Action Spectrum for the Timing of Photo-Induced Cell Division in Adiantum Gametophytes

The photo-induced cell division in single-celled protonemata of the fern Adiantum capillus-veneris was studied. When the protonemata were exposed to monochromatic light at 50 nm intervals between 350 and 750 nm, irradiations in the blue and near-ultraviolet regions effectively induced cell division, while wavelengths longer than 550 nm showed no such effect. As reciprocity between duration and intensity was observed within the range of incident energy used, the action spectrum for the frequency of the photo-induced cell divisions 24 h after irradiation was determined between 360 and 510 nm at 10 nm intervals. Furthermore, the previously known effect of phytochrome on the timing of the cell division was minimized by a short exposure to red light given immediately after the monochromatic irradiation. The resulting action spectra showed a peak in the neighborhood of 460 nm with shoulders and another peak in the near-ultraviolet region.     

APICAL GROWTH OF PROTONEMATA IN ADIANTUM CAPILLUSVENERIS. I. RED FAR-RED REVERSIBLE EFFECT ON GROWTH CESSATION IN THE DARK

Apical growth of individual protonemata in Adiantum capillus-veneris was microphotographically observed before, during and after light treatment. When single-celled protonemata precultured under continuous red light were transferred to darkness, the apical growth continued for the next 24 hr at a rate somewhat slower than that under continuous red light, but the rate significantly decreased thereafter and growth ceased within 72 hr in the dark. The growth in the dark was strongly inhibited by a brief irradiation with far-red light given immediately before the dark period, and the effect of far-red light was fully reversed by subsequent red light. This reversibility was repeatedly observed, suggesting the involvement of a phytochrome system.
The intracellular localization of the phytochrome system in the protonemata was studied, using a narrow-beam irradiator. The results showed that the photoreceptive sites of far-red light are not localized in any particular region of the cell.     

Light-induced synchrony of cell division in the protonema of the fern,Pteris vittata

Summary In protonemata of Pteris vittata grown for 6 days under red light, which brings about a marked depression of mitotic activity, the first division of the cells was synchronously induced by irradiation with blue light, and subsequent cell divisions were also promoted. The peak of the mitotic index reached a maximum of about 70% at 11.5 hrs, and 90% of all protonemata divided between the 11th and 13th hour after exposure to blue light. When the protonemata were continuously irradiated with blue light, synchronism of the next cell division in the apical cells decreased to a mitotic index of about 30%, and further divisions occurred randomly.The synchronization of cell division was found to be a combined effect of red and blue light. Red light maintained the cells in the early G₁ phase of the cell cycle; blue light caused the cells to progress synchronously through the cell cycle, with an average duration of 12 hr. By using ³H-thymidine, the average duration of the G₁, S, G₂ and M phases was determined to be about 3.5, 5, 2.5 and 1 hr, respectively.Synchronous cell division could be induced in older protonemata grown for 6 to 12 days in red light and even in protonemata having two cells. It could be repeated in the same protonema by reexposure to red light for 24 hrs or more before another irradiation with blue light.     

Apical growth of protonemata inAdiantum capillus-veneris

Action spectra for the induction of apical swelling in red-light-grown single-celled protonemata of the fernAdiantum were determined by continuous irradiation with monochromatic light for 5 hr. The resultant action spectra showed a sharp peak at 480 nm with a broad plateau in the region of blue and near ultraviolet light. Wave-lengths longer than 520 nm had no effect. When the tips of filamentous protonemata were irriadiated with a narrow beam (20 μm in width) of blue light for 3 hr, apical swelling and apical growth inhibition obviously took place in all protonemata tested, while no significant effect was observed when any other regions than the tip were irradiated. Polarized blue light vibrating parallel with the developmental axis of protonemata induced apical swelling and also prevented apical growth as effectively as non-polarized light, but that vibrating in a normal direction was significantly less effective.     

PHOTOCONTROL OF THE ORIENTATION OF CELL DIVISION IN ADIANTUM. I. EFFECTS OF THE DARK AND RED PERIODS IN THE APICAL CELL OF GAMETOPHYTES

When protonemata of Adiantum capillus-veneris L. which had been grown filamentously under continuous red light were transferred to continuous white light, the apical cell divided transversely twice, but the 3rd division was longitudinal. An intervening period of darkness lasting from 0 to 90 hr either between the 1st and the 2nd cell division or between the 2nd and the 3rd one did not affect the number of protonemata in which the 3rd cell division was longitudinal. The insertion of red light instead of darkness greatly decreased the percentage of 1st longitudinal divisions occurring at the 3rd division, and increased the number of transverse divisions. Fifty percent reduction of induction of 1st longitudinal division was caused by ca. 50 hr exposure to red light between 1st and 2nd division and by ca. 20 hr between 2nd and 3rd division, and total loss was induced by an exposure of ca. 100 hr or longer to red light in the former and by ca. 40 hr longer in the latter. Thus, by using an appropriate intervening dark period or exposure to red light, the orientation and timing of cell division could be controlled in apical cell of the fern protonemata.     

EFFECT OF CENTRIFUGATION ON THE DEVELOPMENT AND TIMING OF PREMITOTIC POSITIONING OF THE NUCLEUS IN ADIANTUM PROTONEMATA

When single-celled protonemata of Adiantum capillus-veneris L. were centrifuged immediately before transferring to darkness from continuous irradiation with red light, their nuclei were displaced basipetally. Both filamentous and branched protonemata were obtained. The stronger the centrifugal acceleration, the more frequently the branched protonemata were induced.
The effect of centrifugation at 1,300 x g for 15 min on nuclear displacement was different at different stages of the cell cycle. In early G₁ phase, the nucleus was easily displaced by centrifugation, but quickly returned to the original position after centrifugation. In late G₁ phase, the nucleus was displaced, but after centrifugation it never came back to the original position. In late G₂ and M phases, the nucleus was no longer displaced by the centrifugation. Premitotic positioning of the nucleus in cytokinesis took place about 5 hr before cell plate formation in all centrifugal treatments described above.     

Protein Synthesis during Photocontrolled Progression of the Cell Cycle in Single-celled Protonemata of the Fern Adiantum Capillus-veneris

Protein synthesis during photoinduced, synchronous progression of the cell cycle in single-celled protonemata of the fern Adiantum capillus-veneris was studied by tracer techniques. Nuclei of the protonemata were labelled with ³H-thymidine during spore germination so that the amount of ³H incorporated into the TCA-insoluble fraction of the cells could be used as a measure of the cell number in each sample. The rate of the incorporation of ¹⁴C-amino acids into TCA-insoluble materials was not significantly varied at different stages of the cell cycle or by treatment with blue light. Extracts of cells labelled with ³⁵S-methionine at various times after the transfer from red light condition (G₀) to darkness (G₁ to S) were analyzed by two-dimensional gel electrophoresis. At least 3 of about 200 spots showed significant changes in intensity on fluorograms. Spot A (molecular weight 20,000, isoelectric point 6.3) was detectable only in early G₁, whereas spot B (molecular weight 19,500, isoelectric point 6.3) was found only in the late G₁ and S phases. When the cells were exposed to blue light before the dark incubation, the times of disappearance of spot A and appearance of spot B were advanced depending upon the progression of the cell cycle but not upon the clock time.     

Development and germination of rhizoidal gemmae of Bryum violaceum

Approximately four to six weeks after transferring gametophores of Bryum violaceum Crundwell & Nyholm to fresh soil, abundant new rhizoids with stalked gemmae were present at the bases of the gametophores. The development of gemmae on rhizoids was followed from single cell initials to multicellular, three-dimensional forms. Mature gemmae, the vegetative diaspores, were pale orange, purple or reddish brown and each had a uniseriate stalk of two to four cells. While remaining on soil, the rhizoidal gemmae showed no in situ germination. However, following the removal of gametophores with rhizoidal gemmae, or rhizoids alone with gemmae, and their placement upon wet filter paper in Petri dishes in light, the gemmae germinated. During germination, the protonemata emerged consistently from mature gemmae at their distal ends, revealing the existence of gemma polarity. In the case of immature gemmae, on the other hand, the protonemata emerged from any surface cell indicating that gemma polarity had not yet been established.     

Effects of Colchicine and Light on Cell Form in Fern Gametophytes. Implications for a Mechanism of Light-induced Cell Elongation

Spores of the fern, Onoclea sensihilis L., suffer a disruption of normal development when they are cultured on media containing colchicine. Cell division is inhibited, and the spores develop into giant spherical cells under continuous white fluorescent light. In darkness only slight cell expansion occurs. Spherical cell expansion in the light requires continuous irradiation. Photosynthesis does not seem to be involved, since variations in light intensity do not affect the final cell diameter; the addition of sucrose to the medium does not permit cell expansion in darkness; and the inhibitor DCMU does not block the light-induced cell expansion. Continuous irradiation of colchicine-treated spores with blue, red or far-red light produces different patterns of cell expansion. Blue light permits spherical growth, similar to that found under white light, whereas red and far-red light promote the reestablishment of polarized filamentous growth. Although ethylene is unable to induce polarized cell expansion in colchicine-treated spores in darkness or white and blue light, it enhances filamentous growth which already is established by red or far-red irradiation. Both red and far-red light increase the elongation of normal filaments (untreated with colchicine) above that of dark-grown plants, but under all 3 conditions the rates of volume growth are identical. Light, however, does cause a decrease in the cell diameters of irradiated filaments. These data are used to construct an hypothesis to explain the promotion of cell elongation in fern protonemata by red and far-red light. The model proposes light-mediated changes in microtubular orientation and cell wall structure which lead to restriction of lateral cell expansion and enhanced elongation growth.     

Blue light-regulation of cell division in Adiantum protonemata: an approach with pulse stimulation

Abstract Cell division of the single-celled Adiantum protonemata produced by red-light (RL) incubation of germinated spores is induced by transfer to darkness and is stimulated by blue light (BL). It is known that the cellular process leading to this cell division includes one cell cycle and the BL response results from shortening of the Gl phase. The authors studied this BL regulation of cell cycle by giving a pulse of BL after RL termination and measuring changes in the proportion of divided cells. To minimize phytochrome responses arising from BL irradiation, the plants were kept in continuous far-red light instead of total darkness after the RL incubation. The response to a pulse (10–100 s) approached saturation with increasing rluences in a manner that reciprocity is valid. The sensitivity to BL, investigated by measuring the response to a saturating pulse, showed an increase in the first several hours after RL termination, followed by a sustained sensitivity for 20 h. Time courses of the pulse-induced responses showed a lag of about 12 h, which was considerably shorter than in the non-stimulated control; the lag was approximately independent of the strength of BL stimulation or the timing of BL application after RL termination, and the major difference occurred in the slope. It is concluded that the sensitivity to BL is retained during the time span in which the dark-dependent Gl phase progresses, and that the BL response is initiated independently of the reactions involved in the dark-dependent Gl phase. A minimal reaction model of Gl phase is suggested to unify the results.     

Relocalization of the calcium gradient and a dihydropyridine receptor is involved in upward bending by bulging of Chara protonemata, but not in downward bending by bowing of Chara rhizoids

The localization of cytoplasmic free calcium and a dihydropyridine (DHP) receptor, a putative calcium channel, was recorded during the opposite graviresponses of tip-growing Chara rhizoids and Chara protonemata by using the calcium indicator Calcium Crimson and a fluorescently labeled dihydropyridine (FL-DHP). In upward (negatively gravitropically) growing protonemata and downward (positively gravitropically) growing rhizoids, a steep Ca²⁺ gradient and DHP receptors were found to be symmetrically localized in the tip. However, the localization of the Ca²⁺ gradient and DHP receptors differed considerably during the gravitropic responses upon horizontal positioning of the two cell types. During the graviresponse of rhizoids, a continuous bowing downward by differential flank growth, the Ca²⁺ gradient and DHP receptors remained symmetrically localized in the tip at the centre of growth. However, after tilting protonemata into a horizontal position, there was a drastic displacement of the Ca²⁺ gradient and FL-DHP to the upper flank of the apical dome. This displacement occurred after the apical intrusion and sedimentation of the statoliths but clearly before the change in the growth direction became evident. In protonemata, the reorientation of the growth direction started with the appearence of a bulge on that site of the upper flank which was predicted by the asymmetrically displaced Ca²⁺ gradient. With the upward shift of the cell tip, which is suggested to result from a statolith-induced displacement of the growth centre, the Ca²⁺ gradient and DHP receptors became symmetrically relocalized in the apical dome. No major asymmetrical rearrangement was observed during the following phase of gravitropic curvature which is characterized by slower rates of bending. Labeling with FL-DHP was completely inhibited by a non-fluorescently labeled dihydropyridine. From these results it is suggested that FL-DHP labels calcium channels in rhizoids and protonemata. In rhizoids, positive gravitropic curvature is caused by differential growth limited to the opposite subapical flanks of the apical dome, a process which does not involve displacement of the growth centre, the calcium gradient or calcium channels. In protonemata, however, it is proposed that a statolith-induced asymmetrical relocalization of calcium channels and the Ca²⁺ gradient precedes, and might mediate, the rearrangement of the centre of growth, most likely by the displacement of the Spitzenk?rper, to the upper flank, which results in the negative gravitropic reorientation of the growth direction. Received: 13 February 1999 / Accepted: 25 June 1999     

Phytochrome action on the timing of cell division in adiantum gametophytes

When filamentous protonemata of Adiantum capillus-veneris L. precultured under continuous red light were transferred to the dark, the apical cell divided about 24 to 36 hours thereafter. The time of the cell division was delayed for several hours by a brief exposure to far red light given before the dark incubation. The effect of far red light was reversed by a small dose of red light given immediately after the preceding far red light. The effects of red and far red light were repeatedly reversible, indicating that the timing of cell division was regulated by a phytochrome system. When a brief irradiation with blue light was given before the dark incubation, the cell division occurred after 17 to 26 hours in darkness. A similar red far red reversible effect was also observed in the timing of the blue light-induced cell division. Thus, the timing of cell division appeared to be controlled by phytochrome and a blue light-absorbing pigment.     

Hypergravity can reduce but not enhance the gravitropic response ofChara globularis protonemata

Summary The relationship between the position of the statoliths and the direction and rate of tip growth in negatively gravitropic protonemata ofChara globularis was studied with a centrifuge video microscope. Cells placed perpendicularly to the acceleration vector (stimulation angle 90 °) showed a gradual reduction of the gravitropic curvature with increasing accelerations from 1g to 8g despite complete sedimentation of all statoliths on the centrifugal cell flank. It is argued that the increased weight of the statoliths in hypergravity impairs their acropetal transport which is induced when the cell axis deviates from the normal upright orientation. When the statoliths were centrifuged deep into the apical dome at 6g and a stimulation angle of 170 ° the gravitropic curvature after 1 h was identical to that determined for the same cells at 1g and the same stimulation angle. This indicates that gravitropism in Chara protonemata is either independent of the pressure exerted by the statoliths on an underlying structure or is already saturated at 1g. When the statoliths were moved along the apical cell wall at 8g and the stimulation angle was gradually increased from 170 ° to 220 ° the gravitropic curvature reverted sharply when the cluster of statoliths passed over the cell pole. This experiment supports the hypothesis that in Chara protonemata asymmetrically distributed statoliths inside the apical dome displace the Spitzenkörper and thus the centre of growth, resulting in gravitropic bending. In contrast to the positively gravitropic Chara rhizoids, no modifications either in the transport of statoliths during basipetal acceleration (6g, stimulation angle 0 °, 5 h) or in the subsequent gravitropic response could be detected in the protonemata. The different effects of centrifugation on the positioning of statoliths in Chara protonemata and rhizoids indicate subtle differences in the function of the cytoskeleton in both types of cells.Dedicated to Prof. Dr. Zygmunt Hejnowicz on the occasion of his 70th birthday     

Intracellular Photoreceptive Site for Blue Light-Induced Cell Division in Protonemata of the Fern Adiantum--Further Analyses by Polarized Light Irradiation and Cell Centrifugation

The intracellular localization of the photoreceptive site forblue light-induced cell division in single-celled protonemataof Adiantum capillus-veneris L. was investigated using polarizedlight irradiation and protonemal cell centrifugation. The responseto irradiation with polarized blue light showed no dependenceon the direction of light polarization. However, centrifugationof the protonemata followed by microbeam irradiation showedthat the site of blue light perception could be displaced togetherwith the nucleus. Centrifugal treatment changed the distributionof intracellular organelles at the time of light exposure andbasipetally displaced the nucleus about 90µm. This treatmenthad no effect on the induction of cell division with blue lightif the protonemata were centrifuged again acropetally afterthe light treatment. Microbeam (30x30 µm²) irradiationwith blue light of the apical 45–75 ßm region,the receptive site of blue light in non-centrifuged cell, didnot induce cell division. However, cell division was inducedby irradiation of the nucleus-containing region, indicatingthat the photoreceptive site was displaced together with thenucleus by the centrifugation. These results suggest that theblue light receptor regulating cell division in Adiantum protonematais not likely to be located on the plasma membrane. (Received February 20, 1986; Accepted May 27, 1986)     

Photocontrol of the orientation of cell division in Adiantum

Summary Two-dimensional prothallia of Adiantum capillus-veneris always expanded in a plane which was at a right angle to any given direction of irradiation with continuous white light. The expansion began with a longitudinal division of the apical cell, in the filamentous protonema, and the orientation of the mitotic cell plate of this first longitudinal division as well as the subsequent divisions was always parallel to the direction of the incident light. When three irradiations with white light, interrupted by periods of darkness, were given, two transverse and one subsequent longitudinal division were induced. When the last two irradiations were given from the same direction, the cell plate of the first longitudinal division in most protonemata was oriented parallel to the direction of light. However, when the direction of light during the third irradiation was at right angle to that during the second, the frequency of the longitudinal division greatly decreased but that of the third transverse division increased. Thus, the orientation of the first longitudinal division appeared to be controlled in some way not only by the irradiation which actually induced the third division but also by that inducing the preceding transverse division, while the direction of light for the first transverse division had little effect on the orientation of the third division.     

Reorganization of the actin and microtubule cytoskeleton throughout blue-light-induced differentiation of characean protonemata into multicellular thalli

Summary The reorganization of the actin and microtubule (MT) cytoskeleton was immunocytochemically visualized by confocal laser scanning microscopy throughout the photomorphogenetic differentiation of tip-growing characean protonemata into multicellular green thalli. After irradiating dark-grown protonemata with blue or white light, decreasing rates of gravitropic tip-growth were accompanied by a series of events leading to the first cell division: the nucleus migrated towards the tip; MTs and plastids invaded the apical cytoplasm; the polar zonation of cytoplasmic organelles and the prominent actin patch at the cell tip disappeared and the tip-focused actin microfilaments (MFs) were reorganized into a homogeneous network. During prometaphase and metaphase, extranuclear spindle microtubules formed between the two spindle poles. Cytoplasmic MTs associated with the apical spindle pole decreased in number but did not disappear completely during mitosis. The basal cortical MTs represent a discrete MT population that is independent from the basal spindle poles and did not redistribute during mitosis and cytokinesis. Preprophase MT bands were never detected but cytokinesis was characterized by higher-plant-like phragmoplast MT arrays. Cytoplasmic actin MFs persisted as a dense network in the apical cytoplasm throughout the first cell division. They were not found in close contact with spindle MTs, but actin MFs were clearly coaligned along the MTs of the early phragmoplast. The later belt-like phragmoplast was completely depleted of MFs close to the time of cell plate fusion except for a few actin MF bundles that extended to the margin of the growing cell plate. The cell plate itself and young anticlinal cell walls showed strong actin immunofluorescence. After several anticlinal cell divisions, basal cells of the multicellular protonema produced nodal cell complexes by multiple periclinal divisions. The apical-dome cell of the new shoot which originated from a nodal cell becomes the meristem initial that regularly divides to produce a segment cell. The segment cell subsequently divides to produce a single file of alternating internodal cells and multicellular nodes which together form the complexly organized characean thallus. The actin and MT distribution of nodal cells resembles that of higherplant meristem cells, whereas the internodal cells exhibit a highly specialized cortical system of MTs and streaming-generating actin bundles, typical of highly vacuolated plant cells. The transformation from the asymmetric mitotic spindle of the polarized tip-growing protonema cell to the symmetric, higher-plant-like spindle of nodal thallus cells recapitulates the evolutionary steps from the more primitive organisms to higher plants.Abbreviations FITC fluorescein isothiocyanate - MF microfilament - MT microtubule - MSB microtubule-stabilizing buffer - PBS phosphate-buffered saline     

Cytokinin control of growth of Spirodela oligorrhiza in darkness

Summary Continuous heterotrophic growth of Spirodela oligorrhiza cultures following transfer to darkness requires cytokinins, or periodic brief treatment with red light. In the absence of cytokinins or red light growth ceases after 2–3 days. However, growth resumes spontaneously after 3–4 weeks in darkness to produce etiolated plants. The growth rate of these etiolated plants is not stimulated by kinetin.Although the kinetin concentration in treated plants reaches a plateau 30–60 min after adding kinetin to dormant plants in darkness new fronds do not appear for 24 h. Dormant colonies treated with kinetin in darkness for only 6–12 h subsequently grow in darkness at the same rate as plants treated with kinetin for 1, 2 or 3 days. Treatments which inhibit growth in the light, for example cold, chloramphenicol or actidione, eliminate the requirement for cytokinin and allow subsequent growth in darkness. The results suggest that a growth inhibitor may be present but ineffective in Spirodela growing in the light. The inhibitor is active in darkness but slowly decays. Kinetin appears to inactivate the inhibitor in darkness.     

THE RELATIONSHIP BETWEEN THE PROMOTION OF ELONGATION OF FERN PROTONEMATA BY LIGHT AND GROWTH SUBSTANCES

Germinating spores of the fern Onoclea sensibilis L. were grown in darkness, so that they developed as filaments (protonemata). Brief daily exposure of the filaments to red, far-red or blue light increased the rate of filament elongation. Filament elongation was also promoted by indoleacetic acid. When filament elongation was promoted with both indoleacetic acid and exposure to light, the growth promotions caused by red and far-red light were additive to auxin-induced growth. Blue light promoted elongation only at sub-optimal concentrations of auxin. Elongation induced by guanine was additive to red- and far-red-induced elongation. Gibberellic acid had no effect on elongation under any condition. Blue-light-induced elongation resembled auxin-induced elongation in its requirement for exogenous sucrose and sensitivity to inhibition by parachlorophenoxyisobutyric acid. Red and far-red light were active regardless of the presence or absence of sucrose and promoted elongation at a concentration of parachlorophenoxyisobutyric acid which completely inhibited blue-light-induced elongation.     

The changes of nuclear position and distribution of circumferentially aligned cortical microtubules during the progression of cell cycle inAdiantum protonemata

The intracellular positions of the nucleus and of cortical, circumferentially aligned microtubules (CCAM) in filamentous, single-celled protonemata ofAdiantum capillus-veneris were determined throughout the cell cycle in the dark. When apical growth continued at G₁ phase, the nucleus migrated keeping a constant distance from the tip. When the apical growth stopped at late S or G₂ phase, the nucleus stopped moving forward and then slightly moved backward to the site of cytokinesis. The CCAM were found only in the dome of protonemal tip when growing under continuous red light; they increased in number after dark incubation for 12 hr and then decreased after 20th hr in the dark. The CCAM were usually observed in the region between the nucleus and the tip at 28 hr in the dark. They were located around the nuclear region at pre-prophase and prophase, but then totally disappeared at metaphase and thereafter.     

Action Spectra for Light-induced Elongation in Fern Protonemata

The action spectrum for promotion of elongation of protonemata of Onoclea sensibilis has peaks at 400–420, 580–600 and 640–660 nm. The largest growth increments at saturating light doses are produced by yellow and far-red light. Elongation induced by yellow and far-red irradiation persists in old as well as young filaments, while red-light promotion is found only in young filaments. The growth promotion caused by yellow light is partially reversed by red light down to the level of growth produced by red irradiation alone. Elongation of rhizoids is under reversible red, far-red control, while yellow light is inactive. A model is proposed and discussed in which the light-sensitive elongation of filaments is accounted for by the presence of three distinct photoreceptors: phytochrome; a pigment absorbing yellow light. P₅₈₀; and a pigment absorbing blue light, P₄₂₀.