In Vivo Properties of Membrane-bound Phytochrome

After a 3-minute irradiation with red light, which saturates the phototransformation from the red light-absorbing form of phytochrome to the far red light absorbing form of phytochrome, about 40% of the phytochrome extractable from hooks of etiolated squash seedlings (Cucurbita pepo L. cv. Black Beauty) can be pelleted as Pfr at 17,000g after 30 minutes. Dark controls yield only 2 to 4% pelletable phytochrome in the form Pr. If a dark period intervenes between red irradiation and extraction, the bound Pfr gradually loses its photoreversibility. The time course for this destruction parallels the time course for phytochrome destruction in vivo following saturating red irradiation. The soluble fraction of phytochrome remains constant. These results suggest that in squash seedlings phytochrome destruction is related exclusively to the fraction which becomes membrane-bound. The induction of phytochrome binding by red light is not completely reversible by far red. In plants given saturating red followed immediately by saturating far red light, 12% of the phytochrome is found in the bound fraction as Pr if the phytochrome extraction is immediate. If a dark period intervenes between red-far red treatment and extraction, the bound phytochrome is released within 2 hours. A model of the binding properties of phytochrome, based on molecular interaction at the membrane is proposed, and possible consequences for the mechanism of action of phytochrome are discussed.     

Photoregulation of asymmetric cell division followed by rhizoid development in the fern Ceratopteris prothalli

Strap-shaped prothalli of CERATOPTERIS: richardii grown in the dark have an apical meristem, a subapical elongation zone and a basal growth cessation zone [Murata et al. (1997) Plant Cell Physiol. 38: 201]. When the dark-grown prothalli were irradiated with continuous white light, marginal cells of the elongation zone divided asymmetrically, and the resulting smaller cells developed into rhizoids. The asymmetric division was also induced by brief irradiation of red light. The effect of red light was cancelled by subsequent irradiation of far-red light, indicating that the asymmetric division was regulated by phytochrome. Since the response to red light was not observed at 10(1) J m(-2) and saturated at 10(2) J m(-2) and the response is photoreversible by far-red light, the photoresponse was classified as a low-fluence response of phytochrome. Although the asymmetric division was induced by brief irradiation of red light, continuous irradiation of white, blue or red light was necessary to induce rhizoid growth. These results indicate that asymmetric division and subsequent cell growth are independently regulated by light in CERATOPTERIS: prothalli.     

Inhibition of Prechill-induced Dark Germination in Sorghum halepense (L.) Pers. Seeds by Phytochrome Transformations

A 10 C dark prechilling of johnsongrass [Sorghum halepense (L.) Pers.] seeds, when terminated by a 2-hr, 40 C temperature shift, potentiates about 40% germination at 20 C in darkness. Irradiation of the seeds before, during, and at the end of prechilling with far red light reduces the subsequent germination, although red irradiation after the far red can overcome some of the inhibition. However, either brief red or far red irradiation given immediately after the temperature shift inhibits subsequent germination by one-third to one-half. The results suggest that the far red-absorbing form of phytochrome is a factor in the prechill-induced dark germination and that phytochrome participates in the inhibition of germination by irradiations immediately after the temperature shift.     

Amaranthin accumulation in continuous red and blue light by seedlings ofAmaranthus caudatus L.

Four days oldAmaranthus seedlings responded to light treatment with an increase of amaranthin accumulation. With increasing irradiation time, red light caused a saturation effect. Blue light induced a high irradiation response. The blue light effect was reversible to a certain extent by far-red irradiation given at the end of the treatment with blue light. Intermittent red light (3 h red light, 3 h dark, …) caused a higher amaranthin accumulation than 24 h continuous red light. Results obtained with red and blue light are discussed on the basis of the phytochrome system.     

Phytochrome, nitrate movement, and induction of nitrate reductase in etiolated pea terminal buds

The role of phytochrome in the induction of nitrate reductase of etiolated field peas (Pisum arvense L.) was examined. Terminal bud nitrate concentration increased in darkness, and the increase correlated with induction of nitrate reductase following brief exposure of intact plants to red, blue, far red, and white lights. Brief light exposure of intact plants stimulated nitrate uptake and induction of nitrate reductase by terminal buds subsequently excised and incubated on nitrate solution in darkness; exposure of excised buds in contact with nitrate led to less uptake but more induction. Nitrate and nitrate reductase activity both declined during incubation with water, irrespective of light treatment. Nitrate enrichment of intact terminal buds and uptake into excised buds and increases in nitrate reductase activity were all red/far red reversible. Dimethyl sulfoxide (1%, v/v) and sugars (sucrose 0.5%, glucose 1, w/v), although stimulating nitrate uptake into excised tissue in darkness, failed to enhance nitrate reductase activity over dark controls. Phytochrome may regulate nitrate reductase via both nitrate movement and a general mechanism such as enhancement of protein synthesis.     

Model for variable light sensitivity in imbibed dark-dormant seeds

The level of light-induced germination of the seed of common purslane (Portulaca oleracea L.) and curly dock (Rumex crispus L.) changes with dark incubation time prior to brief, low energy, red light treatment. The rate at which phytochrome—far red-absorbing form (Pfr) acts in the light-induced population of seeds was measured by quantitating per cent reversals of the red light effect with saturating far red light exposures at successive times after the red light exposure. A linear positive correlation was found between this rate and the final germination level. These results are compatible with a model involving changing levels, during dark incubation, of a component with which Pfr interacts. In this model, germination is initiated after attainment of a certain level of interaction between Pfr and this component. These findings also support the view that the Pfr to Pr decay rate constant and total phytochrome level are stable during dark incubation.     

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.     

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.     

Phytochrome involvement in stimulation and alleviation of inhibition of plant growth by malformin

Stimulation of stem elongation on green cuttings of Phaseolus aureus by malformin occurred only in red light and was specifically reversible by subsequent treatment with far red radiation. Inhibition of stem elongation of etiolated cuttings by malformin in the dark was alleviated by red light and was repeatedly reversible with far red irradiation. A direct or indirect effect of malformin on phytochrome action was suggested.     

Blue Light Interference in the Phytochrome-controlled Germination of the Spores of Cheilanthes farinosa

Short exposure of the spores of Cheilanthes farinosa to low intensity red light promotes their germination, which is not reversed by a subsequent exposure to far red light. Germination is, however, inhibited by blue light administered before or after red light. Inhibition of germination by blue light is annulled by exposure to a higher intensity of red light, and germination of the repromoted spores is inhibited by far red light. Mutual photoreversibility of germination is also observed in repromoted spores irradiated successively with far red and red light. Although germination appears to be basically under phytochrome control, it is postulated that the presence of a blue light-absorbing pigment interferes with phytochrome transformations in the spores.     

Phytochrome A regulates the intracellular distribution of phototropin 1-green fluorescent protein in Arabidopsis thaliana

It has been known for decades that red light pretreatment has complex effects on subsequent phototropic sensitivity of etiolated seedlings. Here, we demonstrate that brief pulses of red light given 2 h prior to phototropic induction by low fluence rates of blue light prevent the blue light-induced loss of green fluorescent protein-tagged phototropin 1 (PHOT1-GFP) from the plasma membrane of cortical cells of transgenic seedlings of Arabidopsis thaliana expressing PHOT1-GFP in a phot1-5 null mutant background. This red light effect is mediated by phytochrome A and requires approximately 2 h in the dark at room temperature to go to completion. It is fully far red reversible and shows escape from photoreversibility following 30 min of subsequent darkness. Red light-induced inhibition of blue light-inducible changes in the subcellular distribution of PHOT1-GFP is only observed in rapidly elongating regions of the hypocotyl. It is absent in hook tissues and in mature cells below the elongation zone. We hypothesize that red light-induced retention of the PHOT1-GFP on the plasma membrane may account for the red light-induced increase in phototropic sensitivity to low fluence rates of blue light.     

In vivo phytochrome reversion in immature tissue of the alaska pea seedling

Reversion of far red-absorbing phytochrome to red-absorbing phytochrome without phytochrome destruction (that is, without loss of absorbancy and photoreversibility) occurs in the following tissues of etiolated Alaska pea seedlings (Pisum sativum L.): young radicles (24 hours after start of imbibition), young epicotyls (48 hours after start of imbibition), and the juvenile region of the epicotyl immediately subjacent to the plumule in older epicotyls. Reversion occurs rapidly in the dark during the first 30 minutes following initial phototransformation of red-absorbing phytochrome to far red-absorbing phytochrome. If these tissues are illuminated continuously with red light for 30 minutes, the total amount of phytochrome remains unchanged. Beyond 30 minutes after a single phototransformation or after the start of continuous red irradiation, phytochrome destruction commences. In young radicles, sodium azide inhibits this destruction, but does not affect reversion. In older tissues in which far red-absorbing phytochrome destruction begins immediately upon phototransformation, strong evidence for simultaneous far red-absorbing phytochrome reversion is obtained from comparison of far red-absorbing phytochrome loss in the dark following a single phototransformation with far red-absorbing phytochrome loss under continuous red light.     

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.     

Far-Red Reversal of Red Light Effect during Long-Night Induction of Potato (Solanum tuberosum L.) Tuberization

The hypothesis that phytochrome is involved in the regulation of potato (Solanum tuberosum L.) tuberization was tested. When 5 minutes of red light were given in the middle of the 16-hour dark period to which whole plants were exposed daily for 14 days before making cuttings, the percentage of tuberization on cuttings decreased. The effect of red light was significantly reversed by 2 minutes of far-red light given immediately after the red in each of two separate experiments. This supports the hypothesis that phytochrome is at least indirectly involved.     

Ultraviolet action spectrum for anthocyanin formation in broom sorghum first internodes

An action spectrum for anthocyanin formation in dark-grown broom sorghum (Sorghum bicolor Moench, cv Acme Broomcorn and cv Sekishokuzairai Fukuyama Broomcorn) seedlings was determined over the wavelength range from 260 to 735 nanometers. The action peaks were at 290, 650, 385, and 480 nanometers in descending order of height. The action of the 290-nanometer peak was not affected by subsequently given far red light, whereas those of the other three action peaks were nullified completely. The nullification of the 385-nanometer peak action by far red light was reversible. When an irradiation at these action peaks was followed by a phytochrome-saturating fluence of red light irradiation, the action of the 290-nanometer peak remained, whereas that of the 385-nanometer peak as well as those of the 650- and 480-nanometer peaks was masked by the action of the second irradiation. These findings suggested that the 290- and 385-nanometer action peaks involved different photoreceptors, the latter being phytochrome. The blue light-absorbing photoreceptor as reported to be a prerequisite for phytochrome action in milo sorghum was not found to exist in the broom sorghums.

The action spectrum deprived of the involvement of phytochrome was determined in the ultraviolet region by irradiating with far red light following monochromatic ultraviolet light. The spectrum had a single intense peak at 290 nanometers and no action at all at wavelengths longer than 350 nanometers.

     

Phytochrome Action in Oryza sativa L: IV. Red and Far Red Reversible Effect on the Production of Ethylene in Excised Coleoptiles

Excised apical segments of etiolated rice (Oryza sativa L.) coleoptiles produced ethylene. Increasing the number of cut sites per coleoptile increased the rate of ethylene formation. Ethylene produced by an etiolated-intact seedling in the dark was about a half of that by the excised coleoptile segment. Red light of low energy as well as of continuous irradiation inhibited the production of ethylene. The inhibition by a low energy dose of red light was partly relieved, if the red light was followed immediately by a small dose of far red light. The effect of red and far red light was repeatedly reversible, indicating that ethylene production was regulated by a phytochrome system. If the exposure to far red light was preceded by a period of darkness, this photoreversibility disappeared; 50% of the initial reversibility was lost within 5 hours. Applied ethylene (10 microliters per liter) significantly promoted the growth of intact coleoptiles of either totally etiolated or red light-treated seedlings, but had no effect on the excised apical segment of coleoptile.     

Involvement of phytochrome and a blue light photoreceptor in UV-B induced flavonoid synthesis in parsley (Petroselinum hortense Hoffm.) cell suspension cultures

Flavonoid synthesis in cell suspension cultures of parsley (Petroselinum hortense Hoffm.) occurs only after irradiation with ultraviolet light (UV), mainly from the UV-B (280–320 nm) spectral range. However, it is also controlled by phytochrome. A P_fr/P_tot ratio of approximately 20% is sufficient for a maximum phytochrome response as induced by pulse irradiation. Continuous red and far red light, as well as blue light, given after UV, are more effective than pulse irradiations. The response to blue light is considerably greater than that to red and far red light. Continuous red and blue light treatments can be substituted for by multiple pulses and can thus probably be ascribed to a multible induction effect. Continuous irradiations with red, far red and blue light also increase the UV-induced flavonoid synthesis if given before UV. The data indicate that besides phytochrome a separate blue light photoreceptor is involved in the regulation of the UV-induced flavonoid synthesis. This blue light receptor seems to require the presence of P_fr in order to be fully effective.Abbreviations HIR high irradiance response - P_fr far red absorhing form of phytochrome - P_tet total phytochrome - UV ultraviolet light     

Photocontrol of anthocyanin formation in turnip seedlings

Summary The participation of the red/far-red reversible reaction of phytochrome in the control of anthocyanin formation in turnip seedlings has been demonstrated. A brief exposure to red light following a preliminary irradiation period in blue, increased anthocyanin content compared with blue alone; this effect was reversed by a subsequent short exposure to far-red. The sensitivity to red light was largely restricted to 24 hours old seedlings when grown in water at 25°C. Sensitivity was restored in older seedlings when they were grown in phenylalanine and kept in high temperature (35°C) for several hours before light was given; under these conditions, the phytochrome effect was greater in 48 hours old than in 24 hours old seedlings. In the youngest seedlings the largest increase occurred when red followed a preliminary blue exposure of at least 12 hours; in older seedlings the maximum response to red was almost attained after only 4 hours of blue light. Hypocotyl elongation was shown to be hardly affected by the reversible reaction of phytochrome. Possible reasons for these changes in sensitivity to phytochrome are discussed.

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Zusammenfassung Die Beteiligung des reversiblen Hellrot-Dunkelrot-Pigmentsystems Phytochrom wurde bei der Kontrolle der Anthocyansynthese in Keimlingen von Brassica rapa nachgewiesen. Durch kurze Hellrotbestrahlung wird der Anthocyangehalt gegenüber dem der vorausgehenden Bestrahlung mit Blaulicht erhöht. Dieser Effekt wird durch eine nachfolgende kurze Bestrahlung mit Dunkelrot wieder mehr oder weniger aufgehoben. Die Empfindlichkeit gegen Rotlicht ist weitgehend auf 24 Std alte, in Wasser gewachsene Keimlinge beschränkt. Die Empfindlichkeit konnte in älteren Keimlingen wieder hergestellt werden, wenn diese in Phenylalanin aufgezogen und mehrere Stunden vor der Bestrahlung bei hoher Temperatur (35°C) gehalten wurden. Unter diesen Bedingungen war der Phytochrom-Effekt in 48 Std alten Keimlingen größer als in 24 Std alten Keimlingen. In den jüngsten Keimpflanzen wurde die größte Zunahme durch Hellrot erzielt, wenn wenigstens 12 Std mit Blaulicht vorbestrahlt worden war. In älteren Keimlingen wurde die maximale Hellrotwirkung schon nach etwa vierstündiger Vorbelichtung erreicht. Das Hypokotylwachstum war durch das Phytochromsystem kaum beeinflußbar. Mögliche Gründe für die Veränderungen in der Phytochrom-Empfindlichkeit werden diskutiert.

     

Photocontrol of stem elongation in light-grown plants of Fuchsia hybrida

Stems of the caulescent long-day plant, Fuchsia hybrida cv Lord Byron, showed 2 types of response to light. In one, internode length was increased by far-red irradiation given at the end of an 8 h photoperiod: the response was no greater with prolonged exposure and was less when the start of far-red was delayed. The effect of far-red was reversible by a subsequent exposure to red light. Internode length was inversely proportional to the P_fr/P ratio established before entry to darkness and there was no evidence for loss of P_fr during a 16 h dark period. The inhibitory effect of P_fr acted at a relatively late stage of internode growth. With the development of successive internodes a second response appeared in which stems lengthened following prolonged daily exposures to red or far-red light, or mixtures of the two, or to brief breaks with red or white light. In these later internodes, a short exposure to far-red near the middle of the night was not reversible by red because red alone promoted elongation at this time. Internode length increased with increase in the daily duration of light and, when light was given throughout an otherwise dark period of 16 h, with increase in illuminance to a saturation value of 200 lx from tungsten lamps. Elongation increased as a linear function of decrease in photostationary state of phytochrome down to P_fr/P0.3; however, internodes were shorter in far-red light than in 25% red/red+far-red. It was concluded that stem length is a net response to two modes of phytochrome action. An inductive effect of P_fr inhibits a late stage in internode expansion, and a phytochrome reaction which operates only in light (and may involve pigment cycling) promotes an early stage of internode development. Stem elongation is thus a function both of the daily duration of light and its red/red+far-red content. The outgrowth of axillary buds was controlled by the first type of phytochrome action only.Abbreviations and symbols FR far red light - R red light - P phytochrome - P_fr phytochrome in the far-red light absorbing form - SD 8 h short days - LDP long-day plant - SDP short-day plant     

Phytochrome Modifies Blue-light-induced Electrical Changes in Corn Coleoptiles

Unilateral blue light administered to corn coleoptile segments produces no alteration of transmembrane potential on the light side, and only a small and slow hyperpolarization on the dark side. Red light causes a 5-15 millivolt depolarization in cells on the light side causes and somewhat smaller effects on the dark side. Blue given after red causes a rapid hyperpolarization on both sides of the coleoptile. The effect of the potentiating red preirradiation is probably due to phytochrome, being largely abolished by far-red given after red, but before the blue light. The effect of prior red irradiation decays in the dark, showing a half-time of about 45 minutes at room temperature. This rapid cooperativity between phytochrome and the phototropic pigment may indicate a common locale, possibly in a membrane.