Intracellular Photoreceptive Site for Polarotropism in Protonema of the Fern Adiantum capillus-veneris L.

Polarotropic response was induced by short-term irradiationwith polarized red light in single-celled protonemata of thefern Adiantum capillus-veneris L. that had been grown apicallyunder red light for 6 days then for 15 hr in the dark. Sequentialobservation of the apical growth with a time-lapse video systemshowed that the direction of apical growth changed within 30min after the brief irradiation. Microbeam irradiation withpolarized red light of the subapical, dark-grown flank of theapical, 5–15 µm region of the protonema inducedthe polarotropic response most effectively. When both sidesof the flank were irradiated simultaneously with different fluencesof polarized red light with the same vibrating plane of 45°with protonemal axis, polarotropism took place normally, ifthe fluence ratio, B/A (B: fluence given to the side towardwhich the protonema should bend in polarotropism, A: fluencegiven to the other side) was not less than one-half. But, ifthe ratio became less than that, the protonemata no longer showedpolarotropism, they grew toward the side of higher fluence dependingon the difference in fluences between both sides. (Received August 1, 1981; Accepted September 29, 1981)     

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 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.     

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.     

Action spectra for polarotropism and phototropism in protonemata of the fern Adiantum capillus-veneris

The action spectrum for polarotropism was determined, using the Okazaki large spectrograph, by brief irradiation with light between 260 nm and 850 nm in single-celled protonemata of the fern Adiantum capillus-veneris L., which had been cultured for 6 days in red light and then in the dark for 15 h. The action spectrum had a peak at around 680 nm. This effect was nullified by subsequent irradiaton with far-red light, and typical red/far-red reversibility was observed, indicating the involvement of phytochrome. Polarized ultraviolet or blue light had no effect on the direction of apical growth. The action spectrum for phototropism was also determined in the red light region by means of brief microbeam irradiation of a flank of the subapical region of the protonema. This spectrum showed a peak at 662 nm which was consistent with the absorption peak of phytochrome, but not with the peak of the action spectrum for polarotropism.     

Apical Growth of Protonemata in Adiantum capillus-veneris IV. Phytochrome-Mediated Induction in Non-Growing Cells

In non-growing two-celled protonemata of Adiantum capillus-veneris,apical growth was induced most effectively by red light irradiation;half of the samples were induced to grow by 660 nm light ofca. 1.5 J m^–2 and the maximum number by ca. 70 J m^–2.The reciprocity law was valid in this photoinduction. The growthresumption became detectable 6 hr after the light irradiationand reached a plateau within 24 hr irrespective of given fluences.When non-growing samples were irradiated with red light of 4.6W m^–2 for 4 sec or shorter, the effect was fully reversedby a subsequent irradiation with far-red light to the far-redlight control level. But, when the red light was given for 16sec or longer, photoreversibility became partial. An interveningdark period of 2 min between red and far-red light did not significantlyinfluence the photoreversibility so that the escape reactionin the dark may not be attributed to the above-mentioned lossof photoreversibility. By means of a local irradiation with a narrow red light beam(10 µm in width), the apical cell was found to be photosensitivefor the growth induction, but basal cell was not. Photoreceptivesite was not localized in any particular region of the apicalcell, but was rather dispersed in the entire apical cell. (Received January 26, 1981; Accepted March 10, 1981)     

A model system to study the effect of SO2 on plant cells. III. Effects of sulfite on the ultrastructure of fern protonemal cells

The mechanism of the toxic effects on plant cells of sulfite, a product of the air pollutant sulfur dioxide, is not well understood. Therefore, changes in the fine structure and organization of microtubules and microfibrils induced by sulfite were studied by electron and light microscopy in the protonemata of the fernAdiantum capillusveneris L. Under red-light conditions, growing protonemata fumigated with 0.05 or 0.1 μ1/1 SO₂ for 1 to 4 days showed abnormalities, such as apical swelling, and they sometimes burst at the apex. The incidence of abnormalities seemed to be correlated with the concentration of the sulfite dissolved in the culture medium. At an appropriate concentration (3.3–6.6. mM) of sulfite (applied as K₂SO₃), cell swelling at the apical region of protonema was also induced. When the concentration of sulfite was as high as 6.6 mM, more than 60% of protonemata burst at the tip. During the apical swelling, no distinct changes were observed in the fine structure of organelles, such as the chloroplasts, mitochondria, microbodies, Golgi bodies and nucleus. However, the arrangement of cortical microtubules and that of the innermost layer of microfibrils around the subapical region of protonemata were changed from transverse to the cell axis (i.e., circular) to random and the cell wall was thickened. These observations suggest that sulfite may influence the mechanisms that maintain the transverse orientation of microtubules in the subapical region of a protonema and that the resultant random arrangement of microtubules induces the random arrangement of microfibrils and leads to apical swelling.     

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.     

Formation of novel endoplasmic and cortical microtubular arrays inAdiantum protonemal cells induced by blue light irradiation and acropetal centrifugation

Protonemal cells ofAdiantum capillus-veneris were grown under red light conditions over 6 days and exposed to blue light for 8 hr (and then dim green light for 1 hr for technical reasons), before they were centrifuged acropetally over at least 1 hr at 2,000×g. After this treatment, an arrangement of endoplasmic microtubules (MTs) that resembled the shape of a tadpole could be detected some distance below the nucleus in about 40% of the cells. The percentage of protonemata bearing this Mtstructure was dependent on centrifugation time as well as the time of blue light irradiation. The size of the structure was constant at any time of its existence. Additionally, a wide belt of transversally oriented cortical MTs in the upper part of the protonemata was detected in many cells after blue light irradiation and acropetal centrifugation. Its formation rate seemed to be also dependent on the period of blue light irradiation and centrifugation time. None of the endoplasmic and few of the cortical transverse MT patterns could be seen without blue light irradiation. A strict coincidence in the formation of both MT patterns was not seen. Further, a few tadpole-shaped MT arrays remained during mitosis, whereas the cortical transverse MT pattern was found in stages other than metaphase and anaphase.     

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)     

Blue Light-Induced Phototropic Response and the Intracellular Photoreceptive Site in Adiantum Protonemata

Blue light-induced phototropism in Adiantum protonemata wasinvestigated with microbeam irradiation. Brief irradiation withblue light effectively induced a phototropic response when itwas applied to a half-side of the apical 200d µm regionof a protonema. The phototropic response was partly reversedby the subsequent far-red light irradiation but the full reversalof the response was not observed even when the fluence of far-redlight was increased. In the far-red reversible part of the response,blue/far-red photoreversibility was repeatedly observed. Thus,both phytochrome and a blue light-absorbing pigment (other thanphytochrome) seem to be involved in the blue light-induced phototropicresponse in Adiantum protonemata. Furthermore, detailed studiesof the far-red light effect on the fluence-response curve forblue lightinduced phototropism revealed that the concomitantmediation by the two receptors was limited to the response inthe relatively higher fluence range of blue light and that theblue light-absorbing pigment alone was responsible in the lowerfluence range. In the higher fluence range, the response mediatedby the blue light-absorbing pigment became saturated and thephytochrome response became evident, indicating a differencein the sensitivities of the two receptor pigments to blue light. When various regions of half-sides of protonemata were irradiatedwith a blue microbeam of 10 µm width, irradiation at theapical 5–25 µm region was most effective both forphytochrome- and blue light-absorbing pigment-mediated response,indicating that the site of blue light perception is almostidentical for each response. (Received July 14, 1986; Accepted September 26, 1986)     

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.     

The Protonema of Chara fragilis Desv.: Regenerative Formation,Photomorphogenesis, and Gravitropism

When exposed to constant white light for four weeks, isolated nodes of Chara fragilis Desv. regenerated side branches, rhizoids, and multicellular protonemata, the latter being similar to those germinated from oospores. When kept in darkness the nodes developed protonemata exclusively. These were single-celled, colourless, and tip-growing and, with the light microscope, they looked like rhizoids. Upon exposure to blue light, but not to red or far-red, the growth rates of the protonemata rapidly declined, the cell apices swelled, and the nucleus migrated acropetally. Within 24 h the cells went through the first of a series of divisions resulting in the formation of multicellular protonemata. When returned to darkness after a blue light pulse of 5 h the cell divisions proceeded normally, but the protonemata showed etiolated growth. While growth of the internode was drastically promoted, the development of the multicellular apex and the lateral initial were suppressed. Both uni- and multicellular etiolating protonemata showed negative gravitropism but were phototropically insensitive. It is argued that the single-celled protonema is an organ specialized for the penetration of mud covering the nodes or oospores of Chara and thus serves to search for light, comparable to etiolated hypocotyls and stems in seedlings of higher plants.     

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.     

Phytochrome-mediated polarotropism of Adiantum capillus-veneris L. protonemata as analyzed by microbeam irradiation with polarized light

Summary Perception of polarized light inducing phytochrome-mediated polarotropism in protonemata of the fern Adiantum capillus-veneris L. was analyzed using brief microbeam irradiation with polarized red (R) or far-red light (FR). The polarotropic response inducible by irradiation of the subapical 10–30-m part with polarized R vibrating parallel to the cell axis was nullified by subsequently giving R at the apical 0–2.5-m region. This inhibitory effect of R showed an action dichroism, that is, polarized R vibrating normal to the cell axis was effective but the parallel-vibrating R was not. On the other hand, FR irradiation of the extreme tip after irradiation of the whole cell with depolarized R effectively induced a tropic response. This FR effect also showed action dichroism, with parallel-vibrating polarized FR being more effective than FR vibrating normal to the cell axis. When the apical-dome region and the adjacent subapical 10–20-m region were sequentially irradiated with polarized R vibrating obliquely in different directions, polarotropism took place depending on the vibrating direction of the light given to the apical-dome region. Obliquely vibrating polarized FR given to the apical dome after irradiation of the whole cell with depolarized R also induced polarotropism. Thus, the difference in amount (or percent) of the far-redabsorbing form of phytochrome (Pfr) between the extreme tip and the subapical region appears to be crucial in regulating the direction of apical growth; the difference in Pfr level between the two sides of the protonemal apex may occur mainly at the apical dome. Furthermore, the transition moments of the red-absorbing form of phytochrome (Pr) and Pfr seem to be aligned parallel and normal, respectively, to the cell surface at the periphery of the apical hemisphere.Abbreviations FR far-red light - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light     

Blue- and red-light action in photoorientation of chloroplasts in Adiantum protonemata

An action spectrum for the low-fluencerate response of chloroplast movement in protonemata of the fern Adiantum capillus-veneris L. was determined using polarized light vibrating perpendicularly to the protonema axis. The spectrum had several peaks in the blue region around 450 nm and one in the red region at 680 nm, the blue peaks being higher than the red one. The red-light action was suppressed by nonpolarized far-red light given simultaneously or alternately, whereas the bluelight action was not. Chloroplast movement was also induced by a local irradiation with a narrow beam of monochromatic light. A beam of blue light at low energy fluence rates (7.3·10^-3-1.0 W m^-2) caused movement of the chloroplasts to the beam area (positive response), while one at high fluence rates (10 W m^-2 and higher) caused movement to outside of the beam area (negative response). A red beam caused a positive response at fluence rates up to 100 W m^-2, but a negative response at very high fluence rates (230 and 470 W m^-2). When a far-red beam was combined with total background irradiation with red light at fluence rates causing a low-fluence-rate response in whole cells, chloroplasts moved out of the beam area. When blue light was used as background irradiation, however, a narrow far-red beam had no effect on chloroplast distribution. These results indicate that the light-oriented movement of Adiantum chloroplasts is caused by red and blue light, mediated by phytochrome and another, unidentified photoreceptor(s), respectively. This movement depends on a local gradient of the far-red-absorbing form of phytochrome or of a photoexcited blue-light photoreceptor, and it includes positive and negative responses for both red and blue light.Abbreviations BL blue light - FR far-red light - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light - UV ultraviolet     

PHOTOMORPHOGENESIS OF THE GAMETOPHYTES OF LYGODIUM JAPONICUM

Protonemata of Lygodium japonicum turn biplanar in both red and blue light regimes and remain filamentous in far-red light. Biplanar gametophytes formed in red light are longer than broad with long, rectangular cells, whereas in blue light they appear broader than long with short, isodiametric cells. Transfer of protonemata of all ages from far-red regime to red or blue light induces a morphological type of growth characteristic of the new light regime. However, only relatively young biplanar forms transferred from red or blue light are able to resume filamentous type of growth in a subsequent regime of far-red light.     

Photocontrol of the orientation of cell division inAdiantum

The rate of transition from one- to two-dimensional growth of fernAdiantum gametophytes under white light depends on the age of gametophyte cultured under red light. When gametophytes were cultured for longer period under red light, the rate of transition decreased and the number of abnormal gametophytes increased. Although the first step of the transition was the first longitudinal cell division following the two transverse ones (Wada and Furuya, 1970), the time-lapse-video study revealed that the apical cell of protonemata became flattened in the plane perpendicular to the incident ray of white light before the first longitudinal cell division. Analytical study of growing part of the apical cell with grains of activated charcoal as markers revealed that the apical cell flattening occurred evenly throughout the equatorial circumference of the cell even in the shaded side of the protonemata as well as in the side irradiated with white light.     

Negative phototropism in young gametophytes of the fern Schizaea pusilla

Schizaea pusilla is a rare fern that occurs in acidic bogs and is one of the few fern species that maintains a filamentous gametophyte throughout its development. To expand our knowledge of the physiology of this fern, phototropic responses were examined in young gametophytes. In contrast to germ filaments of other fern species, apical protonemata of young gametophytes are negatively phototropic in continuous white, red and blue light at all fluence rates tested. The expression of phototropic curvature is not limited by time since apical protonemata are also negatively phototropic when they are given brief exposures of light and then placed in the dark. In other lower plant groups such as mosses and some algae, the direction of phototropic curvature can change depending on light quality and intensity, but in young gametophytes of Schizaea, negative phototropic curvature was observed in all conditions studied. Blue light is the most effective in promoting the negative phototropic response in Schizaea.     

Blue light reduces photosynthetic efficiency of cyanobacteria through an imbalance between photosystems I and II

Several studies have described that cyanobacteria use blue light less efficiently for photosynthesis than most eukaryotic phototrophs, but comprehensive studies of this phenomenon are lacking. Here, we study the effect of blue (450 nm), orange (625 nm), and red (660 nm) light on growth of the model cyanobacterium Synechocystis sp. PCC 6803, the green alga Chlorella sorokiniana and other cyanobacteria containing phycocyanin or phycoerythrin. Our results demonstrate that specific growth rates of the cyanobacteria were similar in orange and red light, but much lower in blue light. Conversely, specific growth rates of the green alga C. sorokiniana were similar in blue and red light, but lower in orange light. Oxygen production rates of Synechocystis sp. PCC 6803 were five-fold lower in blue than in orange and red light at low light intensities but approached the same saturation level in all three colors at high light intensities. Measurements of 77 K fluorescence emission demonstrated a lower ratio of photosystem I to photosystem II (PSI:PSII ratio) and relatively more phycobilisomes associated with PSII (state 1) in blue light than in orange and red light. These results support the hypothesis that blue light, which is not absorbed by phycobilisomes, creates an imbalance between the two photosystems of cyanobacteria with an energy excess at PSI and a deficiency at the PSII-side of the photosynthetic electron transfer chain. Our results help to explain why phycobilisome-containing cyanobacteria use blue light less efficiently than species with chlorophyll-based light-harvesting antennae such as Prochlorococcus, green algae and terrestrial plants.