Control of the Entry into S Phase by Phytochrome and Blue Light Receptor in the First Cell Cycle of Fern Spores

Phytochrome- and a blue light receptor-dependent pathway antagonisticallyregulate the first mitosis in spores of the fern Adiantum capillus-venerisL. This study focused on determining which phase(s) of the cellcycle is positively regulated by phytochrome and negativelyregulated by a blue light receptor in germinating spores. Incorporationof the radioactivity of ³H-thymidine into the acid-insolublematerial prepared from the spores indicated that phytochromein the PFR form induced the entry into S phase of the firstcell cycle in the spores 20-28 h after irradiation with redlight. Blue light treatment before or after red light treatmenttotally prevented the P_FR-induced DNA synthesis. Brief irradiationwith red, far-red or blue light showed no effects on mitosisif the irradiation was given 28 h after the red light induction,during S and M phases. These results indicate that phytochromeand a blue light receptor regulate the entry into S phase duringthe first cell cycle of fern spores. ( Accepted July 10, 1997)     

Developmental Physiology Apparent Dark Decay of Phytochrome Pfr in Fern Spores

Germination of spores of Dryopteris fllix-mas has been induced by two pulses of saturating red light, separated by a dark period of about 8 to 24 h. By chosing different wavelengths, different P_fr/P_tot levels could be established. Thus, by a “null method” the second pulse could be used as a “test pulse”, determining the actual P_fr level remaining from the “start pulse”, and thus providing information about an apparent Pf_r decay. It cannot be decided yet whether this apparent P_fr decay results from dark destruction or dark reversion. The apparent P_fr decay depends, as expected, on the temperature, being accelerated with increasing temperatures. Moreover, the later after sowing that the decay is tested, the faster it proceeds; a tentative interpretion is that newly synthesized P_r undergoes faster decay after phototransformation than that phytochrome pool present in the resting spores. A third factor that influences the apparent P_fr decay is the Pf_r/P_tot level established by the first pulse (start pulse). The lower this level, the slower the decay kinetics. This could be due to phytochrome biosynthesis partly compensating for P_fr destruction, and the relative contribution of this biosynthesis to the total effect increases with lower P_fr levels. Spores of D. paleacea yield virtually the same results. Whatever the real basis of the observed P_fr decay, i.e. destruction, reversion, or a combination of these reactions with biosynthesis, it can be concluded that modification of this P_fr decay by various factors is the basis of the effect of those factors on light-induced germination.     

Changes in Phytochrome Content during Imbibition in Spores of the Fern Lygodium japonicum

The phytochrome content was determined in intact fern sporesof Lygodium japonicum (Thunb.) Sw. by difference spectrophotometry.The spectral characteristics thus estimated in spores whichhad been imbibed for 9 days in darkness were: far-red maximumat 730?2.5 nm, red maximum at 662?1.5 nm and isosbestic pointat 684.5?1.4 nm. A detectable amount of phytochrome first appearedafter 3 days of dark imbibition, and the level then increasedduring the rest of the imbibition period. On the 7th day, thephytochrome content leveled off. During the dark imbibitionperiod, the phytochrome was revealed to be in the P_R form. (Received February 22, 1982; Accepted July 9, 1982)     

蕨类植物孢子与种子植物花粉萌发的比较

蕨类植物孢子与种子植物花粉在有性生殖过程中都具有重要的作用。花粉作为种子植物的雄配子体, 通过萌发后极性生长的花粉管将精细胞送到胚囊完成受精作用。蕨类植物孢子作为配子体的原始细胞, 通过不对称的有丝分裂产生一大一小两个细胞, 小细胞萌发出极性生长的假根, 大细胞继续分裂发育为原叶体(配子体)。成熟的花粉和蕨类植物孢子都是代谢高度静止的细胞, 两者的萌发过程不仅都受到各种不同环境因子的影响, 而且在信号转导、极性建立和能量代谢等方面可能有着相似的调控机制。本文综述了蕨类植物孢子和种子植物花粉萌发过程的差异和保守性特征。     

蕨类植物孢子与种子植物花粉萌发的比较

蕨类植物孢子与种子植物花粉在有性生殖过程中都具有重要的作用。花粉作为种子植物的雄配子体,通过萌发后极性生长的花粉管将精细胞送到胚囊完成受精作用。蕨类植物孢子作为配子体的原始细胞,通过不对称的有丝分裂产生一大一小两个细胞,小细胞萌发出极性生长的假根,大细胞继续分裂发育为原叶体（配子体）。成熟的花粉和蕨类植物孢子都是代谢高度静止的细胞,两者的萌发过程不仅都受到各种不同环境因子的影响,而且在信号转导、极性建立和能量代谢等方面可能有着相似的调控机制。本文综述了蕨类植物孢子和种子植物花粉萌发过程的差异和保守性特征。     

Variations in the Germination-rate and Development of Fern Spores in Culture

In fern prothalli growing in pure culture, a number of variationshave been observed in the rapidity of the germination of sporesand the subsequent growth of the prothalli. Although these variationshave not all been satisfactorily explained, experiments showthat it is possible to produce considerable changes in the germinationand growth of fern spores by introducing fungal contaminationand prothallial extracts into the media on which they are sown.These changes are considered in relation to variations in thedevelopmental morphology of spores in pure culture and it issuggested that indole-3-acetic acid may be the active substanceinvolved.     

Relationship between Germination and PFR Level in Spores of the Fern Lygodium japonicum

The relationship between germination and P_FR level in sporesof the fern Lygodium japonicum was investigated. Percent P_FRestimated from direct spectrophotometric measurement of sporesincreased with the logarithm of total fluence of 660 nm-light.The transformation from P_R to P_FR was saturated by giving ca.200 Jm^–2 of 660 nm-light and half-saturated by ca. 55J^–2 of 660 nm-light. Clear positive correlation was observedbetween % P_FR levels and germination rates in spores irradiatedwith 660 nm and/or 730 nm-light, or with 686 or 700 nm-light.The P_FR percentage in spores was raised to 16–34% by blue(415 nm) light irradiation. This P_FR level was enough to causesome germination when produced by monochromatic light of redto far-red region, but blue light did not cause any germination. After 660 nm-light irradiation, the P_FR level decreased graduallyin darkness (25±1°C) and P_FR completely disappearedin 8 h, but 730 nm-light given even 16 h after 660 nm-lightirradiation inhibited germination. ⁴Present address: Tropical Botanic Garden and Research Institute,Navaranga Road, Trivandrum 695 011, India. (Received March 15, 1983; Accepted June 4, 1983)     

Spatial Patterns of Phytochrome Expression in Young Leaves of the Fern Adiantum capillus-veneris

The spatial distribution of phytochrome (PHY1) mRNA in the fernAdiantum was investigated by in situ hybridization. PHY1 mRNAsare expressed predominantly in the abaxial rather than the adaxialpart of the petiole of leaf croziers. Moreover, the signalsin light-grown croziers are predominantly nuclear in locationrather than cyto-plasmic. ⁴Present address: Department of Biology, Yale University, 165Prospect Street, New Haven, CT06520-8104, U.S.A.     

Phytochrome Control of Longitudinal Growth and Phytochrome Synthesis in Maize Seedlings

The active, far-red light absorbing, form of phytochrome was found to inhibit growth and phytochrome levels in the mesocotyl and coleoptile of 4- to 5.5-day-old seedlings of Zea mays L. Short, low-irradiance red or far-red light treatments were used to produce different proportions of active phytochrome at the end of highdirradiance white-light periods, which left different levels of total phytochrome in the plants. After light treatments which left relatively high levels of spectrophotometrically assayable phytochrome in the seedlings, apparent phytochrome synthesis in the subsequent dark period was low regardless of the proportions of each form of the pigment present at the beginning of the dark period. In light treatments producing relatively low levels of assayable phytochrome, levels of apparent phytochrome synthesis in both red and far-red treatments and differences between apparent synthesis in red and far-red treatments were maximal. No simple correlation was found between growth and apparent phytochrome synthesis. However, growth and total phytochrome levels were positively correlated in both organs. Using a subtractive method of correlation, in which only phytochrome effects were plotted, strong linear relationships between phytochrome levels or longitudinal growth and P_fr levels were found in those light treatments leaving greater than 8% of dark control levels of phytochrome in the tissues. Using this technique non-linear, inverse relationships between P_fr and apparent phytochrome synthesis was found, indicating that modes of phytochrome control over phytochrome synthesis and growth differ. Our results are consistent with the view that in vivo assays of “bulk’ phytochrome reflect levels and states of the physiologically active phytochrome fraction under our experimental conditions in maize.     

The Effects of Centrifugation on Asymmetric Cell Division and Differentiation of Fern Spores

We have investigated the effects of centrifugation on sporepolarity, asymmetric cell division, and rhizoid differentiationin the sensitive fern Onoclea sensibilis L. Centrifugation at10000 g for 30 min produces a random orientation of spores withstratification of the cell contents. After centrifugation atmost early stages of development, the nucleus retains its normalpattern of migration from the centre of the ellipsoidal sporeto the proximal face and then to an end of the spore, withoutregard to the orientation of stratification. This indicatesthat the polarity of the spore is stable to centrifugation.As long as the nucleus migrates to an end of the spore and asymmetriccell division occurs, the small cell differentiates into a rhizoid.The arrangement of large cytoplasmic organelles appears to haveno influence on nuclear migration, asymmetric cell division,or rhizoid differentiation. The only period during developmentwhen centrifugation blocks asymmetric cell division is immediatelypreceding and during mitosis and cytokinesis. Spores centrifugedat this stage do not complete nuclear migration, and symmetriccell division results, with neither cell differentiating intoa rhizoid. The source of the stable polarity of the spore isdiscussed. cell polarity, rhizoid differentiation, centrifugation, Onoclea sensibilis L., sensitive fern, fern spores     

Phot-LOV1: Photocycle of a Blue-Light Receptor Domain from the Green Alga Chlamydomonas reinhardtii

The “Phot” protein family comprises blue-light photoreceptors that consist of two flavin mononucleotide (FMN)-binding LOV (light, oxygen, and voltage) domains and a serine/threonine kinase domain. We have investigated the LOV1 domain of Phot1 from Chlamydomonas reinhardtii by time-resolved absorption spectroscopy. Photoexcitation of the dark form, LOV1-447, causes transient bleaching and formation of two spectrally similar red-shifted intermediates that are both assigned to triplet states of the FMN. The triplet states decay with time constants of 800 ns and 4 μs with an efficiency of >90% into a blue-shifted intermediate, LOV1-390, that is attributed to a thiol adduct of cysteine 57 to FMN C(4a). LOV1-390 reverts to the dark form in hundreds of seconds, the time constant being dependent on pH and salt concentration. In the mutant C57S, where the thiol adduct cannot be formed, the triplet state displays an oxygen-dependent decay directly to the dark form. We present here a spectroscopic characterization of an algal sensory photoreceptor in general and of a LOV1 domain photocycle in particular. The results are discussed with respect to the behavior of the homologous LOV2 domain from oat.     

Cross-linking Phytochrome to its Receptor in situ using Imidoesters

Phytochrome can be cross-linked to a particulate fraction usingimidoesters, namely dimethy adipimidate (DMA) and dimethyl suberimidate(DMS). DMS was more effective than DMA. Cross-linking of phytochrometo its in situ receptor effected by DMS occurred in red light-irradiatedcoleoptiles. If DMS cross-linking was carried out prior to redlight irradiation there was very little formation of particulatephytochrome. Phytochrome in the particulate fraction obtainedby in situ DMS cross-linking was totally resistant to the solubilizingeffect of washing with solutions of high salt concentrationand high pH and was indistinguishable spectro-scopically fromthe phytochrome in untreated coleoptiles. DMS cross-linkingof phytochrome to its assumed receptor in situ preferentiallyprotected it from destruction following red light irradiationand also prevented it from dissociating from its receptor followingR/FR¹ irradiation when incubated subsequently in the dark. Thesecharacteristics of phytochrome in DMS-treated coleoptiles matchedthose observed using glutaraldehyde as the cross-linking reagent.It is therefore concluded that earlier results obtained usingglutaraldehyde are not peculiar to that reagent and can be duplicatedreadily using more defined bifunctional cross-linkers.     

Control of Light-Induced Carotenoid Synthesis in Raphanus Seedlings by Phytochrome

The effect of short pulses of red and or far-red light on the formation of individual carotenoids was tested in etiolated radish seedlings (Raphanus sativus L.). Red light induces an enhanced synthesis of β-carotene, lutein. violaxanthin and neoxanthin, while the level of antheraxanthin is decreased. Far-red light reverses the red light effects to the level of the far-red light control. The data indicate that in radish seedlings active phytochrome P_fr initiates the light-induced carotenoid synthesis, which is bound to thylakoid formation.     

Phytochrome in the Fern, Adiantum capillus-veneris L.: Spectrophotometric Detection in Vivo and Partial Purification

Spectrophotometric studies of fern phytochrome were performedusing dark-grown leaves of Adiantum. The absorbance differencespectrum between the red- and far-red-light irradiated sampleshowed a photoreversible absorbance change in the far-red region,with a maximum located at 728–730 nm. The concentrationof phytochrome was highest at the leaf tips and decreased graduallyalong the leaf axis. As in the case of angiosperm phytochrome,the level of fern phytochrome decreased under continuous whitelight, and the level increased again when deetiolated tissuewas transferred back to the dark. When the fern tissue was exposedto a pulse of red light, the dark reversion of P_FR to P_R tookplace with almost no destruction of P_FR. Phytochrome could beextracted from light-grown young leaves of the fern with a slightlyalkaline, aqueous buffer that contained 1 M NaCl. The differencespectrum of the partially purified phytochrome from fern wassimilar to that of partially degraded phytochrome from angio-sperms.A polyclonal antibody raised against phytochrome from etiolatedrye seedlings immuno-stained (albeit weakly) a 110-kDa polypeptideafter fractionation by SDS-polyacrylamide gel electrophoresisof the preparation of fern phytochrome. The band was very probablyfern phytochrome since it emitted zinc-induced fluorescence. (Received July 12, 1990; Accepted October 5, 1990)     

Control of Bean Bud ATP Levels by Regulatory Molecules and Phytochrome

This study assessed the involvement of exogenously supplied acetylcholine, dibutyryl cyclic AMP, and various inhibitors of acetylcholine and cyclic AMP metabolism on the rapid phytochrome-mediated alteration in the ATP content of etiolated bean buds (Phaseolus vulgaris L. cv. Red Kidney). ATP was extracted in boiling water and measured by the firefly luciferin-luciferase assay. The depression of ATP content by low levels of either acetylcholine or dibutyryl cyclic AMP was independent of irradiation. Atropine, AMO-1618, and theophylline all altered ATP content, and the effects differed in red light and in darkness. In contrast to other reports, no straightforward model of phytochrome action through either acetylcholine or cyclic AMP could be constructed. Also, the time course of ATP content during the standard 5 min red-irradiation period was determined, and effects of light are observed within 1 min.     

Control of Mitochondrial Activities by Phytochrome in Cotyledons of Dark-Grown Cucumber Seedlings

Mitochondria isolated from cotyledons of dark-grown cucumberseedlings after illumination with white light showed an increasedcapacity for oxidation of malate. This increase is regulatedby phytochrome, since the rate of oxidation was increased bybrief pulses of red light, the effects of which were reversedby brief pulses of far-red light. Similarly, increases in therates of oxidation of citrate and -ketoglutarate were observedin mitochondria isolated from illuminated cotyledons. By contrast,rates of succinate-, exogenous NADH-, durohydroquinone- andascor-bate-dependent consumption of O₂ were little affectedby the light treatment. Exogenous NAD⁺stimulated the oxidationof malate and -ketoglutarate by mitochondria from dark-grown,control cotyledons, while it did not enhance the rates of oxidationof these substrates by mitochondria isolated from light-treatedcotyledons. The activities of mitochondrial enzymes (malateand succinate dehydrogenase, NAD⁺-malic enzyme and cytochromeoxidase) were not different between illuminated and dark-growncotyledons. Neither cycloheximide nor chloramphenicol inhibitedthe increase in the rate of oxidation of malate. (Received October 24, 1988; Accepted May 24, 1989)     

Phytochrome Control of Another Phytochrome-mediated Process

The phytochrome-mediated attachment of root tips of mung bean (Phaseolus aureus) and barley (Hordeum vulgare) to glass is affected by the prior exposure of hydrated seeds or seedlings to red or far red radiation. Prior irradiation of seeds or seedlings of mung bean with red light promotes attachment, while far red light promotes detachment of root tips. Similar exposure of barley seeds and seedlings to red light accentuates detachment, while far red light accentuates attachment of root tips. Red-far red light reversibility of the pretreatments indicates phytochrome control.