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