant (p < 0.001,
Kruskal-Wallis test). The different alphabet letters besides the columns
indicate significant differences as in Figure 4. For values of eand f indexes in (b), see Table 1.
Figure 9.
Surface views of P. patens leaves after 1 h gas | gas
mode experiments treated with the same concentration of
CO2 on both sides of the leaves. The photographs of the
adaxial surfaces are shown with the CO2 concentrations
and the light intensities. O2 was 21%. A bar denotes 50
µm. Different alphabet letters at the left bottom of photos indicate the
significant differences in f shown in Table 2.
Figure 10.
Effects of the CO2 concentrations and DCMU on the gas
| gel mode experiments. The proportion of the number of
chloroplasts in each positional category after the gas | gel
mode experiments for 2 h was shown as in Figure 4. O2was 21%. Samples were illuminated with blue light at 50 µmol
m-2 s-1 (25 µmol
m-2 s-1 from each side) for 2 h. In
(a) the biased chloroplast arrangements induced by the gas |
gel experiment are shown. The same concentration of CO2was supplied to both the samples-attached surface and the opposite
surface of the gel sheet. The respective CO2concentrations are shown in figure. The gel-attached surface of the
samples for each experiment is represented as gel in panel. The
differences in e values were statistically significant (p< 0.001, Kruskal-Wallis test). Different alphabet letters
beside columns indicate significant differences as in Figure 5. (a). The
gas | gel experiment was conducted with 1% DMSO or DCMU
treated samples (b). The CO2 was 700 ppm. The abaxial
surface was attached to the gel sheet. Asterisks indicate the
significant difference in index e of the two experiments atp < 0.001 (Mann-Whitney U test) (b). (c) shows the four
leaves placed on a gellan gum gel sheet and sealed on a window of
aluminum foil. The bar denotes 5 mm.
Figure 11.
A model for CO2 effect on chloroplast relocation in
relation to the light dependency and the motility system in P.
patens leaf lamina cells revealed in this paper.