Heterogeneity of silica and glycan-epitope distribution in epidermal idioblast cell walls in Adiantum raddianum laminae.

Laminae of Adiantum raddianum Presl., a fern belonging to the family Pteridaceae, are characterised by the presence of epidermal fibre-like cells under the vascular bundles. These cells were thought to contain silica bodies, but their thickened walls leave no space for intracellular silica suggesting it may actually be deposited within their walls. Using advanced electron microscopy in conjunction with energy dispersive X-ray microanalysis we showed the presence of silica in the cell walls of the fibre-like idioblasts. However, it was specifically localised to the outer layers of the periclinal wall facing the leaf surface, with the thick secondary wall being devoid of silica. Immunocytochemical experiments were performed to ascertain the respective localisation of silica deposition and glycan polymers. Epitopes characteristic for pectic homogalacturonan and the hemicelluloses xyloglucan and mannan were detected in most epidermal walls, including the silica-rich cell wall layers. The monoclonal antibody, LM6, raised against pectic arabinan, labelled the silica-rich primary wall of the epidermal fibre-like cells and the guard cell walls, which were also shown to contain silica. We hypothesise that the silicified outer wall layers of the epidermal fibre-like cells support the lamina during cell expansion prior to secondary wall formation. This implies that silicification does not impede cell elongation. Although our results suggest that pectic arabinan may be implicated in silica deposition, further detailed analyses are needed to confirm this. The combinatorial approach presented here, which allows correlative screening and in situ localisation of silicon and cell wall polysaccharide distribution, shows great potential for future studies.

An evolutionarily conserved signaling mechanism mediates far-red light responses in land plants.

Phytochromes are plant photoreceptors important for development and adaptation to the environment. Phytochrome A (PHYA) is essential for the far-red (FR) high-irradiance responses (HIRs), which are of particular ecological relevance as they enable plants to establish under shade conditions. PHYA and HIRs have been considered unique to seed plants because the divergence of seed plants and cryptogams (e.g., ferns and mosses) preceded the evolution of PHYA. Seed plant phytochromes translocate into the nucleus and regulate gene expression. By contrast, there has been little evidence of a nuclear localization and function of cryptogam phytochromes. Here, we identified responses to FR light in cryptogams, which are highly reminiscent of PHYA signaling in seed plants. In the moss Physcomitrella patens and the fern Adiantum capillus-veneris, phytochromes accumulate in the nucleus in response to light. Although P. patens phytochromes evolved independently of PHYA, we have found that one clade of P. patens phytochromes exhibits the molecular properties of PHYA. We suggest that HIR-like responses had evolved in the last common ancestor of modern seed plants and cryptogams and that HIR signaling is more ancient than PHYA. Thus, other phytochromes in seed plants may have lost the capacity to mediate HIRs during evolution, rather than that PHYA acquired it.

Red light-induced phytochrome relocation into the nucleus in Adiantum capillus-veneris.

Phytochromes in seed plants are known to move into nuclei in a red light-dependent manner with or without interacting factors. Here, we show phytochrome relocation to the nuclear region in phytochrome-dependent Adiantum capillus-veneris spore germination by partial spore-irradiation experiments. The nuclear or non-nuclear region of imbibed spores was irradiated with a microbeam of red and/or far-red light and the localization of phytochrome involved in spore germination was estimated from the germination rate. The phytochrome for spore germination existed throughout whole spore under darkness after imbibition, but gradually migrated to the nuclear region following red light irradiation. Intracellular distribution of PHY-GUS fusion proteins expressed in germinated spores by particle bombardment showed the migration of Acphy2, but not Acphy1, into nucleus in a red light-dependent manner, suggesting that Acphy2 is the photoreceptor for fern spore germination.

Chloroplasts move towards the nearest anticlinal walls under dark condition.

Chloroplasts change their intracellular positions in response to their light environment. Under darkness, chloroplasts assume special positions that are different from those under light conditions. Here, we analyzed chloroplast dark positioning using Adiantum capillus-veneris gametophyte cells. When chloroplasts were transferred into darkness, during the first 1-5 h, they moved towards the anticlinal cell walls bordering the adjacent cells rather rapidly. Then, they slowed down and accumulated at the anticlinal walls gradually over the following 24-36 h. The chloroplast movements could be roughly classified into two different categories: initial rapid straight movement and later, slow staggering movement. When the chloroplast accumulation response was induced in dark-adapted cells by partial cell irradiation with a microbeam targeted to the center of the cells, chloroplasts moved towards the beam spot from the anticlinal walls. However, when the microbeam was switched off, they moved to the nearest anticlinal walls and not to their original positions if they were not the closest, indicating that they know the direction of the nearest anticlinal wall and do not have particular areas that they migrate to during dark positioning.

Distribution pattern changes of actin filaments during chloroplast movement in Adiantum capillus-veneris.

Chloroplasts change their positions in a cell in response to light intensities. The photoreceptors involved in chloroplast photo-relocation movements and the behavior of chloroplasts during their migration were identified in our previous studies, but the mechanism of movement has yet to be clarified. In this study, the behavior of actin filaments under various light conditions was observed in Adiantum capillus-veneris gametophytes. In chloroplasts staying in one place under a weak light condition and not moving, circular structures composed of actin filaments were observed around the chloroplast periphery. In contrast, short actin filaments were observed at the leading edge of moving chloroplasts induced by partial cell irradiation. In the dark, the circular structures found under the weak light condition disappeared and then reappeared around the moving chloroplasts. Mutant analyses revealed that the disappearance of the circular actin structure was mediated by the blue light photoreceptor, phototropin2.

Photosynthesis-dependent but neochrome1-independent light positioning of chloroplasts and nuclei in the fern Adiantum capillus-veneris.

Chloroplasts change their positions in the cell depending on the light conditions. In the dark, chloroplasts in fern prothallia locate along the anticlinal wall (dark position). However, chloroplasts become relocated to the periclinal wall (light position) when the light shines perpendicularly to the prothallia. Red light is effective in inducing this relocation in Adiantum capillus-veneris, and neochrome1 (neo1) has been identified as the red light receptor regulating this movement. Nevertheless, we found here that chloroplasts in neo1 mutants still become relocated from the dark position to the light position under red light. We tested four neo1 mutant alleles (neo1-1, neo1-2, neo1-3, and neo1-4), and all of them showed the red-light-induced chloroplast relocation. Furthermore, chloroplast light positioning under red light occurred also in Pteris vittata, another fern species naturally lacking the neo1-dependent phenomenon. The light positioning of chloroplasts occurred independently of the direction of red light, a response different to that of the neo1-dependent movement. Photosynthesis inhibitors 3-(3,4 dichlorophenyl)-1,1-dimethylurea or 2,5-dibromo-3-isopropyl-6-methyl-p-benzoquinone blocked this movement. Addition of sucrose (Suc) or glucose to the culture medium induced migration of the chloroplasts to the periclinal wall in darkness. Furthermore, Suc could override the effects of 3-(3,4 dichlorophenyl)-1,1-dimethylurea. Interestingly, the same light positioning was evident for nuclei under red light in the neo1 mutant. The nuclear light positioning was also induced in darkness with the addition of Suc or glucose. These results indicate that photosynthesis-dependent nondirectional movement contributes to the light positioning of these organelles in addition to the neo1-dependent directional movement toward light.

Speed of signal transfer in the chloroplast accumulation response.

Chloroplast photorelocation movement is important for plants to perform efficient photosynthesis. Phototropins were identified as blue-light receptors for chloroplast movement in Arabidopsis thaliana and in the fern Adiantum capillus-veneris, whereas neochrome functions as a dual red/blue light receptor in the latter. However, the signal transduction pathways involved in chloroplast movement remain to be clarified. To investigate the kinetic properties of signalling from these photoreceptors to the chloroplasts, we deduced the speed of signal transfer using Adiantum capillus-veneris gametophytes. When a region of dark-adapted gametophyte cells was subjected to microbeam irradiation, chloroplasts moved towards the irradiated area even in subsequent darkness. We therefore recorded the movement and calculated the speeds of signal transfer by time-lapse imaging. Movement speeds under red or blue light were similar, e.g., about 1.0 microm min(-1) in prothallial cells. However, speeds varied according to cell polarity in protonemal cells. The speed of signal transfer from the protonemal apex to the base was approximately 0.7 microm min(-1), but roughly 2.3 microm min(-1) in the opposite direction. The speed of signal transfer in Arabidopsis thaliana mesophyll cells was approximately 0.8 microm min(-1) by comparison. Surprisingly, chloroplasts located farthest away from the microbeam were found to move faster than those in close proximity to the site of irradiation both in Adiantum capillus-veneris and A. thaliana.

Chloroplasts do not have a polarity for light-induced accumulation movement.

Chloroplast photorelocation movement in green plants is generally mediated by blue light. However, in cryptogam plants, including ferns, mosses, and algae, both red light and blue light are effective. Although the photoreceptors required for this phenomenon have been identified, the mechanisms underlying this movement response are not yet known. In order to analyze this response in more detail, chloroplast movement was induced in dark-adapted Adiantum capillus-veneris gametophyte cells by partial cell irradiation with a microbeam of red and/or blue light. In each case, chloroplasts were found to move toward the microbeam-irradiated area. A second microbeam was also applied to the cell at a separate location before the chloroplasts had reached the destination of the first microbeam. Under these conditions, chloroplasts were found to change their direction of movement without turning and move toward the second microbeam-irradiated area after a lag time of a few minutes. These findings indicate that chloroplasts can move in any direction and do not exhibit a polarity for chloroplast accumulation movement. This phenomenon was analyzed in detail in Adiantum and subsequently confirmed in Arabidopsis thaliana palisade cells. Interestingly, the lag time for direction change toward the second microbeam in Adiantum was longer in the red light than in the blue light. However, the reason for this discrepancy is not yet understood.

Low temperature-induced chloroplast relocation mediated by a blue light receptor, phototropin 2, in fern gametophytes.

Chloroplast movement in response to light has been known more than 100 years. Chloroplasts move towards weak light and move away from strong light. Dark-induced relocation, called dark positioning, has also been shown. However, the effects of other stimuli on chloroplast movement have not been well characterized. Here we studied low temperature-induced chloroplast relocation (termed cold positioning) in prothallial cells of the gametophytes of the fern Adiantum capillus-veneris. Under weak light chloroplasts in prothallial cells accumulated along the periclinal wall at 25 degrees C, but they moved towards anticlinal walls when the prothalli were subsequently transferred to 4 degrees C. A temperature shift from 25 degrees to 10 degrees C or lower was enough to induce cold positioning, and high-intensity light enhanced the response. Nuclei also relocated from the periclinal position (a position along periclinal walls) to the anticlinal position (a position along anticlinal walls) under cold temperature, whereas mitochondria did not. Cold positioning was not observed in mutant fern gametophytes defective of the blue light photoreceptor, phototropin 2.

Phototropins and neochrome1 mediate nuclear movement in the fern Adiantum capillus-veneris.

In gametophytic cells (prothalli) of the fern Adiantum capillus-veneris, nuclei as well as chloroplasts change their position according to light conditions. Nuclei reside on anticlinal walls in darkness and move to periclinal or anticlinal walls under weak or strong light conditions, respectively. Here we reveal that red light-induced nuclear movement is mediated by neochrome1 (neo1), blue light-induced movement is redundantly mediated by neo1, phototropin2 (phot2) and possibly phot1, and dark positioning of both nuclei and chloroplasts is mediated by phot2. Thus, both the nuclear and chloroplast photorelocation movements share common photoreceptor systems.

Negative phototropic response of rhizoid cells in the fern Adiantum capillus-veneris.

In general, phototropic responses in land plants are induced by blue light and mediated by blue light receptor phototropins. In many cryptogam plants including the fern Adiantum capillus-veneris, however, red as well as blue light effectively induces a positive phototropic response in protonemal cells. In A. capillus-veneris, the red light effect on the tropistic response is mediated by phytochrome 3 (phy3), a chimeric photoreceptor of phytochrome and full-length phototropin. Here, we report red and blue light-induced negative phototropism in A. capillus-veneris rhizoid cells. Mutants deficient for phy3 lacked red light-induced negative phototropism, indicating that under red light, phy3 mediates negative phototropism in rhizoid cells, contrasting with its role in regulating positive phototropism in protonemal cells. Mutants for phy3 were also partially deficient in rhizoid blue light-induced negative phototropism, suggesting that phy3, in conjunction with phototropins, redundantly mediates the blue light response.

Accumulation response of chloroplasts induced by mechanical stimulation in bryophyte cells.

Chloroplast movement has been studied in many plants but mainly as a model system for light signaling. However, we recently showed that the avoidance response of chloroplasts is also induced by mechanical stimulation in fern protonemal cells. Here we report the discovery of a mechanically induced accumulation response of chloroplasts in bryophytes. When mechanical stimulation was directly applied with a capillary to a part of a cell, chloroplasts moved towards and accumulated at the pressed site within 30 min after the onset of stimulation in all species tested. The accumulation movement of chloroplasts was inhibited by Cremart but not by cytochalasin B in red-light-grown protonemata of Physcomitrella patens (Hedw.) B., S. & G. To determine the contribution of external Ca(2+) to the response, we examined the effects on the accumulation movement of gadolinium (Ga(3+)), an inhibitor of stretch-activated ion channels, and lanthanum (La(3+)), a potent inhibitor of calcium channels. Mechano-relocation of chloroplasts was abolished by these drugs, but no effects were observed on photo-relocation of chloroplasts, irrespective of light colors and intensity. These results suggest that influx of external Ca(2+) through the plasma membrane is essential for the early steps in signaling of mechano-relocation of chloroplasts whose motility system is dependent on microtubules.