GROWTH REGULATION BY ETHYLENE IN FERN GAMETOPHYTES. IV. INVOLVEMENT OF PHOTOSYNTHESIS IN OVERCOMING ETHYLENE INHIBITION OF SPORE GERMINATION

The inhibitory effects of ethylene on spore germination were investigated. In darkness spore germination was completely inhibited by 10 μ1 · 1⁻¹ ethylene. Light partially overcame this inhibition, and the effect of continuous irradiation with white fluorescent light saturated at about 450 μW · cm⁻². Monochromatic red, blue and far-red light were effective in overcoming ethylene inhibition, whereas green was not. Short periodic exposures to red or far-red light were not sufficient to overcome ethylene inhibition. This suggested that phytochrome was not involved. The photosynthetic inhibitor DCMU blocked the effect of light. Infrared gas analysis showed that photosynthesis saturated at about 450 μW · cm⁻² in white light. Red, blue and far-red light were more efficient photosynthetically than green light; DCMU blocked photosynthesis. Normalized curves of photosynthesis and germination vs. light intensity showed a similar dependence on light energy. It was concluded that light appears to overcome the inhibitory effects of ethylene through some process dependent on photosynthesis.     

PHOTOMORPHOGENESIS IN PTERIS VITTATA: I. PHYTOCHROME-MEDIATED SPORE GERMINATION AND BLUE LIGHT INTERACTION

1. Spores of the fern Pteris vittata did not germinate under totaldark conditions, while an exposure of the spores to continuouswhite light brought about germination. The germination was mosteffectively induced by red light and somewhat by green and far-red,but not at all by blue light. The sensitivity of spores to redlight increased and leveled off about 4 days after sowing at27–28. The promoting effect of red light could be broughtabout by a single exposure of low intensity. Far-red light givenimmediately after red light almost completely reversed the redlight effect, and the photoresponse to red and far-red lightwas repeatedly reversible. The photoreversibility was lost duringan intervening darkness between red and far-red irradiations,and 50% of the initial reversibility was lost after about 6hr of darkness at 27–28. These observations suggest thatthe phytochrome system controls the germination of the fernspore.
2. When the imbibed spores were briefly exposed to a low-energyblue light immediately before or after red irradiation, theirgermination was completely inhibited. The blue light-inducedinhibition was never reversed by brief red irradiation givenimmediately after the blue light. The escape reaction of redlight-induced germination as indicated by blue light given aftervarious periods of intervening darkness was also observed, andits rate was very similar to that determined by using far-redlight. Spores exposed to blue light required 3 days' incubationin darkness at 27–28 to recover their sensitivity tored light. The recovery in darkness of this red sensitivitywas temperature-dependent. It is thus suggested that an unknownbluelight absorbing pigment may be involved in the inhibitionof phytochrome-mediated spore germination.

(Received August 21, 1967; )     

The Effect of the Herbicide Dichlorophen on the Physiology and Growth of Two Bryophytes

Dichlorophen (2, 2'-methylene-bis(4 chlorophenol)) has beenused frequently as a commercial eradicates of bryophytes. Usingthe moss Rhytidiadelphus squarrosus and the thalloid liverwortMarchantia polymorpha, it has been shown that dichlorophen inducesloss of intracellular potassium and magnesium, inhibits photosynthesisand, depending on concentration, either stimulates or depressesthe production of CO₂ in the dark, suggesting that death maybe caused by membrane damage. Sensitivity of the moss appearsto be related to the age of the tissue but to be unaffectedby light before, during or after exposure. Rhytidiadelphus squarrosus, Marchantia polymorpha, dichlorophen, membrane damage, inhibition of growth, photosynthesis and respiration     

Control of senescence in marchantia by phytochrome

Mature green tissue of Marchantia polymorpha L. bleaches markedly when placed in continuous darkness for 4 days but remains green when given daily 1-hour photoperiods of white light. The tissue, however, is induced to bleach when each daily 1-hour photoperiod is terminated with a brief irradiation with far red light. The bleaching does not occur when each irradiation with far red light is followed by a brief irradiation with red light. The bleaching is taken as an index of senescence since the loss of chlorophyll in the bleached tissue is accompanied by a breakdown of cell organelles and cytoplasm. Phytochrome is clearly implicated in the control of senescence by light. It was also found that 5 minutes of red light given once a day was as effective as the 1-hour photoperiods with white light in preventing the bleaching and that bleaching was induced when each daily 5-minute irradiation with red light was followed by a 10-minute irradiation with far red light.     

Microfilaments Anchor Chloroplasts along the Outer Periclinal Wall in Vallisneria Epidermal Cells through Cooperation of PFR and Photosynthesis

In the cytoplasmic layer that faces the outer periclinal wallin epidermal cells of leaves of the aquatic angiosperm Vallisneriagigantea Graebner, we examined a possible interrelationshipamong the configuration of microfilaments, chloroplast motility,and anchoring of chloroplasts. In dark-adapted cells, microfilamentsare arranged in a network array. During a 10-min incubationin darkness 10 to 20 min after irradiation with red light (650nm, 0.41 W m^–2) for 5 min, the number of cells containinga network array decreased substantially while the number ofcells containing microfilaments in a honeycomb array increased.Irradiation with red light rapidly produces an increase in chloroplastmotility, but chloroplast motility declined almost to initiallevels during the 10-min incubation in darkness after the irradiation.Simultaneously, the chloroplasts in these cells became extremelyresistant to centrifugal forces. These effects of red lightwere negated either by far-red light or by the presence of DCMU,and were sensitive to cytochalasin B. It appears, therefore,that microfilaments not only drive the movement of chloroplastsbut also play a crucial role in accumulation of the chloroplastsalong the outer periclinal wall through dynamic changes in theconfiguration under cooperative regulation by P_FR and photosynthesis. (Received July 24, 1998; Accepted September 22, 1998)     

Photocontrol of spore germination in the fern Thelypteris kunthii

The light requirement for germination in spores of the fern Thelypteris kunthii (Desv.) Morton was fully satisfied by a long period of continuous red light or partially by intermittent, short periods of red light. Red light-potentiated spore germination was inhibited by brief far-red light irradiation, indicating phytochrome involvement. Repeated exposure of spores to prolonged red and short far-red irradiations, or exposure of red-potentiated spores to far-red light after an extended period in darkness, led to their escape from inhibition of germination by far-red light. Prolonged irradiation of spores with blue light before or after red light treatment partially antagonized the effect of red light.     

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

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

Action Spectrum between 250 and 800 Nanometers for the Photoinduced Inhibition of Spore Germination in Pteris vittata

An action spectrum between 250 and 800 nm for the inhibitionof red-light-induced germination of spores in the fern Pterisvittata was determined on the Okazaki Large Spectrograph. Theresultant spectrum showed prominent peaks of effectiveness atabout 370, 440 and 730 nm and a minor peak in the neighborhoodof 260 nm. Next, a brief red light irradiation was given immediatelyafter the monochromatic irradiation to cancel the inhibitoryeffect caused by simultaneously formed P_R. This resulted ina complete disappearance of the peak at 730 nm and considerabledecrease of other peaks in the shorter wavelength region exceptat 260 nm. Further correction of the latter spectrum by consideringthe transmission spectrum of a spore coat revealed that 260nm light acted more effectively than lights of 370 and 440 nm.The inhibitory effect of UV light on spore germination was nullifiedby subsequent irradiation with red light for 24 h or darknessfor 48 h followed by a brief red irradiation, indicating thatthe inhibitory action of UV light was ascribable to a blue-ultraviolet light-absorbing pigment. ⁴Present address (KT) and permanent address (MF): Botany Department,Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan. (Received July 30, 1983; Accepted November 21, 1983)     

Migration of prospindle before the first asymmetric division in germinating spore of Marchantia polymorpha

The development of the plant body starts with spore germination in bryophytes. In many cases, the first division of the spore occurs after germination and cell elongation of the spore. In Marchantia polymorpha, asymmetric division occurs upon spore germination to generate two daughter cells: the larger one retains the ability to divide and develops into the thallus via sporeling or protonema, while the smaller one maintains tip growth and differentiates into the first rhizoid, providing a scaffold for initial development. Although spore germination of M. polymorpha was described in the 19th century, the intracellular processes of the first asymmetric division of the spore have not been well characterized. In this study, we used live-cell imaging analyses to elucidate microtubule dynamics during the first asymmetric division concomitantly with germination. In particular, we demonstrated that the preprophase band was not formed in the spore and that the bipolar prospindle, which is a microtubule structure surrounding the nucleus during prophase, migrated from the center to the periphery in the spore, suggesting that it was the earliest visible sign of cell polarity. We also showed that the occurrence of asymmetric division depended on actin filaments. Our findings regarding the first division of the spore in M. polymorpha will lead to a better model for cell-autonomous asymmetric division in plants.     

Glycogen content and nitrogenase activity in Anabaena variabilis

Nitrogenase (=acetylene-reducing activity) was followed during photoautotrophic growth of Anabaena variabilis (ATCC 29413). When cell density increased during growth, (1) inhibition of light-dependent activity by DCMU, an inhibitor of photosynthesis, increased, and (2) nitrogenase activity in the dark decreased. Addition of fructose stabilized dark activity and alleviated the DCMU effect in cultures of high cell density.The resistance of nitrogenase towards oxygen inactivation decreased after transfer of autotrophically grown cells into the dark at subsequent stages of increasing culture density. The inactivation was prevented by addition of fructose. Recovery of acetylene-reducing activity in the light, and in the dark with fructose present, was suppressed by ammonia or chloramphenicol. In the light, also DCMU abolished recovery.To prove whether the observed effects were related to a lack of photosynthetic storage products, glycogen of filaments was extracted and assayed enzymatically. The glycogen content of cells was highest 10 h after inoculation, while light-dependent nitrogenase activity was at its maximum about 24 h after inoculation. Glycogen decreased markedly as growth proceeded and dropped sharply when the cells were transferred to darkness. Thus, when C-supply (by photosynthesis or added fructose) was not effective, the glycogen content of filaments determined the activity of nitrogenase and its stability against oxygen. In cells lacking glycogen, nitrogenase activity recovered only when carbohydrates were supplied by exogenously added fructose or by photosynthesis.Abbreviations Chl chlorophyll a - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Hydrolytic enzyme activities in germinating spores ofAdiantum capillus-veneris L.

Changes in hydrolytic enzyme activities were investigated during spore germination ofAdiantum capillus-veneris L. The spores were incubated for 3 days in the dark at 25 C for imbibition, and then germination of the spores was induced by continuous irradiation with red light. At day 2 after onset of the red light irradiation, rhizoids appeared out of spore coats and protonemal cells became visible on the following day. Lipase occurred in dry spores and its activity decreased during 3 days of dark incubation. The activity started to increase when the spore germination was induced by red light irradiation. On the other hand, amylolytic and aminopeptidase activities which were also detected in dry spores decreased continuously during the dark incubation and following the germination process. RNase activity also decreased during 3 days of dark incubation but the activity was retained thereafter at a constant level with or without red light irradiation. Developmental patterns of these hydrolytic enzymes were classified into two groups: One decreased during imbibition and dark incubation but increased after red light irradiation and the other continuously decreased during dark incubation and germination. These results are discussed in relation to compositional changes of cell constitutions such as lipid, sugars, proteins and amino acids during spore germination.     

The involvement of photosynthesis in inducing bud formation on excised leaf segments of Heloniopsis orientalis (Liliaceae)

The role of photosynthesis in inducing adventitious bud formationon leaf segments of Heloniopsis orientalis was investigated.The effect of white light reached a maximum at about l25 J?m^–2?sec^–1.White, red, blue and far-red light were effective in inducingbud formation, but green light was not. In darkness, bud formationwas induced if sugar was added to the nutrient medium. The photosyntheticinhibitors DCMU and AT blocked the effect of light. Bud formationwas inhibited in CO₂-free air. The requirement of sucrose forbud formation in darkness could be replaced by citrate. It wasconcluded from these results that light appears to induce budson leaf segments through some processes dependent upon photosynthesis. (Received January 11, 1978; )     

Light germination ofAnthoceros miyabeanus spores

The effects of light on the spore germination of a hornwort species,Anthoceros miyabeanus Steph., were investigated. Spores of this species were photoblastic, but their sensitivities to light quality were different. Under either continuous white, red or diffused daylight, more than 80% of the spores germinated, but under blue light none or a few of them germinated. Under continuous far-red light or in total darkness, the spores did not germinate at all.Anthoceros spores required red light irradiation for a very long duration, i.e., over 12–24 hr of red light for saturated germination. However, the spore germination showed clear photo-reversibility by repeated irradiation of red and far-red light. The germination pattern clearly varied with the light quality. There were two fundamental patterns; (1) cell mass type in white or blue light: spores divide before germination, and the sporelings divide frequently and form 1–2 rhizoids soon after germination, and (2) germ tube type in red light: spores germinate without cell division, and the single-cell sporelings elongate without cell division and rhizoid formation.     

SUCCESSIVE PROCESSES INVOLVED IN THE GERMINATION RESPONSE OF NASTURTIUM SEEDS

1. The seeds ofNasturtium palustreDC. do not germinate, eitherin the light or darkness, at various constant temperatures,but require for their full germination a certain period of alow temperature (5°) applied immediately after light irradiation.These results indicate the existance of at least two processes,a light-dependent process and a low temperature-requiring process,in the initiation of germination ofNasturtiumseeds. Experimentalevidence indicated further that the light exposure causes twodifferent processes in the seed germination. 2. When a dark period at 23° was inserted between the lightirradiation and the low temperature treatment the germinationwas suppressed. The inhibitory effect of the inserted dark periodat 23° was eliminated by a short irradiation during thedarkness (light-break). 3. Prolonged exposure ofNasturtium seeds to any concentrationof gibberellin brought about no germination when exposure wasgiven in complete darkness. The germination was promoted onlywhen light irradiation was applied to the seeds. A short applicationof gibberellin at a fairly high concentration was, however,remarkably effective for the germination even in the darkness,and the germination was inhibited as the gibberellin applicationwas lengthened. It was considered that gibberellin could substitutefor the combined effect of light irradiation and low temperaturetreatment to induce the germination of Nasturtium seeds, andthat gibberellin was inhibitive toward the reactions followingthe above treatments which induced the germination (Received October 31, 1996; )     

Enhancement of senescence by far-red light

Summary Far-red light was demonstrated to accelerate senescence in mature, green tissue of intact thalli of Marchantia polymorpha. The red/far-red reversibility of this phenomenon proves the involvement of phytochrome. As a daily exposure to 5 min red light is sufficient to prevent aging of the tissue, photosynthesis does not play a specific role in this response.     

Role of red light in hair-whorl formation induced by blue light in Acetabularia mediterranea

After a pre-treatment with red light, hair formation at the growing tip of the siphonaceous green alga Acetabularia mediterranea Lamour. (= A. acetabulum (L.) Silva) can be induced by a pulse of blue light. Red light is needed again after the inductive blue-light pulse if the new whorl of hairs is to develop within the next 24 h. In order to investigate the role of this red light, the duration of the red irradiation was varied and combined with periods of darkness. The response of hair-whorl formation was dependent on the total amount of red light, regardless of whether the red irradiation followed the blue pulse immediately or was separated from it by a period of darkness. Furthermore, periods of exposure to the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1-1dimethylurea had a similar effect to darkness. Both observations indicate that this red irradiation acts as a light source for photosynthesis. Whether or not the red light had an additional effect via phytochrome was tested in another type of experiment. The dependence of hair-whorl formation on red-light irradiance in the presence of simultaneous far-red irradiation was determined for the pre-irradiation period as well as for the irradiation period after the blue pulse. In both experiments, far-red light caused a small promotion of hair-whorl formation when low irradiances of red light were used. However, these differences were attributable to a low level of photosynthetic activity (which in fact was measurable) caused by red light reflected in the growth chamber. Furthermore, lowering the proportion of active phytochrome by far-red light would be expected to suppress hair-whorl formation. The influence of far-red light was also tested in a strain of Acetabularia mediterranea that developed hair whorls in about 20% of cells even when kept in complete darkness after the blue-light pulse. Far-red irradiation had no effect. These results strongly indicate that phytochrome is not involved in hair-whorl formation. Rather it is concluded that the effects of red light are caused by photosynthesis.Abbreviation DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Factors Controlling Induction of External Carbonic Anhydrase and Change in K?(CO2) of Photosynthesis in Chlorella regularis

Transfer of algal cells of Chlorella regularis from 3% CO₂ inair into ordinary air in the light increased external carbonicanhydrase (CA) activity as well as photosynthetic affinity forCO₂ by several-fold within 2 h. Since no noticeable differencewas observed in CA activity between intact cells and cell homogenates,CA seemed to be mainly localized on the cell surface. Changesin CA activity and K_?(CO₂) of photosynthesis were not observedin the dark. CA induction was 50%-inhibited by incubation with10 µM DCMU during adaptation of high-CO₂ cells to air,whereas it was considerably suppressed when high-CO₂ cells preincubatedwith DCMU in the light for 6 h or without DCMU in the dark for24 h were used. The change in K_?(CO₂) of photosynthesis wasonly slightly affected by DCMU. Uncoupler like carbonylcyanide-m-chlorophenyl-hydrazone(CCCP) and inhibitors of mitochondrial respiration (KCN plussalicylhydroxamic acid) suppressed CA induction during adaptationof high-CO₂ cells to low CO₂ conditions. These results suggest that photosynthesis is not essential forCA induction in Chlorella regularis when some amounts of photosyntheticproducts are previously stored in the cells and respirationis active. A decrease in K_?(CO₂) of photosynthesis during adaptationfrom high to low CO₂ was mostly independent on photosynthesis.However, light is essential for both phenomena. (Received July 16, 1990; Accepted January 21, 1991)     

The involvement of phytochrome in controlling chlorophyll and 5-aminolevulinate formation in a gymnosperm seedling (Pinus sylvestris)

Chlorophyll a (Chl a) accumulation in the cotyledons of Scots pine seedlings (Pinus sylvestris L.) is much higher in the light than in darkness where it ceases 6 days after germination. When these darkgrown seedlings are treated with continuous white light (3,500 lx) a 3 h lag phase appears before Chl a accumulation is resumed. The lag phase can be eliminated by pretreating the seedlings with 7 h of weak red light (0.14 Wm^-2) or with 14 red light pulses separated by relatively short dark periods (<100 min). The effect of 15s red light pulses can be fully reversed by 1 min far-red light pulses. This reversibility is lost within 2 min. In addition, the amount of Chl a formed within 27 h of continuous red light is considerably reduced by the simultaneous application of far-red (RG 9) light. It is concluded that phytochrome (P_fr) is required not only for the elimination of the lagphase but also to maintain a high rate of Chl a accumulation in continuous light. Since accumulation of 5-aminolevulinate (ALA) responds in the same manner as Chl a accumulation to a red light pretreatment it is further concluded that ALA formation is the point where phytochrome regulates Chl biosynthesis in continuous light. No correlation has been found between ALA and Chl a formation in darkness. This indicates that in a darkgrown pine seedling ALA formation is not rate limiting for Chl a accumulation.Abbreviations Chl chlorophyll(ide) - PChl protochlorophyll(ide) - ALA 5-aminolevulinate - P_r the red absorbing form of phytochrome - P_fr the far-red absorbing form of phytochrome - P_tot total phytochrome ([P_r]+[P_fr])     

Photoinduction of Spore Germination in a Fern, Mohria caffrorum

Irradiation of spores of the fern Mohria caffrorum Sw. witheither red light (67.4 µW cm^–2) or far-red light(63.2 µW cm^–2) for a period of 24 h induced maximumlevels of germination. Brief irradiations with blue light (127.6µW cm^–2) administered before or after photoinductioncompletely nullified the effects of red or far-red light; however,with prolonged exposure to blue light, germination levels roseto near maximum. The similar effects of red and far-red lightin promoting spore germination makes the involvement of phytochromein this process questionable. Based on energy requirements,the promotive and inhibitory phases of blue light appear toinvolve independent modes of action. Mohria caffrorum, ferns, spore germination, photoinduction, phytochrome     

Stimulation of Nitrate Reductase by Light and Ammonium in Spirodela oligorrhiza

Light-enhanced nitrate reductase (NR) activity was 8 times greaterthan the dark control. Exogenous application of sucrose, glucoseand fructose increased the induction of NR in the light as wellas in the dark, whereas glycolate had no effect. DCMU [3-(3,4-dichlorophenyl)-1, 1-dimethyl urea] completely inhibited thedevelopment of NR in light. Sucrose, when added with DCMU, reversedthis inhibitory effect NR in vivo was more stable in light thanin darkness, the half-lives being 9.6 h and 6.4 h, respectively.The addition of sucrose did not change the half-life of NR ineither light or darkness. Ammonium, the end product of the inorganicnitrogen assimilatory pathway, stimulated the NR activity whereasamino acids decreased it. Key words: Spirodela oligorrhiza, nitrate reductase, ammonium, light