Growth performance and element concentrations reveal the calcicole-calcifuge behavior of three Adiantum species.

BACKGROUND: The calcicole or calcifuge behavior of wild plants has been related to element deficiency or toxicity. For fern species, however, knowledge about their adaptive differences and responses to soil environmental changes is virtually absent. In the karst regions of southern China, most Adiantum species favor calcareous soils, but A. flabellulatum prefers acidic soils. Such contrasting preferences for soil types in the same genus are interesting and risky because their preferred soils may "pollute" each other due to extreme precipitation events. We mixed calcareous and acidic soils at 1:1 (v/v) to simulate the "polluted" soils and grew three Adiantum species (the calcicole A. capillus-veneris f. dissectum and A. malesianum and the calcifuge A. flabellulatum) on the calcareous, acidic and mixed soils for 120 d and assessed their growth performance and element concentrations. RESULTS: The calcareous soil showed the highest pH, Ca, Mg and P concentrations but the lowest K concentration, followed by the mixed soil, and the acidic soil. After 120 d of growth, the calcifuge A. flabellulatum on the calcareous and mixed soils exhibited lower SPAD and relative growth rate (RGR) than those on the acidic soil, and its leaf and root Ca, Mg and Fe concentrations were higher and K was lower on the calcareous soil than on the acidic soil. The calcicole A. capillus-veneris f. dissectum on the calcareous soil had similar leaf element concentrations and RGR with those on the mixed soil, but their leaf Ca, Fe and Al were lower and leaf P and K concentrations, SPAD and RGR were higher than those on the acidic soil. For the calcicole A. malesianum, leaf Ca, Fe and Al were lowest and leaf P and RGR were highest when grown on the mixed soil, intermediated on the calcareous soil, and on the acidic soil. Compared with A. malesianum, A. capillus-veneris f. dissectum had lower leaf Ca, Fe and Al but higher leaf Mg concentration when grown on the same calcareous or mixed soils. CONCLUSIONS: A. capillus-veneris f. dissectum is a low leaf Ca calcicole species while A. malesianum is an Al accumulating calcicole species. They can effectively take up P and K to leaves and hence can thrive on calcareous soils. In contrast, the calcifuge A. flabellulatum grown on calcareous soils is stunted. Such growth performance may be attributed to the increased leaf Ca and decreased leaf K concentration. If their preferred soils are "polluted", A. flabellulatum can grow worse, A. capillus-veneris f. dissectum can remain almost unaffected while A. malesianum will perform better.

Determination of Phototropism and Polarotropism in Fern Protonemal Cells.

Fern protonemal cells grow at their apices as long, undivided filamentous cells toward red (or weak white) light and change their growth direction if the light direction is changed (i.e., phototropism). When protonemata growing between an agar surface and cover glass are irradiated with polarized red light through the glass on the protonemal side, they start growing at the point where the direction of the vibration plane of polarized light and the transition moment of the photoreceptor, which is parallel to the plasma membrane of the cell's apical part, are equal (i.e., polarotropism). Herein, the methods on how to induce and observe this protonemal phototropism and polarotropism are described.

Cytoskeleton Observation in Phototropism and Polarotropism of Adiantum Protonema.

The distribution patterns of the cytoskeleton, i.e., microtubules and actin filaments, in the apical part of protonemal cells are unique and differ from those of other apical growing cells, such as moss and liverwort protonemata, fungal hyphae, and angiosperm pollen tubes. A ring structure composed of microtubules and actin filaments exists at the basal part of the apical dome of protonemal cells. The structure may control the protonemal diameter and growth direction. Herein, the methods of staining of both microtubules and actin filaments are described.

Temperature-dependent signal transmission in chloroplast accumulation response.

Chloroplast photorelocation movement, well-characterized light-induced response found in various plant species from alga to higher plants, is an important phenomenon for plants to increase photosynthesis efficiency and avoid photodamage. The signal for chloroplast accumulation movement connecting the blue light receptor, phototropin, and chloroplasts remains to be identified, although the photoreceptors and the mechanism of movement via chloroplast actin filaments have now been revealed in land plants. The characteristics of the signal have been found; the speed of signal transfer is about 1 µm min-1 and that the signal for the accumulation response has a longer life and is transferred a longer distance than that of the avoidance response. Here, to collect the clues of the unknown signal substances, we studied the effect of temperature on the speed of signal transmission using the fern Adiantum capillus-veneris and found the possibility that the mechanism of signal transfer was not dependent on the simple diffusion of a substance; thus, some chemical reaction must also be involved. We also found new insights of signaling substances, such that microtubules are not involved in the signal transmission, and that the signal could even be transmitted through the narrow space between chloroplasts and the plasma membrane.

Sporangium Exposure and Spore Release in the Peruvian Maidenhair Fern (Adiantum peruvianum, Pteridaceae).

We investigated the different processes involved in spore liberation in the polypod fern Adiantum peruvianum (Pteridaceae). Sporangia are being produced on the undersides of so-called false indusia, which are situated at the abaxial surface of the pinnule margins, and become exposed by a desiccation-induced movement of these pinnule flaps. The complex folding kinematics and functional morphology of false indusia are being described, and we discuss scenarios of movement initiation and passive hydraulic actuation of these structures. High-speed cinematography allowed for analyses of fast sporangium motion and for tracking ejected spores. Separation and liberation of spores from the sporangia are induced by relaxation of the annulus (the 'throwing arm' of the sporangium catapult) and conservation of momentum generated during this process, which leads to sporangium bouncing. The ultra-lightweight spores travel through air with a maximum velocity of ~5 m s(-1), and a launch acceleration of ~6300 g is measured. In some cases, the whole sporangium, or parts of it, together with contained spores break away from the false indusium and are shed as a whole. Also, spores can stick together and form spore clumps. Both findings are discussed in the context of wind dispersal.

A phytochrome/phototropin chimeric photoreceptor of fern functions as a blue/far-red light-dependent photoreceptor for phototropism in Arabidopsis.

In the fern Adiantum capillus-veneris, the phototropic response of the protonemal cells is induced by blue light and partially inhibited by subsequent irradiation with far-red light. This observation strongly suggests the existence of a phytochrome that mediates this blue/far-red reversible response; however, the phytochrome responsible for this response has not been identified. PHY3/NEO1, one of the three phytochrome genes identified in Adiantum, encodes a chimeric photoreceptor composed of both a phytochrome and a phototropin domain. It was demonstrated that phy3 mediates the red light-dependent phototropic response of Adiantum, and that phy3 potentially functions as a phototropin. These findings suggest that phy3 is the phytochrome that mediates the blue/far-red response in Adiantum protonemata. In the present study, we expressed Adiantum phy3 in a phot1 phot2 phototropin-deficient Arabidopsis line, and investigated the ability of phy3 to induce phototropic responses under various light conditions. Blue light irradiation clearly induced a phototropic response in the phy3-expressing transgenic seedlings, and this effect was fully inhibited by simultaneous irradiation with far-red light. In addition, experiments using amino acid-substituted phy3 indicated that FMN-cysteinyl adduct formation in the light, oxygen, voltage (LOV) domain was not necessary for the induction of blue light-dependent phototropism by phy3. We thus demonstrate that phy3 is the phytochrome that mediates the blue/far-red reversible phototropic response in Adiantum. Furthermore, our results imply that phy3 can function as a phototropin, but that it acts principally as a phytochrome that mediates both the red/far-red and blue/far-red light responses.

Clues to the signals for chloroplast photo-relocation from the lifetimes of accumulation and avoidance responses.

Chloroplast photo-relocation movement is crucial for plant survival; however, the mechanism of this phenomenon is still poorly understood. Especially, the signal that goes from photoreceptor to chloroplast is unknown, although the photoreceptors (phototropin 1 and 2) have been identified and an actin structure (chloroplast actin filaments) has been characterized that is specific for chloroplast movement. Here, in gametophytes of the fern Adiantum capillus-veneris, gametophores of the moss Physcomiterella patens, and leaves of the seed plant Arabidopsis thaliana, we sought to characterize the signaling system by measuring the lifetime of the induced response. Chloroplast movements were induced by microbeam irradiation with high-intensity blue light and recorded. The lifetime of the avoidance state was measured as a lag time between switching off the beam and the loss of avoidance behavior, and that of the accumulation state was measured as the duration of accumulation behavior following the extinction of the beam. The lifetime for the avoidance response state is approximately 3-4 min and that for the accumulation response is 19-28 min. These data suggest that the two responses are based on distinct signals.

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.

Chloroplasts continuously monitor photoreceptor signals during accumulation movement.

Under low light conditions, chloroplasts gather at a cell surface to maximize light absorption for efficient photosynthesis, which is called the accumulation response. Phototropin1 (phot1) and phototropin2 (phot2) were identified as blue light photoreceptors in the accumulation response that occurs in Arabidopsis thaliana and Adiantum capillus-veneris with neochrome1 (neo1) as a red light photoreceptor in A. capillus-veneris. However, the signal molecule that is emitted from the photoreceptors and transmitted to the chloroplasts is not known. To investigate this topic, the accumulation response was induced by partial cell irradiation with a microbeam of red, blue and far-red light in A. capillus-veneris gametophyte cells. Chloroplasts moved towards the irradiated region and were able to sense the signal as long as its signal flowed. The signal from neo1 had a longer life than the signal that came from phototropins. When two microbeams with the same wavelength and the same fluence rate were placed 20 µm apart from each other and were applied to a dark-adapted cell, chloroplasts at an equidistant position always moved towards the center (midpoint) of the two microbeams, but not towards either one. This result indicates that chloroplasts are detecting the concentration of the signal but not the direction of signal flow. Chloroplasts repeatedly move and stop at roughly 10 s intervals during the accumulation response, suggesting that they monitor the intermittent signal waves from photoreceptors.

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.

Chloroplasts can move in any direction to avoid strong light.

Chloroplasts migrate in response to different light intensities. Under weak light, chloroplasts gather at an illuminated area to maximize light absorption and photosynthesis rates (the accumulation response). In contrast, chloroplasts escape from strong light to avoid photodamage (the avoidance response). Photoreceptors involved in these phenomena have been identified in Arabidopsis thaliana and Adiantum capillus-veneris. Chloroplast behavior has been studied in detail during the accumulation response, but not for the avoidance response. Hence, we analyzed the chloroplast avoidance response in detail using dark-adapted Adiantum capillus-veneris gametophyte cells and partial cell irradiation with a microbeam of blue light. Chloroplasts escaped from an irradiated spot. Both duration of this response and the distance of the migrated chloroplasts were proportional to the total fluence irradiated. The speed of movement during the avoidance response was dependent on the fluence rate, but the speed of the accumulation response towards the microbeam from cell periphery was constant irrespective of fluence rate. When a chloroplast was only partially irradiated with a strong microbeam, it moved away towards the non-irradiated region within a few minutes. During this avoidance response two additional microbeam irradiations were applied to different locus of the same chloroplast. Under these conditions the chloroplast changed the moving direction after a lag time of a few minutes without rolling. Taken together, these findings indicate that chloroplasts can move in any direction and never have an intrinsic polarity. Similar phenomenon was observed in chloroplasts of Arabidopsis thaliana palisade cells.

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.

The stomata of the fern Adiantum capillus-veneris do not respond to CO2 in the dark and open by photosynthesis in guard cells.

The stomata of the fern Adiantum capillus-veneris lack a blue light-specific opening response but open in response to red light. We investigated this light response of Adiantum stomata and found that the light wavelength dependence of stomatal opening matched that of photosynthesis. The simultaneous application of red (2 micromol m(-2) s(-1)) and far-red (50 micromol m(-2) s(-1)) light synergistically induced stomatal opening, but application of only one of these wavelengths was ineffective. Adiantum stomata did not respond to CO2 in the dark; the stomata neither opened under a low intercellular CO2 concentration nor closed under high intercellular CO2 concentration. Stomata in Arabidopsis (Arabidopsis thaliana), which were used as a control, showed clear sensitivity to CO2. In Adiantum, stomatal conductance showed much higher light sensitivity when the light was applied to the lower leaf surface, where stomata exist, than when it was applied to the upper surface. This suggests that guard cells likely sensed the light required for stomatal opening. In the epidermal fragments, red light induced both stomatal opening and K+ accumulation in guard cells, and both of these responses were inhibited by a photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The stomatal opening was completely inhibited by CsCl, a K+ channel blocker. In intact fern leaves, red light-induced stomatal opening was also suppressed by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These results indicate that Adiantum stomata lack sensitivity to CO2 in the dark and that stomatal opening is driven by photosynthetic electron transport in guard cell chloroplasts, probably via K+ uptake.

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.

The fern Adiantum capillus-veneris lacks stomatal responses to blue light.

We investigated the responses of stomata to light in the fern Adiantum capillus-veneris, a typical species of Leptosporangiopsida. Stomata in the intact leaves of the sporophytes opened in response to red light, but they did not open when blue light was superimposed on the red light. The results were confirmed in the isolated Adiantum epidermis. The red light-induced stomatal response was not affected by the mutation of phy3, a chimeric protein of phytochrome and phototropin in this fern. The lack of a blue light-specific stomatal response was observed in three other fern species of Leptosporangiopsida, i.e. Pteris cretica, Asplenium scolopendrium and Nephrolepis auriculata. Fusicoccin, an activator of the plasma membrane H(+)-ATPase, induced both stomatal opening and H(+) release in the Adiantum epidermis. Adiantum phototropin genes AcPHOT1 and AcPHOT2 were expressed in the fern guard cells. The transformation of an Arabidopsis phot1 phot2 double mutant, which lost blue light-specific stomatal opening, with AcPHOT1 restored the stomatal response to blue light. Taken together, these results suggest that ferns of Leptosporangiopsida lack a blue light-specific stomatal response, although the functional phototropin and plasma membrane H(+)-ATPase are present in this species.

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.

Chloroplast movement: dissection of events downstream of photo- and mechano-perception.

The study of chloroplast photorelocation movement is progressing rapidly now that mutants for chloroplast movement have become available in Arabidopsis thaliana. However, mechanistic approaches in cell biology still stand to elucidate the mechanisms and regulations of such movement. The fern Adiantum capillus-veneris and the moss Physcomitrella patens are particularly suitable materials for analyzing the kinetics of intracellular chloroplast movement. In these plants, chloroplast movement is induced by red light as well as blue light, mediated by phytochrome and blue light receptor, respectively. In this paper, we review the unique force-generating system for chloroplast motility in P. patens. In addition to light-induced chloroplast movement, we also summarize mechanically induced chloroplast movement in these plants and the motility systems involved. Finally, the different dependency of mechano- and photo-relocation movement on external Ca(2+) is discussed.

Responses of ferns to red light are mediated by an unconventional photoreceptor.

Efficient photosynthesis is essential for plant survival. To optimize photosynthesis, plants have developed several photoresponses. Stems bend towards a light source (phototropism), chloroplasts move to a place of appropriate light intensity (chloroplast photorelocation) and stomata open to absorb carbon dioxide. These responses are mediated by the blue-light receptors phototropin 1 (phot1) and phototropin 2 (phot2) in Arabidopsis (refs 1-5). In some ferns, phototropism and chloroplast photorelocation are controlled by red light as well as blue light. However, until now, the photoreceptor mediating these red-light responses has not been identified. The fern Adiantum capillus-veneris has an unconventional photoreceptor, phytochrome 3 (phy3), which is a chimaera of the red/far-red light receptor phytochrome and phototropin. We identify here a function of phy3 for red-light-induced phototropism and for red-light-induced chloroplast photorelocation, by using mutational analysis and complementation. Because phy3 greatly enhances the sensitivity to white light in orienting leaves and chloroplasts, and PHY3 homologues exist among various fern species, this chimaeric photoreceptor may have had a central role in the divergence and proliferation of fern species under low-light canopy conditions.