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.
6 References
Asada, K. (1999). The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annual review of Plant Physiology and Plant Molecular Biology , 50 , 601-639.
Böhm J. A. (1856). Beiträge zur näheren Kenntnis des Chlorophylls.Sitzungsberichte der Akademie der Wissenschaften mathematisch-naturwissenschaftliche Klasse, 22, 479–512
Dong, X. J., Takagi, S., & Nagai, R. (1995). Regulation of the orientation movement of chloroplasts in epidermal cells ofVallisneria : cooperation of phytochrome with photosynthetic pigment under low-fluence-rate light. Planta , 197, 257-263.
Dong, X. J., Nagai, R., & Takagi, S. (1998). Microfilaments anchor chloroplasts along the outer periclinal wall in Vallisneriaepidermal cells through cooperation of PFR and photosynthesis. Plant and Cell Physiology , 39 , 1299-1306.
Evans, J. R., Caemmerer, S. V., Setchell, B. A., & Hudson, G. S. (1994). The relationship between CO2 transfer conductance and leaf anatomy in transgenic tobacco with a reduced content of Rubisco. Functional Plant Biology , 21, 475-495.
Fujii, Y., Tanaka, H., Konno, N., Ogasawara, Y., Hamashima, N., Tamura, S., … & Kodama, Y. (2017). Phototropin perceives temperature based on the lifetime of its photoactivated state. Proceedings of the National Academy of Sciences of the United States of America , 114, 9206-9211.
Haberlandt G. (1876). Über den Einfluß des Frostes auf die Chlorophyllkörner. Österreichische botanische Zeitschrift , 26, 249–255.
Haberlandt, G. (1886). Über das Assimilationssystem. Berichte der Deutschen botanischen Gesellschaft , Bd.4, S.206.
Haupt, W. (1965). Perception of environmental stimuli orienting growth and movement in lower plants. Annual Review of Plant Physiology , 16, 267-290.
Hill, T. L., & Kirschner, M. W. (1982). Bioenergetics and kinetics of microtubule and actin filament assembly–disassembly.International review of cytology , 78 , 1-125.
Ho, Q. T., Verboven, P., Yin, X., Struik, P. C., & Nicolaï, B. M. (2012). A microscale model for combined CO2 diffusion and photosynthesis in leaves. PloS one , 7, e48376.
Kadota, A., Sato, Y., & Wada, M. (2000). Intracellular chloroplast photorelocation in the moss Physcomitrella patens is mediated by phytochrome as well as by a blue-light receptor. Planta , 210, 932-937.
Kagawa, T., et al. (2001). Arabidopsis NPL1: a phototropin homolog controlling the chloroplast high-light avoidance response.Science , 291, 2138-2141.
Kasahara, M., Kagawa, T., Oikawa, K., Suetsugu, N., Miyao, M., & Wada, M. (2002). Chloroplast avoidance movement reduces photodamage in plants.Nature , 420, 829.
Kasahara, M., Kagawa, T., Sato, Y., Kiyosue, T., & Wada, M. (2004). Phototropins mediate blue and red light-induced chloroplast movements inPhyscomitrella patens . Plant Physiology , 135, 1388-1397.
Kato, Y., Tsukaguchi, T., Yata, I., Yamamura, R., Oi, T., & Taniguchi, M. (2022). Aggregative movement of mesophyll chloroplasts occurs in a wide variety of C4 plant species. Flora , 294, 152133.
Kawai, H., Kanegae, T., Christensen, S., Kiyosue, T., Sato, Y., Imaizumi, T., … & Wada, M. (2003). Responses of ferns to red light are mediated by an unconventional photoreceptor. Nature , 421, 287-290.
Kimura, S., & Kodama, Y. (2016). Actin-dependence of the chloroplast cold positioning response in the liverwort Marchantia polymorphaL. PeerJ , 4, e2513.
Kodama, Y., Tsuboi, H., Kagawa, T., & Wada, M. (2008). Low temperature-induced chloroplast relocation mediated by a blue light receptor, phototropin 2, in fern gametophytes. Journal of Plant Research , 121, 441-448.
Leighton, M. P., & Sivak, D. A. (2022). Performance scaling and trade-offs for collective motor-driven transport. New Journal of Physics , 24 (1), 013009.
Mittmann, F., Brücker, G., Zeidler, M., Repp, A., Abts, T., Hartmann, E., & Hughes, J. (2004). Targeted knockout in Physcomitrellareveals direct actions of phytochrome in the cytoplasm.Proceedings of the National Academy of Sciences of the United States of America , 101, 13939-13944.
Mizokami, Y., Oguchi, R., Sugiura, D., Yamori, W., Noguchi, K., & Terashima, I. (2022). Cost–benefit analysis of mesophyll conductance: diversities of anatomical, biochemical and environmental determinants.Annals of Botany , 130 (3), 265-283.
Mosebach, G. (1958). Zur Phototaxis von Mougeotia (Mesocarpus).Planta , 52, 3-46.
Nishiyama, T., Hiwatashi, Y., Sakakibara, K., Kato, M., & Hasebe, M. (2000). Tagged mutagenesis and gene-trap in the moss,Physcomitrella patens by shuttle mutagenesis. DNA research , 7, 9-17.
Okamoto, G. H., & Lightfoot, E. N. (1992). Energy cost of intracellular organization. Industrial & engineering chemistry research ,31 (3), 732-735.
Sakai, T., et al. (2001). Arabidopsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation.Proceedings of the National Academy of Sciences of the United States of America , 98, 6969-6974.
Sakai, Y., & Takagi, S. (2005). Reorganized actin filaments anchor chloroplasts along the anticlinal walls of Vallisneria epidermal cells under high-intensity blue light. Planta , 221, 823-830.
Sato, Y., Wada, M., & Kadota, A. (2001). Choice of tracks, microtubules and/or actin filaments for chloroplast photo-movement is differentially controlled by phytochrome and a blue light receptor. Journal of Cell Science , 114, 269-279.
Sato, Y., Kadota, A., & Wada, M. (2003). Chloroplast movement: dissection of events downstream of photo-and mechano-perception.Journal of Plant Research , 116, 1-5.
Seitz, K. (1967). Eine Analyse der für die lichtabhängigen Bewegungen der Chloroplasten verantwortlichen Photorezeptorsysteme beiVallisneria spiralis ssp. torta . Zeitschrift für Pflanzenphysiologie , 57, 96-104.
Senn, G. (1908). Die Gestalts-und Lageveränderung der Pflanzen-Chromatophoren . Verlag von Wilhelm Engelmann, Leipzig.
Suetsugu, N., Higa, T., Gotoh, E., & Wada, M. (2016). Light-induced movements of chloroplasts and nuclei are regulated in both cp-actin-filament-dependent and-independent manners in Arabidopsis thaliana . PLoS One , 11, e0157429.
Suetsugu, N., Higa, T., & Wada, M. (2017). Ferns, mosses and liverworts as model systems for light‐mediated chloroplast movements. Plant, Cell & Environment , 40, 2447-2456.
Sugiyama, Y., & Kadota, A. (2011). Photosynthesis-dependent but neochrome 1-independent light positioning of chloroplasts and nuclei in the fern Adiantum capillus-veneris . Plant Physiology , 155, 1205-1213.
Terashima, I., Hanba, Y. T., Tazoe, Y., Vyas, P., & Yano, S. (2006). Irradiance and phenotype: comparative eco-development of sun and shade leaves in relation to photosynthetic CO2 diffusion.Journal of Experimental Botany , 57, 343-354.
Tholen, D., Boom, C., Noguchi, K. O., Ueda, S., Katase, T., & Terashima, I. (2008). The chloroplast avoidance response decreases internal conductance to CO2 diffusion in Arabidopsis thaliana leaves. Plant, Cell & Environment , 31 (11), 1688-1700.
Tholen, D., & Zhu, X. G. (2011). The mechanistic basis of internal conductance: a theoretical analysis of mesophyll cell photosynthesis and CO2 diffusion. Plant physiology , 156 (1), 90-105.
Trebst, A. (1979). Inhibition of photosynthetic electron flow by phenol and diphenylether herbicides in control and trypsin-treated chloroplasts. Zeitschrift für Naturforschung C, 34, 986-991.
Trojan, A., & Gabrys, H. (1996). Chloroplast distribution inArabidopsis thaliana (L.) depends on light conditions during growth. Plant Physiology , 111, 419-425.
Ubierna, N., Sun, W. E. I., Kramer, D. M., & Cousins, A. B. (2013). The efficiency of C4 photosynthesis under low light conditions in Zea mays , Miscanthus x giganteus and Flaveria bidentis . Plant, Cell & Environment , 36, 365-381.
Wada, M., & Kong, S. G. (2018). Actin-mediated movement of chloroplasts. Journal of Cell Science , 131, jcs210310.
Warren, C. R., Löw, M., Matyssek, R., & Tausz, M. (2007). Internal conductance to CO2 transfer of adult Fagus sylvatica: variation between sun and shade leaves and due to free-air ozone fumigation. Environmental and Experimental Botany , 59 , 130-138.
Wen, F., Xing, D., & Zhang, L. (2008). Hydrogen peroxide is involved in high blue light-induced chloroplast avoidance movements in Arabidopsis.Journal of Experimental Botany , 59, 2891-2901.
Williams, T. G., & Flanagan, L. B. (1996). Effect of changes in water content on photosynthesis, transpiration and discrimination against13CO2 and C18O16O in Pleurozium andSphagnum . Oecologia , 108, 38-46.
Yatsuhashi, H., & Wada, M. (1990). High-fluence rate responses in the light-oriented chloroplast movement in Adiantum protonemata.Plant Science , 68, 87-94.
Yamada, M., Kawasaki, M., Sugiyama, T., Miyake, H., & Taniguchi, M. (2009). Differential positioning of C4 mesophyll and bundle sheath chloroplasts: aggregative movement of C4 mesophyll chloroplasts in response to environmental stresses. Plant and Cell Physiology , 50, 1736-1749.
Yi, P., & Goshima, G. (2020). Rho of plants GTPases and cytoskeletal elements control nuclear positioning and asymmetric cell division duringPhyscomitrella patens branching. Current Biology , 30, 2860-2868.
Zurzycki, J. (1962). The action spectrum for the light depended movements of chloroplasts in Lemna trisulca L. Acta Societatis Botanicorum Poloniae , 31, 489-538.
Zurzycki, J. (1967). Properties and localization of the photoreceptor active in displacements of chloroplasts in Funaria hygrometrica. I. Action spectrum. Acta Societatis Botanicorum Poloniae , 36(1), 133-142.
The jamovi project (2022). jamovi. (Version 2.3) [Computer Software]. Retrieved from https://www.jamovi.org.
R Core Team (2021). R: A Language and environment for statistical computing. (Version 4.1) [Computer software]. Retrieved from https://cran.r-project.org. (R packages retrieved from MRAN snapshot 2022-01-01).
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 > 4 samples × 15 cells, df = 14). Different alphabet letters indicate significant differences in f (p< 0.01, post-hoc DSCF pairwise comparisons).
(b) Spearman’s rank correlation ρ for f was evaluated for PFD and CO2 concentration (p < 0.001. N = 1136), or between experimental conditions of PFD and CO2concentration (p = 0.73. N = 1136).
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.