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.     

Intracellular Localization and Dichroic Orientation of Phytochrome in Plasma Membrane and/or Ectoplasm of a Centrifuged Protonema of Fern Adiantum capillus-veneris L.

Protonemata of the fern Adiantum capillus-veneris grown undercontinuous red light for 6 days were kept in darkness for 15h and subsequently centrifuged 3 times in different directions,so that oil droplets and other cytoplasm were removed from theapical region of the protonemata. Electron micrographs clearlydemonstrated that cell wall, plasma membrane, ectoplasm andmicrotubules remained in the apical and subapical regions afterthe centrifugal treatments. A brief local exposure of the flankof the subapical region of the centrifuged protonemata to amicrobeam of red light effectively induced a phototropic responsetoward the irradiated side, suggesting that phytochrome is locatedin the ectoplasm and/or plasma membrane. When the flank of thecentrifuged protonema was irradiated with linearly polarizedred or far-red light, red light with an electrical vector parallelto the cell surface was more effective than that perpendicularto the cell surface. The direction of the electrical vectorof far-red light for reversion of the preirradiated red lighteffect, however, was opposite. These results suggest that differentdichroic orientations of P_R and P_FR exist in the plasma membraneor ectoplasm. (Received May 26, 1983; Accepted September 1, 1983)     

EFFECTS OF PHYTOCHROME AND BLUE-NEAR ULTRAVIOLET LIGHT-ABSORBING PIGMENT ON DURATION OF COMPONENT PHASES OF THE CELL CYCLE IN ADIANTUM GAMETOPHYTES

Single-celled protonemata of the fern Adiantum capillus-veneris, kept under continuous red light, grew with a very low rate of cell division, and the cell cycle was arrested in the early G₁ phase. Cell division was induced by transferring the protonemata to the dark after various light treatments, and the duration of component phases in the cell cycle was determined by a continuous-labelling technique with 3H-thymidine. Blue light irradiation greatly reduced the duration of the G₁ phase but did not affect that of other phases. The greater the fluence of blue light, the shorter was the duration of G₁ phase was observed. In contrast, a brief exposure of red-light-grown protonemata to far-red light given immediately before the dark incubation showed no effect on the duration of G₁ S and M phases but significantly extended that of the G₂ phase. The effect of far-red light on the G₂ phase was reversed by red light, and the effects of red and far-red light were repeatedly reversible. The progression in the M phase was shown by means of a time-lapse video system to be not at all influenced by any pre-irradiation described above.     

Dichroic Orientation of Phytochrome Intermediates in the Pathway from PR to PFR as Analyzed by Double Laser Flash Irradiations in Polarotropism of Adiantum Protonemata

The polarotropic response in protonemata of the fern Adiantumis regulated by phytochrome (Kadota et al. 1984); P_R and P_FRhave been shown to be dichroically oriented parallel and normalto the cell surface, respectively (Kadota et al. 1982). Thischange in the dichroic orientation of phytochrome during photoconversionwas analyzed by a newly-built, polarization plane-rotatabledouble laser flash irradiator. A polarotropic response was effectivelyinduced with a flash of polarized red (640 nm) light (6xl0^–7s) having the vibration plane of the electrical vector parallelto the protonemal cell axis. When a flash of polarized far-red(710 nm) light (6xl0^–7s) was given 30 sec after the redflash, the red flash-induced response was reversed by a far-redflash vibrating normal to the cell axis but not by one vibratingparallel. However, when given 2 µs or 2 ms after the redflash, the polarotropic response was not reversed by a polarizedfar-red flash vibrating normal to the cell axis but was reversedby a parallel-vibrating flash. These results suggest that theorientation of phototransformation intermediates existing 2µs or 2 ms after a red flash is still parallel to thecell surface, and that the change in the orientation of phytochromemolecules occurs between 2 ms and 30 s after the red flash. (Received February 3, 1986; Accepted April 23, 1986)     

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)     

Heart-Shaped Prothallia of the Fern Adiantum capillus-veneris L. Develop in the Polarization Plane of White Light

When red light-precultured filamentous protonemata of Adiantumcapillus-veneris were cultured under linearly polarized whitelight, heart-shaped prothallia developed in the plane parallelto the vibration plane of electrical vector of polarized light,and were directed toward the light source. When the polarizationplane was rotated during the culture, the prothallial wingstwisted correspondingly to develop in the new plane. Continualobservation of the early steps of prothallial development witha time-lapse video system revealed that the apical cell of protonemaafter the first transverse cell division became flattened inthe vibration plane of electrical vector of polarized light,and that the first longitudinal cell division, that is, thefirst step in the transition from one-dimensional to two-dimensionalgrowth, as well as the subsequent cell divisions, occurred perpendicularlyto the electrical vector. (Received February 20, 1986; Accepted May 7, 1986)     

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)     

Polarotropism and Photomovement of Chloroplasts in the Protonemata of the Ferns Pteris and Adiantum: Evidence for the Possible Lack of Dichroic Phytochrome in Pteris

The actions of red and blue light in the photomovement of chloroplastsand the polarotropic response were studied in the protonemataof the homosporous ferns Pteris vittata L. and Adiantum capillus-venerisL. In Pteris, polarotropism could be induced with blue lightbut not with red light, while both colors of light were effectivein Adiantum protonemata. The photomovement of chloroplasts inthe two species studied by both polarized light and microbeamirradiation, also revealed similar responses to red and bluelight as the polarotropism; i.e. both colors of light were effectivein Adiantum but only blue light was active in Pteris. The resultsin Adiantum were consistent with previous results, which ledto the conclusion that both phytochrome and a blue light-absorbingpigment are involved in the two responses (Kadota et al. 1982,1984, Hayami et al. 1986, Yatsuhashi et al. 1985). By contrast,phytochrome is not involved in either polarotropism or chloroplastmovement in Pteris. Since the phytochrome system is evidentlyactive in every other photoresponses so far investigated inPteris as well as in Adiantum, the present study suggests thata phytochrome system specific to polarotropism and to photomovementof chloroplasts is absent in Pteris. Discussions are presentedon the possible involvement of two phytochrome populations ina fern gametophyte cell and on the possible lack of dichroicphytochrome in Pteris. (Received October 7, 1988; Accepted March 8, 1989)     

Phytochrome-Mediated Photoorientation of Chloroplasts in Protonemal Cells of the Fern Adiantum Can be Induced by Brief Irradiation with Red Light

Measuring the ratio of the number of photooriented chloroplaststo the total number of chloroplasts, we found that photoorientationof chloroplasts in protonemata of the fern Adiantum capillus-veneriscould be induced by brief irradiation with polarized red light.After irradiation with red light (R) of 3 or 10 min, orientationalmovement was detected as early as 10 min after the irradiation;it continued during the subsequent dark period for 30–60min, after which chloroplasts gradually dispersed again. WhenR-treated protonemata were irradiated briefly with a second10-min pulse of R, 60 min after the onset of the first irradiation,the orientational response of chloroplasts was again observed.Typical red/far-red photoreversibility was apparent in the response,indicating the involvement of phytochrome. By contrast, irradiationwith polarized blue light for 10 min was ineffective, whileirradiation with blue light (B) at the same fluence for a longerperiod of time clearly induced the photoorientation of chloroplasts.It is likely that longterm irradiation is necessary for theresponse mediated by a blue-light receptor. When protonemata were irradiated with far-red light (FR) immediatelyafter R or after a subsequent dark period of 10 min, the magnitudeof the orientational response was smaller and chloroplasts dispersedmore quickly than those exposed to R alone. When FR was appliedat 50 min, when the response to R had reached the maximum level,chloroplasts again dispersed rapidly to their dark positions.These results indicate that P_FR not only induces the photoorientationmovement of chloroplasts but also fixes the chloroplasts atthe sites to which they have moved as a result of photoorientation. (Received June 2, 1993; Accepted January 11, 1994)     

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.     

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)     

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.     

Change in the rate of organelle movement during progression of the cell cycle inAdiantum protonemata

Summary The rate of organelle movement during progression of the cell cycle in single-celled protonemata of the fernAdiantum capillus-veneris is determined microscopically with a time-lapse video system. Under red light organelle movement is very slow (1.8 m/min) in early G₁ in the apical 100-m region. The rate of organelle movement becomes higher in proportion to distance from the nuclear region, reaching a plateau in the neighborhood of 300 m from the tip. Organelle movement during the progression of G₁ and S phases in the dark does not show a significant difference from that in early G₁ under red light. In M phase, however, organelle movement in the nuclear region slows down a few minutes after nucleolar disappearance and then stops until the beginning of cell plate formation. Organelle movement in the basal region of the protonema slows down, but does not stop, shortly after movement in the nuclear region has ceased. This indicates that a message is sent from the nuclear region to the basal region.     

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.     

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.     

Photoinduction of Spore Germination in Marchantia polymorpha L. is Mediated by Photosynthesis

The effects of light on spore germination (protrusion of protonemata)in the liverwort Marchantia polymorpha L. were examined. Sporegermination was found to be light dependent and light irradiationfor 10 h or longer was necessary. Test using specific wavelengthsshowed that the entire spectrum from near UV to red light waseffective, red light being the most effective. Spore germinationcould be induced by intermittent irradiation with 15-min redlight pulses given every 1 or 2 h for 24 h. The effect of intermittentred light was not reversed by subsequent or simultaneous far-redlight irradiation. However, spore germination was inhibitedby the photosynthesis inhibitor DCMU (100 µM). Completeinhibition of spore germination was found when DCMU was givenduring the light period. When DCMU was applied during the darkperiods, only a slight reduction of germination rate was observed.Further, it was found that Chl formed in the spores during imbibitionin darkness. Light sensitivity increased at nearly the samerate as the appearance of Chl. Moreover, spore germination wasinduced in total darkness by the addition of glucose to themedium. These results clearly indicate that photosynthesis mediatesthe photoinduction of spore germination in Marchantia polymorpha. (Received May 13, 1999; Accepted July 14, 1999)     

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.     

Phytochrome Control of Acid and Alkaline Phosphatases in Etiolated Shoot Apices of Sorghum bicolor

A short pulse of red light or continuous far-red light enhancedthe activities of acid and alkaline phosphatases over the valuesof the dark controls in 5-day-old etiolated seedlings of Sorghumbicolor. For 30 min after the red light pulse 100% of the red/far-redphotoreversibilities was maintained for the acid and 80% forthe alkaline phosphatases. Thereafter, the "photoreversibilityescape reaction" was fast, being completed within 180 min. Cycloheximideas well as 6-methyl purine markedly inhibited red light enhancementof the activities of the phosphatases, but chloramphenicol,lincomiycin and rifamycin SV were ineffective. In spite of photoregulationof both the phosphatases at the time of de novo synthesis, itappears that control of the acid and alkaline phosphatases maybe affected by two independent initial actions of phytochrome. ¹ Present address: Biologisches Institut II, University of Freiburg,FRG. (Received August 4, 1984; Accepted April 3, 1985)     

Inductive and Inhibitory Effects of Light on Cell Division in Chattonella antiqua

The cell division of a red tide flagellate, Chattonella antiqua,was synchronously induced under light and dark regimes of 10L14D(a light period, L, for 10 h followed by a dark period, D, for14 h), 12L12D and l4L10D. In all regimes cell number began toincrease ca. 14 h after the onset of L and almost doubled duringone LD cycle. When the light-off timing of the last L was changedor the whole L was shifted, cells that had been synchronizedunder 12L12D invariably began to divide ca. 14 h after the onsetof L. This shows that the timing of cell division was determinedby the time of the onset of L. When cells were continuously exposed to light after a cell division,the subsequent cell division was inhibited. This effect waslimited to cells that had been synchronized under short-dayconditions. Thus it can be concluded that light has both inductive and inhibitoryeffects on cell division in this alga, the latter effect dependingupon the previously given light and dark regimes. (Received December 21, 1984; Accepted February 28, 1985)     

Photoreversibility of flower initiation in Lemna perpusilla as affected by the light quality of the main light period

Reversible floral responses of Lemna perpusilla to red and far-redlights appeared only at the beginning of the inductive darkperiod when the 8 hr photoperiod consisted of white or red light.When blue or far-red light was given during the 8 hr photoperiod,the far-red given at the beginning of the dark period scarcelyinhibited flowering; red/far-red reversibility newly appearedat the middle of the dark period. This indicates that the photoregulationsystem in the flowering of L. perpusilla can be converted fromthe Pharbitis type to the Xanthium type by changing the lightquality of the main photoperiod from white or red to blue orto far-red, which is known to be effective for the so-calledhigh-energy photoreaction of photomorphogenesis. (Received July 2, 1975; )