i. Title
CO2-induced
chloroplast movement in one cell-layer moss leaves
ii. Authors
Taichi Sugiyama, Ichiro Terashima
iii. Affiliations
Department of Biological Science, School of Science, The University of
Tokyo, Tokyo, Japan
iv. Funding
Ordinary Operating/Management Expenses Grants from The University of
Tokyo.
v. Keywords
Bryophyta, carbon dioxide, chloroplasts, cytoskeletons, light,
orientation (spatial), photosynthesis,
vi. Summary Statement
Chloroplasts in one cell-layered leaf of Physcomitrium patensshowed the CO2-tactic movements in blue and red light.
This CO2 relocation was based on microfilament-motility
system and there were photosynthesis-dependent and -independent
movements.
vii. Abstract
CO2-induced chloroplast movement was reported in the
monograph by Gustav Senn in 1908: unilateral CO2 supply
to the one cell-layered moss leaves induced the positively
CO2-tactic periclinal arrangement of chloroplasts.
However, from the modern criteria, several experimental settings are
unacceptable. Here, using a model moss plant Physcomitrium
patens , we examined basic features of chloroplast
CO2-tactic relocation with a modernized experimental
system. The CO2 relocation was light-dependent and
especially the CO2 relocation in red light was
substantially dependent on photosynthetic activity. Between the
cytoskeletons responsible for chloroplast movement of P. patens ,
the microfilament mainly worked for CO2 relocation, but
the microtubule-based movement was insensitive to CO2.
The CO2 relocation was induced not only by air with and
without CO2 but also by the more realistic difference in
CO2 concentration between the two sides. In the leaves
placed on the surface of a gel sheet, chloroplasts avoided the gel side
and positioned in the air facing surface. This was also shown to be
photosynthesis dependent. Based on these observations, we propose a
working hypothesis that the threshold light intensity between the
light-accumulation and -avoidance responses of the photorelocation would
be increased by CO2, resulting in the
CO2-tactic relocation of chloroplast.
1 Introduction
Chloroplasts in photosynthetic cells move and change positions in
response to environmental and/or endogenous cues, resulting in a
specific arrangement within a cell. Such phenomenon is called
‘chloroplast relocation.’ Chloroplast relocation is supposed to optimize
the chloroplast position for its physiological activities, represented
by photosynthesis. In photosynthesis, pivotal environmental factors are
light, temperature, and CO2. Among chloroplast
relocations in response to these factors, the accumulation and the
avoidance responses in photorelocation and the cold avoidance responses
(Böhm, 1856; Haberlandt, 1876; Haupt, 1965) have been extensively
studied. Studies on chloroplast relocation employing molecular biology
have identified the responsible photoreceptors: phototropin in various
land plants, phytochrome in mosses, and neochrome in ferns and algae
(Sakai et al., 2001; Kawai et al., 2003; Kasahara et al., 2004; Mittmann
et al., 2004; Suetsugu et al., 2017), and furthermore revealed the
mechanism of temperature perception by phototropin (Kodama et al., 2008;
Fujii et al., 2017).
The motility systems for chloroplast relocation have also been attracted
attention. It is widely thought that the photorelocation and the cold
avoidance are solely driven by the actin microfilament (MF) system and
not by the microtubule (MT) system among land plants (Sato et al., 2003;
Kimura & Kodama 2016; Wada & Kong 2018). Several components like
CHUP1, KAC and PMI1 have also been identified to be involved in MF-based
photorelocation (Suetsugu et al., 2016; Wada & Kong 2018). In protonema
cells of Physcomitrium patens , however, not only MF-system but
also MT-based motile system actuates chloroplast movement induced by
blue light. In the blue light response, the chloroplast movement based
on MT system was faster than that by MF-based system. It was further
revealed that the red-light response relied only on MT (Sato et al.,
2001).
It is widely believed that the photorelocation is dependent on the
specific photoreceptors. However, it has been reported that an inhibitor
of the photosynthetic electron transport, DCMU, diminishes the
phytochrome-dependent chloroplast motility, anchorage, and thereby the
consequent positional changes in Vallisneria gigantea , an aquatic
angiosperm (Seitz, 1967; Dong et al., 1995; Dong et al.,1998; Sakai &
Takagi, 2005). DCMU also inhibits the neochrome-independent periclinal
positioning in ferns (Sugiyama & Kadota, 2011). Hydrogen peroxide,
which is partly generated from O2 molecule reduced by
photosystem I especially when chloroplasts are exposed to excess light
under stress conditions (Asada, 1999), is also known to enhance the
photorelocation (Wen et al., 2008). These observations indicate that the
photosynthetic activity is also an important factor for the light
response of chloroplast relocation.
Compared with the studies on the light and temperature effects, there
have been few recent studies directly examining effects of
CO2 concentration on chloroplast relocation. In porous
leaf tissues of land plants, chloroplasts tend to be located along the
cell surfaces facing the intercellular space while the positions
adjacent to neighboring cells are avoided. Such arrangement of
chloroplast is called ‘epistrophe’ (Haberlandt, 1886). Epistrophe of
chloroplasts has been reported broadly from the mesophyll tissues in
angiosperm leaves to the assimilation filaments in liverwort thalli
(Haberlandt, 1886; Senn, 1908; Evans et al., 1994). This chloroplast
position has been argued to facilitate CO2 supply for
photosynthetic carboxylation and to be related to positive
CO2-tactic movement of chloroplast, as it were the
‘CO2 taxis’ (Haberlandt, 1886; Evans et al.,1994).
Another interesting example of chloroplast relocation occurs in C4 plant
leaves. Chloroplasts in mesophyll cells aggregate towards the bundle
sheath cells in response to stresses like high light or drought (Yamada
et al., 2009; Kato et al., 2022). CO2 leak from the
bundle sheath cells would increase under such stress conditions (Ubierna
et al. 2013), which would lead a hypothesis that this chloroplast
aggregation is the result of positive CO2-tactic
movement. However, neither the epistrophe arrangement of chloroplasts
nor the chloroplast aggregation in C4 plants has been proved to be
CO2 relocation. This is due to a difficulty in
distinguishing the CO2-induced relocation from other
chloroplast relocations like photorelocation. Also difficult is to
control the CO2 concentration gradient in one cell in
the complicated and compound tissues to test whether the gradient of
CO2 affects the chloroplast position.
There was an observation of CO2 effect on chloroplast
relocation in a filamentous alga Mougeotia sp. A single
ribbon-shaped chloroplast in a cell takes a horizontal orientation
perpendicular to the light direction in low light and the profile
(parallel) orientation in high light. CO2 elimination
caused an increase in the threshold light intensity that caused the
orientational change from the horizontal to profile orientations
(Mosebach, 1958). However, this plant, in which the chloroplast just
rotates and does not migrate, is not suitable for the study of
CO2-tactic movement.
The phyllid, simple one cell-layered leaf of mosses would enable us to
manipulate CO2 distribution in each cell and provide an
ideal system to study the CO2-tactic movement of
chloroplasts separately from other chloroplast relocation mechanisms.
Indeed, taking this advantage of the moss leaf, Senn (1908) assessed the
CO2-tactic movement of chloroplasts more than a hundred
years ago. He first found an interesting chloroplast position in a leaf
of the Funaria hygrometrica moss placed on the surface of gelatin
gel: chloroplasts were positioned along the surface facing the air,
while there were few chloroplasts along the opposite surface attached to
the gel. This observation reminded him of the chloroplast epistrophe
positioning and the idea of ‘CO2 taxis’ and prompted him
to test the CO2-tactic movement. He devised a
double-chamber system, which was made of two glass half-chambers, each
of which had a funnel-shape throat on one side. A thin mica sheet having
a slit was sandwiched with these half-chambers at their throat parts.
The slit was covered with a F. hygrometrica leaf, the margin of
which was fixed with gelatin. Into each of the half chambers, the
CO2-containing air or the CO2-free air
was introduced, and the leaf was illuminated for enough time. Following
microscopic observation of the leaf revealed the chloroplasts tended to
accumulate to the periclinal surface facing the
CO2-containing air. This description by Senn (1908) was
the first and the only report which showed explicitly the occurrence of
the CO2-tactic movement of chloroplast. In his report,
however, he did not detail the effects of the leaf adaxial-abaxial
polarity, leaf age, direction of the gravity, or the incubation time. He
did not finely control light levels, spectra of light, or the
CO2 concentration, either.
Here, we report characteristics of moss leaf chloroplast
CO2 relocation in leaves of Physcomitriumpatens (syn. Physcomitrella patens ), a model moss plant in
detail according to observations using a modernized double-chamber
system. The system was equipped with monochromatic LED light sources and
mass flow controllers to finely control the spectral properties and PFD
level of light and the CO2 and O2concentrations. Using this system in a gas | gel and a gas
| gas mode, we also examined the photosynthesis dependency and
the responsible cytoskeletal system of CO2-tactic
movement in a pharmacological manner. Based on the results of these
observations, we discuss ecological and physiological aspects of the
chloroplast CO2 relocation.
2 Materials and Methods
2.1 Plant material
Physcomitrium patens (syn. Physcomitrella patens )
Cove-NIBB line (Nishiyama et al., 2000) was cultured on the BCD medium
supplemented with 1 mM CaCl2 and 1.0% [w/v] agar,
which contained 1 mM KNO3, 1.84 mM
KH2PO4, 1.32 mM CaCl2, 1
mM MgSO4, 0.23 µM CoCl2, 0.22 µM
CuSO4, 45 µM FeSO2, 0.1 µM
Na2MoO4, 2 µM MnCl2,
0.19 µM ZnSO4, 1 µM
H3BO3, 0.17 µM KI and 1.0% [w/v]
agar (Hereafter we call this medium simply the BCD agar medium and the
medium without agar as the BCD medium) and was adjusted at pH6.5 with
KOH, in continuous white fluorescent light (FL40SEX-N-HG, NEC Lighting
Ltd., Tokyo, Japan) at PPFD 39~45 µmol
m-2 s-1 with no day/night cycle at
18°C and 80% relative humidity in a growth chamber (LPH-350-SP, Nippon
Medical & Chemical Instruments, Osaka, Japan). A bunch of protonemata
was transplanted on the BCD agar medium and cultured for 9 to 16 days as
a colony. Then several shoots emerged in each colony. From each shoot,
bearing about 10 leaves with midribs reaching to the leaf tips, three
leaves including the largest leaf, and its neighboring younger and older
leaves were collected. Since the leaf was folded at its midrib, one side
of the lamina from each of the leaves was cut off at the midrib and the
half lamina retaining the midrib was used for experiments. The midribs
were utilized for handling. Hereafter, we simply call a half lamina
retaining the midrib ‘a leaf.’ Sufficiently flat leaves were selected
and used in the experiments. Leaves were immersed in the half strength
BCD liquid medium and kept in the dark for one night at 18°C. After the
one-night dark treatment, healthy leaves showed the dark arrangement of
anticlinal chloroplast positioning (Senn, 1908; Suetsugu et al., 2017;
see Before experiment data in Figure 4), and thus, the leaves with
anticlinal chloroplast arrangement were used for the experiments.
2.2 Double-chamber system
A brass double chamber having glass windows on both sides was custom
made (Figure 1a). The volume of each half chamber was 0.7 mL. Mass flow
controllers (MFC) (EL-FLOW Select, Bronkhorst, Ruurlo Netherlands, and
SEC-400MK2, HORIBA, Kyoto, Japan) were used to obtain gas mixture. The
gas was humidified by bubbling in the bottle (Figure 2). The gas was
introduced to each half-chamber at 80 mL min-1. The
concentrations of CO2 and O2 of the gas
were checked with an infrared gas analyzer (LI-840, LI-COR, Lincoln, NE,
U.S.A.) and an oxygen sensor (3080-O2, Walz, Effeltrich,
Germany). The high vacuum grease (Dow Corning Asia Ltd., Hong-Kong,
China) was smeared between the half chambers for airtightness. The
temperature of the double chamber was kept at 19-21°C. To illuminate the
leaf samples, we used blue (λ = 442 nm) or red (λ = 625 nm) LEDs (10W
High Power LED, LED Generic, Yamanashi, Japan) introduced via the
four-branched optic fibers (originally provided for a PAM fluorometer
101 system by Walz). A sheet of tracing paper (STP-B5K-105, SAKAE
TECHNICAL PAPER, Tokyo, Japan) was placed between the half chamber and
the light source for even illumination. Spectra of light sources at the
position of the sample in the chamber measured with a light analyzer
(LA-105, Nippon Medical & Chemical Instruments) are shown in Figure 1b.
The photon flux density (PFD) was measured with a quantum sensor for
photosynthetically active radiation (400-700 nm, LI-250A, LI-COR). When
continuously used, the PFD tended to increase linearly: the increase
after illumination for 3 h was within 5%. The experimental system was
covered with black cloth to eliminate light from other light sources.
The experiments were conducted in two modes: the gas | gas mode
and the gas | gel mode as detailed below. During the
experiment, the leaves were placed vertically with the leaf base upwards
and illuminated equally from both sides to avoid any unilateral effects
of the gravity and light gradient (Figure 2).
2.3 Chloroplast relocation in a gas | gas mode
A piece of aluminum foil (12 µm thickness, UACJ Foil Co., Tokyo, Japan)
was fixed over the aperture of the double chamber using the high vacuum
grease (Dow Corning Asia) (Figure 2a). Four or five rectangular windows
(approx. 300 µm × 600 µm) were opened in the foil. A leaf sample
moistened with the half strength BCD medium was attached over each of
the windows with surface tension of the liquid medium (Figure 2c, e). On
each of the apical and basal ends of the leaves, a piece of 0.8% agar
(FUJIFILM Wako Pure Chemical Co., Osaka, Japan) was placed as water
reservoir to prevent desiccation (*in Figure 2c, e). The leaves were
kept in the dark in the chamber for 30 minutes and subject to the
experimental treatments. Periclinal leaf surfaces were exposed to the
gas mixed separately for each side. The PFD level, the gas
concentrations and treatment period are shown in each figure.
2.4 Chloroplast relocation in a gas | gel mode
A piece of aluminum foil was fixed over the aperture of the double
chamber using the high vacuum grease (Dow Corning Asia Ltd.). A square
window (6 mm × 6 mm) was opened in the foil. A square sheet in 2 mm
thick and 8 mm width of 0.6% [w/v] Gellan gum (FUJIFILM Wako Pure
Chemical Co.) gel containing the half strength BCD medium was placed
over the window (Figure 1d, and 10c). Four leaves were placed on the gel
sheet. Before the experiment, we checked the leaf surface was not
covered with the liquid exuded from the gel sheet. The samples were kept
in the dark for 30 min in the chamber before the onset of experiments.
The same gas mixture was introduced to both half chambers for 2 h. The
PFD level and the gas concentration are shown in each figure.
2.5 Pharmacological treatments
To inhibit photosynthesis, we used
3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) (Sigma Chemical Co.), a
specific inhibitor of electron transport in photosystem II (Trebst
1979), at the final concentration of 100 µM in the half strength BCD
medium containing 1% [v/v] DMSO. The leaves after the one-night
dark treatment were immersed in the half strength BCD medium containing
1% [v/v] DMSO with or without 100 µM DCMU for 1 h. Chlorophyll
fluorescence transients in these leaves were measured with a MINI-PAM-II
(Waltz). Complete inhibition of PSII electron transport was confirmed by
this DCMU treatment (data not shown). The agar gel used for water
reservoir in the gas | gas mode experiment and the gellan gum
sheet used in the gas | gel mode experiment were immersed in
the half strength BCD medium containing 1% [v/v] DMSO with or
without 100 µM DCMU for 1 h.
Oryzalin and latrunculin A (both from FUJIFILM Wako Pure Chemical) were
used to disrupt the polymerization of MT and that of actin MF (Yi &
Goshima, 2020). Stock solutions of oryzalin and latrunculin A were 100
mM and 10 mM in DMSO. In the experiments, oryzalin and latrunculin A
were used at 50 µM and 10 µM in the half strength BCD medium containing
1% [v/v] DMSO. Before the inhibitor treatment, the collected leaves
were immersed in the half strength BCD medium for 5 hours in the dark at
18°C. After this dark treatment, the chloroplasts were in the anticlinal
arrangement, typical of the arrangement in the dark. These leaves were
transferred to the half strength BCD medium containing 1% [v/v]
DMSO with or without inhibitor(s) and incubated in the dark overnight at
18°C. The pieces of agar gel used for water reservoirs were immersed in
the corresponding inhibitor media for 1 h before the experiments.
2.6 Observation on CO2-relocation arrangement in
transverse sections
After the gas | gas mode experiment with one-sided
CO2 exposure, the leaf samples were immediately immersed
in a fixation buffer containing 1% [w/v] glutaraldehyde, 4%
formalin and 50 mM sodium phosphate buffer at pH 7.0 for one h on ice in
the dark and then in a refrigerator for three days at 4°C to harden the
leaves. The samples, being immersed in the fixative, were sectioned
transversely by hand at ca. 50 µm thick with a razor blade (Item No.
99027, FEATHER Safety Razor Co., Osaka, Japan) under a stereo microscope
(SZX16 equipped with SDF PLAPO 1XPF Objective, Olympus, Tokyo, Japan).
Sections were observed under a light microscope equipped with a CCD
camera (BX50 equipped with DP71, Olympus).
2.7 Quantification of distribution of chloroplasts in each cell
To quantitatively examine chloroplast distribution, we recognized five
chloroplast positions: adaxial periclinal position (ad), anticlinal
position (ant), abaxial periclinal position (ab), intermediate between
ad and ant (ad’), and intermediate between ab and ant (ab’) (Figure 3b).
The leaves for quantification were observed under the light microscope
(BX50 and DP71) immediately (< 15 min) after the experimental
treatment. We took seven to ten photos for one leaf, shifting the focal
plane sequentially from the adaxial end to abaxial end (Figure 3c). On
the sequential photos, we counted the numbers of chloroplasts in these
categories for each cell. The number of chloroplasts per cell was 27.6
± 7.6 (Mean ± SD) for 2,381 cells
counted for the data shown in the present paper. Here, the proportion of
chloroplasts in each category to total chloroplasts of the cell was
calculated for each cell:
ri = Ni /(
Nad + Nad’ + Nant +
Nab’ + Nab),
where i is either ad, ad’ ant, ab’ or ab. The proportions thus
obtained are shown in figures.
2.8 Statistical analyses
To analyze the data statistically, the one-sidedness index: e = 2
* rad + rad’ – rab’ –
2 * rab, and the flatness index: f = 2 *
rad + rad’ + rab’ + 2 *
rab were calculated for each cell. e represents
the one-sided periclinal arrangement of chloroplasts to the adaxial
side, while f represents the tendency of periclinal positioning.
Differences in these indexes were statistically examined. When the
results of two conditions were compared, the Mann-Whitney U test was
used. For comparison among more than three conditions, the
Kruskal-Wallis test and thepost hoc pair-wise comparison by the
Dwass-Steel-Critchlow-Fligner (DSCF) multiple comparison method were
employed. Additionally, to see the respective effect of the PFD and the
CO2 concentration, the Spearman’s rank correlation was
also used. The results of analyses are shown in figures. Analyses were
conducted on JAMOVI (Version 2.3, the jamovi project 2022), the software
for statistics based on R packages (Version 4.1, 2021).
3 Results
3.1 Chloroplasts CO2 relocation in Physcomitrium